Synthesis and Antitumor Activity of Diterpenylhydroquinone Derivatives of Natural Ent-Labdanes

Two new compounds 2β-acetoxy-15-phenyl-(22,25-acetoxy)-ent-labda-8(17), 13(E)-diene (9) and 2β-hydroxy-15-phenyl-(22,24,26-trimethoxy)-ent-labda-8(17),13(E)-diene (10) have been prepared by an Electrophilic Aromatic Substitution (EAS) reaction between diterpenyl allylic alcohols and 1,4-hydroquinone or 1,3,5-trimethoxybenzene using BF3.Et2O as a catalyst. These compounds, along with a series of natural ent-labdanes 3-8, have been evaluated for their in vitro cytotoxic activities against cultured human cancer cells of PC-3 and DU-145 human prostate cancer, MCF-7 and MDA-MB-231 breast carcinoma and dermal human fibroblasts (DHF). Some compounds displayed inhibition at µM IC50 values.


Introduction
Terpenylquinones constitute an interesting group of marine natural products, for which a wide variety of biological activities have been described, including anti-inflammatory, antifungal, anti-HIV and most frequently, antineoplastic properties [1,2]. Their cytotoxic activities has been justified by their ability undergo redox cycles and the generation of reactive oxygen species, which could damage tumour cells [3,4]. These compounds are generally characterized for having a sesquiterpene skeleton attached to a (hydro)quinone moiety ( Figure 1). Terpenylquinones are isolated from natural sources in very low yield, and for that reason during the last few decades, considerable research effort has been focused on obtaining these compounds by synthesis. The most recurrent strategies used for synthesizing these compounds involve, as a first step, the separate preparation of the appropriate terpenyl fragments and aromatic nucleus. The crucial step is the attachment of the aromatic synthon to the terpenyl skeleton. There are many publications that report different methods for accomplishing these coupling reactions in the synthesis of sesquiterpenylquinones [5][6][7][8][9][10][11][12]. On the other hand, isolation of natural diterpenylquinones has not been reported and only one work has reported the synthesis of diterpenylnaphthoquinones, which showed an important activity in the inhibition of the growth of cancerigenous cells [1,2].
Recently we reported the synthesis and the structural determination of a series of natural ent-labdane derivatives 3-6 ( Figure 2) and the diterpenylhydroquinones 7 and 8 ( Figure 2) using Electrophilic Aromatic Substitution (EAS) reactions between an arene nucleus and an allylic alcohol as diterpenyl synthon [13].
Using this synthetic protocol and as continuation of that research we describe in this paper the preparation of two new derivatives 9 and 10. Additionally a new compound 11 derived from the natural ent-labdanes was obtained ( Figure 3). Finally the new compounds 9-11 and the previously reported series of natural ent-labdanes derivatives 3-8 were evaluated in vitro against cultured human cancer cells in order to analyse the influence of the molecular structure on the antitumoral activity.

Results and Discussion
Previously we reported the isolation and structural determination of the mixture of ent-labdanes 1-2 from Calceolaria inamoena and the preparation of derivatives 3 and 8 [13,14]. In the saponification reaction used for preparation of diterpenyl synthon 4, a new compound 11 was obtained when acidic conditions were used, presumably due to a transesterification reaction and the presence of ethyl acetate in the workup procedure, nevertheless the target diol 4 could be obtained under partial neutralization conditions (Scheme 1). The next step was the coupling reaction between compound 4 and 1,4-hydroquinone to give diterpenylhydroquinone 8, using the synthetic protocol recently reported by us [13]. A subsequent acetylation reaction, under standard conditions, gave the new compound 9 (Scheme 1). The structural determination of compound 9 was accomplished by 1 H, 13 C, DEPT-135, gs-2D HSQC and gs-2D HMBC NMR techniques and by comparison with the spectral data of compound 8 [13].

Scheme 1. Synthesis of compound 9.
On the other hand, the reaction between the diol 4 and 1,3,5-trihydroxybenzene (12) was not observed and compound 11 was unexpectedly obtained again, presumably for the same reasons given above (Scheme 2). The structure of the new natural ent-labdane derivative 11 was established by comparison with the previously reported spectral data of compound 4 [13].

Bioactivity
The antitumor cytotoxicity of the compounds was evaluated in vitro against three different cancer cell lines: PC-3 and DU-145 human prostate cancer, MCF-7 and MDA-MB-231 breast carcinoma and one non-tumoral cell line, dermal human fibroblasts (DHF). A conventional colorimetric assay was set up to estimate the IC 50 values, which represent the concentration of a drug that is required for 50% inhibition in vitro after 72 h of continuous exposure to the test compounds. Four serial dilutions (from 12.5 to 100 µM) for each sample were evaluated in triplicate. The results obtained from these assays are shown in Table 1

Conclusions
As a conclusion, we have described the synthesis and structural determination of two new diterpenylhydroquinones 9 and 10 from natural ent-labdane derivatives. These compounds were obtained by Electrophilic Aromatic Substitution (EAS) coupling reactions between primary allylic alcohol ent-labdane derivatives with1,4-hydroquinone and 1,3,5-trimethoxybenzene. Furthermore, a new co-compound 11 derived from a natural ent-labdane was obtained. The new compounds 9 and 10 showed cytotoxic bioactivity against cell lines MCF-7 and MDA-MB-231 breast carcinoma. The natural ent-labdane derivatives 3-6 and 11 did not affect the bioactivity of the cells lines studied, however the diterpenylhydroquinones derivatives 7-10 showed IC 50 values with significant inhibitory activity, ranging in the µM level, due to the presence of a hydroquinone moiety. Moreover, compounds 7-9 showed some selectivity for cancer cells versus fibroblast cells, which could be a way to access to compounds potentially less toxicity to normal human cells.

General
All purchased chemical reagents (Merck or Aldrich) were of the highest commercial available purity and were used without previous purification. IR spectra were recorded as thin films on a Nicolet Impact 420 spectrometer and frequencies are reported in cm −1 . Optical rotations were measured with a sodium lamp (λ = 589 nm, D line) on a Perkin Elmer 241 digital polarimeter equipped with 1 dm cells at the temperature indicated in each case. Low resolution mass spectra were recorded on a Shimadzu QP-2000 spectrometer at 70eV ionising voltage and are given as m/z (% rel. int.). 1 H, 13 C (DEPT 135), sel. 1D 1 H NOESY, 2D HSQC and 2D HMBC spectra were recorded in CDCl 3 solutions on a Bruker Avance 400 Digital NMR spectrometer, operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C and are referenced to the residual peaks of CHCl 3 at δ 7.26 ppm and δ 77.0 ppm for 1 H and 13 C, respectively. Chemical shifts are reported in δ ppm and coupling constants (J) are given in Hz. Silica gel (Merck 200-300 mesh) was used for column chromatography and HF-254 silica gel plates for TLC. TLC spots were detected by heating after spraying with 25% H 2 SO 4 in H 2 O.

General Procedure for EAS: Synthesis of Compounds 8, 10
Et 2 O (0.23 g, 1.6 mmol) was gradually added at room temperature to a solution of 1,4-hydroquinone (0.30 g, 2.27 mmol) or 1,3,5-trimethoxybenzene (0.30 g, 1.79 mmol) and compound 3 (0.50 g, 1.6 mmol) in freshly distilled 1,4-dioxane (30 mL). The mixture was stirred at room temperature under a nitrogen atmosphere for 24 h, when the completion of the reaction was verified by TLC. The mixture was poured onto crushed ice (about 30 g) and the organic layer was extracted with EtOAc (3 × 30 mL). The combined organic phase was washed with 5% NaHCO 3 (30 mL), then with water (2 × 20 mL) and dried over anhydrous Na 2 SO 4 , filtered and evaporated. The crude residue was redissolved in CH 2 Cl 2 (5 mL) and chromatographed on silica gel with petroleum ether/EtOAc mixtures of increasing polarity  (9). To diterpenylhydroquinone 8 (48.8 mg, 0.12 mmol) dissolved in dichloromethane (30 mL), acetic anhydride (0.54 g, 0.5 mL, 5.2 mmol) and dimethylaminopyridine (1.5 mg, 0.01 mmol) were added. The mixture was stirred at room temperature during 2 h. The organic phase was washed with water and after it was dried over anhydrous Na 2 SO 4 . The solution was evaporated to dryness to afford the crude reaction product, which gave 45 mg of compound 9 after column chromatography (eluent hexane/ethyl acetate 19. 8

Bioactivity: Cell Growth Inhibition Assay
The colorimetric assay using sulforhodamine B (SRB) was realized following an adaptation of the described method by Skehan et al. [15,16]. Cells were seeded in 96-well microtiter plates, at 5 × 10 3 cells per well in aliquots of 100 µL of DMEM/F-12 medium, and they were allowed to attach to the plate surface by growing in drug-free medium for 18 h. Afterward, compounds samples were added in aliquots (dissolved in EtOH/H 2 O) to achieve a final concentration of 100, 50, 25 12.5 µM. After 72 h exposure, the in vitro cytotoxicity was measured by the SRB dye assay. Cells were fixed by adding cold 50% (wt/vol) trichloroacetic acid (TCA, 25 µL) and incubating for 60 min at 4ºC. Plates were washed with deionized water and dried; SRB solution (0.1% wt/vol in 1% acetic acid, 50 µL) was added to each microtiter well and incubated for 30 min at room temperature. Unbound SRB is removed by washing with 1% acetic acid. Plates were air-dried and bound stain was solubilized with Tris base (100 µL, 10 mM). Optical densities were read on an automated spectrophotometer plate reader at a single wavelength of 540 nm. Values shown are the % viability vs. ctrl + SD, n = four independent experiments in triplicate.