Cenp-m small molecule inhibitor cenpemlin and preparation method and application thereof

Abstract

The application discloses a centromere protein CENP-M small molecule inhibitor cenpemlin and a preparation method and application thereof. The application targets and screens a small molecule compound cenpemlin by analyzing protein site information of a fine structure of CENP-M and CENP-L. Biochemical and cell experimental results show that the cenpemlin inhibits the proliferation of cancer cells by interfering with the interaction between CENP-M and CENP-L, and provides a novel compound for analyzing and interfering with the rapid proliferation of cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to cenpemlin, a small molecule inhibitor of CENP-M, a key centromere protein in mitosis, as well as its preparation method and application. Background Technology

[0002] Precise mitosis ensures genome stability and maintains cellular homeostasis. Mitosis produces two daughter cells with identical genes through the equal separation of sister chromatids. The molecular mechanisms driving chromosome segregation are relatively conserved throughout eukaryotic evolution. The kinetochore is a fundamental element of this mechanism and can be viewed as a layered structure, with its inner and outer layers directly contacting the centromere chromatin and spindle microtubules, respectively. The inner kinetochore layer consists of at least 16 centromere proteins, which assemble into multi-component complexes that interact directly with the centromere chromatin. These complexes include: the CENP-H, I, K, M complex; the CENP-O, P, Q, U, R complex; the CENP-T, W, S, X complex; the CENP-L, N complex; and the CENP-C protein. After assembly, these complexes further recruit the remaining kinetochore proteins. Although the interactions between centromere proteins have been extensively studied, the specific biological functions of some of these proteins still require further investigation.

[0003] Recently, our research group used cryo-electron microscopy to resolve the structure of the CCAN complex. The CCAN complex exhibits a "V" shape, with the CENP-L / N dimer forming an arch through which DNA passes. The CENP-M protein interacts with three other centromere proteins, CENP-H, I, and K, to form a complex that becomes part of the kinetochore inner layer. CENP-M binds near the CENP-L / N dimer at the top of the arch, and has spatial interaction interfaces with both CENP-L and CENP-N. Furthermore, CENP-M exhibits kinetic localization throughout the cell cycle, suggesting a constitutive function, but the specific molecular mechanism remains incompletely understood.

[0004] CENP-M, initially named PANE1 (proliferation-associated nuclear element 1), is conserved only in metazoans. Subsequent studies have demonstrated that CENP-M is closely associated with CENP-A, CENP-L, CENP-N, and CENP-T. The absence of CENP-M leads to the mislocalization of other CCAN proteins, indicating that CENP-M is crucial for the assembly and stability of inner kinetochores. However, despite its structural and evolutionary association with GTP hydrolases, CENP-M is not a true enzyme. It plays a vital role in a range of important biological processes, including recruiting the FACT protein complex for chromatin remodeling, promoting CENP-A nucleosome remodeling, and recruiting spindle checkpoint-associated proteins, through the formation of a quaternary complex with evolutionarily conserved CENP-H, I, and K proteins.

[0005] There are currently no reports on CENP-M inhibitors. Summary of the Invention

[0006] The purpose of this invention is to provide a compound, its isomers, pharmaceutically acceptable salts, their preparation methods, and applications.

[0007] The compound provided by this invention has the structural formula shown in Formula I:

[0008]

[0009] The compound shown in Formula I specifically targets the key centromere protein CENP-M in mitosis, and is named cenpemlin as a CENP-M inhibitor.

[0010] CENP-M is an important member of the inner kinetochore CCAN complex. It was named PANE1 after being identified in rapidly proliferating cells, and subsequent studies revealed that CENP-M is a pseudo-GTP hydrolase. In recent years, numerous studies have been conducted on the inner kinetochore CCAN complex, but its fine structure remains incompletely resolved. We employed a cryo-electron microscopy-based structural biology approach to answer these questions. We expressed and purified the CCAN complex protein using an insect system, followed by in vitro recombination to obtain the complete CCAN complex. Three-dimensional images of the complex were collected using cryo-electron microscopy, followed by 3D reconstruction. Finally, we obtained the structural information of the CCAN complex containing the CENP-M protein. Based on the fine structure of the interaction interface between the CENP-M and CENP-L proteins, we identified a specific CENP-M-targeting small molecule compound, cenpemlin (Formula I), through a combination of molecular docking analysis, virtual screening, cell phenotypic analysis, and protein-protein interaction experiments.

[0011] The compound shown in Formula I is based on Figure 1 The synthetic route shown was prepared by a method including the following steps:

[0012] 1) This allows cyanuric chloride and morpholine to react under alkaline conditions to generate cyanuric chloride intermediate 1 containing a bis(1,4-oxo-nitrogen) heterocycle;

[0013] 2) The intermediate product 1 of chlorocyanide containing a bis(1,4-oxonium-nitrogenous) ring is subjected to a substitution reaction with hydrazine hydrate to generate 1,3(1,4-oxonium-nitrogenous)-5-hydrazine polycyanide (compound 2);

[0014]

[0015] 3) In glacial acetic acid, 1,3(1,4-oxo-nitrogenous heterocyclic)-5-hydrazine cyanurate undergoes a condensation reaction with a hexacarbon monosaccharide to obtain the compound shown in Formula I.

[0016] In step 1) of the above method, the molar ratio of cyanuric chloride to morpholine can be 1:2-2.5, specifically 1:2;

[0017] The reaction is carried out under the action of an ice-water bath, and the reaction time can be 1-1.5 hours, specifically 1 hour.

[0018] The alkaline conditions are provided by sodium bicarbonate; the molar ratio of sodium bicarbonate to morpholine can be 1:1.0-1.2, specifically 1:1;

[0019] In step 2) of the above method, the molar ratio of the chlorocyanide intermediate 1 containing the bis(1,4-oxo-nitrogen heterocycle) to hydrazine hydrate is 1:1-2.

[0020] The reaction temperature is 40℃-60℃, and the time is 3-4 hours, specifically 3 hours.

[0021] In step 3) of the above method, the ratio of 1,3(1,4-oxonitrogen heterocyclic)-5-hydrazine polycyanate to hexacarbon monosaccharide can be 10 mmol: 1.8-2.0 g, specifically 10 mmol: 1.8 g;

[0022] Hexacarbon monosaccharides that can be used as raw materials include, but are not limited to, D-glucose, D-galactose, and D-mannose;

[0023] The reaction is carried out at a temperature of 40℃-60℃ for 4-5 hours, specifically 4 hours.

[0024] The use of the compound shown in Formula I, its isomers, and pharmaceutically acceptable salts in the preparation of products having the following functions is also within the scope of protection of this invention:

[0025] 1) Centromere protein CENP-M inhibitor;

[0026] 2) Reagents that inhibit cell mitosis;

[0027] 3) Products that inhibit the proliferation of tumor cells;

[0028] 4) Products for the prevention and / or treatment of cancer;

[0029] In 2), inhibiting cell mitosis is achieved by interfering with CENP-M-mediated kinetochore assembly, specifically by inhibiting the interaction between CENP-M and CENP-L.

[0030] In 3), the tumor cells may be epithelial cancer cells; specifically, they may be cervical cancer cells, liver cancer cells, breast cancer cells, etc.

[0031] 4) The cancer mentioned can specifically be epithelial cancer, such as cervical cancer, liver cancer, breast cancer, etc.

[0032] The present invention also provides a reagent for inhibiting cell mitosis, said reagent containing a compound of formula I, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0033] The present invention also provides a product for inhibiting tumor cell proliferation, said product containing a compound of formula I, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0034] The present invention also provides a product for the prevention and / or treatment of cancer, said product comprising a compound of formula I, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0035] The CENP-M inhibitory organic small molecule compound provided by this invention, after being added to the culture medium and binding to the CENP-M protein, causes chromosomes to be unable to align correctly and further leads to delayed mitosis and a lagging chromosome phenotype in cells.

[0036] Real-time live-cell imaging revealed that cenpemlin-treated cells exhibited chromosome misalignment and spindle instability, as well as a phenotype of delayed mitosis. These induced mitotic abnormalities were consistent with the phenotype of CENP-M knockdown.

[0037] Cenpemlin at a concentration of 1 μM can inhibit the interaction between CENP-M and CENP-L. The effective inhibition of CENP-M / CENP-L by cenpemlin can be used to further investigate the function of CENP-M in mitotic kinetochore assembly and chromosome alignment.

[0038] The CENP-M small molecule inhibitor cenpemlin of this invention will play an important role in cell biology research, and its efficacy in regulating tumor cell proliferation can lay the foundation for the development of novel chemotherapy drugs.

[0039] This invention utilizes protein surface information derived from the fine structures of CENP-M and CENP-L to target and screen for the small molecule compound cenpemlin. Biochemical and cellular experiments both demonstrate that cenpemlin inhibits cancer cell proliferation by interfering with the interaction between CENP-M and CENP-L, providing a novel compound for elucidating and intervening in the rapid proliferation of cancer cells. Attached Figure Description

[0040] Figure 1 This is a synthetic route diagram of the CENP-M inhibitor shown in Formula 1 of this invention.

[0041] Figure 2 The results are NMR identification of the small molecule compound cenpemlin.

[0042] Figure 3 Phenotypic analysis of the small molecule compound cenpemlin on cell mitosis.

[0043] Figure 4 Real-time imaging analysis of the small molecule compound cenpemlin arresting cell mitosis.

[0044] Figure 5 Elution efficiency analysis of the small molecule compound cenpemlin.

[0045] Figure 6 This indicates that the small molecule compound cenpemlin inhibits the interaction between CENP-M and CENP-L.

[0046] Figure 7 Statistical analysis of the inhibitory effect of the small molecule compound cenpemlin on cancer cell proliferation.

[0047] Figure 8 This study investigated the effect of the small molecule compound cenpemlin on the proliferation of liver cancer cells in mice. Detailed Implementation

[0048] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0050] The CENP-M accession number used in the following embodiments is Accession: NP_076958.1 in the NCBI database (Update: 27-JUN-2022).

[0051] The CENP-L accession number used in the following embodiments is Accession: NP_001164653.1 in the NCBI database (update: 30-JAN-2022).

[0052] Example 1: Synthesis of the small molecule compound cenpemlin

[0053] Cyanurium chloride (10.0 g, 0.054 mol) was dissolved in 150 mL of dichloromethane, and the solution was cooled to below 5 °C in an ice-water bath. Morpholine (9.50 mL, 0.108 mol) and sodium bicarbonate (9.1 g, 0.108 mol) were dissolved in 100 mL of water, and the solution was cooled to below 5 °C in an ice-water bath. In the ice-water bath, the aqueous solution was added dropwise to the dichloromethane solution with rapid stirring, and the reaction was continued for 1 h. The two phases were separated, and the organic phase was washed twice with 50 mL of water. The organic phase was concentrated to 20 mL by rotary evaporation, and 50 mL of petroleum ether was added to the organic phase. The resulting white precipitate was filtered and washed with cold water and cold petroleum ether. After drying, 14.7 g of white solid, i.e., intermediate product 1, was obtained, with a yield of 95%. ¹H NMR: 400 MHz, CDCl₃ δ 3.80–3.68 (m, 16H).

[0054] Intermediate product 1 (5.7 g, 20 mmol) was added to 25 mL of ethanol and 5 mL of hydrazine hydrate. The solution was heated to 40-60 °C and refluxed for 3 hours, then cooled to room temperature. A white solid gradually precipitated out. The product was filtered and washed twice with 20 mL of ethanol. After drying, 5.2 g of white solid product, compound 2, was obtained, with a yield of 93%. 1H NMR spectrum: 400 MHz, CDCl3 δ 3.80-3.68 (m, 16H), 4.77 (d, 2H), 9.39 (t, 1H).

[0055] Compound 2 (2.8 g, 10 mmol) was added to 25 mL of ethanol, followed by 10 mmol of a hexose (1.8 g) and 0.2 mL of glacial acetic acid. The solution was heated to 40-60 °C and refluxed for 4 hours, then cooled to room temperature. A white solid gradually precipitated out. The product was filtered and washed twice with 20 mL of ethanol to obtain a white solid. The product was purified by recrystallization from ethanol / ethyl acetate. The yield of the product ranged from 70-85% depending on the type of hexose used. Suitable hexoses as starting materials include, but are not limited to, D-glucose, D-galactose, and D-mannose.

[0056] The structure of the small molecule was identified using a Bruker AV-500 nuclear magnetic resonance spectrometer. Based on the chemical shift and peak integral area of ​​the proton spectrum, the structure of the small molecule was determined to conform to Formula I.

[0057] Figure 2 The NMR results show the identification of the small molecule compound cenpemlin.

[0058] Example 2: The small molecule compound cenpemlin caused chromosome misalignment.

[0059] 1. Experimental Procedure

[0060] 1) HeLa cells stably expressing GFP-CENP-N were seeded onto round coverslips with a diameter of 12 mm;

[0061] 2) After 24 hours, add Thymidine to a final concentration of 2 mM and treat the cells for 14-16 hours;

[0062] 3) Wash the cells three times with PBS preheated to 37°C to remove Thymidine, then replace with fresh culture medium and continue culturing for 8 hours;

[0063] 4) The experimental group was treated with 1 μM cenpemlin, and the control group was treated with the same volume of DMSO for 1 h.

[0064] 5) Fix cells with PBS buffer containing 3.7% formaldehyde for 10 min;

[0065] 6) After punching with 0.2% Triton X-100 and blocking with 1% BSA, incubate the ACA antibody at room temperature for 1 hour;

[0066] 7) Mount the slide after DAPI staining for 3 minutes;

[0067] 8) Place the slide on the stage of the DV microscope and take a picture under a 60× lens with NA=1.42.

[0068] 2. Results are as follows Figure 3 As shown.

[0069] After cenpemlin treatment of HeLa cells, a large number of chromosomes that were not aligned to the equatorial plate were observed. Using GFP-CENP-N as a characterization of the CENP-L / N complex, kinetochores that were not aligned to the equatorial plate region were observed, indicating that cenpemlin treatment of cells will lead to abnormal chromosome alignment during mitosis.

[0070] Example 3: The small molecule compound cenpemlin caused mitotic arrest.

[0071] 1. Experimental Procedure

[0072] 1) Seed HeLa cells into 35mm live cell culture dishes;

[0073] 2) Transfect plasmid DNA after 24 hours: GFP-tubulin and mCherry-H2B (transfected plasmid cell density is 70%-80%);

[0074] 3) After 6 hours of transfection, replace the medium with fresh medium and treat the cells with 2 mM Thymidine for 14-16 hours;

[0075] 4) Wash the cells three times with PBS preheated to 37°C to release the cells, then replace with fresh culture medium and continue culturing;

[0076] 5) Turn on the Applied Precision Personal DV microscope and the thermostat to stabilize the temperature at 37°C;

[0077] 6) 8-9 h after release, replace the cell culture medium with CO2-independent medium and add cenpemlin to a final concentration of 1 μM or the same volume of DMSO.

[0078] 7) Place the cells on a 37°C constant temperature stage of a DV microscope and take real-time pictures under a 60× lens with NA=1.42, taking one frame every 3 minutes.

[0079] 2. Results are as follows Figure 4 As shown.

[0080] Based on the observation of chromosome movement, cenpemlin treatment caused some chromosomes in the cells to consistently fail to align correctly, and also resulted in a significant delay in mitosis; the cells did not enter anaphase 120 minutes after nuclear membrane rupture. This conclusion also confirms the previous research suggesting that CENP-M has a certain function as a checkpoint for spindle assembly.

[0081] Example 4: Elution effect detection of small molecule compound cenpemlin

[0082] 1. Experimental Procedure

[0083] 1) Seed HeLa cells into 35mm live cell culture dishes;

[0084] 2) Transfect plasmid DNA after 24 hours: GFP-tubulin and mCherry-H2B (transfected plasmid cell density is 70%-80%);

[0085] 3) After 6 hours of transfection, replace the medium with fresh medium and treat the cells with MG132 and cenpemlin or DMSO at a final concentration of 20 μM for 1 hour.

[0086] 4) Turn on the Applied Precision Personal DV microscope and the thermostat to stabilize the temperature at 37°C;

[0087] 5) Wash the cells three times with PBS preheated to 37°C to release the cells, then replace with fresh culture medium and continue culturing;

[0088] 6) Immediately after release, replace the cell culture medium with CO2-independent medium;

[0089] 7) Place the cells on a 37°C constant temperature stage of a DV microscope and take real-time pictures under a 60× lens with NA=1.42, taking one frame every 3 minutes.

[0090] 2. Results are as follows Figure 5 As shown.

[0091] Cells treated with cenpemlin exited mitosis after 45 minutes, but exhibited a multicellular phenotype, i.e., unequal distribution of the genome, due to the multipolar spindle phenomenon observed in cenpemlin-treated cells.

[0092] Example 5: The effect of the small molecule compound cenpemlin on the interaction between CENP-M and CENP-L

[0093] 1. Experimental Procedure

[0094] 1) Seed HEK293T cells into 6cm culture dishes;

[0095] 2) Transfect plasmid DNA after 24 hours: GFP-CENP-M and FLAG-CENP-L (transfected plasmid cell density is 70%-80%);

[0096] 3) Replace with fresh culture medium 4 hours after transfection and continue culturing for 24 hours;

[0097] 4) Treat cells with 1 μM cenpemlin or the same volume of DMSO for 1 h.

[0098] 5) Use a cell scraper to scrape off the cells, collect them in a centrifuge tube, and centrifuge at 1,000 rpm to remove the culture medium;

[0099] 6) Add cell lysis buffer and sonicate to disrupt the cells, then centrifuge at 12,000 rpm for 10 min;

[0100] 7) Take the supernatant and incubate it with FLAG beads for 4 hours;

[0101] 8) Wash the beads three times with cell lysis buffer, boil the sample buffer, and then perform electrophoresis and immunoblotting analysis.

[0102] 2. Results are as follows Figure 6 As shown.

[0103] The interaction between CENP-M and CENP-L was weakened in the cenpemlin-treated (third lane) compared to the control group (second lane), indicating that 1 μM cenpemlin was sufficient to disrupt the interaction between CENP-M and CENP-L. This result echoes the cell phenotype experiment mentioned earlier, suggesting that cenpemlin inhibits tumor cell proliferation by interfering with the interaction between CENP-M and CENP-L.

[0104] Example 6: Broad-spectrum anticancer activity of the small molecule compound cenpemlin

[0105] 1. Experimental Procedure

[0106] 1) HeLa cells (cervical cancer cells), MDA-MB-231 cells (triple-negative breast cancer cells), and HepG2 cells (liver cancer cells) were seeded into 6cm culture dishes;

[0107] 2) Treat cells with either cenpemlin or DMSO at a final concentration of 1 μM for 24 h.

[0108] 3) Digest the cells with trypsin and stain the cell suspension with trypan blue.

[0109] 4) Observe and count the blue-stained cells under a regular optical microscope.

[0110] 2. Results are as follows Figure 7 As shown.

[0111] Cenpemlin treatment resulted in varying degrees of increased trypan blue staining in HeLa, MDA-MB-231, and HepG2 cells. This result echoes the cell phenotype experiments described earlier, indicating that cenpemlin has a broad-spectrum inhibitory effect on cancer cell proliferation.

[0112] Example 8: The small molecule compound cenpemlin inhibits the proliferation of liver cancer cells.

[0113] 1. Experimental Procedure

[0114] 1) Following the published experimental protocol (Chen et al., 2011. Cancer Res), we used MHCC97-H liver cancer cells (5 x 10⁻⁶ cells) that stably expressed luciferase. 6 The drug (in 0.1 mL of physiological saline) was implanted into the liver of 6-week-old female NOD / SCID mice. The experiment consisted of three groups: a DMSO control group, a paclitaxel group, and a cenpemlin group (2 mg / kg), with 10 mice in each group.

[0115] 2) Fourteen days after modeling (successful modeling with fluorescein test), the DMSO control group, paclitaxel group and cenpemlin experimental group (2mg / kg) were injected intraperitoneally (0.2mL per animal; 0.1mL on each side), once a day for three days.

[0116] 3) Inject every other day for four times (on day 11 of administration), and then perform a fluorescein test.

[0117] 2. Results are as follows Figure 8 As shown.

[0118] A shows a fluorescein imaging image on day 11 after drug administration. B shows a statistical analysis graph. The results indicate that the number of liver cancer cells in mice treated with paclitaxel and the small molecule compound cenpemlin was significantly reduced. These results demonstrate that the small molecule compound cenpemlin can effectively inhibit the proliferation of liver cancer cells in mice, providing a new perspective and target for the screening and development of anticancer drugs.

[0119] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of the compound represented by Formula I and its pharmaceutically acceptable salt in the preparation of medicaments for the prevention and / or treatment of cancer, wherein the cancer is cervical cancer, triple-negative breast cancer, or liver cancer. 。

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