Use of compounds for treating tumors

Abstract

The present invention relates to the use of the compound IOWH-032 (DP390). The compound and the pharmaceutical composition containing the compound can be used for preventing and / or treating c-Myc related diseases including tumors.

Description

Technical Field

[0001] The present invention relates to the use of compound IOWH-032 (DP390). The compound and the pharmaceutical composition containing the same can be used for preventing and / or treating c-Myc-related diseases including tumors. Background Art

[0002] The transcription factor c-Myc is a major regulator of normal gene expression, regulating biological processes such as normal cell proliferation, cell cycle, differentiation, metabolism, apoptosis, and angiogenesis (Nat Rev Cancer. 2008 Dec; 8(12):976-90.). However, c-Myc also plays a key role in tumorigenesis, maintenance, and drug resistance. Due to gene amplification, translocation, mRNA upregulation, and abnormal protein stabilization, c-Myc protein is dysregulated in tumors (Cell. 2012 Mar 30; 149(1):22-35.). The c-Myc protein itself has poor stability and only has a stable conformation after binding to Max. After forming the c-Myc-Max complex, it binds to DNA-Ebox to initiate downstream gene transcription (Mol Cancer. 2021 Jan 4; 20(1):3.). Silencing c-Myc expression in various tumor models leads to tumor growth arrest or regression (Science. 2002 Jul 5; 297(5578):102-4., Cancer Res. 65, 4471–4474.). Therefore, targeting dysregulated c-Myc protein has broad therapeutic effects (Cold Spring Harb Perspect Med. 2014 Oct 1; 4(10):a014266.).

[0003] However, c-Myc is an intrinsically disordered protein (IDP) without a stable protein conformation and lacks a defined pocket, and has always been considered a "difficult drug target". For the drug development for treating c-Myc-related diseases, there are currently various development strategies, such as directly targeting the c-Myc-Max interaction in the c-Myc pathway, inhibiting the interaction between the c-Myc-Max complex and DNA, and indirectly regulating the upstream and downstream pathways of c-Myc. Over the years, although researchers have discovered multiple small molecule compounds that directly bind to c-Myc and inhibit c-Myc-Max dimerization and / or the formation of the c-Myc-Max-DNA complex, most of them have weak binding ability to c-Myc, low inhibitory activity on c-Myc-driven tumor cell proliferation, and poor druggability and pharmacokinetic properties, further limiting their in vivo applications (J. Am. Chem. Soc. 2023, 145, 3259-3269). Therefore, there is an urgent need to discover small molecule c-Myc inhibitors with better activity and druggability. Summary of the Invention

[0004] The present invention provides a novel c-Myc inhibitor, which can be used for preventing and / or treating c-Myc-related or mediated diseases, such as tumors.

[0005] The inventors of the present invention have found that the compound shown in the following formula (I) can inhibit c-Myc, bind to the c-Myc protein, interfere with or block the protein-protein interaction of c-Myc-Max, the c-Myc / Max complex interacts with DNA, induces the degradation of c-Myc protein, inhibits the transcriptional function of c-Myc, induces cell cycle arrest, etc., thereby preventing and / or treating c-Myc-related or mediated diseases, such as diseases caused by c-Myc overexpression.

[0006]

[0007] The chemical name of the above compound is 3-(3,5-dibromo-4-hydroxyphenyl)-N-(4-phenoxybenzyl)-1,2,4-oxadiazole-5-carboxamide (3-(3,5-dibromo-4-hydroxyphenyl)-N-(4-phenoxybenzyl)-1,2,4-oxadiazole-5-carboxamide), and the CAS number is: 1191252-49-9. The above compound can be obtained by commercial purchase, and its ID names are IOWH-032 and DP390.

[0008] According to one aspect of the present invention, the present invention provides a method for preventing and / or treating c-Myc-mediated diseases, including administering an effective amount of the compound shown in formula (I) or a pharmaceutically acceptable salt, deuterated compound, solvate, crystal form or prodrug thereof to a subject. The subjects include invertebrates, vertebrates, mammals, especially humans.

[0009] According to another aspect of the present invention, the present invention provides the use of the compound shown in formula (I) or a pharmaceutically acceptable salt, deuterated compound, solvate, crystal form or prodrug thereof for preventing and / or treating c-Myc-mediated diseases.

[0010] According to another aspect of the present invention, the present invention provides the use of the compound shown in formula (I) or a pharmaceutically acceptable salt, deuterated compound, solvate, crystal form or prodrug thereof in the preparation of a drug for preventing and / or treating c-Myc-mediated diseases.

[0011] According to another aspect of the present invention, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt, deuterated compound, solvate, crystal form or prodrug thereof for preventing and / or treating c-Myc-mediated diseases, or a compound represented by formula (I) or a pharmaceutically acceptable salt, deuterated compound, solvate, crystal form or prodrug thereof for preventing and / or treating c-Myc-mediated diseases.

[0012] According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising a compound represented by formula (I) or a pharmaceutically acceptable salt, deuterated compound, solvate, crystal form or prodrug thereof, and a pharmaceutically acceptable carrier.

[0013] According to another aspect of the present invention, the present invention provides a method for preventing and / or treating c-Myc-mediated diseases, comprising administering an effective amount of the above-mentioned pharmaceutical composition to a subject.

[0014] According to another aspect of the present invention, the present invention provides the use of the above-mentioned pharmaceutical composition for preventing and / or treating c-Myc-mediated diseases.

[0015] According to another aspect of the present invention, the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating c-Myc-mediated diseases.

[0016] According to another aspect of the present invention, the present invention provides the above-mentioned pharmaceutical composition for preventing and / or treating c-Myc-mediated diseases, or the above-mentioned pharmaceutical composition for preventing and / or treating c-Myc-mediated diseases.

[0017] According to the present invention, c-Myc-mediated diseases refer to diseases caused by c-Myc overexpression, mutation, abnormal protein stability or abnormally elevated transcriptional activity, such as tumors, such as cancers, including but not limited to lung cancer (including but not limited to small cell lung cancer, non-small cell lung cancer, non-small cell lung cancer such as squamous cell carcinoma, adenocarcinoma, large cell carcinoma, etc.), breast cancer, intestinal cancer (including but not limited to colorectal cancer, colon cancer, rectal cancer, etc.), gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, bladder cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, melanoma, osteosarcoma, liposarcoma, lymphoma, leukemia (including but not limited to acute lymphoblastic leukemia, acute myeloid leukemia including acute promyelocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, etc.), multiple myeloma, neuroendocrine tumors, glioblastoma, neuroblastoma, retinoblastoma, testicular cancer, gallbladder cancer, cholangiocarcinoma, skin cancer, nephroblastoma, lipoma, fibroma, hemangioma, uterine fibroids, meningioma, thyroid adenoma, glioma, pituitary tumor, etc. According to the present invention, the tumor or cancer may be primary or metastatic. Those skilled in the art can understand that the compositions of the present invention can be administered to a subject in any suitable manner, including but not limited to topical (e.g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration. Parenteral administration includes but not limited to subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intra-articular and intraperitoneal injection, etc. In certain embodiments, compositions in forms suitable for oral or parenteral use are preferred.. For intravenous, intramuscular, subcutaneous or intraperitoneal administration, one or more compounds can be mixed with a sterile aqueous solution, which is preferably isotonic with the recipient's blood.

[0018] Those skilled in the art can understand that the compositions of the present invention can be administered to a subject in any suitable dosage form. For example, suitable oral forms include but are not limited to tablets, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs; suitable forms for parenteral administration such as intravenous, intramuscular, subcutaneous, intraperitoneal, etc. include but are not limited to injection solutions, emulsions, suspensions, lyophilized powder injections, etc. Those skilled in the art can select a suitable pharmaceutically acceptable carrier according to the dosage form.

[0019] The compounds of the present invention can be administered to a subject in a prophylactically and / or therapeutically effective amount as required by the specific circumstances of the subject. Typically, the compounds of the present invention are administered in an amount of about 0.001 mg to 1000 mg / kg body weight / day, such as in an amount of about 0.01 to 100 mg / kg body weight / day, about 0.1 to 10 mg / kg body weight / day. The daily dose can be administered as a single dose or as multiple doses.

[0020] Those skilled in the art will understand that the compounds or pharmaceutical compositions of the present invention can be administered to a subject together with one or more other active agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 . The affinity of compound DP390 for MYCi975 and c-Myc protein (361-414) was tested using SPR technology: (a) affinity plot of MYCi975; (b) affinity plot of DP390.

[0022] Figure 2 . STD-NMR confirmed the binding of DP390 to c-Myc protein: (a) molecular structures of DP390 and its soluble homolog DP390-P; (b) affinity plot of DP390-P for c-Myc (353-437) tested by SPR; (c) STD-NMR analysis showed direct binding of DP390-P to c-Myc protein.

[0023] Figure 3 . Inhibition of c-Myc-Max protein-protein interaction and c-Myc / Max complex-DNA interaction by DP390: (a) AlphaLISA experiment to test the effect of DP390 on c-Myc-Max protein-protein interaction, IC 50 = 6.5 μM; (b) HTRF experiment to test the effect of DP390 on c-Myc-Max protein-protein interaction, IC 50 = 17.3 μM; (c) HTRF experiment to test the effect of DP390 on c-Myc / Max complex-DNA interaction, IC 50 = 5.9 μM.

[0024] Figure 4 . Western blot analysis of the change in c-Myc protein level after treating HL-60 cells with DP390 and MYCi975 for 4 h.

[0025] Figure 5 . E-box fluorescence reporter experiment to test the dose-dependent inhibition of the transcriptional function of c-Myc protein by DP390.

[0026] Figure 6. After treating HL-60 cells with DP390 for 48 h, the cell cycle of HL-60 cells was arrested at the G0 / G1 phase.

[0027] Figure 7 . In vivo test of the growth inhibition of DP390 on CT-26 murine tumors (results are shown as mean ± SD, n = 6): (a) experimental procedure; (b) photograph of the final tumor size; (c) tumor growth curve; (d) average tumor weight; (e) change in average body weight; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 vs control.

[0028] Example 1. SPR binding verification

[0029] Surface plasmon resonance (SPR) technology was used to test the affinity of the compound to be tested with c-Myc protein in a steady-state fitting manner. The specific operation was as follows: After activating the CM5 chip with an equal-volume mixed solution of EDC (0.4 M) and NHS (0.1 M), the c-Myc (361-414) protein (25 μg / mL) was conjugated to the CM5 chip, and the conjugation amount was 6192 RU. Then, it was blocked with ethanolamine solution for 10 min. The compound to be tested was dissolved and diluted to a 100 μM test solution with 1×PBST buffer containing 5% DMSO. The test solution was loaded at a flow rate of 30 μl / min for 60 s and dissociated for 180 s, and the instrument automatically recorded the sensing signal to obtain the sensing curve. The Steady state model and 1:1 binding kinetic model mathematical models were used to fit the sensing curve to obtain the KD data.

[0030] Compound DP390 (IOWH-032) has a strong affinity for c-Myc protein (Kd = 14.4 μM) ( Figure 1 b); the affinity of the control compound MYCi975 for c-Myc protein is Kd = 4.0 μM [1] ( Figure 1 a).

[0031] Example 2. STD-NMR confirmation of the binding of DP390 to c-Myc protein

[0032] Saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments were used to detect the binding of DP390 to c-Myc protein. Since the solubility of DP390 is poor, in order to improve the solubility, a phosphate tail was introduced at the hydroxyl end of DP390 to obtain DP390-P ( Figure 2a). The SPR technology of Example 1 was used for testing, and the results showed that DP390-P could also bind to c-Myc protein in a steady state manner and had a dose-dependent response (Kd = 209 μM). ( Figure 2 b), and the reporter experiment of Example 5 verified that it retained the inhibitory activity on the downstream transcriptional function of c-Myc (IC 50 = 2.9 μM).

[0033] The specific operation was as follows: The STD NMR spectrum was recorded by a Bruker Advanced 600 nuclear magnetic resonance spectrometer and processed using Bruker Topspin software. DP390-P was dissolved in PBS buffer (pH 7.4) containing 5% DMSO-d 6 and 95% D 2 O to prepare a test solution with a final concentration of 2 mM (DP390-P) for the reference test experiment; DP390-P and c-Myc (353-437) were respectively dissolved in PBS buffer (pH 7.4) containing 5% DMSO-d 6 and 95% D 2 O to prepare stock solutions with final concentrations of 2 mM (DP390-P) and 60 μM c-Myc (353-437) for the STD NMR experiment. The prepared solutions were vortexed for 30 seconds and then used for the nuclear magnetic resonance experiment. DP390-P was tested separately or in the presence of c-Myc protein. The STD NMR experiment used a Gaussian-shaped saturation pulse sequence with a duration of 50 ms, a power of 200 Hz, and a duration of 3 seconds. The "on" resonance saturation frequency was -0.02 ppm, and the "off" resonance saturation frequency was -4 ppm. The relaxation delay time before the saturation pulse was 4 seconds. The number of scans in the experiment was 128 times, and the spectral width was 16.028 ppm.

[0034] In the STD-NMR experiment, the presence of aromatic hydrogens of the small molecule in the STD-NMR spectrum of the small molecule and the protein showed the binding of DP390-P (2 mM) to c-Myc (60 μM), and mainly the aromatic ring of DP390-P mediated the binding to c-Myc protein. ( Figure 2 c). Thus, we confirmed the direct binding of DP390 to c-Myc protein through the STD-NMR experiment.

[0035] Example 3. DP390 interferes with c-Myc-Max protein-protein interaction and the interaction between c-Myc / Max complex and DNA

[0036] Molecules that bind to the c-Myc protein need to block the protein-protein interaction between c-Myc and Max in order to further block the interaction between the c-Myc / Max complex and DNA and thus exert their biological functions. The effects of DP390 on the protein-protein interaction between c-Myc and Max were tested using two different methods, AlphaLISA and HTRF. The specific operations are as follows:

[0037] AlphaLISA PPI: Using Echo (LABCYTE, 655), add 0.1 μl of compound DP390 diluted with DMSO to a 384-well plate (starting at 100 μM, 3-fold dilution, 9 concentration gradients, double replicates) (cpd well). Add 2.5 μl of c-MYC working solution (ICE, E2210T-H11H) to each well, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 10 min. Add 2.5 μl of MAX working solution (active motif, 81017) to each well, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 60 min. Add 5 μl of Donor (PerkinElmer, AS116D) and acceptor beads (PerkinElmer, AL112C) to each well, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 90 min. Then read the AlphaLISA signal value on a BMG (PHERAstar FSX). The inhibition rate calculation formula is as follows: % inh = 100 * (ave High control – ave cpd well) / (ave High control - ave Low control); where, High control refers to the DMSO control well, and Low control refers to the background well without c-Myc protein; Calculate the IC 50 value;

[0038] HTRF-PPI: Add 0.1 μl of the compound DP390 diluted with DMSO into a 384-well plate using Echo (LABCYTE, 655) (starting from 100 μM, 3-fold dilution, 9 concentration gradients, double replicates) (cpd well). Add 2.5 μl of the c-MYC working solution (ICE, E2210T-H11H) to each well, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 10 min. Add 2.5 μl of the MAX working solution (active motif, 81017) to each well, centrifuge at 1000 rpm for 1 min, add 5 μl of His-Tb (Cisbio, 61HISTLB) and Flag-d2 (Cisbio, 61FG2DLB) to each well, and incubate at 25 °C for 60 min. Then read the HTRF signal value on a BMG (PHERAstar FSX). The inhibition rate calculation formula is as follows: %inh = 100 * (ave High control - ave cpd well) / (ave High control - ave Low control), where High control refers to the DMSO control well, and Low control refers to the background well without c-Myc protein; calculate the IC 50 value;

[0039] HTRF-PPD: Using Echo (LABCYTE, 655), add 0.15 μl of the compound diluted with DMSO to a 384-well plate (starting at 100 μM, 3-fold dilution, 9 concentration gradients, double replicates) (cpd well). Add 2.5 μl of the c-MYC working solution (ICE, E2210T-H11H) to each well, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 10 min. Add 2.5 μl of the MAX working solution (active motif, 81017) to each well, centrifuge at 1000 rpm for 1 min, add 2.5 μl of His-Tb (Cisbio, 61HISTLB) to each well, and incubate at 25 °C for 60 min. Add 5 μl of dsDNA (Genscript) to each well, centrifuge at 1000 rpm for 1 min. Add 2.5 μl of SA-d2 (Cisbio, 610SADLF) to each well, and incubate at 25 °C for 60 min. Then read the HTRF signal value on a BMG (PHERAstar FSX). The inhibition rate calculation formula is as follows: % inh = 100 * (ave High control - ave cpd well) / (ave High control - ave Low control), where High control refers to the DMSO control well, and Low control refers to the background well without c-Myc protein; calculate the IC 50 value;

[0040] The results showed that DP390 (IC 50 = 6.5 / 17.3 μM) was able to inhibit the protein-protein interaction of c-Myc-Max in both tests ( Figure 3 a, 3b). Further HTRF testing of the binding of DP390 to c-Myc / Max complex with DNA showed that DP390 (IC 50 = 5.9 μM) was also able to prevent the formation of the c-Myc / Max-DNA transcription complex ( Figure 3 c). This indicates that the compound DP390 can prevent the c-Myc-Max protein-protein interaction by binding to the c-Myc protein at the protein level, thereby inhibiting the binding of the c-Myc / Max complex to DNA and inhibiting the transcriptional function of c-Myc.

[0041] Example 4. DP390 induces the degradation of c-Myc protein

[0042] The effect of DP390 on the c-Myc protein level was tested at the cellular level. The c-Myc protein is inherently unstable and only has a stable conformation and initiates downstream gene transcription after forming a complex with the Max protein[2] The c-Myc protein that has not formed a complex is easily phosphorylated by upstream proteins such as PLK1, and then ubiquitinated and degraded, with a half-life in vivo of about 30 minutes. [3,4] Therefore, interfering with the interaction between the two proteins can promote the instability and degradation of c-Myc. c-Myc protein was overexpressed in HL-60 cells, and the effect of compound DP390 on the c-Myc protein level was tested by WB experiment in HL60 cells. The specific operation is as follows:

[0043] HL-60 cells were cultured in RPMI-1640 + 10% FBS + 1% P / S medium. The cells were seeded in 6-well plates, with 2×10 6 cells per well; Compound DP390 and MYCi975 were diluted from 10 mM DMSO stock solutions to the corresponding concentrations with the medium. The prepared compounds were transferred to the 6-well plates (the final concentration of DP390 was 10 and 20 μM, and the final concentration of the control molecule MYCi975 was 20 μM). After mixing, the cells were incubated in a 37 °C, 5% carbon dioxide incubator for 4 hours. The cells in the 6-well plates were collected, centrifuged to discard the supernatant, and the cell pellet was retained. The cell pellet was lysed with RIPA lysis buffer, and after measuring the concentration of each sample, the protein concentration was uniformly quantified. 5× protein loading buffer was added, and after boiling at 95 °C for 10 min, it was cooled on ice, and the samples were immediately stored at -80 °C. The obtained protein samples were loaded into the wells of a 10% SDS gel, and electrophoresis was performed with the same loading amount per well. After electrophoresis, the proteins on the SDS gel were transferred to a PVDF membrane and blocked at room temperature for 1 h. After washing with PBST, it was incubated overnight at 4 °C with primary antibodies (c-Myc total protein antibody (CST D84C12, diluted 1:1000) and GAPDH internal reference antibody (Trans, HC301, diluted 1:3000)). The next day, after washing the membrane, it was incubated with a secondary antibody conjugated to HRP, and after washing, the membrane was exposed under a chemiluminescent imager, and the pictures were saved ( Figure 4 ).

[0044] The results showed that DP390 could reduce the c-Myc protein level in a dose-dependent manner after incubating HL-60 cells for 4 hours, and a 20 μM concentration reduced it by about 50%. It was further verified that DP390 could inhibit the binding of c-Myc protein to Max by binding to c-Myc protein, inducing the degradation of c-Myc protein.

[0045] Example 5. DP390 inhibits the transcriptional function of c-Myc

[0046] In cells, after c-Myc forms a heterodimer with Max, it binds to the DNA E-box sequence, promotes the transcription of downstream genes, and regulates biological processes such as cell proliferation, differentiation, cell cycle progression, and metabolism. [5,6]The method of constructing a MYC-dependent E-box luciferase reporter by transfecting HEK293T cells with an exogenous plasmid (Ebox sequence + Luciferase gene) was used to test the effect of compounds on the transcriptional function of c-Myc. The specific operations are as follows:

[0047] (1) HEK293T cells cultured in T25 (purchased from Kebo Biologics) were digested with 1 mL of 0.05% trypsin until the cells detached, 1 mL of complete medium (DMEM + 10% FBS + 1% P / S) was added to terminate the digestion, and the cells were centrifuged at 1000 rpm for 3 min, and the supernatant was discarded;

[0048] (2) Seeding cells in 6-well plates: Resuspend the cells and count them, adjust the cell density to 5×10 5 cells / mL, add 2 mL of cell suspension to each well of the 6-well plate, which contains 1×10 6 cells, and culture the cells in an incubator at 37°C and 5% carbon dioxide for 6 h until the cells adhered to the plate;

[0049] (3) Transfection: Mix 16 μL of transfection reagent + 4 μg of Ebox-Lucifersae plasmid, let it stand at room temperature for 15 min, then drop it into the wells of the 6-well plate, and continue to culture in an incubator at 37°C and 5% carbon dioxide for 18 h;

[0050] (4) Prepare the test compounds: Dilute the test compounds 2-fold with complete medium to the 9th concentration, that is, dilute from 500 μM to 1.95 μM, transfer 10 μL to the 96-well cell plate in sequence, and set three replicates;

[0051] (5) Digest the above transfected cells with 1 mL of 0.05% trypsin until the cells detached, add 1 mL of complete medium to terminate the digestion, centrifuge at 1000 rpm for 3 min, and discard the supernatant; Resuspend the cells and count them, adjust the cell density to 3.3×10 5 cells / mL, add 100 μL of cell suspension to each well of the above 96-well plate containing the test compounds, and statically culture in an incubator at 37°C and 5% carbon dioxide for 24 h;

[0052] (6) Discard all the medium, add 100 μL of lysis buffer to each well, and lyse on ice for 5 min; Take 20 μL and transfer it to a white 96-well enzyme-linked immunosorbent assay (ELISA) plate, add 100 μL of substrate, and detect the luciferase luminescence value; Take another 20 μL of lysis buffer and transfer it to a black 96-well ELISA plate, add 20 μL of CTG2.0, and detect the cell viability;

[0053] (7) Normalize the luciferase luminescence value using the multiple relationship of cell viability. Normalization process: Take the average value A of all the original cell viability data treated with the same compound. Divide the original cell viability data B of each well by the average value to obtain the relative cell viability value of the corresponding well. Then divide the luciferase luminescence value C of each well by the relative cell viability value of the corresponding well to obtain the normalized value L = A * C / B;

[0054] Then, for the value L after normalizing the luciferase luminescence value at each concentration, use GraphPad Prism software to fit the value L after normalizing the luciferase luminescence value with the corresponding concentration to obtain the concentration-response curve and IC 50 value.

[0055] The results showed that DP390 could inhibit E-box luciferase activity in a dose-dependent manner (IC 50 = 15.6 μM), demonstrating that DP390 could inhibit the transcriptional activity of c-Myc protein at the cellular level ( Figure 5 ).

[0056] Example 6. DP390 Induces Cell Cycle Arrest

[0057] The c-Myc protein regulates the normal progression of the cell cycle. Inhibiting the function of c-Myc can arrest the cell cycle at the G0 / G1 phase [7,8] .

[0058] HL-60 cells were cultured in RPMI-1640 + 10% FBS + 1% P / S medium. The cells were seeded in 6-well plates at 1 × 10 6 cells per well; The compound DP390 was diluted from a 10 mM DMSO stock solution to the corresponding concentration with the medium. Transfer the prepared compound into the 6-well plate cells at a final concentration of 3.33 μM. After mixing, incubate in a 37°C, 5% carbon dioxide incubator for 48 hours. Collect the cells in the 6-well plate, centrifuge to discard the supernatant, and retain the cell pellet. Resuspend the cell pellet by dropping and shaking with pre-cooled 75% ethanol, and fix and permeabilize at -20°C for 30 min; After fixing and permeabilizing the cells, centrifuge at 1500 rpm for 5 minutes, and wash twice with pre-cooled 1×PBS; Add PI / RNase staining buffer according to the kit instructions, stain at room temperature for 15 min, and then detect on a flow cytometer (detect at the lowest sample loading speed and collect 15000 events).

[0059] The results are as Figure 6As shown, when HL-60 cells were treated with DP390 at a lower concentration (3.3 μM), it was able to block cell cycle progression, arrest the cell cycle at the G0 / G1 phase, reduce the number of cells in the G2 / M phase, while the number of cells in the S phase remained basically unchanged.

[0060] Example 7. DP390 inhibits the proliferation of multiple cell lines

[0061] c-Myc is overexpressed in tumor cells. We tested the proliferation inhibitory activity of DP390 on multiple cell lines with overexpressed MYC, including HCC, CRC, SCLC, breast cancer cells, human promyelocytic leukemia cells, and murine colon cancer cells CT-26.

[0062] The specific operation for testing the proliferation inhibitory activity of HL-60 cells is as follows:

[0063] 1. Resuscitate HL60 cells (purchased from Hefei Puresheng) with complete medium (RPMI-1640 + 10% FBS + 1% P / S). After passing about two generations, select a cell line with good growth status, collect cells in the logarithmic growth phase and count them. Adjust the cells to an appropriate concentration and inoculate them into a 96-well plate, adding 135 μL of cell suspension to each well, with a seeding density of 2500 cells / well;

[0064] 2. Prepare a stock solution of the test compound (Cpd) with DMSO. Use DMSO to gradually dilute it 2-fold with 15 mM as the highest concentration to obtain 9 concentration gradients;

[0065] 3. Dilute the test compound 30-fold with complete medium. Take 15 μL and add it to a 96-well cell plate containing 135 μL of cells. Add a culture medium without cells (containing 0.33% DMSO) to the Min control well, and add a 15 μL DMSO-cell culture medium mixture (final DMSO concentration is 0.33%) to the Max control. Incubate in an incubator at 37 °C, 5% CO 2 , with a relative humidity of more than 90% for 6 days;

[0066] 4. Add 50 μL / well of CellTiter Glo to end the reaction, incubate at room temperature in the dark for 30 min, gently shake, and then detect on Paradigm to read the fluorescence value RLU of each well. The formula for the cell proliferation inhibition rate (Inhibition Rate) is as follows:

[0067] Inhibition Rate (Inh%) = 100 - (RLUCpd - RLUMin) / (RLUMax - RLUMin) * 100%;

[0068] 5. Calculate the inhibition rate corresponding to compounds at different concentrations in EXCEL, and then fit the inhibition rate curve using GraphPad Prism software and calculate the IC 50 value.

[0069] The specific operation for testing other cell proliferation inhibition activities is as follows:

[0070] After digesting adherent cells, resuspending them with cell culture medium, and counting, dilute the cells to an appropriate concentration. Transfer 100 μl of the resuspended solution to a 96-well plate. The seeding density of HCT116 and Hs578T is 500 cells / well, the seeding density of SK-HEP-1 and MCF10A is 800 cells / well, the seeding density of Hep-G2, JHH-7, Li-7, and MDA-MAB-231 is 1000 cells / well, the seeding density of Huh-7, SNU-387, HT-29, JIMT-1, BT-474, and BT-549 is 1500 cells / well, the seeding density of SNU-398, Hep 3B2.1-7, SNU-423, SNU-182, CW-2, LoVo, SW480, HCC44, and NCI-H446 is 2000 cells / well, the seeding density of COLO-678, COLO 201, CT-26, and T-47D is 3000 cells / well, the seeding density of SW48, SW837, NCI-H847, and MCF7 is 4000 cells / well, and the seeding density of CL-40, NCI-H524, MDA-MB-453, and MDA-MB-361-Luc#1 is 5000 cells / well. After the cells adhere (about 16 h), discard 50 μl of the supernatant culture medium, add 40 μl of fresh culture medium, and add 10 μl of 10× compound (cpd) to the corresponding wells. The final concentration of DMSO is not higher than 1%. Incubate in a 37°C, 5% CO 2 incubator for 6 days. Then, equilibrate the cell culture plate to room temperature. After discarding 50 μl of the culture medium, add 50 μl of Reagent (Promega, G7573) to each well, mix for 2 min, incubate at room temperature for 10 min, and detect the fluorescence value RLU of each well. The formula for calculating the cell proliferation inhibition rate is as follows:

[0071] Inhibition Rate (Inh%) = 100 - (RLUCpd - RLUMin) / (RLUMax - RLUMin) * 100%;

[0072] where RLUCpd is the reading of the compound in each well, RLUMin is the reading of the culture medium, and RLUMax is the reading of the well containing cells and culture medium but no compound.

[0073] The inhibition rate curve was fitted with GraphPad Prism software and the IC 50 value was calculated.

[0074] The results showed that on MYC-overexpressing cells, DP390 could effectively inhibit the growth of respective tumor cells, and the inhibition intensity was basically at the same level as that of the positive control molecule MYCi975. Therefore, the conclusion can be drawn that DP390 can inhibit cell proliferation by binding to c-Myc protein, inducing the degradation of c-Myc protein, and affecting various functions of c-Myc.

[0075] Table 1: DP390 inhibits the proliferation of multiple MYC-overexpressing cells

[0076]

[0077]

[0078] The expression level of Myc protein was derived from THE HUMAN PROTEIN ATLAS database (https: / / www.proteinatlas.org / ). Example 8. PK and pharmacodynamic tests of DP390

[0079] DP390 can inhibit its related functions by binding to c-Myc protein at both the protein level and the cell level. Further, the anti-tumor effect of DP390 was verified in animals. The operation steps of the mouse PK test are as follows:

[0080] (1) Experimental animals: 2 groups of female Balb / c mice, three in each group, SPF grade, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0081] (2) Drug preparation: On the day of administration, a certain amount of compound DP390 was taken and formulated into an intravenous injection solution of 0.2 mg / mL with solvent: 5% DMSO + 10% Solutol HS15

[0082] + 85% Saline, and formulated into a gavage solution of 10 mg / mL with solvent 0.5% CMC-Na + 1% Tween 80.

[0083] (3) Administration dose and administration method: The IV administration dose was 1 mg / kg, and the administration volume was 5 mL / kg. The gavage (po) administration dose was 100 mg / kg, and the administration volume was 10 mL / kg. Weigh the animals before administration, calculate the dosage according to the body weight. Administer by tail vein injection and oral administration. The animals were fasted overnight (10 - 14 hours) before administration and fed 4 hours after administration.

[0084] (4) Blood collection and blood sample processing: Blood was collected at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after drug administration. Blood was collected from the cheek, approximately 0.05 mL was collected for each sample, anticoagulated with EDTA-2K, placed on wet ice after collection, and centrifuged to separate plasma within 1 hour (centrifugation conditions: 6000 g, 3 minutes, 2 - 8 °C). Plasma samples were stored in a -80 °C refrigerator before analysis.

[0085] (5) Plasma sample analysis: After all samples were completely thawed, they were mixed for 10 - 30 seconds and centrifuged at 4000 rpm and 4 °C for 0.5 minutes. 15 μL of plasma sample (15 μL of blank plasma was added to the blank sample and internal standard blank sample) was transferred to a 96-well plate, and 150 μL of internal standard (50% methanol acetonitrile solution (concentration: 100 ng / mL)) solution was added (150 μL of 50% methanol acetonitrile solution was added to the blank sample). The samples were vortexed for 5 minutes and then centrifuged at 4000 rpm and 4 °C for 5 minutes. 100 μL was transferred and added to 100 μL of water, mixed evenly, and submitted for LC-MS / MS analysis.

[0086] (6) Result analysis: Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.0 based on the blood drug concentration data at different time points.

[0087] The PK test showed that intravenous injection (1 mpk) had a higher clearance rate and a half-life of only 0.4 h, while oral administration (100 mpk) had a maximum blood drug concentration of up to 9337 ng / mL (17 μM) and a half-life of 1.77 h, and was able to maintain the blood drug concentration above 11 μM within 4 h.

[0088] The previous cell proliferation inhibition screening experiment demonstrated that DP390 could inhibit the growth of various colorectal cancer cells. Inhibiting the function of MYC can affect the anti-tumor response through multiple mechanisms, including regulating the immune microenvironment. [1,9-11] Therefore, we constructed a colon cancer model based on murine CT26 cells to test the anti-tumor effect of DP390 in vivo.

[0089] CT26 colon cancer cells were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and routinely cultured in RPMI-1640 medium (Adamas Life, C8016) supplemented with 10% fetal bovine serum (Vivacell Biotechnology, C04001-500). To establish a mouse orthotopic tumor model, CT26 cells in the logarithmic growth phase (5×106 cells / mouse) were inoculated subcutaneously into the right axilla of 6-week-old female BALB / c mice (Jiangsu Jicui Yakang Biotechnology Co., Ltd.). On the 8th day after inoculation, when the volume of the transplanted tumor grew to 50-100 mm 3 , the tumor-bearing mice were randomly divided into 3 groups (n = 6) according to body weight and tumor volume. Each group of animals received the following treatments: The control group was given vehicle 0.5% CMC-Na + 1% Tween 80 by gavage daily for 10 consecutive days; the experimental group was given compound DP390 (50 mg / kg body weight / day, vehicle 0.5% CMC-Na + 1% Tween 80) by gavage daily for 5 consecutive days; the positive control group was given MYCi975 (100 mg / kg body weight / day, vehicle 5% DMSO in corn oil) by gavage daily for 10 consecutive days. During the dosing period, the body weight and tumor volume of the mice were monitored every two days. After the last dose, the animals were sacrificed, and the tumor tissues were completely dissected and weighed and recorded. All animal experiments in this study were strictly carried out in accordance with the experimental protocol approved by the Institutional Animal Care and Use Committee (IACUC) of China Pharmaceutical University. The tumor volume was measured using a vernier caliper and calculated according to the formula V(mm 3 ) = 0.5×a×b 2 (a and b represent the long and short diameters of the tumor respectively), and the tumor growth inhibition rate TGI% = [1 - (Vt - V0) / (CVt - CV0)]×100%, (Vt is the average tumor volume measured each time in the treatment group; V0 is the average tumor volume obtained when the treatment group was caged for dosing; CVt is the average tumor volume measured each time in the control group; CV0 is the average tumor volume obtained when the control group was caged for dosing).

[0090] As Figure 7 shown in a, DP390 was administered by gavage (50 mpk po daily) for 5 days and then the administration of DP390 was stopped, while MYCi975 was continuously administered (100 mpk po daily) until the end of the experiment. The experimental results ( Figure 7 b - Figure 7 e) showed that the anti-tumor effect of DP390 was significant (TGI = 90.0%), superior to that of MYCi975 (TGI = 40.7%), and the average tumor weight was also significantly reduced, with little difference in body weight.

[0091] So far, we have verified at the protein level that extracellular DP390 can block the c-Myc-Max protein-protein interaction by binding to the c-Myc protein, thereby inhibiting the formation of the c-Myc / Max-DNA transcription complex, inducing the degradation of c-Myc protein, inhibiting the transcriptional function of c-Myc, arresting the cell cycle at the cellular level, and inhibiting tumor growth by oral administration in vivo, systematically verifying that DP390 can exert anti-tumor biological functions by targeting c-Myc. Moreover, DP390 can inhibit the proliferation of a variety of tumor cells overexpressing Myc, including liver cancer, colorectal cancer, breast cancer, lung cancer, and APL. References:

[0092] 1.1Han H,Jain AD,Truica MI,Izquierdo-Ferrer J,Anker JF,Lysy B,SagarV,Luan Y,

[0093] Chalmers ZR,Unno K,Mok H,Vatapalli R,Yoo YA,Rodriguez Y,Kandela I,Parker JB,

[0094] Chakravarti D,Mishra RK,Schiltz GE,Abdulkadir SA.Small-Molecule MYCInhibitors

[0095] Suppress Tumor Growth and Enhance Immunotherapy.Cancer Cell.2019Nov

[0096] 11;36(5):483-497.e15.

[0097] 2.Madden SK,De Araujo AD,Gerhardt M,et al.Taking the Myc out ofcancer:toward

[0098] therapeutic strategies to directly inhibit c-Myc[J].Molecular Cancer,2021,20(1):3.

[0099] 3.Farrell AS,Sears RC.MYC degradation.Cold Spring Harb PerspectMed.2014Mar

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[0101] 4. Gregory MA, Hann SR. c-Myc proteolysis by the ubiquitin-proteasome pathway:

[0102] stabilization of c-Myc in burkitt’s lymphoma cells. Mol Cell Biol. 2000; 20: 2423–2435.

[0103] 5. Blackwell, T.K., Kretzner, L., Blackwood, E.M., Eisenman, R.N., and Weintraub, H. (1990).

[0104] Sequence-specific DNA binding by the c-Myc protein. Science 250, 1149–1151.

[0105] 6. Evan, G.I., and Vousden, K.H. (2001). Proliferation, cell cycle and apoptosis in cancer.

[0106] Nature 411, 342–348.

[0107] 7. Freytag SO. Enforced Expression of the c-myc Oncogene Inhibits Cell Differentiation by

[0108] Precluding Entry into a Distinct Predifferentiation State in G(0) / G(1)[J]. Mol Cell Biol,

[0109] 1988, 8(4): 1614-1624.

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[0111] Cells via Conditional Gene-Targeted Mutation[J].Immunity,2001,14(1):45-55.

[0112] 9.Casey,S.C.,Tong,L.,Li,Y.,Do,R.,Walz,S.,Fitzgerald,K.N.,Gouw,A.M.,Baylot,V.,

[0113] Gutgemann,I.,Eilers,M.,et al.(2016).MYC regulates the antitumorimmune response

[0114] through CD47 and PD-L1.Science 352,227–231.

[0115] 10.Casey,S.C.,Baylot,V.,and Felsher,D.W.(2018).The MYC oncogene is aglobal regulatorof the immune response.Blood 131,2007–2015.

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[0119] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preventing and / or treating a c-Myc-mediated disease, characterized in that: Administering an effective amount of a compound as represented by formula (I) or a pharmaceutically acceptable salt, deuterated substance, solvate, crystal form or prodrug thereof to a subject 2. Use of the compound represented by formula (I) or its pharmaceutically acceptable salt, deuterated substance, solvate, crystalline form or prodrug in the preparation of a drug for preventing and / or treating c-Myc-mediated diseases 3. A pharmaceutical composition comprising a compound as represented by formula (I) or a pharmaceutically acceptable salt, deuterated product, solvate, crystalline form or prodrug thereof, and a pharmaceutically acceptable carrier 4. A method for preventing and / or treating a c-Myc-mediated disease, characterized in that: An effective amount of the pharmaceutical composition of claim 3 is administered to a subject.

5. Use of the pharmaceutical composition according to claim 3 in the preparation of a medicament for preventing and / or treating c-Myc-mediated diseases.

6. The method according to claim 1 or 4, the use according to claim 2 or 5, or the pharmaceutical composition according to claim 3, characterized in that: The c-Myc-mediated disease is selected from lung cancer, breast cancer, intestinal cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, bladder cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, melanoma, osteosarcoma, liposarcoma, lymphoma, leukemia, multiple myeloma, neuroendocrine tumors, glioblastoma, neuroblastoma, retinoblastoma, testicular cancer, gallbladder cancer, bile duct cancer, skin cancer, Wilms' tumor, lipoma, fibroma, hemangioma, uterine fibroids, meningioma, thyroid adenoma, glioma, pituitary tumor.

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