Design, Synthesis, and Anti-Leukemic Evaluation of a Series of Dianilinopyrimidines by Regulating the Ras/Raf/MEK/ERK and STAT3/c-Myc Pathways

Although the long-term survival rate for leukemia has made significant progress over the years with the development of chemotherapeutics, patients still suffer from relapse, leading to an unsatisfactory outcome. To discover the new effective anti-leukemia compounds, we synthesized a series of dianilinopyrimidines and evaluated the anti-leukemia activities of those compounds by using leukemia cell lines (HEL, Jurkat, and K562). The results showed that the dianilinopyrimidine analog H-120 predominantly displayed the highest cytotoxic potential in HEL cells. It remarkably induced apoptosis of HEL cells by activating the apoptosis-related proteins (cleaved caspase-3, cleaved caspase-9 and cleaved poly ADP-ribose polymerase (PARP)), increasing apoptosis protein Bad expression, and decreasing the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL). Furthermore, it induced cell cycle arrest in G2/M; concomitantly, we observed the activation of p53 and a reduction in phosphorylated cell division cycle 25C (p-CDC25C) / Cyclin B1 levels in treated cells. Additionally, the mechanism study revealed that H-120 decreased these phosphorylated signal transducers and activators of transcription 3, rat sarcoma, phosphorylated cellular RAF proto-oncogene serine / threonine kinase, phosphorylated mitogen-activated protein kinase kinase, phosphorylated extracellular signal-regulated kinase, and cellular myelocytomatosis oncogene (p-STAT3, Ras, p-C-Raf, p-MEK, p-MRK, and c-Myc) protein levels in HEL cells. Using the cytoplasmic and nuclear proteins isolation assay, we found for the first time that H-120 can inhibit the activation of STAT3 and c-Myc and block STAT3 phosphorylation and dimerization. Moreover, H-120 treatment effectively inhibited the disease progression of erythroleukemia mice by promoting erythroid differentiation into the maturation of erythrocytes and activating the immune cells. Significantly, H-120 also improved liver function in erythroleukemia mice. Therefore, H-120 may be a potential chemotherapeutic drug for leukemia patients.


Introduction
Acute erythroid leukemia (AEL) is an uncommon subordinate type of acute myeloid leukemia (AML).Its cytology is characterized by a significant accumulation of erythroid cells with arrested maturation [1,2].Treatment for patients with AEL is generally similar to patients with other types of AML [3].Currently, chemotherapy combined with cytarabine (AraC) and anthracyclines is still the standard treatment for AML [4], including AEL [5].However, there are no prospective clinical trials for AEL patients [6] and no proven effective treatment strategies for AEL [7,8].To improve outcomes for patients with AEL, a concerted focus on research and treatment development for AEL is necessary [9].
The up-regulation of the tumor suppressor p53 has been demonstrated to be significant in leukemia [10].In response to DNA damage, hypoxia, oncogene activation, and various other stimuli, p53 undergoes up-regulation, leading to diverse cellular responses such as growth arrest, apoptosis, and DNA repair [11,12].These responses are orchestrated by multiple factors, among which CDC25C plays a crucial role.CDC25C is a target of the p53 tumor suppressor, and its expression is modulated by p53, either being induced or repressed.It has been established that CDC25C is necessary for G2/M arrest [10].Uncontrolled leukemia proliferation and reduced sensitivity to apoptotic-inducing agents are typically associated with the activation of pro-survival pathways [11].The Ras/Raf/MEK/ERK pathway regulates the activity of many proteins involved in apoptosis [12].Due to its involvement in sensitivity to leukemia therapy, this pathway is frequently implicated in the field.Many of the effects of the pathway on apoptosis are mediated by ERK phosphorylation of key apoptotic effector molecules [13].ERK phosphorylates transcription factors that influence the transcription of the Bcl-2 family in the regulation of apoptosis.Increased expression of Bcl-2 and Bcl-xL is observed in some leukemias [11].Regulating the expression of Bcl-2 and Bcl-xL could improve leukemia therapy and ameliorate human health.As a signal transducer, STAT3 can affect proliferation, differentiation, and apoptosis in cancer cells [14].Structural activation of STAT3 has been shown in many forms of leukemia, including erythroleukemia [15,16].Clinical correlations show that constitutively active STAT3 in patients indicates poor prognosis, in addition to more severe anemia, more proliferation of blasts in the blood, and promoting resistance to chemotherapy [17].Inhibition of abnormal activation of STAT3 has been proven to be effective in the treatment of leukemia [18].Molecular studies indicate that STAT3 mediates transcriptional activation of the transcription factor c-Myc directly [16].c-Myc plays a crucial role in the terminal differentiation of hematopoietic cells via regulating the transcription of downstream target genes [19].A study found that several vital oncogenes in acute myeloid leukemia (AML), such as AML1-ETO, PML/RARa, and PLZF/RARa, induce leukemogenesis by activating c-Myc [20,21].The importance of c-Myc in myeloid leukemogenesis is further demonstrated by the induction of myeloid leukemia using ectopically expressed c-Myc in murine bone marrow (BM) progenitors [20].
Small molecules are highly favored in the development of anti-tumor drugs and the discovery of lead compounds because of their unique chemical and biological properties [22].H-120 is a derivative of N-Phenyl-2-pyrimidinamine. In this study, we evaluated the effect of H-120 on HEL cells and its therapeutic effect in erythroleukemia mice.The results showed that H-120 may achieve an anti-erythroleukemia effect through the Ras/Raf/MEK/ERK signal pathway and STAT3/c-Myc pathway in vitro, and it has a good effect on the treatment of erythroleukemia mice, suggesting that H-120 may be a potential chemotherapeutic drug for patients with erythroleukemia.

Chemistry
The compound H-120 is depicted in Scheme 1.Initially, 2,4-dichloro-5-methylpyrimidine reacted with 2-amino-N-methylthiophene-3-formamide to produce compound A1 with a 54% yield.Subsequently, the 2-chloro group in the pyrimidine ring was substituted by 4-nitroaniline to give compound B1 with a 41% yield.The nitro group of B1 was reduced to an amino group in the presence of Pd/C, and compound C1 was obtained with a 72% yield.At last, compound C1 reacted with propiolic acid, and we obtained the compound H-120 in a 48% yield.The 2,4-dichloropyrimidine is often used as an intermediate compound for the synthesis of various drugs.Its 4-position chlorine atom is more prone to nucleophilic substitution than the 2-position chlorine atom.In this study, compound A1 was synthesized through the nucleophilic substitution reaction between 2,4-dichloro-5-methylpyrimidine and 2amino-N-methylthiophene-3-carboxamide.Then, the structures of A1 were numbered, and each signal in the 1 H-NMR and 13 C-NMR spectra was assigned the corresponding proton and carbon number (Supplementary Figure S2).Additionally, the NOESY spectra of compound A1 revealed a correlation between the C4-N-proton and the C5 (CH 3 )-carbon (pyrimidine ring) (Supplementary Figure S7), suggesting a substitution at the 4-position of 2,4-dichloro-5-methylpyrimidine.
The compounds H-122 are depicted in Scheme 2. Initially, 2,4-dichloro-5-trifluoromethylpyrimidine reacted with 2-amino-N-methylbenzamide to produce A2 with a 55% yield.Subsequently, the 2-chloro group in the pyrimidine ring was substituted by 4nitroaniline to give B2 with a 49% yield.The nitro group of B2 was reduced to an amino group in the presence of Pd/C, and C2 was obtained with a 76% yield.Then, C2 successfully reacted with dimethyl squarate to obtain intermediate D1 in a 62% yield.Finally, D1 reacted with (1R,3R,5S)-adamantan-1-amine, giving the compound H-122 in a 39% yield.

Anti-Leukemia Activities of N-Phenyl-2-Pyrimidinamine Derivatives
To estimate the effects of N-Phenyl-2-pyrimidinamine derivatives (Figure 1A) on HEL, K562, and Jurkat cells, we treated these cells with the compounds at different concentrations (1-20 µM) for 48 h.The MTT assay results show that H-120 and H-122 could selectively inhibit the proliferation of HEL, K562, and Jurkat cells.H-121 could selectively impair the proliferation of HEL and Jurkat cells.H-123 blocked the proliferation of the Jurkat cells.Those four compounds had no toxicity to the HL7702 cell line.Moreover, H-120 was the most effective on HEL cells, and the IC50 was 0.148 ± 0.04 µM (Figure 1B).

H-120 Suppressed HEL Cell Proliferation
To validate the effects of H-120 on the viability of HEL cells, we treated the cells with the compound at different concentrations (1-20 µM) for 24 h, 48 h, and 72 h.The MTT assay results showed that the H-120 prevented the HEL cell proliferation in a doseand time-dependent manner (Figure 1C,D).However, H-120 was not cytotoxic on normal HL7702 (IC50 > 20 µM) (Figure 1B).The morphology of HEL cells treated with H-120 gradually crumpled and appeared fragmented (Figure 1E).These data suggest that H-120 significantly prevented the growth of HEL cells.

H-120 Induces Mitochondrial Damage and Apoptosis in HEL Cells
Apoptosis plays a crucial role in impairing cancer cell viability.To determine whether the H-120 inhibiting HEL cell growth is caused by apoptosis, we applied HEL cells with different concentrations of H-120.The results indicated that the apoptosis rate of HEL cells treated with H-120 (1 µM) for 48 h was 14.28%, and the apoptosis rate increased to 36.45% for 72 h (Figure 2A,B).To explore the mechanism of apoptosis, we used JC-1 staining to detect the effect of H-120 on mitochondrial membrane potential.We found that the relative value of the JC-1 monomer was significantly increased with the H-120 concentration accumulation according to flow cytometry analysis (Figure 2C,D).In addition, the number of JC-1 monomeric cells (green / fluorescence) gradually increased, and the red fluorescence gradually decreased with the concentration increasing (Figure 2E).The results confirmed that H-120 can induce HEL cell apoptosis by impairing mitochondria.

H-120 Leads to Cell Cycle Arrest and DNA Damage in HEL Cells
Cell cycle regulation is critical for cell differentiation and proliferation [23].To investigate whether H-120 affects the cell cycle of HEL, we treated HEL cells with various concentrations of H-120.Flow cytometry results indicated that H-120 increased the proportion of the G2/M phase in HEL cells in a dose-dependent manner (Figure 3A), indicating that HEL cells were arrested in the G2/M phase.Furthermore, H-120 up-regulated p53 and decreased the levels of cell cycle proteins p-CDC25C and CyclinB1 in a dose-dependent manner (Figure 3B,C).The Hoechst staining showed that H-120 caused DNA damage in a dose-dependent manner (Figure 3D), elucidating the apoptotic effect of the H-120.

The Effect of H-120 on Apoptosis Proteins in Mitochondrial
To study the effect of H-120 on apoptosis proteins, we treated HEL cells with various concentrations of H-120.The results showed that Bcl-2 and Bcl-xL were significantly down-regulated in HEL cells treated with H-120.But the Bad, cleaved caspase-9, cleaved caspase-3, and cleaved PARP were up-regulated considerably in HEL cells treated with H-120 (Figure 4A-C).

H-120 Regulates the ERK/Ras/Raf/MEK Signal Pathway in HEL Cells
To assess whether the effect of H-120 on HEL cells is involved in the inactivation of Ras/Raf/MEK/ERK/MAPK signaling pathway, we tracked this pathway bottom-up in HEL cells treated with H-120 using Western blotting.The results showed that H-120 treatment could significantly reduce the expressions of Ras, phosphorylated Raf, MEK, and ERK in HEL cells (Figure 4D-F).

H-120 Inhibits STAT3/c-Myc Signal Pathway
To examine the effect of H-120 on the p-STAT3 and c-Myc, we checked the p-STAT3 and c-Myc levels in HEL cells treated with H-120.The results showed that they were significantly down-regulated in a dose-dependent manner.STAT3 acts through its phosphorylation and enters nuclear.The nuclear-cytoplasmic separation experiment was conducted.The results showed that the phosphorylated form of STAT3 did diminish when cells were treated with H-120 in nuclear.Notably, the expression of c-Myc in the nucleus was also drastically reduced (Figure 5A-E).

Anti-Erythroleukemia Activity of H-120 In Vivo
We used the F-MuLV-induced leukemia mice to determine the anti-leukemia activity of H-120.The results indicated that the spleen weight of the H-120 treatment mice significantly declined compared with the model mice.The effect of H-120 treatment is similar to that in the vincristine (VCR) treatment group (Figure 6A,C).In addition, H&E staining showed a large number of basophilic leukemic cells in the spleen of the model group, and the normal structure was destroyed compared with the normal group.However, the number of leukemic cells in the spleens of the VCR and H-120 groups decreased significantly, and the structures of the spleens were significantly restored (Figure 6B).Encouragingly, the levels of liver index, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were significantly lower in the H-120 treatment group than in the model group (Figure 6D,E).The renal index (URA, CREA, and UA), cardiac index (CK-MB), triglycerides (TG), and total cholesterol (TC) of all groups were within the normal range, and the results suggest that H-120 has no effects on the renal and cardiac functions of erythroleukemia mice (Supplementary Figure S1).The CD71 and Ter119 have been broadly used to reflect mice's dynamic maturation of erythrocytes.We tested CD71 and Ter119 expression in spleen cells and bone marrow cells by using flow cytometry.The result suggested that the maturity of erythrocytes CD71 -Ter119 + significantly increased in spleen cells and bone marrow cells when treated with H-120 compared with the model group; furthermore, the effect of H-120 was close to that of VCR treatment, indicating that H-120 can promote erythroid differentiation into the maturation erythrocytes (Figure 7A,B,E,F).Moreover, the proportions of CD4, CD8a, and B220 cells in erythroleukemia spleen also increased significantly in the H-120 treatment group, indicating that H-120 effectively stimulates the immune cells in erythroleukemia mice (Figure 7A,C,D).

Discussion
Due to the rarity of the AEL (2-5% of all leukemias) with uncertain mutations in genes and abnormal changes at the molecular level, there are no standardized treatment methods in clinical practice [7].Small molecular compounds are the main body of biological agents, and the mechanism of inducing apoptosis of cancer cells has been deeply studied.In this study, we found that H-120 inhibited leukemic HEL cells.
Intact components of cell cycle arrest checkpoints are potential targets for novel antineoplastics [24,25].When cell cycle checkpoints are not completed, cell cycle arrest will occur.The G2/M checkpoint is the final opportunity to repair damaged DNA prior to mitosis [26].The upregulation of p53 expression can effectively impede tumor cell cycle progression, leading to subsequent G2/M arrest and apoptosis [26].CDC25C serves as a crucial regulator in orchestrating the G2 to mitotic transition of the cell cycle and is involved in monitoring DNA damage repair processes [27].Its downregulation through phosphorylation can induce cell arrest in the G2/M phase in response to DNA damage, mediated by p53 signaling.Meanwhile, the deletion of Cyclin B1 has been shown to inhibit tumor cell proliferation and decrease apoptosis.Cyclin B1 forms a complex with CDC2, the activation and maintenance of which are facilitated by CDC25C, thereby remaining active during the G2/M phase transition and progression.Our results indicate that H-120 significantly activates p53, reduces the phosphorylation of CDC25C, and diminishes the expression of Cyclin B1.Consequently, treated HEL cells were arrested in the G2/M phase.
As an essential participant in apoptosis, mitochondria is of great significance for cell division and differentiation as well as participation in apoptosis [28,29].Its apoptotic mechanism is almost a dynamic equilibrium maintained by the Bcl-2 family [27].The anti-apoptotic members of the Bcl-2 family, such as Bcl-2 (B cell leukemia or lymphoma gene number2) and Bcl-xL (B cell lymphoma-extra-large), are frequently overexpressed in neoplasia to inhibit apoptotic cell death during tumorigenesis [27].Our study has proved that H-120 can cause a decrease in mitochondrial membrane potential in HEL cells.In addition, H-120 could down-regulate the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL and upregulate the expression of pro-apoptotic protein Bad.Our results implied that H-120 induced mitochondrial damage of HEL cells, resulting in the release of cytochrome C from the mitochondria into cytosol.Caspases are a family of evolutionary conserved cysteine-dependent endoproteases that hydrolyze their substrates after specific aspartic acid residues [30].The apoptotic caspases can be further sub-categorized into initiator and executioner caspases [31].The caspases-9 have domain architecture akin to the inflammatory caspases.However, their function is to initiate apoptosis by activating the executioner caspases-3 [32,33].PARP is the substrate of caspase-3 and is closely related to the integrity of DNA [33,34].This study showed that H-120 upregulated the cleaved Caspase-9, cleaved caspase-3 and cleaved PARP.These results suggest that the apoptosis of HEL cells may be accomplished by the loss of mitochondrial membrane potential (MMP) caused by H-120, which activates the caspase cascade reaction and hydrolyzes PARP to make DNA fragmentation.
Unrestricted leukemia proliferation and decreased sensitivity to apoptotic-inducing agents and chemoresistance are typically associated with activating pro-survival pathways [11].Ras is a small GTP-binding protein [35][36][37][38]; the activity of its protein is regulated by conformational state.When Ras is active GTP is bound.Active Ras can then bind Raf on the Ras-binding domain (BD) present in Raf.This results in the translocation of Raf to the cell membrane then activated by phosphorylation and dephosphorylation [39].Activated Raf can interact with Ras, resulting in the activation of Raf-1 [40].Raf-1 interacts with BCR-ABL to change the distribution of BAG on the mitochondrial membrane, thus preventing the apoptosis of hematopoietic cells with malignant proliferation.We found that H-120 significantly down-regulated the expression of Ras and p-C-Raf.In addition, Raf-1 can phosphorylate and inactivate Bad, which prevents cancer cells from normal apoptosis [41], H-120 can upregulate the expression of Bad.Therefore, we speculate that H-120 may not only restore the apoptosis-promoting ability of Bad from the mitochondrial pathway but also inhibit Raf-1 by inhibiting the activation of Ras, thus restoring the biological activity of Bad and causing HEL apoptosis.Raf is an effective activator of MEK, and ERK (extracellular signal-regulated kinase) is the main physiological substrate of MEK.Stimulation of Raf activates MEK1 and ERK, resulting in phosphorylation of transcription factors, proliferation, and inhibition of apoptosis [37,39].Our findings showed that H-120 could significantly down-regulate the expression of Ras and the phosphorylated expression of MEK and ERK.Furthermore, Zehua et al. reported that disruption of the MEK/ERK/c-Myc central metabolic axis within tumor ECs is sufficient to halt tumor growth; inhibiting this central metabolic axis has positive implications for tumor ECs treatments [42].c-Myc as a downstream effector of ERK signalling, and ERK may increase the transcriptional activity of c-Myc by directly phosphorylating Ser62 and regulating c-Myc horizontally after transcription [43,44].Our investigation indicated that c-Myc expression was significantly decreased in H-120 tread HEL cells, which suggest that H-120 may disrupt the MEK/ERK/c-Myc biogenetic axis in HEL cell to halt its growth.In addition, the anti-erythroleukemia effect of H-120 may be related to the inhibition of the activation of STAT3/c-Myc signal pathway.Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor [45].When STAT3 was activated, phosphorylated STAT3 dimerized and translocated into the nucleus.Then it bonded to consensus response elements in the promoters of target genes, regulating the transcription of several genes associated with cellular proliferation (such as c-Myc, Bcl-2 and cyclins) and survival (such as Bcl-xL) [46].We found that H-120 could significantly reduce the expression of p-STAT3 and c-Myc.Moreover, the p-STAT3 in the nucleus and cytoplasm of HEL cells treated with H-120 significantly decreased with the increase of H-120 concentration, and the content in the nucleus was substantially lower than that in the cytoplasm.These results indicated that H-120 suppressed STAT3 phosphorylation and activation in the cytoplasm.Obstructing STAT3 dimerization and inability to transfer the nucleus normally, and cannot be involved in activation of the c-Myc gene by directly binding to a site overlapping with the c-Myc E2F binding site in the c-Myc gene P2 promoter.Jihui Guo et al. [47] report that activation of STAT3 also stimulates cyclinB1 expression; it modulates G2/M phase checkpoint by regulating gene expressions of cyclinB1 and Cdc2 via E2F.Therefore, H-120 may also down-regulate the expression of cyclinB1 by inhibiting STAT3, thus arresting HEL cells in the G2 phase.

Cell Viability Assays
The logarithmically grown HEL cells were seeded in a 96-well culture plate at a density of 8000 cells per well and treated with different concentrations of H-120 (0.125 µM, 0.25 µM, 0.5 µM, and 1 µM) or 0.1% DMSO (control).After 24 h, 48 h, and 72 h treatment, the cells were added to 10 µL of MTT reagent for 4 h, and then added to 100 µL of triple cleavage fluid (SDS 10 g, isobutanol 5 mL and 10M HCl 0.1 mL, dissolved with ddH 2 O into 100 mL solution).After using a microscope to observe the completely dissolved crystal, the cells' optical absorbance value was measured at 570 nm using an enzyme-labeling instrument (BioTek, Winooski, VT, United States).Inhibition rate = (OD value of DMSO group-OD value of experimental group)/OD value of DMSO group × 100%.

Cell Growth Curve Determination
The logarithmically grown HEL cells were collected and planted in 96-well plates at a density of 8000 cells per well.After the cell state was stabilized, HEL cells were treated with H-120 solution (0.125 µM, 0.25 µM, 0.5 µM, 1 µM), and then the OD value was measured at 570 nm in different periods of time.The growth curves were plotted.

Apoptosis and Cell Cycle Analyses
For the apoptosis detection assay, the logarithmically grown HEL cells were treated with H-120 solution (0.125 µM, 0.25 µM, 0.5 µM, and 1 µM) for 24 h, 48 h, and 72 h.Subsequently, the cells were collected and rinsed three times with pre-cooled PBS.Based on the instruction of the Annexin V-FITC apoptosis detection kit, the cells were resuspended in 50 µL of 1 × binding buffer and stained for 15 minutes.Then, the apoptosis rate was determined using flow cytometry (ACEA NovoCyte, San Diego, CA, United States).For cell cycle analysis, HEL cells were treated with the different concentrations of H-120 (0.125 µM, 0.25 µM, 0.5 µM, 1 µM) for 24 h.Cells were collected, fixed with pre-cooled 70% ethanol, and kept at 4 • C for 4 h before being transferred to −20 • C overnight.Cells were stained with 0.5 mL of PI (propidium iodide, 50 µg/mL), RNase (5 µg/mL) inhibitor, and Triton × 100 (0.5 µg/mL) for 10 min at room temperature in the dark and then analyzed by flow cytometry.

Hoechst 33258 Staining
After subjecting HEL cells to H-120 solution (0.125 µM, 0.25 µM, 0.5 µM, 1µM) for 24 h, the cells were collected, resuspended, and washed with pre-cooled PBS.Subsequently, the cells were fixed, stained, and washed according to the instructions provided by the Apoptosis-Hoechst 33258 Staining Kit.To prevent fluorescence interference, an appropriate volume of anti-fluorescence quenching sealing solution was added to the cells.The cells were then placed on a spare slide, covered with a coverslip, and immediately observed and photographed using a Nikonfluorescence microscope.

Mitochondrial Membrane Potential Assay
Cells (2 × 10 5 ) were seeded in a 2 mL medium with different concentrations of H-120 (0.125 µM, 0.25 µM, 0.5 µM, 1 µM).After being treated with H-120 for 48 h, the cells were preincubated with JC-1 working solution for 20 min at 37 • C, 5% CO 2 .After incubation, the dye was removed, and the cells were washed two times with JC-1 [48].Subsequently, the change in the membrane potential of treated cells concerning control cells was detected using a fluorescence microscope, and the proportion of cells exhibiting red or green fluorescence was documented.

Extraction of Nuclear Protein and Cytoplasmic Protein
HEL cells were collected after 24 h of H-120 treatment and washed with PBS, and the supernatant was discarded.According to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China), we added cytoplasmic protein extraction reagent A equipped with PMSF (phenylmethanesulfonyl fluoride) and used vigorous vortexing at 2500 rpm for 5 s to suspend and disperse the cell precipitation entirely.After 15 min of an ice bath, we added cytoplasmic protein extraction reagent B. The sample was vigorously vortexed at 2500 rpm for 5 s, then centrifuged at 4 • C for 5 min at 12,000× g, and the supernatant was extracted to leave a precipitate.The supernatant was the cytoplasmic protein.For the precipitate, the cytosolic protein extraction reagent equipped with PMSF was added immediately, mixed, and then placed on ice.The highest-speed vigorous vortex was performed for 20 s every 2 min for 30 min, followed by centrifugation at 12,000× g at 4 • C for 10 min.Finally, the supernatant was taken as the nuclear protein.

Western Blotting Analysis
HEL cells after drug action were collected, added to and mixed with cell lysis buffer, and immunoprecipitated on ice for one hour before protein concentration was detected using a BCA (bicinchoninic acid) kit.Proteins (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF (polyvinylidene fluoride) membrane (0.22 µM, Bio-Rad, Hercules, CA, USA) via wet transfer.Then, 5% bovine serum albumin closed the membrane for 1 h, and the membrane was washed three times with TBS (20 mM Tris-HCl) for 5 min each time.Membranes were allowed to incubate with the primary antibody overnight at 4 • C. The anti-rabbit antibody secondary antibody was incubated at room temperature for 2 h.Then, the membrane was washed with 1× TBST (20 mM Tris-HCl, 0.1% Tween 20) for 15 min.After washing, the fetal membrane was scanned with an infrared imaging scanner.β-actin was used as a loading control.

Animal Experiment
The NIH3T3 cells expressing the F-MuLV clone 57 vector were donated by Professor Ben-David Yaacov, Key Laboratory of Natural Product Chemistry, Chinese Academy of Sciences, Guizhou Province.In order to induce leukemia, BALB/c mice (male and females; Tongxin, Chongqing, China), aged one day, were administered a solitary intraperitoneal injection of F-MuLV.Then, infected neonates were weaned at 4 weeks and randomly divided into 6 groups: normal group (NC), model group (M), positive control group (VCR: 0.25 mg/kg), H-120 low dose group (H-120-L: 1 mg/kg), H-120 medium dose group (H-120-M: 2 mg/kg), and H-120 high dose group (H-120-H: 4 mg/kg).After 5 weeks of virus infection, mice were treated with H-120 intraperitoneally every other day for 3 weeks.The normal and model groups were given saline, and the control group was given vincristine intraperitoneally.Under the supervision of experienced staff, the leukemia mice were sacrificed humanely by cervical dislocation.Serum biochemical indexes of the mice were detected.The spleens of the mice were removed.A portion of the spleen was fixed in formaldehyde for 24 h, and paraffin was embedded and sliced.The effects of H-120 on the normal physiological structure of the spleen were observed by H&E staining.Then, part of the spleen tissue was ground and prepared into a single-cell suspension, which was stained with CD71, Ter119, CD4, CD8a, and B220 antibodies for 30 min, respectively.The expression of markers on the spleen cell surfaces of mice was analyzed by flow cytometry.

Ethics Statement
All animal experiments were approved by the ethics committee of State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou Medical University with the approval number of 2200956.

Statistical Analysis
All data are presented as the mean ± standard deviation (SD) from at least three independent experiments.Statistical analysis was performed using Student's t-test or one-way ANOVA in GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA) and SPSS 26.0 (IBM Corporation, Armonk, NY, USA).p < 0.05 was considered statistically significant.

Conclusions
H-120 exerted excellent anti-leukemia activity both in vitro and in vivo.The compound predominantly displayed the highest cytotoxic potential in HEL cells.It induced apoptosis in HEL cells through the mitochondrial caspase-dependent pathway and arrested HEL cells in G2/M.Furthermore, H-120 inhibited HEL cell proliferation by dual blockade of the Ras/Raf/MEK/ERK and STAT3/c-Myc pathways (Figure 8).Moreover, the compound effectively inhibited the progression of and protected liver function from leukemia in mice.Therefore, H-120 may be a potential chemotherapeutic drug for leukemia patients.

Figure 2 .
Figure 2. H-120 induced apoptosis and mitochondrial damage in HEL cells.(A) HEL cell apoptosis was detected by annexin-V/propidium iodide double staining using flow cytometry.(B) Densitometry of the apoptotic cell percentage at an indicated time interval of H-120 incubation.(C) HEL cells treated with H-120 for 48 h were stained with JC-1 and analyzed using flow cytometry.(D) Graph showing the percentage of depolarized cells in mitochondria at various concentrations of H-120 treatment.(E) Photomicrograph of JC-1 monomers and aggregates after H-120 incubation at 48 h (magnification, ×200; scale bar = 100 µm).Data are represented as mean ± SD (n = 3; * p < 0.05; ** p < 0.001; *** p < 0.001; vs. the DMSO group).

Figure 3 .
Figure 3. H-120 induces DNA damage in HEL cells and arrest in G2/M phase.(A) Cell cycle arrest evaluation by flow cytometry after 24 h, and densitometry plots depicting the variations in the stages of cell cycle arrest.(B) The expression of CDC25C, p-CDC25C, CyclinB1, and p53 was measured by Western blotting.(C) Quantification of relative protein expression levels of CDC25C, p-CDC25C, CyclinB1, and p53, with β-actin used as loading control.(D) Photomicrograph of DNA damage (the pointing of the arrow) at indicated concentrations of H-120 treatment (magnification, ×200; scale bar = 100 µm).Data are represented as mean ± SD (n = 3, * p < 0.05, ** p < 0.01, *** p <0.001, vs. the DMSO group).

Figure 5 .
Figure 5.The effects of H-120 on STAT3/c-Myc pathway.(A) The expression of STAT3, p-STAT3, and c-Myc were measured by Western blotting.(B) Quantification of relative protein expression levels of STAT3, p-STAT3, c-Myc, and β-actin was used as loading control; (C) Isolating cytoplasmic and nuclear proteins and the expression of STAT3, p-STAT3, and c-Myc were measured by Western blotting.(D,E): Quantification of relative protein expression levels of STAT3, p-STAT3, c-Myc, and β-actin was used as loading control for cytoplasmic protein; Lamin B1 was used as loading control for nuclear protein.Data are represented as mean ± SD (n = 3, * p < 0.05, *** p <0.001, vs. the DMSO group).

Figure 7 .
Figure 7. Effect of H-120 on the expression of surface antigens CD71, Ter119, B220, CD4, and CD8a in spleen cells and bone marrow cells of erythroleukemia mice induced by F-MuLV.(A) Flow cytometry was used to analyze the effect of H-120 on the expression of spleen cell surface antigens CD71, Ter119, B220, CD4, and CD8a.(B-D) Statistical map of surface antigens CD71, Ter119, B220, CD4, and CD8a and expression of spleen cells in mice.(E) Flow cytometry was used to analyze the effect of H-120 on the expression of mouse bone marrow cell surface antigens CD71 and Ter119.(F) Statistical map of surface antigens CD71 and Ter119 and expression of bone marrow cells in mice.Data are represented as mean ± SD (n = 3; # p < 0.05, ## p < 0.01, ### p < 0.001, vs. M; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. M).