Small molecule inhibitors targeting CDKL3, HZ1 and its derivative HZ1-X

Abstract

Small molecule inhibitors targeting CDKL3, such as HZ1 and its derivative HZ1-X, belong to the field of biomedicine. This invention provides specific small molecule inhibitors that can effectively inhibit CDKL3 kinase activity, and comprehensively characterizes at the cellular level the key cellular signaling pathways mediated by HZ1 and its derivative HZ1-X that promote the development and progression of various tumors mediated by the protein kinase CDKL3, as well as tumor growth. This invention provides a new method for targeted cancer therapy and a new approach for the clinical treatment of malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the small molecule inhibitor HZ1 targeting CDKL3 and its derivative HZ1-X. Background Technology

[0002] In recent years, research on tumor biomarkers has redefined cancer treatment. Clinical drug use and clinical trials have incorporated biomarker requirements, leading to increasingly widespread research and the development of a number of targeted drugs, including small molecule targeted drugs. Since the launch of imatinib, the first small molecule targeted anticancer drug, in 2002, the golden age of small molecule targeted anticancer drugs has officially begun. Compared to traditional surgery, radiotherapy, and chemotherapy, small molecule targeted anticancer drugs offer significant advantages such as accurate targeting, high specificity, fewer toxic side effects, and convenient administration. Small molecule targeted drugs targeting multiple targets, including EGFR, CDK4 / 6, and HER2, are widely used in clinical treatment.

[0003] CDKL3 belongs to the cyclin-dependent kinase-like (CDKL) kinase subfamily and the CMGC serine / threonine protein kinase superfamily. The CDKL kinase family possesses a conserved α-helix that binds to cyclins, identical to the CDK kinase domain. However, research on the function and mechanisms of the CDKL family is generally insufficient. CDKL5 has been shown to be associated with neurological diseases, and CDKL1 has been reported to regulate cilia formation. CDKL3 was first discovered in 2001 to be involved in cell proliferation and central nervous system development. Recently, CDKL3 has been reported to have an important association with human malignancies, such as... Figure 1 As shown. Furthermore, a novel molecular mechanism and important pathological significance of CDKL3 as a protein kinase directly regulating the cell cycle were discovered, such as... Figure 2 Figure 3 As shown. Therefore, small molecule inhibitors specifically targeting CDKL3 hold promise for playing an important role in cancer treatment. Based on the current research status, designing and developing a targeted small molecule drug that can effectively target CDKL3 is crucial for tumor therapy. Summary of the Invention

[0004] The purpose of this invention is to synthesize a small molecule inhibitor targeting CDKL3, HZ1 and its derivative HZ1-X, wherein HZ1-X includes HZ1-1, HZ1-2, HZ1-3, etc.; the structures are shown below:

[0005]

[0006]

[0007] The first aspect of the present invention is to provide a small molecule inhibitor HZ1 targeting CDKL3 and its derivative HZ1-X, the general structural formula of which is as follows:

[0008]

[0009] Or its pharmaceutically acceptable salt; wherein R 1 R 2 Both "Ring" and "Ring" are derived from the following structures, but are not limited to one of them:

[0010] R 1 Choose from one of the following structures:

[0011]

[0012] R 2 Choose from one of the following structures:

[0013]

[0014] Ring is selected from one of the following structures:

[0015]

[0016] The second aspect of this invention provides a method for preparing the small molecule inhibitor HZ1 targeting CDKL3 and its derivative HZ1-X, the preparation route of which is as follows:

[0017]

[0018] Where X 1 X 2 The leaving groups are selected from any one of Cl, Br, I, OMs and OTs.

[0019] First, compound 1 and N,N-diisopropylethylamine were added to an anhydrous dimethyl sulfoxide solution of compound 2, and the reaction mixture was heated at 60°C for 24 hours. After the reaction was complete, the reaction solution was concentrated, and the concentrate was separated by column chromatography using dichloromethane-methanol as the eluent to obtain compound 3.

[0020] Compound 4 and concentrated hydrochloric acid were added to a n-butanol solution of compound 3. The reaction solution was heated at 100°C for 24 hours. After the reaction was complete, the reaction solution was concentrated. The residue was purified by column chromatography using dichloromethane-methanol as the eluent to obtain HZ1 and its derivative HZ1-X.

[0021] In a third aspect, the invention provides the use of HZ1, a small molecule inhibitor targeting CDKL3, and its derivative HZ1-X in the preparation of a treatment for tumors.

[0022] A fourth aspect of the present invention provides a pharmaceutical composition comprising the small molecule inhibitor HZ1 targeting CDKL3 and its derivative HZ1-X or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0023] A fifth aspect of the invention also provides the use of the above-described pharmaceutical composition in the treatment of tumors.

[0024] The beneficial effects of this invention are:

[0025] This invention discloses a small molecule inhibitor HZ1 and its derivative HZ1-X that can specifically target CDKL3. The preparation process is simple and easy. This compound can inhibit the growth of various tumor cells and has a wider range of applications compared to similar inhibitors. 50 Lower; of great significance for the treatment of tumors. Attached Figure Description

[0026] Figure 1 The clinical relevance of CDKL3 to colorectal cancer;

[0027] A. Analysis of multiple transcriptome databases showed that the expression of CDKL3 protein, targeted by the small molecule targeted drug in this invention, was significantly higher in colon cancer tissues than in normal adjacent non-cancerous tissues;

[0028] High expression of B.CDKL3 is significantly positively correlated with poor prognosis in colorectal cancer patients.

[0029] Figure 2 CDKL3 promotes tumor cell cycle progression;

[0030] A. Flow cytometry results of U2OS cells stained with BrdU-FITC / propidium iodide (PI) showed that after CDKL3 knockout, the cell cycle was significantly arrested at the G0 / G1 phase.

[0031] B, C. Statistical analysis results of Figure A;

[0032] D. Flow cytometry was used to verify the significant cell cycle promoting effect of CDKL3 in DLD-1 cells;

[0033] E. Statistical analysis results of Figure D;

[0034] F. Flow cytometry was used to verify the significant cell cycle promoting effect of CDKL3 in HeLa cells;

[0035] G. Statistical analysis results of Figure F;

[0036] H, I. Immunofluorescence results of BrdU in U2OS cells under conventional serum culture (H) or serum starvation treatment (I) showed that after CDKL3 knockout, the number of BrdU-positive cells was significantly reduced and the cell cycle was significantly inhibited.

[0037] Statistical analysis results of J, K, H, I;

[0038] Immunoblotting results of various cell cycle-related proteins after serum starvation release from L.U2OS cells showed a decrease and delay in changes in pRb and Cyclin D1, indicating a significant inhibition of cell cycle progression.

[0039] MO.MTT cell growth assays showed that the growth of U2OS(M), DLD-1(N), and HeLa(O) cells was significantly inhibited after CDKL3 knockout;

[0040] Statistical analysis of tumor colony numbers in P, Q.U2OS(P), and DLD-1(Q) cell lines under three-dimensional culture conditions showed that colony formation was significantly inhibited after CDKL3 knockout.

[0041] Figure 3 CDKL3 promotes cell cycle progression by directly phosphorylating the cell cycle repressor protein Rb;

[0042] A. Protein immunoprecipitation assays showed that endogenous CDKL3 interacts with endogenous Cyclin A2, B1, D1, E1, and Rb.

[0043] B. In vitro kinase assays showed that CDKL3 directly phosphorylates Rb in the presence of cyclins A2 and E1;

[0044] C. In vitro kinase assays showed that the phosphorylation intensity of CDKL3 on Rb was comparable to that of CDK4 / 6.

[0045] Figure 4 In vitro characterization of HZ1;

[0046] A. Surface plasmon resonance (SPR) technology demonstrates that HZ1 has a strong affinity for CDKL3;

[0047] B. In vitro kinase assays showed that HZ1 can effectively inhibit CDKL3 kinase activity.

[0048] Figure 5 Detection of the killing effect of HZ1 and its derivative HZ1-X on tumor cells;

[0049] Validating the IC50 of HZ1 in DLD-1 (A, B), U2OS (C, D), and HeLa (E, F) cell lines. 50Values ​​(24 hours and 72 hours), and the IC of HZ1 50 The value was lower than the IC50 of Palbociclib (a small molecule CDK4 / 6 inhibitor), a drug used to treat ER+ breast cancer. 50 value;

[0050] G. Validation of the IC50 values ​​of HZ1-1, HZ1-2, and HZ1-3 in the U2OS cell line. 50 Value (72 hours).

[0051] Figure 6 HZ1 directly inhibits cell cycle progression in tumor cells;

[0052] A. Flow cytometry analysis confirmed that HZ1 caused cell cycle arrest in the G0 / G1 phase of the DLD-1 cell line, thus hindering cell cycle progression.

[0053] B. Statistical analysis results of Figure A;

[0054] C. Immunoblotting verification showed that HZ1 could reduce the level of phosphorylated Rb in the DLD-1 cell line;

[0055] D. Flow cytometry analysis confirmed that HZ1 caused cell cycle arrest in the G0 / G1 phase of the U2OS cell line, thus hindering cell cycle progression.

[0056] E. Statistical analysis results of Figure D;

[0057] F. Immunoblotting verification: HZ1 was found to reduce the level of phosphorylated Rb in the U2OS cell line. Detailed Implementation

[0058] The following examples further illustrate the solutions of this invention, which are not intended to limit the invention. Unless otherwise specified, the experimental operations in the examples all employ mature existing techniques in the fields of molecular biology or chemistry, and the raw materials and reagents used are all commercially available.

[0059] Example 1

[0060] The verification of the effect and mechanism of the small molecule targeted drug on tumor growth by CDKL3 is specifically included in this invention:

[0061] Experimental Example 1

[0062] First, we analyzed multiple transcriptome databases (TCGA-COAD RNA-seq database, TNMplot colorectal cancer RNA-seq database, TNMplot colorectal cancer gene chip database) to verify the expression level of CDKL3 in colorectal cancer tissues and normal adjacent tissues, and verified the relationship between CDKL3 and poor prognosis of colorectal cancer patients in the Kaplan Meyer-plotter database to explore the clinical relevance of CDKL3.

[0063] Experimental Example 2

[0064] Following the analysis in Experiment 1 above, specific experiments were conducted to verify the effect of CDKL3 on tumor growth.

[0065] Step 1: Using CRISPR-Cas9 technology, stable CDKL3 knockout cell lines were constructed in U2OS, DLD-1, and HeLa cell lines, as well as stable CDKL3 knockout and complementation cell lines and CDKL3 overexpression cell lines were constructed in U2OS cell line and in U2OS and DLD-1 cell lines.

[0066] First, a target plasmid for knocking out the CDKL3 gene was constructed using lentiCRISPRv2 as a vector.

[0067] (1) In order to clone the target sequence for CDKL3 knockout into the lentiCRISPRv2 vector, primer sequences targeting the CDKL3 gene were first synthesized. After annealing, the primers had the same oligonucleotides as the vector plasmid lentiCRISPRv2 after BsmBI digestion, and could be used to clone into lentiCRISPRv2. The required primers are shown in Table 1 below.

[0068] (2) The CDKL3-targeting sequence was inserted into the lentiCRISPRv2 plasmid vector using a vector digestion-ligation method (using BsmBI restriction endonuclease) to obtain the target plasmid. This target plasmid was then transformed into an *E. coli* DH5α plasmid expression strain using CaCl2 heat-mediated transformation. Because this plasmid carries an ampicillin resistance gene, the DH5α plasmid expression strain was cultured on a medium containing ampicillin resistance, and the correct plasmid was confirmed by Sanger sequencing. The plasmid was thus successfully constructed.

[0069] After the target plasmid required to knock out CDKL3 is successfully constructed, lentivirus packaging is performed to prepare stable cell lines.

[0070] (1) When the HEK293T cells in the culture dish grow to 70%-80%, use the transfection reagent Neofect to transfect the plasmid elements required for packaging lentivirus along with the CDKL3 knockout plasmid.

[0071] (2) Replace with fresh culture medium 17 hours after transfection. Collect the cell culture medium containing the lentivirus at 48 hours and 72 hours respectively. Centrifuge the cell culture medium containing the lentivirus and filter it with a 0.22 μm filter membrane to remove cell residue.

[0072] (3) U2OS, DLD-1, and HeLa cells were cultured in DMEM (+10% FBS + 1% penicillin / streptomycin) medium containing fetal bovine serum (FBS). When the cells in the culture dish reached 60%-70% confluence, the medium was replaced with the filtered medium containing lentivirus, and a final concentration of 10 μg / mL was added. -1 Polybrene is used to infect cells.

[0073] (4) Use 1 μg·mL after 48 hours. -1 Cells that successfully invade the target plasmid are resistant to puromycin and will be screened out when puromycin is used to screen for uninvaded wild-type cells.

[0074] (5) Cells that survived 48 hours after puromycin screening were passaged and cultured, and some cells were lysed. Western blotting was used to verify the successful construction of a stable cell line.

[0075] A CDKL3 expression plasmid was constructed for reintroduction into U2OS, DLD-1 cell lines overexpressing CDKL3, and U2OS cell lines with CDKL3 knocked out, in order to conduct further experiments.

[0076] (1) First, the cDNA sequence of CDKL3 was retrieved from the NCBI database. Using the cDNA library of HEK293T cell line as a template, the cDNA fragment of CDKL3 was obtained by PCR technology. The required primers are shown in Table 1 below.

[0077] (2) Enzyme digestion-ligation (using Xba I and BamHI restriction endonucleases) to insert the CDKL3 cDNA sequence into a self-constructed plasmid, thereby obtaining a CDKL3 expression plasmid containing the CDKL3 cDNA sequence. The CDKL3 expression plasmid was transformed into E. coli DH5α plasmid expression strain using CaCl2 heat-mediated transformation. The CDKL3 expression plasmid carries the ampicillin resistance gene. The transformed DH5α plasmid expression strain was cultured on LB + ampicillin medium, and the correct CDKL3 plasmid carrying the CDKL3 cDNA sequence was obtained by Sanger sequencing.

[0078] (3) After the CDKL3 plasmid was successfully constructed, it was packaged into lentivirus and used to infect different cell lines.

[0079] (4) Use 10 μg·mL after 48 hours. -1 Cells that successfully invaded the target plasmid were screened for budding agents and were resistant to budding agents, thus surviving when screened using budding agents.

[0080] (5) Cells that survived 48 hours after screening with blastomycin were passaged and cultured, and some cells were lysed. Western blotting was used to verify that the stable cell line was successfully constructed.

[0081] The primer set numbers and DNA sequences used in this embodiment are shown in Table 1. The DNA sequences and restriction enzyme sites of the primers used are 5'-3' from left to right.

[0082] Table 1 Primer group numbers and DNA sequences

[0083]

[0084] Step 2: The U2OS, DLD-1 and HeLa cell lines with CDKL3 knocked out in Step 1, as well as the cell lines replenished after CDKL3 knockout in U2OS, were used for flow cytometry analysis to verify the function of CDKL3 in tumor cells.

[0085] (1) Wild-type U2OS, DLD-1 and HeLa cell lines, CDKL3 knockout U2OS, DLD-1 and HeLa cell lines, and U2OS cell lines replenished after CDKL3 knockout were cultured in DMEM (+10% FBS+1% penicilin / streptomycin) and DMEM (+1% penicilin / streptomycin) medium, respectively.

[0086] (2) When the various cells in the culture dish have grown to 60%-70%, add 50μM BrdU to the culture medium and incubate for 1 hour.

[0087] (3) After incubation, wash with PBS buffer and fix with 70% ethanol for 30 minutes at 4°C.

[0088] (4) After fixation, wash the cells twice with cold PBS, then let them stand for 30 minutes in PBS containing 1.5M HCl and 0.5% Triton X-100 at room temperature, and then wash the cells twice again with cold PBS.

[0089] (5) Resuspend the cells in 0.1M NaB4O7 (pH 8.5) for 2 minutes and then wash with cold PBS.

[0090] (6) Immunolabel the cells with anti-BrdU mAb for 1 hour. After washing the cells with cold PBS, incubate them with the fluorescent secondary antibody for 30 minutes.

[0091] (7) Finally, use 200 μg·mL -1 RNase A and 20 mg / mL -1 Cells were treated with propidium iodide (PI) at 37°C for 45 minutes. Cell cycle analysis was performed using flow cytometry to verify the effect of CDKL3 on the cell cycle in tumor cells.

[0092] Step 3: The U2OS CDKL3 knockout cell line constructed in Step 1, as well as the cell line replenished after U2OS CDKL3 knockout, were used for immunofluorescence detection to verify the function of CDKL3 in tumor cells.

[0093] (1) Wild-type U2OS cell lines, CDKL3 knockout U2OS cell lines, and U2OS cell lines replenished after CDKL3 knockout were cultured in DMEM (+10% FBS+1% penicilin / streptomycin) and DMEM (+1% penicilin / streptomycin) medium, respectively.

[0094] (2) When the cells grow to 60%-70%, add 10 μM BrdU to the culture medium and incubate for 1 hour.

[0095] (3) After incubation, wash the cells twice with PBS and fix them with 4% paraformaldehyde at 4°C for 15 minutes. After washing the cells again with PBS, let them stand for 20 minutes at room temperature in PBS containing 1M HCl and 0.2% Triton X-100. Resuspend the cells in 0.1M NaB4O7 (pH 8.5) and let them stand for 30 minutes.

[0096] (4) Wash the cells twice with cold PBS before incubating with the antibody. Immunolabel the cells with anti-BrdU mAb for 1 hour.

[0097] (5) After washing the cells, incubate them with the fluorescent secondary antibody for 30 minutes. Then, capture fluorescence images using a fluorescence microscope. Observe the number of BrdU-positive cells to verify the function of CDKL3 in tumor cells.

[0098] Step 4: The U2OS CDKL3 knockout cell line constructed in Step 1, and the cell line replenished after U2OS CDKL3 knockout, were used for cell synchronization Western Blot detection to verify the function of CDKL3 in tumor cells.

[0099] (1) Wild-type U2OS cell lines, CDKL3 knockout U2OS cell lines, and U2OS cell lines replenished after CDKL3 knockout were cultured in DMEM (+10% FBS+1% penicilin / streptomycin) medium.

[0100] (2) When the cells grow to 40%-50%, gently rinse the cells three times with PBS to remove the FBS components on the cell surface, and replace the culture medium with serum-free DMEM (+1% penicilin / streptomycin) medium and incubate for 24 hours.

[0101] (3) After serum starvation treatment, most cells were synchronized to the G0 phase. At different time points, the cells were replaced with DMEM (+10% FBS+1% penicilin / streptomycin) medium containing serum to release serum. Western blotting was used to examine phosphorylated Rb (pRb), Rb, cyclin A2, cyclin D1, and cyclin E1 proteins to verify the function of CDKL3 in tumor cells.

[0102] Step 5: The U2OS, DLD-1 and HeLa cell lines with CDKL3 knocked out constructed in Step 1, as well as the cell line replenished after CDKL3 knockout in U2OS, were used for MTT cell growth assay to verify the effect of CDKL3 on cell growth in tumor cells.

[0103] (1) Wild-type U2OS, DLD-1 and HeLa cell lines, CDKL3 knockout U2OS, DLD-1 and HeLa cell lines, and U2OS cell lines replenished after CDKL3 knockout were seeded in 96-well plates at a density of 1500 cells per well, in triplicate, and cultured in DMEM (+10% FBS+1% penicilin / streptomycin) medium for 1-8 days respectively.

[0104] (2) On days 2, 4, 6 and 8, the final concentration was increased by adding 0.5 mg·mL⁻¹. -1 Thiazole blue tetrazolium (MTT), incubated at 37°C for 4 hours;

[0105] (3) Remove MTT, add 100 μL DMSO to each well, and incubate for 10 minutes. Measure the effect of CDKL3 on tumor cell growth using an OD 490 microplate reader.

[0106] Step 6: Using the U2OS, DLD-1 cell lines with CDKL3 knocked out and the U2OS, DLD-1 cell lines with CDKL3 overexpression constructed in Step 1, cell colony formation assays were performed to verify the effect of CDKL3 on three-dimensional colony formation in tumor cells.

[0107] (1) Wild-type U2OS and DLD-1 cell lines, CDKL3 knocked-out U2OS and DLD-1 cell lines, and U2OS and DLD-1 cell lines overexpressing CDKL3 were seeded in soft agar in 6-well plates at a density of 1500 cells per well and cultured for 14 days to form colonies.

[0108] (2) After the clones have grown to a certain size and number, the wells are washed with PBS and fixed with 4% paraformaldehyde at 37°C for 20 minutes.

[0109] (3) The cells were stained with 0.5% crystal violet at room temperature for 30 minutes and gently washed several times with ddH2O to remove the background. After the background was removed, the cells were photographed with a digital camera to observe the effect of CDKL3 on the formation of three-dimensional colonies in tumor cells.

[0110] Experiment 3

[0111] After verification by Experiment 2, CDKL3 affects the cell cycle progression of tumor cells, thereby affecting the occurrence and development of tumors. Therefore, specific experiments will be conducted to explore the mechanism by which CDKL3 affects tumor growth.

[0112] Step 1: Using the U2OS cell line, an immunoprecipitation assay was conducted to verify the proteins that interact with CDKL3 in tumor cells.

[0113] (1) Wild-type U2OS cell lines were cultured in DMEM (+10% FBS+1% penicilin / streptomycin) medium. When the cells grew to 80%-90%, the cells were gently washed with PBS and then completely lysed with Passive Lysis Buffer (PLB).

[0114] (2) The total cell lysate was incubated with protein G-agarose resin (after incubation with IgG antibody or CDKL3 antibody) at 4°C overnight.

[0115] (3) The next day, the resin was thoroughly washed 5 times with PLB, and after each incubation at 4°C for 10 minutes with shaking, it was resuspended in SDS loading buffer and boiled at 95°C for 5 minutes for immunoblotting to verify the cell cycle protein that interacts with CDKL3.

[0116] Step 2: Verify, through in vitro kinase experiments, whether the Rb protein that interacts with CDKL3, discovered in Step 1, is a substrate of the kinase CDKL3.

[0117] (1) First, the cDNA sequence of Rb was retrieved from the NCBI database. Due to the large protein content of Rb, bacterial expression of the protein is prone to misfolding. Therefore, a truncated Rb variant with amino acids 792-928, commonly used in in vitro kinase studies, was constructed for subsequent in vitro kinase experiments. Using a cDNA library from the HEK293T cell line as a template, the Rb 792-928 cDNA fragment was obtained using PCR technology. The required primers are shown in Table 2 below.

[0118] (2) The cDNA sequence of Rb 792-928 was inserted into the PGEX4T-1 plasmid using enzyme digestion-ligation (SalI and NotI restriction endonucleases) to obtain a plasmid capable of expressing the Rb 792-928 protein. The plasmid containing the Rb792-928 cDNA sequence was then transformed into the *E. coli* DH5α plasmid expression strain using CaCl2 heat-mediated transformation. Because this plasmid carries an ampicillin resistance gene, the DH5α plasmid expression strain was cultured on ampicillin-resistant culture dishes. Sanger sequencing confirmed the correct plasmid carrying the Rb 792-928 sequence. Thus, the plasmid for expressing Rb 792-928 in bacteria was successfully constructed.

[0119] The primer set numbers and DNA sequences used in this embodiment are shown in Table 1. The DNA sequences and restriction enzyme sites of the primers used are 5'-3' from left to right.

[0120] Table 2 Primer group numbers and DNA sequences

[0121]

[0122] (3) After transforming the Rb 792-928 plasmid into E. coli BL21 competent cells, when the OD value was 0.4-0.6, IPTG was added and expression was induced at 18℃ for 6-10 hours. Then, the bacterial culture was collected by centrifugation at 3000 rpm, and the bacteria were resuspended in 20 mL of lysis buffer (1 mM PMSF, 50 mM Tris, 300 mM NaCl, 10% glycerol). The cells were then sonicated at 50% power for 20 minutes (5 seconds each time, with a 5-second interval). After sonication, the supernatant was collected, and an appropriate amount of GST resin was added to the supernatant. The cells were enriched at 4℃ for 2-3 hours. Then, the GST binding beads were washed 3 times with TBS, and finally, the volume was adjusted with 1 mL PBS. This was used as a substrate for in vitro kinases in subsequent experiments.

[0123] (4) Using the same method as the CDKL3 expression plasmid, CDK4, CDK6, cyclin A2, B1, D1 and E1 expression plasmids were constructed. The constructed plasmids were transfected into HEK293T cells. After 48 hours, the cells were starved for 8 hours in DMEM medium (DMEM + 1% penicilin / streptomycin) without FBS.

[0124] (5) Cells were lysed, and an appropriate amount of resin beads were added to the supernatant to enrich the expressed proteins. Next, the corresponding short peptides (500 μg / mL) were added... -1 Add it to the solution for protein elution.

[0125] (6) Incubate a 30 μL reaction mixture (0.1 mM ATP, 2 mM DTT, 50 mM HEPES, 0.01 M MgCl2, appropriate volume of substrate and kinase) at 37 °C for 30 minutes, and terminate the reaction by incubating at 95 °C for 5 minutes. Then perform Western blotting on the samples to investigate whether CDKL3 uses Rb as a substrate to regulate the cell cycle, and compare CDKL3 with the classical kinases of Rb, CDK4 / 6.

[0126] The results of several experiments in Example 1 demonstrate the clinical association between CDKL3 and human malignant tumors, as well as the novel molecular mechanism and important pathological significance of CDKL3 as a protein kinase directly regulating the cell cycle. Therefore, small molecule inhibitors specifically targeting CDKL3 hold promise for playing a significant role in cancer treatment.

[0127] Example 2: Synthesis of 1-(4-((4-((3-cyclopentyl-1H-pyrazol-5-yl)amino)pyrimidine-(2-yl)amino)phenyl)glycolic acid (HZ1)

[0128]

[0129] Step 1:

[0130] First, 2,4-dichloropyrimidine (447 mg, 3.0 mmol, 1.0 eq) was dissolved in 10 mL of anhydrous DMSO. Then, 3-cyclopentyl-1H-pyrazole-5-triethanolamine (500 mg, 3.3 mmol, 1.1 eq) and N,N-diisopropylethylamine (0.8 mL, 4.5 mmol, 1.5 eq) were added to the solution, and the mixture was heated at 60 °C for 24 hours. After the reaction was complete, the reaction solution was concentrated, and the concentrate was separated by column chromatography using dichloromethane-methanol as the eluent to obtain 650 mg of 2-chloro-N-(3-cyclopentyl-1H-pyrazole-5-yl)pyrimidine-4-amine, with a yield of 82%. 1H NMR (400MHz, DMSO-d6): δ12.17(s,1H),10.27(s,1H),8.15(s,1H),3.01(m,1H),1.99(m,2H),1.51-1.72(m,6H).

[0131] Step 2:

[0132] 1-(4-aminophenyl)ethylenedione (52 mg, 0.38 mmol, 1.0 eq) and concentrated hydrochloric acid (0.05 mL) were added to a solution of 2-chloro-N-(3-cyclopentyl-1H-pyrazol-5-yl)pyrimidin-4-amine (100 mg, 0.38 mmol, 1.0 eq) in n-butanol (4 mL). The solution was heated at 100 °C for 24 hours. After the reaction was complete, the reaction solution was concentrated, and the concentrate was separated by column chromatography using dichloromethane-methanol as the eluent to obtain HZ1, 95 mg, in 70% yield. 1 HNMR (400MHz, CD3OD): δ8.01 (br, 1H), 7.95 (dd, J = 8Hz, 2H), 7.83 (dd, J = 8Hz, 2H), 6. 44(br,1H),6.28(br,1H),3.10(m,1H),2.56(s,3H),2.09(m,2H),1.62-1.80(m,6H). ESI-MS:[MH] - 361.4. The inhibitor HZ1 was dissolved in DMSO as a stock solution for subsequent experiments.

[0133] Example 3: Synthesis of 4-((4-((3-cyclopentyl-1H-pyrazol-5-yl)amino)pyrimidin-2-yl)aminobenzonitrile (HZ1-1)

[0134]

[0135] 4-Aminobenzonitrile (45 mg, 0.38 mmol, 1.0 eq) and concentrated hydrochloric acid (0.05 mL) were added to a solution of 2-chloro-N-(3-cyclopentyl-1H-pyrazol-5-yl)pyrimidin-4-amine (100 mg, 0.38 mmol, 1.0 eq) in n-butanol (4 mL). The solution was heated at 100 °C for 24 hours. After the reaction was complete, the reaction solution was concentrated, and the concentrate was separated by column chromatography using dichloromethane-methanol as the eluent to obtain HZ 245 mg, with a yield of 35%. 1H NMR (400MHz, CD3OD): δ8.02(br,1H),7.89(dd,J=8Hz,2H),7.59(dd,J=8Hz,2H),6.46(br,1H),6.22(br,1H),3.10(m,1H),2.10(m,2H),1.62-1.81(m,6H). ESI-MS:[MH] - 344.4. The inhibitor HZ1-1 was dissolved in DMSO as a stock solution for subsequent experiments.

[0136] Example 4: Synthesis of 2-(4-((4-((3-cyclopentyl-1H-pyrazol-5-yl)amino)pyrimidine-(2-yl)amino)phenyl)ethylene glycol (HZ1-2)

[0137]

[0138] 2-(4-aminophenyl)ethylene glycol (50 mg, 0.38 mmol, 1.0 eq) and concentrated hydrochloric acid (0.05 mL) were added to a solution of 2-chloro-N-(3-cyclopentyl-1H-pyrazol-5-yl)pyrimidin-4-amine (100 mg, 0.38 mmol, 1.0 eq) in n-butanol (4 mL). The solution was heated at 100 °C for 24 hours. After the reaction was complete, the reaction solution was concentrated, and the concentrate was separated by column chromatography using dichloromethane-methanol as the eluent to obtain HZ3 110 mg, with a yield of 80%. 1 H NMR (400MHz, CD3OD): δ11.94(s,1H),9.46(s,1H),8.93(s,1H),7.94(s,1H),7.60(dd,J=8Hz,2H),7.08(dd,J=8Hz,2H),6.42(b r,1H),6.34(br,1H),4.60(t,J=8Hz,1H),3.59(q,J=8Hz,2H),3.01(m,1H),2.66(t,J=8Hz,2H),1.99(m,2H),1.61-1.72(m,6H). ESI-MS:[MH] - 363.4. The inhibitor HZ1-2 was dissolved in DMSO as a stock solution for subsequent experiments.

[0139] Example 5: N 2 -(4-(2-aminoethyl)phenyl)-N 4 Synthesis of -(3-cyclopentyl-1H-pyrazol-5-yl)pyrimidine-2,4-diamine (HZ1-3)

[0140]

[0141] (4-Aminophenethyl)carbamate tert-butyl ester (90 mg, 0.38 mmol, 1.0 eq) and concentrated hydrochloric acid (0.05 mL) were added to a solution of 2-chloro-N-(3-cyclopentyl-1H-pyrazol-5-yl)pyrimidin-4-amine (100 mg, 0.38 mmol, 1.0 eq) in n-butanol (4 mL). The solution was heated at 100 °C for 24 hours. After the reaction was complete, the reaction solution was concentrated, and the concentrate was separated by column chromatography using dichloromethane-methanol as the eluent to obtain HZ 580 mg, with a yield of 58%. 1 HNMR (400MHz, CD3OD): δ11.95(s,1H),9.48(s,1H),8.95(s,1H),7.96(d,J=4Hz,1H),7.63(dd,J=8Hz,2H),7.08(d d,J=8Hz,2H),6.38(br,2H),2.99(m,1H),2.74(t,J=8Hz,2H),2.57(t,J=8Hz,2H),1.99(m,2H),1.60-1.72(m,6H). ESI-MS:[MH] - 362.4. The inhibitor HZ1-3 was dissolved in DMSO as a stock solution for subsequent experiments.

[0142] Example 6: Test of CDKL3 inhibitory activity and antitumor activity of inhibitor HZ1

[0143] Step 1: Verify the affinity between HZ1 and CDKL3 using surface plasmon resonance (SPR) technology. The specific steps are as follows:

[0144] (1) First, the cDNA sequence of CDKL3 was retrieved from the NCBI database. Using the cDNA library of HEK293T cell line as a template, the cDNA fragment of CDKL3 was obtained by PCR technology. The required primers are shown in Table 3 below.

[0145] (2) The CDKL3 cDNA sequence was inserted into the PGEX4T-1 plasmid by enzyme digestion-ligation (using SalI and NotI restriction endonucleases) to obtain a plasmid that can express CDKL3 protein.

[0146] The primer set numbers and DNA sequences used in this embodiment are shown in Table 3. The DNA sequences and restriction enzyme sites of the primers used are from 5' to 3' from left to right.

[0147] Table 3 Primer group numbers and DNA sequences

[0148]

[0149] (3) After transforming CDKL3 plasmid into E. coli BL21 competent cells, when the OD value is 0.4-0.6, IPTG is added and expression is induced at 18℃ for 6-10 hours. Then, the bacterial culture is collected by centrifugation at 3000 rpm, and the bacteria are resuspended in 20 mL of lysis buffer (1 mM PMSF, 50 mM Tris, 300 mM NaCl, 10% glycerol). The cells are then sonicated at 50% power for 20 minutes (5 seconds each time, 5 seconds apart) on a SCIENTZ JY92-IIN sonicator. After sonication, the supernatant is collected, and an appropriate amount of GST resin is added to the supernatant. The cells are enriched at 4℃ for 2-3 hours. Then, the GST-binding beads are washed 3 times with TBS, and finally, the cells are treated with a solution containing 3 mg·mL⁻¹ GST. -1 The protein was eluted with reduced glutathione elution buffer, purified by dialysis, and finally stored in ddH2O for subsequent experiments.

[0150] (4) Surface plasmon resonance (SPR) detection was performed using a Biacore T200 instrument. In the SPR experiment, purified CDKL3 was coupled to the CM5 chip via a standard amine coupling procedure in 10 mM sodium acetate (pH 4.5). After dilution, the compound was injected into the sensor chip at a flow rate of 15 μL / min for 90 seconds (contact phase), followed by a 180-second buffer flow (dissociation phase). KD values ​​were obtained using Biacore T200 evaluation software Version 1.0 (Cytiva) and steady-state analysis of equilibrium data.

[0151] After verifying the affinity between HZ1 and CDKL3, we further verified through in vitro kinase assays whether it could inhibit CDKL3 kinase activity and thus inhibit phosphorylated Rb to exert its function.

[0152] Step two involves using DLD-1, U2OS, and HeLa cell lines to verify the function of HZ1 in inhibiting tumor cell growth. The specific steps are as follows:

[0153] (1) 1500 DLD-1, U2OS, and HeLa cells per well were seeded in 96-well cell culture plates and cultured in DMEM (+10% FBS+1% penicilin / streptomycin) medium. The medium was changed the next day. HZ1 at concentrations ranging from 0-200 μM was added to different wells of different cell lines as the experimental group, and a CDK4 / 6 inhibitor (Palbocilib) at concentrations ranging from 0-30 μM was added as the control group. Three parallel controls were set up for each group. This was to verify the tumor growth inhibitory ability of HZ1 at the cellular level. U2OS cell lines were also used, with HZ1-1, HZ1-2, and HZ1-3 added at concentrations ranging from 0-200 μM in the same manner to verify the tumor growth inhibitory abilities of HZ1-1, HZ1-2, and HZ1-3.

[0154] (2) 24 and 72 hours after drug administration, add 10 μL of thiazolyl blue tetrazolium (MTT) to each well at a dose of 0.5 mg / mL. After incubation at 37°C for 4 hours, remove the MTT and add 100 μL of LDMSO to each well for 10 minutes. Measure the OD490 of each 96-well plate using a microplate reader, plot the growth curve based on the OD490 value, and calculate the IC50. 50 value.

[0155] Step 3 involves further verifying the inhibitory effect of HZ1 on tumor cells using Western blotting and flow cytometry. The specific steps are as follows:

[0156] (1) Flow cytometry was used to verify the inhibitory effect of HZ1 on tumor cell growth.

[0157] a. DLD-1 and U2OS cells were cultured in DMEM (+10% FBS+1% penicilin / streptomycin) medium. After the cells adhered, the small molecule inhibitor HZ1 (100 nM) was added and cultured for 12 hours. The control group was added with an equal amount of DMSO. When the cells in the culture dish grew to 70%-80%, they were incubated in 50 μM BrdU medium for 1 hour. Then they were fixed in 70% ethanol and kept at 4°C for at least 30 minutes.

[0158] b. Wash cells twice with cold PBS, then treat with PBS solution of 1.5M HCl and 0.5% Triton X-100 at room temperature for 30 minutes. Wash cells twice more with cold PBS, then resuspend cells in 0.1M NaB4O7 (pH 8.5) and let stand for 2 minutes. After washing with cold PBS, perform BrdU immunolabeling with anti-BrdU monoclonal antibody for 1 hour. Wash cells twice with cold PBS, then bind with fluorescent secondary antibody for 30 minutes.

[0159] c. Finally, use 200 μg·mL-1 RNase A and 20 mg / mL -1 Cells were treated with propidium iodide (PI) in PBS at 37°C for 45 minutes. Cell cycle analysis was performed using flow cytometry.

[0160] (2) Verification of the inhibitory effect of HZ1 on tumor cells by Western blotting

[0161] a. DLD-1 and U2OS cells were cultured in DMEM (+10% FBS+1% penicilin / streptomycin) medium. When the cells in the culture dish reached 50%-70% confluence, the serum was removed from the medium, and 100 nM HZ1 was added. An equal amount of DMSO was added to the control group cells. After 12 hours of treatment, serum was released over 10 hours in a time gradient.

[0162] b. Lyse the cells and collect the cell lysate for Western blotting.

[0163] Verification results of HZ1 and its derivatives prepared by the method of this invention:

[0164] (1) Functions of CDKL3 targeted by small molecule drugs in tumor cells

[0165] like Figure 1 As shown, analysis of multiple databases revealed that CDKL3 is highly expressed in colorectal cancer tumors and is associated with poor prognosis in these patients. CDKL3 participates in cell cycle regulation in tumor cells, such as... Figure 2 The experimental results show that CDKL3 affects the growth process of tumor cells by influencing the cell cycle. Tumor cells with CDKL3 knocked out exhibited cell cycle arrest, slowed growth, and difficulty in colony formation. CDKL3 plays a crucial role in the occurrence and development of tumors.

[0166] CDKL3 interacts with various cell cycle proteins, such as Figure 3 As shown, CDKL3, with the assistance of cyclin A and cyclin E, can exert its kinase activity by directly phosphorylating Rb as a substrate, thereby directly regulating the cell cycle. Furthermore, its ability to phosphorylate Rb is comparable to that of CDK4 / 6.

[0167] (2) Effects of HZ1 on tumor cells

[0168] The binding affinity between HZ1 and CDKL3 proteins was quantitatively measured using surface plasmon resonance.

[0169] The results showed that HZ1 and CDKL3 have a strong binding interaction. HZ1 and its derivative HZ1-X both exhibited good inhibitory effects on CDKL3 enzyme activity in in vitro kinase system experiments. Figure 4 As shown.

[0170] HZ1 and its derivative HZ1-X can effectively inhibit the growth of various tumor cells, and their IC50 can... 50 Values ​​that are parallel to or lower than those of Palbociclib (a small molecule CDK4 / 6 inhibitor), a clinically used drug for treating ER+ breast cancer by inhibiting the cell cycle, such as... Figure 5 As shown; IC 50 The experimental results show that the small molecule HZ1 targeting CDKL3 has the ability to inhibit tumor cell growth comparable to CDK4 / 6 inhibitors currently used in clinical practice, and can even inhibit the growth of a variety of tumor cells at lower concentrations.

[0171] Treatment with HZ1 and its derivative HZ1-X significantly inhibited the tumor cell cycle, resulting in a significant increase in cells in the G0 / G1 phase, and a significant decrease in phosphorylated Rb protein after treatment. Figure 6 As shown, the addition of HZ1 significantly inhibits the ability of tumor cells to enter the cell cycle. Flow cytometry results also confirm this view, that is, the addition of the HZ1 small molecule inhibitor causes tumor cells to arrest, the cell cycle is blocked, and tumor cell growth is affected.

Claims

1. A small molecule inhibitor compound targeting CDKL3, characterized in that, The structural formula is as follows: 。 2. The use of the small molecule inhibitor compound targeting CDKL3 as described in claim 1 in the preparation of a medicament for treating colorectal adenocarcinoma, osteosarcoma, or cervical cancer.

3. A pharmaceutical composition comprising the small molecule inhibitor compound targeting CDKL3 as claimed in claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

4. Use of the pharmaceutical composition of claim 3 in the preparation of a medicament for treating colorectal adenocarcinoma, osteosarcoma, or cervical cancer.

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