Degradation agent degrading KRAS G12D protein and application thereof

Abstract

The application discloses a degrading agent for degrading KRAS G12D protein and application thereof, and the structure is shown as formula I, which can be used for preparing a pharmaceutical preparation for preventing or treating diseases caused by mediation of KRAS G12D, wherein the diseases caused by mediation of KRAS G12D protein are cancers, including acute myeloid leukemia (AML), pancreatic cancer, colon cancer, cholangiocarcinoma, cervical cancer, breast cancer, lung cancer, liver cancer, malignant glioma, bladder cancer, gastric cancer and prostate cancer. The PROTAC compound disclosed by the application provides a treatment means for treating diseases caused by KRAS G12D overexpression, and provides a solution idea for solving the drug resistance problem caused by KRAS G12D small molecule inhibitors.

Description

Technical Field

[0001] This invention relates to the field of small molecule drugs, and more specifically, to a degrading agent for KRAS G12D protein and its application as a degrading agent targeting KRAS G12D. Background Technology

[0002] Approximately 30% of human cancers are associated with RAS mutations. The RAS family has three subtypes: KRAS, HRAS, and NRAS. The KRAS gene, short for Kirsten Rats Arcomaviral Oncogene Homolog, is one of the most frequently mutated genes in human cancers. KRAS protein upregulation or activation by mutation has been found in various cancers, including pancreatic cancer, lung cancer, and colon cancer. In human cancers, KRAS gene mutations occur in nearly 90% of pancreatic cancers, 30-50% of colorectal cancers, 17% of endometrial cancers, and 15-20% of lung cancers (mostly NSCLC). It also occurs in bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer. KRAS mutations include many different site mutations, among which the main mutation types are G12C, G12D, G12R, G12A, G12S and G12V, especially KRAS G12C and KRAS G12D mutations, which are prevalent in a variety of cancers.

[0003] KRAS was once considered synonymous with "undruggable" targets in the field of oncology drug development. The difficulty in drugging it stems from several factors: First, the KRAS protein has a compact molecular structure and a smooth surface, lacking obvious drug-binding sites, making the design of small-molecule drugs that specifically bind to and inhibit its activity extremely challenging. KRAS has an extremely high affinity for the nucleotide GTP, making it difficult for drugs to compete with it, thus hindering effective inhibition of the KRAS protein. Furthermore, KRAS not only plays a crucial role in tumor development but is also an essential protein for normal cellular function. Therefore, inhibitors targeting KRAS mutation sites often also inhibit the activity of wild-type KRAS, increasing the complexity of drug development and the potential risk of side effects. Adding further complexity, the GTP binding sites differ among different KRAS mutants, further increasing the difficulty of designing specific KRAS inhibitors.

[0004] In recent years, the field of KRAS drug development has achieved significant leaps. In 2021, the approval of the covalent inhibitor sotorasib marked a new treatment option for the previously "undruggable" target KRAS G12C. Following closely, MiratiTherapeutics' adagrasib also received FDA approval, opening up new treatment avenues for non-small cell lung cancer patients carrying KRAS G12C mutations. In addition to these two approved KRAS G12C inhibitors, such as sotorasib and adagrasib, several other candidate drugs are currently in clinical trials, including BI-2852, JAB-21822, and LY3537982.

[0005] However, the G12D mutation is far more frequent than the G12C mutation, making it the most common KRAS mutation subtype and thus a more clinically valuable target. Given that the G12D mutation lacks a nucleophilic cysteine ​​thiol group similar to that found in G12C for covalent reactions, researchers have been forced to explore novel non-covalent binding strategies. Among these, AstraZeneca's next-generation oral selective KRAS G12D inhibitor, AZD0022, has demonstrated superior oral bioavailability and tissue distribution characteristics and has entered clinical development. Furthermore, MRTX1133, as the first inhibitor targeting the KRAS G12D mutation, has shown significant efficacy in preclinical studies. In animal models of pancreatic and colorectal cancer, MRTX1133 at a dose of 3 mg / kg effectively inhibited the growth of KRAS G12D mutant tumors, demonstrating good safety and potential anti-tumor effects, laying the foundation for further clinical research.

[0006] Drug development targeting KRAS G12D is currently in its early stages, with no drugs targeting KRAS G12D mutations yet on the market. Furthermore, the KRAS G12D inhibitor MRTX1133 has been reported to have developed resistance in human pancreatic cancer cells. Simultaneously, there is a lack of effective treatments for pancreatic cancer carrying KRAS G12D mutations. Therefore, there is an urgent need to develop therapeutic drugs targeting KRAS G12D using new technologies.

[0007] However, existing KRAS G12D inhibitors suffer from acquired resistance defects, and KRAS G12D degraders still have drawbacks such as poor efficacy. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by providing a PROTAC compound, its preparation method, and its application as a degrading agent targeting KRAS G12D.

[0009] The objective of this invention is achieved through the following technical solution: a PROTAC compound, or its stereoisomers, tautomers, deuterated derivatives, solvates, prodrugs, metabolites, pharmaceutically acceptable salts, or cocrystals, i.e., a KRAS G12D degrading agent, having the structure shown in formula (I): Formula (I); Where L is the connecting chain, and its structure is shown in the following formula: , Where n is independently selected from integers from 0 to 9; R is selected from the following structures: , , , in, X is C, N, or O. W can be C=O or CH2.

[0010] Furthermore, X is O.

[0011] Furthermore, W is C=O.

[0012] Furthermore, the present invention provides a KRAS G12D-targeting degrader, which is the following compound: This invention also provides a method for preparing a KRAS G12D-targeting degrader, which mainly utilizes a "click reaction" strategy to link the KRAS G12D ligand with the E3 ligase CRBN ligand: First, according to the following reaction route, the important intermediate 6 of KRAS G12D was prepared: ; Next, using 6 as the starting material, after nucleophilic reaction and deprotection, intermediate 8 was obtained: Subsequently, using a mixed solution of tetrahydrofuran and water (v / v = 4:1) as the reaction solvent, and adding sodium ascorbate and copper sulfate as catalysts, intermediate 8 undergoes a Click reaction with an E3 ligase ligand carrying an azide group (N3-) to generate the desired target compound. The reaction equation is as follows: ; in, The Linker here is n is an integer between 0 and 9.

[0013] For the exceptions VII-3 and VII-4, the Click reaction is not used for synthesis; instead, a condensation reaction is performed directly. Taking VII-4 as an example, its synthetic route is as follows: .

[0014] This invention also provides a pharmaceutical application of a KRAS G12D-targeting degrader, specifically its use in the preparation of pharmaceutical formulations for the prevention or treatment of diseases mediated by the KRAS G12D protein, wherein the diseases mediated by KRAS G12D are cancers, including acute myeloid leukemia (AML), pancreatic cancer, colon cancer, bile duct cancer, cervical cancer, breast cancer, lung cancer, liver cancer, malignant glioma, bladder cancer, gastric cancer, prostate cancer, etc.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: Based on the rapidly developing PROTAC (Proteolysis-targeting chimaeras) technology, this invention addresses the common problems of drug resistance and insufficient activity of protein inhibitors through protein degradation. The KRAS G12D-targeting degrader provided by this invention does not require long-term occupation of the active site like inhibitors; instead, it only needs a brief binding to the KRAS G12D protein to induce protein degradation. Since the degraded protein needs to be resynthesized to restore its function, this significantly delays the development of drug resistance; it remains effective even against proteins that have already developed resistance. Therefore, the KRAS G12D-targeting degrader provided by this invention can be used in the preparation of pharmaceutical formulations for the prevention or treatment of diseases mediated by the KRAS G12D protein, such as tumors. Attached Figure Description

[0016] Figure 1 The compound exhibits degradation activity against PANC-1 cells.

[0017] Figure 2The compound exhibits degradation activity against SNU-1 cells.

[0018] Figure 3 The effect of compound VI-1 on the degradation of KRAS protein in different mutant cell lines. Detailed Implementation

[0019] The following examples further illustrate the structure, preparation method, and application of the present invention in the preparation of pharmaceutical formulations for the prevention or treatment of diseases caused by excessive microtubule expression, but do not limit the present invention.

[0020] The analytical data of the samples were determined using the following instruments: The thermometer was not calibrated; Bruker DRX400 NMR spectrometer; Agilent 5975 mass spectrometer; Bruker Vector 22 infrared spectrometer.

[0021] Example 1: Synthesis of key intermediate 6 Step 1: Synthesis of Intermediate 2 Weigh 2.5 g (11.5 mmol) of 7-chloro-8-fluoro-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidine-2,4-dione and 10.1 mL (58 mmol) of DIPEA into a 100 mL sealed tube. After stirring for 5 min in an ice bath, slowly add 24.6 mL (264.2 mmol) of POCl3. Finally, transfer to an oil bath and react at 100 °C. After reacting for 12 h, cool the reaction mixture to room temperature, transfer the reaction solution to a pear-shaped flask, rinse the wall of the flask with redistilled dichloromethane, and transfer the sealed tube into the pear-shaped flask as well. Finally, concentrate and dry to obtain a brownish-yellow solid, which is directly added to the next step.

[0022] Step 2: Synthesis of Intermediate 3 1 g (4 mmol) of compound 2 was dissolved in redistilled DCM, 4.9 mL (28 mmol) of DIPEA was added, and the mixture was stirred at -40 °C for 10 min. Then, 976 mg (4 mmol) of benzyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate was added, and the reaction was continued for 30 min. After the reaction was completed, TLC was performed to confirm that the reaction was complete. The reaction was quenched with an appropriate amount of ice water, extracted three times with DCM (100 mL), and the organic phases were combined and washed three times with saturated brine (200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then loaded onto a dry column with silica gel. Column chromatography yielded 980 mg of yellow solid compound 3, with a yield of 53%. 1H NMR (400MHz, CHLOROFORM-D) δ 8.80 (s, 1H), 7.37-7.30 (m, 5H), 5.18 (s, 2H), 3.69-3.54(m, 4H), 3.10-3.04 (m, 2H), 2.02-1.93 (m, 2H), 1.68(d, J = 8.4 Hz, 2H). 13 CNMR (101 MHz, CHLOROFORM-D) δ 163.75, 161.32, 153.63, 149.82, 148.82, 147.14,144.16, 144.08, 139.19, 139.02, 136.22, 128.73, 128.46, 128.20, 111.09,67.51, 53.70, 41.99, 18.70, 17.44, 12.13. Step 3: Synthesis of Intermediate 4 Weigh 0.22 g (5.4 mmol) of NaH into a three-necked flask, add an appropriate amount of THF to dissolve it, and then use N2 for purging protection. Then add dropwise a solution containing 0.74 g (3.7 mmol) of NaH (2... S A THF solution of 2-(hydroxymethyl)tetrahydropyrrole-1-carboxylic acid-2-methylpropyl-2-yl ester was stirred at 0 °C for 20 min, followed by the addition of a THF solution containing 1 g (2.16 mmol) of compound 3. The mixture was then transferred to room temperature and reacted overnight. The reaction was confirmed to be complete by TLC. The reaction was quenched with a suitable amount of water, extracted three times with EA (100 mL), and the organic phases were combined and washed three times with saturated brine (200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then loaded onto a dry column with silica gel. Column chromatography yielded 623 mg of compound 4, with a yield of 46%. 1 H NMR (400 MHz, CHLOROFORM-D) δ8.69 (s, 1H), 7.39-7.33 (m, 5H), 5.19 (s, 2H), 4.47 (s, 4H), 4.23 (s, 1H),3.66 (s, 2H), 3.36 (d, J = 36.0 Hz, 2H), 1.98 (d, J = 17.7 Hz, 5H), 1.88-1.81(m, 1H), 1.72 (d, J= 7.9 Hz, 2H), 1.44 (s, 9H), 0.92-0.72 (m, 2H). 13 C NMR(101 MHz, CHLOROFORM-D) δ 165.55, 164.23, 154.66, 153.70, 150.11, 143.91,143.83, 136.38, 128.73, 128.41, 128.17, 79.44, 77.35, 68.08, 67.72, 67.42,55.82, 54.08, 46.92, 46.53, 29.78, 29.45, 28.90, 28.58, 28.11, 27.15, 26.49,23.75, 22.91, 22.78, 14.22. Step 4: Synthesis of Intermediate 5 0.5 g (0.8 mmol) of compound 4, 0.23 g (0.8 mmol) of 2-(8-chloronaphthyl-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxoboronylcyclopentane, 0.06 g (0.08 mmol) of methanesulfonyloxy(dadamantyl-n-butylphosphino)-2'-amino-1,1'-biphenyl-2-yl)palladium(II), and 0.78 g (2.4 mmol) of cesium carbonate were weighed into a Schlenk reaction tube. Finally, 9 mL of 1,4-dioxane and 3 mL of purified water were added. The mixture was purged three times with N2 and reacted overnight at 90 °C. The reaction was confirmed to be complete by TLC the next day. The reaction was quenched with an appropriate amount of water, extracted three times with EA (50 mL), and the organic phases were combined and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and loaded onto a dry column with silica gel. Column chromatography yielded 259 mg of a pale yellow solid (5), with a yield of 43%. MS (ESI) calcd for C 41 H 42 ClFN6O5 [M+H] + 753.29; found: 753.28 Step 5: Synthesis of Intermediate 6 Weigh 0.5 g of compound 5 into a single-necked flask, add 2 mL of redistilled dichloromethane, stir at 0 °C for 2 min, then slowly add 1 mL of trifluoroacetic acid dropwise, and react for 1 h. After the reaction is complete, concentrate under reduced pressure, evacuate under vacuum for 0.5 h, and then proceed directly to the next step.

[0023] Step 6: Synthesis of Intermediate 7 0.5 g (0.8 mmol) of compound 6 was weighed into a single-necked flask, dissolved in an appropriate amount of DMF, followed by the addition of 0.36 g (3.6 mmol) triethylamine, 0.14 g (1.44 mmol) penta-4-acetylic acid, and 0.55 g (1.44 mmol) HATU. The mixture was protected with N2 and reacted overnight at room temperature. The reaction was confirmed to be complete by TLC. The mixture was quenched with an appropriate amount of water, extracted three times with EA (50 mL), and the organic phases were combined and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and loaded onto a dry column with silica gel. Column chromatography yielded 370 mg of white solid compound 7. The yield was 63%. MS (ESI) calcd for C 41 H 38 ClFN6O4[M+H] + 733.26; found: 733.89 Step 7: Synthesis of Intermediate 8 Weigh 0.1 g of compound 7 into a single-necked flask, add 2.4 mL of trifluoromethanesulfonic acid, and place at 0 °C. After stirring for 5 min, slowly add 82 μL (0.95 mmol) of trifluoromethanesulfonic acid and react for 2 h. After the reaction is complete, place the flask directly on a stirring table at room temperature and add NaHCO3 solid in portions (the reaction solution will continuously bubble during the addition) until the reaction solution stops bubbling. A small amount of DCM can be added to dissolve the solid. Adjust the pH to 7, wash with DCM, filter, concentrate under reduced pressure, add an appropriate amount of silica gel, and dry-pick the solution onto a column. Column chromatography yields 243 mg of compound 8. 1 H NMR (400 MHz, DMSO- D 6) δ 9.10 (d, J = 4.1 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.65-7.52 (m, 3H), 3.77-3.64 (m, 8H), 3.43-3.39 (m, 2H), 2.73-2.72 (m, 1H), 2.64(dt, J = 9.3, 2.5 Hz, 1H), 2.34 (pd, J= 5.4, 4.2, 2.3 Hz, 2H), 2.06-2.82 (s, 5H), 1.71 (s, 5H), 1.21 (s, 1H). 13 C NMR (101 MHz, DMSO- D 6) δ 169.95, 164.98,163.91, 151.38, 148.83, 147.04, 144.76, 135.99, 131.89, 131.60, 131.06,129.97, 129.29, 128.31, 127.02, 126.49, 111.32, 84.60, 71.65, 66.58, 55.97,54.54, 46.95, 45.92, 33.52, 32.98, 28.63, 27.69, 27.28, 23.94, 21.73, 14.69, 13.96. Example 2: Synthesis of target compound VII-3 Step 1: Synthesis of Intermediate 9 0.5 g (1 mmol) of compound 6 was weighed into a single-necked flask, and then added sequentially: DMF, 0.42 mL (3 mmol) TEA, 0.47 mg (1.2 mmol) [(2-{[2-(2,6-dioxonylhexahydropyridin-3-yl)-1,3-dioxonyl-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]acetic acid, and 0.46 g (1.2 mmol) HATU]. The reaction was carried out under N2 protection at room temperature for 12 h. The reaction was confirmed to be complete by TLC. The mixture was quenched with water and extracted three times with EA (50 mL). The organic phases were combined and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then loaded onto a dry column with silica gel. Column chromatography yielded 573.1 mg of fluorescent green compound 9, with a yield of 56%. 1 H NMR (400 MHz, CHLOROFORM-D)δ 7.98 (dd, J = 7.8, 1.8 Hz, 1H), 7.86 (dd, J = 8.3, 1.3 Hz, 1H), 7.65 - 7.49(m, 3H), 7.47 - 7.31 (m, 8H), 7.03 (ddd, J= 22.8, 7.0, 1.5 Hz, 1H), 6.94 -6.70 (m, 1H), 5.20 (s, 2H), 4.90-4.83 (m, 1H), 4.70 - 4.44 (m, 6H), 3.86 -3.39 (m, 10H), 2.89 - 2.52 (m, 5H), 2.05 - 1.66 (m, 8H). 13 C NMR (101 MHz, CHLOROFORM-D) δ 171.31, 171.00, 170.02, 169.15, 168.50, 167.72, 164.06,153.75, 146.86, 143.37, 136.48, 136.12, 135.98, 132.58, 131.13, 130.75,129.52, 128.73(3C), 128.37, 128.17(2C), 128.16, 126.13, 125.69, 116.84,111.68, 111.52, 110.17, 77.46, 77.35, 77.15, 76.83, 69.34, 67.36, 66.97, 55.86, 54.10, 48.96, 47.55, 45.95, 42.26, 35.43, 34.87, 31.51, 29.79, 27.65, 24.10, 22.90, 21.71, 17.97, 14.51. Step 2: Synthesis of final product VII-3 The procedure is the same as in 4.1.3.7. The yield was 73%. 1 H NMR (400 MHz, DMSO-D6) δ 11.09 (s, 1H), 9.12 (s, 1H), 8.19 (td, J = 7.8, 7.2, 1.9 Hz, 1H), 8.15 - 7.99 (m, 1H), 7.74-7.53 (m, 6H), 7.06 (dd, J = 38.7, 7.8 Hz, 2H), 5.05-4.99 (m, 1H), 4.68-4.11(m, 8H), 3.81 (t, J = 16.5 Hz, 3H), 3.72-3.56 (m, 6H), 1.90 (s, 10H), 1.22(s, 3H).13 C NMR (101 MHz, DMSO- D 6) δ 172.86, 170.13, 169.23, 167.32, 165.13,163.41, 153.11, 146.40, 136.84, 136.23, 135.63, 132.10, 131.11, 130.33,129.47, 128.79, 128.51, 127.95, 127.79, 127.65, 127.64, 126.61, 125.98,125.74, 117.95, 117.43, 110.67, 109.24, 108.37, 69.81, 69.73, 68.87, 66.37, 66.23, 56.00, 51.26, 48.56, 46.70, 44.93, 42.93, 41.68, 34.65, 30.99, 23.31, 22.15. Example 3: Synthesis of target compound VII-4 Step 1: Synthesis of Intermediate 10 0.5 g (1 mmol) of compound 6 was weighed into a single-necked flask, and then added sequentially: DMF, 0.42 mL (3 mmol) TEA, 0.57 mg (1.2 mmol) [(8-{[2-(2,6-dioxonylhexahydropyridin-3-yl)-1,3-dioxonyl-2,3-dihydro-1H-isoindol-4-yl]amino}-3,6-dioxaoctyl-1-yl)oxy]acetic acid, and 0.46 g (1.2 mmol) HATU. The reaction was carried out under N2 protection at room temperature for 12 h. The reaction was confirmed to be complete by TLC. The mixture was quenched with water and extracted three times with EA (50 mL). The organic phases were combined and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then loaded onto a dry column with silica gel. Column chromatography yielded 523 mg of fluorescent green solid compound 10, with a yield of 49%. 1 H NMR (400MHz, CHLOROFORM-D) δ 8.00-7.97 (m, 1H), 7.88-7.85 (m, 1H), 7.62-7.50 (m, 3H), 7.48-7.32 (m, 8H), 7.07 (dd, J= 7.2, 2.3 Hz, 1H), 6.91-6.85 (m, 1H), 5.20(s, 2H), 4.91-4.87 (m, 1H), 4.76-4.06 (m, 8H), 3.81- 3.77 (m, 3H), 3.43 (t, J = 5.4 Hz, 4H), 2.86-2.52 (m, 6H), 2.18-1.79 (m, 13H), 0.96-0.82 (m, 3H). 13 CNMR (101 MHz, CHLOROFORM-D) δ 171.54, 170.38, 169.34, 168.68, 167.76, 165.91,164.10, 153.76, 149.11, 146.86, 143.40, 136.51, 136.09, 136.01, 132.63,131.10, 130.68, 129.50, 128.73(3C), 128.37, 128.16(2C), 126.11, 125.70,116.82, 111.68, 110.42, 77.48, 77.36, 77.16, 76.84, 70.90, 70.63, 70.55, 69.41, 67.59, 67.36, 67.07, 66.81, 55.88, 54.16, 48.97, 47.51, 45.95, 42.42, 35.46, 34.94, 31.53, 29.80, 27.62, 24.10, 22.93, 21.73, 14.26. Step 2: Synthesis of final product VII-4 The procedure is the same as step 2 in Example 3. The yield was 52%. 1 H NMR (400 MHz, DMSO-D6) δ 9.19 - 9.03(m, 1H), 8.20 (dd, J = 8.4, 1.4 Hz, 1H), 8.09 (dt, J = 8.2, 1.7 Hz, 1H), 7.78- 7.45 (m, 6H), 7.18 - 6.92 (m, 2H), 5.05 (dd, J= 12.9, 5.4 Hz, 1H), 4.84 -4.01 (m, 9H), 3.96 - 3.69 (m, 3H), 3.64 - 3.49 (m, 13H), 2.03 - 1.83 (m,11H), 1.22 (s, 2H). 13 C NMR (101 MHz, DMSO-D6) δ 172.98, 170.23, 169.24,167.41, 151.37, 150.47, 148.10, 147.29, 146.52, 138.99, 137.20, 135.57,134.40, 132.16, 131.73, 131.08, 129.54, 128.29, 127.51, 126.43, 125.57,122.37, 119.16, 117.78, 117.29, 115.96, 110.77, 109.30, 102.97, 76.21, 69.87, 69.79, 68.92, 66.42, 58.64, 55.70, 55.30, 54.32, 52.15, 48.64, 46.84, 46.61, 46.50, 41.75, 34.71, 31.06, 25.68, 24.96, 22.23. Example 4: Synthesis of Intermediate 13 1 g (5.6 mmol) of 5-bromopentanoic acid and 2.2 g (5.9 mmol) of HATU were weighed into a single-necked flask. After dissolving in an appropriate amount of DMF, 2.4 mL (13.5 mmol) of DIPEA was added and stirred for 10 min. Then, 2 g (4.5 mmol) of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride was added, and the mixture was reacted overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction was quenched with an appropriate amount of water and extracted three times with DCM (150 mL). The organic phases were combined and washed three times with saturated brine (150 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and loaded onto a dry column with an appropriate amount of silica gel. Column chromatography yielded 1.9 g of white solid compound 13, with a yield of 71%. MS (ESI) calcd for C 27 H 37 BrN4O4S [M+H] + 594.58; found: 594.69 Example 5: Synthesis of Intermediate 14 The procedure was the same as for the synthesis of intermediate 13. The yield was 77%. MS (ESI) calcd for C 28 H 39 BrN4O4S [M+H] + 607.19; found: 607.28 Example 6: Synthesis of Intermediate 15 The procedure was the same as for the synthesis of intermediate 13. The yield was 62%. MS (ESI) calcd for C 29 H 41 BrN4O4S [M+H] + 621.20; found: 621.58 Example 7: Synthesis of Intermediate 16 Weigh 0.5 g (1.8 mmol) of 2-(2,6-dioxanediol-3-yl)-5-fluoroisoindol-1,3-dione and 0.2 g (1.9 mmol) of 2-[(2-aminoethyl)oxy]ethanol-1-ol, dissolve in an appropriate amount of DMF, add 0.94 g (7.2 mmol) of DIPEA, and react overnight at 90 °C. The reaction was confirmed to be complete by TLC. The reaction solution changed from pale yellow to dark green, was quenched with an appropriate amount of water, extracted three times with DCM (150 mL), the organic phases were combined, washed three times with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and loaded onto a dry column with silica gel. Column chromatography yielded 279.7 mg of compound 16, with a yield of 43%. MS (ESI) calcd for C 17 H 19 N3O6 [M+H] + 362.13; found: 362.55 Example 8: Synthesis of Intermediate 17 The procedure was the same as for the synthesis of intermediate 16. The yield was 51%. MS (ESI) calcd for C 19 H 23 N3O7 [M+H] + 405.15; found: 405.32 Synthesis of intermediate 18 The procedure was the same as for the synthesis of intermediate 16. The yield was 56%. MS (ESI) calcd for C 19 H 23 N3O7 [M+H] + 450.18; found: 450.36 Example 9: Synthesis of Intermediate 19 0.5 g (1.2 mmol) of compound 16 and 0.52 mL (3.7 mmol) of TEA were weighed into a single-necked flask. Under ice bath conditions, an appropriate amount of redistilled DCM was added, followed by slow dropwise addition of 0.17 mL (1.9 mmol). The mixture was then transferred to room temperature and reacted for 7 h. The reaction was confirmed to be complete by TLC. The reaction was quenched with a suitable amount of water, and the mixture was extracted three times with DCM (150 mL). The organic phases were combined and washed three times with saturated brine (150 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then loaded onto a dry column with silica gel. Column chromatography yielded 274 mg of compound 19, with a yield of 52%. MS (ESI) calcd for C 18 H 21 N3O8S [M+H] + 440.10; found: 440.68 Example 10: Synthesis of Intermediate 20 The procedure was the same as for the synthesis of intermediate 19. The yield was 42%. MS (ESI) calcd for C 20 H 25 N3O9S [M+H] + 484.13; found: 484.26 Example 11: Synthesis of Intermediate 21 The procedure was the same as for the synthesis of intermediate 19. The yield was 51%. MS (ESI) calcd for C 22 H 29 N3O 10 S [M+H] + 528.16; found: 428.34 Example 12: Synthesis of Intermediate 22 Weigh 0.4 g (3 mmol) of [(2-azidoethyl)oxy]acetic acid into a single-necked flask, add an appropriate amount of DMF, 1 g (3 mmol) of HATU, and 1.1 mL (8 mmol) of TEA. After stirring for 15 min, add 1 g (2 mmol) of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride and react overnight. The reaction was confirmed to be complete by TLC. The reaction was quenched with an appropriate amount of water, extracted three times with EA (150 mL), and the organic phases were combined and washed three times with saturated brine (150 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and loaded onto a dry column with an appropriate amount of silica gel. Column chromatography yielded 808 mg of white crystalline compound 22, with a yield of 72%. 1 H NMR (400 MHz, CHLOROFORM-D) δ 8.66 (s, 1H), 7.32 (d, J = 1.7 Hz, 4H), 4.70 (t, J = 7.9 Hz,1H), 4.53-4.49 (m, 3H), 4.32-4.27 (m, 1H), 3.97 (d, J = 7.3 Hz, 2H), 3.71-3.62 (m, 3H), 3.23-3.16 (m, 1H), 2.49-2.41 (s, 5H), 1.34 (t, J = 7.3 Hz, 2H), 0.95 (s, 9H). 13 C NMR (101 MHz, CHLOROFORM-D) δ 171.22, 171.02, 169.72, 150.45,148.48, 138.20, 131.72, 130.92, 129.55, 128.11, 70.61, 70.29, 70.19, 65.02,58.74, 57.31, 56.81, 50.75, 47.41, 43.24, 36.24, 35.21, 26.43, 16.13, 14.29,8.78. Example 13: Synthesis of Intermediate 23 0.58 g (0.8 mmol) of compound 7 and 0.41 g (0.8 mmol) of compound 22 were weighed into a single-necked flask, dissolved in THF (8 mL) and water (2 mL), and then 0.12 g (0.48 mmol) of CuSO4·5H2O and 0.32 g (1.6 mmol) of sodium ascorbate 70 were added to the flask sequentially. The reaction was incubated at 60 °C for 4 h. The reaction was confirmed to be complete by TLC. The mixture was quenched with an appropriate amount of water, extracted three times with EA (100 mL), and the organic phases were combined and washed three times with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then loaded onto a dry column with silica gel. Column chromatography yielded 632 mg of white crystalline compound 23, with a yield of 64%.

[0024] Example 14: Synthesis of final product VI-1 0.18 g of compound 23 was added to 3.6 mL of TFA and 0.24 mL of TfOH, and the reaction was allowed to proceed for 4 h. The reaction was confirmed to be complete by TLC. The mixture was quenched with an appropriate amount of Na2HCO3 (added until bubbling stopped), and extracted with 60 mL of EA. The organic phases were combined, concentrated, and then separated once by thin-layer chromatography (DCM:MeOH = 10:1). The purified product yielded 70 mg of white solid VI-1, with a yield of 43%. 1 H NMR (400 MHz, DMSO-D6) δ 9.09 (s, 1H), 8.96 (s, 1H), 8.57 (t, J = 6.2 Hz, 1H), 8.19 (d, J = 8.3 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.84-7.79 (m, 1H),7.71 (t, J = 7.6 Hz, 1H), 7.65-7.54 (m, 3H), 7.38 (s, 4H), 7.22 (s, 1H), 6.67(s, 1H), 5.31 (t, J = 5.0 Hz, 3H), 4.53-4.48 (m, 6H), 4.45-4.40 (m, 4H), 4.34(s, 2H), 4.26-4.20 (m, 3H), 3.96-3.91 (m, 2H), 3.87-3.81 (m, 3H), 2.41 (s,5H), 2.02-1.95 (m, 5H), 1.45 (t, J= 7.3 Hz, 3H), 1.23 (s, 9H), 0.85 (t, J =6.4 Hz, 6H). Example 15: Synthesis of other end products Other final products were synthesized using the same method as in Example 14. Their mass spectrometry data are as follows: .

[0025] Example 16: Preparation of VI-1 hydrochloride Take an appropriate amount of compound VI-1, dissolve it in ethanol, and slowly pass HCl gas through it at room temperature until saturation. Cool in an ice bath, and white crystals slowly precipitate out, which are the hydrochloride salt of the compound.

[0026] Example 17: CCK8 assay to detect the effect of PROTAC on cell viability The effects of the compound on cell viability at different time points and in different cell lines were further investigated using the CCK-8 assay, as follows: Cells were seeded into 96-well microplates at a density of 8000–10000 cells / well in 100 μL of complete culture medium. Cells were then treated with DMSO or a serial dilution of the specified compound for 48 h. Cell viability was assessed using CCK-8 (APExBIO). 10 μL of CCK-8 was added to each well, and the plate was incubated at 37 °C and 5% CO2 for 2–4 h. Absorbance was read at 450 nm and 630 nm using a BIO-TEK microplate reader. IC50 was analyzed using a GraphPad Prism 9.5. 50 value.

[0027] The activity results are shown in Table 1: Effects of Series I PROTACs on the Viability of Mia PACA 2, PANC-1, and SNU-1 Cells Note: NT indicates not tested. The experimental results showed that among the series I compounds, I-9 exhibited better activity, significantly reducing cell viability in Mia PACA2 and PANC-1 cell lines, and this reduction in cell viability was time-dependent. 50The values ​​were 35.11 and 18.89 μM and 13.03 and 12.58 μM, respectively.

[0028] Among the series II compounds, compound II-2 has an IC50 in SNU-1 cells. 50 The concentrations were 3.52 μM and 5.53 μM. Compared to PROTACs with different linker lengths of the same type, a 7-carbon chain length may be a better linker length for PROTAC molecules. Comparison with Series I shows that in SNU-1 cells, I-6, I-7 and II-4, II-5 have the same carbon chain length, but they differ in the linker sites at the CRBN linker. Adjacent linker connections may have a better inhibitory effect on cell growth than meta linker connections.

[0029] The series III compounds were linked by a linker derived from the ortho position of the CRBN ligand. Results showed that in SNU-1 cells, compound III-3, with an 8-carbon linker, exhibited the best IC50. 50 The concentrations were 5.36 μM and 3.21 μM, respectively. Compared with I-5 in Series I, both have the same type and length of linker chains and the same linker sites, but the difference lies in the choice of CRBN ligand. For PROTAC with an 8-carbon linker chain length, lenalidomide analogues have a stronger inhibitory effect on cell activity than pomalidomide.

[0030] Series IV are PROTAC molecules with alkyl chains as linkers and CRBN interfaces as imine sites. Among IV-2, IV-3 and IV-4, IV-3 showed the best inhibitory effect, reaching 9.91 μM. Therefore, for PROTACs with imine interfaces, selecting a linker of appropriate length results in better cell inhibition.

[0031] Series V uses a meta-imine linker. Activity assays showed that the cell inhibition effect gradually weakened with increasing PEG linker length. However, compared to compound I-6 in Series I, the inhibitory effect was enhanced. This indicates that for meta-imine linkers, selecting a PEG unit linker length is a good choice.

[0032] Series VI are PROTACs using VHL as an E3 ligase ligand. Their effects on cell antiproliferative activity showed that, considering the type of linker chain, PEG chains had a greater impact on cell proliferation than alkyl chains. Compound VI-1 exhibited the best antiproliferative activity, with an IC50 value of [missing value]. 50 The inhibitory activity was 4.27 μM (PANC-1), and it slightly decreased with increasing PEG group concentration. VI-2, VI-3, and VI-4 achieved the best IC50 values ​​in PANC-1 cells. 50The values ​​were 8.95 μM, 6.66 μM, and 6.16 μM.

[0033] Series VII investigated the effect of the triazole group in the compounds on their degradation activity. The results showed that compound VII-1, which possesses a triazole group, exhibited better anti-proliferative activity compared to compound VII-3, which lacks a triazole group. VII-1 achieved the best IC50 value in PANC-1 cells. 50 The value is 14.82 μM.

[0034] Example 18: Protein degradation effect of PROTAC molecules targeting KRAS G12D Based on the initial screening, we selected seven compounds (I-9, VI-1, VI-2, VI-3, VI-4, II-4, and III-3) exhibiting good anti-proliferative activity as candidate compounds for the next stage of protein degradation experiments. We evaluated the degradation ability of these compounds on KRAS G12D using Western blotting. The degradation effects on KRAS G12D protein in the PANC-1 cell line were measured at different concentrations (mainly 0, 0.1, 1, and 10 μM). The results are shown in the appendix. Figure 1 .

[0035] As can be seen, compound VI-1 achieved a significant degradation effect at a dosage concentration of 10 μM, with a degradation rate of 59%. Compounds VI-2 and VI-3 showed a degradation rate of 20% at a dosage concentration of 10 μM. Compound VI-4 showed a degradation rate of 35% at a dosage concentration of 10 μM. Unfortunately, compounds II-4 and III-3 did not significantly decrease KRAS protein expression in SNU-1 (G12D) cells after 24 h and 48 h of administration. Therefore, for the PANC-1 cell line, PROTAC, which has a PEG unit and triazole as the linker chain, has the best KRAS G12D protein degradation effect.

[0036] The effect of time was determined in the SNU-1 (human gastric cancer) cell line, and the results are shown in the appendix. Figure 2 It is evident that compounds II-2 and III-3 showed virtually no significant degradation effect on G12D protein within 24 hours, but exhibited a significant degradation effect at 48 hours.

[0037] Example 19: KRAS protein degradation effect of VI-1 in different mutant cell lines Next, we determined the degradation effect of VI-1 on KRAS G12D protein in different cell lines. Overall, VI-1 reduced KRAS by 59%, 69%, and 52% at 10 μM in PANC-1, PANC 0203, and SNU-1 cells (KRAS G12D), respectively.

[0038] Furthermore, we also verified the effects of VI-1 degradation in other KRAS mutant cells such as PANC 0327 (G12V), A549 (G12S), NCl-H358 (G12C), and normal pancreatic tissue (HPNE). Results are attached. Figure 3 .

[0039] It is evident that VI-1 has no significant effect on KRAS degradation in other mutant cells such as PANC 0327 (G12V), A549 (G12S), NCl-H358 (G12C), and normal pancreatic tissue (HPNE). Therefore, VI-1 can specifically degrade the KRASG12D mutant protein without affecting protein expression in other KRAS mutants or normal pancreatic tissue.

[0040] In summary, in the in vitro biological evaluation, we first assessed the effects of 37 KRAS G12D PROTAC molecules on cell viability at 24 h and 72 h in MiaPACA 2, PANC-1, and SUN-1 cell lines. The results showed that the IC50 values ​​of seven compounds (I-9, VI-1, VI-2, VI-3, VI-4, II-4, and III-3) were... 50 The concentration was around 10 μM, and its cell viability decreased in a dose-dependent manner over time. Specifically, VI-1 showed an IC50 value of approximately 10 μM in PANC-1 and MIA PACA 2 cells after 72 h. 50 At 4 μM, II-4 and III-3 were IC50 in SNU-1(G12D) cells. 50 At 3-5 μM.

[0041] Subsequently, we investigated the protein degradation effects of these seven compounds. Unfortunately, compounds II-4 and III-3 showed no significant degradation effects. The degradation rates of VI-2, VI-3, and VI-4 in PANC-1 were 20%, 20%, and 35%, respectively. VI-1 at 10 μM degraded KRAS by 59%, 69%, and 52% in PANC-1, PANC 0203, and SNU-1 cells, respectively. Furthermore, we verified that VI-1 had no significant effect on KRAS protein degradation in other mutant cells such as PANC 0327 (G12V), A549 (G12S), and H358 (G12C), as well as in normal pancreatic tissue (HPNE). Therefore, the VHL-designed compound VI-1 can specifically degrade the KRAS G12D mutant protein without affecting protein expression in other KRAS mutants and normal pancreatic tissues.

[0042] This indicates that the compounds of the present invention have good KRAS G12D degradation activity and can be used in the preparation of pharmaceutical formulations for the prevention or treatment of diseases mediated by KRAS G12D protein, including cancers such as acute myeloid leukemia (AML), pancreatic cancer, colon cancer, bile duct cancer, cervical cancer, breast cancer, lung cancer, liver cancer, malignant glioma, bladder cancer, gastric cancer, and prostate cancer.

Claims

1. A degrading agent for KRAS G12D protein, characterized in that, The structure is as follows (I): Formula (I); in, Linker is a connecting chain, with the structure shown in the following formula: ， Where n is independently selected from integers from 0 to 9; E3 Ligand is selected from the following structures: ， ， in, X is CH2, NH or O. W is CH2 or C=O.

2. Stereoisomers, tautomers, deuterated products, solvates, prodrugs, metabolites, pharmaceutically acceptable salts, or cocrystals of the degrading agent for KRAS G12D protein as described in claim 1.

3. The degrading agent for KRAS G12D protein as described in claim 1, characterized in that, The following compounds:

4. The degrading agent for KRAS G12D protein as described in any one of claims 1 to 3, or its stereoisomers, tautomers, deuterated derivatives, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals, used as a KRAS G12D-targeting degrading agent in the preparation of pharmaceutical formulations for the prevention or treatment of diseases mediated by KRAS G12D protein.

5. The application as described in claim 4, characterized in that, The disease mediated by the KRAS G12D protein is cancer.

6. The application according to claim 5, characterized in that, The cancers mentioned include acute myeloid leukemia (AML), pancreatic cancer, colon cancer, bile duct cancer, cervical cancer, breast cancer, lung cancer, liver cancer, malignant glioma, bladder cancer, stomach cancer, and prostate cancer.

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