Structural Optimization and Improving Antitumor Potential of Moreollic Acid from Gamboge

Moreollic acid, a caged-tetraprenylated xanthone from Gamboge, has been indicated as a potent antitumor molecule. In the present study, a series of moreollic acid derivatives with novel structures were designed and synthesized, and their antitumor activities were determined in multifarious cell lines. The preliminary screening results showed that all synthesized compounds selectively inhibited human colon cancer cell proliferation. TH12-10, with an IC50 of 0.83, 1.10, and 0.79 μM against HCT116, DLD1, and SW620, respectively, was selected for further antitumor mechanism studies. Results revealed that TH12-10 effectively inhibited cell proliferation by blocking cell-cycle progression from G1 to S. Besides, the apparent structure–activity relationships of target compounds were discussed. To summarize, a series of moreollic acid derivatives were discovered to possess satisfactory antitumor potentials. Among them, TH12-10 displays the highest antitumor activities against human colon cancer cells, in which the IC50 values in DLD1 and SW620 are lower than that of 5-fluorouracil.


Introduction
Undoubtedly, cancer has become one of the severe diseases that threatens human health and imposes tremendous physical, emotional, and financial stress, and even death, on patients [1,2]. Nowadays, chemotherapeutics remains one of the major strategies for the treatment of cancer. Although plenty of chemotherapy antineoplastic drugs have been discovered, inevitable problems such as selectivity, resistance, and toxicity still linger [3,4]. Thus, seeking novel chemical agents with anticancer features is an urgent mission.
In recent years, we have focused on chemical and pharmacological investigations of natural products, and further uncovered a number of interesting molecules with satisfied antitumor activities [5][6][7][8]. Gamboge, the resin secreted by the Garcinia hanburyi tree, has been used as a natural health product in China for thousands of years [9]. Gamboge is also an effective remedy for treating inflammation, anabrosis, traumatic injury, parasites, tumor, and so on in clinics and folk medicine [10]. A mass of chemical and pharmacological research about gamboge has been carried out, and further indicated that the major components of gamboge are caged xanthones. In particular, gambogic acid (Figure 1) is the most representative caged xanthone in gamboge, and it displays antitumor and anti-proliferative effects, as well as antimicrobial activities [11][12][13]. It is reported that gambogic acid indeed inhibited (TH12-1, Figure 1), an analogue of gambogic acid, was first isolated from gamboge b shii's group and it was demonstrated that it reveals cytotoxicity against cervical c cells [18]. However, there are fewer reports about further pharmacological and che studies of moreollic acid. At present, some problems such as promoting antitumor tivity, enhancing effectiveness, and elucidating the antitumor mechanism of mor acid remain to be solved. On the other hand, the piperidine scaffold played a prom role in the drug design, as it contributed to enhancing the interaction between the and the target. For example, Ferro et al. [19] developed the 4-(4-fluorine phenyl) p dine fragment as a tyrosinase inhibitor to protect against melanoma, in which the p dine ring of the active molecule (compound A, Figure 1) interacted with His263 via t p interaction, thereby promoting its bioactivity. Rui et al. [20] explored a small mol containing the piperidine fragment as a sigma receptor (compound B, Figure 1), in w the piperidine portion played a key role in ligand binding to the receptor subtype showed intensive adhesion towards the S2R receptor. In addition, the introduction peridine fragments into drug molecules is a common strategy for promoting pharm kinetics. The classical instance is that the solubility of the cis-platinum derivative ( pound C, Figure 1) improved remarkably due to decoration by the piperidine ring Significantly, many antitumor drugs approved by the FDA contain piperidine ring Imbruvica, Crizotinib, Ceritinib, and so on [21]. Hence, we considered introducin piperidine fragment (or its isosteres) into moreollic acid and expected to achieve a m cule with potential antitumor activity. Inspired by the above-mentioned discussion, we initially isolated moreollic acid gamboge, and then designed and synthesized 9 moreollic acid derivatives (TH12-2~T 10, Figure 2) via the decoration of the carboxyl to various amides bearing piperidin the piperidine analogue. Subsequently, to go into more detail on the antitumor acti Inspired by the above-mentioned discussion, we initially isolated moreollic acid from gamboge, and then designed and synthesized 9 moreollic acid derivatives (TH12-2~TH12-10,  Figure 2) via the decoration of the carboxyl to various amides bearing piperidine and the piperidine analogue. Subsequently, to go into more detail on the antitumor activities of these compounds, the cytotoxicity in human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116, SW620, DLD1) was assayed. The results indicated that the target compounds displayed stronger cytotoxicity against human colon cancer cell lines than other tested cancer cell types. According to the IC 50 value of the target compounds, TH12-10 with the highest antiproliferation ability against human colon cancer cells was selected for subsequent evaluations of anticancer performance, as well as the superficial mechanism. It was revealed that TH12-10 inhibits tumor cell proliferation by blocking cellcycle progression from the G1 to the S phase. In particular, to our delight, these compounds displayed lower cytotoxicity against NCM460 cells (normal human colonic epithelial cells) than that of HCT-116, SW620, and DLD1.

PEER REVIEW
3 of 13 cell lines than other tested cancer cell types. According to the IC50 value of the target compounds, TH12-10 with the highest antiproliferation ability against human colon cancer cells was selected for subsequent evaluations of anticancer performance, as well as the superficial mechanism. It was revealed that TH12-10 inhibits tumor cell proliferation by blocking cell-cycle progression from the G1 to the S phase. In particular, to our delight, these compounds displayed lower cytotoxicity against NCM460 cells (normal human colonic epithelial cells) than that of HCT-116, SW620, and DLD1. As mentioned above, low selectivity and high toxicity are both the fatal defects of present antineoplastic drugs in the clinic. Here, we found a compound, TH12-10, with selective antitumor activity against colon cancer cells, which displayed more selectivity than the clinical drug 5-fluorouracil (5-Fu). Overall, our study provides a potential lead compound for the design of antineoplastic drugs for colon cancer.

Chemistry
Initially, compound TH12-1 (moreollic acid) was isolated from gamboge as a yellowish solid. Its molecular formula was deduced as C34H40O9 by analysis of its positive highresolution electrospray ionization mass spectroscopy (HRESIMS), nuclear magnetic resonance (NMR). The accurate structure was confirmed by comparing the spectroscopic data with those reported in the literature [18]. As shown in Scheme 1, there was a further condensation reaction between TH12-1 and corresponding amines (Ia-Ii), respectively, in the presence of 2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) to afford TH12-2~TH12-10. As mentioned above, low selectivity and high toxicity are both the fatal defects of present antineoplastic drugs in the clinic. Here, we found a compound, TH12-10, with selective antitumor activity against colon cancer cells, which displayed more selectivity than the clinical drug 5-fluorouracil (5-Fu). Overall, our study provides a potential lead compound for the design of antineoplastic drugs for colon cancer.

Chemistry
Initially, compound TH12-1 (moreollic acid) was isolated from gamboge as a yellowish solid. Its molecular formula was deduced as C 34 H 40 O 9 by analysis of its positive high-resolution electrospray ionization mass spectroscopy (HRESIMS), nuclear magnetic resonance (NMR) (see Supplementary Materials). The accurate structure was confirmed by comparing the spectroscopic data with those reported in the literature [18]. As shown in Scheme 1, there was a further condensation reaction between TH12-1 and corresponding amines (Ia-Ii), respectively, in the presence of 2-(7-Azabenzotriazol-1-yl)-N,N,N',N'tetramethyluronium hexafluorophosphate (HATU) to afford TH12-2~TH12-10.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 µM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 µM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM.

Inhibition of Cell Viability in Tumor Cell Lines
Initially, the target compounds were evaluated for antiproliferative activities against human non-small-cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human esophageal cancer cells (KYSE30), and human colon cancer cells (HCT-116) at 5 μM. All of them were tested by the CCK-8 assay using 5-Fu as the positive control. As shown in Table 1, the target compounds exhibited unequal inhibition activities against different tumor cells and showed more susceptibility to human colon cancer cells (HCT-116). Excitingly, most synthesized compounds possessed a higher inhibition ratio in HCT-116 cells than the positive control drug 5-Fu at 5 μM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC 50 ) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC 50 = 0.62 µM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC 50 values ranging from 1.05 to 2.49 µM, which reveals more potent antitumor activities than 5-Fu (IC 50 = 7.89 µM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC 50 = 0.83, 1.10, and 0.79 µM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC 50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 µM, respectively, while the IC 50 against NCM460 was 4.08 µM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM. To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM.

Th12-10
To further assess the antitumor activities of synthesized compounds against human colon cancer cells, the effects of each synthesized compound on the viability of two additional human colon cancer cells (SW620, DLD1) were evaluated, and normal human colonic epithelial cells (NCM460) were used as a control. The half-maximal inhibitory concentration (IC50) is shown in Table 2. It is obvious that all the synthesized compounds display potential antitumor activities against HCT116, DLD1, and SW620 cells. We observed that the cytotoxicity of most synthesized compounds was similar to 5-Fu (IC50 = 0.62 μM) in HTC116. In addition, for SW620, the activities of most optimized compounds were slightly stronger than that of 5-Fu. Moreover, for DLD1, all target compounds except TH12-4 possess IC50 values ranging from 1.05 to 2.49 μM, which reveals more potent antitumor activities than 5-Fu (IC50 = 7.89 μM). Remarkably, Th12-10 exhibited the most satisfactory antitumor activities (IC50 = 0.83, 1.10, and 0.79 μM in HCT116, DLD1, and SW620, respectively) among all target compounds, of which the anti-proliferation activity was close to that of 5-Fu against HCT116 and much better than that of 5-Fu against DLD1 and SW620 cells. It is worth mentioning that these compounds showed lower cytotoxicity against normal colonic epithelial cells than that of colon cancer cells. Taking TH12-10 as an example, the IC50 values against HCT116, DLD1, and SW620 were 0.83, 1.10, and 0.79 μM, respectively, while the IC50 against NCM460 was 4.08 μM.

Apparent Structure-Activity Relationship
Structurally, the R group was a six-member nitrogenous heterocyclic skeleton with decorating in the 4-position. Based on the results of preliminary screening in human colon cancer cells, different substituent groups in the 4-position of R influence the antitumor activities of these compounds. Apparently, the incremental activity of the atom in the 4-position of R obeyed the rules as C > O > S, for which the cytotoxicity was correlated as follows: TH12-2 > TH12-3 > TH12-4. Interestingly, the antitumor activities of molecules improved visibly when S in the 4-position of R was oxidized to sulfone, as illustrated by the multiplied cytotoxicity of Th12-10 (IC 50 = 0.83, 1.10, and 0.79 µM in HCT116, DLD1, and SW620, respectively) compared to Th12-4 (IC 50 = 7.62, 7.89, and 7.26 µM in HCT116, Molecules 2022, 27, 482 6 of 12 DLD1, and SW620, respectively). Furthermore, when keeping the C atom as constant in the 4-position of R, the substituent in 4-C of R seems to provide a weak contribution to the antitumor activities against HCT116, DLD1, and SW620. For example, TH12-2, TH12-5, TH12-6, TH12-7, and TH12-8 possess similar cytotoxicity levels.

TH12-10 Effectively Inhibited the Cell Proliferation via Modulation of Cell-Cycle Regulatory Proteins
TH-12, one of the most potent anticancer compounds, was selected for further mechanism studies. To gain greater insight into whether the compound inhibits tumor cells' proliferation via inducing tumor cell-cycle arrest, we performed protein analyses for cellcycle-related proteins on SW620 and DLD1. As shown in Figure 3A,B, treating the SW620 and DLD1 cells with indicated concentrations of compound Th12-10 for 48 h significantly decreased the expression of CyclinD1, cyclin-dependent kinase 6 (CDK6), cyclin-dependent kinase 4 (CDK4), and phosphorylated retinoblastoma (p-Rb), which are major components of the G1/S checkpoint. On the other hand, no significant change in the expression of related proteins was observed when treating the NCM460 cells with indicated concentrations of compound Th12-10 ( Figure 4A,B). Additionally, flow cytometric analyses were performed to determine cell-cycle distribution, where 400 ng/mL of nocodazole was used to synchronize cells in G2/M phase. Consistent with the immunoblotting data, 6 h after nocodazole administration, the percentage of DLD1 cells treated with TH12-10 in the G1 phase was significantly higher than that of blank control cells ( Figure 3C,D), while the percentage of NCM460 cells treated with Th12-10 in the G1 phase was similar to that of blank control cells ( Figure 4C,D). Collectively, the compound induced CDK6/CDK4 and CyclinD1 degradation, and subsequently decreased p-Rb levels, thereby blocking colon cancer cell-cycle progression from the G1 to the S phase [22].    (C) Flow cytometric analysis for cell-cycle distribution in DLD1 cells treated with TH12-10 or vehicle and then with nocodazole for 48 h. (D) Quantitative analysis of DLD1 cells in G1 phase as described in (C). All data are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. N.S., not significant. Differences were tested using one-way ANOVA (B) or the unpaired t-test with Welch's correction (D).

Cell Proliferation Assay
To investigate the effects of compounds on tumor cells' proliferation, the cell proliferation assay was performed using the Cell Counting Kit-8 (#40203ES60, Yeasen, Shanghai, China). In brief, cells were seeded in a 96-well plate at 3 × 10 3 cells per well and cultured overnight. Then, the medium was replaced with fresh DMEM medium containing various concentrations of compounds or vehicle. After 48 h of incubation, the medium was exchanged with 100 µL of FBS-free DMEM medium supplemented with 10 µL of CCK8 added to each well and incubated at 37 • C for another 2 h. Absorbance at 450 nm was measured with a microplate reader (Thermo). The experiments were performed in triplicate. The mean ± SD values presented in the figures were calculated from three independent experiments. Comparisons between groups were evaluated using GraphPad Prism version 7.0 software. p < 0.05 was considered statistically significant.

Immunoblot
When 80% confluency was reached, cells were seeded into a 6-well plate at 3 × 10 6 cells per well and allowed to attach overnight. Then, the cells were treated with different concentrations of TH12-10 or vehicle for 48 h. Cells were lysed in RIPA buffer (#89900, Thermo, Waltham, MA, USA) supplemented with protease inhibitor cocktail (#B14002, Bimake, Houston, TX, USA) on ice for 30 min and centrifuged at 12,000 rpm for 10 min at 4 • C. Protein concentration was determined by the BCA kit and equal amounts of lysates were separated by SDS-polyacrylamide gels, and transferred to nitrocellulose membranes (#HATF00010, Millipore, Burlington, MA, USA). Then, the membranes were blocked with 5% milk in TBST for 1 h and probed with the indicated antibodies. Antibodies used for the immunoblot analysis in this study were as follows: Cyclin D1 (55506T, Cell Signaling Technology, Danvers, MA, USA), CDK4 (ab199728, Abcam, Cambridge, UK), CDK6 (13331T, Cell Signaling Technology, Danvers, MA, USA), phospho-Rb (Ser807/811) (8516S, Cell Signaling Technology, Danvers, MA, USA), and β-actin (sc-47778, Santa Cruz Biotechnology, CA, USA)

Conclusions
A series of moreollic acid derivatives, with novel structures designed and synthesized, and their antitumor activities were evaluated. TH12-10 showed the highest antitumor activity against DLD1 and SW620 cells, with IC 50 values of 1.10 and 0.79 µM, respectively, superior to those of 5-Fu. In addition, further mechanism studies recommended that TH12-10 effectively inhibited cell proliferation by blocking cell-cycle progression from G1 to S. In addition, the structure-activity relationship discussion suggested that substituent groups in the 4-position of R affect the bioactivities of title compounds. This work provided useful guidance for the optimization of antitumor molecules from the natural product gamboge.