Arginine methyltransferase PRMT1 inhibitor and application thereof

Abstract

The present invention discloses an arginine methyltransferase PRMT1 inhibitor and its application. The present invention has effectiveness and specificity in inhibiting PRMT1, has extensive and good in vivo and in vitro anti-proliferation ability against tumor cells from various sources such as intestinal adenocarcinoma, esophageal cancer, lung adenocarcinoma, prostate cancer, liver cancer, ovarian cancer, etc., has low killing effect on normal cells in vivo, and has good in vivo safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor proliferation, and specifically relates to an arginine methyltransferase PRMT1 inhibitor and application thereof. Background Art

[0002] PRMT1 is the main enzyme in proteins that catalyzes aDMA, and it prefers to methylate arginine on the RGG / RG motif. PRMT1 mainly participates in different biological processes by regulating different methylated substrates, thereby achieving a variety of functions.

[0003] In breast cancer cell lines and breast tumor samples, BRCA1 (Breast cancer type 1 susceptibility protein), a protein closely related to breast cancer, was identified and proved to be methylated by PRMT1 (PLoS One, 2010.5(6): p.e11379.). As a tumor suppressor protein, BRCA1 is involved in a variety of biological processes such as cell cycle regulation, DNA damage repair and chromatin remodeling. PRMT1-mediated arginine methylation on BRCA1 protein can change BRCA1 binding to specific gene promoter elements, thereby leading to cellular genome instability. In addition, PRMT1 can also methylate MRE11 (Meiotic recombination 11) and 53BP1 (TP53-binding protein 1), key proteins in the DNA damage repair pathway. PRMT1-mediated methylation of MRE11 regulates its exonuclease activity on double-stranded DNA, which is necessary for the checkpoint of DNA damage repair (Genes Dev, 2005.19(6): p.671-6.). Similarly, 53BP1, which is involved in the DNA damage repair pathway, can also be methylated by PRMT1. Its methylation has been shown to be necessary for 53BP1 to function normally. Mutating the arginine on the 53BP1 protein that is methylated by PRMT1 reduces its binding to single-stranded or double-stranded DNA, preventing the recruitment of DNA repair mechanisms. Similarly, by treating cells with methyltransferase inhibitors to inhibit 53BP1 methylation and disrupt the localization of 53BP1 to damaged DNA, fewer γH2AX foci can be formed (Cell Cycle, 2005.4(12): p.1834-41.). In summary, dysregulation of PRMT1 activity may lead to the accumulation of DNA damage, which is a recognized cause of tumorigenesis. Therefore, it is also confirmed from the side that PRMT1 is crucial for the occurrence and development of cancer.

[0004] In addition to the function of DNA damage repair, PRMT1 is also involved in the process of cell signal transduction. For example, in estrogen signal transduction, PRMT1-mediated methylation of the arginine residue 260 of the estrogen receptor ERα (Estrogen receptor) promotes the formation of the ERα / Src / PI3K complex, which is a key step in further activating the Akt pathway (Mol Cell, 2008.31(2): p.212-21.)(Steroids, 2010.75(8-9): p.560-4.). It is worth noting that this pathway is abnormally activated in invasive breast tumors, and the high level of expression of members in this complex is a significant marker of poor prognosis and is associated with a decrease in disease-free survival, which also confirms that high levels of methylation of ERα can trigger abnormal cell proliferation (EMBO MolMed, 2012.4(11): p.1200-13.). In addition, PRMT1 has been shown to regulate Akt (RAC-alphaserine / threonine-protein kinase)-mediated signal transduction and induce cell apoptosis through other mechanisms. One example is that by methylating the arginine residues 248 and 252 on FOXO1 (Forkhead box O1), this methylation modification inhibits Akt recognition of FOXO1, thereby preventing its phosphorylation modified by Akt on the adjacent S253 residue (Mol Cell, 2008.32(2): p.221-31.). Ultimately, FOXO1 is retained in the nucleus and protected from proteasome degradation, thereby increasing its transcriptional activity on target genes. Similarly, PRMT1 binds to and methylates the 94th and 96th arginine residues of the cell death antagonist (BAD) domain on the BCL-2 protein, inhibiting the phosphorylation of Akt on the S99 residue, thereby preventing the binding of 14-3-3 protein and leading to cell apoptosis (Proc Natl Acad Sci US A, 2011. 108 (15): p. 6085-90.). PRMT1 also regulates the Wnt signaling pathway by methylating the R378 residue of Axin, which is a negative regulator of the Wnt pathway (Oncogene, 2011. 30 (20): p. 2379-89.). Methylation of Axin enhances its interaction with glycogen synthase kinase 3β, resulting in reduced degradation. Since abnormal regulation of the Wnt pathway is a driving factor for many different cancers, it is shown that abnormal expression of PRMT1 is related to tumorigenesis.

[0005] PRMT1 is highly expressed in breast cancer (Tumour Biol 32(3):575-582.), colon cancer (Br J Cancer 99(12):2094-2099.), lung cancer (Oncotarget 8(46):80278-80285.), and prostate cancer (Oncol Rep 38(2):1115-1123.), and affects the growth, proliferation, and invasion of acute lymphoblastic leukemia (J Hematol Oncol 5:42.), lung cancer (J Biol Chem 290(21):13479-13489.), breast cancer (Breast Cancer 25(1):74-80.), and colon cancer (J Clin Invest 125(12):4529-4543.) cells through methylation.

[0006] From the above, we can see that PRMT1 has a wide range of cancer-promoting functions and differential expression characteristics, and because it is an enzyme protein, it has the characteristics of being active and easy to intervene, making it one of the excellent potential anti-tumor drug targets. Summary of the invention

[0007] The present invention aims to provide an arginine methyltransferase PRMT1 inhibitor.

[0008] Another object of the present invention is to provide the use of the above-mentioned arginine methyltransferase PRMT1 inhibitor.

[0009] The technical solution of the present invention is as follows:

[0010] An arginine methyltransferase PRMT1 inhibitor, the structural formula of which is

[0011]

[0012] In a preferred embodiment of the present invention, its structural formula is

[0013] Use of the above-mentioned arginine methyltransferase PRMT1 inhibitor in the preparation of anti-tumor cell proliferation drugs.

[0014] In a preferred embodiment of the present invention, the tumor cells are from intestinal adenocarcinoma, esophageal cancer, lung adenocarcinoma, prostate cancer, liver cancer and ovarian cancer.

[0015] An anti-tumor cell proliferation composition, whose effective ingredients include the above-mentioned arginine methyltransferase PRMT1 inhibitor.

[0016] In a preferred embodiment of the present invention, the active ingredient is the above-mentioned arginine methyltransferase PRMT1 inhibitor.

[0017] In a preferred embodiment of the present invention, the tumor cells are from intestinal adenocarcinoma, esophageal cancer, lung adenocarcinoma, prostate cancer, liver cancer and ovarian cancer.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention has effectiveness and specificity in inhibiting PRMT1.

[0020] 2. The present invention has extensive and good in vivo and in vitro anti-proliferation ability against tumor cells from various sources such as intestinal adenocarcinoma, esophageal cancer, lung adenocarcinoma, prostate cancer, liver cancer, ovarian cancer, etc., and has low killing effect on normal cells in vivo, and has good in vivo safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the results of the PRMT1 in vitro methyltransferase activity in Example 3 of the present invention.

[0022] Figure 2 This is a diagram showing the inhibitory effect of ZBD-138 in Example 3 of the present invention on the methyltransferase activity of PRMT1-9.

[0023] Figure 3 is the IC of ZBD-138 in Example 4 of the present invention for in vitro inhibition of PRMT1 50 Measurement result diagram.

[0024] Figure 4 This is a graph showing the results of the in vitro binding ability assay of ZBD-138 and PRMT1 in Example 4 of the present invention.

[0025] Figure 5 is the EC of ZBD-138 in Example 5 of the present invention 50 The measurement result diagram.

[0026] Figure 6 This is a diagram showing the results of the nude mouse tumor formation experiment of MCF7 cells in Example 6 of the present invention.

[0027] Figure 7 This is a diagram showing the results of the nude mouse tumor formation experiment of MDA-MB-231 cells in Example 6 of the present invention.

[0028] Figure 8 This is a graph showing the results of the PRMT1 in vitro methyltransferase activity of the tetrahydroindole[2,1-h]pteridine-6-one compounds in Example 7 of the present invention.

[0029] Fig. 91 is a graph showing the cytotoxicity test results of the tetrahydroindole[2,1-h]pteridine-6-one compounds in Example 8 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0031] The arginine methyltransferase PRMT1 inhibitor of the present invention is a tetrahydroindole [2,1-h] pteridine-6-one compound, and its structural formula is shown in the following table:

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] Synthesis method of each tetrahydroindole [2, 1-h] pteridine-6-one compound in the above table WO 2019126696 A1

[0040] Example 1 Synthesis of ZBD138

[0041] The synthetic routes of the arginine methyltransferase PRMT1 inhibitor of the present invention are all carried out with reference to the technical scheme disclosed in WO 2019126696 A1, taking I-4 (ZBD138) as an example, as follows:

[0042] The main raw materials are shown in the following table:

[0043]

[0044]

[0045] The specific steps are as follows:

[0046] (1) Synthesis of Compound 2: Compound 1 (1142.0 mg, 7.00 mmol) was dispersed in methanol (18.0 mL), and then the reaction system was placed in an ice-water bath, and concentrated sulfuric acid (0.6 mL) was added dropwise under stirring. After the addition was completed, the reaction solution was transferred to a 65°C oil bath and stirred overnight. The reaction was monitored by liquid chromatography-mass spectrometry (LC-MS). After the reaction was completed, ethyl acetate was diluted and saturated sodium bicarbonate solution was neutralized. The aqueous phase was further extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phases were filtered and concentrated to obtain a crude compound 2 (1159.0 mg), which was used for the next step without purification. MS (ESI) m / z: 178.1 [M] + .

[0047] (2) Synthesis of compound 4: Compound 3 (1203.7 mg, 6.21 mmol) was dispersed in 12.0 mL of isopropanol, and the reaction system was placed in an ice-water bath. A mixed solution of compound 2 (1000.0 mg, 5.64 mmol) and DIEA (1.679 mL, 10.16 mmol) in isopropanol (15.0 mL) was added dropwise under stirring. The reaction system was naturally heated and stirred at room temperature overnight. The reaction was monitored by LC-MS. After the reaction was completed, ethyl acetate was added to dilute and saturated sodium bicarbonate solution was used to wash. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 4 (1423.0 mg). MS (ESI) m / z: 335.1 [M] + .

[0048] (3) Synthesis of Compound 5: Compound 4 (1150.0 mg, 3.44 mmol) and iron powder (1154.0 mg, 20.61 mmol) were dispersed in glacial acetic acid (34.0 mL), and then the reaction system was placed in a 50°C oil bath and stirred for 16 h. The reaction was monitored by LC-MS. After the reaction was completed, the remaining iron powder was adsorbed by a magnetic rotor, and the reaction system was concentrated to a solid. CH3OH 30.0 mL and H2O 180.0 mL were added for slurrying for 30 min. The solid was filtered and washed with water. After drying, a crude compound 5 (1012.0 mg) was obtained, which was used for the next step without purification. MS (ESI) m / z: 273.1 [M] + .

[0049] (4) Synthesis of ZBD138: The crude product of compound 5 (290.0 mg, 1.06 mmol), compound 6 (163.4 mg, 0.85 mmol), catalyst Pd2(dba)3 (58.6 mg, 0.06 mmol), phosphine ligand X-Phos (45.8 mg, 0.10 mmol) and K2CO3 (366.8 mg, 2.66 mmol) were dispersed in 10.6 mL of 1,4-dioxane, the reaction system was protected by N2, and the reaction was carried out in an oil bath at 110°C for 4 h. The reaction was monitored by LC-MS. After the reaction was completed, ethyl acetate was added to dilute the mixture, and the mixture was washed with saturated brine and water in turn. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain compound ZBD138 (196.8 mg, 0.46 mmol). 1 H-NMR (600MHz, DMSO-d6): δ10.51 (s, 1H), 8.94 (s, 1H), 8.31 (d, J=8.0Hz, 1H), 7.72 (s, 1H), 7.50 (d, J=8. 6Hz, 2H), 7.29 (d, J=7.3Hz, 1H), 7.20 (t, J=7.7Hz, 1H), 7.00 (t, J=7.4Hz, 1H), 6.89 (d, J=9.0Hz, 2H), 4.96 (t, J=10.7Hz, 1H), 4.66 (d, J=4.2Hz, 1H), 3.59 (dp, J=8.8, 4.3Hz, 1H), 3.44 (dd, J=11.3, 5.6Hz, 2H), 3.3 1 (d, J=11.9Hz, 2H), 2.80-2.70 (m, 2H), 1.83 (dd, J=12.5, 4.3Hz, 2H), 1.50 (ddt, J=13.1, 9.4, 5.0Hz, 2H); 13 C-NMR (150MHz, DMSO-d6): δ164.46, 155.76, 148.08, 146.11, 142.65, 140.09, 132.79, 129.65, 127.38, 12 4.93, 122.61, 120.40, 116.35, 115.07, 113.79, 66.11, 60.24, 47.53, 34.06, 30.66; MS (ESI) m / z: 429.2[M] + .

[0050] Example 2 Construction and purification of prokaryotic expression vector of PRMT1

[0051] (1) On the UCSC Genome Browser website, the human gene PRMT1 was searched based on the GRCh38 / hg38 genome. Based on the mRNA transcript of the NCBI reference sequence NM_001536, the full-length sequence encoding the PRMT1 protein (1-371 amino acids) was determined. The DNA sequence encoding the protein is as follows:

[0052]

[0053] (2) The DNA sequence encoding the human PRMT1 protein (1-371 amino acid residues) was specifically amplified from the human cDNA library using PCR. The 5' and 3' ends of the primers have BamH1 and Xhol restriction sites, respectively. The specific PCR primer sequences are as follows:

[0054] F: 5'-cgggatccatggcggcagccgaggccgc-3' (SEQ ID NO.02)

[0055] R: 5'-ccgctcgagtcagcgcatccggtagtcgg-3' (SEQ ID NO.03)

[0056] The specific PCR amplification system and method are as follows:

[0057] PCR system:

[0058] Reagents volume <![CDATA[5×PrimeSTAR Buffer(Mg 2+ plus)(Takara)]]> 5μL dNTP Mixture(Takara) 2μL Primer F (10mM) 0.5μL Primer R (10mM) 0.5μL cDNA 200ng PrimeSTAR HS DNA Polymerase(2.5U / μL)(Takara) 0.5μL

[0059] Add water to a total volume of 25 μL.

[0060] The PCR procedure is as follows:

[0061] 1) 98℃ for 5 min, 2) 98℃ for 10 s, 3) 56℃ for 30 s, 4) 72℃ for 1 min, 2)-4) for 30 cycles, 5) 72℃ for 10 min, 6) stored at 4℃.

[0062] After the PCR program is completed, the PCR products are separated by agarose gel electrophoresis, and the fragment of the PCR product with a size of about 1.2 kb is found under ultraviolet light, the gel is cut, and the recovered DNA fragment is finally eluted with 20 μL of double distilled water for subsequent molecular cloning connection reaction.

[0063] (3) Use T4 ligase to connect the PCR product obtained in step (2) into the double-digested pET-28A vector to obtain the plasmid pET-28A-PRMT1 that can express the full-length His-tagged PRMT1 protein in Escherichia coli. The specific experimental method is as follows:

[0064] A. Enzyme digestion reaction:

[0065]

[0066] After the enzyme digestion reaction was fully completed for 7 hours, run a DNA gel, find the digested vector fragment under ultraviolet light, cut the gel, and recover it. Finally, the recovered DNA fragment was eluted with 20 μL ultrapure water and used as a vector for subsequent ligation reactions.

[0067] B. Ligation reaction:

[0068] Reagents Addition amount 10×Buffer(TAKARA) 1μL PCR product DNA 4μL Digested vector DNA 4μL

[0069] First, measure the concentration of the inserted fragment and the linearized vector after enzyme digestion, add them to the reaction system at a molar ratio of 5:1, and then add 10×T4 DNA ligase buffer and T4 DNA ligase in sequence. Connect at 25℃ for more than 4 hours. After the connection is completed, add the ligation product to an appropriate amount of competent medium for transformation.

[0070] (5) Transform the prokaryotic expression plasmid pET-28A-PRMT1 into BL21Rosetta of Escherichia coli. TM strain (Novagen) in competent cells. His-tagged PRMT1 protein was purified according to HisPur TM Ni-NTA Resin (Thermo) product manual. The specific bacterial transformation and purification methods are as follows:

[0071] 1) Conversion

[0072] ① Transfer 0.5 μL of plasmid pET-28A-PRMT1 into 50 μL Rosetta and place on ice for 30 min.

[0073] ②Heat shock in 42℃ water bath for 60s.

[0074] ③ Rapidly place in ice bath for 5 minutes.

[0075] ④ Streak on a preheated LB solid culture medium plate with kanamycin resistance.

[0076] ⑤ Invert and culture in a 37℃ incubator overnight.

[0077] 2) Induction

[0078] ① Small-scale culture. Add 20 mL of LB liquid medium to a 50 mL centrifuge tube and then add kanamycin with a final concentration of 1 mM. Pick a single clone and transfer it to a 50 mL centrifuge tube. Incubate in a shaker at 220 rpm and 37°C for 8 h.

[0079] ② Expand the culture. Add 1L LB liquid medium to a 2L conical flask and then add kanamycin with a final concentration of 1mM. Transfer 20mL of the bacterial solution to 1L LB liquid medium and continue to culture in a shaker at 220rpm and 37℃ for 3-4h until the OD value reaches 0.7-0.8. Then add IPTG with a final concentration of 1mM and culture in a shaker at 100rpm and 25℃ overnight (12h).

[0080] 3) Purification

[0081] The pET-28A recombinant plasmid containing the target protein fragment was transformed into the Escherichia coli BL21 competent cells. The specific steps are as described above. The transformation product was spread on a kanamycin-resistant plate and inverted for overnight culture. The next day, a single clone was picked and cultured in 5 mL of LB medium containing kanamycin resistance, and cultured in a 37°C constant temperature shaker at 250 rpm for 6-10 hours. After 6-10 hours, 5 mL of bacterial solution was added to 200 mL of LB medium containing kanamycin resistance, and cultured in a 37°C constant temperature shaker at 250 rpm until OD600 reached the range of 0.8-1.0. Add 200 μL 1M IPTG and culture overnight in a 25°C low temperature shaker at 150 rpm.

[0082] The next day, centrifuge at 8000rpm for 10min to collect the bacterial precipitate. Add 10mL of lysis buffer (50mMNaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0) to the precipitate, and add EDTA-free protease inhibitors. Vortex to mix, and let stand on ice for 15min. Add 10mg lysozyme to a final concentration of 1mg / mL, mix thoroughly, and let stand on ice for 30min, inverting and mixing every few minutes. During the lysis period, prepare the Ni-NTA beads to be used. Take 100μL of Ni-NTA bead suspension and centrifuge to remove the storage solution. Rinse with 1mL of lysis buffer 2-3 times and place on ice for use. After the bacterial solution is lysed, further disrupt the bacteria by ultrasound. When ultrasound, use a large probe, 50% power, and break for 10s, stop for 10s as one cycle, and exceed 20 cycles. After the ultrasound, transfer the bacterial lysate to a high-speed centrifuge tube, centrifuge at 12000rpm for 60-90min, discard the precipitate, pour the supernatant into a clean centrifuge tube, and add the previously prepared Ni-NTA beads. Incubate at 4℃ for 2-4h to allow the target protein to fully bind to the Ni-NTA beads. After the incubation, pour the liquid into a separation tube and rinse the beads with 100-200mL wash buffer (50mMNaH2PO4, 300mM NaCl, 250mM imidazole, pH 8.0) to fully wash away the impurities. 4mL of elution buffer was added to the column four times to elute the protein and collect it in a tube with low protein adsorption, i.e. His-PRMT1 protein.

[0083] Example 3 Establishment of a method for quantitative determination of PRMT1 methyltransferase activity in vitro

[0084] Carry out in vitro methyltransferase enzymatic reaction: mix PRMT protein with known histone substrate short peptide, use SAM as methylation donor, and react at 37°C for 1-2h. When the methyltransferase enzymatic reaction is completed, add MTase-GloTM Reagent to convert SAH into ADP, and then add MTase-GloTM Detection Solution to convert ADP into ATP, and then detect ATP through a pair of luciferase reactions. The luminescent signal can be detected by a plate reader with luminescence detection function, and then correlated with the SAH concentration using the SAH standard curve. The half-life of the luminescent signal is greater than 4h. The longer half-life makes it unnecessary to configure the instrument with an injector during detection, and batch detection can be performed. The results are as follows: Figure 1 The horizontal axis in the figure represents the concentration of PRMT1 (ng / μL), and the vertical axis represents the fluorescence detection intensity.

[0085] In this example, PRMT1-9 protein with FLAG tag was purified from prokaryotic cells HEK293, and the inhibitory effect of 10 μM and 100 μM ZBD-138 on PRMT1-9 methyltransferase activity was determined using a short peptide of H4(1-18) (SGRGKGGKGLGKGGAKRH, SEQ ID NO.04) as a substrate. Figure 2 As shown in the figure, the horizontal axis represents the experimental groups with different PRMTs, and the vertical axis represents the inhibition rate ((fluorescence detection intensity of the control group-fluorescence detection intensity of the experimental group) / fluorescence detection intensity of the control group)×100%).

[0086] Example 4 Determination of IC50 value of ZBD138 against PRMT1 enzyme

[0087] In this example, the initial concentration of ZBD138 was dissolved to 10mM and stored at -20°C. 1μL of 10mM ZBD138 was diluted with ultrapure water in a 2-fold gradient, 20 gradients (from 100μM to 10pM), 1μL was added to each reaction in a PCR tube, and 1μL of ultrapure water was added as a control sample. Subsequently, 2μL of His-PRMT1 protein prepared in Example 2 was added and mixed by pipetting. Let stand at room temperature for 15 minutes to allow the small molecule compound to fully bind to the target protein. Subsequently, 1μL of H4(1-18) short peptide, 4μL of the premix of 5× methylase buffer master solution, and 2μL of 10× MTase-Glo TM Reagent and methyl donor SAM were mixed and incubated in a 37°C incubator for 1 h. 20 μL of 2×MTase-Glo was added to the reaction product. TM DetectionSolution, incubate the reaction in a 37°C incubator for 0.5 h. Finally, fluorescence was used to detect the methylation efficiency. The PRMT1 activity data measured after treatment with different concentrations of compounds were imported into Prism7 software, and the compound concentration required to inhibit PRMT1 activity by half, i.e., IC, was determined based on the fitted dose-response curve. 50 The results are shown in Figure 3. The IC value of ZBD-138 compound for PRMT1 methyltransferase 50 The concentration of ZBD-138 in the methyltransferase of PRMT1 was 270 nM, indicating its effective inhibitory effect on the methyltransferase activity of PRMT1. Next, this example uses BIACORE and in vitro thermodynamic migration experiments to demonstrate the binding ability of ZBD-138 to PRMT1. The results are as follows: Figure 4As shown. In the BIACORE experiment, this embodiment couples the separated and purified His-PRMT1 protein to the chip. During the experiment, inhibitors with different concentration gradients are used as mobile phases to flow through the chip coupled with the His-PRMT1 protein. The inhibitor binds to the PRMT1 protein, which can change the angle of the incident light and ultimately determine its binding constant with the protein. The in vitro thermodynamic migration experiment is to react ZBD138 with the PRMT1 protein and then undergo a temperature gradient treatment. If ZBD138 binds to the PRMT1 protein, the thermal stability of the PRMT1 protein is enhanced. Finally, the content of the PRMT1 protein that has not been denatured is detected to determine the binding of ZBD138 to the PRMT1 protein.

[0088] Example 5 Determination of the EC effect of ZBD138 on tumor cell proliferation 50 value

[0089] In this example, cells were seeded in a 96-well plate, cultured overnight in a 5% carbon dioxide incubator, and then treated for 7 days after adding drugs in gradient dilution. Cell viability was determined using the CellTiter 96 Aqueous Cell Proliferation Assay Kit (MTS, Promega). According to the instructions, specifically, in this example, 20 μL of CellTiter 96 Aqueous reagent was added to 100 μL of culture medium and incubated at 37°C for 1 hour in a 5% CO2 atmosphere. Cell viability was measured at a wavelength of 490 nm on a Spark spectrophotometer (Tecan). The cell activity data measured after treatment with different concentrations of compounds were imported into the Prism7 software, and the compound concentration required to inhibit the activity of cancer cells by half, i.e., EC, was determined based on the fitted dose-response curve. 50 value.

[0090] This example evaluates the killing ability of ZBD-138 on 17 tumor cell lines. Figure 5 As shown, the half maximal effective concentration (EC 50 ) is 0.009 to 25.41 μM. At the same time, ZBD-138 has less cytotoxicity to the normal breast cell line MCF10A than to cancer cells, indicating that ZBD-138 has the ability to effectively and selectively kill tumor cells.

[0091] Example 6 Nude Mouse Tumor Transplant Model

[0092] The nude mouse transplant tumor model experiment was reviewed and approved by the Xiamen University Experimental Animal Management Ethics Committee and was conducted at the Xiamen University Experimental Animal Center. The female BALB / cnu / nu mice used in the experiment were 4-6 weeks old and purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and were raised in a pathogen-free laboratory. The specific experiment is as follows: About 5 million MCF7 or MDA-MB-231 tumor cells were digested with trypsin into a single cell suspension, suspended in PBS, and injected into the subcutaneous area on one side of the mouse's back. When the tumor size reaches about 100mm 3 At the time of the experiment, mice were randomly divided into two groups and injected with solvent control and ZBD-138, respectively. ZBD-138 was administered intraperitoneally every day at the designated dose. Subsequently, the length and width of the tumor were measured with a vernier caliper every day, and the tumor volume (length × width) was calculated. 2 ×0.52). At the same time, the body weight of mice was measured every day. The body weight and major organs of mice were photographed and weighed to evaluate the in vivo efficacy and safety of ZBD-138. The results are as follows Figure 6 (MCF7 transplant tumor experiment) and Figure 7 (MDA-MB-231 transplant tumor experiment) as shown.

[0093] Example 7

[0094] In this example, tetrahydroindole [2,1-h] pteridine-6-one compounds I-1 to I-21 were tested for PRMT1 in vitro methyltransferase activity and cytotoxicity. The results are as follows: Figure 8 and Fig. 9 shown. Figure 8 The horizontal axis represents different compounds, and the vertical axis represents the fluorescence detection intensity. NC is the reaction system without enzyme, and the other groups are all added with PRMT1 protein. Figure 8 It can be seen that the fluorescence intensity of the DMSO group increased significantly compared with the NC group, indicating that the PRMT1 protein has good enzyme activity. If the fluorescence intensity of the drug-added group is reduced compared with the DMSO group, it means that the compound has an inhibitory effect. Fig. 9 For cell proliferation experiments, the effects of inhibitors on cell growth rate were tested. Fig. 9 The horizontal axis represents the experimental groups of different inhibitors, and the vertical axis represents the inhibition rate ((fluorescence detection intensity of the control group-fluorescence detection intensity of the experimental group) / fluorescence detection intensity of the control group)×100%). MS023 is a known inhibitor of PRMT1.

[0095] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An arginine methyltransferase PRMT1 inhibitor, characterized in that: Its structural formula is 2. Use of the arginine methyltransferase PRMT1 inhibitor according to claim 1 in the preparation of an anti-tumor cell proliferation drug, characterized in that: The tumor cells are from breast cancer.

3. An anti-tumor cell proliferation composition, characterized in that: The active ingredient is the arginine methyltransferase PRMT1 inhibitor according to claim 1, and the tumor cells are from breast cancer.

Images