Rhodomollins A and B, two Diterpenoids with an Unprecedented Backbone from the Fruits of Rhododendron molle

Two new grayanoids, rhodomollin A (1) and rhodomollin B (2), possessing an unprecedented D-homo grayanane carbon skeleton, were isolated from the fruits of Rhododendron molle. The structures of 1 and 2 were fully characterized using a combination of spectroscopic analyses and X-ray crystallography. Rhodomollin B (2) exhibited modest activity against influenza virus A/95-359, with an IC50 value of 19.24 μM.

Ring B was elucidated by the HMBC correlations from H-6 to C-1, C-5 and C-10, from H 3 -20 to C-1 and C-10, and from H-2 to C-1, C-5, and C-10. Because the five oxygen atoms in the molecular formula accounted for the six oxygenated carbons in the 13 C-NMR spectrum and H-6 was correlated to C-10 in the HMBC spectrum, ring B was determined to be a furan ring formed through a C6-O-C10 oxygen bridge and fused with ring A at C-1 and C-5. The HMBC correlations from H 3 -20 to C-9 and from H 2 -7 to C-8/C-9 together with spin system b, C(6) H-C(7)H 2 , indicated that ring C shares the C6-O-C10 oxygen bridge with ring B to form a pyran ring.
The HMBC correlations from H-14 to C-8 and C-9 and from H-9 to C-14, together with spin system c elucidated from the 1 H-1 H COSY and HSQC spectra, established a six-membered ring (ring D in Fig. 1). In addition, the HMBC correlations from H 2 -15 to C-8, C-9, and C-14 indicated the linkage of C-15 and C-8. The HMBC correlations from H-12 to C-16 and C-15 confirmed the connectivity of C-16 and C-12. Thus, another six-membered ring (ring E) was established. Subsequently, the HMBC correlations from H 3 -17 to C-12, C-15, and C-16 unambiguously placed CH 3 -17 on C-16.
Consequently, the planar structure of 1 was determined and features a 5/5/6/6/6-fused ring system. Rings D and E compose a bicyclo[2.2.2]octane ring system in which C-8 and C-12 are bridged by C-15 and C-16. This is the first example of a D-homo grayanane carbon skeleton.
The NOESY correlations of OH-5/H-1 and OH-5/H 3 -18 suggested that the two five-membered rings A and B were cis-fused to form a bicyclo[3.3.0]octane. The cis-fusion of rings A and B is preferred in bicyclo[3.3.0] octane because the trans-fusion of two five-membered rings would involve considerable torsional strain, with a very large enthalpy difference (cis to trans 29.6 kJ/mol) 17 . Due to a lack of NOE evidence, the relative configuration of the C6-O-C10 oxygen bridge could not be assigned directly by NOESY. Fortunately, a high-quality single crystal of 1 was obtained from a methanol-water solvent system. The X-ray crystallographic (Cu Kα radiation) data (Table S1-S4) corroborated the planar structure and the relative configuration of 1 and further allowed the assignment of its absolute configuration as 1R, 2R, 3R, 5S, 6R, 8R, 9R, 10S, 12R, 16S [with a Flack parameter of 0.05 (10)] (see Fig. 3). The crystallographic data for 1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition number 1445432.
Rhodomollin B (2) was obtained as white amorphous powder. Its molecular formula, C 20 H 30 O 5 , as deduced from HRESIMS, is identical to that of 1. The UV, IR, and NMR spectral data of 2 also resembled those of 1, except that compared with 1, the 13 C NMR signals for C-15 and C-17 in 2 were downshifted 1.5 ppm and upshifted 2.3 ppm, respectively. Careful analysis of the spectroscopic data allowed us to conclude that its planar structure was identical to that of 1.
The NOESY correlations of H-1/H-3, H-1/H-14, H-9/H-7b, H-9/H-15a, and OH-5/H 3 -18 revealed that the two compounds share the same relative configurations in these positions, except the stereochemistry at C-16. The relative configuration of OH-16 was then confirmed to be α-oriented on the basis of the NOESY correlation between H-9 (β) and H 3 -17, indicating that 2 is a C-16 diastereomer of 1. Thus, the structure of 2 was established and named rhodomollin B.
Rhodomollins A (1) and B (2) represent a new tetracyclic diterpene carbon skeleton with an unprecedented D-homo grayanane (featured a 5/7/6/6-fused ring system) carbon skeleton. We have named this new skeleton "rhodomollane". Compound 1 possesses an unusual β-16-OH. The stereochemistry at C-16 in grayanoids is highly conserved. A likely biosynthetic pathway is proposed in Fig. 4. Compounds 1 and 2 share a common precursor i, which undergoes a C13-alkyl shift to form a carbocation center at C-16 18 15 ). Fraction EM 9 was purified by Sephadex LH-20 column to obtain a terpenoid-containing fraction EM 9 G 1 (9 g), which was further loaded onto an Si gel column and eluted in a gradient of CH 2 Cl 2 :MeOH (20:1-1:2, v/v) to obtain 10 fractions (EM 9 G 1 L 1 -EM 9 G 1 L 10 ). EM 9 G 1 L 6 (0.68 g) was purified by preparative HPLC and semi-preparative HPLC to yield 1 (6.0 mg). EM 9 G 1 L 7 (0.55 g) was purified by preparative HPLC and semi-preparative HPLC to yield 2 (2.2 mg); see supplementary information S5.  For 1 H and 13 C NMR spectroscopic data, see Table 1; HRESI-MS 373.1986 (calcd for C 20 H 30 NaO 5 , 373.1985); see supplementary information S6-S19.  After 2 h of viral adsorption (37 °C), the monolayers were washed by PBS and incubated with or without test compounds in the maintenance medium (37 °C). Cytopathogenic effect (CPE) caused by viral invasion was evaluated when the viral control group reached a level of 4 and the antiviral activity of test compounds was determined according to the Reed and Muench method [19][20][21] .

Anti-influenza A Assays. Madin-Darby Canine Kidney (MDCK) cells and influenza
The cytotoxicity of compounds in the presence of MDCK cells were monitored by CPE. MDCK cells (2.5 × 10 4 /well) were plated into a 96-well plate. A total of 24 h later, the monolayer cells were incubated in the presence of various concentrations of test compounds. The cells were cultured for 48 h at 37 °C and 5% CO 2 in a carbon-dioxide incubator. CPE were counted on the infected MDCK cells. Median toxic concentration (TC 50 ) values of test compounds were calculated by the method of Reed and Muench. Compounds 1 and 2 showed no cytotoxicicy in this assay (TC 50 > 100 μM).