Chinese Chemical Letters  2016, Vol. 27 Issue (7): 1048-1051   PDF    
Two new diterpene alkaloids from the South China Sea Sponge Agelas aff. Nemoechinata
Li Taoa, Wang Binb, J. de Voogd Nicolec, Tang Xu-Lid, Wang Qid, Chu Mei-Jund, Li Ping-Lind, Li Guo-Qiangd     
a Marine Science and Technology College, Zhejiang Ocean University, Zhoushan 316022, China ;
b School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, China ;
c National Museum of Natural History, 2300 RA Leiden, Netherlands ;
d College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China ;
e School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
Abstract: Two new N-methyladenine-containing diterpenes, nemoechines F (1) and G (2), were isolated from the South China Sea sponge Agelas aff. nemoechinata. The structures were assigned on the basis of spectroscopic date. Compound 2 showed weak activity against Jurkat cell lines with IC50 of 17.1 μmol/L.
Key words: South China Sea     Marine sponge     Agelas     Diterpene alkaloids     Cytotoxic    
1. Introduction

Marine organisms produce various secondary metabolites which exhibit characteristic structures features and diverse bioactivities [1]. Marine sponges of the genus Agelas (order Agelasida, family Agelasidae) have been demonstrated to be a rich source of bromopyrrole alkaloids and diterpene alkaloids [2-5]. These unique secondary metabolites displayed a wide range of biological activities, including antileukemic [6], cytotoxic [7], antimicrobial [8, 9], antimalarial [10], antifouling [4] and Na, K-adenosine triphosphatase (ATPase) inhibiting [11] activities. As part of our searching for novel bioactive substances from marine sponges, we investigated Xisha islands (Paracel islands) sponge Agelas aff. nemoechinata, which led to the isolation of two new diterpene alkaloids with a 9-N-methyladenine unit, nemoechines F (1) and G (2) (Fig. 1). Here we report the isolation, structure elucidation and bioactivities of compounds 1 and 2.

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Figure 1. Structures of compounds 1 and 2.

2. Experimental 2.1. General experimental procedures

Optical rotations were measured on a Jasco P-1020 digital polarimeter. UV spectra were measured on a Beckman DU640 spectrophotometer. CD spectra were obtained on a Jasco J-810 spectropolarimeter. IR spectra were recorded on a Nicolet NEXUS 470 spectrophotometer using KBr discs. 1D and 2D NMR spectra were recorded on an Agilent 500-MR DD2 spectrometer using residual solvent signals (for DMSO-d6: δH2.50 and δC 39.52 ppm) as reference standards. HRESIMS was performed on a Micromass Q-TOF Ultima Global GAA076 LC-MS spectrometer. Semi-preparative HPLC utilized an ODS column [YMC-Pack ODS-A, 10 mm × 250 mm, 5 mm, 1.5 mL/min]. MPLC utilized an ODS column [26 mm × 310 mm, 13 mL/min].

2.2. Animal material

The sponge A. aff. nemoechinata was collected from the coralreef regions in the South China Sea, in June 2009, at a depth of about 20 m. The sponge was identified by one of the authors (N.J.D.V.). A voucher specimen (No. XS 2009-26) is deposited in the School of Medicine and Pharmacy, Ocean University of China, People’s Republic of China.

2.3. Extraction and isolation

The frozen specimen (5.0 kg, wet weight) was mined and extracted with MeOH three times (each time, 5 days) at room temperature. The combined solutions were concentrated in vacuum, and the concentrated extract was finally desalted by redissolving with MeOH three times to yield a residue (310.5 g). The crude extract was subjected to silica gel vacuum liquid chromatography (VLC), eluting with a gradient of petroleum/ acetone (from 10:0 to 1:1, v:v) and dichloromethane-methanol (from 50:1 to 1:1, v:v) to obtain seven fractions (FA-FG). FG (10.2 g) was isolated on a silica gel column using dichloromethanemethanol (20:1, v/v) as eluting solvent to afford three fractions (G1-G3) . G2 (4.1 g) was subjected to MPLC (ODS, 26 mm × 310 mm, 13 mL/min, UV detection at 210 nm and 280 nm), eluting with MeOH-H2O-TFA (75:25:0.1) , to give two fractions (G201-G202) . G201 was purified by HPLC (YMC Pack B C-18, 5 mm, 1.5 mL/min, UV detection at 210 nm), eluting with MeOH-H2O-TFA (70:30:0.1) , to give compound 1 (3.8 mg). G202 (18.9 mg) was purified by HPLC (YMC Pack B C-18, 5 mm, 1.5 mL/min, UV detection at 210 nm), eluting with CH3CN-H2O- TFA (50:50:0.1) , to afford compound 2 (3.4 mg).

2.4. Computational

The quantum chemical calculations were performed by using the density functional theory (DFT) as carried out in the Gaussian 09 [12]. The initial structures of compound 1 were built with Spartan 10 software and all trial structures were firstly minimized based on molecular mechanics calculations. Conformational search was performed by Spartan 10 software using MMFF force filed, and conformers occurring within a 10 kcal/mol energy window from the global minimum were chosen for geometry optimization in the gas phase with the DFT method at the B3LYP/6- 31G (d, p) levels. The stable conformations of 1 were calculated for ECD spectra using TD-DFT method with the basis set RB3LYP/ DGDZVP [13]. Solvent effects of methanol solution were evaluated at the same DFT level by using the SCRF/PCM method.

Nemoechine F (1): Yellow oil; [α]D23+9.9 (c 0.1, MeOH); UV (MeOH) (log ε) λmax 209 (3.7) , 270 (3.3) nm; IR (KBr) νmax 3446, 2924, 1733, 1683, 1558, 1540, 1210, 1137, 724 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, see Table 1; ESIMSm/z 438.3 [M]+; HRESIMSm/z 438.3229 [M]+ (calcd. for C26H40N5O, 438.3227) .

Table 1
1H and 13C NMR date for compounds 1 and 2.

Nemoechine G (2): Yellow oil; [α]D23+8.5 (c 0.1, MeOH); UV (MeOH) (log ε) λmax 209 (4.0) , 270 (3.7) nm; IR (KBr) νmax 3402, 2927, 1682, 1557, 1540, 1457, 1209, 1137, 723 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, see Table 1; ESIMSm/z 422.4 [M]+; HRESIMSm/z 422.3282 [M]+ (calcd. for C26H40N5, 422.3278) .

3. Results and discussion

Nemoechine F (1) was obtained as yellow oil. The molecular formular of 1 was determined as C26H40N5O from HRESIMS and 13C NMR date (Table 1). 1H NMR spectrum of 1 showed two aromatic proton ( 9.49, s; 8.47, s) signals, five skeletal methyl (1.82, s; 0.80, d, J = 6.70 Hz; 0.75, d, J = 6.65 Hz; 0.72, s; 0.64, s) signals and one N-methyl (3.88, s) signal, indicating that 1 was a clerodane diterpenoid alkaloid with 9-N-methyladeninium moiety. The 1H and 13C NMR date of 1 were similar to those of 2-oxoagelasine B [14]. Detailed analysis of 1H NMR spectrum of 1 indicated that 1 lacked an olefinic proton (5.70, s) signal and an olefinic methyl (1.94, s) signal compared with 2-oxo-agelasine B [14], instead 1 had one more splitted methyl group (0.75, d, J = 6.65 Hz) and carbonyl group, indicating the difference in the clerodane diterpenoid part structure. The planar structure of compound 1 was speculated with 2D NMR of COSY, HMQC and HMBC spectra (Fig. 2). The COSY spectrum of compound 1 displayed connections from C-10 to C-2, C-4 to C-18, C-6 to C-8, C-8 to C-17, C-11 to C-12 and C-14 to C-15. The HMBC correlations from H3-18 to C-3, C-4 and C-5 suggested 3-oxo-4-methyl moiety in 1. In addition, HMBC correlations from H2-15 to C-13, C-14, C-50 and C-80 revealed that the clerodane unit was attached to N-70 of the 9-N-methyladenine moiety. Therefore, the planar structure of nemoechine F (1) was illuminated as shown in Fig. 2.

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Figure 2. COSY and key HMBC dates for compounds 1 and 2.

The relative stereochemistry of 1 was inferred from NOESY spectrum (Fig. 3). NOESY correlations between H-17 (δH 0.80) /H- 20 (δH 0.72) , H-20 (δH 0.72) /H-19 (δH 0.64) , H-19 (δH 0.64) /H-18 (δH 0.75) , H-10 (1.72) /H-4 (δH 2.32) and H-10 (δH 1.72) /H-8 (δH 1.54) indicated that the four methyl groups were on the same side of the decahydronaphthalene ring and compound 1 possessed a transclerodane moiety. The E-orientation of the double bond at C-13 was established from NOESY correlations between H-12 (δH 2.05) / H-14 (δH 5.49) and H-15 (δH 5.16) /H-16 (δH 1.82) . The circular dichroism (CD) spectrum of 1 showed negative Cotton effect at 220 nm and positive Cotton effect at 288 nm, which are consistent with the negative Cotton effect at 221 nm and positive Cotton effect at 298 nm in the electronic CD (ECD) spectrum of 1 (Fig. 4) predicted from quantum mechanical time dependent density functional theory (TDDFT) calculations [13]. Thus, the absolute figuration was deduced for 1 (4S, 5R, 8R, 9S, 10R).

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Figure 3. Key NOESY correlations of compounds 1 and 2.

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Figure 4. The experimental CD and calculated ECD spectrum of compound 1.

Nemoechine G (2) was isolated as yellow oil. The similarity of UV absorption pattern (λmax 209, 270 nm, MeOH) to that of 1 indicated that 2 was a related secondary metabolite. The molecular form of 2 was revealed to be C26H40N5 by HRESIMS date and 13C NMR date (Table 1). 1H NMR spectrum showed two aromatic proton (δH 9.49, s; δH 8.47, s) signals, five skeletal methyl (δH 1.82, s; 1.55, s; 0.92, s; 0.87, s; 0.81, s) signals and one N-methyl (δH 3.88, s) signal, deducing that 2 was a labdane diterpenoid alkaloid with 9-N-methyladeninium moiety [15]. The planar structure of compound 2 was determined with 2D NMR. Connections from C-1 to C-3, C-5 to C-7, C-11 to C-12 and C-14 to C-15 were clarified by analysis of the 1H-1H COSY of 2. Furthermore, HMBC correlations from H2-15 to C-13, C-14, C-50 and C-80 disclosed that 9-N-methyladenine moiety was attached to C-15 of labdane moiety through N-70. Thus, the planar structure of nemoechine G (2) was elucidated as shown in Fig. 2.

The relative stereochemistry of 2 was deduced from NOESY spectrum (Fig. 3). The trans-configuration of labdane unit of nemoechine G (2) was determined from NOESY correlations between H-19 (δH 0.81) /H-20 (δH 0.92) and H-18 (δH 0.87) /H-5 (δH 1.07) . Moreover, NOESY correlations between H-12 (δH 2.11) /H-14 (δH 5.47) and H-15 (δH 5.16) /H-16 (δH 1.82) indicated the Eorientation of the double bond at C-13. The absolute figuration of natural product 2 (5S, 10S) was established by comparison of its optical rotation [α]D23 +8.5 (c 0.1, MeOH) and the synthesis product (+)-subersic acid [α]D20 +49 (c 0.78, CHCl3) [16].

4. Conclusion

Two new N-methyladenine-containing diterpenes, nemoechines F (1) and G (2), were isolated from the sponge A. aff. nemoechinata. Their structures were elucidated by analysis of 1DNMR, 2D-NMR spectra and HRESIMS. In the cytotoxic assay against human colon carcinoma HCT-116 cells, human lung carcinoma A- 549 cells and human lymphoblastic leukemia Jurkat cells in vitro, only compound 2 showed weak activity against Jurkat cell lines with IC50 of 17.1 mmol/L. Moreover, compounds 1 and 2 did not exhibit antimicrobial activity against Staphyloccocus aureus and Bibrio parahemolyticus.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 41376142 and 41476107) , NSFCShandong Joint Fund for Marine Science Research Centers (No. U1406402) . Special thanks are given to engineer L. Liu (Ocean University of China, Qingdao, China) for the cytotoxicity tests.

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