New 2-Arylbenzofurans from the Root Bark of Artocarpus lakoocha

Three new prenylated 2-arylbenzofurans – artolakoochol, 4-hydroxy-artolakoochol and cycloartolakoochol – have been isolated from the root bark of Artocarpus lakoocha Roxb., Their structures were elucidated through analysis of their spectroscopic data, and their antiherpetic potential was evaluated by the plaque reduction assay.

Compound 2, a white powder, was analyzed for C 29 H 32 O 5 from its [M+H] + ion at m/z 461.2328 (calcd. for 461.2332) in the HR-ESI-MS. Its UV and IR properties were similar to those of 1, suggesting another 2-arylbenzofuran skeleton. Comparison of the molecular formula of compound 2 with that of 1 showed that 2 should be a hydroxy derivative of 1. This hydroxyl group should be located at C-4 of ring A, due to the absence of the H-4 resonance and the appearance of signals for H-5 and H-7, each as a doublet (J = 2.0 Hz) at δ 6.29 and δ 6.49, respectively in the 1 H-NMR spectrum. This was confirmed by the HMBC correlations from C-3a (δ 112.3) to OH-4 (δ 8.83, 1H, br s) and H-3 (δ 6.86, 1H, d, J = 1.0 Hz) ( Table 1).
Compounds 1 and 2 were optically active with levorotation ([α] 20 D −86.1 and −117.6, respectively). Both shared similar CD properties, displaying a negative Cotton effect at 331 -334 nm and a negative peak at 227-230 nm (Figure 4), and therefore should have the same stereochemistry at C-3′′′. ] which should be placed at C-2′ due to the HMBC correlations of C-2′ (δ 120.8) with H-1′′. The 13 C-NMR, HSQC and HMBC spectra of 3 displayed, in addition to the signals for the 2-arylbenzofuran nucleus and the prenyl group, ten carbon signals corresponding to three angular methyls, three methylenes, two methines and two oxygenated quarternary carbons. This indicated that compound 3 also had a monoterpene unit which was attached to C-4′and appeared to form a tricylic structure with the oxygen functionalities on C-3′ and C-5′. The conjugation of a 10carbon moiety to a di-ortho oxygenated aromatic structure to produce a pyran-cyclohexane-pyran system (rings D, E and F) has been recently observed in isorubraine, a monoterpene-chalcone conjugate isolated from the seeds of Alpinia katsumadai [14]. Comparison of the 1 H-and 13 C-NMR data of 3 with those of isorubraine [14] particularly on the tricyclic partial structure revealed their close similarity. Therefore the monoterpene unit should be connected to ring B by a direct linkage between C-4′ (δ 117.2) and C-1′′′ (δ 28.6) with two ether bridges between C-3′ and C-3′′′, and C-5′ and C-8′′′. This was supported by the HMBC correlation between H 2 -2′′′ (δ 1.82 and 2.21) and C-4′. The F ring of 3 appeared to have a chair conformation. Its relative configuration and NMR assignments were obtained from detailed analysis of the COSY, NOESY, HSQC and HMBC spectra. At C-2′′′, the double doublet at δ 1.82 (J = 13.0, 1.5 Hz) was assigned to the axial proton from its NOESY interaction with H-7′′′, whereas the multiplet at δ 2.21 was assigned to the equatorial, consistent with its long-range (W-type) coupling with equatorial H-5′′′ (δ 1.42, m) observed in the COSY spectrum. The axial proton at C-5′′′(δ 1.71, m), as expected, showed NOESY correlation with H 3 -4′′′. The equatorial proton at C-6′′′ (δ 1.25, m) displayed a NOESY cross peak with H-7′′′.
Thus, it was concluded that 3 had the structure as shown in Figure 1, and the trivial name cycloartolakoochol was given to the compound. Regarding its optical activity, 3 was found to be dextrorotatory ([α] 20 D +19.2). In the CD spectrum ( Figure 5), it appeared to show a negative Cotton effect at 243 nm, although two small positive peaks at 320 and 370 nm were observed. These findings reflected the influence of the stereochemistry at C-1′′′, which determined the arrangement of the tricyclic (D/E/F) ring system, on the optical properties of 3 as compared with those of 1 and 2. Biogenetically, the 2-arylbenzofuran nucleus of 1 and 3 might be derived from 4,3′,5′trihydroxystilbene (resveratrol), whereas that of 2 could be originated from 2,4,3′,5′tetrahydroxystilbene (oxyresveratrol) [6][7][8]15]. Compound 3 appears to be a cyclization product of 1. Figure 6. Possible biogenesis of 3 from 1.
As depicted in Figure 6, this reaction could begin with protonation of C-2′′′, resulting in the formation of a carbocation at C-1′′′. This would be followed by bond formation between C-1′′′ and C-7′′′ to give a carbocation at C-8′′′ that would subsequently undergo nucleophilic attack by OH-5′ to produce rings E and F. Compounds 1-3 were evaluated for their inhibitory activity against Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) using the plaque reduction assay [7][8][9], but they were devoid of activity at the concentration of 100 μg/mL.

Plant Material
The root bark of A. lakoocha Roxb. was collected from the Botanical Garden of Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand, in June 2009. Authentication was performed by comparison with herbarium specimens at the Royal Forest Department, Ministry of Agriculture and Co-operatives. A voucher specimen (BS-062552) is on deposit at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University.

Assay of Anti-HSV Activity
Antiviral activity against HSV-1 (Strain KOS) and HSV-2 (Strain 186) was determined using the plaque reduction method, as previously described [7][8][9]. Briefly, virus (30 PFU/25 μL) was mixed with complete medium (25 μL) containing various concentrations of test compound and then incubated at 37 ºC for 1 h. After incubation, the mixtures were added to Vero cells (6 × 10 5 cells/well) in 96-well microtiter plates and incubated at 37 ºC for 2 h. The overlay medium containing the various concentrations of test compound was added to the Vero cells and incubated at 37 ºC in humidified CO 2 incubator for 2 days. Then, virus growth inhibition was evaluated by counting the virus plaque forming on Vero cells compared with the controls. The cells also were stained with 1% crystal violet in 10% formalin for 1 h. The percent plaque inhibition was determined. Acyclovir was used as positive control.

Cytotoxicity Test
Cytotoxicity was evaluated by incubating Vero cell monolayers with completes medium containing various dilutions of sample for 72 h at 37 ºC. Then, cell cytotoxicity was examined by microscopic observation [7][8][9].

Conclusions
Three new 2-arylbenzofurans: artolakoochol (1), 4-hydroxyartolakoochol (2) and cycloartolakoochol (3) were isolated from the root bark of Artocarpus lakoocha Roxb. All of the isolated compounds were evaluated for their anti-HSV effect, but they were devoid of activity.