Novel 9-Methylanthracene Derivatives as p53 Activators for the Treatment of Glioblastoma Multiforme

Glioblastoma multiforme, a highly aggressive and lethal brain tumor, is a substantial clinical challenge and a focus of increasing concern globally. Hematological toxicity and drug resistance of first-line drugs underscore the necessity for new anti-glioma drug development. Here, 43 anthracenyl skeleton compounds as p53 activator XI-011 analogs were designed, synthesized, and evaluated for their cytotoxic effects. Five compounds (13d, 13e, 14a, 14b, and 14n) exhibited good anti-glioma activity against U87 cells, with IC50 values lower than 2 μM. Notably, 13e showed the best anti-glioma activity, with an IC50 value up to 0.53 μM, providing a promising lead compound for new anti-glioma drug development. Mechanistic analyses showed that 13e suppressed the MDM4 protein expression, upregulated the p53 protein level, and induced cell cycle arrest at G2/M phase and apoptosis based on Western blot and flow cytometry assays.


Introduction
The World Health Organization classifies brain tumors into four grades (I-IV) based on histopathological observations and their severity [1].Glioblastoma multiforme (GBM) is one of the most lethal and highly aggressive grade IV brain and spinal cord tumors that usually occur in glial cells of the central nervous system [2].Statistically, GBM accounts for approximately 45.2% of primary malignant brain tumors in adult patients [3].Chemoradiotherapy for GBM has many treatment challenges, such as the rapid proliferation rate of GBM cells, the differentiation of treatment-resistant cell clones, and the difficulty of passing through the blood-brain barrier to access the brain parenchyma, etc. [4].Because of the infiltrative, diffuse, and sporadic nature of GBM, traditional surgical resection and concomitant chemoradiotherapy only extend the median survival rate to 14 months and improve the mean 3-year survival from 1.9% to 16% [5].
Oral imidazotetrazine alkylating agent temozolomide (TMZ) is the most widely used first-line chemotherapeutic drug for GBM patients [6].After entering cells, TMZ is converted to 3-methyl-(triazen-1-yl)imidazole-4-carboxamide and induces methylation of the O6-guanine, N7-guanine, and N3-adenine in DNA, inducing cell cycle arrest in G2/M phase, thereby inhibiting cancer cell proliferation [7].However, the hematological toxicity and strong resistance of TMZ constrain its efficacy in clinical use [8].The high resistance against methylguanine-DNA-methyltransferase, which often occurs during chemotherapy, results in a poor response to TMZ treatment in approximately 50% of patients.More importantly, gliomas obtain TMZ resistance through multiple mechanisms during TMZ treatment, and many patients with high methylguanine-DNA-methyltransferase expression are naturally resistant to TMZ [9].Other first-line anti-glioma drugs, such as carmustine [10], lomustine [11], and the humanized monoclonal antibody bevacizumab [12], result in pul-monary toxicity, hypertension, and leucopenia [13].Therefore, the development of new chemotherapeutic drugs for GBM treatment is still urgent.
As a potent tumor suppressor, the p53 pathway is one of the most promising targets in tumors [14].Restoring p53 function to induce apoptosis or growth arrest has been considered a practical approach to restrain cancer.However, more than half of GBM patients exhibit p53 positivity [15].p53 is prominently regulated by mouse double-minute protein (MDM) 2 and its homolog MDM4 [16], which bind to the N-terminal transactivation domain of p53, blocking its transcriptional function.Downregulation of p53 protein is responsible for the proliferation, invasion, migration, avoidance of apoptosis, and other properties of GBM cells [17].Therefore, reducing MDM4 or MDM2 expression and restoring p53 function represent an attractive GBM treatment strategy.Various MDM intracellular protein-protein interaction inhibitors have been developed to stabilize p53 for cancer treatment [18,19].Although several MDM2 inhibitors have entered clinical trials, increasing evidence suggests that enhanced inhibition of MDM4 remains critical for this class of inhibitors to exert more sensitive and potent activity to release p53 [20].Because the binding domain of MDM4 to p53 contains a peptide sequence and three-dimensional structure that is highly similar to MDM2, the development of selective MDM4 inhibitor remains challenging [21].
As a potent tumor suppressor, the p53 pathway is one of the most promising targets in tumors [14].Restoring p53 function to induce apoptosis or growth arrest has been considered a practical approach to restrain cancer.However, more than half of GBM patients exhibit p53 positivity [15].p53 is prominently regulated by mouse double-minute protein (MDM) 2 and its homolog MDM4 [16], which bind to the N-terminal transactivation domain of p53, blocking its transcriptional function.Downregulation of p53 protein is responsible for the proliferation, invasion, migration, avoidance of apoptosis, and other properties of GBM cells [17].Therefore, reducing MDM4 or MDM2 expression and restoring p53 function represent an attractive GBM treatment strategy.Various MDM intracellular protein-protein interaction inhibitors have been developed to stabilize p53 for cancer treatment [18,19].Although several MDM2 inhibitors have entered clinical trials, increasing evidence suggests that enhanced inhibition of MDM4 remains critical for this class of inhibitors to exert more sensitive and potent activity to release p53 [20].Because the binding domain of MDM4 to p53 contains a peptide sequence and three-dimensional structure that is highly similar to MDM2, the development of selective MDM4 inhibitor remains challenging [21].

In Vitro Antiproliferative Activity Analysis
To determine whether these synthesized XI-011 analogs had desired p53 activation and antiproliferative activities, all compounds were tested for their antiproliferative effect at 10 µM in the human glioblastoma cell line U87.

In Vitro Antiproliferative Activity Analysis
To determine whether these synthesized XI-011 analogs had desired p53 activation and antiproliferative activities, all compounds were tested for their antiproliferative effect at 10 µM in the human glioblastoma cell line U87.
As shown in Table 1, XI-011 and doxorubicin (DOX) were chosen as controls.XI-011 and DOX induced high inhibition rates of U87 cells at 10 µM (91.8% and 81.1%, respectively).DMSO (1%) was used as the vehicle control.SAR analysis of most small sterically hindered analogs, such as 1-naphthyl, 2-naphthyl, and benzyl analogs (12a-12f), showed slight inhibition of U87 cells, and the inhibition rates were maintained at 1.5-15.2%.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.No-tably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.Whether the methyl group was substituted on the naphthalene ring (12a vs. 12b) had little effect on the inhibitory activity.Hydrazinecarbimidothioate analogs 12f and 12g exhibited poor inhibition rates of 13.1% and 14.5% at 10 µM, respectively.The quinolinyl-derived hydrazinecarbimidothioate analog (12k) only had a 10.7% inhibition rate in U87 cells with similar potency to 12g.Notably, after replacing the naphthyl group with a 10-methylanthracenyl group, the resulting compound 13a exhibited approximately 4-fold more potency than 12f, suggesting that the 10-methylanthracenyl was optimal for cell inhibition.a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and py- Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM. a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM. a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM. a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM. a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM.  a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM. a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM.  a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM.  a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.
Next, we explored more substitutions with various thiourea groups for potency while keeping the 10-methylanthracenyl.As indicated in Table 2, moderate U87 inhibitory profiles were observed when the synthesized compounds featured an acetyl (13b) or amidinyl (13c) group.Aryl groups, such as phenyl (13j and 13k), imidazolyl (13g and 13i), and pyrimidine-4,6(1H,5H)-dione-derived (13h) analogs, all showed a significant decrease in the inhibition rate in U87 cells.Compared with these aryl-derived compounds, long-chain alkyl (13l) and imine (13f) analogs exhibited good anti-glioma activity and had a 3-4-fold potency improvement in U87 cells.Remarkably, the compound 13d and methylhydrazine-derived analog 13e showed the highest anti-glioma potency, with 93.3% and 88.6% inhibition rates at 10 µM.Encouraged by the promising anti-glioma effect of analogs 13d and 13e, we performed further optimization of the anthracenyl skeleton.
After replacing the methyl-substituent with an ethyl group at the 10-position of anthracene, seven new analogs (14a-14g) were prepared and evaluated for their anti-glioma activity.As shown in Table 3, analogs 14a-14c exhibited good potency, indicating that introducing a longer hydrophobic substituent on the 10-position might be beneficial for improving their anti-glioma activity.However, the compounds with acetyl (14e), amidinyl (14d), hexyl (14f), and p-CF3-phenyl (14g) groups showed 3~4-fold decreases in antiglioma activity.Further increasing the chain length by one methylene unit (14h) at the 10position of anthracene led to a slight decrease in potency relative to 14a.After removing the alkyl-substituents, compounds 14i-14l exhibited weak to moderate anti-glioma activity.Only analog 14i displayed pronounced good anti-glioma activity (83.0%) at 10 µM.A similar trend was observed when the methyl-substituent was replaced by a bromo-substituent group (14i vs. 14n and 14o vs. 14j).Three acridine analogs (14p-14r) had weak inhibition rates against U87 cells, demonstrating that the heteroatoms in the anthracenyl skeleton were not well tolerated.Notably, compound 14s with a non-rigid structure exhibited approximately 12-fold less potency than XI-011, indicating that the rigid anthracenyl skeleton was necessary.Encouraged by the promising anti-glioma effect of analogs 13d and 13e, we performed further optimization of the anthracenyl skeleton.
After replacing the methyl-substituent with an ethyl group at the 10-position of anthracene, seven new analogs (14a-14g) were prepared and evaluated for their antiglioma activity.As shown in Table 3, analogs 14a-14c exhibited good potency, indicating that introducing a longer hydrophobic substituent on the 10-position might be beneficial for improving their anti-glioma activity.However, the compounds with acetyl (14e), amidinyl (14d), hexyl (14f), and p-CF 3 -phenyl (14g) groups showed 3~4-fold decreases in anti-glioma activity.Further increasing the chain length by one methylene unit (14h) at the 10-position of anthracene led to a slight decrease in potency relative to 14a.After removing the alkylsubstituents, compounds 14i-14l exhibited weak to moderate anti-glioma activity.Only analog 14i displayed pronounced good anti-glioma activity (83.0%) at 10 µM.A similar trend was observed when the methyl-substituent was replaced by a bromo-substituent group (14i vs. 14n and 14o vs. 14j).Three acridine analogs (14p-14r) had weak inhibition rates against U87 cells, demonstrating that the heteroatoms in the anthracenyl skeleton were not well tolerated.Notably, compound 14s with a non-rigid structure exhibited approximately 12-fold less potency than XI-011, indicating that the rigid anthracenyl skeleton was necessary.a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates.
Encouraged by the promising anti-glioma effect of analogs 13d and 13e, we performed further optimization of the anthracenyl skeleton.
After replacing the methyl-substituent with an ethyl group at the 10-position of anthracene, seven new analogs (14a-14g) were prepared and evaluated for their anti-glioma activity.As shown in Table 3, analogs 14a-14c exhibited good potency, indicating that introducing a longer hydrophobic substituent on the 10-position might be beneficial for improving their anti-glioma activity.However, the compounds with acetyl (14e), amidinyl (14d), hexyl (14f), and p-CF3-phenyl (14g) groups showed 3~4-fold decreases in antiglioma activity.Further increasing the chain length by one methylene unit (14h) at the 10position of anthracene led to a slight decrease in potency relative to 14a.After removing the alkyl-substituents, compounds 14i-14l exhibited weak to moderate anti-glioma activity.Only analog 14i displayed pronounced good anti-glioma activity (83.0%) at 10 µM.A similar trend was observed when the methyl-substituent was replaced by a bromo-substituent group (14i vs. 14n and 14o vs. 14j).Three acridine analogs (14p-14r) had weak inhibition rates against U87 cells, demonstrating that the heteroatoms in the anthracenyl skeleton were not well tolerated.Notably, compound 14s with a non-rigid structure exhibited approximately 12-fold less potency than XI-011, indicating that the rigid anthracenyl skeleton was necessary.a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates.
Encouraged by the promising anti-glioma effect of analogs 13d and 13e, we performed further optimization of the anthracenyl skeleton.
After replacing the methyl-substituent with an ethyl group at the 10-position of anthracene, seven new analogs (14a-14g) were prepared and evaluated for their anti-glioma activity.As shown in Table 3, analogs 14a-14c exhibited good potency, indicating that introducing a longer hydrophobic substituent on the 10-position might be beneficial for improving their anti-glioma activity.However, the compounds with acetyl (14e), amidinyl (14d), hexyl (14f), and p-CF3-phenyl (14g) groups showed 3~4-fold decreases in antiglioma activity.Further increasing the chain length by one methylene unit (14h) at the 10position of anthracene led to a slight decrease in potency relative to 14a.After removing the alkyl-substituents, compounds 14i-14l exhibited weak to moderate anti-glioma activity.Only analog 14i displayed pronounced good anti-glioma activity (83.0%) at 10 µM.A similar trend was observed when the methyl-substituent was replaced by a bromo-substituent group (14i vs. 14n and 14o vs. 14j).Three acridine analogs (14p-14r) had weak inhibition rates against U87 cells, demonstrating that the heteroatoms in the anthracenyl skeleton were not well tolerated.Notably, compound 14s with a non-rigid structure exhibited approximately 12-fold less potency than XI-011, indicating that the rigid anthracenyl skeleton was necessary.a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates.
Considering the potent inhibitory activities at 10 µM, the IC50 values of 13d, 13e, 14a, 14b, and 14n were further investigated in human glioblastoma cell line U87, human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, and HHL-5 as a human normal cell line by MTT assays.As shown in Table 4, all compounds showed good in vitro antitumor activities against the three cancer cell lines.Compound 13e showed effective anti-cervical activity against HeLa cells (IC50 = 1.63 µM) and MCF-7 cells (IC50 = 0.97 µM), which was similar to that of Dox (Hela: IC50 = 0.54 µM; MCF-7: IC50 = 1.32 µM).The methyl and ethyl groups in anthracene (14a, 14b, and XI-011) might be crucial for the anti-cervical cancer activity, and substitution with bromo (14n) resulted in a 1-2-fold reduction in ac- a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates.
Considering the potent inhibitory activities at 10 µM, the IC 50 values of 13d, 13e, 14a, 14b, and 14n were further investigated in human glioblastoma cell line U87, human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, and HHL-5 as a human normal cell line by MTT assays.As shown in Table 4, all compounds showed good in vitro antitumor activities against the three cancer cell lines.Compound 13e showed effective anti-cervical activity against HeLa cells (IC 50 = 1.63 µM) and MCF-7 cells (IC 50 = 0.97 µM), which was similar to that of Dox (Hela: IC 50 = 0.54 µM; MCF-7: IC 50 = 1.32 µM).The methyl and ethyl groups in anthracene (14a, 14b, and XI-011) might be crucial for the anti-cervical cancer activity, and substitution with bromo (14n) resulted in a 1-2-fold reduction in activity.Notably, 13e exhibited the best anti-breast cancer cell activity in MCF-7 cells, which was 5-8-fold more potent than that of 13d, 14a, 14b, and XI-011.MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates.
Considering the potent inhibitory activities at 10 µM, the IC50 values of 13d, 13e, 14a, 14b, and 14n were further investigated in human glioblastoma cell line U87, human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, and HHL-5 as a human normal cell line by MTT assays.As shown in Table 4, all compounds showed good in vitro antitumor activities against the three cancer cell lines.Compound 13e showed effective anti-cervical activity against HeLa cells (IC50 = 1.63 µM) and MCF-7 cells (IC50 = 0.97 µM), which was similar to that of Dox (Hela: IC50 = 0.54 µM; MCF-7: IC50 = 1.32 µM).The methyl and ethyl groups in anthracene (14a, 14b, and XI-011) might be crucial for the anti-cervical cancer activity, and substitution with bromo (14n) resulted in a 1-2-fold reduction in activity.Notably, 13e exhibited the best anti-breast cancer cell activity in MCF-7 cells, which was 5-8-fold more potent than that of 13d, 14a, 14b, and XI-011.Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates.
Considering the potent inhibitory activities at 10 µM, the IC50 values of 13d, 13e, 14a, 14b, and 14n were further investigated in human glioblastoma cell line U87, human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, and HHL-5 as a human normal cell line by MTT assays.As shown in Table 4, all compounds showed good in vitro antitumor activities against the three cancer cell lines.Compound 13e showed effective anti-cervical activity against HeLa cells (IC50 = 1.63 µM) and MCF-7 cells (IC50 = 0.97 µM), which was similar to that of Dox (Hela: IC50 = 0.54 µM; MCF-7: IC50 = 1.32 µM).The methyl and ethyl groups in anthracene (14a, 14b, and XI-011) might be crucial for the anti-cervical cancer activity, and substitution with bromo (14n) resulted in a 1-2-fold reduction in activity.Notably, 13e exhibited the best anti-breast cancer cell activity in MCF-7 cells, which was 5-8-fold more potent than that of 13d, 14a, 14b, and XI-011.a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates.
Considering the potent inhibitory activities at 10 µM, the IC50 values of 13d, 13e, 14a, 14b, and 14n were further investigated in human glioblastoma cell line U87, human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, and HHL-5 as a human normal cell line by MTT assays.As shown in Table 4, all compounds showed good in vitro antitumor activities against the three cancer cell lines.Compound 13e showed effective anti-cervical activity against HeLa cells (IC50 = 1.63 µM) and MCF-7 cells (IC50 = 0.97 µM), which was similar to that of Dox (Hela: IC50 = 0.54 µM; MCF-7: IC50 = 1.32 µM).The methyl and ethyl groups in anthracene (14a, 14b, and XI-011) might be crucial for the anti-cervical cancer activity, and substitution with bromo (14n) resulted in a 1-2-fold reduction in activity.Notably, 13e exhibited the best anti-breast cancer cell activity in MCF-7 cells, which was 5-8-fold more potent than that of 13d, 14a, 14b, and XI-011.a Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates.

Mechanistic Analyses of 13e against U87 Cells
Considering the good antiproliferative effect of 13e, the expression of MDM4, p53, and GAPDH was evaluated by Western blot analysis after treating U87 cells with 13e.As shown in Figure 2, after 24 h of treatment with 13e, we observed dose-dependent suppression of MDM4 expression and remarkable stabilization of p53 protein in U87 cells, which was consistent with its cytotoxicity.Notably, compound 13e significantly upregulated p53 protein expression, demonstrating similar effects at 1 µM compared with XI-011.Collectively, these findings provide compelling evidence that compound 13e may exert a strong suppressive effect on MDM4 expression, stabilize p53, and upregulate p53 expression through an MDM4-p53-dependent mechanism, which is similar to the effects of XI-011.

Mechanistic Analyses of 13e against U87 Cells
Considering the good antiproliferative effect of 13e, the expression of MDM4, p53, and GAPDH was evaluated by Western blot analysis after treating U87 cells with 13e.As shown in Figure 2, after 24 h of treatment with 13e, we observed dose-dependent suppression of MDM4 expression and remarkable stabilization of p53 protein in U87 cells, which was consistent with its cytotoxicity.Notably, compound 13e significantly upregulated p53 protein expression, demonstrating similar effects at 1 µM compared with XI-011.Collectively, these findings provide compelling evidence that compound 13e may exert a strong suppressive effect on MDM4 expression, stabilize p53, and upregulate p53 expression through an MDM4-p53-dependent mechanism, which is similar to the effects of XI-011.
Next, we conducted flow cytometric analysis of cell cycle progression in U87 cells treated with 13e to investigate its cellular mechanisms.As shown in Figure 3, 13e at three concentrations arrested U87 cells in G2/M phase and demonstrated a dose-dependent effect similar to XI-011.Compared with the control, treatment with 0.5, 1, and 2 µM 13e decreased the proportion of U87 cells in S phase and increased the proportion of U87 cells in the G2/M fraction.These results indicated that the strong antiproliferative activity of 13e in U87 cells was caused by U87 cell cycle arrest at G2/M phase.Next, we conducted flow cytometric analysis of cell cycle progression in U87 cells treated with 13e to investigate its cellular mechanisms.As shown in Figure 3, 13e at three concentrations arrested U87 cells in G2/M phase and demonstrated a dose-dependent effect similar to XI-011.Compared with the control, treatment with 0.5, 1, and 2 µM 13e decreased the proportion of U87 cells in S phase and increased the proportion of U87 cells in the G2/M fraction.These results indicated that the strong antiproliferative activity of 13e in U87 cells was caused by U87 cell cycle arrest at G2/M phase.
Reducing MDM4 expression restores the p53 function and increases apoptotic cells [25].The apoptotic effects of compound 13e were determined in U87 cells by flow cytometry.As shown in Figure 4, compounds 13e and XI-011 dose-dependently induced U87 cell apoptosis.13e induced 21% apoptosis at 1 µM, which was slightly lower than the 23% apoptosis induced by XI-011.At 2 µM 13e, apoptosis was up to 25.9%.These results provide evidence that compound 13e effectively induces apoptosis in U87 cells.The mechanism by which 13e induces apoptosis in U87 cells may be related to its ability to downregulate MDM4 expression and stabilize p53 protein.Reducing MDM4 expression restores the p53 function and increases apoptotic cells [25].The apoptotic effects of compound 13e were determined in U87 cells by flow cytometry.As shown in Figure 4, compounds 13e and XI-011 dose-dependently induced U87 cell apoptosis.13e induced 21% apoptosis at 1 µM, which was slightly lower than the 23% apoptosis induced by XI-011.At 2 µM 13e, apoptosis was up to 25.9%.These results provide evidence that compound 13e effectively induces apoptosis in U87 cells.The mechanism by which 13e induces apoptosis in U87 cells may be related to its ability to downregulate MDM4 expression and stabilize p53 protein.

Molecular Docking of 13e
In our previous studies, XI-011 disrupted recruitment of the heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) to the promoter and untranslated region of MDM4, leading to inhibition of MDM4 transcription.Therefore, we analyzed the binding

Molecular Docking of 13e
In our previous studies, XI-011 disrupted recruitment of the heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) to the promoter and untranslated region of MDM4, leading to inhibition of MDM4 transcription.Therefore, we analyzed the binding mode of the XI-011 analog 13e and hnRNPA2B1 protein (PDB code: 5HO4) (Figure 5).The results revealed that the anthracenyl skeleton of compound 13e was well accommodated and positioned similarly to that of XI-011 in the cavity of hnRNPA2B1.The -NH 2 and imine groups in 13e formed crucial hydrogen bond interactions with the carboxyl group in Asp-49 and the carboxyl group in Glu-18.Additionally, the anthracenyl group exhibited cation-π interactions with the terminal amino group of Lys-22 and the guanidine group in Arg-99.As anticipated, we observed similar binding modes of 13e and XI-011 to hnRNPA2B1 (Figure 5D).These interactions may explain the effective inhibition of MDM4 expression by 13e.Accumulated evidence has shown that MDM4 inhibition plays a crucial role in reinstating p53 functions.In this study, structural modifications and SAR analysis were conducted based on the reported p53 activator XI-011.Convenient and efficient synthesis is imperative for the effectiveness of new drug development.Here, anthracenyl skeleton compounds were prepared efficiently by two to three linear steps with good yields.SAR analysis indicated that the anthracenyl skeleton analogs exhibited significant potency against glioma cells compared with phenyl and naphthyl analogs.Interestingly, compounds with a small steric hindrance thiourea substitution exhibited more potent antiproliferative activities compared with their counterparts with bulky steric hindrance.Five anthracenyl skeleton compounds were selected and evaluated for their antiproliferative activities in three cancer cell lines, namely U87, Hela, and MCF-7.Remarkably, compound 13e exhibited antiproliferative effects similar to those of doxorubicin and demonstrated slight selectivity of human cancer U87 cells compared with normal HHL-5 cells.These results suggest that 13e is a promising candidate for cancer therapy.Accumulated evidence has shown that MDM4 inhibition plays a crucial role in reinstating p53 functions.In this study, structural modifications and SAR analysis were conducted based on the reported p53 activator XI-011.Convenient and efficient synthesis is imperative for the effectiveness of new drug development.Here, anthracenyl skeleton compounds were prepared efficiently by two to three linear steps with good yields.SAR analysis indicated that the anthracenyl skeleton analogs exhibited significant potency against glioma cells compared with phenyl and naphthyl analogs.Interestingly, compounds with a small steric hindrance thiourea substitution exhibited more potent antiproliferative activities compared with their counterparts with bulky steric hindrance.Five anthracenyl skeleton compounds were selected and evaluated for their antiproliferative activities in three cancer cell lines, namely U87, Hela, and MCF-7.Remarkably, compound 13e exhibited antiproliferative effects similar to those of doxorubicin and demonstrated slight selectivity of human cancer U87 cells compared with normal HHL-5 cells.These results suggest that 13e is a promising candidate for cancer therapy.
To verify whether the antiproliferative mechanism of 13e was similar to that of XI-011, we conducted a series of experiments, including Western blotting, cell cycle assays, and molecular docking.The results provided evidence that analogs of the anthracenyl skeleton compounds inhibited MDM4 expression, stabilized p53 protein, and induced apoptosis in U87 glioma cells.On the basis of these findings and our previous studies, a proposed anti-glioma mechanism of 13e is illustrated in Figure 6.13e might bind to the interaction between hnRNPA2B1 protein and the MDM4 promoter and untranslated region, which was verified by molecular docking analyses.Next, p53 protein expression is upregulated, ultimately resulting in apoptosis of U87 cells.Several limitations of this study should be highlighted.This study relied on in vit experiments, restricting insights into the drugs' ability to permeate the blood-brain ba rier, which is crucial for GBM treatment.Future studies should validate these findin through experiments involving the blood-brain barrier and xenograft models.Addition experimental evidence is also necessary to fully elucidate the antitumor mechanism these anthracene skeleton compounds.Moreover, further exploration of combining a thracenyl skeleton compounds, which demonstrate the potential for p53 activation, wi other antitumor drugs is warranted.

Cytotoxicity Evaluation
U87, Hela, MCF-7, and HHL-5 cells were provided by Chinese Academy of Medic Sciences (Beijing, China).The cancer cells were cultured in DMEM media containing 10 heat-inactivated fetal calf serum.All cells were incubated at 37 °C in a 5% CO2 humidifie atmosphere and harvested in the exponential growth phase for assays.

MTT Assay
Cells were seeded in a 96-well plate and treated with the test substance at the desire concentration for 72 h.In cytotoxicity experiments, cells were treated with 10 µM of th test compound or 1 µM XI-011 and doxorubicin as positive controls.DMSO (1%) was use as a negative control.In the IC50 analysis, doxorubicin, XI-011, 13d, 13e, 14a, 14b, and, 14 were applied to HHL-5 at 50, 25, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 µM and U cells at 10, 7.5, 5, 25, 1.25, 0.625, 0.3125, and 0.15625 µM, whereas 30, 10, 3, 0.5, and 0.1 µ were applied to Hela and MCF-7 cells.Treated cells were incubated with an MTT solutio for 2 h and then with DMSO to dissolve the crystals.Absorbance was determined at 5 nm using a microplate reader (BioTek, Seattle, DC, USA).IC50 values were calculated usin Several limitations of this study should be highlighted.This study relied on in vitro experiments, restricting insights into the drugs' ability to permeate the blood-brain barrier, which is crucial for GBM treatment.Future studies should validate these findings through experiments involving the blood-brain barrier and xenograft models.Additional experimental evidence is also necessary to fully elucidate the antitumor mechanism of these anthracene skeleton compounds.Moreover, further exploration of combining anthracenyl skeleton compounds, which demonstrate the potential for p53 activation, with other antitumor drugs is warranted.

Cytotoxicity Evaluation
U87, Hela, MCF-7, and HHL-5 cells were provided by Chinese Academy of Medical Sciences (Beijing, China).The cancer cells were cultured in DMEM media containing 10% heat-inactivated fetal calf serum.All cells were incubated at 37 • C in a 5% CO 2 humidified atmosphere and harvested in the exponential growth phase for assays.

Cell Cycle Analysis
Cells were cultured for 24 h in a 6-well plate, and then the test compound (0.5, 1, and 2 µM) was added to the culture medium.After treatment for 24 h, the cells were harvested and fixed at 4 • C overnight in an ethanol solution (80%).The cells were washed with PBS and stained with a PI solution (20 mg/mL PI and 20 mg/mL RNase in PBS).After 30 min, the cells were subjected to flow cytometry (C6; BD, Franklin Lakes, NJ, USA).The recorded cell fluorescence was used to analyze the cell cycle distribution.

Data and Statistical Analysis
Data are presented as the mean ± SD.Statistical analysis was performed using Graph-Pad Prism 8.0.2Software.Differences among groups were assessed by the t-test.p < 0.05 was considered statistically significant.

Synthesis of Compound 4
In an argon atmosphere, 2 (50 mmol) was dissolved in 350 mL of dry tetrahydrofuran.A solution of methylmagnesium bromide (100 mL, 1.5 M/L, 150 mmol) was slowly added dropwise to the solution at 50 • C. The reaction mixture was heated and stirred at 50 • C for 3 h.Subsequently, the reaction was quenched with 100 mL water and extracted with dichloromethane (3 × 80 mL).The organic phase was combined, dried with anhydrous sodium sulfate, filtered, and evaporated in a vacuum to yield crude 9,10-dimethyl-9,10dihydroanthracene-9,10-diol 3 without further purification.
Next, 3 (10 mmol) was dissolved in 250 mL tetrahydrofuran, and 50 mL of 48% hydrobromic acid was added to the reaction mixture, followed by stirring for 2 h at room temperature.The yellow precipitate was filtered and collected to recover compound 4 (69% yield).

Synthesis of Compound 7
Compound 5 (50 mmol) was dissolved in 50 mL anhydrous toluene.Subsequently, ethylmagnesium bromide (100 mL, 1.5 M, 150 mmol) was added dropwise to the solution at rt.The reaction mixture was heated to 50 • C and stirred for 5 h.Then, 50 mL of 6N HCl was added to quench the reaction.After removing the toluene in a vacuum, CH 2 Cl 2 (150 mL) was added to dissolve the residue.The residue was then extracted with a 10% NaOH solution five times (5 × 20 mL each).The combined organic phase was dried over sodium sulfate and concentrated in a vacuum to yield the crude product of 9-ethylanthracene 6 without further purification.
Paraformaldehyde (62.5 mmol) was dissolved in dried glacial acetic acid (70 mL), followed by slow addition of dry HCl gas to the reaction until the paraformaldehyde was completely dissolved.9-Ethylanthracene 6 (25 mmol) was added to the reaction mixture, followed by stirring at 25 • C overnight.H 2 O (150 mL) was added to quench the reaction, followed by filtering out the yellow solid 7 without further purification, yielding 60%.

Synthesis of Compound 9
Compound 9 was synthesized following a similar synthetic procedure to that for 7 using compound 5 and propyl magnesium bromide to produce 9-propylanthracene.This was followed by generation of 9-(chloromethyl)-10-propylanthracene 9 with a yield of 62%.

Synthesis of Compound 11
PPh 3 (5 mmol) was dissolved in anhydrous acetonitrile (15 mL).The solution was purged with nitrogen for 20 min, followed by gradual addition of 0.5 mL bromine (10 mmol) to the solution using an airtight syringe.Commercially available 10 (2.5 mmol) was added to the reaction, followed by stirring at room temperature for 1 h.The precipitate was filtered, collected at 0 • C, and recrystallized from chloroform to recover 9-bromo-10-(bromomethyl)anthracene 11 with an 80% yield.

Computational Modeling Methods
The X-ray crystal structure of hnRNPA2B1 (PDB:5HO4) was obtained from the Protein Data Bank and used as the receptor for docking analysis.Protein geometry was generated with a Dreiding-like force field by the Discovery Studio 2018 toolbox standard procedure.The protein complex was prepared by monitoring bad valency, removing water molecules, and adding hydrogen.The CHARMm force field was then applied to the receptor with 13e and XI-011.Active site spheres (10 Å diameter) centered on the cognate ligand were automatically generated.The remaining parameters were the default settings.

Conclusions
Forty-three analogs of p53 activator XI-011 were designed and synthesized as potent inhibitors of U87 cells.Among them, five new compounds (13d, 13e, 14a, 14b, and 14n) exhibited good anti-glioma activity at the cellular level.Remarkably, compound 13e exhibited excellent in vitro anti-glioma activity, with an IC 50 value of 0.53 µM.Additionally,

a
Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.Each experiment was carried out using triplicates.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control. 13c 13d 13k

Figure 5 .
Figure 5. Overview of 13e and XI-011 binding to the hnRNPA2B1 protein pocket (PDB code: 5HO4).(A) Three-dimensional view of 13e (green) with hnRNPA2B1 protein (gray).The carbon, sulfur, and nitrogen atoms of 13e are displayed in green, brown, and blue, respectively.The carbon, oxygen, and nitrogen atoms of amino acid residues in hnRNPA2B1 are displayed in light blue, red, and blue, respectively.(B) Two-dimensional view of 13e with hnRNPA2B1 protein.(C) hnRNPA2B1 protein shown by gray surface representation and 13e (green) shown as sticks.(D) Overlap of 13e (green) and XI-011 (yellow).

Figure 5 .
Figure 5. Overview of 13e and XI-011 binding to the hnRNPA2B1 protein pocket (PDB code: 5HO4).(A) Three-dimensional view of 13e (green) with hnRNPA2B1 protein (gray).The carbon, sulfur, and nitrogen atoms of 13e are displayed in green, brown, and blue, respectively.The carbon, oxygen, and nitrogen atoms of amino acid residues in hnRNPA2B1 are displayed in light blue, red, and blue, respectively.(B) Two-dimensional view of 13e with hnRNPA2B1 protein.(C) hnRNPA2B1 protein shown by gray surface representation and 13e (green) shown as sticks.(D) Overlap of 13e (green) and XI-011 (yellow).

Table 1 .
In vitro anti-proliferative activities of

Table 1 .
In vitro anti-proliferative activities of

Table 1 .
In vitro anti-proliferative activities of

Table 1 .
In vitro anti-proliferative activities of

Table 1 .
In vitro anti-proliferative activities of

Table 1 .
In vitro anti-proliferative activities of

Table 1 .
In vitro anti-proliferative activities of

Table 2 .
Cont.Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates. a

Table 3 .
In vitro anti-proliferative activities of XI

-011 and 14a-14s a .
Inhibition rate (%) of U87 cells treated with 10 µM of a compound for 72 h, as determined by MTT assays.p < 0.05 compared with the control group.AlogP is the Ghose-Crippen octanol-water partition coefficient of a compound.Doxorubicin (DOX; 1 µM) was used as the positive control.DMSO (1%) was used as the vehicle control.Each experiment was carried out using triplicates. a

Table 3 .
In vitro anti-proliferative activities of XI

Table 4 .
In vitro antiproliferative activities of XI

Table 4 .
In vitro antiproliferative activities of XI

Table 4 .
In vitro antiproliferative activities of XI
a IC50 values were calculated based on at least three independent experiments using MTT assays.Doxorubicin (Dox) was used as the positive control.Cells were treated with XI-011, Dox, 13d, 13e, 14a, 14b, and 14n for 72 h.Data are represented as the mean ± SD (n = 3).