Cytotoxic Sesquiterpenoids From Soft Soral Capnella imbricata

Abstract

A new sesquiterpene, capgermacrene H (1) was isolated together with 7 other compounds, capgermacrenes A-G (2-8), from a population of soft coral Capnella imbricata collected from Mantanani Island, Sabah, Malaysia. The structure of this metabolite was elucidated based on spectroscopic data such as nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry. Capgermacrene A (2) showed potent cytotoxicity against adult T-cell leukemia, S1T cells at half-maximal inhibitory concentration of 0.79 µg/mL.

Soft corals of the genus Capnella is known to be a rich source of sesquiterpenes. ¹ Isolated terpenes often exhibited profound biological activities such as antitumor, antiviral, antifouling, and anti-inflammation. ² In the past, we discovered and reported bicyclogermacrene-related compounds from the Bornean soft coral genus Capnella, which showed anti-inflammatory and antibacterial potentials. ^{3 -5} As part of our search for cytotoxic substances, a new germacrane, capgermacrene H (1), together with another 7 known bicyclogermacrenes, capgermacrenes A-G (2-8), ^{3 -5} was isolated from this population of Capnella imbricata (Figure 1). Here, we report the isolation, structure elucidation and cytotoxic activity of these compounds against adult T-cell leukemia, S1T cells.

Figure 1

Structure of compounds 1 to 8.

Compound 1 was isolated as colorless oil. Its molecular formula was determined as C₁₅H₂₂O₂ based on high-resolution electrospray ionization mass spectrometry (HRESIMS) ions at m/z 257.1509 [M + Na]⁺ (calcd for C₁₅H₂₂O₂Na, 257.1512). The IR spectrum suggested the presence of a hydroxy and conjugated carbonyl groups (3400 and 1683 cm⁻¹). The ¹³C NMR data (Table 1) showed 15 distinct signals implying a sesquiterpene, while the presence of a carbonyl group at δ _C 207.3, one trisubstituted olefin at δ _C 138.1 and 136.7; δ _H 5.40, two disubstituted double bonds at δ _C 145.9, 139.5, 124.1, and 110.3; δ _H 5.33, 5.31, 4.79, and 4.78, a hydroxy-bearing carbon at δ _C 73.9, and hydroxy methyl group at δ _C 29.1; δ _H 1.27 within the molecule was indicated by both ¹H and ¹³C NMR spectra (Table 1). The presence of two oxygen atoms was consistent with HRESIMS measurement.

Two spin systems were determined based on the correlations of H-1/H₂-2/H₂-3 and H-5/H-6/H-7/H₂-8 from ¹H-¹H correlation spectroscopy (COSY) spectrum (bold lines in Figure 2a). These substructures and key heteronuclear multiple bond correlation (HMBC) experiment (arrows in Figure 2a) of H₃-12 to C-7, C-11, and C-13; H₃-14 to C-1, C-9, and C-10; H₃-15 to C-3, C-4, and C-5; and H₂-8 to C-9 permitted the establishment of a 10-membered ring germacrane-type sesquiterpenoid with a hydroxy group at C-4, an isopropenyl moiety at C-7, and a ketone group at C-9. The β relative configuration was arbitrary assigned at hydroxy methyl carbon in 1 (δ _C 29.1, C-15) because of large difference in ¹³C NMR to those of structurally related germacrene, lochmolin G with α relative configuration at hydroxy methyl carbon (δ _C 23.4, C-15). ⁶ Based on this finding, the β relative configuration at H₃-15 has permitted the assignment of α relative configuration for an isopropenyl unit at C-7 due to nuclear Overhauser effect (NOE) correlations (double arrows in Figure 2b) between H₃-15/H-5 and H-5/H-7. Furthermore, both the coupling constant and splitting pattern of H-6 in 1 having dd (15.1, 10.3 Hz) being identical to those of lochmolin G with dd (16.0, 10.0 Hz) suggested that H-7 in 1 must be in axial-like position as identical to those of lochmolin G [6]. However, ¹H NMR signals of H-5 (δ _H 5.31) and H-6 (δ _H 5.33) have a poor resolution because their signals were nearly superimposed in CDCl₃. Therefore, different shift-inducing pulsing power (C₆D₆) was used to resolve these signals (δ _H 5.05, H-5; δ _H 5.55, H-6), thus further confirmed above NOE correlations. The geometry of the double bond at C-1/C-10 was determined to be Z from the ¹³C NMR of the olefinic methyl carbon at C-14 (δ _C 20.0). ^7,8 This was further confirmed by NOE correlation between H-1/H₃-14, while the E configuration was assigned for a pair of double bond at C-5/C-6 based on large coupling constants between H-5 and H-6 (J = 15.1 Hz). ⁹ Upon examination of the molecular modeling, comparison with molecular modeling of lochmolin G and the above findings, the relative configuration of 1 was established.

Figure 2

(a) ¹H-¹H COSY and key HMBC and (b) selected NOE correlations of 1.

Cytotoxic effects of compounds 1 to 8 against adult T-cell leukemia, S1T cells were investigated by WST-8 assay except 4 due to insufficient yield. Compound 1 was inactive (half-maximal inhibitory concentration [IC₅₀] > 30.0 µg/mL) against S1T cells. The most potent compound was capgermacrene A (2) with IC₅₀ of 0.79 µg/mL, while the positive control etoposide had IC₅₀ of 0.048 µg/mL. Capgermacrenes B (3), D (5), E (6), F (7) and G (8) exhibited IC₅₀ of 7.79, 6.19, 4.75, 2.39, and 3.97 µg/mL, respectively.

Experimental

General

¹H NMR (600 MHz) and ¹³C NMR (150 MHz) spectra were recorded on a JEOL ECA 600 FT-NMR using CDCl₃ with TMS as an internal standard. The high-resolution mass spectrum was acquired via Liquid Chromatography-Mass Spectrometry Ion Trap Time Of Flight (Shimadzu). The AUTOPOL IV automatic polarimeter (Rudolph Research Analytic) and Fourier transform infrared spectrophotometer (Thermo Nicolet) were used.

Biological Material

A specimen of Capnella imbricata was collected from Mantanani Island, Sabah, Malaysia (6^o43.059″N, 116^o20.189″E), in June 2013. The voucher specimen (BORMI0006) was deposited in the BORNEENSIS Collection of the Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah.

Extraction and Isolation

Soft coral (0.9 kg wet weight) was extracted in MeOH at room temperature. The MeOH extract was concentrated and partitioned between EtOAc/H₂O. The EtOAc fraction was further partitioned with hexane/90% MeOH. The hexane crude (600.0 mg) was subjected to column chromatography eluted in hexane-EtOAc (increasing polarity) to obtain 6 fractions. A total of 175.0 mg of fraction 4 (hexane-EtOAc (1:1)) was purified by HPLC to yield compound 1 (2.0 mg). HPLC conditions: C18 column, 40°C of oven temperature, 1 mL/min for flow rate, 220 nm for UV detector wavelength, isocratic mode with H₂O-MeCN (35:65 (v/v)).

Compound 1

Colorless oil.

[α]_D ²⁵: -70.0 (c 0.02, CHCl₃).

IR (KBr) λmax: 3400 and 1683 cm^-1.

¹H and ¹³C NMR: Table 1.

Table 1

¹H and ¹³C NMR Data (600 and 150 MHz) of 1 (δ in ppm).

No	a δ C	a δ H (multiple, J in Hz)	b δ C	b δ H (multiple, J in Hz)
1	136.7	5.40 dd (11.0, 5.1)	136.4	5.08 dd (10.3, 4.8)
2	23.7	2.28 dt (14.6, 11.0)	24.1	2.61 m
		1.98 m		1.94 m
3	41.9	1.75 m	42.1	1.49 dd (14.4, 8.9)
		1.51 dd (14.6, 11.0)		1.16 dd (14.4, 11.0)
4	73.9		73.4
5	139.5	5.31 d (15.1)	139.7	5.05 d (15.1)
6	124.1	5.33 dd (15.1, 10.3)	124.5	5.55 dd (15.1, 10.3)
7	47.4	3.02 m	47.8	2.94 m
8	44.5	2.84 dd (12.4, 5.5)	44.7	2.69 dd (12.4, 5.5)
		2.38 t (12.4)		2.51 t (12.4)
9	207.3		205.0
10	138.1		138.2
11	145.9		146.3
12	20.9	1.76 s	20.9	1.69 s
13	110.3	4.79 s	110.2	4.89 s
		4.78 s		4.85 s
14	20.0	1.95 s	19.9	1.65 s
15	29.1	1.27 s	29.3	1.12 s

^aSpectra recorded in CDCl₃.

^bSpectra recorded in C₆D_6.

HRESIMS m/z 257.1509 [M + Na]⁺ (calcd for C₁₅H₂₂O₂Na, 257.1512) and 217.1591 [M − H₂O + H]⁺ (calcd for C₁₅H₂₁O, 217.1587).

Cytotoxic Assay

The adult T-cell leukemia cell line S1T was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamate. The cytotoxic assay was carried out as known procedure. ¹⁰

Supplemental Material

Supporting Information - Supplemental material for Cytotoxic Sesquiterpenoids From Soft Soral Capnella imbricata

Supplemental material, Supporting Information, for Cytotoxic Sesquiterpenoids From Soft Soral Capnella imbricata by Chin-Soon Phan, Takashi Kamada, Takahiro Ishii, Toshiyuki Hamada, and Charles Santhanaraju Vairappan in Natural Product Communications