Proteolytic Targeted Chimera for the Treatment of Multiple Myeloma, its Preparation Method and Application

Abstract

This invention belongs to the field of heterocyclic compound technology, specifically relating to a protein hydrolysis-targeted chimera for the treatment of multiple myeloma, its preparation method, and its application. Compounds 2, 3, and HATU are dissolved in THF, then DIPEA is added and the reaction is stirred. Water is added to terminate the reaction, followed by extraction, washing, drying, and column chromatography to obtain product 4. Under argon protection, product 4 is dissolved in dichloromethane, then trifluoroacetic acid is added and the reaction is carried out. Toluene is added, and the mixture is distilled under reduced pressure to obtain product 5. Product 5, compound 6, HATU, and DIPEA are dissolved in THF and the reaction is stirred. Water is added to terminate the reaction, followed by extraction, washing, drying, and column chromatography to obtain the protein hydrolysis-targeted chimera for the treatment of multiple myeloma. The protein hydrolysis-targeted chimera for the treatment of multiple myeloma in this invention exhibits significant anti-proliferative activity against RPMI-8266 multiple myeloma cells. The preparation process is simple and suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of heterocyclic compound technology, specifically relating to a protein hydrolysis-targeted chimera for the treatment of multiple myeloma, its preparation method, and its application. Background Technology

[0002] Proteolysis-targeting chimeras (PROTACs) are a class of heterobifunctional molecules that can induce the degradation of target proteins. The concept was first proposed by RJ Deshaies and CM Crews, among others. The chemical structure of PROTACs comprises three key parts: a target protein ligand, a linker, and an E3 ubiquitin ligase ligand. The mechanism of action of PROTACs differs from the "occupation-driven" model of traditional small molecule inhibitors; instead, it operates through an "event-driven" model. The target protein ligand in the PROTAC molecule binds to the target protein, while the E3 ubiquitin ligase ligand recruits the E3 ubiquitin ligase, forming a target protein-PROTACs-E3 ubiquitin ligase ternary complex. Through protein-protein interactions, PROTACs achieve ubiquitination of the target protein, enabling it to be recognized and specifically degraded by the ubiquitin-proteasome system in the body. Ultimately, this modulates the expression level of abnormal proteins to achieve therapeutic effects. Compared to traditional small molecule inhibitors, PROTACs have significant advantages, including high catalytic activity, low toxicity, low dependence on target affinity, effective overcoming of drug resistance caused by target mutations, and the ability to clear abnormal accumulation of pathogenic proteins. After more than 20 years of research and development, PROTACs have been widely used in multiple fields such as oncology, neurodegenerative diseases, inflammation, and immunology, with several candidate drugs entering clinical research stages, demonstrating broad application prospects.

[0003] Multiple myeloma is a common hematologic malignancy caused by the malignant proliferation of plasma cells. Its relapse and refractory nature present a major clinical challenge, thus necessitating the development of therapeutic strategies that overcome drug resistance or target novel mechanisms of action. Proteolytic targeted chimeras can induce target protein ubiquitination by recruiting E3 ubiquitin ligases, thereby enabling the target protein to be degraded by the proteasome. This provides a novel paradigm for targeted therapy of traditionally "undruggable" targets. Compared to the sustained target occupancy of traditional inhibitors, proteolytic targeted chimeras offer advantages such as high catalytic activity, low dosage requirements, and effective overcoming of target resistance. However, current proteolytic targeted chimeras for multiple myeloma treatment suffer from limitations such as limited availability, large molecular weight, high preparation costs, and low activity. These issues significantly increase the difficulty of screening candidate drugs and hinder the clinical translation of proteolytic targeted chimeras for multiple myeloma treatment.

[0004] PT-2385 is a selective hypoxia-inducible factor-2 (HIF-2) inhibitor that effectively inhibits the expression of tumor-derived VEGFA protein. Treatment with PT-2385 (10 mg / kg) significantly reduced the expression levels of the proliferation marker Ki67 and the angiogenesis marker CD-31. PT-2799, a clinical investigation compound further optimized from the PT-2385 structure, has successfully completed clinical trials, met its primary endpoint, and been approved for marketing. It is used to treat VHL-related renal cell carcinoma, central nervous system hemangioblastoma, and pancreatic neuroendocrine tumors. Chinese patent CN107973754A discloses a small molecule inhibitor and its preparation method, along with its application in the treatment of multiple myeloma. This patented small molecule inhibitor can inhibit the activity of Bruton's tyrosine kinase, and therefore can be applied to the treatment of multiple myeloma, such as IgE-type multiple myeloma. However, the mechanism of action of small molecule inhibitors such as PT-2385 and its derivatives relies on the traditional "occupation-driven" mode, exerting their inhibitory effect by continuously occupying the target active site. After long-term use, patients inevitably develop problems such as target resistance. Summary of the Invention

[0005] The purpose of this invention is to provide a proteolytic targeted chimera for the treatment of multiple myeloma, which can induce ubiquitination of target proteins, thereby promoting the degradation of target proteins by the proteasome. This invention also provides a method for preparing the proteolytic targeted chimera for the treatment of multiple myeloma and its application.

[0006] The structural formula of the protein hydrolysis-targeting chimera for the treatment of multiple myeloma described in this invention is as follows:

[0007] .

[0008] The method for preparing the protein hydrolysis-targeted chimera for the treatment of multiple myeloma according to the present invention includes the following steps:

[0009] (1) Compound 2, compound 3 and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were dissolved in tetrahydrofuran (THF), and N,N-diisopropylethylamine (DIPEA) was added and stirred. The reaction was terminated by adding water, and the product was extracted, washed, dried and subjected to column chromatography to obtain product 4.

[0010] (2) Under argon protection, product 4 was dissolved in dichloromethane (DCM), and then trifluoroacetic acid (TFA) was added to react; toluene was added, and the mixture was distilled under reduced pressure to obtain product 5;

[0011] (3) Under argon protection, product 5, compound 6, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were dissolved in tetrahydrofuran and stirred to react; water was added to terminate the reaction, and the mixture was extracted, washed, dried and column-chromatographically analyzed to obtain a protein hydrolysis-targeted chimera for the treatment of multiple myeloma.

[0012] The structural formula of compound 2 in step (1) is as follows:

[0013] ;

[0014] The structural formula of compound 3 is as follows:

[0015] ;

[0016] The structural formula of product 4 is as follows:

[0017] ;

[0018] The ratio of compounds 2, 3, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and tetrahydrofuran was 1:0.9-1.2:1-1.4:3-5, wherein compounds 2, 3, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were expressed in mmol, and tetrahydrofuran in mL; the molar ratio of N,N-diisopropylethylamine to compound 2 was 3-5:1; the volume ratio of tetrahydrofuran to water was 1:3.8-4.5, and extraction was performed using ethyl acetate.

[0019] The endpoint of the stirring reaction in step (1) is the complete disappearance of compound 2 as monitored by thin-layer chromatography (TLC). The developing solvent for TLC is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 8-12:1. The detection method for TLC is ultraviolet detection. The temperature of the stirring reaction is 20-30℃. The washing is performed using sodium chloride solution.

[0020] The structural formula of product 5 in step (2) is as follows:

[0021] ;

[0022] The ratio of product 4, dichloromethane, and trifluoroacetic acid is 1:6-7.1:2.5-3.5, where product 4 is expressed in mmol, and dichloromethane and trifluoroacetic acid are expressed in mL; the volume ratio of dichloromethane to toluene is 1.3-1.7:1.

[0023] In step (2), trifluoroacetic acid is added at -5 to 5°C, and the reaction temperature is -5 to 5°C. The endpoint of the reaction is when product 4 completely disappears as monitored by thin-layer chromatography. The developing solvent for thin-layer chromatography is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 1.8 to 2.2:1. The detection method for thin-layer chromatography is ultraviolet detection or iodine fuming detection. The pressure of vacuum distillation is -0.1 to -0.06 MPa, and the temperature of vacuum distillation is 55 to 65°C.

[0024] The structural formula of compound 6 in step (3) is as follows:

[0025] ;

[0026] The ratio of product 5, compound 6, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, and tetrahydrofuran was 1:0.89-1:1.2-1.8:0.6-1.0:7-9, where product 5, compound 6, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were all expressed in mmol, and N,N-diisopropylethylamine and tetrahydrofuran were expressed in mL.

[0027] The endpoint of the stirring reaction in step (3) is the complete disappearance of compound 6 as monitored by thin-layer chromatography. The developing solvent for thin-layer chromatography is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 8-12:1. The detection method for thin-layer chromatography is ultraviolet detection or iodine fuming detection. The temperature of the stirring reaction is 20-30℃.

[0028] In step (3), the volume ratio of tetrahydrofuran to water is 1:11-13.5. Extraction is performed using ethyl acetate, and washing is performed sequentially using hydrochloric acid, saturated sodium bicarbonate solution, and sodium chloride solution.

[0029] The application of the protein hydrolysis-targeted chimera for the treatment of multiple myeloma described in this invention is its application in the preparation of drugs for the treatment of multiple myeloma.

[0030] The beneficial effects of this invention are as follows:

[0031] (1) The proteolytic targeted chimera for the treatment of multiple myeloma in this invention, based on the existing E3 ubiquitin ligase ligand, introduces the structure of a natural chiral amino acid (tyrosine) into the connection site between the E3 ubiquitin ligase ligand and the linker chain. The α-chiral carbon of the natural chiral amino acid provides a fixed spatial orientation for the connection site between the E3 ubiquitin ligase ligand and the linker chain, transforming the originally flexible and random linker structure into a rigid node with a precise conformation. This change significantly reduces the ineffective conformation of PROTACs molecules and reduces the formation of target protein-PROTACs-E3 ubiquitin links. The conformational entropy loss during the formation of the ternary enzyme complex improves the efficiency of effective ternary complex formation. In addition, the introduction of chiral amino acid structures optimizes the proteolytic targeting chimera. The phenolic hydroxyl group of tyrosine can form additional hydrogen bonds with E3 ubiquitin ligase and polar amino acid residues on the surface of the target protein, further stabilizing the binding between PROTACs and the target protein and E3 ubiquitin ligase, improving the stability of the target protein-PROTACs-E3 ubiquitin ligase ternary complex, promoting the ubiquitination of the target protein, and further promoting the degradation of the target protein by the proteasome. The C=N double bond, with its rigid planar structure, locks the spatial orientation of the target protein ligand module, preventing conformational disorder caused by free rotation. This precisely matches the spatial shape of the target protein binding pocket, ensuring the specific recognition and binding of PROTACs to the target protein. The C=N double bond and chiral amino acid structure are covalently linked by a linker chain and together form a stable ligand-binding region through hydrogen bonds, π-π stacking interactions, and dipole-dipole interactions. The C=N double bond and chiral amino acid structures enhance the binding affinity of PROTACs to the target protein and E3 ubiquitin ligase, respectively. These two bindings form a positive synergistic binding effect, meaning that after PROTACs bind to one protein, their affinity for the other protein is significantly increased. The synergistic effect of the C=N double bond and chiral amino acid structures further enhances the stability and formation efficiency of the target protein-PROTACs-E3 ubiquitin ligase ternary complex, efficiently inducing ubiquitination labeling of the target protein, and ultimately accelerating the recognition and degradation of the target protein by the proteasome.

[0032] (2) Existing inhibitors, such as the inhibitor PT2385, are in an "occupation-driven" mode. Their inhibitory activity depends on the continuous occupation of the target at a high concentration. They need to maintain a high blood drug concentration to take effect. Moreover, they can only inhibit the transcriptional activity of the target protein but cannot clear the target protein. Long-term use can easily lead to acquired drug resistance due to mutations in the target binding site. The protein hydrolysis-targeting chimera used in the present invention for the treatment of multiple myeloma is an "event-driven" catalytic cycle mode of action. It can bind to the target protein and cause the target protein to be degraded by the proteasome. After completing a single target protein degradation, it can participate in the next degradation cycle without the need for continuous administration of high doses. Furthermore, existing inhibitors, such as the inhibitor PT2385, possess chiral structures, requiring additional asymmetric catalysis or chiral resolution steps during preparation, resulting in complex synthetic routes and high production costs. In contrast, the chiral structural units of the proteolytic targeted chimera used in the treatment of multiple myeloma in this invention are all commercially available, high-optical-purity raw materials: the core chiral linker is N-tert-butoxycarbonyl-L-tyrosine (a protected derivative of natural L-tyrosine), and the E3 ubiquitin ligase binding module is a known optically pure VHL ligand. All of the above raw materials are commercially available single optical isomer products, eliminating the need for additional asymmetric catalytic synthesis or chiral resolution steps during preparation. This effectively simplifies the chiral control process, significantly shortens the synthetic route, reduces production costs, and facilitates large-scale preparation.

[0033] (3) The protein hydrolysis targeted chimera used in the present invention for the treatment of multiple myeloma has significant anti-proliferative activity against multiple myeloma RPMI-8266 cells, has good application prospects, and has a simple preparation process that is suitable for industrial production. Attached Figure Description

[0034] Figure 1 This is the 1H NMR spectrum of the protein hydrolysis-targeting chimera used for the treatment of multiple myeloma in Example 1.

[0035] Figure 2 The image shows the 1H NMR spectrum of the protein hydrolysis targeting chimera in Comparative Example 1. Detailed Implementation

[0036] The present invention will be further described below with reference to embodiments.

[0037] Example 1

[0038] (1) At room temperature, 3.00 mmol of compound 2, 3.00 mmol of compound 3 and 3.90 mmol of HATU were dissolved in 12 mL of tetrahydrofuran in a flask, and then 12.0 mmol of N,N-diisopropylethylamine was added. After stirring at 25 °C for 12 hours, the reaction was stopped by TLC (using a mixture of dichloromethane and methanol in a volume ratio of 10:1 as the developing solvent and UV detection as the detection method). Compound 2 disappeared. The reaction was stopped by adding 50 mL of water. The product was extracted with ethyl acetate (3 times × 50 mL) to obtain an organic phase. The organic phases were combined and washed with saturated sodium chloride solution (2 times × 20 mL). The product was then dried with anhydrous NaSO4 for 30 minutes to obtain a crude product. The crude product was purified by column chromatography to obtain a white solid, i.e., product 4. The gradient elution program for column chromatography purification was as follows:

[0039] 0 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 99:1)

[0040] 40 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 20:1);

[0041] (2) Under argon protection, 0.470 mmol of product 4 was dissolved in 3 mL of dichloromethane, and 1.5 mL of trifluoroacetic acid was slowly added at 0 °C. After reacting at 0 °C for 1.5 hours, TLC (using a 2:1 mixture of dichloromethane and methanol as the developing solvent and UV detection) showed that product 4 disappeared. 2 mL of toluene was added, and the mixture was distilled under reduced pressure at -0.1 MPa and 65 °C to remove toluene, dichloromethane, and unreacted trifluoroacetic acid, yielding product 5.

[0042] (3) Under room temperature and argon protection, 0.250 mmol of compound 6, 0.280 mmol of product 5, 0.340 mmol of HATU and 0.190 mmol of LDIPEA were dissolved in 2 mL of THF. After stirring at 25 °C for 10 hours, TLC (using a 10:1 mixture of dichloromethane and methanol as the eluent and UV detection) showed that compound 6 disappeared. The reaction was terminated by adding 25 mL of water and extracted with ethyl acetate (3 times × 25 mL) to obtain the organic phase. The organic phases were combined and washed sequentially with 0.5 N hydrochloric acid (2 times × 10 mL), saturated sodium bicarbonate solution (15 mL), and saturated sodium chloride solution (2 times × 10 mL). The mixture was then dried with anhydrous NaSO4 for 30 minutes to obtain the crude product. The crude product was purified by column chromatography to obtain product 1, which is the protein hydrolysis-targeted chimera for the treatment of multiple myeloma. The gradient elution program for column chromatography purification was as follows:

[0043] 0 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 99:1)

[0044] 40 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 32:1).

[0045] The synthetic route for the protein hydrolysis-targeted chimera used in the treatment of multiple myeloma is as follows:

[0046] .

[0047] The 1H NMR spectrum of product 4 was analyzed and is shown below: 1 H NMR (400 MHz, CDCl3): δ 9.01(t, J=5.4Hz, 1H), 8.98(s, 1H), 8.60 (t, J=5.6 Hz, 1H), 8.30(s, 1H), 8.23 ​​(t, J=5.4 Hz,1H), 7.96(d, J=8.4 Hz, 1H), 7.90(d, J=9.2 Hz, 1H), 7.63(d, J=9.3 Hz, 1H), 7.53(s, 1H), 7.46-7.35(m, 4H), 7.33(d, J=8.4 Hz, 1H), 5.16(d, J=2.4 Hz, 1H),4.53(d, J=9.3 Hz, 1H), 4.47-4.39(m, 2H), 4.35(s, 1H), 4.22(dd, J=15.5, 5.2Hz, 1H), 3.89(d, J=5.3 Hz, 2H), 3.85-3.77(m, 2H), 3.72-3.57(m, 2H), 3.41(s,3H), 3.15-3.07(d, J=4.9 Hz, 2H), 2.88-2.78(m, 2H), 2.43 (s, 3H), 2.08-2.00(m,1H), 1.95-1.85(m, 1H), 0.93(s, 9H); HRMS (ESI) [M+H] + C 43 H 48 FN6O 10 S2 + The calculated value is 891.2852, and the measured value is 891.2855.

[0048] The 1H NMR spectrum of the proteolytic-targeting chimera used for the treatment of multiple myeloma is shown below. Figure 1 , 1H NMR (400MHz, CDCl3): δ 8.65(s, 1H), 8.05(d, J=8.4 Hz, 1H), 7.36-7.22(m, 5H), 7.21(d,J=7.3 Hz, 1H), 7.15(s, 1H), 7.06(d, J=8.7 Hz, 1H), 6.98(d, J=8.5 Hz, 1H), 6.91(d, J=7.9 Hz, 2H), 6.57(d, J=7.8 Hz, 2H), 4.88-4.76(m, 1H), 4.68-4.57(s,3H), 4.46(dd, J=15.0, 5.9 Hz, 1H), 4.37-4.27(m, 2H), 3.80-3.56(m, 3H), 3.32(s, 3H), 3.19-3.11(m, 2H), 3.05-2.91(m, 5H), 2.44(s, 3H), 2.29-2.18(m, 1H),2.16-2.00(m, 1H); HRMS (ESI) [M+H] + C 44 H 42 FN6O9S2 + The calculated value is 881.2433, and the measured value is 881.2437.

[0049] Example 2

[0050] (1) At room temperature, 3.00 mmol of compound 2, 2.70 mmol of compound 3 and 3.00 mmol of HATU were dissolved in 10 mL of tetrahydrofuran in a flask, and then 10.0 mmol of N,N-diisopropylethylamine was added. After stirring at 20 °C for 12 hours, the reaction was stopped by TLC (the developing solvent was a mixture of dichloromethane and methanol in a volume ratio of 8:1, and the detection method was ultraviolet detection). Compound 2 disappeared. 38 mL of water was added to terminate the reaction. The product was extracted with ethyl acetate (3 times × 50 mL) to obtain an organic phase. The organic phases were combined and washed with saturated sodium chloride solution (2 times × 20 mL). The product was then dried with anhydrous NaSO4 for 30 minutes to obtain a crude product. The crude product was purified by column chromatography to obtain a white solid, namely product 4. The gradient elution program for column chromatography purification was as follows:

[0051] 0 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 99:1)

[0052] 40 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 20:1);

[0053] (2) Under argon protection, 0.450 mmol of product 4 was dissolved in 2.70 mL of dichloromethane, and 1.2 mL of trifluoroacetic acid was slowly added at -5 °C. After reacting at -5 °C for 1.5 hours, the product 4 disappeared as monitored by TLC (the developing solvent was a mixed solution of dichloromethane and methanol in a volume ratio of 1.8:1, and the detection method was ultraviolet detection). 2 mL of toluene was added, and the product 5 was obtained by vacuum distillation at -0.06 MPa and 55 °C to remove toluene, dichloromethane and unreacted trifluoroacetic acid.

[0054] (3) Under room temperature and argon protection, 0.230 mmol of compound 6, 0.250 mmol of product 5, 0.330 mmol of HATU and 0.150 mmol of LDIPEA were dissolved in 2 mL of THF. After stirring at 20 °C for 10 hours, TLC (using a mixture of dichloromethane and methanol in a volume ratio of 8:1 as the developing solvent and UV detection) showed that compound 6 disappeared. The reaction was terminated by adding 27 mL of water and extracted with ethyl acetate (3 times × 25 mL) to obtain the organic phase. The organic phases were combined and washed sequentially with 0.5 N hydrochloric acid (2 times × 10 mL), saturated sodium bicarbonate solution (15 mL), and saturated sodium chloride solution (2 times × 10 mL). The mixture was then dried with anhydrous NaSO4 for 30 minutes to obtain the crude product. The crude product was purified by column chromatography to obtain product 1, which is the protein hydrolysis-targeted chimera for the treatment of multiple myeloma. The gradient elution program for column chromatography purification was as follows:

[0055] 0 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 99:1)

[0056] 40 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 32:1);

[0057] The synthetic route for the protein hydrolysis-targeted chimera used in the treatment of multiple myeloma is the same as in Example 1.

[0058] Example 3

[0059] (1) At room temperature, 3.00 mmol of compound 2, 3.60 mmol of compound 3 and 4.20 mmol of HATU were dissolved in 15 mL of tetrahydrofuran in a flask, and then 15.0 mmol of N,N-diisopropylethylamine was added. After stirring at 30 °C for 12 hours, the reaction was stopped by TLC (using a mixture of dichloromethane and methanol in a volume ratio of 12:1 as the developing solvent and UV detection as the detection method). Compound 2 disappeared. The reaction was terminated by adding 67 mL of water. The product was extracted with ethyl acetate (3 times × 50 mL) to obtain an organic phase. The organic phases were combined and washed with saturated sodium chloride solution (2 times × 20 mL). The product was then dried with anhydrous NaSO4 for 30 minutes to obtain a crude product. The crude product was purified by column chromatography to obtain a white solid, i.e., product 4. The gradient elution program for column chromatography purification was as follows:

[0060] 0 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 99:1)

[0061] 40 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 20:1);

[0062] (2) Under argon protection, 0.470 mmol of product 4 was dissolved in 3.30 mL of dichloromethane, and 1.6 mL of trifluoroacetic acid was slowly added at 5 °C. After reacting at 5 °C for 1.5 hours, TLC (using a mixed solution of dichloromethane and methanol in a volume ratio of 2.2:1 as the developing solvent and iodine fumigation as the detection method) showed that product 4 disappeared. 2 mL of toluene was added, and the mixture was distilled under reduced pressure at -0.08 MPa and 60 °C to remove toluene, dichloromethane, and unreacted trifluoroacetic acid, yielding product 5.

[0063] (3) Under room temperature and argon protection, 0.280 mmol of compound 6, 0.280 mmol of product 5, 0.500 mmol of HATU and 0.280 mmol of LDIPEA were dissolved in 2.50 mL of THF. After stirring at 30 °C for 10 hours, TLC (using a 12:1 mixture of dichloromethane and methanol as the eluent and iodine fuming as the detection method) showed that compound 6 disappeared. The reaction was terminated by adding 28 mL of water and extracted with ethyl acetate (3 times × 25 mL) to obtain the organic phase. The organic phases were combined and washed sequentially with 0.5 N hydrochloric acid (2 times × 10 mL), saturated sodium bicarbonate solution (15 mL), and saturated sodium chloride solution (2 times × 10 mL). The mixture was then dried with anhydrous NaSO4 for 30 minutes to obtain the crude product. The crude product was purified by column chromatography to obtain product 1, which is the protein hydrolysis-targeted chimera for the treatment of multiple myeloma. The gradient elution program for column chromatography purification was as follows:

[0064] 0 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 99:1)

[0065] 40 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 32:1);

[0066] The synthetic route for the protein hydrolysis-targeted chimera used in the treatment of multiple myeloma is the same as in Example 1.

[0067] Comparative Example 1

[0068] (1) At room temperature, 3.00 mmol of compound 2, 3.00 mmol of compound 7 and 3.90 mmol of HATU were dissolved in 12 mL of tetrahydrofuran in a flask, and then 12.0 mmol of N,N-diisopropylethylamine was added. After stirring at 25 °C for 12 hours, the reaction was stopped by TLC (using a mixture of dichloromethane and methanol in a volume ratio of 10:1 as the developing solvent and UV detection as the detection method). Compound 2 disappeared. The reaction was terminated by adding 50 mL of water. The product was extracted with ethyl acetate (3 times × 50 mL) to obtain an organic phase. The organic phases were combined and washed with saturated sodium chloride solution (2 times × 20 mL). The product was then dried with anhydrous NaSO4 for 30 minutes to obtain a crude product. The crude product was purified by column chromatography to obtain a white solid, i.e., product 8. The gradient elution program for column chromatography purification was as follows:

[0069] 0 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 99:1)

[0070] 40 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 20:1);

[0071] (2) Under argon protection, 0.660 mmol of product 8 was dissolved in 3 mL of dichloromethane, and 1.5 mL of trifluoroacetic acid was slowly added at 0 °C. After reacting at 0 °C for 1.5 hours, TLC (using a 2:1 mixture of dichloromethane and methanol as the developing solvent and UV detection) showed that product 8 disappeared. 2 mL of toluene was added, and the mixture was distilled under reduced pressure at -0.1 MPa and 65 °C to remove toluene, dichloromethane, and unreacted trifluoroacetic acid, yielding product 9.

[0072] (3) Under room temperature and argon protection, 0.300 mmol of compound 6, 0.330 mmol of product 9, 0.450 mmol of HATU and 1.50 mmol of LDIPEA were dissolved in 6 mL of THF. After stirring at 25 °C for 8 hours, TLC (using a 10:1 mixture of dichloromethane and methanol as the eluent and UV detection) showed that compound 6 disappeared. The reaction was terminated by adding 30 mL of water and extracted with ethyl acetate (3 times × 25 mL) to obtain the organic phase. The organic phases were combined and washed sequentially with 0.5 N hydrochloric acid (2 times × 10 mL), saturated sodium bicarbonate solution (15 mL), and saturated sodium chloride solution (2 times × 10 mL). The mixture was then dried with anhydrous NaSO4 for 30 minutes to obtain the crude product. The crude product was purified by column chromatography to obtain the protein hydrolysis-targeted chimera, i.e., product 11. The gradient elution program for column chromatography purification was as follows:

[0073] 0 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 99:1)

[0074] 40 min, dichloromethane-methanol (dichloromethane to methanol volume ratio 32:1);

[0075] The structural formula of the protein hydrolysis-targeting chimera is as follows:

[0076] ;

[0077] The synthetic route for protein hydrolysis-targeted chimeras is as follows:

[0078] ;

[0079] The proton NMR spectrum of the protein hydrolysis-targeting chimera is shown below. Figure 2 , 1H NMR (400 MHz, DMSO-d6): δ 8.97(s, 1H), 8.47(t, J=5.5 Hz, 1H), 8.00-7.92(m, 2H), 7.76(d, J=8.0 Hz, 1H),7.62-7.50(m, 2H), 7.48-7.39(m, 2H), 7.38-7.30(m, 2H), 7.18(d, J=8.3 Hz, 1H),4.70(s, 2H), 4.43-4.22(m, 4H), 4.02-3.86(m, 2H), 3.69-3.45(m, 2H), 3.40(s,1H), 3.38(s, 3H), 3.12-2.96(m, 4H), 2.44(s, 3H), 2.10-2.01(m, 1H), 1.94-1.85(m, 1H); HRMS (ESI) [M+H] + C 37 H 36 FN6O8S2 + The calculated value is 775.2015, and the measured value is 775.2018.

[0080] In vitro antiproliferative activity study of proteolytic-targeting chimeras for the treatment of multiple myeloma

[0081] Example 1: Cells (RPMI-8226 multiple myeloma cells) were seeded at a density of 10,000 cells / well (100 μL of culture medium) in 96-well plates. After the cells adhered, different concentrations of the proteolytic targeted chimera used in Example 1 for the treatment of multiple myeloma (concentrations of 100 μM, 50 μM, 10 μM, 5 μM, 1 μM and 0.1 μM) were added to each well and incubated for 72 hours. Then, 10 μL of LTT solution was added to each well and incubated for another 4 hours. The supernatant in the wells was discarded, and 100 μL of dimethyl sulfoxide was added to each well. The absorbance was measured using a Multiskan GO 1510 microplate reader (Thermo Scientific).

[0082] Control group:

[0083] The protein hydrolysis-targeted chimera used for the treatment of multiple myeloma in Example 1 was not added, and all other operations were the same as in Example 1 group;

[0084] Comparative Example 1:

[0085] The protein hydrolysis-targeting chimera from Comparative Example 1 was used instead of the protein hydrolysis-targeting chimera used in Example 1 for the treatment of multiple myeloma, and all other operations were the same as in Example 1.

[0086] All experiments were performed in triplicate. The absorbance of the control group was used as a standard, and the absorbance of Example 1 and Comparative Example 1 groups was standardized to calculate the half-maximal inhibitory concentration (IC50). 50 Value); The calculated half-maximal inhibitory concentration (IC50) of the proteolytic-targeting chimera used in Example 1 for the treatment of multiple myeloma against RPMI-8226 multiple myeloma cells was... 50 The half-maximal inhibitory concentration (IC50) of the proteolytic-targeting chimera in Comparative Example 1 against RPMI-8226 multiple myeloma cells was 9.5 ± 1.8 μM, exhibiting significant cytotoxicity; the half-maximal inhibitory concentration (IC50) of the proteolytic-targeting chimera in Comparative Example 1 against RPMI-8226 multiple myeloma cells was 9.5 ± 1.8 μM, 50 Value) > 100μM.

Claims

1. A protein hydrolysis-targeting chimera for the treatment of multiple myeloma, characterized in that... The structure is as follows: 。 2. A method for preparing the protein hydrolysis-targeting chimera for the treatment of multiple myeloma as described in claim 1, characterized in that... Includes the following steps: (1) Compounds 2, 3 and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were dissolved in tetrahydrofuran, and N,N-diisopropylethylamine was added and stirred to react. The reaction was terminated by adding water, extracted, washed, dried and column chromatography were performed to obtain product 4. (2) Under argon protection, product 4 was dissolved in dichloromethane, and then trifluoroacetic acid was added to react; toluene was added and the mixture was distilled under reduced pressure to obtain product 5. (3) Under argon protection, product 5, compound 6, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were dissolved in tetrahydrofuran and stirred to react; water was added to terminate the reaction, and the mixture was extracted, washed, dried and subjected to column chromatography to obtain a protein hydrolysis-targeted chimera for the treatment of multiple myeloma. The structural formula of compound 2 in step (1) is as follows: ； The structural formula of compound 3 is as follows: ； The structural formula of compound 6 in step (3) is as follows: 。 3. The method for preparing the protein hydrolysis-targeting chimera for the treatment of multiple myeloma according to claim 2, characterized in that... The structural formula of product 4 in step (1) is as follows: ； The ratio of compounds 2, 3, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and tetrahydrofuran was 1:0.9-1.2:1-1.4:3-5, wherein compounds 2, 3, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were expressed in mmol, and tetrahydrofuran in mL; the molar ratio of N,N-diisopropylethylamine to compound 2 was 3-5:1; the volume ratio of tetrahydrofuran to water was 1:3.8-4.5, and extraction was performed using ethyl acetate.

4. The method for preparing the protein hydrolysis-targeting chimera for the treatment of multiple myeloma according to claim 2, characterized in that... The endpoint of the stirring reaction in step (1) is the complete disappearance of compound 2 as monitored by thin-layer chromatography. The developing solvent of thin-layer chromatography is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 8-12:

1. The detection method of thin-layer chromatography is ultraviolet detection. The temperature of the stirring reaction is 20-30℃. The washing is done with sodium chloride solution.

5. The method for preparing the protein hydrolysis-targeting chimera for the treatment of multiple myeloma according to claim 2, characterized in that... The structural formula of product 5 in step (2) is as follows: ； The ratio of product 4, dichloromethane, and trifluoroacetic acid is 1:6-7.1:2.5-3.5, where product 4 is expressed in mmol, and dichloromethane and trifluoroacetic acid are expressed in mL; the volume ratio of dichloromethane to toluene is 1.3-1.7:

1.

6. The method for preparing the protein hydrolysis-targeting chimera for the treatment of multiple myeloma according to claim 2, characterized in that... In step (2), trifluoroacetic acid is added at -5 to 5°C, and the reaction temperature is -5 to 5°C. The endpoint of the reaction is when product 4 completely disappears as monitored by thin-layer chromatography. The developing solvent for thin-layer chromatography is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 1.8 to 2.2:

1. The detection method for thin-layer chromatography is ultraviolet detection or iodine fuming detection. The pressure of vacuum distillation is -0.1 to -0.06 MPa, and the temperature of vacuum distillation is 55 to 65°C.

7. The method for preparing the protein hydrolysis-targeting chimera for the treatment of multiple myeloma according to claim 2, characterized in that... In step (3), the ratio of product 5, compound 6, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine and tetrahydrofuran is 1:0.89-1:1.2-1.8:0.6-1.0:7-9, where product 5, compound 6 and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate are all expressed in mmol, and N,N-diisopropylethylamine and tetrahydrofuran are all expressed in mL.

8. The method for preparing the protein hydrolysis-targeting chimera for the treatment of multiple myeloma according to claim 2, characterized in that... The endpoint of the stirring reaction in step (3) is the complete disappearance of compound 6 as monitored by thin-layer chromatography. The developing solvent for thin-layer chromatography is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 8-12:

1. The detection method for thin-layer chromatography is ultraviolet detection or iodine fuming detection. The temperature of the stirring reaction is 20-30℃.

9. The method for preparing the protein hydrolysis-targeting chimera for the treatment of multiple myeloma according to claim 2, characterized in that... In step (3), the volume ratio of tetrahydrofuran to water is 1:11-13.

5. Extraction is performed using ethyl acetate, and washing is performed sequentially using hydrochloric acid, saturated sodium bicarbonate solution, and sodium chloride solution.

10. The application of the proteolytic targeted chimera of claim 1 for the treatment of multiple myeloma, characterized in that... Application in the preparation of drugs for the treatment of multiple myeloma.

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