Application of compound N2 in preparation of erythroleukemia induced differentiation treatment medicine

Abstract

The invention discloses an application of a compound N2 in preparation of an erythroleukemia induced differentiation treatment drug.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a compound N2 in the preparation of a drug for inducing differentiation treatment of erythroleukemia. Background Art

[0002] Leukemia is a malignant clonal disease of hematopoietic stem cells. Abnormal primitive cells and immature cells in the bone marrow proliferate in large numbers and widely infiltrate various organs such as the liver, spleen, and lymph nodes, inhibiting normal hematopoiesis. There are many types of leukemia, among which erythroleukemia is a rare and aggressive subtype of acute myeloid leukemia. Erythroleukemia is difficult to treat, has a high recurrence rate and a high mortality rate. Traditional chemotherapy drugs mainly induce leukemia cell death through cytotoxic effects, which can alleviate the progression of leukemia to a certain extent, but it will also damage normal cells.

[0003] Differentiation therapy refers to inducing tumor cells to differentiate into normal mature cells through differentiation inducers. It is a therapeutic strategy to reverse the uncontrolled proliferation of tumor cells. Differentiation therapy has broad prospects in hematological malignancies. By targeting specific molecules and signaling pathways, it is possible to achieve a more effective treatment strategy with fewer side effects. At present, the development of drugs that can induce differentiation of erythroleukemia has become one of the research directions.

[0004] Compound N2 is a nootkatone acylhydrazone derivative with antibacterial activity. Summary of the invention

[0005] The object of the present invention is to provide the use of compound N2 in the preparation of erythroleukemia inducing differentiation therapeutic drugs, wherein the compound has anti-erythroleukemia cell activity, can inhibit the proliferation of erythroleukemia cells, and promote the megakaryotic differentiation of erythroleukemia cells.

[0006] The compound N2 of the present invention is used in the preparation of a drug for inducing differentiation and treating erythroleukemia, and the chemical structure of the compound N2 is shown in Formula I:

[0007]

[0008] The use of the above-mentioned compound N2 in the preparation of a drug for inducing differentiation therapy of erythroleukemia, wherein the active ingredient of the drug is compound N2 and also includes pharmaceutically acceptable excipients.

[0009] Compared with the prior art, the present invention has obvious beneficial effects. From the above technical scheme, it can be seen that the compound N2 of the present invention inhibits the proliferation of HEL cells and K562 cells by inducing G2 cycle arrest in HEL cells and K562 cells, and experiments have shown that it has anti-erythroleukemia cell HEL and K562 activity. The compound N2 of the present invention promotes the transformation of HEL cells and K562 cells from malignant proliferating cells to normal megakaryocytes by inducing the enlargement of cell particle size and the significant increase of multinucleated megakaryocytes, while promoting the expression of surface marker molecules of megakaryocyte differentiation, and exerts an anti-erythroleukemia effect. Further experiments show that compound N2 promotes the transcription of megakaryocyte-specific genes by upregulating megakaryocyte differentiation-related transcription factors GATA1, FOS, FOSL2, JUN, JUNB, JUND and ETS2, thereby inducing HEL cells and K562 cells to differentiate into megakaryocytes, transforming malignant cells into benign normal cells, and exerting an anti-erythroleukemia effect.

[0010] The active ingredient of the present invention includes compound N2, which is a drug for inducing differentiation treatment of erythroleukemia. Drug experiments have shown that the size and weight of the spleen of leukemia mice can be significantly reduced after treatment, and the hematocrit and platelet count of mice are significantly improved. In addition, the compound N2 treatment has no obvious effect on the weight of mice, and the safety is high. In vivo experiments show that the drug can induce megakaryotic differentiation of erythroleukemia in mice and play an anti-leukemia role, laying a foundation for the development and research of differentiation treatment drugs for erythroleukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The actual pictures of cell morphology of HEL cells treated with compound N2 at different concentrations;

[0012] Figure 2 The cell morphology of K562 cells treated with different concentrations of compound N2;

[0013] Figure 3 It is a cell growth curve diagram of HEL cells treated with different concentrations of compound N2;

[0014] Figure 4 It is a cell growth curve of K562 cells treated with different concentrations of compound N2;

[0015] Figure 5 The cell cycle distribution diagram of HEL cells treated with different concentrations of compound N2;

[0016] Figure 6 The cell cycle distribution diagram of K562 cells treated with different concentrations of compound N2;

[0017] Figure 7Wright-Giemsa staining of HEL cells treated with different concentrations of compound N2;

[0018] Figure 8 Wright-Giemsa staining of K562 cells treated with different concentrations of compound N2;

[0019] Fig. 9 Flow cytometry detection of megakaryocyte surface marker molecules of HEL cells treated with different concentrations of compound N2;

[0020] Fig.10 Flow cytometry detection of megakaryocyte surface marker molecules in K562 cells treated with different concentrations of compound N2;

[0021] Fig.11 The figure shows the qRT-PCR detection results of megakaryocyte differentiation-related transcription factors in HEL cells treated with different concentrations of compound N2;

[0022] Fig.12 The qRT-PCR detection results of megakaryocyte differentiation-related transcription factors in K562 cells treated with different concentrations of compound N2;

[0023] Fig.13 This is a graph showing the effect of compound N2 on the basic indicators of leukemia mice;

[0024] Fig.14 This is a diagram showing the effect of compound N2 on megakaryocyte surface marker molecules in mouse spleen cells;

[0025] Fig.15 This graph shows the effect of compound N2 on the body weight of leukemia mice. DETAILED DESCRIPTION

[0026] Example 1: Cell viability test experiment

[0027] (1) Preparation of Compound N2 solution: Compound N2 solutions with concentrations of 20, 10, 5, 2.5 and 1.25 mM were prepared. The solvent was DMSO. The dosage was calculated using the AbMole molar concentration calculator. The formula was: mass (mg) = concentration (mM) × volume (mL) × molecular weight (g / mol).

[0028] Wherein: the synthesis method of compound N2 refers to Liang Q, Gao F, Jian J, Yang J, Hao X, Huang L. Design, Synthesis and Antifungal Activity of Nootkatone Derivatives Containing Acylhydrazone and Oxime Ester. Chem Biodivers. 2024; 21(5): e202400355.doi:10.1002 / cbdv.202400355.

[0029] Its chemical structure is shown in Formula I:

[0030]

[0031] (2) Test method: HEL and K562 cells were cultured in RPMI-1640 and DMEM medium, respectively. A mixture of 10% fetal bovine serum and 1% penicillin-streptomycin was added to the cell culture medium and cultured in an incubator at 37°C, saturated humidity, and 5% CO2. When the cells grew to the logarithmic phase, HEL and K562 cells were collected and seeded in a 96-well plate (HEL cells: 10,000 cells / well; K562 cells: 8,000 cells / well). 90 μL of sterile water was added around the well plate and cultured in an incubator at 37°C for 4 h. After the HEL cells were stabilized, they were divided into groups, each with 5 replicates, and five groups were selected to add different concentrations (10, 5, 2.5, 1.25, 0.625 mM) of N2 as the treatment group, so that the final concentrations were 10, 5, 2.5, 1.25, 0.625 μM, respectively, and another group was added with 0.1% DMSO as the control group; after the K562 cells were stabilized, they were divided into groups, each with 5 replicates, and five groups were selected to add different concentrations (20, 10, 5, 2.5 and 1.25 mM) of N2 as the treatment group, so that the final concentrations were 20, 10, 5, 2.5, 1.25 μM, respectively, and another group was added with 0.1% DMSO as the control group. After the drug was applied for 72 hours (37°C), 10 μL of 5 mg / mL MTT solution was added to each well and incubated at 37°C for 4 hours. After the formazan is completely formed, add 100 μL of triple solution to each well (preparation of triple solution: add 100 g SDS, 50 ml isobutanol, 1 ml concentrated hydrochloric acid to a clean glass beaker, add purified water to 1 L, stir until completely dissolved, then divide and store), and place at 37°C overnight.

[0032] (3) Test results: After the formazan was completely dissolved, the absorbance OD value (570 nm) was measured using an ELISA instrument and the inhibition rate was calculated using the following formula.

[0033] Inhibition rate = (OD value 570nm of control group - OD value 570nm of treatment group) / OD value 570nm of control group.

[0034] The control group was 0.1% DMSO, and the treatment groups were different concentrations of N2 (20, 10, 5, 2.5 and 1.25 μM).

[0035] The half inhibition rate (IC 50 ) calculation method: According to the inhibition rate, the forecast function is used to obtain the IC corresponding to the compound 50 .

[0036] The results showed that different concentrations of N2 could inhibit the cell viability of HEL and K562 in a concentration-dependent manner. 50 The IC value for K562 was 4.58±0.15μM. 50 It is 6.54±0.27μM.

[0037] Example 2: Cell morphology observation experiment

[0038] (1) Preparation of Compound N2 solution: Compound N2 solutions with concentrations of 2, 4 and 8 mM were prepared using the same method as in Example 1;

[0039] (2) Experimental method: According to the culture method of Example 1, when the cells grow to the logarithmic phase, HEL and K562 cells are collected and planted in 96-well plates (HEL cells: 10,000 cells / well; K562 cells: 8,000 cells / well), and cultured in an incubator at 37°C for 4 hours. After the cells are stable, different concentrations (2, 4 and 8 mM) of compound N2 solution are added to HEL cells for treatment, so that the final concentrations are 2, 4, and 8 μM, respectively, with 0.1% DMSO as the control group, and the treatment is carried out for 72 hours and 96 hours, respectively. Different concentrations (2, 4 and 8 mM) of compound N2 are added to K562 cells for treatment, so that the final concentrations are 2, 4, and 8 μM, respectively, with 0.1% DMSO as the control group, and the treatment is carried out for 72 hours and 96 hours, respectively. The morphological changes of each group of cells are observed by taking pictures with an inverted microscope, magnification: 200 times, scale: 100 μm, as shown in Figure 1 and Figure 2 shown.

[0040] (3) Observation results: Figure 1 and Figure 2 It can be seen that with the increase of the concentration of compound N2 and the extension of the action time, the cell number of HEL and K562 cells decreased, the cell size became larger, an irregular morphology appeared, and the suspension cells could be transformed into an adherent morphology.

[0041] Example 3: Cell growth curve measurement experiment

[0042] (1) Preparation of Compound N2 solution: Compound N2 solutions with concentrations of 2, 4 and 8 mM were prepared using the same method as in Example 1;

[0043] (2) Determination method: HEL and K562 cells in the logarithmic growth phase were collected according to the method of Example 1, and planted in a 96-well plate (HEL cells: 10,000 cells / well; K562 cells: 8,000 cells / well). 90 μL of sterilized water was added around the well plate, and cultured in an incubator for 4 hours. After the cells were stabilized, different concentrations (2, 4, and 8 mM) of compound N2 were added to the HEL cells for treatment, so that the final action concentrations were 2, 4, and 8 μM, respectively. The OD value (570 nm) was measured at 0 h, 24 h, 48 h, 72 h, and 96 h, respectively. Different concentrations (2, 4, and 8 mM) of compound N2 solution were added to the K562 cells for treatment, so that the final action concentrations were 2, 4, and 8 μM, respectively. 0.1% DMSO was used as the control group, and the OD value (570 nm) was measured at 0 h, 24 h, 48 h, 72 h, and 96 h, respectively. The cell growth curve was drawn, as shown in FIG. Figure 3 and Figure 4 shown.

[0044] (3) Measurement results: Figure 3 and 4 It can be seen that compared with the DMSO group, different concentrations of compound N2 can inhibit the proliferation of these two cell lines, and with the increase of concentration, the growth rate of the two cell lines gradually slowed down, indicating that compound N2 can inhibit HEL( Figure 3 ) and K562( Figure 4 )’s proliferation.

[0045] Example 4: Cell cycle detection experiment

[0046] (1) Preparation of Compound N2 solution: Compound N2 solutions with concentrations of 2, 4 and 8 mM were prepared using the same method as in Example 1;

[0047] (2) Detection method: HEL and K562 cells were cultured at 3×10 5100 cells / well were planted in a cell culture dish with a diameter of 60 mm. Different concentrations (2, 4 and 8 mM) of compound N2 solution were added to HEL cells to make the final concentrations 2, 4 and 8 μM, respectively. 0.1% DMSO was used as the control group. After 72h and 96h at 37°C, the cells were collected. Different concentrations (2, 4 and 8 μM) of compound N2 were added to K562 cells. 0.1% DMSO was used as the control group. After 72h and 96h at 37°C, the cells were collected. The cells were collected in a 15 mL centrifuge tube, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS in a 1.5 mL EP tube, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in pre-cooled 70% ethanol and fixed at -20°C overnight. Centrifuge at 1300rpm for 10min, discard the supernatant, resuspend and wash the cells with PBS, centrifuge at 1300rpm for 5min, discard the supernatant, and then resuspend the cells with 500μL PBS. Add 5μL RNase A, 25μL propidium iodide and 0.25μL Triton X-100 to each sample, mix well, incubate in a 37℃ water bath in the dark for 30min, centrifuge at 1000rpm for 5min, discard the dye, resuspend with 200μL PBS, filter with a 300-mesh sieve, and finally analyze by flow cytometry. The results are shown in Figure 5 (A is the effect of different concentrations of compound N2 on the HEL cell cycle after 72h and 96h, B is the statistical diagram of the cell cycle distribution ratio (72h), and C is the statistical diagram of the cell cycle distribution ratio (96h)) and Figure 6 (A is the effect of different concentrations of compound N2 on the K562 cell cycle after 72h and 96h, B is the statistical graph of cell cycle distribution ratio (72h), and C is the statistical graph of cell cycle distribution ratio (96h)).

[0048] (3) Test results: Figure 5 It can be seen that after HEL cells were treated with high concentration of compound N2 for 96 hours, the proportion of cells in G2 / M phase increased from 18.85%±1.21% to 30.88%±0.48% compared with the DMSO group.

[0049] Depend on Figure 6 It can be seen that after K562 cells were treated with high concentration of compound N2 for 96 h, the proportion of cells in the G2 / M phase increased from 16.80%±0.67% to 41.64%±0.96% compared with the DMSO group.

[0050] The results showed that the proportion of HEL and K562 cells in the G2 / M phase increased significantly after treatment with compound N2, and this increasing trend was concentration-dependent, indicating that compound N2 can induce G2 / M phase cell cycle arrest in HEL and K562 cells.

[0051] Example 5: Wright-Giemsa staining experiment

[0052] (1) Preparation of Compound N2 solution: Compound N2 solutions with concentrations of 2, 4 and 8 mM were prepared using the same method as in Example 1;

[0053] (2) Experimental method: HEL and K562 cells were cultured at 3×10 5 Cells were planted in a cell culture dish with a diameter of 60 mm per well. Different concentrations (2, 4 and 8 mM) of compound N2 solution were added to HEL cells and K562 cells for treatment, so that the final concentrations were 2, 4 and 8 μM, respectively. 0.1% DMSO was used as the control group. After 72 h and 96 h at 37 ° C, the cells were collected. The cells were collected in a 15 mL centrifuge tube, centrifuged at 1500 rpm for 5 min, discarded the supernatant, resuspended in PBS in a 1.5 mL EP tube, centrifuged at 1500 rpm for 5 min, and resuspended in 100 μL PBS. Take a clean glass slide, scrape 10 μL of the cell suspension onto the glass slide, place it horizontally for 30 minutes, and dry it. Add Wright-Giemsa stain on the glass slide until the cells are completely covered, let it stand for 1 minute, add an equal amount of PBS to dilute the stain, let it stand for 4 minutes, slowly rinse the stain with water, and observe it under a microscope after drying. The results are as follows Figure 7 and Figure 8 shown.

[0054] (3) Experimental results: Figure 7 and 8 It can be seen that compared with DMSO, the HEL treated with compound N2 ( Figure 7 ) and K562( Figure 8 ) The cell size became larger, and the percentage of multinucleated cells increased significantly, and it was time- and concentration-dependent. HEL and K562 cells are megakaryotic progenitor cells and can differentiate into megakaryotic cells. These phenomena are typical phenomena of megakaryotic differentiation.

[0055] Example 6: Megakaryocyte surface marker molecule detection experiment

[0056] (1) Preparation of Compound N2 solution: Compound N2 solutions with concentrations of 2, 4 and 8 mM were prepared using the same method as in Example 1;

[0057] (2) Detection method: HEL and K562 cells were cultured at 3×10 5100 μL of cells / well were planted in a cell culture dish with a diameter of 60 mm. Different concentrations (2, 4 and 8 mM) of compound N2 were added to HEL cells and K562 cells for treatment, so that the final concentrations were 2, 4 and 8 μM, respectively. 0.1% DMSO was used as the control group. After 72 h and 96 h at 37 ° C, the cells were collected. The cells were collected in a 15 mL centrifuge tube, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS in a 1.5 mL EP tube, centrifuged at 1500 rpm for 5 min, and 100 μL of PBS was added to resuspend. 0.5 μL of CD41a-FITC and CD61-APC antibodies were added to each tube, mixed, incubated at 4 ° C in the dark for 40 min, filtered with a 300 mesh sieve, and finally analyzed by flow cytometry. The results are as follows Fig. 9 (A and B are the effects of different concentrations of compound N2 on the proportion of CD41a and CD61 positive cells in HEL cells after 72h and 96h, C is a statistical graph of the proportion of CD41a positive cells, and D is a statistical graph of the proportion of CD61 positive cells) and Fig.10 (A and B are the effects of different concentrations of compound N2 on the proportion of CD41a and CD61 positive cells in K562 cells after 72h and 96h of action, C is a statistical graph of the proportion of CD41a positive cells, and D is a statistical graph of the proportion of CD61 positive cells).

[0058] (3) Test results: Fig. 9 It can be seen that after HEL cells were treated with high concentration (8 μM) of compound N2 for 96 h, the proportion of CD41a-positive cells increased from 40.79%±3.83% to 81.51%±0.73%, and the proportion of CD61-positive cells increased from 48.99%±1.50% to 77.65%±0.99% compared with the DMSO group.

[0059] Depend on Fig.10 It can be seen that after K562 cells were treated with high concentration (8 μM) of compound N2 for 96 h, the proportion of CD41a-positive cells increased from 7.38%±0.55% to 10.88%±0.33%, and the proportion of CD61-positive cells increased from 9.61%±0.34% to 46.04%±0.20% compared with the DMSO group.

[0060] The expression levels of megakaryotic differentiation surface marker molecules were detected by Wright-Giemsa staining and flow cytometry, and it was found that compound N2 could induce megakaryotic differentiation of HEL and K562 cells.

[0061] Example 7: Experiment on detecting mRNA levels of transcription factors related to megakaryocyte differentiation after the action of compound N2 using real-time fluorescence quantitative PCR (qRT-PCR)

[0062] (1) Extraction of total RNA

[0063] HEL and K562 cells were cultured at 3×10 5 Cells were planted in a cell culture dish with a diameter of 60 mm per well. Compound N2 solution (prepared in the same manner as in Example 1) of different concentrations (2, 4 and 8 mM) was added to HEL cells and K562 cells to make the final concentrations 2, 4 and 8 μM, respectively. 0.1% DMSO was used as the control group. After 48 hours of exposure at 37°C, the cells were collected. Total RNA of the cells was extracted according to the instructions of the TRIzol kit. An appropriate amount of dissolved total RNA was taken, and its RNA quality was detected by electrophoresis. The RNA concentration was determined using a NanoDrop 2000 ultra-micro spectrophotometer. Store at -80°C for later use.

[0064] (2) Reverse transcription to synthesize cDNA: TaKaRa reverse transcription kit was used to synthesize cDNA.

[0065] (3) According to the instructions of the qRT-PCR kit, the reaction system was prepared and qRT-PCR was performed. The genes detected were differentiation-related genes GATA1, FOS, FOSL2, JUN, JUNB, JUND and ETS2, and the relative mRNA level data were obtained. The results are as follows Fig.11 and Fig.12 shown.

[0066] Depend on Fig.11 and Fig.12 It can be seen that after compound N2 acted on HEL and K562 cells, the transcription factors GATA1, FOS, FOSL2, JUN, JUNB, JUND and ETS2 related to megakaryotic differentiation showed a significant upregulation trend.

[0067] Experimental Example 1: Establishment of mouse leukemia model, compound N2 treatment and data collection

[0068] (1) Establishment of mouse leukemia model

[0069] Mouse erythroleukemia CB3 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. The cells were collected when the cell state was stable and the density was close to 80%, and the cells were resuspended in physiological saline to a cell density of 8×10 5 100 μL of CB3 cell suspension was implanted into 6-week-old BALB / c mice via tail vein injection.

[0070] (2) Dosage group

[0071] The mice after modeling were divided into a model group and a drug administration group. The drug administration group was divided into a low-dose group (10 mg compound N2 / kg mouse) and a high-dose group (20 mg compound N2 / kg mouse), and the formulation conditions were 95% sesame oil and 5% DMSO. At the same time, a group of unmodeled mice was set as a normal group, with 6 mice in each group.

[0072] (3) Treatment with Compound N2

[0073] After modeling, the mice were given drugs according to the grouping on the second day. The drug administration method was intraperitoneal injection. The injection volume for each mouse was 200 μL. The normal group was not treated, the model group was injected with an equal volume of drug solvent, the low-dose group was injected with 10 mg compound N2 / kg mouse, and the high-dose group was injected with 20 mg compound N2 / kg mouse. The preparation conditions were 95% sesame oil and 5% DMSO. The drugs were administered every other day.

[0074] (4) Data Collection

[0075] After 24 days of treatment, the mice were anesthetized and sacrificed for sampling and subsequent experiments:

[0076] 1) Determination of mouse spleen size and weight

[0077] The size of the mouse spleen was measured with a ruler and the weight was measured with an electronic balance. Fig.13 (Among them, A is the change in spleen size of mice in different groups, B is the change in spleen weight of mice in different groups, C is the change in hematocrit of mice in different groups, and D is the change in platelet number of mice in different groups) as shown in A and B.

[0078] 2) Hematocrit determination

[0079] After 24 days of treatment, a heparinized hematocrit capillary tube was inserted into the posterior ocular venous plexus to collect blood, which was centrifuged at 3500rpm for 15min. The percentage of the volume of red blood cells in the capillary tube in the whole blood was measured and calculated, which was the hematocrit. The changes in hematocrit of each group were compared. The results are as follows: Fig.13 As shown in C.

[0080] 3) Platelet count measurement

[0081] After the treatment, an appropriate amount of anticoagulated whole blood was collected using an EDTA anticoagulation tube, and the platelet count of each group of mice was detected using a fully automatic five-category blood analyzer. The results were as follows: Fig.13 As shown in D.

[0082] 4) Detection of megakaryocyte surface markers CD41 and CD61 by flow cytometry

[0083] Spleen tissues of mice in different groups were collected after grinding and filtration. Spleen cells were collected into 15mL centrifuge tubes, centrifuged at 1500rpm for 5min, supernatant was discarded, resuspended in PBS into 1.5mL EP tubes, centrifuged at 1500rpm for 5min, and resuspended in 100μL PBS. 0.5μL CD41-FITC and CD61-FITC antibodies were added to each tube, mixed, incubated at 4℃ in the dark for 40min, filtered through a 300-mesh sieve, and analyzed by flow cytometry. The results are shown in Figure 2. Fig.14 (A is the effect of compound N2 on megakaryocyte surface marker molecules CD41 and CD61 in mouse spleen cells, B is a statistical graph of the proportion of CD41 positive cells, and C is a statistical graph of the proportion of CD61 positive cells).

[0084] 5) Mouse body weight measurement

[0085] Weigh your body weight with an electronic balance. Fig.15 shown.

[0086] Each experiment was repeated three times independently, and all data were expressed as mean ± SD. GraphPad Prism6 was used for analysis. Independent sample t-test was used for comparison between two samples, and one-way analysis of variance was used for comparison between multiple samples. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001.

[0087] Depend on Fig.13 As can be seen in A and B, the spleen size and weight of mice can be seen. Compared with the normal group, the spleen of the model group is significantly enlarged, indicating that the leukemia mouse model was successfully established. In addition, the doses of 10 mg compound N2 / kg mouse and 20 mg compound N2 / kg mouse can significantly reduce the spleen volume and spleen weight of leukemia mice. Fig.13 As shown in Figure C, compared with the normal control group, the hematocrit of the model group was significantly reduced, and the hematocrit of the mice in the compound N2 treatment group was significantly increased compared with the model group. At the same time, compared with the normal control group, the platelet count of the model group was reduced. After treatment with 10 mg compound N2 / kg mouse and 20 mg compound N2 / kg mouse, the platelet count of leukemia mice was significantly increased ( Fig.13 These results indicate that compound N2 can improve the symptoms of leukemia mice and has a strong anti-leukemia effect in vivo.

[0088] Depend on Fig.14It can be seen that the flow cytometry results showed that compared with the normal control group, the proportion of CD41-positive cells and CD61-positive cells in the spleen tissue of the model group was significantly reduced. When treated with compound N2, the proportion of CD41-positive cells and CD61-positive cells in the spleen tissue of mice could be significantly increased.

[0089] Depend on Fig.15 It can be seen that compared with the model group, the compound N2 treatment group had no significant effect on the weight of mice. Body weight is one of the physiological indicators of mice, which indirectly reflects the health status of mice.

[0090] It can be seen that the size of the spleen of leukemia mice and the weight of the spleen can be reduced after treatment with the compound N2 provided by the present invention, and the hematocrit and platelet count of the mice have been significantly improved. At the same time, the proportion of CD41-positive cells and CD61-positive cells in the spleen of mice can be significantly increased after treatment with compound N2, and the compound N2 treatment group has no significant effect on the weight of mice, and has high safety. Based on the above results, compound N2 can be used as a potential small molecule compound for the treatment of erythroleukemia, providing a research basis for the development of differentiation therapy drugs for erythroleukemia.

[0091] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. Use of a compound N2 in the preparation of a drug for inducing differentiation therapy of erythroleukemia, wherein the chemical structure of the compound N2 is shown in Formula I:

2. Use of the compound N2 as claimed in claim 1 in the preparation of a drug for inducing differentiation therapy of erythroleukemia, wherein the active ingredient of the drug is the compound N2 and further comprises pharmaceutically acceptable excipients.

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