A New Bioactive Cembranolide Sarcophytonolide V from Bornean Soft Coral Genus Sarcophyton

Abstract

A new cembranolide diterpene, sarcophytonolide V (1), along with 6 known compounds, isosarcophytonolide D (2), (4Z,8S*,9R*,12E,14E)-9-hydroxy-1-(prop-1-en-2-yl)-8,12-dimethyl-oxabicyclo[9.3.2]-hexadeca-4,12,14-trien-18-one (3), (7E,11E)-3,4-epoxy-7,11,15-cembratriene (4), (1S*,3S*,4S*,7E,11E)-3,4-epoxy-13-oxo-7,11,15-cembratriene (5), (-)-eunicenone (6), and 2-[(E,E,E)-7′,8′-epoxy-4′,8′,12′-trimethylcyclotetradeca-1′,3′,11′-trienyl]propan-2-ol (7) were isolated from the Bornean soft coral Sarcophyton sp. Their structures were elucidated based on spectroscopic data, such as nuclear magnetic resonance (NMR) and high resolution electron spray ionization mass spectroscopy (HRESIMS). These compounds were evaluated for their biological activity against marine pathogenic fungi.

Soft corals genus Sarcophyton (Alcyoniidae) are known to be a rich source of diterpenes, where cembrane represent the most commonly encountered structural type. ^1,2 In addition, some of them have exhibited very interesting biological activities such as anticancer and antibacterial. ^2,3 In the course of our recent investigation, one population of Sarcophyton sp. was collected from the coastal waters in Sepanggar Bay, Sabah, North Borneo and has led to the isolation of a new cembranolide, sarcophytonolide V (1), along with 6 known compounds (2-7), as shown in Figure 1. Here, we report the isolation, structure elucidation, and antifungal activities of these secondary metabolites.

Figure 1

Structures of compounds 1 to 7.

Sarcophytonolide V (1) was obtained as a colorless oil with ${\displaystyle [\alpha {]}_{\mathrm{D}}^{25}}$ −60.8 (c 0.32, CHCl₃). Its molecular formula, C₂₀H₂₈O₄, was deduced based on high resolution electron spray ionization mass spectroscopy (HRESIMS) [M + H]⁺ ion at m/z 333.2067 (calculated for C₂₀H₂₉O₄, 333.2060). Thus, seven degrees of unsaturation were determined for compound 1. Infrared spectroscopy (IR) absorptions at ν _max 1762 and 1703 cm⁻¹, suggested the presence of carbonyl functionality. The presence of α, β-unsaturated γ-lactone system was obvious with the presence of δ _C 172.5, 150.4, 127.7, and 80.3; δ _H 7.02 and 5.20, 2 ketone carbonyls at δ _C 208.1 and 205.7, 2 trisubstituted double bonds at δ _C 150.4, 131.0, 128.0, and 127.7; δ _H 7.02 and 5.22, and isopropyl group at δ _C 28.9, 20.3, and 19.3; δ _H 2.28, 1.13, and 1.04 (Table 1). The two ketone carbonyls, ester carbonyl, and two pairs of double bonds accounted for five degrees of unsaturation. The remaining two degrees of unsaturation were attributed to the bicyclic system in compound 1.

Table 1

¹H-/¹³C-NMR Data (600 MHz/150 MHz, CDCl₃) for Compound 1 (δ in ppm, J in Hz).

Position	δ C	δ H
1	58.5	2.84 m
2	80.3	5.20 dd (9.6, 1.4)
3	150.4	7.02 d (1.4)
4	127.7
5	40.2	3.42 d (15.8), 3.16 d (15.8)
6	205.7
7	49.7	2.32 m, 2.31 m
8	28.1	1.72 m
9	35.7	1.42 m
10	24.3	2.21 m, 2.00 m
11	131.0	5.22 m
12	128.0
13	56.2	3.26 d (12.4), 2.82 m
14	208.1
15	28.9	2.28 m
16	20.7	1.13 d (6.9)
17	19.3	1.04 d (6.9)
18	172.5
19	20.3	0.94 d (6.9)
20	16.6	1.68 s

Two ¹H–¹H correlation spectroscopy (COSY) spin systems were present; H-15/H-1/H-2 and H₂-7/H-8/H₂-9/H₂-10/H-11. Key heteronuclear multiple bond correlation spectroscopy (HMBC) were H₃-20 to C-11, C-12, and C-13; H₃-19 to C-7, C-8, and C-9; H₃-17 to C-1, C-15, and C-16; H₃-16 to C-1, C-15, and C-17; H₂-13 to C-14; H₂-7 to C-6; H₂-5 to C-3, C-6, and C-18; H-15 to C-14; H-2 to C-4, these correlations lead to the establishment of the cembranolide-type skeleton of compound 1. Therefore, planar structure of compound 1 was established as shown in Figure 2. Position of α, β-unsaturated γ-lactone at C-4 (α), C-3 (β), C-2 (γ), and C-18 (carbonyl carbon) was deduced from HMBC between H-3 and C-2, C-4, C-5, and C-18 and between H-2 and C-1. Vinyl methyl group attached at C-12 was observed by HMBC between H₃-20 and C-11, C-12, and C-13 and between H₂-10 and C-11 and C-12. The last methyl group was assigned at C-8 based on HMBC between H₃-19 and C-7, C-8, and C-9, biogenetic considerations were also taken into account in this placement. Geometry of carbon–carbon for H₃-20 was defined as E based on its chemical shift that was <20 ppm. The ¹H and ¹³C NMR data (Table 1) of compound 1 were near identical to sarcophytonolide C, where methylene at C-14 in sarcophytonolide C was replaced by a carbonyl in compound 1. ⁴ Presence of carbonyl at C-14 resulted in a downfield shifted in chemical shifts at C-1 and C-13 in compound 1 when compared to sarcophytonolide C. The relative stereochemistry of compound 1 was assigned on the basis of a 2D NOESY experiment (Figure 2). Correlations were observed between H-3, H-8 and H₂-13, and between H-2, H₃-16 and H₃-17. These correlations suggested H-2 and H-8 are on the same face, while H-1 is on the opposite face. These relative configurations were identical to those closely related analogs, sarcophytonolides C, E and J. ⁴

Figure 2

¹H–¹H COSY (bold), and key HMBC (single-headed arrows) and NOE (double-headed arrows) correlations of compound 1.

Known compounds were identified as isosarcophytonolide D (2), (4Z,8S*,9R*,12E,14E)-9-hydroxy-1-(prop-1-en-2-yl)-8,12-dimethyl-oxabicyclo[9.3.2]-hexadeca-4,12,14-trien-18-one (3), (7E,11E)-3,4-epoxy-7,11,15-cembratriene (4), (1S,3S,4S,7E,11E)-3,4-epoxy-13-oxo-7,11,15-cembratriene (5), (-)-emicenone (6), and 2-[(E,E,E)-7′,8′-epoxy-4′,8′,12′-trimethylcyclotetradeca-1′,3′,11′-trienyl]propan-2-ol (7) by comparing their spectroscopic data with those reported in the literature. ^{5 -9} These compounds were tested for their antifungal activities against 8 strains of marine fungi (Exophiala sp. NJM 1551, Ochroconis humicola NJM 1503, Haliphthoros milfordensis IPMB 1603, Lagenidium thermophilum IPMB 1401, Fusarium moniliforme NJM 8995, Fusarium oxysporum NJM 0179, Haliphthoros sabahensis IPMB 1402, and Fusarium solani NJM 8996). Compound 1 exhibited inhibition against hyphal growth of O. humicola and H. milfordensis at minimum inhibitory concentration (MIC) 6.25 µg/mL.

Experimental

General

NMR (ECA 600 MHz, Jeol, Japan); liquid chromatography electrospray ionisation ion trap time of flight mass spectrometry (LC-ESI-IT-TOF-MS) (Shimadzu, Japan); fourier transform infrared spectroscopy (Nicolet, Thermo Fisher Scientific, America); AUTOPOL IV automatic polarimeter (Rudolph Research Analytical, America). All instruments are located at the Laboratory of Natural Products Chemistry, Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah.

Biological Material

A specimen of Sarcophyton sp. was collected from Sepanggar Bay, North Borneo, (6^o4.683′N, 116^o4.710′E) on July 2016. The voucher specimen (BORMI0054) was deposited in the BORNEENSIS Collection of the Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Malaysia.

Extraction and Isolation

Fresh soft coral (0.6 kg wet weight) was extracted in methanol (MeOH) at room temperature for 3 days. The resulting raw crude extract was concentrated in vacuo and partitioned between ethyl acetate (EtOAc)/distilled water (H₂O), which EtOAc fraction was further partitioned with n-hexane/90 % MeOH. The resulting crude extracts were subjected to column chromatography (CC) eluting with a gradient of n-hexane and EtOAc with increasing polarity. Fraction 1 obtained in n-hexane-EtOAc (9:1) gave compound 4 (5.7 mg; 2.7%) after purification by preparative thin layer chromatography (TLC) using n-hexane. The fraction 2, obtained in n-hexane-EtOAc (8:2), was subjected to preparative TLC with n-hexane-EtOAc (9:1) and toluene-EtOAc (9:1) to yield compounds 5 (27.0 mg; 12.9%) and 6 (12.3 mg; 5.9%). The fraction 3 obtained in n-hexane-EtOAc (7:3) gave compounds 1 (9.7 mg; 4.6%), 2 (9.0 mg; 4.3%), and 7 (21.3 mg; 10.1%) after purification by preparative TLC using n-hexane-EtOAc (8:2), toluene-EtOAc (8:2), and CHCl₃. The fraction 4, obtained in n-hexane-EtOAc (5:5), was subjected to preparative TLC with n-hexane-EtOAc (7.5:2.5) to yield compound 3 (22.1 mg; 10.5%). Percentages of compounds were the average of the respective compounds in 90% MeOH crude.

Sarcophytonolide V (1)

Colorless oil.

${\displaystyle [\alpha {]}_{\mathrm{D}}^{25}}$ : −60.8 (c 0.32, CHCl₃).

IR: λ _max 2961, 2927, 1762, 1703, and 1373 cm⁻¹.

¹H and ¹³C NMR: Table 1.

HRESIMS: m/z 333.2067 [M + H]⁺ (calculated for C₂₀H₂₉O₄, 333.2060).

Antifungal Activity

The MIC against 8 strains of marine fungi was performed by incorporating the compound solutions (100, 50, 25, 12.5, and 6.25 µg/mL) onto peptone yeast glucose sea water agar in petri dish. ¹⁰ The MIC was determined visually as the lowest concentration showing no hyphal growth when they were incubated at 25°C for 7 days.