C Rings from

Note pubs.acs.org/jnp

Xylomexicanins I and J: Limonoids with Unusual B/C Rings from Xylocarpus granatum Yi-Bing Wu,†,§ Ya-Zhen Wang,‡ Zhi-Yu Ni,§,¶ Xia Qing,†,§ Qing-Wen Shi,*,†,§,¶ Françoise Sauriol,⊥ Christopher J. Vavricka,∥ Yu-Cheng Gu,▽ and Hiromasa Kiyota*,# †

School of Pharmaceutical Sciences, §Hebei Key Laboratory of Forensic Medicine, and ¶Collaborative Innovation Center of Forensic Medical Molecular Identification, Hebei Medical University, Shijiazhuang, Hebei Province 050017, People’s Republic of China ‡ Hebei General Hospital, Medical Examination Center, Heping Xilu, No. 348, Shijiazhuang, Hebei Province 050051, People’s Republic of China ⊥ Department of Chemistry, Queen’s University, Kingston, K7L 3N6, Ontario Canada ∥ Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan ▽ Syngenta Jealott’s Hill International Research Centre, Berkshire, RG42 6EY, United Kingdom # Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan S Supporting Information *

ABSTRACT: Two tetranortriterpenoids with new skeletons, xylomexicanins I and J (1 and 2), were isolated during the investigation of chemical constituents from seeds of the Chinese mangrove, Xylocarpus granatum. Xylomexicanin I (1) is an unprecedented limonoid with bridged B- and C-rings. A biosynthesis pathway for 1 from xylomexicanin F is proposed.

Xylocarpus granatum Koenig is a marine mangrove of the family Meliaceae that is mainly distributed in Southeast Asia and along the Indian Ocean. The plant is used as a folk medicine in Southeast Asia for the treatment of cholera, diarrhea, fever diseases such as malaria, and also as an antifeedant.1 Accordingly, over 50 X. granatum limonoid derivatives have been reported.2,3 Further investigation of the seeds from the same plant4 led to the isolation of two new tetranortriterpenoids, xylomexicanins I and J (1 and 2) (Figure 1). Notably, 1 represents an unprecedented limonoid with a bridged skeleton

between the B- and C-rings, contrasting with analogues possessing bridged A- and B-rings. In this report, the isolation and structural elucidation of these two new limonoids are described. Natural product isolation was performed according to the reported procedure.4 Ethanol extracts of dried X. granatum seeds (10 kg) were concentrated and further extracted with petroleum ether and CH2Cl2. The CH2Cl2 fraction was concentrated and loaded on an open silica gel column with petroleum ether/acetone as the mobile phase. Fractions containing limonoid-like compounds were subjected to further semipreparative HPLC separation with a mobile phase of MeOH/H2O to yield 1 (5 mg) and 2 (7 mg). Xylomexicanin I (1) was isolated as a white powder. HRTOFMS and 13C NMR data analysis showed the molecular formula of 1 to be C27H30O8 with 13 indices of hydrogen deficiency. The 13C NMR spectroscopic data revealed that 1 contains eight olefinic and four carbonyl carbons. Accordingly, the five remaining indices of hydrogen deficiency required 1 to be pentacyclic. The 1H NMR, 13C NMR, and HSQC data (Table 1 and Figure S4, Supporting Information) showed the presence of five methyl, two methylene, 10 methine (five

Figure 1. Xylomexicanins I and J.

Received: April 8, 2017 Published: August 22, 2017

© 2017 American Chemical Society and American Society of Pharmacognosy

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DOI: 10.1021/acs.jnatprod.7b00305 J. Nat. Prod. 2017, 80, 2547−2550

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Table 1. NMR Spectroscopic Data (Methanol-d4) for Xylomexicanin I (1) and J (2) xylomexicanin I (1) position

δC

δH (mult., J, Hz)

1 2 3 4 5 6a 6b 7 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16 17 18 19 20 21 22 23 28 29a 29b 30 7OMe 1′ 2′

201.3 149.2 58.5 38.4 49.1 35.2

--2.91(t, 3.2) -2.12 (ddd, 11.4, 5.5, 4.8) 2.63 (dd, 16.5, 5.5) 2.23 (dd, 16.5, 4.8) ---2.26 (dq, 11.4, 7.1) 3.30 (m)

175.4 77.9 210.7 46.7 47.2 34.3 39.6 170.1 115.0

1.74 (dd, 13.7, 9.7) 2.13 (m) --6.28 (s)

165.8 81.9 17.9 16.4 120.9 142.6 110.5 144.3 18.4 25.4

-5.35 1.02 1.15 -7.58 6.48 7.55 0.90 1.02

141.7 52.2

6.59 (d, 3.2) 3.68 (s)

xylomexicanin J (2) δC

NOESY

11, 12β, 29 6a, 6b, 19, 28 5, 6b, 28, 29 5, 6a, 28

98.2 42.6 76.8 37.9 36.4 32.5 175.8 60.9 48.4 42.0 19.1

19 3, 12α, 29 11, 12β, 18 3, 12α, 17

34.6 36.9 47.1 34.2

30

(s) (s) (d, 7.1)

12β, 18, 21, 22 12α, 17, 21, 22 5, 10

(m) (m) (t, 1.6) (s) (s)

17, 18 17, 18, 23 22 5, 6a, 6b, 29 3, 6a, 11, 28 15

174.6 80.6 26.7 14.5 121.8 142.5 111.0 144.2 15.0 68.2 60.2 52.3 171.7 20.5

olefinic), and 10 nonprotonated carbons (one oxygenated tertiary, two keto and two ester carbonyls, three olefinic, and two sp3 carbons). In addition, three tertiary methyls [δH 1.02 (s), 0.90 (s), and 1.02 (s); δC 17.9, 18.4, and 25.4], one methoxy (δH 3.68; δC 52.2), and a β-substituted furanyl moiety [δH 7.58 (br s), 6.48 (br s), and 7.55 (t); δC 120.9, 142.6, 110.5, and 144.3] were assigned according to the 1H and 13C NMR data. These spectroscopic data indicated the limonoid nature of 1. The constitutions of rings A, B, C, D, and E (Figure 2) were deduced by analysis of 1H−1H COSY, HMBC, and HSQC data. The A-ring proton spin spin coupling system from H2-6 to H3-19 through H-5 and H-10 was observed via 1H−1H COSY correlations. HMBC cross-peaks (H-6/C-4, H-6/C-5, H-6/C-7, and methoxy protons at δH 3.68 to C-7) suggested that C-6 is connected to a methoxycarbonyl group [δH 3.68 (s); δC 52.2, 175.4]. The HMBC cross-peak of H3-19 to C-1 of the α,βunsaturated carbonyl carbon (δC 201.3) indicated the connection of C-10 to C-1. Furthermore, H-3 [δH 2.91 (t)] and two geminal methyl singlets resonating at δH 0.90 and 1.02 all exhibited HMBC cross-peaks to the C-4 quaternary carbon atom and the C-5 sp3 methine carbon. Assignment of ring A as a cyclohexanone with branch points at C-11, C-12, and C-30

δH (mult., J, Hz) -2.90 5.23 -2.75 2.46 ---2.09 -1.84 1.68 1.30 2.01 -1.60 2.91 3.30 -5.32 1.01 1.04 -7.64 6.54 7.53 0.62 3.96 3.44 3.18 3.70

NOESY

(dd, 10.2, 2.3) (d, 10.2)

3 2, 28, 29b

(m, 8.5) (m)

6, 11β, 15β, 28 5, 19, 28, 29a

(dd, 13.6, 4.7)

11α, 12α, 19

(br.dq, 12.8, 4.0) (br.qd, 13.6, 2.4) (m, 3.0) (dt, 14.3)

9, 11β, 12β, 19 5, 11α, 17 9, 12β, 18 11α, 12α, 17, 21, 22

(dd, 13.1, 5.4) (o) (o)

30, 15α, 18 14, 15β, 30 5, 15α, 17

(s) (s) (s)

11β, 12β, 15β, 18, 21, 22 12α, 14, 17, 21, 22 6, 9, 11α, 29a

(br.s) (br.s) (t, 1.7) (s) (d, 9.7) (dd, 9.7, 1.8) (d, 2.3) (s)

12β, 17, 18 12β, 17, 18, 23 22 3, 5, 6, 29a, 29b 6, 19, 28, 29b 3, 28, 29a 14, 15α

-2.23 (s)

Figure 2. Key HMBC (H→C) correlations of 1 and 2. The bold bonds denote 1H−1H COSY correlations.

was indicated by the 1H−1H COSY correlations of H2-12 to H11, H-11 to H-3, and H-3 to H-30. The α,β-unsaturated δ-lactone structure of ring D was indicated by analysis of NMR chemical shifts [δH 6.28 (s), 5.35 (s); δC 39.6, 170.1, 115.0, 165.8, 81.9], and further supported by the HMBC cross-peaks (H-15/C-13, H-15/C-16, and H-17/ C-14) (Figure 2). The HMBC cross-peaks of H-17 to C-20, C2548

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21, and C-22 showed that the furanyl moiety E is connected to C-17. HMBC cross-peaks also indicated that the 18-methyl and 12-methylene groups are attached to C-13, thus establishing that ring D is connected with ring B via the C-11−C-12 bond. In addition, the connection of the oxygenated tertiary C-8 to C14 was elucidated by the HMBC cross-peak of H-15 to C-8. The remaining ketocarbonyl C-9 was located between C-8 and C-11, as shown by an H-12α to C-9 HMBC cross-peak. Finally, C-8 and C-30 was connected to complete the carbon architecture, and a hydroxy group connected to C-8 satisfies the molecular formula. Thus, rings B and C had a bicyclo[3.3.1]nonane core bridged through C-8, C-9, and C11. In the 1H NMR spectra, the 3.3 ppm region was occupied by a large methanol-d4 solvent peak. After changing the solvent to CDCl3, the proton resonating at 3.3 ppm could be assigned as H-11. NOESY analysis was used to elucidate the relative configuration of 1. A series of NOE correlations between H6a/H3-29, H3-29/H-3, H-3/H-12β, and H-12β/H-17 revealed that these protons are cofacial. This was also confirmed by the observation of NOE associations between H3-19/H-5, H-5/H328, H-11/H-12α, and H-12α/H3-18. The orientation of the 8OH group must be α, cis-to H-11, because the alternative highly strained “in−out” trans intrabridged structure is incompatible with a bicyclo[3.3.1]nonane skeleton. According to the above results, the relative configuration of 1 was determined as shown in the MM2 generated 3D structure (Figure 3). Xylomexicanin I (1) is the first limonoid with bridged B and C rings. The natural precursor of 1 might be xylomexicanin F, which is also present in Xylocarpus granatum.4d Following C-30 oxygenation of xylomexicanin F (3 in Scheme 1), an acyloin rearrangement of the α-hydroxycarbonyl moiety would afford 4. Enolate addition to the allylic alcohol moiety furnishes 1.

Scheme 1. Plausible Biosynthetic Formation of 1

Xylomexicanin J (2) was isolated as a white powder. HRTOFMS and 13C NMR data analysis showed the molecular formula of 2 to be C29H36O10 with 12 indices of hydrogen deficiency. 13C NMR analysis of 2 revealed four olefinic and three carbonyl carbons. Thus, the structure of 2 must contain seven rings. The 1H NMR and 13C NMR spectroscopic data (Table 1) revealed the presence of five methyl, five methylene, 10 methines (three oxygenated and three olefinic), and four nonprotonated carbons (one hemiacetal, three ester carbonyl, and one olefinic). Moreover, three tertiary methyl groups [δH 1.01 (s), 1.04 (s) and 0.62 (s); δC 26.7, 14.5, 15.0], a methoxy group (δH 3.70; δC 52.3), and a β-substituted furanyl moiety [δH 6.54 (br. s), 7.53 (t), and 7.64 (br. s); δC 111.0, 144.2, 142.5, and 121.8] were distinguished by the 1H and 13C NMR data. The presence of an acetoxy group at C-3 was confirmed by the resonances at δH 2.23 (3H, s, 2′-Me); δC 20.5 and 171.7 (C-1′) and HMBC cross-peak between H-3/C-1′. Furthermore, the HMBC cross-peaks between H-3/C-4, H-3/C-30, H3/OCOCH3, H-29a/C-1, H-29b/C-4, H3-28/C-29, H3-28/C-5, and H3-28/C-3 indicated that the A ring was as shown in Figure 2. The HMBC cross-peaks between H-17/C-22, H-17/ C-23, H3-18/C-8, H3-18/C-14, H-15β/C-8, and H-15β/C-13 indicated the connectivity of the B, C, D, and E rings. Compound 2 showed significant NOESY cross-peaks between H-29b/H-3, H3-28/H-5, H-5/H-15β, H-15α/H-30, H-9/H-19, and H-15β/H-17 (Figure 3). According to the above information, the structure and relative configuration of 2 was elucidated as shown in Figure 1. Xylomexicanin J (2) is the 3de-O-tigloyl-3-O-acetyl congener of xyloccensin L (xylolactone) isolated from the stem bark of X. granatum.5

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Jasco DIP-370. NMR analysis was performed using a Bruker AV-600; at 600 MHz (1H) and 151 MHz (13C) in methanold4 (δ in ppm rel. to Me4Si as an internal standard, J in Hz). MS analysis was performed using a QStar XL QqTOF from Applied Biosystems. Chromatography was carried out with silica gel 200−300 mesh (Qingdao Marine Chemical Factory, China). Semipreparative HPLC was performed using a Waters Delta Prep 3000 pump with a

Figure 3. Relative configurations of 1 and 2. The arrows denote selected NOESY correlations. The 3D structures were calculated with MM2 [Chem3D program version 10.0, CambridgeSoft, MS (USA)]. 2549

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UV 2487 detector, and a Whatman Partisil 10 ODS-2 column (9.4 × 250 mm). Plant Material. X. granatum seeds were collected in March 2012 at Hainan Island, Southern China and dried. Identification was performed by Dr. Wen-Qing Wang, School of Life Sciences, XiaMen University, People’s Republic of China. Several voucher specimens (No. HEBNMC-2012-1) were deposited in the herbarium of the School of Pharmaceutical Sciences, Hebei Medical University, China. Extraction and isolation. Dried seeds (10 kg) of X. granatum were extracted with 95% EtOH (30 L × 3) at ambient temperature. The EtOH extract was evaporated in vacuo, and the residue (1 kg) was suspended in H2O (6 L) and successively extracted with petroleum ether (5 L × 3) and CH2Cl2 (5 L × 3). The CH2Cl2 extract (421 g) was purified by silica gel column chromatography using a petroleum/ acetone gradient (10:1 to 1:3) to yield 66 fractions. Fraction 36 (500 mg) was further separated on a semipreparative HPLC column with MeOH/H2O (53:47) as a mobile phase to yield 1 (5 mg, tR = 26.5 min) and 2 (7 mg, tR = 21.7 min). Xylomexicanin I (1). White powder, [α]24D +14 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 220 (2.56) nm; IR (KBr) νmax 3327, 1734, 1709, 1640, 1257 cm−1; HRTOFMS m/z 482.1937 (calcd for C27H30O8, 482.1935). Xylomexicanin J (2). White powder, [α]24D −47 (c 0.1, CHCl3); UV (CHCl3) λmax nm 214; IR (KBr) 3600−3200, 1740−1715 cm−1; HRTOFMS m/z 544.2302 (calcd for C29H36O10, 544.2303).

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682−685. (f) Cui, J.; Wu, J.; Deng, Z.; Proksch, P.; Lin, W. J. Nat. Prod. 2007, 70, 772−778. (4) (a) Shen, L. R.; Dong, M.; Guo, D.; Yin, B. W.; Zhang, M. L.; Shi, Q. W.; Huo, C. H.; Kiyota, H.; Suzuki, N.; Cong, B. Z. Naturforsch. 2009, 64C, 37−42. (b) Huo, C. H.; Guo, D.; Shen, L. R.; Yin, B. W.; Sauriol, F.; Li, L. G.; Zhang, M. L.; Shi, Q. W.; Kiyota, H. Tetrahedron Lett. 2010, 51, 754−757. (c) Ni, Z. Y.; Wu, Y. B.; Zhang, M. L.; Wang, Y. F.; Dong, M.; Sauriol, F.; Huo, C. H.; Shi, Q. W.; Gu, Y. C.; Kiyota, H.; Cong, B. Biosci. Biotechnol. Biochem. 2013, 77, 736−740. (d) Wu, Y. B.; Qing, X.; Huo, C. H.; Yan, H. M.; Shi, Q. W.; Sauriol, F.; Gu, Y. C.; Kiyota, H. Tetrahedron 2014, 70, 4557−4562. (e) Wu, Y. B.; Liu, D.; Liu, P. Y.; Yang, X. M.; Liao, M.; Lu, N. N.; Sauriol, F.; Gu, Y. C.; Shi, Q. W.; Kiyota, H.; Dong, M. Helv. Chim. Acta 2015, 98, 691−698. (5) (a) Wu, J.; Zhang, S.; Xiao, Q.; Li, Q. X.; Huang, J. S.; Long, L. J.; Huang, L. M. Tetrahedron Lett. 2004, 45, 591−593. (b) Wu, J.; Xiao, Q.; Li, Q. X. Biochem. Syst. Ecol. 2006, 34, 838−841.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00305. Detailed experimental procedures, 1D and 2D NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hiromasa Kiyota: 0000-0002-1330-6522 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (81602978), Hebei Province Outstanding Youth Science Fund Project (H2015206482), Hebei Medical University Development Project (2016-kyfz111), Syngenta Ltd (2016-Hebei Medical University-Syngenta-03), JSPS KAKENHI Grant Numbers 19580120, 22560112, 25450144 and 17K07772), and the JSPS Postdoctoral Fellowship for Foreign Researchers (P14392).

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