Methoxylated Cinnamic Esters with Antiproliferative and Antimetastatic Effects on Human Lung Adenocarcinoma Cells

Lung cancer is the leading cause of cancer mortality worldwide, and malignant melanomas are highly lethal owing to their elevated metastatic potential. Despite improvements in therapeutic approaches, cancer treatments are not completely effective. Thus, new drug candidates are continuously sought. We synthesized mono- and di-methoxylated cinnamic acid esters and investigated their antitumor potential. A cell viability assay was performed to identify promising substances against A549 (non-small-cell lung cancer) and SK-MEL-147 (melanoma) cells. (E)-2,5-dimethoxybenzyl 3-(4-methoxyphenyl)acrylate (4m), a monomethoxylated cinnamic acid derivative, was identified as the lead antitumor compound, and its antitumor potential was deeply investigated. Various approaches were employed to investigate the antiproliferative (clonogenic assay and cell cycle analysis), proapoptotic (annexin V assay), and antimigratory (wound-healing and adhesion assays) activities of 4m on A549 cells. In addition, western blotting was performed to explore its mechanism of action. We demonstrated that 4m inhibits the proliferation of A549 by promoting cyclin B downregulation and cell cycle arrest at G2/M. Antimigratory and proapoptotic activities of 4m on A549 were also observed. The antitumor potential of 4m involved its ability to modulate the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway once phosphorylated-ERK expression was considerably reduced in response to treatment. Our findings demonstrate that 4m is a promising anticancer drug candidate.


Introduction
Cancer is one of the leading causes of human deaths worldwide, and thus remains a public health problem [1,2]. It is projected that with the growth and aging of the population, the global burden will reach 30.3 million new cancer cases and 16.3 million cancer deaths by 2040 [3].
Cancer treatment involves different therapeutic approaches, which depend on the type and stage of cancer [4]. Systemic treatment is widely used [5]; however, it causes severe side effects in patients [6]. Besides this, drug resistance acquisition in cancer cells is a crucial problem in cancer therapy [7,8]. Thus, the research for and development of new drugs that overcome the aforementioned problems are relevant.
Cutaneous melanoma is an aggressive skin cancer with high mortality rates [1, 22,23]. Classical chemotherapy, and immune and targeted therapies, are generally used for metastatic melanoma. Although these treatments have improved the clinical outcome of many patients, there are still either refractory cases or those exhibiting side effects [24][25][26][27][28].
Lung cancer is the most common cause of cancer-related death in men and the second most common in women [29]. Among the diagnosed cases, ~85% are classified as non-small-cell lung cancer (NSCLC), a group of histological subtypes in which lung adenocarcinoma is the most prevalent (~40% of cases) [30][31][32]. Systemic treatment for NSCLC uses different substances, including platinum-based drugs, molecular-targeted The antitumor potential of cinnamic acid and its derivatives [19][20][21] has been demonstrated. Niero et al. (2013) demonstrated cinnamic acid-induced apoptosis in HTT-144 human melanoma cells, enacted by disrupting the cytoskeleton [19]. Yen et al. (2011) reported that both cisand trans-cinnamic acids could inhibit the phorbol-12-myristate-13-acetate-stimulated invasive behavior of lung adenocarcinoma A549 cells [20].  demonstrated that cinnamic acid derivatives, such as caffeic acid, ferulic acid, and chlorogenic acid, inhibited the invasive behavior of A549 cells by modulating oncogenic signaling pathways, including mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) and phosphoinositide-3-kinase/protein kinase [21].
Cutaneous melanoma is an aggressive skin cancer with high mortality rates [1, 22,23]. Classical chemotherapy, and immune and targeted therapies, are generally used for metastatic melanoma. Although these treatments have improved the clinical outcome of many patients, there are still either refractory cases or those exhibiting side effects [24][25][26][27][28].
Lung cancer is the most common cause of cancer-related death in men and the second most common in women [29]. Among the diagnosed cases,~85% are classified as non-smallcell lung cancer (NSCLC), a group of histological subtypes in which lung adenocarcinoma is the most prevalent (~40% of cases) [30][31][32]. Systemic treatment for NSCLC uses different substances, including platinum-based drugs, molecular-targeted agents, and immune checkpoint agents [33][34][35][36][37][38]. Although therapeutic improvements have been achieved by introducing targeted immunotherapy, the mortality rate for lung cancer remains high. Based on the exposed scenario, it is imperative to advance the engineering of new chemical structures that can serve as starting structures for developing new chemotherapeutic agents, especially for cancers that are aggressive and resistant to available drugs.
We have been interested in synthesizing and biologically investigating compounds inspired by natural products that may have a promising antitumor effect against solid tumors, such as melanoma and lung adenocarcinoma [39][40][41][42]. Thus, herein, we describe the preparation of methoxylated cinnamic acid esters and cinnamides, and their cytotoxic profile against melanoma SK-MEL-147 and lung adenocarcinoma A549 cells. In addition, the antiproliferative and antimetastatic activities and proapoptotic effects of lead compound 4m on A549 cells were investigated.
White solid, purified by silica gel column chromatography, eluting with ethyl acetate To a 50 mL round bottom flask, benzyl alcohol (3a) (0.117 g, 1.08 mmol), dichloromethane (10.0 mL), (E)-3,4-dimethoxy cinnamic acid (2) (0.205 g, 0.986 mmol), DIC (0.124 g, 0.986 mmol), and DMAP (0.0120 g, 0.982 mmol) were added. The reaction mixture was magnetically stirred at room temperature for 45 min. After the reaction was completed, as TLC analysis confirmed, the reaction mixture was washed with distilled water (15.0 mL) and saturated sodium chloride solution (30.0 mL). The organic phase was reserved and the aqueous solution was extracted with dichloromethane (3 × 20.0 mL). The organic phases were combined, and the resulting organic layer was dried over sodium sulfate, filtered, and concentrated under residue pressure. The residue was submitted to purification using silica gel column chromatography, eluting with hexane/ethyl acetate (3:2 v/v). This procedure afforded compound 5a with 62% yield (0.182 g, 0.611 mmol). The following data support the structure of 5a.
White solid, purified by silica gel column chromatography, eluting with ethyl acetate

Cell Lines, Culture Cell Conditions and Sample Preparation
Herein, human tumor cells (lung adenocarcinoma-A549, and melanoma cell-SK-MEL-147) and normal cells (primary dermal fibroblast cell-CCD-1059Sk) were used. These cell lines were purchased from the Rio de Janeiro Cell Bank. Cells were maintained in DMEM/F12 (Dulbecco's Modified Eagle's Medium plus F12, Sigma Aldrich, Saint Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Vitrocell, Campinas, Brazil). Cells were grown in a humidified atmosphere of 95% air and 5% CO 2 under 37 • C.
The thirty-two synthesized compounds (monomethoxylates, 4a-4p and dimethoxylates, 5a-5p) and cinnamic acids (1 and 2) were dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich, Saint Louis, MO, USA), and the stock solution (20 mM) was stored at −20 • C until use. The compounds were solubilized in a fresh culture medium at final concentrations before experiments.

Screening Strategy and Cell Viability Assay
Synthetic compounds (4a−4p and 5a−5p) and cinnamic acids (1 and 2) were subjected to a screening assay that tested cell viability to select the most active compound(s) for subsequent assays.
Initially, cell viability was investigated using the sulforhodamine B (SRB) method [50]. For this purpose, A549 and SK-MEL-147 cells were seeded at a density of 1 × 10 4 per well in 96-well plates. After attachment (24 h), cells were treated for 48 h with various compounds at a concentration of 40 µM (concentration used for preliminary analysis of cell viability). Cell monolayers were fixed with 10% (w/v) trichloroacetic acid (Sigma Aldrich, Saint Louis, MO, USA) at 4 • C for 1 h and stained with SRB (0.4% in 1% acetic acid) for 30 min. Samples were washed repeatedly with 1% acetic acid to remove unbound SRB. The protein-bound dye was dissolved in 10 mM Tris base solution, and the optical density was determined at 540 nm with a reference of 690 nm using a microplate reader. Subsequently, the concentration capable of inhibiting 50% of cell viability (IC 50 ) was determined for the most active compound against A549, SK-MEL-147 and CCD-1059Sk. The concentrations used for dose-response curves were 0, 0.1, 1, 10, 100, and 1000 µM, and cell viability rates were determined using the SRB method, as previously described. DMSO (0.4% v/v) was used as a negative control and cisplatin as a positive control.

The Colony Formation Assay
A549 lung adenocarcinoma cells were used for the clonogenic assay in 35 mm plates (200 cells/plate). After cell attachment (24 h), cells were treated with compound 4m (20 and 40 µM) and DMSO (0.4%) for 24 h and then recovered in a drug-free medium for 12 days. Colonies were then fixed with methanol for 30 min and stained with crystal violet (1%) for 20 min. Only colonies with >50 cells were counted via direct visual inspection with a stereomicroscope at 20× magnification [51].

Apoptosis Detection using Annexin V/7-AAD
According to the manufacturer's instructions, a Guava Nexin ® kit (Merck Millipore, Darmstadt, Germany) was used to determine phosphatidylserine externalization. A549 cells were seeded in 24-well plates at a density of 1 × 10 5 cells/well. Cell cultures were treated with 20 and 40 µM 4m or 25 µM cisplatin for 24 or 48 h. Cells were then harvested via enzymatic digestion (Trypsin/EDTA, Sigma Aldrich, Saint Louis, MO, USA), and samples were centrifuged under 200× g for 5 min at 4 • C, washed with ice-cold PBS, and then 2 × 10 4 cells were suspended in 100 µL DMEM. Next, 100 µL of a mixed solution of buffered Annexin V-PE (apoptosis detection kit) and 7-aminoactinomycin D (7-AAD, viability staining solution) was added. Samples were read after 20 min of incubation at room temperature in a dark chamber. Analysis was performed via flow cytometry using GuavaSoft 2.7 software.

Metastatic Behavior Assays Cell Migration Assay
The wound healing assay was performed to evaluate the ability of compound 4m to inhibit cell migration using a modified previously published methodology [42]. A549 cells were seeded at a concentration of 1.6 × 10 5 cells/well on 24-well plates and allowed to reach confluence (90%) after overnight incubation at 37 • C and 5% CO 2 atmosphere. The monolayers were then injured with a sterile 2000 µL pipette tip. Cells were washed twice with PBS to remove detached cells and then treated with 5, 10 and 20 µM of compound 4m. DMSO (0.4% v/v) was used as a control. Photomicrographs were taken using a camera connected to an inverted microscope (40× magnification, Zeiss, Oberkochen, Germany). Wound closure rates were then quantitatively calculated as the difference between wound width at 0 h and 36 h. The area of the wound was quantified using the public program ImageJ (NIH, Bethesda, ML, USA). Results are expressed as a percentage of cell migration.

Cell Matrix Adhesion Assay
First, trypsinized A549 cells were treated with 5, 10, and 20 µM of compound 4m or vehicle DMSO (0.4% v/v) for 30 min. Cells were then plated in 96-well plates (3 × 10 4 cells/well), coated with matrigel (60 µL/well), and incubated at 37 • C for 4 h for adhesion. Then, the cells were washed with PBS, and the adherent cells were stained with toluidine blue (1% v/v, Sigma Aldrich, Saint Louis, MO, USA) and solubilized with SDS (1% w/v) at 37 • C for 30 min. The absorbance was measured at 540 nm. Photomicrographs were taken using a camera coupled to an inverted microscope (40× magnification; Oberkochen, Germany) [42].

Transwell Invasion Assays
This assay was performed using transwell inserts with a pore diameter of 8 µm (Millicell, Ireland) based on protocols previously described [42]. Briefly, 60 µL of matrigel (BD Biosciences, São Paulo, São Paulo State, Brazil) diluted in serum-free medium (1:5) was added to the upper chambers, and incubated at 37 • C for 1 h. Subsequently, A549 cells were resuspended with serum-free DMEM/F12, treated with 5, 10, and 20 µM 4m, and inoculated into a transwell chamber coated with matrigel (8 × 10 4 cells, 400 µL/well). DMSO vehicle treatment (0.4% v/v) was used as a control. The lower chamber was filled with 500 µL of culture medium containing 20% v/v FBS as the chemoattractant. After 24 h, the chambers were fixed in methanol for 30 min, washed, and stained with toluidine blue (1% v/v) for 15 min. Cells were counted using an inverted microscope (BEL Photonics, Piracicaba, São Paulo State, Brazil).

Statistical Analysis
The results were tested for significance using a t-test or one-way analysis of variance (ANOVA) followed by Dunnett's posttest using GraphPad Prism ® 6.0. p values < 0.05 were considered statistically significant. Values are expressed as mean ± standard deviation.

Preparation of Methoxylated Cinnamic Esters
A set of compounds derived from methoxylated cinnamic acids 1 and 2 was prepared, as depicted in Scheme 1, via Steglich esterification [53] of the carboxylic acids with different hydroxylated compounds, following the reported procedures described by Sova et al. [54]. Under the mild reaction conditions, thirty cinnamic acid derivatives were prepared with yields ranging from 57% to 86%. The compounds were characterized using nuclear 1 H and 13 C NMR and infrared (IR) spectroscopic techniques. The coupling constants of the hydrogens in the aliphatic double bonds (shown in red in Scheme 1) were 15. 3-16.4 Hz, which agree with the trans configuration for the aliphatic double bonds. Carbony stretchings of the cinnamic acid derivatives shown in Scheme 1 were observed at ∼1700 cm −1 . Besides this, carbonyl functionality was confirmed from the signal observed at δC ≈170 ppm.
We also attempted to conduct the Steglish reaction between the hydroxylated nat-Scheme 1. Preparation of cinnamic ester derivatives 4a-4o and 5a-5o.
Under the mild reaction conditions, thirty cinnamic acid derivatives were prepared with yields ranging from 57% to 86%. The compounds were characterized using nuclear 1 H and 13  the cinnamic acid derivatives shown in Scheme 1 were observed at~1700 cm −1 . Besides this, carbonyl functionality was confirmed from the signal observed at δ C ≈ 170 ppm.
We also attempted to conduct the Steglish reaction between the hydroxylated natural product bisabolol and the acids 1 and 2 (Scheme 2). However, the expected esters were not synthesized. Instead, the cinnamides 4p and 5p were obtained owing to the steric hindrance around the hydroxy group in bisabolol, which prevented ester formation. Once synthesized, the compounds 4a-4p and 5a-5p were submitted to a screening assay, using cell viability as a parameter to select the most active compound against lung adenocarcinoma cell lines (A549) and melanoma cells (SKMEL-147).

Cytotoxicity of Compound 4m against Lung Cancer Cells and Dermal Fibroblast
The cytotoxic activity of compounds (4a-4p) and (5a-5p) derived from methoxylated cinnamic acids was investigated against two human tumor cell lines, lung cancer (A549) and melanoma (SK-MEL-147). The cell viability assay results show that at 40 μM the monomethoxylated cinnamic acid esters 4b, 4c, 4m, 4n, and 4o decreased the relative viability of A549 cells compared to the DMSO-treated cells (Figure 2A). In addition, compound 4m also decreased the viability of the SK-MEL-147 cells compared with those treated with DMSO ( Figure 2A). As for the dimethoxylated compounds (5a-5p), none of them showed remarkable cytotoxicity (Figure 2A). These results show that the monomethoxylated compound 4m was the most active in decreasing the cell viability in both studied cell lines. However, its effect was more pronounced on the A549 cell line (cell viability = 44.83%) than on the SK-MEL-147 cell line (cell viability = 58.90%).
We constructed the dose-response curves to determine the IC50 values of 4m on A549 and SK-MEL-147 cell lines ( Figure 2B,C). The IC50 results confirm that 4m was more effective against the A549 cell line (IC50 = 40.55 ± 0.41 μM) than the SK-MEL-147 cell line (IC50 = 62.69 ± 0.70 μM), thus showing better cytotoxic activity on lung cancer. However the compound 4m did not show an IC50 value lower than that of cisplatin on both cells tested. We also investigated the cytotoxicity of compound 4m against normal cells (dermal fibroblast cells); no relevant results were observed ( Figure 2C). These findings support the selection of compound 4m for future studies against the A549 cell line.
A clonogenic assay was performed to examine the effect of 4m on the proliferation Once synthesized, the compounds 4a-4p and 5a-5p were submitted to a screening assay, using cell viability as a parameter to select the most active compound against lung adenocarcinoma cell lines (A549) and melanoma cells (SKMEL-147).

Cytotoxicity of Compound 4m against Lung Cancer Cells and Dermal Fibroblast
The cytotoxic activity of compounds (4a-4p) and (5a-5p) derived from methoxylated cinnamic acids was investigated against two human tumor cell lines, lung cancer (A549) and melanoma (SK-MEL-147). The cell viability assay results show that at 40 µM, the monomethoxylated cinnamic acid esters 4b, 4c, 4m, 4n, and 4o decreased the relative viability of A549 cells compared to the DMSO-treated cells (Figure 2A). In addition, compound 4m also decreased the viability of the SK-MEL-147 cells compared with those treated with DMSO ( Figure 2A). As for the dimethoxylated compounds (5a-5p), none of them showed remarkable cytotoxicity (Figure 2A). These results show that the monomethoxylated compound 4m was the most active in decreasing the cell viability in both studied cell lines. However, its effect was more pronounced on the A549 cell line (cell viability = 44.83%) than on the SK-MEL-147 cell line (cell viability = 58.90%).
We constructed the dose-response curves to determine the IC 50 values of 4m on A549 and SK-MEL-147 cell lines ( Figure 2B,C). The IC 50 results confirm that 4m was more effective against the A549 cell line (IC 50 = 40.55 ± 0.41 µM) than the SK-MEL-147 cell line (IC 50 = 62.69 ± 0.70 µM), thus showing better cytotoxic activity on lung cancer. However, the compound 4m did not show an IC 50 value lower than that of cisplatin on both cells tested. We also investigated the cytotoxicity of compound 4m against normal cells (dermal fibroblast cells); no relevant results were observed ( Figure 2C). These findings support the selection of compound 4m for future studies against the A549 cell line.

Cell Cycle Arrest and Apoptosis Analysis of Compound 4m in A549 Cells
Cell cycle analysis showed that the progression dynamic was not altered in A549 cells treated with 20 μM 4m, but an increase in the G2/M population was observed in A549 cells treated with 40 μM 4m, indicating its ability to induce cell cycle arrest. Concurrent with the increase in the G2/M population treated with 40 μM 4m, there was a considerable increase in the sub-G1 population, suggesting the cytotoxic activity of A clonogenic assay was performed to examine the effect of 4m on the proliferation dynamics of A549 cells. Our results show that 4m has an antiproliferative effect on this cell line, as the abundance of colonies was lower in the groups treated with 20 and 40 µM compared to the cells treated with vehicle (0.4% v/v DMSO) ( Figure 2D).

Cell Cycle Arrest and Apoptosis Analysis of Compound 4m in A549 Cells
Cell cycle analysis showed that the progression dynamic was not altered in A549 cells treated with 20 µM 4m, but an increase in the G2/M population was observed in A549 cells treated with 40 µM 4m, indicating its ability to induce cell cycle arrest. Concurrent with the increase in the G2/M population treated with 40 µM 4m, there was a considerable increase in the sub-G1 population, suggesting the cytotoxic activity of compound 4m at this concentration ( Figure 3A,B). Since treatment with 40 µM 4m showed a considerable result in the cell cycle analysis, this concentration was selected to evaluate the expression profile of cyclin B at protein levels. Thus, it was observed that cell cycle arrest was accompanied by a decrease in cyclin B expression in A549 cells treated with 40 µM 4m for 24 and 48 h ( Figure 3C). compound 4m at this concentration ( Figure 3A,B). Since treatment with 40 μM 4m showed a considerable result in the cell cycle analysis, this concentration was selected to evaluate the expression profile of cyclin B at protein levels. Thus, it was observed that cell cycle arrest was accompanied by a decrease in cyclin B expression in A549 cells treated with 40 μM 4m for 24 and 48 h ( Figure 3C).
Considering the significant increase in G2/M population in A549 cells treated with 40 μM 4m previously observed in cell cycle analysis ( Figure 3A,B), we investigated the proapoptotic potential of 4m in A549 cells using the Annexin V assay ( Figure 3D,E). In this experimental approach, cisplatin was used as a positive control to validate the data obtained using flow cytometry. We found an increased frequency of Annexin V-positive cells in cells treated with 20 and 40 μM 4m for 48 h (Figure 3D,E).  Considering the significant increase in G2/M population in A549 cells treated with 40 µM 4m previously observed in cell cycle analysis ( Figure 3A,B), we investigated the proapoptotic potential of 4m in A549 cells using the Annexin V assay ( Figure 3D,E). In this experimental approach, cisplatin was used as a positive control to validate the data obtained using flow cytometry. We found an increased frequency of Annexin V-positive cells in cells treated with 20 and 40 µM 4m for 48 h (Figure 3D,E).

Effect of Compound 4m on the Metastatic Behavior of A549 Cells
To investigate the effect of compound 4m on A549 cell migration, we performed a wound-healing assay with 5, 10, and 20 µM 4m, and evaluated cell motility. We found that compound 4m at concentrations of 10 and 20 µM could inhibit cell migration compared to DMSO-treated cells ( Figure 4A,B). In addition, we investigated the effect of compound 4m on the invasion and adhesion of A549 cells. The invasion of A549 cells decreased after 24 h of treatment with 5 (70.4%), 10 (91%), and 20 (96.6%) µM of compound 4m compared with DMSO-treated cells ( Figure 4B,C). Similarly, cell adhesion was significantly decreased at 5 (20.8%), 10 (24.3%), and 20 µM (27%) 4m compared to DMSO-treated cells ( Figure 4E,F).

Discussion
The antitumor potential of cinnamic acid and its derivates have been previously reported [14,55,56]; however, few studies explore methoxylated cinnamic esters as antitumor prototypes. Thus, this study aimed to synthesize two series of compounds (monomethoxylated and dimethoxylated cinnamic esters) to evaluate their antitumor potential. Thirty cinnamate esters (compounds 4a-4o obtained from 4-methoxy cinnamic acid and 5a-5o derived from 3,4-dimethoxy acid, Figure 1) were obtained. Two cinnamides (compounds 4p and 5p) were also prepared when attempting to synthesize corresponding esters by reacting the methoxylated cinnamic acids and bisabolol. These substances were submitted to in vitro screening tests [57], and compound 4m was eligible for further investigations based on its cytotoxic profiles on tumor and normal cells. Herein, the IC 50 values obtained for 4m in this study were much lower than other di-or trimethoxylated cinnamic acid derivatives previously reported [58,59].
The cancer cells used as a study model in this investigation harbor mutations in the RAS oncogene, responsible for the hyperactivation of RAS signaling [60,61] and downstream signaling pathways, including MAPK/ERK. The SK-MEL-147 cells have a mutation in NRAS (Q61R) [60], whereas the A549 cells cause a mutation in KRAS (G12S) [61]. The A549 cells also harbor an inactivation mutation in the tumor suppressor gene CDKN2A, which encoded p16 INK4 and p14 ARF proteins by alternative splicing. These proteins negatively regulate cell cycle progression via retinoblastoma protein (pRB) and p53 gene activity, respectively. Considering that A549 cells were more responsive to 4m treatment than SK-MEL-147 cells, they were used for further investigations.
The antiproliferative activity of 4m on A549 cells was demonstrated using clonogenic assay and cell cycle analysis. There was a considerable reduction in colony frequencies in samples treated with 4m, suggesting that the treatment drastically affected the clonogenic capacity of A549 cells. Apparently, the size of the colonies in 4m-treated samples was smaller than the colonies in the control group, suggesting that the cells exposed to the tested compound had fewer cycles of division than the non-exposed samples. In fact, 4m inhibited the cell cycle progression in A549 cells, as demonstrated by a considerable increase in the G2/M population in A549-treated cultures. The effects of cinnamic acid or its derivatives on the cell cycle of lung adenocarcinoma cells have not been studied yet; however, our results are in accordance with Anantharaju et al. (2017), [57] who showed the inhibitory effect of dihydroxy cinnamic acid (caffeic acid) on the cell cycle progression of human colon cancer cells (HCT116 and HCT-15). They showed that cancer colon cells were arrested in S and G2/M phases in response to treatment with caffeic acid. The pharmacological agents that can interrupt the cell cycle progression are recognized as potential anticancer agents [55].
The G2/M transition is highly regulated by the orchestrated activity of kinase and phosphatase proteins. Importantly, the activation of cyclin-dependent kinase 1 (CDK1)cyclin B complexes represents a critical event in the transition from G2 to the M phase [62,63]. Thus, the expression levels of cyclin B were assessed using Western blotting. Compound 4m at 40 µM considerably reduced the cyclin B expression in A549 cells. This finding is very promising and indicates that the inhibitory effect of 4m on the proliferation of A549 is associated with cyclin B downregulation. Recently, it was reported that cyclin B is highly expressed in patients with lung adenocarcinoma, which was associated with a poor prognosis [64]. In addition, Wang et al. (2019) [65] demonstrated that cyclin B is not involved in the apoptosis induction, migration, and invasion of NSCLC, but is directly associated with proliferation cycles. The reduction in cyclin B levels promoted cell cycle arrest at G2/M, diminishing the dimension of xenograft tumors.
Cell cycle analysis also revealed an accumulation of cells in the SubG1 phase in A549 cultures treated for 48 h with 4m, suggesting that this compound can induce cell death. Cell cycle arrest may trigger signals culminating in apoptosis [66]. Thus, the proapoptotic activity of 4m on A549 cells was investigated. The results show an increased frequency of cells positive for annexin V in treated cultures when compared to controls, suggesting that the cytotoxic activity of 4m on A549 cells, at least in part, involves apoptosis induction. The eduction of cyclin B, cell cycle arrest at G2/M, and apoptosis induction were reported in A549 cells treated with natural diterpenoids [67,68].
Considering that MAPK signaling pathway is associated with cell proliferation and survival [69] and that A549 cell harbor activating mutation in KRAS, the influence of 4m on the MAPK/ERK signaling pathway was investigated. The Western blotting analysis revealed that 4m considerably reduced p-ERK expression, suggesting that RAS/RAF/MEK/ERK signaling was attenuated in A549 cells in response to treatment, reinforcing the antitumor activity of 4m. Cell cycle arrest at G2/M, apoptosis induction, and reduced ERK activation levels were also observed in NSCLC cell lines (H2122, H358, H460) containing KRAS mutation in response to small molecule 0375-0604 treatment, a KRAS inhibitor according to docking studies [70]. Similar results were found by Li et al. (2021) [71], who treated NSCLC (H358) with stapled peptide-based RAS inhibitors. In addition,   [21] reported that cinnamic acid (cis-CA and trans-CA) could inhibit the MAPK/ERK signaling in A549 cells. Therefore, the findings obtained in this work suggest that the antiproliferative activity of 4m on A549 cells is related to its ability to modulate the MAPK/ERK signaling pathway and regulate the cell cycle.
Since metastasis is the general cause of cancer deaths, including lung cancer [72], some in vitro parameters such as adhesion, migration, and invasion were evaluated to predict the ability of 4m to inhibit lung cancer metastasis. The results show that 4m could inhibit the adhesion, migration, and invasion capacity of A549 cells. Furthermore, in vivo studies should be performed to validate these in vitro findings; however, our data are in accordance with those of   [56], who described the antimetastatic potential of cinnamic acid derivatives using A549 cell line as study model. We have previously reported that phenyl 2,3-dibromo-3-phenylpropanoate, a cinnamic acid derivative compound, has antimetastatic potential against melanoma cells [42].
The synthesis of cinnamate esters was planned to assess the effects of different R groups (benzyl, phenyl and alicyclic groups) against the aforementioned tumor cell lines. Compound 4m was derived from 4-methoxy cinnamic acid, in which R corresponds to the 4-methoxy benzyl. Thus, herein, combining the 4-methoxy cinnamoyl portion with the 4-methoxy-benzyloxy group resulted in the most effective compound, 4m, on the tumor cell lines.

Conclusions
Herein, thirty-two cinnamic acid derivatives were synthesized and characterized using spectroscopic methods. Compound 4m, a monomethoxylated cinnamate ester derivative, was identified as the lead compound based on cytotoxicity studies against SK-MEL-147 (melanoma) and A549 (lung adenocarcinoma) cells. It effectively inhibited the proliferation, migration, and invasion of A549 cells. The antiproliferative activity of 4m on A549 cells was associated with its ability to reduce cyclin B expression and ERK activation, leading to cell cycle arrest at G2/M and, ultimately, apoptosis. Our findings indicate that combining a 4-methoxy cinnamoyl portion with the 4-methoxy-benzyloxy group may improve the antitumor activity of methoxylated cinnamic acid derivatives.  Table S1: Crystallographic data and refinements for compound 5o; Figure S112: Asymetric unit of compound 5o.