Xylomexicanins K-N: limonoids from the leaves and twigs of Xylocarpus granatum

Abstract Six new compounds, xylomexicanins K-N (1–4), granasteroid (5) and 5-methoxy-2-pentylbenzofuran-7-ol (6), along with nine known compounds were isolated from the leaves and twigs of Xylocarpus granatum. Among them, 1 was a biogenetic precursor of 1,8,9-phragmalin limonoid, and 4 represent the first example of degraded A-ring limonoid. The structures of them were elucidated on the basis of one- and two-dimensional NMR spectroscopic data (including 1H, 13C-NMR, DEPT, 1H-1H COSY, HSQC, HMBC, and NOESY) and confirmed by high-resolution mass spectrometry.


Results and discussion
Xylomexicanin K (1) was obtained as a white power. The molecular formula was deduced as C 33 H 40 O 14 with fourteen degrees of unsaturation by HRTOFMS. The 13 C-NMR spectral data (Table S1) revealed that 1 contains three C ¼ C and five C ¼ O groups. Therefore, the remaining six unsaturations indicated that 1 contains six rings. The 1 H-and 13 C-NMR spectral data exhibited signals of seven Me, three CH 2 , ten CH groups (four O-bearing and four olefinic ones), and thirteen quaternary C-atoms.  9, 110.2, 142.5, 120.6] were assigned. The 13 C-NMR spectra implied a C ¼ C bond at C14/15 (d C 120.5, 169.9). These spectroscopic data of 1 indicated the limonoid nature, thus the connectivity of the tetra carbocyclic core structure was elucidated following the literature data and confirmed by 2 D NMR analyses ( Figure S2), as detailed for compound 3. The HMBC correlations from Me protons to the respective 13 C resonances explained the three tertiary Me groups located at C-18, C-19, and C-28.  (Cui et al. 2007), probably due to a ring current effect by the furan ring. Detailed analysis of the 1 H-1 H COSY and HMBC correlations further established the structure of 1 is a typical limonoid with three rarely unbonded OH groups at C-1, C-8 and C-9.
The relative configuration of 1 was defined on the basis of the NOESY spectrum. H-12 exhibited a NOE with H-17, but not with Me-18; Me-18 displayed a NOE with H-22, indicating that the furan ring, Me-18 and 12-OAc were on the same side. The NOE between H-11b/H-18, H-11b/1-OH, H-11b/9-OH, H-19/1-OH, H-19/9-OH, 8-OH/9-OH, but without NOE between H-30/1-OH, H-30/9-OH confirmed that 1-OH, 8-OH, 9-OH, Me-19 and 30-OAc were on the same side. The NOE correlations from H-2 to H-29b, from H-3 to H-29b and from H-5 to H-12 evidenced H-29, H-2, H-3 were on the same side and H-5 on the other side. Based on the above results, the relative stereochemistry of 1 was elucidated as shown in Figure 1.
Xylomexicanin L (2) was obtained as a white power. The molecular formula was deduced as C 35 H 42 O 16 with fifteen degrees of unsaturation by HRTOFMS. The 13 C-NMR spectral data (Table S1) revealed that 2 contains one C ¼ C and six C ¼ O groups. Therefore, the remaining eight unsaturations indicated that 2 consisted eight rings. The 1 H-NMR and 13 C-NMR spectra exhibited signals of eight Me (one MeO and four tertiary Me), four CH 2 , nine CH groups (five O-bearing and one olefinic), and fourteen quaternary C-atoms (four O-bearing, six ester, and one olefinic C-atoms). The spectroscopic data showed it is a phragmalin orthoester, characterized by a methyl singlet at d H 1.63 (s) and the HMBC correlation with a quaternary carbon at d C 119.0 for a 1,1,1trioxyethyl moiety. Typical oxygenated carbon resonances at d C 83.4, 84.3, and 86.5 further substantiated this orthoester identification. The HMBC correlations well supported the limonoid core framework ( Figure S7). The 1 H-NMR spectra exhibited three acetyl Me groups at d H 2.17 (s), 1.90 (s), 2.01 (s) as deduced by the HMBC correlation from Me protons to the respective C ¼ O resonances located AcO groups at C-3, C-12, and C-30, respectively. The above analyses of the 1 H-NMR, 13 C-NMR spectra suggested that 2 was a phragmalin orthoester limonoid with an a,b-unsaturated lactone ring [d H 6.25 (br. s), 7.24 (s); d C 97.0, 147.9, 134.6, 167.1] at C-17 by the HMBC correlation from H-17 to C-20. Detailed analysis of the 1 H-1 H COSY and HMBC correlations further established the connectivities of 2.
The relative configuration of 2 was defined on the basis of the NOESY spectrum. The NOE correlations from H-6b to H-19 and H-28, from H-28 to H-3, from H-3 to H-29b, from H-29a to H-19 evidenced the configuration of the bicyclo[2.2.1]heptane ring. The NOE between H-17/H-12, H-17/H-30, H-17/H-23 indicated that the a,b-unsaturated lactone ring, 23-OH, 12-OAc and 30-OAc were on the same side. Based on the above results, the relative stereochemistry of 2 was elucidated as shown in Figure 1.
Xylomexicanin M (3) was obtained as a white power. The molecular formula was deduced as C 35 H 42 O 14 with fifteen degrees of unsaturation by HRTOFMS. The 13 C-NMR spectral data (Table S1) revealed that 3 contains two C ¼ C and five C ¼ O groups. Therefore, the remaining eight unsaturations indicated that 3 consisted eight rings. The 1 H-NMR and 13 C-NMR spectra exhibited signals of eight Me, four CH 2 , ten CH groups (four O-bearing and three olefinic ones), and thirteen quaternary C-atoms (four O-bearing, five ester, and one olefinic C-atoms). In addition, three Me groups (d H 1.21, 1.05, 0.92; d C 13.9, 15.5, 14.2), one MeO group (d H 3.74, d C 51.6), and a b-substituted furan ring [d H 6.43 (br. s), 7.38 (br. t), 7.46 (br. s); d C 109.8, 142.9, 140.9, 120.6)] were assigned. The spectroscopic data indicated a phragmalin 1,1,1-trioxyethyl derivative as in 2. Typical oxygenated carbon resonances at d C 83.0, 84.5, and 86.2 further substantiated this orthoester identification. The structure information of the limonoid core was obtained as following according to 2 D NMR analyses ( Figure S12 Compound 3 showed significant NOESY cross-peaks from H-28 to H-3, from H-29a to H-19, from H-29b to H-3, from H-5 to H-12 and H-30, from H-12 to H-17, to H-21, from H-17 to H-21 ( Figure S12). Based on the above information, the relative stereochemistry of 3 was elucidated as shown in Figure 1.
Xylomexicanin N (4) was obtained as a colourless oil. The molecular formula was deduced as C 15 H 24 O 5 with four degrees of unsaturation by HRTOFMS. The 13 C-NMR spectrum revealed that 4 contains one C ¼ C and two C ¼ O carbons. Therefore, the remaining one unsaturation required 4 to be monocyclic. The 1 H NMR, and 13 C NMR spectra (Table S2) showed the presence of six Me, one CH 2 , four CH groups (one oxygenated and one olefinic), and four quaternary carbons (one keto, one ester, and one olefinic carbon). In addition, two tertiary Me [d H 1.19 (s) and 1.05 (s); d C 28.1 and 20.3], three MeO (d H 3.69, 3.35 and 3.34; d C 51.9, 54.3 and 54.2), and one keto C ¼ O (d C 199.0) groups were also observed in the 13 C NMR spectrum. Furthermore, two geminal Me singlets resonated at d H 1.19 and 1.05 exhibited HMBCs to the quaternary carbon atom (C-4) and a saturated CH carbon C-5, which implied that C-4 was located between C-3 and C-5, bearing two Me groups. The mentioned spectroscopic data implied an A-ring of limonoid type feature of 4, the same numbering as limonoid were kept to show the similarity.
The relative stereochemistry of 4 was elucidated as 5,10-trans, considering the biogenetic origin and that no NOE correlation was observed between H 2 -6ab to H-19. ( Figure S18).
The biogenetic precursor of 4 might be xylomexicanin F (B-ring seco-limonoid), found also in this plant by us (Wu et al. 2014). The C-9-C-30 bond of B-ring may be ruptured to form 4.
Granasteroid (5) was obtained as a white power. The molecular formula was deduced as C 29 H 44 O 3 with eight degrees of unsaturation by HRTOFMS. The 13 C-NMR spectrum revealed that 5 contains two C ¼ C and two C ¼ O groups. Therefore, the remaining four unsaturations demonstrated that 5 consisting of four rings. The 1 Hand 13 C-NMR spectral data (Table S2) C 199.5, 171.2, 123.8, 38.5, 35.7, and 33.9, elucidated 5 was to contain an a,b-unsaturated carbonyl structural unit in ring A. The presence of a COOH group was also determined from the 13 C-NMR chemical shifts d C 176.3. The HMBC spectra from H-20 to C-21 ( Figure S23) allowed us to determine the position of the COOH group to be at C-20. The HMBCs from 18-Me to C-12, to C-13, to C-14 and to C-17; from 19-Me to C-1, to C-5, to C-9 and to C-10; from 26-Me to C-24 and to C-25, from 27-Me to C-24, to C-25 and C-26; indicated that 5 was a typical steroid. On the basis of above findings and other detailed NOE correlations, the structure of 5 was fully established as a rare 21-carboxy steroid (Su et al. 2007, Chao et al. 2008. 5-Methoxy-2-pentylbenzofuran-7-ol (6) was obtained as a yellow viscous liquid. The molecular formula was deduced as C 14 H 18 O 3 with six degrees of unsaturation by HRTOFMS. The 1 H-NMR and 13 C-NMR spectra (Table S2)  .13 (s) to C-7a and C-6 implied that the OH was located at C-7; from H-1 0 to C-1, C-2, C-10 and from H-5 0 to C-3 0 , C-4 0 implied that the pentyl group was assigned to C-2 ( Figure S28). The NOSEY correlations from 5-OMe to H-6 and to H-4 allowed an assignment to a 5-OMe group.
On the basis of above findings and other detailed NOE correlations, the structure of 6 was fully established as a 5-methoxy-2-pentylbenzofuran-7-ol.

General
Optical rotations were measured with a Jasco DIP-370. NMR analysis was performed using a Bruker AV-600; at 600 MHz ( 1 H) and 151 MHz ( 13 C) (d in ppm rel. to Me 4 Si as an internal standard, J in Hz). MS analysis was performed using a QStar XL QqTOF (ESI) 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 UV 2487 detector, and a Whatman Partisil 10 ODS-2 column (9.4 Â 250 mm).

Plant material
Twigs and leaves of X. granatum were collected in March 2012 at Hainan Island, Southern China, dried at ambient temperature, and identified by Dr. Wen-Qing Wang, School of Life Sciences, Xia-Men University, China. Several voucher specimens (No. HEBNMC-2012-2) have been deposited in the herbarium of School of Pharmaceutical Sciences, Hebei Medical University, China.

Disclosure statement
No potential conflict of interest was reported by the authors