Ceramicines M–P from Chisocheton ceramicus: isolation and structure–activity relationship study

Ceramicines are a series of limonoids which were isolated from the bark of Malaysian Chisocheton ceramicus (Meliaceae) and show various biological activities. Ceramicine B, in particular, has been reported to show a strong lipid droplet accumulation (LDA) inhibitory activity on a mouse pre-adipocyte cell line (MC3T3-G2/PA6). With the purpose of discovering compounds with stronger activity than ceramicine B, we further investigated the constituents of C. ceramicus. As a result, from the bark of C. ceramicus four new ceramicines (ceramicines M–P, 1–4) were isolated, and their structures were determined on the basis of NMR and mass spectroscopic analyses in combination with NMR chemical shift calculations. LDA inhibitory activity of 1–4 was evaluated. Compounds 1–3 showed LDA inhibitory activity, and 3 showed better selectivity than ceramicine B while showing activity at the same order of magnitude as ceramicine B. Since 3, which possess a carbonyl group at C-7, showed better selectivity than 5, which possess a 7α-OH group, while showing activity at the same order of magnitude as 5, we also investigated the effect of the substituent at C-7 by synthesizing several derivatives and evaluating their LDA inhibitory activity. Accordingly, we confirmed the importance of the presence of a 7α-OH group to the LDA inhibitory activity.

With the purpose of discovering compounds with stronger LDA inhibitory activity than ceramicine B (5), we further investigated the constituents of C. ceramicus. As a result, four new ceramicines (ceramicines M-P, 1-4, Fig. 1) were isolated and their structures were determined on the basis of NMR and mass spectroscopic analyses in combination with NMR chemical shift calculations. In addition, on the basis of the LDA inhibitory activity of the isolated compounds, we further investigated the effect of the substituent at C-7 of 5. IR absorptions implied the presence of a,b-unsaturated ketone (1690 cm -1 ) and hydroxy (3350 cm -1 ) groups. 1 H and 13 C NMR data (Table 1) revealed 24 carbon resonances due to one carbonyl, two sp 2 quaternary carbons, four sp 3 quaternary carbons, six sp 2 methines, five sp 3 methines, three sp 3 methylenes, and three methyls. Among them, three sp 3 carbons (d C 73.5, 92.4, and 101.7) and two sp 2 methines (d C 141.1 and 143.8) were ascribed to those bearing an oxygen atom.
Analyses of the HSQC and 1 H-1 H COSY spectra (Fig. 2) revealed the presence of four partial structures: a (C-2 and C-3), b (C-9, C-11, and C-12), c (C-15 to C-17), and d (C-22 and C-23). HMBC correlations of H 3 -18 to C-12, C-13, C-14, and C-17 suggested the connectivity of b, c, C-14, and C-18 through C-13. HMBC correlations of H-17 to C-20, C-21, and C-22, and H-23 to C-20 and C-21 suggested the presence of b-furyl at C-17, and the correlation of H 2 -16 to C-14 completed the structure of ring D. The presence of ring C was deduced from the HMBC cross-peaks of H 3 -30 to C-7, C-8, C-9, and C-14, and the connectivity of b, C-1, C-5, and C-19 through C-10 was suggested by the HMBC correlations of H 3 -19 to C-1, C-5, C-9, and C-10. HMBC correlations of H-2 to C-10 and C-4, and H-3 to C-1 and C-5 suggested the presence of ring A. Finally, HMBC correlations of H-6 to C-4, C-5, and C-7, and H-7 to C-4, and the chemical shift of C-4 (d C 101.7) suggested the planar structure of 1 to be as shown in Fig. 2.
The relative configuration of 1 was assigned by analyses of the 1 H-1 H coupling constant data and the NOESY correlations (Fig. 3). First, H-6, H-17, CH 3 -19, and CH 3 -30 were assigned to be b-axially oriented from the NOESY correlations of H-6/H 3 -19 and H 3 -30, and H-12a/H-17 and H 3 -30, while H-9 and CH 3 -18 were deduced to possess aorientation from the NOESY correlations of H 3 -18/H-9 and H-12b. Both H-5 and H-7 should possess b-orientation since C-4 and C-7 can only be connected through an ether linkage, and the multiplicity pattern of H-6 (br s) further supports this assumption. The orientation of the epoxy group was assumed to be a on the basis of the NOESY correlation of H-15/H 3 -30 and the multiplicity of H-15 (d, 2.8 Hz). In the case of a b-oriented epoxy group, the multiplicity of H-15 would be a triplet. This assumption was further supported by DFT NMR chemical shift calculations of the two possible isomers, 2a with a-oriented epoxy group and 2b with b-oriented epoxy group. As can be seen in Table 2, among the two possible isomers 2a gave the smallest mean average difference (MAD) and root-mean-square difference (RMSD) between the calculated and experimental chemical shifts, indicating the 2a as the more likely structure.
Ceramicine O (3) was obtained as an optically active, IR absorptions implied the presence of ketone (1730 and 1680 cm -1 ) and hydroxy (3480 cm -1 ) groups. 1 H and 13 C NMR data (Table 1) of 3 were highly similar to those of 5 [2]. In comparison to 5, the 1 H and 13 C NMR data of 3 showed a carbonyl signal (d C 205.6) in place of an oxymethine (d H 4.23, d C 72.5), and downfield shifts of CH-6 and C-8 signals. These data suggested 3 to be the 7-oxo derivative of 5. The presence of a carbonyl at C-7 in 3 was   [3]. In contrast to ceramicine E, the 1 H and 13 C NMR data of 4 showed two olefinic methine signals (d H 6.01, d C 130.9 and d H 6.24, d C 146.8) instead of two oxymethine signals (d H 3.01, d C 57.5 and d H 3.35, d C 52.9), indicating the presence of an a,b-unsaturated ketone in 4 in place of an a,b-epoxy ketone in ceramicine E. In addition, the chemical shifts of H-6 (d H 4.18) and H-7 (d H 5.38) of 4 indicate the presence of an acetyl at C-7. The structure of 4 was further confirmed by analyses of its 2D NMR data. In particular, HMBC correlations of H 2 -29 to d C 146.8 (C-3), H 3 -30 with d C 75.0 (C-7), and H-7 to d C 170.8 (COMe) confirmed the presence of an a,b-unsaturated ketone and the acetyl position. The relative configurations of 4 were deduced to be similar to those of ceramicine E on the basis of the 1 H-1 H coupling constant data and NOESY correlations.
The absolute configurations of the isolated compounds were assumed to be similar to those of the previously reported ceramicines. The similarities of the CD spectra of 2 and 3 with those of ceramicine B, and 4 with ceramicine E further support this assumption.
The isolated compounds were tested for LDA inhibitory activity on MC3T3-G2/PA6 cells. As can be seen in Table 3, 1, 2, and 4 are less potent than 5. However, 3 showed better selectivity than 5 while showing activity at the same order of magnitude as 5.

Syntheses of ceramicine B derivatives
We have previously reported that the etherification or esterification of the 7a-OH group resulted in the increase of the cytotoxicity or decrease of LDA inhibitory activity [5]. In this work, we found that 3 with a carbonyl moiety at C-7 showed better selectivity than 5 while showing activity at the same order of magnitude as 5. Thus, we decided to further investigate the effect of the substituent at C-7 of 5.
First, we synthesized 7-dehydroxyceramicine B (6) and 7-epi-ceramicine B (7). Compound 6 was readily obtained through Barton-McCombie deoxygenation of 5 (Scheme 1) [8]. Since inversion of the configuration at C-7 could not be achieved through Mitsunobu reaction, we examined the feasibility of obtaining 7 through reduction of compound 3, which can be easily obtained from 5 after oxidation pyridinium chlorochromate (PCC). We used the Meerwein-Ponndorf-Verley (MPV) reduction to selectively reduce the carbonyl at C-7 [9], and as can be seen in Table 4, we obtained 7, albeit in a low yield. Interestingly, a ring-contracted side product 8 can also obtained using the MPV reduction (Table 4 entry 2). After obtaining 7, we synthesized its derivatives 9-11 (Scheme 2) to study the effects of etherification and esterification of the 7b-OH on the LDA inhibitory activity.  The LDA inhibitory activities of the synthesized compounds are shown in Table 3. Compounds 6 and 7 have lower LDA inhibitory and cytotoxic activities than 5. Thus, the absence of an a-oriented hydroxy group at C-7 led to a decrease of both the LDA inhibitory and cytotoxic activities.
The effects of esterification and etherification of 7-OH group on the LDA inhibitory activity are as follow. On the basis of the IC 50 values of 7 and 9-11, for the 7b-OH derivatives, esterification and etherification did not significantly change the LDA inhibitory activities. In contrast, on the basis of the IC 50 values of 5 and 12-14, for the 7a-OH derivatives, esterification and etherification led to lower LDA inhibitory activities.
In addition, compound 8, with a contracted B-ring, showed no LDA inhibitory activity at 50 lM.

Experimental section General experimental procedures
Optical rotations were measured on a JASCO DIP-1000 polarimeter. UV spectra were recorded on a Shimadzu UVmini-1240 spectrophotometer and IR spectra on a JASCO FT/IR-4100 spectrophotometer. High-resolution ESI MS were obtained on a LTQ Orbitrap XL (Thermo Scientific). 1 H and 2D NMR spectra were measured on a 400-MHz or 600-MHz spectrometer at 300 K, while 13 C NMR spectra were measured on a 100-MHz or 150-MHz spectrometer. The residual solvent peaks were used as internal standards (d H 7.26 and d C 77.0 for CDCl 3 , d H 3.31 and d C 49.0 for CD 3 OD). Standard pulse sequences were used for the 2D NMR experiments. Merck silica gel 60 (40-63 lm) was used for the column chromatography, and the separations were monitored by Merck silica gel 60 F 254 , or Merck silica gel RP C-18 F 254 TLC plates.

Extraction and isolation
The bark of C. ceramicus (8 kg) was extracted with MeOH to obtain 1.43 kg of extract. The MeOH extract was successively partitioned with n-hexane, EtOAc, n-BuOH, and water. The n-hexane-soluble materials were further separated by silica gel column chromatography (nhexane/EtOAc 1:

Synthesis of 6
Compound 5 (40 mg, 0.1 mmol) was dissolved in 8 mL of THF, put under argon, and cooled to 0°C. To the solution of 5, NaH (76 mg, 3.2 mmol) was added and the solution was stirred. After 2 h, CS 2 (800 lL, 13.2 mmol) was added, and the solution was further stirred. After 1.5 h, the solution was returned to rt before adding MeI (400 lL, 6.4 mmol) and stirred for another 4 h. Finally, cold water was added to the reaction mixture and partitioned with Et 2 O. The Et 2 O layer was dried over anhydrous Na 2 SO 4 before being dried under reduced pressure to afford solid residues. The residues were separated by silica gel column chromatography (n-hexane/EtOAc, 10:1 ? 3:1) to afford 15 (30 mg, 62%).

Synthesis of 3
Compound 5 (100 mg, 0.25 mmol) was dissolved in 10 mL of CH 2 Cl 2 , put under argon, and cooled to 0°C. To the solution of 5, PCC (181 mg, 0.84 mmol) was added, and the solution was stirred at rt. After 2 h, more PCC (125 mg, 0.58 mmol) was added, and the solution was further stirred at rt for 2 h. To the resulting mixture, diethyl ether was added, and the solids were filtered through Celite. The filtrates were then dried under reduced pressure to afford solid residues. The residues were separated by silica gel column chromatography (nhexane/EtOAc = 1:1) to afford 3 (70 mg, 70%).
Preparation of i Bu 2 AlO i Pr i Bu 2 AlH (1.0 M in toluene, 5.0 mmol, 5.0 mL) was put under argon and cooled to 0°C. Isopropanol (390 lL, 5.0 mmol) was then added, and the mixture was stirred for 1 h at rt before being used in the reactions below.
Preparation of i Bu 2 AlOH i Bu 2 AlH (1.0 M in toluene, 5.0 mmol, 5.0 mL) was put under argon and cooled to 0°C. Water (90 lL, 5.0 mmol) was then added, and the mixture was stirred for 1 h at rt before being used in the reaction below. To a solution of 3 (5 mg, 0.012 mmol) in toluene (0.5 mL) under argon, i BuAlO i Pr (72 lL, 0.072 mmol) was added at rt. The mixture was then stirred at 70°C for 5 h, and cooled to rt before adding 72 lL of water and stirred for another hour. To the resulting mixture, EtOAc was added, and the solids were filtered through Celite. The filtrates were then dried under reduced pressure to afford solid residues. The residues were separated by ODS HPLC (Shiseido ODS MGII 4.6 9 250 mm, MeOH/H 2 O, 75:25 at 0.5 mL/min, UV detection at 210 nm) to afford 5 (1.7 mg, 33%, t R = 28.0 min) and 7 (0.15 mg, 3%, t R = 26.4 min). To a solution of 3 (5 mg, 0.012 mmol) in toluene (0.5 mL) under argon, i BuAlO i Pr (24 lL, 0.024 mmol) was added at rt. The mixture was then refluxed for 0.5 h, and cooled to rt before adding 72 lL of water and stirred for another hour. To the resulting mixture, EtOAc was added, and the solids were filtered through Celite. The filtrates were then dried under reduced pressure to afford solid residues. The residues were separated by ODS HPLC (Shiseido ODS MGII 4.6 9 250 mm, MeOH/H 2 O, 75:25 at 0.5 mL/min, UV detection at 210 nm) to afford 5 (1.8 mg, 36%, t R = 28.0 min), 7 (0.6 mg, 12%, t R = 26.4 min), and 8 (0.4 mg, 8%, t R = 29.6 min). To a solution of 3 (20 mg, 0.049 mmol) in toluene (2.0 mL) under argon, i BuAlOH (390 lL, 0.39 mmol) was added and stirred at rt. After 20 h, the mixture was cooled to 0°C before adding 1 N HCl (5 mL). The resulting mixture was then partitioned with EtOAc, and the EtOAc layer was dried with anhydrous Na 2 SO 4 before being dried under reduced pressure to afford solid residues. The residues were separated by preparative TLC (benzene/EtOAc, 4:1) to afford 5 (7.2 mg, 36%) and 7 (3.0 mg, 15%).  13

LDA inhibitory activity and cytotoxicity
MC3T3-G2/PA6 murine pre-adipocytes (Riken Cell Bank, Ibaraki, Japan) were maintained in basal medium [alpha minimum essential medium (a-MEM) (Wako, Osaka, Japan) supplemented with 10% FBS (Cell Culture Bioscience, Tokyo, Japan)]. LDA inhibitory activity and cytotoxicity on MC3T3-G2/PA6 were measured using the same methods as in our previous report [5,6]. Briefly, the LDA inhibitory activity of the samples was measured on the basis of the amount of LDA after 6 days of incubation with a mixture of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin (MDI inducer), and was expressed as IC 50 value (the concentration of the sample causing 50% inhibition of LDA relative to an untreated control). The cytotoxicity was evaluated indirectly via MTT assay which is based on mitochondrial succinate dehydrogenase activity and confirmed via microscopic observation. The cytotoxicity was expressed as CC 50 value which was defined as the concentration of the sample causing 50% cell viabilities relative to an untreated control.