Discovery of Dihydropyrrol-2-ones as Novel G0/G1-Phase Arresting Agents Inducing Apoptosis

A series of dihydropyrrol-2-ones (DHPs) were designed and synthesized via an efficient multicomponent reaction at room temperature for evaluation of their bioactivities against four human cancer lines (MCF-7, RKO, HeLa, and A549) in vitro. Preliminary structure–activity relationship studies showed that R4 = 3-MeO-4-OH-Ph is a crucial group for increasing cytotoxicities against RKO cells and the influences of R1–R3 depend on their combination. It was found that DHPs 5a, 5q, and 5s showed the best antiproliferative activities against A549, RKO, and all four studied cell lines, respectively (IC50 = 1.9, 0.8, and 0.9–2.4 μM). They can be used as new lead compounds for developing potentially selective or broad spectrum anticancer agents. 5q proves as a potent G0/G1-phase arresting agent inducing cell apoptosis by increasing/decreasing the levels of p53 and p21/cyclin D1.


■ INTRODUCTION
Cancer, the second leading cause of global death, was responsible for 8.8 million deaths in 2015, amounting to nearly 1 in 6 of all deaths in the world. 1 Therefore, many different fields have devoted much effort to exploring anticancer therapeutics. In cell cycles, there are two important transitions, the transitions of the G1 to S phase and G2 to M phase. These transitions are very important for regulating cellular multiplication processes and cell apoptosis, and are easily affected by environmental conditions. The tumorigenesis of many cancers is closely related to the improper signaling of cell cycle regulators. G0/G1-phase arresting agents can arrest the cellular proliferating cycle progressing from the G0/G1 to S phase. Palbociclib (I, Figure 1), 2 first approved as a CDK4/6 inhibitor for breast cancer in 2015, arrests tumorigenesis by interfering with S and M phases of the cell cycle and withdraws the cell cycle back into the G0/G1-phase. Two other CDK4/6 inhibitors, abemaciclib (Lilly) (II) 3 and ribociclib (III), 4 are approved for the treatment of multiple types of cancers. Compound IV exhibits potent activity against breast cancer cells, arresting cells in the G0/G1-phase and decreasing the cellular levels of cyclin D1. 5 G0/G1-phase arresting agents V 6 and VI 7 show good anticancer activity against SH-SY5Y cells and HCT-116 colon cancer cells, respectively.
Multicomponent reaction (MCR) has advantages of atom economy, high efficiency, and fast building structural diversity and complexity of compound libraries 8−10 and becomes a powerful tool in drug synthesis and discovery. 9,11,12 Considering that heterocycles have diverse bioactivities, we are interested in their MCR synthesis 13−17 and bioactivities. 18 Pyrrolidone rings are important heterocycles with a wide range of pharmacological activities, such as G0/G1-phase arresting agents VI. In this work, we report novel G0/G1-phase arresting agents pentasubstituted dihydropyrrol-2-ones (DHPs) (Table 1), the products of a convenient MCR that we developed. 13

■ RESULTS AND DISCUSSION
In the experimental screening of the bioactivities against MCF-7 (human breast cancer), RKO (human colon cancer), HeLa (human cervical cancer), and A549 (human lung cancer) cell lines of small organic compounds, it was found that DHP 5a has good activity against the four cell lines, especially A549. Then, 19 DHPs (5b−5t) were designed and synthesized to further study their bioactivities using 5-fluorouracil (5-Fu) as a positive control. Their antiproliferative activities were expressed as IC 50 values and are summarized in Table 1. As shown in Table 1, most of the DHPs exhibited moderate to satisfactory antiproliferative activities against the four human cancer cell lines. Among these DHPs, 5a displayed higher cytotoxicities against A549 cells (IC 50 = 1.9 μM) than the other three human cancer cells, with the IC 50 value being 7.7fold higher than that of 5-Fu (IC 50 =14.7 μM). DHP 5b showed higher cytotoxicities against RKO, HeLa, and A549 cells than MCF-7, with IC 50 values one to 6-fold higher than that of 5-Fu. Moreover, DHP 5q demonstrated the best antiproliferative activity (IC 50 = 0.8 μM) against the RKO cell line. DHPs 5c and 5r−5t showed satisfactory antiproliferative activities against all of the tested cancer cells.
Based on the above primary antiproliferative activities, the structure−activity relationship of 5 was discussed. As for R 4 , 4-CF 3 Ph is in favor of the antiproliferative activities against the four cell lines (2.7−4.6 μM) than other groups (comparing 5c and 5a−5h except 5c). Very interestingly, the combination of 3-MeO and 4-OH functional groups at phenyl shows excellent selectivity to the RKO cell line (5h, 2.4 μM). However, 4-OH (5d, 91.5 μM), 3-MeO (5e, >100 μM), 4-MeO (5f, 34.6 μM), and the combination of 4-MeO and 3-OH (5g, 60.7 μM) show much lower or almost no activities to the RKO cell line. Therefore, using R 4 = 3-MeO-4-OHPh and R 1 = Et, the influences of R 2 and R 3 on the selectivity were investigated (5i−5q). It can be seen that except 5o and 5p with alkyl R 2 and R 3 , others with aryl R 2 and R 3 show much higher activity against the RKO cell line than the other three cell lines, with the combination of R 2 = Ph and R 3 = 3-ClPh optimal (5q) against RKO cells. Unexpectedly, only changing R 1 from Et (5i, 5l, and 5n) to Me (5r−5t) leads to a significant decrease of the selectivity with the increase of the activities against all cell lines. This means that R 1 is also a very important group. The obtained experimental results indicate that R 4 = 3-MeO-4-OHPh is crucial for the antiproliferative activities against RKO cells and the influences of R 1 −R 3 depend on their combinations.
To understand the primary mechanism of action, the most potent DHP 5q against RKO cells was used to investigate the effects on the cell cycle distribution and apoptosis in RKO cells via flow cytometry. As shown in Figure 2A, DHP 5q arrested the RKO cells at the G0/G1 phase in a dose-dependent manner and induced the increase of RKO cells at the G0/G1 DHPs were synthesized by the MCR that we developed, 13 and all are new compounds except 5a. b Antiproliferative activity (half maximal inhibitory concentration (IC 50 )) determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after 72 h of incubation. Data expressed as mean ± standard deviation from at least three independent experiments. c Synthesized as other DHPs except using ethanol as solvent and under 70°C. phase from 31.33% (the blank control group) to 59.81% (5 μM). Concurrently, a reduction of RKO cells at G2/M and S phases was observed. The apoptotic effect of DHP 5q was investigated by Annexin V-FITC/propidium iodide analyses, using dimethyl sulfoxide (DMSO) as the control. As shown in Figure 2B, DHP 5q induced 30% apoptosis at higher concentrations.
It is well known that D-type cyclins (cyclins D1, D2, and D3) are involved in the regulation of apoptosis and cell cycle progression from the G0/G1-phase into the S phase. 19 Overexpression of Cyclin D1 has been reported to be associated with shorter survival in patients, and its high expression generally increases migration and invasion of tumors. 20 The tumor suppressor, p53 protein, could induce tumor apoptosis and cell cycle arrest. 21 p21 is considered as an independent prognostic parameter for colorectal cancer, and its overexpression is positively correlated with the survival time of patients, which may be related to the inhibition of tumor cell proliferation by blocking the cell cycle in the G1 phase. 22 Therefore, the effect of DHP 5q on p53, p21, and cyclin D1 was investigated to explore the mechanism of 5q that induced the G0/G1-phase arrest. As shown in Figure 3, the expression of p53 and p21 proteins was increased by the presence of DHP 5q (5 μM), while the expression of cyclin D1 was inhibited (5 μM), which is consistent with the results of the cell cycle and apoptosis assay.

■ CONCLUSIONS
Twenty DHPs 5a−5t (all are new compounds except 5a) were designed and synthesized for their bioactivity evaluation against four human cancer cell lines (MCF-7, RKO, HeLa, and A549). It was found that R 4 = 3-MeO-4-OH-Ph is a crucial group for increasing cytotoxicities against RKO cells and the influences of R 1 −R 3 depend on their combination, with the most potent DHP 5q against RKO cells (IC 50 = 0.8 μM) and three potent DHPs (5r−5t) against four studied cancer cell lines (IC 50 = 0.9−3.9 μM) containing 3-MeO-4-OH-Ph as the R 4 group. Interestingly, the study on the primary mechanism of 5q against RKO cells proves 5q as a G0/G1-phase arresting agent inducing cell apoptosis by decreasing the levels of cyclin D1 but increasing the levels of p53 and p21 in RKO cells. The advantages of an efficient synthetic method, simple molecular structure, and potential antiproliferative activity against multiple cancer cell lines are expected to make the most potent anticancer DHPs 5a (1.9 μM), 5q (0.8 μM), and 5s (0.9−2.4 μM) against A549, RKO, and all studied four cell  lines, respectively, attractive lead compounds for developing potential selective or broad spectrum anticancer agents.

■ EXPERIMENTAL SECTION
General Procedures for the Synthesis of Pentasubstituted 2,5-Dihydro-1H-pyrrol-2-ones 5a−5t. Pentasubstituted 2,5-dihydro-1H-pyrrol-2-ones 5a−5t were synthesized as reported in our previous work. 13 The reactions were run with the following steps: (a) but-2-ynedioates (1 mmol) and amines (R 2 NH 2 , 1 mmol) were added into test tube A with 3 mL of MeOH and kept at room temperature for 30 min; (b) amines (R 3 NH 2 , 1.6 mmol), aldehydes (3.5 mmol), salicylic acid (0.3 mmol), and Cu(OAc) 2 ·H 2 O (0.4 mmol) were added into test tube B with 3 mL of MeOH, and then the mixture in tube B was added into the above mixture in tube A and stirred at room temperature for desired time. After completion of the reactions, the product mixture was purified by preparative thin layer chromatography with petroleum ether/ethyl acetate (15:1−3:1) as the eluent to afford the desired products. It is worth mentioning that the procedures for the synthesis of 5o and 5p are the same as those mentioned above except EtOH and 70°C, instead of MeOH and room temperature, were used as solvent and temperature, respectively.
Cell Culture. The human breast cancer MCF-7, colon cancer RKO, cervical cancer HeLa, and nonsmall cell lung cancer A549 were obtained from American Type Culture Collection (ATCC). All cells were maintained in RPMI-1640 medium (Gibco) with 10% fetal bovine serum (Capricorn Scientific GmbH, Germany) at 37°C in a humidified incubator with 5% CO 2 .
Cell Proliferation Assay. MTT assay is a quantitative colorimetric method for the determination of cell survival and proliferation. 23,24 The assessed parameter is the metabolic activity of viable cells. Metabolically active cells reduce pale yellow tetrazolium salt (MTT) (Sigma) to a dark blue waterinsoluble formazan, which can be directly quantified after solubilization with DMSO. The absorbance of the formazan directly correlates with the number of viable cells. Cells were plated at a density of 2 × 10 3 cells/well in 96-well plates. On the following day, the cells were incubated with tested compounds for at least three cell doublings. At the end of the treatment period, 10 μL of MTT solution (5 mg/mL) was added and incubated for another 4 h. After the supernatant was carefully removed, 150 μL of DMSO was added for solubilization for about 10 min and the absorbance at 570 nm was measured using a microplate spectrophotometer (Benchmark Plus, Bio-Rad Laboratories, CA). 25 25 Cell Cycle Analysis. Flow cytometry analysis was performed as a reported method. 26 Generally, after 24 h incubation, the cells were treated with compound 5q for different concentrations, respectively. Following treatment, the cells were harvested, washed with cold phosphate-buffered saline (PBS) twice, and fixed in 70% ethanol at −20°C for appropriate time. The cells were then washed twice with cold PBS and incubated with RNAase and propidium iodide (50 μg/mL) for 30 min at 37°C in the dark. The percentages of cell cycle distribution were analyzed on a BD LSRFortessa Flow Cytometer (BD Accuri Cytometers Inc.; Ann Arbor, MI).

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b02543.

Notes
The authors declare no competing financial interest.