Novel N-Substituted 2-(2-(Adamantan-1-yl)-1H-Indol-3-yl)-2-Oxoacetamide Derivatives: Synthesis and Biological Evaluation

In this study, a series of novel N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide derivatives were synthesized, and evaluated for their cytotoxicity in human cell lines including Hela (cervical cancer), MCF7 (breast cancer ) and HepG2 (liver cancer). Several compounds were found to have potent anti-proliferative activity against those human cancer cell lines and compound 5r showed the most potent biological activity against HepG2 cells with an IC50 value of 10.56 ± 1.14 μΜ. In addition, bioassays showed that compound 5r induced time-dependent and dose-dependent cleavage of poly ADP-ribose polymerase (PARP), and also induced a dose-dependent increase in caspase-3 and caspase-8 activity, but had little effect on caspase-9 protease activity in HepG2 cells. These results provide evidence that 5r-induced apoptosis in HepG2 cell is caspase-8-dependent.


Introduction
Despite continued research efforts, cancer remains one of the biggest threats to human health, and it was estimated to be responsible for 15% of all global deaths in 2010 [1]. Current treatment for tumors generally involves surgical resection (if possible), followed by radiotherapy and chemotherapy, with the most common chemotherapies, according to Cancer Research UK, being temozolomide, procarbazine, carmustine, lomustine and vincristine [2][3][4][5]. However these drugs alone simply aren't sufficient for long-term treatment because of the rapid chemoresistance developed by many cancers [6]. Multidrug resistance (MDR) is a major obstacle to successful cancer treatment [7,8]. This has driven the development of a variety of new anticancer agents with more potent, high specific and low cytotoxic properties [9].
The indole moiety has been described as a privileged structure as it appears extensively in many unrelated areas of biology and medicine, and depending on the substituents, can have a diverse range of effects [10]. The indole ring system as one of the most ubiquitous heterocycles in Nature, and has been becoming an important structural component in many pharmaceutical agents, such as antidepressant [11], anticonvulsant [12], antifungal [13], antiviral [14] and anti-inflammatory [15], and particularly new antitumor agents [16][17][18][19]. Indole-3-glyoxylamide compounds, as a new class of Table 1. Inhibition of humor cancer cell lines (Hela, MCF7 and HepG2) by N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide derivatives.              Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 35  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 40  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 36  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 16  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 34  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 37  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 47  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 78  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 32  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 42  Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC50 values of over 100 μM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC50 value of 17.65 ± 1.54 μM. These data suggested the presence of alkyl ring at the N′-position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH3), trifluoromethyl (CF3), methoxy (OCH3), cyano (CN), nitro (NO2) and/or methoxy formyl (COOCH3) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) 31 Interestingly, compound 5a with a N-cyclopropyl group exhibited the lowest cytotoxic activity against these three cancer lines, with IC 50 values of over 100 µM, however introduction of single ring aryl group at the N-position such as in compounds 5c, 5d and 5f caused an increase in cytotoxic activity, and among them compound 5f with a N-benzene substituent had the strongest cytotoxic activity against the Hela cell line, with an IC 50 value of 17.65˘1.54 µM. These data suggested the presence of alkyl ring at the N 1 -position had an important relationship with anti-proliferative activity. To investigate the impact of the substituents on the N-phenyl ring, halogen (F, Cl, Br or I), methyl (CH 3 ), trifluoromethyl (CF 3 ), methoxy (OCH 3 ), cyano (CN), nitro (NO 2 ) and/or methoxy formyl (COOCH 3 ) was introduced (compounds 5g-u). Substitution at the N-phenyl ring was unhelpful, except for compound 5r with m-Cl and p-F on the N-phenyl ring as shown in Table 1. In addition, replacement of phenyl with pyridyl (5v and 5w), pyrimidine (5x) or isoxazole (5y) resulted in similar cellular anti-proliferative activity and the introduction of a fused aromatic ring group (compound 5e) or alkyl group (5b) between nitrogen atom and phenyl group was generally unfavorable for anti-proliferative properties.
Based on data collected from three independent experiments, compound 5r showed the most cytotoxic activity against Hela, MCF-7 and HepG2 cell lines with IC 50 values of 16.12˘1.54, 12.54˘1.15 and 10.56˘1.14 µM, respectively, so we used compound 5r for further biological activity studies.

Growth Inhibitory Activity of 5r in HepG2
As cell proliferation depends on cell division which is regulated by the cell cycle, absence of normal cell-cycle control is a hallmark of cancer [30]. Cell cycle-related proteins have been as the therapeutic targets against cancer and lots of small molecules were developed as potent antitumor agents, such as microtubule-targeting agents, and cyclin-dependent kinases, aurora kinases and polo-like kinases inhibitors [31][32][33][34]. The present study sought to determine how compound 5r inhibited HepG2 cell growth. Microscopic analysis indicated that the colonies of HepG2 cells decreased after compound 5r treatment in a dose dependent manner, compared to the control group ( Figure 1A). Furthermore, flow cytometry was performed to examine cell cycle inhibition after 12 h of compound 5r treatment. The results revealed a significant accumulation of cell-cycle arrest, with a decrease in G0/G1 phase and an increase in G2/M phase arrest at 12 h, indicating that inhibitory activity of compound 5r was associated with disruption of cell cycle ( Figure 1B). Meanwhile, the effects of compound 5r on colony-formation and cell-cycle distribution in Hela and MCF-7 cells were also examined (see Supplementary Materials). or alkyl group (5b) between nitrogen atom and phenyl group was generally unfavorable for anti-proliferative properties. Based on data collected from three independent experiments, compound 5r showed the most cytotoxic activity against Hela, MCF-7 and HepG2 cell lines with IC50 values of 16.12 ± 1.54, 12.54 ± 1.15 and 10.56 ± 1.14 μM, respectively, so we used compound 5r for further biological activity studies.

Growth Inhibitory Activity of 5r in HepG2
As cell proliferation depends on cell division which is regulated by the cell cycle, absence of normal cell-cycle control is a hallmark of cancer [30]. Cell cycle-related proteins have been as the therapeutic targets against cancer and lots of small molecules were developed as potent antitumor agents, such as microtubule-targeting agents, and cyclin-dependent kinases, aurora kinases and polo-like kinases inhibitors [31][32][33][34]. The present study sought to determine how compound 5r inhibited HepG2 cell growth. Microscopic analysis indicated that the colonies of HepG2 cells decreased after compound 5r treatment in a dose dependent manner, compared to the control group ( Figure 1A). Furthermore, flow cytometry was performed to examine cell cycle inhibition after 12 h of compound 5r treatment. The results revealed a significant accumulation of cell-cycle arrest, with a decrease in G0/G1 phase and an increase in G2/M phase arrest at 12 h, indicating that inhibitory activity of compound 5r was associated with disruption of cell cycle ( Figure 1B). Meanwhile, the effects of compound 5r on colony-formation and cell-cycle distribution in Hela and MCF-7 cells were also examined (see Supplementary Materials).

Figure 1. Effects of compound 5r on colony-formation and cell-cycle distribution in HepG2 cells. (A)
Compound 5r inhibits the colony-forming activity of HepG2 cells. Cells were grown in 6-well plates for 6 days and treated with compound 5r (1, 5 and 10 μM). Colonies of HepG2 cells decreased after compound 5r treatment in a dose dependent manner (** p < 0.01 and *** p < 0.001 compared with the control, t-test). (B) A flow cytometry assay was performed to examine cell cycle arrest. HepG2 cells were treated with 10 μM compound 5r for 12 h. Compound 5r reduced a significant accumulation of cell-cycle arrest (*** p < 0.001 compared with the control, t-test).

Inducing Apoptosis in HepG2 Cells
Apoptosis, as a fundamental biological process, plays an important role in cell growth, development and tissue homeostasis [35][36][37]. Deregulation in apoptotic cell contributes to many diseases, including cancer, neurodegenerative disorders and cardiovascular diseases [38,39]. Apoptosis can be further characterized as cell death accompanied by the activation of a unique family of cysteine-dependent specific proteases called caspases [40]. Two major signaling pathways induce apoptotic cell death: the mitochondrial pathway and the death receptor pathway [41]. The former relies on mitochondrial depolarization and permeability increase in response to a variety of  5 and 10 µM). Colonies of HepG2 cells decreased after compound 5r treatment in a dose dependent manner (** p < 0.01 and *** p < 0.001 compared with the control, t-test); (B) A flow cytometry assay was performed to examine cell cycle arrest. HepG2 cells were treated with 10 µM compound 5r for 12 h. Compound 5r reduced a significant accumulation of cell-cycle arrest (*** p < 0.001 compared with the control, t-test).

Inducing Apoptosis in HepG2 Cells
Apoptosis, as a fundamental biological process, plays an important role in cell growth, development and tissue homeostasis [35][36][37]. Deregulation in apoptotic cell contributes to many diseases, including cancer, neurodegenerative disorders and cardiovascular diseases [38,39]. Apoptosis can be further characterized as cell death accompanied by the activation of a unique family of cysteine-dependent specific proteases called caspases [40]. Two major signaling pathways induce apoptotic cell death: the mitochondrial pathway and the death receptor pathway [41]. The former relies on mitochondrial depolarization and permeability increase in response to a variety of cellular stresses, including DNA damage, growth factor deprivation, ER stress, thus resulting in the release of cytochrome C, then initiating formation of an APAF-1/caspase-9 complex and activation of downstream executionary caspases, including caspase-3, caspase-6, and caspase-7, and finally leading to cell death [42,43]. The second pathway is activated predominantly by the binding of death receptor ligands, including tumor necrosis factor-α (TNF-α), fas ligand (CD95) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) to their respective death receptors, then initiating the assembly of large macromolecular complexes that recruit and activate caspase-8, which further cleave and activate caspase-3 for apoptosis [44]. In our work, it was demonstrated that compound 5r could induce poly ADP-ribose polymerase (PARP) cleavage, which served as a marker of cells undergoing apoptosis, in a time-and dose-dependent manner (Figure 2A,B).
Molecules 2016, 21, 530 6 of 15 cellular stresses, including DNA damage, growth factor deprivation, ER stress, thus resulting in the release of cytochrome C, then initiating formation of an APAF-1/caspase-9 complex and activation of downstream executionary caspases, including caspase-3, caspase-6, and caspase-7, and finally leading to cell death [42,43]. The second pathway is activated predominantly by the binding of death receptor ligands, including tumor necrosis factor-α (TNF-α), fas ligand (CD95) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) to their respective death receptors, then initiating the assembly of large macromolecular complexes that recruit and activate caspase-8, which further cleave and activate caspase-3 for apoptosis [44]. In our work, it was demonstrated that compound 5r could induce poly ADP-ribose polymerase (PARP) cleavage, which served as a marker of cells undergoing apoptosis, in a time-and dose-dependent manner (Figure 2A,B). The PARP is one of the important targets of caspase-3, which is also downstream of capase-8 and caspase-9 [45]. Therefore, the activation of caspase-3, caspase-8 and caspase-9 was examined in HepG2 cells using a caspase activity assay kit after treatment with compound 5r. It was shown that compound 5r significantly stimulated caspase-3 and caspase-8 protease activities in HepG2 cells, yet had little effect on caspase-9 protease activity ( Figure 3). The results suggested that compound 5r resulted in the caspases-3 activation and PARP cleavage by activating caspase-8, finally leading to cell death.  The PARP is one of the important targets of caspase-3, which is also downstream of capase-8 and caspase-9 [45]. Therefore, the activation of caspase-3, caspase-8 and caspase-9 was examined in HepG2 cells using a caspase activity assay kit after treatment with compound 5r. It was shown that compound 5r significantly stimulated caspase-3 and caspase-8 protease activities in HepG2 cells, yet had little effect on caspase-9 protease activity ( Figure 3). The results suggested that compound 5r resulted in the caspases-3 activation and PARP cleavage by activating caspase-8, finally leading to cell death.
Molecules 2016, 21, 530 6 of 15 cellular stresses, including DNA damage, growth factor deprivation, ER stress, thus resulting in the release of cytochrome C, then initiating formation of an APAF-1/caspase-9 complex and activation of downstream executionary caspases, including caspase-3, caspase-6, and caspase-7, and finally leading to cell death [42,43]. The second pathway is activated predominantly by the binding of death receptor ligands, including tumor necrosis factor-α (TNF-α), fas ligand (CD95) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) to their respective death receptors, then initiating the assembly of large macromolecular complexes that recruit and activate caspase-8, which further cleave and activate caspase-3 for apoptosis [44]. In our work, it was demonstrated that compound 5r could induce poly ADP-ribose polymerase (PARP) cleavage, which served as a marker of cells undergoing apoptosis, in a time-and dose-dependent manner (Figure 2A,B). The PARP is one of the important targets of caspase-3, which is also downstream of capase-8 and caspase-9 [45]. Therefore, the activation of caspase-3, caspase-8 and caspase-9 was examined in HepG2 cells using a caspase activity assay kit after treatment with compound 5r. It was shown that compound 5r significantly stimulated caspase-3 and caspase-8 protease activities in HepG2 cells, yet had little effect on caspase-9 protease activity ( Figure 3). The results suggested that compound 5r resulted in the caspases-3 activation and PARP cleavage by activating caspase-8, finally leading to cell death.

General Information
All reagents were commercially available and used without further purification unless otherwise indicated. Reaction mixtures were magnetically stirred and monitored by thin-layer chromatography (TLC) on Yantai Wish chemical products Co., Ltd. (Yantai, China) silica gel 60F-254 by fluorescence quenching under UV light. All of the final compounds were purified by column chromatography. 1 H-NMR and 13 C-NMR spectra were recorded on an AV2 600 MHz spectrometer (Bruker Biospin, Swiss). Chemical shifts (δ) were given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Multiplicities were abbreviated as follows: single (s), doublet (d), doublet-doublet (dd), doublet-triplet (dt), triplet (t), triplet-triplet (tt), triplet-doublet (td), quartet (q), quartet-doublet (qd), multiplet (m), and broad signal (br s). Positive mode high-resolution mass spectral (HRMS) data were acquired using electrospray ionization (ESI) on a Q Exactive LC-MS/MS instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA) with UV detection at 254 nm. (2) A mixture of o-toluidine (10.0 mmol), adamantane-1-carbonyl chloride (10.0 mmol) and anhydrous potassium carbonate (7 mmol) in toluene (50 mL) was stirred at room temperature for 4 h. The resulting solids was filtered off and stirred at room temperature for 1.5 h with H 2 O (50 mL). After completion of the reaction, the solid was filtered off and recrystallized from the appropriate solvent. White solid product, yield 75.5%. 1

Synthesis of 2-Adamantane-1H-indole (3)
A stirred solution of compound 2 (10 mmol) in 50 mL of tetrahydrofuran (THF) under a N 2 atmosphere was maintained at an internal temperature of -5 to 5˝C and treated dropwise with 0.1-0.15 mol of 2.5 M n-BuLi in hexane. The mixture was stirred at ambient temperature for 3 h, cooled in an ice bath, and treated dropwise with 2 M HCl (12 mL). Then, the organic layer was separated and the aqueous layer washed with C 6 H 6 . The combined organic layer was dried with anhydrous MgSO 4 , filtered, and concentrated in vacuo. The residue was recrystallized from the appropriate solvent. White solid product, yield 56.5%. 1  3.4. General Procedure for Synthesis of 5a-y [23,46] A dry 50 mL capacity carousel reaction tube was charged with compound 3 (1.5 mmol), and this starting material was dissolved in dry ether (12 mL). Oxalyl chloride (1.65 mmol) was added and the mixture stirred at room temperature for 2 h, the solid was filtered off. Then compound 4 was used, without purification, for the next reaction. To a solution of 4 (1 mmol) and a substituted amine (1 mmol) in toluene (10 mL) was added K 2 CO 3 (1.0 mmol), and the mixture stirred at room temp. After completion of the reaction, the mixture was concentrated and purified by column chromatography using appropriate mixtures of CH 2 Cl 2 /MeOH to give compounds 5a-y.  13

Cytotoxicity against Cancer Cell Lines
Confluent cancer cells in good state were cultured in 96-well plates (5-6ˆ10 3 cells/well) and treated with various concentrations of compounds at 37˝C for 24 h. Then, the cells were incubated with 20µL of 5 mg/mL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT, (Sigma-Aldrich, Saint Louis, MO, USA) reagent at 37˝C for 4 h. The supernatant was removed, and cells were dissolved in 150 µL dimethyl sulfoxide and shaken for 5 min. Finally, the light absorption (OD) of the dissolved cells was measured at 490 nm.

Western Blot Analysis
Equal amounts of the lysates were electrophoresed on 8% SDS-PAGE gel and transferred onto PVDF membranes (Roche, Shanghai, China). After blocked with 5% nonfat milk in TBST (20 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.1% Tween 20) for 1 h, The membranes were incubated with various primary antibodies overnight and secondary antibodies for 2 h, finally detected using ECL system. Proteins were detected with the following antibodies: Rabit anti-Parp mAb (46D11, Cell Signaling Technology, Shanghai, China), Mouse anti-β-actin mAb (sc-8432, Santa Cruz, Shanghai, China).

A Flow Cytometry Assay
Hela cells were cultured in 6-well plate and treated with various compounds in serum free medium for 24 h. Then, the cells were detached by trypsin and fixed in 70% cold ethanol overnight at 4˝C. The next day, the cells were centrifuged in 3000 rpm for 53 min, washed twice in PBS, and incubated with DNase A (100 µg/mL) and propidium iodide (PI) solution (50 µg/mL) at room temperature for 30 min. The cell cycle was detected by flow cytometry (Beckman Coulter, Pasadena, CA, USA).

Colony Formation Assay
Hela cells were cultured in 6-well plate (200 cells/well) and treated with various compounds in 1% serum medium for 6 days. Then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.

Caspase Activity Assay
The activities of caspase-3, caspase-8 and caspase-9 were measured using the caspase activity kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. Briefly, testis lysates were prepared after treatment. 50 µL testis lysate, 50 µL reaction buffer and 5 µL caspases substrate were added, incubated at 37˝C for 3 h. Samples were measured with an ELISA Reader (Bio-Rad instrument Group, Hercules, CA, USA) at an absorbance of 405 nm. All the experiments were carried out in triplicates.

Conclusions
In this paper, we synthesized and conducted a biological evaluation of a new series of N-substituted 2-(2-(adamantan-1-yl)-1H-indol-3-yl)-2-oxoacetamide derivatives as potential anticancer agents. The synthetic method was relatively simple, and the compounds were produced in high yields and easily purified. Compound 5r showed more significant inhibitory activity against HepG2 cells than other compounds, with an IC 50 value of 10.56˘1.14 µM, as well as excellent selectivity toward HepG2 over HeLa and MCF-7 cells. Western blot analysis and flow cytometry assay demonstrated that compound 5r could arrest the cell cycle, activate caspase-8 and caspase-3 and induce cell apoptosis. However, determining its roles in preventing cancer still require further intensive study.