BTK protein degradation agent with silicon-containing group as hydrophobic label as well as preparation method, pharmaceutical composition and application of BTK protein degradation agent

Abstract

The invention belongs to the field of chemical drugs, and discloses a BTK protein degradation agent taking a silicon-containing group as a hydrophobic label, and a preparation method, a pharmaceutical composition and application thereof. The structure of the BTK protein degradation agent with the silicon-containing group as the hydrophobic label is shown in a formula I, and linker is a saturated aliphatic chain, an unsaturated aliphatic chain, a connecting structure composed of an aromatic ring and a saturated aliphatic chain or a connecting structure composed of an aromatic ring and an unsaturated aliphatic chain; r is a specific silicon-containing group as a hydrophobic label. The BTK protein degradation agent with the silicon-containing group as the hydrophobic label is obtained, the preparation process is simple and easy to implement, and the obtained BTK protein degradation agent has high liver microsome metabolism stability and considerable oral bioavailability, shows an efficient treatment effect in an in-vivo experiment, has a certain inhibition effect on proliferation of various tumor cells, and has a good application prospect. The compound is suitable for developing drugs for treating leukemia and other cancers. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicines, and specifically relates to a BTK protein degrader with a silicon-containing group as a hydrophobic tag, a preparation method thereof, a pharmaceutical composition and application thereof. Background Art

[0002] Bruton's tyrosine kinase (BTK) is a member of the TEC family of intracytoplasmic non-receptor tyrosine kinases. It plays an important role in the growth, development, proliferation and differentiation of B cells, and is also a therapeutic target for B cell malignancies. Although mainstream BTK inhibitors (ibrutinib, acalabrutinib, zanubrutinib) have excellent clinical therapeutic effects, like other targeted drugs, acquired drug resistance is gradually shortening the application range of this type of molecules, especially mutations in the key site C481. The development of BTK degraders may be a solution.

[0003] The hydrophobic tag (Hyt) bifunctional molecule consists of three parts: target protein ligand, linker, and hydrophobic group. By connecting a large and hydrophobic group to a small molecule that can bind to the target, such a two-headed molecule will be mistakenly identified as a misfolded part of the target protein by the intracellular protein repair mechanism after binding to the target, and then it can be folded by the chaperone protein and then degraded by the proteasome. The hydrophobic group in the Hyt molecule often has a small molecular weight, so it may have higher solubility and drugability. At present, the research and development of degraders based on hydrophobic tags is still in the exploratory stage. On the one hand, there are few reported hydrophobic tag fragments, and there is still a lot of room for optimization in degradation activity and physicochemical properties. On the other hand, the exact degradation mechanism has not yet been clarified. Therefore, it is very important to explore more hydrophobic fragments with high activity and excellent physicochemical properties and clarify their corresponding degradation mechanisms for the development of hydrophobic tag fragments towards clinical applications.

[0004] The hydrophobic fragments used in the hydrophobic tag degraders that have been reported so far include adamantane, menthol, norbornene, Boc arginine, carborane, fluorenyl, etc. However, the degradation effects of these conventional hydrophobic tag degraders are relatively low, their metabolic stability in liver microsomes is poor, and their solubility is poor and difficult to be taken orally, making them difficult to be used for further clinical treatment and development. The development of new hydrophobic tag degraders with better liver microsome stability and oral administration is needed to meet clinical needs. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide a BTK protein degrader with a silicon-containing group as a hydrophobic tag, which has high metabolic stability in liver microsomes and can be orally administered, and a preparation method, a pharmaceutical composition and application thereof. Based on this, the first aspect of the present invention provides a BTK protein degrader with a silicon-containing group as a hydrophobic tag, and its structure is shown in Formula I:

[0006]

[0007] Wherein, linker is a saturated fatty chain, an unsaturated fatty chain, a linking structure consisting of an aromatic ring and a saturated fatty chain, or a linking structure consisting of an aromatic ring and an unsaturated fatty chain; R is trimethylsilyl, triethylsilyl, tri-n-propylsilyl, triisopropylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, cyclohexyldimethylsilyl, vinyldimethylsilyl, trimethoxysilyl, triethoxysilyl, phenylvinylmethylsilyl or triphenylsilyl.

[0008] The present invention provides a BTK protein degrader with a silicon-containing group as a hydrophobic tag. In a bifunctional molecule, the silicon-containing group is used as a hydrophobic tag, and the ibrutinib core is used as a target protein ligand, thereby obtaining a protein degrader that can effectively degrade BTK. The BTK protein degrader prepared by the present invention can effectively induce the degradation of BTK in MOLM-13 (human acute myeloid leukemia cells) in a dose-dependent and time-dependent manner, has considerable liver microsome stability and bioavailability, and exhibits excellent in vivo anticancer effects.

[0009] Among them, Linker is a saturated fatty chain, an unsaturated fatty chain, a connection structure composed of an aromatic ring and a saturated fatty chain, or a connection structure composed of an aromatic ring and an unsaturated fatty chain. By selecting ideal linkers of different types and lengths, the purpose of not affecting the binding of the two proteins in space and maintaining their binding is achieved. Based on this, in some more preferred implementations, the structure of the BTK protein degrader with a silicon-containing group as a hydrophobic tag is shown in Formula II or Formula III:

[0010]

[0011] wherein m and n are independently selected from integers in the range of 1-10.

[0012] Further preferably, the structure of the BTK protein degrader with a silicon-containing group as a hydrophobic tag is as shown in Formula IV:

[0013]

[0014] The compound shown in Formula IV can effectively induce the degradation of BTK in human B lymphocytoma Ramos cell line and human diffuse large B lymphoma TMD8 cell line in a dose-dependent and time-dependent manner, has considerable liver microsomal stability and bioavailability, and exhibits excellent in vivo anticancer effects.

[0015] The second aspect of the present invention provides a method for preparing the above-mentioned BTK protein degrader with a silicon-containing group as a hydrophobic tag. When the structure of the above-mentioned BTK protein degrader with a silicon-containing group as a hydrophobic tag is shown in Formula II, its preparation method is shown in Scheme 1; when the structure of the above-mentioned BTK protein degrader with a silicon-containing group as a hydrophobic tag is shown in Formula III, its preparation method is shown in Scheme 2;

[0016] Route 1: tert-Butyldiphenylsilyl chloride (compound B) and azidoethanol (compound A) are reacted with imidazole to obtain intermediate C. The ibrutinib core (compound D) and acetylenic acids of different lengths are condensed with N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate and N,N-diisopropylethylamine to obtain intermediates E1-E6. Intermediate C undergoes click reaction with intermediates E1-E6 to obtain final products 1-6.

[0017]

[0018] Route 2: tert-Butyldiphenylsilyl chloride (compound B) and bromohydrins of different lengths (compounds F1-F4) are reacted with imidazole to obtain intermediates G1-G4, which are then substituted with compound D in the presence of cesium carbonate to obtain the final products 7-10.

[0019]

[0020] The preparation method of the invention has a simple route, the raw materials used are cheap and easily available, and the overall reaction yield is high.

[0021] The BTK protein degrader or a pharmaceutically acceptable salt thereof using a silicon-containing group as a hydrophobic tag can be used to prepare a drug for treating related cancers with BTK abnormalities. The above-mentioned related cancers include: leukemia, lung cancer, gastric cancer, breast cancer, colorectal cancer, and pancreatic cancer. More preferably, the related cancer is leukemia.

[0022] The third aspect of the present invention provides a pharmaceutical composition, including the above-mentioned BTK protein degrader with a silicon-containing group as a hydrophobic tag or a pharmaceutically acceptable salt thereof (or, the above-mentioned BTK protein degrader with a silicon-containing group as a hydrophobic tag or a pharmaceutically acceptable salt thereof as the main active ingredient). In some preferred implementations, the above-mentioned pharmaceutical composition may also include at least one of an excipient, a solvent and a pharmaceutical carrier. Among them, the excipient is at least one of gum arabic, syrup, lanolin and starch; it has no incompatibility with the main drug, does not produce side effects, does not affect the efficacy, is not easy to deform, crack, mildew, or be worm-eaten at room temperature, is harmless to the human body, has no physiological effect, does not produce chemical or physical effects with the main drug, and does not affect the content determination of the main drug. The solvent can be water, glycerol or ethanol.

[0023] The beneficial effects of the present invention are as follows: the present invention obtains a BTK protein degrader with a silicon-containing group as a hydrophobic tag, the preparation process is simple and easy, the obtained BTK protein degrader has high liver microsome metabolic stability, has considerable oral bioavailability, shows a high therapeutic effect in in vivo experiments, has a certain inhibitory effect on the proliferation of various tumor cells, and is suitable for the development of cancer drugs for the treatment of leukemia and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The graphs show the concentration-dependent degradation of BTK in MOLM-13 cell line by compound 2; (A) is a Western Blot graph, (B) is a DC 50 picture;

[0025] Figure 2 Shown is the time-dependent degradation effect of compound 2 on BTK in the MOLM-13 cell line. DETAILED DESCRIPTION

[0026] The following will be combined with the embodiments and drawings to clearly and completely describe the concept and technical effects of the present invention, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0027] Example 1

[0028] A BTK protein degrader (denoted as compound 1-6) with a silicon-containing group as a hydrophobic tag, and its structures are shown below:

[0029]

[0030]

[0031] The structures of the intermediate compound A, compound B, compound C, compound D, compound acetylic acid, and compounds E1-E6 used are shown below:

[0032]

[0033] The specific preparation method is:

[0034] 1) Preparation of compound C:

[0035] Under an ice bath, compound A (174 mg, 2.00 mmol) was dissolved in dichloromethane (12 mL), and compound B (825 mg, 3.00 mmol) and imidazole (408 mg, 6.00 mmol) were added respectively. The ice bath was removed after 30 minutes, and the mixture was stirred at room temperature overnight. The solid was filtered out, and the remaining organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel flash column chromatography (petroleum ether: ethyl acetate = 60:1) to obtain compound C.

[0036] Compound C was tested, and the test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.72–7.66(m,4H),7.41(tt,J=8.1,5.7Hz,6H),3.81(dd,J=5.5,4.6Hz,2H),3.29(t,J=5.0Hz,2H),1.07(s,9H). 13 C NMR (100MHz, CDCl3) δ135.6,133.1,129.9,127.8,63.3,53.2,26.7,19.1.

[0037] 2) Preparation of Compounds E1-E6:

[0038] Preparation of compound E1:

[0039] Compound 3-butynoic acid (168 mg, 1.50 mmol) was dissolved in N,N-dimethylformamide (3 mL), NN-diisopropylethylamine (330 μL, 2.00 mmol) was added, the reaction system was cooled to 0°C, and then N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (570 mg, 1.50 mmol) was added. Stirring was carried out at 0°C for 10 minutes, and then compound D (463 mg, 1.20 mmol) was added, and the reaction was allowed to proceed overnight. After the reaction was completed, the mixture was extracted with ethyl acetate (3×10 mL), and the organic phases were combined and washed with saturated sodium bicarbonate solution (10 mL), 1N hydrochloric acid solution (10 mL), and saturated NaCl solution (10 mL), respectively. The organic phase was dried with anhydrous sodium sulfate and concentrated, and the obtained crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain compound E1.

[0040] Compound E1 was tested, and the test results were as follows: 1 H NMR(400MHz, CDCl3)δ8.30(s,1H),7.83–7.70(m,2H),7.41–7.29(m,2H),7.25(d,J=8.1Hz,1H),7.19(d,J=8.0Hz,1H),7.15–7.06(m,4H),7.03– 6.96(m,3H),4.88(dt,J=5.4,2.7Hz,1H),3.88(d,J=2.8Hz,2H),3.65–3 .44(m,5H),2.26(t,J=3.0Hz,1H),2.18–1.99(m,1H),2.00–1.82(m,4H).

[0042] Preparation of compound E2:

[0043] According to the same method as that for preparing compound E1, compound E2 was obtained by replacing compound 3-butynoic acid with compound 4-pentynoic acid (147 mg, 1.50 mmol).

[0045] Compound E2 was tested, and the test results were as follows: 1H NMR (400MHz, CDCl3) δ8.34(d,J=13.5Hz,1H),7.63(d,J=8.1Hz,2H),7.38(t,J=7.4Hz, 2H),7.16(t,J=6.9Hz,3H),7.08(d,J=7.7Hz,2H),5.81(s,2H),4.90–4.50(m,2H),4.0 8–3.68(m,1H),3.74–3,26(m,1H),2.97(dt,J=144.1,12.6Hz,1H),2.70–2.53(m,3H), 2.36(dt,J=19.4,10.1Hz,1H),2.24(s,1H),2.00(d,J=20.4Hz,3H),1.78–1.62(m,1H).

[0047] Preparation of compound E3:

[0048] According to the same method as that for preparing compound E1, compound E3 was obtained by replacing compound 3-butynoic acid with compound 5-hexynoic acid (168 mg, 1.50 mmol).

[0050] Compound E3 was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ8.35(d,J=9.4Hz,1H),7.64(dd,J=8.6,3.0Hz,2H),7.47–7.33(m,2H),7.16(ddd,J=12.1,8.0,2.9Hz,3H),7.08(d,J= 8.1Hz,2H),5.69(s,2H),4.98–4.45(m,2H),4.15–3.84(m,1H),3.72– 3.25(m,1H),3.19–2.74(m,1H),2.69–2.17(m,5H),2.06–1.78(m,6H).

[0051] Preparation of compound E4:

[0052] According to the same method as that for preparing compound E1, compound E4 was obtained by replacing compound 3-butynoic acid with compound 6-heptynoic acid (189 mg, 1.50 mmol).

[0053] Compound E4 was tested, and the test results are as follows: 1H NMR (400MHz, CDCl3) δ8.35(d,J=11.1Hz,1H),7.64(dd,J=8.7,2.9Hz,2H),7.46–7.33(m,2H),7.15( dt,J=8.4,3.5Hz,3H),7.08(d,J=8.0Hz,2H),5.71(s,2H),4.99–4.49(m,2H),4.17–3.82(m,1H),3. 78–3.22(m,1H),3.18–2.72(m,1H),2.40(tq,J=11.7,4.4,3.8Hz,2H),2.35–2.28(m,1H),2.21(dtd ,J=13.9,7.1,2.7Hz,3H),1.97–1.88(m,3H),1.77(tdd,J=12.7,8.2,5.9Hz,2H),1.65–1.51(m,2H).

[0054] Preparation of compound E5:

[0055] Compound E5 was obtained by the same method as that for preparing compound E1 except that compound 3-butynoic acid was replaced by compound 7-octynoic acid (210 mg, 1.50 mmol).

[0056] Compound E5 was tested, and the test results are as follows: 1 H NMR(400MHz, CDCl3)δ8.32(d,J=18.0Hz,1H),7.67–7.59(m,2H),7.37(t,J=7.3H z,2H),7.14(d,J=6.0Hz,3H),7.07(d,J=7.8Hz,2H),5.99(s,2H),4.83–4.57(m,2 H),4.06–3.86(m,1H),3.67–3.22(m,1H),3.15–2.70(m,1H),2.47–2.27(m,3H), 2.27–2.13(m,3H),1.93(d,J=22.2Hz,2H),1.72–1.61(m,3H),1.59–1.36(m,4H).

[0057] Preparation of compound E6:

[0058] According to the same method as that for preparing compound E1, compound E6 was obtained by replacing compound 3-butynoic acid with compound 8-nonynoic acid (231 mg, 1.50 mmol).

[0059] Compound E6 was tested, and the test results are as follows: 1H NMR(400MHz, CDCl3)δ8.30(s,1H),7.91–7.56(m,2H),7.41–7.31(m,2H),7.25(d,J=8.1Hz,1H ),7.19(d,J=8.0Hz,1H),7.16–7.06(m,4H),7.04–6.98(m,3H),4.91(dt,J=5.4,2.7Hz,1H),3 .82(d,J=2.7Hz,2H),3.64–3.41(m,3H),2.38(t,J=7.9Hz,3H),2.12(td,J=6.1,3.1Hz,3H),2 .08–1.99(m,2H),2.00–1.86(m,4H),1.63–1.52(m,2H),1.52–1.41(m,3H),1.42–1.31(m,4H).

[0060] 3) Preparation of compound 1-6:

[0061] Preparation of compound 1:

[0062] E1 (104 mg, 0.230 mmol) and compound C (91.1 mg, 0.280 mmol) were dissolved in a mixed solvent of t-BuOH / H2O (5 mL, 1:1), and CuSO4·5H2O (18.6 mg, 0.116 mmol) and sodium L-ascorbate (38.4 mg, 0.194 mmol) were added. The mixture was reacted at room temperature overnight. After the reaction was completed, CuSO4·5H2O was filtered out, and the filtrate was concentrated in vacuo and separated by silica gel flash column chromatography (dichloromethane: methanol = 50:1) to obtain compound 1.

[0063] Compound 1 was tested, and the test results were as follows: 1 H NMR(400MHz, CDCl3)δ8.30(s,1H),7.84–7.75(m,3H),7.65(s,1H),7.64–7.58(m,5H),7.41 –7.32(m,11H),7.25(d,J=8.1Hz,1H),7.19(d,J=8.1Hz,1H),7.15–7.07(m,4H),7.03–6.95 (m,3H),4.90(dt,J=5.6,2.7Hz,1H),4.31(td,J=4.1,1.1Hz,3H),4.18(t,J=4.1Hz,3H),3. 90–3.77(m,3H),3.67(d,J=2.4Hz,3H),3.56–3.45(m,2H),2.16–1.82(m,5H),1.03(s,9H).

[0065] Preparation of compound 2:

[0066] Compound 2 was obtained by the same method as that for preparing compound 1 except that compound E1 was replaced by compound E2 (107 mg, 0.230 mmol).

[0067] Compound 2 was tested, and the test results were as follows: 1 H NMR (400MHz, CDCl3) δ8.34(s,1H),7.63(d,J=8.2Hz,2H),7.51(q,J=7.6,5.8Hz,4H),7.38(dp, J=15.6,7.2Hz,9H),7.16(dd,J=13.2,7.7Hz,3H),7.08(d,J=8.0Hz,2H),5.67(s,2H),4.88–4. 55(m,2H),4.43(dt,J=16.8,5.3Hz,2H),4.14–3.91(m,3H),3.70–3.22(m,1H),3.08(q,J=7.0H z,2H),2.85–2.68(m,2H),2.34–2.17(m,2H),1.95(s,2H),1.66(s,1H),0.99(d,J=14.0Hz,9H). 13 CNMR (100MHz, CDCl3) δ170.6,158.7,157.9,156.4,155.9,155.8,154.3,146.8,135.5,132.8,132.8,130.1,127.9, 124.2,124.2,123.0,122.9,119.7,119.7,119.3,62.8,53.5,52.7,52.4,45.9,33.0,30.4,26.8,25.2,24.2,21.3.

[0071] Preparation of compound 3:

[0072] Compound 3 was obtained by the same method as that for preparing compound 1 except that compound E1 was replaced by compound E3 (111 mg, 0.230 mmol).

[0073] Compound 3 was tested, and the test results were as follows: 1H NMR(400MHz, CDCl3)δ8.35(d,J=7.0Hz,1H),7.63(dd,J=8.6,3.5Hz,2H),7.53–7.47(m,4H),7.44–7 .32(m,9H),7.15(dt,J=8.5,3.3Hz,3H),7.08(d,J=8.0Hz,2H),5.70(s,2H),4.94–4.55(m,2H),4.4 3(dt,J=18.7,5.2Hz,2H),4.12–3.81(m,3H),3.66–3.20(m,1H),3.13–2.65(m,3H),2.43(dp,J=24. 3,9.1,8.2Hz,2H),2.12–1.88(m,5H),1.67(ddt,J=19.6,15.3,7.6Hz,1H),0.99(d,J=11.6Hz,9H). 13 C NMR (100MHz, CDCl3) δ171.3,158.6,158.5,157.9,156.3,155.8,155.6,154.3,147.3,144.1,143.9,135.4,132.7,130 .0,127.9,124.1,124.1,122.3,119.6,119.2,62.7,53.5,52.4,52.3,44.9,41.1,32.6,30.3,26.7,25.2,25.0,19.1.

[0076] Preparation of compound 4:

[0077] Compound 4 was obtained by the same method as that for preparing compound 1 except that compound E1 was replaced by compound E4 (114 mg, 0.230 mmol).

[0078] Compound 4 was tested, and the test results were as follows: 1H NMR (400MHz, CDCl3) δ8.35(d,J=8.8Hz,1H),7.64(dd,J=8.5,3.5Hz,2H),7.51(t,J=7.0Hz,4H),7.4 5–7.33(m,9H),7.16(dd,J=13.2,7.7Hz,3H),7.08(d,J=8.0Hz,2H),5.66(s,2H),4.92–4.52(m,2H) ,4.43(dt,J=13.9,5.2Hz,2H),4.15–3.49(m,4H),3.28–3.11(m,1H),2.75(dt,J=15.4,6.2Hz,2H), 2.39(q,J=8.9,8.3Hz,2H),2.28(dt,J=33.8,8.1Hz,2H),1.88–1.61(m,6H),1.00(d,J=7.4Hz,9H). 13 C NMR (100MHz, CDCl3) δ171.6,158.7,158.6,158.0,157.9,156.4,155.9,155.8,154.4,147.7,135.5,132.8,130.1,128.0 ,124.2,124.2,122.3,122.2,119.7,119.3,62.9,53.6,52.4,50.0,41.8,33.2,30.4,30.1,29.4,26.8,25.6,24.9,19.2.

[0080] Preparation of compound 5:

[0081] Compound 5 was obtained by the same method as that for preparing Compound 1 except that Compound E1 was replaced by Compound E5 (117 mg, 0.230 mmol).

[0082] Compound 5 was tested, and the test results are as follows: 1H NMR (400MHz, CDCl3) δ8.35(d,J=7.7Hz,1H),7.63(dd,J=8.5,3.8Hz,2H),7.50(t,J=7.4Hz,4H),7.42(d,J=14.9Hz,3H),7.3 9–7.30(m,6H),7.15(t,J=6.8Hz,3H),7.07(d,J=8.0Hz,2H),5.79(s,2H),4.87–4.55(m,2H),4.43(dt,J=10.9,5.1Hz,2H), 3.96(dt,J=9.5,5.3Hz,2H),3.87–3.60(m,1H),3.16(dt,J=60.2,12.4Hz,1H),2.76–2.69(m,2H),2.34(t,J=7.5Hz,2H),2. 28–2.18(m,2H),1.94(d,J=13.9Hz,2H),1.69(dt,J=14.9,7.7Hz,5H),1.40(dt,J=22.2,7.6Hz,2H),0.99(d,J=11.4Hz,9H). 13 C NMR (100MHz, CDCl3) δ171.8,158.6,158.5,157.9,156.4,155.8,155.6,154.3,154.1,147.9,144.1,135.4,132.6,130.0,12 7.9,124.1,124.1,122.1,119.6,119.2,62.8,53.5,52.3,49.9,45.7,41.7,33.4,29.4,29.1,26.7,25.5,25.1,24.1,19.1.

[0088] Preparation of compound 6:

[0089] Compound 6 was obtained by replacing Compound E1 with Compound E6 (120 mg, 0.230 mmol) in the same manner as for preparing Compound 1.

[0090] Compound 6 was tested, and the test results were as follows: 1H NMR(400MHz, CDCl3)δ8.30(s,1H),7.83–7.74(m,3H),7.67–7.57(m,5H),7.48(s,1H),7.41–7.30(m,11H),7.25( d,J=8.1Hz,1H),7.19(d,J=8.1Hz,1H),7.15–7.05(m,4H),7.04–6.95(m,3H),4.92(dq,J=5.4,2.8Hz,1H),4.31( td,J=4.2,0.9Hz,3H),4.18(t,J=4.1Hz,3H),3.82(d,J=2.7Hz,2H),3.58–3.33(m,3H),2.66(t,J=8.0Hz,2H),2. 38(t,J=7.9Hz,2H),2.13–1.88(m,5H),1.79(p,J=8.1Hz,3H),1.63–1.51(m,3H),1.48–1.27(m,6H),1.03(s,9H).

[0093] Example 2

[0094] A BTK protein degrader (denoted as compound 7-10) with a silicon-containing group as a hydrophobic tag, the structures of which are shown below:

[0095]

[0096] The structures of the intermediate compounds F1, F2, F3, F4, B, G1, G2, G3, G4 and D are shown below:

[0097]

[0098] The specific preparation method is:

[0099] 1) Preparation of compounds G1-G4:

[0100] Preparation of compound G1:

[0101] According to the same method as that for preparing compound C in Example 1, compound A was replaced with compound F1 (278 mg, 2.00 mmol) to obtain compound G1.

[0102] Compound G1 was tested, and the test results are as follows: 1H NMR (400MHz, CDCl3) δ 7.74–7.49 (m, 4H), 7.46–7.29 (m, 6H), 3.83 (t, J = 5.4Hz, 2H), 3.44 (t, J = 4.9Hz, 2H), 2.03 (p, J = 5.2Hz, 2H), 1.03 (s, 9H).

[0103] Preparation of compound G2:

[0104] According to the same method as that for preparing compound C in Example 1, compound A was replaced with compound F2 (306 mg, 2.00 mmol) to obtain compound G2.

[0105] Compound G2 was tested, and the test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.77–7.51(m,4H),7.50–7.24(m,6H),3.71(t,J=5.9Hz,2H),3.44(t,J=4.5Hz,2H),1.98–1.55(m,4H),1.03(s,9H).

[0106] Preparation of compound G3:

[0107] According to the same method as that for preparing compound C in Example 1, compound A was replaced with compound F3 (334 mg, 2.00 mmol) to obtain compound G3.

[0108] Compound G3 was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ7.75–7.54(m,4H),7.50–7.27(m,6H),3.68(t,J=6.0Hz,2H),3.45(t,J =4.6Hz,2H),1.87–1.67(m,2H),1.64–1.56(m,2H),1.52(qd,J=7.0,1.2Hz,2H),1.03(s,9H).

[0109] Preparation of compound G4:

[0110] According to the same method as that for preparing compound C in Example 1, compound A was replaced with compound F4 (362 mg, 2.00 mmol) to obtain compound G4.

[0111] Compound G4 was tested, and the test results are as follows: 1H NMR (400MHz, CDCl3) δ7.84–7.57(m,4H),7.51–7.23(m,6H),3.68(t,J=6.0Hz,2H),3.45(t,J=4.6Hz, 2H), 1.84 (tt, J=7.6, 4.6Hz, 2H), 1.57 (dtd, J=7.1, 6.5, 5.8Hz, 2H), 1.50–1.26 (m, 4H), 1.03 (s, 9H).

[0112] 2) Preparation of compound 7-10:

[0113] Preparation of compound 7:

[0114] Compound D (155 mg, 0.400 mmol) was dissolved in acetonitrile (5 mL), and then compound G1 (226 mg, 0.600 mmol) and cesium carbonate (391 mg, 1.20 mmol) were added. The reaction was refluxed overnight, then cooled to room temperature and concentrated, diluted with water (20 mL), and extracted with ethyl acetate (3×10 mL). The organic phases were then combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 40:1) to obtain compound 7.

[0115] Compound 7 was tested, and the test results are as follows: 1 H NMR(400MHz, CDCl3)δ8.30(s,1H),7.85–7.75(m,3H),7.65–7.55(m,5H),7.41–7.32(m,11H),7.25(d,J=8.1Hz,1H), 7.19(d,J=8.1Hz,1H),7.15–7.07(m,4H),6.99(dd,J=7.8,1.4Hz,2H),4.72(dddd,J=5.5,4.2,3.1,1.4Hz,1H),3.74( t,J=6.5Hz,3H),2.96(dd,J=12.4,1.3Hz,1H),2.84(dd,J=12.5,4.0Hz,1H),2.68–2.56(m,3H),2.03(dddd,J=12.3,7 .7, 6.3, 3.0Hz, 1H), 1.93 (ddt, J=12.3, 8.3, 5.7Hz, 1H), 1.86–1.78 (m, 2H), 1.73 (pd, J=6.6, 3.7Hz, 3H), 1.03 (s, 9H).

[0116] Preparation of compound 8:

[0117] Compound 8 was obtained by the same method as that for preparing compound 7 except that compound G1 was replaced by compound G2 (235 mg, 0.600 mmol).

[0118] Compound 8 was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ8.30(s,1H),7.90–7.73(m,2H),7.67–7.56(m,5H),7.42–7.31(m,11H),7.25(d,J=8.1Hz, 1H),7.19(d,J=8.1Hz,1H),7.17–7.06(m,4H),7.03–6.92(m,3H),4.76(dddd,J=5.5,4.1,3.0,1.3Hz,1H),3.85 –3.54(m,3H),2.96(dd,J=12.4,1.3Hz,1H),2.84(dd,J=12.4,3.9Hz,1H),2.72–2.56(m,3H),2.55–2.45(m,2H) ,2.03(dddd,J=12.3,7.7,6.3,3.0Hz,1H),1.93(ddt,J=12.3,8.3,5.7Hz,1H),1.70–1.50(m,5H),1.03(s,9H).

[0119] Preparation of compound 9:

[0120] Compound 9 was obtained by the same method as that for preparing compound 7 except that compound G1 was replaced by compound G3 (243 mg, 0.600 mmol).

[0121] Compound 9 was tested, and the test results were as follows: 1H NMR(400MHz, CDCl3)δ8.30(s,1H),7.86–7.71(m,3H),7.66–7.58(m,5H),7.43–7.29(m,10H),7.25(d,J=8.1Hz,1H),7.19( d,J=8.1Hz,1H),7.15–7.08(m,4H),7.04–6.95(m,3H),4.76(dddd,J=5.5,4.1,3.0,1.3Hz,1H),3.67(t,J=6.0Hz,3H),2.9 6(dd,J=12.4,1.3Hz,1H),2.84(dd,J=12.4,3.9Hz,1H),2.73–2.54(m,3H),2.03(dddd,J=12.3,7.7,6.3,3.0Hz,1H),1.93 (ddt,J=12.3,8.3,5.7Hz,1H),1.83(tddt,J=6.8,4.4,3.0,2.1Hz,3H),1.65–1.45(m,5H),1.45–1.32(m,3H),1.03(s,9H).

[0122] Preparation of compound 10:

[0123] Compound 10 was obtained by the same method as that for preparing compound 7 except that compound G1 was replaced by compound G4 (252 mg, 0.600 mmol).

[0124] Compound 10 was tested, and the test results were as follows: 1 H NMR(400MHz, CDCl3)δ8.30(s,1H),7.82–7.72(m,3H),7.67–7.55(m,5H),7.41–7.32(m,11H),7.25(d,J=8.1Hz,1H),7.1 9(d,J=8.1Hz,1H),7.16–7.07(m,4H),7.04–6.95(m,3H),4.76(dddd,J=5.5,4.1,3.0,1.3Hz,1H),3.68(t,J=6.0Hz,3H) ,2.96(dd,J=12.4,1.3Hz,1H),2.84(dd,J=12.4,3.9Hz,1H),2.70–2.54(m,3H),2.49(td,J=6.4,2.8Hz,3H),2.03(dddd ,J=12.3,7.7,6.3,3.0Hz,1H),1.98–1.88(m,1H),1.88–1.70(m,2H),1.65–1.43(m,5H),1.43–1.24(m,6H),1.03(s,9H).

[0125] Comparative Example 1

[0126] Compound 11 is used as a comparative example of a non-silicon-containing hydrophobic tag. Its structure is shown below:

[0127]

[0128] Its synthetic route is as follows:

[0129]

[0130] The structures of the intermediate compound H, compound I, compound J, and compound E2 used are shown below:

[0131]

[0132] The specific preparation method is:

[0133] 1) Preparation of Compound J:

[0134] At 0°C, compound I (456 mg, 3.00 mmol) was dissolved in tetrahydrofuran (15 mL), and then sodium hydride (180 mg, 4.50 mmol, the content of sodium hydride used was 60%) was added. After stirring at this temperature for ten minutes, compound H (1.45 g, 6.00 mmol) was added, and the reaction was carried out at room temperature overnight. After the reaction was completed, ethyl acetate (40 mL) was added, and the organic phase was washed with saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain compound J.

[0135] Compound J was tested, and the test results are as follows: 1 H NMR (400MHz, CDCl3) δ3.68 (t, J = 6.8 Hz, 2H), 3.41 (t, J = 6.7 Hz, 2H), 2.32 (dt, J = 10.8, 5.3 Hz, 3H), 2.11 (d, J = 5.1 Hz, 6H), 1.70 (t, J = 5.6 Hz, 6H).

[0136] 2) Preparation of compound 11:

[0137] Compound 11 was obtained by replacing compound C with compound J (62.0 mg, 0.280 mmol) in the same manner as for preparing compound 1.

[0138] Compound 11 was tested, and the test results were as follows: 1H NMR(400MHz, CDCl3)δ8.30(s,1H),7.89–7.71(m,2H),7.49(s,1H),7.39–7.31(m,2H),7. 25(d,J=8.1Hz,1H),7.19(d,J=8.1Hz,1H),7.15–7.06(m,3H),7.02–6.95(m,2H),4.92(dq ,J=5.4,2.8Hz,1H),4.29(t,J=4.0Hz,2H),3.90–3.75(m,4H),3.60–3.37(m,2H),3.02–2 .90(m,2H),2.83–2.69(m,2H),2.15–2.01(m,4H),2.00–1.89(m,3H),1.80–1.65(m,12H).

[0139] Compound performance test

[0140] 1) The above-mentioned BTK protein degraders with silicon-containing groups as hydrophobic tags (BTK protein degraders 1-10 prepared in Examples 1-2) and the comparative examples of BTK protein degraders with non-silicon-containing groups as hydrophobic tags (BTK protein degraders 11 prepared in Comparative Example 1) were evaluated for their ability to degrade BTK in MOLM-13 (human acute myeloid leukemia cells) by Western blotting, and the concentration of the compounds used was 5 μM. The degradation efficiency results are shown in Table 1. At the same time, the IC values ​​of each compound on the MOL M-13 cell line (human acute myeloid leukemia cells) were evaluated. 50 An evaluation was conducted and the results are shown in Table 1.

[0141] Table 1 BTK degradation efficiency and IC of compounds in MOLM-13 (human acute myeloid leukemia cell line) 50

[0142]

[0143] It can be seen from Table 1 that each compound has a certain ability to degrade BTK, and is better than the comparative example compound 11 of the BTK protein degrader with a non-silicon-containing group as a hydrophobic tag. Among them, compound 2 has the best effect. Compound 2 was selected for further degradation effect evaluation. The MOLM-13 cell line was selected and eight concentration gradients of 0, 0.15, 0.3, 0.6, 1.25, 2.5, 5, and 10 μM were set to determine whether the compound can degrade BTK in a concentration-dependent manner. The results are shown in Figure 1 The results showed that compound 2 could degrade BTK in MOLM-13 cell line in a concentration-dependent manner and degrade DC 50The MOLM-13 cell line was selected and six time gradients of 0, 3, 6, 9, 12, and 24 h were set to determine whether these preferred compounds can degrade BTK in a time-dependent manner. Figure 2 The results showed that compound 2 could degrade BTK in MOLM-13 cell line in a time-dependent manner.

[0144] 2) The metabolic stability of compound 2 in liver microsomes was evaluated by comparing compound 11 which is a hydrophobic tag without silicon group and using Diclofenac as a positive control.

[0145] The specific method for evaluating the metabolic stability of liver microsomes is as follows: take out the liver microsomes from the -80°C refrigerator, place them in a 37°C water bath constant temperature oscillator for pre-incubation for 3 minutes, and melt them for use. Then weigh a certain amount of NADPH, add an appropriate amount of magnesium chloride solution to dissolve it into a 2mM solution for use. According to the composition ratio of the experimental incubation system (3Mm MgCl2-PB solution, 1μM test compound, 0.5mg / mL liver microsomes), prepare the incubation system mixed solution (without NADPH) and distribute it in 40μL / tube.

[0146] 240 μL of internal standard working precipitant was added to the 0min sample, and then 40 μL of NADPH solution was added (40 μL of magnesium chloride solution was added to the negative control group), and 40 μL of NADPH solution was added to other samples to start the reaction (40 μL of magnesium chloride solution was added to the negative control group), and 240 μL of internal standard precipitant was added after incubation in a 37°C water bath for 5min, 15min, 30min, and 60min, and 40 μL of NADPH solution was added to the positive control group to start the reaction, incubated in a 37°C water bath for 5min, and 240 μL of internal standard precipitant was added after 15min. All samples were vortexed and centrifuged to take 150 μL of supernatant, vortexed and mixed after adding 150 μL of water, and analyzed by LC-MS / MS injection. It was found through analysis and calculation that compound 2 showed medium clearance efficiency in two species, such as humans and mice. And they were significantly stable in BTK protein degrader comparative example 11 with non-silicon-containing groups as hydrophobic tags, and the results are shown in Table 2.

[0147] Table 2 Metabolic stability of liver microsomes

[0148]

[0149] The oral bioavailability of compound 2 was evaluated.

[0150] The specific method for evaluating oral bioavailability is as follows: after fasting overnight, three rats were gavaged and administered (10 mg / kg), and three rats were administered (1 mg / kg) by tail vein. Then, blood was collected at 5min, 15min, 30min, 1h, 2h, 4h, 6h, and 8h, respectively, and placed in a pre-heparinized sodium tube. The blood and sodium heparin were fully mixed by flicking several times, and then centrifuged (4°C, 3000rpm, 10min) to obtain plasma. 50μL of plasma sample was taken, 50μL of diluent (50% methanol / water) and 250μL of methanol precipitant were added, vortexed and centrifuged (4°C, 12000rpm, 10min), and the supernatant was sealed and sent to LC-MS / MS for detection. The results (Table 3) show that compound 2 has a good oral bioavailability of 17.9%.

[0151] Table 3 Kinetic parameters

[0152]

[0153] From Table 2 and the evaluation test results of the oral bioavailability of compound 2, it can be seen that the present invention has made optimizations in terms of the hydrophobic tag type and the linker type, and the selected compound 2 can exert higher metabolic stability and in vivo bioavailability.

[0154] 3) In vivo pharmacodynamic PD experiments were evaluated in a xenograft model, and mice received a single oral dose of compound 2 (10 mg / kg). The mice were euthanized at the specified time points (0 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours) after oral administration, and tumor tissues were harvested for Western blotting analysis. The data are shown in Table 4, and the results show that compound 2 significantly reduced BTK expression within 6 hours, and the effect lasted up to 48 hours. These findings indicate that degrader 2 can maintain BTK degradation for a long time in vivo.

[0155] Table 4 PD data

[0156]

[0157] 4) To evaluate the in vivo efficacy of compound 2, MOLM-13 cells were used to establish a xenograft model.

[0158] The specific method is as follows: MOLM-13 cells were injected into NOD / SCID mice. Two days after cell injection, the mice were divided into three groups: group 1 was orally administered with compound 2 (5 mpk, 6 mice, daily), group 2 was orally administered with compound 2 (10 mpk, 6 mice, daily), and control group (6 mice).

[0159] After 18 days, there was no significant fluctuation in the body weight of mice in the treatment group and the control group, and there was no obvious abnormality in the state of the mice, indicating that compound 2 had good safety. The tumor weight and tumor volume of mice in the treatment group were significantly lower than those in the control group. The tumor inhibition rate ((1-tumor weight of the treatment group / tumor weight of the control group)*100%) is shown in Table 5. The results showed that compound 2 had a strong in vivo therapeutic effect.

[0160] Table 5 MOLM-13 cell xenograft model TGI

[0161]

[0162] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. A BTK protein degrader with a silicon-containing group as a hydrophobic tag, characterized in that: Its structure is shown in Formula I: Wherein, linker is a saturated fatty chain, an unsaturated fatty chain, a linking structure consisting of an aromatic ring and a saturated fatty chain, or a linking structure consisting of an aromatic ring and an unsaturated fatty chain; R is trimethylsilyl, triethylsilyl, tri-n-propylsilyl, triisopropylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, cyclohexyldimethylsilyl, vinyldimethylsilyl, trimethoxysilyl, triethoxysilyl, phenylvinylmethylsilyl or triphenylsilyl.

2. The BTK protein degrader with a silicon-containing group as a hydrophobic tag according to claim 1, characterized in that: Its structure is shown in Formula II or Formula III: wherein m and n are independently selected from integers in the range of 1-10.

3. The BTK protein degrader with a silicon-containing group as a hydrophobic tag according to claim 2, characterized in that: Its structure is shown in Formula IV:

4. A method for preparing a BTK protein degrader with a silicon-containing group as a hydrophobic tag as claimed in claim 2, characterized in that: When the structure of the BTK protein degrader with a silicon-containing group as a hydrophobic tag is shown in Formula II, its preparation method is shown in Scheme 1; when the structure of the BTK protein degrader with a silicon-containing group as a hydrophobic tag is shown in Formula III, its preparation method is shown in Scheme 2; Route 1: Route 2:

5. Use of the BTK protein degrader having a silicon-containing group as a hydrophobic tag or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a drug for treating cancers related to BTK abnormalities.

6. The use according to claim 5, characterized in that: The related cancer is leukemia.

7. A pharmaceutical composition, characterized in that A BTK protein degrader comprising a silicon-containing group as a hydrophobic tag as described in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, characterized in that Pharmaceutically acceptable excipients are also included.

9. The pharmaceutical composition according to claim 8, characterized in that The excipient is at least one of gum arabic, syrup, lanolin and starch.

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