Monastrol

Fatty-monastrol derivatives and its cytotoxic effect against melanoma cell growth

Milene Medeiros de Moraes¹, Tamara Germani Marinho Treptow², Wystan Kreisly Othon Teixeira2, Luciana Almeida Piovesan2,3, Marcelo Gonçalves Montes D’Oca²,
Ana Paula de Souza Votto¹*

1Laboratório de Cultura Celular, Programa de Pós-graduação em Ciências

Fisiológicas, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande –

FURG, Rio Grande do Sul, Brazil

2Laboratório Kolbe de Síntese Orgânica, Escola de Química e Alimentos, Universidade

Federal do Rio Grande – FURG, Rio Grande, Rio Grande do Sul, Brazil

3Nanobusiness Informação e Inovação Ltda, Incubadora de Projetos, Instituto Nacional de Metrologia, Qualidade e Tecnologia – INMETRO, Duque de Caxias,
Rio de Janeiro, Brazil.

*Corresponding author: Instituto de Ciências Biológicas – Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brazil, Zip Code: 96203-900, Rio Grande, RS, Brazil. E-mail address: [email protected] (Ana Paula de Souza Votto).
Phone/Fax: +55 53 32935192/ + 55 53 32336848.

Abstract

Melanoma is the most dangerous type of skin cancer due to the occurrence of metastases. This work is aimed at studying the effects of the insertion of palmitic and oleic acid chain into monastrol in the melanoma cell line, B16F10. Cells were treated with monastrol, palmitic-monastrol or oleic-monastrol for periods of 0, 24, 48 and 72 hours, and the cytotoxic effect was observed for palmitic-monastrol and oleic-monastrol after 24 hours. For monastrol the effects were observed in 48 hours on B16F10 cells, and in 24 hours for a non-tumour cell line, melan-a. In this cell line, fatty-monastrol derivatives were cytotoxic after 24 hours of exposure in the same concentrations as B16F10. However, oleic-monastrol inhibited cell growth at 20 µM only after 72 hours, in contrast to the B16F10 cell line, in which oleic-monastrol inhibited cell growth at 48 hours, showing that at least in this structural modification, melan-a was less sensitive than B16F10. The ability of compounds to induce apoptosis and/or necrosis was measured, and it was observed that monastrol induces apoptosis within 24 hours. However, the cells treated with fatty-monastrol derivatives did not remain adhered on the well plate after 3 hours of treatment. At this time point, these cells still emitted fluorescence indicating viable cells, suggesting a possible effect of palmitic- and oleic- monastrol in the adhesion proteins found on the cell membrane.
Keywords: dihydropyrimidine; fatty acids; hybrid molecules; tumoural cells; cytoxicity; palmitic-monastrol; oleic-monastrol.
1.Introduction

Melanoma is the most serious skin cancer due to the possibility of metastasis occurrence [1]. Treatments may include chemotherapy, radiotherapy and surgery. In addition to these conventional treatments, new ones are being studied, e.g. the use of synthetic molecules such as the novel 3,4-dihydropyrimidin-2(1H)-ones (DHPM) [2]. The DHPM

compounds were synthetically created and have shown therapeutic and pharmacological properties, such as anticarcinogenic, antihypertensive, and calcium channel modulator properties [2-6]. Among these molecules, the 3,4-dihydropyrimidine-based compound monastrol was identified to be a molecule with potential for development of new drugs for cancer treatment as it shows mitotic activity and is capable of inhibiting the mitotic kinesin Eg5 [7].
Monastrol is a reversible, cell-permeable, small molecule that selectively inhibits the mitotic kinesin Eg5 without attacking tubulin; its activity is based on the specific and reversible inhibition of motility of the mitotic kinesin Eg5, a microtubule-associated motor protein required for bipolar spindle formation during mitosis [7,8]. Monastrol causes a cell cycle arrest in mitosis by forming monoastral spindles, which are comprised of a radial array of microtubules surrounded by a ring chromosome [7,9]. Peters et al. [9] tested monastrol in the LS180 cell line, human colon adenocarcinoma, and found that monastrol shows a time- and concentration-dependent effect on mRNA expression of glycoprotein P (Pgp), an efflux pump responsible for chemotherapeutic extrusion from multidrug resistant (MDR) cells. The field of chemotherapy exhibits great advances, but there are still two major problems associated with cytostatic treatment: severe side effects, such as neurotoxicity; and the development of multidrug resistance. The discovery of the kinesin Eg5 inhibitors such as monastrol, which specifically target mitotic spindle assembly, may be an opportunity to develop antitumour drugs with fewer side effects and the potential to overcome multidrug resistance.
Sashidhara et al. [10] tested coumarin-monastrol hybrids in different cell lines, MCF-7, T47D, MDA-MB-231 (breast cancer), A549 (lung cancer), PC-3, DU-145 (human prostate), HepG2 (human hepatocellular liver carcinoma), NIH3T3 (murine embryonic

fibroblast), HEK-293 (human kidney) and isolated primary mouse skin fibroblasts. Their results showed that one of the coumarin-monastrol hybrids, coumarin- thiodihydropyrimidone with tertiary butyl group on the benzene ring of the coumarin segment, significantly inhibited the proliferation of MCF-7, T47D and MDA-MB-231 breast cancer cell lines at all concentrations (2.5 µM, 5 µM, 10 µM, 20 µM) in a time- dependent manner. Additionally, there was no activity (inactive) in the non-cancer cells (NIH/3T3, HEK-293 and isolated primary mouse skin fibroblasts). However, this hybrid did not show significant anticancer activity in the other cancer cells.
Leizerman et al. [11], found that at 50 µM, after 72 hours, monastrol inhibited cell growth in the AGS (human gastric adenocarcinoma) cell line, but in HT29 (human colon adenocarcinoma), cell growth was inhibited only in high concentrations, 150 µM, showing that AGS is more sensitive to monastrol. In the AGS cell line, cell arrest caused by monastrol at 24 hours was reversible, but that at 48 hours it was not. Following the removal of monastrol most of the cells remained arrested in the G2/M cycle and the number of cells containing sub-G1 DNA content increased, indicating that cells irreversibly arrested in the G2/M cycle by monastrol started apoptosis. In HT29, at 48 hours, the cycle arrest was partly reversible, consistent with less sensitivity. According to literature, appendages to the central nucleus of DHPMs have been proposed. Hybrids of DHPMs with others molecules, such as sugar [12], peptoids [13]
or oxadiazole-chloroquine [14], have been reported.

Sośnicki et al. [15] verified that the presence of an aryl substituent on the DHPM was important for enhancement of the anticancer activity, showing that the 5-aryl substituted compounds were more potent inhibitors than monastrol against some cell lines, including human melanoma (A375 cell line). In previous work, we described the first synthesis of fatty acid analogues of DHPM- derived from fatty acetoacetates by the

Biginelli multicomponent protocol [16,17]. Antiproliferative activity of new fatty- DHPM was evaluated against two glioma cell lines (C6 rat and U-138-MG human) showing ability to reduce its cell viability[17a]. Compared to temozolamide, these compounds showed better potency against glioma cell lines. According to results the presence of a hydroxyl group on the aromatic ring and a fatty alkyl chain are essential for superior antiproliferative activity of hybrid fatty-DHPM against gliomas. However, even being cytotoxic to glioma cells, the DHPM-fatty acids showed a large safety range to neural cells, represented by the organotypic hippocampal culture, since the results showed that the fatty-DHPM did not increase the neural cell death by at least 20-fold and 4-fold the active glioma concentration (25 µM).
In this context, this study investigated the effect of insertion of palmitic and oleic chain to monastrol in the melanoma cell line B16F10, which is extremely resistant and, when in vivo, causes an extremely aggressive tumour, also verifying its effects in the non- tumour melanocyte cell line, melan-a.

2.Material and methods

2.1.Apparatus and Chemistry

The methyl acetoacetate, and 3-hydroxy benzaldehyde, thiourea were purchased from Aldrich Chemical Co. and were used without purification. The fatty acetoacetate derived from palmitic (C16:0) and oleic (cis-C18:1) acids were synthesized by transesterification of methyl acetoacetate with the respective alcohols in accordance with previous work [17b]. All organic solvents used for the synthesis were of analytical grade. Melting points were obtained on a Fisatom 430D apparatus and are reported as uncorrected values. The NMR spectra were recorded using Bruker AVANCE III 400 spectrometer (1H at 400 MHz and 13C at 100 MHz) in CDCl3 or

DMSO-d6 as the solvent. The chemical shifts are reported in δ(ppm) downfield from the tetramethylsilane (TMS) internal standard. Coupling constants (J) are reported in Hz and refer to apparent peak multiplicities.
2.2.Synthesis

2.2.1.General procedure for the synthesis of monastrol

To a round-bottom flask equipped with a magnetic stir bar and reflux condenser were added methyl acetoacetate (1 mmol), 3-hydroxy benzaldehyde (1 mmol), thiourea (1.3 mmol) in methanol (10 mL) using InCl3 (10 mol%) as catalyst. The reaction mixture was stirred at 70 ºC for 5 hours. The reaction mixture was cooled to 0 ºC and the precipitate was separated by filtration, then purified by recrystallization from acetonitrile [16].
2.2.2.Monastrol [16]

C13H14N2O3S, MW 278.32 g.mol-1. White solid. Yield: 90%. Mp 219–212 °C. Log P1.23.1H NMR (400 MHz, DMSO-d6): δ(ppm) 10.31 (s, 1H, NH), 9.61 (s, 1H, NH), 9.44 (s, 1H, OH), 7.12 (m, 1H, Ph), 6.65 (m, 3H, Ph), 5.10 (s, 1H, CH), 3.57 (m, 3H, CH3–O), 2.29 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ(ppm) 174.6, 166.1, 157.9, 145.5, 145.0, 130.0, 117.3, 115.1, 113.6, 100.9, 54.2, 51.5, 17.6.
2.2.3.General procedure for the synthesis of fatty-monastrol derivatives [17a]

To a round-bottom flask equipped with a magnetic stir bar and reflux condenser, were added acetoacetate derived from palmitic (C16:0) or oleic (cis-C18:1) acids (1 mmol), 3-hydroxy benzaldehyde (1 mmol), thiourea (1.3 mmol) in acetonitrile (10 mL) using InCl3 (10 mol%) as a catalyst. The reaction mixture was stirred constantly at 90 °C for 24 hours. The reaction mixture was cooled to 0 °C and the precipitate was separated by filtration. The products were purified by recrystallization from acetonitrile.

2.2.4.Palmitic-monastrol [Hexadecyl 4-(3-hydroxyphenyl)-6-methyl-2-thioxo- 1,2,3,4-tetrahydropyrimidine-5-carboxylate] [17b]
C28H44N2O3S, MW 488.31 g.mol-1. White solid. Yield: 77%. Mp 62–64 °C. Log P7.48.1H NMR (400 MHz, DMSO-d6): δ(ppm) 10.28 (s, 1H, NH), 9.58 (s, 1H, NH), 9.40 (s, 1H, OH), 7.10 (m, 1H, Ph), 6.65 (m, 3H, Ph), 5.08 (s, 1H, CH), 3.96 (m, 2H, CH2–O), 2.29 (s, 3H, CH3), 1.47 (t, 2H, J=7.5 Hz, CH2–CH2–O), 1.24 (m, 26H, 13CH2),
0.85 (t, 3H, J=6 Hz, CH3). 13C NMR (100 MHz, DMSO-d6): δ(ppm) 174.5, 165.6,

157.9, 145.4, 145.1, 129.8, 117.4, 115.0, 113.7, 101.0, 63.9, 54.4, 31.7, 29.5(2C), 29.4 (2C), 29.3 (4C), 29.1, 29.0, 28.5, 25.8, 22.5, 17.5, 14.4.
2.2.5.Oleic-monastrol [(Z)-octadec-9-enyl 4-(3-hydroxyphenyl)-6-methyl-2-tioxo- 1,2,3,4-tetrahydropyrimidine-5-carboxylate] [17a]
C30H46N2O3S, MW 514.76 g.mol-1. White solid. Yield: 70%. Mp 82–84 °C. Log P8.0.1H NMR (400 MHz, CDCl3): δ(ppm) 8.27 (s, 1H, NH), 7.87 (s, 1H, NH), 7.16 (m, 1H, Ph), 6.82 (m, 3H, Ph), 5.36 (m, 2H, CH), 5.34 (s, 1H, CH), 4.05 (m, 2H, CH2–O), 2.34 (s, 3H, CH3), 2.02 (m, 4H, CH2), 1.55 (t, 2H, J=6 Hz, CH2–CH2–O), 1.29 (m, 22H, 11CH2), 0.90 (t, 3H, J=6 Hz, CH3,). 13C NMR (100 MHz, CDCl3): δ(ppm) 174.0, 165.6, 157.3, 143.7, 143.8, 130.1, 130.0, 129.7, 118.8, 115.6, 113.7, 102.8, 64.8, 55.8, 31.9, 29.7, 29.6, 29.5, 29.3 (2C), 29.2(3C), 28.5, 27.2(2C), 25.9, 22.6, 18.3, 14.2.
2.3.Lipophilicity calculations

The physicochemical parameter, Log P (the logarithm of n-octanol/water partition coefficient P) based on established chemical interactions was calculated using ChemDraw, Level: Ultra, Version: 12.0.2.1076 (CambridgeSoft, Cambridge, MA, USA).

2.4.Cell Culture

The murine melanoma cell line, B16F10, was obtained from Rio de Janeiro Cell Bank, and the murine melanocyte cell line, melan-a, was obtained from the Laboratory of Experimental Oncology at Instituto do Câncer do Estado de São Paulo (ICESP). Both cell lines were maintained in Dulbecco’s Modified Essential Medium (DMEM) supplemented with sodium bicarbonate (0.2 g/L), L-glutamine (0.3 g/L), Hepes (3 g/L), 10% foetal bovine serum, 1% antibiotic and antimycotic-streptomycin (100 µg/mL), penicillin (100 U/mL) and amphotericin B (0.25 µg/mL) in cell culture flasks at 37 °C. The melan-a cells were maintained with 200 nM of phorbol 12-myristate 13-acetate (PMA) for induction of mitotic activity.
2.5.Cells treatments

The cells were trypsinized, centrifuged, suspended in supplemented DMEM medium, seeded in 96-well plates (B16F10 = 105 cells/mL and melan-a = 2×105 cells/mL), cultured for 24 hours for adhesion and then treated with each molecule for 72 hours. The control cells received the same volume of solvent of the highest concentrations of compounds (0.5% of ethanol). For the melan-a cell line, PMA was removed during the experiments.
2.6.Cell viability

The viability of B16F10 and melan-a was measured by the MTT (3-(4,5-2-yl)-2,5- ditetrazolium bromide) assay, at 0, 24, 48 and 72 hours after treatment with monastrol, palmitic-monastrol, oleic-monastrol, palmitic acid, oleic acid, monastrol + palmitic acid or monastrol + oleic acid in the following concentrations: 20 µM, 80 µM, 140 µM and 200 µM. At the end of each incubation time, molecule-containing medium was removed, and the cells were washed once with PBS and incubated for 3 hours with 20 µL of MTT (5 mg/mL) dissolved in 200 µL of DMEM medium at 37 °C. After 3 hours,

the medium containing MTT was removed and the formazan crystals were dissolved in 200 µL of dimethylsulfoxide (DMSO). The absorbance values were recorded at 490 nm on a multiwell plate reader (ELX 800 Universal Microplate Reader, Bio-TEK). The IC50 (concentration causing 50% inhibition) was estimated for the two cell lines after 72 hours of exposure for cell viability assessed by MTT assay.
2.7.Quantitative analysis of apoptosis and necrosis

The evaluation of apoptosis and necrosis was performed according to Ribble et al. [18]

modified with the addition of 2 µL of working solution composed of PBS, with 100 µg/mL of acridine orange and 100 µg/mL of ethidium bromide after the treatment of B16F10 (104 cells) for 24, 48 and 72 hours with 80 µM and 140 µM of monastrol and for 0, 3 and 6 hours with the same concentrations of palmitic-monastrol or oleic- monastrol. The analysis was performed from areas captured from the well plate (20X) with an epifluorescence microscope (Olympus IX81). All cells were analyzed and the data were expressed as the percentage related to the total cell number visualized in the captured area. The cells were classified according to Kosmider et al. [19] and modified as follows: those showing a green fluorescence cytoplasm and a green nucleus were considered viable cells; those presenting an orange nucleus with fragmented chromatin were considered apoptotic, those with uniformly orange-stained nuclei were considered necrotic.
2.8.Statistical analysis

Each experiment was repeated at least three independent times using five replicate samples. The results were expressed as the means ± SEs. Significant differences among groups were analyzed using one-way ANOVA followed by post-hoc comparisons (Tukey’s test). Normality and homoscedasticity were tested. Statistical significance was

accepted at p < 0.05.Values of IC50 and their respective 95% confidence intervals were estimated according to the Trimmed Spearman Karber method. 3.Results and Discussion 3.1.Chemistry The monastrol (Figure 1) was synthesized using the Biginelli multicomponent protocol between methyl acetoacetate, 3-hydroxy benzaldehyde, thiourea in methanol using InCl3 as catalyst. The presented spectroscopic data are in agreement with the literature [16]. The hybrids fatty-monastrol (Figure 1) were synthesized according to previous work [17a]. The Biginelli multicomponent reaction was carried between acetoacetate derived from palmitic (C16:0) or oleic (cis-C18:1) acids, 3-hydroxy benzaldehyde, thiourea in acetonitrile using InCl3 as a catalyst. The products were purified by recrystallization from acetonitrile and analyzed by proton and carbon NMR. The presented spectroscopic data are in agreement with previous work [17a]. Figure 1. Monastrol, palmitic- and oleic-monastrol derivatives 3.2.Cell Viability To investigate B16F10 cell proliferation effects by monastrol, palmitic-monastrol- and oleic-monastrol (Figure 1), a MTT viability assay was performed. The assay demonstrated that monastrol, after 48 hours exposure, inhibits cell growth in 80 µM, 140 µM and 200 µM concentrations (Figure 2A). In the melan-a cell line, monastrol inhibited growth after 24 hours, showing a higher effect when compared with B16F10, in 80 µM, 140 µM and 200 µM concentrations (Figure 3A). This different sensitivity also was shown in the IC50 values calculated for two cell lines after 72 hours of treatment (Table 1). In relation to the structural modification of monastrol, palmitic-monastrol and oleic- monastrol were cytotoxic after 24 hours exposure in 80 µM, 140 µM and 200 µM concentrations and were capable of inhibiting cell growth in 20 µM after 72 hours and 48 hours, respectively, for B16F10 cells (Figure 2B, C). In the melan-a cell line, palmitic- and oleic-monastrol were also cytotoxic after 24 hours of exposure in the same concentrations as B16F10. However, both of the molecules inhibited cell growth at 20 µM only after 72 hours, in contrast to the B16F10 cell line, in which oleic-monastrol inhibited cell growth at 48 hours, showing that at least in this structural modification, melan-a was less sensitive than B16F10 (Figure 3B, C). The IC50 values also indicate that higher concentrations were necessary for melan-a cells inhibition (Table 1). To verify the efficiency of the chemical modification of monastrol, experiments were conducted only with palmitic and oleic acids, as well as the combination of each with monastrol. Palmitic acid showed no effect in the B16F10 cell line (Figure 2D), and oleic acid inhibited cell growth after 48 hours at all concentrations (Figure 2E). These data contrasted with the results of Sousa Andrade et al.[20], where, for B16F10 at 200 µM palmitic acid induced loss of membrane integrity after 24 hours and oleic acid had no effect on cell viability. However, for the S91 murine melanoma cell line, 200 µM of oleic acid caused loss of membrane integrity and DNA fragmentation after 24 hours of treatment, and palmitic acid showed no effect. For human melanoma cell lines, SK-Mel 23 and SK-Mel 28, palmitic acid caused a loss of membrane integrity after 48 hours and DNA fragmentation after 24 hours and 48 hours treatment, respectively. Oleic acid had no effect on human melanoma cell lines. Hawkins et al.[21] showed that micromolar (µM) concentrations of fatty acids are toxic to cancer cells both in vitro and in vivo; however, our study showed no cytotoxicity for both acids and showed inhibited growth only for oleic acid. For the non-tumour melanocyte cell line, we found that palmitic acid had no effect (Figure 3D), and oleic acid inhibited cell growth at all concentrations after 72 hours treatment (Figure 3E), therefore showing that melan-a is more resistant than B16F10, which could also be observed in the IC50 values (Table 1). However, several studies have demonstrated the toxic effect of palmitic acid, as a result of apoptosis or necrosis, on normal cells, such as pancreatic islets, [22] rat cardiomyocytes [23, 24] and granulotic cells [25]. The combination of monastrol with palmitic acid or oleic acid for B16F10 (Figure 2F, G) and melan-a (Figure 3F, G) showed an effect similar to that of monastrol alone, presenting IC50 values equal or even higher that observed for monastrol (Table 1), indicating that the enhanced effect in palmitic- and oleic-monastrol is the result of the modification of the monastrol structure and not the combination of monastrol and fatty acids. Figure 2. Cell proliferation, measured by MTT assay. B16F10 cells were treated with different concentrations of monastrol (A), palmitic-monastrol (B), oleic- monastrol (C), palmitic acid (D), oleic acid (E), monastrol + palmitic acid (F) and monastrol + oleic acid (G) at 0, 24, 48 and 72 hours. Data are expressed as the mean ± SE. Similar letters indicate absence of significant differences at each time point (p> 0.05).

Figure 3. Cell proliferation, measured by MTT assay. Melan-a cells were treated with different concentrations of monastrol (A), palmitic-monastrol (B), oleic-monastrol (C), palmitic acid (D), oleic acid (E), monastrol + palmitic acid (F) and monastrol + oleic acid (G) at 0, 24, 48 and 72 hours. Data are expressed as the mean ± SE. Similar letters indicate absence of significant differences at each time point (p> 0.05).

Table 1. Estimated compounds concentrations (µM) that caused 50% of inhibition (IC50) in B16F10 and melan-a cell lines after 72 hours of exposure, according to MTT assay.
Compounds B16F10 Melan-a
Monastrol 95.64 (67.04-136.43) 43.68 (38.27-49.87)
Palmitic-monastrol 24.30 (19.76-29.87) 28.39 (24.29-33.18)
Oleic-monastrol 20.00 26.27 (22.00-31.37)
Palmitic acid – –
Oleic acid 200.00 –
Monastrol + Palmitic acid 108.90 (62.45-189.91) 41.79 (31.37-55.66)
Monastrol + Oleic acid 148.85 (114.68-193.19) 68.66 (53.32-88.40)

Values in parentheses represent the 95% confidence interval for each IC50 estimated. – represents not calculable IC50 values.

3.3.Apoptosis and/or Necrosis

Leizerman et al. [11] found 50% of cells in the AGS cell line and 20% of the Ht29 cells to be in apoptosis after 48 hours of treatment with 100 µM of monastrol. Sashidhara et al. [10] reported that the coumarin–monastrol hybrid induces apoptosis in primary and metastatic breast cancer cell lines.
According to ours results, monastrol showed apoptosis in B16F10 cells after 24 hours of exposure at 80 µM and 140 µM. At the same time point, no necrosis was found. At 48 hours and 72 hours, there was anincrease in apoptosis for 140 µM and necrosis was found, but it was significantly different from the control at only 72 hours (Figure 4).

Figure 4. Visual field of cells in the control group demonstrating viable cells (A) and cells treated with 140 µM of monastrol after 72 hours of exposure (B), showing viable cells, in apoptosis or necrosis captured by fluorescence microscopy. Percentage of viable, apoptotic and necrotic B16F10 cells at 24 hours (C), 48 hours (D) and 72 hours (E) after treatment with 80 µM and 140 µM of monastrol from the visual field captured by fluorescence microscopy. Data are expressed as the mean ± SE. Similar letters indicate the absence of significant differences in each group (p> 0.05).

However, in our present study, it was not possible to quantify apoptotic cells under palmitic-monastrol and oleic-monastrol treatment because, after 24 hours, there were no more cells attached to the well. For this reason, we quantified apoptosis and necrosis at 0, 3 and 6 hours after exposure to 80 µM (data not shown) and 140 µM of palmitic- monastrol and oleic-monastrol (Figure 5). Immediately after treatment at both concentrations, only viable cells were observed, but after 3 hours, the cells started detaching from the well. However, they still emitted green fluorescence, indicating viable cells. This effect suggests that palmitic-monastrol and oleic-monastrol might affect the adhesion proteins found on the cell membrane.

Figure 5. Visual field of cells treated with palmitic-monastrol (A, C, E) and oleic- monastrol (B, D, F) after 0, 3 and 6 hours after exposure with 140 µM, respectively, captured by fluorescence microscopy.

Cell adhesion to the extracellular matrix (ECM) plays an essential role in the regulation of fundamental cellular processes, such as cell survival, proliferation and migration. Disruption of these cellular processes is involved in initiation and/or progression of

various diseases. Integrin, a family of cell adhesion molecules, serves as a key determinant for adhesion-dependent cell regulation [26].
Velasco-Velázquez et al. [27] analyzed the effects of 4-hydroxycoumarin (4-HC), one minor biotransformation product of coumarin, and found that the 4-HC produced a concentration-dependent reduction in the adhesion of B16F10 cells to ECM proteins. Their experiments described selective effects of 4-HC on metastatic melanoma cells on several parameters: cytoskeleton stability, adhesion to components of the ECM, motility, and phosphorylation on tyrosine residues. Efficient adhesion to ECM needs, besides integrin activation, adequate spatial organization of the cytoskeleton [28,29] and cytoskeletal contractility [30,31] for the recruitment of signaling molecules.
Thus, these results lead us to conclude that by modifying the molecular structure of monastrol, its effect was also modified, indicating a change in its mechanism of action.

4.Conclusions

This work describes the synthesis of fatty-monastrol derivatives using a multicomponent Biginelli reaction. The effect of the insertion of palmitic acid or oleic acid onto monastrol in the melanoma cell line, B16F10, was investigated. According to the results demonstrated by this study, monastrol inhibits cell growth in B16F10 and melan-a cell lines, with melan-a demonstrating greater sensitivity than B16F10. The structural modifications are shown to be effective because palmitic-monastrol and oleic- monastrol were cytotoxic for both cell lines. This effectiveness is further supported by the lack of cytotoxic effect when palmitic and oleic acids were tested individually as well as when these fatty acids were used only in combination with monastrol. Despite these changes had been effective not only on tumoural cells, the lowest concentration of oleic-monastrol inhibited the proliferation of B16F10 cells in less time than that in

melan-a cells. Besides, IC50 values demonstrate that hybridization of the molecules renders melanoma cells more sensible than non-tumour melanocytes, especially for oleic-monastrol. For apoptosis and/or necrosis, monastrol caused apoptosis after 24 hours of exposure. Additionally, at 48 hours and 72 hours, there was an increase in apoptosis for 140 µM and necrosis was observed. However, palmitic-monastrol and oleic-monastrol showed a different effect: after 3 hours of treatment, cells detached from the well, indicating that they might affect the adhesion proteins found on the cell membrane. Further studies must be developed to elucidate the mechanisms involved in this process and the possibility to study the effects of these new molecules in other cell lines.

5.Acknowledgements

This work was financed by Capes (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) – Ministério da Educação and Programa de Pós-Graduação em Ciências Fisiológicas: Fisiologia Animal Comparada, FURG.

6.References

[1]World Health Organization (WHO), http://www.who.int/en/ (accessed in November 25, 2014).
[2]S. Terracciano, A. Foglia, M.G. Chini, M.C. Vaccaro, A.Russo, F. Dal Piaz, C. Saturnino, R. Riccio, G. Bifulco, I. Bruno, New dihydropyrimidin-2(1H)-one based Hsp90 C-terminal inhibitors, RSC Adv. 6(2016) 82330-82340.
[3]K.S. Atwal, B.N. Swanson, S.E. Unger, D.M. Floyd, S. Moreland, A. Hedberg, B.C. O’Reilly, Dihydropyrimidine calcium channel blockers. 3.¹ 3-Carbamoyl-4-aryl-1,2,3,4-

tetrahydro-6-methyl-5-pyrimidinecarboxylic acid esters as orally effective antihypertensive agents. J. Med. Chem. 34 (1991) 806-811.
[4]G.C. Rovnyak, K.S. Atwal, A. Hedberg, S.D. Kimball, S. Moreland, J.Z. Gougoutas, B.C. O’Reilley, J. Schwartz, M.F. Malley, Dihydropyrimidine Calcium Channel Blockers. 4. Basic 3-Substituted-4-aryl-1,4-dihydropyrimidine-5-carboxyAliccid Esters. Potent ntihypertensive Agents. J. Med. Chem. 35 (1992) 3254-3263.
[5]C.O. Kappe, 4-Aryldihydropyrimidines via the Biginelli condensation: Aza-analogs of nifedipine-type calcium, Molecules. 3 (1998) 1–30.
[6]B. Jauk, T. Pernat, C.O. Kappe, Design and synthesis of a conformationally rigid mimic of the dihydropyrimidine calcium channel modulator SQ 32,926, Molecules. 5 (2000) 227–239.
[7]T.U. Mayer, T.M. Kapoor, S.J. Haggarty, R.W. King, S.I. Schreiber, T.J. Mitchison, Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen, Science. 268 (1999) 971–974.
[8]J.C. Cochran, J.E. Gatial, T.M. Kapoor, S.P. Gilbert, Monastrol inhibition of the mitotic kinesin Eg5, J. Biol. Chem. 280 (2005) 12658–12667.
[9]T. Peters, H. Lindenmaier, W.E. Haefeli, J. Weiss, Interaction of the mitotic kinesin Eg5 inhibitor monastrol with P-glycoprotein, Arch. Pharmacol. 372 (2006) 291–299.
[10]K.V. Sashidhara, S.R. Avula, K.Sharma, G.R. Palnati, S.R. Bathula, Discovery of coumarin–monastrol hybrid as potential antibreast tumor-specific agent, Eur. J. Med. Chem. 60 (2013) 120-127.
[11]R. Leizerman, M. Avunie-Masala, M. Elkbets, A. Fich, L. Gheber, Differential effects of monastrol in two human cell lines. L. Cell. Mol. Life. Sci. 61 (2004) 2060– 2070.

[12]A. Dondoni, A. Massi, S. Sabbatini, V. Bertolasi, Three-component Biginellicyclocondensation reaction using C-glycosylated substrates. Preparation of a collection of dihydropyrimidinone glycoconjugates and the synthesis of C-glycosylated monastrol analogues, J. Org. Chem. 67 (2002) 6979–6994.
[13]C.M. Wright, R.J. Chovatiya, N.E. Jameson, D.M. Turner, G. Zhu, S. Werner, D.M. Huryn, J.M. Pipas, B.W. Day, P. Wipf, J.L. Brodsky, Pyrimidinone-peptoid hybrid molecules with distinct effects on molecular chaperone function and cell proliferation, Bioorg. Med. Chem. 16 (2008) 3291–3301.
[14]A. Shaikh, J. Meshram, Synthesis and pharmacological activity evaluation of oxadiazoles containing substituted dihydropyrimidinone and chloroquinoline moities, Int. J. Pharm. Sci. Res. 4 (2013) 4607–4614.
[15]J.G. Sośnicki, Ł. Struk, M. Kurzawski, M. Perużyńska, G. Maciejewska, M. Droździk, Regioselective synthesis of novel 4,5-diaryl functionalized 3,4- dihydropyrimidine-2(1H)- thiones via a non-Biginelli-type approach and evaluation of their in vitro anticancer activity, Org. Biomol. Chem. 12 (2014) 3427-3440.
[16]M.N. Godoi, H.S. Costenaro, E. Kramer, P.S. Machado, M.G.M. D’Oca, D. Russowsky, Síntese do Monastrol e Novos Compostos de Biginelli Promovida por In(OTf)3, Quim. Nova 28 (2005) 1010–1013.
[17](a) T.G.M. Treptow, F. Figueiró, E.H.F. Jandrey, A.M.O. Battastini, C.G. Salbego, J.B. Hoppe, P.S. Taborda, S.B. Rosa, L.A. Piovesan, C.R.M. D’Oca, D. Russowsky, M.G.M. D’Oca, Novel hybrid DHPM-fattyacids: Synthesis and activity against glioma cell growth in vitro, Eur. J. Med. Chem. 95 (2015) 552-562.(b) A.C.H. Weber, T.C. Batista, B. Gonçalves, C.R.L. Hack, L.M. Porciuncula, T.G.M. Treptow, C.R.M. D’Oca, D. Russowsky, M.G.M. D’Oca, Synthesis of Fatty Acetoacetates Under Microwave

Irradiation Catalysed by Sulfamic Acid in a Solvent-Free System, J. Am. Oil Chem. Soc. 93(2016) 1399-1406.
[18]D. Ribble, N.B. Goldstein, D.A. Norris, Y.G. Shellman, A simple technique for quantifying apoptosis in 96-well plates, BMC Biotechnol. 5 (2005) 12-18.
[19]B. Kosmider, E. Zyner, R. Osiecka, J. Ochocki, Induction of apoptosis ans necrosis in A549 cells by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP, Mutat. Res. 563 (2004) 61-70.
[20]L.N. Sousa Andrade, T.M. Lima, R. Curi, A.M.L. Castrucci, Toxicity of fatty acids on murine and human melanoma cell lines, Toxicol. in Vitro. 19 (2005) 553–560.
[21]R.A. Hawkins, M.J. Sangster, M.J. Arends, Apoptotic death of pancreatic cancer cells induced by polyunsaturated fatty acids varies with double bond number and involves Na oxidative mechanism, J. Pathol. 185-1 (1998) 61–70.
[22]M. Cnop, J.C. Hannaert, A. Hoorens, D.L. Eizirik, D.G. Pipeleers, Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation, Diabetes 50 (2001) 1771–1777.
[23]G. Sparagna, D. Hickson-Bick, L. Buja, J.B. McMillin, A metabolic role for mitochondria in palmitate-induced cardiac myocyte apoptosis, Am. J. Physiol. Heart Circ. Physiol. 279-5 (2000) H2124–H2132.
[24]D.B. Ostrander, G.C., Sparagna, A.A Amoscator,. J.B. McMillin, W. Dowhan, Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate- induced cardiomyocyte apoptosis, J. Biol. Chem. 41-12 (2001) 38061–38067.
[25]Y.M. Mu, T. Yanase, Y. Nishi, A. Tanaka, M. Saito, C.H. Jin, C. Mukasa, T.Okabe, M. Nomura, K. Goto, H. Nawata, Saturated FFAs, palmitic acid and stearic

acid, induce apoptosis in human granulosa cells, Endocrinology. 142-8 (2001) 3590– 3597.
[26]T. Matsunaga, T. Iyoda, F. Fukai, Chapter 12 – Adhesion-dependent cell Regulation via Adhesion molecule, integrin: Therapeutic application of integrin activation-modulating factors, Colloid and Interface Science in Pharmaceutical Research and Development, in: H. Ohshima, K. Makino (Eds.), Colloid and Interface Science in Pharmaceutical Research and Development, Elsevier, Oxford, 2014, pp. 243–260.
[27]M.A. Velasco-Velázquez, J. Agramonte-Hevia. D. Barrera, A. Jiménez-Orozco, M.J. García-Mondragón, N. Mendoza-Patiño, A. Landa, J. Mandoki, 4- Hydroxycoumarin disorganizes the actin cytoskeleton in B16–F10 melanoma cells but not in B82 fibroblasts, decreasing their adhesion to extracellular matrix proteins and motility. Cancer Lett. 198 (2003) 179–186.
[28]S.M. Schoenwaelder, K. Burridge, Bidirectional signaling between the cytoskeleton and integrins, Curr. Opin. Cell. Biol. 11 (1999) 274–286.
[29]G. Pawlak, D.M. Helfmann, Cytoskeletal changes in cell transformation and tumorigenesis, Curr. Opin. Genet. Dev. 11 (2001) 41–47.
[30]D.E. Ingber, Transegrity II. How structural networks influence cellular information processing networks, J. Cell. Sci. 116 (2003) 1397–1408.
[31]E. Zamir, B. Kats, S. Aota, K. Yamada, B. Geiger, Z. Kam, Molecular diversity of cell-matrix adhesion, J. Cell. Sci. 112 (1999) 1655–1669.

Highlights

•Synthesis and cytotoxicity of new fatty-DHPM in melanoma cells

•Fatty-monastrol were cytotoxic and inhibited proliferation in both cell lines

•Hybridization of the monastrol increase its effects in melamona cells

•Oleic-monastrol was more effective in melamona cells than in non-tumour melanocytes
•Fatty-monastrol had a possible effect in the adhesion proteins

Graphical abstract