Design, Synthesis and Structure-Activity Relationship Studies of Meridianin Derivatives as Novel JAK/STAT3 Signaling Inhibitors

Hyperactivation of Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling is an attractive therapeutic target for tumor therapy. Herein, forty-eight novel meridianin derivatives were designed and synthesized, and their antitumor activity was evaluated in vitro both for activity optimization and structure–activity relationship (SAR) study. The results indicated that most derivatives exhibited significantly improved antitumor activity, especially for compound 6e. The compound 6e contains an isothiouronium linked by an alkyl chain consisting of six carbon atoms with IC50 ranging from 1.11 to 2.80 μM on various cancer cell lines. Consistently, the 6e dose dependently induced the apoptosis of A549 and DU145 cells, in which STAT3 is constitutively active. Western blotting assays indicated that the phosphorylation levels of JAK1, JAK2 and STAT3 were inhibited by 6e at 5 μM without significant change in the total STAT3 level. Moreover, 6e also suppressed the expression of STAT3 downstream genes, including c-Myc, Cyclin D1 and Bcl-XL at 10 μM. An additional in vivo study revealed that 6e at the dose of 10 mg/kg could potently inhibit the DU145 xenograft tumor without obvious body weight loss. These results clearly indicate that 6e could be a potential antitumor agent by targeting the JAK/STAT3 signaling pathway.


Introduction
The Janus kinase (JAK) and Signal transducer and activator of transcription (STAT) signaling pathway is essential in the regulation of various biological processes, including immune responses, cell division, hematopoiesis and tumor formation [1][2][3][4]. Various cytokines and growth factors transmit signals through the JAK/STAT signaling pathway, which consists of tyrosine kinase-associated receptors, JAKs and downstream transcription factor STATs [5,6]. After stimulated by cytokines, such as type I and type II interferons or IL-6, the JAKs phosphorylate each other at tyrosine residues and then phosphorylate and activate STAT proteins, which themselves dimerize and translocate to the nucleus, where they regulate gene transcription. Aberrant activation of the JAK/STAT signaling pathway has been closely associated with many diseases. Four members of the JAK family have been identified in mammals, including JAK1, JAK2, JAK3 and TYK2. The mammalian STAT family has seven members, comprising STAT1-4, STAT5(a/b) and STAT6 [1]. Among The reaction sequence employed to synthesize the target compounds is outlined in Schemes 1-4.
The marine natural products indole alkaloids (meridianin A, C, D and G) were synthesized in four steps starting from commercially available indoles [27]. Firstly, the indolic nitrogen was protected by the reaction with tosyl chloride in the presence of NaOH and in acetonitrile, leading to the formation of compounds 2, 17 and 21 in 53-94% yields. Then, the C-3 position of indoles was acetylated using acetic anhydride and aluminum chloride inmethylene chloride to give derivatives 3, 18 and 22 in 73-79% yields. The enaminone intermediates proceeded with DMF/dimethylformamide-dimethylacetal (DMF-DMA) in 69-79% yield. Finally, compounds 1a-g, 3a-g and 4a-g were obtained from enaminone intermediates using 5-11 in 2-methoxyethanol in the presence of potassium carbonate in considerable yields (Scheme 1). As depicted in Scheme 2, the indolic nitrogen and 4-hydroxyl of compound 12 were tosyl chloride-protected to get compounds 13 in 85% yield. Then, the preparation of the corresponding 2a-g derivatives was undertaken using a similar synthetic pathway in 38-66% yields. As shown in Schemes 3 and 4, isothiouronium derivatives 5a-g, 6a-g and 6e-2-6 were synthesized in medium yield by introducing

Cell Viability Assay and SAR Analysis
To evaluate the antitumor activities of meridianin A, C, D, G and their derivatives, four JAK/STAT3 overactivated human cancer cell lines: HeLa, MDA-MB-231, A549 and DU145 were examined. Initially, the meridianins (A, C, D and G) and their derivatives 1a-g, 2a-g, 3a-g and 4a-g were prepared and evaluated for cell growth inhibitory activities against the cancer cell lines ( Table 1). The results indicated that meridianins A, C, D and G displayed weak cancer cell growth inhibition in the four tested cell lines. By comparison, meridianin derivatives 1a-g, 2a-g, 3a-g and 4a-g showed significantly increased inhibitory activities. With the aim of improving the antitumor activity, compounds 5a-g and 6a-g were obtained by incorporating isothiourea groups at the C1 position of meridianins D and G with different lengths of carbon alkyl chains. Notably, the antitumor effects of the compounds were significantly enhanced, almost all compounds had a IC50 less than 10 μM (Table 2). Among them, the most potent compound 6e inhibited the growth of HeLa, MDA-MB-231, A549 and DU145 cells with IC50 values of 1.11, 1.22, 2.80 and 1.13 μM, which exhibited better activity than the positive control (Gefitinib), respectively. Based on the above results, the analogs of compound 6e with different carbon chain lengths or without isothiourea were obtained, and the antitumor activity results are shown in Table 3. The structure-activity relationship suggests that reducing or increasing

Biological Activity Assessments 2.2.1. Cell Viability Assay and SAR Analysis
To evaluate the antitumor activities of meridianin A, C, D, G and their derivatives, four JAK/STAT3 overactivated human cancer cell lines: HeLa, MDA-MB-231, A549 and DU145 were examined. Initially, the meridianins (A, C, D and G) and their derivatives 1a-g, 2a-g, 3a-g and 4a-g were prepared and evaluated for cell growth inhibitory activities against the cancer cell lines ( Table 1). The results indicated that meridianins A, C, D and G displayed weak cancer cell growth inhibition in the four tested cell lines. By comparison, meridianin derivatives 1a-g, 2a-g, 3a-g and 4a-g showed significantly increased inhibitory activities. With the aim of improving the antitumor activity, compounds 5a-g and 6a-g were obtained by incorporating isothiourea groups at the C1 position of meridianins D and G with different lengths of carbon alkyl chains. Notably, the antitumor effects of the compounds were significantly enhanced, almost all compounds had a IC 50 less than 10 µM (Table 2). Among them, the most potent compound 6e inhibited the growth of HeLa, MDA-MB-231, A549 and DU145 cells with IC 50 values of 1.11, 1.22, 2.80 and 1.13 µM, which exhibited better activity than the positive control (Gefitinib), respectively. Based on the above results, the analogs of compound 6e with different carbon chain lengths or without isothiourea were obtained, and the antitumor activity results are shown in Table 3. The structure-activity relationship suggests that reducing or increasing the number of carbon atoms will lead to decreased antiproliferative activity. On the other hand, compound 6e-1 was found to be completely inactive with IC 50 values greater than 100 µM, indicating that the substitution of the isothiourea group significantly contributed to the antitumor activity. The inhibitory effect of compound 6e on the proliferation of the four normal cell lines HUVEC, L02, L929 and MCF10A was determined by the MTT assay. The data showed that 6e had low toxicity compared to normal cells, predicting that it may be relatively safe in vivo (Table 4).

Compound 6e Inhibited Cancer Cell Proliferation and Induced Cell Apoptosis
To evaluate the antiproliferative activities of compound 6e in the cell models, its effects on DU145 and A549 colony survival were evaluated. The results of the CFA analysis showed that 6e significantly inhibited the proliferation of both cells, and the effect was enhanced with the increasing 6e concentration. The effect of compound 6e on inducing tumor cell apoptosis was analyzed in Figure 1B. A549 and DU145 cells were incubated with 6e at different concentrations for 24 h. Annexin V-FITC/PI staining was carried out, and the percentage of apoptotic cells was further determined using flow cytometry. The results showed that the 6e dose dependently induced the apoptosis of the A549 and DU145 cells. As shown in Figure 1B, in A549, the induced apoptosis rates at 0, 1, 5 and 10 µM were 2.59%, 4.30%, 14.78% and 45.40%, respectively. In DU145, the induced apoptosis rates at 0, 1, 5 and 10 µM were 2.10%, 5.19%, 5.32% and 19.25%, respectively.

Molecular Docking
Molecular docking was performed for understanding the interaction mechanisms between compound 6e with JAK1 (PDB ID:4I5C), JAK2 (PDB ID:5CF5) and STAT3 (PDB ID:1BG1) [28], respectively. For JAK1, the results showed that the hydrophobic fatty chain was accommodated at a hydrophobic pocket mainly defined by residues Leu881, Val889, Ala906, Val938, Phe958, Leu959 and Leu1010 (Figure 2A). The hydrogen in the imine of 6e engages H-bond formation with Glu957 and Gly1020. For JAK2, the docking poses suggested that the imine group of 6e interacts with a carboxyl group of Asp994 and carbonyl group of Gly861 by forming two hydrogen bonds ( Figure 2B). In addition, the nitrogen of  The ability of compound 6e to inhibit the phosphorylation of JAK/STAT3 was determined in A549 and DU145 cells. As shown in Figure 3A,B, after 24 h of treatment with 5-µM 6e, the decreased levels of JAK1, JAK2 and STAT3 were observed in both A549 and DU145 cells, but no significant change was seen with the total STAT3 protein expression. Moreover, compound 6e significantly inhibited the expression of JAK/STAT3 downstream genes c-Myc, Cyclin D1 and Bcl-XL at 10 µM after 24 h of treatment ( Figure 3C). Therefore, pretreatment with compound 6e suppressed the JAK/STAT3 signaling pathway and its downstream gene expressions, which were consistent with the above results obtained in vitro. and the percentage of apoptotic cells was further determined using flow cytometry. The results showed that the 6e dose dependently induced the apoptosis of the A549 and DU145 cells. As shown in Figure 1B, in A549, the induced apoptosis rates at 0, 1, 5 and 10 μM were 2.59%, 4.30%, 14.78% and 45.40%, respectively. In DU145, the induced apoptosis rates at 0, 1, 5 and 10 μM were 2.10%, 5.19%, 5.32% and 19.25%, respectively. Compound 6e induced A549 and DU145 cancer cells apoptosis in vitro. A549 and DU145 cells were incubated with 6e at different concentrations (0-10 μM) for 24 h. Annexin V/PI staining was carried out, and the percentage of apoptotic cells was further determined using flow cytometry. * Statistically significant (n = 2, p < 0.05).

Molecular Docking
Molecular docking was performed for understanding the interaction mechanisms between compound 6e with JAK1 (PDB ID:4I5C), JAK2 (PDB ID:5CF5) and STAT3 (PDB ID:1BG1) [28], respectively. For JAK1, the results showed that the hydrophobic fatty chain was accommodated at a hydrophobic pocket mainly defined by residues Leu881, Val889, Ala906, Val938, Phe958, Leu959 and Leu1010 (Figure 2A). The hydrogen in the imine of 6e engages H-bond formation with Glu957 and Gly1020. For JAK2, the docking poses suggested that the imine group of 6e interacts with a carboxyl group of Asp994 and carbonyl group of Gly861 by forming two hydrogen bonds ( Figure 2B). In addition, the nitrogen of imine on 6e also forms a salt bridge interaction with Asp994. While, for STAT3, Phe710 forms π-π interactions with the indole ring of 6e, Glu652 forms a hydrogen bond and a salt bridge with the H atom and the N atom on imine, respectively ( Figure 2C) [29].

Compound 6e Inhibits the Expression of JAK/STAT3 Target Genes
The ability of compound 6e to inhibit the phosphorylation of JAK/STAT3 was determined in A549 and DU145 cells. As shown in Figure 3A,B, after 24 h of treatment with 5-μM 6e, the decreased levels of JAK1, JAK2 and STAT3 were observed in both A549 and DU145 cells, but no significant change was seen with the total STAT3 protein expression. Moreover, compound 6e significantly inhibited the expression of JAK/STAT3 downstream genes c-Myc, Cyclin D1 and Bcl-XL at 10 μM after 24 h of treatment ( Figure 3C). Therefore, pretreatment with compound 6e suppressed the JAK/STAT3 signaling pathway and its downstream gene expressions, which were consistent with the above results obtained in vitro.

Compound 6e Inhibits the Expression of JAK/STAT3 Target Genes
The ability of compound 6e to inhibit the phosphorylation of JAK/STAT3 was determined in A549 and DU145 cells. As shown in Figure 3A,B, after 24 h of treatment with 5-μM 6e, the decreased levels of JAK1, JAK2 and STAT3 were observed in both A549 and DU145 cells, but no significant change was seen with the total STAT3 protein expression. Moreover, compound 6e significantly inhibited the expression of JAK/STAT3 downstream genes c-Myc, Cyclin D1 and Bcl-XL at 10 μM after 24 h of treatment ( Figure 3C). Therefore, pretreatment with compound 6e suppressed the JAK/STAT3 signaling pathway and its downstream gene expressions, which were consistent with the above results obtained in vitro. To further investigate the antitumor potential in vivo, we evaluated the effects of compound 6e in a nude mice tumor model ( Figure 4). After the solid tumor was established, twenty-four nude mice were randomly divided into four groups, which were the vehicle control group, 6e groups (5 mg/kg and 10 mg/kg) and Gefitinib-positive control group. All the compounds were taken by intragastric gavage. Compound 6e at the dose

Compound 6e Inhibited Tumor Growth in a Mouse Breast Cancer Model
To further investigate the antitumor potential in vivo, we evaluated the effects of compound 6e in a nude mice tumor model ( Figure 4). After the solid tumor was established, twenty-four nude mice were randomly divided into four groups, which were the vehicle control group, 6e groups (5 mg/kg and 10 mg/kg) and Gefitinib-positive control group. All the compounds were taken by intragastric gavage. Compound 6e at the dose of 10 mg/kg could significantly inhibit tumor growth, and the tumor inhibition rate of 6e was over 40%, which was comparable to that of the positive control ( Figure 4A-C). Subsequently, Ki67 and Tunel staining were performed on tumor sections, which showed that tumor proliferation marker Ki67 was significantly inhibited, and the proportion of apoptotic cells that were marked by Tunel-positive staining also significantly increased with 6e treatment at a dose of 10 mg/kg ( Figure 4D,E). Moreover, during the administration period, the weight of the nude mice did not increase or decrease significantly, indicating that there was no obvious biological toxicity of 6e ( Figure 4F). H&E staining was performed on the liver and kidneys of each group of mice to observe the hepatic and renal toxicity of 6e. The results showed that no significant liver and kidney damage was observed in all the groups of mice, suggesting that 6e was less toxic at therapeutic doses ( Figure 4G). 6e treatment at a dose of 10 mg/kg ( Figure 4D,E). Moreover, during the administration period, the weight of the nude mice did not increase or decrease significantly, indicating that there was no obvious biological toxicity of 6e ( Figure 4F). H&E staining was performed on the liver and kidneys of each group of mice to observe the hepatic and renal toxicity of 6e. The results showed that no significant liver and kidney damage was observed in all the groups of mice, suggesting that 6e was less toxic at therapeutic doses ( Figure 4G).

Immunohistochemical (IHC) Analysis
To further test the inhibitory effects of 6e on JAK/STAT3 signaling, the IHC analysis of nude mice inoculated with DU145 tumor cells was performed. As shown in Figure 5, after 6e treatment, the intratumoral staining of p-STAT3, cyclin D1 and c-Myc in DU145-

Immunohistochemical (IHC) Analysis
To further test the inhibitory effects of 6e on JAK/STAT3 signaling, the IHC analysis of nude mice inoculated with DU145 tumor cells was performed. As shown in Figure 5, after 6e treatment, the intratumoral staining of p-STAT3, cyclin D1 and c-Myc in DU145inoculated mice was significantly lower than those in the NC group, and their staining levels decreased while the dose of 6e increased. Therefore, compound 6e may exert antitumor effects by inhibiting the JAK/STAT3 signaling pathway both in vitro and in vivo.

Conclusions
In summary, a novel series of meridianin derivatives was obtained and biologically evaluated. Initially, the meridianins (A, C, D and G) and their four series derivatives of compounds 1a-g, 2a-g, 3a-g and 4a-g were prepared, and the results indicated that the meridianins (A, C, D and G) displayed weak inhibitory activity on four JAK/STAT3 overactivated human cancer cell lines: HeLa, MDA-MB-231, A549 and DU145, whereas most of the meridianin derivatives exerted promising inhibitory activity on the tested cell lines. To improve the antitumor activity, meridianin derivatives 5a-g and 6a-g were designed and synthesized by incorporating isothiourea groups at the N1 position with different lengths of carbon alkyl chains. Surprisingly, the antitumor effects of the isothiouroniummodified compounds were significantly enhanced, with IC50 less than 10 μM. Among them, the most potent compound, 6e with an alkyl chain of six carbon atoms, had an IC50 that ranged from 1.11 to 2.80 μM in various cancer cell lines, which was superior to the positive control, Gefitinib. The structure-activity relationship (SAR) study indicated that isothiouronium modified by N-alkylation with 6C alkyl chains may contribute the most

Conclusions
In summary, a novel series of meridianin derivatives was obtained and biologically evaluated. Initially, the meridianins (A, C, D and G) and their four series derivatives of compounds 1a-g, 2a-g, 3a-g and 4a-g were prepared, and the results indicated that the meridianins (A, C, D and G) displayed weak inhibitory activity on four JAK/STAT3 overactivated human cancer cell lines: HeLa, MDA-MB-231, A549 and DU145, whereas most of the meridianin derivatives exerted promising inhibitory activity on the tested cell lines. To improve the antitumor activity, meridianin derivatives 5a-g and 6a-g were designed and synthesized by incorporating isothiourea groups at the N1 position with different lengths of carbon alkyl chains. Surprisingly, the antitumor effects of the isothiouronium-modified compounds were significantly enhanced, with IC 50 less than 10 µM. Among them, the most potent compound, 6e with an alkyl chain of six carbon atoms, had an IC 50 that ranged from 1.11 to 2.80 µM in various cancer cell lines, which was superior to the positive control, Gefitinib. The structure-activity relationship (SAR) study indicated that isothiouronium modified by N-alkylation with 6C alkyl chains may contribute the most to antitumor activity. It is worth noting that 6e had low toxicity to normal cells. The Western blotting assays suggested that treatment with compound 6e could decrease the phosphorylation level of JAK1, JAK2 and STAT3 at 5 µM but did not affect the total STAT3 level. Moreover, 6e also suppressed the expression of STAT3 downstream genes, including c-Myc, cyclin D1 and Bcl-XL. Consistently, 6e dose-dependently inhibited the proliferation and induced the apoptosis of A549 and DU145 cells. Molecular docking studies demonstrated that an H-bond is the main type of interaction between compound 6e and the JAK1/JAK2 kinases, and 6e could also tightly bind to the STAT3 SH2 domain. An additional in vivo study revealed that the application of 6e at a dose of 10 mg/kg could significantly inhibit the DU145 xenograft tumor growth without an obvious body weight loss, which was comparable to that of the positive control. Taken together, these results clearly indicated that 6e could be a highly potent antitumor agent by targeting the JAK/STAT3 signaling pathway. In addition, the pharmacokinetic properties of compound 6e will be further investigated in the future.

Chemistry
All commercially available starting materials and solvents were purchased from commercial vendors and used without further purification. Reactions were monitored using analytical thin-layer chromatography (TLC) on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, China) and visualized under ultraviolet light (254 nm and 365 nm). Column chromatography was performed on silica gel (200-300 mesh). Melting points were determined on a Mitamura-Riken micro-hot stage and uncorrected. 1 H and 13 C NMR spectra were recorded on the Broker AVANCE NEO and Agilent DD2 500 with 400 or 500 MHz for proton ( 1 H NMR) and 100 or 125 MHz for carbon ( 13 C NMR), respectively. The chemical shifts (δ) were expressed in parts per million (ppm) downfield, and the coupling constant (J) values were described as hertz. High-resolution (ESI) MS spectra were recorded using a QTOF-2 Micromass spectrometer. The purity of the final compounds for biological evaluation was higher than 95% by analytical HPLC analysis with the Primaide 1210 system. To the solution of intermediates 4, 19 and 23 (1.0 equiv.) in 2-methoxyethanol (5 mL) was added 5-11 (2.5 equiv.) and potassium carbonate (2.0 equiv.), respectively. The reaction mixture was stirred at 120 • C for 20 h under a nitrogen atmosphere. Then, the mixture was poured into ice water and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine and dried over magnesium sulfate anhydrous. After filtration, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) to give the final target compounds 1a-g, 3a-g and 4a-g.  13 163.45, 162.20, 160.00,  157.16, 139.90, 138.58, 130.96, 130.26, 124.71, 124.30, 123.76, 120.49, 117.11, 115.47, 115.

General Procedure for Synthesis of 2a-g Meridianin Analogs
To a stirring solution of 4-hydroxyindole (12) in dry DMF (10 mL) was added sodium hydride (5.0 equiv.) at 0 • C, and the mixture was stirred for 30 min. Then, p-toluenesulfonyl (3.0 equiv.) was added. After stirring for 4 h at room temperature. the reaction was quenched with saturated NaHCO 3 solution and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine and dried over magnesium sulfate anhydrous. After filtration, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) to give compound 13.
1-tosyl-1H-indol-4-yl 4-methylbenzenesulfonate (13) To a stirring solution of acetic anhydride (2.0 equiv.) in dry dichloromethane (8 mL) was added aluminum chloride (5.0 equiv.) at 0 • C. Then, compound 13 in dry dichloromethane (8 mL) was added dropwise, and the mixture was stirred for 2 h at room temperature. The reaction was quenched with saturated aqueous NH 4 Cl and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine and dried over magnesium sulfate anhydrous. After filtration, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 8:1) to give compound 14.
3-acetyl-1-tosyl-1H-indol-4-yl 4-methylbenzenesulfonate (14).  To a solution of compound 14 in DMF (5 mL) was added DMF-DMA (1.5 equiv.). The reaction mixture was stirred at 110 • C for 5 h under a nitrogen atmosphere. Then, the mixture was poured into ice water and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine and dried over magnesium sulfate anhydrous. After filtration, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 2:1) to give intermediate 15.  To a solution of intermediate 15 in 2-methoxyethanol (5 mL) was added 5-11 (2.5 equiv.) and potassium carbonate (2.0 equiv.), respectively. The reaction mixture was stirred at 120 • C for 20 h under a nitrogen atmosphere. Then, the mixture was poured into ice water and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine and dried over magnesium sulfate anhydrous. After filtration, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) to give get the final target compounds 2a-g.  163.34, 161.31, 160.10, 157.75, 152.13, 139.92, 138.74, 130.73, 130.52, 125.24, 120.57, 117.21,  114.70, 113.50, 113.47, 106.46, 103.47    To the solution of 1a-g in DMF (5 mL) was added 1,6-dibromohexane (5.0 equiv.), and the mixture was stirred at 50 • C for 5 h. Then, the reaction mixture was removed under vacuum, and the residue was poured into ice water and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine, dried over magnesium sulfate anhydrous and concentrated to give intermediates 23-29 and used in the next step without further purification. To a stirring solution of compounds 23-29 in ethanol was added thiocarbamide (2.0 equiv.), and the mixture was stirred at 65 • C for 3 h. Then, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (dichloromethane/methanol 10:1) to give the final target compounds 5a-g.  To the solution of 4a-g in DMF (5 mL) was added 1,6-dibromohexane (5.0 equiv.), and the mixture was stirred at 50 • C for 5 h. Then, the reaction mixture was removed under vacuum, and the residue was poured into ice water and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine, dried over magnesium sulfate anhydrous and concentrated to give intermediates 30-36 and used in the next step without further purification. To a stirring solution of compounds 30-36 in ethanol was added thiocarbamide (2.0 equiv.), and the mixture was stirred at 65 • C for 3 h. Then, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (dichloromethane/methanol 10:1) to give the final target compounds 6a-g.
In vitro inhibitory activity. The resazurin indicator was used to evaluate the cell viability. HeLa, MDA-MB-231, A549, DU145, HUVEC, L02, L929 and MCF10A cells were seeded in 96-well plates in 50 µL at plating densities ranging from 4000 to 8000 cells/well, depending on the doubling time of individual cell lines. After incubation for 24 h, different concentrations of compounds were added, and then, the cells were further cultured for 72 h with 0.5% DMSO as the solvent control group, and 10 µL of resazurin solution (1 mg/mL) was directly added to each well as a redox indicator. Plates were incubated for 3 h to measure the absorbance of a SpectraMax@i3 (Molecular Devices, Madison, WI, USA) of each well at a 595-nm emission wavelength (549-nm excitation wavelength). Each treatment was performed in triplicate to reduce the experimental error. Results were analyzed with GraphPad Prism 6, and the data were shown as the mean ± SD. Molecular Docking. All calculations were performed using the Molecular Docking program of MOE (version MOE 2020.09). The crystal structures of the proteins involved in this article were retrieved from the Protein Data Bank (PDB). Firstly, all compounds were treated through energy minimization. The parameters and charges were assigned with the MMFF94x force field. Secondly, after removing water molecules, each selected protein structure was treated by adding hydrogen atoms. Finally, the small molecules were docked into the pockets of the proteins defined by the originally bound ligands in the crystal structures, respectively. The poses were ranked by the scores from the GBVI/WSA-binding free energy calculations, and the results were analyzed using Pymol (1.8) (https://pymol.org/2/, accessed on 23 November 2021).
Colony formation assay. The colony formation assay was performed to examine the effect of compound 6e on cell colony survival. DU145 and A549 cells were seeded in 6-well plates with 500~1000 cells/well. The second day, various concentrations of 6e were added. After that, the cell culture medium was changed, and the corresponding concentration of 6e was added every 2 days until the colonies were visible. About 14 days later, the cells were fixed using 4% paraformaldehyde fix solution (Beyotime, Shanghai, China) and stained with crystal violet (Beyotime). Then, we observed and calculated the number of colonies.
Flow cytometry analysis of apoptotic cells. An Annexin V-FITC/PI apoptosis kit (Invitrogen) was used to detect cell apoptosis. A549 and DU145 cells were cells at a density of 5 × 10 5 per well cultured in regular growth medium in 6-well plates for 24 h and disposed induplicate with various concentrations of compound 6e for 24 h. After 48 h later, A549 and DU145 were trypsinized, centrifuged and washed with precooled PBS twice with an Annexin V-FITC/PI apoptosis kit (Invitrogen) following the manufacturer's instructions.
Western blot analysis. A549 and DU145 cells were plated in 6-well plates and cultured overnight, respectively, and different concentrations of compound 6e were added for 2 h. The corresponding cells were collected, washed with PBS and lysed with cell lysis buffer to extract the total proteins. The extracted protein was loaded and subjected to SDS-PAGE electrophoresis, and then, the protein was transferred to a PDVF membrane and incubated in the corresponding primary antibody overnight. The next day, the primary antibody was recovered and labeled, and the corresponding secondary antibody was incubated. The immune complexes were detected using chemiluminescence HRP substrate (Millipore) and visualized by the Tanon 5200 Chemiluminescence Imaging System (Biotanon, Shanghai, China).
In vivo studies. Six-week-old male nude mice (SPF degree, 17-20 g weight, nu/nu) were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Nude mice were injected into the back with DU145 tumor cells (about 15 × 10 6 ). After 2 weeks, the mice were randomly divided into four groups: blank control group (NC, DMSO), positive control Gefitinib group (PC, 100 mg/kg), compound 6e group (5 mg/kg) and compound 6e group (10 mg/kg), with 6 mice per group. The compound 6e groups and the PC group were intraperitoneally injected or intragastric-administered every two days until the mice were sacrificed. The body weights of the nude mice were recorded every three days, and the tumor weights were recorded on the day of death of the nude mice All of the procedures were approved by the Committee of Experimental Animals of the Ocean University of China and conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Immunohistochemistry (IHC) analyses. The mouse tumor tissues were collected, fixed in 4% PFA for 72 h at 4 • C, embedded in paraffin and cut into sections. The sections were deparaffinized in xylene, rehydrated in graded ethanol, boiled in antigen retrieval solution [31] and then incubated with fresh 3% H 2 O 2 to inactivate endogenous peroxidase. After PBS washing, the slides were blocked with fatty free milk and incubated with the primary antibody at 4 • C overnight, followed by incubation with the HRP-conjugated secondary antibody at room temperature (Boster, Wuhan, China), according to the manufacturer's instructions. Finally, DAB color developing solution was added dropwise. A brown color in the cell membrane indicated positive staining. Images were captured using an upright fluorescence microscope (Olympus BX53, Tokyo, Japan). For hematoxylin and eosin staining (H&E staining), the tumor sections were incubated in hematoxylin solution and then counterstained with eosin.
Statistical analysis. Data were reported as the mean ± SEM. Statistical analyses and significance as measured by repeated measures ANOVA (followed by Dunnett's posttest or Friedman test) were obtained using GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego, CA, USA). p < 0.05 was considered significant.