Synthesis and Biological Evaluation of a Fused Structure of Indolizine and Pyrrolo[1,2-c]pyrimidine: Identification of Its Potent Anticancer Activity against Liver Cancer Cells

A highly efficient approach to a new indolizine scaffold fused with pyrrolo[1,2-c]pyrimidine was achieved via one-pot three-component coupling followed by an oxidative cyclization reaction. The simple two-step sequence allowed rapid access to various tetracyclic compounds from commercially available starting materials with the formation of five new bonds. Here, we observed the effects of these compounds on cell viability in HepG2, H1299, HT29, AGS, and A549 cancer cell lines. Interestingly, this fused scaffold had more potent anticancer activity in hepatocellular carcinoma HepG2 and Huh7 cells than other cancer cells. In particular, 5r strongly decreased cell viability in HepG2 and Huh7 cells with an IC50 value of 0.22 ± 0.08 and 0.10 ± 0.11 µM, respectively, but had a very weak inhibitory effect on the cell viability of other cancer cell lines. In addition, 5r significantly inhibited cell migration and induced apoptosis in HepG2 and Huh7 cells via the activation of caspase-3 and cleavage of PARP in a dose-dependent manner. Notably, the co-treatment of 5r with gemcitabine resulted in the significant additional inhibition of cell viability in HepG2 and Huh7 cells. Our results suggest that 5r could be used to develop new chemotype anticancer agents against liver cancers.


Introduction
Polyheterocycles are widely found in both natural products and artificial materials while exhibiting a wide range of biological and optophysical properties. Owing to their versatile usages in many different fields, efforts to expand the polyheterocyclic chemical space via design and synthesis are highly valuable [1,2]. As part of our research interest on novel polyheterocyclic structures, we designed several new heterocyclic scaffolds having two or more pharmacophores, hoping that these new fused polycycles might have intriguing properties that had not been previously discovered with each heterocyclic ring system. Along this line, we developed several efficient synthetic strategies of novel fused heterocycles [3][4][5], which enabled us to investigate their biological and/or photophysical activities ( Figure 1) [6].
Liver cancer is one of the most common causes of cancer death worldwide and the fifth most common cancer in the United States [7]. Based on the announcement of Globocan 2020, 905,677 new liver cancer patients occurred in 2020 and 830,180 deaths were confirmed. The incidence of liver cancer is generally higher in developing countries and risk factors include hepatitis B virus, hepatitis C virus, fatty liver disease, smoking, alcoholic cirrhosis, obesity, and diabetes [8,9]. With respect to the surgical treatment of liver cancer, only In 2017, Hulme and co-workers reported a direct one-pot three-component assembly route to 1-cyano-3-amino-2-arylindolizines 1 (Scheme 1a) [12]. Inspired by this work, we came up with the general idea that a new indolizine-fused heteroaromatic system 3 would be constructed via 2 by using a (hetero)aryl aldehyde in the three-component coupling process followed by oxidative cyclization (Scheme 1b). In this context, we planned to employ pyrrole-2-carboxaldehyde in the first step to gain access to 4. The subsequent oxidative cyclization of 4 with an aldehyde leads to an indolizine skeleton fused with pyrrolo[1,2-c]pyrimidine 5. As indolizine [13] and pyrrolo [1,2-a]pyrimidine [14,15] have been frequently employed in material and medicinal sciences, we anticipated to discover new biochemical functions with this new skeleton. Here, we wish to describe the synthesis and biological evaluation of this tetracyclic fused scaffold. While we were preparing this manuscript, the Hulme group disclosed the synthesis and photophysical properties of indolizino [3,2-c]isoquinoline 7 via the intermediate 6 [16].
Thus, more derivatives were prepared in a similar way and their anticancer activities against HepG2 and Huh7 cells were evaluated (Table 3). In general, heterocycles or aryl groups with ortho-or meta-substituent(s) at the C5 position seemed beneficial for enhanced anticancer activity. We were pleased to find that 5r bearing a 2-hydroxyphenyl at the C5 site had more potency than 5n. Interestingly, 5s with a 3-hydroxyphenyl moiety at the C5 position exhibited less cytotoxicity than 5r, indicating that the orientation of the hydroxyl in this scaffold is critical for potent activity. Under the optimal conditions, various aldehydes were allowed to react with 4 to furnish derivatives 5 (Table 1). Electron-poor as well as electron-rich aryl moieties were installed at the C5 position of this heterocyclic system in good yields. Indolizine-pyrrolo [1,2-c]pyrimidine hybrids bearing heteroaromatic rings such as pyridine, indole, furan, and thiophene at the C5 site were readily accessed as well.    The preliminary biological screening of these compounds revealed that this class of compounds exhibited strong inhibitory activity against HepG2 and H1299 cells out of the five cancer cells evaluated (Table 2). In particular, stronger anticancer activities were observed with compounds 5k, 5l, and 5n in HepG2 cells, having pyridine, indole, and thio- More analogs of 5r were synthesized to examine the effects of the substituent(s) of the 2-hydroxyphenyl moiety on inhibitory activity (Table 4). Unfortunately, an additional methoxyl or chlorine on the ring did not increase the potency. cell lung carcinoma, and A549 lung carcinoma cell lines. As shown in Figure 2A,B, 5r potently reduced cell viability in HepG2 and Huh7 cells with an IC 50 value of 0.22 ± 0.08 and 0.10 ± 0.11 µM, respectively. However, 5r had a weak effect on the other cancer cell lines, AGS, H1299, and A549 ( Figure 2C-E). In addition, 5r also weakly decreased cell viability in non-cancerous cells including HaCaT, NIH3T3, and HEK293T cells ( Figure 2F-H). These results suggest that 5r can be developed as a selective anticancer agent against liver cancer.  Thus, more derivatives were prepared in a similar way and their anticancer activities against HepG2 and Huh7 cells were evaluated (Table 3). In general, heterocycles or aryl groups with ortho-or meta-substituent(s) at the C5 position seemed beneficial for enhanced anticancer activity. We were pleased to find that 5r bearing a 2-hydroxyphenyl at the C5 site had more potency than 5n. Interestingly, 5s with a 3-hydroxyphenyl moiety at the C5 position exhibited less cytotoxicity than 5r, indicating that the orientation of the hydroxyl in this scaffold is critical for potent activity. Thus, more derivatives were prepared in a similar way and their anticancer activities against HepG2 and Huh7 cells were evaluated (Table 3). In general, heterocycles or aryl groups with ortho-or meta-substituent(s) at the C5 position seemed beneficial for enhanced anticancer activity. We were pleased to find that 5r bearing a 2-hydroxyphenyl at the C5 site had more potency than 5n. Interestingly, 5s with a 3-hydroxyphenyl moiety at the C5 position exhibited less cytotoxicity than 5r, indicating that the orientation of the hydroxyl in this scaffold is critical for potent activity.   More analogs of 5r were synthesized to examine the effects of the substituent(s) of the 2-hydroxyphenyl moiety on inhibitory activity (Table 4). Unfortunately, an additional methoxyl or chlorine on the ring did not increase the potency.    To investigate the inhibitory effect of 5r on cell migration, we performed an in vitro wound-healing assay in HepG2 and Huh7 cells. As shown in Figure 3, 5r significantly inhibited cell migration in a dose-dependent manner in both HepG2 and Huh7 cells. , and HEK293T cells were cultured with 5r at the indicated concentrations for 72 h, and medium was replaced every 24 h with freshly added 5r. Cell viability was measured by MTS analysis (mean ± S.D., n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001. In vitro woundhealing assay was performed in HepG2 and Huh7 cells (mean ± S.D., n = 3). HepG2 and Huh7 cells were treated with the indicated concentrations of 5r, and time-lapse images were acquired every 2 h after wound infliction. (C,D) Representative images were taken at 0, 48, or 72 h following administration of 5r at the indicated concentrations.

5r Induces Activation of Caspase-3 and PARP Cleavage in HepG2 and Huh7 Cells
To investigate whether 5r induces apoptosis in HepG2 and Huh7 liver cancer cells, we observed caspase-3 activation and the cleavage of PARP, a hallmark of apoptosis, in HepG2 and Huh7 cells. Interestingly, 5r potently increased caspase-3 activity (green) in both HepG2 and Huh7 cells ( Figure 4A,B). In addition, caspase-3 activity was significantly increased by 5r in a dose-dependent manner, and the increased activity of caspase-3 was fully inhibited by Ac-DEVD-CHO, a specific caspase-3 inhibitor ( Figure 4C,D). In the case of PARP cleavage, 5r increased the expression level of cleaved PARP in a dose-dependent manner in HepG2 and Huh7 cells ( Figure 4E-H). These results suggest that 5r showed a significant anticancer effect by inducing apoptosis via the activation of caspase-3 and cleavage of PARP in HepG2 and Huh7 cells.

5r Dose Not Significantly Affect Cell Cycle of HepG2 Cells
To determine the effect of 5r on the cell cycle of HepG2 cells, we performed a flow cytometry analysis using propidium iodide staining. As shown in Figure 5, 5r significantly increased the ratios in the Sub-G1 (apoptotic peak) phase but did not affect the ratio in the G2/M phase in HepG2 cells. In the case of 5r, the G0/G1 phase decreased from 64.6% to 29.3% and the Sub-G1 phase increased from 24.1% to 56.9% compared with the control. These results suggest that 5r strongly increases apoptosis without a significant effect on cell cycle arrest.

Combination Effect of 5r with Gemcitabine on Cell Viability in HepG2 and Huh7 Cells
Gemcitabine exhibits broad activity in a variety of solid tumors and has shown potent activity against hepatocellular carcinoma cells in preclinical studies [19,20]. However, gemcitabine alone showed a response rate of 0-20% in phase 2 clinical trials [21][22][23][24][25][26]. To investigate the combinational effect of 5r with gemcitabine, a cell viability analysis was performed in HepG2 and Huh7 cells. As shown in Figure 6, interestingly, the co-treatment of 5r with gemcitabine showed an additional effect rather than a synergistic effect. These results suggest that 5r-based drug development could potentially increase the therapeutic effect in liver cancer.    for 72 h, and medium was replaced every 24 h with freshly added 5r and gemcitabine. Cell viability was determined by MTS analysis (mean ± S.D., n = 5). ** p < 0.01, *** p < 0.001.

General Methods
Unless specified, all reagents and starting materials were purchased from commercial sources and used as received without purification. "Concentrated" refers to the removal of volatile solvents via distillation using a rotary evaporator. "Dried" refers to pouring onto, or passing through, anhydrous magnesium sulfate followed by filtration. Flash chromatography was performed using silica gel (230−400 mesh) with hexane, ethyl acetate, and dichloromethane as the eluents. All reactions were monitored by thin-layer chromatography on 0.25 mm silica plates (F-254) visualized with UV light. Melting points were measured using a capillary melting point apparatus. Then, 1 H and 13 C NMR spectra were recorded on a 400 MHz NMR spectrometer and were described in terms of chemical shifts, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constant in hertz (Hz), and number of protons (see Supplementary Materials). HRMS was measured with an electrospray ionization (ESI) and Q-TOF mass analyzer.

Synthesis of 10
Compounds 10 were prepared with 9 (30.0 mg, 0.11 mmol, 1.0 equiv) by following the same procedures for the synthesis of 5.

Cell Viability Assay
Cell viability assay was conducted using Cell Titer 96 ® AQueous One Solution Cell proliferation Assay kit (Promega, Madison, WI, USA). Five cancer cells and HaCaT cells were plated in 96-well microplates with growth medium for 24 h. When cell confluency reached~30%, cells were treated with DMSO (vehicle) and test compounds. The medium and test compounds were replaced every 24 h. To measure cell viability, the cells were incubated with MTS solution for 1 h. The absorbance was measured by using Infinite M200 microplate reader (Tecan, Männedorf, Switzerland) at 490 nm. for C17H13N4 273.1135, found 273.1140.

Synthesis of 10
Compounds 10 were prepared with 9 (30.0 mg, 0.11 mmol, 1.0 equiv) by following the same procedures for the synthesis of 5.   Compounds 10 were prepared with 9 (30.0 mg, 0.11 mmol, 1.0 equiv) by following the same procedures for the synthesis of 5.

In Vitro Wound-Healing Assay
The suppressive effect of 5r on cell migration was assessed through in vitro woundhealing assay. HepG2 and Huh7 cells were grown to approximately 100% confluence to form a monolayer in a 96-well microplate. Wounds were formed through 96-well wound maker (Essen BioScience, Ann Arbor, MI, USA). Then, growth medium was removed and washed out twice with phosphate-buffered saline (PBS) and incubated with 200 µL of DMEM and RPMI 1640 medium containing 5r or vehicle (DMSO). Images of the wound area were taken by using IncuCyte ZOOM (Essen BioScience, Ann Arbor MI, USA), and the percentage of wound closure was measured by using IncuCyte software.

Caspase-3 Activity Assay
HepG2 and Huh7 cells were plated in 96-black-well plate to about 40% confluence, and then 5r was treated for 24 h. To assess activity of caspase-3, the growth medium was changed with 100 µL of PBS containing 1 µM caspase-3 substrate, NucView 488, and incubated at room temperature. After 20 min, 1 µM Hoechst 33342 was treated to stain the cells. Activity of caspase-3 was suppressed by Ac-DEVD-CHO, a selective caspase-3 inhibitor. FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) was used to assess the fluorescence of NucView 488 and Hoechst, and Lionheart FX Automated Microscope (BioTek, Winooski, VT, USA) was used to acquire the fluorescence microscopy images.

Western Blot Analysis
For Western blot analysis, HepG2 and Huh7 cells were lysed with RIPA buffer (50 mM Tris-HCl, PH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na 3 VO 4 , and protease inhibitor). Lysed samples were centrifuged at 13,000 RPM for 20 min at 4 • C to remove the cell debris, and equal amounts (60 µg protein/lane) of supernatant protein were divided by 4-12% Tris Glycine Precast Gel (KOMA BIOTECH, Seoul, Korea). Then, PVDF membranes (Millipore, Billerica, MA, USA) were used to transfer the separated proteins. Membrane blocking was performed by Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% bovine serum albumin (BSA) at room temperature for 50 min. The membranes were incubated with primary antibodies overnight at 4 • C with the indicated primary antibodies: anti-cleaved PARP (BD Biosciences) and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After incubating overnight, the membranes were washed out three times in 0.1% TBST and incubated with horseradish-peroxidaseconjugated secondary antibodies for 1 h. After 1 h, membranes were detected by using ECL Plus Western blotting detection system (GE Healthcare, Piscataway, NJ, USA).

Flow Cytometry Analysis
HepG2 cells were grown to approximately 40% confluence in a 6-well plate and then 5r was treated for 24 h. After 24 h, HepG2 cells were washed out twice with PBS and centrifuged at 1000 RPM for 2 min at room temperature. The cells were stained with propidium iodide for 15 min and then cell cycle phases were measured by using FACS (Beckman Coulter, Fullerton, CA, USA).

Conclusions
In summary, a new indolizine fused with pyrrolo[1,2-c]pyrimidine was designed and synthesized via one-pot three-component coupling followed by oxidative cyclization. This two-step protocol enabled the rapid construction of a novel tetracyclic heteroaromatic chemical library. The biological investigation of these compounds in five cancer cells revealed that some reduced the cell viability of HepG2 and Huh7 cells more strongly than those of the other three cancer cells. The structure-activity relationship study of these compounds led us to identify 5r showing potent and selective anticancer activity against liver cancer cells (HepG2 and Huh7). Notably, 5r had a weak inhibitory effect on the cell viability in nontumorigenic human keratinocytes (HaCaT) cells. Moreover, 5r strongly inhibited cell migration and induced apoptosis through increase of caspase-3 activity and cleavage of PARP in a dose-dependent manner in HepG2 and Huh7 cells. In addition, co-treatment of 5r with gemcitabine showed additional effect on cell viability in HegG2 and