Unusual Antifeedant Spiro-Sesterterpenoid from The

Tetrahedron Letters 54 (2013) 235–237

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Tetrahedron Letters

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Unusual antifeedant spiro-sesterterpenoid from the ﬂowers of Leucosceptrum canum ⇑ Shi-Hong Luo a,b, Juan Hua a, Chun-Huan Li a,b, Yan Liu a, Xiao-Nian Li a, Xu Zhao a, Sheng-Hong Li a, a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China b University of Chinese Academy of Sciences, Beijing 100039, PR China article info abstract

Article history: Leucosceptroid O (1), an unusual sesterterpenoid possessing a spiro a,b-unsaturated c-lactone moiety, Received 12 July 2012 was discovered from the ﬂowers of a large woody Labiatae, Leucosceptrum canum. Its structure including Revised 23 October 2012 absolute stereochemistry was determined by comprehensive NMR, MS, and single-crystal X-ray diffrac- Accepted 2 November 2012 tion (with copper radiation) analyses. The obvious antifeedant activity of 1 against the generalist Available online 12 November 2012 plant-feeding insect Helicoverpa armigera suggested that it to be a new defensive sesterterpenoid of Leucosceptrum canum. Keywords: Ó 2012 Elsevier Ltd. All rights reserved. Leucosceptrum canum Labiatae Sesterterpenoid Leucosceptroid O Antifeedant activity

Terpenoids are not only the largest group of natural products recently, Horne’s group has synthesized the core structure of leu- with highly diversified structures, but also have a variety of roles cosceptroids A–D.8 Our further investigation on the defensive sest- in mediating antagonistic and beneficial interactions among organ- erterpenoids in the flowers of L. canum led to the discovery of an isms in the natural world.1 Among them, sesterterpenoids, a spe- additional intriguing novel sesterterpenoid, leucosceptroid O (1) cial group of pentaprenyl terpenoids that are originated from (Fig. 1), containing a spiro a,b-unsaturated c-lactone moiety. geranylfarnesyl diphosphate, have been reported from widespread Herein, we report its isolation, structure determination including sources including various marine organisms especially sponges, absolute stereochemistry and antifeedant activity against the terrestrial fungi, lichens, higher plants, and insects.2 However, their generalist plant-feeding insect Helicoverpa armigera. distribution and natural functions in plants have not been well The flowers of L. canum (25.0 kg) were air-dried and extracted investigated. with petroleum ether, and the extract was separated with various Leucosceptrum canum Smith, is perhaps the highest plant column chromatographies as described in detail in the supporting belonging to the family Labiatae (also Lamiaceae)3 that distributed information, and finally purified with reversed-phase semiprepar- from the Himalayas to the southwest of China,4 and so far the only ative HPLC to yield 17.2 mg of compound 1. colored nectar plant4 of all Labiatae species. The species locally Compound 1 was obtained as colorless blocks, having a molec- known as Bhusure is used as an insecticidal agent in remote areas ular formula of C25H34O6, with nine units of unsaturation, as deter- of Nepal,5 and is rarely attacked by herbivores and only occasionally mined by its EI-MS and high resolution EI-MS. The IR spectrum by pathogens. Interested in the relationship between the secondary indicated the presence of hydroxy (3386 cm1) and carbonyl metabolites of L. canum and its above special traits, we have found groups (1771 and 1703 cm1).9 In the 1H NMR spectrum (Table 1), that the glandular trichomes of this plant harbor unique defensive two secondary methyls at dH 0.84 (d, J = 7.3 Hz) and 0.92 (d, sesterterpenoids leucosceptroids A and B possessing a furan- J = 6.7 Hz) and four tertiary methyls at dH 1.31 (s), 1.74 (s), 1.77 6 containing tetracyclic C25 skeleton. Since the trichomes mainly ex- (s), and 2.24 (s) were clearly shown. Two olefinic protons at dH ist on the surface of the leaves and flowers of L. canum, a continuing 5.60 (d, J = 9.0 Hz) and 5.84 (s) indicated the presence of two work on the whole leaves resulted in the discovery of two tri-substituted double bonds. Two singlets at dH 3.49 and 4.46 additional interesting defensive sesterterpenoids leucosceptroids and one doublet at dH 5.23 (J = 9.0 Hz) were ascribable to either C and D which contain unusual antipodal cyclopentenones.7 Very oxygenated methine or free hydroxy groups. Other signals oc- curred in a relatively highfield region (between 1.39 and 2.46 ppm) and mostly overlapped, resonating from either methines ⇑ Corresponding author. Tel./fax: +86 871 5223035. or methylenes. Twenty-five carbon resonances were resolved in 13 E-mail address: [email protected] (S.-H. Li). the C NMR spectrum (Table 1), and were further classified by

1 OH H O 2 19 3 H 20 21 O O O O 18 25 22 HMBC H C 4 H O O O 8 H OH 17 6 O 5 7 H OH 16 O 13 11 9 O 12 15 10 14 H OH O O 23 24 22 Leucosceptroid A Leucosceptroid O (1)

4 7 Figure 1. Chemical structures of leucosceptroid O (1) and the known leucoscept- 6 roid A. ROESY H H 13 11 DEPT experiments as six methyls, four methylenes, seven 10 methines, and eight quaternary carbons. These data were consis- 24 23 tent with the above molecular formula and signals observed in the 1H NMR spectrum, suggesting that 1 was a highly oxygenated sesterterpenoid. Figure 2. Key HMBC and ROESY correlations of leucosceptroid O (1). The resemblance in the NMR spectra of 1 (Table 1) and leucosceptroid A6 indicated that 1 was structurally similar to leucosceptroid A (Fig. 1). The major difference was that the furan ring in of leucosceptroid A revealed that 5-OH was disappeared in 1 and leucosceptroid A was replaced by an a,b-unsaturated c-lactone one of the oxygenated quaternary carbons (C-5) of 1 was dramat- moiety in 1, which was supported by the HMBC correlations ically shifted downﬁeld (from dC 84.8 to dC 91.1). This fact

(Fig. 2) from H-19 (dH 5.84) to an ester carbonyl carbon at dC combined with the existence of a ketal group (C-17) suggested 170.4 (C-20), an oleﬁnic quaternary carbon at dC 170.7 (C-18), a ke- an oxygen bridge between C-5 and C-17 in 1, forming a spiro tal carbon at dC 110.3 (C-17), and a methyl carbon at dC 13.1 (Me- a,b-unsaturated c-lactone framework. Therefore, the planar 25). Methylation at C-18 of the a,b-unsaturated c-lactone moiety structure of 1 was deduced as shown in Figure 1. was evident from the HMBC correlations from Me-25 to C-17, C- In the ROESY spectrum of 1 (Fig. 2), the correlations of H-7 with 18, and C-19. Further comparison of the NMR data of 1 with those H-4 and Me-22, and 11-OH with H-10 and Me-24, indicated that H- 4, H-7, 11-OH, Me-22, and Me-24 were b-oriented. ROESY correlations of H-6 with H-13 and Me-23 demonstrated that H-6, H-13, Table 1 and Me-23 were a-oriented. However, it is not possible to use 1H, 13C NMR and 1H–1H COSY data of leucosceptroid O (1) in acetone-d (d in ppm, J in 6 ROESY experiment to identify the stereochemistry of the chiral Hz)a center C-17. Therefore, an X-ray diffraction was necessary to clarify 1 1 Position dH dC H– H COSY its structure especially the stereochemistry of the spiro lactone. A 1 1.74 s (3H) 18.9 q H-3 single crystal of 1 was obtained from a mixture of MeOH/water 2 — 137.9 s — (5:1), and X-ray crystallographic analysis with copper radiation 3 5.60 d (9.0) 122.1 d H-4, Me-1, 21 was successfully performed (CCDC 866045), which unambiguously 4 5.23 d (9.0) 78.9 d H-3 5 — 91.1 s — determined the complete structure of 1 as deduced (Fig. 3), with 6 1.71 m 44.0 d H-7, Me-22 the absolute stereochemistry of 4R,5R,6S,7S,10S,11R,13S,14S,17R. 7 1.97 m 51.3 d H-6, H2-8 The antifeedant activity of leucosceptroid O (1) against a gener- 8a 1.56 m 31.4 t H-8b,H2-9 alist insect, cotton bollworm (H. armigera), was assayed as de- 8b 2.12 m H-8a,9b 6 9a 1.39 m 30.9 t H-9b,H-8 scribed previously. It showed obvious antifeedant activity, with 2 2 9b 1.99 m H-9a, 10, H2-8 antifeedant index (AI%) of 45.81 ± 2.78% at 15.73 lg/cm , though 10 2.30 m 45.8 d H-9b, Me-23 less active than a commercial neem oil (1% azadirachtin: 11 — 85.1 s — AI% = 76.17 ± 1.95%) produced by Kunming Rixin Dachuan Tech- 12 — 208.0 s — nology Co., which suggested that 1 maybe the component respon- 13 3.49 s 65.3 d H-15a 14 — 84.1 s — sible for the defense of L. canum against herbivore enemies.

15a 1.70 m 38.9 t H-13, 15b, H2-16

15b 1.90 m H-15a, H2-16 Acknowledgments

16a 1.78 m 31.1 t H2-15 16b 2.46 m This research was supported ﬁnancially by the NSFC-Yunnan 17 — 110.3 s — 18 — 170.7 s — Joint Fund (U1202263), the National Basic Research Program of 19 5.84 s 116.3 d Me-25 China (973 Program) on Biological Control of Key Crop Pathogenic 20 — 170.4 s — Nematodes (2013CB127505), the National Natural Science Founda- 21 1.77 s (3H) 26.2 q H-3 tion of China (31070320 and 31100222), and the ‘Hundred Talents 22 0.92 d (3H, 6.7) 14.3 q H-6 Program’ of the Chinese Academy of Sciences (CAS). 23 0.84 d (3H, 7.3) 16.8 q H-10 24 1.31 s (3H) 27.0 q — 25 2.24 s (3H) 13.1 q H-19 Supplementary data 11-OH 4.46 s — —

a1H NMR spectrum was recorded at 500 MHz and 13C NMR spectrum at Supplementary data (this data include experimental proce- 125 MHz. dures, plant material, and 1D and 2D NMR spectra of 1) associated S.-H. Luo et al. / Tetrahedron Letters 54 (2013) 235–237 237

Figure 3. X-ray crystallographic structure of leucosceptroid O (1) showing the absolute stereochemistry.

13 with this article can be found, in the online version, at http:// 9. Leucosceptroid O (1): colorless blocks, mp 198–200 °C; ½aD + 80.1 (c = 0.2, dx.doi.org/10.1016/j.tetlet.2012.11.010. MeOH); UV (MeOH) kmax (loge): 202 (4.01) nm; IR (KBr) mmax: 3386, 2966, 2926, 2865, 1771, 1703, 1449, 1380, 1223, 1175, 1082, 907, 850 cm1; EI-MS m/z (%): 430 (9) [M]+, 346 (54), 328 (45), 273 (51), 236 (61), 208 (80), 83 (100); HR-EI- + + References and notes MS: m/zobsd 430.2349 [M] (m/zcalcd [C25H34O6] = 430.2355). Crystal data of 1 leucosceptroid O (1): C25H34O6, M = 430.52 g mol , colorless blocks, size 3 1. (a) Gershenzon, J.; Dudareva, N. Nat. Chem. Biol. 2007, 3, 408–414; (b) 0.60 0.19 0.09 mm , orthorhombic, space group P2(1)2(1)2(1), a = 10.8208 (4) Å, b = 12.3058 (5) Å, c = 17.5139 (7) Å, a =90°, b =90°, c =90°, V = 2332.1 Degenhardt, J.; Köllner, T.; Gershenzon, J. Phytochemistry 2009, 70, 1621– 3 3 1637. (16) Å , T = 173 °C, Z =4, d = 1.226 g cm , l(Mo-Ka) = 1.54178 Å, 2. Liu, Y.; Wang, L.; Jung, J. H.; Zhang, S. Nat. Prod. Rep. 2007, 24, 1401–1429. F(000) = 928, 16791 reflections in h(13/11), k(12/14), l(17/20), measured 6 6 3. Peng, H.; Wu, Z. Y. Acta Bot. Yunnan. 2001, 23, 278–286. in the range 4.39° h 68.2°, completeness hmax = 95.1%, 3964 independent 4. Hansen, D. M.; Olesen, J. M.; Mione, T.; Johnson, S. D.; Muller, C. B. Bio. Rev. 2007, reflections, Rint = 0.0402, 3964 reflections with Fo>4r(Fo), 288 parameters, 0 82, 83–111. restraint, R1obs = 0.0285, wR2obs = 0.0720, R1all = 0.0287, wR2all = 0.0722, GOF = 1.050, Absolute structure parameter 0.09(13), largest difference peak 5. (a) Choudhary, M. I.; Ranjit, R.; Atta-ur-Rahman; Shrestha, T. M.; Yasin, A.; 3 Parvez, M. J. Org. Chem. 2004, 69, 2906–2909; (b) Choudhary, M. I.; Ranjit, R.; and hole = 0.170 and 0.143 e Å . The crystal structure of 1 was solved by Atta-ur-Rahman; Hussain, S.; Devkota, K. P.; Shrestha, T. M.; Parvez, M. Org. Lett. direct method using the program SHELXS-97 (G. M. Sheldrick, SHELXS97 and 2004, 6, 4139–4142. SHELXL97, University of Gottingen, Germany, 1997) and subsequent Fourier difference techniques, and refined anisotropically by fullmatrix least-squares on 6. Luo, S. H.; Luo, Q.; Niu, X. M.; Xie, M. J.; Zhao, X.; Schneider, B.; Gershenzon, J.; Li, 2 S. H. Angew. Chem., Int. Ed. 2010, 49, 4471–4475. F using SHELXL-97 (G. M. Sheldrick, SHELXTL, Version 6.10, Bruker AXS Inc., 7. Luo, S. H.; Weng, L. H.; Xie, M. J.; Li, X. N.; Hua, J.; Zhao, X.; Li, S. H. Org. Lett. 2011, Madison, Wisconsin, USA, 2000). Crystallographic data for the structure of 1 13, 1864–1867. with copper radiation has been deposited in the Cambridge Crystallographic 8. Xie, J.; Ma, Y. L.; Horne, D. A. J. Org. Chem. 2011, 76, 6169–6176. Data Centre (deposition number: CCDC 866045).