Application of compound in preparation of medicament for treating leukemia

Abstract

The invention discloses application of a compound in preparation of a medicament for treating leukemia. The invention relates to application of a compound with a structure as shown in a formula (I) or a salt thereof in preparation of a medicament for treating leukemia. # imgabs0 #

Description

Technical Field

[0001] The invention relates to use of a compound or a salt thereof in preparing a drug for treating leukemia. Background Art

[0002] Leukemia is a malignant clonal disease originating from hematopoietic stem cells. Its complex pathogenesis is associated with multiple factors such as biology, physics, chemistry, genetics and other blood diseases. The uncontrolled proliferation of leukemia cells in the bone marrow and other hematopoietic tissues leads to the suppression of normal hematopoiesis and infiltration into other organs.

[0003] According to the degree of cell differentiation and maturity and the natural course of the disease, leukemia can be divided into acute leukemia (AL) and chronic leukemia (CL). According to the main affected cell series, leukemia can be divided into lymphocytic leukemia and non-lymphocytic (myeloid) leukemia. AL can be divided into acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). CL can be divided into chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) and rare types of leukemia such as hairy cell leukemia and prolymphocytic leukemia.

[0004] CN116655605A discloses a phenylpyrazole compound, a preparation method and an application thereof. Provided is a phenylpyrazole compound as shown in formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof. Such compounds can effectively and selectively inhibit the key protein MCL-1 in the process of apoptosis at the molecular level, or they have a significant killing effect and high selectivity on cancer cells, especially human plasma cell leukemia cells H929 and human myelomonocytic leukemia cells MV-4-11, and have the potential to be prepared into a new anti-acute myeloid leukemia drug.

[0005] CN118615282A discloses a clematis alkaloid compound and its application, and the application of the clematis alkaloid compound in preparing a pharmaceutical composition for preventing or treating cancer, wherein the cancer is leukemia, and the clematis alkaloid compound is clematisindolin A. Summary of the invention

[0006] The object of the present invention is to provide a compound as shown in formula (I) or a salt thereof for use in preparing a drug for treating leukemia.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned purpose.

[0008] The present invention provides a compound having a structure as shown in formula (I) or a salt thereof for use in preparing a drug for treating leukemia:

[0009]

[0010] According to the use of the present invention, preferably, the drug forms a pharmaceutical preparation for treating leukemia; the pharmaceutical preparation comprises a compound as shown in formula (I).

[0011] According to the use of the present invention, preferably, the leukemia is acute leukemia or chronic leukemia.

[0012] According to the use of the present invention, preferably, the drug forms a pharmaceutical preparation for inhibiting the proliferation of acute myeloid leukemia cell lines.

[0013] According to the use of the present invention, preferably, in a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 4 μM and the experiment is performed for 48 hours, the cell proliferation inhibition rate of human acute myeloid leukemia cell MOLM-13 is greater than 99%.

[0014] According to the use of the present invention, preferably, the drug forms a pharmaceutical preparation for inhibiting the proliferation of promyelocytic leukemia cell lines.

[0015] According to the use of the present invention, preferably, in a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 4 μM and the experiment is carried out for 24 hours, the cell proliferation inhibition rate of human promyelocytic leukemia cells HL60 is greater than 97%.

[0016] According to the use of the present invention, preferably, the drug forms a pharmaceutical preparation for inhibiting the proliferation of myelomonocytic leukemia cell lines.

[0017] According to the use of the present invention, preferably, in a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 2 μM and the experiment lasts for 72 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cell MV4-11 is greater than 96%.

[0018] According to the use of the present invention, preferably, the drug forms a pharmaceutical preparation for inhibiting the proliferation of chronic myeloid leukemia cell lines.

[0019] The compound of the present invention as shown in formula (I) has a good cell proliferation inhibitory effect on leukemia, has substantially no drug toxicity to normal non-tumor cells, and has high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the IC of YL-939 in Example 7 for tumor stem cells from different acute myeloid leukemia patients 50 value.

[0021] Figure 2 The IC of YL-939 in Example 8 for tumor stem cells derived from patients with acute myeloid leukemia resistant to venetola 50 value. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0023] The present invention provides a compound having a structure represented by formula (I) or a salt thereof for use in preparing a drug for treating leukemia:

[0024]

[0025] In the present invention, the molecular formula of the compound represented by formula (I) is C 25 H 26 N6O, molecular weight is 426.51. The compound represented by formula (I) is a known compound, CAS No.: 3023925-68-7, and can be purchased commercially.

[0026] In the prior art, the compound shown in formula (I) is also called YL-939. YL-939 is an effective ferroptosis inhibitor. YL-939 inhibits ferroptosis by targeting the PHB2 / ferritin / iron axis and is a PHB2 inhibitor. It is generally believed that the expression of PHB2 is related to the proliferation, metastasis and metabolism of various solid tumors such as melanoma, breast cancer, and lung cancer; however, there is no literature report on whether PHB2 is involved in regulating immune function, especially tumor immune function. So far, no reports have been found on the use of YL-939 for the preparation of drugs for the treatment of leukemia. The present invention finds that the compound shown in formula (I) (also called YL-939) has a good cell proliferation inhibitory effect on leukemia, and has basically no drug toxicity to normal non-tumor cells, and has high safety.

[0027] In the present invention, the drug forms a pharmaceutical preparation for treating leukemia; the pharmaceutical preparation comprises a compound as shown in formula (I). The pharmaceutical preparation may also comprise other known active ingredients for treating leukemia, and pharmaceutically acceptable excipients. According to a preferred embodiment of the present invention, the active ingredients in the pharmaceutical preparation only comprise the compound as shown in formula (I) of the present invention.

[0028] In the present invention, the dosage form of the pharmaceutical preparation is not limited, and can be tablets, granules, capsules, pills, oral liquids, injections, etc. The pharmaceutical preparation may also contain pharmaceutically acceptable excipients. The types of the pharmaceutically acceptable excipients are not limited. Excipients may be fillers, flavoring agents, lubricants, etc. Fillers are also called diluents, such as wheat starch, tapioca starch, corn starch, potato starch, dextrin, lactose, etc. Examples of flavoring agents include but are not limited to sucralose, isomaltulose, aspartame, acesulfame potassium, etc. Examples of lubricants include but are not limited to magnesium stearate, talc, micro powder silica gel, magnesium lauryl sulfate, etc.

[0029] In the present invention, the leukemia is acute leukemia or chronic leukemia.

[0030] According to one aspect of the present invention, the drug forms a pharmaceutical preparation for inhibiting the proliferation of acute myeloid leukemia cell lines. In a cell experiment, when the concentration of the compound shown in formula (I) is greater than or equal to 4 μM and the experiment is 48h, the cell proliferation inhibition rate of human acute myeloid leukemia cell MOLM-13 reaches greater than 99%.

[0031] According to a specific embodiment of the present invention, in a cell experiment, when the concentration of the compound represented by formula (I) is 4 μM and the experiment is carried out for 24 hours, the cell proliferation inhibition rate of human acute myeloid leukemia cells MOLM-13 reaches 83.54%; at 48 hours of the experiment, the cell proliferation inhibition rate of human acute myeloid leukemia cells MOLM-13 reaches 99.57%.

[0032] In the present invention, the drug forms a pharmaceutical preparation for inhibiting the proliferation of acute myeloid leukemia cell lines. In a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 4 μM and the experiment is carried out for 48 hours, the cell proliferation inhibition rate of human acute myeloid leukemia cells OCL-AML3 reaches greater than 99%.

[0033] According to a specific embodiment of the present invention, in a cell experiment, when the concentration of the compound represented by formula (I) was 4 μM and the experiment lasted for 48 hours, the cell proliferation inhibition rate of human acute myeloid leukemia cells OCL-AML3 reached 99.93%.

[0034] According to a specific aspect of the present invention, the drug forms a pharmaceutical preparation for inhibiting the proliferation of promyelocytic leukemia cell lines. In a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 4 μM and the experiment is carried out for 24 hours, the cell proliferation inhibition rate of human promyelocytic leukemia cells HL60 reaches greater than 97%.

[0035] According to another specific embodiment of the present invention, in a cell experiment, when the concentration of the compound represented by formula (I) was 4 μM and the experiment lasted for 24 hours, the cell proliferation inhibition rate of human promyelocytic leukemia cells HL60 reached 97.58%; at 48 hours of the experiment, the cell proliferation inhibition rate of human promyelocytic leukemia cells HL60 reached 99.89%.

[0036] According to another specific aspect of the present invention, the drug forms a pharmaceutical preparation for inhibiting the proliferation of myelomonocytic leukemia cell lines. In a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 2 μM and the experiment is carried out for 72 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cell MV4-11 reaches greater than 96%.

[0037] According to a specific embodiment of the present invention, in a cell experiment, when the concentration of the compound shown in formula (I) is 2 μM and the experiment is 72 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cell MV4-11 reaches 96.68%. When the concentration of the compound shown in formula (I) is 4 μM and the experiment is 24 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cell MV4-11 reaches 90.09%; at 48 hours of the experiment, the cell proliferation inhibition rate of human myelomonocytic leukemia cell MV4-11 reaches 99.71%.

[0038] According to another aspect of the present invention, the drug forms a pharmaceutical preparation for inhibiting the proliferation of chronic myeloid leukemia cell lines. In a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 4 μM and the experiment is carried out for 48 hours, the cell proliferation inhibition rate of human chronic myeloid leukemia cells K562 reaches greater than 99%.

[0039] According to a specific embodiment of the present invention, in a cell experiment, when the concentration of the compound represented by formula (I) was 4 μM and the experiment lasted for 48 hours, the cell proliferation inhibition rate of human chronic myeloid leukemia cells K562 reached 99.74%.

[0040] Furthermore, the compound represented by formula (I) of the present invention (i.e., YL-939) has an effective inhibitory effect on leukemia cells at low doses, and does not show drug toxicity to normal non-tumor cells at effective doses, and has very good safety, making it suitable for further research and development as a potential anti-tumor drug.

[0041] Furthermore, in the present invention, the compound represented by formula (I) (or referred to as YL-939) of the present invention has an inhibitory effect on the proliferation of acute myeloid leukemia tumor stem cells. After 48 hours of intervention, YL-939 has a significant inhibitory effect on acute myeloid leukemia tumor stem cells at a low dose, IC 50The range is around 1.7-2.3 μM. This result shows that YL-939 can inhibit the proliferation of tumor stem cells in patients with acute myeloid leukemia and has the potential to become a drug for the treatment of leukemia.

[0042] In addition, the present invention also found that the compound shown in formula (I) (or referred to as YL-939) has a significant inhibitory effect on venetola-resistant acute myeloid leukemia tumor stem cells. After 48 hours of intervention, YL-939 has a significant inhibitory effect on venetola-resistant acute myeloid leukemia tumor stem cells at a low dose, IC 50 The range is about 1.7-2.4 μM. The above results show that YL-939 can inhibit the proliferation of acute myeloid leukemia tumor stem cells, especially for leukemia stem cells resistant to venetola, and is expected to become a tumor treatment drug that overcomes venetola resistance.

[0043] Example 1 - Inhibitory effect of YL939 on proliferation of human promyelocytic leukemia cells (HL60 cells)

[0044] 1.1 Experimental Materials:

[0045] YL-939 (ie, the compound represented by formula (I)) was purchased from Taoshu (Shanghai) Biotechnology Co., Ltd., Cat#T73513. Human promyelocytic leukemia cell line HL60 was purchased from CyBio (Shanghai) Biotechnology Co., Ltd., Cat#iCell-h098. Luminescent cell viability assay kit was purchased from Promega (Beijing) Biotechnology Co., Ltd., Cat#G7571.

[0046] 1.2 Experimental methods:

[0047] Human promyelocytic leukemia HL60 cells were seeded in a white 96-well plate, 5000 cells / well, and cultured in 100μl IMDM + 10% FBS. The experiment was divided into 4 groups, and YL-939 was added to the cells in the well plate at concentrations of 0μM, 1μM, 2μM, and 4μM, respectively. Each group had 6 replicate wells. After 24h, 48h, and 72h of culture, the cells were treated with Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, mix on an orbital shaker for 2 min, incubate the plate at room temperature for 10 min, and read using a microplate luminometer ( Navigator, Promega) to detect luminescent signals. CellTiter-Glo is a cleavage-based detection method based on ATP. Luciferin is oxidized by luciferase with the participation of ATP to produce light. The ATP in the luciferase reaction comes entirely from the ATP of living cells, so the light intensity produced is proportional to the number of living cells.

[0048] 1.3 Calculation of cell proliferation inhibition rate:

[0049] Cell proliferation inhibition rate (%) = 1-[A(drug addition)-A(blank)] / [A(0 drug addition)-A(blank)]×100%, where:

[0050] A (dosing): with cells, The optical signal intensity of the wells with Reagent and drug solutions;

[0051] A (blank): with culture medium and The light signal intensity of the wells with Reagent but no cells;

[0052] A(0 drug addition): with cells, Reagent but no drug solution.

[0053] The drug solution refers to the YL-939 solution.

[0054] 1.4 Experimental results:

[0055] The experimental results are shown in Table 1.

[0056] Table 1 Cell proliferation inhibition rate of YL-939 on human promyelocytic leukemia cells HL60 (%, M±SD)

[0057] 0μM 1μM 2μM 4μM 24h 0±2.61 19.44±1.81 26.60±1.97 97.58±0.23 48h 0±2.71 34.37±1.34 36.29±2.35 99.89±0.01 72h 0±6.11 35.94±1.03 40.56±4.31 99.95±0.01

[0058] As can be seen from Table 1, YL-939 significantly inhibited the proliferation of HL60 cells. After different concentrations of YL-939 (1μM, 2μM, 4μM) intervened in HL60 cells, cell proliferation inhibition results were shown after 24h, 48h and 72h of intervention, respectively. Among them, the cell proliferation inhibition rate reached 97.58% after 24h of intervention using a concentration of 4μM.

[0059] The above results indicate that YL-939 can inhibit the proliferation of human promyelocytic leukemia cell lines and has the potential to become a drug for the treatment of leukemia.

[0060] Example 2 - Inhibitory effect of YL-939 on proliferation of human acute myeloid leukemia cells (MOLM-13 cells)

[0061] 2.1 Experimental Materials:

[0062] The source of YL-939 is the same as above. Human acute myeloid leukemia cell line MOLM-13 was purchased from Synuclein (Shanghai) Biotechnology Co., Ltd., Cat# iCell-h423. The source of the Luminescent Cell Viability Assay Kit is the same as above.

[0063] 2.2 Experimental methods:

[0064] Human acute myeloid leukemia MOLM-13 cells were inoculated into a white 96-well plate, 5000 cells / well, and cultured in 100μl RPMI1640+10% FBS. The experiment was divided into 4 groups, and YL-939 was added to the cells in the well plate at concentrations of 0μM, 1μM, 2μM, and 4μM, respectively. Each group had 6 replicate wells. After 24h, 48h, and 72h of culture, the cells were treated with Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, mix on an orbital shaker for 2 min, incubate the plate at room temperature for 10 min, and read using a microplate luminometer ( Navigator, Promega) to detect luminescent signals. CellTiter-Glo is a cleavage-based detection method based on ATP. Luciferin is oxidized by luciferase with the participation of ATP to produce light. The ATP in the luciferase reaction comes entirely from the ATP of living cells, so the light intensity produced is proportional to the number of living cells.

[0065] 2.3 Calculation of cell proliferation inhibition rate:

[0066] The calculation method is the same as above.

[0067] 2.4 Experimental results:

[0068] The experimental results are shown in Table 2.

[0069] Table 2 Cell proliferation inhibition rate of YL-939 on human acute myeloid leukemia cell MOLM-13 (%, M ± SD)

[0070] 0μM 1μM 2μM 4μM 24h 0±2.41 5.07±0.33 38.51±0.49 83.54±0.78 48h 0±9.30 52.16±2.91 80.19±2.66 99.57±0.03 72h 0±4.60 50.62±4.68 95.10±1.08 99.96±0.00

[0071] As can be seen from Table 2, YL-939 significantly inhibited the proliferation of MOLM-13 cells. After different concentrations of YL-939 (1μM, 2μM, 4μM) intervened in MOLM-13 cells, cell proliferation inhibition results were shown after 24h, 48h and 72h of intervention, respectively. Among them, the cell proliferation inhibition rate reached 80.19% after 48h of intervention using a smaller concentration of 2μM.

[0072] The above results indicate that YL-939 can inhibit the proliferation of human acute myeloid leukemia cell lines and has the potential to become a drug for the treatment of leukemia.

[0073] Example 3 - Inhibitory effect of YL-939 on proliferation of human myelomonocytic leukemia cells (MV4-11 cells)

[0074] 3.1 Experimental Materials:

[0075] The source of YL-939 is the same as above. Human myelomonocytic leukemia line MV4-11 was purchased from Synbio (Shanghai) Biotechnology Co., Ltd., Cat# iCell-h381. The source of the Luminescent Cell Viability Assay Kit is the same as above.

[0076] 3.2 Experimental methods:

[0077] Human myelomonocytic leukemia cells MV4-11 (or MV-4-11) were inoculated into a white 96-well plate, 5000 cells / well, and cultured in 100μl IMDM + 10% FBS. The experiment was divided into 4 groups, and YL-939 was added to the cells in the well plate at concentrations of 0μM, 1μM, 2μM, and 4μM, respectively. Each group had 6 replicate wells. After 24h, 48h, and 72h of culture, the cells were treated with Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, mix on an orbital shaker for 2 min, incubate the plate at room temperature for 10 min, and read using a microplate luminometer ( Navigator, Promega) to detect luminescent signals. CellTiter-Glo is a cleavage-based detection method based on ATP. Luciferin is oxidized by luciferase with the participation of ATP to produce light. The ATP in the luciferase reaction comes entirely from the ATP of living cells, so the light intensity produced is proportional to the number of living cells.

[0078] 3.3 Calculation of cell proliferation inhibition rate:

[0079] The calculation method is the same as above.

[0080] 3.4 Experimental results:

[0081] The experimental results are shown in Table 3.

[0082] Table 3 Inhibitory effect of YL-939 on proliferation of human myelomonocytic leukemia cell line MV4-11 (%, M ± SD)

[0083] 0μM 1μM 2μM 4μM 24h 0±5.93 11.33±4.73 42.79±2.83 90.09±0.31 48h 0±2.08 17.63±3.27 79.99±0.99 99.71±0.04 72h 0±6.63 14.59±3.53 96.68±0.59 99.98±0.00

[0084] As can be seen from Table 3, YL-939 significantly inhibited the proliferation of MV4-11 cells. After different concentrations of YL-939 (1μM, 2μM, 4μM) intervened in MV4-11 cells, cell proliferation inhibition results were shown after 24h, 48h and 72h of intervention, respectively. Among them, the cell proliferation inhibition rate reached 79.99% after 48h of intervention using a smaller concentration of 2μM.

[0085] The above results indicate that YL-939 can inhibit the proliferation of human myelomonocytic leukemia cell lines and has the potential to become a drug for the treatment of leukemia.

[0086] Example 4 - Inhibitory effect of YL-939 on proliferation of human acute myeloid leukemia cells (OCL-AML3 cells)

[0087] 4.1 Experimental Materials:

[0088] The source of YL-939 is the same as above. Human acute myeloid leukemia cells OCL-AML3 were purchased from Pronocell (Wuhan) Biotechnology Co., Ltd., Cat# CL-0837. The source of the Luminescent Cell Viability Assay Kit is the same as above.

[0089] 4.2 Experimental methods:

[0090] Human acute myeloid leukemia cells OCL-AML3 were inoculated into a white 96-well plate, 5000 cells / well, and cultured in 100μl IMDM + 15% FBS. The experiment was divided into 4 groups, and YL-939 was added to the cells in the well plate at concentrations of 0μM, 1μM, 2μM, and 4μM, respectively. Each group had 6 replicate wells. After 24h, 48h, and 72h of culture, the cells were treated with Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, mix on an orbital shaker for 2 min, incubate the plate at room temperature for 10 min, and read using a microplate luminometer ( Navigator, Promega) to detect luminescent signals. CellTiter-Glo is a cleavage-based detection method based on ATP. Luciferin is oxidized by luciferase with the participation of ATP to produce light. The ATP in the luciferase reaction comes entirely from the ATP of living cells, so the light intensity produced is proportional to the number of living cells.

[0091] 4.3 Calculation of cell proliferation inhibition rate:

[0092] The calculation method is the same as above.

[0093] 4.4 Experimental results:

[0094] The experimental results are shown in Table 4.

[0095] Table 4 Inhibitory effect of YL-939 on proliferation of human acute myeloid leukemia cells OCL-AML3 (%, M±SD)

[0096] 0μM 1μM 2μM 4μM 24h 0±4.84 18.19±2.73 19.05±1.87 87.79±1.02 48h 0±1.79 55.47±1.51 61.41±1.20 99.93±0.01 72h 0±1.51 63.12±2.08 72.73±0.71 99.92±0.01

[0097] As can be seen from Table 4, YL-939 significantly inhibited the proliferation of OCL-AML3 cells. After different concentrations of YL-939 (1 μM, 2 μM, 4 μM) intervened in OCL-AML3 cells, cell proliferation inhibition results were shown after 24 h, 48 h and 72 h of intervention, respectively. Among them, the cell proliferation inhibition rate reached 72.73% after 72 h of intervention with 2 μM.

[0098] The above results indicate that YL-939 can inhibit the proliferation of human acute myeloid leukemia cell lines and has the potential to become a drug for the treatment of leukemia.

[0099] Example 5 - Inhibitory effect of YL-939 on proliferation of human chronic myeloid leukemia cells (K562 cells)

[0100] 5.1 Experimental Materials:

[0101] The source of YL-939 is the same as above. Human chronic myeloid leukemia cell line K562 cells were purchased from Synbio (Shanghai) Biotechnology Co., Ltd. Cat# iCell-h118. The source of the Luminescent Cell Viability Assay Kit is the same as above.

[0102] 5.2 Experimental methods:

[0103] Human chronic myeloid leukemia K562 cells were inoculated into a white 96-well plate, 5000 cells / well, and cultured in 100μl IMDM + 10% FBS. The experiment was divided into 4 groups, and YL-939 was added to the cells in the well plate at concentrations of 0μM, 1μM, 2μM, and 4μM, respectively. Each group had 6 replicate wells. After 24h, 48h, and 72h of culture, the cells were treated with Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, mix on an orbital shaker for 2 min, incubate the plate at room temperature for 10 min, and read using a microplate luminometer ( Navigator, Promega) to detect luminescent signals. CellTiter-Glo is a cleavage-based detection method based on ATP. Luciferin is oxidized by luciferase with the participation of ATP to produce light. The ATP in the luciferase reaction comes entirely from the ATP of living cells, so the light intensity produced is proportional to the number of living cells.

[0104] 5.3 Calculation of cell proliferation inhibition rate:

[0105] The calculation method is the same as above.

[0106] 5.4 Experimental results:

[0107] The experimental results are shown in Table 5.

[0108] Table 5 Cell proliferation inhibition rate of YL-939 on human chronic myeloid leukemia cells K562 (%, M±SD)

[0109] 0μM 1μM 2μM 4μM 24h 0±4.04 10.27±2.74 14.78±0.53 91.67±0.63 48h 0±2.60 32.99±3.26 51.55±3.05 99.74±0.02 72h 0±1.78 69.52±0.39 78.14±1.03 99.94±0.01

[0110] As can be seen from Table 5, YL-939 significantly inhibited the proliferation of K562 cells. After different concentrations of YL-939 (1 μM, 2 μM, 4 μM) intervened in K562 cells, cell proliferation inhibition results were shown after 24h, 48h and 72h of intervention, respectively. Among them, the cell proliferation inhibition rate reached 78.14% after 72h of intervention using a lower concentration of 2 μM.

[0111] The above results indicate that YL-939 can inhibit the proliferation of human chronic myeloid leukemia cell lines and has the potential to become a drug for the treatment of leukemia.

[0112] Example 6 - Safety Experimental Study of YL-939

[0113] 6.1 Experimental Materials:

[0114] The source of YL-939 is the same as above. The sources of human promyelocytic leukemia cell line HL60 cells, human chronic myeloid leukemia cell line K562 cells, human acute myeloid leukemia cell line MOLM-13 cells, human myelomonocytic leukemia cell line MV4-11 cells and human acute myeloid leukemia cell line OCI-AML3 cells are the same as above. Human umbilical vein endothelial cell line HUVEC cells were purchased from Synbio (Shanghai) Biotechnology Co., Ltd., Cat#iCell-h110. Human histiocytic lymphoma cell line U937 cells were purchased from Synbio (Shanghai) Biotechnology Co., Ltd., Cat#iCell-h267. Luminescent cell viability assay kit was purchased from Promega (Beijing) Biotechnology Co., Ltd., Cat#G7571.

[0115] 6.2 Experimental methods:

[0116] The above-mentioned human histiocytic lymphoma cell line U937, human promyelocytic leukemia cell line HL60, human chronic myeloid leukemia cell line K562, human acute myeloid leukemia cell line MOLM-13, human myelomonocytic leukemia cell line MV4-11, human acute myeloid leukemia cell line OCI-AML3 and human umbilical vein endothelial cell line HUVEC were seeded in 96-well plates, 5000 cells / well, and cultured in 100 μl RPMI1640+10% FBS. YL-939 was added to each cell in the well plate at concentrations of 0 μM, 0.234375 μM, 0.46875 μM, 0.9375 μM, 1.875 μM, 3.75 μM, 7.5 μM, 15 μM, 30 μM and 60 μM, respectively, with 6 replicate wells in each group. After 48 h of culture, the cells were cultured using Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, mix on an orbital shaker for 2 min, incubate the plate at room temperature for 10 min, and read using a microplate luminometer ( Navigator, Promega) to detect luminescent signals. CellTiter-Glo is a cleavage-based detection method based on ATP. Luciferin is oxidized by luciferase with the participation of ATP to produce light. The ATP in the luciferase reaction comes entirely from the ATP of living cells, so the light intensity produced is proportional to the number of living cells.

[0117] 6.3 Cell IC 50 Calculation of value:

[0118] IC 50 The values ​​were calculated using GraphPad software.

[0119] 6.4 Experimental results:

[0120] The experimental results are shown in Table 6.

[0121] Table 6 IC of each cell line 50 value

[0122] Cell lines U937 HL60 K562 MOLM-13 <![CDATA[IC 50 Value (μM)]]> 1.83 2.94 1.71 1.78 Cell lines MV4-11 OCL-AML3 HUVEC <![CDATA[IC 50 Value (μM)]]> 1.22 1.16 5.11

[0123] It can be seen that after 48 hours of intervention, YL-939 at a low dose can significantly inhibit the proliferation of multiple leukemia cell lines, while the IC 50 The value reached 5.11 μM, which is about 2-4 times the drug concentration in tumor cell lines.

[0124] The above results show that YL-939 has an effective inhibitory effect on leukemia cells at low doses, and has no drug toxicity to normal non-tumor cells at effective doses. It has very good safety and is suitable for further research and development as a potential anti-tumor drug.

[0125] Example 7 - Inhibitory effect of YL-939 on proliferation of acute myeloid leukemia tumor stem cells 7.1 Experimental Materials:

[0126] The source of YL-939 was the same as above. Bone marrow specimens from patients with acute myeloid leukemia were obtained from the Cancer Center of Sun Yat-sen University. The human CD34+ stem cell magnetic bead sorting kit was purchased from Miltenyi Biotech (Germany) Co., Ltd. Cat#130-046-702. Luminescent cell viability detection kit was purchased from Promega (Beijing) Biotechnology Co., Ltd., Cat#G7571. Human stem cell culture medium was purchased from STEMCELL Technologies (Canada), Cat#09720.

[0127] 7.2 Experimental methods:

[0128] Mononuclear cells were isolated from bone marrow specimens of patients with acute myeloid leukemia, and CD34+ leukemia stem cells were sorted from bone marrow mononuclear cells using a human CD34+ stem cell magnetic bead sorting kit. Human CD34+ leukemia stem cells were seeded in a white 96-well plate, 5000 cells / well, and cultured in 100μl of human stem cell culture medium. YL-939 was added to each cell in the well plate at concentrations of 0μM, 0.234375μM, 0.46875μM, 0.9375μM, 1.875μM, 7.5μM, 15μM, 30μM and 60μM, with 6 replicate wells in each group. After 48h of culture, the cells were cultured using Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, mix on an orbital shaker for 2 min, incubate the plate at room temperature for 10 min, and read using a microplate luminometer ( Navigator, Promega) to detect luminescent signals. CellTiter-Glo is a cleavage-based detection method based on ATP. Luciferin is oxidized by luciferase with the participation of ATP to produce light. The ATP in the luciferase reaction comes entirely from the ATP of living cells, so the light intensity produced is proportional to the number of living cells.

[0129] 7.3 Cell IC 50 Calculation of value:

[0130] IC 50 The values ​​were calculated using GraphPad software.

[0131] 7.4 Experimental results:

[0132] The experimental results are shown in Figure 1 . Figure 1 In the figure, #Z017, #Z016, and #Z022 are numbers, representing tumor stem cells from different AML patients, respectively. AML stands for acute myeloid leukemia (AML).

[0133] Depend on Figure 1 It can be seen that after 48 hours of intervention, YL-939 at low doses had a significant inhibitory effect on acute myeloid leukemia tumor stem cells, IC 50 The range is around 1.7-2.3μM.

[0134] The above results indicate that YL-939 can inhibit the proliferation of tumor stem cells in patients with acute myeloid leukemia and has the potential to become a drug for the treatment of leukemia.

[0135] Example 8 - Inhibitory effect of YL-939 on proliferation of venetolazine-resistant acute myeloid leukemia tumor stem cells

[0136] 8.1 Experimental Materials:

[0137] The source of YL-939 is the same as above. Bone marrow specimens from patients with acute myeloid leukemia were obtained from the Cancer Center of Sun Yat-sen University. Human CD34+ stem cell magnetic bead sorting kit was purchased from Miltenyi Biotech (Germany), Cat#130-046-702. Luminescent cell viability detection kit was purchased from Promega (Beijing) Biotechnology Co., Ltd., Cat#G7571. Human stem cell culture medium was purchased from STEMCELL Technologies (Canada), Cat#09720.

[0138] 8.2 Experimental methods:

[0139] Mononuclear cells were isolated from bone marrow specimens of patients with acute myeloid leukemia, and CD34+ leukemia stem cells were sorted from bone marrow mononuclear cells using a human CD34+ stem cell magnetic bead sorting kit. Human CD34+ leukemia stem cells were seeded in a white 96-well plate, 5000 cells / well, and cultured in 100μl of human stem cell culture medium. YL-939 was added to each cell in the well plate at concentrations of 0μM, 0.234375μM, 0.46875μM, 0.9375μM, 1.875μM, 7.5μM, 15μM, 30μM and 60μM, with 6 replicate wells in each group. After 48h of culture, the cells were cultured using Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, mix on an orbital shaker for 2 min, incubate the plate at room temperature for 10 min, and read using a microplate luminometer ( Navigator, Promega) to detect luminescent signals. CellTiter-Glo is a cleavage-based detection method based on ATP. Luciferin is oxidized by luciferase with the participation of ATP to produce light. The ATP in the luciferase reaction comes entirely from the ATP of living cells, so the light intensity produced is proportional to the number of living cells.

[0140] 8.3 Cell IC 50 Calculation of value:

[0141] IC 50 The values ​​were calculated using GraphPad software.

[0142] 8.4 Experimental results:

[0143] The experimental results are shown in Figure 2 . Figure 2 In the figure, #Z019 and #Z020 are numbers, which respectively represent tumor stem cells derived from AML patients with different venetoclax resistance. AML stands for acute myeloid leukemia (AML).

[0144] Depend on Figure 2 It can be seen that after 48 hours of intervention, YL-939 at low doses had a significant inhibitory effect on venetolazepine-resistant acute myeloid leukemia tumor stem cells, IC 50 The range is around 1.7-2.4μM.

[0145] The above results indicate that YL-939 can inhibit the proliferation of acute myeloid leukemia tumor stem cells, especially for leukemia stem cells that are resistant to venetola, and is expected to become a tumor treatment drug that overcomes venetola resistance.

[0146] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. Use of a compound having a structure as shown in formula (I) or a salt thereof in the preparation of a drug for treating leukemia:

2. The use according to claim 1, characterized in that The drug forms a drug preparation for treating leukemia; the drug preparation contains the compound shown in formula (I).

3. The use according to claim 2, characterized in that: The leukemia is acute leukemia or chronic leukemia.

4. The use according to claim 2, characterized in that: The medicament forms a pharmaceutical preparation for inhibiting the proliferation of acute myeloid leukemia cell lines.

5. The use according to claim 4, characterized in that In a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 4 μM and the experiment is carried out for 48 hours, the cell proliferation inhibition rate of human acute myeloid leukemia cell MOLM-13 is greater than 99%.

6. The use according to claim 2, characterized in that: The medicament forms a pharmaceutical preparation for inhibiting the proliferation of promyelocytic leukemia cell lines.

7. The use according to claim 6, characterized in that In a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 4 μM and the experiment lasts for 24 hours, the cell proliferation inhibition rate of human promyelocytic leukemia cell HL60 is greater than 97%.

8. The use according to claim 2, characterized in that: The medicament forms a pharmaceutical preparation for inhibiting the proliferation of myelomonocytic leukemia cell lines.

9. The use according to claim 8, characterized in that In a cell experiment, when the concentration of the compound represented by formula (I) is greater than or equal to 2 μM and the experiment is carried out for 72 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cell MV4-11 is greater than 96%.

10. The use according to claim 2, characterized in that: The drug forms a pharmaceutical preparation for inhibiting the proliferation of chronic myeloid leukemia cell lines.

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