Facile Synthesis and Cytotoxic Activity of the First Ferrocene-Resveratrol Conjugate

The bioorganometallic III containing trimethylene chain between ferrocene and resveratrol (3,5,4'-trihydroxystilbene, RSV) moieties connected via ester bond has been synthesized. The novel bioconjugate was characterized using IR and NMR (1H, 13C, COSY, NOESY, HMBC) spectroscopy, ESI-MS and HRMS. The RSV and ferrocene-RSV conjugate III were screened in vitro for their inhibitory effects against proliferation of hepatoblastoma (Hep G2) cells by MTT assay. Also, possible cytotoxicity towards normal ovary cells (CHO-K1) was evaluated. The obtained data revealed profound effects in biological/cytotoxic activity of III vs. RSV in Hep G2 cell line. Lower cytotoxicity of III was observed in normal ovary cells as compared to hepatoblastoma cells.


INTRODUCTION
WING to its therapeutic and protective role, resveratrol [3,5,4'-trihydroxystilbene (RSV) (Figure 1a)] came into focus of research in recent years.−3] In order to enhance its activity, RSV was derivatized by: (i) modification of the number and position of the phenolic groups, (ii) insertion of a long alkyl chains or functionalized chains [4] and (iii) the addition of acyl chains to free hydroxyl groups. [5]The improved biological activity of the so-obtained RSV derivatives in comparison to those of the parent molecule is likely to be due to their lipophilicity and facilitated transport through cell membrane.Recently, RSV-derivatives I and II containing lipophilic ferrocene moiety instead of one benzene ring were synthesized (Figure 1b). [4]The heteroannularly disubstituted derivative IIb, containing additional aromatic unit linked by an ester bond, was found to exhibit more than 10-fold higher inhibitory activity in SW480 and HepG2 cell lines compared to those of RSV.Prompted by these results, we have prepared and tested conjugate III containing trimethylene alkyl chain between ferrocene and RSV connected via ester linkage (Figure 1c).

EXPERIMENTAL
The material and methods data are provided in Supplementary Materials.

Chemistry
The trans-resveratrol (RSV) (95 mg, 0.42 mmol) was dissolved in dry THF (7 ml) and added to a solution of ferrocene butyric acid 1 [6] (374 mg, 1.37 mmol) in the same solvent (10 ml), followed with addition of DMAP (7.6 mg, 0.06 mmol) and NEt3 (0.35 ml, 2.49 mmol) [7] (Scheme 1).The reaction mixture was cooled to −15 °C and solu on of Boc2O (354 mg, 1.62 mmol) in dry THF (8 ml) was added.After 5 minutes, the reaction mixture was allowed to warm to room temperature and then stirred at ambient temperature during 5 days.After the total consumption of the starting material, as monitored by TLC, the mixture was diluted with EtOAc (10 ml) and washed with 2M HCl (30 ml), 5 % NaHCO3 (50 ml) and saturated NaCl.The organic layer was dried over Na2SO4 and evaporated in vacuo to leave the crude product.Since the numerous yellow and UV visible spots were seen on TLC plate, the TLC purification of the crude material was repeated for several times using different solvent systems (petroleum ether/ diethyl ether = 2/1; petroleum ether/ diethyl ether = 5/1; CH2Cl2 /hexane = 3/1) until satisfactory purity of the compound III was obtained and confirmed by ESI-LC method.Yellow-orange oil; 43 mg (21 %); Rf = 0.68 in petroleum ether/ diethyl ether = 2/1; IR (CH2Cl2) ṽmax/cm −1 : 3676 w (OH), 1759 s (C=O), 1137 s (CO); 1

Cytotoxic Activity
Treatment: Stock solutions of RSV and ferrocene-RSV conjugate III were prepared as 20 mM solutions in ethanol (EtOH) and stored at 4° C. Prior to use in cytotoxicity assay, the stock solutions were further diluted with culture medium to obtain final concentrations (10-100 μM).
Cytotoxic effects in Hep G2 and CHO-K1 cells were evaluated after 48 h of exposition.Samples with ethanol without test compounds were used as controls.
MTT Cytotoxicity Assay: For experimental purposes cells were seeded in multiwell plates (5  10 4 cells/mL), then treated with RSV and III at a range of concentrations (10− 100 μM) and after 48 h cell viability was determined by MTT assay. [8]The experiments were performed two times with at least three parallels for each concentration and data were expressed as the means ± SEM.Cell viability was expressed as percentage of treated cells vs. control cells.The IC50 values, defined as the concentrations of the tested compound that result in 50 % cell growth inhibition, were derived from the equations of related trend lines.
Statistical Analysis: A two-tailed Student's t-test was applied to evaluate the significant differences between control and treated cells.The results are reported as means ± SEM, p < 0.05 was considered significant.

RESULTS AND DISCUSSION
The conjugate III was prepared by esterification [7] of ferrocene butyric acid (1) [6] with C3-OH group of trans-RSV in the presence of DMAP, NEt3 and Boc2O (Scheme 1).Although the reaction conditions were adjusted to provide the simultaneous formation of mono-(III), di-and three esterified conjugates, we were able to isolate and confirm the presence of only one pure compound, i.e. monoester III in yield of 21 %.However, TLC-plate indicated the presence of a several yellow-coloured ferrocene-containing compounds that had proved to be difficult to purify to the required level.
The NMR assignments in the present study are in a good agreement with previously published data for RSV [9,10] and its 3-substituted RSV-glycoside [11] (Table 1).
The 1 J(H,H) coupling between two alkene hydrogens α and α' of 16.5 Hz is consistent with the trans orientation of the phenolic rings.MS and NMR data unambiguously confirmed the presence of one single ferrocene moiety, introduced by esterification of C3-OH group of RSV.As it was shown for 3-substituted RSV-glycoside [11] , the clearly separated NMR signals of C2 and C6, C3 and C5 and H2 and H6 in comparison to magnetically equivalent assignments for H2'/6', H3'/5', C2'/6' and C3'/5' indicate the alteration of their chemical environment due to the esterification of Scheme 1.The synthesis of ferrocene-RSV conjugate III.
The six protons from alkyl chain were observed as two triplets at δ 2.85 and 2.48 ppm and one multiplet at 1.96 ppm.Multiple resonances detected for the same proton during the course of NMR analysis are attributed to the presence of 5:95 cis-trans mixture of RSV.
The RSV and ferrocene-RSV conjugate III were evaluated in vitro as inhibitors of hepatoblastoma (Hep G2) cell proliferation.Also, possible cytotoxicity towards normal ovary cells (CHO-K1) was evaluated.The concentration-dependent analysis of the cytotoxicity was evaluated by the widely used MTT method.The summarized results of cytotoxicity evaluation in Hep G2 and CHO-K1 cell lines with MTT bioassay are presented in Figure 3. IC50 values shown in Table 2 were derived from the equations of related polynomial trend lines for RSV and III, in both cell lines.
Presented data reveal profound effects in biological activity of ferrocene-RSV conjugate III vs. RSV in hepatoblastoma cell line.III in concentrations of 35, 75 and 100 μM significantly (p<0.001-p<0.5)decreased Hep G2 cell proliferation (Figure 3a) and in all tested concentrations inhibitory effect is more pronounced for III compared to the same doses of RSV.This finding is also supported by comparison of IC50 values in Hep G2 cells: IC50 value for III is 20 % lower than IC50 value for RSV [4] (Table 2).The results in normal CHO-K1 cells (Figure 3b), as opposite to those obtained in Hep G2 carcinoma cells, indicate much lower cytotoxicity of III to normal cells -in fact, there was no statistically significant cytotoxicity observed in normal cells, except for the highest tested concentration (100 µM) and IC50 value was out of the applied concentration range.

CONCLUSION
The previously described ferrocene-RSV derivative IIb, containing two benzene, one ferrocene and one ester group, displayed higher inhibitory activity in SW480 and HepG2 cell lines as compared to RSV, owing to its enhanced lipophilicity.With that in mind, we have synthesized conjugate III comprised of the same elements but with different structural and spatial patterns and equipped with additional trimethylene alkyl chain in order to improve its lipophilicity as a crucial requirement for biological activity.Although the novel conjugate III is found to be less active than IIb, it is more potent in comparison to RSV.Therefore, the herein presented preliminary results hold potential for further studies of ferrocene-RSV conjugates based on multi-method approach in order to obtain more detailed information.Table 1.NMR (δ in ppm) spectroscopic data of RSV, [9,10] RSV-glycoside [11] and ferrocene-RSV conjugate III (c = 110 −3

OFigure 1 .
Figure 1.The chemical structures of (a) resveratrol (RSV), (b) ferrocene-RSV analogs I and II and (c) ferrocene-RSV conjugate III.The numbering of the carbon atoms is according to IUPAC nomenclature.

Figure 3 .
Figure 3.In vitro anti-proliferative effect of RSV and ferrocene-RSV conjugate III against (a) hepatoblastoma (Hep G2) cells and (b) normal ovary cells (CHO-K1), obtained with MTT assay after 48 h exposure.Data are presented as percentage of control: mean  SEM of 2 experiments with at least three measurements within each experiment for each concentration.Statistical significance vs. control: a p < 0.001; b p < 0.025; c p < 0.05.

Table 2 .
mol dm −3 ) IC50 n.d.-non determinated IC value for the corresponding incubation period, cannot be calculated from the equations of related polynomial trend lines.