A small molecule inhibitor, synthetic method and application

Abstract

The present application relates to the technical field of pharmacy, more particularly to a small molecule inhibitor, a synthetic method and an application. The small molecule inhibitor is a chemical structural formula as shown in formula (I) or a pharmaceutically acceptable salt comprising the chemical structural formula as shown in formula (I); the small molecule inhibitor has biological activity and can effectively inhibit cell mitosis, which is of great significance for preventing abnormal cell proliferation and the occurrence of cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to a small molecule inhibitor, its synthesis method, and its application. Background Technology

[0002] The cell cycle refers to the series of ordered physiological and biochemical processes a cell undergoes from the end of one division to the end of the next. The cell cycle can be divided into two main phases: interphase and mitosis (MPhase). Mitosis is a crucial process in the cell cycle, responsible for the equal distribution of genetic material from one parent cell to two daughter cells. This process is essential for maintaining normal tissue growth and repair. Mitosis involves several complex steps, including chromosome condensation, spindle formation, chromosome separation, and cytoplasmic division. Precise regulation of these steps is crucial for preventing abnormal cell proliferation and the development of cancer.

[0003] Under normal circumstances, each stage of the cell cycle is strictly regulated, ensuring that cells divide and proliferate at the appropriate times. However, in cancer, this regulatory mechanism is often disrupted, leading to uncontrolled cell cycle progression, with cancer cells dividing and proliferating uncontrollably. Many types of cancer exhibit abnormal cell cycle regulation, resulting in uncontrolled cell proliferation. Inhibiting mitosis can prevent the division and spread of cancer cells, thus achieving therapeutic goals and representing an important strategy for treating various diseases, especially cancer. Therefore, there is a need to develop a small-molecule inhibitor that can suppress cell mitosis. Summary of the Invention

[0004] To address the above problems, this invention provides a small molecule inhibitor, its synthesis method, and its application.

[0005] This invention is achieved through the following technical solution:

[0006] A small molecule inhibitor, said small molecule inhibitor being a chemical structure of formula (I) or a pharmaceutically acceptable salt including the chemical structure of formula (I);

[0007]

[0008] R1 can be hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, trifluoromethyl, or trifluoromethoxy.

[0009] R2 can be hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, trifluoromethyl, or trifluoromethoxy.

[0010] R3 is methyl, ethyl, or allyl.

[0011] R4 is either methoxy or ethoxy.

[0012] R5 can be hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, trifluoromethoxy, or trifluoromethyl.

[0013] The method for synthesizing the small molecule inhibitor specifically includes the following steps:

[0014] (1) Compound 1 was alkylated with alkyl iodine under the action of an alkaline catalyst to prepare compound 2.

[0015] (2) The first step reaction involves the condensation reaction of compound 2 with acetaldehyde under the action of an alkaline catalyst to obtain the reaction product; the second step reaction involves the dehydration reaction of the reaction product under acidic conditions to prepare compound 3.

[0016] (3) Compound 3 was reacted with compound 4, methanol, 2,6-bis[(4S)-4-phenyl-2-oxazolinyl]pyridine and (5aS,10bR)-2-metrimethyl-4,5a,6,10b-tetrahydro-2H-indeno[2,1-b][1,2,4]triazolo[4,3-d][1,4]oxazine-11-onium tetrafluoroborate in the presence of a copper catalyst to prepare the small molecule inhibitor.

[0017]

[0018] In step 1, the molar ratio of compound 1, alkyl iodine, and basic catalyst is 1:1.1-1.3:2.5-3.5.

[0019] In step 2, the molar ratio of compound 2, acetaldehyde, and alkaline catalyst is 1:4-6:0.1-0.12.

[0020] In step 3, the molar ratio of compound 3 to compound 4, methanol, 2,6-bis[(4S)-4-phenyl-2-oxazolinyl]pyridine and (5aS,10bR)-2-trimethylyl-4,5a,6,10b-tetrahydro-2H-indeno[2,1-b][1,2,4]triazolo[4,3-d][1,4]oxazine-11-onium tetrafluoroborate and copper catalyst is 1.4–1.6:1:4–6:0.09–0.11:0.04–0.06:0.04–0.06.

[0021] Preferably, the solvent used in step 1 is acetonitrile or N,N-dimethylformamide; the alkaline catalyst used in step 1 is sodium carbonate, potassium carbonate, cesium carbonate, or rubidium carbonate; the reaction temperature in step 1 is 80℃~100℃, and the reaction time is 10h~15h.

[0022] Preferably, the solvent used in step 2 is tetrahydrofuran; the alkaline catalyst used in step 2 is DBU; the first reaction temperature in step 2 is -30℃ to -20℃, and the reaction time is 14h to 18h; the second reaction temperature is 90℃ to 110℃, and the reaction time is 20min to 40min.

[0023] Preferably, the solvent used in step 3 is tetrahydrofuran, toluene, or dichloromethane; the copper catalyst used in step 3 is copper tetraacetonitrile hexafluorophosphate; the reaction temperature in step 3 is 10℃~30℃, and the reaction time is 10h~15h.

[0024] Preferably, the alkyl iodine is iodomethane, iodoethane, or allyl iodine.

[0025] Preferably, the acidic conditions in step 2 are achieved by adding a mixed solution of sulfuric acid, acetic acid and water to the reaction products.

[0026] Preferably, the volume ratio of sulfuric acid, acetic acid and water is 1:15:5.

[0027] The small molecule inhibitor is used in the preparation of reagents that inhibit cell mitosis or in antitumor drugs.

[0028] Preferably, the anti-tumor tumor cells are HeLa cells.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention provides a small molecule inhibitor. This small molecule inhibitor possesses biological activity and can effectively inhibit cell mitosis, exhibiting high efficiency, specificity, low toxicity, and broad applicability. Experimental results show that this inhibitor can effectively interfere with the mitotic process, leading to metaphase arrest and exit failure in tumor cells, thereby inhibiting their proliferation. Compared with existing technologies, the small molecule inhibitor of this invention has higher antitumor activity and lower toxicity to normal cells, while its preparation process is simple and inexpensive, suitable for large-scale industrial production. Furthermore, its good biocompatibility and environmental friendliness further enhance its potential for clinical application. This invention provides a novel, efficient, and safe strategy for tumor treatment, possessing significant scientific value and broad application prospects. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a schematic diagram of the experimental procedure for verifying the effects of small molecules on cells according to the present invention.

[0033] Figure 2 This is a schematic diagram illustrating the effect of the small molecule inhibitor of the present invention on cells.

[0034] Figure 3 This is a statistical result graph showing the mitotic phenotypes of cells after treatment with the small molecule inhibitor of this invention.

[0035] Figure 4 This is a diagram showing the verification results of the A5 NMR spectrum of this invention.

[0036] Figure 5 This is a diagram showing the verification results of the B5 NMR spectrum of this invention.

[0037] Figure 6 This is a diagram showing the C5 NMR spectrum verification results of this invention.

[0038] Figure 7 This is a diagram showing the verification results of the D5 NMR spectrum of this invention. Detailed Implementation

[0039] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0041] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0042] This invention is the first to discover a small molecule inhibitor having the chemical structure described in formula (I) that can significantly inhibit tumor cell division;

[0043]

[0044] R1 can be hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, trifluoromethyl, or trifluoromethoxy.

[0045] R2 can be hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, trifluoromethyl, or trifluoromethoxy.

[0046] R3 is methyl, ethyl, or allyl.

[0047] R4 is either methoxy or ethoxy.

[0048] R5 can be hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, trifluoromethoxy, or trifluoromethyl.

[0049] The method for synthesizing the small molecule inhibitor is as follows:

[0050] (1) Compound 1 was alkylated with alkyl iodine under the action of an alkaline catalyst to prepare compound 2.

[0051] (2) The first step reaction involves the condensation reaction of compound 2 with acetaldehyde under the action of an alkaline catalyst to obtain the reaction product; the second step reaction involves the dehydration reaction of the reaction product under acidic conditions to prepare compound 3.

[0052] (3) Compound 3 was reacted with compound 4, methanol, 2,6-bis[(4S)-4-phenyl-2-oxazolinyl]pyridine and (5aS,10bR)-2-trimethylyl-4,5a,6,10b-tetrahydro-2H-indeno[2,1-b][1,2,4]triazolo[4,3-d][1,4]oxazine-11-onium tetrafluoroborate in the presence of a copper catalyst to prepare a small molecule inhibitor.

[0053]

[0054] In step 1, the molar ratio of compound 1, alkyl iodine, and basic catalyst is 1:1.1-1.3:2.5-3.5.

[0055] In step 2, the molar ratio of compound 2, acetaldehyde, and alkaline catalyst is 1:4-6:0.1-0.12.

[0056] In step 3, the molar ratio of compound 3 to compound 4, methanol, 2,6-bis[(4S)-4-phenyl-2-oxazolinyl]pyridine and (5aS,10bR)-2-trimethylyl-4,5a,6,10b-tetrahydro-2H-indeno[2,1-b][1,2,4]triazolo[4,3-d][1,4]oxazine-11-onium tetrafluoroborate and copper catalyst is 1.4–1.6:1:4–6:0.09–0.11:0.04–0.06:0.04–0.06.

[0057] In one specific embodiment, the solvent for the reaction in step 1 is acetonitrile or N,N-dimethylformamide.

[0058] The alkaline catalyst in step 1 is sodium carbonate, potassium carbonate, cesium carbonate, or rubidium carbonate.

[0059] The reaction temperature in step 1 is 80℃~100℃, and the reaction time is 10h~15h.

[0060] In one specific embodiment, the solvent used in step 2 is tetrahydrofuran.

[0061] The alkaline catalyst in step 2 is DBU.

[0062] The first step of step 2 has a reaction temperature of -30℃ to -20℃ and a reaction time of 14h to 18h. The second step has a reaction temperature of 90℃ to 110℃ and a reaction time of 20min to 40min.

[0063] In one specific embodiment, the solvent for the reaction in step 3 is tetrahydrofuran, toluene, or dichloromethane.

[0064] The copper catalyst in step 3 is copper tetraacetonitrile hexafluorophosphate.

[0065] The reaction temperature in step 3 is 10℃~30℃, and the reaction time is 10h~15h.

[0066] In one specific embodiment, the alkyl iodine is iodomethane, iodoethane, or allyl iodine.

[0067] In one specific embodiment, the acidic condition in step 2 is achieved by adding a mixed solution of sulfuric acid, acetic acid, and water to the reaction products.

[0068] Example 1: A method for synthesizing a small molecule inhibitor

[0069] Small molecule inhibitors are The chemical structural formula is used for synthesis.

[0070] (1) 1.0 equivalent of A1, 1.1 equivalent of iodomethane, and 2.5 equivalent of sodium carbonate were alkylated in acetonitrile at 80°C for 10 h. A2 was obtained by chromatographic separation.

[0071]

[0072] (2) In the first step, 1.0 equivalent of A2, 4.0 equivalent of acetaldehyde, and 0.1 equivalent of DBU were mixed with tetrahydrofuran as solvent and stirred at -20°C for 14 h to carry out a condensation reaction to obtain the reaction product. The solvent was evaporated to dryness to carry out the second step reaction. A mixture of sulfuric acid, acetic acid and water was added to the reaction product. The volume ratio of sulfuric acid, acetic acid and water was 1:15:5. The dehydration reaction was carried out at 90°C for 20 min. A3 was obtained by chromatographic separation.

[0073]

[0074] (3) 1.4 equivalents of A3, 1.0 equivalents of 4, 4.0 equivalents of methanol, 0.04 equivalents of copper hexafluorophosphate tetraacetonitrile, 0.09 equivalents of 2,6-bis[(4S)-4-phenyl-2-oxazolinyl]pyridine, and 0.04 equivalents of (5aS,10bR)-2-mesotrimethyl-4,5a,6,10b-tetrahydro-2H-indeno[2,1-b][1,2,4]triazolo[4,3-d][1,4]oxazine-11-onium tetrafluoroborate were subjected to an asymmetric conjugate addition reaction at 10 °C for 10 h in tetrahydrofuran as solvent. The compound A5 was obtained by chromatographic separation.

[0075]

[0076] The NMR spectrum of compound A5 was used for verification, and the results are as follows: Figure 4 As shown, this indicates the synthesis of a chemical structure with the following structural formula:

[0077] Example 2: A method for synthesizing a small molecule inhibitor

[0078] Small molecule inhibitors are The chemical structural formula is used for synthesis.

[0079] (1) 1.0 equivalent of B1, 1.2 equivalent of iodomethane, and 3.0 equivalent of sodium carbonate were alkylated in acetonitrile at 90°C for 12 h. B2 was obtained by chromatographic separation.

[0080]

[0081] (2) In the first step, 1.0 equivalent of B2, 5.0 equivalent of acetaldehyde, and 0.11 equivalent of DBU were mixed with tetrahydrofuran as solvent and stirred at -25°C for 15 h to carry out a condensation reaction to obtain the reaction product. The solvent was evaporated to dryness to carry out the second step reaction. A mixture of sulfuric acid, acetic acid and water was added to the reaction product. The volume ratio of sulfuric acid, acetic acid and water was 1:15:5. The dehydration reaction was carried out at 100°C for 30 min. B3 was obtained by chromatographic separation.

[0082]

[0083] (3) 1.5 equivalents of B3, 1.0 equivalents of 4 and 5.0 equivalents of methanol, 0.05 equivalents of copper hexafluorophosphate tetraacetonitrile, 0.1 equivalents of 2,6-bis[(4S)-4-phenyl-2-oxazolinyl]pyridine, and 0.05 equivalents of (5aS,10bR)-2-mesotrimethyl-4,5a,6,10b-tetrahydro-2H-indeno[2,1-b][1,2,4]triazolo[4,3-d][1,4]oxazine-11-onium tetrafluoroborate were subjected to an asymmetric conjugate addition reaction at 20 °C for 13 h in tetrahydrofuran as solvent. The compound B5 was obtained by chromatographic separation.

[0084]

[0085] The NMR spectrum of compound B5 was used for verification, and the results are as follows: Figure 5 As shown, this indicates the synthesis of a chemical structure with the following structural formula:

[0086] Example 3: A method for synthesizing a small molecule inhibitor

[0087] Small molecule inhibitors are The chemical structural formula is used for synthesis.

[0088] (1) 1.0 equivalent of Al, 1.3 equivalent of iodoethane, and 3.5 equivalent of sodium carbonate were alkylated in acetonitrile at 100°C for 15 h. C2 was obtained by chromatographic separation.

[0089]

[0090] (2) In the first step, 1.0 equivalent of C2, 6.0 equivalent of acetaldehyde, and 0.12 equivalent of DBU were mixed with tetrahydrofuran as solvent and stirred at -30°C for 18 h to carry out a condensation reaction to obtain the reaction product. The solvent was evaporated to dryness to carry out the second step reaction. A mixture of sulfuric acid, acetic acid and water was added to the reaction product. The volume ratio of sulfuric acid, acetic acid and water was 1:15:5. The dehydration reaction was carried out at 110°C for 40 min. C3 was obtained by chromatographic separation.

[0091]

[0092] (3) 1.6 equivalents of C3, 1.0 equivalents of 4, 6.0 equivalents of methanol, 0.06 equivalents of copper hexafluorophosphate tetraacetonitrile, 0.11 equivalents of 2,6-bis[(4S)-4-phenyl-2-oxazolinyl]pyridine, and 0.06 equivalents of (5aS,10bR)-2-mesotrimethyl-4,5a,6,10b-tetrahydro-2H-indeno[2,1-b][1,2,4]triazolo[4,3-d][1,4]oxazine-11-onium tetrafluoroborate were subjected to an asymmetric conjugate addition reaction at 30 °C for 15 h in tetrahydrofuran as solvent. The compound C5 was obtained by chromatographic separation.

[0093]

[0094] The NMR spectrum of compound C5 was used for verification, and the results are as follows: Figure 6 As shown, this indicates the synthesis of a chemical structure with the following structural formula:

[0095] Example 4: A method for synthesizing a small molecule inhibitor

[0096] Small molecule inhibitors are The chemical structural formula is used for synthesis.

[0097] (1) 1.0 equivalent of A1, 1.3 equivalent of iodomethane, and 3.5 equivalent of sodium carbonate were alkylated in acetonitrile at 100°C for 15 h. A2 was obtained by chromatographic separation.

[0098]

[0099] (2) In the first step, 1.0 equivalent of A2, 6.0 equivalent of acetaldehyde, and 0.12 equivalent of DBU were mixed with tetrahydrofuran as solvent and stirred at -30°C for 18 h to carry out a condensation reaction to obtain the reaction product. The solvent was evaporated to dryness to carry out the second step reaction. A mixture of sulfuric acid, acetic acid and water was added to the reaction product. The volume ratio of sulfuric acid, acetic acid and water was 1:15:5. The dehydration reaction was carried out at 110°C for 40 min. A3 was obtained by chromatographic separation.

[0100]

[0101] (3) 1.6 equivalents of A3, 1.0 equivalents of 4 and 6.0 equivalents of ethanol, 0.06 equivalents of copper hexafluorophosphate tetraacetonitrile, 0.11 equivalents of 2,6-bis[(4S)-4-phenyl-2-oxazolinyl]pyridine, and 0.06 equivalents of (5aS,10bR)-2-mesotrimethyl-4,5a,6,10b-tetrahydro-2H-indeno[2,1-b][1,2,4]triazolo[4,3-d][1,4]oxazine-11-onium tetrafluoroborate were reacted with tetrahydrofuran as solvent and subjected to an asymmetric conjugate addition reaction at 30 °C for 15 h. The compound D5 was obtained by chromatographic separation.

[0102]

[0103] The NMR spectrum of compound D5 was used for verification, and the results are as follows: Figure 7 As shown, this indicates the synthesis of a chemical structure with the following structural formula:

[0104] It should be noted that in all embodiments, the solvent used in step 1 is acetonitrile or N,N-dimethylformamide; the alkaline catalyst used in step 1 is sodium carbonate, potassium carbonate, cesium carbonate, or rubidium carbonate; and the solvent used in step 3 is tetrahydrofuran, toluene, or dichloromethane, all of which can be used to synthesize the small molecule inhibitor.

[0105] Experimental Example 1: The Effect of Small Molecule Inhibitors on Cell Mitosis

[0106] Experimental steps, such as Figure 1 As shown:

[0107] 1. Seed HeLa cells onto a glass coverslip with a diameter of 12 mm.

[0108] 2. Add thymidine to a final concentration of 2 mM to synchronize HeLa to the G1 / S phase.

[0109] 3. After 16 hours of treatment with Thymidine, the cells were released and washed three times with pre-warmed PBS for 5 minutes each time.

[0110] 4. Release for 9 hours to allow cells to enter the G2 phase.

[0111] 5. Dilute the small molecule in Opti-MEM medium, mix well by pipetting, and then treat HeLa cells with medium containing the small molecule for 1 hour.

[0112] 6. After small molecule treatment, HeLa cells were fixed with preheated PBS buffer containing 3.7% formaldehyde for 10 minutes.

[0113] 7. After fixation, HeLa was perforated with PBS buffer containing 0.1% Triton X-100 for 3 min.

[0114] 8. After washing once with PBS, block with a final concentration of 1% BSA solution for 30 minutes.

[0115] 9. After blocking, label the microtubules in the cells with DM1A-FITC antibody and incubate for 30 minutes.

[0116] 10. Use DAPI to label DNA in cells to characterize chromatin (interphase DNA staining during the cell cycle) and chromosomes (DNA staining during mitosis).

[0117] Experimental results are as follows Figure 2 As shown in the figure, the left side of the image represents the DMSO negative control group and the small molecule inhibitor treatment groups from Examples 1 to 4. Images were randomly taken using a DV microscope, and the figure shows three fields of view: field 1, field 2, and field 3. It can be seen that after DMSO treatment, HeLa cells could normally enter and exit mitosis, the chromosome queue in metaphase was normal (blue), and the spindle apparatus composed of microtubules was normal (green). However, after small molecule treatment, varying degrees of abnormal metaphase mitosis were observed, and mitotic exit failed. Fifty cells were counted in each group, and the statistical data are shown below. Figure 3 As shown in the diagram, green represents the percentage of cells in normal late mitotic phase, indicating that mitosis can exit normally; red represents the percentage of cells in mitotic metaphase with errors, indicating mitotic arrest; and blue represents the percentage of cells in normal metaphase. A blue percentage below 50% indicates abnormal mitosis. Therefore, the small molecule inhibitor of this invention can significantly inhibit tumor cell mitosis.

[0118] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0119] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a small molecule inhibitor in the preparation of a reagent for inhibiting HeLa cell mitosis, characterized in that, The small molecule inhibitor is a pharmaceutically acceptable salt of the chemical structure of formula (I) or including the chemical structure of formula (I); Formula (I); Where R1 is hydrogen or bromine; R2 is either hydrogen or bromine; R3 is methyl or ethyl; R4 is either methoxy or ethoxy; R5 stands for fluorine.

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