Design, Synthesis and Biological Evaluation of [1,2,4]Triazolo[1,5-a]pyrimidine Indole Derivatives against Gastric Cancer Cells MGC-803 via the Suppression of ERK Signaling Pathway

[1,2,4]Triazolo[1,5-a]pyrimidine and indole skeletons are widely used to design anticancer agents. Therefore, in this work, a series of [1,2,4]triazolo[1,5-a]pyrimidine indole derivatives were designed and synthesized by the molecular hybridization strategy. The antiproliferative activities of the target compounds H1–H18 against three human cancer cell lines, MGC-803, HCT-116 and MCF-7, were tested. Among them, compound H12 exhibited the most active antiproliferative activities against MGC-803, HCT-116 and MCF-7 cells, with IC50 values of 9.47, 9.58 and 13.1 μM, respectively, which were more potent than that of the positive drug 5-Fu. In addition, compound H12 could dose-dependently inhibit the growth and colony formation of MGC-803 cells. Compound H12 exhibited significant inhibitory effects on the ERK signaling pathway, resulting in the decreased phosphorylation levels of ERK1/2, c-Raf, MEK1/2 and AKT. Furthermore, compound 12 induced cell apoptosis and G2/M phase arrest, and regulated cell cycle-related and apoptosis-related proteins in MGC-803 cells. Taken together, we report here that [1,2,4]triazolo[1,5-a]pyrimidine indole derivatives, used as anticancer agents via the suppression of ERK signaling pathway and the most active compound, H12, might be a valuable hit compound for the development of anticancer agents.

Considering the important roles of [1,2,3]Triazolo[1,5-a]pyrimidine and indole skeletons in the development of anticancer agents, we linked an indole fragment to the [1,2,3]Triazolopyrimidine scaffold through the principle of molecular hybridization strategy, and then introduced amino fragments or benzothiazole groups to obtain novel [1,2,3]Triazolo[1,5a]pyrimidine indole derivatives ( Figure 3). Although surgery procedures and chemotherapy have improved greatly in recent years, it is highly desirable to develop newly targeted therapy for human cancers with minimal side-effects and new classes of anti-cancer agents with excellent selectivity between cancer and normal cells. Therefore, the antiproliferative activities of target compounds against three human cancer cell lines, MGC-803, HCT-116 and MCF-7, were tested, and the potential antitumor mechanisms of the most active compound were further explored.  Figure 3). Although surgery procedures and chemotherapy have improved greatly in recent years, it is highly desirable to develop newly targeted therapy for human cancers with minimal side-effects and new classes of anti-cancer agents with excellent selectivity between cancer and normal cells. Therefore, the antiproliferative activities of target compounds against three human cancer cell lines, MGC-803, HCT-116 and MCF-7, were tested, and the potential antitumor mechanisms of the most active compound were further explored.

Biological Evaluation
Antiproliferative Activities of Compounds H1-H18 The MTT assay was carried out to explore the in vitro antiproliferative activities of compounds H1-H18 against MGC-803 cells (human gastric cancer cells), HCT-116 cells (human colorectal carcinoma cells) and MCF-7 cells (human breast cancer cells) using 5-Fu as the positive drug. The following Table 1 summarized the results of the antiproliferative activities of compounds H1-H18.

Biological Evaluation
Antiproliferative Activities of Compounds H1-H18 The MTT assay was carried out to explore the in vitro antiproliferative activities of compounds H1-H18 against MGC-803 cells (human gastric cancer cells), HCT-116 cells (human colorectal carcinoma cells) and MCF-7 cells (human breast cancer cells) using 5-Fu as the positive drug. The following Table 1 summarized the results of the antiproliferative activities of compounds H1-H18.
As shown in Table 1, among the compounds H1-H18, compound H12 exhibited the most active antiproliferative activities against MGC-803, HCT-116 and MCF-7 cells, with IC50 values of 9.47, 9.58 and 13.1 μM, respectively, which were more potent than that of the positive drug 5-Fu. On the whole, most of compounds displayed moderate antiproliferative activities against three cancer cells, with IC50 values < 80 μM. The antiproliferative activities of compounds H1-H18 varied with its substituent groups of R. Additionally, most of compounds were more sensitive to HCT cells than MGC-803 and MCT-cells, excepting compounds H5, H9, H12 and H18. As for compounds H3-H11, the inhibitory activities varied with its substituent groups at phenyl groups of R. When the para position at phenyl of R were electron donating group ethyl substituent group (compound H5), its activity against HCT-116 cells was better than the para position at phenyl of R with electron withdrawing groups F (compound H6), Cl (compound H9) and Br (compound H11). In addition, the relationships between the electron withdrawing groups and the antiproliferative activities against HCT-116 cells were 4-Cl > 3-F > 4-Br > 3-Cl > 2-F > 4-F. Compared to compounds H3-H11, the change of arylamines to cyclopropylmethanamine (compound H12) improved inhibitory efficacy on MGC-803, HCT-116 and MCF-7 cells, Scheme 1. Synthesis of the target compounds H1-H18. As shown in Table 1, among the compounds H1-H18, compound H12 exhibited the most active antiproliferative activities against MGC-803, HCT-116 and MCF-7 cells, with IC 50 values of 9.47, 9.58 and 13.1 µM, respectively, which were more potent than that of the positive drug 5-Fu. On the whole, most of compounds displayed moderate antiproliferative activities against three cancer cells, with IC 50 values < 80 µM. The antiproliferative activities of compounds H1-H18 varied with its substituent groups of R. Additionally, most of compounds were more sensitive to HCT cells than MGC-803 and MCT-cells, excepting compounds H5, H9, H12 and H18. As for compounds H3-H11, the inhibitory activities varied with its substituent groups at phenyl groups of R. When the para position at phenyl of R were electron donating group ethyl substituent group (compound H5), its activity against HCT-116 cells was better than the para position at phenyl of R with electron withdrawing groups F (compound H6), Cl (compound H9) and Br (compound H11). In addition, the relationships between the electron withdrawing groups and the antiproliferative activities against HCT-116 cells were 4-Cl > 3-F > 4-Br > 3-Cl > 2-F > 4-F. Compared to compounds H3-H11, the change of arylamines to cyclopropylmethanamine (compound H12) improved inhibitory efficacy on MGC-803, HCT-116 and MCF-7 cells, indicating the appropriate volume of substituents of R is conducive to the maintenance of the activities. However, the introduction of six membered nitrogen-containing heterocyclic groups or the benzothiazole group of R had little effect on the activities, compared with compounds H5 and H12.

Inhibitory Effects of Compound H12 on MGC-803 Cells
To illustrate the inhibitory effect of compound H12 on gastric cancer cells MGC-803, MGC-803 cells were treated with different concentrations of compound H12 and then detected by MTT assay. As shown in Figure 4A,B, compound H12 could inhibit MGC-803 cells in dose-and time-dependent manners. Next, the growth status of MGC-803 under the effects of compound H12 were monitored by Real Time Cell Analysis (RTCA). The results are shown in Figure 4C: compound H12 at different concentrations inhibited cell growth to varying degrees. The morphological changes of the MGC-803 cells after treatment with compound H12 could be observed, and it was found that compound H12 caused a decrease in the density and cell rupture in MGC-803 cells. These results indicated that compound H12 could dose-and time-dependently inhibit gastric cancer cells MGC-803.

Inhibition of the ERK Signaling Pathway by Compound H12
The ERK signaling pathway plays an important role in regulating cell growth and is highly activated in cancers, and, therefore, has been identified as a promising therapeutic target for the treatment of human cancers [31,32]. Through the screening of various signaling pathways, we found that compound H12 exhibited significant inhibitory effects on the ERK signaling pathway. As shown in Figure 5, the activation (phosphorylation) levels of ERK1/2, the core protein of the ERK signaling pathway, and its upstream proteins c-Raf and MEK1/2, were significantly inhibited in MGC-803 cells in a dose-dependent manner after treatment with compound H12. The levels of FoxO3 and c-Myc, which are regulated by the ERK signaling pathway [33], were also significantly down-regulated. In addition, compound H12 could effectively reduce the phosphorylation level of AKT, indicating that the AKT signaling pathway, a related pathway to the ERK signaling pathway, was also inhibited. The above results suggested that compound H12 might be an effective inhibitor of the ERK signaling pathway. Molecules 2022, 27, x FOR PEER REVIEW 6 of 17

Inhibition of the ERK Signaling Pathway by Compound H12
The ERK signaling pathway plays an important role in regulating cell growth and is highly activated in cancers, and, therefore, has been identified as a promising therapeutic target for the treatment of human cancers [31,32]. Through the screening of various signaling pathways, we found that compound H12 exhibited significant inhibitory effects on the ERK signaling pathway. As shown in Figure 5, the activation (phosphorylation) levels of ERK1/2, the core protein of the ERK signaling pathway, and its upstream proteins c-Raf and MEK1/2, were significantly inhibited in MGC-803 cells in a dose-dependent manner after treatment with compound H12. The levels of FoxO3 and c-Myc, which are regulated by the ERK signaling pathway [33], were also significantly down-regulated. In addition, compound H12 could effectively reduce the phosphorylation level of AKT, indicating that the AKT signaling pathway, a related pathway to the ERK signaling pathway, was also inhibited. The above results suggested that compound H12 might be an effective inhibitor of the ERK signaling pathway.

Effects of Compound H11 on the Proliferation of MGC-803 Cells
The ERK signaling pathway could regulate cell proliferation [34]. Therefore, the effects of compound H12 on cell proliferation were next examined. As shown in Figure 6B

Effects of Compound H11 on the Proliferation of MGC-803 Cells
The ERK signaling pathway could regulate cell proliferation [34]. Therefore, the effects of compound H12 on cell proliferation were next examined. As shown in Figure 6B, the percentage of the G2/M phase of MGC-803 cells was concentration-dependently increased after treatment with compound H12. At concentrations of 8, 16 and 24 µM, the percentages of G2/M phase induced by compound H12 were 31.88, 39.32 and 50%, respectively. Meanwhile, the percentages of G2/M phase in the control group were 22.52%. The effects of compound H12 on the levels of cycle-related proteins were explored using Western blotting assay. As shown in Figure 6C, the levels of cell cycle-related proteins p-Cdc2 and CyclinB1 decreased and the levels of M-phase marker protein p-Histone H3 increased after treatment with compound H12 in a dose-dependent manner. The decrease in cell proliferation capacity was also manifested in the cell colony formatting activity ( Figure 6D). The results of the colony formatting assay suggested that a low concentration of compound H12 could significantly inhibit the colony formatting activity of MGC-803 cells. The above results indicated compound H12 had significant inhibitory effects on the cell proliferation of MGC-803 cells.

Effects of Compound H12 on the Apoptosis of MGC-803 Cells
The ERK signaling pathway also affects apoptosis in cancer cells [35]. Therefore, the effects of compound H12 on cell apoptosis were next explored using the flow cytometry assay. As shown in Figure 7A,B, the percentage of apoptotic cells in MGC-803 cells increased after treatment with compound H12 for 48 h. At concentrations of 8, 16 and 24 μM, the percentages of apoptotic cells induced by compound H12 were 9.92, 25.62 and 45.92%, respectively. Meanwhile, the percentages of apoptotic cells in the control group were 5.50%. We also used the Western blotting assay to explore the effects of compound H12 on the levels of apoptosis-related proteins. As shown in Figure 7C, apoptosis-like changes were also observed at the protein levels. The level of the pro-apoptotic protein Bax was increased and the levels of the anti-apoptotic proteins Mcl-1 and Bcl-2 were decreased. In addition, compound H12 could up-regulate the level of cleaved-Caspase7. In conclusion, compound H12 could induce cell apoptosis and regulate apoptosis-related proteins in MGC-803 cells.

Effects of Compound H12 on the Apoptosis of MGC-803 Cells
The ERK signaling pathway also affects apoptosis in cancer cells [35]. Therefore, the effects of compound H12 on cell apoptosis were next explored using the flow cytometry assay. As shown in Figure 7A,B, the percentage of apoptotic cells in MGC-803 cells increased after treatment with compound H12 for 48 h. At concentrations of 8, 16 and 24 µM, the percentages of apoptotic cells induced by compound H12 were 9.92, 25.62 and 45.92%, respectively. Meanwhile, the percentages of apoptotic cells in the control group were 5.50%. We also used the Western blotting assay to explore the effects of compound H12 on the levels of apoptosis-related proteins. As shown in Figure 7C, apoptosis-like changes were also observed at the protein levels. The level of the pro-apoptotic protein Bax was increased and the levels of the anti-apoptotic proteins Mcl-1 and Bcl-2 were decreased. In addition, compound H12 could up-regulate the level of cleaved-Caspase7. In conclusion, compound H12 could induce cell apoptosis and regulate apoptosis-related proteins in MGC-803 cells.

Conclusions
In conclusion, [1,2,4]triazolo[1,5-a]pyrimidine indole derivatives were designed and synthesized by the molecular hybridization strategy and their antiproliferative activities against three human cancer cell lines, MGC-803, HCT-116 and MCF-7, were tested. Among these compounds, compound H12 exhibited the most active antiproliferative activities against MGC-803, HCT-116 and MCF-7 cells, with IC50 values of 9.47, 9.58 and 13.1 μM, respectively, which were more potent than that of the positive drug 5-Fu. Further antitumor mechanisms suggested that compound H12 dose-dependently inhibited the growth and colony formation of MGC-803 cells. After screening different signaling pathways, it was found that compound H12 inhibited the activity of the ERK signaling pathway, resulting in decreased phosphorylation levels of ERK1/2, c-Raf, MEK1/2 and AKT. The ERK signaling pathway is closely related to both cell proliferation and apoptosis. Therefore, it could be found that compound H12 could arrest gastric cancer cells MGC-803 in the G2/M phase and induce apoptosis by regulating related proteins. In conclusion, compound H12 could inhibit the ERK signaling pathway, and inhibit the proliferation of and induce apoptosis in MGC-803 cells.

Materials and Methods
All the chemical reagents were purchased from commercial suppliers (Energy chemical Company and Aladdin reagent, Shanghai, China). NMR and HRMS spectral data were recorded with a Bruker spectrometer (Karlsruhe, Baden-Wurttemberg, Germany).

Synthesis of Compound C
A solution of commercially available compound 1H-1,2,4-triazol-5-amine (A) (1.0 mmol, 1.0 eq) and ethyl 4-chloro-3-oxobutanoate (B) (1.0 mmol, 1.2 eq) were added into 20 mL acetic acid at 25 °C. Then, the reaction mixture was stirred at 120 °C for 6 h. After 8 h, the reaction mixture was evaporated to give crude products and 20 mL ethyl acetate was added, giving a white solid. The white solid was filtered and dried (C) without further purification.

Conclusions
In conclusion, [1,2,3]Triazolo[1,5-a]pyrimidine indole derivatives were designed and synthesized by the molecular hybridization strategy and their antiproliferative activities against three human cancer cell lines, MGC-803, HCT-116 and MCF-7, were tested. Among these compounds, compound H12 exhibited the most active antiproliferative activities against MGC-803, HCT-116 and MCF-7 cells, with IC 50 values of 9.47, 9.58 and 13.1 µM, respectively, which were more potent than that of the positive drug 5-Fu. Further antitumor mechanisms suggested that compound H12 dose-dependently inhibited the growth and colony formation of MGC-803 cells. After screening different signaling pathways, it was found that compound H12 inhibited the activity of the ERK signaling pathway, resulting in decreased phosphorylation levels of ERK1/2, c-Raf, MEK1/2 and AKT. The ERK signaling pathway is closely related to both cell proliferation and apoptosis. Therefore, it could be found that compound H12 could arrest gastric cancer cells MGC-803 in the G2/M phase and induce apoptosis by regulating related proteins. In conclusion, compound H12 could inhibit the ERK signaling pathway, and inhibit the proliferation of and induce apoptosis in MGC-803 cells.

Materials and Methods
All the chemical reagents were purchased from commercial suppliers (Energy chemical Company and Aladdin reagent, Shanghai, China). NMR and HRMS spectral data were recorded with a Bruker spectrometer (Karlsruhe, Baden-Wurttemberg, Germany).

Synthesis of Compound C
A solution of commercially available compound 1H-1,2,4-triazol-5-amine (A) (1.0 mmol, 1.0 eq) and ethyl 4-chloro-3-oxobutanoate (B) (1.0 mmol, 1.2 eq) were added into 20 mL acetic acid at 25 • C. Then, the reaction mixture was stirred at 120 • C for 6 h. After 8 h, the reaction mixture was evaporated to give crude products and 20 mL ethyl acetate was added, giving a white solid. The white solid was filtered and dried (C) without further purification.

Synthesis of Compound D
A solution of compound C (1.0 mmol, 1.0 eq) was added into 20 mL phosphorus oxychloride at 25 • C. Then, the reaction mixture was stirred at 90 • C for 6 h. After 6 h, the reaction mixture was evaporated to give crude product, and then crude product was purified to give compound D by column chromatography.

Synthesis of Compound F
A solution of compound C (1.0 mmol, 1.1 eq), 1-methyl-1H-indole (E) (1.0 mmol, 1.0 eq) and Bis(trifluoromethane sulfonimide) (0.2 mmol, 0.2 eq) was added into 20 mL HIFP at 25 • C. Then, the reaction mixture was stirred at 100 • C for 6 h. After 6 h, the reaction mixture was evaporated to give crude product, and then crude product was purified to give compound F by column chromatography.

Synthesis of Compounds H1-H18
A solution of compound F (1.0 mmol, 1.1 eq), 1 substituted amines/2-mercaptobenzothiazole (E) (1.0 mmol, 1.0 eq) and NaH (0.2 mmol, 0.2 eq) was added into 10 mL DMF. Then, the reaction mixture was stirred at 25 • C for 3 h. After 3 h, the reaction mixture was added to 10 mL H 2 O and then extracted three times using ethyl acetate. The organic phases were combined and dried with anhydrous magnesium sulfate, which then were evaporated to give crude products. The products were purified to obtain compounds H1-H18 by column chromatography.  13 13 13

Cell Culture
All the human cancer cells were purchased from were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China).The MGC-803 cells (human gastric cancer cells), HCT-116 cells (human colorectal carcinoma cells) and MCF-7 cells (human breast cancer cells) used were cultured in humidified incubator at 37 • C and 5% CO 2 . The RPMI-1640 medium was supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (0.1 mg/mL) [36].

Colony Formation Assay
5000 per well MGC-803 cells were seeded in a 6-well plate and incubated at 37 • C in 5% CO 2 for 24 h, then treated with different concentrations of 10e. After 7 days, the culture medium was removed, the cells were washed with PBS twice, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells' images were captured with camera [37,38].

Western Blotting Assay
Gastric cancer cells MGC-803 were treated with different concentrations of compound H12 (0 µM, 8 µM, 16 µM and 24 µM) for 48 h and then collected. Then the cells were lysed. The obtained protein samples were separated by SDS-PAGE electrophoresis, followed by constant current 200 mA transfer for 60 min to nitrocellulose membranes; membranes were then blocked with 5% skimmed milk for 1 h. The membranes were incubated with primary antibody overnight, washed 5 times with TBST, incubated with secondary antibody for 2 h at room temperature. Finally, membranes were subjected to luminescence and imaging using ECL.

Cell Cycle Distribution Assay
MGC-803 cells were treated with different concentrations of compound H12 (0 µM, 8 µM, 16 µM and 24 µM) for 48 h, then collected and fixed in 70% cold ethanol for 24 h. A total of 100 µL of RNase A solution was added to the samples, which were incubated at 37 • C for 30 min. Then, 400 µL of PI staining solution was added to the samples, mixed and incubated at 4 • C for 30 min. The samples were then analyzed by flow cytometry after filtering.

Real-Time Cell Analyzer (RTCA) Cell Proliferation Detection Assay
The RTCA software (Agilent Technologies Inc, Santa Clara, CA, USA) was used to set up the program and test if the culture plate was available. Then, 50 µL of medium was added to each well of the culture plate and the monitoring time set. Following this, 4000 cells per well were seeded into the culture plate, equilibrated at room temperature for 30 min and the program started. When the growth curve was stable, the drug was added to treat the cells. We stopped the program after continuing the incubation for 48 h and exported the data to obtain the growth curve.

Statistical Analysis
The data of three independent experiments were expressed as mean ± SD and calculated by SPSS version 20 (IBM, Almonk, New York, NY, USA).