A method for preparing a compound targeting degradation of ERK5 protein

Abstract

The application belongs to the field of organic compound preparation, and discloses a preparation method of a compound for targeted degradation of ERK5 protein. First, a reactant one is used as a starting material, and is dissolved into an amide compound solvent under acidic conditions together with an amine compound. Then, an organic weak base and a condensation reagent are added, and stirring is performed for reaction. After the reaction is completed, an intermediate one is obtained through purification. Then, the intermediate one is dissolved in an ether compound, the reaction temperature is reduced, and then an inorganic reducing agent is added for reaction. After the reaction is completed, an intermediate two is obtained through purification. Finally, the intermediate two is dissolved in an organic mixed solvent together with a reactant two under acidic conditions, an acid binding agent and a catalyst are added respectively for stirring reaction, and then an organic reducing agent is added for continuous stirring reaction. After the reaction is completed, the compound for targeted degradation of ERK5 protein is obtained through purification. The compound for targeted degradation of ERK5 protein has the effects of anti-proliferation, anti-inflammation, and pharmacological results, and can target degradation of ERK5 protein.

Description

Technical Field

[0001] This invention relates to the field of organic compound preparation, and more particularly to a method for preparing a compound that targets and degrades ERK5 protein. Background Technology

[0002] ERK5 cascade signaling plays a crucial role in various diseases, including cancer and inflammation. Studies have shown overexpression or constitutive activation of MEK5 or ERK5 in various malignant tumors such as prostate cancer and hepatocellular carcinoma, and ERK5 knockdown inhibits proliferation in various tumor models. Research on ERK5 gene knockout or knockdown suggests that ERK5 may be a highly promising therapeutic target, leading to the development of several ATP-competitive ERK5 inhibitors. The well-known ERK5 inhibitor XMD8-92 has demonstrated good anti-tumor efficacy and anti-inflammatory activity in vitro and in vivo. However, further research indicates that the biological effects of XMD8-92 and its analogue XMD17-109 stem from their off-target activity against the BET protein family. Subsequently developed selective ERK5 inhibitors AX15836 and BAY-885 did not exhibit anti-proliferative or anti-inflammatory effects. This contrasts with the phenotype observed in ERK5 gene knockdown, suggesting that ERK5 may play other crucial roles in signaling that require further investigation, and that this mechanism of action is independent of its kinase activity. Furthermore, some ERK5 inhibitors, such as XMD17-109 and AX15836, can drive the translocation of ERK5 protein into the cell nucleus, leading to abnormal activation of ERK5 transcriptional activity. Therefore, the complex biological functions of ERK5 still require further investigation, and new chemical strategies need to be developed for drug discovery targeting this target.

[0003] Molecular chaperones, as a class of molecular machines, also play a crucial role in protein degradation. Currently, over 100 E3 ligases are capable of interacting with molecular chaperones. Examples of existing technologies include: CHIP-induced chaperone-dependent c-ErbB2 / Ne degradation; the crystal structure of CHIP-HSP90 binding and its ubiquitin degradation mechanism; and HECTD3-mediated HSP90-dependent client protein degradation. These findings suggest that molecular chaperone-mediated protein degradation may involve multiple E3 ligases other than PROTAC, and that the E3 ligases selected by molecular chaperones are cell-driven rather than artificially designed, potentially improving protein degradation efficiency and preventing drug resistance.

[0004] Protein degradation-targeting chimeras (PROTACs) recruit E3 ligases to the vicinity of target proteins, thereby inducing ubiquitination and subsequent proteasome degradation. Unlike traditional small-molecule inhibitors, these degraders can rapidly and pharmacologically deplete the target protein. Therefore, the development of ERK5 degraders will enable the chemical knockout of the ERK5 protein, which is closer to the ERK5 gene knockout or deletion event and the study of ERK5's non-enzymatic functions—something that ATP-competitive inhibitors cannot achieve. Although PROTAC-ERK5 (INY-06-061) is a highly selective and potent ERK5 degrader, the acute pharmacological degradation of ERK5 induced by INY-06-061 treatment did not produce an anti-proliferative effect compared to ERK5 gene deletion; that is, INY-06-061 treatment did not lead to any pharmacological results. Summary of the Invention

[0005] This application provides a method for preparing a compound that has anti-proliferative, anti-inflammatory, and pharmacologically effective properties for the targeted degradation of ERK5 protein.

[0006] This invention discloses a method for preparing a compound that targets and degrades ERK5 protein. The method is characterized by the following steps: First, using reactant one as a starting material, it is dissolved in an amide solvent under acidic conditions along with an amine compound. Then, an organic weak base and a condensing agent are added, and the mixture is stirred to carry out the reaction. After the reaction, the mixture is diluted with water, extracted, washed, dried, filtered, concentrated, and purified by silica gel column chromatography to obtain intermediate one. Second, intermediate one is dissolved in an ether compound, and the reaction temperature is lowered. An inorganic reducing agent is then added to carry out the reaction. After the reaction, the mixture is quenched, extracted, washed, dried, filtered, concentrated, and purified by silica gel column chromatography to obtain intermediate two. Third, intermediate two is dissolved in an organic mixed solvent with reactant two under acidic conditions. An acid-binding agent and a catalyst are added, and the mixture is stirred to carry out the reaction. Then, an organic reducing agent is added, and the reaction is continued with stirring. After the reaction, the mixture is quenched, extracted, washed, dried, filtered, concentrated, and purified by silica gel column chromatography to obtain the compound that targets and degrades ERK5 protein. The compound that targets and degrades ERK5 protein of this application has the ability to degrade ERK5 protein and also exhibits anti-proliferative and anti-inflammatory effects.

[0007] Furthermore, in the first step, the amine compound is CONC.

[0008] Furthermore, in the first step, the solvent for the amide compound is DMF, the weak organic base is DIEA, and the condensing agent is HATU. DMF is a commonly used aprotic polar solvent with good solubility and stability. Using DMF as a solvent can dissolve reactant one and the amine compound in the first step, ensuring thorough mixing in the solution and increasing the contact area between them, thereby improving the reaction rate. DIEA, as a weak organic base, can promote the reaction between carboxylic acids and amines, forming amide bonds and promoting the synthesis of amide compounds. DIEA can also act as a catalyst, accelerating the reaction and improving the reaction yield and selectivity. HATU, as a condensing agent, can promote the condensation reaction between carboxylic acids and amines, forming amide bonds. HATU can also act as a catalyst, accelerating the reaction and improving the reaction yield and selectivity. In this application, DIEA is used as the weak organic base, and HATU is used as the condensing agent. The combined action of DIEA and HATU catalyzes and condenses the reaction, accelerating the reaction rate.

[0009] Furthermore, in the second step, the ether compound is dry tetrahydrofuran, and the inorganic reducing agent is LiAlH4. Tetrahydrofuran is a polar aprotic solvent with good solubility for many organic and inorganic substances. It can dissolve many sparingly soluble compounds, such as some polymers, aliphatic compounds, and aromatic compounds. The use of tetrahydrofuran in the second step effectively dissolves intermediate one. Tetrahydrofuran is chemically stable, but its low boiling point makes it easily volatile at room temperature. It can be purified and separated by methods such as distillation or drying. LiAlH4 is a strong reducing agent that can reduce many compounds. It can effectively reduce intermediate one, exhibiting high selectivity and varying reduction selectivity for different functional groups. Under specific temperature and pH conditions, it can selectively reduce certain functional groups without affecting others. The reduction products of LiAlH4 are usually relatively singular and do not produce other byproducts, which is beneficial for product separation and purification. Using LiAlH4 as an inorganic reducing agent can effectively reduce the functional groups in intermediate one to prepare intermediate two.

[0010] Furthermore, in the third step, the organic mixed solvent is DCE / MeOH, the acid-binding agent is Et3N, the catalyst is Ti(Oi-Pr)4, and the organic reducing agent is NaBH(OAc)3. Both DCE and MeOH are polar solvents, capable of dissolving many organic and inorganic substances, and exhibit good solubility for intermediate two and reactant two. Using DCE / MeOH as the organic mixed solvent ensures that intermediate two and reactant two are fully dissolved and mixed in the DCE / MeOH solvent, increasing the contact area between them and improving the reaction rate. DCE and MeOH are chemically stable and do not readily react with other substances. DCE / MeOH can adjust the reaction conditions; by adjusting the ratio of DCE to MeOH, the polarity and solubility of the solvent can be altered, thereby regulating the rate of the third-step reaction and the selectivity of the products. The acid-binding agent is usually a basic substance that can neutralize acidic substances, thus stabilizing the pH of the reaction system and promoting the reaction. The acid-binding agent can also be used to protect sensitive functional groups in the reaction. Et3N is a strong basic acid-binding agent that can effectively adjust the pH of the third step reaction and promote its progress. Furthermore, Et3N exhibits different reactivity selectivity for different functional groups, allowing it to selectively neutralize certain acidic substances without affecting other functional groups. Et3N is chemically stable and does not readily react with other substances, so using Et3N as an acid-binding agent will not affect the third step reaction.

[0011] Furthermore, in the second step, the temperature range after lowering the reaction temperature is greater than or equal to -5℃ and less than or equal to 5℃. After adding the inorganic reducing agent, the reaction temperature is less than or equal to -73℃ and greater than or equal to -83℃. The reaction time after adding the inorganic reducing agent is greater than or equal to 55 min and less than or equal to 65 min. In the second step, intermediate one is dissolved in an ether compound, then the reaction temperature is lowered, and then an inorganic reducing agent is added to carry out the reaction. The selectivity of the inorganic reducing agent for reducing functional groups varies under different temperature conditions. In order to prepare intermediate two, it is necessary to ensure that the inorganic reducing agent selectively reduces the functional groups of intermediate one. In summary, this application sets the reaction temperature after adding the inorganic reducing agent to less than or equal to -73℃ and greater than or equal to -83℃. A reaction temperature of less than or equal to -73℃ and greater than or equal to -83℃ can ensure that the functional groups in intermediate one are selectively reduced to generate intermediate two. After adding the inorganic reducing agent, a certain reaction time is required to ensure a complete reaction. After the reaction is complete, the next step can be carried out. In this application, the reaction time after adding the inorganic reducing agent is greater than or equal to 55 min and less than or equal to 65 min to ensure that the reduction reaction is complete.

[0012] Furthermore, in the third step, the preparation of the compound requires a certain temperature and time. The reaction temperature after adding the acid-binding agent and catalyst and stirring is set to be greater than or equal to 15°C and less than or equal to 30°C, and the reaction time is greater than or equal to 50 min and less than or equal to 70 min.

[0013] Furthermore, ethyl acetate was used for extraction in steps one, two, and three; water and brine were used for washing in steps one, two, and three; and anhydrous sodium sulfate was used for drying in steps one, two, and three. Ethyl acetate is a commonly used extraction solvent that can effectively extract the reaction product and is readily available. Similarly, anhydrous sodium sulfate is also a commonly used drying agent and is readily available.

[0014] Furthermore, in the second step, quenching is performed using an aqueous solution of potassium sodium tartrate, and in the third step, quenching is performed using an aqueous solution of saturated sodium bicarbonate. Potassium sodium tartrate is used as a quenching agent in the second step primarily because it can rapidly terminate the reaction. Potassium sodium tartrate is a strong alkaline substance that can quickly neutralize acidic substances in the reaction system, thereby stopping the reaction. Potassium sodium tartrate does not interfere with subsequent analysis; it does not react with other substances in the reaction system and does not interfere with subsequent analysis and detection. Potassium sodium tartrate is a common chemical reagent, easy to prepare and use, and relatively inexpensive. The main reason for using an aqueous solution of saturated sodium bicarbonate as a quenching agent in the third step is that it does not react with other substances in the reaction system and is readily available.

[0015] Furthermore, the acidic conditions in both the first and third steps are adjusted using HCl. HCl is a common chemical reagent that is easy to prepare and use, and is relatively inexpensive. Detailed Implementation

[0016] To enable those skilled in the art to better understand the present invention, the technical solutions in the specific embodiments of the present invention will be clearly and completely described below.

[0017] In this application:

[0018] Reactant 1: ;

[0019] Reactant 2: ;

[0020] Intermediate 1: ;

[0021] Intermediate 2: ;

[0022] Compounds that target and degrade ERK5 protein: ;

[0023] Reference compound: .

[0024] Preparation of compounds that target and degrade ERK5 protein:

[0025] Example 1: Preparation of compounds targeting the degradation of ERK5 protein

[0026] Step 1: Using reactant 1 as the starting material, 2.0 g of reactant 1 (3.53 mmol) and 0.38 g of CONC (3.88 mmol) were dissolved in 20 mL of DMF under acidic conditions. Then, 1.37 g of DIEA (10.5 mmol) and 1.48 g of HATU (3.88 mmol) were added, and the mixture was stirred at room temperature for 1 hour to allow the reaction to proceed. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate (50 mL * 2). The combined organic phases were washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column filtered through silica gel (PE:EA = 1:1) to obtain intermediate 1 (3.28 mmol) with a yield of 93%.

[0027] Step 2: 2.0 g of intermediate one (3.28 mmol) was dissolved in 50 mL of dry tetrahydrofuran solution. The reaction temperature was then lowered to 0°C. Next, 249 mg of LiAlH4 (6.56 mmol) was added, and the reaction was carried out at -78°C for 1 hour. After the reaction was complete, the reaction was quenched with potassium sodium tartrate aqueous solution. The reaction solution was extracted with ethyl acetate (30 mL * 2). The combined organic phases were washed with water and brine, respectively, and then dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to obtain the crude product. Finally, the crude product was obtained by silica gel column chromatography (PE:EA = 1:1) to yield 1.2 g of intermediate two (2.18 mmol), with a yield of 66%.

[0028] Step 3: Dissolve 1.15 g of intermediate II (2.04 mmol) and 1.05 g of reactant II (2.65 mmol) in 50 mL of DCE / MeOH at a ratio of 4:1. Then add 4.1 g of Et3N (4.08 mmol) and 2.32 g of Ti(Oi-Pr)4 (8.15 mmol), and stir at room temperature for 1 hour. Then add 2... The reaction was continued with 16 g of NaBH(OAc)3 and 10.18 mmol of NaBH(OAc)3 under stirring. The reaction was quenched with saturated sodium bicarbonate aqueous solution, and the reaction solution was extracted with ethyl acetate (50 mL * 2). The combined organic phases were washed with water and brine, respectively, dried with anhydrous sodium sulfate, filtered, concentrated, and finally passed through a silica gel column (DCM:MeOH = 40:1) to obtain a compound with a mass of 1.3 g and a molar amount of 1.40 mmol that targets and degrades ERK5 protein, with a yield of 69%.

[0029] Example 2: Preparation of compounds targeting the degradation of ERK5 protein

[0030] Step 1: Using reactant 1 as the starting material, 4.0 g of reactant 1 (7.06 mmol) and 0.76 g of CONC (7.76 mmol) were dissolved in 40 mL of DMF under acidic conditions. Then, 2.74 g of DIEA (21.0 mmol) and 2.96 g of HATU (7.76 mmol) were added, and the mixture was stirred at room temperature for 1.5 hours to carry out the reaction. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate (100 mL * 2). The combined organic phases were washed with water and brine, dried with anhydrous sodium sulfate, filtered, concentrated, and column filtered through silica gel (PE:EA = 1:1) to obtain intermediate 1 (6.89 mmol) with a yield of 97%.

[0031] Step 2: 4.0 g of intermediate one (6.56 mmol) was dissolved in 100 mL of dry tetrahydrofuran solution. The reaction temperature was then lowered to 0°C. 498 mg of LiAlH4 (13.12 mmol) was added, and the reaction was carried out at -78°C for 1.5 hours. After the reaction was complete, the reaction was quenched with potassium sodium tartrate aqueous solution. The reaction solution was extracted with ethyl acetate (60 mL * 2). The combined organic phases were washed with water and brine, respectively, and then dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to obtain the crude product. Finally, column chromatography (PE:EA = 1:1) yielded 2.5 g of intermediate two (4.54 mmol), with a yield of 69%.

[0032] Step 3: Dissolve 2.3 g of intermediate II (4.08 mmol) and 2.1 g of reactant II (5.30 mmol) in 100 mL of DCE / MeOH at a ratio of 4:1. Then add 8.2 g of Et3N (8.16 mmol) and 4.64 g of Ti(Oi-Pr)4 (16.30 mmol), respectively, and stir at room temperature for 1.5 hours. Then add 4 g of... The reaction was continued with 0.32 g of NaBH(OAc)3 and 20.36 mmol of NaBH(OAc)3 under stirring. The reaction was quenched with saturated sodium bicarbonate aqueous solution, and the reaction solution was extracted with ethyl acetate (100 mL * 2). The combined organic phases were washed with water and brine, respectively, dried with anhydrous sodium sulfate, filtered, concentrated, and finally passed through a silica gel column (DCM:MeOH = 40:1) to obtain a compound with a mass of 2.8 g and a molar amount of 3.01 mmol that targets and degrades ERK5 protein, with a yield of 74%.

[0033] Example 3: Preparation of compounds targeting the degradation of ERK5 protein

[0034] Step 1: Using reactant 1 as the starting material, 6.0 g of reactant 1 (10.59 mmol) and 1.14 g of CONC (11.64 mmol) were dissolved in 50 mL of DMF under acidic conditions. Then, 4.11 g of DIEA (31.5 mmol) and 4.44 g of HATU (11.64 mmol) were added, and the mixture was stirred at room temperature for 2 hours to carry out the reaction. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate (150 mL * 2). The combined organic phases were washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column filtered through silica gel (PE:EA = 1:1) to obtain intermediate 1 (6.3 g, 10.34 mmol), with a yield of 97%.

[0035] Step 2: 6.0 g of intermediate one (9.84 mmol) was dissolved in 100 mL of dry tetrahydrofuran solution. The reaction temperature was then lowered to 0°C. 747 mg of LiAlH4 (19.68 mmol) was added, and the reaction was carried out at -78°C for 2 hours. After the reaction was complete, the reaction was quenched with potassium sodium tartrate aqueous solution. The reaction solution was extracted with ethyl acetate (80 mL * 2). The combined organic phases were washed with water and brine, respectively, and then dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to obtain the crude product. Finally, column chromatography (PE:EA = 1:1) yielded 3.8 g of intermediate two (6.90 mmol), with a yield of 70%.

[0036] Step 3: Dissolve 3.45 g of intermediate II (6.12 mmol) and 3.15 g of reactant II (7.95 mmol) in 150 mL of DCE / MeOH at a ratio of 4:1. Then add 12.3 g of Et3N (12.24 mmol) and 6.96 g of Ti(Oi-Pr)4 (24.45 mmol), respectively, and stir at room temperature for 2 hours. Then add... The reaction was continued with 6.48 g of NaBH(OAc)3 and 30.54 mmol of substance, and the reaction was quenched with saturated sodium bicarbonate aqueous solution. The reaction solution was extracted with ethyl acetate (150 mL * 2). The combined organic phases were washed with water and brine, respectively, dried with anhydrous sodium sulfate, filtered, concentrated, and finally passed through a silica gel column (DCM:MeOH = 40:1) to obtain a compound with a mass of 4.3 g and a substance amount of 4.62 mmol of substance that targets and degrades ERK5 protein, with a yield of 75%.

[0037] Comparative Example 1: Preparation of the control compound

[0038] This invention also provides a control compound. The preparation of the control compound, similar to that of the compound targeting the degradation of ERK5 protein, begins with reactant one. Reactant one and CONC are dissolved in DMF under acidic conditions. Then, DIEA and HATU are added, and the mixture is stirred at room temperature for 1 hour. After the reaction, the mixture is diluted with water, extracted with ethyl acetate, and the combined organic phases are washed with water and brine, dried with anhydrous sodium sulfate, filtered, concentrated, and column-coated on silica gel (PE:EA = 1:1) to obtain intermediate one. Intermediate one is then dissolved in a dry tetrahydrofuran solution, and the reaction temperature is lowered to 0°C. LiAlH4 is then added, and the reaction is carried out at -78°C for 1 hour. After the reaction, the reaction is quenched with potassium sodium tartrate aqueous solution, and the reaction solution is extracted with ethyl acetate. The combined organic phases are washed with water and brine, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Finally, the crude product is obtained by column chromatography on silica gel (PE:EA = 1:1) to obtain intermediate two. Intermediate 2 and reactant 2 were dissolved in DCE / MeOH at a concentration ratio of 4:2. Et3N and Ti(Oi-Pr)4 were then added separately, and the mixture was stirred at room temperature for 1 hour. NaBH(OAc)3 was then added, and the reaction was continued with stirring. The reaction was quenched with saturated sodium bicarbonate aqueous solution, and the reaction solution was extracted with ethyl acetate. The combined organic phases were washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated, and finally column-coated with silica gel (DCM:MeOH = 40:1) to obtain the control compound. .

[0039] I. Determination of the binding affinity between the compound targeting and degrading ERK5 protein and the control compound and HSP90 protein.

[0040] Experimental steps:

[0041] Sample processing

[0042] 1. Prepare the premix: The components of the premix are assay buffer (provided in the kit, Hsp90α5X buffer1, BPS#50311, diluted with double-distilled water to 1X), DTT, BSA, and galdromycin-FITC. The final concentrations of each component in the premix are as follows: assay buffer (1X), DTT final concentration is 4mM, BSA final concentration is 0.2mg / ml, and galdromycin-FITC final concentration is 20nM.

[0043] 2. Prepare an Hsp90 solution by diluting the Hsp90 protein to 14.6 ug / ml in the assay buffer.

[0044] 3. Transfer the prepared premix to a 384-well plate, with a volume of 10 μl / well.

[0045] 4. Add the Hsp90 solution to the 384-well plate. The volume of Hsp90 solution in the 384-well plate is 9.6 μl / well. Stir well until homogeneous.

[0046] Incubate at 5.25℃ for 2 hours.

[0047] 6. The compound that targets and degrades ERK5 protein and the control compound were added to the 384-well plate and the binding ability of the compound that targets and degrades ERK5 protein and the control compound to Hsp90 was tested.

[0048] 7. The first two wells in each row are positive controls, using assay buffer instead of the compound that targets and degrades ERK5 protein and Hsp90.

[0049] 8. The last two wells in each row are negative controls, using DMSO instead of the compound that targets and degrades the ERK5 protein.

[0050] Incubate at 9.25℃ for 2 hours.

[0051] 10. Data collection and processing.

[0052] II. Evaluation of the inhibitory effect of compounds targeting ERK5 protein degradation on Erk5 kinase activity

[0053] 1. Dilute XMD17-109 (10mM stock solution) 5-fold with 100% DMSO. Dilute the compound that targets and degrades ERK5 protein 5-fold. Perform a 4-fold serial dilution in a 96-well plate. Add 1 μL of the compound that targets and degrades ERK5 protein to 49 μL of kinase reaction buffer and shake on a microplate shaker for 20 min.

[0054] 2. Transfer 2 μL of 2XErk5 kinase to a 384 reaction plate, add 1 μL of the compound to be tested that targets and degrades ERK5 protein (prepared in step 1) to the 384 reaction plate, centrifuge at 1000 rpm / min for 1 min, and then incubate at 25°C for 10 min.

[0055] 3. Transfer 1 μL of the 4X substrate mixture to a 384 reaction plate, centrifuge at 1000 rpm for 1 min, and then incubate at 25 °C for 60 min.

[0056] 4. Add 1 μL of a mixture of MBP and ATP (final concentration of 10 μM) to each well, seal the plate with sealing film, centrifuge at 1000g for 30 seconds, and incubate at room temperature for 60 minutes.

[0057] 5. Transfer 4uLADP-Glo ​​to a 384 reaction plate, centrifuge at 1000 rpm / min for 1 min, and then incubate at 25°C for 40 min.

[0058] 6. Transfer 8 μL of detection solution to a 384 reaction plate, centrifuge at 1000 rpm / min for 1 min, and then incubate at 25 °C for 40 min.

[0059] 7. Use a multi-functional plate reader to read the RLU (Relative Luminescence Unit) signal. The signal intensity is used to characterize the activity level of the kinase.

[0060] 8. Data analysis.

[0061] 8.1 Calculate the inhibition rate:

[0062] Inhibition rate (%) = (1 - (lum targeted ERK5 protein degradation compound - lum positive control) / lum negative control - lum positive control) * 100%.

[0063] 8.2 Calculate IC50 and plot inhibition curves of compounds targeting the degradation of ERK5 protein:

[0064] The IC50 (half-maximal inhibitory concentration) of compounds targeting ERK5 protein degradation was obtained using the following nonlinear fitting formula for data analysis: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where X is the log value of the concentration of the compound targeting ERK5 protein degradation, and Y is the inhibition rate.

[0065] III. Determination of the degradation activity of the target ERK5 protein compound and control compound on ERK5 protein in various cell lines.

[0066] The following cancer cell lines were used for cell culture: Molp-8 myeloma cells, HCC1187, Hs578T and MDA-MB-231 breast cancer cells were used to prepare the culture medium.

[0067] ERK5 protein degradation experiment:

[0068] 1. Molp-8, HCC1187, Hs578T and MDA-MB-231 were seeded into 12-well plates (2.5 x 10⁵ cells / well / 0.5 ml).

[0069] 2. Add the compound solution that targets and degrades ERK5 protein to the wells of the 12-well plate to achieve the designed concentration (10 nM, 100 nM, 300 nM, 1000 nM, etc.), mix thoroughly and incubate for 48 hours.

[0070] 3. Wash the wells with cold PBS once each for Molp-8, HCC1187, Hs578T and MDA-MB-231 cells. After washing, aspirate the supernatant and add 120 μL of pre-chilled RIPA lysis buffer and protease / phosphatase inhibitor directly to ice to lyse Molp-8, HCC1187, Hs578T and MDA-MB-231 cells respectively. After 30 minutes, prepare cell lysates for Molp-8, HCC1187, Hs578T and MDA-MB-231 cells respectively.

[0071] 4. Centrifuge the lysates of Molp-8, HCC1187, Hs578T and MDA-MB-231 cells at 12000 rpm / min and 4°C for 10 minutes. Collect the supernatant and transfer 20 μL to a new BCA tube. Quantify the lysate sample using the BCA method.

[0072] 5. Add loading buffer to the lysates of Molp-8, HCC1187, Hs578T, and MDA-MB-231 cells to prepare loading samples for Molp-8, HCC1187, Hs578T, and MDA-MB-231 cells, respectively. Heat the loading samples of Molp-8, HCC1187, Hs578T, and MDA-MB-231 cells at 100°C for 10 minutes and then cool to room temperature.

[0073] 6. Adjust the final concentration of Molp-8, HCC1187, Hs578T and MDA-MB-231 cell loading samples to 2.75 ug / ul using RIPA and loading buffer.

[0074] 7. Add the Molp-8, HCC1187, Hs578T and MDA-MB-231 cell samples to the gel electrophoresis apparatus, run gel electrophoresis at 80V for 20 minutes, and then increase the voltage to 120V and run gel electrophoresis for 120 minutes.

[0075] 8. Electrotransfer the protein to the NC membrane using a wet transfer method with a current of 250 m, and block the membrane with TBST and BSA for 1 hour.

[0076] 9. Wash the membrane three times with 1XTBST for 5 minutes each time. Incubate the primary antibody with the primary antibody prepared in blocking buffer containing 1XTBST and 5% BSA overnight at 4°C.

[0077] 10. Wash the membrane three times with 1XTBST for 5 minutes each time. Incubate with secondary antibody at room temperature for 1 hour. (Anti-rabbit IgG (Licor, 926-32211) 1:5000, anti-mouse IgG (LI-COR, 926-68070) 1:5000).

[0078] 11. Clean the membrane three times with 1XTBST, 5 minutes each time. Then clean with ROH2O. Measure the signal using LiCOR.

[0079] 12. Data processing.

[0080] The data is shown in Table 1.

[0081]

[0082] Table 1

[0083] IV. Determine the cytotoxicity of compounds that target and degrade ERK5 protein and control compounds in various cell lines.

[0084] Cell culture was performed using the following cancer cell lines: SW48 colorectal cancer cells (ATCC), Molp-8 myeloma cells (Cobioer), HCC1187, Hs578T, and three triple-negative breast cancer (ATCC) cells, MDA-MB-231.

[0085] Cancer cell proliferation experiment:

[0086] SW48, Molp-8, HCC1187, Hs578T, and MDA-MB-231 cells were seeded at 1000 cells per well in 384-well tissue culture plates and incubated at 37°C, 5% CO2, and 30 μL of culture medium for 24 to 48 hours. Each test compound was prepared in 3-fold serial dilutions. Each cell line was then treated with different concentrations of the test compound, with a final concentration of 0.1% DMSO / well, and incubated at 37°C and CO2 for 24 to 72 hours. 30 μL of CellTiter-Glo® reagent was added to each well and treated according to the reagent's experimental protocol. The results were then analyzed, and the IC50 value was calculated.

[0087] Data processing:

[0088] The analysis results and IC50 values ​​are shown in Table 2.

[0089]

[0090] Table 2

[0091] Results analysis:

[0092] 1. The compound targeting the degradation of ERK5 protein showed good binding affinity to both ERK5 protein (less than 100 nM) and HSP90 protein (less than 300 nM), while the control compound only showed binding affinity to HSP90 protein (less than 500 nM) but no significant binding affinity to ERK5 protein (greater than 50,000 nM) (see Table 1).

[0093] 2. Compounds that target and degrade ERK5 protein with binding forces at both ends showed the ability to degrade ERK5 protein in various cell lines, while control compounds that only bind to one end of HSP90 failed to show a significant ability to degrade ERK5 protein under the same conditions (see Table 1).

[0094] 3. Compounds that target and degrade ERK5 protein with binding forces at both ends showed good cytotoxicity in a variety of cell lines, while control compounds that only bind to one end of HSP90 showed relatively weak cytotoxicity under the same conditions (see Table 2).

[0095] The above results validate that simultaneous binding to the target protein ERK5 and the molecular chaperone HSP90 is key to the degradation of the target protein. Compounds targeting ERK5 protein degradation demonstrated good ERK5 protein degradation ability and cytotoxicity in in vitro experiments, showing potential for further development.

[0096] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing a compound that targets and degrades ERK5 protein, characterized in that: Step 1: Using reactant one Using this as the starting material, it and an amine compound were dissolved in an amide compound solvent under acidic conditions. Then, a weak organic base and a condensing agent were added, and the mixture was stirred to initiate the reaction. After the reaction was complete, the intermediate was obtained by dilution with water, extraction, washing, drying, filtration, concentration, and silica gel column chromatography. ; Step 2: Dissolve intermediate one in an ether compound, then lower the reaction temperature, and then add an inorganic reducing agent to carry out the reaction. After the reaction is complete, obtain intermediate two by quenching, extraction, washing, drying, filtration, concentration, and silica gel column chromatography. ; Step 3: React intermediate 2 with reactant 2 under acidic conditions. Dissolved in a mixed organic solvent, an acid-binding agent and a catalyst were added separately and stirred to react. Then, an organic reducing agent was added and the reaction was continued with stirring. After the reaction was completed, the compound that targets and degrades ERK5 protein was obtained by quenching, extraction, washing, drying, filtering, concentration, and silica gel column chromatography. ; In the first step, the solvent for the amide compound is DMF, the weak organic base is DIEA, and the condensation reagent is HATU; In the second step, the ether compound is dry tetrahydrofuran, and the inorganic reducing agent is LiAlH4; In the third step, the organic mixed solvent is DCE / MeOH, the acid-binding agent is Et3N, the catalyst is Ti(Oi-Pr)4, and the organic reducing agent is NaBH(OAc)3; In the second step, the temperature range after lowering the reaction temperature is greater than or equal to -5℃ and less than or equal to 5℃. After adding the inorganic reducing agent, the reaction temperature is less than or equal to -73℃ and greater than or equal to -83℃. After adding the inorganic reducing agent, the reaction time is greater than or equal to 55min and less than or equal to 65min.

2. The method for preparing the compound for targeted degradation of ERK5 protein according to claim 1, characterized in that: In the third step, after adding the acid-binding agent and catalyst, the reaction temperature is greater than or equal to 15℃ and less than or equal to 30℃, and the reaction time is greater than or equal to 50min and less than or equal to 70min.

3. The method for preparing the compound for targeted degradation of ERK5 protein according to claim 1, characterized in that: Ethyl acetate was used for extraction in steps one, two, and three. Water and salt water are used for washing in the first, second and third steps; Anhydrous sodium sulfate was used for drying in the first, second and third steps.

4. The method for preparing the compound for targeted degradation of ERK5 protein according to claim 1, characterized in that: In the second step, quenching is performed using an aqueous solution of potassium sodium tartrate. In the third step, quenching is performed using a saturated sodium bicarbonate aqueous solution.

5. The method for preparing the compound for targeted degradation of ERK5 protein according to claim 1, characterized in that: The acidic conditions in both the first and third steps were adjusted using HCl.

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