Bioactive Terpenoids from the Fruits of - American Chemical Society

Jun 17, 2013 - Syngenta, Jealott,s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom. •S Supporting Information...
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Bioactive Terpenoids from the Fruits of Aphanamixis grandifolia Yao Zhang,† Jun-Song Wang,*,† Dan-Dan Wei,† Yu-Cheng Gu,‡ Xiao-Bing Wang,† and Ling-Yi Kong*,† †

State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China ‡ Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom S Supporting Information *

ABSTRACT: From the fruits of the tropical tree Aphanamixis grandifolia, five new evodulone-type limonoids, aphanalides I−M (1−5), one new apo-tirucallane-type triterpenoid, polystanin E (6), and three new chain-like diterpenoids, nemoralisins A−C (7−9), along with 12 known compounds were identified. The absolute configurations were determined by a combination of single-crystal X-ray diffraction studies, Mo2(OAc)4-induced electronic circular dichroism (ECD) data, the Mosher ester method, and calculated ECD data. The cytotoxicities of all the isolates and the insecticidal activities of the limonoids were evaluated.

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C26H33O7, 457.2232), indicative of 10 indices of hydrogen deficiency. The strong IR absorptions implied the presence of hydroxy (3466 cm−1), carbonyl (1729 cm−1), and olefinic (1683 cm−1) functionalities. The UV absorption at 230 nm featured an α,β-unsaturated ester carbonyl moiety. In accordance with its molecular formula, all 26 carbons were observed in the 13C NMR spectrum (Supporting Information, Table S1), which were further classified by DEPT and HSQC experiments as five methyl, two methylene, 12 methine (three oxygenated and five olefinic), and seven quaternary carbons (two carbonyls, one oxygenated, and one olefinic). These functionalities took five out of the 10 indices of hydrogen deficiency, and the remaining five indices suggested compound 1 to be pentacyclic. Its 1H NMR spectrum displayed signals for five tertiary methyl groups (δH 1.55, 1.37, 1.30, 1.23, and 0.66, each 3H, s), a β-substituted furan ring (δH 7.58, 7.58, and 6.58, each 1H, s), and an α,β-unsaturated ester carbonyl moiety [δH 6.75 (1H, d, J = 12.0 Hz) and 5.79 (1H, d, J = 12.0 Hz)] (Supporting Information, Table S2). The aforementioned data indicated that 1 was an evodulone-type limonoid,20 structurally similar to aphanagranin A.12 The key HMBC correlations from H-1 to C-3, C-5, and C10, from H-2 to C-3, from gem-dimethyl Me-28 and Me-29 to C-4, and from Me-19 to C-1 established an α,β-unsaturated seven-membered lactone in the A-ring. Three one-proton doublets at δH 5.22, 4.88, and 4.72 were ascribed to three hydroxy groups since no cross-peaks to any carbons were observed in the HSQC spectrum. Their positions at rings B and C were determined by the 2J HMBC correlations from the

eliaceous plants have attracted a broad range of interests from researchers in the fields of both organic chemistry1 and agrochemistry.2 Limonoids are characteristic components of the Meliaceae family and are well known for their insecticidal activity. The commercial application of limonoids in agriculture has experienced significant growth in recent decades.3 Aphanamixis grandifolia Bl. is a wild timber tree distributed mainly in the tropical and subtropical areas of South and Southeast Asia. The roots and leaves of this plant are utilized to relieve rheumatoid joint pain and numbness of limbs in some regions of China. Triterpenoids,4−6 limonoids,7−9 and other kinds of secondary metabolites10,11 have been isolated from its stem bark and seeds. Noteworthily, amoorastatin and aphanastatin, two limonoids possessing a 14,15-oxirane moiety, isolated from the seeds of the title plant, showed strong growth inhibitory effects against murine P388 cells with an ED50 value less than 0.065 μg/mL.7,8 Therefore, a study of the bioactive components from the fruits of A. grandifolia was undertaken. As a result, five new evodulone-type limonoids, aphanalides I−M (1−5), one new apo-tirucallane-type triterpenoid, polystanin E (6), and three new chain-like diterpenoids, nemoralisins A−C (7−9), along with 12 known compounds, aphanagranin A (10),12 aphanalide C (11),13 polystanin A (12),14 meliasenins S (13) and T (14),15 agladupols E (15) and D (16),16 polystanins C (17) and D (18),14 21α-methoxy-21,23-epoxy24α-hydroxytirucall-7-en-3-one (19),17 23,26-dihydroxytirucall7,24-dien-3-one (20),18 and nemoralisin (21),19 were isolated from the fruits of A. grandifolia. Herein, the details of the structural elucidation of these isolates and their selective bioactivities are presented. Aphanalide I (1) was obtained as colorless crystals (MeOH), and its molecular formula of C26H34O7 was established by the negative HRESIMS ion at m/z 457.2234 [M − H]− (calcd for © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 20, 2013

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Figure 1. X-ray crystal structure of aphanalide I (1).

Aphanalide J (2), a white, amorphous powder, showed an ion at m/z 493.3 [M + Cl]− in the negative ESIMS mode. The molecular formula was established as C26H34O7 by the HRESIMS ion at m/z 457.2229 [M − H]− (calcd for C26H33O7, 457.2232), the same molecular formula as that of 1. The 1H and 13C NMR spectra of 2 were similar to those of 1, with the exception of the D-ring. The one-proton doublet at δH 4.80 (J = 3.5 Hz) was assigned to 15-OH based on the same coupling constant as that of H-15 and its HMBC correlation with the oxymethine at δC 75.4 (C-15). The quaternary oxygenated carbon was assigned to C-14 by its HMBC correlations with Me-18 and Me-30. The remaining index of hydrogen deficiency required an additional ring in 2. The HMBC correlations between H-7 and C-14 established an oxetane ring moiety. The relative configurations in 2 were similar to those of 1 according to its ROESY spectrum. The cross-peaks of H-7/Me-30 and Me-30/15-OH determined the oxetane ring moiety to be α-oriented, consistent with those for aphanalides A and C,13 determined by single-crystal X-ray diffraction analyses. The threo configuration of the vicinal diol (11-OH and 12OH) moiety was established by the key ROESY correlations of 11-OH/Me-19β and 12-OH/Me-18α. The absolute configuration of the vic-diol was assigned by using the in situ Mo2(OAc)4-induced electronic circular dichroism (ECD) method, developed by Frelek.22 According to the empirical rule proposed by Snatzke,23,24 the induced ECD curve at around 310 nm (band IV) showing the same sign with the O− C−C−O torsion angle in the favored conformation allowed the assignment of the absolute configuration. The metal complexes of compound 2 in DMSO gave a significant induced ECD spectrum (Supporting Information, Figure S2), in which the negative Cotton effect (CE) at 308 nm permitted the assignment of 11R and 12R for 2. The structure of compound 2 was thus established as shown. Aphanalide K (3) was also obtained as crystals (MeOH). The HRESIMS data of 3 exhibited an ion at m/z 517.2437 [M − H]− (calcd for C28H37O9 517.2443), suggesting a molecular formula of C28H38O9, 60 mass units more than that of 1. Its NMR spectra were in general similar to those of 1, especially in rings B−E. The α,β-unsaturated ester carbonyl moiety in the Aring of 1 was absent in 3, as evidenced by the lack of the pair of cis-olefinic doublets and the downfield-shifted ester carbonyl carbon resonance at δC 170.1 (Δδ +3.1, C-3). Moreover, signals

protons at δH 5.22, 4.88, and 4.72 to C-11, C-7, and C-12, respectively. The HMBC correlations from H-14 and H2-16 to a downfield ketocarbonyl at C-15 revealed the cyclopentenone nature of the D-ring. The C-17 and C-20 linkage between the terminal furan ring and the core skeleton was established by the key HMBC correlations from H-17 to C-20, C-21, and C-22 and from H2-16 to C-20. In the 1H NMR spectrum of 1, a large coupling constant between H-5 and one of the C-6 methylene protons at δH 1.93 (JH5−H6ax = 13.0 Hz) indicated that the B-ring was in a chair conformation and that this proton was in an axial position and β-oriented. The double triplet of the other methylene proton at δH 1.45 (dt, J = 13.0, 3.5 Hz) and the singlet of H-7 suggested that the two protons were nearly orthogonal and that H-7 was in an equatorial position and β-oriented. The strong ROESY cross-peaks (Supporting Information, Figure S1) of H-6β/Me19, Me-19/11-OH, Me-19/Me-29, Me-19/Me-30, Me-30/H-7, Me-30/H-12, and H-12/H-17 indicated that H-7, 11-OH, H12, H-17, Me-19, Me-29, and Me-30 were cofacial and βoriented. Thus, the ROESY correlations of H-6α/H-5, H-5/ Me-28, H-5/H-9, H-9/H-14, H-14/7-OH, H-14/Me-18, and Me-18/12-OH revealed that they were α-oriented. To resolve any ambiguity in structure and also its absolute configuration, compound 1 was crystallized from MeOH to afford crystals for an X-ray crystallography study at low temperature (150 K). On the basis of seven oxygen atoms in the molecule, the final refinement on the Cu Kα data allowed unambiguous assignment of the absolute configuration with a Flack parameter value x = −0.02(17).21 The configurations of the 10 stereogenic centers were thus established as 5R, 7R, 8R, 9R, 10R, 11R, 12R, 13S, 14S, and 17S. In its crystal structure (Figure 1), the A-ring adopted a boat conformation, the B- and C-rings chair conformations, and the D-ring an envelope conformation. The absolute configuration of such an evodulone-type limonoid containing a cyclopentenone D-ring was determined for the first time via the single-crystal X-ray diffraction method. B

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for an O-acetyl group [δH 1.90 (3H, s); δC 169.7 (C), 20.9 (CH3)] were observed. The HMBC correlations from H-1 to C-2 and the two carbonyl carbons at δC 169.7 (OAc) and 170.1 (C-3) located the O-acetyl group at C-1. The relative configurations of 3 were determined by the ROESY experiment in a similar way to that of 1, in which the cross-peak between H-1 and Me-19 determined the O-acetyl group to be αoriented. The absolute configuration of 3 was confirmed by single-crystal X-ray diffraction analysis (Supporting Information, Figure S4), which finally established the absolute configuration of 3 as 1S, 5R, 7R, 8R, 9S, 10R, 11R, 12R, 13S, 14S, and 17S by the anomalous dispersion method with a Flack parameter of −0.10(14)21 using Cu Kα radiation. Aphanalide L (4) was obtained as a white powder. Its HRESIMS showed a negative ion at m/z 595.2307 [M + Cl]− (calcd for C30H40ClO10 595.2315), corresponding to a molecular formula of C30H40O10, indicating 11 indices of hydrogen deficiency. Its 1D NMR spectra resembled those of 3. The major differences between them were in the D-ring and the presence of an additional O-acetyl group [δH 2.10 (s); δC 169.5 (C), 21.1 (CH3)] in 4. This additional O-acetyl group was located at C-7 by the HMBC cross-peak between its ester carbonyl carbon at δC 169.5 and H-7 (δH 4.42). The remaining index of hydrogen deficiency and one unused oxygen atom necessitated the formation of a 14,15-oxirane moiety [δH 3.31 (H-15); δC 72.4 (C-14), 56.1 (C-15)], which was confirmed by the HMBC correlations from H-15, Me-18, and Me-30 to C-14. The β-orientation of H-7 was determined by ROESY correlation of H-7/Me-30. Due to the absence of ROESY cross-peaks from H-15 to neighboring proton signals, it was difficult to define the configuration of the 14,15-oxirane moiety. However, limonoids incorporating a 14,15-oxirane moiety have been structurally established by the X-ray crystallographic analysis.25 The similar chemical shifts of C-14 and C-15 of 5 to those of analogous limonoids favored the β-orientation of the 14,15-oxirane moiety. The significant negative CE observed at 308 nm in the induced ECD spectrum allowed the assignment of the 11R and 12R for 4. Aphanalide M (5) was obtained as a white powder with [α]25 D +43.6. The molecular formula of C34H41NO10, with 15 indices of hydrogen deficiency, was determined from the positive HRESIMS ion at m/z 646.2617 [M + Na]+ (calcd for C34H41NO10Na, 646.2623). The 1H NMR spectrum showed the characteristic signals of a nicotinoyl moiety [δH 8.77 (1H, d, J = 2.0 Hz, H-3′), 8.79 (1H, dd, J = 5.0, 2.0 Hz, H-5′), 7.54 (1H, ddd, J = 8.0, 5.0, 2.0 Hz, H-6′), and 8.00 (1H, dt, J = 8.0, 2.0 Hz, H-7′)]26 and an O-acetyl group [δH 1.83 (3H, s)]. Except for signals belonging to these substituents, the 1D NMR data resembled those of 4. The nicotinoyl group was located at C-12 according to the HMBC correlation between H-12 (δH 5.29) and C-1′ (δC 164.5). The relative configuration of 5 was analyzed via a ROESY spectrum in the same procedure as for 1. On the basis of these results and biosynthetic considerations,13 5 was assigned the same configurations as 1−4. Polystanin E (6) was isolated as a white powder. Its molecular formula of C34H52O10 was determined by the positive ion at m/z 643.3448 [M + Na]+ (calcd for C34H52O10Na, 643.3453) in the HRESIMS data. The 1H NMR spectrum showed the presence of nine tertiary methyl groups (δH 2.08, 2.01, 1.55, 1.37, 1.32, 1.22, 1.21, 1.21, and 1.09, each 3H, s), one olefinic proton (δH 5.27), and five oxygenated carbon protons (δH 5.13, 4.83, 3.87, 3.80, and 3.56). In addition to two O-acetyl groups, the 13C NMR spectrum exhibited seven

methyl, seven methylene (one oxygenated), eight methine (three oxygenated and one olefinic), and eight quaternary carbons (one hemiacetal and two oxygenated). Such data indicated that 6 was an apo-tirucallane-type protolimonoid27 with a modified side chain, which was established mainly by the HMBC experiment. The two O-acetyl groups were located at C-1 and C-7 according to the HMBC correlations from H-1 and H-7 to two ester carbonyl signals at δC 171.5 and 172.0, respectively. The HMBC correlations from H-1 and H2-2 to an ester carbonyl at δC 173.6 indicated that the A-ring comprised a seven-membered lactone moiety as in 3. Furthermore, the HMBC correlations from Me-19 to C-5 and C-9, from Me-30 to C-7, C-9, and C-14, from Me-18 to C-12, C-14, and C-17, and from an olefinic proton H-15 to C-8, C-13, and C-17 established the rings B−D. The HMBC correlations from the C-21 oxymethylene protons to the C-24 hemiacetal carbon (δC 96.7) revealed the existence of an oxygen bridge between C-21 and C-24 in the side chain. The cross-peaks from Me-26 and Me-27 protons to the hemiacetal carbon positioned a 2hydroxyisopropyl group at C-24. The HMBC correlations from H-23 to C-22 and C-24 located a hydroxy group at C-23. The relative configuration of 6 was established mainly by the ROESY experiment. ROESY correlations of H-1/Me-19, Me19/Me-29, Me-19/Me-30, Me-30/H-7, and Me-30/H-17 indicated that H-1, H-7, Me-19, Me-29, and Me-30 were cofacial and were randomly assigned as β-oriented. The ROESY cross-peaks of Me-18/H-9, H-9/H-5, and H-5/Me-28 thus indicated that they were α-oriented. The NOE associations via a ROESY spectrum from ring to side chain protons could lead to erroneous configurational conclusions. The relative configurations of C-20, C-23, and C-24 were determined by analysis of the preferred conformation of 6 and by comparison of carbon resonances. The small coupling constant between H-23 and H2-22 (JH23−H22ax = JH23−H22eq = 3.0 Hz) indicated that the tetrahydropyran ring at C-17 was in a chair conformation and that H-23 was in an equatorial position and β-oriented. The chemical shifts of C-20 (δC 31.1, Δδ 0.0), C-23 (δC 69.2, Δδ +0.1), and C-24 (δC 96.7, Δδ +0.2) of 6 were similar to those of chisopanin A, which were confirmed by the single-crystal Xray diffraction analysis,28 suggesting that H-20 and 23-OH were α-oriented and 24-OH was β-oriented. Therefore, the structure of 6 was assigned as depicted. Nemoralisin A (7) was obtained as an oil, and its molecular formula was determined to be C20H28O5 by the positive HRESIMS ion at m/z 371.1835 [M + Na]+ (calcd for C20H28O5Na, 371.1829), suggesting seven indices of hydrogen deficiency. The 1H NMR spectrum displayed signals for three typical olefinic protons (δH 5.78, 5.55, and 5.31) and five methyl groups (δH 1.96, 1.66, 1.32, 1.32, and 1.19), similar to that of nemoralisin, isolated first from Polyalthia nemoralis (Annonaceae).19 The observed 20 carbon resonances were well resolved in the 13C NMR spectrum, which, aided by HSQC, revealed the presence of one α,β-unsaturated ketone, one α,βunsaturated δ-lactone, three trisubstituted double bonds, and 12 sp3 carbon resonances. Deducting five indices of hydrogen deficiency accounted for two carbonyls and three double bonds, indicating a bicyclic system in 7. The α,β-unsaturated δ-lactone in the A-ring, the 3-oxofurano B-ring, and the aliphatic chain between rings A and B were established by HMBC correlations. The ROESY correlations of H-5/Me-19, H-6/H-4a, and H6/H-4b indicated an E-geometry of the Δ6(7) double bond. The ROESY cross-peak between H-13 and Me-18 inferred the spatial proximity of these protons. The configurations at C-5, C

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7721, compared to the positive control doxorubicin. The primary structure−activity relationships of these terpenoids indicated that the presence of the multiple double bonds in these isolates was important for their activities. Limonoids 1−5, 10, and 11 were further screened for their insecticidal activities against a panel of insects. As shown in Table S5 (Supporting Information), compounds 4 and 5 showed strong insecticidal activities against Diabrotica balteata, and 5 also showed strong activity against Sitobion avenae compared to the positive controls, while 1 and 2 exhibited weak to moderate activities against the four insects. The initial structure−activity relationships of limonoids 1−5, 10, and 11 indicated that the presence of the 14,15-oxirane moiety was important for their insecticidal activities and that the nitrogen atom enhanced such activity.

C-8, and C-11 could not be assigned from the ROESY spectrum because of the flexibility of the aliphatic chain. Therefore, the Mosher ester method29 combined with calculated ECD data established the absolute configuration of 7. Treatment of 7 with (R)-(−)- and (S)-(+)-α-methoxy-α(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) gave the (S)- and (R)-MTPA esters 7a and 7b, respectively. The 1H NMR signals of the two MTPA esters were assigned unambiguously based on 1H−1H COSY spectra, and the ΔδH(S−R) values were then calculated (Figure 2). The results

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-4 instrument and are uncorrected. Optical rotations were measured with a JASCO P-1020 polarimeter. ECD spectra were recorded on a JASCO J-810 spectrometer. UV spectra were recorded on a UV-2450 UV/vis spectrophotometer. IR spectra (KBr disks, in cm−1) were recorded on a Bruker Tensor 27 spectrometer. NMR spectra were recorded on a Bruker Avance III NMR instrument using standard pulse sequences (1H: 500 MHz, 13C: 125 MHz) with TMS as internal standard. Mass spectra were obtained on an Agilent 1100 Series LC/MSD ion-trap mass spectrometer (ESIMS) and an Agilent 6520B UPLC-Q-TOF spectrometer (HRESIMS), respectively. Preparative HPLC was carried out using a Shimadzu LC-8A equipped with a Shim-pack RP-C18 column (20 × 200 mm, i.d.) with a flow rate of 10.0 mL/min, detected by a binary channel UV detector. Silica gel (100−200 and 200−300 mesh) and YMC RP-C18 (50 μm) were used for column chromatography (CC). Fractions obtained from CC were monitored by TLC with precoated silica gel GF254 plates. Spots were visualized by heating silica gel plates sprayed with vanillin−sulfuric acid. All solvents used were of analytical grade. Plant Material. The fruits of Aphanamixis gradifolia Bl. were collected from Xishuangbanna, Yunnan Province, China, in April 2010, and authenticated by Prof. Jing-Yun Cui of Xishuangbanna Tropical Garden, Chinese Academy of Sciences. A voucher specimen (No. AGFruits-2010-04-ZY) has been deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The air-dried and powdered fruits of A. grandifolia (2.8 kg) were extracted with 95% aqueous EtOH (3 × 10 L) under reflux. After removal of the solvent under reduced pressure, the crude extract (405.3 g) was suspended in H2O (1 L) and successively partitioned with petroleum ether (3 × 1 L) and EtOAc (3 × 1 L). The EtOAc extract (110.2 g) was subjected to a silica gel column, eluted with a gradient of CH2Cl2−acetone (20:1 to 0:1), to give seven fractions (A−G). Fraction B (12.8 g) was chromatographed over a silica gel column, eluted with a gradient of petroleum ether− acetone (6:1 to 2:1), to give 6 (4.3 mg), 13 (13.5 mg), 14 (5.4 mg), 15 (41.8 mg), 16 (20.0 mg), 17 (58.3 mg), 18 (15.1 mg), and 19 (17.8 mg). Fraction C (9.6 g) was subjected to a silica gel column, eluted with a gradient of CHCl3−MeOH (100:1 to 10:1), to yield 7 (30.6 mg), 8 (2.3 mg), 9 (5.3 mg), 12 (200.6 mg), 20 (18.3 mg), and 21 (120.1 mg). Fraction D (10.0 g) was chromatographed over a silica gel column, eluted with a gradient of petroleum ether−EtOAc (2:1 to 1:2), to give 4 (13.1 mg), 5 (2.8 mg), 10 (30.5 mg), and 11 (6.3 mg). Fraction F (12.5 g) was subjected to a silica gel column, eluted with a gradient of petroleum ether−EtOAc (1:1 to 0:1), to yield 1 (24.2 mg), 2 (10.6 mg), and 3 (20.5 mg) (detailed procedure for extraction and isolation, see Supporting Information).

Figure 2. Values of ΔδH(S−R) (measured in CDCl3) for the MTPA esters of 7.

indicated the 8S absolute configuration. All four possible isomers (5S,8S,11S)-7, (5S,8S,11R)-7, (5R,8S,11S)-7, and (5R,8S,11R)-7 were then concluded and ECD spectra calculated (Supporting Information, Figures S5 and S6). The experimental ECD spectrum of 7 was in accordance with the calculated ECD spectrum for (5S,8S,11R)-7, thus establishing the assignment of the absolute configuration of 7 as depicted. Nemoralisin B (8) was obtained in a minute amount as a colorless oil. The positive ion at m/z 349.2008 [M + H]+ indicated a molecular formula of C20H28O5, the same as 7. Their NMR data (Supporting Information, Table S3) were similar, which combined with their HMBC spectra revealed that they shared the same carbon skeleton and functionalities. The major difference existed in the A-ring. Compound 8 might be a C-5 epimer of 7 according to the large chemical shift discrepancy (ca. Δδ +10.2) at C-5, due to the anisotropic effects of the Δ6(7) double bond, which was also supported by the absence of the ROESY cross-peak between H-5 and Me-19. The calculated ECD spectrum of (5R,8S,11R)-8 matched well with the experimental ECD spectrum of 8, thus establishing the absolute configuration of 8 as depicted (Supporting Information, Figure S7). Nemoralisin C (9) had the molecular formula C20H28O5, as determined by the HRESIMS. Its NMR data suggested that 9 was structurally related to 7. The major differences were the disappearance of a methyl doublet in 9 and its replacement by a downfield-shifted methyl singlet at δH 1.45, correlating with a quaternary oxygenated carbon signal at δC 72.8 in the HMBC spectrum. The experimental ECD spectrum of 9 was almost the same as that of 7, thus defining the absolute configuration of 9 as shown. Compounds 1−21 were tested in vitro for their cytotoxicities against the human tumor cell lines HL-60, Bel-7402, HepG2, SMMC-7721, A549, and MCF-7. Compounds with IC50 values over 10 μM were deemed inactive (Supporting Information, Table S4). The ranges of IC50 values were 1.80−8.72 μM for 8, 12, 13, 15, and 17−21 against HL-60, 1.53−8.50 μM for 6, 12, 13, and 21 against Bel7402, 3.62−9.31 μM for 7, 8, 12, 20, and 21 against HepG2, and 0.67−7.84 μM for 6−8, 12−16, and 19−21 against SMMC-7721. Compounds 20 and 21 showed the most potent cytotoxicities against HepG2 and SMMCD

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(17) Mitsui, K.; Saito, H.; Yamamura, R.; Fukaya, H.; Hitotsuyanagi, Y.; Takeya, K. Chem. Pharm. Bull. 2007, 55, 1442−1447. (18) Luo, X. D.; Wu, S. H.; Ma, Y. B.; Wu, D. G. Phytochemistry 2001, 57, 131−134. (19) He, X. F.; Wang, X. N.; Fan, C. Q.; Gan, L. S.; Yin, S.; Yue, J. M. Helv. Chim. Acta 2007, 90, 783−791. (20) Sondengam, B. L.; Kamga, C. S.; Connolly, J. D. Tetrahedron Lett. 1979, 20, 1357−1358. (21) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881. (22) Gorecki, M.; Jablonska, E.; Kruszewska, A.; Suszczynska, A.; Urbanczyk-Lipkowska, Z.; Gerards, M.; Morzycki, J. W.; Szczepek, W. J.; Frelek, J. J. Org. Chem. 2007, 72, 2906−2916. (23) Snatzke, G. Angew. Chem., Int. Ed. Engl. 1979, 18, 363−366. (24) Di Bari, L.; Pescitelli, G.; Pratelli, C.; Pini, D.; Salvadori, P. J. Org. Chem. 2001, 66, 4819−4825. (25) Ndung’u, M.; Hassanali, A.; Hooper, A. M.; Chhabra, S.; Miller, T. A.; Paul, R. L.; Torto, B. Phytochemistry 2003, 64, 817−823. (26) González, A. G.; Rodríguez, F. M.; Bazzocchi, I. L.; Ravelo, A. G. J. Nat. Prod. 2000, 63, 48−51. (27) Luo, X. D.; Wu, S. H.; Wu, D. G.; Ma, Y. B.; Qi, S. H. Tetrahedron 2002, 58, 6691−6695. (28) Yang, M. H.; Wang, J. S.; Luo, J. G.; Wang, X. B.; Kong, L. Y. Bioorg. Med. Chem. 2011, 19, 1409−1417. (29) Huang, H. L.; Wang, C. M.; Wang, Z. H.; Yao, M. J.; Han, G. T.; Yuan, J. C.; Gao, K.; Yuan, C. S. J. Nat. Prod. 2011, 74, 2235−2242.

ASSOCIATED CONTENT

S Supporting Information *

Details for extraction and isolation, X-ray crystallographic analyses of aphanalide I (1) and aphanalide K (3), absolute configuration determination (Snatzke’s method, Mosher ester method, and quantum chemical ECD calculation), and bioassays (cytotoxicity and insecticidal activity assays). Copies of HRESIMS, 1D and 2D NMR, ECD, and IR spectra of 1−9 and induced ECD spectra of 2 and 4. CIF files of 1 and 3. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-25-8327-1405. E-mail: [email protected] (L.-Y.K.) or [email protected] (J.-S.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research work was financially supported by the National Natural Science Foundation of China (21272275), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193), and the Fundamental Research Funds for the Central Universities (JKY2011011, 3092013112014). The Syngenta Postgraduate Fellowship (SPF-048) awarded to Y.Z. is also appreciated.

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REFERENCES

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