FAK protein targeting chimera as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to an FAK protein targeting chimera as well as a preparation method and application thereof. The FAK protein targeted chimera compound disclosed by the invention plays the roles of resisting tumors and reversing multidrug resistance of tumors by degrading FAK protein. Experimental results show that the compound provided by the invention can well degrade FAK protein to play a role in reversing tumor multidrug resistance, can be further used for preparing a tumor multidrug resistance reversal agent, and also has certain antitumor activity and drug sensitization ability.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a FAK protein-targeting chimera and its preparation method and use. Background Art

[0002] As an emerging protein degradation technology, proteolysis targeting chimeras (PROTACs) have received extensive attention in recent years. PROTACs utilize the intracellular ubiquitin-proteasome system to achieve targeted degradation of target proteins. It mainly consists of three parts: one is the ligand part that binds to the E3 ligase, the second is the ligand that binds to the target protein, and the third is the linker that connects the above two parts. Its mechanism of action is to form a ternary complex by binding to the target protein and the E3 ligase, tagging the target protein with ubiquitin, so that the ubiquitinated protein can be recognized and degraded by the proteasome in the cell.

[0003] Focal adhesion kinase (FAK) is a classical target in the field of cancer research. It is a key regulator of signals mediated by growth factor receptors and integrins, and functions as a non-receptor cytoplasmic tyrosine kinase and a scaffold protein. The levels of FAK protein and mRNA are elevated in a variety of solid tumors and are associated with poor clinical outcomes. Currently developed FAK inhibitors are mainly FAK kinase inhibitors, relatively neglecting the function of the FAK scaffold protein. Therefore, the development of PROTAC molecules targeting FAK is quite promising. Among the FAK inhibitors entering clinical trials, Defactinib is currently in phase II clinical trials. It is the most classical FAK inhibitor, and its clinical indications are non-hematological malignancies and KRAS mutant non-small cell lung cancer. The drug GSK-2256098 is currently undergoing clinical trials for recurrent glioblastoma multiforme.

[0004] It is worth mentioning that, looking at the numerous clinical trials of FAK inhibitors, the clinical trial results of single FAK kinase inhibitors are not very optimistic, but as a clinical combination strategy, targeting FAK is relatively effective. Currently, a phase I / II clinical trial of Defactinib, carboplatin, and paclitaxel in combination for recurrent platinum-resistant ovarian cancer is underway.

[0005] Therefore, the research on the development of novel PROTAC compounds targeting FAK may bring new hopes and opportunities for the development of tumor multidrug resistance reversers. Summary of the Invention

[0006] The object of the present invention is to provide novel PROTAC compounds with good ability to degrade FAK protein, and particularly relates to a pyrimidine compound containing a thalidomide-like flexible side chain.

[0007] Another object of the present invention is to provide a method for preparing the novel PROTAC compound targeting FAK as described above.

[0008] The present invention also provides the use of the novel PROTAC compound targeting FAK in anti-tumor, reversing tumor multi-drug resistance and drug sensitization.

[0009] To achieve the object of the present invention, the following technical solutions are adopted in the present invention:

[0010] In the first aspect, the present invention provides a compound targeting FAK or a pharmaceutically acceptable salt thereof, and the compound has a general formula structure as shown below:

[0011]

[0012] wherein R1 is selected from hydrogen, halogen, -OR a , -NR a R a , -CF3, -CN, -OCN, -SCN, -NO, -NO2, -C 1-3 alkyl, -C 1-3 haloalkyl or -C 1-3 haloalkoxy;

[0013] Each R a is independently selected from hydrogen or selected from optionally substituted -C 1-6 alkyl, -C 3-10 cycloalkyl, -C 4-11 cycloalkylalkyl, -C 6-10 aryl, -C 7-16 arylalkyl, 2-6 membered heteroalkyl, 3-8 membered heterocycloalkyl, 4-14 membered heterocycloalkylalkyl, 5-12 membered heteroaryl or 6-18 membered heteroarylalkyl;

[0014] A is a linker fragment required in PROTAC technology;

[0015] B is a short-chain alkyl or a substituted thalidomide fragment targeting the ubiquitin ligase required in PROTAC technology;

[0016] C is a molecular fragment targeting the FAK protein.

[0017] As an alternative, in the above-mentioned compound or its pharmaceutically acceptable salt, the compound or its pharmaceutically acceptable salt is a PROTAC molecular compound targeting FAK.

[0018] As an alternative, in the above-mentioned compound or its pharmaceutically acceptable salt, wherein R1 is selected from hydrogen, halogen, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -C 1-3 alkyl, -C1-3 haloalkyl or -C 1-3 haloalkoxy group.

[0019] As an alternative, in the above compound or its pharmaceutically acceptable salt, wherein R1 is selected from -CF3.

[0020] As an alternative, in the above compound or its pharmaceutically acceptable salt, A is selected from nitrogen-containing alkyl chains: -NH(CH2) n NH-, -NHCO(CH2) n NH-, where n = 2 - 5.

[0021] Alternatively, A is selected from aliphatic heterocyclic chains:

[0022] Preferably, m = 1 and n = 2.

[0023] As an alternative, in the above compound or its pharmaceutically acceptable salt, B is selected from

[0024] Preferably, B is selected from

[0025] As an alternative, in the above compound or its pharmaceutically acceptable salt, the compound is selected from the following:

[0026]

[0027] In a second aspect, the present invention provides a method for preparing the compound or its pharmaceutically acceptable salt described in the first aspect above, characterized in that: the preparation method comprises the following steps:

[0028] Firstly, starting materials are used to prepare a key intermediate through multiple-step reactions

[0029] Secondly, the thalidomide fragment targeting the ubiquitin ligase is combined with the required Linker linking fragment through multiple-step reactions to obtain a key intermediate

[0030] Finally, the key intermediate is combined with through a reaction to obtain the compound, and the compound reacts with common inorganic or organic acids to obtain its corresponding pharmaceutically acceptable salt.

[0031] In a third aspect, the present invention provides a drug composition targeting FAK, which comprises the compound described in the first aspect above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0032] Preferably, the compound or its pharmaceutically acceptable salt is a PROTAC molecular compound targeting FAK.

[0033] In a fourth aspect, the present invention provides the use of the compound described in the first aspect above or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0034] As an alternative, in the above use, the tumor is a solid tumor. Preferably, the solid tumor is breast cancer, lung cancer, colon cancer, liver cancer, gastric cancer, pancreatic cancer or prostate cancer.

[0035] In a fifth aspect, the present invention provides the use of the compound described in the first aspect above or a pharmaceutically acceptable salt thereof in the preparation of a drug for reversing tumor multi-drug resistance or having drug sensitization ability.

[0036] As an alternative, in the above use, the tumor is a solid tumor. Preferably, the solid tumor is breast cancer, lung cancer, colon cancer, liver cancer, gastric cancer, pancreatic cancer or prostate cancer.

[0037] As an alternative, in the above use, having drug sensitization ability means having the ability to sensitize commonly used clinical chemotherapeutic drugs.

[0038] Preferably, having drug sensitization ability means having the ability to sensitize paclitaxel, doxorubicin, vinorelbine, vincristine or tamoxifen.

[0039] The present invention has the following beneficial effects compared with the prior art:

[0040] In vitro and in vivo experimental results show that the compound of the present invention can promote the sensitivity of tumors to chemotherapeutic drugs, indicating that the PROTAC compound has the effect of reversing tumor multi-drug resistance. In addition, the compound also exhibits certain anti-tumor activity and the ability to promote the sensitivity of tumor cells to tamoxifen (Tamoxifen, TAM) in vitro. Therefore, the compound of the present invention can be applied to the treatment of tumor multi-drug resistance-related diseases and is an ideal drug for assisting chemotherapeutic drugs in treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Statistical analysis of the degradation of phosphorylated FAK (A) and FAK (B) by the PROTAC molecule F2 at gradient concentrations in MDA-MB-231 cells in Example 1.

[0042] Figure 2 Statistical analysis of the degradation of phosphorylated FAK (A) and FAK (B) by 1 μM PROTAC molecule F2 in MDA-MB-231 cells at gradient time periods in Example 2.

[0043] Figure 3 Statistical analysis of the recovery of FAK and phosphorylated FAK after drug withdrawal of PROTAC molecule F2 in MDA-MB-231 cells in Example 3.

[0044] Figure 4 Statistical analysis of the effect of inhibitor F1, thalidomide, and proteasome inhibitor MG132 on the degradation of FAK by PROTAC molecule F2 in MDA-MB-231 cells in Example 4.

[0045] Figure 5 In vitro antitumor effects of PROTAC molecule F2 on 4T1 cells (A), MDA-MB-231 cells (B), MDA-MB-468 cells (C), and MDA-MB-435 cells (D) in Example 5.

[0046] Figure 6 Statistical analysis of the effect of PROTAC molecule F2 on the migration ability of 4T1 cells in Example 6.

[0047] Figure 7 Statistical analysis of the effect of PROTAC molecule F2 on the migration ability of MDA-MB-231 cells in Example 6.

[0048] Figure 8 Statistical analysis of the effect of PROTAC molecule F2 on the invasion ability of 4T1 cells (A) and MDA-MB-231 cells (B) in Example 7.

[0049] Figure 9 Effect of PROTAC molecule F2 on the adhesion ability of 4T1 cells (A) and MDA-MB-231 cells (B) in Example 8.

[0050] Figure 10 Statistical analysis of the effect of PROTAC molecule F2 alone or in combination with PTX on the migration ability of HCT8 / T cells (A) and alone or in combination with DOX on the migration ability of MCF-7 / ADR cells (B) in Example 10.

[0051] Figure 11 Statistical analysis of the effect of PROTAC molecule F2 alone or in combination with DOX on the invasion ability of MCF-7 / ADR cells (A), alone or in combination with PTX on the invasion ability of HCT8 / T cells (B), and alone or in combination with PTX on the invasion ability of A549 / T cells (C) in Example 11.

[0052] Figure 12 Statistical analysis of the effect of the PROTAC molecule F2 alone or in combination with PTX on the adhesion ability of HCT8 / T cells (A) and the effect of the PROTAC molecule F2 alone or in combination with PTX on the adhesion ability of A549 / T cells (B) in Example 12.

[0053] Figure 13 To show the effect of the PROTAC molecule F2 on the sensitivity of MCF-7 cells (A) and MCF-7 / TAM cells (B) to TAM in Example 13.

[0054] Figure 14 Tumor growth curves of the PROTAC molecule F2 alone or in combination with PTX for the treatment of HCT8 / T xenografts in vivo in Example 14.

[0055] Figure 15 Tumor weights of the PROTAC molecule F2 alone or in combination with PTX for the treatment of HCT8 / T xenografts in vivo in Example 14. Detailed implementation manners

[0056] The present invention will be further described below with reference to specific examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0057] For those technical or conditions not specified in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0058] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0059] Preparation examples:

[0060] Overall synthesis route:

[0061] 1. Synthesis routes of intermediates IM-7 and IM-7':

[0062]

[0063] 2. Synthesis routes of intermediates IM-11, IM-13 and IM-15:

[0064]

[0065]

[0066] 3. Synthesis routes of the target compounds ND-F-2, ND-F-3, ND-F-4 and ND-F-5:

[0067]

[0068] Specific synthesis steps examples:

[0069] 1. Synthesis of IM-1:

[0070]

[0071] Take a 500 mL round-bottom flask, add 8.8 g of SM-1, dissolve it with 125 mL of pyridine, add 9.2 mL of Ac2O, react at room temperature for 8.0 h, monitor the reaction progress by TLC (PE:EA = 10:1), and the reaction is complete. Adjust the pH to 2.0 with 1.0 M HCl, wash with water, extract with DCM three times, combine the organic phases, dry, concentrate, and purify by column chromatography to obtain the product IM-1 (8.0 g, 73.5%).

[0072] 2. Synthesis of IM-2:

[0073]

[0074] Take a 250 mL round-bottom flask, add 6.1 g of IM-1, dissolve it with 120 ml of CCl4, add 5.1 g of NBS and 450 mg of AIBN, displace N2 three times, and reflux for 36 h. Monitor by TLC (PE:EA = 5:1), and there is still raw material remaining. Extending the time has no obvious improvement. Concentrate the reaction solution and directly perform column chromatography to obtain 8.4 g of the product of the mixed raw materials for the next step.

[0075] 3. Synthesis of IM-3:

[0076]

[0077] Take a 100 mL reaction flask, add 8.4 g of the crude IM-2, add a methanol solution of 2.0 M methylamine, and reflux for 8.0 h. Monitor the reaction by TLC (PE:DCM:EA = 3:1:1) until the reaction is complete. Concentrate and purify by column chromatography (PE:DCM:EA = 3:1:1) to obtain 2.95 g of the product, with a two-step yield of 67%.

[0078] 4. Synthesis of IM-4:

[0079]

[0080] Take a 100 mL reaction flask, add 600 mg of IM-3, dissolve it with 30 mL of acetone, add 1.02 g of potassium carbonate, place it in an ice bath, add 800 mg of SM-2, allow it to warm up to room temperature naturally, react for 4.0 h, monitor the reaction progress by TLC (PE:EA = 1:1, repeat the operation continuously for 3 times), and the reaction is complete. Concentrate, wash with water, extract with DCM, combine the organic phases, dry, concentrate, and purify by column chromatography to obtain IM-4 and IM-4', a total of 950 mg, with a yield of 75%. The polarities of the two are similar and it is difficult to purify, and the ratio is close to 1:1.

[0081] 5. Synthesis of IM-5:

[0082]

[0083] Take 500 mg of SM-3, dissolve it with 15 mL of ethanol, add 1.0 mL of concentrated sulfuric acid, reflux and react for 10 h, and TLC shows that the reaction is complete. Quench with saturated NaHCO3, concentrate, wash with water, extract with DCM, combine the organic phases, dry, concentrate, and purify by column chromatography (PE:EA = 5:1) to obtain 496 mg of the product, with a yield of 86%.

[0084] 6. Synthesis of IM-6 and IM-6':

[0085]

[0086] Take 100 mg of IM-5, add a mixture of 242 mg of IM-4 and IM-4', add 65 mg of Pd2(dba)3, 67 mg of X-Phos and 195 mg of K2CO3, and finally dissolve it with 4.0 mL of tert-butanol. Replace the gas with nitrogen 5 times and react at an external temperature of 95 °C for 8.0 h. Detect the reaction situation by TLC (PE:EA = 5:1), and the raw materials disappear completely. Concentrate the reaction solution and separate and purify it by PTLC with PE:EA = 1:1 (repeat the operation continuously for 2 times) to obtain 77 mg of the product IM-6 with small polarity and 43 mg of the product IM-6' with large polarity, and the yields are 31.4% and 17.6% respectively.

[0087] 7. Synthesis of IM-7:

[0088]

[0089] Take 139 mg of the compound IM-6, dissolve it with 4.0 mL of THF and 2.0 mL of MeOH. Take another reaction flask, add 22 mg of NaOH, dissolve it with 2.0 mL of H2O, add this solution to the reaction of IM-6, and react at room temperature for 5.0 h. Detect by TLC, the raw materials disappear completely, adjust the pH of the solution to 5 with 1.0 M HCl, concentrate the reaction solution to obtain the crude product IM-7, which is directly used for the next step.

[0090] 8. Synthesis of IM-7':

[0091]

[0092] Take 43 mg of compound IM-6', dissolve it in 2.0 mL of THF and 1.0 mL of MeOH. Take another reaction flask, add 7 mg of NaOH, dissolve it in 1.0 mL of H2O, add this solution to the reaction of IM-6', and react at room temperature for 3.0 h. Detect by TLC, the raw materials disappear completely, adjust the pH of the solution to 5 with 1.0 M HCl, concentrate the reaction solution to obtain the crude product IM-7', and directly use it for the next step.

[0093] 9. Synthesis of IM-8:

[0094]

[0095] At room temperature, put 500 mg of SM-4 and 617 mg of SM-5 into a single-necked flask, add 10 mL of DCM to dissolve, then add 1.05 g of NaBH(AcO)3 and stir for 10 h. Detect the completion of the reaction by TLC (DCM:MeOH = 10:1), add saturated sodium bicarbonate solution to quench the reaction, separate the liquid, concentrate the solvent, and directly use it for the next step.

[0096] 10. Synthesis of IM-9:

[0097]

[0098] Dissolve 1.07 g of IM-8 in 15 mL of methanol, add 0.2 g of Pd(OH)2 / C. Replace with H2 three times, raise the temperature to 65 °C and stir for 10 h. Detect the complete reaction of the raw materials by TLC (DCM:MeOH = 10:1). Filter using diatomaceous earth and spin-dry the filtrate to obtain the product.

[0099] 11. Synthesis of IM-10:

[0100]

[0101] Dissolve 138 mg of IM-9 in 10 mL of DMSO, add 128 mg of SM-6 and 120 mg of DIPEA. React at 100 °C for 5 h. Detect the complete reaction of the raw materials by TLC (DCM:MeOH = 20:1), add water to quench the reaction, add 30 mL of DCM, wash with saturated sodium chloride three times, separate the liquid, spin-dry the solvent, and obtain 200 mg of the product by column chromatography (DCM:MeOH = 30:1) with a yield of 78%.

[0102] 12. Synthesis of IM-11:

[0103]

[0104] At room temperature, 200 mg of IM-10 was dissolved in 5 mL of DCM, 0.63 mL of 4M HCl / EA solution was added, and the mixture was stirred for 4 h. LCMS detected that the raw material reaction was complete, and the IM-11 product was obtained directly by filtration.

[0105] 13. Synthesis of IM-12:

[0106]

[0107] 92 mg of SM-7 was dissolved in 5 mL of DMSO, 128 mg of SM-6 and 120 mg of DIPEA were added. The reaction was carried out at 100 °C for 5 h. TLC (DCM:MeOH = 20:1) detected that the raw material reaction was complete, water was added to quench the reaction, 30 mL of DCM was added, and the mixture was washed with saturated sodium chloride three times. After liquid separation, the solvent was evaporated, and column chromatography (DCM:MeOH = 30:1) gave 120 mg of the product with a yield of 57%.

[0108] 14. Synthesis of IM-13:

[0109]

[0110] At room temperature, 120 mg of IM-12 was dissolved in 5 mL of DCM, 0.33 mL of 4M HCl / EA solution was added, and the mixture was stirred for 4 h. LCMS detected that the raw material reaction was complete, and the IM-13 product was obtained directly by filtration.

[0111] 15. Synthesis of IM-14:

[0112]

[0113] 100 mg of IM-8 was dissolved in 5 mL of DMF, 74 mg of SM-9, 281 mg of HATU and 120 mg of DIPEA were added. The reaction was carried out at room temperature for 10 h. TLC (DCM:MeOH = 20:1) detected that the raw material reaction was complete, water was added to quench the reaction, 30 mL of DCM was added, and the mixture was washed with saturated sodium chloride three times. After liquid separation, the solvent was evaporated, and column chromatography (DCM:MeOH = 30:1) gave 110 mg of the product with a yield of 65%.

[0114] 16. Synthesis of IM-15:

[0115]

[0116] At room temperature, 110 mg of IM-14 was dissolved in 5 mL of DCM. 0.38 mL of 4 M HCl / EA solution was added, and the mixture was stirred for 4 h. LCMS detected that the raw material had completely reacted, and the IM-15 product was directly obtained by filtration.

[0117] 17. Synthesis of ND-F-2:

[0118]

[0119] 283 mg of IM-7 was dissolved in 6.0 mL of DMF. 486 mg of IM-11 and 500 μL of DIPEA were added, and the mixture was stirred for 5 min. 330 mg of HATU was added, and the reaction was carried out at room temperature for 6.0 h. TLC (DCM:MeOH = 20:1) detected that the raw material had completely reacted. The reaction solution was concentrated, washed with water, extracted with CH2Cl2, the organic phases were combined, dried, concentrated, and purified by column chromatography (DCM:MeOH = 30:1) to obtain 501 mg of the product ND-F-2 with a yield of 93%. MS: m / z 928.30 (M+H) + 。 1 1H-NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 8.77 (d, J = 7.2 Hz, 2H), 7.92 (s, 1H), 7.75–7.51 (m, 3H), 7.34–7.26 (m, 2H), 7.27–7.11 (m, 2H), 7.07 (d, J = 6.5 Hz, 1H), 5.07 (dd, J = 12.9, 5.4 Hz, 1H), 4.48 (s, 2H), 4.04 (d, J = 12.7 Hz, 2H), 3.79 (s, 3H), 3.65 (d, J = 47.2 Hz, 1H), 2.95 (d, J = 11.8 Hz, 5H), 2.86 (d, J = 20.9 Hz, 2H), 2.68–2.53 (m, 2H), 2.14 (s, 2H), 2.06–1.95 (m, 2H), 1.79 (d, J = 12.5 Hz, 4H), 1.54 (s, 2H), 1.29–1.13 (m, 5H).

[0120] 18. Synthesis of ND-F-3:

[0121]

[0122] Dissolve 15 mg of IM-7 in 1.0 mL of DMF, add 20 mg of IM-13 and 27 μL of DIPEA, stir for 5 min, add 18 mg of HATU, and react at room temperature for 4.0 h. TLC (DCM:MeOH = 10:1) detected that the raw materials had completely reacted. Concentrate the reaction solution, wash with water, extract with CH2Cl2, combine the organic phases, dry, concentrate, and purify by PTLC (DCM:MeOH = 10:1) to obtain 9 mg of the product ND-F-3 with a yield of 36%. MS: m / z 831.20 (M+H) + 。 1 1H-NMR (400 MHz, chloroform-d) δ 8.45 (d, J = 13.2 Hz, 2H), 8.06 (d, J = 23.8 Hz, 2H), 7.70 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 7.0 Hz, 1H), 7.32 (d, J = 2.4 Hz, 1H), 7.09 (dd, J = 8.6, 2.4 Hz, 1H), 6.74 (dd, J = 15.3, 7.5 Hz, 1H), 4.95 (dd, J = 12.3, 5.3 Hz, 1H), 4.31 (s, 1H), 4.11 (s, 3H), 4.00 (s, 3H), 3.94 (d, J = 13.8 Hz, 2H), 3.22 (t, J = 12.3 Hz, 2H), 2.98–2.67 (m, 4H), 2.25–2.08 (m, 3H), 1.65 (d, J = 12.6 Hz, 3H).

[0123] 19. Synthesis of ND-F-4:

[0124]

[0125] Dissolve 18 mg of IM-7’ in 1.0 mL of CH2Cl2, add 5 mg of SM-10 and 28 μL of DIPEA. After dissolution, add 31 mg of HATU and react at room temperature for 3.0 h. TLC (DCM:MeOH 10:1) detected that the raw materials had completely reacted. Wash the reaction solution with water, extract with CH2Cl2, combine the organic phases, dry, concentrate, and purify by PTLC (DCM:MeOH = 10:1) to obtain 11 mg of the product ND-F-4 with a yield of 46%. MS: m / z 589.30 (M+H)+. 1 1H-NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H), 8.24 (s, 1H), 7.82 (s, 1H), 7.01-7.51 (m, 5H), 4.22 (m, 1H), 3.85 (s, 3H), 3.59 (m, 1H), 3.27 (s, 3H), 2.15–2.54 (m, 7H), 1.85-1.91 (m, 4H).

[0126] 20. Synthesis of ND-F-5:

[0127]

[0128] Dissolve 15 mg of IM-7 in 1.0 mL of DMF, add 18 mg of IM-15 and 28 μL of DIPEA, stir for 5 min, add 18 mg of HATU, and react at room temperature for 4.0 h. TLC (DCM:MeOH = 10:1) detected that the raw materials had completely reacted. Concentrate the reaction solution, wash with water, extract with CH2Cl2, combine the organic phases, dry, concentrate, and perform column chromatography (DCM:MeOH = 10:1) to obtain 10 mg of the product ND-F-5 with a yield of 40%. MS: m / z 847.20 (M+H)+. 1 1H-NMR (400 MHz, methanol-d4) δ 8.64 (s, 1H), 7.97 (d, J = 2.0 Hz, 1H), 7.75 (ddd, J = 7.8, 4.4, 2.7 Hz, 2H), 7.58 (dd, J = 10.5, 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 6.7 Hz, 2H), 5.02 (dd, J = 12.8, 5.4 Hz, 1H), 3.79–3.67 (m, 5H), 3.52 (q, J = 6.6 Hz, 2H), 3.39 (t, J = 7.0 Hz, 1H), 3.25–3.17 (m, 2H), 3.02 (s, 3H), 2.89–2.60 (m, 3H), 2.49 (t, J = 6.8 Hz, 2H), 2.28 (t, J = 8.0 Hz, 1H), 2.16–2.11 (m, 1H).

[0129] Effect examples:

[0130] The 4T1 cells, MDA-MB-231 cells, MDA-MB-468 cells, MDA-MB-cells, HCT8 / T cells, A549 / T cells, MCF-7 / ADR cells, MCF-7 cells, and Balb / c nude mice used in the following experiments can be obtained by those skilled in the art through commercially available channels. The MCF-7 / TAM cells were obtained by self-inducing MCF-7 cells with the drug TAM for two months.

[0131] All data (cell viability, IC 50Values, tumor volume, weight, etc. were analyzed using GraphPad Prism 8.0 software and presented as mean ± SEM or mean ± SD. The Student's two-tailed t-test was used to compare two groups, and one-way ANOVA and Tukey's test were used to compare more than two groups. p < 0.05 was considered statistically significant and represented as *p < 0.05, **p < 0.01, ***p < 0.001.

[0132] Example 1: Degradation of FAK protein and phosphorylated FAK protein by PROTAC molecule F2 at gradient concentrations (i.e., ND-F-2 in the preparation example, the same below) in MDA-MB-231 cells

[0133] MDA-MB-231 cells were seeded in six-well plates at a density of 2x10 5 cells / well. After overnight adherent growth, the culture medium was discarded, and the culture medium containing 10 nM, 100 nM, 1 μM, 3 μM, 10 μM of PROTAC molecule F2 or inhibitor F1 (Ifebemtinib, BI853520, CAS No. 1227948-82-4) was added respectively. The culture plates were pre-cultured in a 37 °C incubator with 5% CO2 (volume percentage). After 48 hours, 200 μL of cell lysate was added to each well, and the cells were lysed on ice and the proteins were collected for Western Blot assay. Finally, the target bands were developed and analyzed using a chemiluminescent developing solution. Statistical analysis was as Figure 1 shown in Figure 1 A and

[0134] Example 2: Degradation of FAK and phosphorylated FAK by 1 μM PROTAC molecule F2 in MDA-MB-231 cells at gradient time periods

[0135] MDA-MB-231 cells were seeded in six-well plates at a density of 2x10 5 cells / well. After overnight adherent growth, the culture medium was discarded, and the culture medium containing 1 μM PROTAC molecule F2 was added. The culture plates were pre-cultured in a 37 °C incubator with 5% CO2 (volume percentage). Samples were collected at 6, 12, 24, 48, and 72 hours respectively. 200 μL of cell lysate was added to each well, and the cells were lysed on ice and the proteins were collected for Western Blot assay. Finally, the target bands were developed and analyzed using a chemiluminescent developing solution. Statistical analysis was as Figure 2 shown in Figure 2As shown in Figure B. The PROTAC molecule F2 of the present invention has an obvious degradation effect on FAK protein and phosphorylated FAK protein at the cellular level. After 6 hours of action, obvious degradation of phosphorylated FAK can be seen, and after 12 hours of action, obvious degradation of FAK can be seen.

[0136] Example 3: Recovery of FAK and phosphorylated FAK after withdrawal of the PROTAC molecule F2 in MDA-MB-231 cells

[0137] Seed MDA-MB-231 cells at a density of 2x10 5 cells / well in a six-well plate. After overnight adherent growth, discard the culture medium, and add the culture medium containing 1 μM PROTAC molecule F2 or 1 μM inhibitor F1. Incubate the culture plate in an incubator at 37 °C with 5% CO2 (v / v). After 48 hours, discard the culture medium and replace it with fresh culture medium for continued culture. Harvest samples at 12, 24, and 48 hours respectively. Add 200 μL of cell lysis buffer to each well, lyse on ice and collect the proteins, and perform Western Blot experiments. Finally, use a chemiluminescent developing solution to develop and analyze the target bands for plotting. Statistical analysis is as Figure 3 shown. In the present invention, the degradation effect of the PROTAC molecule F2 on FAK protein and phosphorylated FAK protein at the cellular level recovers more slowly and to a lower extent than inhibitor F1 after drug withdrawal.

[0138] Example 4: Effects of inhibitor F1, thalidomide, and the proteasome inhibitor MG132 on the degradation of FAK by the PROTAC molecule F2 in MDA-MB-231 cells

[0139] Seed MDA-MB-231 cells at a density of 2x10 5 cells / well in a six-well plate. After overnight adherent growth, discard the culture medium, and add 3 μM PROTAC molecule F2 alone, 3 μM inhibitor F1, 5 μM thalidomide, 5 μM MG132, or add 3 μM PROTAC molecule F2 together with one of the other three drugs. Incubate the culture plate in an incubator at 37 °C with 5% CO2 (v / v). After 24 hours, add 200 μL of cell lysis buffer to each well, lyse on ice and collect the proteins, and perform Western Blot experiments. Finally, use a chemiluminescent developing solution to develop and analyze the target bands for plotting. Statistical analysis is as Figure 4 shown. The degradation effect of the PROTAC molecule F2 in the present invention on is achieved through the proteasome pathway by binding to FAK protein or phosphorylated FAK protein and an E3 ubiquitin ligase.

[0140] Example 5: In vitro anti-tumor effects of PROTAC molecule F2 on 4T1 cells, MDA-MB-231 cells, MDA-MB-468 cells and MDA-MB-435 cells

[0141] Add 180 μL of 4T1, MDA-MB-231, MDA-MB-468 or MDA-MB-435 cell suspension with a density of 3000 cells / well to a 96-well plate, and pre-culture the culture plate in an incubator at 37 °C with a volume percentage concentration of 5% CO2 or an air incubator. The next day, add a medium containing gradient concentrations of PROTAC molecule F2 to the culture plate and incubate in the incubator. After 72 hours, add 10 μL of CCK-8 solution to each well. After incubating the culture plate in the incubator for 1 hour, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader. Cell viability (%) = [OD value (experimental well) - OD value (blank well)] / [OD value (control well) - OD value (blank well)] × 100%. The results are as shown in Figure 5 Figures 5A to 5D. The PROTAC molecule F2 in the present invention has certain inhibitory activities against 4T1 cells, MDA-MB-231 cells, MDA-MB-468 cells and MDA-MB-435 cells and is superior to inhibitor F1.

[0142] Example 6: Effects of PROTAC molecule F2 on the migration ability of 4T1 cells and MDA-MB-231 cells

[0143] Seed the cells at a density of 1×10 6 cells / well in a six-well plate. After overnight adherent growth, discard the culture medium, use a pipette tip to draw uniform straight lines vertically in each well, rinse twice with 1×PBS, and add a low-serum concentration culture medium containing 1 μM inhibitor F1 or PROTAC molecule F2. Observe and photograph the scratch healing at 0, 24, 48 and 72 hours respectively under a microscope. The results are as shown in Figure 6 and Figure 7 Figures 6A and 6B. The PROTAC molecule F2 in the present invention can significantly inhibit the migration ability of 4T1 and MDA-MB-231 cells and is superior to inhibitor F1.

[0144] Example 7: Effects of PROTAC molecule F2 on the invasion ability of 4T1 cells and MDA-MB-231 cells

[0145] Dilute Matrigel matrix with serum-free cell culture medium at a ratio of 1:8 at 4 °C, take 100 μL and evenly coat it on the surface of the polycarbonate membrane in the upper chamber, and place it at 37 °C for 1 h to solidify. Seed the cells at 5×10 5Seed the cells at a density of [number] / well in a six-well plate. Discard the culture medium the next day and add culture medium containing 1 μM inhibitor F1 or PROTAC molecule F2. Digest the cells after 48 hours, wash them with 1×PBS, resuspend the cells in medium containing 5% FBS, and adjust the cell density to 5x10 5 cells / mL. Add 650 μL of medium containing 20% FBS to the lower chamber of a 24-well plate. Then, using forceps, place a Transwell insert into the 24-well plate. Take 150 μL of the cell suspension and add it to the upper chamber. Finally, place the plate in an incubator. After 48 hours, remove the insert, aspirate the medium, and gently wipe the Matrigel and the cells in the upper chamber with a cotton swab. Take a new 24-well plate and add 600 μL of 4% paraformaldehyde to each well. Place the insert in and fix for 20 min. Discard the fixative, stain with 0.5% crystal violet for 15 min, wash 3 times with 1×PBS, air dry, and observe and photograph the cells under a microscope. The results are shown in Figure 8 A and Figure 8 B. The PROTAC molecule F2 in the present invention can significantly inhibit the invasion ability of 4T1 and MDA-MB-231 cells and is superior to inhibitor F1.

[0146] Example 8: Effect of PROTAC molecule F2 on the adhesion ability of 4T1 cells and MDA-MB-231 cells

[0147] Dilute FN to 10 μg / mL with serum-free medium. Add 50 μL of 10 μg / mL FN to each well of a 96-well plate and incubate overnight at 4°C. Aspirate the coating solution, add 200 μL of 1% BSA to each well, incubate at 37°C for 2 h, and rinse the 96-well plate 3 times with serum-free medium. Seed the cells at a density of 5x10 5 cells / well in a six-well plate. Discard the culture medium the next day and add culture medium containing 1 μM inhibitor F1 or PROTAC molecule F2. Digest the cells after 48 hours, wash them with 1×PBS, and seed them at a density of 5x10 5 cells / well into the 96-well plate coated with FN. Add 100 μL of the cell suspension to each well. At the same time, set up blank control wells with only culture medium without cells. Set up 3 replicate wells for each group of cells. After culturing in a 37°C, 5% CO2 incubator for 2 h, take out the cell culture plate, discard the culture medium, rinse 3 times with 1×PBS, add 100 μL of 10% CCK8 solution diluted with serum-free medium to each well, and incubate at 37°C for 3 h. Measure the OD value of the sample wells with an enzyme-linked immunosorbent assay (ELISA) reader. Cell adhesion rate = [(ODexperimental group - ODblank group) / (ODcontrol group - ODblank group)] × 100%. The results are shown in Figure 9 A and Figure 9 B. The PROTAC molecule F2 in the present invention can significantly inhibit the adhesion ability of 4T1 and MDA-MB-231 cells and is superior to inhibitor F1.

[0148] Example 9: Reversal of multidrug resistance of PROTAC molecule F2 in HCT8 / T cells, A549 / T cells and MCF-7 / ADR cells

[0149] HCT8 / T cells, A549 / T cells and MCF-7 / ADR cells are human colorectal cancer paclitaxel-resistant cells, human non-small cell lung cancer paclitaxel-resistant cells and human breast cancer doxorubicin-resistant cells respectively, all of which are multidrug-resistant cells with high expression of P-glycoprotein. Add 180 μL of HCT8 / T cell, A549 / T cell, and MCF-7 / ADR cell suspensions with a density of 3000 cells / well into a 96-well plate, and pre-culture the culture plate in an incubator at 37 °C with a volume percentage concentration of 5% CO2 or an air incubator. The next day, add a medium containing gradient concentrations of PTX, DOX, NVB or VCR to the culture plate, and add inhibitor F1 or PROTAC molecule F2 with a final concentration of 1 μM, and incubate in the incubator. After 72 hours, add 10 μL of CCK-8 solution to each well. After incubating the culture plate in the incubator for 1 hour, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader. Cell viability (%) = [OD value (experimental well) - OD value (blank well)] / [OD value (control well) - OD value (blank well)] × 100%. Use the software GraphPad Prism to calculate the IC 50 value. The results are shown in Table 1. The PROTAC molecule F2 in the present invention has an obvious effect of reversing tumor multidrug resistance on HCT8 / T cells, A549 / T cells and MCF-7 / ADR cells and is superior to inhibitor F1.

[0150] Table 1 Reversal of multidrug resistance of F2 in MCF-7 / ADR, HCT8 / T and A549 / T cells

[0151]

[0152]

[0153] Example 10: Effect of PROTAC molecule F2 on the migration ability of HCT8 / T cells and MCF-7 / ADR cells

[0154] Seed the cells at 1x10 6Cells were seeded at a density of [number] / well in a six-well plate. After overnight adherent growth, the culture medium was discarded. Uniform straight lines were vertically scratched in each well with a pipette tip, and the wells were rinsed twice with 1×PBS. Then, culture medium containing drugs was added: For HCT8 / T cells: (i) control group; (ii) 1 μM PTX; (iii) 1 μM F1; (iv) 1 μM F2; (v) 1 μM PTX and 1 μM F1; (vi) 1 μM PTX and 1 μM F2; For MCF-7 / ADR cells: (i) control group; (ii) 3 μM DOX; (iii) 1 μM F1; (iv) 1 μM F2; (v) 3 μM DOX and 1 μM F1; (vi) 3 μM DOX and 1 μM F2. The wound healing was observed and photographed under a microscope at 0, 24, and 48 hours respectively. The results are as Figure 10 A to Figure 10 shown in B. The PROTAC molecule F2 in the present invention can enhance the inhibitory effect of chemotherapeutic drugs on the migration ability of HCT8 / T cells and MCF-7 / ADR cells.

[0155] Example 11: Effect of PROTAC molecule F2 on the invasion ability of HCT8 / T cells and MCF-7 / ADR cells

[0156] Matrigel was diluted with serum-free cell culture medium at a ratio of 1:8 at 4°C. 100 μL was taken and evenly coated on the surface of the polycarbonate membrane in the upper chamber, and placed at 37°C for 1 h to solidify. Cells were seeded at a density of 5×10 5 cells / well in a six-well plate. The next day, the culture medium was discarded, and culture medium containing drugs was added. For HCT8 / T cells: (i) control group; (ii) 1 μM PTX; (iii) 1 μM F1; (iv) 1 μM F2; (v) 1 μM PTX and 1 μM F1; (vi) 1 μM PTX and 1 μM F2; For MCF-7 / ADR cells: (i) control group; (ii) 3 μM DOX; (iii) 1 μM F1; (iv) 1 μM F2; (v) 3 μM DOX and 1 μM F1; (vi) 3 μM DOX and 1 μM F2; For A549 / T cells: (i) control group; (ii) 3 μM PTX; (iii) 1 μM F1; (iv) 1 μM F2; (v) 3 μM PTX and 1 μM F1; (vi) 3 μM PTX and 1 μM F2. After 48 hours, the cells were digested, washed with 1×PBS, and then resuspended in medium containing 5% FBS, and the cell density was adjusted to 5×10 5cells / mL. Add 650 μL of medium containing 20% FBS to the lower chamber of a 24-well plate. Then, use forceps to place the Transwell insert into the 24-well plate. Take 150 μL of the cell suspension and add it to the upper chamber. Finally, place it in an incubator for culture. After 48 hours, remove the insert, aspirate the medium, and gently wipe the Matrigel and the cells in the upper chamber with a cotton swab. Take a new 24-well plate and add 600 μL of 4% paraformaldehyde to each well. Place the insert in and fix for 20 - 30 min. Discard the fixing solution, stain with 0.5% crystal violet for 15 min, wash 3 times with 1×PBS, air dry, and then observe the cells under a microscope and take pictures. The results are as Figure 11 A to Figure 11 shown in C. The PROTAC molecule F2 in the present invention can enhance the inhibitory effect of chemotherapeutic drugs on the invasion ability of HCT8 / T, MCF-7 / ADR, and A549 / T cells.

[0157] Example 12: Effect of PROTAC molecule F2 on the adhesion ability of HCT8 / T cells and A549 / T cells

[0158] Dilute FN to 10 μg / mL with serum-free medium. Add 50 μL of 10 μg / mL FN to each well of a 96-well plate and incubate overnight at 4°C. Aspirate the coating solution, add 200 μL of 1% BSA to each well, incubate at 37°C for 2 h, and rinse the 96-well plate 3 times with serum-free medium. Seed the cells in a six-well plate at a density of 5×10 5 cells / well. Discard the culture medium the next day and add the culture medium containing the drug. For HCT8 / T cells: (i) control group; (ii) 1 μM PTX; (iii) 1 μM F1; (iv) 1 μM F2; (v) 1 μM PTX and 1 μM F1; (vi) 1 μM PTX and 1 μM F2. For A549 / T cells: (i) control group; (ii) 3 μM PTX; (iii) 1 μM F1; (iv) 1 μM F2; (v) 3 μM PTX and 1 μM F1; (vi) 3 μM PTX and 1 μM F2. Digest the cells after 48 hours, wash with 1×PBS, and seed them into the 96-well plate coated with FN at a density of 5×10 5 cells / well. Add 100 μL of the cell suspension to each well. At the same time, set up blank control wells with only culture medium without cells. Set 3 replicate wells for each group of cells. Culture in an incubator at 37°C and 5% CO2 for 2 h. Take out the cell culture plate, discard the culture medium, rinse 3 times with 1×PBS, add 100 μL of 10% CCK8 solution diluted with serum-free medium to each well, and incubate at 37°C for 3 h. Measure the OD value of the sample wells with an enzyme-linked immunosorbent assay (ELISA) reader. Cell adhesion rate = [(ODexperimental group - ODblank group) / (ODcontrol group - ODblank group)] × 100%. The results are as Figure 12 A and Figure 12As shown in Figure B. The PROTAC molecule F2 in the present invention can enhance the ability of chemotherapeutic drugs to inhibit the adhesion of HCT8 / T and A549 / T cells.

[0159] Example 13: The PROTAC molecule F2 increases the sensitivity of MCF-7 and MCF-7 / TAM cells to TAM

[0160] Add 180 μL of MCF-7 or MCF-7 / TAM cell suspension with a density of 3000 cells / well to a 96-well plate, and place the culture plate in a 37 °C incubator with a volume percentage concentration of 5% CO2 or an air incubator for pre-incubation. The next day, add a medium containing a gradient concentration of TAM to the culture plate, and add an inhibitor F1 with a final concentration of 1 μM, 1 μM PROTAC molecule F2, or 3 μM PROTAC molecule F2, and incubate in the incubator. After 72 hours, add 10 μL of CCK-8 solution to each well. After incubating the culture plate in the incubator for 1 hour, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The results are as shown in Figure 13 Figure A and Figure 13 Figure B. Cell viability (%) = [OD value (experimental well) - OD value (blank well)] / [OD value (control well) - OD value (blank well)] × 100%. The PROTAC molecule F2 in the present invention can promote the sensitivity of both MCF-7 and MCF-7 / TAM cells to TAM.

[0161] Example 14: Animal model experiment of the PROTAC molecule F2 reversing the drug resistance of HCT8 / T cells to PTX

[0162] Nude mice (n = 3) were subcutaneously inoculated with HCT8 / T cells on both the left and right sides, 1 × 10 6 cells per side. After 2 weeks, the well-grown xenografts were cut into fragments and subcutaneously transplanted to the right side of the nude mice. When the tumors reached an average volume of 10 mm 3 , the mice were randomly divided into 6 groups (n = 6) and received 14 days of treatment: (i) vehicle (intraperitoneal injection); (ii) PTX (20 mg / kg, intraperitoneal injection, q3d); (iii) F1 (10 mg / kg, intraperitoneal injection, qd); (iv) F2 (15 mg / kg, intraperitoneal injection, qd); (v) F1 (10 mg / kg, intraperitoneal injection, qd) and PTX (20 mg / kg, intraperitoneal injection, q3d); (vi) F2 (15 mg / kg, intraperitoneal injection, qd) and PTX (20 mg / kg, intraperitoneal injection, q3d). The tumor volume was measured separately every two days. The results are as shown in Figure 14 Figure and Figure 15As shown. On the day of ending drug treatment, the average tumor volume (TV), relative tumor volume (RTV), average tumor growth inhibition rate (TGI), and average tumor weight (TW) of each group are shown in Table 2. The calculation formulas are as follows: TV = (width 2 × length) / 2; TGI = (1 - average tumor weight of the treatment group / average tumor weight of the control group) × 100%; RTV = Vt / V0, where V0 is the tumor volume measured when the animals are caged for drug administration, and Vt is the tumor volume at each measurement. The PROTAC molecule F2 in the present invention can reverse the drug resistance of tumor multidrug-resistant cells HCT8 / T cells to the chemotherapeutic drug PTX in vivo.

[0163] Table 2: Tumor volume, relative tumor volume, tumor weight, and tumor growth inhibition rate of HCT8 / T xenografts before and after treatment

[0164]

[0165] The above is the preferred implementation manner of the present invention. It should be noted that for those of ordinary skill in the technical field covered by the present invention, without departing from the method of the present invention, several supplements and improvements can be made, and these supplements and improvements should also be regarded as the protection scope of the present invention.

Claims

1. A compound targeting FAK or a pharmaceutically acceptable salt thereof, characterized in that: The compound has a general formula structure as shown below: wherein R1 is selected from hydrogen, halogen, -OR a , -NR a R a , -CF3, -CN, -OCN, -SCN, -NO, -NO2, -C 1-3 alkyl, -C 1-3 haloalkyl or -C 1-3 haloalkoxy; Each R a is independently selected from hydrogen or selected from optionally substituted -C 1-6 alkyl, -C 3-10 cycloalkyl, -C 4-11 cycloalkylalkyl, -C 6-10 aryl, -C 7-16 arylalkyl, 2-6 membered heteroalkyl, 3-8 membered heterocycloalkyl, 4-14 membered heterocycloalkylalkyl, 5-12 membered heteroaryl or 6-18 membered heteroarylalkyl; A is a linker fragment required in PROTAC technology; B is a short-chain alkyl group or a substituted thalidomide fragment of a target ubiquitin ligase required in PROTAC technology; C is a target FAK protein molecular fragment.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound or its pharmaceutically acceptable salt is a PROTAC molecular compound targeting FAK.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: wherein R1 is selected from hydrogen, halogen, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -C 1-3 alkyl, -C 1-3 haloalkyl or -C 1-3 haloalkoxy.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: A is selected from nitrogen-containing alkyl chains: -NH(CH2) n NH-, -NHCO(CH2) n NH-, where n = 2 - 5; alternatively, A is selected from aliphatic heterocyclic chains: n = 1 - 2, m = 1 - 3, n = 1 - 2, preferably, m = 1, n = 2.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: B is selected from Preferably, B is selected from 6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from the following:

7. A process for preparing the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, characterized in that: The preparation method comprises the following steps: First, starting materials are used and the key intermediate is prepared through multiple-step reactions Secondly, the thalidomide fragment targeting the ubiquitin ligase is combined with the required Linker linking fragment through multiple-step reactions to obtain the key intermediate Finally, the key intermediate is reacted with to obtain the said compound through reaction combination, and the said compound is reacted with common inorganic or organic acids to obtain its corresponding pharmaceutically acceptable salts.

8. A drug composition targeting FAK, characterized in that: The pharmaceutical composition comprises the compound or its pharmaceutically acceptable salt according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier or excipient. Preferably, the compound or its pharmaceutically acceptable salt is a PROTAC molecular compound targeting FAK.

9. Use of the compound or its pharmaceutically acceptable salt according to any one of claims 1 to 6 in the preparation of an anti-tumor drug.

10. Use of the compound or its pharmaceutically acceptable salt according to any one of claims 1 to 6 in the preparation of a drug for reversing tumor multi-drug resistance or having drug sensitization ability.

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