New Cytotoxic Sesquiterpenoids from Siegesbeckia glabrescens

Abstract

Two new sesquiterpenoids, siegenolides A (1) and B (2), and two known sesquiterpenes 3 and 4 were isolated from Siegesbeckia glabrescens. Their structures were elucidated by spectroscopic analyses, and they were further evaluated for their cytotoxic activities against human cancer cells (MCF-7, AsPC-1, SW480, HCT 116, HepG2, HeLa). Compounds 1–4 showed differential cytotoxic effects on the target cancer cells with IC₅₀ values in the range of 0.9–33.3 μM.

1. Introduction

Siegesbeckia glabrescens Makino (Compositae) is an annual herb that has been used as a Chinese medicine to treat inflammatory disease, asthma, paralysis and allergic disorders [1]. Flavonoids [1], sesquiterpene [2] and kaurane diterpenes [3] have been isolated from this plant in previous phytochemical studies. It has been reported that the extracts of S. glabrescens exhibit antioxidative, anti-inflammatory [1], antiallergic [4], antibacterial [5] and anti-tumor activities [6,7]. In our screening program to discover new antitumor agents from medicinal herbs, two new compounds 1–2 and two known sesquiterpenes 3–4 were isolated from the ethyl acetate (EtOAc)-soluble fraction of the methanolic extract of S. glabrescens by chromatographic procedures. In this study, we present the structural elucidation of these two new compounds, as well as their cytotoxic activities against several human cancer cell lines including MCF-7, AsPC-1, SW480, HCT 116, HepG2 and HeLa.

2. Results and Discussion

Dried S. glabrescens was extracted with methanol. The crude methanol extract was subjected to liquid-liquid partition as well as a combination of several column chromatography steps to yield two new sesquiterpenoids 1 and 2 together with two known sesquiterepnoids 3 and 4 (Figure 1).

Figure 1. Chemical structures of compounds 1–4 and key HMBC (→) and NOESY (bold ↔) correlations for compound 1.

Figure 1. Chemical structures of compounds 1–4 and key HMBC (→) and NOESY (bold ↔) correlations for compound 1.

Compound 1 was obtained as an amorphous solid. The HREIMS spectrum suggested a molecular formula of 1 as C₂₁H₂₆O₇. The ¹³C-NMR, DEPT, and HSQC spectra showed twenty-one carbon signals including three carbonyl carbons, eight olefinic carbons, three methylene carbons, four methine carbons, one methoxyl and two methyl groups. In ¹H-¹H COSY spectrum, H-4 (δ 5.26) correlated with H-3 (δ 2.82) and H-5 (δ 5.09), and also H₂-8 (δ 2.62~2.73) correlated with methylene protons of H₂-7 (δ 2.06 and 2.78) and H-9 (δ 6.97). This data suggested that this compound has AMX and A₂M₂X spin systems. In HMBC spectrum, oxy-methylene protons H₂-12 (δ 4.36 and δ 4.39) correlated with C-5 (δ 129.4), C-6 (δ 142.3) and C-7 (δ 33.3), and H-3 (δ 2.82) correlated with C-9 (δ 159.7) and C-10 (δ 141.5) (Figure 1 and Table 1). These data indicated that compound 1 has eight-membered ring in the structure with two double bonds. The correlation between terminal methylene H₂-11 (δ 5.75 and 6.15) and a carbonyl carbon C-1 (δ 171.2) and C-3 (δ 51.9) in HMBC indicated that compound 1 is a bicycle [6.3.0]-γ-lactone having an exocyclic double bond in lactone ring. We also found the HMBC correlations between oxy-methine H-4 and C-6, and between H-5 and C-3. In ¹H-¹H COSY spectrum, we found another AMX spin system from the correlations of H-14 (δ 3.94) with H-13 (δ 6.63) and H-15 (δ 9.43). From the coupling constant of H-15 (J = 2.0 Hz) and chemical shift of C-15 (δ 196.8) and the HMBC correlations between H-15 (δ 9.43) and C-14 (δ 79.4), we identified the presence of an aldehyde group that is linked to C-14. In HMBC spectrum, we also found correlation between a methoxyl protons (δ 3.10) and C-14 (δ 79.4), and correlation between H-13 (δ 6.63) and C-3 (δ 51.9). The long range allylic coupling was observed between H-13 (dd, 8.4, 1.2 Hz) and H-9. These results indicated that an oxy-carbon C-13 is linked to the eight-membered ring at C-10. From the coupling constant between H-3 and H-4 (J = 10.0 Hz,) we postulated the configurations of H-3 and H-4 based on Karplus relationship and the reported values of several bicycle [6.3.0]-γ-lactone derivatives (Figure 1 and Table 1) [8]. Furthermore, the presence of 2-methylbut-2-enoyl group was recognized by ¹H-¹H COSY correlation of an olefinic methine H-3' (δ 6.10) with methyl protons (δ 1.93) and the HMBC correlations of methyl protons (δ 1.88) with C-2' (δ 128.9) and C-1' (δ 168.4), and the correlations of methyl protons (δ 1.93) with C-2' (δ 128.9) and C-3' (δ 138.9). The HMBC correlation of an oxy-proton H-13 with carbonyl ester C-1' confirmed the esterification of 2-methylbut-2-enoyl group at C-13. We found NOESY correlations of H-13 (δ 6.63) with H-3 (δ 2.82) and H-11a (δ 6.15) that indicates the orientation of H-13 as β that is located close to H-3 and H-11a. NOESY correlations was also observed between H-9 (δ 6.97) and H-15 (δ 9.43) implying the orientation of methoxyl group as β. This orientation was confirmed by the coupling constant between H-13 and H-14 as 8.2 Hz. Thus, compound 1 was identified as 2-methylbut-2-enoic acid 1-(8-hydroxymethyl-3-methylene-2-oxo-2,3,3a,6,9a-hexahydro-cycloocta[b]furan-4-yl)-2-methoxy-3-oxo-propyl ester. This structure is new and we named compound 1 as siegenolide A.

Compound 2 was obtained as an amorphous solid. The HREIMS spectrum suggested a molecular formula as C₂₀H₂₄O₇. The ¹³C-NMR, DEPT, and HSQC spectra showed similar signals as those of compound 1 except showing one terminal olefinic methylene signals of H₂-3' (δ 5.65, 6.14) instead of the methyl protons (H₃-4') of compound 1. The relative stereochemistry of compound 2 was determined by the analysis of NOESY spectra and coupling constants, which was same as compound 1. Thus, the structure of compound 2 was determined as 2-methyl-acrylic acid 1-(8-hydroxymethyl-3-methylene-2-oxo-2,3,3a,6,9a-hexahydro-cycloocta[b]furan-4-yl)-2-methoxy-3-oxo-propyl ester, which was a new structure and named as siegenolide B.

Compounds 3 and 4 were identified as 2-methylbut-2-enoic acid,2,3,3a,4,5,8,9,10,11,11a-decahydro-6,10-bis(hydroxymethyl)-3-methylene-2-oxocyclodeca[b]furan-4-yl ester (3) and 2-methylacrylic acid, 2,3,3a,4,5,8,9,10,11,11a-decahydro-6,10-bis(hydroxymethyl)-3-methylene-2-oxocyclodeca[b]-furan-4-yl ester (4), respectively, by comparison with the reported spectral data (Figure 1) [2,9].

The four sesquiterpenoids 1–4 were evaluated for their cytotoxic activity on human cancer cell lines such as MCF-7, AsPC-1, SW480, HCT 116, HepG2 and HeLa cells. Compounds 1–4 showed differential cytotoxic effects on these cancer cell lines (Table 2). All of them showed significant cytotoxicity against SW480 cell line, with IC₅₀ values of 1.8, 0.9, 5.2 and 3.8 μM, respectively. The cytotoxicity of compounds 3 and 4 against AsPC-1 cells was more potent (IC₅₀ values of 7.3 and 4.9 μM, respectively) than that of compounds 1 and 2 (IC₅₀ values 14.5 and 12.1 μM, respectively).

Table 1. NMR Spectroscopic data (400 MHz, CD₃OD) for siegenolides A (1) and B (2).

Position	Siegenolide A (1)			Siegenolide B (2)
	δC	δH (J in Hz)	HBMC a	δC	δH (J in Hz)	HBMC a
1	171.2			171.3
	136.8			136.7
3	51.9	2.82, m	9, 10	52.0	2.83, m	9
4	75.7	5.26, t (10.0)	6	75.7	5.33, t (10.0)
5	129.4	5.09, d (10.0)	3	129.5	5.09, d (10.0)	7, 12
6	142.3			142.2
7a	33.3	2.78, m		33.5	2.78, m
7b		2.06, td (12.4, 2.0)			2.06, td (12.4, 2.0)
8	28.4	2.62~2.73, m		28.5	2.74~2.68, m
9	159.7	6.97, dd (10.4, 7.6)		159.6	6.97, dd (10.4, 7.6)
10	141.5			141.6
11a	121.5	6.15, d (3.2)	1, 3	121.4	6.13, d (3.2)	1, 3
11b		5.75, d (3.2)	1, 3		5.73, d (3.2)	1
12a	60.8	4.39, brd (13.2)	5, 6, 7	61.0	4.39, brd (13.2)	5, 6, 7
12b		4.36, brd (13.2)	5, 6, 7		4.33, brd (13.2)	5, 6, 7
13	70.6	6.63, dd (8.4, 1.2)	3, 1'	71.6	6.56, dd (8.4, 1.6)	1'
14	79.4	3.94, dd (8.4, 2.0)		79.4	3.92, dd (8.4, 2.0)	15
15	196.8	9.43, d (2.0)	14	196.8	9.44, d (2.0)	14
1'	168.4			167.7
2'	128.9			137.4
3'	138.9	6.10, m		126.8	5.65, dd (3.2, 1.6)
					6.14, dd (3.2, 1.6)
14-OCH3	56.9	3.10, s	14	57.0	3.09, s	14
2'-Me	20.7	1.88, pentet (1.6)	1', 2'	18.5	1.94, brs	1', 2'
3'-Me	15.9	1.93, dq (7.2, 1.6)	2', 3'

Note: ^a HMBC correlations start from proton(s) to the indicated carbon.

Table 1. NMR Spectroscopic data (400 MHz, CD₃OD) for siegenolides A (1) and B (2).

Position	Siegenolide A (1)			Siegenolide B (2)
	δC	δH (J in Hz)	HBMC a	δC	δH (J in Hz)	HBMC a
1	171.2			171.3
	136.8			136.7
3	51.9	2.82, m	9, 10	52.0	2.83, m	9
4	75.7	5.26, t (10.0)	6	75.7	5.33, t (10.0)
5	129.4	5.09, d (10.0)	3	129.5	5.09, d (10.0)	7, 12
6	142.3			142.2
7a	33.3	2.78, m		33.5	2.78, m
7b		2.06, td (12.4, 2.0)			2.06, td (12.4, 2.0)
8	28.4	2.62~2.73, m		28.5	2.74~2.68, m
9	159.7	6.97, dd (10.4, 7.6)		159.6	6.97, dd (10.4, 7.6)
10	141.5			141.6
11a	121.5	6.15, d (3.2)	1, 3	121.4	6.13, d (3.2)	1, 3
11b		5.75, d (3.2)	1, 3		5.73, d (3.2)	1
12a	60.8	4.39, brd (13.2)	5, 6, 7	61.0	4.39, brd (13.2)	5, 6, 7
12b		4.36, brd (13.2)	5, 6, 7		4.33, brd (13.2)	5, 6, 7
13	70.6	6.63, dd (8.4, 1.2)	3, 1'	71.6	6.56, dd (8.4, 1.6)	1'
14	79.4	3.94, dd (8.4, 2.0)		79.4	3.92, dd (8.4, 2.0)	15
15	196.8	9.43, d (2.0)	14	196.8	9.44, d (2.0)	14
1'	168.4			167.7
2'	128.9			137.4
3'	138.9	6.10, m		126.8	5.65, dd (3.2, 1.6)
					6.14, dd (3.2, 1.6)
14-OCH3	56.9	3.10, s	14	57.0	3.09, s	14
2'-Me	20.7	1.88, pentet (1.6)	1', 2'	18.5	1.94, brs	1', 2'
3'-Me	15.9	1.93, dq (7.2, 1.6)	2', 3'

Note: ^a HMBC correlations start from proton(s) to the indicated carbon.

Table 2. Cytotoxicity of compounds 1–4 against cancer cell lines.

Compounds	IC50 (μM)
	MCF-7	AsPC-1	SW480	HCT116	HepG2	HeLa
1	9.5 ± 0.3	14.5 ± 0.9	1.8 ± 0.1	5.9 ± 0.2	20.2 ± 1.1	33.3 ± 2.3
2	8.7 ± 0.4	12.1 ± 0.2	0.9 ± 0.1	3.2 ± 0.3	9.9 ± 0.4	23.9 ± 1.2
3	9.7 ± 0.7	7.3 ± 0.5	5.2 ± 0.4	9.2 ± 0.6	14.4 ± 1.0	12.3 ± 0.7
4	12.7 ± 0.7	4.9 ± 0.2	3.8 ± 0.1	11.4 ± 0.8	27.8 ± 1.4	24.7 ± 0.9
cisplatin	13.0 ± 0.6	2.3 ± 0.2	4.8 ± 0.4	3.6 ± 0.1	5.9 ± 0.7	0.89 ± 0.1

Table 2. Cytotoxicity of compounds 1–4 against cancer cell lines.

Compounds	IC50 (μM)
	MCF-7	AsPC-1	SW480	HCT116	HepG2	HeLa
1	9.5 ± 0.3	14.5 ± 0.9	1.8 ± 0.1	5.9 ± 0.2	20.2 ± 1.1	33.3 ± 2.3
2	8.7 ± 0.4	12.1 ± 0.2	0.9 ± 0.1	3.2 ± 0.3	9.9 ± 0.4	23.9 ± 1.2
3	9.7 ± 0.7	7.3 ± 0.5	5.2 ± 0.4	9.2 ± 0.6	14.4 ± 1.0	12.3 ± 0.7
4	12.7 ± 0.7	4.9 ± 0.2	3.8 ± 0.1	11.4 ± 0.8	27.8 ± 1.4	24.7 ± 0.9
cisplatin	13.0 ± 0.6	2.3 ± 0.2	4.8 ± 0.4	3.6 ± 0.1	5.9 ± 0.7	0.89 ± 0.1

Against HCT116 and HepG2 cells, compound 2 showed relatively high cytotoxicity, and against MCF-7 cells all compounds showed moderate cytotoxicity. All of these compounds displayed weak cytotoxicity against HeLa cells, with IC₅₀ values (12.3–33.3 μM) compared to that of cisplatin (0.9 μM). Cytotoxicity of sesquiterpenes with α, β-unsaturated lactone structure was well known. Compounds 1 and 2 are uncommon sesquiterpenoids with eight-membered ring and they also showed same type of cytotoxicity as reported [10].

3. Experimental Section

3.1. General Experimental Procedures

UV spectra were recorded using an Ultraspec 4000 double beam spectrophotometer (Pharmacia Biotech, Cambridge, UK). 1D- and 2D-NMR spectra were obtained on a UNITY INOVA 400 spectrometer (Varian, Palo Alto, CA, USA). Mass spectra were determined on a JMS-AX505WA mass spectrometer (JEOL, Tokyo, Japan). Column chromatography was carried out over silica gel (40–60 μm, Merck, Merck, Kenilworth, NJ, USA), LiChroprep RP-C18 (40–60 μm, Merck) and µ-Bondapak C₁₈ column (10 μm, 10 i.d. × 300 mm) (Waters Co., Milford, MA, USA). Fractions obtained from column chromatography were monitored by thin layer chromatography (TLC) (RP-C18 F_254S and silica gel 60 F₂₅₄, Merck).

3.2. Plant Material

The whole plant of Siegesbeckia glabrescens Makino (Compositae) was collected from Wan-Do, Jeolla-Namdo Province, Korea in November 2005 and authenticated by Prof. K. S. Yang at the College of Pharmacy, Sookmyung Women’s University (SMU). A voucher specimen (No. SPH 2005007) was deposited in the herbarium of SMU.

3.3. Extraction and Isolation

The air-dried material (5 kg) was reflux extracted with methanol (6 × 2 L) to yield after solvent removal a crude methanol extract (578 g), which was successively partitioned twice with EtOAc (3 L) and H₂O (3 L). The EtOAc soluble fraction (368 g) was subjected to silica gel column chromatography (CC) (13 × 26 cm, 0.063–0.2 mm) eluting with a gradient mixture of CHCl₃–MeOH (100:1, 70:1, 30:1, 20:1, 12:1, 5:1; 2 L each) to give 26 fractions. Fraction 9 (45 g, V_R 5.5–6.0 L) was further fractionated by silica gel column with a gradient elution of CHCl₃–MeOH (40:1 to 36:1, 2 L each) to afford Fr. 9-4 (3.4 g, V_R 1.2–1.6 L). Fr. 9-4 was subjected to an ODS column (5 × 8 cm, 0.040–0.063 mm) eluting with MeOH–H₂O (1:1) to afford cytotoxic Fr. 9-4-1 (400 mg, V_R 1.2–1.4 L) which was subjected to Sephadex LH-20 CC (0.018–0.111 mm) eluted with 70% MeOH to yield pure compound 1 (18 mg, V_R 230–290 mL). Fr. 9-4-1-1 (113.6 mg) was subjected to ODS column chromatography eluting with MeOH–H₂O (1:1.6) to yield compound 2 (11.2 mg, V_R 80–120 mL). Fr. 11 (4.0 g, V_R 9.1–10.8 L) was separated by a silica gel column chromatography eluting with CHCl₃–MeOH (40:1) to obtain cytotoxic Fr. 11-9 (234 mg, V_R 2.8–3.2 L). Fr. 11-9 (234 mg) was purified by ODS column chromatography eluting with MeOH–H₂O (1:3) to yield compounds 3 (40 mg, V_R 120–145 mL) and 4 (11 mg, V_R 210–240 mL). The purities of compounds 1–4 were higher than 95% as confirmed by HPLC chromatogram and ¹H-NMR spectra.

Compound 1: amorphous solid; ${\left[\mathrm{\alpha }\right]}_D^{24}$: −96.4, (c 0.005, MeOH), UV (MeOH) λ_max (log ε) 244 (3.11), 235 (3.10) nm; IR (CaF₂, cm⁻¹) 3476, 2925, 1764, 1720, 1686. ¹H- and ¹³C-NMR data, see Table 1; HREIMS m/z 390.1685 [M]⁺ (calcd for C₂₁H₂₆O₇, 390.1678); EIMS m/z 390 [M]⁺.

Compound 2: amorphous solid; ${\left[\mathrm{\alpha }\right]}_D^{27}$: −5.7, (c 0.006, MeOH), UV (MeOH) λ_max (log ε) 253 (3.20), 245 (3.18) nm; IR (CaF₂, cm⁻¹) 3500, 2931, 1766, 1722, 1686, 1157. ¹H- and ¹³C-NMR data, see Table 1; HREIMS m/z 376.1518 [M]⁺ (calcd for C₂₀H₂₄O₇, 376.1522); EIMS m/z 376 [M]⁺.

3.4. Cytotoxicity Assay

The cytotoxicity of compounds 1–4 against human breast cancer (MCF-7), pancreatic cancer (AsPC-1), colon cancers (SW480 and HCT 116), hepatoma (HepG2), and cervical carcinoma (HeLa) were assessed by the MTT method [11]. Cells were plated at a density of 3000 cells/well in a 96 well plate. Cells were incubated with various concentrations of compounds 1–4 for 3 days, and then treated with MTT (5 mg/mL) solution for 4 h and lysed with DMSO. Absorbance at 540 nm was measured by using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cisplatin (purity > 98%) (Sigma, St. Louis, MO, USA) was used as a positive control.