Synthesis and Biological Evaluation of (S)-2-(Substituted arylmethyl)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide Analogs and Their Synergistic Effect against PTEN-Deficient MDA-MB-468 Cells

A series of twenty-six compounds of furfuryl or benzyl tetrahydropyrazino[1,2-a]indole analogs were synthesized and evaluated for cytotoxic activity against the estrogen receptor (ER)-positive breast cancer cell line (MCF-7) and the epidermal growth factor receptor (EGFR) over-expressed triple-negative breast cancer cell line (MDA-MB-468). Among them, compounds 2b, 2f and 2i showed more potent activity and selectivity against MDA-MB-468 cells than gefitinib, as an EGFR- tyrosine kinase inhibitor. In addition, it was confirmed by means of isobologram analysis of combinational treatment with gefitinib that they have a synergistic effect, especially compounds 2b and 2f, which inhibit Akt T308 phosphorylation. Moreover, it was confirmed that 2-benzyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs (2b, 2f, and Ref 2) tend to selectively inhibit PI3Kβ, which is involved in the phosphorylation of Akt.


Introduction
Cancer is a leading cause of death worldwide and is caused by the uncontrolled growth of cells in an organ of the body. Over 200 types of cancers tend to develop invasion and metastasis into other tissues [1]. In 2020, there were 19.3 million new cancer cases and almost 10.0 million cancer deaths worldwide. The incidence of cancer in men is highest in the order of lung, prostate, colorectum, stomach, and liver, and the highest percentage of cancer types in women occurs in the breast, colorectum, lung, cervix uteri, and thyroid. Breast and lung cancers are the most common types of cancer of both sexes [2]. For the treatment of cancer, small-molecule drugs have been approved by the Food and Drug Administration (FDA) and have been developed to control various therapeutic targets, including hormone receptors and oncogenic kinases, or as traditional chemotherapy [3][4][5][6].
In targeted therapy against lung cancer, many studies have focused on the more common non-small cell lung cancer (NSCLC) and involve inhibitors related to the epidermal growth factor receptor (EGFR) pathway, including the PI3K/AKT/mTOR and RAS-MAPK pathways, anaplastic lymphoma kinase (ALK), proto-oncogene ROS1, and programmed death 1 (PD-1) [7][8][9][10][11]. Among them, the EGFR pathway is considered an attractive target; thus, anti-EGFR therapies such as epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors and monoclonal antibodies have been developed [12,13]. Targeted therapy against breast cancer involves the overexpression of specific receptors and their downstream signaling pathways. Therefore, there are various breast cancer subtypes produced
In conclusion, the introduction of substituents into the N2-benzyl ring altered its growth inhibitory activity and tumor selectivity. In particular, it was interesting to observe that they improved in series 2 with the substituents that had heteroatoms, which generally decreased in series 1. In addition, the growth inhibitory activity of the synthesized analogs showed higher sensitivity in MDA-MB-468 cells than in MCF-7 cells, suggesting that it may act on the activated pathway only in MDA-MB-468 cells. Compounds 2b, 2f and 2i, with NO 2 , CN and OCF 3 groups at the para-position of the N2-benzyl ring, respectively, greatly increased tumor selectivity and inhibitory activity. Therefore, we performed additional biological studies to confirm the mechanism of action.

Flow Cytometric Analysis
To confirm the apoptosis-inductive effect of the MDA-MB-468 cell line, compounds 2b, 2f and 2i, as the groups with the most potent and tumor-selective activity, were analyzed by flow cytometric analysis with annexin V-FITC and propidium iodide (PI) ( Figure 2). The rates of apoptosis induction activated by compounds 2b and 2i were 24.0% and 21.0%, respectively, which were similar to gefitinib (21.1%) as a positive control at a concentration of 10 µM for 24 h. Notably, compound 2f showed 37.1% apoptosis induction, which was 1.76 times more potent than gefitinib. These results imply that compounds 2b, 2f and 2i, which all showed cytotoxic activity, induce apoptosis in the form of programmed cell death in MDA-MB-468 cells.

Combinational Treatment of Compounds 2b, 2f and 2i with Gefitinib
In cancer research, combinational therapy is used a treatment to overcome single drug resistance [41,42]. Combination with inhibitors of the PI3K/AKT/mTOR pathway and the EGFR-TK inhibitor increases therapeutic effects by synergism against TNBC and NSCLC with resistance to EGFR-TK inhibitors [31,43]. In addition, our previous results confirmed that compounds Ref 1 and Ref 2 have synergistic effects in the combinational treatment of MDA-MB-468 cells with gefitinib [40]. Therefore, the synergistic effects were investigated to confirm the targeted pathway. In this study, compounds 2b, 2f, and 2i, which showed potent cytotoxic activity against the MDA-MB-468 cell line as an EGFR-overexpressed cell line, were tested to confirm their specific target by combination treatment with gefitinib as an EGFR-TK inhibitor. Compounds 2b, 2f, and 2i exhibited cytotoxicity at various concentrations (0, 1, 3, and 10 µM) in a dose-dependent manner against the MDA-MB-468 cell line. In addition, their combination treatment with the use of several doses (0, 1, 5, and 20 µM) of gefitinib resulted in the MDA-MB-468 cell viability graphs, as shown in Figure 3a. Generally, the addition of gefitinib decreases cell viability in a dose-dependent manner and is more cytotoxic than single treatment with compounds 2b, 2f and 2i. Based on the cell viability of the combination treatment, we performed several isobologram analyses to assess the interaction between gefitinib and the compound at concentrations of 30%, 40%, 50%, and 60% of the maximal inhibition of cell proliferation, referred to as GI 30 , GI 40 , GI 50 , and GI 60 , respectively (Figure 3b). Compounds 2b, 2f, and 2i showed synergistic effects with gefitinib in all analyzed GI values. In a combinational analysis using the Chou and Talalay equation (CI = (D) 1 /(D χ ) 1 + (D) 2 /(D χ ) 2 ) [44], the combination index (CI) at GI 60 was 0.57, 0.58, and 0.42 by compounds 2b, 2f, and 2i, respectively, and they had more synergism than GI 30 , GI 40 , and GI 50 . At the GI 50 values, compounds 2b, 2f, and 2i with 1 µM gefitinib had the best combination index (CI) values of 0.71, 0.83, and 0.71, respectively, which indicates that they had a synergistic effect with a CI of less than 1. gefitinib, as the EGFR-TK inhibitor, against MDA-MB-468 cells, as EGFR-overexpressing TNBC. MDA-MB-468 cells have been reported as cancer possessing overexpressed EGFR without mutation, but also with p53 mutation and PTEN deletion [32,33]. In the reported studies, it was shown that a synergistic effect with EGFR-TK inhibitor may occur in the case of using inhibitors of the PI3K/AKT/mTOR pathway because the PI3K/AKT/mTOR signaling pathway is hyper-activated by the deletion of PTEN [30,31]. Therefore, we evaluated their activity in the downstream signaling pathway of EGFR.

Effects of the Compounds on Downstream Signaling of EGFR
To gain a better insight into the mechanisms underlying the activity of these compounds, the inhibitory effects of compounds 2b, 2f, and 2i were tested and compared with gefitinib (GE), compound Ref1, and Ref2 in the downstream signaling pathway of EGFR in MDA-MB-468 cells, as an EGFR-overexpressed basal-like breast cancer cell line. Interestingly, they induced increased apoptosis and showed synergistic effects with gefitinib, as the EGFR-TK inhibitor, against MDA-MB-468 cells, as EGFR-overexpressing TNBC. MDA-MB-468 cells have been reported as cancer possessing overexpressed EGFR without mutation, but also with p53 mutation and PTEN deletion [32,33]. In the reported studies, it was shown that a synergistic effect with EGFR-TK inhibitor may occur in the case of using inhibitors of the PI3K/AKT/mTOR pathway because the PI3K/AKT/mTOR signaling pathway is hyper-activated by the deletion of PTEN [30,31]. Therefore, we evaluated their activity in the downstream signaling pathway of EGFR.

Effects of the Compounds on Downstream Signaling of EGFR
To gain a better insight into the mechanisms underlying the activity of these compounds, the inhibitory effects of compounds 2b, 2f, and 2i were tested and compared with gefitinib (GE), compound Ref 1, and Ref 2 in the downstream signaling pathway of EGFR in MDA-MB-468 cells, as an EGFR-overexpressed basal-like breast cancer cell line.
In western blotting of compounds after 6 h, gefitinib, a known EGFR-TK inhibitor, reduced the phosphorylation of both ERK and Akt. However, compounds 2b and 2i did not suppress ERK phosphorylation or Akt Ser473 phosphorylation. Ser473 phosphorylation, as reported in a previous study. However, it was confirmed that compounds 2b, 2f, and 2i did not inhibit Akt Ser473 phosphorylation. Among them, only compounds 2b, and 2f showed potent inhibitory activity on Akt Thr308 phosphorylation, as shown in Figure 4. In addition, compounds 2f and Ref 2 inhibited ERK phosphorylation, although not as dramatically as gefitinib.
Ref1, and Ref2 showed inhibition of ERK phosphorylation. After 18 h, gefitinib exhibited potent inhibitory activity on the ERK and Akt pathways. Compounds Ref1 and Ref2 inhibit Akt Ser473 phosphorylation, as reported in a previous study. However, it was confirmed that compounds 2b, 2f, and 2i did not inhibit Akt Ser473 phosphorylation. Among them, only compounds 2b, and 2f showed potent inhibitory activity on Akt Thr308 phosphorylation, as shown in Figure 4. In addition, compounds 2f and Ref2 inhibited ERK phosphorylation, although not as dramatically as gefitinib.

Combinational Treatment of Compounds 2b, 2f, and 2i with Gefitinib
Based on the results of western blotting, it was confirmed that they have inhibitory activity for the generation of phosphorylated Akt in the EGFR-overexpressed breast cancer cell line MDA-MB-468. To identify the targets of compounds that inhibit Akt phosphorylation, we tested their enzyme activity against 18 kinases including PI3K (phosphatidylinositol-3-kinase), PDK (phosphoinositide-dependent protein kinase), mTOR (mechanistic target of rapamycin), DNA-PK (DNA-dependent protein kinase), and TBK1 (TANK-binding kinase-1). These kinases directly phosphorylate Akt or induce the upstream activation of the Akt signaling pathway. Activation of PI3Ks produces PIP3 (phosphatidylinositol 3,4,5-triphosphate) through phosphorylation of PIP2 (phosphatidylinositol 4,5-bisphosphate), and PIP3 regulates PDK1 and sequentially induces Akt T308 phosphorylation. Akt S473 phosphorylation is also regulated by DNA-PK, PDK2, and mTORC2. In addition, TBK1 promotes the phosphorylation of Akt at S473 and T308 in a manner that is dependent on PI3K signaling [45][46][47].

Combinational Treatment of Compounds 2b, 2f, and 2i with Gefitinib
Based on the results of western blotting, it was confirmed that they have inhibitory activity for the generation of phosphorylated Akt in the EGFR-overexpressed breast cancer cell line MDA-MB-468. To identify the targets of compounds that inhibit Akt phosphorylation, we tested their enzyme activity against 18 kinases including PI3K (phosphatidylinositol-3-kinase), PDK (phosphoinositide-dependent protein kinase), mTOR (mechanistic target of rapamycin), DNA-PK (DNA-dependent protein kinase), and TBK1 (TANK-binding kinase-1). These kinases directly phosphorylate Akt or induce the upstream activation of the Akt signaling pathway. Activation of PI3Ks produces PIP 3 (phosphatidylinositol 3,4,5-triphosphate) through phosphorylation of PIP 2 (phosphatidylinositol 4,5-bisphosphate), and PIP 3 regulates PDK1 and sequentially induces Akt T308 phosphorylation. Akt S473 phosphorylation is also regulated by DNA-PK, PDK2, and mTORC2. In addition, TBK1 promotes the phosphorylation of Akt at S473 and T308 in a manner that is dependent on PI3K signaling [45][46][47].
As shown in Table 2, 2-arylmethyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3carboxamide analogs, except compound 2i, exhibited higher inhibitory activity on PI3Kβ than other kinases at 10 µM. Compounds 2b, 2f, and Ref 2 showed the most potent activities on the PI3Kβ enzyme, with 71.8%, 70.9%, and 55.3% enzyme activity, respectively. Interestingly, compounds Ref 1 and Ref 2, which showed inhibitory activity of phosphorylation on both T308 and S473 in the western blot, exhibited more potent activity on PI3Kβ and DNA-PK than other kinases. Thus, the various effects of compounds, such as growth inhibitory activity, apoptosis, and inhibition of Akt phosphorylation may cautiously suggest that the inhibition of PI3Kβ is involved.
In the analysis of apoptosis, compound 2f exhibited 37.1% apoptosis induction, and was 1.76 times more potent than that of gefitinib. On MDA-MB-468 cells, which were resistant to EGFR-TK inhibitors because of PTEN mutation, combination treatment with gefitinib induced synergistic effects, and the combination index at GI 50 showed the best effect with 1 µM gefitinib. These results are due to their activity in relation to the inhibition of Akt T308 phosphorylation, unlike the inhibition of EGFR-TK by gefitinib. It was confirmed that benzyl 2-benzyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs with 4-NO 2 (2b), 4-CN (2f), or H (Ref 2) on the N2-benzyl ring tend to inhibit PI3Kβ among the kinase activities that are involved in the phosphorylation of Akt.
Recently, it has been reported that inhibition of the PI3Kβ isoform is very important for the progression of PTEN-null tumors [48,49]. In the PTEN-deficient TNBC cell line MDA-MB-468, a new potential therapeutic strategy for combination targeting of both EGFR and PI3Kβ was suggested to induce anti-tumor activity [50]. Therefore, these results, regarding 2-arylmethyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs, are expected for the development of anticancer agents against PTEN-deficient and EGFRoverexpressing cancer.