PROTAC compound for targeted degradation of ALK and application of PROTAC compound

Abstract

The invention discloses a PROTAC compound for targeted degradation of ALK protein and application of the PROTAC compound, the structural formula of the compound is # imgabs0 #, and Linker is a linker. The PROTAC compound capable of degrading the ALK protein in a targeted manner is applied to preparation of drugs for treating ALK positive tumors. The compound can effectively solve the problem that an existing small-molecule inhibitor cannot degrade ALK protein, and the compound can inhibit proliferation of an ALK drug-resistant mutation cell strain.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a PROTAC compound for targeted degradation of ALK protein and an application thereof. Background Art

[0002] Anaplastic lymphoma kinase (ALK) was first discovered in anaplastic large cell lymphoma (ALCL) and is the product of the NMP-ALK fusion gene, which is caused by the chromosomal translocation t(2;5)(p23;Q35). Subsequent studies have found more ALK gene rearrangements, including EML4-ALK in non-small cell lung cancer (NSCLC), CARS-ALK in inflammatory myofibroblastic tumor (IMT), CLTC-ALK in diffuse large B-cell lymphoma (DLBCL), etc. In the case of full-length ALK, ALK is activated by ligand-induced homodimerization. However, when chromosomal rearrangements occur in the ALK domain, oligomerization of the ALK fusion protein mediated by the oligomerization domain of the fusion partner induces spontaneous activation of the kinase. These oncogenic tyrosine kinases play a key role in the occurrence and development of various cancers by activating downstream signaling pathways such as PI3K / AKT, Ras / ERK, JAK / STAT, etc. ALK is an important target for the treatment of non-small cell lung cancer, and most ALK-positive patients experience significant remission after treatment with ALK inhibitors. A variety of ALK inhibitors have been used clinically, including crizotinib, alectinib, ceritinib, brigatinib, and lorlatinib. Compared with traditional chemotherapy, targeted drugs have significantly improved progression-free survival (PFS) and objective response rate (ORR). However, drug-resistant mutations will inevitably occur during the use of kinase inhibitors, leading to treatment failure. In addition, kinase inhibitors can only inhibit enzyme activity, which is not enough to completely inhibit the biological function of the target. Therefore, it is of great significance to develop new ALK targeted treatment strategies. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention provides a PROTAC compound for targeted degradation of ALK protein and its application, which can effectively solve the problem that existing small molecule inhibitors cannot degrade ALK protein, and the compound can inhibit the proliferation of ALK resistant mutant cell lines.

[0004] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0005] A PROTAC compound that targets and degrades ALK protein, the structural formula of the compound is:

[0007]

[0008] Among them, Linker is a connector.

[0009] Furthermore, the structure of Linker is

[0010] Where n1 = 1, 2, 3, 4, 5, n2

[0011] =0, 1, n3 = 4, 5, 6, n4 = 4, 5, 6, n5 = 0, 1, n6 = 4, 5, 6, n7

[0012] =0,1,n8=4,5,6.

[0013] Further, the PROTAC compound comprises at least one of the following structures:

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020] The use of the above-mentioned PROTAC compound that targets and degrades ALK protein in the preparation of drugs for treating ALK-positive tumors.

[0021] The beneficial effects produced by the present invention are:

[0022] The PROTAC compound of the present invention can target and degrade ALK protein in tumor cells through the ubiquitin proteasome system, downregulate the level of ALK protein, and achieve complete inhibition of ALK biological function. The compound inhibits the proliferation of ALK resistant mutant cell lines, and can therefore be used to treat ALK-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the results of PROTAC compound degradation of ALK. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments.

[0025] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0026] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0027] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0028] Example 1

[0029]

[0030] The above reaction process is the synthesis process of flexible PROTACs compounds 3CC-7CC. Reagents and reaction conditions: (a) DCE, NaBH(OAc)3, AcOH, 60℃, 3h; (b) DCM, TEA, 0℃, 2h; (c) DMF, DIPEA, 80℃, 4h; (d) DCM, TFA, 2h; (e) DMSO, DIPEA, 90℃, 6h.

[0031] Take 3CC as an example: Ceritinib (111.7 mg, 0.20 mmol, 1 eq) and compound 2a (n = 1) (69.1 mg, 0.22 mmol, 2 eq) were dissolved in DCE (6 mL), reacted at room temperature for 20 min, then NaBH (OAc) 3 (127.2 mg, 0.6 mmol, 3 eq) was added, the reaction temperature was adjusted to 60 ° C, the reaction was reacted for 3 h, and the reaction process was monitored by thin layer chromatography. The reaction mixture was cooled to room temperature and quenched by adding sodium bicarbonate (NaHCO3). Ethyl acetate (15 mL * 3) was added and extracted 3 times. The organic phase was combined, washed with saturated brine, and dried over anhydrous sodium sulfate. After the organic phase was concentrated, the crude product was purified by silica gel column chromatography (DCM: MeOH = 40: 1). A yellow solid powder 3CC was obtained: (yield 22.1%): 1 H NMR(400MHz,Chloroform-d)δ9.50(s,1H),8.59(d,J=8.3Hz,1H),8.16(s,1H),8.00(s,1H),7.92 (d,J=7.7Hz,1H),7.67–7.51(m,3H),7.25(m,2H),6.93(d,J=2.0Hz,1H),6.86(s,1H),6.70(d,J=7 .9Hz,1H),4.93(dd,J=11.2,4.8Hz,1H),4.62(hept,J=6.0Hz,1H),3.38–3.12(m,5H),3.02–2.33( m,7H),2.25–2.06(m,6H),1.88(d,J=32.4Hz,6H),1.38(d,J=5.7Hz,6H),1.32(d,J=6.7Hz,6H).MS calcd forC 44 H 51 ClN8O7S[M+H + ]871.3, found 871.3.

[0032] 4CC (yellow powder, yield 5.4%): 1H NMR (400 MHz, CDCl3) δ 9.52 (s, 1H), 8.60 (d, J = 8.3 Hz, 1H), 8.18 (s, 1H), 8.02 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.66–7.56 (m, 3H), 7.26 (m, 2H), 6.99 (d, J = 2.0 Hz, 1H), 6.85 (s, 1H), 6.77 (d, J = 8.3 Hz, 1H), 4.95 (dd, J = 12.3, 5.3 Hz, 1H), 4.56 (hept, J = 6.0 Hz, 1H), 3.34–3.12 (m, 5H), 2.94–2.71 (m, 4H), 2.50 (s, 2H), 2.18 (s, 3H), 2.14 (d, J = 5.4 Hz, 1H), 1.84–1.62 (m, 9H), 1.53–1.47 (m, 2H), 1.37 (d, J = 6.0 Hz, 6H), 1.34 (d, J = 6.9 Hz, 6H). MS calcd for C45H53ClN8O7S [M+H+] 885.3, found 885.2.

[0033] 5CC (yellow powder, yield 2.1%): 1H NMR (400 MHz, CDCl3) δ 9.52 (s, 1H), 8.60 (d, J = 8.3 Hz, 1H), 8.17 (s, 1H), 8.01 (s, 1H), 7.95 (d, J = 9.2 Hz, 1H), 7.66–7.56 (m, 3H), 7.28 (m, 2H), 6.99 (d, J = 1.9 Hz, 1H), 6.85 (s, 1H), 6.75 (d, J = 8.3 Hz, 1H), 4.95 (dd, J = 12.2, 5.3 Hz, 1H), 4.56 (hept, J = 6.0 Hz, 1H), 3.32–3.11 (m, 5H), 2.95–2.69 (m, 4H), 2.48 (s, 2H), 2.21–2.08 (m, 6H), 1.88–1.60 (m, 9H), 1.50 (q, J = 6.9 Hz, 2H), 1.37 (d, J = 6.1 Hz, 6H), 1.34 (d, J = 6.9 Hz, 6H). MS calcd for C46H55ClN8O7S [M+H+] 899.4, found 899.4.

[0034] Example 2

[0035]

[0036] The above reaction process is the synthesis of rigid PROTACs compounds S1-S6, L1-L3. Reagents and reaction conditions: (a) DCE, NaBH(OAc)3, AcOH, 60℃, 3h; (b) DCM, TFA, 2h; (c) DMSO, DIPEA, 90℃, 6h.

[0037] S6 is taken as an example: Ceritinib (111.7 mg, 0.20 mmol, 1 eq) and compound 9c (n2 = 6) (49.8 mg, 0.44 mmol, 2 eq) are dissolved in DCE (6 mL), reacted at room temperature for 20 min, then NaBH (OAc) 3 (127.2 mg, 0.6 mmol, 3 eq) is added, the reaction temperature is adjusted to 60 ° C, the reaction is reacted for 3 h, and the reaction process is monitored by thin layer chromatography. After the reaction is completed, the reaction mixture is cooled to room temperature and quenched by adding sodium bicarbonate. Ethyl acetate (15 ml) is added and extracted three times, the organic phase is washed with saturated brine, dried over anhydrous sodium carbonate, and purified by column chromatography (DCM: MeOH = 40: 1) to obtain compound 11a (n1 = 4), (111.3 mg, yield 78%). The Boc protecting group of 11a was removed with trifluoroacetic acid, and after purification by column chromatography (DCM: MeOH = 20: 1), DMSO (5 mL) was mixed with DIPEA (41.4 mg, 0.32 mmol, 2 eq) and 5-fluorothalidomide (49.7 mg, 0.18 mmol, 1.1 eq) solution, stirred at 90 ° C for 6 h, and monitored by TLC. The reaction mixture was cooled to room temperature, washed with saturated brine, and extracted with ethyl acetate (20 mL) 3 times. The organic phase was dried and concentrated with anhydrous sodium carbonate, and the residue was purified with silica gel (DCM: MeOH = 40: 1) to obtain compound S6 (yellow powder, yield 14.8%): 1 H NMR (400MHz, CDCl3) δ9.50 (s, 1H), 8.59 (d, J = 8.3Hz, 1H), 8.43 (s, 1H), 8.16 (s, 1H), 8.00 (s, 1H), 7.9 3(d,J=7.9Hz,1H),7.70–7.55(m,3H),7.27–7.22(m,2H),7.05(d,J=8.2Hz,1H),6.83(s,1H),4.94(d d,J=12.0,5.1Hz,1H),4.63–4.52(m,3H),3.97(d,J=12.4Hz,2H),3.26(dt,J=13.6,6.8Hz,1H),3.18 –2.60(m,9H),2.36–2.10(m,7H),2.02–1.62(m,9H),1.37(d,J=6.0Hz,6H),1.32(d,J=6.8Hz,6H).MS calcd forC 47 H55 ClN8O7S[M+H + ]911.36, found 911.6.

[0038] L1 (yellow powder, yield 21.6%): 1 H NMR(400MHz, CDCl3)δ9.43(s,1H),8.51(d,J=7.6Hz,1H),8.39(s,1H),8.09(s,1H),7.93(s,1H),7.85(d,J =7.1Hz,1H),7.59–7.48(m,3H),7.19–7.14(m,1H),6.77(s,1H),6.69(s,1H),6.43(d,J=7.1Hz,1H),4.86( dd,J=10.7,3.6Hz,1H),4.47(s,1H),4.00(s,2H),3.92(s,2H),3.44–3.37(m,2H),3.21–3.14(m,1H),2.54 (s,9H),2.09(s,3H),2.06–2.02(m,1H),1.83–1.60(m,6H),1.29(d,J=4.8Hz,6H),1.25(d,J=6.1Hz,6H).MS calcd for C 47 H 53 ClN8O7S[M+H + ]909.4,found 909.5.

[0039] Example 3

[0040]

[0041] The above reaction is the synthesis process of rigid PROTACs compounds 4A-6F. Reagents and reaction conditions: (b) DCM, TFA, 2h; (c) DMSO, DIPEA, 90℃, 6h.

[0042] 4B is taken as an example: Ceritinib (111.7 mg, 0.20 mmol, 1 eq) and compound 8a (37.7 mg, 0.22 mmol, 2 eq) are dissolved in DCE (6 mL), reacted at room temperature for 20 min, then NaBH(OAc)3 (127.2 mg, 0.6 mmol, 3 eq) is added, the reaction temperature is adjusted to 60 ° C, the reaction is reacted for 3 h, and the reaction process is monitored by TLC. After the reaction is completed, the reaction mixture is cooled to room temperature and sodium bicarbonate is added to quench. Ethyl acetate (15 ml) is added and extracted three times, the organic phase is washed with saturated brine, dried over anhydrous sodium carbonate, and purified by column chromatography (DCM: MeOH = 40: 1) to obtain compound 11a, (111.3 mg, yield 78%). The Boc protecting group of 11a was removed with trifluoroacetic acid, purified by column chromatography (DCM: MeOH = 20: 1), and then reduced by amination with 8c (n3 = 6) (35.1 mg, 0.18 mmol, 1.1 eq) under the above conditions to obtain 4AM (101.9 mg, 82% yield). The Boc protecting group of 4AM was removed with trifluoroacetic acid, purified by column chromatography (DCM: MeOH = 20: 1), and then DMSO (5 mL) and DIPEA (33.6 mg, 0.26 mmol, 2 eq) and 5-fluorothalidomide (39.5 mg, 0.14 mmol, 1.1 eq) solution were stirred at 90 ° C for 6 h and monitored by TLC. The reaction mixture was cooled to room temperature, washed with saturated brine, and extracted with ethyl acetate (20 mL) three times. The organic phase was dried and concentrated with anhydrous sodium carbonate, and the residue was purified with silica gel (DCM: MeOH = 40: 1) to obtain compound 4B (yellow powder, yield 18.9%): 1 H NMR (400MHz, CDCl3) δ9.43(s,1H),8.86(s,1H),8.51(d,J=8.3Hz,1H),8.09(s,1H),7.91(s,1H),7.85(d,J=7.9Hz, 1H),7.60–7.48(m,3H),7.19–7.14(m,1H),6.96(d,J=8.3Hz,1H),6.73(s,1H),4.86(dd,J=12.0,5.2Hz,1H),4.44(h ept,J=6.3Hz,1H),3.74(d,J=12.9Hz,2H),3.52(s,2H),3.19(hept,J=7.0Hz,1H),3.02–2.53(m,12H),2.34(s,1H), 2.08(s,3H),2.05–1.99(m,1H),1.93–1.85(m,2H),1.79–1.51(m,7H),1.28(d,J=6.0Hz,6H),1.24(d,J=6.8Hz,6H). 13C NMR (101MHz, CDCl3) δ171.37,168.59,168.05,167.29,157.50,155.33,144.80,138.5 0,134.65,134.36,131.26,127.48,126.86,125.40,124.86,123.67,123.09,120.79,1 18.74,117.85,110.83,108.70,105.73,71.41,63.41,57.27,55.45,55.36,53.46,51 .21,49.15,45.90,37.88,32.20,31.52,28.13,22.78,22.26,18.94,15.37.HRMScalcd for C 49 H 58 ClN9O7S[M+H + ]952.3868, found 952.3872.

[0043] 4C (yellow powder, yield 23.2%): 1 H NMR (400MHz, CDCl3) δ9.44(s,1H),9.24(s,1H),8.50(d,J=8.3Hz,1H),8.09(s,1H),7.93(s,1H),7.85(d,J=9 .2Hz,1H),7.61–7.48(m,3H),7.20–7.13(m,2H),6.95(d,J=8.6Hz,1H),6.72(s,1H),4.88(dd,J=11.8,5.5Hz ,1H),4.44(hept,J=6.3Hz,1H),4.28(s,1H),3.84(d,J=12.3Hz,2H),3.72–3.30(m,3H),3.19(hept,J=7.0Hz ,1H),3.01–2.29(m,12H)),2.07(s,3H),2.06–1.48(m,11H),1.29(d,J=6.0Hz,6H),1.24(d,J=6.8Hz,6H).MS calcd for C 50 H 60 ClN9O7S[M+H + ]966.4,found966.6.

[0044] 5A (yellow powder, yield 17.9%): 11H NMR (400 MHz, CDCl3) δ 9.43 (s, 1H), 8.50 (d, J = 8.0 Hz, 2H), 8.09 (s, 1H), 7.93 (s, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.59–7.49 (m, 3H), 7.19–7.13 (m, 1H), 6.78 (s, 1H), 6.71 (s, 1H), 6.45 (d, J = 8.3 Hz, 1H), 4.84 (dd, J = 11.1, 5.0 Hz, 1H), 4.49 (s, 1H), 4.03 (t, J = 7.3 Hz, 2H), 3.88–3.83 (m, 2H), 3.25–3.13 (m, 2H), 2.98–2.84 (m, 2H), 2.84–2.58 (m, 8H), 2.21–2.02 (m, 7H), 1.87–1.59 (m, 6H), 1.28 (d, J = 5.2 Hz, 6H), 1.24 (d, J = 6.3 Hz, 6H). 13 13C NMR (101 MHz, CDCl3) δ 171.35, 168.65, 167.83, 167.44, 157.41, 155.31, 154.85, 138.49, 134.62, 134.25, 131.26, 125.27, 124.85, 123.60, 123.10, 120.56, 114.31, 110.93, 105.10, 55.47, 55.35, 49.09, 40.98, 31.46, 22.78, 22.21, 19.00, 15.36, 15.31. MS calcd for C 48 H 56 ClN9O7S [M+H + 938.4, found 938.6.

[0045] 5D (yellow powder, yield 19.4%): 11H NMR (400 MHz, CDCl3) δ 9.43 (s, 1H), 9.20 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 8.08 (s, 1H), 7.91 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.59–7.49 (m, 3H), 7.19–7.15 (m, 1H), 6.77 (s, 1H), 6.70 (s, 1H), 6.43 (d, J = 8.3 Hz, 1H), 4.82 (dd, J = 11.9, 5.2 Hz, 1H), 4.55–4.45 (m, 1H), 4.05–3.99 (m, 2H), 3.91–3.85 (m, 2H), 3.49–3.43 (m, 1H), 3.24–3.10 (m, 2H), 2.86–2.14 (m, 15H), 2.11–1.99 (m, 6H), 1.80–1.44 (m, 5H), 1.28 (d, J = 6.0 Hz, 6H), 1.24 (d, J = 6.8 Hz, 6H). MS calcd for C 49 H 58 ClN9O7S [M+H + 952.4, found 952.6.

[0046] 6A (yellow powder, yield 19.7%): 1 1H NMR (400 MHz, CDCl3) δ 9.42 (s, 1H), 8.80 (s, 1H), 8.51 (d, J = 8.3 Hz, 1H), 8.08 (s, 1H), 7.90 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.59–7.47 (m, 3H), 7.19–7.11 (m, 1H), 6.76 (s, 1H), 6.71 (s, 1H), 6.45 (d, J = 8.3 Hz, 1H), 4.84 (dd, J = 11.7, 5.1 Hz, 1H), 4.47 (dt, J = 11.2, 5.5 Hz, 1H), 4.03 (t, J = 7.2 Hz, 2H), 3.86–3.77 (m, 2H), 3.34–3.24 (m, 1H), 3.22–3.14 (m, 1H), 3.03 (s, 2H), 2.93 (d, J = 9.9 Hz, 2H), 2.83–2.59 (m, 4H), 2.51–2.24 (m, 3H), 2.08 (s, 3H), 2.05–2.00 (d, J = 6.8 Hz, 1H), 1.96–1.61 (m, 10H), 1.27 (d, J = 6.0 Hz, 6H), 1.24 (d, J = 6.8 Hz, 6H). 13C NMR (101MHz, CDCl3) δ171.38,168.64,167.88,167.48,157.51,155.32,154.9 2,144.85,138.50,134.66,134.27,131.24,125.26,124.83,123.67,123.08,1 20.74,118.03,114.15,110.90,105.69,105.06,71.38,55.52,55.45,54.80,5 3.46,50.08,49.86,49.09,31.48,22.79,22.25,18.96,18.96,15.37.MScalcd for C 49 H 58 ClN9O7S[M+H + ]952.4, found 952.5.

[0047] 6D (yellow powder, yield 20.3%): 1 H NMR (400MHz, CDCl3) δ9.42 (s, 1H), 8.70 (s, 1H), 8.51 (d, J = 8.3Hz, 1H), 8.08 (s, 1H), 7.90 (s, 1H), 7.85 (d, J = 7.9Hz, 1H),7.58–7.47(m,3H),7.17–7.20(m,1H),6.72(d,J=11.0Hz,2H),6.44(d,J=8.3Hz,1H),4.84(dd,J=11.7,5.0Hz, 1H),4.47(hept,J=6.0Hz,1H),4.03(t,J=7.2Hz,2H),3.87–3.79(m,2H),3.30–3.19(m,2H),3.07–2.45(m,9H),2.2 3–2.13(m,2H),2.12–1.94(m,6H),1.88–1.56(m,10H),1.28(dd,J=6.1,1.7Hz,6H),1.24(dd,J=6.9,1.8Hz,6H).MS calcd for C 50 H 60 ClN9O7S[M+H + ]966.4, found 966.6.

[0048] Test example

[0049] The anti-Karpas299 cell line proliferation activity and ALK degradation activity of the PROTAC compounds targeted for degradation of ALK protein prepared in Examples 1, 2, and 3 were tested respectively. The specific test methods are as follows, and the specific test results are shown in Tables 1-2.

[0050] 1. Anti-Karpas299 cell line proliferation activity test

[0051] Collect cells in logarithmic growth phase, resuspend and count with a cell counter. Take an appropriate amount of cell suspension according to 9000 cells per well of 96-well plate, mix with culture medium and plate. After 72 hours of action of gradient concentration bifunctional PROTAC compounds, add 10 μL of CCK8 to each well, incubate in the dark for 2 to 4 hours, and detect the absorbance value corresponding to each well at a wavelength of 450 nm with an enzyme reader;

[0052] Cell inhibition rate = 100% - (average absorbance of the drug-added group / average absorbance of the blank control group) × 100%, and the half inhibition concentration IC is obtained by analysis and calculation. 50 , the specific results are shown in Table 1-2.

[0053] Table 1: Antiproliferative activity of PROTAC compounds 3CC-7CC

[0054]

[0055] Table 2: Antiproliferative activity of PROTAC compound S1-6F

[0056]

[0057]

[0058] The data in Tables 1 and 2 show that all PROTAC molecules have good anti-proliferation activity against Karpas299 cell lines. Among them, PROTAC molecules 4B, 5A and 6A have the best activity, which is comparable to that of ceritinib (IC 50 <5nM).

[0059] 2. ALK degradation activity test

[0060] Karpas299 cells in the logarithmic growth phase were plated in a 6-well plate and treated with ALK bifunctional PROTAC compounds S6, L1, 4B, 4C, 5A, 5D, 6A, and 6D for 48 hours. The cells were washed three times with pre-cooled PBS, and then collected with a scraper. An appropriate amount of RIPA lysis buffer containing protease inhibitors was added to lyse the cells on ice for 15 minutes. After low-temperature ultrasound, the supernatant was collected by centrifugation (4°C, 13000 rpm, 15 minutes), and the supernatant was mixed with SDS-PAGE protein loading buffer (sodium dodecyl sulfate-polypropylene glycol). After mixing with 1% 2-amino-4-nitropropene amide gel, the mixture was placed in boiling water and boiled for 10 minutes. The protein sample was separated by 10% SDS-PAGE electrophoresis and then transferred to a PVDF (polyvinylidene fluoride) membrane. The membrane was blocked with skim milk at room temperature for one hour. β-actin was used as the internal reference protein. The corresponding primary antibody and PVDF membrane were incubated at 4°C overnight. After further labeling with the corresponding horseradish enzyme-labeled secondary antibody, the immunoblot was developed by chemiluminescence. The activity of the ALK bifunctional PROTAC compound in degrading ALK was determined by comparing the grayscale of the corresponding protein immunoblot. The specific results are shown in Figure 1 .

[0061] Figure 1 The results showed that PROTAC molecules S6, L1, 4B, 4C, 5A, 5D, 6A, and 6D can induce a significant decrease in ALK protein levels, thereby effectively inhibiting ALK protein function and exerting anti-tumor activity.

Claims

1. A PROTAC compound targeting degradation of ALK protein, characterized in that: The structural formula of the compound is: Among them, Linker is a connector.

2. The PROTAC compound targeting ALK protein degradation according to claim 1, characterized in that The structure of Linker is Among them, n1=1, 2, 3, 4, 5, n2=0, 1, n3=4, 5, 6, n4=4, 5, 6, n5=0, 1, n6=4, 5, 6, n7=0, 1, n8=4, 5, 6.

3. The PROTAC compound targeting degradation of ALK protein according to claim 1, characterized in that The PROTAC compound comprises at least one of the following structures:

4. Use of the PROTAC compound targeting the degradation of ALK protein according to any one of claims 1 to 3 in the preparation of a drug for treating ALK-positive tumors.

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