Crystal Structure , Synthesis and Biological Activity of Ether and Ester Trans-Ferulic Acid Derivatives

The structures of methoxymethyl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate, 2; (E)-3-(3-methoxy-4-(methoxymethoxy)phenyl)acrylic acid, 3; methyl (E)3-(4-(benzyloxy)-3-methoxyphenyl)acrylate, 4; benzyl (E)-3-(4-(benzyloxy)3-methoxyphenyl)acrylate, 6; and (E)-3-(4-(benzyloxy)-3-methoxyphenyl)acrylic acid, 7; were established by spectroscopic and X-ray diffraction studies. Structure 2 is a new compound. Compounds with free phenolic hydroxyls v.gr. methyl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate 1, 2 and benzyl(E)-3-(4-hydroxy-3-methoxyphenyl)acrylate 5, showed scavenging freeradical and antioxidant activity while moderate scavenging free-radical was observed in compound 3. Moderate inhibition of lipid peroxidation was observed for 7. Compound 5 exerted significant inhibition of cell growth in PC-3, K562 tumor cell lines and 4 exhibited the largest cytotoxic effect upon the K562 cell line.


Introduction
Ferulic acid (FA) is a phenolic derivative of cinnamic acid, which exhibits a wide spectrum of biomedical activities [1].Many staple foods such as grain bran, whole-grain foods, citrus fruits, banana, coffee, orange juice, eggplant, bamboo shoots, beetroot, cabbage, spinach and broccoli are among the richest sources of FA [2].This compound is present in seeds and leaves, both in its free form and in its covalent-conjugated form, in plant cell-wall polysaccharides, glycoproteins, polyamides, lignin and hydroxy fatty acids.FA confers rigidity to the cell-wall and it is a precursor to other important organic compounds such as diferulic acid and curcumin [3].Several reports have described the biological effects of FA, for example, as an antioxidant [4], anti-inflammatory and anticarcinogenic [5].
The well-known antioxidant activity of FA has been related to its phenolic nucleus and extensive chain conjugation that forms a resonance-stabilized phenoxy radical ion [4].Since the hydroxyl and phenoxy groups of FA donate electrons to quench free radicals [6], FA can be used as a renewable, aromatic scaffold for conversion into novel, and useful products in the food, pharmaceutical and cosmetic industries.The ability of FA to inhibit peroxidation of fatty acids finds uses such as preservative for oranges or to inhibit the autoxidation of linseed oil.It is also employed in the industry of cosmetic skin lotions [4].FA is used during oxidative-reductive depolymerization (ORD) of polysaccharides to protect both guar gum and cassava starch from thermal degradation [7].
The synthesis of new derivatives of FA in general has been aimed to improve its chemical, physicochemical and biological properties, in particular in its esterified form at the acidic moiety, which increases both radical scavenging activity and partition coefficient [8].However, it is known that the blockade of the phenolic groups of FA can limit its antioxidant potential.
In the present report, the synthesis, spectroscopic characterization, X-ray analysis and biological activity of ether and ester derivatives of FA is described and it is depicted in Scheme 1.We also highlight the importance of the free hydroxyl and phenolic group in relation to the antioxidant and cytotoxic activity of FA and how the protection of these functional groups reduces their biological activity.

Physical Measurements
All chemicals were reagent grade and were used as purchased.Solvents were purified by standard methods [14].
Melting points were determined on an Electrothermal IA9100 digital melting point apparatus and are uncorrected.IR absorption spectra were recorded in the 4000 -400 cm −1 range as KBr pellets on a Perkin Elmer 283-B spectrophotometer. 1 H and 13 C NMR spectra were recorded in CDCl 3 on a Bruker 500 MHz spectrometer using TMS as an internal reference.NMR spectra were processed with MestReNOVA 12.0.0software.International Journal of Organic Chemistry Scheme 1. Synthetic pathway for FA derivatives.
The mass spectrometry spectra were recorded in equipment MStation JMS-700 or Jeol, SX 102A using electron ionization impact or fast atom bombardment (FAB) mode.
X-ray data were collected on a Bruker APEX2 CCD area detector with a graphite monochromator and Mo Kα radiation (λ = 0.71073 Å) at room temperature with ω scan mode.All collected data were reduced using SAINTV8.27B[15].These data are shown in Table 1.

Scavenging Activity of DPPH Free Radicals
The free radical scavenging activity was measured using a modified method from Mellors and Tappel [16].The test was carried out on 96-well microplates.A 50 µL aliquot of the solution of the test compound was mixed with 150 µL of an ethanol solution of DPPH (final concentration 100 µM).This mixture was incubated at 37˚C for 30 min, and the absorbance was then measured at 515 nm using a BioTek microplate reader SYNERGY HT.The inhibition percent for each compound was determined by comparison with a 100 µM DPPH ethanol blank solution.

Inhibition of Lipid Peroxidation on Rat Brain
Inhibition of lipid peroxidation of FA derivatives was assayed following previous described method [17] [18].

Cytotoxicity Assay
All synthetized compounds were screened in vitro at doses of 50 μg/mL against International Journal of Organic Chemistry

Refinement Details
All reflections were defined based on F 2 .The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 .The threshold expression of F 2 > σ (F 2 ) is used only for calculating R-factors (gt) etc., and is not relevant to the choice of reflections for refinement.R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on all data will be even larger.
There is an intramolecular hydrogen bond and one intermolecular hydrogen bond between the hydroxyl group at C7 and O5 with a symmetry-related molecule forming sheets along the a-axis.In structure 3, the bond lengths and angles are similar to those found in 3-( 4

Geometric Details
All estimation of standard deviation (esd's) (except the esd in the dihedral angle between two least square planes) are estimated using the full covariance matrix.
The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry.An approximate (isotropic) treatment of cell esds was used for estimating esds involving least square planes.

Structural X-Ray Data
All structures were solved by direct methods [20], and then refined by full-matrix least-squares technique; the position and anisotropic parameters of all non-hydrogen atoms were obtained.Table 1 shows the data collection and handling for compounds 2, 3, 4, 6, and 7. Table 2 shows the crystal and refinement parameters for compounds 2, 3, 4, 6 and 7. Figure 8 contains the molecular structures of 2, 3, 4, 6 and 7.This figure was drawn with 50% displacement ellipsoids using ORTEP-3 for Windows [21].The geometry of the molecule was calculated using PARST [22] [23] software.In all structures, the bond distances and bond angles are in good agreement with the corresponding values and are within normal ranges.The molecular structure of 2 is not planar, with a fully extended methoxymethyl group of the acrylate residue as shown in Figure 8.The dihedral angle between the side chain at C4 and the phenyl ring is 32.7(2)˚.The hydroxyl group at C7 has an intramolecular hydrogen with O3 and one intermolecular hydrogen bond between the hydroxyl group at C7 and O5 with a symmetry related molecule (−x − 1, +y, +z) forming sheets along the a-axis.There are no stacking interactions between phenyl rings.The crystal packing diagram is shown in Figure 9.International Journal of Organic Chemistry Figure 8 shows the molecular structure of 3. Bond lengths and angles are similar to those found in 3-(4-Hydroxy-3-methoxyphenyl)-2-propenoic acid (FA) [24].The dihedral angles between the side chain at C4, the acrylate and the phenyl ring are 48.31(5) and 9.32 (5)˚, respectively.The molecular packing of 3 in the crystal shows an intermolecular O-H…O hydrogen-bonded carboxylic International Journal of Organic Chemistry acid dimers that together with several C-H…O interactions form a sheet-like structure (Figure 9).
The molecular structure of 4 corresponds to a new benzoyloxy and acrylate derivative of ferulic acid.There are no significant molecular distortions in terms of bond lengths and angles.The dihedral angles between benzene ring C4 to C9 and the side chains at C4 and C7 are 21.25 (5) and 8.84 (5)˚, respectively.However, the benzene ring C4 to C9 makes and angle of 86.00 (5)˚ with the benzene ring C11 to C16.The molecule 4 in the crystal are linked by one C-H…O intermolecular interaction involving an H atom of the phenyl ring C4-C9 and carbonyl oxygen of the acrylate group.This interaction linked the molecules in the crystal and packed at normal van der Waals distances (Figure 9).
Figure 8 shows the molecular structure of compound 6.There are two independent molecules 4 in the crystal.Molecule 6 contains three substituents mainly at C4 a methoxyphenyl acrylate moiety; at C6 a methoxy group and C7 a benzoyloxy moiety.The dihedral angles between phenyl rings C11-C16 and C19-C24 with the central phenyl ring C4-C9 are 70.50 (12) and 69.61 (14), respectively.The dihedral angles between phenyl rings C35-C40 and C43-C48 with the central phenyl ring C28-C33 are 77.00 (14) and 73.92 (16), respectively.The dihedral angle between the phenyl rings C4-C9 and C28-C33 is only 4.76 (12)˚.There are seven C-H…O interactions linked the two independent molecules in the crystal packed at normal van der Waals distances (Figure 9).
The molecular structure of 7 is shown in Figure 8.The molecule contains three substituents mainly at C4 an acrylic acid group; at C6, a methoxy group and at C7 a benzoyloxy moiety.The dihedral angles between phenyl ring C12-C17; the linker C7-O4-C11-C12 and acrylic acid group at C4 with the central phenyl ring C4-C9 are 74.62 (6), 12.17 (9) and 7.37 (5)˚, respectively.There is one intermolecular hydrogen bond O-H…O with a symmetry related molecule (+2 − x, +2 − y, +2 − z) forming sheets along the b-axis and two C-H…O interactions linked the molecules in the crystal and packed at normal van der Waals distances (Figure 9).
For geometrical details and notations of all of these hydrogen bonds and intermolecular interactions are given in Table 3.

Biological Assay
Interestingly, compounds 1, 2 and 5, with free hydroxyl group, showed to have DPPH free radicals scavenging activity in vitro in moderate to high level (see Table 4).These results are consistent with a previous report, where it is postulated that antioxidant property of FA and its derivatives is associated with the number of free hydroxyl groups on the benzene ring and ortho substitution with the electron donor methoxy group, due to stabilization of the phenoxy radical [25].FA derivatives, especially those with etherified phenolic groups, did not show effect upon the free radical essay.International Journal of Organic Chemistry Moreover, compounds 1, 2 and 5 also inhibited lipoperoxidation induced by ferric sulphate in the rat brain, mainly due to preservation of phenolic group and a similar mechanism of action of antioxidant effect described above.Remarkably, compound 7 showed moderate inhibition of lipid peroxidation, despite the fact of it has the phenol group blocked probably due to higher lipophilicity, which aids to penetrate the cell membrane where lipid peroxidation takes place International Journal of Organic Chemistry [26].
The FA derivatives showed low cytotoxic activity in the preliminary screening in different cancer cell lines (all compounds were tested at 50 μM).As seen in Table 4, only compound 5 inhibited cancer cell lines PC-3 and K562 by 59% and 48% respectively, while to healthy cell line COS-7 it did so by 53%.Compounds 4 and 6 also inhibited the growth of K562 cell line by 72% and 32% respectively.
Since there were not relevant cytotoxic agents, it is remarkable that addition of benzyl groups to the FA improved cytotoxic activity in vitro, possibly by the increment of lipophilicity of compounds and a more efficient molecular action within the cell, as it has been reported previously [27] [28].Our findings do not coincide with Jayaprakasam et al. (2006), who reported anti-inflammatory, anticancer and antioxidant activity of FA esters at low concentrations [29].New refined analyses are required in order to elucidate molecular effects of FA and its derivatives.

Conclusion
The synthesis with moderate to good yields and unambiguous assignment of
-Hydroxy-3-methoxyphenyl)-2-propenoic acid (ferulic acid).Molecular packing of molecules in the crystal shows an intermolecular O-H…O hydrogen-bonded carboxylic acid dimers that together with several C-H…O interactions form a sheet-like structure.In structure 4 is a new benzoyloxy and acrylate derivative of FA.The molecules in the crystal are linked by one C-H…O intermolecular interaction involving H atom of the phenyl ring C4-C9 and carbonyl oxygen of the acrylate group.This interaction links crystal and packed molecules at normal van der Waals distances.In structure 6, there are two independent molecules in the crystal.Structure 6 contains three substituents mainly at C4 a methoxyphenylacrylate moiety; at C6 a methoxy group and C7 a benzoyloxy moiety.The dihedral angle between phenyl rings C4-C9 and C28-C33 is only 4.76 (12)˚.There are seven C-H…O interactions that link two independent molecules in the crystal and packed structures at normal van der Waals distances.Structure 7 contains three substituents mainly at C4 an acrylic acid group; at C6 a methoxy group and C7 a benzoyloxy moiety.There is one intermolecular hydrogen bond O-H…O with a symmetry related molecule (+2 − x, +2 − y, +2 − z) forming sheets along the b-axis and two C-H…O interactions linked the molecules in the crystal and packed at normal van der Waals distances.
2 ml of H 2 SO 4 were added and the mixture was refluxed for 6 hours.The reaction was quenched by the addition of NaHCO 3 until it reached pH 6 and the mixture was extracted with ethyl acetate (3 × 20 mL).The combined extracts were dried over anhydrous Na 2 SO 4 , then filtered and the solvent removed in vacuo, 82% yield, yellowish liquid.