Cenpenlin, a targeting inhibitor of cell division regulation important kinetochore protein cenp-n, and application thereof

Abstract

The application discloses a cell division regulation important centromere protein CENP-N targeting inhibitor Cenpenlin and an application thereof. The compound shown in a formula I is applied to the preparation of products with the following functions: 1) a centromere protein CENP-N targeting inhibitor; 2) an agent for affecting normal arrangement of chromosomes and delaying mitosis process; 3) a product for inhibiting tumor cell proliferation; and 4) a product for preventing and / or treating cancer. Through immunofluorescence detection, it is confirmed that the small molecule plays an inhibitory effect by interfering with the interaction between CENP-N and other components in the CCAN complex.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to Cenpenlin, a target inhibitor of CENP-N, an important centromere protein for cell division regulation, and its applications. Background Technology

[0002] The kinetochore complex in the centromere region carries chromosomal genetic information. The centromere-associated network protein CCAN is a sixteen-membered complex responsible for the connection between the CENP-A nucleosome and the centromere spindle, with CENP-N playing a crucial role in the complex. The cryo-electron microscopy structure of CCAN reveals the interactions between CENP-N and other components of the CCAN complex. CENP-N binds to CNEP-A in a pattern different from the classic octamer nucleosome. Kinetochores mediate the connection between chromosomes and spindle microtubules. Centromeres aggregate in a specific chromatin region, and CENP-A recruits the 16-subunit CCAN complex. CENP-C and CENP-N specifically recognize CENP-A; CENP-C binds to the CENP-A nucleosome through its central region and CENP-C motif, while CENP-N recognizes the L1 (RG loop) of CENP-A. It provides an anchoring region for the Knl1-Mis12-Ndc80 (KMN) complex, mediating the attachment and sliding regulation of cell cycle microtubules. During cell mitosis, genetic material is equally distributed to two daughter cells, and CENP-A is replenished to compensate for its reduction during DNA replication. The protein machinery based on the CENP-A nucleosome and CCAN kinetochore complex has evolved from lower organisms to various eukaryotes such as humans, but its composition has been conserved in metazoans.

[0003] In yeast, the centromere is established on a 125-base-pair DNA segment, limited to the Cse4 (CENP-A) nucleosome, and is called the dot centromere. Most CCAN subunits are recognized in the organism and are collectively referred to as the Ctf19 complex. Recent cryo-electron microscopy resolution of the Ctf19-Cse4 nucleosome complex structure in Saccharomyces cerevisiae revealed the interaction patterns between the subunits.

[0004] Unlike Saccharomyces cerevisiae, most eukaryotes possess regional centromeres, controlled by repetitive DNA sequences, such as the AT-rich, 171-base-pair alpha satellite DNA repetitive sequences in the human centromere. However, the assembly and inheritance of regional centromeres may be relatively independent of the DNA sequence. The CENP-A-associated CCAN protein machinery recruits CCAN complex components through cell cycle regulation of CENP-A. The conservation of CCAN indicates the functional partitioning of regional centromeres. Summary of the Invention

[0005] The object of this invention is to provide novel pharmaceutical uses for the compound of Formula I, its isomers, and pharmaceutically acceptable salts thereof, wherein the compound of Formula I is named Cenpenlin.

[0006]

[0007] The novel pharmaceutical uses of the compound of Formula I, its isomers, and pharmaceutically acceptable salts provided by this invention are applications of the compound of Formula I in the preparation of products having the following functions:

[0008] 1) Centromere protein CENP-N targeting inhibitor;

[0009] 2) Reagents that affect the normal alignment of chromosomes and delay the process of mitosis;

[0010] 3) Products that inhibit the proliferation of tumor cells;

[0011] 4) Products for the prevention and / or treatment of cancer.

[0012] Specifically, 1) affects the interaction between CENP-N and CENP-W and / or CENP-L, and targets and inhibits the assembly of centromere protein CENP-N into the CCAN complex;

[0013] In the third step, the tumor cells are osteosarcoma cells, cervical cancer cells, and gastric cancer cells;

[0014] In the context of 4), the cancer may specifically be osteosarcoma, cervical cancer, or gastric cancer.

[0015] The spatiotemporal sequence of CENP-N assembly into the CCAN complex remains unresolved. If an inhibitor of CENP-N can halt the assembly of the CCAN complex, we will be able to elucidate the function of CENP-N assembly into the CCAN complex and its spatiotemporal control mechanism within a short period.

[0016] Among the reported CCAN complex structures, the full-length structure of human CENP-N remains unresolved. Our collaborating team, based on cryo-electron microscopy-resolved structures of the human CENP-N complex, designed inhibitors to intervene in CENP-N. We screened several compounds potentially affecting CENP-N from a drug-like molecule library using molecular docking, and further identified phenotypes influencing mitotic processes through phenotypic screening. Immunofluorescence assays confirmed that the small molecules exert their inhibitory effect by interfering with the interactions between CENP-N and other components of the CCAN complex. Attached Figure Description

[0017] Figure 1 This is the synthetic route for the small molecule Cenpenlin.

[0018] Figure 2 The image shows the NMR spectrum of the synthesized Cenpenlin small molecule.

[0019] Figure 3 The cryo-electron microscopy structure and analysis of the human centromere protein machine CCAN are shown in Figure A. The cryo-electron microscopy structure of the human centromere protein machine CCAN complex is shown in Figure B. The gray solid structure shows the resolved CENP-N structure. The pink rod structure shows the small molecule of Cenpenlin and its interaction space.

[0020] Figure 4 A diagram showing chromosome misalignment caused by the small molecule compound Cenpenlin. The addition of 1 μM Cenpenlin showed some inhibitory effect on the proliferation of human osteosarcoma cell line U2OS(A) and human cervical cancer cell line HeLa(B); however, it did not have a significant inhibitory effect on the proliferation of normal human retinal pigment epithelial cell line RPE1(C). Scalebars = 10 μm.

[0021] Figure 5 A diagram showing the mitotic arrest caused by the small molecule compound Cenpenlin. The chromosome movement data indicates that Cenpenlin treatment resulted in some chromosomes failing to align correctly and caused significant mitotic delay; the cells did not enter anaphase 120 minutes after nuclear membrane rupture. This conclusion also confirms the previous research suggesting that CENP-N may function as a checkpoint for spindle assembly.

[0022] Figure 6 This figure shows the effect of the small molecule compound Cenpenlin on centromere proteins CENP-N and CENP-W. Using DMSO as a control, the final concentration of Cenpenlin added was 300 nM. Based on GFP-CENP-N and GFP-CENP-W, the results indicate that the small molecule has a certain effect on CENP-N and CENP-W, causing the production of lagging chromosomes. Scalebars = 10 μm.

[0023] Figure 7 Phenotypic analysis of Cenpenlin on cell growth and division. Knockout of CENP-N led to an increase in the distance between the centrioles of the spindle; however, incubation with a final concentration of 500 nM Cenpenlin did not induce an increase in the distance between the spindle poles in CENP-N knockout cell lines, and they were more prone to forming multipolar spindles. Scalebar = 10 μm.

[0024] Figure 8This study analyzed the phenotypic effects of the small molecule compound Cenpenlin on mitosis in CENP-A knockout cell lines. The addition of Cenpenlin shortened the distance between the centrioles of the spindle fibers. Incubation with a final concentration of 500 nM Cenpenlin significantly affected the microtubule stability of the CENP-A knockout cell lines and resulted in delayed chromosome formation. Scale bar = 10 μm.

[0025] Figure 9 Phenotypic representation of the biotin-labeled small molecule compound Bio-Cenpenlin leading to multipolar spindle formation during mitosis. Adding biotin to Cenpenlin resulted in the formation of multipolar spindles during mitosis in cells.

[0026] Figure 10 The inhibitory effect of Cenpenlin on the growth of gastric cancer organoid cells was investigated. A. Gastric cancer organoids were grown in Matrigel for 7 days, and then 150 nM Cenpenlin was added. B. The survival rate of gastric cancer organoid cells was determined by PI staining. Cenpenlin inhibited the growth of gastric cancer organoid cells by 90%. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] The small molecule compound Cenpenlin used in the following examples is in accordance with Figure 1 The synthesis route shown is used for synthesis.

[0030] The specific operating steps are as follows:

[0031] Synthesis of compound 2:

[0032] 7.6 mmol of p-methylthiophenol and 7.0 mmol of ethyl 4-chloroacetoacetate were dissolved in 50 mL of dichloromethane. The two solutions were cooled to 0°C in an ice-water bath, then mixed and stirred. 10.8 mmol of triethylamine was added dropwise to the mixture, resulting in a white precipitate. The mixture was stirred in an ice-water bath for 30 minutes until the reaction was complete. 100 mL of water was added, and the organic phase was separated. The aqueous phase was extracted with 10 × 3 mL of ethyl acetate. The combined organic phases were washed successively with saturated sodium bicarbonate solution, 0.25 M dilute hydrochloric acid, and saturated brine. The organic phase was then concentrated by rotary evaporation and eluented with ethyl acetate:petroleum ether = 1:9 using a silica gel column to prepare compound 1, a pale yellow oil. Compound 1 was immediately dissolved in 20 mL of ethanol, and an equivalent amount of hydrazine hydrate (containing 70% NH2) was added. The mixture was stirred overnight at room temperature, resulting in a white precipitate. The precipitate was filtered, washed with cold ethanol, and dried to give a white solid, which was compound 2. The overall yield of the two-step reaction was 69%. The 1H NMR spectrum of compound 2 was as follows: 1 H NMR (500MHz, DMSO-d6): 11.55(brs,1H),9.47(brs,1H),7.09(d,2H),7.22(d,2H),5.31(s,1H),4.08(s,2H),2.32(s,3H).

[0033] Synthesis of compound 3:

[0034] 50 mL of malononitrile was dissolved in 25 mL of ethanol, and 50 mL of 2,3-dimethoxybenzaldehyde was added to the above solution. The mixture was stirred at room temperature for 4 hours until the reaction was complete. The product was a precipitate, which was filtered and washed with cold ethanol to obtain a solid powder with a yield of 92%. The 1H NMR data of compound 3 were as follows (500 MHz, CDCl3): 3.91 (s, 3H, CH3O), 3.95 (s, 3H, CH3O), 7.15–7.21 (m, 2H, ArH), 7.85 (dd, J = 7.6 Hz, J′ = 2.8 Hz, 1H, ArH), 8.26 (s, 1H, CH=).

[0035] Synthesis of compound 4:

[0036] Compound 3 (10 mmol) and compound 2 (11 mmol) were added to 20 mL of ethanol, followed by 0.5 mL of triethylamine. The mixture was heated under reflux for 4 hours, filtered while hot to remove insoluble matter, and cooled to room temperature. The product gradually crystallized out. The product was filtered and washed with cold ethanol and cold petroleum ether to give a white solid product in 92% yield. The 1H NMR data for compound 4 were as follows (500 MHz, DMSO-d6): 2.29 (3H, s, CH3); 3.59 (1H, d, J = 14.4, SCH2); 3.90 (1H, d, J = 14.4, SCH2); 4.60 (1H, s, CH); 6.89–7.42 (9H, m, NH2+ArH); 12.42 (1H, br.s, NH).

[0037]

[0038] The structural formula of the compound Cenpenlin is shown above, and its 600MHz... 1 HNMR spectrum ( Figure 2 The ownership status of () is as follows:

[0039] 1 H NMR(600MHz,DMSO-d6)δ7.09(13-17,d,2CH),δ7.04(14-16,d,2CH),δ6.99(22,t,CH),δ6.92(21,d,CH),δ6.85(26,s,N H2), δ6.58(23,d,CH), δ6.54(4,s,NH), δ4.57(7,s,CH), δ3.79(10,s,CH2), δ2.55(27,s,CH3), δ2.28(29-31,s,2CH3).

[0040] The CENP-N accession number used in the following embodiments is NP_001094094.2 in the NCBI database (update: 09-JUN-2022);

[0041] The CENP-L accession number used in the following examples is NP_001164653.1 in the NCBI database (update: 30-JAN-2022).

[0042] Example 1: Molecular docking simulation of the small molecule compound Cenpenlin

[0043] 1. Experimental Procedure

[0044] 1) Using DOCK software, the structure of CENP-N was selected as the target protein, the largest pocket was selected, and candidate small molecules were attached to the binding pocket;

[0045] 2) The docking site was visualized using Pymol software. Based on molecular docking simulations, the interface between CENP-L and CENP-N was demonstrated, showing the binding of Cenpenlin.

[0046] 2. Results are as follows Figure 3 As shown.

[0047] Based on molecular docking simulation analysis, it was found that Cenpenlin binds at the interface between CENP-L / CENP-N, suggesting that it disrupts the interaction between the complexes through allosteric reactions.

[0048] Example 2: The small molecule compound Cenpenlin causes chromosome misalignment.

[0049] 1. Experimental Procedure

[0050] 1) HeLa, U2OS, and RPE1 cells that stably express GFP-CENP-N were seeded onto round coverslips with a diameter of 12 mm;

[0051] 2) After 24 hours, add Thymidine to a final concentration of 2 mM and treat the cells for 16 hours.

[0052] 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;

[0053] 4) The experimental group was treated with 1 μM Cenpenlin, and the control group was treated with the same volume of DMSO for 1 h.

[0054] 5) Fix cells with PBS buffer containing 3.7% formaldehyde for 10 min;

[0055] 6) After punching with 0.2% Triton X-100 and blocking with 1% BSA, incubate the ACA antibody at room temperature for 1 hour;

[0056] 7) Mount the slide after DAPI staining for 3 minutes;

[0057] 8) Place the slide on the stage of the DV microscope and take a picture under a 60× lens with NA=1.42.

[0058] 2. Results are as follows Figure 4 As shown.

[0059] The addition of 1 μM Cenpenlin inhibited the proliferation of human osteosarcoma cell line U2OS(A) and human cervical cancer cell line HeLa(B); however, it did not significantly inhibit the proliferation of normal human retinal pigment epithelial cell line RPE1(C). Scalebars = 10 μm.

[0060] Example 3: The small molecule compound Cenpenlin causes mitotic arrest.

[0061] 1. Experimental Procedure

[0062] 1) Seed HeLa cells into 35mm live cell culture dishes;

[0063] 2) Transfect plasmid DNA after 24 hours: GFP-tubulin and mCherry-H2B (transfected plasmid cell density is 70%-80%);

[0064] 3) After 6 hours of transfection, replace the medium with fresh medium and treat the cells with Thymidine at a final concentration of 2 mM for 16 hours.

[0065] 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;

[0066] 5) Turn on the Applied Precision Personal DV microscope and the thermostat to stabilize the temperature at 37°C;

[0067] 6) Eight hours after release, replace the cell culture medium with CO2-independent medium and add Cenpenlin to a final concentration of 300 nM or the same volume of DMSO.

[0068] 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;

[0069] 8) Add Reversine to a final concentration of 250 nM and continue real-time shooting, taking one frame every 3 minutes.

[0070] 2. Results are as follows Figure 5 As shown.

[0071] With DMSO as a control (A), HeLa cells treated with 300 nM cenpenlin showed a large number of chromosomes that were not aligned to the equatorial plate. Judging from the chromosome movement, cenpenlin treatment caused some chromosomes in the cells to be unable to align correctly. The proportion of multipolar spindles formed was statistically analyzed (B). It also caused a significant delay in mitosis. The cells did not enter the anaphase stage even 120 min after the nuclear membrane ruptured. The addition of Reversine at a final concentration of 250 nM alleviated the mitotic arrest caused by cenpenlin and enabled the cells to enter the anaphase stage smoothly.

[0072] Example 4: Effect of the small molecule compound Cenpenlin on centromere proteins CENP-N and CENP-W

[0073] 1. Experimental Procedure

[0074] 1) Place a circular coverslip into the 24-well plate and apply a layer of Poly-Lysine solution to the coverslip to increase adhesion;

[0075] 2) Wash 3 times with 500μL PBS, remove the PBS, open the 24-well plate and place it in a clean bench, then irradiate with UV for 15 min.

[0076] 3) Divide the cells into wells containing coverslips and let them grow overnight until they adhere to the walls;

[0077] 4) Transfect with GFP-CENP-N and GFP-CENP-W plasmids;

[0078] 5) After 24 hours, add Thymidine to a final concentration of 2 mM and treat the cells for 16 hours.

[0079] 6) 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;

[0080] 7) Cenpenlin was added to a final concentration of 300 nM, while an equal volume of DMSO was added to the control group;

[0081] 8) Paraformaldehyde fixation; fixation and permeabilization were performed simultaneously. The culture medium in the wells was removed, and 500 μL of preheated PTEM containing 3.7% formaldehyde was added for fixation for 10 min. PBST was added for washing 3 times, 5 min each time.

[0082] 9) Use pointed tweezers to remove the sealing film that has been flattened by the coverslip (with the cell side facing up), add 50 μL of PBST containing 1% BSA, and incubate at room temperature for 30 min;

[0083] 10) Use absorbent paper to blot away the liquid along the edge of the coverslip, add 50 μL of diluted primary antibody solution, and incubate at room temperature for 50 min;

[0084] 11) Remove the primary antibody, add 50 μL of PBST and incubate for 5 min. Repeat 3 times to wash away unbound antibody.

[0085] 12) Add 50 μL of diluted fluorescent secondary antibody solution and incubate at room temperature for 30 min; add 50 μL of PBST and incubate for 5 min, repeat 3 times, and wash away unbound antibody;

[0086] 13) Remove the secondary antibody, add 50 μL of diluted DAPI dye, and incubate at room temperature for 1 min;

[0087] 14) Place 1 μL of antiquenching agent on a clean, dry glass slide, place a coverslip upside down on the slide (with the cell side facing down), apply nail polish along the edge of the coverslip to seal it, and let it dry before observing it under a microscope.

[0088] 2. Results are as follows Figure 6 As shown.

[0089] Using DMSO as a control, and adding Cenpenlin to a final concentration of 300 nM, based on GFP-CENP-N and GFP-CENP-W display, it was found that the small molecule had a certain effect on CENP-N and CENP-W, causing the production of delayed chromosomes.

[0090] Example 5: Effects of the small molecule compound Cenpenlin on centromere protein CENP-N and CENP-A knockout cell lines

[0091] 1. Experimental Procedure

[0092] 1) Place a circular coverslip into the 24-well plate and apply a layer of Poly-Lysine solution to the coverslip to increase adhesion;

[0093] 2) Wash 3 times with 500μL PBS, remove the PBS, open the 24-well plate and place it in a clean bench, then irradiate with UV for 15 min.

[0094] 3) Separate the CENP-N and CENP-A knockout cell lines into wells containing coverslips and let them grow overnight until they adhere to the surface;

[0095] 4) Doxycycline induced the knockout of CENP-N and CENP-A, respectively;

[0096] 5) After 24 hours, add Thymidine to a final concentration of 2 mM and treat the cells for 16 hours.

[0097] 6) 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;

[0098] 7) Cenpenlin was added to a final concentration of 500 nM, while an equal volume of DMSO was added to the control group;

[0099] 8) Paraformaldehyde fixation; fixation and permeabilization were performed simultaneously. The culture medium in the wells was removed, and 500 μL of preheated PTEM containing 3.7% formaldehyde was added for fixation for 10 min. PBST was added for washing 3 times, 5 min each time.

[0100] 9) Use pointed tweezers to remove the sealing film that has been flattened by the coverslip (with the cell side facing up), add 50 μL of PBST containing 1% BSA, and incubate at room temperature for 30 min;

[0101] 10) Use absorbent paper to blot away the liquid along the edge of the coverslip, add 50 μL of diluted primary antibody solution, and incubate at room temperature for 50 min;

[0102] 11) Remove the primary antibody, add 50 μL of PBST and incubate for 5 min. Repeat 3 times to wash away unbound antibody.

[0103] 12) Add 50 μL of diluted fluorescent secondary antibody solution and incubate at room temperature for 30 min; add 50 μL of PBST and incubate for 5 min, repeat 3 times, and wash away unbound antibody;

[0104] 13) Remove the secondary antibody, add 50 μL of diluted DAPI dye, and incubate at room temperature for 1 min;

[0105] 14) Place 1 μL of antiquenching agent on a clean, dry glass slide, place a coverslip upside down on the slide (with the cell side facing down), apply nail polish along the edge of the coverslip to seal it, and let it dry before observing it under a microscope.

[0106] 2. Results are as follows Figure 7 As shown.

[0107] Phenotypic analysis of Cenpenlin on cell growth and division. Knockout of CENP-N led to an increase in the distance between the two poles of the centrioles of the spindle; however, incubation with a final concentration of 500 nM Cenpenlin did not induce an increase in the distance between the two poles of the spindle in CENP-N knockout cell lines, and they were more prone to forming multipolar spindles.

[0108] The results are as follows Figure 8 As shown.

[0109] Analysis of the phenotypic effects of the small molecule compound Cenpenlin on mitosis in CENP-A knockout cell lines. The addition of Cenpenlin shortened the distance between the poles of the centrioles in the spindle. Furthermore, incubation with a final concentration of 500 nM Cenpenlin significantly affected the microtubule stability of the CENP-A knockout cell lines and resulted in delayed chromosome formation.

[0110] Example 6: Biotin-labeled small molecule compound Bio-Cenpenlin induces a mitotic multipolar spindle phenotype.

[0111] 1. Experimental Procedure

[0112] 1) Place a circular coverslip into the 24-well plate and apply a layer of Poly-Lysine solution to the coverslip to increase adhesion;

[0113] 2) Wash 3 times with 500μL PBS, remove the PBS, open the 24-well plate and place it in a clean bench, then irradiate with UV for 15 min.

[0114] 3) Divide the cells into wells containing coverslips and let them grow overnight until they adhere to the walls;

[0115] 4) After 24 hours, add Thymidine to a final concentration of 2 mM and treat the cells for 16 hours.

[0116] 5) 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;

[0117] 6) Biotin-labeled Bio-Cenpenlin was added to a final concentration of 1 μM, while an equal volume of DMSO was added to the control group.

[0118] 7) Paraformaldehyde fixation; fixation and permeabilization were performed simultaneously. The culture medium in the wells was removed, and 500 μL of preheated PTEM containing 3.7% formaldehyde was added for fixation for 10 min. PBST was added for washing 3 times, 5 min each time.

[0119] 8) Use pointed tweezers to remove the sealing film that has been flattened by the coverslip (with the cell side facing up), add 50 μL of PBST containing 1% BSA, and incubate at room temperature for 30 min;

[0120] 10) Use absorbent paper to blot away the liquid along the edge of the coverslip, add 50 μL of diluted primary antibody solution, and incubate at room temperature for 50 min;

[0121] 11) Remove the primary antibody, add 50 μL of PBST and incubate for 5 min. Repeat 3 times to wash away unbound antibody.

[0122] 12) Add 50 μL of diluted fluorescent secondary antibody solution and incubate at room temperature for 30 min; add 50 μL of PBST and incubate for 5 min, repeat 3 times, and wash away unbound antibody;

[0123] 13) Remove the secondary antibody, add 50 μL of diluted DAPI dye, and incubate at room temperature for 1 min;

[0124] 14) Place 1 μL of antiquenching agent on a clean, dry glass slide, place a coverslip upside down on the slide (with the cell side facing down), apply nail polish along the edge of the coverslip to seal it, and let it dry before observing it under a microscope.

[0125] 2. Results are as follows Figure 9 As shown.

[0126] Adding biotin to the small molecule Cenpenlin resulted in cells exhibiting the ability to form multipolar spindles during mitosis.

[0127] Example 7: Inhibitory effect of the small molecule compound Cenpenlin on the growth of gastric cancer organoid cells.

[0128] 1. Experimental Procedure

[0129] 1) Add an appropriate amount of matrix gel to the isolated gastric cancer tissue cells, and then separate them into 10cm culture dishes;

[0130] 2) Place the petri dish at 37℃ for 10-30 minutes. After the substrate gel has fully solidified, add the complete culture medium to the petri dish.

[0131] 3) Change the culture medium every 3 days;

[0132] 4) After growing in the matrix gel for 7 days, Cenpenlin with a final concentration of 150 nM was added, and the control group was given an equal volume of DMSO.

[0133] 5) Every 24 hours, the survival rate of gastric cancer organoid cells was determined and statistically analyzed by staining with PI (the fluorescent dye is propidium iodide, abbreviated as PI).

[0134] 2. Results are as follows Figure 10 As shown.

[0135] A. Gastric cancer organoids were grown in Matrigel for 7 days, then 150 nM Cenpenlin was added. B. The survival rate of gastric cancer organoid cells was determined by PI staining; Cenpenlin inhibited the growth of gastric cancer organoid cells by 90%.

[0136] 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 salts in the preparation of products for the prevention and / or treatment of cancer: The cancers mentioned are osteosarcoma, cervical cancer, and stomach cancer.

2. The application according to claim 1, characterized in that: The compounds represented by Formula I and their pharmaceutically acceptable salts prevent and / or treat cancer by interfering with the interaction between CENP-N and other components in the CCAN complex.

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