Clionaterpene, a New Cadinene Sesquiterpene from the Marine Sponge Cliona sp.

Abstract

Clionaterpene (1), a new cadinene sesquiterpene, along with six known compounds 2–7 were isolated from the marine sponge Cliona sp. The structure of 1 with absolute configuration was determined by the spectroscopic data (UV, IR, MS, and NMR) and quantum chemical calculation. Compound 1 was the first cadinene sesquiterpene metabolite isolated from this genus. In addition, the cytotoxic and antibacterial activities of these compounds were evaluated. However, none of them exhibited significant inhibition effects.

1. Introduction

The marine sponges of genus Cliona belong to the family Clionidae, order Hadromeride, which were mainly discovered in Canada [1,2], Italy [3], Portugal [4], Spain [5], and Thailand [6]. Thus far, about 78 species of this genus had been described. Scientific study of this genus could be traced back to 1910s. In recent years, a variety of secondary metabolites had been isolated, mainly including sterols [7], peptides [8], alkaloids [5], lipids [9], and a small amount of terpenes [10]. Most of these compounds represented plentiful biological activities, such as antibacterial [11], cytotoxic [7], anti-inflammatory and antioxidant [4]. Cadinene sesquiterpenes originated from FPP (Farnesyl diphosphate) as a precursor and were catalyzed by STS (Sesquiterpene synthase) [12]. Most of these sesquiterpenes had been isolated from plants, rarely discovered from fungi and bacteria, and only a few separated from animals [13]. However, it had never been separated from this genus yet.

In the process of our exploration of marine natural products [14,15,16,17], a new cadinene sesquiterpene, clionaterpene (1), together with six known compounds (2–7), had been isolated from the marine sponge Cliona sp. (Figure 1). Compound 1 was the first cadinene sesquiterpene metabolite isolated from this genus. Compounds 2–7 were isolated from the genus of Cliona for the first time. Herein, we discussed the isolation process, structural identification and biological activities of these compounds.

2. Materials and Methods

2.1. Materials

The marine sponge was collected from Xuwen Country, Zhanjiang City, Guangdong Province in May 2017, which was identified by the researcher Liu Cui as Cliona sp. The specimen (2017-3) had been deposited in the Department of Chemistry, College of Chemistry and Materials Science, Jinan University.

2.2. Extaction and Isolation

The marine sponge Cliona sp. (20.0 kg) was cold soaked and extracted with 95% EtOH at room temperature to get the crude extract (516.0 g). The extract was suspended in water and partitioned with petroleum ether, EtOAc and n-butanol successively to afford three corresponding portions. The petroleum ether extract (101.9 g) was applied to silica gel column chromatography with gradient elution using petroleum ether/EtOAc (100:0~0:100) to gain 10 fractions (Frs. 1~10). Compound 1 (1.1 mg) was isolated by semi-preparative reversed-phase HPLC (2.5 mL/min, CH₃OH/H₂O = 85:15, t_R = 68.0 min) from Fr. 7 (3.9 g). The EtOAc extract (8.3 g) was applied to silica gel column chromatography with gradient elution using CH₂Cl₂/acetone (100:0~0:100) to gain 8 fractions (Frs. 1~8). Fr. 4 (444.2 mg) was subjected to a silica gel column eluted with petroleum ether/ethyl acetate to provide 9 fractions (Frs. 4–1~4–9). Compounds 2 (2.6 mg) and 3 (1.8 mg) were isolated by semi-preparative reversed-phase HPLC (2.5 mL/min, CH₃CN/H₂O = 40:60, t_R = 22.0 min; 2.5 mL/min, CH₃OH/H₂O = 40:60, t_R = 16.2 min) from Fr. 4–6 (90.1 mg), respectively. Compound 4 (12.7 mg) was purified by semi-preparative reversed-phase HPLC (2.5 mL/min, CH₃OH/H₂O = 50:50, t_R = 8.7 min) from Fr. 4–8 (56.8 mg). Compound 5 (3.1 mg) was separated by preparative reversed-phase HPLC (5 mL/min, CH₃OH/H₂O = 40:60, t_R = 23.1 min) from Fr. 7 (152.0 mg). Compound 7 (3.1 mg) was purified by preparative reversed-phase HPLC (5 mL/min, CH₃OH/H₂O = 25:75, t_R = 11.3 min) from Fr. 8 (141.3 mg). Fr. 9 (575.0 mg) was subjected to a silica gel column eluted with CH₂Cl₂/acetone to provide 8 fractions (Frs. 9–1~9–8). Compound 6 (3.4 mg) was obtained after recrystallization of crystals precipitated from Fr. 9–2 (7.7 mg).

3. Results

Clionaterpene (1) was isolated as yellow amorphous powder. The molecular formula of 1 was established as C₁₅H₂₄O₂ by HR-ESI-MS at m/z 237.1842 [M + H]⁺ (Figure S1). The UV absorption maxima were at 201 and 223 nm (Figure S2). The IR bands revealed the presence of hydroxyl (3446 cm⁻¹) and carbonyl (1748 and 1646 cm⁻¹) groups (Figure S3). The 1D NMR spectra (Figures S4 and S5) of 1 demonstrated that 1 included a carbonyl (δ_C 199.7), a pair of double bond carbons (δ_H 6.87 (q, J = 1.2 Hz, H-5); δ_C 153.5 and 133.7), a quaternary carbon (δ_C 74.9), four methines (δ_H 2.11 (m, H-10), 2.03 (m, H-12), 2.01 (H-1) and 1.53 (H-7); δ_C 49.0, 46.3, 29.2 and 27.0), three methylenes (δ_H 2.41 (dd, J = 18.0, 6.0 Hz, H-2a), 2.31 (dd, J = 18.0, 13.8 Hz, H-2b), 1.58 (H-9a), 1.52 (H-8) and 1.18 (m, H-9b); δ_C 34.7, 28.8 and 20.4), and four methyls (δ_H 1.80 (d, J = 1.2 Hz, H-11), 0.99 (d, J = 7.2 Hz, H-13), 0.90 (d, J = 7.2 Hz, H-14) and 0.87 (d, J = 7.2 Hz, H-15); δ_C 23.8, 18.6, 18.4 and 15.6). These data were analogous to those of 6α,9α-dihydroxycadinan-4-en-3-one [18], which suggested that 1 was also a cadinene sesquiterpene. The distribution of protons and carbon signals (Figure S6) were displayed in

Table 1. ¹H and ¹³C NMR data of 1 (in CDCl₃ δ, J in Hz) ^a.

No.	δH	δC
1	2.01	49.0
2	a 2.41 dd (18.0, 6.0) b 2.31 dd (18.0, 13.8)	34.7
3	-	199.7
4	-	133.7
5	6.87 q (1.2)	153.5
6	-	74.9
7	1.53	46.3
8	1.52	20.4
9	a 1.58	28.8
	b 1.18 m
10	2.11 m	29.2
11	1.80 d (1.2)	15.6
12	2.03 m	27.0
13	0.99 d (7.2)	18.6
14	0.90 d (7.2)	23.8
15	0.87 d (7.2)	18.4

^a Overlapped signals are reported without designating multiplicity.

.

The ¹H-¹H COSY (Figure S7) indicated a group of spin coupling systems, which are shown in blue and bold in Figure 2. In the HMBC spectrum (Figure S8), the correlations from H-1 to C-7, from H-2 to C-3/C-6, from H-5 to C-1/C-3/C-7, from H-8/H-12 to C-6 as well as from H-11 to C-3/C-5 revealed the basic skeleton of cadinene sesquiterpene. The presence of the 6-OH group was defined by the molecular formula and the chemical shifts of C-6. Thus, the planar structure of 1 was determined (Figure 2).

In the NOESY spectrum of 1 (Figure S9), the NOE correlations between H-1 and H-13/H-15 revealed that these protons were at the same orientation. The NOE correlations between H-7 and H-10 demonstrated that these protons were at the oppositive side to those mentioned above (Figure 3). However, due to the lack of sufficiently convincing NOE correlations, the relative configuration of the remaining stereocenter (C-6) could not be assigned.

In order to demonstrate the relative configuration of 1, DFT-NMR calculations (Figure S10 and Table S1) and DP4+ probability (Table S2) were carried out with the two possible configurations, 1A (1R*, 6S*, 7S*, 10S*) and 1B (1R*, 6R*, 7S*, 10S*). The results exhibited that the calculated ¹³C NMR data for 1A showed a good agreement with the measured one (Figure 4). In addition, the plausible candidate 1A was determined to have 1R*, 6S*, 7S*, and 10S* configuration with 99.92% probability (

Table 2. DP4+ probability of ¹³C NMR of 1.

Possible Isomer	1A	1B
sDP4+	96.70%	3.30%
uDP4+	97.84%	2.16%
DP4+	99.92%	0.08%

). Therefore, the relative configuration of 1 could be ascertained as 1A.

Furthermore, to determine the absolute configuration of 1, a quantum chemical ECD calculation of 1 was carried out. Conformational searches for (1R*, 6S*, 7S*, 10S*)-1A were performed using the MMFF94s force-field (Figure S11). From this, we determined one stable conformer (Table S3). The optimized conformer was used for the quantum-chemical ECD calculations with the two possible absolute configurations, (1R, 6S, 7S, 10S)-1 and (1S, 6R, 7R, 10R)-1. As a result, the calculated ECD curve of (1S, 6R, 7R, 10R)-1 showed a good agreement with the experimental one, which established the absolute configuration of 1 as 1S, 6R, 7R, 10R (Figure 5).

The six known compounds were identified as 3S,5R-loliolide (2) [19], 3R,5R-loliolide (3) [19], benzeneacetamide (4) [20], catechol (5) [21], 4-hydroxybenzal (6) [22], and 4-aminophenylacetic acid (7) [23] by comparing their spectroscopic data with those of literatures. Additionally, the cytotoxic and antibacterial activities were evaluated (Biological assay). However, none of them showed obvious cytotoxic and antibacterial activities on the K562 cell lines and the five bacterial strains with IC₅₀ values of >50 μM and MIC values of >128 μg/mL, respectively.

4. Conclusions

In conclusion, a new cadinene sesquiterpene, clionaterpene (1), together with six known compounds (2–7), were isolated from the marine sponge Cliona sp. Compound 1 was the first cadinene sesquiterpene metabolite separated from this genus. Compounds 2–7 were isolated from this genus for the first time. However, none of them displayed significant cytotoxic and antibacterial activities.