Design, Synthesis, and Biological Evaluation of N14-Amino Acid-Substituted Tetrandrine Derivatives as Potential Antitumor Agents against Human Colorectal Cancer

As a typical dibenzylisoquinoline alkaloid, tetrandrine (TET) is clinically used for the treatment of silicosis, inflammatory pulmonary, and cardiovascular diseases in China. Recent investigations have demonstrated the outstanding anticancer activity of this structure, but its poor aqueous solubility severely restricts its further development. Herein, a series of its 14-N-amino acid-substituted derivatives with improved anticancer effects and aqueous solubility were designed and synthesized. Among them, compound 16 displayed the best antiproliferative activity against human colorectal cancer (HCT-15) cells, with an IC50 value of 0.57 μM. Compared with TET, 16 was markedly improved in terms of aqueous solubility (by 5-fold). Compound 16 significantly suppressed the colony formation, migration, and invasion of HCT-15 cells in a concentration-dependent manner, with it being more potent in this respect than TET. Additionally, compound 16 markedly impaired the morphology and motility of HCT-15 cells and induced the death of colorectal cancer cells in double-staining and flow cytometry assays. Western blot results revealed that 16 could induce the autophagy of HCT-15 cells by significantly decreasing the content of p62/SQSTM1 and enhancing the Beclin-1 level and the ratio of LC3-II to LC3-I. Further study showed that 16 effectively inhibited the proliferation, migration, and tube formation of umbilical vein endothelial cells, manifesting in a potent anti-angiogenesis effect. Overall, these results revealed the potential of 16 as a promising candidate for further preclinical studies.


Introduction
The burden of cancer incidence and mortality is growing rapidly worldwide [1], and more than 19.3 million new cases and 10 million cancer deaths were estimated according to GLOBOCAN 2020 [2]. Colorectal cancer, also known as large bowel cancer, is one of the leading cause of cancer-related morbidity and mortality [3]. It is very heterogeneous, with a high variability in terms of patient prognosis and treatment response [4]. Approximately half of patients with colorectal cancer eventually succumb to the disease after the surgical resection of the primary tumor, predominantly due to metastases [5]. Therefore, metastasis

Chemistry
The synthesis of TET derivatives (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) is depicted in Scheme 1. Since optimizations on the C14 position of TET have commonly provided derivatives with better anticancer activities than the parent compound [32,33], herein, a series of L-amino acids were attached to this position through an amide bond. The C14 position of TET was selectively nitrified at a mild condition to afford 14-nitro-TET (1) with a high yield, the nitro of which was then reduced to an amino group (2) to facilitate the introduction of amino acids. An amide condensation reaction then proceeded between compound 2 and nine Boc-protected amino acids to afford 3-11 in the presence of condensing agent N,N'-dicyclohexylcarbodiimide (DCC). Then, the N-protected groups of 3-11 were removed smoothly with trifluoroacetic acid (TFA) to provide derivatives 12-20. All the synthesized compounds were characterized by IR, 1 H NMR, 13 C NMR, and high-resolution mass spectrum (HRMS). To date, few structural modifications have been performed on TET, with these mainly focusing on the C 5 and C 14 positions, and experimental studies have shown that optimizations on the C 14 position generally provides derivatives with better activities than TET against several cancer cells [31][32][33][34][35]. Nevertheless, fewer investigations on the enhancement of the solubility of TET have been reported, and most of the derivatives achieved the improvement through the nano-encapsulation strategy [36][37][38]. Large polymer amounts are required in these pharmaceutical applications, and these strategies cannot improve the druggability of TET itself. Natural and synthetic amino acids, which are commercially available to medicinal chemists [39], provide wide structural diversity and physicochemical properties in drug development [40]. Amino acids are excellent moieties with superior aqueous solubility and permeability, and a number of studies have successfully increased the solubility of chemical entities with the aid of amino acid fragments [41][42][43][44][45]. Several drugs (such as valacyclovir and valganciclovir) were also reported to have improved pharmaceutical properties by using these moieties [46][47][48]. In addition, as the building blocks of proteins, amino acids are generally regarded as safe molecular fragments. However, the introduction of most amino acid moieties increases the challenge of synthesis and purification because of the addition of an additional chiral center in the molecule [49]. Furthermore, some amino acids, such as L-glutamic acid and L-cysteine, have the potential to be converted into other amino acids (L-pyroglutamic acid and dimer cysteine, respectively) in solution, and moderate reaction conditions should be cultivated to take this into account [50]. Thus, in this study, a series of amino acids were attached to the C14 amino of 14-amino TET with a mild reaction condition (Figure 1), which was derived from TET with a moderate anticancer effect [32], with the aim of improving its solubility and biological activity. Encouragingly, consistent with the above hypothesis, a number of TET derivatives were found with outstanding anticancer activities and aqueous solubilities.

Chemistry
The synthesis of TET derivatives (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) is depicted in Scheme 1. Since optimizations on the C 14 position of TET have commonly provided derivatives with better anticancer activities than the parent compound [32,33], herein, a series of L-amino acids were attached to this position through an amide bond. The C 14 position of TET was selectively nitrified at a mild condition to afford 14-nitro-TET (1) with a high yield, the nitro of which was then reduced to an amino group (2) to facilitate the introduction of amino acids. An amide condensation reaction then proceeded between compound 2 and nine Boc-protected amino acids to afford 3-11 in the presence of condensing agent N,N'-dicyclohexylcarbodiimide (DCC). Then, the N-protected groups of 3-11 were removed smoothly with trifluoroacetic acid (TFA) to provide derivatives 12-20. All the synthesized compounds were characterized by IR, 1 H NMR, 13 C NMR, and high-resolution mass spectrum (HRMS).

Aqueous Solubility Analysis
To better understand the improvement in terms of solubility as a result of introducing L-amino acids, three polar amino acids (Ser, Thr, and Tyr) and six non-polar amino acids (Gly, Ala, Val, Leu, Pro, and Phe) were selected in this study, and the water-solubility of TET derivatives was measured by HPLC according a reported procedure [51]. As presented in Figure 2, the solubility of TET was very poor in water (less than 30 μg·mL -1 ),

Aqueous Solubility Analysis
To better understand the improvement in terms of solubility as a result of introducing L-amino acids, three polar amino acids (Ser, Thr, and Tyr) and six non-polar amino acids (Gly, Ala, Val, Leu, Pro, and Phe) were selected in this study, and the water-solubility of TET derivatives was measured by HPLC according a reported procedure [51]. As presented in Figure 2, the solubility of TET was very poor in water (less than 30 µg·mL −1 ), which was consistent with the reported study [52]. By contrast, except for 10, 11, and 18-20, most of the amino acid derivatives exhibited higher solubilities than TET (p < 0.05). Most notably, which was consistent with the reported study [52]. By contrast, except for 10, 11, and 18-20, most of the amino acid derivatives exhibited higher solubilities than TET (p < 0.05). Most notably, the solubility of Boc-protected glycine derivative 3 significantly increased approximately 16-fold compared to TET. Among the amino acid derivatives, compound 16 displayed a marked improved aqueous solubility (>5-fold), indicating that the incorporation of amino acids can effectively improve the water solubility of TET. Figure 2. Aqueous solubility analysis of TET derivatives in neutral water. *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs. TET.

Cytotoxicity Analysis
The antiproliferative effect of the synthesized TET derivatives was evaluated by MTT assay against five common cancer cell lines, including human lung cancer cells (A549), colon cancer cells (HCT-15), liver cancer cells (HepG2), pancreatic cancer cells (BxPC-3), and breast cancer cells (MCF-7). Meanwhile, the toxicity and anti-angiogenesis activity of the title compounds were evaluated with respect to human normal hepatocyte cells (L-02) and umbilical vein endothelial cells (HUVEC), respectively. TET was evaluated as a comparison. As shown in Table 1, except for 8 and 9, most of the prepared compounds significantly inhibited the proliferation of cancer cells, with IC50 values comparable to or lower than that of TET. These TET derivatives exhibited better inhibitory activities on HCT-15, HepG2, and BxPC-3 than on A549 and MCF-7, and these derivatives displayed the best anticancer effect on HCT-15 cells, with IC50 values ranging from 0.57 to 5.28 μM. Specifically, compared with TET (IC50 = 6.12 μM), the introduction of Boc-protected glycine to the C14 amino of TET 3 improved the antiproliferative activity by about 2-fold against HCT-15 cells. Increasing the substituent size on the methylene of glycine, from methyl 4 to isopropyl 5 and isobutyl 6, gradually impaired the inhibitory activity, which was most likely attributed to the reduction in terms of aqueous solubility. However, the attachment of Boc-protected proline to the C14 amino of TET 7 enhanced the anticancer effect (with an IC50 of 1.15 μM). By contrast, the introduction of Boc-protected serine 8 and threonine 9 markedly weakened the activity. Interestingly, for Boc-protected phenylalanine derivative of TET 10, the antiproliferative effect was increased by more than 6-fold compared to TET, with IC50 decreased to 0.91 μM, whereas the Boc-protected tyrosine derivative 11 only exhibited a retained activity.
The protecting groups of 3-11 were removed to provide derivatives 12-20, respectively, with generally improved anticancer effects against HCT-15 cells (Table 1). The inhibitory activity trend of 12-20 were almost in line with the parent compounds 3-11.

Cytotoxicity Analysis
The antiproliferative effect of the synthesized TET derivatives was evaluated by MTT assay against five common cancer cell lines, including human lung cancer cells (A549), colon cancer cells (HCT-15), liver cancer cells (HepG2), pancreatic cancer cells (BxPC-3), and breast cancer cells (MCF-7). Meanwhile, the toxicity and anti-angiogenesis activity of the title compounds were evaluated with respect to human normal hepatocyte cells (L-02) and umbilical vein endothelial cells (HUVEC), respectively. TET was evaluated as a comparison. As shown in Table 1, except for 8 and 9, most of the prepared compounds significantly inhibited the proliferation of cancer cells, with IC 50 values comparable to or lower than that of TET. These TET derivatives exhibited better inhibitory activities on HCT-15, HepG2, and BxPC-3 than on A549 and MCF-7, and these derivatives displayed the best anticancer effect on HCT-15 cells, with IC 50 values ranging from 0.57 to 5.28 µM. Specifically, compared with TET (IC 50 = 6.12 µM), the introduction of Boc-protected glycine to the C 14 amino of TET 3 improved the antiproliferative activity by about 2-fold against HCT-15 cells. Increasing the substituent size on the methylene of glycine, from methyl 4 to isopropyl 5 and isobutyl 6, gradually impaired the inhibitory activity, which was most likely attributed to the reduction in terms of aqueous solubility. However, the attachment of Boc-protected proline to the C 14 amino of TET 7 enhanced the anticancer effect (with an IC 50 of 1.15 µM). By contrast, the introduction of Boc-protected serine 8 and threonine 9 markedly weakened the activity. Interestingly, for Boc-protected phenylalanine derivative of TET 10, the antiproliferative effect was increased by more than 6-fold compared to TET, with IC 50 decreased to 0.91 µM, whereas the Boc-protected tyrosine derivative 11 only exhibited a retained activity. The protecting groups of 3-11 were removed to provide derivatives 12-20, respectively, with generally improved anticancer effects against HCT-15 cells (Table 1). The inhibitory activity trend of 12-20 were almost in line with the parent compounds 3-11. Among them, the L-proline derivative 16 exhibited the best antiproliferative activity, and with IC 50 values that increased to higher than 10-fold (0.57 µM) the values for TET. Moreover, compound 16 also effectively inhibited the proliferation of the other four cancer cells, with IC 50 s lower than 1.5 µM. Regrettably, no enhancement in terms of antiproliferative activity was observed for the deprotected product of 10 (19).
Considering its improved aqueous solubility and anticancer activity, compound 16 was then selected for the further biological evaluation. Notably, during the cytotoxicity assay, the morphology of the HCT-15 cells was altered after the treatment of 16, and cell shrinkage and nuclear karyorrhexis were observed, indicating the disorganization of the internal architecture ( Figure S1 in Supplementary Materials). To determine the safety of the synthesized compounds, the toxicity of 16 was then evaluated on human hepatic cells L-02 (Table 1). Compared with their inhibitory activity against the cancer cells, lower cytotoxicities were observed for TET derivatives 3-20 on the normal cell, and the IC 50 values of 16 were >20 µM, suggesting that the compound had a lower toxicity.

Colony Formation Assay
The HCT-15 cell is a widely used cell line for studying the tumor biology and experimental therapy of human colorectal cancer in vitro [53], and this cell was used to investigate the anticancer effect of 16 in the following experiments. To evaluate the ability of 16 to undergo unlimited division and form colonies, the colony formation assay was performed on HCT-15 cells. As shown in Figure 3A, compound 16 effectively inhibited the colony formation of HCT-15 cells in a concentration-dependent manner, which was superior to TET at 1.25 and 2.5 µM (p < 0.05, Figure 3B). Specially, very few colonies were found in the plate treated with 16 even at 0.625 µM. These results indicated that compound 16 markedly inhibited the colony formation of HCT-15 cells.

Migration Assay
The capacity of 16 to inhibit migration, invasion, and angiogenesis was then assessed to investigate its antimetastatic potential. The wound healing assay was carried out to evaluate the effect of compound 16 on the migration of HCT-15 cells. The results revealed that the scratched areas in the control groups were approximately 46% covered after 24 h of culture ( Figure 4A,B). However, the recovery of the scratched areas was suppressed with the treatment of 16 μM in a concentration-dependent manner (p < 0.01). Compared with TET, derivative 16 exhibited much lower migration rates at all the concentrations (p < 0.05). Specifically, the scratched areas decreased to 14.1% and 10.5% after the addition of 5 and 10 μM of 16, respectively, indicating its strong suppressing effect on the migration of HCT-15 cells.

Migration Assay
The capacity of 16 to inhibit migration, invasion, and angiogenesis was then assessed to investigate its antimetastatic potential. The wound healing assay was carried out to evaluate the effect of compound 16 on the migration of HCT-15 cells. The results revealed that the scratched areas in the control groups were approximately 46% covered after 24 h of culture ( Figure 4A,B). However, the recovery of the scratched areas was suppressed with the treatment of 16 µM in a concentration-dependent manner (p < 0.01). Compared with TET, derivative 16 exhibited much lower migration rates at all the concentrations (p < 0.05). Specifically, the scratched areas decreased to 14.1% and 10.5% after the addition of 5 and 10 µM of 16, respectively, indicating its strong suppressing effect on the migration of HCT-15 cells.

Invasion Assay
During the metastasis of cancer, cell invasion through the extracellular matrix is a significant process, and the detached cancer cells can move to distant organs for metastasis [54]. To investigate the ability of 16 to inhibit invasion, a transwell assay was then carried out on HCT-15 cells. After the treatment of 16, the invasions of colorectal cancer cells were significantly weakened in a concentration-dependent manner ( Figure 5A), with the effect being more potent than that of TET at all the concentrations (p < 0.05, Figure 5B). Moreover, almost no cancer cells were observed for 16 at 5 and 10 µM.

Invasion Assay
During the metastasis of cancer, cell invasion through the extracellular matrix is a significant process, and the detached cancer cells can move to distant organs for metastasis [54]. To investigate the ability of 16 to inhibit invasion, a transwell assay was then carried out on HCT-15 cells. After the treatment of 16, the invasions of colorectal cancer cells were significantly weakened in a concentration-dependent manner ( Figure 5A), with the effect being more potent than that of TET at all the concentrations (p < 0.05, Figure 5B). Moreover, almost no cancer cells were observed for 16 at 5 and 10 μM.

Morphological Analysis
Since the migration and invasion of HCT-15 cells were inhibited by 16, the morphological changes in the cancer cells induced by this compound were further evaluated. The F-actin filaments and nuclei of HCT-15 cells were stained with Phalloidin-FITC and DAPI, respectively. Microfilaments are the major components which maintain the normal architecture of cells and play an important role in the motility, differentiation division, and membrane organization of cancer cells. As depicted in Figure 6, HCT-15 cells in the control

Morphological Analysis
Since the migration and invasion of HCT-15 cells were inhibited by 16, the morphological changes in the cancer cells induced by this compound were further evaluated. The F-actin filaments and nuclei of HCT-15 cells were stained with Phalloidin-FITC and DAPI, respectively. Microfilaments are the major components which maintain the normal architecture of cells and play an important role in the motility, differentiation division, and membrane organization of cancer cells. As depicted in Figure 6, HCT-15 cells in the control group exhibited a regular array of F-actin filaments present along the cells. By contrast, a loss in cell volume and cell shrinkage were observed after treatment with 16 for 24 h, with these effects being more obvious than those in the TET groups. Additionally, the cells displayed a reduced amount of F-actin and a disorganization of actin filaments after the treatment of 16, indicating that the cytoskeleton of HCT-15 cells was damaged and the motility significantly impaired. As a result, the migration and invasion of HCT-15 cells were severely suppressed.  F-actin proteins and nuclei were stained with FITC-phalloidin (green) and DAPI (blue), respectively. White arrows represent cytoskeleton disruption and yellow arrows nuclear damage.

Cell Death Analysis
To better understand the cytotoxicity of compound 16 against HCT-15 cells, double staining with fluorescein diacetate (FDA)/propidium iodide (PI) was conducted to analyze the cell death induced by TET derivatives. Very few dead cells stained with PI (red) were detected in the control group ( Figure 7A), whereas the amount of dead cells was markedly increased after the addition of 16. Only a small number of vital cells that took up the fluorogen FDA (green) were observed for 16 at 5 and 10 µM. Statistical analysis revealed that the percentage of dead cells was enhanced with an increase in the concentration of 16 compared with the control group (p < 0.01, Figure 7B), and only 18.3% and 17.6% of viable cells were retained at 5 and 10 µM, respectively. Meanwhile, the cell death rate of 16 was much higher than that of TET at the same concentration (p < 0.05).  Hoechst 33258 staining was performed to investigate the effect of 16 on the nuclear morphology of colorectal cancer cells. As illustrated in Figure 7C, the nucleus of HCT-15 cells in the control groups were stained in blue and were uniform in shape. By contrast, the nuclear morphology of cancer cells was altered after treatment with 16. The chromatin was obviously swollen in the nucleus of cancer cells incubated with 16, with it being much brighter than in the control groups, particularly at concentrations higher than 1.25 µM. Moreover, nuclear fragmentation was also observed, indicating characteristics that differed from the typical apoptosis [55,56].

Flow Cytometry Assay
In order to investigate the characteristics of compound 16, flow cytometry analysis was used to analyze cell apoptosis and the cell cycle. The results revealed that 16 effectively increased the apoptotic percentage of HCT-15 cells in a concentration-dependent manner (p < 0.05), with apoptotic rates of 25.4% and 52.7% in the case of 16 at concentrations of 2.5 and 5 µM, respectively ( Figure 8). By contrast, only small percentages of apoptotic cells (lower than 14%) were detected in the TET treatment groups. Additionally, cell necrosis was also observed in the HCT-15 cells with the treatment of 16 at concentrations of 2.5 and 5 µM, with necrotic rates of 11.0% and 35.5%, respectively. Necrosis is commonly recognized as a side effect of anticancer agents [57]; however, as a genetically programmed form of necrotic cell death, necroptosis is also associated with the progression, metastasis, and immunosurveillance of cancer cells [58]. Thus, further study of this mechanism study is required to clarify the anticancer effect of 16.
The effect of 16 on cell cycle progression was then investigated ( Figure S2), but no significant influence on cell cycle redistribution was observed at concentrations of 1.25, 2.5, and 5 µM. These results suggested that, despite the induction of significant apoptosis in HCT-15 cells, the anticancer activity of 16 is independent of cell cycle control [59,60], and in this respect it diverges from commonly reported anticancer agents. metastasis, and immunosurveillance of cancer cells [58]. Thus, further study of this mechanism study is required to clarify the anticancer effect of 16.
The effect of 16 on cell cycle progression was then investigated ( Figure S2), but no significant influence on cell cycle redistribution was observed at concentrations of 1.25, 2.5, and 5 μM. These results suggested that, despite the induction of significant apoptosis in HCT-15 cells, the anticancer activity of 16 is independent of cell cycle control [59,60], and in this respect it diverges from commonly reported anticancer agents.

Western Blot Analysis
To further study the anticancer mechanism of 16 in HCT-15 cells, the expression of key apoptosis-related proteins (Bax, Bcl-2, and caspase-3) was investigated by Western blot analysis [61,62]. As shown in Figure 9, the ratio of Bax/Bcl-2 was enhanced (p < 0.05) only after treatment with a high concentration of 16 (5 μM). Additionally, compound 16 significantly decreased the caspase-3 level at 0.625, 1.25, and 2.5 μM (p < 0.05), and no concentration dependence was observed. These results indicated that the anticancer mechanism of 16 should be distinguished from typical apoptosis.
Several investigations have demonstrated that TET induces autophagy in cancer cells

Western Blot Analysis
To further study the anticancer mechanism of 16 in HCT-15 cells, the expression of key apoptosis-related proteins (Bax, Bcl-2, and caspase-3) was investigated by Western blot analysis [61,62]. As shown in Figure 9, the ratio of Bax/Bcl-2 was enhanced (p < 0.05) only after treatment with a high concentration of 16 (5 µM). Additionally, compound 16 significantly decreased the caspase-3 level at 0.625, 1.25, and 2.5 µM (p < 0.05), and no concentration dependence was observed. These results indicated that the anticancer mechanism of 16 should be distinguished from typical apoptosis.

Molecular Docking Study
Molecular docking is a powerful tool which is used to predict the binding mode of a small molecule to a protein target, and several binding modes of TET have been reported so far. Among them, TET was proved to promote autophagy mediated-cell death in cancer cells by inhibiting PKC-α [28], which is consistent with the above observation in TET derivative 16. Therefore, the binding pose of 16 with PKC-α (PDB ID: 4RA4) was investigated by Autodock 4.2. Beforehand, the PKC-α inhibitor 28 [65] was docked into the binding site in order to validate the docking method ( Figure 10A). Results showed that the redocked 28 (yellow carbon) was almost superimposed with that of the co-crystallized ligand (green carbon), indicating the reliability of the docking method. Then, 16 was docked into the active cavity. As depicted in Figure 10B, 16 was well anchored to the binding site of PKC-α and interacted with amino acid residues including Phe350, Val353, Met470, Asp467, Ala480, and Asp 481. Further structural analyses revealed that a pi-pi stacking interaction was observed between Phe350 and the phenyl group of 16, and strong hydrophobic forces were also involved between 16 and Val353 and Ala480. Moreover, two strong hydrogen bonds were formed between the C14 proline and Asp 481, which was speculated as the main cause of the strong anticancer effect of 16.  Several investigations have demonstrated that TET induces autophagy in cancer cells [63,64]; thus, the autophagic related proteins p62/SQSTM1 (P62), Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3) were further evaluated to identify the activity of 16 in terms of autophagy induction in HCT-15 cells. As depicted in Figure 9, P62 contents were significantly lessened (p < 0.05) after the treatment of 16 (1.25, 2.5, and 5 µM) in a concentration-dependent manner. By contrast, the levels of Beclin-1 were markedly enhanced with an increase in the concentration of 16 (p < 0.01). Also, the ratios of LC3-II to LC3-I were increased for 16 (0.625, 1.25, 2.5, and 5 µM, p < 0.05). The above results demonstrated that 16 effectively induced the death of HCT-15 cells through autophagy.

Molecular Docking Study
Molecular docking is a powerful tool which is used to predict the binding mode of a small molecule to a protein target, and several binding modes of TET have been reported so far. Among them, TET was proved to promote autophagy mediated-cell death in cancer cells by inhibiting PKC-α [28], which is consistent with the above observation in TET derivative 16. Therefore, the binding pose of 16 with PKC-α (PDB ID: 4RA4) was investigated by Autodock 4.2. Beforehand, the PKC-α inhibitor 28 [65] was docked into the binding site in order to validate the docking method ( Figure 10A). Results showed that the re-docked 28 (yellow carbon) was almost superimposed with that of the co-crystallized ligand (green carbon), indicating the reliability of the docking method. Then, 16 was docked into the active cavity. As depicted in Figure 10B, 16 was well anchored to the binding site of PKC-α and interacted with amino acid residues including Phe350, Val353, Met470, Asp467, Ala480, and Asp 481. Further structural analyses revealed that a pi-pi stacking interaction was observed between Phe350 and the phenyl group of 16, and strong hydrophobic forces were also involved between 16 and Val353 and Ala480. Moreover, two strong hydrogen bonds were formed between the C 14 proline and Asp 481, which was speculated as the main cause of the strong anticancer effect of 16. into the active cavity. As depicted in Figure 10B, 16 was well anchored to the binding site of PKC-α and interacted with amino acid residues including Phe350, Val353, Met470, Asp467, Ala480, and Asp 481. Further structural analyses revealed that a pi-pi stacking interaction was observed between Phe350 and the phenyl group of 16, and strong hydrophobic forces were also involved between 16 and Val353 and Ala480. Moreover, two strong hydrogen bonds were formed between the C14 proline and Asp 481, which was speculated as the main cause of the strong anticancer effect of 16.

Anti-Angiogenesis Activity Analysis
As a widely studied human endothelial cell type, HUVECs (human umbilical vein endothelial cells) are useful for studying the physiological and pathological processes of the vasculature in vitro [66,67]; thus, the antiproliferative activity of 16 was assessed on HUVECs (Table 1). The results showed that 16 also exhibited a strong anti-proliferation effect on HUVECs, with an IC 50 of 1.48 µM, indicating the potential of anti-angiogenesis activity. Double staining with FDA/PI was then performed to evaluate the cytotoxicity of compound 16 against HUVECs. As shown in Figure 11A, with an increase in the concentration of 16, the number of vital cells decreased (green), whereas the dead cells stained with red increased, resulting in enhanced cell death relative to the untreated control cells. Further statistical analysis demonstrated that the percentage of dead cells was significantly boosted with increasing concentrations of 16 (p < 0.01, Figure 11B), with this effect being more potent than in the case of TET (p < 0.01). Specifically, at 5 and 10 µM, very few viable cells were detected in the groups treated with 16, with viable cell percentages of 1.9% and 0.1%, respectively.
The inhibitory activity of 16 against the migration of vascular endothelial cell, which is a significant process during angiogenesis, was then investigated. The results demonstrated that, in the control group, the scratched area was almost completely covered after 24 h (Figure 12A), with a migration rate of 85% ( Figure 12B). However, compound 16 significantly delayed the recovery of the scratched areas (p < 0.05), and few cells covered the blank areas at 10 µM, with a migration rate of 10.0%. Moreover, the migration rate of 16 was much lower than that of TET (p < 0.05), suggesting that 16 effectively inhibited the migration of HUVECs.
Neovascularization is a crucial process during cancer growth, and tube formation assay on HUVEC is commonly used to evaluate the ability of endothelial cells to form capillarylike structure [68]. Thus, this model was then used to analyze the anti-angiogenesis effect of 16. As illustrated in Figure 13A, integrated tubes on the Matrigel matrix were observed in the control groups, whereas the tubes were fragmentary with the treatment of 16. The tube length, tube area and number of branch points of endothelial cells were obviously reduced with increasing concentration of 16. Compared with the control group, the HUVEC tube forming capacity was significantly inhibited by 16 at 5 µM (p < 0.01, Figure 13B), with almost no tube structure was observed.
tration of 16, the number of vital cells decreased (green), whereas the dead cells stained with red increased, resulting in enhanced cell death relative to the untreated control cells. Further statistical analysis demonstrated that the percentage of dead cells was significantly boosted with increasing concentrations of 16 (p < 0.01, Figure 11B), with this effect being more potent than in the case of TET (p < 0.01). Specifically, at 5 and 10 μM, very few viable cells were detected in the groups treated with 16, with viable cell percentages of 1.9% and 0.1%, respectively. The inhibitory activity of 16 against the migration of vascular endothelial cell, which is a significant process during angiogenesis, was then investigated. The results demonstrated that, in the control group, the scratched area was almost completely covered after 24 h (Figure 12A), with a migration rate of 85% ( Figure 12B). However, compound 16 significantly delayed the recovery of the scratched areas (p < 0.05), and few cells covered the blank areas at 10 μM, with a migration rate of 10.0%. Moreover, the migration rate of 16 was much lower than that of TET (p < 0.05), suggesting that 16 effectively inhibited the migration of HUVECs. Neovascularization is a crucial process during cancer growth, and tube formation assay on HUVEC is commonly used to evaluate the ability of endothelial cells to form capillary-like structure [68]. Thus, this model was then used to analyze the anti-angiogenesis effect of 16. As illustrated in Figure 13A, integrated tubes on the Matrigel matrix were

Instruments and Materials
All the reagents and chemicals were purchased from commercial sources. Bicinchoninic acid (BCA) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-B-cell lymphoma 2 (Bcl-2) antibody, anti-Bcl-2 associated X protein (Bax) antibody, cysteine protease-3 antibody (caspase3), and β-actin antibody were purchased from ProteinTech (WuHan, China). Anti-Beclin-1 and anti-p62/SQSTM1 (P62) were purchased from Abcam (Cambridge, ENG), and anti-microtubule-associated protein 1 light chain 3 (LC3) was purchased from Cell Signaling Technology (Danvers, MA, USA). IR spectra were measured by the Perkin-Elmer Spectrum One FT-IR spectrometer (as KBr pieces; in cm −1 ) (Perkin-Elmer, Boston, MA, USA). 1 H NMR and 13 C NMR spectra were recorded on a JEOL spectrometer (400 MHz) using CDCl3 or CD3OD as solvents with an internal standard of tetramethylsilane. HRMS was recorded on a Thermo Scientific Q Exactive Plus Orbitrap liquid chromatograph triple quadrupole mass spectrometer (LC-MS/MS). All prepared compounds were purified to >96% purity as determined through HPLC (Thermo Scientific Dionex Ultimate 3000, Waltham, MA, USA) analysis using the following methods. A SuperLu C18(2) (particle size = 5 μm, pore size = 4.6 nm, dimensions = 250 mm) column was used, the injection volume was 10 μL, and the mobile phase consisted of methyl alcohol and 0.1% trimethylamine (85:15) with a gradient elution at a flow rate of 1.0 mL/min. Each analysis lasted for 20 min. The detection wavelength was 280 nm. The retention times (RT,HPLC) and purity data (%) are listed in the analytical data of each compound.

Instruments and Materials
All the reagents and chemicals were purchased from commercial sources. Bicinchoninic acid (BCA) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-B-cell lymphoma 2 (Bcl-2) antibody, anti-Bcl-2 associated X protein (Bax) antibody, cysteine protease-3 antibody (caspase3), and β-actin antibody were purchased from ProteinTech (Wuhan, China). Anti-Beclin-1 and anti-p62/SQSTM1 (P62) were purchased from Abcam (Cambridge, ENG), and anti-microtubule-associated protein 1 light chain 3 (LC3) was purchased from Cell Signaling Technology (Danvers, MA, USA). IR spectra were measured by the Perkin-Elmer Spectrum One FT-IR spectrometer (as KBr pieces; in cm −1 ) (Perkin-Elmer, Boston, MA, USA). 1 H NMR and 13 C NMR spectra were recorded on a JEOL spectrometer (400 MHz) using CDCl 3 or CD 3 OD as solvents with an internal standard of tetramethylsilane. HRMS was recorded on a Thermo Scientific Q Exactive Plus Orbitrap liquid chromatograph triple quadrupole mass spectrometer (LC-MS/MS). All prepared compounds were purified to >96% purity as determined through HPLC (Thermo Scientific Dionex Ultimate 3000, Waltham, MA, USA) analysis using the following methods. A SuperLu C18(2) (particle size = 5 µm, pore size = 4.6 nm, dimensions = 250 mm) column was used, the injection volume was 10 µL, and the mobile phase consisted of methyl alcohol and 0.1% trimethylamine (85:15) with a gradient elution at a flow rate of 1.0 mL/min. Each analysis lasted for 20 min. The detection wavelength was 280 nm. The retention times (R T,HPLC ) and purity data (%) are listed in the analytical data of each compound.

Methods of Synthesis
3.2.1. General Procedure for the Preparation of 14-Nitrotetrandrine (1) The synthesis of compound 1 followed the reported method [69]. Concentrated HNO 3 (0.6 mL, 9.6 mmol) was slowly added dropwise to a solution of (CH 3 CO) 2 O (1.5 mL, 16.0 mmol) at -10 • C under the protection of a nitrogen atmosphere. Then, 10 min later, the reaction mixture was warmed up to 0 • C and stirred for 20 min, and TET (0.5 g, 0.8 mmol) dissolved in CH 2 Cl 2 (20 mL) was added dropwise. Upon completion, the mixture was quenched with water (50 mL), extracted with CH 2 Cl 2 (50 mL × 3), dried over by anhydrous magnesium sulfate, and filtered. The solvent was concentrated under reduced pressure, and the syrup was purified by silica gel chromatography from CH 2 Cl 2 /MeOH (60/1 v/v) to provide compound 1, with a yield of 94%. (2) The preparation of compound 2 was performed according to the reported literature [33]. Compound 1 (1.0 g, 1.5 mmol) was added to a mixture of SnCl 2 •2H 2 O (1.70 g, 7.5 mmol) in EtOAc (50 mL), and the reaction was stirred at 80 • C for 4 h. The mixture was then cooled to room temperature, brought to a pH of 8 by adding anhydrous sodium carbonate, and concentrated under reduced pressure to provide the crude product. The residue was purified by silica gel chromatography from CH 2 Cl 2 /MeOH (50/1 v/v, 0.1% TEA) to afford compound 2, with a yield of 45%.

Aqueous Solubility Determination
The water-solubility of the title compounds was measured according to the reported procedure by reversed-phase HPLC (column: Superlu C 18, 250 × 4.6 mm, 5 µM; isocratic elution with a mobile phase of 85% CH 3 OH/15% H 2 O; injection volume, 20 µL; flow rate: 1.0 mL/min; column temperature, 30 • C; UV detection at the wavelength of the maximum absorbance of each compound) [51]. Briefly, each tested compound (10 mg) was dissolved in 10 mL of CH 3 OH, and the standard solution (0.5 mL) was used to determine the wavelength of maximum absorbance by HPLC. The solution was diluted as necessary, and the solubility was determined by linear regression with R 2 > 0.999. Each tested compound (5 mg) was ultrasound dissolved in 3 mL of pure water for 1 h at room temperature, and the mixture was then concentrated at 3000 rpm for 15 min. The saturated supernatants were determined by HPLC at the wavelength of the maximum absorption, and the absorbances were obtained. The solubility was calculated according to the standard curve.

Colony Formation Assay
HCT-15 cells were diluted in 2 mL of culture medium and plated in six-well plates at 37 • C in 5% CO 2 . After an overnight incubation, compound 16 (0.625, 1.25, 2.5, 5, and 10 µM) was added, and the cells were cultured for 10 d. Afterwards, the medium was replaced. The cells were washed by cold PBS and fixed with 4% paraformaldehyde. Finally, the cell colonies were stained with a 1% crystal violet solution for 20 min. The plates were recorded, and the colonies were counted digitally using ImageJ software with customized macros.

Wound Healing Assays
HCT-15 cells or HUVECs were seeded in six-well culture plates and allowed to grow to 80-90% confluence. Subsequently, a cell-free line was manually created by scratching the confluent cell monolayers with a 200 µL sterile pipette tip, and the detached cells were washed with PBS. The cells were then incubated with 10% FBS and different concentrations of 16 (0.625, 1.25, 2.5, 5, and 10 µM). Then, 24 h later, images of the same location were obtained using a microscope (Nikon ECLIPSE TE2000-U, Tokyo, Japan). Each experiment was carried out at least three times.

Invasion Assay
The cell motility inhibitory effect of 16 on HCT-15 cells was evaluated by invasion assay. Briefly, cells were harvested and resuspended in serum-free medium that contained 0.625, 1.25, 2.5, 5, and 10 µM of 16, and were seeded into the upper wells of a transwell chamber (Millicell, 8 mm pore size, 12-mm diameter Millipore) coated with 50 µL of Matrigel (1:3 dilution in serum-free medium, Corning/BD Biosciences) at a density of 2 × 10 5 cells/mL. Meanwhile, 600 µL medium containing 10% FBS and corresponding samples were added into the lower chambers. Then, 20 h later, the invading cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet for 30 min. Afterwards, the chambers were washed twice with PBS and left to dry. Cells which had migrated to the lower chamber of the transwell were photographed using a digital camera with an inverted microscope (Nikon ECLIPSE TE2000-U, Tokyo, Japan), and the crystal violet positive cells were counted.

F-Actin Phalloidin Staining
The effect of compound 16 on the morphology of HCT-15 cells was evaluated by F-actin staining. Briefly, the cancer cells were seeded in 6-well plates with glass bottoms overnight at 37 • C and were incubated with 0.625, 1.25, 2.5, 5, 10 µM of 16 for 24 h. Then, the medium was removed, and the cells were fixed in 4% paraformaldehyde for 5 min and washed thrice with PBS before staining. A solution (200 µL) of 100 nM in MeOH FITC-phalloidin (Molecular Probes, Eugene, OR, USA) containing 1% BSA was added and treated for 30 min in total darkness. Afterwards, the cells were washed thrice with PBS and stained with 200 µL of DAPI (10 mg/mL, Solarbio Science & Technology, Beijing, China) for 5 min at 37 • C. Photos were acquired by a Nikon ECLIPSE TE2000-U inverted epifluorescent microscope using appropriate filters.

Live/Dead Cell Analysis
HCT-15 or HUVEC cells were cultured in six-well plates for 24 h. The cells were washed with PBS and incubated with medium containing 0.625, 1.25, 2.5, 5, and 10 µM of 16 for 48 h. Afterwards, the medium was replaced, and the cells were washed twice with PBS, resuspended in the solution containing 100 µL FDA (0.02 mg/mL, Sigma, St. Louis, MO, USA) and 30 µL PI (0.02 mg/mL, Sigma, St. Louis, MO, USA), and treated in the dark at room temperature for 10 min. The cells were imaged with a fluorescence microscope (Nikon ECLIPSE TE2000-U, Tokyo, Japan).

Hoechst 33258 Staining
HCT-15 cells were cultured in six-well plates for 24 h. Then, the cells were incubated with medium containing different concentrations of 16 (0.625, 1.25, 2.5, 5, 10 µM) for 48 h. After incubation, the medium was removed, and the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, stained in 200 µL of buffer Hoechst 33258 (10 mg/mL, Sigma, St. Louis, MO, USA), and incubated in the dark at room temperature for 10 min. The cells were then imaged using a fluorescence microscope.

Flow Cytometry Analysis
HCT-15 cells were cultured in complete medium in six-well plates for 24 h, and 1.5. 2.5, 5 µM of 16 was added in triplicate for 48 h incubation. The cells were digested with trypsin without EDTA, washed twice with cold PBS, and resuspended in 500 µL binding buffer. The cell suspension was stained successively with 5 µL Annexin V-FITC and 5 µL PI (Keygen Biotech, Nanjing, China), and cultured for 15 min and 5 min in total dark, respectively. Afterwards, the percentages of apoptotic cells were determined by a flow cytometry. Data were analyzed by FlowJo software (Version 10, FlowJo, LLC, Ashland, OR, USA).

Cell Cycle Analysis
HCT-15 cells were cultured in six-well plates overnight at 37 • C. The cells were treated with 1.25, 2.5, 5 µM of 16 or DMSO and incubated for 24 h. Afterwards, the cells were harvested, and 70% ice cold ethanol was used to fix cells at 4 • C. Subsequently, the cells were centrifuged to remove the fixative solution, washed twice with PBS, and exposed to 500 µL of PI/RNase working solution (Keygen Biotech, Nanjing, China) at 4 • C for 30 min in the dark. The cell cycle distribution was analyzed by flow cytometry.

Molecular Docking
PKC-α was chosen as the target receptor, and the 3D structure of the receptor was obtained from the Protein Data Bank (PDB ID: 4RA4). The ligand (16) was prepared for docking as described in our previous work [71]. Docking studies were performed with the AutoDock (4.2) program suite. The 3D affinity map was a cube with 60 Å × 60 Å × 60 Å grid points separated by 0.375 Å, and the docking parameters were identical to our previous work [71]. The resulting docked orientations within a root mean square deviation of 2 Å were clustered together.

Tube Formation Assay
Briefly, a 96-well plate was pre-coated with 50 µL Matrigel per well and incubated at 37 • C for 30 min. Then, 100 µL of HUVECs suspension (2 × 10 4 ) were added to each well. The cells were cultured with 1.25, 2.5, and 5 µM of 16 for 8 h. Subsequently, the tube formation was observed, randomly selected, and recorded by an inverted microscope.

Statistical Analysis
The experimental results were collected from at least three independent experiments and expressed as mean ± SEM. Data were tested for normal distribution (Kolmogorov-Smirnoff test) and subsequently analyzed with one-way ANOVA and t-test using GraphPad Prism 8.0, with p < 0.05 indicating a significant difference.

Conclusions
In summary, a group of 14-N-amino acid-substituted derivatives of TET were designed and synthesized, with improved aqueous solubility and anticancer activity compared to TET. Among them, compound 16 exhibited an outstanding antiproliferative activity against HCT-15 cells, with an IC 50 of 0.57 µM. Moreover, the aqueous solubility of 16 was markedly improved to higher than 5-fold over TET. Compound 16 significantly inhibited the colony formation, migration, and invasion of HCT-15 cells in a concentration-dependent manner. The flow cytometry assay demonstrated that 16 induced the death of HCT-15 cells; however, no significant effect on the cell cycle redistribution was observed for this compound. Further Western blot studies revealed that 16 induced the death of cancer cells through autophagy rather than apoptosis. Compound 16 markedly inhibited the content of P62 and enhanced the Beclin-1 level and the ratio of LC3-II to LC3-I. In addition, the proliferation, migration, and tube formation of HUVECs were significantly inhibited by 16, suggesting strong antiangiogenesis activity. Thus, all these results indicated the potential of 16 as a promising anticancer candidate for further preclinical studies.