Ganoderic Acid A and Its Amide Derivatives as Potential Anti-Cancer Agents by Regulating the p53-MDM2 Pathway: Synthesis and Biological Evaluation

The mechanisms of action of natural products and the identification of their targets have long been a research hotspot. Ganoderic acid A (GAA) is the earliest and most abundant triterpenoids discovered in Ganoderma lucidum. The multi-therapeutic potential of GAA, in particular its anti-tumor activity, has been extensively studied. However, the unknown targets and associated pathways of GAA, together with its low activity, limit in-depth research compared to other small molecule anti-cancer drugs. In this study, GAA was modified at the carboxyl group to synthesize a series of amide compounds, and the in vitro anti-tumor activities of the derivatives were investigated. Finally, compound A2 was selected to study its mechanism of action because of its high activity in three different types of tumor cell lines and low toxicity to normal cells. The results showed that A2 could induce apoptosis by regulating the p53 signaling pathway and may be involved in inhibiting the interaction of MDM2 and p53 by binding to MDM2 (KD = 1.68 µM). This study provides some inspiration for the research into the anti-tumor targets and mechanisms of GAA and its derivatives, as well as for the discovery of active candidates based on this series.


Introduction
Ganoderma lucidum is the dry fruiting body of Ganoderma lucidum Karst and Ganoderma sinensis, which belong to the genus Ganoderma of the family Polyporaceae. The chemical composition of Ganoderma lucidum is complex. There are currently about 400 known compounds, the majority of which are triterpenoids, polysaccharides, nucleosides, sterols, and other compounds, of which more than 300 are triterpenoids. Ganoderic acid A (GAA, Figure 1), one of the most prominent and highly concentrated triterpenes from Ganoderma lucidum, exhibits a variety of biological properties, including anti-tumor [1], anti-inflammatory [2][3][4], anti-depressant [5,6], neuroprotection [7,8], anti-fibrosis [9], liver protection [10,11], improvement of glucose and lipid metabolism and myocardial protection [12][13][14], etc., which can be used as a potential resource for drug development. The anti-tumor activity is one of the earliest discovered activities of GAA, which has received the most attention since then [1]. Many researchers have investigated the anti-tumor activities of Ganoderma lucidum triterpenoids and predicted their anti-tumor pathway. Studies have shown that GAA can inhibit tumor growth through a variety of signaling pathways. For example, GAA has good cytotoxicity on human glioblastoma by inducing apoptosis, autophagy and inhibiting PI3K/AKT signaling pathways [15]; it can inhibit the expression of KDR mRNA and protein, induce apoptosis of human glioma cell U251 cell, and inhibit its proliferation and invasion [16]. However, there is no relevant literature that clearly indicates the possible anti-tumor target of GAA. Cancer and the MDM2-p53 signaling pathway are closely related. p53 is a tumor suppressor gene. When cells are damaged by a variety of causes such as DNA damage, ribosomal stress, the expression of the p53 protein is activated to repair damaged cells or to directly induce apoptosis if the DNA damage is already too severe. p53 is essential for a number of processes that occur throughout life, including DNA damage repair, cell cycle arrest, metabolism, senescence, and apoptosis [17]. If too much p53 protein is produced during certain physiological processes, cell function is impaired or the tendency to form tumors is increased. Therefore, the expression of murine double minute 2 (MDM2) protein in the downstream signaling pathway will increase when p53 protein accumulates in normal cells. To achieve the balance and stability of p53 protein levels in cells, MDM2 can interact with the transcriptional activation domain of the p53 to form the p53-MDM2 complex, which suppresses the transcriptional activity of p53. When a cell is stressed, MDM2 expression decreases, p53 expression increases, and the increase in p53 induces MDM2 expression at the transcriptional level, creating a negative feedback regulatory loop (Figure 2) [18]. MDM2 can also act as an E3 ubiquitin ligase, targeting p53 protein and inducing its ubiquitination and degradation to maintain low levels of p53 protein [19]. p53 has long been an intriguing cancer target [20]. Individuals carrying certain inherited loss-offunction mutations in p53 have a 50% chance of developing cancer by the age of 30 and a 90% chance of developing cancer by the age of 70 [21]. Mice knocked out of p53 quickly develop tumors. Up to 50% of cancers have mutations in both copies of p53 [22]. Drugs that can reactivate the tumor suppressing ability of p53 may therefore have a powerful anti-cancer effect. However, it is more difficult to activate proteins than to inhibit them, so the interaction of MDM2 with p53 provides an opportunity to activate p53 by inhibiting the interaction of MDM2 to exert anti-tumor effects.  Cancer and the MDM2-p53 signaling pathway are closely related. p53 is a tumor suppressor gene. When cells are damaged by a variety of causes such as DNA damage, ribosomal stress, the expression of the p53 protein is activated to repair damaged cells or to directly induce apoptosis if the DNA damage is already too severe. p53 is essential for a number of processes that occur throughout life, including DNA damage repair, cell cycle arrest, metabolism, senescence, and apoptosis [17]. If too much p53 protein is produced during certain physiological processes, cell function is impaired or the tendency to form tumors is increased. Therefore, the expression of murine double minute 2 (MDM2) protein in the downstream signaling pathway will increase when p53 protein accumulates in normal cells. To achieve the balance and stability of p53 protein levels in cells, MDM2 can interact with the transcriptional activation domain of the p53 to form the p53-MDM2 complex, which suppresses the transcriptional activity of p53. When a cell is stressed, MDM2 expression decreases, p53 expression increases, and the increase in p53 induces MDM2 expression at the transcriptional level, creating a negative feedback regulatory loop ( Figure 2) [18]. MDM2 can also act as an E3 ubiquitin ligase, targeting p53 protein and inducing its ubiquitination and degradation to maintain low levels of p53 protein [19]. p53 has long been an intriguing cancer target [20]. Individuals carrying certain inherited lossof-function mutations in p53 have a 50% chance of developing cancer by the age of 30 and a 90% chance of developing cancer by the age of 70 [21]. Mice knocked out of p53 quickly develop tumors. Up to 50% of cancers have mutations in both copies of p53 [22]. Drugs that can reactivate the tumor suppressing ability of p53 may therefore have a powerful anti-cancer effect. However, it is more difficult to activate proteins than to inhibit them, so the interaction of MDM2 with p53 provides an opportunity to activate p53 by inhibiting the interaction of MDM2 to exert anti-tumor effects. Cancer and the MDM2-p53 signaling pathway are closely related. p53 is a tumor suppressor gene. When cells are damaged by a variety of causes such as DNA damage, ribosomal stress, the expression of the p53 protein is activated to repair damaged cells or to directly induce apoptosis if the DNA damage is already too severe. p53 is essential for a number of processes that occur throughout life, including DNA damage repair, cell cycle arrest, metabolism, senescence, and apoptosis [17]. If too much p53 protein is produced during certain physiological processes, cell function is impaired or the tendency to form tumors is increased. Therefore, the expression of murine double minute 2 (MDM2) protein in the downstream signaling pathway will increase when p53 protein accumulates in normal cells. To achieve the balance and stability of p53 protein levels in cells, MDM2 can interact with the transcriptional activation domain of the p53 to form the p53-MDM2 complex, which suppresses the transcriptional activity of p53. When a cell is stressed, MDM2 expression decreases, p53 expression increases, and the increase in p53 induces MDM2 expression at the transcriptional level, creating a negative feedback regulatory loop (Figure 2) [18]. MDM2 can also act as an E3 ubiquitin ligase, targeting p53 protein and inducing its ubiquitination and degradation to maintain low levels of p53 protein [19]. p53 has long been an intriguing cancer target [20]. Individuals carrying certain inherited loss-offunction mutations in p53 have a 50% chance of developing cancer by the age of 30 and a 90% chance of developing cancer by the age of 70 [21]. Mice knocked out of p53 quickly develop tumors. Up to 50% of cancers have mutations in both copies of p53 [22]. Drugs that can reactivate the tumor suppressing ability of p53 may therefore have a powerful anti-cancer effect. However, it is more difficult to activate proteins than to inhibit them, so the interaction of MDM2 with p53 provides an opportunity to activate p53 by inhibiting the interaction of MDM2 to exert anti-tumor effects.   promoted the apoptosis-promoting genes bad and p53 [23]. Tang Wen et al. found that 95-D cells expressing wild-type p53 protein were 3.3 times more sensitive to ganoderic acid T than H1299 cells that did not express p53 protein [24]. Other studies suggest that GAA and sterols with similar structures may have some affinity for the MDM2. Froufe et al. found that some Ganoderma lucidum triterpenoids have potential affinities with MDM2 protein through virtual screening prediction, including ganoderic acid A (K i = 147 nM) and ganoderic acid F (K i = 212 nM) [25]. Staszczak et al. summarized the role of secondary metabolites in fungi on the ubiquitin-protesome system, in which sterols have certain interactions with MDM2, indicating that such structure has advantages in interactions with MDM2 [26]. All these results suggest that GAA is likely to be related to the p53-MDM2 pathway. However, considering the low anti-tumor effect of GAA and the size of the pocket of MDM2, and there is no relevant literature highlighting the anti-cancer activities of synthetic GAA derivatives on potential MDM2-p53 interaction inhibitions, we decided to simply modify the structure of GAA at the carboxyl group to improve its anti-tumor activity and reduce possible pharmacokinetic problems caused by the free carboxyl group, and investigated the effects of the different GAA amide derivatives on the MDM2-p53 pathway.
In this study, GAA was modified to determine the in vitro anti-tumor activity of these derivatives on different tumor cell lines, and compound A2 (Figure 1), which has good activity in different cell lines and low toxicity to normal cells, was selected to investigate the relevant mechanism. First, we investigated the effect of A2 on cell apoptosis and the expression of proteins related to the MDM2-p53 pathway by flow cytometry and Western blot experiment. Next, in silico target fishing and molecular docking was performed to investigate the binding potential of A2 and MDM2. We then used a surface plasmon resonance (SPR) experiment to show that A2 has a certain binding affinity with MDM2 in vitro. It was speculated that A2 might play a role in increasing p53 protein levels by binding to MDM2 to inhibit the interaction of MDM2 and p53. This work is valuable in further demonstrating the potential of GAA and its amide derivatives as MDM2-p53 binding inhibitors and in developing candidates with anti-tumor activity.

Chemistry
Based on the structure of GAA, we retained its core structure of tetracyclic triterpenoids, and introduced a series of amino groups to modify GAA at the carboxyl site. As shown in Scheme 1, GAA was treated with amino compounds, 2-(1H-benzotriazole-1yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and N,N-diisopropylethylamine (DIPEA) to obtain GAA derivatives [27,28]. A1-A12 refers to the amide derivatives formed with fatty amine, aniline, benzylamine, phenylethylamine and other different types of primary amine compound. A13-A15 refers to the derivatives formed with piperazine compounds.
Except for the low yield of substituted aniline, the yields of the other compounds are 70~98%, which is easy to obtain. See Methods and Materials for the detailed synthesis and purification methods of all compounds. After being substituted by different amine fragments, the hydrogen signal of amide bond appears at 7-5 ppm. The methylene peak of amine fragments is mostly distributed at 4.5-3 ppm. The chemical shift of hydrogen signal in GAA itself does not change very much. All new compounds were identified by 1 H-NMR, 13 C-APT and HRMS spectroscopy. The corresponding spectra are presented in the Supplementary Materials. Except for the low yield of substituted aniline, the yields of the other compounds are 70~98%, which is easy to obtain. See Methods and Materials for the detailed synthesis and purification methods of all compounds. After being substituted by different amine fragments, the hydrogen signal of amide bond appears at 7-5 ppm. The methylene peak of amine fragments is mostly distributed at 4.5-3 ppm. The chemical shift of hydrogen signal in GAA itself does not change very much. All new compounds were identified by 1 H-NMR, 13 C-APT and HRMS spectroscopy. The corresponding spectra are presented in the Supplementary Materials.

In Vitro Anti-Proliferation Activity
2.2.1. The Anti-Proliferation Activity on MCF-7 MCF-7 is a commonly used tumor cell line. Previous studies have shown that GAA has some anti-tumor activity against MCF-7, and there is a high expression of MDM2-p53 in MCF-7. The anti-tumor activities of GAA derivatives on MCF-7 were tested for 48 h, and the results were shown in Table 1 and Figure 3. The results showed that compounds A2, A6, A7, A8, A9, A15 had significant anti-proliferation activities on MCF-7 cell line compared with GAA. Among all derivatives, A6 has the strongest anti-proliferation effect, and its inhibition rate of MCF-7 at 50 µM can reach 63.64%.
Overall, among aliphatic amines, anilines, benzylamines, phenylethylamines, and (hetero) cyclic amines, benzylamine derivatives (A6, A7, A8) were significantly more potent than other substituted compounds. Among the aliphatic amines (A1, A2), the chain length of six carbon atoms is better than that of four carbon atoms. Among benzylamine compounds, the activities of electron withdrawing groups on benzene ring (A6, A7, A8) (cell viability at 50 µM less than 50%) are better than that of electron donating group (A5), and 3,5-diCl double substitution is better than 4-Cl single substitution, indicating that the position and amount of electron withdrawing groups can affect the activities of GAA derivatives. Compared with anilines (A3, A4), benzylamines (A6, A7, A8) and phenylethylamines (A10, A11, A12) substituted compounds, the activities of benzylamines are better, which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the antiproliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. which also indicated that the chain length of substituents may affect their activities. same time, the introduction of the common anti-tumor fragment indene can impro anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl p zine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N nyl piperazine can improve activity (A13, A14, A15), indicating that the anti-prolife activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the ant liferation activity of the derivatives. The results were shown in Table 2 and which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and

A3
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and 116.4 ± 4.8 120.7 ± 2.0 108.9 ± 1.1

A5
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and

A8
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and

A9
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The N N 87.5 ± 7.6 53.3 ± 6.9 40.3 ± 7.8

A10
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and

A11
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The N N 100.5 ± 2.5 101.8 ± 0.2 58.4 ± 5.5

A12
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The N N 119.0 ± 3.4 110.5 ± 6.0 102.9 ± 3.8

A13
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The N N 113.6 ± 2.1 107.3 ± 9.1 102.7 ± 3.5

A14
which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The which also indicated that the chain length of substituents may affect their activities. At the same time, the introduction of the common anti-tumor fragment indene can improve the anti-proliferative activity of GAA (A9). The introduction of N-methyl or N-ethyl piperazine with strong hydrophilicity can't improve the anti-tumor activity of GAA, but N-phenyl piperazine can improve activity (A13, A14, A15), indicating that the anti-proliferation activities of GAA derivatives may have certain requirements for hydrophobicity. To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3

A2 Induces Apoptosis in SJSA-1 Cells
We next examined the effect of A2 (24 h incubation, at concentrations of 12.5, 25, 50 µM) on the SJSA-1 which A2 showed the highest anti-proliferation potency among all the cell lines. Cells were stained with Annexin V-FITC and propidium iodide. The results are shown in the Figure 4. The results showed that different concentrations of A2 could induce different degrees of apoptosis in SJSA-1 cells. At low concentrations, the proportion of cells undergoing early apoptosis increased slightly from 11.6% (12.5 µM) to 12.3% (25 µM). However, the proportion of apoptosis cells increased significantly at 50 µM (18.7%), while the proportion of late apoptosis remained essentially unchanged with increasing of concentration. The results indicated that A2 can induce cell apoptosis in a dose-dependent manner.

The Anti-Proliferation Activity on SJSA-1, HepG2 and HK2
To investigate the selectivity of these derivatives towards different tumor cell lines, we also selected HepG2 and osteosarcoma cell line SJSA-1 cells to evaluate the anti-proliferation activity of the derivatives. The results were shown in Table 2 and Figure 3. The results showed that the inhibitory effect of this series of derivatives on HepG2 was overall better than that on MCF-7 on the whole. Except for compounds A2 and A11, the effects of other compounds on SJSA-1 were not strong. In HepG2 cell line, compounds A2, A7, A8 and A9 still have potent anti-proliferation activity, whereas A6 and A15, which were better in MCF-7, have weaker anti-proliferation effect on HepG2. However, A12 had strong selectivity on HepG2, and the inhibition rate of this cell below 50 µM can reach 74.37%. In SJSA-1 cell line, compound A2 still showed potent inhibition, whereas A11 showed some selectivity for SJSA-1, and it was found that GAA had better anti-tumor activity for SJSA-1 than for HepG2 and MCF-7.

A2 Induces Apoptosis in SJSA-1 Cells
We next examined the effect of A2 (24 h incubation, at concentrations of 12.5, 25, 50 µM) on the SJSA-1 which A2 showed the highest anti-proliferation potency among all the cell lines. Cells were stained with Annexin V-FITC and propidium iodide. The results are shown in the Figure 4. The results showed that different concentrations of A2 could induce different degrees of apoptosis in SJSA-1 cells. At low concentrations, the proportion of cells undergoing early apoptosis increased slightly from 11.6% (12.5 µM) to 12.3% (25 µM). However, the proportion of apoptosis cells increased significantly at 50 µM (18.7%), while the proportion of late apoptosis remained essentially unchanged with increasing of concentration. The results indicated that A2 can induce cell apoptosis in a dose-dependent manner. In the introduction section, we introduced that the concept that the MDM2-p53 pathway can induce cell apoptosis by up-regulating the expression of p53 protein to inhibit the proliferation of tumor cells, and by blocking the interaction of MDM2 and p53, the activation of p53 results in transcription of MDM2 mRNA, leading to robust MDM2 protein accumulation [29][30][31]. In order to verify the effect of A2 on this pathway, we examined the effect of A2 on the protein level of MDM2, p53 protein and Bcl-2/Bax related to apoptosis, as shown in Figure 5. The results showed that after treatment of MCF-7 cells with A2 for 24 h, both MDM2 and p53 protein showed an increasing trend at 50 µM. The level of Bcl-2/Bax decreased which was consistent with the apoptosis of MCF-7 cells induced by A2. We also investigated the effect of A2 on the SJSA-1 cell line which overexpresses MDM2. Compared with MCF-7, the expression of MDM2 and p53 protein in this cell line increased in a dose-dependent manner which may be the reason for the best anti-proliferation effect on SJSA-1 among all three cell lines. This experiment demonstrated that A2 can affect the MDM2-p53 pathway to induces apoptosis.  In the introduction section, we introduced that the concept that the MDM2-p5 way can induce cell apoptosis by up-regulating the expression of p53 protein to the proliferation of tumor cells, and by blocking the interaction of MDM2 and p activation of p53 results in transcription of MDM2 mRNA, leading to robust MDM tein accumulation [29][30][31]. In order to verify the effect of A2 on this pathway, we ex the effect of A2 on the protein level of MDM2, p53 protein and Bcl-2/Bax related t tosis, as shown in Figure 5. The results showed that after treatment of MCF-7 ce A2 for 24 h, both MDM2 and p53 protein showed an increasing trend at 50 µM. T of Bcl-2/Bax decreased which was consistent with the apoptosis of MCF-7 cells i by A2. We also investigated the effect of A2 on the SJSA-1 cell line which overex MDM2. Compared with MCF-7, the expression of MDM2 and p53 protein in this increased in a dose-dependent manner which may be the reason for the best anti-p ation effect on SJSA-1 among all three cell lines. This experiment demonstrated can affect the MDM2-p53 pathway to induces apoptosis. In order to speculate whether A2 effects the p53-MDM2 pathway by binding with MDM2 to inhibit the interaction between MDM2 and p53, we carried out in silico and in vitro binding experiments. First, we performed computer simulation to conduct target fishing of GAA and found that MDM2 interacts with GAA in silico (FitValue 0.79). We used the S-value to evaluate the binding degree of the compound and MDM2 in the molecular docking experiments. The higher the absolute value of this number, the stronger the binding force. Molecular docking (see Figure 6) revealed that the hydroxyl-H of GAA In order to speculate whether A2 effects the p53-MDM2 pathway by binding with MDM2 to inhibit the interaction between MDM2 and p53, we carried out in silico and in vitro binding experiments. First, we performed computer simulation to conduct target fishing of GAA and found that MDM2 interacts with GAA in silico (FitValue 0.79). We used the S-value to evaluate the binding degree of the compound and MDM2 in the molecular docking experiments. The higher the absolute value of this number, the stronger the binding force. Molecular docking (see Figure 6) revealed that the hydroxyl-H of GAA interacts with Met58 in MDM2 (S-value: −6.49). When A2 was docked to MDM2, it was found that, the core of A2 was in the opposite direction compared to GAA. In addition to the interaction with Met58 similar to GAA, the n-hexyl is well anchored in the hydrophobic pocket and the methylene has some hydrophobic interaction with His92 (S-value: −7.22). To verify whether GAA and A2 have a certain binding ability with MDM2 in vitro, we used surface plasmon resonance (SPR) experiment to investigate the interaction between GAA, A2 and MDM2 (see Figure 7). The K D of GAA and MDM2 is 12.73 µM, indicating that they do have some affinity. At the same time, A2 which has a stronger anti-proliferation activity has a stronger binding affinity with MDM2 than GAA, with a K D of 1.68 µM. These results demonstrated that A2 can affect the MDM2-p53 pathway to induces apoptosis probably by inhibiting the interaction of MDM2 and p53.

GAA and A2 have in vitro binding affinity with MDM2
In order to speculate whether A2 effects the p53-MDM2 pathway by binding with MDM2 to inhibit the interaction between MDM2 and p53, we carried out in silico and in vitro binding experiments. First, we performed computer simulation to conduct target fishing of GAA and found that MDM2 interacts with GAA in silico (FitValue 0.79). We used the S-value to evaluate the binding degree of the compound and MDM2 in the molecular docking experiments. The higher the absolute value of this number, the stronger the binding force. Molecular docking (see Figure 6) revealed that the hydroxyl-H of GAA interacts with Met58 in MDM2 (S-value: −6.49). When A2 was docked to MDM2, it was found that, the core of A2 was in the opposite direction compared to GAA. In addition to the interaction with Met58 similar to GAA, the n-hexyl is well anchored in the hydrophobic pocket and the methylene has some hydrophobic interaction with His92 (S-value: −7.22). To verify whether GAA and A2 have a certain binding ability with MDM2 in vitro, we used surface plasmon resonance (SPR) experiment to investigate the interaction between GAA, A2 and MDM2 (see Figure 7). The KD of GAA and MDM2 is 12.73 µM, indicating that they do have some affinity. At the same time, A2 which has a stronger antiproliferation activity has a stronger binding affinity with MDM2 than GAA, with a KD of 1.68 µM. These results demonstrated that A2 can affect the MDM2-p53 pathway to induces apoptosis probably by inhibiting the interaction of MDM2 and p53.  A2 (B). The pose of GAA in MDM2 is that the carboxyl term exposed to the solvent, the ring core is located in the pocket, and the hydroxyl-H has hydrogen bond interaction with Met58. The pose of A2 in MDM2 is opposite to that of GAA. The longer hydrophobic n-hexyl anchors into the pocket and has hydrophobic interaction with His92. The ring part faces out of the solvent and retains the interaction with Met58.  A2 (B). The pose of GAA in MDM2 is that the carboxyl term exposed to the solvent, the ring core is located in the pocket, and the hydroxyl-H has hydrogen bond interaction with Met58. The pose of A2 in MDM2 is opposite to that of GAA. The longer hydrophobic n-hexyl anchors into the pocket and has hydrophobic interaction with His92. The ring part faces out of the solvent and retains the interaction with Met58.

Chemistry
Unless otherwise stated, all reagents and solvents were obtained from commercial sources were used without further purification. GAA were purchased from Biopurify (Chengdu, China). Flash column chromatography was performed on Biotage Isolera Four (Sweden). 1 H NMR and 13 C-APT spectra were recorded on a Bruker AvanceIII 600MHz spectrometer (Germany). HRMS was performed on a Thermo Fisher LTQ Orbitrap XL (United States).
3.1.1. Synthesis of (n-butyl)-(7β,15α,25R)-7,15-Dihydroxy-3,11,23-Trioxolanost-8-en-26- We then investigated the effects of compounds with significantly higher activity than GAA in three cell lines on HK2, which is a normal cell line used to assess cytotoxicity. The results are shown in Table 2 and Figure 3. The results showed that at high concentration, benzylamine compounds A6, A7 and A9 with anti-tumor fragments had some toxicity to HK2 cells, whereas the other compounds with stronger activity had lower cytotoxicity to HK2 cells. To sum up, this series of GAA derivatives showed some selectivity in different cell lines, and have the potential to be developed as various tumor inhibitors. Given the strong anti-proliferation effect of derivative A2 in various cell lines and its low effect on normal cells, A2 was selected to investigate its anti-proliferation mechanism.

Chemistry
Unless otherwise stated, all reagents and solvents were obtained from commercial sources were used without further purification. GAA were purchased from Biopurify (Chengdu, China). Flash column chromatography was performed on Biotage Isolera Four (Sweden). 1 H NMR and 13 C-APT spectra were recorded on a Bruker AvanceIII 600MHz spectrometer (Germany). HRMS was performed on a Thermo Fisher LTQ Orbitrap XL (United States).

Target Fishing and Molecular Docking by In Silico Approaches
The binding targets of GAA were predicted using Discovery Studio 2016 v16.1 (BIOVIA Software Inc., San Diego, CA, USA), a software suite for the computational analysis of data relevant to Life Sciences research. To predict the probable targets of GAA, we used Ligand Profiler protocol which maps a set of pharmacophores, including Pharma DB by default. The ligand GAA was prepared by the Specifying Ligands parameter protocol. After setting parameters, the job was run, and the results were gained for three days. To explore the potential binding mode of GAA and A2 with MDM2 protein (PDB code: 4j3e), a molecular modeling research was performed with docking program named Induced-Fit, a refinement method in another software MOE. To eliminate any bond length and bond angle biases, the ligands (GAA and A2) were subjected to the "energy minimize" prior to docking. The binding affinities (S-values) in MOE were used to evaluate the interactions between MDM2 and ligands. The scores (binding affinities) were obtained based on the virtual calculation of various interactions of ligands with the targeted receptor.

Surface Plasmon Resonance (SPR) Assay
GAA derivatives bound to MDM2 protein were assayed with a molecular interaction analyzer (PALL FORTEBIO, USA). MDM2 protein (5 mg/mL, Protintech) was immobilized on a PCH sensor chip (Octet) and preactivated with EDC/NHS mixture for 420 s at a flow rate of 10 µL/min. A2 was diluted to 100, 50, 25, 12.5, 6.25, 3.13, 0 µM with PBST buffer containing 1% DMSO. The binding time was 600 s, and the flow rate was 20 µL/min. The dissociation time was 180 s, and the affinity constant K D value was obtained by computer fitting and steady-state analysis.

Flow Cytometric Analysis of the Apoptosis Rate with Annexin V-FITC/PI Staining
To determine the apoptosis rate, an Annexin V-FITC/PI double staining apoptosis assay kit (Beyotime) was used to detect apoptotic cells by flow cytometry (BD FACSA-LOBUR), according to the manufacturer's instructions. Briefly, SJSA-1 cells were treated with 0.1% DMSO, 12.5, 25, and 50 µM of A2 for 24 h. After harvesting, the cells were incubated with 5 µL Annexin V-FITC for 15 min and 10 µL PI for 5 min at 4 • C under dark conditions. Flow cytometry was then performed to analyze the apoptosis rate. Data were analyzed by using BD FACSDiva 8.0.1.

Western Blot Analysis of Protein Expression
For Western blot analysis, MCF-7 and SJSA-1 cells were treated with different concentrations of A2 for 24 h. The total cell protein was extracted, and proteins were isolated using 10% SDS-PAGE gel system. The proteins on the gel were transferred to PVDF membrane, blocked in 5% BSA at room temperature for 2 h, incubated in primary antibody dilution at 4 • C overnight, and washed with TBST for 3 times, 10 min each. Then, they were transferred to dilute release solution of secondary antibody and incubated at room temperature for 2 h. ECL chemiluminescence development solution (Beyotime, BeyoECL star) was added uniformly and detected on gel imaging system (Clinx ChemiScope, China). Antibodies for blotting were MDM2 (abcam, ab16895), P53 (Proteintech, 10442-1-AP), Bcl-2 (CST, 15071S), Bax (CST, 2772T) and β-actin (abcam, ab8226).

Statistical Analysis
The results are expressed as the means ± standard deviation. A one-way AVONA and t-test were used for comparison of differences between groups, and GraphPad Prism 8.0 software was used for graph and statistical analysis. Statistical significance was set at p < 0.05.

Conclusions
Natural products are rich in beneficial scaffolds that have been used in anti-tumor, antiinflammatory, neuroprotective and other aspects. However, these natural products have the problem of unclear targets and weak activity. Therefore, if we can determine the relevant mechanism of the action of natural products and identify specific pathways and targets, we can improve their activity based on adding appropriate interaction with binding amino acid residues in the active pocket of the target. In this study, we modified Ganoderma lucidum triterpenoid compound GAA and evaluated anti-proliferative effects of these derivatives in different tumor cell lines. Finally, compound A2 was selected for further investigation of its mechanism. The results showed that A2 could induce apoptosis by interfering with the MDM2-p53 pathway. Target fishing and SPR experiments suggested that A2 might play a role by binding to MDM2 and blocking its inhibition of p53. Although these compounds may have weaker anti-tumor activity than other small molecule anti-tumor drugs, this study may provide insights into finding the target of GAA and developing new natural product anti-cancer compounds. If we can confirm the specific targets of GAA in different diseases, we can carry out target-based rational design of GAA to greatly improve its efficacy and provide an excellent scaffold for the development of new drugs.

Patents
In order to protect the structure and activity of compounds in a timely manner, the patent Preparation method of Ganoderic A amide derivatives useful as anti-tumor drugs, China CN112574272 A 2021-03-30, refers to the synthesis of the derivatives and simple in vitro cell anti-proliferation screening. In subsequent studies, the activity of the derivatives in other cell lines was found and the mechanism was investigated. The relevant experimental results are presented in this article.