Wightianines A−E, Dihydro-β-agarofuran Sesquiterpenes fromParnassia wightiana, and Their Antifungal and Insecticidal ActivitiesDong-Mei Wang,†,∥ Cheng-Chen Zhang,†,∥ Qiang Zhang,† Nusrat Shafiq,‡ Gennaro Pescitelli,‡

Deng-Wu Li,† and Jin-Ming Gao*,§

†College of Forestry, and §Shaanxi Engineering Center of Bioresource Chemistry and Sustainable Utilization, College of Science,Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China‡Dipartimento di Chimica e Chimica Industriale, Universita di Pisa, Via Risorgimento 35, 56126 Pisa, Italy

*S Supporting Information

ABSTRACT: Five new sesquiterpene polyol esters with a dihydro-β-agarofuran skeleton, designated as wightianines A−E (1−5), besides two known compounds, were isolated from the methanolic extract of the whole plant of the traditional herbalmedicine Parnassia wightiana Wall. The structures of the isolated compounds were elucidated on the basis of spectroscopicanalyses, including two-dimensional nuclear magnetic resonance techniques (correlation spectroscopy, heteronuclear multiple-quantum coherence, nuclear Overhauser effect spectrometry, and heteronuclear multiple-bond correlation) and electroniccircular dichroism studies. The antifungal and insecticidal activities of five compounds were evaluated against several plantpathogenic fungi and armyworm larvae (Mythimna separata Walker). Among the test metabolites, compounds 2 and 7 bothexhibited potent antifungal activity against the phytopathogenic fungus Cytospora sp. with minimum inhibitory concentrationvalues of 0.78 μg/mL, which are equal to the two positive controls, hymexazol and carbendazim. However, no insecticidal activityof the test compounds was observed in the present study. Compounds 2 and 7 could be promising leads for developing newfungicides against agriculturally important fungus Cytospora sp.

KEYWORDS: natural products, structural elucidation, antifungal activity, absolute configuration, TDDFT CD calculations

■ INTRODUCTIONThe genus Parnassia (family Saxifragaceae) comprises about 50species in the north temperate zones. Among the 36 speciesfound in China, 4 of them grow in the Qinling Mountains.Parnassiae wightiana W. (“cang er qi” in Chinese) is aherbaceous perennial and evergreen species distributed mainlyin Guangdong, Yunnan, Shaanxi, Gansu, and Fujian provincesof China.1 The dried whole herb of P. wightiana is used as atraditional medicine for the treatment of cough, hemoptysis,traumatic injury, abnormal leukorrhea, eczema, sores, andcarbuncles.1 Previous studies have shown that the genusParnassia is rich in flavonoids,2,3 and little is known aboutstudies of chemical constituents of this plant.As part of our ongoing search for new bioactive natural

products from the traditional Chinese herbal medicines in theQinba Mountains,4−7 chemical studies of the whole plant of P.wightiana was carried out, leading to the discovery of five newdihydro-β-agarofuran-type sesquiterpene polyol esters, namely,wightianines A−E (1−5), together with two known congeners(6 and 7). Their antifungal and insecticidal activities of somecompounds were examined. Herein, we report the isolation andstructure elucidation of these compounds, including theirabsolute configuration as well as their antifungal and insecticidalactivities of some compounds.

■ MATERIALS AND METHODSGeneral Experimental Procedures. Optical rotations were

measured on a Rudolph Autopol III automatic polarimeter. Circulardichroism (CD) spectra were run on a JASCO J-715 spectropolarim-eter. Infrared (IR) spectra were recorded on a Bruker Tensor 27 FT/

IR spectrophotometer, and ultraviolet (UV) spectra were obtained ona UV−vis Evolution 300 spectrometer. Nuclear magnetic resonance(NMR) spectra were acquired on Bruker AVANCE III 400 or 500MHz UltraShield-Plus digital NMR spectrometer. Electrosprayionization−mass spectrometry (ESI−MS) was recorded on a ThermoFisher LTQ Fleet Thermo-Finnigan system equipped with a hot ESIsource, and high-resolution (HR)-ESI−MS was taken on an Agilent6520 accurate mass quadrupole time-of-flight (Q-TOF) liquidchromatography/mass spectrometry (LC/MS) spectrometer. Thin-layer chromatography (TLC) was performed on precoated silica gel 60F254 plates (0.25 mm thick, Qingdao Marine Chemical Plant, Qingdao,China). Preparative high-performance liquid chromatography(HPLC): Shimadzu SPD-10Avp, FRC-10Avp, SCL-10Avp, LC-8Asystem, UV detector, Agela Vennsil XBP-C18 column (50 × 250 mm,10 μm). Preparative HPLC: Gilson-281 system, UV detector,Shimadzu C18 column (21.2 × 250 mm, 15 μm). PreparativeHPLC: Gilson-215 system, UV detector, Agela Durashell C18 column(21.2 × 50 mm, 5 μm). Preparative HPLC: Waters 2545 system, UVdetector, Waters Xbridge Prep C18 column (30 × 150 mm, 5 μm).

Plant Material. The whole herbs of P. wightiana W. were collectedin the Qinling Mountains, at 2000−2150 m, from 33° 25′ to 33° 29′ Nand from 108° 25′ to 108° 30′ E, in September 2010, Shaanxiprovince, China. The plant material was identified by the authors andhas been deposited at the College of Forestry, Northwest A&FUniversity, China, under the acquisition number 10-H-16.

Extraction, Isolation, and Purification. The air-dried powder of theherbal material (1.5 kg) was extracted 5 times with 95% EtOH at room

Received: April 14, 2014Revised: June 5, 2014Accepted: June 19, 2014Published: June 19, 2014

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temperature (each for 12 h). The extracts were combined andevaporated in vacuum. The resulting crude extract (9.52 g) wassequentially dispersed in water and extracted by petroleum ether (PE),EtOAc, and n-BuOH. The EtOAc extract (9.5 g) was dissolved inMeOH/tetrahydrofuran (THF) (50:50) and subjected to preparativeHPLC on a column (50 × 250 mm, 10 μm) of 10 μm Agela VennsilXBP C18, at a flow rate of 100 mL/min and detection wavelength of214/254 nm, using a CH3CN−10 mM NH4HCO3 gradient as theeluant (0−15 min, 45−85% CH3CN; 15−20 min, 95% CH3CN; 20−24 min, 45% CH3CN) to give six fractions (fractions 1−6). Fraction 4eluting at 12.0−15.0 min was collected and further separated byGilson-281 preparative HPLC on a Shimadzu C18 (21.2 × 250 mm, 15μm) column, at a flow rate of 30 mL/min and detection wavelength of214/254 nm, using a CH3CN−0.1% HCO2H−H2O gradient as theeluant (0−20 min, 54−64% CH3CN; 20−25 min, 95% CH3CN) toafford six subfractions. Each subfraction was further purified repeatedlyon a Gilson-215 preparative HPLC on an Agela Durashell C18 (21.2 ×50 mm, 5 μm) column, at a flow rate of 20 mL/min and detectionwavelength of 214/254 nm, using a CH3CN−10 mM NH4HCO3−H2O gradient as the eluant (0−8 min, 65% CH3CN; 8−11 min, 95%CH3CN) to yield pure compounds 6 (3.8 mg), 3 (3.7 mg), 4 (110mg), and 2 (10 mg). The PE extract (36.4 g) was chromatographed onGilson-281 preparative HPLC on a Shimadzu C18 column (21.2 × 250mm, 15 μm), eluting with a mixture of CH3CN−0.1% FA−watergradient (0−16 min, 45−75% CH3CN; 16−21 min, 95% CH3CN;21−25 min, 45% CH3CN) to afford four fractions (CP-1−CP-4). CP-2 and CP-3 were further purified by the same chromatographicconditions to yield compounds 1 (29.6 mg), 5 (1.5 mg), and 7 (1.5mg).Wightianine A (1). Amorphous white powder; [α]D

28.6 +17.3 (c 0.33,MeOH); UV (MeOH) λmax (log ε), 201 (4.62), 234 (4.20), 263(4.37), 300 (3.72) nm; CD λ (MeOH) nm (Δε), 220 (+4.65), 247(+6.85), 236 (−2.14), 280 (−4.27); IR (KBr) ν, 3506, 1718, 1602,1452, 1387, 1276, 1107, 1025 cm−1; 1H and 13C NMR (CDCl3), seeTables 1 and 2, respectively; HRMS (ESI) m/z calcd for [C31H36O8 +Na]+, 559.2302 [M + Na]+; found, 559.2321.

Wightianine B (2). Amorphous white powder; [α]D25.2 +42.4° (c

0.69, CHCl3); UV (MeOH) λmax (log ε), 204 (4.28), 232 (4.42), 275(3.30), 282 (3.22) nm; IR (KBr) νmax, 3448, 1718, 1276, 1108, 1027cm−1; 1H and 13C NMR (CDCl3), see Tables 1 and 2, respectively;MS (ESI) m/z, 559.79 [M + Na]+; HRMS (ESI) m/z calcd for[C31H36O8 + Na]+, 559.2302 [M + Na]+; found, 559.2305.

Wightianine C (3). Amorphous white powder; UV (MeOH) λmax(log ε), 212 (4.62), 232 (4.20), 263 (4.37), 289 (3.72) nm; IR (KBr)νmax, 3438, 1725, 1108, 1021 cm−1; 1H and 13C NMR (CDCl3), seeTables 1 and 2, respectively; MS (ESI) m/z, 575.31 [M + Na]+;HRMS (ESI) m/z calcd for [C31H36O9 + Na]+, 575.2252 [M + Na]+;found, 575.2242.

Wightianine D (4). Amorphous white powder; [α]D15.4 +0.86 (c 0.21,

acetone); UV (MeOH) λmax (log ε), 210 (4.62), 234 (4.21), 266(4.37); CD λ (MeOH) (Δε), 249 (+12.0), 291 (−29.7); IR (KBr) ν,3471, 1753, 1725, 1601, 1452, 1370, 1315, 1272, 1231, 1178, 1107cm−1; 1H and 13C NMR (CDCl3), see Tables 1 and 2, respectively;HRMS (ESI) m/z calcd for [C31H34O9 + Na]+, 573.2095 [M + Na]+;found, 573.2115.

Wightianine E (5). Amorphous white powder; IR (KBr) νmax, 3435,1744, 1718, 1109 cm−1; 1H and 13C NMR (CDCl3), see Tables 1 and2, respectively; MS (ESI) m/z, 557.82 [M + Na]+; HRMS (ESI) m/zcalcd for [C31H34O8 + Na]+, 557.2146 [M + Na]+; found, 557.2154.

Compound 6. Amorphous white powder; 1H and 13C NMR were inagreement with published values;8 MS (ESI) m/z, 649.2 [M + H]+;HRMS (ESI) m/z calcd for [C32H40O14 + Na]+, 671.2211 [M + Na]+;found, 671.2215.

Compound 7 (Ejap-4). Amorphous white powder; 1H and 13CNMR were in agreement with published values;9 MS (ESI) m/z, 633.3[M + H]+; HRMS (ESI) m/z calcd for [C32H41O13 + H]+, 633.3119[M + H]+; found, 633.3114.

Computation. Merck molecular force field (MMFF) and densityfunctional theory (DFT) calculations were run with Spartan’10 (WaveFunction, Inc., Irvine, CA), with standard parameters and convergencecriteria. Time-dependent density functional theory (TDDFT)calculations were run with Gaussian’09,10 with default grids andconvergence criteria. Conformational searches were run with the

Table 1. 1H NMR Spectroscopic Data (400 MHz, CDCl3) of Compounds 1−5a

1 2 3 4 5

position δH mult (J in Hz) δH mult (J in Hz) δH mult (J in Hz) δH mult (J in Hz) δH mult (J in Hz)

1 4.61 d (3.6) 5.57 d (4.0) 5.35 d (3.5) 5.98 s 6.01 s2 5.34 m 4.47 m 4.57 m3 2.39 m 2.40−2.44 m 2.40−2.44 m 3.40 dd (12.8, 7.4) 3.42 dd (12.8, 7.4)

1.87 dd (15.1, 1.4) 1.90 m 1.92 m 2.31 dd (12.8, 1.4) 2.28 dd (12.8, 1.4)4 2.51 m 2.49−2.60 m 2.52 m 3.02 m 3.02 m6 5.66 s 5.67 s 6.26 s 6.24 s 5.62 s7 2.39 m 2.40−2.44 m 2.64 d (3.4) 2.69 d (3.2) 2.49 t (2.9)8 2.61 ddd (16.1, 7.0, 3.3) 2.49−2.60 m 4.45 m 2.57 ddd (16.3, 7.3, 3.4)

2.28 dd (16.4, 3.1) 2.23 dd (16.4, 2.9) 4.45 t (3.2) 2.35 dd (16.3, 2.9)9 5.08 d (6.8) 5.04 d (6.8) 4.97 s 4.93 s 5.07 d (6.8)12 1.21 d (7.6) 1.31 d (7.6) 1.23 d (7.6) 1.41 d (7.5) 1.01 d (7.5)13 1.42 s 1.60 s 1.57 s 1.46 s 1.34 s14 1.53 s 1.45 s 1.43 s 1.48 s 1.54 s15 1.49 s 1.44 s 1.49 s 1.58 s 1.54 sAc 2.05 s 1.73 s 2.06 s 1.72 s 1.73 s6-Bz2′/6′ 8.06 d (7.5) 8.07 d (7.5) 8.06 d (7.5) 8.02 d (7.5) 8.06 dd (7.5, 2.5)3′/5′ 7.47 t (7.5) 7.45 t (7.5) 7.49 t (7.5) 7.42 t (7.5) 7.45 t (7.5)4′ 7.58 t (7.5) 7.57 t (7.5) 7.59 t (7.5) 7.60 t (7.5) 7.57 t (7.5)9-Bz2′/6′ 8.09 d (7.5) 8.09 d (7.5) 8.08 d (7.5) 8.06 d (7.5) 8.04 dd (7.5, 2.5)3′/5′ 7.50 t (7.5) 7.49 t (7.5) 7.50 t (7.5) 7.50 t (7.5) 7.50 t (7.5)4′ 7.62 t (7.5) 7.61 t (7.5) 7.63 t (7.5) 7.63 t (7.5) 7.63 t (7.5)

aAssignments were performed by one-dimensional (1D) and two-dimensional (2D) [correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC)] NMR experiments.

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Monte Carlo algorithm implemented in Spartan’10 using MMFF. Allstructures thus obtained were optimized with the DFT method usingB3LYP functional and 6-31G(d) basis set.10 The most stable B3LYP/6-31G(d) structures within 2 kcal/mol were re-optimized usingB3LYP functional and 6-311+G(d,p) basis set.10

Preliminary TDDFT calculations were run using B3LYP and CAM-B3LYP functionals and Ahlrichs’ SVP and TZVP basis sets.11 TheB3LYP/SVP combination was then employed for the final calculations,including 60 excited states (roots). CD spectra were generated usingSpecDis12 by applying a Gaussian band shape with 0.3 eV exponentialhalf-width, from dipole-length rotational strengths. The difference withdipole velocity values was checked to be minimal for all relevanttransitions.Antifungal Bioassay. The test phytopathogenic fungi used in this

study were Fusarium solani, Fusarium oxysporum f. sp. vasinfectum,Cytospora sp., and Fusarium graminearum. All of the fungi were isolatedfrom infected plant organs at the Northwest A&F University.Antifungal activity was examined by the microbroth dilution

method in 96-well culture plates using a potato dextrose (PD)medium.13,14 The test compounds was made up to 2 mg/mL indimethyl sulfoxide (DMSO). Two commercial fungicides, carbenda-zim and hymexazol (Aladdin Chemistry Co., Ltd.), were used as apositive control, and the solution of equal concentration of DMSO wasused as a negative control. The tested fungi were incubated in the PDmedium for 18 h at 28 ± 0.5 °C at 150 rpm, and the spores of differentmicroorganism concentrations were diluted to approximately 1 × 106

colony forming units (CFU)/mL with PD medium. Test compounds(10 μL) were added to 96-well microplates, and 90 μL of PD mediumwas added. Serial dilutions were made in the 96-well round-bottomsterile plates in triplicate in 50 μL of PD medium, and then 50 μL of

the fungal suspension was added. After incubation for 48 h at 28 ± 0.5°C, minimum inhibitory concentration (MIC) was taken as the lowestconcentration of the test compounds in the wells of the 96-well plate,in which no microbial growth could be observed.

Insecticidal Assay. An insecticidal assay was performed by themethod previously reported,15 in which Mythimna separata was used asthe test target. All samples for the insecticidal test were dissolved inacetone.

■ RESULTS AND DISCUSSION

Structural Elucidation. A 95% ethanolic extract of thedried whole plant of P. wightiana was successively partitionedbetween PE, EtOAc, n-BuOH, and water. The PE- and EtOAc-soluble extracts were separated and purified repeatedly bypreparative reversed-phase HPLC to afford five newsesquiterpene compounds, wightianines A−E (1−5), alongwith two known compounds (6 and 7) (Scheme 1). Structuralelucidation of the compounds was achieved by spectroscopicanalysis and comparison to closely related compounds.Compound 1 was obtained as an amorphous white powder

with the molecular formula C31H36O8, which was deduced fromthe HR-ESI−MS molecular ion at m/z 559.2321 ([M + Na]+,calcd 559.2302). The IR spectrum shows the presence ofhydroxyl (3506 cm−1) and carbonyl (1718 cm−1) groups, andthe UV spectrum reveals the presence of an aromatic moiety(234 and 263 nm). The 1H and 13C NMR spectroscopic data ofcompound 1 (Tables 1 and 2) suggest that it belongs to a class

Table 2. 13C NMR Spectroscopic Data (100 MHz, CDCl3) of Compounds 1−5a

1 2 3 4 5

position δC δC δC δC δC

1 68.9, CH 74.1, CH 68.8, CH 77.6, CH 77.5, CH2 73.8, CH 69.1, CH 73.8, CH 204.5, C 204.9, C3 31.2, CH2 32.7, CH2 31.3, CH2 44.0, CH2 43.9, CH2

4 34.1, CH 34.3, CH 33.9, CH 38.8, CH 38.9, CH5 90.0, C 90.3, C 90.7, C 89.8, C 89.0, C6 80.2, CH 80.0, CH 76.3, CH 76.0, CH 80.0, CH7 49.1, CH 49.0, CH 55.7, CH 55.7, CH 49.0, CH8 31.6, CH2 31.8, CH2 74.8, CH2 74.7, CH2 32.5, CH2

9 73.3, CH 73.5, CH 80.4, CH 80.6, CH 73.3, CH10 51.5, C 50.1, C 50.7, C 54.7, C 55.9, C11 82.8, C 82.7, C 81.7, C 82.6, C 83.7, C12 18.8, CH3 19.2, CH3 18.7, CH3 18.1, CH3 18.2, CH3

13 19.2, CH3 21.4, CH3 19.5, CH3 20.7, CH3 20.0, CH3

14 26.1, CH3 26.1, CH3 25.7, CH3 25.6, CH3 26.0, CH3

15 30.9, CH3 30.6, CH3 31.3, CH3 31.2, CH3 30.7, CH3

AcCO 171.5, C 169.8, C 171.5, C 169.4, C 169.4, CCH3 21.4, CH3 20.7, CH3 21.5, CH3 20.0, CH3 20.0, CH3

6-BzCO 165.6, C 165.7, C 165.7, C 165.4, C 165.6, C1′ 129.7, C 130.1, C 130.0, C 129.7, C 129.6, C2′/6′ 129.6, CH 129.7, CH 129.7, CH 129.6, CH 129.5, CH3′/5′ 128.5, CH 128.6, CH 128.7, CH 128.8, CH 128.5, CH4′ 133.4, CH 133.3, CH 133.3, CH 133.6, CH 133.5, CH9-BzCO 165.7, C 165.7, C 165.7, C 165.4, C 165.7, C1′ 128.7, C 130.1, C 130.0, C 128.7, C 129.2, C2′/6′ 129.6, CH 129.7, CH 129.7, CH 129.9, CH 129.8, CH3′/5′ 128.7, CH 128.6, CH 128.7, CH 128.6, CH 128.8, CH4′ 133.3, CH 133.3, CH 133.3, CH 133.8, CH 133.6, CH

aAssignments were made by a combination of 1D and 2D (COSY, HSQC, and HMBC) NMR experiments.

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of sesquiterpene polyol esters with a dihydro-β-agarofuranskeleton found in the Celastraceae plants.16,17 The 1H NMRspectrum (Table 1) shows the presence of one acetyl group (δH2.05), two benzoate groups between δH 7.47 and 8.09, fourmethyl groups, including three tertiary methyl groups (δH 1.49,1.53, and 1.42) and a secondary methyl group [δH 1.21 (d, J =7.6 Hz)], two sets of methylene protons [δH 2.39 (1H, m), 1.87(1H, dd, J = 15.1 and 1.4 Hz); 2.61 (1H, ddd, J = 16.1, 7.0, and3.3 Hz), 2.28 (1H, dd, J = 16.4 and 3.1 Hz)], and six methineprotons (δH 5.66, 4.61, 5.08, 5.34, 2.61, and 2.39), includingone bearing an acetoxy group at δH 5.34 (m, H-2). The 13CNMR and distortionless enhancement by polarization transfer(DEPT) spectra (Table 2) in combination with HSQC dataindicate that compound 1 has an agarofuran skeleton16 with 15carbons, including four methyl carbons at δC 18.8 ppm (C-12),19.2 (C-13), 26.1 (C-14), and 30.9 (C-15), two methylenecarbons at δ 31.2 (C-3) and 31.6 (C-8), six methine carbons atδC 68.9 (C-1), 73.8 (C-2), 34.1 (C-4), 80.2 (C-6), 49.1 (C-7),and 73.3 (C-9), and three quaternary carbons at δC 90.0 (C-5),51.5 (C-10), and 82.8 (C-11). The 13C NMR quaternarysignals at δ 90.0 (C-5) and 82.8 (C-11) are characteristic ofdihydro-β-agarofuran.8 All of these data indicate thatcompound 1 is a polyester sesquiterpene with a 1,2,6,9-tetrasubstituted dihydro-β-agarofuran skeleton.The substitution pattern of compound 1 was established by a

HMBC experiment (Figure 1). Correlations were found of H-1(δH 4.61) with C-2, C-10, and C-13, H-2 (δH 5.34) with thecarbonyl signal (δC 171.5) of the acetate group, and H-6 (δH5.66) and H-9 (δH 5.08) with the carbonyls (δC 165.6 and165.7) of the two benzoate moieties, respectively. The relativestereochemistry was established on the basis of the couplingconstants and confirmed by a nuclear Overhauser effectspectrometry (NOESY) experiment. In this class of dihydro-β-agarofuran sesquiterpenes, the stereochemistry of the ringjunctions is generally trans and H-6 is axial,18−20 which iscorroborated by the NOESY cross-peaks observed between H-1(δH 4.61) and H-13 (δH 1.42) and between H-13 and H-9(Figure 2). Furthermore, in the COSY experiment, the vicinalcoupling constant of H-1/H-2 (J1,2 = 3.6 Hz) indicates a cisrelationship between H-1 and H-2. This evidence proves thatthe relative configuration of the substituents is 1β, 2β, 6β, and9β.Because compounds 1−5 contain two benzoate moieties

each, their absolute configuration could be in principle assigned

using the dibenzoate chirality method, the most commonextension of the electronic CD exciton chirality protocol.21 Theexciton-coupled CD (ECCD) protocol is based on the sign ofthe bisignate CD bands (exciton couplet) observed in the majorabsorption region of the benzoates (π−π*1La transition around235 nm). The sign of the couplet is generally related to theabsolute sense of twist between the electric transition dipolemoments, which are oriented respectively along the long axes ofthe benzoate chromophores (C-1′/C-4′ direction). In com-pounds 1−5, however, the dibenzoate 1,4-arrangement makestheir π−π* transition dipoles oriented almost parallel to eachother. For exactly parallel arrangement, the predicted excitoncoupling is zero. For slight deviations from the parallel, ECCDcouplets are usually weak and difficult to predict.21,22 Therefore,to assign the absolute configuration of compounds 1 and 4, wepreferred to apply quantum mechanic full CD calculations. Thistreatment, described in the next section, allowed us to establishcompound 1 as (1S,2R,4R,5S,6R,7R,9S,10S)-2β-acetoxy-6β,9β-dibenzoyloxy-1β-hydroxydihydro-β-agarofuran and namedwightianine A.Compound 2 was also obtained as an amorphous white

powder with the same molecular formula C31H36O8 ascompound 1. The HR-ESI−MS molecular ion is at m/z559.2305 [M + Na]+ (calcd for C31H36NaO8, 559.2302). Its IR

Scheme 1. Structures of Compounds 1−7

Figure 1. Key COSY and HMBC correlations of compounds 1−4.

Figure 2. Key nuclear Overhauser effects (NOEs) of compounds 1−4.

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spectrum shows absorption bands for hydroxyl (3448 cm−1)and ester carbonyl (1718 cm−1) groups, and the UV spectrumreveals the presence of an aromatic moiety (232 and 275 nm).The 1H NMR spectrum (Table 1) shows the presence of one

acetyl group (δH 1.73), two benzoate groups between δH 7.45and 8.09, four methyl groups, including three tertiary methylgroups (δH 1.44, 1.45, and 1.60) and a secondary methyl group[δH 1.31 (d, J = 7.6 Hz)], two sets of methylene protons [δH2.40−2.44 (1H, m), 1.90 (1H, m); 2.49−2.60 (1H, m), 2.23(1H, dd, J = 16.4 and 2.9 Hz)], and six methine protons (δH5.67, 5.57, 5.04, 4.47, 2.49−2.60, and 2.40−2.44), including onebearing an acetoxy group at δH 4.47 (m, H-2). The 1H and 13CNMR spectra of compound 2 (Tables 1 and 2) closelyresemble those of compound 1, suggesting that compound 2should be an isomer of compound 1. A comparison of thesimilar 1H and 13C NMR spectra of compounds 1 and 2 revealsthe presence of an acetyl group at C-1 (δC 74.1) in compound2 because of the shifts of the signals of the H-1 proton from δH4.61 in compound 1 to δH 5.57 in compound 2. Thus, it isconcluded that compound 2 was a 1-acetyl-2-deacetyl derivativeof compound 1. This is further confirmed by the HMBCcorrelations of H-1 (δH 5.57) with C-10 (δC 50.1), C-13 (δC21.4), and the carbonyl signal (δC 169.8) of the acetate group(Figure 1). Unambigous assignments of the 1H and 13C NMRdata and the relative configuration are deduced from the COSY,HMBC, and NOESY experiments (Figures 1 and 2). Itsabsolute configuration can be assigned from the biosyntheticpoint of view. Therefore, compound 2 is identified as(1S,2R,4R,5S,6R,7R,9S,10R)-1β-acetoxy-6β,9β-dibenzoyloxy-2β-hydroxydihydro-β-agarofuran and named wightianine B.Compound 3 was isolated as an amorphous white powder

with the molecular formula C31H36O9, as evidenced by the HR-ESI−MS molecular ion at m/z 575.2242 ([M + Na]+, calcd575.2252). The molecular formula indicates that compound 3contains one more oxygen atom than compound 2. The IRspectrum shows the presence of hydroxyl (3438 cm−1) andcarbonyl (1725 cm−1) groups, while the NMR spectra showone acetyl group (δH 2.06, s; δC 171.5, s, and 21.5, q) and twobenzoyl groups (δH 7.49−8.08; δC 165.7, s, and 128.7−133.3).Further comparison of the 1H and 13C NMR spectroscopic dataof compound 3 (Tables 1 and 2) to those of compound 2reveals that compound 3 contains one less methylene groupand one more methine bearing a hydroxyl group. The positionof the hydroxyl group was determined to be at C-8 (δC 74.8) bythe evidence of downfield shifts of the carbon signals from δC49.0 (C-7) and δC 73.5 (C-9) in compound 2 to δC 55.7 (C-7)and δC 80.4 (C-9) in compound 3, respectively. Thisconclusion is further supported by the HMBC correlations ofH-6, H-7, and H-9 with C-8 (Figure 1). Analysis of thecoupling constants for H-1/H-2 (J1/2 = 3.5 Hz) and H-7/H-8(J7/8 = 3.4 Hz) reveals their cis configuration. Therefore, thehydroxyl and ester groups are located on the β face, asconfirmed by the NOESY correlations shown in Figure 2. Thelocations and relative configurations of the esters and hydroxylgroups in compound 3 were deduced from analysis of itsCOSY, HMBC, and NOESY spectra (Figures 1 and 2).Similarly, its absolute configuration was assigned from thebiosynthetic point of view. Thus, compound 3 is identified as(1S,2R,4R,5S,6R,7R,8S,9R,10S)-2β-acetoxy-6β,9β-dibenzoy-loxy-1β,8β-dihydroxydihydro-β-agarofuran and named wight-ianine C.Compound 4 gives the molecular formula C31H34O9 based

on the HR-ESI−MS molecular ion at m/z 573.2115 [M + Na]+

(calcd for C31H34NaO9, 573.2095), indicating that compound 4contains two less hydrogens than compound 3. The IRspectrum shows the presence of hydroxyl (3471 cm−1) andcarbonyl (1753 and 1725 cm−1) groups. The NMR spectraprove the presence of one acetyl group (δH 1.72, s; δC 169.4, s,and 20.0, q) and two benzoyl groups (δH 7.42−8.06; δC 165.4and 128.6−133.8). Further comparison of the NMR data ofcompound 4 (Tables 1 and 2) to those of compound 3 revealsthat they are very similar, except for the presence of one ketoniccarbonyl at δC 204.5 (s, C-2) in compound 4, instead of onehydroxyl methine [δC 73.8 (d, C-2); δH 4.57 (m)] incompound 3. The position of the ketone group was determinedto be at C-2, as evidenced by the downfield shifts of the carbonsignals from δC 68.8 (C-1), 31.3 (C-3), and 33.9 (C-4) incompound 3 to δC 77.6 (C-1), 44.0 (C-3), and 38.8 (C-4) incompound 4, respectively. This is further supported by theHMBC correlations of H-1, H-3, and H-4 with C-2 (δC 204.5)(Figure 1). The coupling constant (J7,8 = 3.2 Hz) for H-7 andH-8 indicates their cis relationship. This suggests that thehydroxyl group at C-8 is β-orientated. The locations andrelative configurations of the ester groups in compound 4 werefurther assigned from its COSY, HMBC, and NOESY spectra(Figures 1 and 2). In addition, the absolute configuration ofcompound 4 was determined by quantum mechanic CDcalculations (see the next section). Therefore, compound 4 isidentified as (1S,4R,5S,6R,7R,8S,9R,10S)-1β-acetoxy-6β,9β-di-benzoyloxy- 8β-hydroxy-2-oxo-dihydro-β-agarofuran andnamed wightianine D.Compound 5 gives the molecular formula C31H34O8, as

evidenced by the HR-ESI−MS molecular ion at m/z 557.2154([M + Na]+, calcd 557.2146). The molecular formula indicatesthat compound 5 contains one less oxygen than compound 4.Further comparison of the NMR data of compound 5 (Tables 1and 2) to those of compound 4 reveals that they are verysimilar, except for the presence of a methylene [δC 32.5 (t, C-8); δH 2.57 (ddd, J = 16.3, 7.3, and 3.4 Hz, H-8α), 2.35 (dd, J =16.3 and 2.9 Hz, H-8β)] in compound 5, instead of onehydroxyl methine [δC 74.7 (d, C-8); δH 4.45 (t, J = 3.2 Hz)] incompound 4. The locations and relative configurations of theesters in compound 5 were assigned on the basis of its HMBCand NOESY spectra (Figures 1 and 2). From the prospective ofbiosynthesis, its absolute configuration can be proposed. Thus,the st ructure of compound 5 i s e luc idated as(1S,4R,5S,6R,7R,9S,10R)-1β-acetoxy-6β,9β-dibenzoyloxy-2-oxo-dihydro-β-agarofuran and named wightianine E.When their physical and spectral data were compared to

those reported in the literature, two known dihydro-β-agarofurans 6 and 7 were identified as 1α,2α,6β,8β,13-pentaacetoxy-9β-benzoyloxy-4β-hydroxy-β-dihydroagarofuran,8

isolated from Celastrus angulatus and Ejap-4 from Euonymusjaponicus,9 respectively.To date, 462 dihydro-β-agarofuran sesquiterpenes have been

reported from the Celastraceae plants.23 These metaboliteshave usually been described as the primary characteristicconstituents of the family. Interestingly, 2-oxo-dihydro-β-agarofuran-type sesquiterpenes are very rare natural products,as examplied by triptogelin A-4, which had been describedpreviously in Tripterygium wilfordii var. Regelii.24 To ourknowledge, the present study is the second report of thesesquiterpenoids of this class from the alternative familySaxifragaceae. Importantly, the sesquiterpenes with a dihydro-β-agarofuran scaffold as “privileged structures” have attractedconsiderable attention from synthetic chemists and pharmacol-

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ogists because of their complex structures and biologicalactivities, such as immunosuppressive,25,26 cytotoxic,17,18 anti-HIV,25,27 antiherpetic,28 reversing multi-drug-resistance(MDR) phenotype,29,30 antitumor,31,32 and insecticidal8

activities.Conformational Analysis and Absolute Configuration

Assignment. Compounds 1 and 4 were selected asrepresentatives of the two classes of wightianines (with 2-hydroxy/2-acetoxy and 2-oxo substituents, respectively) toassign their absolute configuration by quantum mechanic CDcalculations.33 First, the conformation of compounds 1 and 4was studied by means of a well-established protocol based on apreliminary conformational analysis based on molecularmechanics, followed by geometry optimizations with DFT.22

Starting from a structure of compounds 1 and 4 with arbitrary(1S,2R,4R,5S,6R,7R,9S,10S)-1 and (1S,4R,5S,6R,7R,8S,9R,10S)-4 configurations, respectively, the conformational analysis wasrun using a Monte Carlo algorithm and the MMFF. All MMFFminima within 10 kcal/mol were optimized with DFT at theB3LYP/6-31G(d) level. Finally, the relevant DFT minima(within 2 kcal/mol) were re-optimized at the B3LYP/6-311G+(d,p) level. For compound 1, four low-energy conformerswere obtained with relative internal energies within 2 kcal/mol;they differed in the conformation of 9-benzoate and 2-acetategroups (Figure 3). For compound 4, only two low-energy

conformations were obtained, differing in the conformation ofthe 6-benzoate group (Figure 4). Both of them have thecyclohexanone ring in a twist conformation, probably to relievethe steric hindrance between 1β-acetate and 9β-benzoategroups, with the latter hydrogen-bonding to 8-OH. Thisconformation is confirmed by the strong NOE between H-1αand H-3α (see Figure 2).The CD spectra of compounds (+)-1 and (+)-4 were

recorded in methanol (Figures 5 and 6). They show several

bands between 200 and 350 nm, because of the benzoate and(for compound 4) carbonyl chromophore. In both cases, thespectra are quite weak and there is no clear-cut exciton coupletwith crossover (zero value) around 235 nm, where thebenzoate π−π* 1La transition occurs. This demonstrates thatthe ECCD protocol (see the previous section) cannot beapplied directly.21,22 Rather, we calculated the CD spectra ofcompounds 1 and 4 with TDDFT33 using DFT-optimizingstructures as input geometries and averaging the calculatedspectra for the various low-energy conformations with therespective Boltzmann factors at 300 K (using internal energies).The calculated averages with B3LYP/SVP, shown in Figures 5and 6, reproduce well the experimental CD profiles. Therefore,the absolute configurations of compounds 1 and 4 areestablished as (+)-(1S,2R,4R,5S,6R,7R,9S,10S)-1 and(+)-(1S,4R,5S,6R,7R,8S,9R,10S)-4.

Biological Activity. Five compounds selected (because of alimited quantity of samples) were examined for their antifungalactivity toward phytopathogenic fungi (F. solani, F. oxysporum f.sp. vasinfectum, Cytospora sp., and F. graminearum) in vitro usinga mycelial growth inhibitory rate method.13,14 As shown in

Figure 3. DFT-optimized [B3LYP/6-31G(d) level] low-energystructures for (+)-(1S,2R,4R,5S,6R,7R,9S,10S)-1 and relative internalenergies.

Figure 4. DFT-optimized [B3LYP/6-31G(d) level] low-energystructures for (+)-(1S,4R,5S,6R,7R,8S,9R,10S)-4 and relative internalenergies.

Figure 5. CD spectra of (+)-(1S,2R,4R,5S,6R,7R,9S,10S)-1 recorded inmethanol (1.9 mM, 0.1 cm cell, solid line) and calculated withTDDFT (B3LYP/SVP level, dotted line) as the Boltzmann average at300 K for the four structures shown in Figure 3. The calculatedspectrum was obtained as the sum of Gaussians with 0.3 eVbandwidth, and it is scaled and red-shifted by 30 nm for comparison.

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Table 3, of these isolated compounds tested, only compounds 2and 7 both inhibited mycelial growth of Cytospora sp. withminimum inhibitory concentration (MIC) values of 0.78 μg/mL, which are the same as the two positive controls hymexazoland carbendazim, demonstrating that these compounds weregood fungicides. Additionally, compound 2 also showedmarked antifungal effects on F. graminearum and F. oxysporumwith their respective MIC data of 12.5 and 50 μg/mL. Incontrast, the other compounds were almost inactive at 100 μg/mL of MIC data. A preliminary structure−antifungal activityrelationship of these test compounds is given as follows. Whena hydroxyl group attached to C-2 instead of an acetoxy group atC-2 of compound 1, as seen in compound 2, the antifungalactivity against F. graminearum and Cytospora sp. was largelyincreased, as compared to that of compound 1. However,conversion of OH at C-2 to a ketone group can lead to a loss ofactivity (compound 1 versus compound 4). When theree is oneH at C-4 instead of a tertiary OH at C-4 of compound 6, asseen in compound 7, the activity against Cytospora sp. washighly increased, as compared to that of compound 6. Fromthis evidence (Table 3), it can be concluded that the presenceof the number, nature, and position of substituent groups ofthese test compounds could influence antifungal activity to thephytopathogens tested in this study.Insecticidal activity of compounds 1−7 was tested against the

fourth instar larvae of M. separata. However, in the presentstudy, none of these tested compounds was found to displayinsecticidal activity to M. separata.

In conclusion, seven sesquiterpene esters (1−7) wereisolated from the whole plant of P. wightiana, and theirstructures were elucidated by spectroscopic analyses andelectronic CD studies. Compounds 2 and 7 were both foundto display high antifungal property against the plant pathogenCytospora sp.

■ ASSOCIATED CONTENT

*S Supporting Information1D and 2D NMR and HR-ESI−MS spectra of compounds 1−5(Figures S1−S30). This material is available free of charge viathe Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Telephone: +86-29-87092515. E-mail: jinminggao@nwsuaf.edu.cn.

Author Contributions∥D.M.W. and C.C.Z. contributed equally to this work.

FundingThis work was financially supported by the Forestry ResearchFoundation for the Public Service Industry of China(200904004) and the Program for Youth Barebone Talentsof Northwest A&F University (Z111020902).

NotesThe authors declare no competing financial interest.

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Figure 6. CD spectra of (+)-(1S,4R,5S,6R,7R,8S,9R,10S)-4 recorded inmethanol (1.5 mM, 0.02 cm cell, solid line) and calculated withTDDFT (B3LYP/SVP level, dotted line) as the Boltzmann average at300 K for the two structures shown in Figure 4. The calculatedspectrum was obtained as the sum of Gaussians with 0.3 eVbandwidth, and it is scaled and red-shifted by 20 nm for comparison.

Table 3. Antifungal Data of Some Compounds against Four Plant Pathogens in Vitroa

compound F. solani CFCC86805 F. oxysporum ACCC30026 F. graminearum ACCC36249 Cytospora sp. ACCC30052

1 >100 100 >100 1002 >100 50 12.5 0.784 >100 100 >100 1006 >100 >100 >100 >1007 >100 >100 >100 0.78hymexazol 100 50 3.12 0.78carbendazim 0.78 100 1.57 0.78

aResults are expressed as MIC values in μg/mL.

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