A new diketopiperazine from the gorgonian coral Menella kanisa

Chemical investigation on the gorgonian Menella kanisa collected from Beibu Gulf led to the isolation of a new diketopiperazine, named menazepine A (1), as well as three known diketopiperazines, namely cyclo(4-hydroxyprolylleucyl) (2), cyclo(Pro-Leu) (3) and cyclo(4-hydroxyprolylphenylalanyl) (4). The structure of the new diketopiperazine was elucidated on the basis of extensive spectroscopic analysis and by comparing the data with those of related metabolites. Compounds 1–4 were also evaluated for brine shrimp lethality.


Results and discussion
Compound 1, a white needle crystal, was established as C 14 H 19 N 3 O 5 (seven units of unsaturation) based on the HR-ESI-MS ([M þ H] þ at m/z 310.1401, calcd for C 14 H 20 N 3 O 5 , 310.1403). The 13 C NMR data for 1 confirmed the presence of 14 carbon signals, characterised by DEPT as a methyl, four sp 3 methylenes, four sp 3 methines, a sp 2 methine, four sp 2 quaternary carbons. 1 H NMR chemical shifts of two a-methine protons at d H 4.56 (1H, dd, J ¼ 7.1, 3.0 Hz, H-9) and 4.60 (1H, dd, J ¼ 6.3, 3.1 Hz, H-6), and 13 C NMR chemical shifts of two carbonyl carbons at d C 169.0 (C-7) and 167.5 (C-1), supported the presence of a peptide fragment. The fact that compound 1 was negative to the ninhydrin test suggested a cyclic or an N-terminusblocked peptide (Shigemori et al. 1998;Huang et al. 2010b).
The absolute configuration of compound 1 was determined by optical rotation and NOESY spectrum. In the NOESY spectrum ( Figure S2), H-9 exhibited a correlation with H-11, not with H-6, and H-6 did not display a correlation with H-4, which indicated that H-4, H-9 and H-11 were situated on the same face and assigned as b protons. The sign for [a] D for prolinecontaining diketopiperazines is either negative or positive, depending only on the absolute configuration of Pro (Adamczeski et al. 1995;Huang et al. 2010b). On the basis of ½a 20 D (2 28) and by comparing the NMR data of the proline residue with those of proline-containing diketopiperazines (Adamczeski et al. 1995;Jayatilake et al. 1996;Huang et al. 2010b), which suggested C-6 has (S)-configuration, Pro in 1 has, therefore, (S)-configuration, and the abovementioned data approved the absolute configuration of 1 as 4R, 6S, 9R and 11R.
The results of brine shrimp lethality of compounds 1 -4 are shown in Table S1. The toxicity of compound 3 was significantly stronger than those of the other three compounds (Figure 1).
3. Experimental 3.1. General experimental procedures 1 H and 13 C NMR spectra were recorded on a Bruker AC 600 NMR spectrometer with TMS as an internal standard at 600 and 150 MHz, respectively. HR-ESI-MS data were obtained from Bruker Maxis mass spectrometer. Waters-2695 HPLC system, was performed on a Sunfire TM C 18 column (250 £ 10 mm i.d., 10 mm) coupled to a Waters 2998 photodiode array detector. Optical rotation data were measured by Perkin-Elmer Model 341 polarimeter. The silica gel GF 254 used for CC was supplied by the Qingdao Marine Chemical Factory, Qingdao, China. Spots were detected on TLC under UV light or by heating after spraying with 5% H 2 SO 4 in EtOH (v/v).

Brine shrimp lethality bioassay
The brine shrimp lethality bioassay was carried out by the reported method (Lou et al. 2009).

Conclusion
In conclusion, our investigation on the chemical constituents of gorgonian M. kanisa led to the identification of four diketopeperazines (1-4). Among these compounds, 3 showed significant toxicity towards brine shrimp larvae, while the others displayed moderate toxicity. The results suggest that 3 is a potential toxic natural product.

Supplementary material
Experimental details relating to this article are available online alongside Figures S2-S8 and Table S1.