A class of protac molecules with crizotinib as a target head, preparation method and application

Abstract

The application discloses a kind of PROTAC molecules with crizotinib as target head, preparation method and application, belong to the field of biological medicine;Preparation method includes: chloro carboxylic acid, thionyl chloride and anhydrous DMF are mixed, after heating reflux reaction, remove thionyl chloride, obtain acyl chloride;Pomalidomide, acyl chloride, organic solvent are mixed and reacted, then filtered to obtain intermediate M;Intermediate M, crizotinib, Na2CO3 are mixed and reacted, then filtered to obtain the PROTAC molecule with crizotinib as target head.The in vitro biological evaluation shows that the new PROTAC molecule can efficiently and selectively induce ubiquitination and degradation of target protein, and exhibits significant antiproliferative activity in drug-resistant tumor cell models and animal models;The application proves that the PROTAC molecule has great potential in overcoming drug resistance of traditional inhibitors, and provides a promising lead compound for developing new therapies for treating refractory diseases such as cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a class of PROTAC molecules targeting crizotinib, their preparation methods, and applications. Background Technology

[0002] Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor superfamily, and its gene rearrangements and fusions are key drivers in various malignancies. In non-small cell lung cancer, EML4-ALK is the most common fusion type, while in human degenerative large cell lymphoma, NPM-ALK is the predominant oncogenic form. Tyrosine kinase inhibitors targeting ALK, such as first-generation crizotinib, second-generation alectinib and ceritinib, and third-generation loratinib, have significantly improved the prognosis of patients with ALK-positive tumors.

[0003] The clinical application of ALK TKIs has consistently been limited by acquired resistance. The resistance mechanisms mainly fall into two categories: first, secondary point mutations in the ALK kinase domain (such as G1202R, L1196M, F1174L, etc.), which directly weaken the affinity of TKIs for the ATP-binding pocket; and second, activation of bypass signaling (such as upregulation of epidermal growth factor receptor EGFR) or transformation of tumor cell types. Although next-generation TKIs can overcome some older mutations, they can also select for new resistance mutations (such as the ALK G1202R / L1196M complex mutation resistant to lorlatinib), leading to treatment failure. Furthermore, traditional "site-driven" inhibition requires sustained high drug concentrations to block kinase activity and cannot eliminate the scaffold function of ALK fusion proteins, rendering them ineffective against bypass resistance mechanisms. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a class of PROTAC molecules targeting crizotinib, their preparation method and applications, thus solving the problems in the prior art.

[0005] The objective of this invention can be achieved through the following technical solutions: The general structural formula of a PROTAC molecule targeting crizotinib is: Where n is a positive integer from 1 to 5.

[0006] Furthermore, the synthetic route of the PROTAC molecule targeting crizotinib is as follows: Furthermore, the method for preparing the PROTAC molecule targeting crizotinib includes the following steps: S1, chlorocarboxylic acid, thionyl chloride and anhydrous DMF are mixed, heated under reflux and reacted to remove thionyl chloride, yielding acyl chloride; S2, pomalidomide, acyl chloride, and organic solvent are mixed and reacted, and then filtered to obtain intermediate M; S3 involves mixing intermediate M, crizotinib, and Na2CO3, reacting the mixture, and then filtering to obtain a PROTAC molecule targeting crizotinib.

[0007] Furthermore, the structural formula of the chlorocarboxylic acid is: The structural formula of the acyl chloride is: The structural formula of the intermediate M is: In this context, the values ​​of n in the chlorocarboxylic acid, acyl chloride, intermediate M, and the PROTAC molecule are the same.

[0008] Furthermore, the process for removing thionyl chloride includes: removing excess thionyl chloride by rotary evaporation under reduced pressure, and removing residual thionyl chloride by adding anhydrous toluene.

[0009] Furthermore, the molar ratio of pomalidomide to acyl chloride is 18.3:25.

[0010] Furthermore, the organic solvent is tetrahydrofuran.

[0011] Furthermore, the molar ratio of intermediate M, crizotinib, and Na2CO3 is 4.3:3.5:14.

[0012] The aforementioned PROTAC molecules targeting crizotinib are used in the preparation of drugs for the treatment of human degenerative large cell lymphoma or lung adenocarcinoma.

[0013] A drug comprising the aforementioned PROTAC molecule targeting crizotinib.

[0014] The beneficial effects of this invention are: This invention designs and prepares a novel PROTAC molecule that simultaneously binds a target protein ligand and an E3 ubiquitin ligase ligand via an optimized linker, forming a stable ternary complex. This design utilizes the intracellular natural ubiquitin-proteasome system to achieve "event-driven" degradation of specific disease-driving proteins, rather than traditional inhibition. The preparation process employs a modular synthesis strategy for efficient connection of functional units. In vitro biological evaluations show that this novel PROTAC molecule can efficiently and selectively induce ubiquitination and degradation of the target protein, and exhibits significant antiproliferative activity in drug-resistant tumor cell models and animal models. This invention demonstrates the great potential of this PROTAC molecule in overcoming resistance to traditional inhibitors, providing a promising lead compound for the development of novel therapies for cancer and other refractory diseases. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The inhibitory activity of the target compound on NCI-H3122 and Karpas299 cells at different concentrations was measured. Figure 2 It represents the expression level of a specific protein in NCI-H3122 cells; Figure 3 It represents the expression level of a specific protein in Karpas299 cells. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The general structural formula of a PROTAC molecule targeting crizotinib is: The value of n ranges from 1 to 5.

[0019] The synthetic route for PROTAC molecules targeting crizotinib is as follows: .

[0020] The preparation method of PROTAC molecules targeting crizotinib includes the following steps: S1, chlorocarboxylic acid, sulfoxide (SOCl2) and anhydrous DMF (catalyst) are mixed, heated under reflux and reacted to remove sulfoxide, yielding acyl chlorides with different carbon chain lengths; S2, pomalidomide, acyl chloride and tetrahydrofuran are mixed and reacted, and then filtered to obtain intermediate M; S3, intermediate M, crizotinib, and Na2CO3 are mixed and reacted, and then filtered to obtain PROTAC molecule Y with crizotinib as the target; The structural formula of chlorocarboxylic acids is: The structural formula of acyl chloride is: The structural formula of intermediate M is: Among them, the values ​​of n in chlorocarboxylic acid, acyl chloride, intermediate M, and PROTAC molecule Y are the same, ranging from 1 to 5.

[0021] The technical solution of the present invention will be described below through the following embodiments; wherein the sources of each raw material in the embodiments are as follows: Chloroacetic acid: Manufacturer is Anaiji Chemical, CAS: 79-11-8; 3-Chloropropionic acid: Manufacturer: Anaiji Chemical, CAS: 107-94-8; 4-Chlorobutyric acid: Manufacturer: Anaiji Chemical, CAS: 627-00-9; 5-Chlorovalerate: Manufacturer: Anaiji Chemical, CAS: 1119-46-6; 6-Chlorohexanoic acid: Manufacturer: Anaiji Chemical, CAS: 4224-62-8; Thionyl chloride: Manufacturer is Anaiji Chemical, CAS: 7719-09-7; Anhydrous DMF: Manufacturer: Anaiji Chemical, CAS: 68-12-2; Pomalidomide: Manufacturer: Anaiji Chemical, CAS: 19171-19-8; Crizotinib: Manufacturer: Anaiji Chemical, CAS: 877399-52-5.

[0022] Example 1 This embodiment describes the preparation process of acyl chlorides C1~C5; 1) Preparation of C1: Chloroacetic acid (4.7 g, 50 mmol), 10 mL of thionyl chloride, and 0.1 mL of anhydrous DMF were used as catalysts. The reaction was stopped after heating under reflux for 5 h. Excess thionyl chloride was removed by rotary evaporation under reduced pressure, and 10 mL of anhydrous toluene was added to remove the remaining trace amount of thionyl chloride, yielding acyl chloride C1.

[0023] 2) Preparation of C2: 3-chloropropionic acid (5.4 g, 50 mmol), 10 mL of thionyl chloride, and 0.1 mL of anhydrous DMF were used as catalysts. The reaction was stopped after heating under reflux for 5 h. Excess thionyl chloride was removed by rotary evaporation under reduced pressure, and 10 mL of anhydrous toluene was added to remove the remaining trace amount of thionyl chloride, yielding acyl chloride C2.

[0024] 3) Preparation of C3: 4-chlorobutyric acid (6.1 g, 50 mmol), 10 mL of thionyl chloride, and 0.1 mL of anhydrous DMF were used as catalysts. The reaction was stopped after heating under reflux for 5 h. Excess thionyl chloride was removed by rotary evaporation under reduced pressure, and 10 mL of anhydrous toluene was added to remove the remaining trace amount of thionyl chloride, yielding acyl chloride C3.

[0025] 4) Preparation of C4: 5-Chlorovalerate (6.8 g, 50 mmol), 10 mL of thionyl chloride and 0.1 mL of anhydrous DMF were used as catalysts. The reaction was stopped after heating under reflux for 5 h. Excess thionyl chloride was removed by rotary evaporation under reduced pressure, and 10 mL of anhydrous toluene was added to remove the remaining trace amount of thionyl chloride, yielding acyl chloride C4.

[0026] 5) Preparation of C5: 6-Chlorohexanoic acid (7.5 g, 50 mmol), 10 mL of thionyl chloride and 0.1 mL of anhydrous DMF were used as catalysts. The reaction was stopped after heating under reflux for 5 h. Excess thionyl chloride was removed by rotary evaporation under reduced pressure, and 10 mL of anhydrous toluene was added to remove the remaining trace amount of thionyl chloride, yielding acyl chloride C5.

[0027] In the structural formulas of acyl chlorides C1~C5, the value of n is 1~5 respectively.

[0028] Example 2 In this embodiment, the preparation process of intermediates M1-M5 is described; 1) Preparation of intermediate M1; The structural formula for M1 is: .

[0029] The preparation process includes: Pomalidomide (5.0 g, 18.3 mmol), acyl chloride C1 (2.8 g, 25 mmol), and tetrahydrofuran 30 mL were placed in a single-necked flask and reacted at 60 °C for 6 h. After cooling to room temperature, the mixture was filtered under reduced pressure. The filter cake was washed with an appropriate amount of tetrahydrofuran and dried to obtain 4.4 g of white solid, with a yield of 65.7%.

[0030] The proton NMR spectrum of intermediate M1 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm):δ = 11.18 (s,1H), 10.32 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H), 7.91 – 7.87 (m, 1H), 7.69 (d, J =4.0 Hz, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 4.54 (s, 2H), 2.94~2.85 (m, 1H), 2.67 - 2.54 (m, 2H), 2.09 - 2.05 (m, 1H). 13 C NMR (100 MHz, DMSO- d6 , ppm): δ =173.26, 170.24, 168.28, 167.06, 166.18, 136.90, 136.04, 131.92, 126.00,119.51, 117.74, 49.43, 43.63, 31.40, 22.42. 2) Preparation of intermediate M2; The structural formula for M2 is: .

[0031] The preparation process includes: Pomalidomide (5.0 g, 18.3 mmol), acyl chloride C2 (3.1 g, 25 mmol.), and tetrahydrofuran 30 mL were placed in a single-necked flask and reacted at 60 °C for 6 h. After cooling to room temperature, the mixture was filtered under reduced pressure. The filter cake was washed with an appropriate amount of tetrahydrofuran and dried to obtain 4.6 g of white solid, with a yield of 69.7%.

[0032] The proton NMR spectrum of intermediate M2 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm):δ = 11.16 (s,1H), 9.9 (s, 1H), 8.43 (d, J= 8.0 Hz, 1H), 7.87 – 7.83 (m, 1H), 7.66 (d, J = 8.0Hz, 1H), 5.18-5.13 (m, 1H), 3.91-3.88 (m, 2H), 3.02~2.99 (m, 2H), 2.95-2.86(m, 1H), 2.64- 2.55 (m, 2H), 2.1-2.06 (m, 1H). 13 C NMR (100 MHz, DMSO- d6 , ppm): δ = 173.28, 170.30, 169.51, 167.78, 167.10, 136.55, 136.42, 132.07, 127.34,119.22, 118.01, 49.37, 40.75, 31.40, 22.44. 3) Preparation of intermediate M3; The structural formula for M3 is: .

[0033] The preparation process includes: Pomalidomide (5.0 g, 18.3 mmol), acyl chloride C3 (3.5 g, 25 mmol), and tetrahydrofuran (30 mL) were placed in a single-necked flask and reacted at 60 °C for 6 h. After cooling to room temperature, the mixture was filtered under reduced pressure. The filter cake was washed with an appropriate amount of tetrahydrofuran and dried to obtain 4.9 g of white solid, with a yield of 71.0%.

[0034] The proton NMR spectrum of intermediate M3 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm):δ = 11.15 (s,1H), 9.80 (s, 1H), 8.40 (d, J = 8.3 Hz, 1H), 7.85-7.81 (m, 1H), 7.63 - 7.61 (m,1H), 5.17 - 5.12 (m, 1H), 3.72 (t, J = 8.0 Hz, 2H), 2.95 - 2.86 (m, 1H), 2.64 -2.52 (m, 4H), 2.10 - 2.03 (m, 3H). 13 C NMR (100 MHz, DMSO- d6, ppm): δ = 173.27,171.52, 170.29, 167.91, 167.13, 136.71, 136.52, 131.98, 127.23, 119.01,117.98, 49.36, 45.18, 34.02, 31.40, 28.21, 22.44. 4) Preparation of intermediate M4; The structural formula for M4 is: .

[0035] The preparation process includes: Pomalidomide (5.0 g, 18.3 mmol), acyl chloride C4 (3.9 g, 25 mmol), and tetrahydrofuran (30 mL) were placed in a single-necked flask and reacted at 60 °C for 6 h. After cooling to room temperature, the mixture was filtered under reduced pressure. The filter cake was washed with an appropriate amount of tetrahydrofuran and dried to obtain 5.1 g of white solid, with a yield of 70.8%.

[0036] The proton NMR spectrum of intermediate M4 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm):δ = 11.15 (s,1H), 9.73 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H), 7.85 – 7.81 (m, 1H), 7.63 (d, J =8.0 Hz, 1H), 5.17 (dd, J = 12.0, 4.0 Hz, 1H), 3.70-3.67 (m, 2H), 2.95~2.86 (m,1H), 2.64 - 2.52 (m, 4H), 2.10-2.05 (m, 1H), 1.81-1.71 (m, 4H); 13 C NMR (100MHz, DMSO- d6 , ppm): δ = 173.28, 172.19, 170.30, 168.05, 167.14, 136.88,136.56, 131.95, 126.94, 118.87, 117.63, 49.35, 45.52, 35.90, 31.83, 31.40,22.56, 22.44. 5) Preparation of intermediate M5; The structural formula for M5 is: .

[0037] The preparation process includes: Pomalidomide (5.0 g, 18.3 mmol), acyl chloride C5 (4.2 g, 25 mmol), and tetrahydrofuran (30 mL) were placed in a single-necked flask and reacted at 60 °C for 6 h. After cooling to room temperature, the mixture was filtered under reduced pressure. The filter cake was washed with an appropriate amount of tetrahydrofuran and dried to obtain 5.1 g of white solid, with a yield of 68.9%.

[0038] The proton NMR spectrum of intermediate M5 is as follows: 1 H NMR (400 MHz, CDCl3, ppm): δ = 9.41 (s, 1H),8.83 (d, J = 8.0 Hz, 1H), 8.25 (s, 1H)7.74-7.70 (m, 1H), 7.56 (d, J = 8.0 Hz,1H), 4.98-4.93 (m, 1H), 3.57-3.54 (m, 2H), 2.95~2.91 (m, 1H), 2.83 - 2.72 (m,2H), 2.50 - 2.47 (m, 2H), 2.18-2.15 (m, 1H), 1.87-1.75 (m, 4H), 1.58-1.51 (m,2H); 13 C NMR (100 MHz, CDCl3, ppm): δ = 172.00, 170.78, 169.20, 167.90, 166.70,137.81, 136.54, 131.07, 125.32, 118.56, 115.28, 49.26, 44.77, 37.69, 32.24,31.39, 26.39, 24.45, 22.69. Example 3 This embodiment describes the preparation of PROTAC molecule Y with crizotinib as the target; 1) Preparation of Y1; The structural formula of Y1 is: Preparation methods include: M1 (1.5 g, 4.3 mmol), crizotinib (1.6 g, 3.5 mmol), and Na2CO3 (1.5 g, 14 mmol) were placed in a single-necked flask and reacted at 60 °C for 5 h. After cooling to room temperature, a white solid precipitated. The solid was filtered, washed three times with an appropriate amount of methanol, and dried to obtain 1.37 g of white solid Y1, with a yield of 51.3%.

[0039] The proton NMR spectrum of Y1 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm):δ = 1 H NMR (400 MHz, DMSO) δ= 11.14 (s, 1H), 10.99 (s, 1H), 8.81 (d, J = 12.0 Hz, 1H), 7.96 (d, J =4.0 Hz, 1H), 7.88-7.84 (m, 1H), 7.76 (s, 1H), 7.61 – 7.55 (m, 3H), 7.46-7.42(m, 1H), 6.90 (s, 1H), 6.11-6.08 (m, 1H), 5.67 (s, 2H), 5.17-5.13 (m, 1H), 4.18 (brs, 1H), 3.27-3.22 (m, 2H), 2.99-2.89 (m, 4H), 2.67 – 2.56 (m, 4H),2.20-2.10 (m, 4H), 1.81 (d, J = 4.0 Hz, 3H). 13 C NMR (100 MHz, CDCl3, ppm): δ =173.24, 170.63, 170.39, 168.60, 167.32, 149.92, 139.24, 137.27, 136.89,136.82, 135.85, 135.10, 131.81, 131.07, 129.25, 124.77, 124.15, 121.61,119.61, 118.45, 118.05, 117.82, 117.74, 116.32, 114.87, 72.38, 61.73, 58.07,52.65, 49.41, 32.23, 32.10, 31.38, 22.35, 19.09. 2) Preparation of Y2; The structural formula for Y2 is: Preparation methods include: M2 (1.6 g, 4.3 mmol), crizotinib (1.6 g, 3.5 mmol), and Na2CO3 (1.5 g, 14 mmol) were placed in a single-necked flask and reacted at 60 °C for 5 h. After cooling to room temperature, a white solid precipitated. The solid was filtered, washed three times with an appropriate amount of methanol, and dried to obtain 1.1 g of white solid Y2, with a yield of 40.7%.

[0040] The proton NMR spectrum of Y2 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm): δ = 11.14 (s, 1H), 10.47 (d, J = 3.1 Hz, 1H), 8.56 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.85 – 7.81(m, 1H), 7.74 (d, J = 4.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.57 (dd, J = 8.0, 4.0Hz, 1H), 7.50 (s, 1H), 7.46-7.41 (m, 1H), 6.89 (d, J = 4.0 Hz, 1H), 6.10 - 6.05(m, 1H), 5.66 (s, 2H), 5.15-5.10 (m, 1H), 4.14-4.09 (m, 1H), 3.10-3.04 (d, J= 12.9 Hz, 2H), 2.69 – 2.66 (m, 4H), 2.54 - 2.52 (m, 4H), 2.19-2.13 (m, 2H), 2.06-1.99 (s, 4H), 1.80 (d, J = 8.0 Hz, 3H). 13 C NMR (100 MHz, DMSO- d6 , ppm): δ =173.19, 171.91, 170.29, 167.90, 167.18, 149.90, 139.24, 137.28, 136.92,136.48, 135.91, 134.99, 132.04, 129.18, 126.94, 124.08, 121.59, 121.40,119.47, 118.70, 118.01, 117.87, 117.78, 117.29, 114.84, 72.40, 58.93, 53.54,52.09, 49.40, 36.25, 34.61, 31.90, 31.42, 22.41, 19.06. 3) Preparation of Y3; The structural formula for Y3 is: Preparation methods include: M3 (1.6 g, 4.3 mmol), crizotinib (1.6 g, 3.5 mmol), and Na2CO3 (1.5 g, 14 mmol) were placed in a single-necked flask and reacted at 60 °C for 5 h. After cooling to room temperature, a white solid precipitated. The solid was filtered, washed three times with an appropriate amount of methanol, and dried to obtain 1.25 g of white solid Y3, with a yield of 45.1%.

[0041] The proton NMR spectrum of Y3 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm): δ = 11.17 (s, 1H), 9.72 (s, 1H), 8.50 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.84 – 7.80 (m, 1H), 7.73 (d, J = 4.0 Hz, 1H), 7.60 – 7.56 (m, 2H), 7.47-7.42 (m, 2H), 6.87 (d, J = 1.6 Hz, 1H), 6.09 (d, J = 8.0 Hz, 1H), 5.67 (s, 2H), 5.17-5.12 (m, 1H), 4.05 – 4.00 (m,1H), 2.93 (d, J = 8.0 Hz, 2H), 2.61 – 2.56 (m, 4H), 2.39-2.35 (m, 2H), 2.06-2.00 (m, 4H), 1.95-1.92 (m, 2H), 1.85-1.79 (m, 7H). 13 C NMR (100 MHz, DMSO- d6,ppm): δ = 173.24, 172.57, 170.27, 168.27, 167.16, 149.89, 139.24, 137.29,137.15, 136.59, 135.89, 134.92, 131.93, 129.22, 126.62, 123.88, 121.60,121.41, 119.48, 118.64, 118.03, 117.84, 117.80, 117.28, 114.84, 72.39, 60.23,59.00, 57.27, 52.39, 49.36, 35.24, 32.49, 31.39, 26.80, 22.81, 22.44, 19.07. 4) Preparation of Y4; The structural formula for Y4 is: Preparation methods include: M4 (1.7 g, 4.3 mmol), crizotinib (1.6 g, 3.5 mmol), and Na2CO3 (1.5 g, 14 mmol) were placed in a single-necked flask and reacted at 60 °C for 5 h. After cooling to room temperature, a white solid precipitated. The solid was filtered, washed three times with an appropriate amount of methanol, and dried to obtain 1.33 g of white solid Y4, with a yield of 47.2%.

[0042] The proton NMR spectrum of Y4 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm): δ = 11.17 (s, 1H), 9.72 (s, 1H), 8.50 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.84 – 7.80 (m, 1H), 7.73 (d, J = 4.0 Hz, 1H), 7.60 – 7.56 (m, 2H), 7.47-7.42 (m, 2H), 6.87 (d, J = 1.6 Hz, 1H), 6.09 (d, J = 8.0 Hz, 1H), 5.67 (s, 2H), 5.17-5.12 (m, 1H), 4.05 – 4.00 (m,1H), 2.93 (d, J= 8.0 Hz, 2H), 2.61 – 2.56 (m, 4H), 2.39-2.35 (m, 2H), 2.06-2.00 (m, 4H), 1.95-1.92 (m, 2H), 1.85-1.79 (m, 7H). 13 C NMR (100 MHz, DMSO- d6 ,ppm): δ = 173.24, 172.57, 170.27, 168.27, 167.16, 149.89, 139.24, 137.29,137.15, 136.59, 135.89, 134.92, 131.93, 129.22, 126.62, 123.88, 121.60,121.41, 119.48, 118.64, 118.03, 117.84, 117.80, 117.28, 114.84, 72.39, 60.23,59.00, 57.27, 52.39, 49.36, 35.24, 32.49, 31.39, 26.80, 22.81, 22.44, 19.07. 5) Preparation of Y5; The structural formula for Y5 is: Preparation methods include: M5 (1.7 g, 4.3 mmol), crizotinib (1.6 g, 3.5 mmol), and Na2CO3 (1.5 g, 14 mmol) were placed in a single-necked flask and reacted at 60 °C for 5 h. After cooling to room temperature, a white solid precipitated. The solid was filtered, washed three times with an appropriate amount of methanol, and dried to obtain 1.65 g of white solid Y5, with a yield of 46.9%.

[0043] The proton NMR spectrum of Y5 is as follows: 1 H NMR (400 MHz, DMSO- d6 , ppm): δ = 11.17 (s, 1H), 9.72 (s, 1H), 8.50 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.84 – 7.80 (m, 1H), 7.73 (d, J = 4.0 Hz, 1H), 7.60 – 7.56 (m, 2H), 7.47-7.42 (m, 2H), 6.87 (d, J = 1.6 Hz, 1H), 6.09 (d,J = 8.0 Hz, 1H), 5.67 (s, 2H), 5.17-5.12 (m, 1H), 4.05 – 4.00 (m,1H), 2.93 (d, J = 8.0 Hz, 2H), 2.61 – 2.56 (m, 4H), 2.39-2.35 (m, 2H), 2.06-2.00 (m, 4H), 1.95-1.92 (m, 2H), 1.85-1.79 (m, 7H). 13 C NMR (100 MHz, DMSO- d6 ,ppm): δ = 173.24, 172.57, 170.27, 168.27, 167.16, 149.89, 139.24, 137.29,137.15, 136.59, 135.89, 134.92, 131.93, 129.22, 126.62, 123.88, 121.60,121.41, 119.48, 118.64, 118.03, 117.84, 117.80, 117.28, 114.84, 72.39, 60.23,59.00, 57.27, 52.39, 49.36, 35.24, 32.49, 31.39, 26.80, 22.81, 22.44, 19.07. Example 4 In this embodiment, the effects of compounds (Y0 and Y1-Y5 prepared in Example 3) on the viability of NCI-H3122 cells and Karpas299 cells and the expression levels of ALK and p-ALK (Y1507) proteins were studied using a CCK8 kit and Western blotting experiments.

[0044] Y0 represents crizotinib, manufactured by Anaiji Chemical, CAS: 877399-52-5; 1. Experimental reagents include: Fetal bovine serum (12003C), RPMI 1640 medium (R8758) (Sigma-Aldrich, Darmstadt, Germany); NCI-H3122 special medium (ZM0754) (ZQXZBIO, Shanghai, China); trypsin-EDTA digestion solution (25200056) (Gibco, Grand Island, NY, USA); CCK8 kit (C0037) (Beyotime, ShangHaier, China); PVDF membrane (0.2 μm) (ISEQ00010) (Millipore, Schwalbach, Germany); StarSignal Western Protein Marker (10-200 kDa) (M227-01) (GenStar, Beijing, China); Ponceau S (97063-650), Tween 20 (97063-872), PMSF (97064-898) (Amresco, VWR International, OH, USA); Protein blot membrane regeneration solution (ZN1923, Biolab, Beijing, China); 30% acrylamide (ST003), Ammonium persulfate substitute (APSsubstitute) (ST005), 1.0 mol / L Tris pH 6.8 (ST768), 1.5 mol / L Tris pH 8.8 (ST789) (Beyotime, Shanghai, China); BCA protein quantification kit (KGB2101), RIPA lysis buffer (KGB5203) (KeyGEN Biotech, Nanjing, China); Loading buffer (WB2001) (NCM Biotech, Suzhou, China); 10% SDS (BL517A) (Biosharp, HeFei, China); TEMED (AR1165) (BOSTER, WuHan, China); Anhydrous ethanol (10009257), diethanol (10014118) (SCRC, Shanghai, China); Transfer powder (G2017), electrophoresis powder (G2018), TBST powder (G0001) (Servicebio, WuHan, China); ECL luminescence solution (32209) (Thermo Fisher Scientific, Pittsburgh, PA). USA); Anti-p-ALK (Y1507) Antibody (AF3489) (Affinity, Changzhou, China); Anti-ALK Antibody (24184-1-AP) and Anti-β-actin Antibody (20536-1-AP) were purchased from Proteintech (Proteintech, Wuhan, China); Goat Anti-Rabbit IgG H&L (HRP) (ab205718) was purchased from Abcam (Abcam, Cambridge, UK).

[0045] 2. The experimental apparatus includes: Ultra-low temperature freezer (FORMA 700), Thermo Fisher Scientific; pharmaceutical storage cabinet (YC-300L), Zhongke Meiling Cryogenic Technology Co., Ltd.; ultrapure water system (Direct-Q with pump), Millipore; ultra-clean workbench (SW-CJ-2FD), Suzhou Purification Equipment Co., Ltd.; low-temperature high-speed centrifuge (3K15), Sigma; electronic balance (BS224), Beijing Sartorius Instrument Systems Co., Ltd.; water-jacketed CO2 incubator (Forma 3111), Thermo Electron, USA; full-wavelength microplate reader (HBS-SCANX), Nanjing Detie Biotechnology Co., Ltd.; inverted microscope (TS2), Nikon, Japan; Western blotting system (model: Criterion™ electrophoresis tank, Trans-Blot® transfer tank), Bio-Rad; luminescence imaging workstation (Tanon 5200), Tanon. Company; Decolorizing Shaker (Xk-8), Jiangsu Xinkang Medical Equipment Co., Ltd.

[0046] 3. The experimental cells were selected as follows: NCI-H3122 cells (ZQ0754) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (ZQXZBIO, Shanghai, China).

[0047] Karpas299 cells (06072604) were purchased from Sigma-Aldrich (Sigma-Aldrich, Darmstadt, Germany).

[0048] 4. Experimental methods: 4.1 Cell Culture Conditions NCI-H3122 cells were passaged and cultured in NCI-H3122-specific medium. When the cells reached 90% confluence, the old medium was discarded, and the cells were washed twice with 2 mL PBS. After discarding the PBS, 1 mL of trypsin-EDTA digestion solution was added. The cells were observed under a microscope for about 30 seconds. Once the cells became rounded, 2 mL of complete medium was quickly added to stop the digestion. The cells were gently pipetted and collected. The cells were centrifuged at 800 rpm for 5 min at 4°C. The supernatant was discarded, and the cells were resuspended in complete medium and cultured in an incubator at 37°C with 5% CO2. The cells were then divided into flasks and cultured, with the medium changed every 2 to 3 days.

[0049] Karpas299 cells were passaged and cultured in RPMI 1640 medium containing 20% ​​FBS. When the cells reached 90% confluence, the old medium was discarded, and the cells were washed twice with 2 mL PBS. The PBS was discarded, and the cells were collected and centrifuged at 800 rpm for 5 min at 4°C. The supernatant was discarded, and the cells were resuspended in complete medium and cultured in an incubator at 37°C with 5% CO2. The cells were then divided into flasks and cultured, with the medium changed every 2 to 3 days.

[0050] 4.2 Experimental groupings are as follows; NC group: NCI-H3122 cells and Karpas299 cells were cultured normally for 72 h. (N=3) Y0 groups (100, 500, 1000 nM): NCI-H3122 cells and Karpas299 cells were incubated for 72 h in a medium containing Y0 (final concentrations of 100, 500, and 1000 nM, respectively). (N=3) Y1 group (1, 5, 10 μM): NCI-H3122 cells and Karpas299 cells were incubated for 72 h in medium containing Y1 (final concentrations of 1, 5, and 10 μM, respectively). (N=3) Y2 groups (1, 5, 10 μM): NCI-H3122 cells and Karpas299 cells were incubated for 72 h in medium containing Y2 (final concentrations of 1, 5, and 10 μM, respectively). (N=3) Y3 group (1, 5, 10 μM): NCI-H3122 cells and Karpas299 cells were incubated for 72 h in medium containing Y3 (final concentrations of 1, 5, and 10 μM, respectively). (N=3) Y4 groups (1, 5, 10 μM): NCI-H3122 cells and Karpas299 cells were incubated for 72 h in medium containing Y4 (final concentrations of 1, 5, and 10 μM, respectively). (N=3) Y5 groups (1, 5, 10 μM): NCI-H3122 cells and Karpas299 cells were incubated for 72 h in medium containing Y5 (final concentrations of 1, 5, and 10 μM, respectively). (N=3) After incubation, cell viability was assessed using the CCK8 assay kit, and Western blotting was performed on each group. Expression levels of ALK and p-ALK (Y1507) in cells.

[0051] 4.3 CCK8 Experiment Cells from each group were collected by centrifugation at a concentration of 2 × 10⁻⁶. 3 Cells were seeded at a density of 100 cells / well into 96-well plates, with 3 replicates per group. Appropriate culture medium was added according to the grouping. After 72 h of incubation, CCK-8 assay was performed: old culture medium was aspirated, the plates were washed three times with PBS, and 100 μL of medium containing 10% CCK-8 working solution was added to each well. The plates were gently shaken and incubated in a cell culture incubator. After 2 h of incubation, the absorbance values ​​were measured at 450 nm using a microplate reader, and comparative analyses were performed.

[0052] 4.4 Western Blot 4.4.1 Extraction of total cellular protein Cell suspensions prepared for each group were seeded into 6-well plates at a density of 5 × 10⁵ cells per well. When cell confluence reached 90%, cells were treated according to their respective experimental groups. After treatment, cells were collected using trypsin, centrifuged, and the supernatant was discarded. The cell samples were washed twice with pre-chilled PBS. For every 100 μL compressed volume of cell sample, 1 mL of RIPA containing PMSF was added. After complete lysis, the cells were centrifuged at 12000 g for 5 min at 4 °C. The supernatant was immediately transferred to a pre-chilled Eppendorf tube, yielding the extracted cell protein, which was then frozen at -80 °C for later use. Protein quantification was performed using the BCA method. Finally, 5 × loading buffer was added, and the sample was incubated in a boiling water bath for 10 min. The sample preparation was complete and the sample could be stored at -20 °C.

[0053] 4.4.2 Gel electrophoresis and membrane transfer Depending on the molecular weight of the protein to be tested, prepare 10% separating gel and 5% stacking gel, and pour SDS-PAGE gels as shown in Tables 1 and 2: Table 1. SDS-PAGE Electrophoretic Separation Gel Formulation Table Add 5 mL of the prepared separating gel to each gel plate, then add isopropanol and press the gel. After the gel line forms (gelation is complete), lay the gel rack horizontally and remove the isopropanol with filter paper.

[0054] Table 2 SDS-PAGE Electrophoresis Stacking Gel Formulation Table After preparing and mixing the stacking gel, pour 2 mL of the prepared stacking gel into the top of the separating gel, and immediately insert the comb vertically, keeping the comb horizontal. After the stacking gel solidifies, remove the comb and place the gel in an electrophoresis tank containing electrophoresis buffer.

[0055] After adding an appropriate amount of pre-cooled 1× electrophoresis buffer, begin sample loading (add 5 μL of marker and sample to each well from left to right). Adjust the sample loading volume to 60 μg. Electrophoresis is performed at 80 V for approximately 30 min. Once the sample enters the separating gel, adjust the voltage to 120 V and continue electrophoresis. Electrophoresis ends when the target band reaches the appropriate position (refer to the position of the pre-stained protein marker). Cut a PVDF membrane to the size of the gel, activate it in methanol for 1 min, and then soak it in transfer buffer. Filter paper is also soaked in transfer buffer for 15 min. Prepare a transfer "sandwich" according to the principle of PVDF membrane ≥ gel ≥ filter paper, ensuring air bubbles are removed before starting constant voltage transfer. After transfer, stain the membrane with Ponceau S, then wash twice with TBST and observe the proteins on the membrane.

[0056] 4.4.3 Development After wetting the membrane with TBS from bottom to top, transfer it to a petri dish containing blocking solution (5% skim milk powder TBST solution). Block on a decolorizing shaker at room temperature for 1 hour to block the immunoglobulin binding sites of the PVDF membrane. Wash off any residual liquid with TBST, then place the membrane in a resealable bag using a sealing machine. Seal three sides, add primary antibody diluted to an appropriate concentration with TBST (see Table 3 for specific primary antibody and dilution ratios), remove as many air bubbles as possible, seal the bag, and incubate overnight at 4°C. Cut open the resealable bag and wash the membrane three times with TBST for 10 minutes each time. Then place the membrane in a sealing bag, add an appropriate amount of secondary antibody at an appropriate concentration (see Table 3 for specific secondary antibody and dilution ratios), seal the bag, and incubate at room temperature for 1 hour. Cut open the resealable bag and wash the membrane three times with TBST for 10 minutes each time. Equal volumes of chemiluminescence reagents A and B were mixed, and the membrane protein side down was brought into full contact with the mixture. After 5 min, the membrane was detected using a Tanon 5200 luminescence imaging workstation. When the same PVDF membrane required multiple exposures, it was washed with strip solution (protein blot membrane regeneration solution) at room temperature with shaking for 45 min, followed by washing three times with TBST. The blocking step was then restarted, and subsequent steps were the same as before. Protein expression levels were analyzed using Image Pro Plus 6.0 software to measure optical density values. The relative protein expression level was calculated as the gray value of the target protein / the gray value of the internal reference protein.

[0057] Table 3 Antibodies and Dilution Factors 5. Statistical processing Data were analyzed and plotted using GrapHpad Prism 9 (Version 9.4.0), and processed and combined using Adobe Illustrator 2022 (Version 26.3.1). All data are expressed as mean ± SD. Statistical differences between groups were analyzed using a one-way ANOVA test, with a p-value less than 0.05 considered statistically significant.

[0058] Figure 1 In this context, A represents the inhibitory activity of compounds Y0~Y5 against NCI-H3122 cells. Figure 2 In this context, B represents the inhibitory activity of compounds Y0-Y5 against Karpas299 cells; from Figure 1 The results show that, compared with the NC group, the cell viability of compound Y0 was significantly reduced at concentrations of 100 nM, 500 nM, and 1000 nM, and the cell viability of compounds Y1, Y2, Y3, Y4, and Y5 was significantly reduced at concentrations of 1 μM, 5 μM, and 10 μM, indicating that the test drugs had inhibitory effects on NCI-H3122 cells and Karpas299 cells at the corresponding concentrations.

[0059] Figure 2 In the table, A, C, and D represent the inhibition of ALK and p-ALK (Y1507) protein expression in NCI-H3122 cells by compounds Y0~Y3. Figure 2 B, E, and F in the table represent the inhibition of ALK and p-ALK (Y1507) protein expression in NCI-H3122 cells by compounds Y0, Y4, and Y5, respectively. Figure 2 It can be seen that, compared with the NC group, the protein expression level of p-ALK (Y1507) in NCI-H3122 cells of the Y0 group (100, 500, 1000 nM), Y1 group (1, 5, 10 μM), Y2 group (1, 5, 10 μM), Y3 group (1, 5, 10 μM), Y4 group (1, 5, 10 μM) and Y5 group (1, 5, 10 μM) was significantly reduced.

[0060] Figure 3 In the figure, A, C, and D represent the effects of compounds Y0-Y3 on the expression of ALK and p-ALK (Y1507) proteins in Karpas299 cells. Figure 3 B, E, and F in the figure represent the inhibition of ALK and p-ALK (Y1507) protein expression in Karpas299 cells by compounds Y0, Y4, and Y5, respectively. Figure 3It can be seen that, compared with the NC group, the protein expression level of p-ALK (Y1507) in Karpas299 cells was significantly reduced in the Y0 group (100, 500, 1000 nM), Y1 group (1, 5, 10 μM), Y2 group (1, 5, 10 μM), Y3 group (1, 5, 10 μM), Y4 group (1, 5, 10 μM), and Y5 group (1, 5, 10 μM).

[0061] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0062] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A PROTAC molecule targeting crizotinib, characterized in that, Its general structural formula is: Where n is a positive integer from 1 to 5.

2. The method for preparing the PROTAC molecule targeting crizotinib as described in claim 1, characterized in that, The synthetic route for the PROTAC molecule targeting crizotinib is as follows: 。 3. The method for preparing the PROTAC molecule targeting crizotinib as described in claim 1, characterized in that, The method for preparing the PROTAC molecule targeting crizotinib includes the following steps: S1, chlorocarboxylic acid, thionyl chloride and anhydrous DMF are mixed, heated under reflux and reacted to remove thionyl chloride, yielding acyl chloride; S2, pomalidomide, acyl chloride, and organic solvent are mixed and reacted, and then filtered to obtain intermediate M; S3 involves mixing intermediate M, crizotinib, and Na2CO3, reacting the mixture, and then filtering to obtain a PROTAC molecule targeting crizotinib.

4. The method for preparing a PROTAC molecule targeting crizotinib according to claim 3, characterized in that, The structural formula of the chlorocarboxylic acid is: The structural formula of the acyl chloride is: The structural formula of the intermediate M is: In this context, the values ​​of n in the chlorocarboxylic acid, acyl chloride, intermediate M, and the PROTAC molecule are the same.

5. The method for preparing a PROTAC molecule targeting crizotinib according to claim 3, characterized in that, The process for removing thionyl chloride includes: removing excess thionyl chloride by rotary evaporation under reduced pressure, and removing residual thionyl chloride by adding anhydrous toluene.

6. The method for preparing a PROTAC molecule targeting crizotinib according to claim 3, characterized in that, The molar ratio of pomalidomide to acyl chloride is 18.3:

25.

7. The method for preparing a PROTAC molecule targeting crizotinib according to claim 3, characterized in that, The organic solvent is tetrahydrofuran.

8. The method for preparing a PROTAC molecule targeting crizotinib according to claim 3, characterized in that, The molar ratio of intermediate M, crizotinib, and Na2CO3 is 4.3:3.5:

14.

9. The use of the PROTAC molecule targeting crizotinib as described in claim 1 in the preparation of a drug for treating human degenerative large cell lymphoma or lung adenocarcinoma.

10. A drug, characterized in that, Includes the PROTAC molecule with crizotinib as the target as described in claim 1.

Images