Novel synthetic derivatives of cinnamic and p-coumaric acids with antiproliferative effect on breast MCF-7 tumor cells

Abstract p-Coumaric acid is derived from cinnamic acid and is one of the major compounds in the Brazilian green propolis extract. Studies have shown that both p-coumaric acid and cinnamic acid have promising antiproliferative effects. In this context, aiming to increase the complexity of these active natural products and their activities, we performed coupling reactions with propargylamine and benzylamine, as well as with threonine, phenylalanine and lysine amino acids, aiming to enhance their antiproliferative effects towards the hormone-dependent breast cancer MCF-7 cells. Overall, the p-coumaric acid coupling with L-threonine amino acid (compound 15) had the best selectivity index (SI = 5.1), with half-maximal inhibitory concentration of 39.6 ± 1 μM, showing a high selectivity against hormone-dependent breast cancer cell lines MCF-7 and low cytotoxicity against the normal breast cell lines MCF-10A. Thus, this new natural product derivative may represent a prototype for the future development of antiproliferative agents, especially against hormone-dependent breast cancer. Graphical Abstract


Introduction
Propolis is a resinous material made by honeybees from different plant materials, composed mainly of vegetative fragments and plant exudates.The composition of propolis is closely related to the flora of each region, and the called 'green propolis' or Brazilian propolis is produced mainly by Baccharis dracunculifolia, a shrub widely distributed in the Brazilian cerrado (Rodrigues et al. 2020;Sepúlveda et al. 2020).The p-coumaric acid, a phenolic compound derived from cinnamic acid, is one of the majority compounds in the Brazilian green propolis extract.p-coumaric acid is also found in several plants, vegetables, fruits and cereals.The biological activities of both p-coumaric and cinnamic acid are already well-known in the literature, showing several anti-mutagenic, anti-inflammatory, anti-oxidant, anti-proliferative and anti-microbial effects (Janicke et al. 2005;endo et al. 2012;Ferreira et al. 2021;Mariadoss et al. 2022).
According to the study carried out by Serafim et al. six caffeic acid derivatives were tested against three breast cancer cell lines (MCF7, MDA-MB-231 and hS578T) and it was reported that these derivatives were able to inhibit cell proliferation and induce cell cycle changes and death, especially in MCF-7 cell lines (Serafim et al. 2011).Another study evaluated the activity of phenolic acids found in green propolis: caffeic, dihydroxycinnamic and p-coumaric acids on four triple-negative breast cancer cell lines (BT-20, BT-549, MDA-MB-231 and MDA-MB-436).The results showed that propolis was able to reduce the viability of BT-20 and BT-549 cells, while p-coumaric acid induced cytotoxic effects in four analyzed triple-negative breast cancer cell lines (Assumpção et al. 2020).
Several chemical processes are based on the privileged structural scaffolds of amino acids and amides for improving physicochemical properties, metabolic stability and biological activities of natural precursors.In this context, coupling reactions with different amines are a simple and easy way in organic chemistry to increase the complexity of active natural products and their activities (de Castro et al. 2020).Recently, Rodrigues and et al. performed coupling reactions with L-amino acids and prenylated compounds from green propolis showing that they enhanced the antiproliferative effects against MCF-7 when compared to their natural precursors (Rodrigues et al. 2021).Following this trend, herein we describe the synthesis of eleven derivatives (Scheme 1) from cinnamic 1 and p-coumaric acids 2 by coupling reactions with propargylamine and benzylamine, as well as with threonine, phenylalanine and lysine amino acids, aiming to enhance their biological activities, especially their antiproliferative effects, towards the hormone-dependent breast cancer MCF-7 cells.

Isolation of p-coumaric acids 2
Pure p-coumaric acid 2 was isolated from the green propolis extract.About 300 g of green propolis was first pulverized, extracted with a hydroalcoholic solution (9:1) and after fractioned by classic chromatographic column with hexane/ethyl acetate (95:05 to 85:15 v/v) gradient elution (Ferreira et al. 2021).each obtained fraction was analyzed by hPLC to pick an assignment with the authentic standards, as well as after purification by preparative hPLC-uV (Rodrigues et al. 2020).The purity of p-coumaric acid, as estimated by hPLC-PDA, was higher than 97% and was confirmed by 1 h and 13 C NMR analyses.

Synthesis of novel derivatives from cinnamic and p-coumaric acids
The synthesis of thirteen new compounds derived from cinnamic 1 (commercial reagent, details in section 3.1) and p-coumaric 2 (isolated compound, details in section 2.1) acids was carried out by coupling reactions of these natural products with distinct amines and amino acids.
Thus, the coupling reactions of cinnamic acid 1 and protected p-coumaric acid 3 with benzylamine and propargylamine gave the corresponding compounds 10-11 and 12-13 in yields varying from 49% to 65% (Scheme 1A B and B), whilst coupling of 1 and 3 with the amino acids 7, 8 and 9 afforded the compounds 14-19 in yields varying from 59% to 75% (Scheme 1C).The structures of the novel thirteen derivatives were confirmed by spectroscopic ( 1 h NMR and 13 C NMR) and spectrometric analyses (high resolution MS).In general, the 1 h NMR of all compounds showed characteristic signals of the α and β cinnamic acid hydrogens (doublets at δ 7.6 and δ 6.3 ppm, J = 15.1 hz), from the natural precursor, and the benzyl ester group (multiplet at δ 5.2), as well as to others specific amino acid hydrogen, detailed in 3.3 section.

In vitro antiproliferative assays
Based on what was previously reported in the literature (Rodrigues et al. 2021), phenolic compounds derived from green propolis can act against breast and prostate hormone-dependent cancer tumor cells.Thus, herein we tested the natural precursor 1-2 and synthetic derivatives 3, 10-19 against breast tumor cell lines.
The natural compounds cinnamic 1 and p-coumaric 2 acids as well as their derivatives 3, 10-19 were tested against MCF-7 breast tumor cells and MCF-10A breast normal cells to determine the half-maximal inhibitory concentration (IC 50 ) (Table 1).Based on the obtained results, the best antiproliferative effects were observed for the amino acid derivatives 14-19, which showed IC 50 values in MCF-7 ranging from 13.9 to 51.8 μM, considerably better than their natural precursors 1 and 2 (IC 50 > 200 μM).These results are in agreement with what was reported previously (Rodrigues et al. 2021), since the coupling with amino acids L-threonine, L-lysine and L-phenylalanine increased the activity of the precursors cinnamic 1 and p-coumaric 2 acids.
We also noticed that acetylated amino acid derivatives 15, 17 and 19 had better IC 50 values in MCF-7 than their precursors 14, 16 and 18, which may demonstrate a better interaction with the possible target.endo et al. (2012) demonstrated that green propolis compounds, such as p-coumaric acid, can interact with the active site of the enzyme AKR1C3, which is overexpressed in hormone-dependent breast cancer.Further studies are necessary, such as molecular docking and QSAR, to demonstrate the action mode of these cinnamic and p-coumaric derivatives.
In addition, the selectivity index (SI) was calculated for all derivatives, by division between the value of IC 50 in breast normal cells by breast tumor cells.SI values higher than two demonstrate selectivity of the compounds against breast cancer cells compared to normal cancer cells.Among all tested derivatives, it is worth noting compound 15 (p-coumaric acid-Thr) that showed the best selectivity index value, SI 5.1, as assessed by its improved antiproliferative activity against MCF-7 cells (IC 50 39.6 μM), comparable to its precursor p-coumaric 2 acid (IC 50 > 200 μM), and desirable low cytotoxic activity against normal MCF-10A cells (IC 50 > 200 μM), (Table 1, Figure 34S-35S, supplementary material).With this work, we also have demonstrated that the synthetic derivatives 17, 18 and 19 are statistically more actives than Cisplatin.In contrast, compounds 14, 15, 16 and 17 are less cytotoxic to MCF-10A cells, suggesting a protective effect when compared to positive control.however, we expected to obtain higher selective compounds since future translational studies are expected.Our findings demonstrated that compound 15 is more active than cisplatin and no cytotoxic to healthy cells, indicating a high specific effect in cancer cells.In this sense, compound 15 represent a strong candidate for future studies.

General
The reagents for synthesis including cinnamic acid, PyBOP, hOBt, DIPeA, benzylamine and propargylamine were purchased from Sigma-Aldrich.The solvents were purchased as reagent grade, and the silica gel was flash (40-63 μm).
The nuclear magnetic resonance spectra of hydrogen ( 1 h NMR) and carbon ( 13 C NMR) were recorded on a Bruker Advance DRX 300 (300 Mhz) or DPX 400 (400 Mhz) spectrometer.The chemical shifts (δ) are reported in parts per million (ppm), the coupling constant (J) given in hertz (hz), and the number of hydrogens was deduced from the relative integral.The assignments described were performed with the aid of two-dimensional analyzes of COSY and hMQC.In addition, mass spectrometry differences between active compounds to the positive control were evaluated using one-way aNoVa followed by post-hoc Bonferroni tests; *p < 0.01, **p < 0.001 and ***p < 0.0001 versus the cisplatin drug.ns, not significant.
analyses by eSI-MS (electrospray Ionization) were performed using a positive ionization mode in a Bruker DaltonicsultrOTOF-Q -eSI-TOF (uSP) device.

Isolation of p-coumaric acids (2)
The green propolis´ crude extract was first analyzed by hPLC-DAD to confirm the presence of p-coumaric acid 2. After that, this extract was fractioned by classic chromatographic column with hexane/ethyl acetate gradient elution (95:05 to 85:15 v/v) furnishing approximately one hundred fractions, which were further purified by hPLC-uV (Rodrigues et al. 2021).Thus, the pure p-coumaric acid 2 (50 mg) was obtained and confirmed by 1 h and 13 C NMR analyses (Ferreira et al. 2021).

General procedure for coupling reactions
The phenolic compounds, cinnamic 1 and acetylated p-coumaric 3 acids (1 equiv each), and the reagents benzylamine, propargylamine and the O-benzylated amino acids (2 equiv each) were treated with PyBOP, hOBt (1 equiv each) and DIPeA (2 equiv) in DMF.The reaction mixtures were stirred at room temperature for 16 h, concentrated under reduced pressure, and purified by flash chromatography, according to each product, and by preparative hPLC-uV (section 4.2.), to afford the pure compounds with relative peak purity above 95%, (Marchiori et al. 2015).

In vitro assays with the synthetic derivatives against breast cancer cell lines (MCF-7)
The breast tumor and normal cell lines, MCF-7 and MCF-10A, respectively, were cultured in DMeM (Dulbeccós modified eaglés medium), supplemented with fetal bovine serum 10% (FBS), penicillin/streptomycin 1% v/v and incubated at 37 °C with 5% CO 2 .The synthetic derivatives 3, 10-19 and the natural precursors cinnamic 1 and p-coumaric acids 2 were evaluated using the MTT method (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide) to measure cell viability.For this, in a 96-well plate were added 100 µL of 5 × 10 4 cells/mL (MCF-7 and MCF-10A) and after 24 h the cells were treated with each compound (200-1.56µM) solubilized in medium with 0.5% (v/v) DMSO.After, the cells were incubated for 72 h and the absorbance at 570 nm was read on the Biotek Synergy hT equipment.The data are expressed as means of at least two independent experiment and later evaluated using the GraphPad Prism v.5., using ANOVA with p value less than 0.05 was considered statistically significant.

Conclusion
In summary, novel eleven synthetic amide derivatives were synthesized from the natural products cinnamic and p-coumaric acids as precursors through coupling reactions using the PyBOP/hOBt/DIPeA system.Such derivatives were submitted to antiproliferative assays against the hormone dependent breast cancer MCF-7 cells.
Our results showed that only amino acid derivatives had excellent to good IC 50 results against MCF-7 cells, with all these derivatives 14-19 displaying better IC 50 values than the natural precursors 1 and 2, indicating that the amino acid coupling is promising to increase the activity of these natural products against MCF-7 cells.
Overall, the p-coumaric acid coupling with L-threonine amino acid (compound 15) had the best selectivity index (SI = 5.1), with half-maximal inhibitory concentration of 39.6 ± 1 μM, showing a high selectivity against hormone-dependent breast cancer cell lines MCF-7 and low cytotoxicity against the normal breast cell lines MCF-10A.Thus, this new natural product derivative may represent a prototype for future development of antiproliferative agents, especially against hormone-dependent breast cancer.

Table 1 .
half-maximal inhibitory concentration (Ic 50 ) and selectivity index determination (sI) for the treatments of breast tumor and normal cells with the derivatives compounds from cinnamic and p-coumaric acids.Note: the Ic 50 values are expressed as the means ± standard deviation of duplicated experiments on alternate days.the cell lines were treated with increasing concentrations (1.56-200 μM) of each compound for 72 h.cell viability was determined using the Mtt assay.McF-7: breast adenocarcinoma; McF-10a: breast epithelial (non-tumor cell line).nd, not determined.sI, selectivity index provided by ratio between Ic 50 of McF-10a and Ic 50 McF-7 cells.