Synthesis and Cytotoxic Activity of Some

3-Benzyl-furan-2(5H)-one (2a) and 3-(4-bromobenzyl)-furan-2(5H)-one (2b) were treated with TBDMSOTf and converted into the corresponding tert-butyldimethyl-silylfuran ethers. These furans were further condensed with several aromatic aldehydes affording compounds 5-14 with general 3-benzyl-5-arylidene-furan-2(5H)-one structures in 31% to 98% yields. Such compounds are analogues of the naturally occurring nostoclide lactones, reported to present moderate cytotoxic activity. Compounds 5-14 were submitted to an in vitro bioassay against the HL-60, HCT-8, SF295 and MDA-MB-435 cancer cell lines using the MTT cytotoxicity assay.


Introduction
α,β-Unsaturated lactones possessing an alkylidene appendage group at the γ-position, are frequently termed γ-alkylidenebutenolides [1][2][3].Over the past few decades, an increasing number of these compounds has been isolated from various natural sources [4].The members of this class of compounds vary greatly in structural complexity, as well as functionality.Besides that, many of them have been shown to display a wide range of biological activities, such as the antibiotic activity displayed by protoanemonin [5][6][7], the cytotoxicity associated with the goniobutenolides A and B [8][9][10][11][12], and the inhibition of cholesterol biosynthesis observed with xerulin and xerulinic acid [13][14].Two other interesting examples of γ-alkylidenebutenolides are the compounds 3-methyl-2H-furo [2,3c]pyran-2-one and peridinin.The former is known to activate seed germination [15] while the latter corresponds to one of the most complex γ-alkylidenebutenolides known to date [16].Peridinin plays an important role in the photosynthesis of sea plankton (Figure 1).In 1993, Yang and co-workers reported the isolation of two-chlorinated metabolites, named nostoclides I and II (Figure 2), from the culture of a symbiotic blue-green alga, Nostoc sp., in Peltigera canina, a common lichen [17].Since their discovery, these γ-alkydenebutenolides have attracted the attention of synthetic organic chemists, which has resulted in the development of different approaches to achieve the total synthesis of these secondary metabolites [18][19][20].Although the nostoclides have been known since 1993, their biological properties have not been fully investigated.It has been suggested that these compounds may be alleopathic agents, since it was observed that the lichen Peltigera canina is able to support an unusually clean, contamination-free culture.Besides that, both compounds showed moderate cytoxicity against the cell lines Neuro-2a CCL 131 and KB CCL 17 [17].
As part of our ongoing efforts to develop bioactive compounds [21][22][23][24][25], and taking into consideration that there is no systematic investigation regarding the cytotoxic activity of nostoclides analogues, we describe herein the preparation and evaluation of antitumour activity of ten new lactone analogues of the nostoclides.

Preparation of lactones
The vinologous aldol reaction with the silyloxy diene furan synthon and the relevant aldehydes was the strategy utilized to prepare compounds 5-14 [18,20,[26][27][28][29].Thus, treatment of lactones 2a or 2b with tert-butyldimethylsilyltrifluoromethanesulfonate (TBDMSOTf) and diisopropylethylamine (DIPEA) resulted in the formation of the furan 3, which was not isolated.This compound, in turn, reacted with suitable aldehydes to give the adducts presenting the general structure 4. The addition of DBU to 4 under refluxing conditions led to an elimination process with concomitant formation of the nostoclide lactone analogues 5-14 in yields ranging from 31% to 98% (Scheme 1).It is important to note that in the case of compounds 12-14, the removal of the tert-butyldimethylsilyl (TBDMS) protecting group was accomplished by utilizing a 1:1 mixture of MeCN/HF [30].
Although reaction conditions were not optimized, in general the reactions were complete within three hours after the addition of DBU under refluxing conditions.For the production of the 3nitrobenzylidene derivative, the reaction mixture was refluxed for 1 hour, a procedure that afforded compound 11 in moderate yield (31%).It was observed that extended refluxing times led to the formation of a complex mixture.For not well understood reasons, a moderate yield was also obtained for compound 6.The synthetic route described above allowed the synthesis of a variety of compounds presenting different substitution patterns with respect to the arylidene moiety (Scheme 1).
The lactones 5-14 were fully characterized based on NMR, IR spectroscopic and MS spectrometric data.In all cases, the presence of molecular ion peaks was observed in the mass spectra, which correlated with the corresponding molecular formulas of the compounds.Scheme 1. Synthetic route involved in the preparation of lactones 5-14.The IR spectra of these compounds revealed intense absorption bands ranging from 1721-1764 cm -1 assigned to carbonyl groups.The frequency of these absorptions varies according to the substitution pattern on the arylidene ring.As expected, a decrease in the carbonyl absorption frequencies was observed for compounds having electron-donating groups such as OH, OCH 3 and methylenedioxy attached to the benzylidene ring (Scheme 1).
A combination of two dimensional NMR analyses (HSQC and HMBC) of the lactones synthesized not only confirmed the presence of a five membered α,β-unsaturated lactone ring moiety, substituted by both the benzyl and benzylidene functionalities, but also allowed complete hydrogen and carbon assignments.Taking compound 10 into consideration, some of the major long-range correlations (J 2 and J 3 ) observed in the HMBC contour plot are depicted in Scheme 2. The stereochemistry of the exocyclic double bound in lactones 5-14 was confirmed by 2-D NOESY experiments.With the exception of compound 7, all lactones synthesized exhibited the (Z) configuration (Scheme 1).This configuration assignment was supported by the observation of a NOE cross-peak between H-4 and H-6, as exemplified in Scheme 3 for compound 10, where other correlations observed in the NOESY contour plot are also shown.
One interesting aspect found in the NOESY contour plot of compound 14 deserves comment.The expected NOE cross-peak between H-2" and H-6 was not observed.This fact was interpreted in terms of a preferential conformation attained by the compound.In order to alleviate the non-bonding steric repulsion between the O-1 electronic pairs and the OH group (conformation A), compound 14 preferentially attains conformation B (Scheme 4).
H HO H

Conformation B
3" In this conformation, the distance between the aforementioned hydrogens is longer when compared with conformation A. Thus, the cross-peak between H-2" and H-6 is not observed.To support this proposal, semi-empirical calculations, using the AM1 algorithm [31], were performed to evaluate the difference in energy between the two conformations and also to get insight about the distances between the hydrogen atoms H-2 " and H-6 in the different conformations.The calculations revealed that conformation A is 5.81 kcal mol -1 higher in energy than conformation B.Moreover, in conformation A the distance between H-2" and H-6 is 2.23 Å, while in conformation B the spatial separation between the hydrogen atoms is more pronounced and equal to 3.84 Å (Scheme 5).With regards to compound 7, the exocyclic double bond presented an E configuration.Since the ortho positions are both substituted by methoxy groups, the compound attains the E configuration to alleviate the non-bonding destabilizing steric interaction between O-1 in the lactone ring and the methoxy groups.In fact, density functional theory calculations (B3LYP/6-31+G* level) using the Gaussian 03 program demonstrated that the E configuration of substance 7 is more stable than the corresponding Z one [32].
The opposite situation was observed for the other nostoclide analogues synthesized.In this case, AM1 calculations revealed that the Z isomer is more stable than the E isomer, as exemplified in Scheme 6, which shows the values of heat of formation (∆H f º) found for different stereoisomers of compounds 8 and 14.The most stable conformation of each stereoisomer is depicted.
Thus, the reactions involved in the preparation of the nostoclide analogues probably predispose formation of the stereoisomers depicted in Scheme 1 and the isomer selection forces appear to be thermodynamic rather than kinetic in origin [32].

Biological Evaluation
The screening program of the U.S. National Cancer Institute (NCI), which tests more than 10,000 samples per year [33], uses cytotoxic analyses by the MTT method.It is a fast, sensitive and cheap methodology, described for the first time by Mosman [34] in 1983 and subsequently modified in 1996 by Alley et al. [35].This evaluation allows one to easily determine the cytotoxicity of a particular compound, but it does not provide any insight into the mechanism of action [36].
In order to carry out a preliminary structure-activity relationship study, ten lactones 5-14 were prepared containing a variation on the substituting groups on both aromatic rings.Compounds 5, 7, 8, 12, 13 and 14 have a benzyl group linked at carbon 3 of the lactone ring, while compounds 6, 9, 10 and 11 have a 4-bromobenzyl group at the same position.We have chosen the bromine group arbitrarily, as we did for the groups present on the benzylidene ring, once we had no indication of the influence of such groups on the biological activity.The effects of lactones 5-14 were then evaluated against four tumour cell lines (HL-60, HCT-8, MDA/MB-435 and SF295), using the MTT assay.The results of the IC 50 data (µM) for the antitumour activities are presented in Table 1, along with the data obtained for doxorubicin, used as a positive control.
Compound 5 caused no effect on cell lines HL-60, MDA/MB-435 and SF295, but for the cell line HCT-8 a modest inhibition was observed (IC 50 = 101.5 µM).A modest activity was also observed for compound 7 regarding MDA-MB-435 line cells (IC 50 = 119.5 µM).The data obtained for compound 6, an analogue of compound 5 containing a bromine atom at carbon 4' of the benzyl ring, revealed no effect in any of the four cell lines tested, suggesting that the bromine had a negative influence on the bioactivity.The same negative results were observed for compound 11.The other two compounds (9 and 10) with a bromine atom on the benzyl ring had no activity against line cells HL-60 and SF295.Compounds 9 and 10 had small effects on cell lines HCT-8 (IC 50 = 114.4µM ) and MDA/MB-435 (IC 50 = 82.8µM), respectively.All other compounds having the benzyl group at carbon 3 of the lactone ring showed moderate activity against at least one line cell tested.Compound 13, having an OH group at meta position in the benzylidene ring caused inhibition on all four line cells (IC 50 = 17.6 to 62.2 µM).The most potent proliferation inhibitors in this series was compound 14 with IC 50 = 8.9 µM for HL-60 line cell.This compound caused no effect on the other cell lines.It is important to be pointed out that for the best of our knowledge it is the first time that nostoclides analogues are screened with respect to their cytotoxic activity.

Conclusions
In summary, we have demonstrated that several analogues of the natural product nostoclides, lacking the isopropyl group at carbon 4, exhibit moderate cytotoxicity against at least one of the cell lines tested (HL-60, HCT-8, MDA/MB-435 and SF295).In general, the presence of a bromine atom at the benzyl group linked at carbon 3 of the lactone ring results in decreased activity.The nature of the groups linked to the benzylidene ring influences the biological activities in this series of compounds.It appears that more polar groups like OH are associated with increased bioactivity.Further detailed investigation on the structure-activity relationship should consider the substitution pattern on both aromatic rings as a means to lead to the discovery of a more potent and selective cytotoxic compound.

Scheme 3 .
Scheme 3. Correlations observed in the NOESY contour plot of compound 10.

Scheme 5 .
Scheme 5. Conformations A and B for lactone 14 and the corresponding heats of formation (∆H f º) obtained from AM1 semi-empirical calculations.Distances (in Å) between H2" and H-6 in the conformations are also shown.Oxygen, hydrogen and carbon atoms are, respectively, red, white and gray.

Scheme 6 .
Scheme 6. Structures of the Z and E minimum energy conformers for compounds 8 and 14.Oxygen, hydrogen, bromine, and carbon atoms are, respectively, red, white, orange and gray.
presence of concentrations of pure compounds (0.39 -25 µg/mL).Each concentration was tested in triplicate and the analyses were performed in duplicate.b Data are presented as IC 50 (µM) values and 95% confidence interval (given in parentheses) obtained from at least three independent experiments.

Scheme 2 .
Some long range correlations found in the HMBC contour plot of compound 10.