A Tri-O-Bridged Diels-Alder Adduct from Cortex Mori Radicis

Sanggenon X, an unusual tri-O-bridged Diels-Alder adduct, was isolated from Cortex Mori Radicis. Its structure was established by spectroscopic analysis, including NMR and HR-MS (High Resolution Mass Spectrometry). Sanggenon X contained three O-bridged rings, where the oxygenated bridgeheads were all quaternary carbons. Chemical methylation was carried out to deduce the linkages of the three O-bridges. The absolute configuration was determined by calculating the ECD (Electronic Circular Dichroism) using the TDDFT (Time-Dependent Density Functional Theory) method. Sanggenon X showed significant antioxidant activity against Fe2+-Cys-induced lipid peroxidation in rat liver microsomes, and was as effective as the positive control, curcumin.

Given the structures of 1a and 1b, the three O-bridges in 1 were suggested to be at C-1 /C-16 , C-2 /C-10 , and C-4 /C-5 , depending on the proposed reaction mechanism. In the methylation of 1, there were two reactive centers: the hemiketal at C-2 and its adjacent benzyl proton H-3 . In pathway A, deprotonation at C-3 under alkali conditions formed a ketone from the hemiketal. Subsequently, the two O-bridges at C-2 and C-4 were broken to form a 1,4-butenedione. In pathway B, the hydroxyl group at C-2 hemiketal was first methylated before deprotonation at C-3 under alkali conditions. A double bond was formed as the O-bridge at C-2 migrated to C-1 with an intramolecular 1,2-rearrangement, and the O-bridge between C-1 /C-16 was broken. Meanwhile, a configuration inversion of the C-7 methyl group was observed from 1 to 1b. This phenomenon was further confirmation of the intramolecular O-bridge migration from C-2 to C-1 . Because the two bridged rings on C1 /C5 and C2 /C4 were adjacent to each other, they must be on opposite sides of the hexane plane. Thus, the orientation of C-3 yielded two sets of epimers-cis-trans or all-trans, in agreement with the biosynthesis pathway [17] of the D-A adducts in the genus Morus. Although the benzyl carbonyl of 1 was coplanar with the aromatic ring, its CD (Circular Dichroism) spectrum did not show the split Cotton effects typical of non-O-bridged D-A adducts, such as mulberrofurans C and J [16]. Two positive Cotton effect peaks at 349 nm and 308 nm were observed, in accordance with the calculated ECD (Electronic Circular Dichroism) spectrum ( Figure 4) of one 3 H-α epimer-i.e., (3 R, 4 S, 5 R)-by using the TDDFT (Time-Dependent Density Functional Theory) method. Therefore, the absolute configuration of 1 was determined to be (1 R, 2 R, 3 R, 4 S, 5 R). α epimer-i.e., (3″R, 4″S, 5″R)-by using the TDDFT (Time-Dependent Density Functional Theory) method. Therefore, the absolute configuration of 1 was determined to be (1″R, 2″R, 3″R, 4″S, 5″R). The genus Morus is a plant source with rich D-A adducts. More than 50 D-A adducts have been found in the previous studies [18]. However, a natural product with a highly oxygenated D-A skeleton is rarely reported [19]. A plausible biosynthetic pathway for 1 was postulated in Figure 5, based on the KEGG pathway prediction. Kuwanon Y, a D-A adduct found in genus Morus [16], afforded 1 through three oxidization steps. First, the double bond of the D-A skeleton was oxidized to an epoxide by an oxidase [20] or putative Cyt P450 monooxygenase [19], then the epoxide was attacked by 16″-OH at C-1″, and 2″-OH was formed. Sequentially, the newly formed 2″-OH was oxidized to a carbonyl by an oxidoreductase [21] and was attracted by 10″-OH to form a hemiketal [22]. Finally, the α-position of the 8″-carbonyl was oxidized to form an electrophilic center and was trapped by 5′-OH [23] to afford 1.  The genus Morus is a plant source with rich D-A adducts. More than 50 D-A adducts have been found in the previous studies [18]. However, a natural product with a highly oxygenated D-A skeleton is rarely reported [19]. A plausible biosynthetic pathway for 1 was postulated in Figure 5, based on the KEGG pathway prediction. Kuwanon Y, a D-A adduct found in genus Morus [16], afforded 1 through three oxidization steps. First, the double bond of the D-A skeleton was oxidized to an epoxide by an oxidase [20] or putative Cyt P450 monooxygenase [19], then the epoxide was attacked by 16 -OH at C-1 , and 2 -OH was formed. Sequentially, the newly formed 2 -OH was oxidized to a carbonyl by an oxidoreductase [21] and was attracted by 10 -OH to form a hemiketal [22]. Finally, the α-position of the 8 -carbonyl was oxidized to form an electrophilic center and was trapped by 5 -OH [23] to afford 1. α epimer-i.e., (3″R, 4″S, 5″R)-by using the TDDFT (Time-Dependent Density Functional Theory) method. Therefore, the absolute configuration of 1 was determined to be (1″R, 2″R, 3″R, 4″S, 5″R). The genus Morus is a plant source with rich D-A adducts. More than 50 D-A adducts have been found in the previous studies [18]. However, a natural product with a highly oxygenated D-A skeleton is rarely reported [19]. A plausible biosynthetic pathway for 1 was postulated in Figure 5, based on the KEGG pathway prediction. Kuwanon Y, a D-A adduct found in genus Morus [16], afforded 1 through three oxidization steps. First, the double bond of the D-A skeleton was oxidized to an epoxide by an oxidase [20] or putative Cyt P450 monooxygenase [19], then the epoxide was attacked by 16″-OH at C-1″, and 2″-OH was formed. Sequentially, the newly formed 2″-OH was oxidized to a carbonyl by an oxidoreductase [21] and was attracted by 10″-OH to form a hemiketal [22]. Finally, the α-position of the 8″-carbonyl was oxidized to form an electrophilic center and was trapped by 5′-OH [23] to afford 1.  In in vitro bioassays, sanggenon X (1) showed significant antioxidant activity against Fe 2+ -Cys-induced lipid peroxidation in rat liver microsomes with 81.25% inhibition of malondialdehyde (MDA) release, similar to the positive control, curcumin, with an 81.75% inhibition ratio.

General Experimental Procedures
Melting points were determined on an XT5B melting point apparatus (Beijing Keyi Electric Light Instrument Factory, Beijing, China) and were uncorrected. Optical rotations were measured with a P-2000 polarimeter (Jasco, Tokyo, Japan). ECD spectra were recorded at room temperature with a J-815 spectropolarimeter (Jasco, Tokyo, Japan). UV spectra were collected in MeOH on a V-650 spectrophotometer (Jasco, Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 spectrometer (Thermo, Madison, WI, USA) by the FT-IR transmission electron microscopy method. 1 H-and 13 C-NMR spectra were acquired using an AVIIIHD 600 spectrometer (Bruker, Billerica, MA, USA).

Plant Material
The

Extraction and Isolation
The powdered Cortex Mori Radicis (50 kg) were soaked with 50% EtOH for 24 h and percolated with 300 L 50% EtOH. Then evaporation of the solvent under reduced pressure gave a liquid extract, which was suspended in H 2 O and partitioned with EtOAc. The EtOAc extract (ca. 1 kg) was applied to a silica gel (100-200 mesh, 2 kg) column, eluting with a gradient of increasing MeOH concentration (0-100%) in CHCl 3 , to yield 22 fractions A-V. Fraction M-O (50 g) was applied to a Sephadex LH-20 (3 L) column, using 90% MeOH as eluent, to give subfractions MO-1 to 13. Fraction MO-11 (8 g) was loaded on a silica gel (100-200 mesh, 160 g) column and eluted with a gradient of increasing MeOH concentration (0-100%) in CH 2 Cl 2 to yield five subfractions. The second fraction (3.2 g) was chromatographed over Sephadex LH-20 (400 mL, eluted by MeOH), MPLC over C 18 Figure S3a-h, Table S1).

Lipid Peroxidation Assay
Antioxidative activity was evaluated as the inhibitory activity of compounds against Fe 2+ -Cys-induced lipid peroxidation in rat liver microsomes by the formation of malondialdehydethiobarbituric acid (MDA-TBA) adduct. Microsomes were isolated from SD rat livers and suspended in 100 mM TMS buffer (pH 7.4). The microsomal suspension (1 mg protein/mL), different concentrations of compound or vehicle, and 0.2 mM cysteine in 0.1 M PBS (pH 7.4) were incubated at 37 • C for 15 min, 50 µM FeSO 4 was added, and the reaction mixture was then incubated at 37 • C for 15 min again. An equal volume of 20% (w/v) TCA (Trichloroacetic Acid) and 0.6% (w/v) TBA were added and kept in a boiling water bath for 10 min. After the mixture was centrifuged at 3000× g for 10 min, the absorbance of supernatant was measured at 532 nm and the concentration of MDA was calculated as C = (OD − 0.006)/0.07 × 10 nmol/mL. Lipid peroxidation inhibitory activity was calculated as follows: [1 − (T − B)/(C − B)] × 100%, in which T, C, and B are MDA concentrations of the sample treated, the control without sample, and the zero time control, respectively. Curcumin (10 −4 M) was used as the positive control.

Conclusions
In this paper, a tri-O-bridged D-A adduct, sanggenon X (1), was isolated from a 55% alcohol extract of Cortex Mori Radicis. Given its complex structure with several quaternary carbons in the bridgeheads, it was fortunate for us to determine the exact structure with the help of chemical methylation and calculated ECD. The structure of 1, with highly oxygenated D-A skeleton, adds a new skeletal entity to the natural D-A adducts and provides a new framework for synthesis and biological evaluation in the future.