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Phragmalin-type limonoids from the roots of Trichilia sinensis †

Twelve new phragmalin-type limonoids, including trichisintons A – D ( 1 – 4 ), which contain an unusual bicyclo[5.2.1 4,10 ]decane motif in the tetranortriterpenoid core, and trisinenmalins A – C and E – I ( 5 – 7 and 10 – 14 ), together with the known compounds 15-acetyltrichagmalin C ( 8 ) and trisinenmalin D ( 9 ), were isolated from the roots of Trichilia sinensis . Their structures were determined on the basis of the analysis of MS and NMR spectroscopic data, as well as by comparison with the literature. Compounds 5 – 7 and 10 – 14 displayed weak inhibitory activity against selected tumor cell lines. Compounds 2 – 4 , 6 – 9 and 12 – 14 exhibited weak acetylcholinesterase-inhibiting activity in vitro .


Introduction
Natural products play an important role in the discovery of new drugs, because the great structural diversity of natural products with their various signicant biological activities has always been an important source of inspiration for medicinal chemists in their search for new drug leads with pharmacological activities. [1][2][3][4] Limonoids, which are a class of tetranortriterpenoids with a b-substituted furan ring, are secondary metabolites that are mainly found in the Rutaceae and Meliaceae families 5 and have attracted continuous attention owing to their diverse structures and various pharmacological activities, including antitumor, antimicrobial, antiprotozoal 6,7 and neuroprotective effects. 8 Rearranged limonoids with three uncommon carbon frameworks (mexicanolide-type, phragmalin-type, and trijugintype) have been found in the Trichilia genus, 7 among which the phragmalin-type and modied phragmalin-type limonoids possess characteristic tricyclo[3.3.1 2,10 .1 1,4 ]decane and bicyclo [5.2.1 4,10 ]decane rings. 7 Trichilia sinensis Bentv., which is a member of the Trichilia genus (Meliaceae) known to be rich in limonoids, [9][10][11][12][13][14][15] triterpenoids, 16,17 diterpenoids 18,19 and steroids, 20,21 is a shrub that is mainly distributed in China and Vietnam 22 of which the fruits, leaves and roots have historically been used as folk medicines to treat abdominal pain caused by Ascaris lumbricoides, chronic osteomyelitis, scabies, eczema, 23 rheumatism and traumatic injury. 24 A previous investigation of the chemical constituents of T. sinensis revealed a series of mexicanolide-type limonoids, 25 whereas phragmalin-type limonoids have never been reported from this species.

Results and discussion
Structural determination of new compounds 1-4, 5-7 and 10-14 Trichisinton A (1) was isolated as a white, amorphous powder, and its molecular formula C 37 H 46 O 13 , which was established by HREIMS (m/z 698.2935 [M] + , calcd as 698.2938), requires 15 degrees of unsaturation. The IR absorption at 1750 cm À1 showed the presence of a carbonyl group. The 13 C NMR data ( Table 2) displayed 37 carbon resonances, which were classied as nine methyl (one O-methyl), four methylene, eleven methine (three olenic and four oxygenated), and 13 quaternary (three olenic and seven carbonyl) carbons, which was supported by the DEPT and HSQC spectra. A typical b-substituted furan ring (d H 6.40, 7.41, and 7.43; d C 109.8, 120.3, 142.0 and 143.5), two keto carbonyls (d C 219. 2, 199.9), and ve ester carbonyls (d C 167.6, 168.7, 173.9, 175.3, and 176.1) were evident from the NMR data (Tables 1 and 2). Furthermore, one acetoxy and two isobutyryloxy groups were inferred according to the 1 H-1 H COSY and HMBC correlations (Fig. 2). The above observations accounted for 11 of the 15 degrees of unsaturation and suggested that 1 is a limonoid analogue with four additional rings.  -29 were assigned by the HMBC  correlations with the ketone carbonyl C-1 (d C 219.2), C-3 (d C 83.8), C-4 (d C 45.7), C-5 (d C 40.9), C-10 (d C 54.1) and C-28 (d C 21.9). The key HMBC correlations from H-5 (d H 3.62) to C-1, C-3, C-4, C-6 (d C 33.1), C-9 and C-10, as well as from the singlet methyl protons at d H 0.95 (H 3 -19) to C-1, C-5 and C-10, suggested that a cyclopentanone ring was constructed by C-1, C-29, C-4, C-5 and C-10, which was characteristic of trichiliton-type limonoids with an unusual bicyclo [5.2.1 4,10 ]decane motif in the tetranortriterpenoid core. A comparison of the NMR data revealed great similarity to those of trichiliton A. 26 Detailed analysis of the 1D ( 1 H NMR, 13 C NMR and DEPT) and 2D NMR data ( 1 H-1 H COSY, HSQC and HMBC) indicated that both compounds shared the same skeleton, and the major differences were the substituents at C-3, C-15 and C-30, as well as the carbonyl carbon at C-2 instead of the double bond between C-2 and C-3 in the known compound trichiliton A, which was supported by the downeld chemical shi of C-2 (d C 199.9) and the HMBC correlations from H-3 (d H 5.33) and H-30 (d H 5.77) to C-2. In addition, the two isobutyryloxy groups at C-3 and C-15, respectively, and the acetoxy substituent at C-30 were assigned  13 Na 749.3149). The NMR spectroscopic data indicated that 2 had a similar skeleton to that of 1, except for the different substitution patterns at C-3 and C-15. Instead of isobutyryloxy groups, compound 2 possessed two 2-methylbutanoate groups at C-3 (d C 83.8) and C-15 (d C 64.4), respectively, on the basis of the corresponding 1 H-1 H COSY correlations of H-2 0 (H-2 00 )/H-3 0 (H-3 00 ), H-3 0 (H-3 00 )/H-4 0 (H-4 00 ) and H-2 0 (H-2 00 )/H-5 0 (H-5 00 ), as well as the HMBC correlations from H-2 0 (d H 2.67; H-2 00 , d H 2.28) to C-1 0 (d C 175.5; C-1 00 , d C 174.7). The attachment of these two moieties to C-3 and C-15 was deduced from the HMBC correlations from H-3 (d H 5.36) to C-1 0 (d C 175.5) and from H-15 (d H 6.62) to C-1 00 (d C 174.7), respectively (Fig. 2). The relative conguration was found to be the same as that of 1 by an ROESY experiment (ESI Fig. S92 †).
Trichisinton C (3), which was a white, amorphous powder, had a molecular formula of C 38 H 46 O 13 on the basis of its prominent HRESIMS ion peak at m/z 749.2578 ([M + K] + , C 38 H 46 O 13 K calcd as 749.2570). A comparison between the NMR data (Tables 1 and 2) of 1 and 3 showed that the tiglyloxy group at C-3 in 3 was not present in 1, which was conrmed by the HMBC correlations from H-5 0 (d H 1.98) to C-1 0 (d C 166.7), C-2 0 (d C 129.4) and C-3 0 (d C 140.1), and from H-4 0 (d H 1.82) to C-3 0 . The attachment of this moiety to C-3 was determined by the crosspeak of H-3 (d H 5.00)/C-1 0 in the HMBC spectrum. Similarly, instead of a 2-methylbutanoate group, an isobutyryloxy moiety was found to be connected to C-15 as a result of the HMBC correlations from H-2 00 (d H 2.42) to C-1 00 (d C 174.7) (Fig. 2). Therefore, the structure of trichisinton C (3) was determined, as shown in Fig. 1.
Trichisinton D (4), which was a white, amorphous powder, displayed a peak for [M + K] + at m/z 763.2726 (calcd for C 39 H 48 O 13 K 763.2726) in the HRESIMS spectrum, which was 14 mass units greater than that of 3. Detailed analysis of the 1 H and 13 C NMR data of 4 revealed a structure that was closely related to that of 3, and the major difference was the replacement of the isobutyryloxy group at C-15 by a 2-methylbutanoate group in 4. The 2-methylbutanoate group was conrmed by the 1 H-1 H COSY correlations of H-2 00 /H-3 00 , H-3 00 /H-4 00 and H-2 00 /H-5 00 , as well as the HMBC correlations from H-2 00 (d H 2.24) to C-1 00 (d C 174.1). The connection with C-15 was conrmed as a result of the HMBC correlations from H-15 (d H 6.41) to C-13 (d C 40.4), C-14 (d C 141.9), C-16 (d C 167.6) and C-1 00 (d C 174.1) (Fig. 2). Therefore, the structure of 4 was established, as shown in Fig. 1, with the same relative conguration as that of 1 according to the ROESY spectrum.
Trisinenmalin A (5) was isolated as a white, amorphous powder, and its molecular formula of C 40 H 50 O 14 , which was established by the HRESIMS spectrum (m/z 777.3099 [M + Na] + , calcd as 777.3098), requires 16 degrees of unsaturation. Its IR absorption bands showed the presence of hydroxy (3444 cm À1 ) and carbonyl groups (1729 cm À1 ). The 13 C NMR and DEPT spectroscopic data of 5 displayed 40 carbon signals, which was in agreement with the molecular formula, including ten methyl (one methoxy group), four methylene, eleven methine (four oxygenated and four olenic), and een quaternary carbons (six carbonyl and four olenic). Furthermore, one tiglyloxy, one isobutyryloxy, and two acetoxy groups were inferred according to the HMBC correlations and 1 H-1 H COSY correlations (Fig. 2). In addition, the typical b-furan moiety (d H 6.46, 7.39 and 7.57; d C 110.0, 120.6, 142.1 and 143.1) was deduced. The above fragments, together with the remaining 23 carbons, revealed that 5 was a limonoid with six rings, which was consistent with the   orientation of the corresponding protons. Therefore, the structure of compound 5 was nally established, as shown in Fig. 1 The relative conguration of 6 was assigned by an ROESY experiment (ESI Fig. S92 †) and was found to be the same as that of 5. Therefore, the structure of 6 was conrmed, as shown in Fig. 1.
Trisinenmalin C (7), which was obtained as a white, amorphous powder, displayed a molecular formula of C 39 H 50 O 13 , as determined by the prominent HRESIMS ion peak at m/z 765.2888 ([M + K] + , calcd as 765.2883), which was 42 mass units more than that of 6. MS and NMR (Tables 3 and 4) analysis indicated that 7 was an acetylated derivative of 6. On the basis of the coherent HMBC correlation from H-30 (d H 5.42) to the carbon resonance at d C 171.1, the acetoxy group was determined to be located at C-30. Thus, 7 was demonstrated to be the 30acetyl derivative of 6, and its relative conguration was found to be the same as that of 6 by an ROESY experiment. Therefore, the structure of 7 was established, as shown in Fig. 1.
Trisinenmalin E (10) was obtained as a white, amorphous powder with a molecular formula of C 41 H 52 O 15 (prominent HRESIMS ion peak at m/z 807.3208 [M + Na] + , calcd as 807.3204). The 1 H and 13 C NMR data of 10 indicated that its structure was closely related to that of trisinenmalin D (9), and the only difference lay in the presence of an isobutyryloxy group in 10 instead of the tiglyloxy moiety in 9, which was conrmed by the 1 H-1 H COSY correlations of H-2 0 /H-3 0 and H-2 0 /H-4 0 and the HMBC correlation from H-2 0 (d H 2.62) to C-1 0 (d C 176.3). The attachment of the isobutyryloxy group to C-3 was deduced from the HMBC correlation from H-3 (d H 5.31) to the carbon resonance at d C 176.3. Accordingly, the structure of trisinenmalin E (10) was established, as shown in Fig. 1, and the relative conguration was found to be the same as that of 9 according to the respective ROESY correlations.
Trisinenmalin F (11), which was a white amorphous powder, had the molecular formula of C 39 H 50 O 14 , as determined by the prominent HRESIMS ion peak at m/z 765.3109 ([M + Na] + , calcd as 765.3098). Analysis of the MS and NMR data of 11 indicated that its structure was closely related to that of trisinenmalin E (10), except for the absence of signals for acetoxy groups and the presence of a hydroxyl group in 11, which was consistent with the reduction of 42 amu in molecular weight, as was observed. Moreover, the chemical shi of C-1 (d C 84.1) in 11 was shied upeld (Dd C 4.5) in comparison with that in trisinenmalin E (10), which further conrmed that C-1 was substituted by a hydroxy group instead of an acetoxy moiety. The relative conguration was found to be the same as that of 10 by an ROESY experiment (ESI Fig. S92 †). Accordingly, the structure of trisinenmalin F (11) was determined, as shown in Fig. 1 13 C NMR data revealed that 12 was a structural congener of 11.
Detailed analysis of the 1D and 2D NMR data indicated that the major differences between these two compounds were the substituent groups at C-2 and C-3. The hydroxy group at C-2 and the 2-methylbutanoate group at C-3 were inferred from the corresponding HMBC correlations, respectively. On the basis of extensive analysis of all the data, compound 12, which was given the trivial name of trisinenmalin G, was established, as shown in Fig. 1, and assigned the same relative conguration as that of 11 by analysis of the ROESY spectrum.
Trisinenmalin H (13) was isolated as a white, amorphous powder. The molecular formula of C 39 H 50 O 14 was deduced from the positive prominent HRESIMS ion peak at m/z 781.2833 ([M + K] + , calcd for C 39 H 50 O 14 K 781.2832). By comparison, the 1 H and 13 C NMR data (Tables 3 and 4) of 13 were similar to those of 3) and C-2 were observed; moreover, this conclusion was consistent with the reduction of 42 amu in the molecular weight in comparison with that of 10. The ROESY spectrum displayed signals for the same relative conguration as that of 10. Thus, the structure of 13 was identied, as shown in Fig. 1. Trisinenmalin I (14) had an adduct ion peak at m/z 739.2727 [M + K] + in the HRESIMS spectrum, which was 42 amu less than that of 13, which indicated that 14 was a deacetylated derivative of 13. Moreover, the chemical shi of C-1 (d C 83.6) in 14 was shied upeld (Dd C 5.7) in comparison with that of C-1 (d C 89. 3) in trisinenmalin H (13), which indicated that C-1 was substituted by a hydroxy group. By extensive analysis of the 1D and 2D NMR data, the structure of 14 was established, as shown in Fig. 1.
Two known phragmalin-type limonoids were also isolated from the same extract. Compound 8 was identied as 15-acetyltrichagmalin C 11 on the basis of a comparison of its NMR and ESI-MS data with those of the reported compound, and compound 9 was conrmed to be trisinenmalin D 28 by spectroscopic data (UV, IR, 1D and 2D NMR) and HRESIMS analysis, as well as by comparison with the literature.
Trichisintons A-D were assigned to the class of limonoids that contain the unusual bicyclo[5.2.1 4,10 ]decane ring system. So far, compounds with this type of skeleton have only been found in four species, namely, Trichilia connaroides, 13,26 Xylocarpus granatum, X. moluccensis 29 and Chisocheton ceramicus. 30 Fang et al. 26 proposed a biosynthetic pathway of this type of compound, of which the direct precursors were 1,2dihydroxyphragmalin-type limonoids. However, they did not obtain any phragmalin-type limonoids during their isolation experiments. In this study, ve 1,2-dihydroxyphragmalin-type limonoids (6-8, 12, and 14) were identied, which provided a crucial factor to prove the abovementioned hypothesis of the biosynthesis of these types of compound. Accordingly, 7 and 14 were the direct precursors of 4 and 1, respectively, and were processed by cleavage of the carbon bond in the 1,2-diol.
In summary, 12 new and 2 known phragmalin-type limonoids were isolated from the roots of T. sinensis for the rst time.

Experimental section
General Optical rotations were measured in CHCl 3 with a Rudolph Autopol III polarimeter (Rudolph Research Analytical, USA) at 25 C. UV spectra were recorded using a Shimadzu UV-2550 spectrometer (Beckman, USA) and a DU800 spectrophotometer (Beckman, Brea, CA, USA). IR absorption was measured with a Nicolet 380 FT-IR instrument (Thermo, USA) using KBr pellets. 1D and 2D NMR spectra were recorded on Bruker Avance 500 NMR spectrometers (Bruker, Germany) using TMS as an internal standard. HREIMS spectra were recorded with an AutoSpecPremier spectrometer (Waters, Milford, MA, USA). HRESIMS spectra were recorded with an API QSTAR Pulsar mass spectrometer (Bruker, Germany). Column chromatography was performed with silica gel (60-80, 200-300 mesh,

Extraction and isolation
Air-dried and powdered roots of Trichilia sinensis (13.2 kg) were extracted three times with 95% EtOH at room temperature to afford a crude extract (

Cytotoxicity assay
A human chronic myelogenous leukemia cell line (K562), a human gastric carcinoma cell line (SGC-7901), and a human hepatocellular carcinoma cell line (BEL-7402) were bought from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology. An MTT assay was used to determine inhibition of the growth of K562, SGC-7901, and BEL-7402 cells in vitro, and taxol was used as a positive control. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU mL À1 penicillin, and 100 mg mL À1 streptomycin at 37 C under a 5% CO 2 atmosphere with 90% humidity. Cells in the logarithmic phase were used for experiments. The specic experimental procedures were the same as those described previously. 31

Bioassay of AChE inhibitory activity
The AChE inhibitory activity of these compounds was assayed by the spectrophotometric method developed by Ellman 32 with slight modications. S-Acetylthiocholine iodide, 5,5 0 -dithiobis(2-nitrobenzoic acid) (DNTB, Ellman's reagent) and AChE were purchased from Sigma Chemicals. The compounds were dissolved in DMSO. The reaction mixture (total 200 mL) containing phosphate buffer (pH 8.0), the test compound (50 mg mL À1 ), and acetylcholinesterase (0.02 U mL À1 ) was incubated for 20 min (30 C). The reaction was initiated by the addition of 20 mL DNTB (2.48 mg mL À1 ) and 20 mL acetylthiocholine iodide (1.81 mg mL À1 ) for the assay of AChE inhibitory activity. The hydrolysis of acetylthiocholine was monitored at 405 nm for one hour. Tacrine (Sigma-Aldrich, 99%) was used as a positive control with a nal concentration of 0.08 mg mL À1 , and DMSO was used as a negative control with a nal concentration of 0.1%. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition ¼ (E À S)/E Â 100 (where E is the absorbance at 405 nm of the solution without the test compound and S is the absorbance at 405 nm of the solution with the test compound). The values are expressed as the mean AE SD of triplicate experiments.