Use of compounds for the manufacture of a medicament for the treatment of leukemia

Abstract

The application discloses use of a compound in preparation of a medicine for treating leukemia. The application discloses use of a compound with a structure as shown in formula (I) or a salt thereof in preparation of a medicine for treating leukemia.

Description

TECHNICAL FIELD

[0001] The present application relates to a compound or a salt thereof for use in the preparation of a medicament for treating leukemia. BACKGROUND

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

[0003] According to the degree of cell differentiation and natural course, leukemia can be divided into acute leukemia (AL) and chronic leukemia (CL). According to the main affected cell series, leukemia can be divided into lymphocyte leukemia and non-lymphocyte (myelocyte) leukemia. AL can be divided into acute lymphoblastic 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, prolymphocytic leukemia, etc.

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

[0005] CN118615282A discloses a clematis indolin A and its use, and the application of the clematis indolin A in the preparation of a pharmaceutical composition for preventing or treating cancer, wherein the cancer is leukemia. SUMMARY

[0006] The purpose of the present application is to provide a compound as shown in formula (I) or a salt thereof for use in the preparation of a medicament for treating leukemia.

[0007] The application achieves the above-mentioned purpose by adopting the following technical scheme.

[0008] The application provides use of a compound or a salt thereof with a structure as shown in the formula (I) in preparation of a drug for treating leukemia.

[0009]

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

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

[0012] According to the use of the application, preferably, the drug forms a drug preparation for inhibiting proliferation of an acute myeloid leukemia cell line.

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

[0014] According to the use of the application, preferably, the drug forms a drug preparation for inhibiting proliferation of a promyelocytic leukemia cell line.

[0015] According to the use of the application, preferably, in a cell experiment, when the concentration of the compound as shown in the formula (I) is greater than or equal to 4 mu M, and the experiment is performed 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 application, preferably, the drug forms a drug preparation for inhibiting proliferation of a myelomonocytic leukemia cell line.

[0017] According to the use of the application, preferably, in a cell experiment, when the concentration of the compound as shown in the formula (I) is greater than or equal to 2 mu M, and the experiment is performed for 72 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cells MV4-11 is greater than 96%.

[0018] According to the use of the application, preferably, the drug forms a drug preparation for inhibiting proliferation of a chronic myeloid leukemia cell line.

[0019] The compound as shown in the formula (I) has good cell proliferation inhibition effect on leukemia, and has no drug toxicity on normal non-tumor cells, and is high in safety. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0022] The present invention will be further described below with reference to 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 has a molecular weight of 426.51. The compound represented by formula (I) is a known compound, CAS No.: 3023925-68-7, and is commercially available.

[0026] In the prior art, the compound shown in formula (I) is also known as 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, there has been no report on the use of YL-939 for the preparation of drugs for the treatment of leukemia. The present invention found that the compound shown in formula (I) (also known as YL-939) has a good cell proliferation inhibitory effect on leukemia, and has basically no drug toxicity to normal non-tumor cells, and is highly safe.

[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, as well as pharmaceutically acceptable excipients. According to a preferred embodiment of the present invention, the active ingredients in the pharmaceutical preparation comprise only the compound as shown in formula (I) of the present invention.

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

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

[0030] According to one aspect of the present application, the drug forms a pharmaceutical preparation for inhibiting the proliferation of an acute myeloid leukemia cell line. 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 48 h, the cell proliferation inhibition rate of human acute myeloid leukemia cells MOLM-13 reaches more than 99%.

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

[0032] In the present application, the drug forms a pharmaceutical preparation for inhibiting the proliferation of an acute myeloid leukemia cell line. 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 48 h, the cell proliferation inhibition rate of human acute myeloid leukemia cells OCL-AML3 reaches more than 99%.

[0033] According to one specific embodiment of the present application, in a cell experiment, when the concentration of the compound represented by formula (I) is 4 μM, and the experiment is 48 h, the cell proliferation inhibition rate of human acute myeloid leukemia cells OCL-AML3 reaches 99.93%.

[0034] According to one specific aspect of the present application, the drug forms a pharmaceutical preparation for inhibiting the proliferation of an acute myeloid leukemia cell line. 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 48 h, the cell proliferation inhibition rate of human acute myeloid leukemia cells OCL-AML3 reaches more than 99%.

[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 formulation 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 line MV4-11 reaches greater than 96%.

[0037] According to one embodiment of the present invention, in a cell experiment, when the concentration of the compound represented by formula (I) was 2 μM and the experiment was continued for 72 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cell line MV4-11 reached 96.68%. When the concentration of the compound represented by formula (I) was 4 μM and the experiment was continued for 24 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cell line MV4-11 reached 90.09%. At 48 hours of the experiment, the cell proliferation inhibition rate of human myelomonocytic leukemia cell line MV4-11 reached 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 K562 cells 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 shows no drug toxicity to normal non-tumor cells at effective doses. It has excellent safety and is suitable for further development as a potential anti-tumor drug.

[0041] Furthermore, in the present invention, the compound represented by formula (I) (or YL-939) 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 represented by 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 around 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 venetula, and is expected to become a tumor treatment drug that overcomes venetula resistance.

[0043] Example 1 - Inhibition of proliferation of human promyelocytic leukemia cells (HL60 cells) by YL939

[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 into a white 96-well plate at 5000 cells / well and cultured in 100μl of IMDM + 10% FBS. The experiment was divided into 4 groups. YL-939 was added to the cells at concentrations of 0μM, 1μM, 2μM, and 4μM, with 6 replicates in each group. 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, use orbital shaker to mix for 2 minutes, incubate the culture plate at room temperature for 10 minutes, and use a microplate luminometer ( Luminescence signals were detected using a luciferase assay (Navigator, Promega). CellTiter-Glo is an ATP-cleavage assay. Luciferin is oxidized by luciferase in the presence of ATP, generating light. Since the ATP used in the luciferase reaction comes entirely from the ATP of living cells, the intensity of the light generated 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 added)-A(blank)] / [A(0 drug added)-A(blank)]×100%, where:

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

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

[0052] A(0 drug addition): with cells, The optical signal intensity of the wells containing Reagent but no drug solution.

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

[0054] 1.4 Experimental results:

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

[0056] Table 1 Inhibitory effect of YL-939 on the proliferation of human promyelocytic leukemia cells HL60 (%, M ± SD)

[0057] 0 μM 1 μM 2 μM 4 μM 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 shown in 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 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 - Inhibition of proliferation of human acute myeloid leukemia cells (MOLM-13 cells) by YL-939

[0061] 2.1 Experimental Materials

[0062] The source of YL-939 was the same as above. The human acute myeloid leukemia cell line MOLM-13 was purchased from Subcon (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 seeded into a white 96-well plate at 5000 cells / well and cultured in 100μl of RPMI1640 + 10% FBS. The experiment was divided into 4 groups. YL-939 was added to the cells at concentrations of 0μM, 1μM, 2μM, and 4μM, with 6 replicates in each group. 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, use orbital shaker to mix for 2 minutes, incubate the culture plate at room temperature for 10 minutes, and use a microplate luminometer ( Luminescence signals were detected using a luciferase assay (Navigator, Promega). CellTiter-Glo is an ATP-cleavage assay. Luciferin is oxidized by luciferase in the presence of ATP, generating light. Since the ATP used in the luciferase reaction comes entirely from the ATP of living cells, the intensity of the light generated 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 cells MOLM-13 (%, M ± SD)

[0070] 0 μM 1 μM 2 μM 4 μM 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 shown in Table 2, YL-939 significantly inhibited the proliferation of MOLM-13 cells. After different concentrations of YL-939 (1 μM, 2 μM, 4 μM) intervened in MOLM-13 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 80.19% after 48 h of intervention using a lower concentration of 2 μM.

[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 - Inhibition of proliferation of human myelomonocytic leukemia cells (MV4-11 cells) by YL-939

[0074] 3.1 Experimental Materials

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

[0076] 3.2 Experimental method:

[0077] Human myelomonocytic leukemia cells MV4-11 (also known as MV-4-11) were inoculated in white 96-well plates, 5000 cells / well, 100 μl of IMDM + 10% FBS culture. The experiment was divided into 4 groups, and YL-939 with concentrations of 0 μM, 1 μM, 2 μM and 4 μM were added to the cells in the plate, respectively, each group had 6 repeated wells. After 24 h, 48 h and 72 h of culture, the cell viability was detected by using Luminescent Cell Viability Assay Kit, 100 μl Reagent was added to each well, mixed for 2 min using an orbital shaker, and the culture plate was incubated at room temperature for 10 min. The luminescent signal was detected by using a microplate luminescence detector (Navigator, Promega). CellTiter-Glo is an ATP-based lysis detection method. Luciferin is oxidized by luciferase to produce light under the participation of ATP. All ATP for luciferase reaction comes from ATP of living cells, so the intensity of light 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 Cell proliferation inhibition rate (%) of YL-939 on human myelomonocytic leukemia cells MV4-11 (M ± SD)

[0083] 0 μM 1 μM 2 μM 4 μM 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 (1 μM, 2 μM, 4 μM) were used to intervene MV4-11 cells, the cell proliferation inhibition results were shown after 24 h, 48 h and 72 h of intervention, respectively. Among them, the cell proliferation inhibition rate could reach 79.99% after 48 h of intervention with a smaller concentration of 2 μM.

[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 - Inhibition of proliferation of human acute myeloid leukemia cells (OCL-AML3 cells) by YL-939

[0087] 4.1 Experimental Materials

[0088] The source of YL-939 was the same as above. Human acute myeloid leukemia cells OCL-AML3 were purchased from Pronose (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 seeded into a white 96-well plate at 5000 cells / well and cultured in 100μl of IMDM + 15% FBS. The experiment was divided into 4 groups. YL-939 was added to the cells at concentrations of 0μM, 1μM, 2μM, and 4μM, with 6 replicates in each group. 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, use orbital shaker to mix for 2 minutes, incubate the culture plate at room temperature for 10 minutes, and use a microplate luminometer ( Luminescence signals were detected using a luciferase assay (Navigator, Promega). CellTiter-Glo is an ATP-cleavage assay. Luciferin is oxidized by luciferase in the presence of ATP, generating light. Since the ATP used in the luciferase reaction comes entirely from the ATP of living cells, the intensity of the light generated 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 the proliferation of human acute myeloid leukemia cells OCL-AML3 (%, M ± SD)

[0096] 0 μM 1 μM 2 μM 4 μM 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 shown in Table 4, YL-939 significantly inhibited the proliferation of OCL-AML3 cells. After intervention with different concentrations of YL-939 (1 μM, 2 μM, 4 μM), OCL-AML3 cells showed cell proliferation inhibition results 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 - Inhibition of proliferation of human chronic myeloid leukemia cells (K562 cells) by YL-939

[0100] 5.1 Experimental Materials

[0101] The source of YL-939 was the same as above. Human chronic myeloid leukemia cell line K562 cells were purchased from Subcon (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 seeded into a white 96-well plate at 5000 cells / well and cultured in 100μl of IMDM + 10% FBS. The experiment was divided into 4 groups. YL-939 was added to the cells at concentrations of 0μM, 1μM, 2μM, and 4μM, with 6 replicates in each group. 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, use orbital shaker to mix for 2 minutes, incubate the culture plate at room temperature for 10 minutes, and use a microplate luminometer ( Luminescence signals were detected using a luciferase assay (Navigator, Promega). CellTiter-Glo is an ATP-cleavage assay. Luciferin is oxidized by luciferase in the presence of ATP, generating light. Since the ATP used in the luciferase reaction comes entirely from the ATP of living cells, the intensity of the light generated 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 Inhibitory effect of YL-939 on K562 cells (%, M ± SD)

[0109] 0 μM 1 μM 2 μM 4 μM 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 shown in 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 24 h, 48 h and 72 h of intervention, respectively. Among them, the cell proliferation inhibition rate reached 78.14% after 72 h 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 study of YL-939

[0113] 6.1 Experimental Materials

[0114] The source of YL-939 was 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 were the same as above. Human umbilical vein endothelial cell line HUVEC cells were purchased from ProBio (Shanghai) Biotechnology Co., Ltd., Cat# iCell-h110. Human histiocytic lymphoma cell line U937 cells were purchased from ProBio (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 at a density of 5000 cells / well and cultured in 100 μl of 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 treated with YL-939. Luminescent Cell Viability Assay Kit was used to detect cell viability. 100 μl of Reagent, use orbital shaker to mix for 2 minutes, incubate the culture plate at room temperature for 10 minutes, and use a microplate luminometer ( Luminescence signals were detected using a luciferase assay (Navigator, Promega). CellTiter-Glo is an ATP-cleavage assay. Luciferin is oxidized by luciferase in the presence of ATP, generating light. Since the ATP used in the luciferase reaction comes entirely from the ATP of living cells, the intensity of the light generated 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 line U937 HL60 K562 MOLM-13 IC 50 values (μM) 1.83 2.94 1.71 1.78 Cell line 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 can significantly inhibit the proliferation of multiple leukemia cell lines at low doses, 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 - Inhibition of proliferation of acute myeloid leukemia tumor stem cells by YL-939 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 isolation kit was purchased from Miltenyi Biotec GmbH (Germany), Cat# 130-046-702. Luminescent cell viability assay 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 at 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, use orbital shaker to mix for 2 minutes, incubate the culture plate at room temperature for 10 minutes, and use a microplate luminometer ( Luminescence signals were detected using a luciferase assay (Navigator, Promega). CellTiter-Glo is an ATP-cleavage assay. Luciferin is oxidized by luciferase in the presence of ATP, generating light. Since the ATP used in the luciferase reaction comes entirely from the ATP of living cells, the intensity of the light generated 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 derived 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 has 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 - Inhibition of proliferation of venetoclax-resistant acute myeloid leukemia tumor stem cells by YL-939

[0136] 8.1 Experimental Materials:

[0137] YL-939 was obtained from the same source 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 isolation kit was purchased from Miltenyi Biotec GmbH (Germany), Cat# 130-046-702. Luminescent cell viability assay 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 at 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, use orbital shaker to mix for 2 minutes, incubate the culture plate at room temperature for 10 minutes, and use a microplate luminometer ( Luminescence signals were detected using a luciferase assay (Navigator, Promega). CellTiter-Glo is an ATP-cleavage assay. Luciferin is oxidized by luciferase in the presence of ATP, generating light. Since the ATP used in the luciferase reaction comes entirely from the ATP of living cells, the intensity of the light generated 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 representing tumor stem cells derived from different venetolac-resistant AML patients. 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 venetola-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. Any modification, improvement, or substitution that can be conceived by those skilled in the art without departing from the essential content of the present invention 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 medicine forms a pharmaceutical preparation for treating leukemia; the pharmaceutical 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 lasts for 48 hours, the cell proliferation inhibition rate of human acute myeloid leukemia cell line 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 cells 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 lasts for 72 hours, the cell proliferation inhibition rate of human myelomonocytic leukemia cells 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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