A Novel Trimeric Triterpene From Chisocheton ceramicus Miq.

Abstract

A novel trimeric triterpene, bismoronic ceramicine (1), was isolated from the bark of Chisocheton ceramicus Miq. The structure was elucidated based on spectroscopic data and chemical correlations. Bismoronic ceramicine (1) showed moderate antimalarial activity against Plasmodium falciparum strain 3D7.

The family Meliaceae has been known to produce various types of limonoids with medicinal properties.^1–6 In the course of our continuing search for bioactive natural products from tropical plants,^7–12 we have reported ceramicines from the bark of Malaysian Chisocheton ceramicus (Meliaceae).^13–17 Ceramicine B showed a strong lipid droplet accumulation inhibitory activity on a mouse pre-adipocyte cell line (MC3T3-G2/PA6), and anti-melanin deposition activity against B16-F10 melanoma cells.^18–20

In this paper, we report the isolation and structure elucidation of a novel trimeric triterpene, bismoronic ceramicine (1) from the bark of Chisocheton ceramicus Miq., and its antimalarial activity.

Bismoronic ceramicine (1) was obtained as an optically active colorless amorphous solid, [α] + 28 (c 1.0, CHCl₃). The IR spectrum showed important absorptions at 3450, 1735, and 1703 cm⁻¹ for hydroxyl, ester carbonyl, and ketone groups, respectively. The ESIMS (pos.) of 1 showed a molecular ion peak at m/z 1379 and the molecular formula of 1 was established as C₈₇H₁₂₆O₁₃ from HRESIMS (pos.). The ¹H and ¹³C NMR data (Supplemental Figures S1 and S2) (Table 1) and HSQC spectrum (Supplemental Figure S5) of 1 revealed the presence of 87 carbon signals due to 15 sp³ methines, 23 sp³ methylenes, 19 methyls, 17 sp³ quaternary carbons, five sp² methines, three sp² quaternary carbons, and five carbonyl carbons. Among them, six sp³ methines (δ_C 72.2; δ_H 4.15, δ_C 75.1; δ_H 4.35, δ_C 77.3; δ_H 3.73, δ_C 97.6; δ_H 6.50, δ_C 102.0; δ_H 6.19, δ_C 111.4; δ_H 4.68), one sp³ quaternary carbon (δ_C 78.3), and one methyl (δ_C 55.3; δ_H 3.40) were attributed to those attached to an oxygen atom. From the above data, 1 was presumed to be a trimeric triterpene (Figure 2; units A∼C).

Figure 1.

Structure of bismoronic ceramicine (1).

Figure 2.

Selected 2D NMR correlations of 1.

Table 1.

¹H (600 MHz) and ¹³C (150 MHz) NMR Data of Bismoronic Ceramicine (1) in CDCl₃ ( δ in ppm, J in Hz).

no.	δ H	δ C	no.	δ H	δ C	no.	δ H	δ C
1		202.6	1′a	1.42 (1H, m)	39.8	1’’a	1.42 (1H, m)	39.8
			1’b	1.96 (1H, m)		1’’b	1.96 (1H, m)
2	5.82 (1H, d, 9.8)	130.4	2’a	2.44 (1H, m)	34.1	2’’a	2.44 (1H, m)	34.1
			2’b	2.49 (1H, m)		2’’b	2.49 (1H, m)
3	6.91 (1H, d, 9.8)	150.5	3’		218.1	3’’		218.5
4		45.9	4’		47.2	4’’		47.2
5	3.00 (1H, d, 12.8)	44.2	5’	1.33 (1H, m)	55.0	5’’	1.33 (1H, m)	54.8
6	4.35 (1H, dd, 12.8, 2.8)	75.1	6’	1.44 (1H, m)	19.6	6’’	1.44 (1H, m)	19.5
7	4.15 (1H, d, 2.8)	72.2	7’a	1.33 (1H, m)	33.8	7’’a	1.33 (1H, m)	33.7
			7’b	1.46 (1H, m)		7’’b	1.48 (1H, m)
8		46.6	8’		40.6	8’’		40.5
9	2.46 (1H, m)	35.6	9’	1.33 (1H, m)	50.5	9’’	1.33 (1H, m)	50.4
10		46.6	10’		36.9	10’’		36.9
11a	1.67 (1H, m)	17.9	11’a	1.35 (1H, m)	21.3	11’’a	1.49 (1H, m)	21.2
11b	2.54 (1H, m)		11’b	1.53 (1H, m)		11’’b	1.55 (1H, m)
12a	1.58 (1H, m)	35.2	12’a	1.25 (1H, m)	26.1	12’’a	1.29 (1H, m)	26.1
12b	2.13 (1H, m)		12’b	1.61 (1H, m)		12’’b	1.55 (1H, m)
13		47.3	13’	2.27 (1H, brd, 12.6)	41.4	13’’	2.13 (1H, m)	41.3
14		158.4	14’		42.6	14’’		42.6
15	5.51 (1H, brs)	120.0	15’a	1.22 (1H, m)	29.1	15’’a	1.16 (1H, m)	29.0
			15’b	1.65 (1H, m)		15’’b	1.56 (1H, m)
16a	2.08 (1H, m)	30.2	16’a	1.34 (1H, m)	33.4	16’’a	1.37 (1H, m)	33.2
16b	2.68 (1H, dd, 12.9, 12.9)		16’b	2.18 (1H, m)		16’’b	2.11 (1H, m)
17	2.07 (1H, m)	57.4	17’		48.3	17’’		47.8
18	1.23 (3H, s)	23.3	18’		137.4	18’’		136.3
19	1.13 (3H, s)	14.9	19’	5.18 (1H, s)	133.1	19’’	5.09 (1H, s)	132.9
20		78.3	20’		32.0	20’’		31.9
21	6.50 (1H, s)	97.6	21’	1.39 (2H, m)	33.5	21’’	1.36 (2H, m)	33.4
22	3.73 (1H, d, 6.4)	77.3	22’a	1.63 (1H, m)	33.1	22’’a	1.57 (1H, m)	33.0
			22’b	2.00 (1H, m)		22’’b	1.90 (1H, m)
23	6.19 (1H, s)	102.0	23’	1.03 (3H, s)	21.0	23’’	1.03 (3H, s)	20.9
			24’	1.07 (3H, s)	26.6	24’’	1.07 (3H, s)	26.8
			25’	0.98 (3H, s)	16.5	25’’	0.97 (3H, s)	16.4
			26’	1.01 (3H, s)	15.6	26’’	0.98 (3H, s)	15.9
			27’	0.77 (3H, s)	14.7	27’’	0.75 (3H, s)	14.8
28	4.68 (1H, s)	111.4	28’		174.6	28’’		174.3
29	1.25 (3H, s)	18.3	29’	0.98 (3H, s)	30.3	29’’	0.94 (3H, s)	30.3
30	1.09 (3H, s)	26.2	30’	0.98 (3H, s)	29.1	30’’	0.92 (3H, s)	29.0
31	3.40 (3H, s)	55.3

The gross structure of 1 was elucidated by analysis of 2D NMR data, including ¹H-¹H COSY, HSQC, and HMBC spectra (Supplemental Figures S3, S5, and S6) in CDCl₃ (Figure 2). The ¹H-¹H COSY spectrum of 1 exhibited 15 spin systems, as shown in bold lines in Figure 2. In the HMBC spectrum, the correlations of H₃-29 to C-3/C-4/C-5/C-28, H₃-19 to C-1/C-5/C-9/C-10, H₃-30 to C-7/C-8/C-9/C-14, H₃-18 to C-12/C-13/C-14/C-17, H-28 to C-6, as well as H-16a to C-14 led to the fused 5-rings moiety of unit A. In addition, the HMBC cross-peaks of H-21 to C-17/C-20/C-22, OH-22 to C-20/C-22, and H-23 to C-21 revealed the presence of a tetrahydrofuran moiety at C-17. In unit B, the oleanane skeleton was verified by the HMBC correlations of H₃-24′ to C-3′/C-4′/C-5′/C-23′, H₃-25′ to C-1′/C-5′/C-9′/C-10′, H₃-26′ to C-7′/C-8′/C-9′/C-14′, H₃-27′ to C-8′/C-13′/C-14′/C-15′, H₃-29′ to C-19′/C-20′/C-21′/C-30′, H-19′ to C-13′/C-17′/C-18′, H-16′a to C-17′/C-28′, H-22′a to C-17′/C-28′ as well as H-1′b to C-3. The planned structure of unit C was also elucidated in similar ways to unit B.

Finally, the HMBC correlations of H-21/C-28′ and H-23/C-28′′ provided that units A and B, as well as units A and C were connected via C-21－C-28′ and C-23－C-28′′ bonds, respectively.

The relative configuration of 1 was elucidated by ROESY (Supplemental Figure S4) correlations and ³J coupling constants.

In unit A, the ROESY correlations of H-9 and H-5/H₃-18 suggested that these protons were cofacial, while the correlations of H₃-29 and H₃-19/H-28, as well as H-6 and H₃-19/H₃-30, indicated that C-19, C-29, C-30, H-6, and H-28 were β-oriented (Figure 3). Also, the ³J coupling constants between H-5 and H-6 (12.8 Hz), as well as between H-6 and H-7 (2.8 Hz), supported the β-orientation of H-7. Furthermore, the ROESY correlations of H-21 and H-12b/H₃-18, H-16b and H₃-18/H-22, as well as H₃-18 and OH-20, and coupling constants between H-16b and H-17 (12.9 Hz), as well as H-22 and H-23 (0 Hz) confirmed the stereochemistry of the tetrahydrofuran moiety.

Figure 3.

Selected ROESY correlations and ³J_H,H coupling constants for unit A of 1.

In unit B, C-24′, C-25′, C-26′, and H-13′ were assigned to be β-axially oriented from the ROESY correlations of H₃-25′ and H₃-24′/H₃-26′, as well as H₃-26′ and H-13′, while H-5′, H-9′, C-27′, H-16′a, H-22′a, and C-29′ were deduced to possess α-orientation from the ROESY correlations of H-5′ and H₃-23′/H-9′, H₃-27′ and H-9′/H-16′a, as well as H-22′ and H-16′a/H₃-29′ (Figure 4). Thus, the relative configuration of unit B was identified as the ester form of moronic acid. ²¹ Similar ROESY correlations were observed in unit C.

Figure 4.

Selected ROESY correlations for unit B of 1.

Finally, the ROESY correlations of H-23 and H-13′/H₃-26′, as well as OH-20 and H₃-26′′, confirmed the stereochemistry of each unit of 1 (Figure 5).

Figure 5.

Selected ROESY correlations of 1.

Treatment of 1 with LiOH gave a hydrolysate, whose spectral data and [α]_D value {[α] : + 68 (c 1.0, CHCl₃)} were identical to those of moronic acid {[α]_D: + 29 (c 0.41, CHCl₃)}. ²¹ Thus, the absolute stereochemistry of 1 was assigned as shown in Figure 1.

The antimalarial activity of 1 was evaluated in vitro against Plasmodium falciparum 3D7 strain. The result showed that 1 possessed moderate antimalarial activity, with an IC₅₀ value of 2.6 μM.

Experimental

General experimental procedures: Optical rotations were measured on a JASCO P-1030 polarimeter, UV spectra on a Shimadzu UVmini-1240 spectrophotometer, and IR spectra on a JASCO FT/IR-230 using Zn/Se cell. High-resolution ESI MS were obtained on a Waters Xevo G2-XS QTof LC/MS spectrometer using a sample dissolved in MeOH. ¹H and 2D NMR spectra were measured on a 600 MHz spectrometer at 300 K, while ¹³C NMR spectra were measured on a 150 MHz spectrometer. The residual solvent peaks were used as internal standards (δ_H 7.26 and δ_C 77.0 for CDCl₃). Standard pulse sequences were used for the 2D NMR experiments. Column chromatography was performed using silica gel 60 (230-400 mesh; Merck KGaA, Darmastadt, Germany), ODS (Cosmosil 140C₁₈-OPN; Nacalai tesque, Inc., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Sweden).

Plant materials: The barks of Chisocheton ceramicus Miq. were collected in July 2013 from Terengganu, Malaysia. Botanical identification was made by Prof. A. Hamid A. Hadi, University of Malaya. A voucher specimen (No. Hoshi₁₃CCB) was deposited in the Department of Pharmacognosy, Hoshi University.

Extraction and Isolation: The dried barks of C. ceramicus (8 kg) were extracted with MeOH, and the extract (1247 g) was successively partitioned with n-hexane, EtOAc, n-butanol, and water. The hexane-soluble materials were subjected to column chromatograph (CC) over silica gel and eluted with hexane/EtOAc (1:0 to 1:1) followed by CHCl₃/MeOH (1:0 to 0:1) to give 10 fractions (A-J). Fraction H was further separated by Sephadex LH-20 with CHCl₃/MeOH (1:1) to afford 5 fractions (HA-HE). Fraction HC was separated by ODS CC with MeOH/H₂O (9:1) continued with MeOH/Acetone (1:0 to 0:1) to give 7 fractions (HCA-HCG). Fraction HCE was then successively purified using a silica gel column with CHCl₃/MeOH (1:0 to 0:1) and a silica gel column with benzene/EtOAc (9:1 to 4:1) to yield bismoronic ceramicine (1, 17.7 mg, 0.00022%).

Bismoronic ceramicine (1)

Colorless amorphous solid.

[α]: + 28 (c 1.0, CHCl₃).

IR ν_max (film): 3450, 1735, and 1703 cm⁻¹.

UV (MeOH) λ_max 203 (ε 15500) nm.

CD (MeOH) λ_max 321 (Δε -0.49), 278 (Δε + 0.79), and 222 (Δε −7.79) nm.

¹H and ¹³C NMR (CDCl₃): see Table 1 and Supplemental Figures S1 and S2).

MS (ESI): m/z: 1379 [M + H]⁺.

HRESIMS m/z: 1379.9288 [M + H]⁺ (calcd. for C₈₇H₁₂₇O₁₃, 1379.9277).

Hydrolysis of 1 : To a solution of 0.5 mg of 1 in THF (0.5 mL) was added 0.5 mL LiOH aq. (2 mM). The mixture was stirred at 40 °C for 1 h. The mixture was added to n-hexane and partitioned. The water layer was extracted with CHCl₃. After evaporation of the CHCl₃ layer, moronic acid (1a) was obtained. 1a: Colorless amorphous solid; [α] : + 68 (c 1.0, CHCl₃); ¹H NMR (CDCl₃): see Supplemental Figure S7; ESIMS m/z: 455 [M + H]⁺.

Parasite strain and culture : P. falciparum laboratory strain 3D7 was obtained from Prof. Masatsugu Kimura (Osaka City University, Osaka, Japan). For the assessment of antimalarial activity of the compound in vitro, the parasites were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 0.5 g/L L-glutamine, 5.96 g/L HEPES, 2 g/L sodium bicarbonate (NaHCO₃), 50 mg/L hypoxanthine, 10 mg/L gentamicin, 10% heat-inactivated human serum, and red blood cells (RBCs) at a 3% hematocrit in an atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 37 °C, as previously described. ²² RBCs infected with ring-form parasites were collected using the sorbitol synchronization technique. ²³ Briefly, the cultured cells were collected by centrifugation at 840 g for 5 min at room temperature, suspended in a 5-fold volume of 5% D-sorbitol (Nacalai Tesque, Kyoto, Japan) for 10 min at room temperature, and then washed twice with RPMI 1640 medium to remove the D-sorbitol. The utilization of blood samples of healthy Japanese volunteers for the parasite culture was approved by the institutional review committee of the Research Institute for Microbial Diseases (RIMD), Osaka University (approval number: 22-3).

Antimalarial activity : Ring-form-synchronized parasites were cultured with 1 at sequentially decreasing concentrations (50, 15, 5, 1.5, 0.5, and 0.15 µM) for 48 h. Parasitemia was measured by flow cytometric analysis using an automated hematology analyzer, XN-30. The analyzer was equipped with a prototype algorithm for cultured falciparum parasites (software version: 01-03, build 16) and used specific reagents (CELLPACK DCL, SULFOLYSER, Lysercell M, and Fluorocell M) (Sysmex, Kobe, Japan).^24,25 Approximately 100 µL of the culture suspension diluted with 100 µL phosphate-buffered saline was added to a BD Microtainer MAP Microtube for Automated Process K₂ EDTA 1.0 mg tube (Becton Dickinson and Co., Franklin Lakes, NJ, USA) and loaded onto the XN-30 analyzer with an auto-sampler, as described in the instrument manual (Sysmex). The parasitemia (MI-RBC%) was automatically reported. ²⁴ DMSO (0.5%) alone or containing 5 µM artemisinin were used as the negative and positive controls, respectively. The growth inhibition (GI) rate was calculated from the MI-RBC% according to the following equation:

GI (%) = 100 – (test sample – positive control)/(negative control – positive control) × 100

The IC₅₀ was calculated from GI (%) using GraphPad Prism version 9.0 (GraphPad Prism Software, San Diego, CA, USA). ²⁶

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211053202 - Supplemental material for A Novel Trimeric Triterpene From Chisocheton ceramicus Miq.

Supplemental material, sj-docx-1-npx-10.1177_1934578X211053202 for A Novel Trimeric Triterpene From Chisocheton ceramicus Miq. by Alfarius Eko Nugroho, Marika Okabe, Yusuke Hirasawa and Chin Piow Wong, Toshio Kaneda, Takahiro Tougan, Toshihiro Horii, Hiroshi Morita in Natural Product Communications