A g4 stabilizer and a preparation process thereof

Abstract

The application relates to the technical field of biological medicine, in particular to a G4 stabilizer and a preparation process thereof, which comprises the following steps: S1, mixing raw material A and a solvent in a mixing device to obtain a mixed solution; the raw material A is at least one selected from Tmpyp4 and CX5461; the solvent is at least one selected from methanol, ethanol, N, N dimethylformamide and dimethyl sulfoxide; S2, adding a catalyst into the mixed solution and heating the reaction under the protection of inert gas; the catalyst is at least one selected from triethylamine, potassium carbonate and palladium chloride; the reaction temperature is 60 DEG C to 90 DEG C, and the reaction time is 4 h to 8 h; S3, after the reaction is completed, cooling to room temperature, removing insoluble substances through filtration, collecting the organic phase, and vacuum drying at 40 DEG C to 50 DEG C to obtain a crude product; S4, purifying the crude product through silica gel column chromatography, and using a mixed solvent of dichloromethane and methanol as an eluent to obtain the G4 stabilizer; and the method guarantees the yield and batch stability of the crude product.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a G4 stabilizer and its preparation process. Background Technology

[0002] Hepatocellular carcinoma (HCC) ranks 6th in incidence and 2nd in mortality among malignant tumors worldwide, accounting for 85% of primary liver cancers and seriously threatening human life and health. The occurrence and development of liver cancer are closely related to the silencing of tumor suppressor gene expression and abnormal transcriptional regulation. KLF6 is a widely expressed nuclear transcriptional regulator, but it is significantly underexpressed in liver cancer tissues. Its expression level is positively correlated with patient survival. Overexpression of KLF6 can effectively inhibit the proliferation and migration of liver cancer cells, making it a key tumor suppressor gene that regulates the progression of liver cancer. Chromatin binding peak distribution analysis showed that KLF6 binding sites were mainly enriched in the proximal region of the promoter (62.1%), indicating that its biological function is highly dependent on promoter-level transcriptional regulation.

[0003] G Quadruplex (G4) is a secondary nucleic acid structure formed by the pairing and stacking of guanine-rich sequences through Hoogsteen hydrogen bonds. It is widely distributed in key genomic regions such as gene promoters and can precisely regulate gene transcription. Using G4 structure as a target to regulate the expression of tumor-related genes with small molecule stabilizers has become an important frontier direction in anti-tumor drug development. Sequence analysis and functional experiments confirmed that the KLF6 promoter region contains G-rich sequences and has the potential to form G4 structures. Mutating this G4 to form a motif can significantly enhance promoter activity, while the G4 stabilizer Tmpyp4 can inhibit its promoter activity, proving that the G4 structure is a key cis-acting element regulating KLF6 transcription.

[0004] Although G4 stabilizers have significant potential applications in regulating KLF6 expression and fighting liver cancer, existing G4 stabilizers generally suffer from insufficient targeting, poor stability, and complex preparation processes. Currently, there is still a lack of specific stabilizers targeting the G4 structure of the KLF6 promoter and their standardized preparation processes. Therefore, developing highly targeted and stable G4 stabilizers and establishing efficient preparation processes are of great theoretical and clinical significance for promoting the research and development of novel anti-liver cancer drugs. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a G4 stabilizer and its preparation process.

[0006] The present invention provides a G4 stabilizer preparation process, comprising the following steps: S1. Add raw material A and solvent to a mixing device and mix to obtain a mixture; the raw material A is selected from at least one of Tmpyp4 and CX5461; the solvent is selected from methanol, ethanol, N,N At least one of dimethylformamide or dimethyl sulfoxide; S2. Add a catalyst to the mixture and heat it under inert gas protection; the catalyst is selected from at least one of triethylamine, potassium carbonate or palladium chloride; the reaction temperature is 60℃~90℃ and the reaction time is 4h~8h. S3. After the reaction is complete, cool to room temperature, filter to remove insoluble matter, extract the filtrate with ethyl acetate and saturated sodium chloride solution, collect the organic phase, and dry under vacuum at 40℃~50℃ to obtain the crude product; S4. The crude product is purified by silica gel column chromatography using a mixed solvent of dichloromethane and methanol as the eluent. The target fraction is collected, concentrated, and then vacuum dried to obtain the G4 stabilizer. This process only requires four steps, S1 to S4, and is simple to operate and easy to scale up for industrial production. In step S2, the reaction temperature is controlled at 60℃ to 90℃ and the reaction time is 4h to 8h. The reaction conditions are mild, energy consumption is low, and equipment requirements are not high, which significantly reduces production costs. In step S3, extraction with ethyl acetate and saturated sodium chloride solution, combined with vacuum drying at 40℃ to 50℃, effectively ensures the yield and batch stability of the crude product. In step S4, purification by silica gel column chromatography using a mixed solvent of dichloromethane and methanol as the eluent yields a G4 stabilizer with a purity of over 95%. Meanwhile, the raw materials A (Tmpyp4, CX5461), solvents, and catalysts used are all commonly used commercially available reagents, with a wide range of raw material sources and controllable costs.

[0007] Preferably, the mixing equipment includes a mixing tank, a controller, a support frame, a feed pipe, a filter cover, and a discharge pipe. The controller is mounted on the mixing tank, which is installed on the support frame. A feed pipe is located at the top of the mixing tank. A filter cover is installed inside the mixing tank, directly below the feed pipe. The bottom of the mixing tank is connected to the input end of the discharge pipe. The equipment also includes: A stirring assembly, installed on a mixing tank, is used to form a mixture of materials and solvents; The reflux assembly, installed between the discharge pipe outlet and the mixing assembly, is used for conveying the mixture. The controller is electrically connected to the reflux assembly and the stirring assembly. Both the material and the solvent are injected into the mixing tank through the feed pipe. The filter hood filters out large particles in the material. Then, the reflux assembly is activated, which refluxes the mixture back into the mixing tank. At the same time, the stirring assembly is activated to quickly mix the material and the solvent, improving the stirring efficiency and saving time for subsequent processing.

[0008] Preferably, the return liquid assembly includes a transfer pump, a three-way pipe, a discharge pipe, a discharge valve, a return pipe, and a return valve. The input end of the transfer pump is connected to the output end of the discharge pipe, the output end of the transfer pump is connected to the input end of the three-way pipe, the first set of output ends of the three-way pipe is connected to the input end of the discharge pipe, a discharge valve is installed on the discharge pipe, the second set of output ends of the three-way pipe is connected to the input end of the return pipe, a return valve is installed on the return pipe, and the transfer pump is electrically connected to the controller. In use, the material and solvent are inside the mixing tank. The discharge valve is closed and the return valve is opened. The transfer pump is started by the controller, so that the mixture inside the mixing tank drives the stirring assembly through the discharge pipe and the return pipe. This causes the stirring assembly to mix and stir the material and solvent inside the mixing tank, and the material and solvent driven by the stirring assembly return to the mixing tank, realizing rapid mixing of the material and solvent.

[0009] Preferably, the stirring assembly includes a fixed base, a water motor, a hollow rotating shaft, hollow stirring rods, a No. A sprocket, a No. B sprocket, a No. A chain, an infusion pipe, a rotary joint, and a branch pipe. A fixed base is located at the top of the mixing tank, and a water motor is mounted on the fixed base. A hollow rotating shaft is rotatably mounted inside the mixing tank, and multiple sets of hollow stirring rods are mounted on the hollow rotating shaft, communicating with it. The top of the hollow rotating shaft extends above the mixing tank. A No. A sprocket is located at the output end of the water motor, and a No. B sprocket is mounted on the hollow rotating shaft. The No. A sprocket and the No. B sprocket are driven by a No. A chain. The output end of the return pipe is connected to the inlet end of the water motor, and the outlet end of the water motor is connected to the inlet end of the infusion pipe. The output end of the infusion pipe is rotatably connected to the top of the hollow rotating shaft via a rotary joint. A branch pipe is mounted on the infusion pipe, and the output end of the branch pipe extends into the mixing tank and is close to the filter cover. In use... The delivery pump is started, and the mixture enters the hydraulic motor through the return pipe. The hydraulic motor drives sprocket A to rotate, which in turn drives sprocket B via chain A. This, in turn, causes the hollow shaft to rotate multiple sets of hollow stirring rods inside the mixing tank. The hollow stirring rods mix and agitate the mixture inside the mixing tank. The mixture inside the hydraulic motor enters the hollow shaft through the delivery pipe and rotary joint, and is then redistributed back into the mixing tank by the multiple sets of hollow stirring rods. Simultaneously, another set of mixture flows through a branch pipe to flush the filter cover, allowing the mixture to enter the filter cover and dissolve larger impurities in the filtered material. The delivery pump provides delivery pressure for the mixture, which then enters the hydraulic motor to drive the hollow shaft. The rotation of the hollow shaft and the hollow stirring rods further disperses the mixture into the mixing tank, achieving mixing and improving stirring efficiency.

[0010] Preferably, it also includes a tapping component, which is mounted on the mixing tank and used to tap the side wall of the filter cover to prevent the filter cover from clogging; The striking assembly includes a second sprocket A, a second sprocket B, a second chain, a rotating shaft, a cam, a fixed plate, a spring, and a striking block. The second sprocket A is mounted on the hollow rotating shaft. A rotating shaft is rotatably mounted at the top of the mixing tank, extending to the top of the mixing tank. A second sprocket B is mounted at the top of the rotating shaft. The second sprocket A and the second sprocket B are driven by the second chain. A cam is mounted at the bottom of the rotating shaft. A fixed plate is mounted at the top of the mixing tank, and a spring is mounted on the fixed plate. A striking block is mounted at the end of the spring near the filter cover. When the water motor drives the hollow shaft to rotate, the hollow shaft rotates through the cooperation of sprockets No. 2A, No. 2B, and No. 2 chain. This rotation further drives the cam to rotate. When the cam contacts the spring, the spring bends and deforms, causing the striking block to move away from the filter cover. When the cam separates from the bent spring, the spring elastically recovers, causing the striking block to strike the outer wall of the filter cover. This knocks down the particles remaining inside the filter holes, further preventing clogging and extending the service life.

[0011] Preferably, it also includes a sampling valve tube and a sampling valve. A sampling tube is installed on the discharge pipe, and a sampling valve is fixedly installed on the sampling tube. After mixing and stirring for a period of time, 209 is opened, and the staff uses a sampling tool to sample and test the mixture flowing out through the sampling valve, thereby improving convenience.

[0012] Preferably, it also includes a solenoid valve, which is provided on the branch pipe and electrically connected to the controller; the controller controls the opening and closing degree of the solenoid valve to control the flow rate of the mixture in the branch pipe, thereby improving flexibility.

[0013] Preferably, the filter cover is made of stainless steel.

[0014] This invention provides a G4 stabilizer, which is prepared by the process described above. The G4 stabilizer specifically binds to and stabilizes the G4 in the promoter region of the KLF6 gene. The four-strand structure can inhibit the transcriptional activity of the KLF6 gene promoter.

[0015] Preferably, the G4 stabilizer is a small molecule organic compound or a metal complex.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: the process can be completed in only four steps, S1 to S4, which is simple, easy to operate, and easy to scale up for industrial production; in step S2, the reaction temperature is controlled at 60℃ to 90℃ and the reaction time is 4h to 8h, the reaction conditions are mild, the energy consumption is low, and the equipment requirements are not high, which significantly reduces the production cost; in step S3, ethyl acetate and saturated sodium chloride solution are used for extraction, combined with vacuum drying at 40℃ to 50℃, which effectively ensures the yield and batch stability of the crude product; in step S4, silica gel column chromatography is used for purification, with a mixed solvent of dichloromethane and methanol as the eluent, and the purity of the obtained G4 stabilizer can reach more than 95%; at the same time, the raw materials A (Tmpyp4, CX5461), solvents and catalysts used are all commonly used commercially available reagents, the raw material sources are wide and the cost is controllable. Attached Figure Description

[0017] Figure 1 This is a process flow diagram of the present invention; Figure 2 This is a first isometric structural schematic diagram of the mixing device of the present invention; Figure 3 This is a side view of the mixing device of the present invention; Figure 4 It is along Figure 3 A sectional view of line A-A in the middle; Figure 5 This is an exploded structural diagram of the mixing device of the present invention; Figure 6 This is an enlarged structural diagram of the mixing tank and controller, etc. Figure 7 This is an enlarged structural diagram of the transfer pump and tee pipe, etc. Figure 8 It is an enlarged structural diagram of structures such as hollow rotating shaft and water motor; Figure 9 It is an enlarged structural diagram of components such as cams and springs; Figure 10 This is a second isometric structural schematic diagram of the mixing device of the present invention.

[0018] In the attached diagram, the following are labeled: 101, mixing tank; 102, controller; 103, support frame; 104, feed pipe; 105, filter cover; 106, discharge pipe; 201, transfer pump; 202, tee pipe; 203, discharge pipe; 204, discharge valve; 205, return pipe; 206, return valve; 207; 208, sampling pipe; 209, sampling valve; 301, mounting base; 302, hydraulic motor; 303. Hollow rotating shaft; 304, hollow stirring rod; 305, sprocket A; 306, sprocket B; 307, chain; 308, infusion tube; 309, rotary joint; 310, branch pipe; 311, solenoid valve; 401, sprocket A; 402, sprocket B; 403, chain; 404, rotating shaft; 405, cam; 406, fixed plate; 407, spring; 408, striking block. Detailed Implementation

[0019] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0020] Example 1 A process for preparing G4 stabilizer according to the present invention, such as Figures 1 to 10 As shown, it includes the following steps: S1. Add raw material A and solvent to a mixing device and mix to obtain a mixture; the raw material A is selected from at least one of Tmpyp4 and CX5461; the solvent is selected from methanol, ethanol, N,N At least one of dimethylformamide or dimethyl sulfoxide; S2. Add a catalyst to the mixture and heat it under inert gas protection; the catalyst is selected from at least one of triethylamine, potassium carbonate or palladium chloride; the reaction temperature is 60℃~90℃ and the reaction time is 4h~8h. S3. After the reaction is complete, cool to room temperature, filter to remove insoluble matter, extract the filtrate with ethyl acetate and saturated sodium chloride solution, collect the organic phase, and dry under vacuum at 40℃~50℃ to obtain the crude product; S4. The crude product was purified by silica gel column chromatography with dichloromethane and methanol as the eluent. The target fraction was collected, concentrated, and dried under vacuum to obtain the G4 stabilizer. The mixing equipment includes a mixing tank 101, a controller 102, a support frame 103, a feed pipe 104, a filter cover 105, and a discharge pipe 106. The controller 102 is mounted on the mixing tank 101 and is used to coordinate the timing of the operation of each electrical component. The mixing tank 101 is fixedly mounted on the support frame 103, which provides a stable support foundation for the mixing tank. The feed pipe 104 is located at the top of the mixing tank 101 for injecting raw material A and solvent. The filter cover 105 is located inside the mixing tank 101, directly below the feed pipe 104, for... The filter hood 105 is used to receive materials falling from the feed pipe and intercept large particles. It is important to note that the installation position of the filter hood 105 is directly on the feed path, so that large particles are separated as soon as the material enters the mixing tank, avoiding the difficulty of handling large particles after they settle to the bottom. As an alternative, the filter hood 105 can also adopt a conical structure to increase the filtration area and reduce the risk of clogging. The material of the filter hood 105 is preferably stainless steel to improve corrosion resistance and structural strength. The bottom end of the mixing tank 101 is connected to the input end of the discharge pipe 106, and the mixed liquid is discharged through the discharge pipe.

[0021] like Figure 2 , Figure 5 , Figure 7 As shown, the mixing equipment also includes a stirring assembly and a reflux assembly; the stirring assembly is installed on the mixing tank 101 and is used to drive the material and solvent to form a uniform mixture; the reflux assembly is installed between the output end of the discharge pipe 106 and the stirring assembly and is used to draw the mixture from the bottom of the mixing tank and transport it to the stirring assembly; the controller 102 is electrically connected to the reflux assembly and the stirring assembly to realize automatic control; it should be understood that the setting of the reflux assembly enables the mixture to form a circulating flow outside the mixing tank, thereby providing a power source for the stirring assembly while realizing multiple mixing of the material.

[0022] like Figure 5 , Figure 7 As shown, the reflux assembly includes a delivery pump 201, a three-way pipe 202, a discharge pipe 203, a discharge valve 204, a reflux pipe 205, and a reflux valve 206. The input end of the delivery pump 201 is connected to the output end of the discharge pipe 106, and the output end of the delivery pump 201 is connected to the input end of the three-way pipe 202. The first set of output ends of the three-way pipe 202 is connected to the input end of the discharge pipe 203, and the discharge pipe 203 is equipped with a discharge valve 204 to control the on / off state of product discharge. The second set of output ends of the three-way pipe 202 is connected to the input end of the reflux pipe 205, and the reflux pipe 205 is equipped with a reflux valve 206 to control the on / off state of the reflux circuit. The delivery pump 201 is electrically connected to the controller 102, and its start / stop and speed are controlled by the controller.

[0023] It is important to understand that the reflux assembly has two operating modes: when the discharge valve 204 is closed and the reflux valve 206 is open, the delivery pump 201 pumps all the mixture from the bottom of the mixing tank to the reflux pipe 205, which then enters the stirring assembly; when the reflux valve 206 is closed and the discharge valve 204 is open, the mixture is discharged through the discharge pipe 203 to the subsequent process. As an alternative, the three-way pipe 202 can be replaced with an electric three-way valve, which allows the controller to directly switch the flow direction, simplifying the pipeline layout and improving the response speed. like Figure 4 , Figure 5 , Figure 8 As shown, the stirring assembly includes a fixed base 301, a water motor 302, a hollow rotating shaft 303, a hollow stirring rod 304, a first A sprocket 305, a first B sprocket 306, a first chain 307, an infusion pipe 308, a rotary joint 309, and a branch pipe 310; the fixed base 301 is provided at the top of the mixing tank 101, and the water motor 302 is fixedly installed on the fixed base 301; the water motor 302 is a power element that converts liquid pressure energy into rotational mechanical energy. It does not require an external motor and can output torque solely by the flow of the mixture itself; a hollow rotating shaft 303 is rotatably mounted inside the mixing tank 101 via bearings, and the hollow rotating shaft is vertically arranged along the axis of the mixing tank; multiple sets of hollow stirring rods 304 are mounted on the hollow rotating shaft 303, and the internal channels of the multiple sets of hollow stirring rods 304 are connected to the internal cavity of the hollow rotating shaft 303 to form a continuous fluid passage; the top of the hollow rotating shaft 303 extends above the mixing tank 101.

[0024] The output end of the water motor 302 is equipped with a No. A sprocket 305, and a No. B sprocket 306 is mounted on the hollow shaft 303. The No. A sprocket 305 and the No. B sprocket 306 are driven by a No. A chain 307. It should be understood that using chain drive instead of gear drive allows for a certain installation position deviation between the water motor and the hollow shaft, reducing the requirements for machining accuracy. As an alternative, synchronous belt drive can also be used to achieve smoother operation.

[0025] The output end of the return pipe 205 is connected to the inlet end of the water motor 302, and the outlet end of the water motor 302 is connected to the input end of the delivery pipe 308. The output end of the delivery pipe 308 is rotatably connected to the top end of the hollow shaft 303 through a rotary joint 309. The function of the rotary joint 309 is to achieve a sealed connection between the delivery pipe 308 and the hollow shaft 303 while ensuring that the hollow shaft 303 can rotate freely, so as to prevent leakage of the mixed liquid. A branch pipe 310 is also provided on the delivery pipe 308. The output end of the branch pipe 310 extends into the mixing tank 101 and is close to the outer wall of the filter cover 105.

[0026] It is important to note that in this embodiment, the mixing liquid is driven by the delivery pump 201. The mixing liquid enters the water motor 302, which drives its output shaft to rotate. This, in turn, drives the hollow rotating shaft 303 and the hollow stirring rod 304 to rotate via chain transmission. At the same time, the mixing liquid is divided into two paths from the outlet of the water motor 302 through the delivery pipe 308: the first path enters the hollow rotating shaft 303 and the hollow stirring rod 304 through the rotary joint 309, and is evenly dispersed into the mixing tank 101 through the dispersion holes or dispersion nozzles provided on the hollow stirring rod 304; the second path is transported to the outside of the filter cover 105 through the branch pipe 310, and dissolves and washes away the large particles trapped in the inner cavity of the filter cover from the outside to the inside.

[0027] The advantage of this design is that the transfer pump simultaneously performs the functions of "providing stirring power" and "providing flushing liquid flow," eliminating the need for an additional independent power source or flushing device. Alternatively, a solenoid valve 311 (such as...) can be installed on the branch pipe 310. Figure 8 As shown), the solenoid valve 311 is electrically connected to the controller 102. The controller 102 controls the flow rate of the branch pipe 310 by adjusting the opening of the solenoid valve 311, thereby flexibly adjusting the flushing intensity. like Figure 4 , Figure 5 , Figure 9 As shown, the mixing device also includes a tapping component, which is installed on the mixing tank 101 and is used to periodically tap the side wall of the filter cover 105 to shake off the particles remaining in the filter holes of the filter cover and prevent the filter cover 105 from clogging. The striking assembly includes a second sprocket A 401, a second sprocket B 402, a second chain 403, a rotating shaft 404, a cam 405, a fixing plate 406, a spring 407, and a striking block 408; the second sprocket A 401 is fixedly mounted on the hollow rotating shaft 303; the rotating shaft 404 is rotatably mounted on the top of the mixing tank 101 via a bearing, and the top of the rotating shaft 404 extends above the mixing tank 101; the top of the rotating shaft 404 is equipped with a second sprocket B 402, and the second sprocket A 401 and the second sprocket B 402 are connected by a bearing. The second chain 403 drives the transmission; a cam 405 is provided at the bottom end of the rotating shaft 404; a fixed plate 406 is fixedly provided at the top of the inside of the mixing tank 101, and a spring piece 407 is fixedly provided on the fixed plate 406. A striking block 408 is fixedly provided at one end of the spring piece 407 near the filter cover 105; it should be noted that the spring piece 407 is made of a metal material with good elastic recovery ability, which can quickly rebound after being bent under force; the material of the striking block 408 is preferably polytetrafluoroethylene or rubber to avoid damage to the filter cover 105.

[0028] It is important to understand that the working process of the striking component is as follows: When the hollow rotating shaft 303 rotates, the rotating shaft 404 is driven to rotate synchronously through the transmission of the second sprocket A 401, the second chain 403, and the second sprocket B 402, thereby causing the cam 405 to rotate accordingly; when the protrusion of the cam 405 contacts the spring 407, the spring 407 undergoes elastic bending deformation, at which point the striking block 408 moves away from the filter cover 105; when the cam 405 continues to rotate until the protrusion disengages from the spring 407, the spring 407 elastically resets, driving the striking block 408 to move rapidly toward the filter cover 105, thereby applying a strike to the outer wall of the filter cover 105; this process is repeated periodically to achieve continuous striking of the filter cover 105; it should be further noted that the striking direction of the striking block 408 on the filter cover 105 is approximately perpendicular to the outer wall surface of the filter cover, which allows the vibration generated by the striking to be effectively transmitted to the inside of the filter pores, shaking off the particles attached to the filter pores; It should be noted that the rotational power required for the striking component comes entirely from the hollow shaft 303, without the need for an additional drive motor or pneumatic device, thus achieving the anti-blocking function while maintaining the compactness and low energy consumption of the overall structure.

[0029] It also includes a sampling valve tube 208 and a sampling valve 209. A sampling tube 208 is provided on the discharge tube 203, and a sampling valve 209 is fixedly provided on the sampling tube 208. like Figure 7 As shown, a sampling tube 208 is installed on the discharge pipe 203, and a sampling valve 209 is fixedly installed on the sampling tube 208. During the mixing process, the operator can open the sampling valve 209 and take out a sample of the mixture through the sampling tube 208 for testing to determine whether the mixing meets the requirements.

[0030] It should be noted that in this embodiment, the stirring assembly, reflux assembly, and tapping assembly are all modularly arranged around the mixing tank 101. The components are connected by pipelines and transmission components, which facilitates independent disassembly and maintenance, and allows for combination or replacement according to process requirements. For example, in scenarios where the tapping function is not required, the tapping assembly and its related transmission components can be omitted without affecting the normal operation of the stirring and flushing functions. In this embodiment, the reaction temperature in step S2 is controlled at 60℃~90℃ and the reaction time is 4h~8h. The reaction conditions are mild, energy consumption is low, and equipment requirements are not high, significantly reducing production costs. Step S3 uses ethyl acetate and saturated sodium chloride solution for extraction, combined with vacuum drying at 40℃~50℃, effectively ensuring the yield and batch stability of the crude product. Step S4 uses silica gel column chromatography for purification, using a mixed solvent of dichloromethane and methanol as the eluent, and the purity of the obtained G4 stabilizer can reach over 95%. Meanwhile, the raw materials A (Tmpyp4, CX5461), solvent, and catalyst used are all commonly used commercially available reagents, with wide-ranging sources and controllable costs. The mixing equipment is used for mixing... Inside the mixing tank 101, the discharge valve 204 is closed and the return valve 206 is opened. The transfer pump 201 is started via the controller 102. The mixture enters the hydraulic motor 302 through the return pipe 205. The hydraulic motor 302 drives sprocket A 305 to rotate, which in turn drives sprocket B 306 via chain 307. This, in turn, causes the hollow shaft 303 to drive multiple sets of hollow stirring rods 304 to rotate inside the mixing tank 101. The multiple sets of hollow stirring rods 304 mix and stir the mixture inside the mixing tank 101. The mixture inside the hydraulic motor 302 enters the hollow shaft 303 through the cooperation of the infusion pipe 308 and the rotary joint 309. While the mixture is redistributed back into the mixing tank 101 by multiple sets of hollow stirring rods 304, another batch of mixture flushes the filter shroud 105 through branch pipe 310, allowing the mixture to enter the filter shroud 105 and dissolve larger impurities in the filtered material. The delivery pump 201 provides delivery pressure for the mixture, which then enters the water motor 302 to drive the hollow rotating shaft 303. The rotation of the hollow rotating shaft 303 and the hollow stirring rods 304 further disperses the mixture into the mixing tank 101, achieving mixing. When the water motor 302 drives the hollow rotating shaft 303 to rotate, the hollow rotating shaft 303, through the second A sprocket 401 and the second... The cooperation of sprocket 402 and chain 403 drives shaft 404 to rotate, which in turn drives cam 405 to rotate. When cam 405 contacts spring 407, spring 407 bends and deforms, causing striking block 408 to move away from filter cover 105. When cam 405 separates from the bent and deformed spring 407, spring 407 elastically recovers, causing striking block 408 to strike the outer wall of filter cover 105, thereby knocking down particles remaining inside the filter holes of filter cover 105. After mixing and stirring for a period of time, sampling valve 209 is opened, and staff use sampling tools to sample the mixture flowing out through sampling tube 208 for testing.

[0031] Example 2 The present invention provides a G4 stabilizer, wherein the G4 stabilizer specifically binds to and stabilizes the G4 region of the KLF6 gene promoter. The G4 stabilizer has a four-strand structure and can inhibit the transcriptional activity of the KLF6 gene promoter. The G4 stabilizer is a small molecule organic compound or a metal complex.

[0032] The main functions achieved by this invention are: 1. Through a four-step process (S1-S4, mixing → reaction → extraction and drying → column chromatography purification), combined with the synergistic effects of efficient circulating mixing, in-situ dispersion, self-cleaning filter hood, and mechanical knocking to prevent clogging, a high-purity (≥95%) and batch-stability G4 stabilizer was prepared. This G4 stabilizer can specifically bind to and stabilize the G-quadruplex structure of the KLF6 gene promoter region, inhibiting the transcriptional activity of the KLF6 gene promoter, thus providing a high-quality compound basis for the development of anti-liver cancer drugs.

[0033] 2. The mixture at the bottom of the mixing tank 101 is extracted by the transfer pump 201 and sent back to the mixing tank 101 through the return pipe 205 to form a closed-loop circulation system, so that the material and solvent are repeatedly circulated and mixed in the tank, improving the mixing uniformity and providing a uniform reaction system for subsequent reactions.

[0034] 3. The hydraulic motor 302 is driven to rotate by the flow pressure of the mixture itself. The hydraulic motor 302 drives the hollow shaft 303 and the hollow stirring rod 304 to rotate through the chain drive, thereby realizing the mechanical stirring of the material in the mixing tank 101. No external motor is required, the structure is more compact, and the energy consumption is lower. The mixture enters the hollow stirring rod 304 through the hollow shaft 303 and is evenly sprayed out at multiple points in the mixing tank through the dispersion holes or dispersion nozzles on the stirring rod, thereby realizing the in-situ dispersion of the mixture, avoiding local uneven concentration, and ensuring that the raw material A and the solvent are in full contact.

[0035] 4. Branch pipe 310 delivers part of the mixture to the outside of the filter cover, so that the mixture flows from the outside to the inside, continuously dissolving and flushing the large particles trapped on the inner wall of the filter cover 105, preventing the filter cover 105 from clogging, and ensuring the continuous circulation of the mixture and filtration efficiency.

[0036] 5. When the hollow shaft 303 rotates, it drives the cam 405 to rotate through the chain drive. The cam periodically pushes the spring 407. When the spring 407 rebounds, it drives the striking block 408 to strike the outer wall of the filter cover. The mechanical vibration shakes off the particles remaining in the filter holes. This, together with the flushing effect of the liquid flow, further enhances the anti-clogging effect and ensures the long-term stable operation of the equipment.

[0037] The present invention discloses a G4 stabilizer and its preparation process. The installation, connection, or setting methods are all common mechanical methods; any method that achieves the beneficial effects can be implemented. Wear protection: The striking block 408 and the filter cover 105 are periodically contacted and struck, posing a risk of wear. Therefore, the striking block 408 is made of polytetrafluoroethylene or rubber, possessing good wear resistance and cushioning performance. Simultaneously, the striking block 408 and the spring 407 employ a detachable connection structure for easy periodic replacement. Sealing and lubrication: A mechanical seal is provided at the rotating connection between the hollow rotating shaft 303 and the mixing tank 101 to prevent leakage of the mixture along the outer wall of the rotating shaft. The moving parts inside the water motor 302 are pre-lubricated during manufacturing, and a filter screen (not shown in the diagram) is provided at the inlet end of the water motor to prevent particles in the mixture from entering the motor and causing wear. Heat dissipation considerations: The transfer pump 201 and the water motor 302 will generate some heat during long-term operation. However, since the mixture itself is a liquid at room temperature or heated to 60℃~90℃, and it circulates continuously, it can remove the heat from the surface of the components. Therefore, no additional heat dissipation device is required. Electrical protection: The controller 102 is installed on the outer wall of the mixing tank 101. Its shell adopts a sealed structure with a protection level of not less than IP54 to prevent electrical failures caused by splashing of the mixture or dust intrusion. The controller 102, filter cover 105, transfer pump 201, reflux valve 206, water motor 302, spring 407, and striking block 408 of the G4 stabilizer and its preparation process of this invention are commercially available. Those skilled in the art only need to install and operate them according to the accompanying instruction manual, without requiring any creative effort from those skilled in the art.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A process for preparing G4 stabilizer, characterized in that, Includes the following steps: S1. Add raw material A and solvent to a mixing device and mix to obtain a mixture; the raw material A is selected from at least one of Tmpyp4 and CX5461; the solvent is selected from methanol, ethanol, N,N At least one of dimethylformamide or dimethyl sulfoxide; S2. Add a catalyst to the mixture and heat it under inert gas protection; the catalyst is selected from at least one of triethylamine, potassium carbonate or palladium chloride; the reaction temperature is 60℃~90℃ and the reaction time is 4h~8h. S3. After the reaction is complete, cool to room temperature, filter to remove insoluble matter, extract the filtrate with ethyl acetate and saturated sodium chloride solution, collect the organic phase, and dry under vacuum at 40℃~50℃ to obtain the crude product; S4. The crude product was purified by silica gel column chromatography with dichloromethane and methanol as the eluent. The target fraction was collected, concentrated, and then dried under vacuum to obtain the G4 stabilizer.

2. The G4 stabilizer preparation process as described in claim 1, characterized in that, The mixing equipment includes a mixing tank (101), a controller (102), a support frame (103), a feed pipe (104), a filter cover (105), and a discharge pipe (106). The controller (102) is installed on the mixing tank (101). The mixing tank (101) is mounted on the support frame (103). The feed pipe (104) is installed at the top of the mixing tank (101). The filter cover (105) is installed inside the mixing tank (101) and is located directly below the feed pipe (104). The bottom end of the mixing tank (101) is connected to the input end of the discharge pipe (106). The equipment also includes: A stirring assembly, which is installed on a mixing tank (101), is used to form a mixture of materials; A reflux assembly, installed between the outlet end of the discharge pipe (106) and the stirring assembly, is used for conveying the mixture. The controller (102) is electrically connected to the reflux assembly and the stirring assembly.

3. The G4 stabilizer preparation process as described in claim 2, characterized in that, The return liquid assembly includes a transfer pump (201), a three-way pipe (202), a discharge pipe (203), a discharge valve (204), a return pipe (205), and a return valve (206). The input end of the transfer pump (201) is connected to the output end of the discharge pipe (106), the output end of the transfer pump (201) is connected to the input end of the three-way pipe (202), the first set of output ends of the three-way pipe (202) is connected to the input end of the discharge pipe (203), the discharge pipe (203) is equipped with a discharge valve (204), the second set of output ends of the three-way pipe (202) is connected to the input end of the return pipe (205), the return pipe (205) is equipped with a return valve (206), and the transfer pump (201) is electrically connected to the controller (102).

4. The G4 stabilizer preparation process as described in claim 3, characterized in that, The mixing assembly includes a fixed base (301), a water motor (302), a hollow rotating shaft (303), hollow stirring rods (304), a No. A sprocket (305), a No. B sprocket (306), a No. A chain (307), an infusion pipe (308), a rotary joint (309), and a branch pipe (310). The fixed base (301) is provided at the top of the mixing tank (101), and the water motor (302) is provided on the fixed base (301). The hollow rotating shaft (303) is rotatably arranged inside the mixing tank (101), and multiple sets of hollow stirring rods (304) are provided on the hollow rotating shaft (303). The multiple sets of hollow stirring rods (304) are connected to the hollow rotating shaft (303), and the top of the hollow rotating shaft (303) extends to the mixing tank (101). Above, a No. A sprocket (305) is provided at the output end of the water motor (302), and a No. B sprocket (306) is mounted on the hollow shaft (303). The No. A sprocket (305) and the No. B sprocket (306) are driven by a No. A chain (307). The output end of the return pipe (205) is connected to the liquid inlet end of the water motor (302), and the liquid outlet end of the water motor (302) is connected to the input end of the delivery pipe (308). The output end of the delivery pipe (308) is rotatably connected to the top of the hollow shaft (303) through a rotary joint (309). A branch pipe (310) is provided on the delivery pipe (308), and the output end of the branch pipe (310) extends into the mixing tank (101) and is close to the filter cover (105).

5. The G4 stabilizer preparation process as described in claim 4, characterized in that, It also includes a tapping assembly, which is mounted on the mixing tank (101) and is used to tap the side wall of the filter cover (105) to prevent the filter cover (105) from clogging. The striking assembly includes a second A sprocket (401), a second B sprocket (402), a second chain (403), a rotating shaft (404), a cam (405), a fixing plate (406), a spring (407), and a striking block (408). The second A sprocket (401) is mounted on the hollow rotating shaft (303). The rotating shaft (404) is rotatably mounted at the top of the mixing tank (101), and the top of the rotating shaft (404) extends above the mixing tank (101). The top of the rotating shaft (404) is provided with a second B sprocket (402), and the second A sprocket (401) and the second B sprocket (402) are driven by the second chain (403). The bottom of the rotating shaft (404) is provided with a cam (405). The top of the inside of the mixing tank (101) is provided with a fixing plate (406), and a spring piece (407) is provided on the fixing plate (406). A striking block (408) is provided at the end of the spring piece (407) near the filter cover (105).

6. The G4 stabilizer preparation process as described in claim 3, characterized in that, It also includes a sampling valve tube (208) and a sampling valve (209). A sampling tube (208) is provided on the discharge tube (203), and a sampling valve (209) is fixedly provided on the sampling tube (208).

7. The G4 stabilizer preparation process as described in claim 4, characterized in that, It also includes a solenoid valve (311), which is provided on the branch pipe (310) and is electrically connected to the controller (102).

8. The G4 stabilizer preparation process as described in claim 2, characterized in that, The filter cover (105) is made of stainless steel.

9. A G4 stabilizer, characterized in that, The G4 stabilizer is prepared by any one of the preparation processes described in claims 1 to 8. The G4 stabilizer can specifically bind to and stabilize the G4 in the promoter region of the KLF6 gene. The four-strand structure can inhibit the transcriptional activity of the KLF6 gene promoter.

10. The G4 stabilizer and its preparation process as described in claim 9, characterized in that, The G4 stabilizer is a small molecule organic compound or a metal complex.

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