Meroterpenoid Hydroquinones from Cordia globosa

Two new meroterpenoid hydroquinones, rel-(4bE,6Z,8E,9aS,10S)-1,4-dihydroxy-9a,10dihydro-10,12-epoxy-5-methylbenzo[a]azulen-12-one and rel-(4bZ,6Z,8E,9aS,10S)-1-hydroxy9a,10-dihydro-4,11:10,12-diepoxy-benzo[a]azulen-11,12-dione, along with the known peptide derivative (S)-N-benzoylphenylalanine-(S)-2-benzamide-3-phenylpropyl ester, were isolated from the roots of Cordia globosa. Their structures were determined by 1D and 2D nuclear magnetic resonance (NMR) spectrometry, Fourier transform infrared (FTIR) spectroscopy and high resolution atmospheric pressure chemical ionization mass spectrometry (HRAPCIMS) data analysis. The new compounds were tested against three human cancer cell lines (colon adenocarcinoma, ovarian carcinoma and glioblastoma), but none of them exhibited any activity.


Introduction
1][12] In the present work the EtOH extract from roots of C. globosa, an annual and aromatic shrub native to the northeast of Brazil, was investigated, which led to the isolation and characterization of two new terpenoid hydroquinones (1 and 2), and a known peptide derivative (3) (Figure 1).

General experimental procedures
The Fourier transform infrared (FTIR) spectra were recorded on a Perkin-Elmer spectrum 100 equipped with a universal attenuated total reflectance (UATR) accessory.Optical rotations were measured on a Perkin-Elmer 341 digital polarimeter.One-dimensional [ 1 H, 13  heteronuclear multiple-bond correlation (HMBC) and nuclear Overhauser effect spectroscopy (NOESY)] were recorded on a Bruker DRX-500 spectrometer operating at 500 MHz for 1 H and 125 MHz for 13 C, using standard pulse sequences supplied by the manufacturer.The high resolution mass spectrometry (HRMS) using atmospheric pressure chemical ionization (HRAPCIMS) was performed on a liquid chromatography-mass spectrometry ion trap and time-of-flight (LCMS-IT-TOF, Shimadzu) spectrometer.
The positive ion mass spectra were recorded in the m/z 200-700 range, using a potential of 4.0 kV on the capillary and He as the collision gas.The high performance liquid chromatography (HPLC) analysis was carried out using an ultra-fast liquid chromatography (UFLC, Shimadzu) system equipped with a SPD-M20A

Cytotoxicity evaluation
Cytotoxicity was evaluated against three human cancer cell lines provided by the National Cancer Institute (Bethesda, MD, USA): colon adenocarcinoma (HCT-116), ovarian carcinoma (OVCAR-8) and glioblastoma (SF-295).Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mmol L -1 glutamine, 100 U mL -1 penicillin, 100 µg mL -1 streptomycin at 37 °C under a 5% CO 2 atmosphere.For all experiments, cells were plated in 96-well plates (10 5 cells per well for adherent cells or 0.3 × 10 5 cells per well for suspended cells in 100 µL of medium).After 24 h, all the compounds (0.048-5.0 µg mL -1 ) dissolved in 1% dimethylsulfoxide (DMSO) were added to each well using a high throughput screening system (Biomek 3000, Beckman Coulter, Inc.), and the cultures were incubated for 72 h.Doxorubicin (Zodiac) was used as a positive control.Control groups received the same amount of DMSO.Tumor cell growth was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) to a purple formazan product as previously described. 13t the end of the incubation, the plates were centrifuged and the medium was replaced with fresh medium (150 µL) containing MTT (0.5 mg mL -1 ).Three hours later, the plates were centrifuged, the MTT formazan product was dissolved in 150 µL DMSO, and the absorbance was measured using a multiplate reader (Spectra Count, Packard).The drug effect was quantified as the percentage of the control absorbance of the reduced dye at 550 nm.The concentration values that inhibit growth in 50% (IC 50 ) were calculated, along with the respective 95% of confidence interval (CI), by non-linear regression using the software GraphPad Prism 5.0.

Results and Discussion
Compound 1, a yellow powder, showed IR absorption bands for hydroxyl groups (3317 cm -1 ), conjugated carboxyl of γ-lactone moieties (1718 cm -1 ), carbon-carbon double bonds (1684 and 1639 cm -1 ) and carbon-oxygen bonds (1215-1260 cm -1 ).The molecular formula of C 16 H 12 O 4 (11 degrees of unsaturation) was determined by HRAPCIMS through the molecule protonated peak [M + H] + at m/z 269.0808 (calcd.m/z 269.0808).The 1 H NMR spectrum (Table 1) exhibited signals for an aromatic ring at d H 6.85 (d, J 8.5 Hz, H-2) and 6.82 (d, J 8.5 Hz, H-3), indicating an AB system similar to those of a 1,4-hydroquinone moiety.In addition, signals at d H 7.03 (dd, J 5.8 and 1.6 Hz, H-8), 7.02 (d, J 11.4 Hz, H-6) and 6.77 (dd, J 11.4 and 5.8 Hz, H-7) were related to a coupling system of olefinic protons, while the signals at d H 6.17 (d, J 8.0 Hz, H-10) and 2.96 (d, J 8.0 Hz, H-9a) were associated with methines, one of which corresponding to an oxymethine proton.Finally, a singlet at d H 2.48 (s, Me-11), was compatible with a vinyl methyl.Besides the vicinal correlations for the protons H-2/H-3, H-6/H-7, and H-9a/H-10, the COSY spectrum exhibited the allylic coupling for H-9a and H-8, as well as the homoallylic coupling of H-9a and the Me-11.The 13 13 C NMR spectra revealed eight non-hydrogenated carbon atoms, one characteristic of a γ-lactone carboxyl at d C 167.3 (C-12), as well as the signals at d C 149.4 (C-1) and 147.6 (C-4) related to the oxygenated carbons of the 1,4-hydroquinone moiety.In addition, signals for three olefinic double bonds were observed, which, after COSY and HMBC analyses (Figure 2), were shown to make part of an extensive conjugated system involving the lactone Compound 2 was also isolated as a yellow powder.Its FTIR spectrum showed absorption bands for hydroxyl groups (3466 cm -1 ), conjugated carboxyl groups (1750 cm -1 ), carbon-carbon double bonds (1628 and 1465 cm -1 ) and carbon-oxygen bonds (1188 and 1209 cm -1 ).The molecular formula of C 16 H 8 O 5 (13 degrees of unsaturation) was determined by HRAPCIMS analysis through the molecule protonated peak [M + H] + at m/z 281.0469 (calcd.m/z 281.0444).Despite the 1 H NMR spectrum of 2 (Figure 1) being run in a different solvent (C 5 D 5 N) than that used for 1 ((CD 3 ) 2 CO), it showed the same number of protons and splitting pattern, except for the disappearance of methyl group, revealing the same backbone structure of 1.
The 13 C and DEPT NMR spectra of 2 were also similar to those of 1 (Table 1).The main difference was the appearance of an additional d-lactone carboxyl group at d C 161.6 (C-11) in compound 2, in substitution of the Me-11 of 1.The HMBC correlation of the proton signal at d H 7.62 (H-6) with the carboxyl at d C 161.6 supported the lactonization between C-4 and C-5 (Figure 2).Additional long range correlations, depicted in Figure 2, corroborated the structure of 2. The structures of compounds 1 and 2 show a high degree of similarity, and one could then speculate on the biogenetic formation of 2 simply by the oxidation of Me-11 of 1 to the correspondent carboxyl acid followed by an intramolecular nucleophilic substitution reaction yielding the d-lactone moiety.Thus, the structure of 2 was established as rel-(4bZ,6Z,8E,9aS,10S)-1-hydroxy-9a,10dihydro-4,11:10,12-diepoxy-benzo[a]azulen-11,12-dione.

Conclusions
In this work two new meroterpenoid hydroquinones (1 and 2) were isolated from the EtOH extract of roots of Cordia globosa, in addition to a peptide derivative ( 3) not yet reported for this genus.Compounds 1 and 2 are 1,4-hydroquinones fused to a monoterpene moiety, a structural feature that is frequently found in Cordia species, particularly in roots.
C NMR spectrum (Table 1) displayed signals for 16 carbon atoms, 13 of which corresponding to sp 2 hybridized carbons.The DEPT spectrum revealed a methyl group at d C 23.6 (C-11), an oxymethine at d C 79.8 (C-10) and another methine at d C 47.7 (C-9a), in addition to five monohydrogenated sp 2 carbon atoms at d C 118.8-144.2.Comparison of DEPT with