Carbamate derivatives and sesquiterpenoids from the South China Sea gorgonian Melitodes squamata

Five carbamate derivatives, obtucarbamates C and D (1, 2), dimethyl ((carbonylbis(azanediyl))bis(2-methyl-5,1-phenylene))dicarbamate (3), obtucarbamates A and B (4, 5), and four aromadendrane-type sesquiterpenoids, (+)-4β-N-methenetauryl-10β-methoxy-1β,5α,6β,7β-aromadendrane (6), (−)-4β-N-methenetauryl-10β-methoxy-1β,5β,6α,7α-aromadendrane (7), (−)-4α,10β-aromadendranediol (8), (+)-4β,10β-aromadendranediol (9) were obtained from the South China Sea gorgonian coral Melitodes squamata Nutting. Compounds 1, 2, 6, and 7 were new, and their structures were established by spectroscopic analyses. Compounds 6 and 7 contained a taurine group that was rarely found in marine natural compounds, and 7 showed moderate antibacterial activity. The possible biosynthesis routes of 1–5 were conjectured.


Introduction
Gorgonians are recognized to mainly produce acetogenins, sesquiterpenoids, diterpenoids, prostanoids, and steroids [1,2]. However, nitrogen-containing compounds were relatively few obtained from gorgonians. Gorgonian Melitodes squamata Nutting belonging to Melithaea family is a kind of pharmaceutical coral that has the efficacy of relieving cough, bleeding, and diarrhea, soothing nerves, and calming scare, etc [3]. A previous study on the chemical constituents of Melithaea family gorgonians led to the isolation of four new steroids melithasterols A-D from Melithaea ocracea [4].
Urea derivatives are closely related in structure to carbamates. Urea is synthesized in the body of many organisms as part of the urea cycle, which is namely a cycle of biochemical reactions occurring in many animals that produces urea ((NH 2 ) 2 CO) from ammonia (NH 3 ). According to the reactions of the urea cycle [5], we made conjectures about the possible biosynthesis routes of compounds 2-4 as shown ( Figure 3). The possible biosynthesis routes to 1, 5 and 2-4 are essentially the same, except starting from 4-methylbenzene-1,3-diamine in place of 2-methybenzene-1,3-diamine. This is the first time that carbamate derivatives from gorgonians have been reported.    (Table 2) showed similarities to those of (-)-4α,10β-aromadendranediol (8), (+)-4β,10β-aromadendranediol (9) and other analogues [7 -11], which suggests that 6 is an aromadendrane-type sesquiterpenoid with a methoxy group and a side chain that contained three methylene carbons.
This suggestion was proved by the HMBC correlations ( Figure 4). In the HMBC spectrum of 6, correlation of MeO-   [12,13]. The IR spectrum of 6 contains two strong bands at 1217 and 1041 cm −1 , which supports the presence of a sulfonic acid group. The -NHCH 2 CH 2 SO 3 H group has rarely been found in marine natural compounds, such as N-methyltaurine, taurine, and spongidine D from sponges [12,13].
The relative stereochemistry of H-1, H-5, H-6, H-7, Me-14 and Me-15 in 6 was determined by NMR data comparison with literature data and NOESY spectral analysis. The chemical shifts (δ C 16.6, 28.7) of the methyl groups of Me-12 and Me-13 correlate well with those previously assigned to the corresponding methyl groups in 8, 9 and other analogues [7][8][9][10][11], which suggests the cis-orientation of the cyclopropane ring  (Table 2) showed similarities to those of 6, except for some obvious differences in the chemical shift data of H-5 (from δ H 0.98 to δ H 1.72), C-3 (from δ C 37.0 to δ C 33.4), C-5 (from δ C 46.0 to δ C 41.7), C-9 (from δ C 37.2 to δ C 32.8), and C-15 (from δ C 17.8 to δ C 24.4), which were caused by the stereochemistry change. In the 1 H-1 H COSY spectrum, no correlation between H-1 and H-5 is observed, which suggests a small coupling constant and the cis relationship between H-1 and H-5. The coupling constant (J = 9.5 Hz) between H-5 and H-6 indicates the trans-relationship of H-5 with H-6. In the NOESY spectrum, H-16 shows correlations with H-1/H-5, which suggests that H-16, H-1, and H-5 are in β-orientation; meanwhile, correlations of H-6 with H-7/Me-14/Me-13 indicate that H-6, H-7, Me-14 and Me-13 are in α-orientation. The chemical shift of Me-15 (δ C 24.4) appears at a slightly lower field reflecting that Me-15 is at the same α-orientation as H-6 and H-7 [7]. Thus, the structure of 7 was elucidated to be as shown and named as (−)-4β-N-methenetauryl-10β-methoxy-1β,5β,6α,7α-aromadendrane.
The cytotoxicity of compounds 1-9 against human malignant melanoma A735 and cervical carcinoma HeLa cell lines was evaluated by MTT assay. The results show that 1-9 does not exhibit cytotoxicity against A735 and HeLa cell lines with IC 50 > 200 μg/mL. Antibacterial activity of compounds 1-5 at a concentration of 25 μg/disc (diameter 6 mm) was measured against bacteria Escherichia coli, Bacillus subtilis, and Micrococcus luteus strains by using standard disc-diffusion assay.
The results show that 1-5 exhibit no inhibitory effects towards all tested bacteria at 25 μg/disc, 7 can inhibit the growth of B. subtilis and M. luteus with inhibition zones of 6.3 mm and 6.2 mm, respectively, at 25 μg/disc, while 7 has no effect towards E. coli at 25 μg/disc. It was reported that taurine and its derivatives have a number of physiological functions, including interference with GABA and glycine receptors, antinociceptic effects, anticonvulsive actions, neuroprotective actions, etc. [14]. In this study, because of the limited quantity of compound, we did not further test the other bioactivities of 6 and 7.

Experimental
General experimental procedures