Antitumor Activity of Some Prenylated Xanthones

Pyranoxanthones 6-8 were obtained by dehydrogenation of the respective dihydropyranoxanthones 3-5 with DDQ in dry dioxane. Two prenylated xanthones 10,11 were obtained from the reaction of 1-hydroxyxanthone (9) with prenyl bromide in alkaline medium, or by condensation of xanthone 9 with isoprene in the presence of orthophosphoric acid. The structural elucidation of the two new compounds 6,11, as well as an update of data for the already described prenylated derivatives 7,8,10 were accomplished by IR, UV, HRMS and NMR (1H, 13C, HSQC and HMBC) techniques. The effect of the prenylated xanthone derivatives on the in vitro growth of human tumor cell lines MCF-7 (breast adenocarcinoma) and NCI-H460 (non-small cell lung cancer) is also reported. Compounds 10 and 11 have been found to exhibit a moderate growth inhibitory activity against the MCF-7 cell line.


Introduction
Many naturally occurring xanthones and their prenylated derivatives are found to exhibit significant biological and pharmacological properties, such as antibacterial, antifungal and antitumor activities and it can be inferred that the presence of prenyl groups can be associated with an improvement of potency and selectivity for some of these properties [1,2]. As a large number of biologically active xanthone derivatives with pyran and dihydropyran rings are commonly found in Nature, we were interested in obtaining this type of compounds to evaluate their antitumor activity. For this purpose, molecular modifications of the hit compounds, 1,3-dihydroxy-2-methylxanthone (1) and 1,3-dihydroxyxanthone (2) (Figure 1) were carried out [3].  Prenylation of xanthones 1 and 2 with prenyl bromide, followed by cyclisation of the respective monoprenylated products furnished dihydropyranoxanthones 3-5 [3] (Figure 1), which were evaluated for their effects on the in vitro growth of three human tumour cell lines (MCF-7, NCI-H460 and SF-268). These compounds were found to be more selective, showing their growth inhibitory effects only against the breast cancer MCF-7 cells when compared with their building blocks, respectively 1 and 2 [3].
The fact that naturally occurring pyranoxanthones are more active than dihydropyranoxanthones in many biological activity assays [1] has led us to resort to a rigidification strategy to improve the antitumor activity of the xanthone derivatives. Thus, unsaturation strategy was applied to the dihydropyran ring of dihydropyranoxanthones 3-5 to give pyranoxanthones 6-8, respectively ( Figure 1).
The second approach is to introduce the prenyl side chain to the xanthone nucleus, using a C-prenylation strategy. Thus, two C-prenylated derivatives, 10 and 11 were synthesized by prenylation of xanthone 9 ( Figure 1). Though C-prenylated derivatives are not as common in nature as the O-prenylated analogues, they show very interesting properties [1]. Based on this observation, xanthone 9 ( Figure 1) was submitted to a C-prenylation strategy to furnish compounds 10 and 11.
The xanthone derivatives 6-8, 10 and 11, were then evaluated for their capacity to inhibit the in vitro growth of MCF-7 (breast adenocarcinoma) and NCI-H460 (non-small cell lung cancer) cells, and their effects were compared with those of their building blocks [3,4].
The prenylated derivative 11 was obtained by condensation of 1-hydroxyxanthone (9) with isoprene, in the presence of catalytic amounts of orthophosphoric acid [8] (Scheme 2). The acidcatalysed condensation of isoprene with the phenol moiety of the xanthonic scaffold may be regarded as the chemical equivalent of the proposed biogenetic pathways [8].

Structural elucidation of the prenylated xanthones
The structures of compounds 6-8 and 10, 11 were established by IR, UV, HRMS and NMR ( 1 H-, 13 C-, HSQC and HMBC) techniques, while the spectroscopic data of compounds 1-5 and 9 are in agreement with those reported in the literature [3,[9][10][11][12]. Although the spectroscopic data of pyranoxanthones 7 and 8, as well as of prenylated xanthone 10 have been previously described [7,[13][14], here we provide an updated and complete structure elucidation of these compounds.
The EI-HRMS of compound 6 gave the accurate molecular mass at 308.1049 and the corresponding molecular formula C 19 H 16 O 4 , indicating that there were two hydrogen atoms less than in its dihydropyranoxanthone precursor 3. The 1 H-NMR spectrum of compound 6 was very similar to that of compound 3, except for the two doublets of the olefinic protons at δ H 5.62 (J = 10.0 Hz) and δ H 6.86 (J = 10.0 Hz), instead of the triplets of the protons of two methylene groups at δ H 1.88 (J = 6.8 Hz) and δ H 2.89 (J = 6.8 Hz) of the dihydropyran ring [3]. The protons of the geminal methyl groups of the pyran ring in compound 6 appeared as a singlet at δ H 1.50. The 13 C-NMR spectrum of compound 6 was also similar to that of dihydropyranoxanthone 3 [3], except for the substitution of the two methylene carbons at δ C 16.4 and 31.7 with the two olefinic carbon signals at δ C 115.3 and δ C 126.8.
In turn, the EI-HRMS of compound 7 indicated the accurate molecular mass at 294.0886, corresponding to the molecular formula C 18 H 14 O 4 . The 1 H-and 13 C-NMR spectra of compound 7 were very similar to those of compound 6, except for the presence of a singlet of the aromatic proton at C-2 at δ H 6.28 instead of the singlet of the methyl group at δ H 2.12. As in compound 6, the presence of the pyran ring in compound 7 was confirmed by the two doublets of the olefinic protons at δ H 5.62 (J = 10.0 Hz) and δ H 6.85 (J = 10.0 Hz) in the 1 H-NMR spectrum which showed cross peaks with the olefinic carbons at δ C 127.2 and δ C 115.0, respectively in the HSQC spectrum.
The EI-HRMS of compound 8 gave the accurate molecular mass at 294.0898 and the molecular formula C 18 H 14 O 4 . As expected, the 1 H-and 13 C-NMR spectra of compound 8 were similar to those of its dihydropyranoxanthone precursor 5 [3], except for the signals of the olefinic protons (δ H 6.74, d, Finally, the EI-HRMS of compounds 10 and 11 indicated their accurate molecular masses at 280.1099 and 280.1096, respectively, and thus, a molecular formula C 18 H 16 O 3 for both compounds. This molecular formula confirmed the prenylation of xanthone 9. In turn, the 1 H-NMR spectra of compounds 10 and 11 showed, besides, the proton signals corresponding to the non substituted aromatic ring of the xanthone nucleus, the signals of another two ortho coupled aromatic protons

Biological Activity studies
Though the number of compounds prepared was small, some basic structure-activity relationship trends can be observed. When the effects of the prenylated xanthones 6-8 on the growth of MCF-7 cells are compared with those of their respective xanthonic building blocks 3-5, it was found that the presence of the unsaturation in the pyran ring was associated with a loss of inhibitory activity against MCF-7 (Table 1). It can be presumed that the lack of activity of compounds 6 and 8 could be a consequence of the rigidification of the dihydropyran ring. On the other hand, C-prenylation of the inactive xanthone 9 [4] was found to be associated with the growth of the inhibitory effect against MCF-7 of the prenylated derivatives 10 and 11 ( Table 1). The introduction of the lipophilic prenyl group in C-2 of the xanthonic scaffold is probably the reason for the appearance of this activity for xanthones 10 and 11.
Results are given in concentrations that were able to cause 50% of cell growth inhibition

General
Purification of compounds were performed by flash chromatography using Merck silica gel 60 (0.040-0.063 mm) and preparative thin layer chromatography (TLC) using Merck silica gel 60 (GF 254 ) plates. Reactions were monitored by TLC. Melting points were obtained in a Köfler microscope and are uncorrected. IR spectra were measured on an ATI Mattson Genesis series FTIR (software: WinFirst v. 2.10) spectrophotometer in KBr microplates (cm -1 ). UV spectra were taken in ethanol [15] and were recorded on a Varian CARY 100 spectrophotometer: λ max in nm (software: Cary Win UV v. 3.0). 1 H and 13 C NMR spectra were taken in CDCl 3 at room temperature, on a Bruker Avance 300 instrument. Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference. 1 H-NMR spectra were measured at 300.13 MHz and assignment abbreviations are the following: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), doublet of doublets (dd), and double doublet of doublets (ddd). 13 C-NMR spectra were measured at 75.47 MHz. 13 C-NMR assignments were made by 2D HSQC and HMBC experiments (long-range C, H coupling constants were optimized to 7 Hz). HRMS spectra were recorded as EI (electronic impact) mode on a VG Autospec M spectrometer (m/z) at CACTI, Vigo, Spain. Prenyl bromide, Isoprene and DDQ were purchased from Sigma Aldrich. Compounds 1-5 and 9 were obtained and characterized according to the described procedures [3,[9][10][11][12]. The following materials were synthesized and purified by the described procedures.

Synthesis of prenylated xanthone 11
A solution of isoprene (200 μL; 2.00 mmol) in xylene (1 mL) was added to a stirred mixture of 1hydroxyxanthone (9, 0.20 mg; 0.96 mmol), orthophosphoric acid (85%, 1 mL) and xylene (4 mL), with constant stirring at 31ºC during 2 h. The mixture was stirred for a further 28 h and then neutralised growth of these cell lines was evaluated in all experiments by exposing untreated control cells to the maximum concentration (0.25%) of DMSO used in each assay.

Conclusions
In contrast to their dihydropyranoxanthone precursors 3-5, the pyranoxanthones 6-8 did not exhibit growth inhibitory effect against the breast adenocarcinoma MCF-7 cells. On the other hand, Cprenylation of the inactive hydroxyxanthone 9, led to prenylated derivatives 10 and 11 which exhibited moderate growth inhibitory activity against the MCF-cells. From these results, we can conclude that introduction of an unsaturation on the extra ring was not effective in improving the biological activity of these compounds. On the contrary, the introduction of the prenyl side chain on an appropriate position of the xanthonic scaffold was found to improve the antitumor activity of compounds 10 and 11. The increase of the lipophilicity of the molecule and/or an extra molecular motif to interact with biological targets furnished by the prenyl group can be a key to explain the improvement this activity.