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Article

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

by
Yuxin Feng
1,
Yingjie Wang
1,
Xiaoxue Li
2,
Ziqiang Sun
2,
Sihan Qiang
2,
Hongbo Wang
1,* and
Yi Liu
2,*
1
Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, Yantai University, Yantai 264005, China
2
School of Chemistry and Chemical Engineering, Yantai University, Yantai 264005, China
*
Authors to whom correspondence should be addressed.
Molecules 2024, 29(10), 2396; https://doi.org/10.3390/molecules29102396
Submission received: 26 March 2024 / Revised: 28 April 2024 / Accepted: 14 May 2024 / Published: 19 May 2024
(This article belongs to the Special Issue Anticancer Drug Discovery and Development II)
Browse Figures
Versions Notes

Abstract

:
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.

1. 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 pulmonary 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].
In previous studies, we discovered a small molecule, (10-methylanthracen-9-yl)methyl carbamimidothioate (XI-011) (Figure 1), which effectively inhibits MDM4 expression in cancer cells [22,23]. Recent studies have demonstrated that XI-011 effectively binds to the oncogenic driver heterogeneous nuclear ribonucleoprotein A2B1 and disrupts the heterogeneous nuclear ribonucleoprotein A2B1/MDM4 promoter and untranslated region interaction, thereby activating and stabilizing p53, and inhibiting cancer cell growth [24]. Here, 43 new analogs of XI-011 were designed, synthesized, and evaluated for their anti-glioma activities.

2. Results and Discussion

2.1. Chemistry

The general synthetic routes of targeted 12a–12l, 13a–13l, and 14a–14s are outlined in Scheme 1. Synthesis of targeted 12a–12l, 13a–13l, and 14a–14s is first required for the synthesis of halogenated derivatives (4, 7, 9, and 11). 9-(Bromomethyl)-10-methylanthracene 4 was prepared from anthracene-9,10-dione 2 by treatment with CH3MgBr at 55 °C, followed by aromatization and bromination in the presence of a 40% HBr solution to generate 4 with a 69% yield. Synthesis of ethyl 7 and propyl 9 analogs started from anthracene-9(10H)-one 5, which were, respectively, reacted with CH3CH2MgBr and CH3CH2CH2MgBr at 60 °C to produce 9-substituted anthracenes (6 and 8). Next, targets 7 and 9 were prepared from synthesized 9-substituted anthracenes (6 and 8) via the Mannich reaction with paraformaldehyde/HCl, resulting in 60–62% yields.
The bromination reaction of anthracen-9-ylmethanol 10 proceeded smoothly in the presence of PPh3/Br2 at room temperature to yield 9-bromo-10-(bromomethyl)anthracene 11 with an 80% yield. Using 4, 7, 9, and 11, various thiourea derivatives finally substituted the chloro or bromo group of anthracene to yield the corresponding compounds 12a–12l, 13a–13l, and 14a–14s.

2.2. 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. 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.
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 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 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.
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.
As anticipated, compound 13e also exhibited the best anti-glioma activity (IC50 = 0.53 µM), which was similar to those of the controls XI-011 (IC50 = 0.69 µM) and Dox (IC50 = 0.44 µM). Notably, 13e exhibited more potential cytotoxicity to U87 cells (IC50 = 0.53 µM) and 2-fold lesser toxicity to human normal HHL-5 cells (IC50 = 1.11 µM). Other analogs (13d, 14a, 14b, and 14n) also had cell growth inhibitory IC50 values of less than 2 µM in U87 cells, demonstrating that these anthracenyl skeleton compounds were efficient and promising for developing new anti-glioma drugs.

2.3. 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.
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.

2.4. 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 -NH2 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.
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 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.

3. Materials and Methods

3.1. 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% CO2 humidified atmosphere and harvested in the exponential growth phase for assays.

3.2. MTT Assay

Cells were seeded in a 96-well plate and treated with the test substance at the desired concentration for 72 h. In cytotoxicity experiments, cells were treated with 10 µM of the test compound or 1 µM XI-011 and doxorubicin as positive controls. DMSO (1%) was used as a negative control. In the IC50 analysis, doxorubicin, XI-011, 13d, 13e, 14a, 14b, and, 14n were applied to HHL-5 at 50, 25, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 µM and U87 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 µM were applied to Hela and MCF-7 cells. Treated cells were incubated with an MTT solution for 2 h and then with DMSO to dissolve the crystals. Absorbance was determined at 570 nm using a microplate reader (BioTek, Seattle, DC, USA). IC50 values were calculated using the Hill model in GraphPad Prism 8.0.2 software.

3.3. 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.

3.4. Western Blotting

Protein levels in U87 cells treated with test compound 13e (0.5, 1, and 2 µM) for 24 h were measured by Western blot analysis as described previously [26]. Briefly, after 13e treatment, cells were lysed in RIPA buffer. Proteins were separated by 8% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was incubated with an anti-MDM4 antibody (A300-287A, Bethyl Laboratories, Montgomery, TX, USA), anti-p53 antibody (sc-126, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or anti-GAPDH antibody (AG019, Beyotime, Shanghai, China) followed by an HRP-linked secondary antibody (A0216, Beyotime) for 2 h. ECL reagent was used for chemiluminescent development. Densitometric analysis of protein bands was performed using ImageJ V.1.8.0 software.

3.5. Data and Statistical Analysis

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

3.6. Chemistry Section

3.6.1. General Methods

All reagents were purchased from commercial suppliers. Reactions under standard conditions were monitored by thin-layer chromatography on F254 gel plates. Flash chromatography was performed using a silica gel (200–300 mesh). High-resolution mass spectrometric data were acquired using a Q-TOF analyzer (Thermo Fisher Scientific, BRE, German). 1H and 13C NMR spectra were recorded using 500 MHz/125 MHz Bruker spectrometers (Billerica, MA, USA) or 400 MHz/100 MHz JEOL JNM-ECZ400S spectrometers (Akishima, Japan). The coupling constant (J) was Hz. Signals were reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), ddt (doublet of doublets of triplets), dq (doublet of quartets), br s (broad singlet), and m (multiplet). The purity of 13e was high (≥99.2%). 1H and 13C nuclear magnetic resonance (NMR) spectroscopy data of compounds 12a–12l, 13a–13l, and 14a–14s, and HPLC spectra of 13e are shown in Supplementary Materials.

3.6.2. 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-9,10-dihydroanthracene-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).

3.6.3. 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, CH2Cl2 (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. H2O (150 mL) was added to quench the reaction, followed by filtering out the yellow solid 7 without further purification, yielding 60%.

3.6.4. 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%.

3.6.5. Synthesis of Compound 11

PPh3 (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.

3.6.6. General Synthesis Procedure for XI-011, 12a–12l, 13a–13l, and 14a–14s

A halogenated compound (4, 7, 9, or 11) (5 mmol) was dissolved in an acetonitrile solution and then treated with various commercially available thiourea compounds (7.5 mmol). After stirring for 2 h at room temperature, the precipitate was filtered out and sequentially washed with 50 mL petroleum ether, 50 mL dichloromethane, and 50 mL ethyl acetate. After drying, the desired products were obtained with various yields.

3.7. 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.

4. 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 IC50 value of 0.53 µM. Additionally, 13e showed effective anti-cancer activities against HeLa cells (IC50 = 1.63 µM) and MCF-7 cells (IC50 = 0.97 µM). Antitumor mechanistic analyses indicated that 13e efficiently suppressed the expression of MDM4 and promoted p53 activation to induce U87 cell cycle arrest at G2/M phase, inducing cancer cell apoptosis. Taken together, our study showcases a series of new p53 activators featuring an anthracenyl skeleton that are promising for future development for GBM treatment.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29102396/s1. The characterization of synthesized compounds (XI-011, compounds 12a–12l, compounds 13a–13l, compounds 14a–14s). Figures S1–S88: NMR spectra for 44 products (XI-011, compounds 12a–12l, compounds 13a–13l, compounds 14a–14s). The HPLC spectra of 13e (Figure S89). Dose–response curves of compounds (XI-011, 13d, 13e, 14a, 14b, 14n, and DOX) on the cytotoxicity of U87-MG, Hela, MCF-7, and HHL-5 cells (Figures S90–S96). The origin western blots results of 13e and XI-011 in U87 cells ( Figures S97–S99).

Author Contributions

Conceptualization, H.W. and Y.L.; Data curation, Y.F., Y.W., X.L. and S.Q.; Formal analysis, Y.F.; Funding acquisition, H.W. and Y.L.; Investigation, Y.W., X.L., Z.S. and H.W.; Methodology, Y.L.; Project administration, H.W. and Y.L.; Writing—original draft, Y.F. and Y.L.; Writing—review and editing, H.W. and Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partially supported by Taishan Scholar Project (tsqn202211112), National Natural Science Foundation of China (82273969, 82073888), and Shandong Provincial Natural Science Foundation (ZR2020MB105, ZR2021LSW011).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The author declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Molecules 29 02396 g001
Figure 1. Structures of representative MDM4 inhibitors and XI-011.
Figure 1. Structures of representative MDM4 inhibitors and XI-011.
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Molecules 29 02396 sch001
Scheme 1. General synthetic procedure of compounds 12a–12l, 13a–13l, and 14a–14s.
Scheme 1. General synthetic procedure of compounds 12a–12l, 13a–13l, and 14a–14s.
Molecules 29 02396 sch001
Molecules 29 02396 g002
Figure 2. (A) Western blot analysis of p53 and MDM4 expression in U87 cells after 24 h of treatment with XI-011 (1.0 µM) and 13e (0.5, 1, and 2 µM). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (B) Effect of 13e on MDM4 expression in U87 cells (n = 3). (C) Effect of 13e on p53 expression in U87 cells (n = 3). Data are represented as the mean ± SD (error bars). * p < 0.05, ** p < 0.01.
Figure 2. (A) Western blot analysis of p53 and MDM4 expression in U87 cells after 24 h of treatment with XI-011 (1.0 µM) and 13e (0.5, 1, and 2 µM). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (B) Effect of 13e on MDM4 expression in U87 cells (n = 3). (C) Effect of 13e on p53 expression in U87 cells (n = 3). Data are represented as the mean ± SD (error bars). * p < 0.05, ** p < 0.01.
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Molecules 29 02396 g003
Figure 3. (A) Flow cytometric analysis of the cell cycle distribution of U87 cells in the presence of XI-011 (1.0 µM) and 13e (0.5, 1, and 2 µM) for 24 h. (B) Cell cycle distribution of U87 cells treated with 13e and XI-011. The experiments were repeated three times. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group.
Figure 3. (A) Flow cytometric analysis of the cell cycle distribution of U87 cells in the presence of XI-011 (1.0 µM) and 13e (0.5, 1, and 2 µM) for 24 h. (B) Cell cycle distribution of U87 cells treated with 13e and XI-011. The experiments were repeated three times. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group.
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Figure 4. (A) Flow cytometric analysis of apoptosis induction in U87 cells in the presence of XI-011 (1.0 µM) and 13e (0.5, 1, and 2 µM) for 24 h. (B) Histograms of apoptotic cells (%). Data are shown as the mean ± SD of three independent experiments.
Figure 4. (A) Flow cytometric analysis of apoptosis induction in U87 cells in the presence of XI-011 (1.0 µM) and 13e (0.5, 1, and 2 µM) for 24 h. (B) Histograms of apoptotic cells (%). Data are shown as the mean ± SD of three independent experiments.
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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. 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).
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Figure 6. Proposed anti-glioma mechanism of 13e.
Figure 6. Proposed anti-glioma mechanism of 13e.
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Table 1. In vitro anti-proliferative activities of 12a–12l, and 13a a.
Table 1. In vitro anti-proliferative activities of 12a–12l, and 13a a.
CmpdStructureU87 (% Inhib. @ 10 µM)AlogPCmpdStructureU87 (% Inhib. @ 10 µM)AlogP
XI-011Molecules 29 02396 i00191.84.6512hMolecules 29 02396 i0029.94.17
12aMolecules 29 02396 i00315.23.6512iMolecules 29 02396 i0042.73.58
12bMolecules 29 02396 i0059.03.1612jMolecules 29 02396 i0066.82.58
12cMolecules 29 02396 i00711.82.6512kMolecules 29 02396 i00810.72.65
12dMolecules 29 02396 i00914.81.4412lMolecules 29 02396 i01011.33.19
12eMolecules 29 02396 i0111.53.1613aMolecules 29 02396 i01257.94.62
12fMolecules 29 02396 i01313.13.62DOX81.1
12gMolecules 29 02396 i01414.53.14DMSO4.7
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.
Table 2. In vitro antiproliferative activities of 13a–13l a.
Table 2. In vitro antiproliferative activities of 13a–13l a.
CmpdStructureU87 (% Inhib. @ 10 µM)AlogPCmpdStructureU87 (% Inhib. @ 10 µM)AlogP
13aMolecules 29 02396 i01557.94.6213hMolecules 29 02396 i0160.74.56
13bMolecules 29 02396 i01719.34.5813iMolecules 29 02396 i01831.25.18
13cMolecules 29 02396 i01935.44.9313jMolecules 29 02396 i02013.36.83
13dMolecules 29 02396 i02193.35.1713kMolecules 29 02396 i02228.18.73
13eMolecules 29 02396 i02388.64.9213lMolecules 29 02396 i02486.77.24
13fMolecules 29 02396 i02565.15.47DOX81.1
13gMolecules 29 02396 i02641.24.97DMSO4.7
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.
Table 3. In vitro anti-proliferative activities of XI-011 and 14a–14s a.
Table 3. In vitro anti-proliferative activities of XI-011 and 14a–14s a.
CmpdStructureU87 (% Inhib. @ 10 μM)AlogPCmpdStructureU87 (% Inhib. @ 10 μM)AlogP
XI-011Molecules 29 02396 i02791.84.6514kMolecules 29 02396 i02814.24.99
14aMolecules 29 02396 i02979.05.0614lMolecules 29 02396 i0300.74.09
14bMolecules 29 02396 i03194.75.5814mMolecules 29 02396 i03236.84.99
14cMolecules 29 02396 i03383.64.7214nMolecules 29 02396 i03486.55.51
14dMolecules 29 02396 i03517.35.3514oMolecules 29 02396 i03650.74.96
14eMolecules 29 02396 i03719.35.0014pMolecules 29 02396 i03819.03.64
14fMolecules 29 02396 i03943.67.6614qMolecules 29 02396 i04012.53.67
14gMolecules 29 02396 i04128.08.1714rMolecules 29 02396 i0422.16.27
14hMolecules 29 02396 i04364.65.4814sMolecules 29 02396 i0446.94.35
14iMolecules 29 02396 i04576.44.68DOX81.1
14jMolecules 29 02396 i04611.84.13DMSO4.7
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.
Table 4. In vitro antiproliferative activities of XI-011, 13d, 13e, 14a, 14b, and 14n in cancer cell lines U87, Hela, and MCF-7, and HHL-5 as a normal cell line a.
Table 4. In vitro antiproliferative activities of XI-011, 13d, 13e, 14a, 14b, and 14n in cancer cell lines U87, Hela, and MCF-7, and HHL-5 as a normal cell line a.
CmpdIC50 (μM)
U87HelaMCF-7HHL-5
Molecules 29 02396 i047 (XI-011)0.69 ± 0.021.51 ± 0.064.87 ± 0.300.55 ± 0.03
Molecules 29 02396 i048 (13d)1.62 ± 0.025.66 ± 0.294.74 ± 0.200.85 ± 0.10
Molecules 29 02396 i049 (13e)0.53 ± 0.031.63 ± 0.250.97 ± 0.111.11 ± 0.08
Molecules 29 02396 i050 (14a)1.24 ± 0.243.64 ± 0.274.56 ± 0.031.51 ± 0.16
Molecules 29 02396 i051 (14b)1.35 ± 0.102.66 ± 0.194.55 ± 0.271.62 ± 0.13
Molecules 29 02396 i052 (14n)1.84 ± 0.127.74 ± 0.817.60 ± 1.371.67 ± 0.16
Dox0.44 ± 0.060.54± 0.051.32 ± 0.160.32 ± 0.02
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).
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MDPI and ACS Style

Feng, Y.; Wang, Y.; Li, X.; Sun, Z.; Qiang, S.; Wang, H.; Liu, Y. Novel 9-Methylanthracene Derivatives as p53 Activators for the Treatment of Glioblastoma Multiforme. Molecules 2024, 29, 2396. https://doi.org/10.3390/molecules29102396

AMA Style

Feng Y, Wang Y, Li X, Sun Z, Qiang S, Wang H, Liu Y. Novel 9-Methylanthracene Derivatives as p53 Activators for the Treatment of Glioblastoma Multiforme. Molecules. 2024; 29(10):2396. https://doi.org/10.3390/molecules29102396

Chicago/Turabian Style

Feng, Yuxin, Yingjie Wang, Xiaoxue Li, Ziqiang Sun, Sihan Qiang, Hongbo Wang, and Yi Liu. 2024. "Novel 9-Methylanthracene Derivatives as p53 Activators for the Treatment of Glioblastoma Multiforme" Molecules 29, no. 10: 2396. https://doi.org/10.3390/molecules29102396

APA Style

Feng, Y., Wang, Y., Li, X., Sun, Z., Qiang, S., Wang, H., & Liu, Y. (2024). Novel 9-Methylanthracene Derivatives as p53 Activators for the Treatment of Glioblastoma Multiforme. Molecules, 29(10), 2396. https://doi.org/10.3390/molecules29102396

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