Synthesis and Biological Evaluation of Novel Synthetic Indolone Derivatives as Anti-Tumor Agents Targeting p53-MDM2 and p53-MDMX

A series of novel indolone derivatives were synthesized and evaluated for their binding affinities toward MDM2 and MDMX. Some compounds showed potent MDM2 and moderate MDMX activities. Among them, compound A13 exhibited the most potent affinity toward MDM2 and MDMX, with a Ki of 0.031 and 7.24 μM, respectively. A13 was also the most potent agent against HCT116, MCF7, and A549, with IC50 values of 6.17, 11.21, and 12.49 μM, respectively. Western blot analysis confirmed that A13 upregulated the expression of MDM2, MDMX, and p53 by Western blot analysis. These results indicate that A13 is a potent dual p53-MDM2 and p53-MDMX inhibitor and deserves further investigation.


Introduction
Tumor suppressor p53 plays a crucial role in the regulation of DNA repair, cell cycle, apoptosis, and differentiation [1,2]. It is inactivated in the majority of human cancers, resulting from mutations or the overexpression of its negative regulators, murine double minute 2 (MDM2) and/or murine double minute X (MDMX) [3]. Approximately 50% of human cancers have a p53 gene mutation and may be combated using gene therapy. For the remaining cancer types, the function of p53 can be reactivated by disrupting the interaction with MDM2 and MDMX proteins using small-molecule inhibitors [4]. Therefore, the interfaces of p53-MDM2 and p53-MDMX have emerged as important targets for the development of novel chemotherapeutic agents.
Studies have verified that dual MDM2/MDMX inhibitors can activate p53 better than selective inhibitors that only target MDM2 or MDMX [25]. Currently, some progress has been made in designing dual inhibitors of MDM2 and MDMX [26][27][28][29][30]. ALRN-6924 (Figure 1), a dual inhibitor of MDM2 and MDMX, has gone into a phase I clinical trial (NCT04022876) in patients with solid tumors [31]. However, there are many challenges that need to be overcome before use in clinical practice and novel inhibitors need to be developed.
Molecules 2022, 27, x FOR PEER REVIEW 2 of 17 ( Figure 1), a dual inhibitor of MDM2 and MDMX, has gone into a phase I clinical trial (NCT04022876) in patients with solid tumors [31]. However, there are many challenges that need to be overcome before use in clinical practice and novel inhibitors need to be developed. Indole, as a heterocyclic molecule, is a significant source of the pharmacologically active compound. Due to its chemical reactivity, indole could be modified to obtain many indole derivatives, exhibiting a variety of bioactivities. Especially as anti-cancer agents, indoles are effective against a variety of cancers including acute myeloid leukemia [32,33], lung cancer [34], cervical cancer [35], colon cancer, breast cancer [36], and others. The targets of indoles as anti-cancer agents were also reported such as the Mcl-1, Pim, HDAC, SIRT, tubulin [32,37], RAF-MEK-ERK, and PI3K-PDK1-AKT pathways [34], and so on. Some indole derivatives have even been identified as very promising candidates for phototherapy application against highly aggressive and resistant cancers [38].
As shown in Figure 2, some indole derivatives, which are reported as anti-cancer agents targeting MDM2 and MDMX (1b, RO-2443) [36,39], have been modified to improve the activities and explore new mechanisms. In the present study, novel indolone compounds were identified and the derivatives were synthesized and evaluated for their affinities toward MDM2 and MDMX. The most active compounds were tested for their in vitro anti-tumor activity against the MCF7, HCT116, and A549 cell lines to verify their antiproliferative activity. Indole, as a heterocyclic molecule, is a significant source of the pharmacologically active compound. Due to its chemical reactivity, indole could be modified to obtain many indole derivatives, exhibiting a variety of bioactivities. Especially as anti-cancer agents, indoles are effective against a variety of cancers including acute myeloid leukemia [32,33], lung cancer [34], cervical cancer [35], colon cancer, breast cancer [36], and others. The targets of indoles as anti-cancer agents were also reported such as the Mcl-1, Pim, HDAC, SIRT, tubulin [32,37], RAF-MEK-ERK, and PI3K-PDK1-AKT pathways [34], and so on. Some indole derivatives have even been identified as very promising candidates for phototherapy application against highly aggressive and resistant cancers [38].
As shown in Figure 2, some indole derivatives, which are reported as anti-cancer agents targeting MDM2 and MDMX (1b, RO-2443) [36,39], have been modified to improve the activities and explore new mechanisms. In the present study, novel indolone compounds were identified and the derivatives were synthesized and evaluated for their affinities toward MDM2 and MDMX. The most active compounds were tested for their in vitro anti-tumor activity against the MCF7, HCT116, and A549 cell lines to verify their antiproliferative activity. s 2022, 27, x FOR PEER REVIEW 3 of 17 Figure 2. The design strategy of the target compounds.

Chemistry
Compounds A1-A30 and B1-B10 were synthesized according to Scheme 1. A condensation reaction was carried out to obtain intermediate compounds 1-5 with the commercially available substituted oxindole and benzaldehyde materials. Next, compounds 1-5 were sulfonylated or acylated to obtain A1-A30 and B1-B10, with corresponding benzene sulfonyl chloride or benzoyl chloride in the presence of sodium hydroxide in tetrahydrofuran. In total, 40 derivatives were synthesized and the compound structures are listed in Table 1. The 1 H NMR spectrum, 13 C NMR spectrum, and mass spectrum of compounds 1-5, A1-A30, and B1-B10 were listed in the Supplementary Materials.

Chemistry
Compounds A1-A30 and B1-B10 were synthesized according to Scheme 1. A condensation reaction was carried out to obtain intermediate compounds 1-5 with the commercially available substituted oxindole and benzaldehyde materials. Next, compounds 1-5 were sulfonylated or acylated to obtain A1-A30 and B1-B10, with corresponding benzene sulfonyl chloride or benzoyl chloride in the presence of sodium hydroxide in tetrahydrofuran. In total, 40 derivatives were synthesized and the compound structures are listed in Table 1. The 1 H NMR spectrum, 13 C NMR spectrum, and mass spectrum of compounds 1-5, A1-A30, and B1-B10 were listed in the Supplementary Materials.

Chemistry
Compounds A1-A30 and B1-B10 were synthesized according to Scheme 1. A condensation reaction was carried out to obtain intermediate compounds 1-5 with the commercially available substituted oxindole and benzaldehyde materials. Next, compounds 1-5 were sulfonylated or acylated to obtain A1-A30 and B1-B10, with corresponding benzene sulfonyl chloride or benzoyl chloride in the presence of sodium hydroxide in tetrahydrofuran. In total, 40 derivatives were synthesized and the compound structures are listed in Table 1. The 1 H NMR spectrum, 13 C NMR spectrum, and mass spectrum of compounds 1-5, A1-A30, and B1-B10 were listed in the Supplementary Materials.  that was three-fold higher. Surprisingly, the introduction of a chlorine group to R 3 of A8 (A12-A14, R 3 = 2-Cl, 3-Cl, 4-Cl) contributed to an obvious enhancement in activity, with a K i of 0.19, 0.031 and 0.082 µM, respectively. A13 and A14 especially showed a greater potency than nutlin-3a. However, the results were not satisfactory when the methyl group was introduced to the same position (A9-A11). This phenomenon indicates that an electron-withdrawing group might be needed to increase the enzyme affinity. Other electron-withdrawing groups were introduced in subsequent experiments including the bromine, fluorine, trifluoromethyl, and nitro groups. Compounds A16, A19, and A21 with the 3-substituted electron-withdrawing R 3 group exhibited more potency than the compound with 2-substituted or 4-substituted group (A15, A17, A18, and A20). A16, A19, and A21 exhibited an even higher potency than nutlin-3a. Compared to A1 and A8, when the 2-Cl or 3-Cl group of R 2 was replaced with 4-Cl, A28 exhibited a lower activity than A8, but greater than A1. Next, structure modification was carried out at the position of R 3 (A23-A27) with K i values of >1 µM. A29 and A30 showed a lower activity than A13 with the introduction of the chlorine atom to R 1 (5-Cl and 6-Cl). Among the B1-B10 compounds with benzoyl groups, B3 showed the most potency with a K i of 7.86 µM, which was lower than the A series with benzene sulfonyl groups.
Potency analysis of the compounds toward MDMX was carried out in the following assays. A13 was the most potent MDMX binder with a K i of 7.24 µM. The K i values of some compounds (A5-A8, A12-A24, and A27-A30) ranged from 10.33 to 43.18 µM, while the rest of the compound values were >50 µM. Despite the significant homology of MDM2 and MDMX, the affinities of the same compound were notably different.
In general, the results listed in Table 1 showed that the activity of compounds A1-A30 was better than that of compounds B1-B10, demonstrating that the introduction of the benzene sulfonyl chloride group led to a greater potency than the benzoyl chloride group. Compound A13 showed the most potent affinity toward MDM2 and MDMX, with a K i of 0.031 and 7.24 µM, respectively.

In Vitro Anti-Tumor Activities
The anti-tumor activities of the most active compounds with K i < 1 µM toward MDM2 in the fluorescence polarization assay were evaluated against the MCF7, HCT116, and A549 cell lines in vitro using the standard MTT assay, with nutlin-3a as a positive control.
As illustrated in Table 2, all compounds showed anti-tumor activities. Except for A15, A16, and A17, all of the other tested compounds showed a greater potency against HCT116 than nutlin-3a. A13, in particular, exhibited satisfactory potency, with an IC 50 of 6.17 µM, which was about three-fold greater compared to that of nutlin-3a. The IC 50 values of the tested compounds against MCF7 ranged from 11.21 to 37.70 µM. Twelve compounds showed a greater activity than nutlin-3a. A13 and A21 were the most potent agents against A549, with IC 50 values of 12.49 and 13.48 µM, respectively, which were superior to the positive control.
In general, all of the tested compounds showed a better potency against HCT116 than MCF7 and A549. A13 was the most potent compound against HCT116, MCF7, and A549, with IC 50 values of 6.17, 11.21, and 12.49 µM, respectively. Surprisingly, A13 exhibited the best enzymatic affinities as well as anti-tumor activities. Further research on A13 was carried out in the following experiment.

Western Blotting Analyses
The dual inhibition of the p53-MDM2 and p53-MDMX interactions is expected to activate p53, increasing its level in the cells with wild-type p53. In addition, p53 activation can also result in an increase in the levels of the MDM2 and MDMX proteins. To verify these predictions, Western blotting was used to assess the effects of compound A13 in the HCT 116 cells. The cells were treated with compound A13 for 48 h. Subsequently, the cells were lysed and protein extracts were analyzed for the levels of p53, MDM2, and MDMX. As shown in Figure 3, compound A13 caused a dose-dependent increase in the level of p53 proteins in HCT116 cells, indicating a good activation of p53. Expression of MDM2 and MDMX as well as the p53 downstream gene was also induced in a dose-dependent manner, which is consistent with the prediction. According to these results, it can be concluded that compound A13 might exhibit anti-cancer activity through interrupting the p53-MDM2 and p53-MDMX interactions.

Western Blotting Analyses
The dual inhibition of the p53-MDM2 and p53-MDMX interactions is ex tivate p53, increasing its level in the cells with wild-type p53. In addition, p can also result in an increase in the levels of the MDM2 and MDMX prote these predictions, Western blotting was used to assess the effects of compou HCT 116 cells. The cells were treated with compound A13 for 48 h. Subseque were lysed and protein extracts were analyzed for the levels of p53, MDM2, As shown in Figure 3, compound A13 caused a dose-dependent increase i p53 proteins in HCT116 cells, indicating a good activation of p53. Expressi and MDMX as well as the p53 downstream gene was also induced in a dos manner, which is consistent with the prediction. According to these results, cluded that compound A13 might exhibit anti-cancer activity through int p53-MDM2 and p53-MDMX interactions.

Molecular Docking Analyses
Molecular docking was carried out and analyzed in order to observe the binding modes of compound A13 in the active site of MDM2 and MDMX. The binding mode of compound A13 in the MDM2 active site is shown in Figure 4A. The phenyl ring connected with the sulfonyl formed a non-classical H-bond with Tyr67, resulting in a hydrophobic interaction with Val93. The chlorine atom had σ-π and σ-σ hydrophobic interactions with His73 and Val93, respectively. The benzimidazole group formed a σ-π hydrophobic interaction with Vai93 and σ-σ hydrophobic interactions with Val93 and Ile99. The phenyl connected with a double bond formed a σ-π hydrophobic interaction with His96, and the chlorine atom made σ-π, σ-σ, and σ-π hydrophobic interactions with His96, Ile99, and Tyr100, respectively. The binding mode of compound A13 in the MDMX active site is shown in Figure 4B. The chlorobenzene group connected with sulfonyl formed σ-π and σ-σ hydrophobic interactions with Val92 and Met53. The benzimidazole group made σ-π hydrophobic interactions with Val92 and Ile60. The chlorobenzene connected with a double bond made σ-σ and σ-π hydrophobic interactions with Met61 and Tyr66, respectively. The connected with a double bond formed a σ-π hydrophobic interaction with His96, and the chlorine atom made σ-π, σ-σ, and σ-π hydrophobic interactions with His96, Ile99, and Tyr100, respectively. The binding mode of compound A13 in the MDMX active site is shown in Figure 4B. The chlorobenzene group connected with sulfonyl formed σ-π and σ-σ hydrophobic interactions with Val92 and Met53. The benzimidazole group made σπ hydrophobic interactions with Val92 and Ile60. The chlorobenzene connected with a double bond made σ-σ and σ-π hydrophobic interactions with Met61 and Tyr66, respectively. The predicted Ki values of compound A13 binding to MDM2 and MDMX were 0.018 and 5.18 μM, consistent with the test values. From the docking results exhibited in Figure 4, the interactions between compound A13 and MDM2 were obviously stronger than those of A13 and MDMX, which might explain the different enzymatic activities of A13 targeting MDM2 and MDMX.

General Procedures
The mass spectra were acquired using an Agilent 6210 ESI/TOF or ThermoFisher Q Exactive mass spectrometer. 1 H and 13 C NMR spectra were recorded on a Bruker BioSpin GmbH spectrometer at 400 or 600 MHz. Column chromatography was performed on silica gel (200-300 mesh) from Qingdao Ocean Chemical. Unless specified, all reagents were obtained from commercially available sources.

General Procedure for Preparation of Intermediates (1-5)
A mixture of indolone (7.51 mmol), 2-chlorobenzaldehyde (7.51 mmol), and sodium hydroxide (7.51 mmol) in ethanol (30 mL) was stirred at 60 °C for 4 h. Then, the reaction mixture was allowed to cool to room temperature and added to the ice water. The precipitate was collected by filtration and purified using recrystallization with methanol to obtain the intermediates 1-5.  From the docking results exhibited in Figure 4, the interactions between compound A13 and MDM2 were obviously stronger than those of A13 and MDMX, which might explain the different enzymatic activities of A13 targeting MDM2 and MDMX.

General Procedures
The mass spectra were acquired using an Agilent 6210 ESI/TOF or ThermoFisher Q Exactive mass spectrometer. 1 H and 13 C NMR spectra were recorded on a Bruker BioSpin GmbH spectrometer at 400 or 600 MHz. Column chromatography was performed on silica gel (200-300 mesh) from Qingdao Ocean Chemical. Unless specified, all reagents were obtained from commercially available sources.

MTT Assay in Vitro
All compounds with K i values of <1 µM toward MDM2 in the fluorescence polarization assay were evaluated against the MCF7, HCT116, and A549 cell lines (ATCC, Manassas, VA, USA) in vitro using a standard MTT assay, with nutlin-3a as a positive control. Compounds were tested at six concentrations (0.001-100 µM).
The cells were seeded into a 96-well plate and allowed to attach overnight, after which various concentrations of test compounds or DMSO were added to the culture medium and the cells were incubated for 48 h at 37 • C in a 5% CO 2 humidified incubator. Then, 20 µL of 0.5% the fresh MTT solution was added to each well. After incubation for another 4 h, the culture medium was removed and 150 µL of DMSO was added to dissolve the formazan crystals. The absorbance at 540 nm was measured using a microplate reader. The results, expressed as IC 50 values, were the average of three determinations and were calculated using nonlinear regression analysis.

Western Blotting Assay
The HCT116 cells with wild type p53 were treated with compound A13 (0, 5, 10, and 20 µM) for 48 h. The cells were harvested and lysed with RIPA buffer (Sigma, St. Louis, MI, USA) supplemented with a protease inhibitor cocktail (Sigma), followed by centrifugation at 12,000× g for 10 min. The supernatants were heated to 100 • C with loading buffer and the total protein concentrations were determined using a Bio-Rad protein assay. Protein samples were subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk, and then incubated with the corresponding primary antibody (MDM2, MDMX, p53, and GAPDH) at 4 • C overnight. After binding with an appropriate secondary antibody conjugated with peroxidase, proteins were visualized using the ECL chemiluminescence system. GAPDH was used as a loading control.

Molecular Docking
Molecular docking analysis was carried out using Sybyl-X2.0. The crystal structures of MDM2 (PDB code: 3LBL) and MDMX (PDB code: 2N0W) were processed. The ligand and the water molecules were first removed and the hydrogen atoms were added. The biopolymer was protonated at pH 7.4 and the Amber7 FF99 force field was applied. Other parameters were set at the default. The minimized protein and compound A13 were subjected to the docking protocol using the Surflex-Dock Geom mode of Sybyl-X2.0.

Conclusions
In this study, a library of new indolone derivatives was synthesized and evaluated for their biological activity. The preliminary structure-activity relationship study showed that the compounds with benzene sulfonyl groups demonstrated a higher potency than the benzene sulfonyl groups. The 3-substituted electron-withdrawing group at the R 3 position is necessary (3-Cl > 3-F > 3-Br > 3-CF 3 > 3-NO 2 ) to increase the affinity to the enzyme. All of the compounds exhibited better activities to MDM2 than MDMX. Compound A13 was identified as having the most potent affinity toward MDM2 and MDMX through the fluorescence polarization assay, with a K i of 0.031 and 7.24 µM, respectively. The docking studies demonstrated that compound A13 formed hydrophobic interactions with Leu54, Tyr67, His73, Val93, His96, Ile99, and Tyr100 in the MDM2 active site, and Met53, Ile60, Met61, Tyr66, and Val92 in the MDMX active site. A13 also exhibited a greater potency than nutlin-3a against the HCT116, MCF7, and 143B cell lines. The Western blot analysis showed that A13 induced accumulations of MDM2, MDMX, and p53, suggesting the activation of the p53 pathway.
Author Contributions: Y.W. carried out the experiments and wrote the paper; B.J. and Z.C. synthesized the compounds; L.Z., Y.C., and Y.L. evaluated the biological activities of the target compounds; J.R. contributed to the data analysis of the biological tests; W.L. contributed to the data analysis of the NMR spectrum; Y.M. contributed to the analysis of molecular docking. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.