Application of small molecule compound QC6352

Abstract

The invention discloses application of a small molecule compound QC6352, the small molecule compound QC6352 can be used for preparing related drugs for treating AML, and the small molecule compound QC6352 can effectively act on AML cells, inhibit proliferation of the AML cells and induce apoptosis of the AML cells at a low concentration, can up-regulate expression of NKG2D ligands on the surfaces of tumor cells and enhance the killing effect of immune cells, and can be used for preparing drugs for treating AML. The obvious curative effect is achieved on AML treatment. In addition, the QC6352 has no obvious toxicity to bone marrow blood mononuclear cells of healthy people, and is high in safety. The application provides a new thought and scheme for clinical treatment of AML, and has important significance.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to the application of the small molecule compound QC6352. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant disease originating from myeloid hematopoietic stem cells. It is characterized by the differentiation blockage and abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. The prognosis is poor, and the five-year survival rate is only about 30%. Currently, the main treatments for AML include chemotherapy (such as cytarabine combined with anthracyclines), targeted therapy (FLT3 inhibitors such as Gilteritinib, IDH1 / IDH2 inhibitors such as Ivosidenib, BCL-2 inhibitor Venetoclax, etc.) and hematopoietic stem cell transplantation, but the treatment effect is still unsatisfactory.

[0003] Specifically, although the existing chemotherapy regimen can achieve complete remission in patients, they often relapse in a short period of time. According to statistics, the relapse rate of patients after chemotherapy is as high as 60% to 70%, and the relapse rate of high-risk patients is even as high as 80% or more, and relapsed leukemia will be resistant to previous treatment methods. In addition, according to existing research results, the commonly used chemotherapy drugs often damage the function of patients' immune cells, or cause AML cells to downregulate immune activation molecules and upregulate inhibitory molecules to achieve immune escape, thereby further exacerbating the difficulty of treatment. Furthermore, hematopoietic stem cell transplantation is the only treatment that can completely cure acute myeloid leukemia, but more than 40% of patients will still relapse after transplantation; and the transplantation operation is expensive, and transplant-related deaths caused by complications such as graft-versus-host disease, infection, implantation failure and long-term organ toxicity limit its application in all patients, especially elderly patients.

[0004] Therefore, there is an urgent need to research and develop new drugs to treat acute myeloid leukemia. Summary of the invention

[0005] In view of this, through a large number of experimental studies and verifications in the early stage of this application, it was innovatively discovered that the small molecule compound QC6352 has significant therapeutic effects on acute myeloid leukemia, and has no obvious toxicity to other normal blood cells. It is highly safe and has significant efficacy, providing a new solution for the development of acute myeloid leukemia drugs.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions:

[0007] One aspect of the present application provides the use of a small molecule compound QC6352 in the preparation of a drug, wherein the drug has at least one of the following effects:

[0008] a: Treatment of AML;

[0009] b: Inhibit the proliferation of AML cells;

[0010] c: Induce apoptosis and / or differentiation of AML cells;

[0011] d: Inhibit the immune escape of AML cells;

[0012] e: Enhance the killing of AML cells by NK cells.

[0013] After a large number of preliminary experimental studies and verifications, this application has determined that the small molecule compound QC6352 can inhibit the proliferation of AML cells, induce apoptosis and differentiation of AML cells, inhibit the immune escape of AML cells, and enhance the killing ability of NK cells against AML cells, thereby achieving the effect of treating AML.

[0014] Another aspect of the present application provides a drug containing the small molecule compound QC6352.

[0015] Beneficial effects of this application:

[0016] The present invention reveals for the first time that the small molecule compound QC6352 can be used as a new drug for targeted AML treatment; QC6352 can effectively act on AML cells at a low concentration (starting from 15nM), and has no obvious toxic effect on the bone marrow mononuclear cells of healthy subjects; QC6352 can inhibit the proliferation of AML cells, and can also induce AML cell apoptosis, upregulate the expression of NKG2D ligands on the surface of tumor cells, and enhance the killing effect of immune cells; compared with existing drugs, QC6352 can reverse the immune escape characteristics of AML cells, making them more susceptible to killing by effector immune cells.

[0017] In general, the small molecule compound QC6352 can be used as a new drug for targeted treatment of AML, providing a new solution for the targeted treatment of AML in clinical practice. It can also be combined with immunotherapy to improve the treatment effect of AML. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The proliferation results of AML cells detected by CCK-8 experiment in Example 2, wherein: Figure 1 a is the experimental result of AML cell Thp-1, Figure 1 b shows the experimental results of AML cells U937.

[0019] Figure 2 The experimental results of AML cell lines Thp-1 and U937 treated with different concentrations of QC6352 for 48 hours in Example 3, wherein: Figure 2 a is the flow cytometric diagram of Thp-1, Figure 2b is the proportion of live cells counted by Thp-1, Figure 2 c is the flow cytometry image of U937, Figure 2 d is the percentage of live cells counted by U937, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0020] Figure 3 The apoptosis results of primary cells from AML patients treated with different concentrations of QC6352 for 48 hours in Example 3, wherein: Figure 3 a is the flow cytometry diagram, Figure 3 b is the statistical proportion of apoptotic cells, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0021] Figure 4 The apoptosis results of healthy donor bone marrow mononuclear cells treated with different concentrations of QC6352 for 48 hours in Example 3, wherein: Figure 4 a is the flow cytometry diagram, Figure 4 b is the statistical percentage of apoptotic cells, ns indicates no statistically significant difference.

[0022] Figure 5 The experimental results of AML cell line Thp-1 treated with different concentrations of SD49-7 for 48 hours in Example 3, wherein: Figure 5 a is the flow cytometric diagram of Thp-1, Figure 5 b is the percentage of live cells counted by Thp-1, ns indicates no statistically significant difference.

[0023] Figure 6 The cell morphology of AML cells treated with 30 nM QC6352 for 48 hours in Example 4.

[0024] Figure 7 The expression of myeloid differentiation marker CD11b on the cell surface after being treated with different concentrations of QC6352 for 48 hours in Example 4, wherein: Figure 7 a is the flow cytometry diagram, Figure 7 b is the statistical percentage of cells expressing the myeloid differentiation marker CD11b on the cell surface, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

[0025] Figure 8 The expression of MICA / B and ULBP3 on the surface of Thp-1 and U937 cells after being treated with different concentrations of QC6352 for 48 hours in Example 4, wherein: Figure 8 a is the flow cytometry peak diagram of MICA / B expression levels on the surface of Thp-1 and U937 cells, Figure 8b is the flow cytometry peak diagram of ULBP3 expression level on the surface of Thp-1 and U937 cells, Figure 8 c is a statistical graph of MICA / B expression levels on the surface of Thp-1 and U937 cells, Figure 8 d is a statistical graph of the expression levels of ULBP3 on the surface of Thp-1 and U937 cells, **** indicates P < 0.0001.

[0026] Fig. 9 The killing results of NK cells on AML cells treated with QC6352 in Example 5, wherein: Fig. 9 a is the flow cytometry diagram, Fig. 9 b is a statistical chart of Thp-1 killing level, Fig. 9 c is a statistical chart of the killing level of U937. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the implementation methods of the present application. The technical solutions in the implementation methods described below are exemplary and are only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can fully combine the implementation methods of the present application to obtain other implementation methods without creative work, and these implementation methods are also within the scope of protection of the present application.

[0028] The first aspect of the present application discloses the use of a small molecule compound QC6352 in the preparation of a drug. Based on this, a drug is provided, wherein the drug contains the small molecule compound QC6352.

[0029] The structural formula of the small molecule compound QC6352 is as follows:

[0030]

[0031] Although it has anti-tumor and anti-proliferative effects, it is currently mainly used in the treatment of breast cancer and colon cancer. No studies have shown that it has significant effects in the treatment of AML, and there are no previous cases of its application in the treatment of AML. In the early stage of this application, a large number of experimental verifications have confirmed that small molecule compounds have significant therapeutic effects in the treatment of AML.

[0032] In the present application, the drug has at least one of the following effects:

[0033] a: Treatment of AML;

[0034] b: Inhibit the proliferation of AML cells;

[0035] c: Induce apoptosis and / or differentiation of AML cells;

[0036] d: Inhibit the immune escape of AML cells;

[0037] e: Enhance the killing of AML cells by NK cells.

[0038] In this application, it is found through in vitro experiments that the small molecule compound QC6352 can not only inhibit the proliferation of AML cells, but also induce AML cell apoptosis and / or differentiation, and inhibit the immune escape of AML cells; in this application, it is also found that it can enhance the killing of AML cells by NK cells. The small molecule compound QC6352 can exert an excellent effect in treating AML.

[0039] In some examples of the present application, the AML cells may be AML cell lines, and specific examples include but are not limited to Thp-1 or U937. In other examples of the present application, the AML cells are mononuclear cells isolated from peripheral blood or bone marrow blood samples of patients with acute myeloid leukemia. It is understood that the specific separation method can be well known to those skilled in the art without special requirements. In some specific embodiments of the present application, density gradient centrifugation is used for separation and acquisition.

[0040] It is understood that the drug of the present application contains not only the small molecule compound QC6352 but also at least one pharmaceutically acceptable auxiliary material and / or carrier as required.

[0041] In this application, the pharmaceutically acceptable excipients and / or carriers refer to inactive substances that do not have harmful interactions with the active ingredient (small molecule compound QC6352 in this application) when co-formulated with the active ingredient and can ensure the stability, safety, manufacturability and convenience of administration of the pharmaceutical composition. The selection of excipients and / or carriers usually varies depending on the dosage form and the mode of administration. Those skilled in the art have such ability. When configuring or selecting, the selection of these excipients and / or carriers must comply with the requirements of relevant international or regional pharmaceutical standards.

[0042] In some examples, the auxiliary materials and / or carriers include but are not limited to excipients, disintegrants, binders, lubricants, glidants, coating agents, solvents / dispersion media, preservatives, antioxidants, pH regulators, osmotic pressure regulators, thickeners, and flavoring / coloring agents. For the specific types of auxiliary materials and / or carriers, conventional types in the art can be selected. For example, for excipients, it can be at least one of lactose, microcrystalline cellulose, mannitol, and calcium hydrogen phosphate; for disintegrants, it can be at least one of cross-linked carboxymethyl cellulose sodium, cross-linked polyvinylpyrrolidone, and low-substituted hydroxypropyl cellulose; for binders, it can be at least one of hydroxypropyl methylcellulose, polyvinyl pyrrolidone, and starch slurry; for lubricants, it can be at least one of magnesium stearate, talc, and sodium lauryl sulfate; for coating agents, it can be at least one of hydroxypropyl methylcellulose, ethyl cellulose, and polyvinyl alcohol; for solvents / dispersing media, it can be at least one of water for injection, ethanol, and propylene glycol; for preservatives, it can be at least one of sodium benzoate, parabens, and benzalkonium chloride; for antioxidants, it can be at least one of ascorbic acid, α-tocopherol, and sodium thiosulfate; for pH adjusters, it can be at least one of citrate buffer, phosphate buffer, and sodium hydroxide. It can be understood that the above excipients and / or carriers are merely examples made to make the technical solution clearer. The specific types of excipients and / or carriers that can be applied to the drugs in this application are not limited to the above examples. Those skilled in the art can select corresponding excipients and / or carriers according to the drug dosage form, administration method, etc. without special restrictions.

[0043] In the present application, the preferred mode of administration is oral administration or injection administration. According to the different modes of administration, the corresponding drug dosage form is designed. For oral administration, the dosage form of the drug can be tablets, capsules or granules, etc., but is not limited thereto. For injection administration, the drug is prepared into an injection, preferably a subcutaneous injection. In some specific examples, the drug dosage form is preferably a tablet or an injection.

[0044] In the drug of the present application, the content of the small molecule compound QC6352 is an effective amount. Here, "effective amount" refers to the amount of the active ingredient in the drug, which is sufficient to produce the expected pharmacological effect under specific administration methods and treatment conditions, thereby achieving disease prevention, treatment or symptom relief. There is no special limitation on the specific amount, and those skilled in the art can determine it through the pharmacokinetic and pharmacodynamic properties of common drugs in the field and clinical trial results. For example, in some examples, in an in vitro experiment, at a density of 2×10 5 / mL is used as the measurement standard, and the effective concentration of the small molecule compound QC6352 is 15-60nM, and can be any concentration of 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM or the range between any two concentrations. The dosage of other excipients and / or carriers can also be specifically configured according to the corresponding drug standards, according to the specific dosage form and administration method, etc., without any special limitation.

[0045] The following are specific embodiments of the present application. It should be noted that the following specific embodiments are only for illustrative purposes and do not limit the scope of the present application in any way.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0047] In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used are all commercially available.

[0048] Example 1 Acquisition of mononuclear cells

[0049] In this example, bone marrow and peripheral blood samples from healthy subjects and acute myeloid leukemia patients were collected from the laboratory of Anhui Provincial Hospital. The specific steps are as follows:

[0050] Peripheral blood or bone marrow blood samples were diluted with PBS at a volume ratio of 1:1, and the diluted blood samples were added to human mononuclear cell separation solution (Ficoll solution) with a Basai pipette, and the volume ratio of Ficoll solution to diluted peripheral blood was 1:1. Centrifuged at 800g for 30 minutes at room temperature, and the lifting and lowering speeds were all 0. The buffy coat layer between the upper serum layer and the Ficoll solution is the peripheral blood mononuclear cells (PBMC) / bone marrow blood mononuclear cells (BMMC) obtained. 10mL PBS was added, and 400g was centrifuged for 10 minutes at room temperature to fully wash the PBMC / BMMC, and the obtained cells were resuspended in complete culture medium (RPIM 1640 culture medium + 10% fetal bovine serum + 1% antibiotics) for subsequent experiments. Wherein, culture medium information in this embodiment: RPIM 1640 culture medium (BL303A, Biosharp), fetal bovine serum (F7524, Sigma), antibiotics (BL142A, Biosharp).

[0051] Example 2 CCK-8 assay to detect proliferation of AML cells

[0052] 1. Experimental methods

[0053] (1) Thp-1 (H3-0901, Saiye Biotechnology) and U937 cells (H5-0101, Saiye Biotechnology) in the logarithmic growth phase were collected and centrifuged at 300 g for 5 minutes at room temperature. The supernatant was discarded and the cells were resuspended in complete culture medium (same as in Example 1) and the cells were counted.

[0054] (2) According to the counting results, the cell density was adjusted to 2×10 5 / mL, mix the cell suspension thoroughly, and inoculate the cells into a 96-well plate with a volume of 100 μL per well.

[0055] (3) The small molecule compound QC6352 (HY-104048, MedChemExpress) was dissolved in DMSO, and the corresponding concentration of the small molecule drug compound QC6352 was added to the corresponding well plate according to the pre-designed drug concentration gradient (0; 15nM; 30nM; 60nM), and the cells were cultured in a cell culture incubator. Among them, 0nM means adding an equal volume of DMSO.

[0056] (4) On days 0, 1, 2, 3, and 4, 10 μL of CCK-8 reagent (K1018, APExBIO) was added to each well of 100 μL of culture system, gently shaken evenly, and incubated at 37°C in the dark for 3 h.

[0057] (5) Use an ELISA reader to measure the absorbance of each well at 450 nM.

[0058] 2. Experimental results

[0059] Figure 1 The results of CCK-8 experiment on AML cell proliferation are shown in Figure 2. It can be seen that QC6352 treatment with as low as 15 nM can significantly inhibit the proliferation of AML cells. Compared with AML cells in the control group, the growth rate of AML cells treated with QC6352 is significantly slowed down.

[0060] Example 3 Annexin-V / 7-AAD staining flow cytometry detection of cell apoptosis.

[0061] In this example, in vitro experiments were performed to investigate the apoptosis of AML cell lines and primary cells of AML patients at different concentrations of the small molecule compound QC6352. In order to preliminarily explore the safety of the small molecule compound QC6352, different concentrations of QC6352 were used to treat bone marrow mononuclear cells of healthy donors in this example.

[0062] 1. Experimental methods

[0063] (1) Apoptosis of acute myeloid leukemia cell lines

[0064] Take the human acute myeloid leukemia cell lines Thp-1 and U937 in the logarithmic growth phase, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, resuspend and mix with 1mL complete culture medium (RPIM 1640 culture medium + 10% fetal bovine serum + 1% antibiotics), and count the number of cells under a microscope. According to the counting results, adjust the number of cells and inoculate the cells into a 48-well plate, with 2×10 cells per well. 5 Cells were treated with 500 μL system per well. Drug QC6352 was added according to the concentration gradient (0; 15 nM; 30 nM; 60 nM; 120 nM; 240 nM) for 48 h.

[0065] After the treatment time is reached, the cells are transferred from the 48-well plate to a 1.5 mL EP tube, centrifuged at 300 g for 5 minutes to remove the culture medium, and then washed twice with PBS, resuspended with 100 μL Annexin V Binding Buffer, and 2 μL PE-Annexin V antibody and 2 μL 7-AAD dye are added thereto, shaken and mixed, and incubated at room temperature in the dark for 15 minutes. After 15 minutes, 100 μL Annexin V Binding Buffer is added thereto, and the cells are transferred to a flow tube and detected using a BD flow cytometer. The apoptosis kit used above is the PE-Annexin V apoptosis kit (BD, Cat#559763).

[0066] (2) Primary cells from patients with acute myeloid leukemia

[0067] Take the BMMC of the acute myeloid leukemia patient obtained in Example 1, count the number of cells under a microscope and adjust the number of cells according to the counting result, and inoculate the cells into a 48-well plate, with 2×10 5 Cells were treated with 500 μL system per well. Drug QC6352 was added according to the concentration gradient (0; 15 nM; 30 nM; 60 nM) for 48 h.

[0068] After the treatment time is reached, the cells are transferred from the 48-well plate to a 1.5 mL EP tube, centrifuged at 400 g for 8 minutes to remove the culture medium, washed twice with PBS, resuspended with 100 μL PBS, and 0.5 μL anti-CD45 (304014, Biolegend) and anti-CD33 antibodies (303408, Biolegend) are added to label AML cells, and incubated at room temperature in the dark for 25 minutes. After the time is reached, 1 mL PBS is added and centrifuged at 400 g for 10 minutes to wash away excess antibodies. The supernatant is discarded, the cells are resuspended with 100 μL Annexin V Binding Buffer, and 2 μL PE-Annexin V antibody and 2 μL 7-AAD dye are added thereto, shaken and mixed, and incubated at room temperature in the dark for 15 minutes. After 15 minutes, 100 μL Annexin V Binding Buffer is added thereto, and the cells are transferred to a flow tube and detected using a BD flow cytometer.

[0069] (3) Bone marrow mononuclear cells from healthy donors

[0070] Take the BMMC of the healthy donor obtained in Example 1, count the number of cells under a microscope and adjust the number of cells according to the counting result, and inoculate the cells into a 48-well plate, with 2×10 5 Cells were treated with 500 μL system per well. Drug QC6352 was added according to the concentration gradient (0; 15 nM; 30 nM; 60 nM) for 48 h.

[0071] When the treatment time is reached, transfer the cells from the 48-well plate to a 1.5 mL EP tube, centrifuge at 400 g for 8 minutes to remove the culture medium, wash twice with PBS, resuspend with 100 μL PBS, add 0.5 μL anti-CD45 antibody to label leukocytes, and incubate at room temperature in the dark for 25 minutes. After the time is up, add 1 mL PBS and centrifuge at 400 g for 10 minutes to wash away excess antibodies. Discard the supernatant, resuspend the cells with 100 μL Annexin V Binding Buffer, add 2 μL PE-Annexin V antibody and 2 μL 7-AAD dye, shake and mix, and incubate at room temperature in the dark for 15 minutes. After 15 minutes, add 100 μL Annexin VBinding Buffer, transfer to a flow tube, and detect with a BD flow cytometer.

[0072] (4) In addition, in order to demonstrate the significant advantages of the small molecule compound QC6352 in the present application in treating AML, the same apoptosis experiment of acute myeloid leukemia cell line was carried out in this example using SD49-7 as a control agent. The specific steps are as follows:

[0073] Take the human acute myeloid leukemia cell line Thp-1 in the logarithmic growth phase, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, resuspend and mix with 1mL complete culture medium (RPIM 1640 culture medium + 10% fetal bovine serum + 1% antibiotics), and count the number of cells under a microscope. According to the counting results, adjust the number of cells and inoculate the cells into a 48-well plate, with 2×10 cells per well. 5 Cells were treated with 500 μL system per well. Drug SD49-7 (Selleck, E1335) was added according to the concentration gradient (0; 250 nM; 500 nM) for 48 h.

[0074] After the treatment time, the cells were transferred from the 48-well plate to a 1.5 mL EP tube, centrifuged at 300 g for 5 minutes to remove the culture medium, and then washed twice with PBS. The cells were resuspended with 100 μL Annexin V Binding Buffer, and 2 μL PE-Annexin V antibody and 2 μL 7-AAD dye were added, shaken and mixed, and incubated at room temperature in the dark for 15 minutes. After 15 minutes, 100 μL Annexin V Binding Buffer was added, and the cells were transferred to a flow tube and detected using a BD flow cytometer.

[0075] 2. Experimental results

[0076] Figure 2 The results show the proportion of live cells in AML cell lines Thp-1 and U937 treated with different concentrations of the small molecule compound QC6352 in vitro after 48 hours. Figure 3 The results show the proportion of apoptotic cells in primary cells of AML patients treated with small molecule compound QC6352 at different concentrations in vitro after 48 hours. It can be seen that the small molecule compound QC6352 can induce apoptosis in AML cell lines Thp-1 and U937, as well as primary cells of AML patients at very low concentrations (starting from 15nM).

[0077] Figure 4 The results show the percentage of apoptotic cells in healthy donor bone marrow mononuclear cells treated with small molecule compound QC6352 at different concentrations after 48 hours. It can be seen that the small molecule compound QC6352 has no significant effect on the viability of mononuclear cells in healthy human bone marrow and is safe.

[0078] Figure 5 The results of apoptosis induction of AML cell line Thp-1 by SD49-7 at different concentrations are shown. It was found that after the same treatment for 48 hours, SD49-7 at a higher concentration (250 nM or 500 nM) still had no apoptosis induction effect on AML cells compared with QC6352.

[0079] The above results indicate that the small molecule compound QC6352 can specifically target AML cells at a lower concentration without affecting other normal blood cells in the body and has good safety.

[0080] Example 4 Small molecule compound QC6352 induces AML cell differentiation

[0081] Since one of the pathogenesis of AML is the abnormal blockage of differentiation of hematopoietic stem cells, promoting the differentiation of AML cells is a treatment for acute myeloid leukemia. The current clinical differentiation treatment regimen is retinoic acid combined with arsenic, which is used to treat M3 promyelocytic leukemia. According to previous clinical and preclinical research results, in non-M3 acute myeloid, promoting the differentiation of AML cells can significantly inhibit the self-renewal and proliferation of AML cells and reduce the stemness of AML cells. Therefore, inducing differentiation is a new way to treat acute myeloid leukemia. In this example, the small molecule compound QC6352 was used to observe the differentiation of AML cells induced by Giemsa staining, and the cell surface protein level was detected by flow cytometry to investigate the differentiation of AML cells induced by the small molecule compound QC6352.

[0082] 1. Experimental methods

[0083] (1) Giemsa staining

[0084] Thp-1 and U937 cell suspensions (500 μL / well) were inoculated in 48-well plates, with approximately 2 × 10 cells per well. 5 cells. 0.5 μL of DMSO (1084ML500, BioFroxx) or 30 nM QC6352 was added to the cell suspension and cultured in a 37°C incubator with complete medium (same as in Example 1) for 48 hours. After 48 hours, the cell suspension was transferred to a 1.5 mL EP tube, centrifuged at 300 g for 5 minutes at room temperature, the supernatant medium was discarded, and PBS was added to wash twice. The cells were resuspended with 100 μL PBS and counted under a microscope, and the appropriate density was adjusted to about 8×10 5 / mL. Take 100μL of cell suspension and use a spinner to prepare a spin slide. After the spin slide is completed, dry the residual water. Add GIEMSA dye solution (BA4017, Beso Biological) A solution to the glass slide to cover the entire cell area and stain at room temperature for 1 minute. After 1 minute, add 3 times the volume of GIEMSA dye solution B solution to fully blend it with A solution and stain at room temperature for 5 minutes. After 5 minutes, rinse the dye with running water and let the glass slide dry naturally. The dried glass slide is used to observe cell morphology under a microscope later.

[0085] (2) Flow cytometry to detect cell surface protein levels

[0086] Take the human acute myeloid leukemia cell lines Thp-1 and U937 in the logarithmic growth phase, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, resuspend and mix with 1mL complete medium, and count the number of cells under a microscope. According to the counting results, adjust the number of cells and inoculate the cells into a 48-well plate, with 2×10 cells per well. 5 Cells, 500 μL system per well. Add drug QC6352 to treat cells according to concentration gradient (0; 15nM; 30nM; 60nM), and the treatment time is 48h. After the treatment time is reached, the cells are transferred from the 48-well plate to a 1.5mL EP tube, centrifuged at 300g for 5 minutes to remove the culture medium, and then washed twice with PBS, resuspended with 100μL PBS, and added with 1μL APC-CY7-CD11b antibody (Biolegend, Cat#301342) / APC-CY7-MICA / B antibody (Biolegend, Cat#320908) / PE-ULBP3 antibody (R&D, Cat#FAB1517P), shaken and mixed, and incubated at room temperature in the dark for 25 minutes. After the time is reached, add 1mL PBS to wash once, resuspend with 200μL PBS, and transfer to a flow tube for detection using a BD flow cytometer.

[0087] 2. Experimental results

[0088] Figure 6 The staining results of the cells after the addition of the small molecule compound QC6352 to the AML cell line for 48 hours are shown. It can be seen that compared with the untreated group (DMSO treatment), the AML cells treated with the small molecule compound QC6352 showed a reduced nuclear-cytoplasmic ratio and a horseshoe-shaped nucleus, which resembled differentiated myeloid cells.

[0089] Figure 7 The flow cytometry results after 48 hours of treatment with the small molecule compound QC6352 are shown. Figure 5 It can be seen that after 48 hours of QC6352 treatment, the proportion of cells expressing the myeloid differentiation marker CD11b on the cell surface increased significantly, indicating that QC6352 induced more cells to differentiate. Figure 8 The results of the expression of MICA / B and ULBP3 on the surface of Thp-1 and U937 cells after being treated with different concentrations of QC6352 for 48 hours are shown. It can be seen that after being treated with different concentrations of QC6352 for 48 hours, the expression of MICA / B and ULBP3 on the surface of Thp-1 and U937 cells increased, indicating that QC6352 not only induced apoptosis and differentiation of AML cells, but also increased the expression of MICA / B and ULBP3, the ligands of NK cell activating receptor NKG2D on the surface of tumor cells.

[0090] The above results indicate that the small molecule compound QC6352 can induce AML cell differentiation.

[0091] Example 5 PBMC and AML cell in vitro co-culture model

[0092] 1. Experimental methods

[0093] Take the human acute myeloid leukemia cell lines Thp-1 and U937 in the logarithmic growth phase, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, resuspend and mix with 1mL complete medium, and count the number of cells under a microscope. According to the counting results, adjust the number of cells and inoculate the cells into 6-well plates, with 2×10 cells per well. 6 AML cells were treated with 30 nM QC6352 or an equal volume of DMSO for 48 h. The drug was washed off after the time, and the AML cells were stained for live cells with CellTrace Violet dye (Invitrogen, Cat#C34571), and the cells were counted.

[0094] After the human PBMC obtained by density gradient centrifugation was incubated overnight at 37°C, the cells were counted. PBMC were co-cultured with AML cells at an effector-target ratio of E:T = 2.5:1 / 5:1 / 10:1 for 4 hours. Flow cytometry was used to detect the apoptosis of AML cells.

[0095] 2. Experimental results

[0096] Fig. 9 The results of PBMC killing against blank control and AML cell lines treated with 30nM QC6352 for 48h are shown, among which Annexin-V+ cells are apoptotic cells, and their proportion in total cells reflects the degree of killing of AML cells by NK cells. It can be seen that AML cells treated with QC6352 are more easily killed by NK cells.

[0097] Based on the above experimental results, it can be concluded that the small molecule compound QC6352 can be used as a new drug for the targeted treatment of AML. Low concentrations of QC6352 can effectively act on AML cells and have no obvious toxicity to the bone marrow mononuclear cells of healthy people. In addition, QC6352 can not only induce apoptosis of AML cells, but also upregulate the expression of NKG2D ligands on the surface of tumor cells, thereby enhancing the killing effect of immune cells.

[0098] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and the same effect as the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. The use of the small molecule compound QC6352 in the preparation of medicines, characterized in that: The drug has at least one of the following effects: a: Treatment of AML; b: Inhibit the proliferation of AML cells; c: Induce apoptosis and / or differentiation of AML cells; d: Inhibit the immune escape of AML cells; e: Enhance the killing of AML cells by NK cells.

2. The use according to claim 1, characterized in that The AML cells are Thp-1 or U937.

3. The use according to claim 1, characterized in that The AML cells are peripheral blood or bone marrow mononuclear cells from patients with acute myeloid leukemia.

4. A drug, characterized in that The medicine contains the small molecule compound QC6352.

5. The drug according to claim 4, characterized in that The drug has at least one of the following effects: a: Treatment of AML; b: Inhibit the proliferation of AML cells; c: Induce apoptosis and / or differentiation of AML cells; d: Inhibit the immune escape of AML cells; e: Enhance the killing of AML cells by NK cells.

6. The drug according to claim 5, characterized in that The AML cells are Thp-1 or U937.

7. The drug according to claim 5, characterized in that The AML cells are peripheral blood or bone marrow mononuclear cells from patients with acute myeloid leukemia.

8. The drug according to claim 4, characterized in that The drug further comprises at least one pharmaceutically acceptable auxiliary material and / or carrier.

9. The drug according to claim 4, characterized in that The dosage form of the medicine is tablet or injection.

10. The drug according to any one of claims 4 to 9, characterized in that The AML cell density was 2×10 5 / mL is used as the measurement standard, and the effective concentration of the small molecule compound QC6352 is 15-60nM.

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