Synthesis of Novel Cinnamides and a Bis Cinnamate Bearing 1,2,3-Triazole Functionalities with Antiproliferative and Antimetastatic Activities on Melanoma Cells

The present investigation describes the synthesis of novel cinnamides and a bis cinnamate bearing 1,2,3-triazole functionalities and investigation of their antiproliferative and antimetastatic effects on melanoma cells. The necessity for the development of new chemotherapeutic agents for melanoma treatment motivated this work. Sixteen derivatives were obtained with yields ranging from 23-81% and fully characterized by spectroscopic (H and C nuclear magnetic resonance, infrared) and spectrometric high resolution mass spectrometry (HRMS) techniques. The derivatives were in vitro evaluated against B16-F10 murine melanoma cell line. The most effective compound (a bis cinnamate) (6b) reduced the melanoma cell viability, generated cell cycle arrest, and influenced the metastatic behavior of melanoma cells by decreasing migration, invasion, and colony formation. Based on these findings, it is believed that compound 6b may represent an interesting scaffold to be explored toward the development of new antimelanoma agents.


Introduction
Melanoma is the most serious type of skin cancer. 1,2 It is originated from uncontrolled growth of melanocytes that are dendritic-like cells responsible for skin pigmentation. 3,4 Melanoma accounts for the highest number of skin cancer deaths worldwide, and its incidence rate is increasing over the last years. [5][6][7] In 2020, new melanoma cases in the United States were estimated to be about 100,350 with 6,850 deaths, whereas in Brazil approximately 8,450 new cases were projected. 5,8 Genetic mutations, excessive ultraviolet radiation exposure, severe sunburn, outdoor training, advanced age, and gender are relevant factors related to melanoma susceptibility. 2,9 Additionally, melanoma has a high tendency to spread to other parts of the body. This metastatic behavior increases the challenge to treat this disease. Altogether, these facts evidence the melanoma severity. [10][11][12][13] Surgical resection is the main option available for patients with early stage of melanoma. Once present the metastatic form, systemic treatment is the mainstay of therapy, which includes radiotherapy, cytotoxic chemotherapy, immunotherapy, and targeted therapies. 5,12 Particularly, the chemotherapy has been using several compounds over the years, such as dacarbazine, dabrafenibe, binimetinib, vemurafenib, encorafenib, trametinib, and cobimetinib. 12,[14][15][16][17][18] However, these drugs still exhibit important side effects and low efficacy when used individually. 19 These facts justify the need for the development of new chemotherapeutic agents to be used in the treatment of metastatic melanoma.
In the search for new antimelanoma agents, natural products have been an extraordinary source of compounds with great chemical variability and biological activities, [20][21][22][23] including antimelanoma. 24, 25 Newman and Cragg 26 reported that 83% of anticancer drugs approved between 1981 and 2014 were either natural products per se or were based thereon. For instance, paclitaxel is an antimelanoma drug from natural sources derived from the bark of the Pacific yew tree (Taxus brevifolia). 12 Cinnamic acid and its derivatives are natural plantderived compounds that present antitumor and other biological activities. They have been used as templates for designing and arriving at newly compounds with antitumor activities. 27,28 Our research group reported the preparation of a series of twenty-six cinnamic acid derivatives resulting from the connection of cinnamic acid with 1,2,3-triazole functionalities. 28 In the latter, B16-F10 cell line was used in in vitro assays to evaluate the antimelanoma activity of these compounds. The most potent cinnamate 3-(1-benzyl-1H-1,2,3-triazol-4-yl)propyl showed significant antiproliferative and antimetastatic activities against B16-F10 cells by interacting with matrix metalloproteinase 9 (MMP-9) and MMP-2, which are directly involved in melanoma progression. 28 Indeed, compounds bearing the 1,2,3-triazole ring present a variety of therapeutic effects including antitumor activity. 29,30 Due to this fact, this fragment is relevant to medicinal chemistry 29 and used as pharmacophore. 31 Likewise, cinnamides are cinnamic acid derivatives found in nature. [32][33][34] Also known as cinnamamides and cinnamic acid amides, they present a broad range of pharmacological activities, which include antitubercular, anti-trypanosomal, anti-diabetic, anti-microbial, antiviral, anti-inflammatory, anti-malarial, nervous disorders, and antitumor. 35 In our ongoing efforts to find useful compounds for the treatment of melanoma, 28,36,37 and considering antitumor activity linked to cinnamides, cinnamates, and compounds displaying the 1,2,3-triazole functionality, it is herein described the synthesis and antimelanoma evaluation of a series novel cinnamides and a bis cinnamate bearing 1,2,3-triazole fragment(s).

Synthesis
The steps involved in the synthesis of cinnamides 3a-3n are outlined in Scheme 1.
The synthesis of cinnamides 3a-3n required the preparation of twelve benzyl azides and two 3-azidocoumarins. The benzyl azides were prepared via conversion of benzyl alcohols to the corresponding ester sulfonates, followed by the treatment of these esters with sodium azide, as previously reported. 43 The 3-azidocoumarins, in turn, were obtained from the substituted salicylaldehyde and N-acetylglycine or ethyl nitroacetate through routes involving two and three steps, according to methodology previously described. 44 One aspect deserves comment at this point. In our previous work, we synthesized a series of cinnamates bearing 1,2,3-triazole functionalities. 28 Indeed, these cinnamates presented different degrees of efficiency against the melanoma B16-F10 cell line. Their efficiency depended on the benzyl groups present in the triazole functionality of the cinnamates. These benzyl groups, in turn, came from the benzyl azides. Herein, benzyl azides were also used in the preparation of cinnamides in order to make possible a comparison between the antimelanoma activity of cinnamates previously published by us 28 and the cinnamides herein investigated.
The structures of the new cinnamides 3a-3n were confirmed based on infrared (IR), 1 H, 13 C nuclear magnetic resonance (NMR) spectroscopic techniques and highresolution mass spectrometry (HRMS) analyses. The IR spectrum showed bands for the N-H stretching within the 3230-3279 cm -1 range. Also, carbonyl stretching vibrations for the amide groups were noticed within the interval 1615-1670 cm -1 . In the 1 H NMR, the coupling constants for the hydrogens of aliphatic double bonds were approximately equal to 16 Hz, which confirms the trans stereochemistry of these bonds. The singlet typically observed around 8 ppm confirmed the presence of the 1,2,3-triazole functionality in the structures of the cinnamides. The presence of the amide group was also confirmed by 13 C NMR; the signals for the carbonyl groups of this functionality were noticed within the 164.8-168.4 ppm range. The molecular formulas of the cinnamides were confirmed via HRMS analyses.
Our research group has demonstrated important cytotoxic effects for symmetrical 1,4-disubstituted bis-1,2,3-triazoles. 45 Based on that, we decided to prepare the bis cinnamide 6a and cinnamate 6b as shown in Scheme 2. The synthesis of bis azide 4 has been previously reported. 45 The compound 6b was prepared to compare the biological response of the bis ester cinnamate in relation to bis cinnamide 6a (Scheme 2). The ester 5 was prepared via condensation of cinammic acid (1) and propargyl alcohol promoted by EDAC as previously published by us. 28 The spectroscopic data that confirmed the structures of cinnamic acid derivatives 3a-3n, 6a and 6b are available in the Supplementary Information (SI) section.
Although metastatic B16-F10 is known to be very resistant to antitumor agents, 46 the compounds 3j and 6b displayed superior cytotoxic activity (IC 50 values of 153.4 and 57.66 µM, respectively) than cinnamic acid (> 200.0 µM, data not shown). In contrast, the cinnamides 3c, 3e, and 3f showed IC 50 values greater than 200.0 µM. The cytotoxic effects presented by compounds 3j and 6b corroborate with previous studies, in which cinnamic acid derivatives also have presented relevant cytotoxic effects on the metastatic melanoma cell line. 28,47 In the study of Sova et al. 47 cytotoxic effects of representative cinnamic acid esters and amides were seen in different types of cancer in vitro, including melanoma. Besides that, the compounds tested showed selectivity of these cytotoxic effects on the malignant cell lines versus the peripheral blood mononuclear cells. 47  Effect of the compounds 3c, 3e, 3f, 3j, and 6b on nontumoral cell viability The cytotoxicity of compounds in non-tumoral cells was also evaluated by means of Vero fibroblast-like kidney cells treated with the compounds 3c, 3e, 3f, 3j, and 6b. Vero cells showed sensitivity for the compounds 3e, 3f, and 6b at 100.0 µM, being 3e the most cytotoxic ( Figure 2). This assay is relevant to compare the effect of compounds on non-tumoral cells, once that novel cancer chemotherapy relies on the selection of malignant-cell specific drugs and non-toxic to normal cells. 48 Meantime, cytotoxic chemotherapy can kill more cancer cells than normal tissue, as seen in cytotoxic drugs used to treat cancer. 49 It was the case of the results observed for the compound 6b, which presented certain cytotoxicity in non-tumor cells, but it was the most effective in B16-F10 tumor cells. Taking the findings together, we selected the derivative 6b for the subsequent assays due to its activity against the melanoma cell line B16-F10 (lowest IC 50 ). .31% (at 50.0 µM; p < 0.01). Thus, these data expressed a B16-F10 cells accumulation in the G0/G1 phase and fewer cells in the S-phase (phase of duplication of genetic material), resulting in growth inhibition/cell cycle arrest. Drugs that affect the tumor cell cycle are promising, as they negatively influence the proliferation of cancer cells. 50 In the case of cinnamic acid derivatives, previous studies 47, 51 have reported their capacity to induce cell cycle arrest in cancer cells. Therefore, our data corroborate the studies with cinnamic acid derivatives, since the compounds inhibited cell proliferation by disruption of cell cycle.
Effect of compound 6b on the metastatic behavior of melanoma cells Metastasis involves a series of progressive stages which include cell migration, invasion of blood and lymph vessels, cell colonization, and the ability of these cells to survive in other organs. 52,53 In order to analyze the cell migration, we evaluated the cell migration capacity through the wound healing assay using concentrations of 12.5, 25.0 and 50.0 µM of the compound 6b for 24 h, all concentrations below the IC 50 value. The compound 6b significantly reduced in approximately 42% the cell migration at the concentration of 50.0 µM, in relation to the DMSO control ( Figure 4).
Cinnamic acid and its derivatives normally interfere with cell dynamics, decreasing cell migration. Niero and Machado-Santelli 54 observed that the treatment with cinnamic acid on melanoma cells caused cytoskeleton disruption. Any change in the cell cytoskeleton interferes with cell locomotion, since these filaments are crucial for cell movement. Therefore, our data confirm the hypothesis that cinnamic acid derivatives interfere with cell migration.
Once that 6b interfered with cell migration and cell invasion is a key step of metastasis, invasion assay was performed. B16-F10 cells were treated with 12.5, 25.0 and  Compounds with properties to reduce cellular invasion of cancer cells are interesting. 55 The ability of these synthetic cinnamic acid derivatives to inhibit the invasive capacity of melanoma cells in vitro, shows once again that cinnamic acid and its derivatives have antiinvasive properties against cancer cells, an effect also observed against colon carcinoma cells, human lung, adenocarcinoma, and even against melanoma cells. 28,56,57 This anti-invasion action of compound 6b may be related to the activity of metalloproteinases (MMPs), since previous studies 28,56,57 have shown that cinnamic acid derivatives are potent inhibitors of MMPs.
Finally, the colony formation was also assessed to evaluate the long-term effects of the compound 6b. This compound significantly reduced colony formation, with a reduction in the number of colonies at the concentrations of 12.5 (26.5%), 25.0 (41.6%) and 50.0 µM (53.3%) when compared to vehicle-treated cells ( Figure 6). This important result is probably due to a set of factors, such as the negative action of compound 6b on cell proliferation and its impact on cell mobility and invasion, as seen in previous in vitro assays. 58 Taken together, all the in vitro experiments performed suggested that the cinnamic acid derivative would render antiproliferative and antimetastatic effect in melanoma cells.

Conclusions
In summary, a series of novel triazole cinnamides and a hitherto unknown bis-triazole ester cinnamate were prepared and had their antiproliferative and antimetastastic activities evaluate in vitro on B16-F10 murine cell line. It was demonstrated that the derivative 6b, the most effective compound, reduced the melanoma cell viability, generated cell cycle arrest, and influenced the metastatic behavior of melanoma cells, by decreasing migration, invasion, and colony formation. Taken together, these results clearly showed the cytotoxic, antiproliferative and antimetastatic potential of compound 6b against melanoma    N-((1-benzyl-1H-1,2,3

N-(prop-2-yn-1-yl)cinnamamide
(1) (0.185 g, 1.00 mmol) and CuSO 4 ·5H 2 O (0.100 g, 0.400 mmol). The reaction mixture was vigorously stirred at room temperature for 30 min. Subsequently, water (10.0 mL) was added and the resulting aqueous phase was extracted with dichloromethane (3 × 20.0 mL). The organic extracts were combined, and the resulting organic phase was dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure. The compound 5a was purified from the residue by silica gel column chromatography eluted with hexane/ethyl acetate/methanol (5:3:1 v/v). The described procedure gave compound 5a with 58% yield (0.185 g, 0.580 mmol). White solid; mp 165. 8 Compounds 3b-3n were prepared from the corresponding alkyne 2 and azides as described for compound 5a. All the compounds were fully characterized by IR and NMR ( 1 H and 13 C) as well as high resolution mass spectrometry. Structures of the compounds are supported by the following data.   Cell viability assay and cytotoxicity B16-F10 cells were plated in 96-well at a concentration of 1.0 × 10 4 cells per well in a 96-well flat bottom microplate. The cells grew for 24 h and were treated with the concentration of 100 µM of each synthesized compound derived from cinnamic acid 3a-3n, 6a and 6b. DMSO (0.4% v/v) and RPMI-1640 were used as control. After 48 h of treatment, the cell viability was determined by MTT (3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, US) metabolization. The MTT solution was added to each well (final concentration 5 mg mL -1 ) and the plate was incubated for 3 h. Finally, 100 μL of DMSO were added to each well and the absorbance was measured in a plate reader (Sinergy HT, Biotek) at 540 nm. Results were normalized considering the cultures treated with 0.4% DMSO (control). The half-maximal inhibitory concentration (IC 50 ) of the most active compounds was also analyzed using the MTT method, after treating the B16-F10 cells with increasing doses (0-200.0 μM) of these compounds. The IC 50 was calculated as previously reported. 59 Cell viability on non-tumor cell line

Cell migration assay
The wound-healing assay was conducted to evaluate the ability of the compound 6b to inhibit cell migration. B16-F10 cells were seeded onto 24-well plate at a concentration of 1.0 × 10 5 cells per well and allowed to reach full confluence after incubation overnight at 37 °C under 5% CO 2 atmosphere. Monolayers were then wounded using a sterile 200 μL pipette tip. Cells were washed twice with PBS to remove detached cells and then treated with the compounds at the concentrations of 12.5, 25.0 and 50.0 μM. The DMSO vehicle treatment (0.4% v/v) was used as control. Photos of the wound were taken using an inverted microscope (Life Technologies, Carlsbad, California, USA). Wound closure rates were then calculated quantitatively as the difference between wound width at 0 and 24 h. Results were expressed as a percentage of cell migration.

Cell invasion assay
The matrigel matrix (BD Biosciences, Franklin Lakes, New Jersey, US) was diluted with serum-free RPMI-1640 culture medium at 1:12 ratio. Subsequently, the upper chamber of the transwell (8.0 μm polycarbonate membrane, Corning) was coated with 35 μL diluted Matrigel matrix and incubated at 37 °C, for 2 h, for full condensation. Then, the B16-F10 cells were re-suspended with serum-free RPMI-1640, treated with 6b at 12.5, 25.0, and 50.0 μM for 60 min, and inoculated into the upper chamber Matrigel-precoated (5.0 × 10 4 cells, 100 μL per well). The DMSO-vehicle treatment (0.4% v/v) was used as control. The well was filled with 650 μL of culture medium containing 10% v/v FBS as a chemoattractant. After 24 h, the chambers were fixed in methanol for 30 min, washed and stained with toluidine blue (1% v/v, Sigma-Aldrich, St. Louis, MO, US) for 15 min. Images from 10 fields were chosen at random/group, captured using an inverted microscope (Leica Microsystems, Wetzlar, Germany) and the cells were counted using the ImageJ software. 60 The results were expressed as a percentage of cell invasion.
Cell colony assay B16-F10 cells were seeded in 6-well plates in triplicate at the density of 1.0 × 10 3 cells per well. After 24 h, the cells were treated with the compound 6b at 12.5, 25.0, and 50.0 μM for 24 h. The complete medium was exchanged for complete medium with 2% FBS, and the cells were cultured for 7 days. The colonies formed were then fixed and stained with toluidine blue solution (1% v/v, Sigma-Aldrich, St. Louis, MO, US) and methanol (20% v/v). Colonies were counted by using ImageJ software and the results were expressed as a percentage of the untreated control cultures. 60

Statistical analysis
All numeric data were obtained from three independent experiments, each experiment with triplicate, and are shown as mean ± standard error of the mean (SEM). The analyses were performed using Microsoft Excel (Microsoft Office Software System) and GraphPad Prism (GraphPad Software Inc.). 61,62 The statistical analyses were carried out by oneway analysis of variance (ANOVA) followed by Dunn's or Dunnett's tests. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0001 were considered significant.