Aphapolynins A and B, two new limonoids from the fruits

of Aphanamixis polystachya

Yao Zhang, Jun-Song Wang, Xiao-Bing Wang, Dan-Dan Wei, Jian-Guang Luo, Jun Luo,Ming-Hua Yang, Ling-Yi Kong ⇑

Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China

a r t i c l e i n f o

Article history:

Received 7 January 2011Revised 27 February 2011Accepted 10 March 2011Available online 17 March 2011

Keywords:

Aphanamixis polystachya

Meliaceae

A,B-seco limonoidsCu Ka X-ray crystallographyCytotoxicity

a b s t r a c t

Two new highly oxidized A,B-seco limonoids, aphapolynins A (1) and B (2), were isolated from the fruitsof Aphanamixis polystachya. Their structures were elucidated by spectroscopic analysis, in particular, theabsolute configuration of aphapolynin A was determined by a single-crystal X-ray study using a mirrorCu Ka radiation. Aphapolynin A exhibit moderate cytotoxicities when tested against a panel of tumor celllines.

Ó 2011 Elsevier Ltd. All rights reserved.

The species ofMeliaceaewere shown to be a rich source of char-acteristic limonoids (tetranortriterpenoids),1 which have beenthoroughly studied due to their significant biological activitiessuch as antifeedant, insecticidal, antitumor, and antimalarial activ-ities,2 and also their interesting structures with diverse skeletons.3

The plant Aphanamixis polystachya (Wall.) R.N. Parker is a timertree mainly distributed in the tropical areas of Asia such as South-ern China, India, Malaysia, and Indonesia.4 For its repellent, anti-proliferative, antifeedant and contact toxicity to pests,5 its seedextracts have been intensively investigated which abound in ringsA,B-seco limonoids, mainly of the prieurianin type.6 We previouslystudied the stem barks of Aphanamixis grandifolia grown in thetropical rainforest of Xishuangbanna, Southern Yunnan, China,and reported many novel C24, C26, and C29 nortriterpenoids as wellas their cytotoxicities.7 The aerial parts of A. grandifolia collectedfrom Hainan province, China, were also investigated, leading tothe isolation of some cycloartane-type triterpenoids.8 The seedsof A. polystachya were rich in oxidized limonoids,6f,6g and recently,aphanamolide A9 with a novel C3–C6 carbon skeleton was isolatedfrom them. In a continuing phytochemical study of species of theMeliaceae, the fruits of A. polystachya were examined, and twonew oxidized limonoids, aphapolynins A (1) and B (2) wereisolated.

Aphapolynin A features an unusual A,B-seco rearranged limo-noid skeleton with an unprecedented conjugated b-hydroxy-

a,b:c,d-dienoate moiety. Their structures were elucidated byspectroscopic analysis and the absolute configuration of 1 wasdetermined by a single-crystal X-ray study using a mirror Cu Karadiation. Herein, we describe the isolation and structure elucida-tion of 1 and 2 (Fig. 1) and the cytotoxic evaluation on theseisolates.

Air-dried fruits of the plant material were extracted with 95%ethanol under refluxing three times. The EtOH extract was filteredand concentrated in vacuo to afford a residue, which was thendefatted by petroleum ether and partitioned with CHCl3. The CHCl3extract, was then submitted to a series of chromatography on silicagel, ODS, and Sephadex LH-20 to yield compounds 1 and 2.

0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.tetlet.2011.03.047

⇑ Corresponding author. Tel./fax: +86 025 8327 1405.

E-mail address: cpu_lykong@126.com (L.-Y. Kong).

O

O

O

OH

OO

O

O

O

OHO

HO

O

OH

O

OH O

HO

O

O

OO

OOH

H

H

H

1 2

18

1

2

3 284S

5S

6

9R

17S

15

14S

10R

7

11R

8

12R

13R16

20

19

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1'

2'R

30

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4'5'

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28

45

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9

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15

14

10

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8

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1316

20

19

21

23

22

29

30

1'

2'3'

4'5'

6'

HH

A

C D

E

Figure 1. The chemical structure of 1 and 2.

Tetrahedron Letters 52 (2011) 2590–2593

Contents lists available at ScienceDirect

Tetrahedron Letters

journal homepage: www.elsevier .com/ locate / tet let

Aphapolynin A (1)10 was obtained as a crystal (in MeOH–H2O)with a molecular formula of C33H40O12 as determined by HRESIMSat m/z 651.2415 [M+Na]+ (calcd for C33H40O12Na, 651.2412), sug-gesting 14 degrees of unsaturation. The IR spectrum showed thepresence of hydroxyl (3440 cmÿ1) and carbonyl (1740, 1710 and1659 cmÿ1) groups. In accordance with the molecular formula, 33carbon resonances were resolved in the 13C NMR spectrum, andwere further classified by DEPT and HSQC experiments into thecategories of 5 methyls, 4 methylenes (an olefinic carbon), 12methines (5 olefinic ones), and 12 quaternary carbons (4 carbonyland 4 olefinic ones). The five carbon–carbon double bonds and fourcarbonyl groups accounted for nine degrees of unsaturation. Theremaining five degrees of unsaturation suggested that the mole-cule contained five rings. Comprehensive analysis of the 1H and13C NMR data (Table 1) and 2D NMR spectra of 1 suggested that1 was an A,B-seco limonoid.6b,6e,11

The down-field shifted proton resonances at dH 7.29 (s, H-21),6.38 (s, H-22), and 7.40 (s, H-23) were typical of a b-substitutedfuranyl moiety. A typical ABX coupling system was found for ringD, composed of a broad triplet for H-17 at dH 3.91 (t, J = 9.0 Hz)and two characteristic downfield shifted methylene protons ofH2-16 [dH 2.86 (dd, J = 19.0, 9.0 Hz), 2.32 (dd, J = 19.0, 9.0 Hz)].These assignments were confirmed by the observed HMBC correla-

tions (Fig. 2A) from H-17 to C-13 (dC 50.8), C-14 (dC 80.7), C-18(dC 13.5), and C-20 (dC 125.0), C-21 (dC 142.3), C-22 (dC 112.1) ofthe b-furanyl ring and from H2-16 to C-13, C-14 and C-15 (dC209.2). The 1H–1H COSY (Fig. 2A) spectrum revealed the existenceof another ABX coupled protons H(9)-H(11)-H(12) in ring C, andthe HMBC cross-peaks arised from them to the neighboringcarbons allowed the assignments of signals for ring C with a char-acteristic exocyclic methylene group (dH 6.07 and 5.38, both sin-glet) at C-8. A 2-hydroxy-3-methylpentanoyl moiety was alsoestablished by 1H–1H COSY and its linkage to C-12 was determinedby the observed HMBC cross-peak of a doublet proton signal at dH5.98 (H-12) to a carbonyl carbon at dC 175.3; a low-field singlet sig-nal at dH 7.59 characteristic of a formate group was present in the1H NMR, which correlated with another carbonyl carbon signal atdC 162.4, allowed its attachment to C-11. Rings C–E were thusestablished.

The remaining 11 carbons comprised rings A and B including anunusual conjugated b-hydroxy-a,b:c,d-dienoate moiety which wassuggested by the presence of a cis-double bond with coupling con-stant at 10.5 Hz and a large chemical shift difference between thetwo carbons C-3 and C-6 (DdC 77.4), a low-field exchangeable pro-ton signal at dH 11.88 when recorded in CDCl3 (Supplementarydata, Fig. S17). This moiety was also supported by the observedmaximum absorption at 325 nm (log e 1.8) in the UV spectrumand further confirmed by the HMBC 3JHC correlations from onedoublet olefinic proton at dH 7.12 (d, J = 10.5 Hz) to C-3 at dC171.0, and from another doublet olefinic proton at dH 6.02(d, J = 10.5 Hz) to C-6 at dC 93.6. The singlet methyl at dH 1.44was assignable to H3-19 by its correlations to C-1, C-5, and C-10;likewise, another singlet methyl at dH 1.50 was ascribed to H3-28due to its HMBC correlations with C-4, C-5, and C-29. The linkageof rings A and C between C-9 and C-10 was revealed by the ob-served HMBC correlation from H-9 to C-10, thus established theplanar structure of 1 as an unprecedented rearranged rings A,B-seco limonoid.

In the 1H NMR spectrum of 1, the large coupling constant be-tween H-11 and H-12 (J11,12 = 11.0 Hz) indicated that ring C wasin a chair conformation and these protons adopted an 11,12-diaxialorientation. The ROESY cross-peaks (Fig. 2B) of H-9a and H-11, H-17b and H-12, H-11 and Me-18, H-17 and H-16b revealed that theyhad the same configurations as other reported rings A,B-seco limo-noids.6 However, the assignments of relative configurations ofrings A and B encountered difficulties due to the rotatable natureof C9–C10 bond. Fortunately, after many attempts with differentsolvents, a single crystal of compound 1 was successfully obtainedwith MeOH/H2O (5:1), which were analyzed on an X-ray (Fig. 3)diffractometer with a mirror Cu Ka (k = 1.54184 Å) radiation (xscans, 2hmax = 139.2972°).12 By anomalous dispersion methodswith Flack x = 0.02 (15), the absolute configuration of 1 was deter-mined to be 4S, 5S, 9R, 10R, 11R, 12R, 13R, 14S, 17S, 20R and 30R,

Table 11H and 13C NMR data of compounds 1 and 2 (J in Hz)

No. 1a 2b

dC dH dC dH

1 150.6 7.12 (d, 10.5) 83.3 4.19 (dd, 10.5, 8.0)2 123.1 6.02 (d, 10.5) 32.8 2.52 (dd, 18.0, 10.5)2 2.73 (dd, 18.0, 8.0)3 171.0 175.54 69.1 89.95 52.7 3.00 (s) 42.0 2.88 (dd, 10.5, 8.0)6 93.6 34.6 2.44 (dd, 14.0, 10.5)6 2.48 (dd, 14.0, 8.0)7 174.7 171.28 143.2 140.09 52.3 4.47 (d, 8.0) 49.9 3.47 (d, 10.0)

10 42.9 — 48.411 69.6 5.49 (dd,11.0,8.0) 80.1 4.06 (dd, 10.0, 7.0)12 73.9 5.98 (d, 11.0) 78.0 5.87 (d, 7.0)13 50.8 48.214 80.7 80.115 209.2 209.7

16a 43.2 2.32 (dd,19.0, 9.0) 42.1 2.36 (dd,19.5, 10.5)16b 2.86 (dd,19.0, 9.0) 2.89 (dd, 19.5, 9.0)17 36.3 3.91 (t, 9.0) 34.9 3.89 (t, 9.5)18 13.5 0.98 (s) 13.5 0.85 (s)19 35.2 1.44 (s) 19.7 1.12 (s)20 125.0 122.621 142.3 7.29 (s) 140.5 7.19 (s)22 112.1 6.38 (s) 110.5 6.21 (s)23 144.1 7.40 (s) 142.9 7.36 (s)28 25.7 1.50 (s) 20.1 1.47 (s)

29a 78.8 4.09 (d, 11.0) 67.8 3.77 (d, 12.5)29b 3.82 (d, 11.0) 3.55 (d, 12.5)30a 121.7 6.07 (s) 120.5 6.06 (s)30b 5.38 (s) 5.35 (s)10 175.3 174.920 76.2 3.14 (d, 2.5) 74.2 3.44 (d, 3.0)30 39.1 1.41 (m) 39.2 1.62 (m)

40a 23.4 1.14 (m) 23.9 1.26 (m)40b 1.03 (m) 1.14 (m)50 11.9 0.75 (t, 7.5) 11.9 0.81 (t, 7.5)60 15.9 0.81 (d, 6.5) 14.9 0.89 (d, 7.0)10 0 162.4 7.59 (s)

OMe 52.2 3.70 (s)

a 1H and 13C NMR data were recorded in CD3OD-d4.b 1H and 13C NMR data were recorded in CDCl3. The NMR experiments were

carried out at 298 K.

O

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OH O

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OOH

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11 18

28

4

9

17

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3'

ROESYHMBC1H-1H COSY

A B

Figure 2. Key 1H–1H COSY and HMBC (H?C) correlations (A), and selective ROESYcorrelations (B) of 1.

Y. Zhang et al. / Tetrahedron Letters 52 (2011) 2590–2593 2591

which represented, for the first time, the definite establishment ofthe absolute configurations of A,B-seco limonoids including thecommonly occurred 2-hydroxy-3-methylpentanoyl group, by sin-gle-crystal X-ray diffraction analysis using a mirror Cu Karadiation.

Aphanamolide A, a compound also found from the seeds of thesame plant, has been recently reported with the same carbon skel-eton but with the reservation of a commonly occured seven-mem-bered lactone and without such an unusual conjugated b-hydroxy-a,b:c,d-dienoate moeity. Ester condensation is the key process inthe proposed biosynthesis of this novel skeleton,9 In this process,the previous d-lactone ring rotated to nearly 180°, and as a result,the relative configurations of H-5 and Me-28 reversed, from a-ori-entation in common rings A,B-seco limonoid6,11 to b-orientation.The two parts joined through C-9 and C-10, due to their bulky nat-ure, adopted a nearly perpendicular orientation, which made thereported assignment of an a configuration to Me-28 based on itsobserved ROESY correlation with H-9 unreliable, since both in aor b configuration, Me-28 is close enough to H-9, permitting aROESY correlation between them.

Aphapolynin B (2),13 a white amorphous powder, displayed amolecular formula of C33H44O12 as determined by HRESIMS at m/z 655.2726 [M+Na]+ (calcd for 655.2725), indicating 12 degreesof unsaturation. Its IR spectrum showed the presence of hydroxy(3434 cmÿ1) and carbonyl (1743 cmÿ1) groups. Careful comparisonof its NMR data with those of 1 indicated that they shared the samerings C–E except for the absence of the formate group at C-11 pre-sented in the NMR spectra of 1.

Compound 2 displayed a typical ABX coupling system in ring A,which composed of H-1 at dH 4.19 (dd, J = 10.5, 8.0 Hz) and H2-2 atdH 2.73 (dd, J = 18.0, 8.0 Hz) and 2.52 (dd, J = 18.0, 10.5 Hz), re-vealed also by 1H–1H COSY spectrum (Fig. 4A). The HMBC 3JHCstrong correlations from H-11 to C-1 and H-1 to C-11 suggestedthat C-1 and C-11 were connected via an oxygen atom, forming atetrafuran ring. Moreover, the absence of HMBC cross-peaks fromthe two protons of H2-29 (dH 3.77 and 3.55) to the ester carbonylcarbon at dC 171.2 (C-7), and the presence of the methoxyl protonsat dH 3.70 with its correlation indicated that the six-membered lac-tone cleaved, forming a methyl ester. The relative configuration ofcompound 2 was mainly determined by ROESY spectrum (Fig. 4B),in which the correlations of H-17/H-12, H-17/H-16b, and H-12/H-1indicated that they were co-facial, and were arbitrarily assigned tobe in b-orientation. The ROESY cross-peaks of Me-18/H-11, Me-18/H-16a and H-11/H-9 indicated that H-9, H-11 and Me-18 were a-

orientated. Accordingly, the structure of 2 was assigned asdepicted.

All the isolates were evaluated for their cytotoxicity against apanel of cell lines in vitro, including Bel-7402, SGC-7901, BGC-823, HepG2, HeLa, and MCF-7 with taxol as positive control, usingthe MTT method.14 Compound 2 showed no cytotoxicity (IC50

>50 lM) against any of the tested tumor cell lines, while 1 showedmoderate effects with IC50 at 23.7 and 25.6 lM for Bel-7402 andBGC-823, respectively, which might be due to the conjugated b-hy-droxy-a,b:c,d-dienoate moiety in it.

Acknowledgments

This research work was supported by Project of National Natu-ral Science Foundation of China (Grant No. 21072230), and theScaling Project for Innovation Scholars, Natural Science Foundationof Jiangsu Province, China (Grant No. BK2008039).

Supplementary data

Supplementary data (general experimental procedures, plantmaterial, extraction and isolation, X-ray analysis, CD exciton chi-rality, bioassay, 1D and 2D NMR spectra of 1 and 2, and the CDspectrum for 1) associated with this article can be found, in the on-line version, at doi:10.1016/j.tetlet.2011.03.047.

References and notes

1. (a) Liao, S. G.; Chen, H. D.; Yue, J. M. Chem. Rev. 2009, 109, 1092–1140; (b) Yin,S.; Wang, X. N.; Fan, C. Q.; Liao, S. G.; Yue, J. M. Org. Lett. 2007, 9, 2353–2356; (c)Luo, J.; Wang, J. S.; Luo, J. G.; Wang, X. B.; Kong, L. Y. Org. Lett. 2009, 11, 2281–2284.

2. (a) Carpinella, M. C.; Defago, M. T.; Valladares, G.; Palacios, S. M. J. Agric. FoodChem. 2003, 51, 369–374; (b) Abdelgaleil, S. A. M.; Okamura, H.; Iwagawa, T.;Sato, A.; Miyahara, I.; Doe, M.; Nakatani, M. Tetrahedron 2001, 57, 119–126; (c)Nugrohoa, B. W.; Edrada, R. A.; Wray, V.; Witte, L.; Bringmann, G.; Gehling, M.;Proksch, P. Phytochemistry 1999, 51, 367–376.

3. Mohamad, K.; Hirasawa, Y.; Lim, C. S.; Awang, K.; Hadi, A. H. A.; Takeya, K.;Morita, H. Tetrahedron Lett. 2008, 49, 4276–4278.

4. Flora Reipublicae Popularis Sinicae (Zhongguo Zhiwu Zhi); Chen, S. K., Chen, B. Y.,Li, H., Eds.; Science Press: Beijing, China, 1997; 43, pp 239–240.

5. (a) Talukder, F. A.; Howse, P. E. J. Stored Prod. Res. 1995, 31, 55–61; (b) Rabi, T.;Gupta, R. C. Int. J. Pharm. 1995, 33, 359–361; (c) Jagetia, G. C.; Venkatesha, V. A.K. Biol. Pharm. Bull. 2005, 28, 69–77.

6. (a) Connolly, J. D.; Okorie, D. A.; Wit, L. D.; Taylor, D. A. H. J. Chem. Soc., Chem.

Commun. 1976, 22, 909–910; (b) Brown, D. A.; Taylor, D. A. H. Phytochemistry1978, 17, 1995–1999; (c) King, T. J.; Taylor, D. A. H. Phytochemistry 1983, 22,307; (d) Maclachlan, L. K.; Taylor, D. A. H. Phytochemistry 1982, 21, 2426–2427;(e) Mulholland, D. A.; Naidoo, N. Phytochemistry 1999, 51, 927–930; (f) Zhang,H. P.; Wu, S. H.; Luo, X. D.; Ma, Y. B.; Wu, D. G. Chin. Chem. Lett. 2002, 13, 341–342; (g) Zhang, H. P.; Chen, F.; Wang, X.; Wu, D. G.; Chen, Q.Magn. Reson. Chem.2007, 45, 189–192.

7. (a) Zhang, Y.; Wang, J. S.; Wei, D. D.; Wang, X. B.; Luo, J.; Luo, J. G.; Kong, L. Y.Phytochemistry 2010, 71, 2199–2204; (b) Zhang, Y.; Wang, J. S.; Luo, J.; Kong, L.Y. Chem. Pharm. Bull. 2011, 59, 282–286.

8. Liu, Q.; Chen, C. J.; Shi, X.; Zhang, L.; Chen, H. J.; Gao, K. Chem. Pharm. Bull. 2010,58, 1431–1435.

Figure 3. X-ray crystal structure of 1.

O

O

O

OH

OO

O

O

O

OHO

HO

ROESYHMBC1H-1H COSY

128

11 12

18

16

17

19

5 9

A B

Figure 4. Key 1H–1H COSY and HMBC (H?C) correlations (A), and selective ROESYcorrelations (B) of 2.

2592 Y. Zhang et al. / Tetrahedron Letters 52 (2011) 2590–2593

9. Yang, S. P.; Chen, H. D.; Liao, S. G.; Xie, B. J.; Miao, Z. H.; Yue, J. M. Org. Lett. 2011,13, 150–153.

10. Aphapolynin A (1): Colorless crystal; mp 191–192 °C; a½ �26D +91.2 (c 0.23, CHCl3);UV (acetonitrile): kmax (log e) 194 (2.4), 325 (1.8) nm; CD (acetonitrile) kmax

(De) = 203 (ÿ34.4), 236 (+48.4), 262 (+0.1), 332 (+48.0) nm; IR (KBr) mmax cmÿ1:

3440, 2964, 2878, 1740, 1710, 1659, 1595, 1468, 1410, 1383, 1296, 1243, 1223,1135, 1028, 991, 874, 805, 669, 601;1H and 13C NMR data, see Table 1; ESI–MS:m/z 629.2 [M+H]+, 646.3 [M+NH4]

+, 627.1 [MÿH]ÿ, and 663.1 [M+Cl]ÿ; HR-ESI–MS: m/z 651.2415 (calcd for C33H40O12Na 651.2412).

11. Jolad, S. D.; Hoffmann, J. J.; Schram, K. H.; Cole, J. R.; Tempesta, M. S.; Bates, R. B.J. Org. Chem. 1981, 46, 641–644.

12. X-ray analysis: Colorless crystal, asymmetric unit C33H40O12�H2O, P21212,a = 20.09840(10) Å, b = 17.44210(10) Å, c = 9.32170(10) Å, a = b = c = 90°,V = 3267.80(4) Å3, Z = 4, dx = 1.314 Mg/m3, F(0 0 0) = 1376. Data were used formeasurements on an Oxford Diffraction Gemini S Ultra CCD diffractometerwith a mirror Cu Ka (k = 1.54184 Å) radiation at 100 K (x scans,2hmax = 139.2972°). The crystal structures were solved by direct methodsusing SHELXS-97 (Sheldrick, G.M. SHELXS-97: Program for Crystal StructureResolution; University of Göttingen: Göttingen, Germany, 1997.) and expandedusing difference Fourier techniques, refined by SHELXL-97 (Sheldrick, G. M.SHELXL-97: Program for Crystal Structure Refinement; University ofGöttingen: Göttingen, Germany, 1997.) and full-matrix least-squarescalculations. The absolute configuration was determined by anomalous

dispersion effects in diffraction measurements on the crystal. H atoms on Oatoms were fixed geometrically and constrained to ride on its parent atomswith O–H = 0.82 Å and with Uiso (H) = 1.2 Ueq (O). The remaining H atoms on Catoms were positioned geometrically with C–H = 0.93, 0.96, 0.97, and 0.98 Åfor aromatic, methyl, methylene, and methine H atoms, respectively, andconstrained to ride on their parent atoms with Uiso (H) = x Ueq (C), wherex = 1.2 for aromatic, methylene, and methine H atoms and x = 1.5 for methyl Hatoms. The total number of independent reflections measured was 6018, ofwhich 5820 were observed. Final indices (|F|2P 2r|F|2): R1 = 0.0402,wR2 = 0.1284, S = 1.111, (D/r)max = 0.181, (Dq)min = ÿ0.258 e/Å3,(Dq)max = 0.405 e/Å3. Flack x = 0.02 (15). The CCDC-804447 (aphapolynin A)contains the supplementary crystallographic data for this Letter. These datacan be obtained free of charge from The Cambridge Crystallographic DataCentre via www.ccdc.cam.ac.uk/data_request/cif.

13. Aphapolynin B (2): White amorphous powder; a½ �26D ÿ124.5 (c 0.15, CHCl3); UV(acetonitrile): kmax (log e) 194 (2.8) nm; IR (KBr) mmax cm

ÿ1: 3434, 2963, 2930,1743, 1632, 1462, 1392, 1272, 1211, 1136, 1061, 1028, 963, 899, 874, 792, 667,603; 1H and 13C NMR data, see Table 1; ESI–MS: m/z 633.3 [M+H]+, 650.3[M+NH4]

+, 631.3 [MÿH]ÿ, and 667.3 [M+Cl]ÿ; HR-ESI–MS: m/z 655.2726 (calcdfor C33H44O12Na 655.2725).

14. Huang, Z. J.; Zhang, Y. H.; Zhao, L.; Jing, Y. W.; Lai, Y. S.; Zhang, L. Y.; Guo, Q. L.;Yuan, S. T.; Zhang, J. J.; Chen, L.; Peng, S. X.; Tian, J. D. Org. Biomol. Chem. 2010, 8,632–639.

Y. Zhang et al. / Tetrahedron Letters 52 (2011) 2590–2593 2593