Novel and Neuroprotective Tetranortriterpenoids from Chinese Mangrove Xylocarpus granatum Koenig

Eight new tetranortriterpenoids (1–8) were isolated from the twigs and leaves of the Chinese mangrove plant Xylocarpus granatum, together with four related known ones (9–12). The structures of new compounds were elucidated by detailed spectroscopic analysis. The absolute configuration of 9-epixylogranatin A (1) was determined by time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) calculations of the solution conformers. Xylogranatumin A (2) represents the first example of the 9, 10-seco limonoid with an unprecedented oxygen-bridged B ring (2,7-dioxabicyclo[2.2.1]-heptane). All the isolates were evaluated for the in vitro neuroprotective activity, both compounds 11 and 12 displayed moderate effects against H2O2-induced neurotoxicity in PC12 cells at the concentration of 10 μM, with an increase in cell viability of 12.0% and 11.6%, respectively.

compounds. Four known of them were readily identified by comparison of their spectroscopic data with those reported in the literature to be xylogranatin A (9) 4 , xylocarpin D (10) 17 , xylocarpin B (11) 17 , and xylocarpin G (12) (Fig. 1). 17 On the basis of careful analysis of NMR data, and by comparison with the known compounds, the eight new natural products were determined to be tetranortriterpenoids. Among them, 1-6 all showed characteristic signals of a α-furyl ring at C-17 position, whereas compound 7 possessed a γ-hydroxybutenolide group instead. At the same position, while differed from 1-7, compound 8 exhibited a γ-butyrol lactone moiety. Accordingly, the detailed structure elucidation of these new molecules is described as follows.
9-epixylogranatin A (1) was isolated as a colorless gum. The molecular formula of 1 was determined to be C 34 H 42 O 12 12 , which is the same as that of the co-occurring tetranortriterpenoid, xylogranatin A (9), previously isolated from the seeds of the same species. 4 The NMR data of 1 showed diagnostic signals of a furan ring [δ C 109.8, 119.7, 141.2, and 142.9; δ H 6.34 (1H, d, J = 0.9 Hz), 7.41 (1H, s), and 7.41 (1H, t, J = 0.9 Hz)], an α, β-unsaturated lactone group (δ C 117.7, 163.7, and 165.5; δ H 6.12 s), and a tetrasubstituted double bond (δ C 119.7, C-10 and 136.6, C-1). Detailed analysis of 1D (Tables 1 and 2) and 2D NMR (Fig. 2) spectra revealed that the planar structure of 1 was identical to that of 9. 4 In fact, the 13 C NMR data from C-3 to C-7 and C-16 to C-23 of 1 are almost the same as those of 9, and the main differences at C-1, C-2, C-8, C-9, C-10, C-12, C-15 and C-30, indicated the different stereochemistry between 1 and 9 in certain chiral centers. The relative configurations of six stereocenters at C-2, C-3, C-5, C-13, C-17, and C-30 were elucidated to be the same as those of 9 by the comparison of their ROESY spectral results, as well as by analysis of the coupling constants and splitting patterns of H-2 (δ H 3.30, dd, J = 11.1, 3.8 Hz), H-3 (δ H 5.02, d, J = 3.8 Hz), and H-30 (δ H 5.10, d, J = 11.1 Hz). NOE correlations observed of H-2/H-3, H-2/CH 3 -28, H-3/CH 3 -28, and H-3/Ac-30 ( Fig. 2) suggested that both H-2 and H-3 were α-oriented. On the contrary, the β-orientation of H-5 and H-30 was determined by the significant NOE cross-peak of H-5/H-30. The coupling constants of H-2/H-3 (J = 3.8 Hz) and H-2/H-30 (J = 11.1 Hz) indicated that both H-2 and H-30 were axial orientation, whereas H-3 was equatorial orientation, which further supported the relative configurations of H-2, H-3, and H-30. The diagnostic NOE correlations of H-30/H 2 -11 and H-30/H 2 -12 revealed that rings B and C were cis-fused, and both 8-OH and 9-OH were α-oriented. Thus, compound 1 was established as 9-epimer of xylogranatin A (9). With the aim to determine the absolute configuration of 1, time-dependent density functional theory electronic circular dichroism calculations (TDDFT-ECD) of its solution conformers were carried out. The ECD spectrum of 1 was recorded in acetonitrile (MeCN), which showed positive, negative and positive Cotton effects (CEs) at 270, 243 and 225 nm, respectively. The initial Merck Molecular Force Field (MMFF) conformational analysis of the arbitrarily chosen (2R, 3R, 5S, 8S, 9R, 13R, 17R, 30R) absolute configuration of 1 resulted in 151 conformers, which were re-optimized at B3LYP/6-31G(d) level of theory in vacuo, as well as at B3LYP/TZVP level with Polarizable Continuum Model (PCM) solvent model for MeCN. The gas-phase re-optimization afforded 9 conformers with Boltzmann population above 2%, while the PCM one yielded 14 geometries above 2% (see S46 in supporting information). These structures were used as input for ECD calculations. The C-3 tigloyl group is more suitable for an axial orientation in the low-energy conformers (conformers A-H), which is in accordance with the coupling constant between H-2 and H-3 ( 3 J 2-Hax,3-Heq = 3.8 Hz). Ring B possesses a twist-boat conformation with equatorial 30-OAc group in most of the conformers, which is in agree with the coupling constants between H-3 and H-30 ( 3 J 3-Hax,30-Hax = 11.1 Hz). As to prevent the side effects of the solvent, the ECD spectra were also calculated for the (2R, 3R, 5S, 8S, 9R, 13R, 17R, 30R) enantiomer with various functions (B3LYP, BH&HLYP, PBE0) and TZVP basis set for both the in vacuo and the solvent model re-optimized structures. The computed ECD curves of the individual conformers were quite similar to each other, and as well as to the experimental ECD spectrum, since their rings D and E share the same orientation. Moreover, all the Boltzmann-weighted ECD spectra reproduced well the experimental ECD curve with BH&HLYP/TZVP PCM/MeCN giving the most agreement (Fig. 3). Thus, the absolute configuration of 1 was unambiguously determined as (2R, 3R, 5S, 8S, 9R, 13R, 17R, 30R). To our best knowledge, this is the first report on the configurational assignment of limonoids with 9, 10-seco skeleton by TDDFT ECD calculations. The related xylogranatin A (9) was only reported for its relative configuration by the X-ray diffraction analysis 4 , while as it shared the same planar structure as that of 9-epixylogranatin A (1), the absolute configuration of 9 could then be determined by comparing their ECD spectra, which rely upon the unsaturated δ-lactone and furan chromophores of ring D governed by the C-13 and C-17 chirality centers. The similar ECD pattern of 9 [225 nm (Δ ε + 23.66), 269 nm (Δ ε + 1.57) in MeCN] and 1 indicated the same (13R, 17R) absolute configuration for both compounds. Thus the absolute configuration of xylogranatin A (9) can be deduced as (2R, 3R, 5S, 8S, 9S, 13R, 17R, 30R), which could further allow the absolute configurational assignment of the related xylogranatins B-D 4 .
The aforementioned data implied that compound 2 bore a similar skeleton as that of compound 1. The comparison of the 1D and 2D NMR data of 2 with those of the co-occurring limonoid 1 indicated that they shared the similar rings C, D, and E (Fig. 4). Correspondingly, the construction of rings A and B was important for the structure determination of 2. Two proton-bearing partial structures of C-19 → C-10 → C-5 → C-6 and C-3 → C-2 → C-30 were readily recognized from the 1 H-1 H COSY spectrum (Fig. 5). These two structural segments were connected to the quaternary carbons, C-1 and C-4, respectively, by the observation of HMBC correlations from Me-19 (δ 1.02, 3H, d, J = 6.6 Hz) to C-1/C-5, H-3 (δ H 5.52, 1H, d, J = 3.5) to C-1/C-30, H-2 (δ H 2.08, 1H, t, J = 3.5) to C-1, Me-28 to C-4/C-5 and Me-29 to C-3/C-4. In addition, the HMBC correlations of OH-9 to C-9 and C-8, as well as the typical hemiketal chemical shift of C-9 (99.8), revealed that the hydroxyl group (δ H 3.30, OH-9) linked to C-9, which was further connected to C-8. The methoxyl group was located at C-7 on the basis of the HMBC cross-peak from MeO-to C-7, and the assignment of the two acetoxyl groups at C-3 and C-30 were clearly indicated by HMBC correlations from H-3 (δ 5.52, 1 H, d, J = 3.5 Hz) to δ C 169.8 and H-30 (δ 5.30, 1 H, d, J = 3.5 Hz) to δ C 170.2 (Fig. 4).
In terms of ring B, the presence of one acetal carbon at δ C 109.2 (C-1, sp 3 ), one oxygenated quaternary carbon signal at δ C 89.6 (C-8, sp 3 ), a typical hemiacetal carbon at δ C 99.8 (C-9), and a tertiary oxygenated carbon at δ C 71.3 (C-30) suggested that ring B was constructed via the C-8 → C-30 → C-2 → C-1 → O → C-9 → C-8 bond to form a tetrahydropyran ring with an oxygen bridge between C-8 and C-1. The linkage of C-30 to C-8 was also confirmed by the distinct HMBC correlation from H-30 to C-9. A planar structure of 2 was thus proposed as depicted in Fig. 1, which was consistent with its molecular composition and degrees of unsaturation.
The relative configuration of compound 2 was elucidated by the analysis of ROESY spectrum and proton coupling constants, as well as by analogy with that of 1. The same relative stereochemistry of C-2, C-3, C-5, C-13 and C-17 in 2 was deduced from the similar carbon chemical shifts, proton coupling constants, and ROESY correlations with those of 1 and the known compounds 10-16 (for C-13 and C-17), as well as by a biogenetic consideration of such limonoids in Nature 2,17 . In addition, the NOE correlations between H-5 and CH 3 -19, H-10 and CH 3 -28, CH 3 -28 and H-3, H-3 and H-2, H-14 and CH 3 -18, H-14 and H-30, H-30 and OH-9 (Fig. 4) further confirmed the relative configuration of 2R*, 3R*, 5S*, 9S*, 10R*, 13R*, 14R*, 17R*, 30S*. Although the relative configurations of C-1 and C-8 cannot be determined by distinct ROE correlations, the correlation between H-30 and OH-9 implied the β -orientation of the oxygen bridge due to the smaller transannular strain.
Biogenetically, this interesting molecule might be derived from hainangranatumin D (Fig. 5), a limonoid previously isolated from X. granatum with absolute configuration established 18 , by a first plausible weak acid promoted nucleophilic addition of acetoxyl group at C-3 position, which allowed the double bond migration and the epoxidation from C-1 to C-9. The resulting intermediate then underwent a C-1 hydration, followed by a second acid promoted epoxidation from C-1 to C-8 with the elimination of H 2 O. Finally, a C-30 epimerization, which possibly occurred during the previous epoxidation to lower the energy of the molecule, allowed the production of compound 2. Since the relative stereochemistry has been established, the common biosynthetic origin of 2, compound 1, the known compounds 9-16 and hainangranatumin D 2,4,17,18 , suggested the corresponding chiral centers, especially C-13 and C-17 adjacent to the furan core should be the same. Therefore, the absolute configuration of compound 2 was deduced as (1S, 2R, 3R, 5S, 8S, 9S, 13R, 17R, 30S). Based on the above information, xylogranatumin A (2) was determined as a novel limonoid characterized by a 9, 10-seco skeleton bearing an oxygen-bridge between C-1 and C-8 (Fig. 1). and the discovery of xylogranatumin A provided a new example to the extremely diverse and complex family of limonoids.
6-O-acetyl xylocarpin D (3) gave a HRESIMS pseudomolecular ion peak at m/z 769.2634 [M + Na] + , a plus of 42 mass units on that of the co-occurring xylocarpin D (10), which was previously isolated from the fruits of X. granatum with absolute configuration determined 17 , indicating the presence of an additional acetyl group in 3, which was further confirmed by the careful comparison of their 1 H and 13 C NMR data (Tables 1 and 2), with an observation of additional peaks of δ H at 2.19 and δ C at 169.6 and 21.0 on 3. The location of the acetyl group at C-6 was established by the expected downfield shifted 1 H NMR resonance of H-6 (from δ H 4.29 to 5.30). Therefore, compound 3 was determined as the 6-acetyl derivative of xylocarpin D (10).
The HRMS data for 14-hydroxy-14,15-dihydrogranatumin C (4) displayed a pseudomolecular ion peak at 584.2643 [M] + (calcd 584.2621), consistent with the molecular formula C 32 H 40 O 10 . Detailed analysis of the 1 H and 13 C NMR data of 4 were reminiscent of those of 13, which was previously isolated from the seeds of a Krishna mangrove, X. granatum 19 . Their main differences were an oxygenated quaternary carbon at C-14 (δ C 62.7) and a     The relative configuration of 4, except C-14 position, was determined to be the same as that of 13 due to the similar 13 C NMR shifts and coupling constants in 1 H NMR and further confirmed by ROESY experiments. Unfortunately, the absence of the proton signal of OH-14 in 4, which were usually presented in the NMR spectra of the limonoids when measuring in CDCl 3 , such as Granaxylocarpin C 20 , bearing the similar substructure as compound 4, prevented us from assigning the configuration of C-14 position via the available NMR data.
The molecular formula of 9-O-methyl xylogranatin R (6), C 28 H 34 O 9 , was deduced by HRESIMS (m/z 537.2100, calcd for [M+ Na] + 537.2101). The 1 H and 13 C NMR spectra of 6 were almost identical to those of xylogranatin R (15), which was isolated as an antifeedant from the seeds of the same species with the absolute configuration established 5 , except for the presence of an additional methoxy group (δ H 3.68; δ C 51.8), suggesting that 6 was an O-methyl derivative of 15, in agreement with an addition of 14 mass units in 6 to that of 15. The chemical shift of C-9 (δ 172.8) in 6 was upfield shifted Δ δ 2.7 from that of 15, indicating the carboxylic acid at C-9 was esterified to methyl ester, which was further confirmed by the HMBC correlation from 9-OCH 3 (δ H 3.67) to the carbonyl carbon at C-9 (δ C 172.8). The complete assignments of the 1 H and 13 C NMR of 6 were achieved by a comprehensive analysis of 2D NMR spectra including HSQC, COSY, HMBC, and ROESY. Compound 6 was thus determined as the methyl ester of xylogranatin R (15). In view of the presence of methyl ester moiety in a great number of limonoids previously isolated from this species, such as xylogranatins A-D 4 , and hainangranatumins A-J 18 , the authors believe that compound 6 is an original natural product rather than an artifact.
The molecular formula of 30-O-acetylhainangranatumin E (7) (Tables 1 and 2). The detailed NMR data analysis reminded us those of hainangranatumin E (16), previously isolated from the seeds of Hainan mangrove X. granatum 18 . The only differences were the presence of the acetyl group (δ H 2.09 s; δ C 169.9/169.8 qC, 21.0 CH 3 ) at C-30 in 7 instead of the methylbutyryl group in 16. In addition, two sets of carbon resonances at δ 208.2/208.0 (C-9), 38.6/38.5 (C-23) in 13 C NMR spectrum suggested compound 7 was a mixture of unseparated C-23 epimers as depicted in Fig. 1. 1,2-dihydro-3α -hydroxy-turranolide (8) was isolated as a colorless gum. Its molecular formula, C 28 H 42 O 5 , was deduced by HREIMS at m/z 481.2892, a plus of two mass units to that of turranolide (17), previously isolated from the root bark of Turraea robusta collected from Awasi, Kisumu District, Kenya 21 . The NMR spectra of 8 were similar to those of 17, except for the presence of oxymethine signal at C-3 (δ H 3.42, t, J = 2.8 Hz) in 1 H NMR. A reduction of the ketone at C-3 in 17 to the hydroxyl group in 8 was then easily recognized, which further confirmed by the upfield 13 C NMR signal at 75.9 in 8 in replace of that at δ 216.2 in 17. In addition, the α-orientation of the hydroxyl at C-3 of 8 was confirmed by the ROESY correlations of H-3/CH 3 -29 and H-5/CH 3 -28. The structure of 1,2-dihydro-3α -hydroxy-turranolide (8) was thus determined as the C-3 reductive derivative of turranolide (17).
Similar as that of compound 2, since the relative stereochemistry of the new compounds 3, 5, 6, and 8 has been established, the common biosynthetic origin of such furan limonoids 2,4,17,18 suggested the corresponding chiral centers, such as C-13 and C-17 should be the same R configuration. Thus the absolute configuration of the above mentioned new compounds could be arbitrary determined as showed in Fig. 1.
In summary, eight new tetranortriterpenoids (1-8) together with four related known compounds (9-12) were isolated from the twigs and leaves of Chinese mangrove plant X. granatum. The structures of new compounds were elucidated by extensive spectroscopic analysis. The absolute configuration of 9-epixylogranatin A (1) was determined by TDDFT ECD calculations, which incidentally allowed the elucidation of the absolute configurations of xylogranatin A (9) by comparison with their ECDs, solving a puzzle of the previously reported natural products. The discovery of the 9, 10-seco limonoid 2 with a characteristic 2,7-dioxabicyclo [2.2.1]-heptane ring system added a novel skeleton to the family of tetranortriterpenoids, revealing the high diversity and complexity of such beautiful molecules.
Neuroprotective activity evaluation. In the light of a wide range of biological activities and pharmacological properties of limonoids 2,8 , we performed in vitro investigation of neuroprotective activity of compounds 1-12 on PC12 cells, since the isolated protolimoloids by us from Toona ciliata var. pubescens displayed significant cell protecting activity 11 . Both compounds 11 and 12 showed moderate neuroprotective effects against H 2 O 2 -induced neurotoxicity in PC12 cells at the concentration of 10 μM, with an increase in cell viability of 12.0% and 11.6%, respectively. N-Acetyl-L-cysteine (NAC) was used as the positive control with the increase in cell viability of 22.0% at 10 μM. In comparison with the tested structures, it is possible that the variation of rings A and B of these limonoids play an important role for the neuroprotective activity.

Methods
General experimental procedures. Optical rotations were measured on a Perkin-Elmer polarimeter