Preparation, Spectral Characterization and Anticancer Potential of Cinnamic Esters

Cinnamic acid and its derivatives show a remarkable variety of biological activities and are often studied in search of the development of new and highly effective drugs. This work aims to synthesize, characterize and evaluate the cytotoxic activity of esters derived from cinnamic acid. Eighteen esters were synthesized through Steglich’s esterification, of which eleven were not reported in the literature. All compounds were fully characterized by Fourier transform infrared epectroscopy (FTIR), nuclear magnetic resonance (H and C NMR) and high-resolution mass spectrometry (HRMS) data. The cytotoxic activity of esters obtained was evaluated using four human tumor cell lines: SNB-19 (astrocytoma), HCT-116 (colon carcinoma, human), PC3 (prostate) and HL60 (promyelocytic leukemia) through the 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium (MTT) colorimetric assay. These studies showed that the compound 3-methoxybenzyl (E)-3-(4-methoxyphenyl)acrylate (12) is the most potent against HCT-116, PC3 and SBN-19 cells, with the lowest half maximal inhibitory concentration (IC50) value of 16.2 μM in the HCT-116 strain. The derivatives were obtained in good yields (76.6-95%), except for compounds 5-isopropyl-2-methylphenyl (E)-3-(3-hydroxy-4-methoxyphenyl)acrylate (17) (18.6%) and 2-isopropyl-5-methylphenyl (E)-3-(3-hydroxy-4-methoxyphenyl)acrylate (18) (15.5%).


Introduction
Among the countless diseases that affect humanity, cancer is that one that affects millions of people, being the second cause of death worldwide, with an estimated number of 9.6 million in the year of 2018. 1 Treatment of the disease includes chemotherapy, which uses drugs that destroy cancer cells. Several chemotherapeutic agents are used such as doxorubicin, epirubicin and cyclophosphamide, among many others. However, these chemical agents often cause serious side effects. 2 In this context, numerous researches around the world are related to the development of new drugs to fighting cancer, many of them related to works involving derivatives of natural products. 3,4 In addition, cinnamic acid and similar such as acids caffeic and ferulic, are important nutrients present in human food. Several food-stuffs (coffee, chocolate, almonds, among others) that are part of the diet of many people are potentially rich of this type of constituents. 5,6 There are reports in the literature on the cytotoxic activity of cinnamic acid (1a) and some of its analogs: acid p-methoxycinnamic (2a), ferulic acid (3a), isoferulic acid (4a), p-hydroxycinnamic acid (5a) and caffeic acid (6a), Figure 1, against some cancer cell lines: MCF-7 (breast carcinoma), PC3 (prostate) and SW480 (human colon). 7 It is worth mentioning that 1a has attracted the attention of researchers for a long time, due to its anti-cancer properties. 6 Research has shown that synthetic derivatives of phenylpropanoid acids have several biological activities: hypolipidemic, 8 hypoglycemic, 9 acetylcholinesterase inhibitor, 10 antioxidant, 11 antimicrobial, 12 antimalarial, 13 antifungal, 14 and anticancer. 15 Others studies have also revealed anticancer properties of cinnamates in specific human tumor cell lines: HeLa 127 (cervix), MCF-7 (breast), PC3 (prostate) and K562 (myeloid leukemia). 15,16 In these studies, cinnamates had shown promising anticancer activity, presenting a high level of cytotoxicity and selectivity.
An effective and simple method for forming cinnamates is to use the esterification of Steglich, 17,18 for not wanting high temperatures or using acyl halide, and in milder reaction media and forming very reactive intermediates, being possible to apply to different reaction systems. The Steglich reaction is a modification of an esterification in which N,N'-dicyclohexylcarbodiimide (DCC) acts as a coupling reagent and 4-(N,N'-dimethylamino)pyridine (DMAP) as a catalyst. Initially, the DCC acts as a base and gives rise to the carboxylate anion which, in turn, attacks the protonated DCC imidic carbon forming the O-acylisourea. This, with reactivity similar to acid anhydride, is protonated, indirectly activating its carbonyl carbon to attack the hydroxyl group of alcohol. After deprotonation, precipitation of dicyclohexylurea (DCU) occurs with formation of the ester (Figure 2). 19 In order to evaluate the anticancer activity of cinnamates, the present work describes the synthesis via esterification of Steglish and characterization of esters of cinnamic acid, p-methoxycinnamic acid and ferulic acid. The cytotoxic activity of esters (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) was assessed using four human tumor cell lines: SNB-19 (astrocytoma), HCT-116 (colon carcinoma, human), PC3 (prostate) and HL60 (promyelocytic leukemia), in addition to of a healthy L929 cell (murine fibroblast).

Cytotoxicity
The cytotoxic activity of cinnamates was evaluated in SNB-19 (astrocytoma), HCT-116 (colon carcinoma), PC3 (prostate carcinoma), HL60 (promyelocytic leukemia), which were obtained from the National Cancer Institute (Washington, USA). All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640, except for L929, which was cultivated in Dulbecco's Modified Eagle Medium (DMEM), obtained from the Rio de Janeiro Cell Bank (BCRJ) (Rio de Janeiro, Brazil). All cell culture experiments were performed at 37 °C. Cells were supplemented with 10% fetal calf serum and 1% of antibiotics, in a 5% CO 2 humidified atmosphere. The L929 cell line was used to evaluate the selectivity of the extracts and these assays, the anticancer drug doxorubicin was used as positive control.

Statistical analysis
All experiments were performed in duplicate and repeated three times. For samples that showed > 70% inhibitory, activity the selectivity index (SI) was calculated. The calculation of this index corresponds to the division between the IC 50 value of each test compound in the nontumor cell line L929 and the IC 50 value of each compound in the tumor cell line (SI = neoplastic cells IC 50 L929/IC 50 ). 28 The results obtained were analyzed using the GraphPad Prism 5.01 software and expressed as mean ± standard deviation. 29 IC 50 values were obtained by interpolation from non-linear regression analysis with a 95% confidence level. IC 50 was defined as the concentration sufficient to obtain 50% of the maximum inhibitory effect on cell viability.

Results and Discussion
As previously described, derivatives of phenylpropanoid acids exhibit various biological activities, including anticancer. Considering these important aspects, in the present study eighteen esters derived from cinnamic (1a), p-methoxycinnamic (2a) and ferulic (3a) acids were synthesized. It is interesting to note that the relationship between molecular structure and pharmacological activity is studied according to several parameters, including electronic, steric and stereochemical. The esters derived here synthesized contain the cinnamoyl residue, cited as a biologically active molecular fragment, while the alcohol/ phenol part gives the structure a steric and/or stereogenic dimension.
Among all the synthesized cinnamates, compounds 1, 2, 3, 8, 9, 10, 11, 12, 14, 15 and 16, no synthesis and biological studies were reported, being unpublished. However, compounds 4, 5, 6, 7, 13, 17 and 18 have been reported and exhibited antimicrobial activity, 17,20-24 but there are no cytotoxicity test studies on any cell line. Thus, the cytotoxic activity of all esters derived from cinnamic acids against four human cancer cell lines was evaluated: SNB-19 (astrocytoma), HCT-116 (colon carcinoma, human), PC3 (prostate) and HL60 (promyelocytic leukemia). We use the MTT colorimetric method developed by Mosmann. 25 All the compounds have been characterized by IR, HRMS and NMR ( 1 H and 13 C) spectral data. Some spectral data were characteristic and common to all, as expected. Thus, for all esters, the spectra: IR showed absorption bands in the range of 1726-1707 cm -1 due to stretches of carbonyl groups, as expected for stretches of conjugated ester carbonyl groups; 1 H NMR showed a characteristic spin system consisting of trans olefinic protons arranged in a polarized carbon-carbon double bond (d H 7.19 and 7.86, J 16 Hz); 13 C NMR, signals around d C 166.00, also consistent with conjugated esters carbonyl carbons. The 1 H and 13 C NMR assignments were performed taking into account aspects such as chemical shifts, multiplicity and coupling constants, displayed by the signals in the respective spectra. In addition, by comparison with data recorded in the literature for compounds of the same nature. 17,20,23,30 The 1 H and 13 C NMR data of these compounds are given in the Experimental section.
Initially, the esters were screened using a program from the National Cancer Institute (NCI), 31 which easily allows a qualitative or semi-quantitative analysis to determine cytotoxicity. 25 An intensity scale was used to assess the cytotoxic potential of products, according to the following results: 1-50% (low or medium), 50-75% (moderate) and 75-100% (high). 32,33 According to this program, of the eighteen synthesized compounds, eleven exhibited cytotoxic potential with cell growth inhibition above 50% and six with 75-100% inhibition (high activity). Thus, based on the initial screening, compounds 6, 8, 12, 14, 15 and 18 showed promising cytotoxicity, with > 70% inhibition of cells proliferation in at least one of the lines tested. Only those with an inhibition percentage above 70% were evaluated for the mean inhibitory concentration (IC 50 = concentration causing a 50% inhibition) ( Table 1). For the other compounds, the results were not satisfactory, with the value < 70% inhibition. The results were summarized in the cell inhibition (Figures S1-S4) which can be found in the Supplementary Information section.
As summarized in Table 1, the six compounds demonstrated a general (non specific) cytotoxic response. The comparison between the activity in relation to neoplastic cell lines and normal cells (L929) was made to calculate the selectivity index (SI), as an indication of the potential of using the compounds for future clinical tests. Ideally, the drug should only kill patient cancer cells without significantly affecting healthy cells. SI is considered significant when it has a value greater than or equal to 2.0, that is, this value means that the compound has activity twice in the lineages of neoplastic cells than in normal cells. 28 For the compounds 6, 8, 12, 14, 15 and 18 the SI was calculated, which can be seen in Table 2.
For all strains tested, SI values were > 2 showing that the compounds are selective between neoplastic and normal cells. Combining SI with antiproliferative activity, the substances become candidates for drugs for future studies. However, a notable exception was the compound 6, with SI values of 0.84, 1.4 and 0.7 for the SNB-19, HCT-116 and PC3 strains, mutually. It is important to note that the compound 6 showed a high toxicity, preferentially inhibiting normal cells than neoplastic cells SNB-19 and PC3.
It is important to remember that the influence of α,β-unsaturated part of cinnamic acid and its derivatives relative to biological activity, was studied 34 when comparing compounds (E)-3-(3,4-dihydroxyphenyl)acrylate (α,β-unsaturated) and 3-methyl- (3,4-dihydroxyphenyl) propanoate (α,β-saturated). It was observed that the compound α,β-unsaturated contributes positively to the action against breast cancer cells (T-47D) and colon (WiDr), with IC 50 values 64 and 59 μM, respectively, while the saturated compound is inactive. On the other hand, in their studies Sova et al. 35 compared the inhibition effect in relation to the phenol/alcohol part of the ester. The (E)-phenyl cinnamate derivative inhibited the growth of HeLa (cervical adenocarcinoma), K562 (myeloid leukemia), Fem-x (malignant melanoma) and MCF-7 (breast cancer) cell lines, with an IC 50 of 75.6 ± 12, 52.6 ± 3, 69.0 ± 4 and 58.6 ± 4 μM, respectively, presenting cytotoxic effects superior to the (E)-cyclohexyl cinnamate derivative, with IC 50 >180 μM in all cells. 35 Analyzing the results from the point of view of the structures of the products, there was not a sufficiently coherent answer, however, allows some considerations. For example, 7 showed toxicity < 50% on the NCI scale, while its analog 15, with a methoxy group in the para position on the cinnamate part and with toxicity > 50% on the same scale, exhibited very low IC 50 against all cell lines. In another example, 4 exhibited toxicity < 50% on the NCI scale, while its 12 analogue, with a methoxy group in the para position in the cinnamate part, affected SNB-19 and PC3 cells growth with IC 50 values of 42.1 and 41.9 µM, respectively, and showed a potent antiproliferative effect against HTC-116 cells with an IC 50 value of 16 µM. Finally, 18 showed remarkable toxicity to HTC-116 cells  (IC 50 15 µM), whereas 17 with a similar structure (inversion of methyl and isopropyl substituents in the aromatic ring of the phenol part) was not even detectable for IC 50 . Studies involving cinnamic acids and analgesics have shown that inhibition targets in several cancer cell lines occur through the inhibitory action on the deoxyribonucleic acid (DNA) synthesis of growing cells. 36 In general, the data indicate that cinnamates inhibit cell growth by selective induction of cell death and cycle disruption. [37][38][39] Thus, the inhibitory action of compounds that showed activity in front of cancer cells in this work, was also suspected of involving inhibition in the synthesis of DNA and guaranteeing an interruption of the cell cycle.

Conclusions
In this study, eighteen esters were obtained through the Stiglich esterification. The MTT test shows that the activities of compounds with an aromatic ring in the cinnamoyl fraction are more active than cyclohexyl. In comparison between the esters obtained, this study showed that the compound 12 is the most potent against HCT-116, PC3 and SNB-19 cells, with the lowest IC 50 value of 16.2 μM in the HCT-116 strain. The compound 18 also has a low IC 50 value in HCT-116 (15.38 μM). The compound 8 was the only one that showed the highest cytotoxicity in HL60 (IC 50 = 25.2 μM). The compounds 8, 12 and 18 showed selectivity against normal cells (L929). According to some examples observed, there was an apparent increase in biological activity with increased conjugation in the cinnamate fraction, provided by electron donating substituents, such as methoxy and hydroxyl groups. This research indicates that the tested cinnamic acid derivatives present good initial performance for the development of candidates for antineoplastic drugs, bringing new perspectives for the structurally modified natural substances under study, contributing to the knowledge and elaboration of new bioactive compounds, more effective against cancer.

Supplementary Information
Supplementary information ( 1 H and 13 C NMR, IR, HRMS and potential for cell inhibition) is available free of charge at http://jbcs.sbq.org.br, as PDF file.