Highly Oxygenated Grayanane Diterpenoids from Flowers of Pieris

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Highly Oxygenated Grayanane Diterpenoids from Flowers of Pieris japonica and Structure−Activity Relationships of Antifeedant Activity against Pieris brassicae Xuan-Qin Chen, Ling-Huan Gao, Yan-Ping Li, Hong-Mei Li, Dan Liu, Xia-Li Liao, and Rong-Tao Li* School of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, China S Supporting Information *

ABSTRACT: Six new highly oxygenated grayanane diterpenoids, neopierisoids G−L, 1−6, together with 10 known related compounds, 7−16, were identified from the flowers of the poisonous plant Pieris japonica. The structures were elucidated on the basis of comprehensive NMR spectroscopy and mass analysis. The relative configurations of 1−6 were elucidated by analysis of ROESY spectra and comparison of NMR data with the analogues. The absolute configurations of 1−6 were established by the Xray diffraction analysis of 1 and comparison of the CD spectra of 1−6. Compared with the skeleton of the normal grayanane diterpenoids, compounds 1−6 shared an unusual seco A ring moiety. The antifeedant activities of compounds 1−16 against Pieris brassicae were evaluated by using a dual-choice bioassay, and compounds 1−10 with a normal grayanane skeleton showed potent antifeedant activity against P. brassicae. The structure−activity relationships of antifeedant activities of 1−16 against P. brassicae are discussed. KEYWORDS: Pieris japonica, highly oxygenated grayanane diterpenoids, neopierisoids G-L, antifeedant activity, structure−activity relationships

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INTRODUCTION

General Experimental Procedures. A DIP-370 digital polarimeter (JASCO Corp., Tokyo, Japan) was utilized to measure the optical rotations. UV data were recorded on a UV-2401A spectrophotometer (Shimadzu, Kyoto, Japan). IR spectroscopy with KBr pellets was performed using an FtS-135 spectrophotometer (BioRad Corp., Hercules, CA, USA). CD spectra were tested on a Chirascan Q100 instrument (Applied Photophysics Ltd., Surrey County, UK). 1D- and 2D-NMR spectra were measured via a DRX500 spectrometer (Bruker BioSpin Group, Karlsruhe, Germany). Unless otherwise specified, chemical shifts (δ) are conveyed in parts per million referred to an internal standard (tetramethylsilane, TMS). Column chromatography was run with silica gel (100−200 mesh) (Qingdao Marine Chemical, Inc., Qingdao, China), Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden), and LiChroprep RP18 (40−63 μM) (Merck KGaA, Darmstadt, Germany). EI-MS and HREIMS (70 eV) were measured using an Auto Spec-3000 spectrometer (VG PRIMA, Birmingham, UK). Semipreparative HPLC was carried out on a model 1200 liquid chromatograph (Agilent, Palo Alto, CA, USA) equipped with a 250 mm × 9.4 mm i.d. Zorbax SB-C18 column monitored at 198 nm. Fractions were surveyed by TLC (Si gel GF254) (Qingdao Marine Chemical, Inc.). Silica gel plates sprinkled with a chromogenic agent (10% H2SO4 in EtOH) were heated by an electric stove to visualize the spots. All solvents before the use were distilled. The purity of all compounds was measured by HPLC, NMR, and ESI-MS as >95%. Plant Material. The flowers of P. japonica were gathered from Jindian Mountain, Kunming city, Yunnan province, China, in September 2014. The materials were authenticated by Professor Haizhou Li, and voucher specimens (KM20140915) have been

Diterpenoids in poisonous plants belonging to the Ericaceae family comprise a few specialized carbon frameworks usually decorated with highly oxygenated chemical groups. The four main compound types are grayanane, leucothane, ent-kaurane, and kalmane.1−7 The grayanane-type diterpenoids, derived biogenetically from the ent-kanrane skeleton, predominate in Ericaceae plants. Nearly 130 grayanoids have been identified from the family Ericaceae, and some of them have exhibited remarkable biological activities, such as antifeedant, growth inhibitory, and insecticidal activities.2,8−10 The grayanane-type diterpenoids are believed to play a mainly defensive role in poisonous plants of Ericaceae family by exhibiting antifeedant and insecticidal activities. Previously, we reported the isolation of highly acylated 9,10-seco- and 3,4-seco-grayanoids from the fruits of Pieris formosa.11,12 More recently, two chlorinated 3,4seco-grayanane diterpenoids with significant antifeedant activity were identified from Pieris japonica flowers collected in Xundian county by our group. 13 In the continuous chemical investigation on the poisonous plants, 6 new grayanoids were now isolated and identified from the flowers of P. japonica, together with 10 known compounds.6,14−19 Pieris brassicae is a pernicious and oligophagous insect, with the larvae feeding dominantly on members of the Brassicaceae family.20 The adults feed on the sap of a variety of crop and vegetable plants, thus resulting in great economic loss to farmers.21 Herein, we report the isolation and structural elucidation of the new compounds and activities of all of the isolated compounds, as well as the structure−activity relationships to antifeedant activity against P. brassicae. © 2017 American Chemical Society

MATERIALS AND METHODS

Received: Revised: Accepted: Published: 4456

April 3, 2017 May 12, 2017 May 12, 2017 May 12, 2017 DOI: 10.1021/acs.jafc.7b01500 J. Agric. Food Chem. 2017, 65, 4456−4463

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of compounds 1−16.

Table 1. 1H NMR Spectroscopic Data of Compounds 1−6a position 1 2a 2b 6 7 9 11a 11b 12a 12b 13 14 15 17 18a 18b 19 20 6-OAc 7-OAc/OPr 11-OAc 14-OPr 15-OAc 16-OAc 3-OCH3 18-OCH3 a

1

2

3.71 2.87 3.27 6.90 6.31 3.49 4.73

(dd, 10.3, 6.4) (dd, 18.0, 6.4) (dd, 18.0, 10.3) (d, 4.8) (br s) (d, 5.2) (m)

3.68 2.84 3.24 6.88 6.27 3.59 4.73

(dd, 10.8, 7.0) (dd, 18.2, 7.0) (dd, 18.2, 10.8) (d, 3.8) (br s) (br s) (m)

2.29 2.44 3.46 6.72 5.04 1.99 1.68

(m) (m) (d, 8.5) (br s) (br s) (s) (s)

2.31 (m) 2.44a (m) 3.37 (d, 9.1) 6.78 (br s) 5.07 (br s) 2.00 (s) 1.67 (s)

1.66b 1.66b 2.26 (s) 2.49 (m) 1.23 (t, 7.4)

1.66 1.64 2.25 2.34

2.34 (s)

2.44a (m), 2.53 (m) 1.27 (t, 7.4) 2.04 (s) 2.14 (s)

2.17 (s) 2.05 (s)

(s) (s) (s) (s)

3

4

5

6

3.89 (dd, 13.4, 7.6) 3.52b 3.13 (dd, 17.6, 7.6) 5.72 (d, 9.7) 6.51 (d, 9.7) 3.45 (d, 4.5) 5.75 (m) 2.48 (m) 2.18 (m) 3.52b 7.02 (br s) 5.43 (br s) 1.78 (s) 3.34 (d, 5.6) 2.56 (d, 5.6) 1.67 (s) 2.30 (s) 2.06 (s) 2.18 (s)

3.58 (m) 2.93 (dd, 9.3, 14.7) 3.03 (m) 6.64 (d, 7.3) 5.70 (d, 7.3) 2.83 (br t) 2.17b 2.27 (br s) 1.63b 2.17b 3.64 (m) 6.77 (br s) 5.38 (br s) 1.69 (s) 5.74 (br s) 5.54 (br s) 4.70 (d, 11.4) 4.6 (d, 11.4) 1.35 (s) 2.17b (s) 1.95 (s)

3.51 (d, 10.8) 2.77 (dd, 17.5, 10.8) 2.58 (d, 17.5) 5.09b (br s) 4.76 (d, 5.8) 1.94 (s) 1.46 (m) 1.48 (m) 1.68 (m) 1.77 (m) 2.26 (br s) 4.66 (m) 5.09b (br s) 1.42 (s) 4.37 (d, 11.3) 4.22 (d, 11.3) 1.95 (s) 1.28 (s)

3.52 (d, 10.7) 2.74 (dd, 17.5, 10.7) 2.60 (d, 17.5) 5.18 (d, 1.9) 4.77b 1.94 (t, 5.7) 1.79 (m) 1.69 (m) 1.68 (m) 1.75 (m) 2.28 (br s) 4.73 (br s) 3.78 (br s) 1.42 (s) 4.77b 4.59 (d, 12.2) 2.09 (s) 1.28 (s)

2.22 2.75 1.37 2.25 2.33

2.62 1.30 2.15 2.13

3.69 (s) 3.25 (s)

3.68 (s)

(s) (m), 2.46 (m) (t, 7.4) (s) (s)

(m), 2.54 (m) (t, 7.5) (s) (s)

Recorded in pyridine-d5, δ in ppm, J in Hz. bSignals were overlapped.

deposited at the Faculty of Life Science and Technology, Kunming University of Science and Technology. Extraction and Isolation. The air-dried P. japonica flowers (3.3 kg) were powdered and extracted with Me2CO/H2O (75:25, 3 × 12 L, 48 h each) at room temperature. The extract was evaporated under a

reduced pressure distillation to get a crude residue (640 g), which was then partitioned between H2O and EtOAc. The soluble EtOAc fraction (452 g) was separated over silica gel column eluted with a gradient system of CHCl3/Me2CO (1:0, 9:1, 8:2, 7:3, and 6:4, each 20 L) to afford five fractions (Fr 1−5). Fr 3 (CHCl3/Me2CO, 8:2, 15.7 g) 4457

DOI: 10.1021/acs.jafc.7b01500 J. Agric. Food Chem. 2017, 65, 4456−4463

Article

Journal of Agricultural and Food Chemistry Table 2. 13C NMR Spectroscopic Data of Compounds 1−6a no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 6-OAc 7-OAc/OPr

1

2

3

4

5

6

50.6 (d) 34.7 (t) 175.9 (s) 77.3 (s) 93.0 (s) 75.0 (d) 73.2 (d) 54.5 (s) 51.9 (d) 77.0 (s) 66.5 (d) 35.2 (t) 42.8 (d) 80.3 (d) 89.3 (d) 89.6 (s) 22.5 (q) 26.2 (q) 27.8 (q) 32.9 (q) 169.9 (s) 21.7 (q) 173.6 (s) 28.5 (t) 9.2 (q)

50.9 (d) 34.8 (t) 175.9 (s) 77.3 (s) 92.9 (s) 74.6 (d) 73.6 (d) 54.6 (s) 51.8 (d) 77.0 (s) 66.5 (d) 35.4 (t) 43.3 (d) 80.2 (d) 89.3 (d) 89.8 (s) 23.6 (q) 26.3 (q) 27.9 (q) 33.1 (q) 170.4 (s) 21.9 (q) 170.6 (s) 21.0 (q)

43.9 (d) 30.5 (t) 174.0 (s) 59.9 (s) 73.2 (s) 73.7 (d) 67.0 (d) 53.4 (s) 60.1 (d) 88.6 (s) 68.4 (d) 30.8 (t) 42.4 (d) 76.1 (d) 91.4 (d) 88.4 (s) 19.9 (q) 49.8 (t) 18.5 (q) 29.5 (q) 170.2 (s) 20.4 (q) 169.6 (s) 21.6 (q)

55.2 (d) 32.5 (t) 174.9 (s) 152.3 (s) 90.6 (s) 70.8 (d) 67.4 (d) 55.8 (s) 48.4 (d) 77.1 (s) 19.9 (t) 25.1 (t) 44.5 (d) 79.0 (d) 87.5 (d) 88.1 (s) 18.8 (s) 112.4 (t) 63.1 (t) 33.5 (q) 169.1 (s) 21.0 (q) 169.1 (s) 20.5 (q)

45.4 (d) 36.3 (t) 173.5 (s) 127.8 (s) 143.3 (s) 78.4 (d) 76.3 (d) 53.6 (s) 54.4 (d) 85.7 (s) 18.3 (t) 25.5 (t) 51.7 (d) 82.7 (d) 89.3 (d) 79.2 (s) 22.7 (q) 74.9 (t) 17.8 (q) 21.6 (s)

45.3 (d) 36.2 (t) 173.6 (s) 131.3 (s) 140.7 (s) 78.3 (d) 76.3 (d) 53.7 (s) 54.4 (d) 85.5 (s) 25.5 (t) 18.3 (t) 51.8 (d) 82.5 (d) 89.2 (d) 79.2 (s) 22.7 (q) 64.8 (t) 17.9 (q) 21.6 (q)

174.1 (s) 28.7 (t) 9.3 (q) 170.6 (s) 22.7 (q) 169.9 (s) 21.8 (q)

168.9 (s) 22.8 (q) 174.6 (s) 28.4 (t) 9.3 (q) 171.7 (s) 21.4 (q) 170.3 (s) 20.8 (q)

174.1 (s) 28.4 (t) 9.3 (q) 169.9 (s) 22.9 (q) 172.0 (s) 22.0 (q) 51.7 (q) 57.5 (s)

51.8 (q)

11-OAc 14-OPr/OAc

170.3 (s) 20.9 (q)

15-OAc

170.6 (s) 21.8 (q) 170.8 (s) 23.2 (q)

16-OAc 3-OCH3 18-OCH3 a

Recorded in pyridine-d5, 125 MHz, δ in ppm. Neopierisoid G, 1 (see Figure 1 for structure): colorless needles, = −27.3 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 206.8 [α]23.3 D (2.26) nm; IR (KBr) νmax 3432, 2985, 2946, 1736, 1634, 1428, 1373, 1267, 1234, 1126, 1063, 1023, 953, 893, 689, 607 cm−1; 1H (500 MHz) and 13C (125 MHz) NMR data, Tables 1 and 2; ESIMS (pos) m/z 656 [M + Na]+; HREIMS (pos) m/z 656.2699 [M + Na]+ (calcd for C31H44O15Na, 656.2680). = −28.2 (c Neopierisoid H, 2: white amorphous powder, [α]23.3 D 0.07, MeOH); UV (MeOH) λmax (log ε) 201 (2.47) nm; IR (KBr) νmax 3573, 3563, 3471, 3443, 1642, 1633, 1022, 719, 688 cm−1; 1H (500 MHz) and 13C (125 MHz) NMR data, Tables 1 and 2; ESIMS (pos) m/z 656 [M + Na]+; HREIMS (pos) m/z 656.2693 [M + Na]+ (calcd for C31H44O15Na, 656.2680). Neopierisoid I, 3: white amorphous powder, [α]23.3 D = −0.2 (c 0.1, MeOH−CHCl3 1:1); 1H (500 MHz) and 13C (125 MHz) NMR data, Tables 1 and 2; ESIMS (pos) m/z 696 [M + Na]+; HREIMS (pos) m/ z 696.2605 [M + Na]+ (calcd for C33H44O16Na, 696.2629). Neopierisoid J, 4: white amorphous powder, [α]23.3 D = +10.9 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 202 (2.63) nm; IR (KBr) νmax 3439, 2984, 2943, 1782, 1734, 1633, 1427, 1237, 1057, 896, 808, 687 cm−1;1H (500 MHz) and 13C (125 MHz) NMR data, Tables 1 and 2; ESIMS (pos) m/z 638 [M + Na]+; HREIMS (pos) m/z 638.2571 [M + Na]+ (calcd for C31H42O14Na, 638.2575). = −96.9 (c, Neopierisoid K, 5: white amorphous powder, [α]23.3 D 0.07, MeOH); IR (KBr) νmax 3441, 3426, 1735, 1639, 1631 cm−1; 1H

was then chromatographed over silica gel eluted with gradient CHCl3/ Me2CO (10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1 3:1, 2:1, 1:1, each 1 L) to give three subfractions (SF 3.1−3.3). SF 3.1 (CHCl3/Me2CO, 8:1 → 6:1, 3.4 g) was subjected to a repeated silica gel column chromatography eluted with CHCl3/MeOH (10:1, 3 L) to get a diterpenoid mixture (65 mg). This mixture was then purified by semipreparative HPLC (MeCN/H2O, 23:77, flow rate = 2 mL/min) to yield 5 (4.6 mg, tR = 11.1 min), 10 (4.3 mg, tR = 13.3 min), 9 (5.7 mg, tR = 22.7 min), and 6 (4.3 mg, tR = 27.4 min). Compound 7 (65 mg) was crystallized from SF 3.2 (CHCl3/Me2CO, 5:1, 237 mg), and then the mother liquor of SF 3.2 (165 mg) was applied to silica gel column chromatography (petroleum ether/Me2CO, 6:1, 1 L) to afford 2 (7.6 mg), 7 (12.1 mg), 12 (7.7 mg), 14 (5.5 mg), and 16 (14.0 mg). Fr 4 (CHCl3/Me2CO, 7:3, 22.5 g) was further purified by MPLC (MeOH/H2O, 30:70, 50:50, 70:30, and 90:10, each 2 L) to give four main subfractions (SF 4.1−4.4). SF 4.1 (MeOH/H2O, 30:70, 2.8 g) was purified by Sephadex LH-20 (MeOH/H2O, 8:2, 2 L) and semipreparative HPLC (MeCN/ H2O, 20:80, flow rate = 2 mL/min) to yield 3 (10.1 mg, tR = 12.3 min), 1 (6.6 mg, tR = 19.5 min), 4 (5.7 mg, tR = 27.1 min), and 8 (4.9 mg, tR = 31.3 min). Compounds 13 (20.8 mg) and 15 (9.1 mg) were separated from SF 4.2 (MeOH/H2O, 50:50, 3.0 g) by RP-18 (MeOH/ H2O, 40:60, 1.0 L). SF 4.3 (MeOH/H2O, 70:30, 2.2 g) was subjected to Sephadex LH-20 (CHCl3/MeOH, 1:1, 0.8 L) and silica gel eluted with CHCl3/MeOH (10:1, 0.4 L) to afford 11 (7.5 mg). 4458

DOI: 10.1021/acs.jafc.7b01500 J. Agric. Food Chem. 2017, 65, 4456−4463

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Journal of Agricultural and Food Chemistry (500 MHz) and 13C (125 MHz) NMR data, Tables 1 and 2; ESIMS (pos) m/z 426; HREIMS (pos) m/z 426.2323 (Calcd for C22H34O8, 426.2254). Neopierisoid L, 6: white amorphous powder, [α]23.3 D = −7.8 (c, 0.21, MeOH); 1H (500 MHz) and 13C (125 MHz) NMR data, Tables 1 and 2; ESIMS (pos) m/z 430 [M + Na]+; HREIMS (pos) m/z 430.2223 (calcd for C21H34O9, 430.2203). Antifeedant Assay. Antifeedant activity was tested using a modified dual-choice bioassay.22 Briefly, the P. brassicae larvae were fed a man-made diet in an incubator under a manipulated photoperiod (12/8 h, light/dark) and temperature (25 ± 2 °C). Before the start of each bioassay, the third-instar larvae of P. brassicae were starved for 4− 5 h. Leaf disks (1.8 cm in diameter) were prepared from the fresh Brassica chinensis leaves using a cork borer. Leaf disks, which were brushed with the tested compounds at concentrations of 16, 8, 4, 1, and 0.25 μg/cm2 (dissolved in 20 μL acetone), were defined as the treated group. Leaf disks painted with 20 μL of acetone served as a control group. Then, a Petri dish (150 mm in diameter) was selected and covered with a piece of moist filter paper at its bottom. After airdrying, the leaf disks (both pieces) in treated and control groups were alternatively placed on the Petri dish and then one-third of the larvae were laid aside at the middle of the dish. After 24 h of feeding, the percentage of leaf disk area consumed by the insects was calculated.13 Each treatment was repeated 10 times. Azadirachtin (Sigma Inc., Santa Clara, CA, USA) was used as the positive control. The antifeedant activity of the tested compounds was represented as the EC50 value, which was calculated according to a previous method.13 X-ray Crystallographic Analysis of Neopierisoid G, 1. Neopierisoid G, 1: C31H44O15, M = 656.27, colorless needles, size 0.53 × 0.56 × 0.33 mm3; crystal data for 1, C31H44O15·H2O, M = 674.68, monoclinic, a = 10.1897(5) Å, b = 17.7300 (8) Å, c = 17.8528 (8) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 3225.3(3) Å3, T = 100(2) K, space group P21, Z = 4, μ (Mo Kα) = 0.953 mm−1, 17473 reflections measured, 5297 independent reflections (Rint = 0.0308). The final R1 value was 0.0322 [I > 2σ(I)]. The final wR(F2) values was 0.0833 [I > 2σ(I)]. The final R1 values were 0.0332 (all data). The final wR(F2) values were 0.0833 (all data). The goodness of fit on F2 was 1.099. Flack parameter = 0.06(7). Crystallographic data of neopierisoid G, 1 have been deposited in the database of Cambridge Crystallographic Data Center (CCDC code: 1536367). Copies of the data are available free of charge via the Internet at www.ccdc.cam.ac.uk.

1 due to the reappearance of a methyl group (C-19) and the molecular formula of 1, which was responsible for the transformation of one methylene in neopierisoid A to the methyl group (C-19) in 1. One hydroxyl group was inferred to be attached at C-11 in 1 on the basis of the oxygenated nature of C-11 concluded from the HSQC spectrum. This deduction was further proved by the HMBC correlation of H-9, H-12, and H-13 with C-11 and the 1H−1H COSY spin system of H-14/H13/H-12/H-11/H-9 (Figure 2).

Figure 2. Key HMBC and 1H−1H COSY of neopierisoid G, 1.

The relative stereochemistry of 1 was elucidated by the ROESY spectrum. The ROESY correlations between H-1/H-6, H-6/H-14, H-14/H-13, and H-12α/H-14 demonstrated that H-1, H-6, H-13, and H-14 all possessed an α-orientation. However, β-orientations of H-7, H-9, H-11, H-15, Me-17, and Me-20 were established from the ROESY correlations of Me20/H-9, Me-20/H-11, H-9/H-15, H-15/H-7, H-15/Me-17, and Me-17/H-12β. The 2-hydroxyisopropyl group attached at C-5 was deduced to be an α-orientation on the basis of the ROESY correlation of H-1/Me-18 (Figure 3). Finally, the absolute

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RESULTS AND DISCUSSION Analysis of HREIMS revealed that compound 1 has the molecular formula C31H44O15, requiring 10 degrees of unsaturation. The IR spectrum indicated the presence of hydroxyl (3432 cm−1) and carbonyl (1736 cm−1) groups. The 1 H NMR of 1 showed resonances ascribed to the signals for a propionyl group at δH 1.23 (3H, t, J = 7.4 Hz) and 2.49 (2H, m), four acetyl methyls at δH 2.26, 2.34, 2.17, and 2.05 (s, each 3H), and four tertiary methyls at δH 1.99 (Me-17), 1.68 (Me18), 1.66 (Me-19), and 1.66 (Me-20), as well as four oxygenated methines at δH 6.90 (d, J = 4.8 Hz, H-6), 6.31 (br s, H-7), 4.73 (m, H-11), 6.72 (br s, H-14), and 5.04 (br s, H-15) (Table 1). Besides 1 propionyl and 4 acetyl units, the 13 C NMR and DEPT spectra of 1 showed 20 carbon resonances for the core skeleton (Table 2), including 4 methyls, 2 methylenes, 8 methines (5 oxygenated), and 6 quaternary carbons (a carbonyl and 4 oxygenated). The spectroscopic data suggested compound 1 to be a highly acylated diterpenoid with a different parent nucleus from the normal grayanane or kalmane skeleton. The NMR data of 1 were similar to those of neopierisoid A,13 and the main difference was that the signals of two methylenes in neopierisoid A were replaced by those of an oxygenated methine and one methyl in 1. The chlorine atom attached at C-19 in neopierisoid A was deduced to be absent in

Figure 3. Key ROESY of neopierisoid G, 1.

configuration of 1 was established from X-ray diffraction analysis (Figure 4). Consequently, compound 1 (neopierisoid G) was fully identified as shown in Figure 1.

Figure 4. X-ray diffraction of neopierisoid G, 1. 4459

DOI: 10.1021/acs.jafc.7b01500 J. Agric. Food Chem. 2017, 65, 4456−4463

Article

Journal of Agricultural and Food Chemistry

The relative configurations of 3 at C-16, C-15, C-14, C-13, C-11, C-10, C-9, C-8, C-7, C-6, and C-1 were deduced to be identical to those of 1 due to the similarity in the ROESY spectra. The Me-19 was assigned as α-orientation by the NMR data comparison of C-4, C-18, and C-19 with those of pierisoids A and B, the absolute configurations of which have been confirmed by X-ray crystallography.15 The 5-OH was inferred to be β-oriented due to the ROESY correlation of H-6/ Me-19. Consequently, compound 3 was identified as shown and named neopierisoid I. An analysis of HREIMS indicated that compound 4 possessed the molecular formula of C31H42O14, indicating 11 degrees of unsaturation. Compound 4 displayed IR, UV, and NMR spectroscopic features similar to those of secorhodomollolide B, 9.14 The only structural difference between the two compounds was that the Me-19 in 9 was replaced by a hydroxymethyl in 4. This assignment was supported by a significant downshift of C-19 (δC 63.1, t) in 4 as compared with that (δC 18.2, q) in 9. The HMBC correlations of H-19(H-18)/ C-4, H-19(H-18)/C-5, H-18/C-19, and H-19/C-18 further supported the attachment of the hydroxyl at C-19. The relative configuration of 4 was determined to be identical to that of 9 by comparison of the key ROESY correlations and chemical shifts between them. Accordingly, the structure of 4 was established. The molecular formula of 5 was inferred to be C22H34O8 from HREIMS, demanding 6 degrees of unsaturation. The typical absorptions of hydroxyl (3441 cm−1), ester carbonyl (1735 cm−1), and double bond (1639 and 1631 cm−1) functional groups were observed in the IR spectrum. In the 1 H NMR spectrum, the resonances for three tertiary methyls at δH 1.28, 1.42, and 1.95 (s, each 3H), two methoxys at δH 3.25 and 3.69 (s, each 3H), and an AB methylene at δH 4.22 (d, J = 11.3 Hz) and 4.37 (d, J = 11.3 Hz) were distinguished. The low-field region displayed four oxygenated methines at δH 5.09 (overlapped, br s, H-6), 4.76 (d, J = 5.8, H-7), 4.66 (m, H-14), and 5.09 (overlapped, br s, H-15) (Table 1). Analyses of the 13 C NMR and DEPT spectra of 5 showed 22 carbons, including 5 methyls (2 methoxy groups), 4 methylenes, 7 methines (4 oxygen-bearing), and 6 quaternary carbons (1 carboxyl group, 2 olefinic, and 2 oxygenated) (Table 2). Compound 5 was also speculated to be a 3,4-seco-grayanoid diterpene as hinted by the above spectroscopic feature. By comparison of the NMR data of 5 with those of 1−4, the upfield chemical shift of ester carbonyl (C-3) of 5 suggested that the five-membered lactone moiety in 1−4 would have been ring-opened in 5. The entire absence of those acyls in 5 indicated that 5 was one of the deacylated derivatives of 1−4 at C-16, C-15, C-14, C-7, and C-6. The NMR data of 5 showed some similarities to those of 4, except the difference in upshift of the ester carbonyl (C-3) and absence of five acyls in 5; the others included the presence of two methoxyls, one methyl, and one olefinic quaternary carbon in 5 and the absence of the olefinic terminal methylene (C-18) and oxgentated sp3 quaternary carbon (C-4) in 4. Following the δ-lactone opening in 4, one carboxymethyl was deduced to form at C-3 in 5 as confirmed by HMBC correlations of one methoxyl with the ester carbonyl (C-3), and the pair of double bonds was inferred to migrate to C-4 and C-5 in 5 instead of C-4 and C-18 in 4, according to the HMBC correlation from H-18, H-19, and H-1 to C-4 and from H-1, H-2, H-6, and H-7 to C-5. The other methoxyl was inferred to attach at C-19 in 5 rather than the OH in 4 due to the oxygenated nature of C-19 and the HMBC correlation of the methoxyl with C-19. Except for 2 degrees of

Compound 2 has the same molecular formula of C31H44O15 as 1, as concluded from HREIMS. The 1H and 13C NMR data of 2 and 1 look alike except for the differences in the signals of H-6 (C-6), H-7 (C-7), H-13 (C-13), H-14 (C-14), and the propionyl carbonyl. The chemical shifts of H-6, H-7, H-13, and H-14 in 2 were shifted by ΔδH −0.02, −0.04, −0.09, and +0.06, as compared with those in 1, whereas those resonances of C-6, C-7, C-13, C-14, and the propionyl carbonyl were in turn shifted by ΔδC −0.04, −0.06, + 0.05, −0.01, and +0.05. In addition, the HMBC correlations of 2 from H-14 to the propionyl carbonyl (δC 174.1, s) and from H-7 to an acetyl carbonyl (δC 170.6, s) revealed that the propionyloxy group at C-7 and the acetyloxy at C-14 in 1 were reversed in 2. Because 2 was the isomer of 1 and may be derived from 1 by intramolecular acyl interchange like secorhodomollolides A and B,14 it is understandable that no significant difference was observed in comparison of 1D NMR data between 1 and 2. A ROESY experiment showed that 2 had the same relative configuration as 1. Therefore, the structure of compound 2 was determined as neopierisoid H. Compound 3 had the molecular formula C33H44O16, deduced from HREIMS. The 1H NMR of 3 showed the presence of a propionyl unit at δH 1.37 (3H, t, J = 7.4 Hz), 2.46 (1H, m), and 2.75 (1H, m) as well as five acetyl methyls at δH 2.06, 2.18, 2.22, 2.25, and 2.33 (s, each 3 H). Three tertiary methyls at δH 1.78 (Me-17), 1.67 (Me-19), and 2.30 (Me-20) together with an AB methylene at δH 2.56 (d, J = 5.6 Hz) and 3.34 (d, J = 5.6 Hz) ascribed to the core skeleton moiety were observed in the relatively high-field region. Five protons in oxygenated methines at δH 5.72 (d, J = 9.7 Hz, H-6), 6.51 (d, J = 9.7 Hz, H-7), 5.75 (m, H-11), 7.02 (br s, H-14), and 5.43 (br s, H15) were present at relatively low field (Table 1). Besides one propionyl and five acetyl units, the 13C NMR spectrum of 3 showed 20 carbon resonances for the carbon skeleton (Table 2), which were classified into three methyls, three methylenes (an oxygenated), eight methines (five oxygenated), and six quaternary carbons (a carbonyl and five oxygenated) in the DEPT spectrum. The protons at δ H 3.34 and 2.56, corresponding to the methylene at δC 49.8 (C-4) in the HSQC spectrum, showed a HMBC correlation with the oxygenated quaternary carbon at δC 55.9, which suggested that the molecular structure of 3 contains a 2,2-disubstituted oxirane moiety. The NMR data of 3 (Tables 1 and 2) and pierisoid A, 8, were quite similar to each other and possess the same 3,4-secograyanane skeleton.15 The most obvious distinction was the emergence of a fifth acetyloxy moiety and an oxygenated methine in 3 and the absence of one methylene in 8. The fifth acetyloxy group was assigned at C-11 due to the HMBC correlation of H-11 with the acetyl carbonyl, together with the 1 H−1H COSY spin system of H-14/H-13/H2-12/H-11/H-9. However, when the NMR data of 3 were carefully assigned according to the 2D NMR spectra, the HMBC correlation of H-14 with one propionyl carbonyl suggested that the propionyloxy group was connected to C-14 in 3 rather than C-6 in 8. Three acetyloxy groups were inferred to be at C-15, C-7, and C-6, respectively, as supported by the HMBC correlations from H-15, H-7, and H-6 to the three acetyl carbonyls (δC 171.7, 169.6, and 170.2), respectively, and the 1 H−1H COSY system of H-6/H-7. The last acetyloxy group was assigned at C-16 on the basis of the oxidized property of this carbon and the structural formula of 3. 4460

DOI: 10.1021/acs.jafc.7b01500 J. Agric. Food Chem. 2017, 65, 4456−4463

Article

Journal of Agricultural and Food Chemistry

Compounds 1−6 are structural analogues that may be derived from normal grayanane diterpenoids. Therefore, the absolute configurations of the carbon skeleton of compounds 2−6 were assumed to be identical to that of 1. This speculation was also supported by an analysis of the CD spectra of compounds 1−6, which showed similar positive Cotton effects in the range of 200−220 nm (compounds 1−4, approximately 220 nm; compounds 5−6, approximately 200 nm) (Figure 6). It is well-known that secondary metabolites are important defensive substances in plant defense against natural enemies. The fruits and flowers are the most vital organs for the plants to achieve survival of generations. Therefore, such defensive substances can be distributed in the fruits and flowers to protect them from damage by natural enemies.23,24 Grayanane diterpenoids have been recognized as major toxins to mammals.25 Besides these grayanane diterpenoids, several novel 3,4-secograyanane diterpenoids have been also identified from the flowers or fruits of plants of the Ericaceae family.14,15 Although the poisonous mechanism of grayanotoxins to insects is not yet clear, several studies have revealed that grayanotoxins exhibited antifeedant and insecticidal activities against P. brassicae and Helicoverpa armigera.13,15 In the present study, six new grayanotoxins, 1−6, were identified from the flowers of the poisonous plant P. japonica. The new compounds, 1−6 as well as the known ones, 7−9, shared an unusual 3,4-seco A ring compared with those grayananoids, 10−16, with the usual skeleton. The antifeedant activities of 1−9 (EC50> 10 μg/cm2) against P. brassicae were much weaker than those of 10−16, which showed EC50 values of 10.0 0.028a 2.18f 0.16b 0.39c 0.66d 1.18e 0.11b 0.008

0.016−0.04 0.98−3.43 0.05−0.22 0.17−0.53 0.39−0.82 0.65−1.78 0.03−0.20 0.003−0.014

ASSOCIATED CONTENT

S Supporting Information *

b

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01500.

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1D- and 2D-NMR and HR-MS spectra of 1−6 and X-ray data of 1 (PDF)

AUTHOR INFORMATION

Corresponding Author

*(R.-T.L.) E-mail: [email protected]. Phone: +86-87165920569. Fax: +86-871-6592570.

a

The same letters indicate that EC50 was at the same statistical level (P > 0.05). bCI, confidential interval.

ORCID

Rong-Tao Li: 0000-0002-6172-0568 Funding

important role than those with 3,4-seco skeletons, at least in plant defense against P. brassicae. Although the new compounds presented here did not exhibit significant antifeedant activity, the preliminary SAR analysis may provide new clues for the development of grayanane diterpenoids as a highly efficient insecticide against P. brassicae.

We thank the National Natural Science Foundation of China (No. 21262021, 21572082) and the Open Foundation of the State Key Laboratory of Phytochemistry and Plant Resources in West China (P2015-KF01) for financial support. Notes

The authors declare no competing financial interest. 4462

DOI: 10.1021/acs.jafc.7b01500 J. Agric. Food Chem. 2017, 65, 4456−4463

Article

Journal of Agricultural and Food Chemistry

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(19) Katai, M.; Fujiwara, M.; Terai, T.; Meguri, H. Studies on the constituents of the leaves of Pieris japonica D. Don. Chem. Pharm. Bull. 1980, 28, 3124−3126. (20) Hansan, F.; Ansari, M. S. Effects of different brassicaceous host plants on the fitness of Pieris brassicae (L.). Crop Prot. 2011, 30, 854− 862. (21) Ferreres, F.; Sousa, C.; Valentao, P.; Pereira, J. A.; Seabra, R. M.; Andrade, P. B. Tronchuda cabbage flavonoids uptake by Pieris brassicae. Phytochemistry 2007, 68, 361−367. (22) Isman, M. B.; Koul, O.; Luczynski, A.; Kaminski, J. Insecticidal and antifeedant bioactivities of neem oils and their relationship to azadirachtin content. J. Agric. Food Chem. 1990, 38, 1406−1411. (23) Zhang, Y.; Wang, J. S.; Wang, X. B.; Gu, Y. C.; Wei, D. D.; Guo, C.; Yang, M. Y.; Kong, L. Y. Limonoids from the fruits of Aphanamixis polystachya (Meliaceae) and their biological activities. J. Agric. Food Chem. 2013, 61, 2171−2182. (24) Defago, M.; Valladares, G.; Banchio, E.; Carpinella, C.; Palacios, S. Insecticide and antifeedant activity of different plant parts of Melia azedarach on Xanthogaleruca luteola. Fitoterapia 2006, 77, 500−505. (25) Puschner, B.; Holstege, D. M.; Lamberski, N.; Le, T. Grayanotoxin poisoning in three goats. J. Am. Vet. Med. Assoc. 2001, 218, 573−575. (26) Hikino, H.; Ohta, T.; Ogura, M.; Ohizumi, Y.; Konno, C.; Takemoto, T. Structure-activity relationship of ericaceous toxins on acute toxicity in mice. Toxicol. Appl. Pharmacol. 1976, 35, 303−310.

ACKNOWLEDGMENTS We thank Dr. Susan L. Morris-Natschke in the Natural Products Research Laboratories, University of North Carolina, for help with linguistic polishing.

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REFERENCES

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DOI: 10.1021/acs.jafc.7b01500 J. Agric. Food Chem. 2017, 65, 4456−4463