New Antifeedant Grayanane Diterpenoids from the Flowers of Pieris formosa

Three new grayanane diterpenoids, pierisoids C‒E (1–3), as well as 10 known ones (4–13), were evaluated from the flowers of Pieris formosa, which is used as an insecticide in rural areas of China. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analyses. Significant antifeedant activity of 1, 3 and 10 against the beet armyworm (Spodoptera exigua) was found, indicating that these diterpenoids might also be involved in the plant defense against insect herbivores.

Detailed analysis of the 1D ( 1 H and 13 C) and 2D ( 1 H-1 H COSY, HSQC, and HMBC) NMR spectra (Figures S1-S6 in supplementary data) of 1 revealed that its structure closely resembled that of asebotoxin VIII [19,26], a known grayanane diterpenoid previously isolated from both P. japonica and P. formosa. The obvious difference between the two compounds was that the acetoxy group located at C-6 in asebotoxin VIII migrated to C-15 in 1, as indicated by the HMBC correlations from H-15 (δ H 4.93, s) to the acetoxy carbonyl group at δ C 170.3 and from H 2 -6 (δ H 2.05, m) to C-5 (δ C 84.1) and C-7 (δ C 72.4) (Figure 3a). In the ROESY spectrum, the correlations of H-15 with Me-18 as well as Me-20 suggested that H-15, Me-18, and Me-20 were all in the same β-orientation ( Figure 3d). In addition, the correlations of 3-OH with Me-18, and of 15-H with 7-H and Me-17 suggested that 3-OH, H-7 and Me-17 were also in β-orientation ( Figure 3d). Further analysis of the ROESY spectrum indicated the configurations of the remaining functional groups in 1 were the same as those in asebotoxin VIII, namely 3β, 5β, 7α, 10α, 16α-pentahydroxy, and 14β-propionyloxy. Accordingly, the structure of 1 was deduced as shown in Figure 2, and was named pierisoid C.
Compound 2 was obtained as colorless oil with a molecular formula of C 23 H 36 O 8 , as determined by a combination of HR-EI-MS and NMR spectra (including 1 H, 13 C, and DEPT) (Figures S7-S2 in supporting information). The resemblance of the NMR spectra of 2 ( Table 1) with those of 1 disclosed that 2 was another grayanane diterpenoid structurally similar to 1. The major difference was the replacement of a methylene carbon in 1 by an oxygen-occurring methine in 2 (δ C 78.2), suggesting that either C-6, or C-11, or C-12 of 2 was oxygenated. In the HMBC spectrum of 2, the HMBC correlations from 5-OH to the methine carbon at δ C 78.2 indicated that this methine was ascribable to C-6 ( Figure 3b). Carefully comparison of 13 C-NMR spectral data of 2 with those of 1 ( Table 1) obviously found that the upfield-shift of C-15 (δ C 68.6) and C-16 (δ C 61.3) in 2, indicated an oxygen bridge, was formed between C-15 and C-16; this was supported by the HR-EI-MS spectrum. In the ROESY spectrum of 2, the correlations of Me-17 with H-15; of 3-OH and 5-OH with Me-18; and of 5-OH with 6-OH and H-7 indicated that 3-OH, 5-OH, 6-OH, H-7, H-15, and Me-17 were in the same β-orientation (Figure 3e). Consequently, the structure of 2 was determined as shown in Figure 2 and was named pierisoid D.  13 C-(100 MHz) NMR spectroscopic data of compounds 1-3 in acetone-d 6 (δ (ppm), J (Hz)). Compound 3, colorless crystals, has a molecular formula of C 31 H 42 O 14 , as determined by a combination of HR-EI-MS and NMR spectra (including 1 H-, 13 C-, and DEPT) (Figures S13-S18 in supplementary data). Its spectroscopic data were very similar to those of secorhodomollolide B, a 3,4-secograyanane diterpenoid also isolated from P. formosa [27]. The only difference between them was that the terminal double bond between C-4 (δ C 146.0) and C-18 (δ C 116.7) in secorhodomollolide B was replaced by a 4,18-oxirane group (δ C 62.5 and 52.8) in 3, which was confirmed by the HMBC correlations from Me-19 (δ H 1.34, s) to C-4, C-5 (δ C 87.5) and C-18 (Figure 3c). Such an oxirane moiety has also been found in pierisoid A, another 3,4-secograyanane diterpenoid we reported from the flowers of P. formosa [20]. In the ROESY spectrum of 3, the correlations of Me-19 with H-1, and of Me-20 with H-7 indicated that Me-19 and H-1 were in α-orientation and Me-20 coupled with H-7 were in β-orientation (Figure 3f). Therefore, compound 3 was identified as shown in Figure 2 and was named pierisoid E.

Antifeedant Activity of Compounds 1, 3, 4, and 10
The antifeedant activity of 1, 3, 4 and 10 against the generalist insect herbivore, beet armyworm (Spodoptera exigua), was assayed as previously described [31][32][33]. Compounds 1, 3 and 10 were found to be potential deterrents of the beet armyworm, with EC50 values of 10.91, 33.89 and 6.58 g/cm 2 , respectively. It seems that the antifeedant activity of grayanane diterpenoids may be reduced with the increase of degree of esterification. Although less active than the commercial neem oil containing 1% azadirachtin (EC50 = 3.71 g/cm 2 ) ( Table 2), the significant antifeedant activity of these individual compounds and the overall effect they might have suggest a defensive role of grayanane diterpenoids for P. formosa against insect herbivores.

Discussion
Compounds 1-3 are highly oxygenated grayanane diterpenoids, which occur extensively in the plants of Ericaceae and exhibit remarkable biological activities, such as antifeedant and insecticidal activities. Compound 2 is a grayanane-type diterpenoid with an unprecedented 15,16-epoxy group in the

Antifeedant Activity of Compounds 1, 3, 4, and 10
The antifeedant activity of 1, 3, 4 and 10 against the generalist insect herbivore, beet armyworm (Spodoptera exigua), was assayed as previously described [31][32][33]. Compounds 1, 3 and 10 were found to be potential deterrents of the beet armyworm, with EC 50 values of 10.91, 33.89 and 6.58 µg/cm 2 , respectively. It seems that the antifeedant activity of grayanane diterpenoids may be reduced with the increase of degree of esterification. Although less active than the commercial neem oil containing 1% azadirachtin (EC 50 = 3.71 µg/cm 2 ) ( Table 2), the significant antifeedant activity of these individual compounds and the overall effect they might have suggest a defensive role of grayanane diterpenoids for P. formosa against insect herbivores.

Discussion
Compounds 1-3 are highly oxygenated grayanane diterpenoids, which occur extensively in the plants of Ericaceae and exhibit remarkable biological activities, such as antifeedant and insecticidal activities. Compound 2 is a grayanane-type diterpenoid with an unprecedented 15,16-epoxy group in the grayanoids family. Compound 3, a 3,4-seco-grayanane diterpenoid, possesses a 4,18-epoxy substituent, which is also unusual in nature. Compared to compounds 1 and 3, 10 displayed more significant antifeedant activity against S. exigua, which was possibly attributed to the integrity of the A ring.

General Experimental Procedures
Melting points were recorded on an Aisey YLD-6000 instrument and are uncorrected. Column chromatography was performed on 200-300 mesh silica gel (Qingdao Marine Chemical Factory, Qingdao, China). Optical rotations were measured on a Horiba-SEAP-300 spectropolarimeter (Horiba, Tokyo, Japan). UV spectral data were obtained on a Shimadzu-210A double-beam spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra were recorded on a Bruker-Tensor-27 spectrometer with KBr pellets (Bruker Optics, Ettlingen, Germany). NMR experiments were carried out on either a Bruker AV-400 or a DRX-500 spectrometer with tetramethyl silane (TMS) as an internal standard (Bruker, Karlsruhe, Germany). MS were recorded on a VG-Auto-Spec-3000 spectrometer (Waters Corp., Milford, MA, USA). TLC (Thin Layer Chromatography) spots were visualized under UV light, by dipping into 10% H 2 SO 4 in EtOH followed by heating. All solvents including petroleum ether (60−90 • C) were distilled before use.

Plant Materials
The flowers of P. formosa were collected at Qu Jing, Yunnan province, China, in March 2008. The plant material was identified by Dr. Sheng-Hong Li.

Antifeedant Activity
Beet armyworms (Spodoptera exigua) were purchased from the Pilot-Scale Base of Bio-Pesticides, Institute of Zoology, Chinese Academy of Sciences. A modified dual-choice bioassay was performed for an antifeedant test as previously described [20,[31][32][33]. The larvae were reared on an artificial diet under a controlled photoperiod (light:dark, 12:8 h) and temperature (25 ± 2 • C). The larvae were starved for 3−4 h before each bioassay. Fresh leaf disks were cut from Brassica chinensis, using a cork borer (1.1 cm in diameter). The treated leaf disks were painted with 10 µL of the test compound in acetone, and control leaf disks were treated with the same amount of acetone. After air drying, the tested and control leaf disks were set in alternating position in the same Petri dish (90 mm in diameter), with moistened filter paper at the bottom. Two-thirds of the instars were placed at the center of the Petri dish. Five replicates were run for each treatment. After feeding for 24 h, the areas of leaf disks consumed were measured. The antifeedant index (AFI) was calculated according to the following formula: AFI = [(C − T)/(C + T)] × 100, where C and T represent the control and treated leaf areas consumed by the insect. The insect antifeedant potency of the test compound was evaluated in terms of the EC 50 value, which was determined by probit analysis for each insect species.

Conclusions
Terpenoids play an important role in natural product chemistry and biology, such as antifungal and insecticidal activities [34]. Secondary metabolites, such as the grayanane diterpenoids that occur extensively in the plants of Ericaceae, are fascinating for their remarkable toxicity, as well as their significant antifeedant and insecticidal activity. In the current study, three new grayanane diterpenoids, pierisoids C-E (1-3), as well as 10 known ones (4-13), were identified from the flowers of P. formosa via their extensive 1D and 2D NMR spectroscopic data analyses. Notably, compounds 1, 3 and 10, especially 10, exhibited obvious antifeedant activity against the beet armyworm (S. exigua), suggesting that these diterpenoids were important defensive substances in P. formosa against natural enemies.