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

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].

Results and Discussion

In order to further obtain new bioactive compounds from gorgonians, we studied the chemical constituents of the South China Sea gorgonian M. squamata, which led to the obtainment of five carbamate derivatives, obtucarbamates C and D (1, 2), ((carbonylbis(azanediyl))bis(2-methyl-5,1-phenylene))dicarbamate (3) [5], obtucarbamate A (4) [6], obtucarbamate B (5) [6], 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) [7], (+)-4β,10β-aromadendranediol) (9) [7] (Figure 1) from the EtOH/CH₂Cl₂ extracts of M. squamata. Compounds 1, 2, 6, and 7 have not been previously reported. The cytotoxicity of 1–9 against human malignant melanoma A735 and cervical carcinoma HeLa cell lines, and the antibacterial activity of 1–5 and 7 towards bacteria Escherichia coli, Bacillus subtilis and Micrococcus luteus were evaluated. The possible biosynthesis routes of 1–5 were conjectured.

Compound 1 showed the same molecular formula of C₁₉H₂₂N₄O₅ as 3, which was determined by its HRMS–ESI (m/z 409.1568 [M + Na]⁺) and NMR spectra. The ¹H NMR spectrum of 1 (Table 1) exhibited signals for three ABX system aromatic protons at δ_H 7.50 (s, 1H), 7.19 (dd, J = 2.0, 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), three ABC system aromatic protons at δ_H 7.57 (d, J = 8.0 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), two methyl groups at δ_H 2.12 (s, 3H), 2.07 (s, 3H), and two methoxy groups at δ_H 3.63 (s, 3H), 3.64 (s, 3H). It also showed four –NH protons at δ_H 8.90 (s), 8.78 (s) and 8.52 (s, 2H). The ¹³C NMR and DEPT 135 spectral data (Table 1) showed the presences of 19 carbons, including three carbonyl carbons (δ_C 154.5, 154.6, 152.7), 12 aromatic carbons, two methyl carbons (δ_C 16.9, 12.4), and two methoxy carbons at δ_C 51.5. The NMR data of 1 were very similar to those of compounds 3–5 [5,6]. Compound 3 is a symmetric urea derivative that was formed from the amidation of two molecules of 4 [6]. Comparison of the NMR data of 1 with 3–5 suggested that 1 was an asymmetric urea derivative that was formed from the amidation of 4 and 5 [5,6].

The suggestion was supported by the HMBC correlations (Figure 2). In the HMBC spectrum of 1, correlations of Me-7 (s, 2.12) with δ_C 130.1 (d, C-5), 124.3 (s, C-6), 136.5 (s, C-1) and Me-7’ (s, 2.07) with δ_C 124.0 (s, C-2’), 137.1 (s, C-1’), 137.1 (s, C-3’) indicate a methyl group attached at both C-6 and C-2’. The HMBC correlations of δ_H 3.64 (s) with δ_C 154.5 (s) and δ_H 3.63 (s) with δ_C 154.6 (s) revealed the presence of two –NHCOOMe groups. The HMBC correlations of δ_H 8.78 (NH) with δ_C 124.3 (C-6), 114.0 (C-2), δ_H 8.90 (NH) with δ_C 124.0 (C-2’), 119.2 (C-4’)_, and δ_H 8.52 (2NH) with δ_C 114.0 (C-2), 114.5 (C-4), 121.8 (C-6’), 124.0 (C-2’), suggest that the four –NH groups are attached at C-1, C-3, C-1’, C-3’ of two aromatic rings. Based on the above data analysis, the structure of 1 was elucidated to be as shown above and named as obtucarbamate C.

Compound 2 also exhibited the molecular formula of C₁₉H₂₂N₄O₅ as deduced from NMR and HRMS–ESI (m/z 409.1576 [M + Na]⁺). The ¹H NMR data of 2 (Table 1) exhibited signals for two ABX aromatic systems, including six aromatic protons at δ_H 7.54 (s, 1H), 7.19 (br d, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 7.93 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H). The ¹H and ¹³C NMR spectral data of 2 show similarities to those of 3 [5] (Table 1). Comparison of the NMR data of 2 and 3 suggests that 2 is an asymmetric urea derivative that was formed from the amidation of two molecules of 4 [5,6], and the only difference between 2 and 3 is the amidation position of the two molecules of 4. Based on the ¹H NMR, ¹³C NMR HSQC, HMBC and ¹H–¹H COSY spectral data analysis, the structure of 2 was elucidated to be as shown above and named as obtucarbamate D.

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 ((NH₂)₂CO) from ammonia (NH₃). 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.

Compound 6 was assigned the molecular formula of C₁₉H₃₅NSO₄ as deduced from NMR spectra and HRMS–ESI (m/z 372.2170 [M − H]⁻). The ¹H NMR data (Table 2) indicate an aromadendrane skeleton with two cyclopropyl protons resonating at δ_H 0.44 (t, J = 10.0 Hz, 1H) and 0.67 (ddd, J = 6.5, 10.0, 11.5 Hz, 1H) [7-11]. The ¹H NMR spectrum also showed four singlet methyl groups (δ_H 0.93, 1.02, 1.11, 1.11) and a methoxy group (δ_H 3.16). The ¹³C NMR spectrum (Table 2) displayed 19 carbon signals, including five methyl carbons (δ_C 16.6, 28.7, 17.6, 17.8, 48.3), seven methylene carbons (δ_C 24.0, 37.0, 19.5, 37.2, 45.8, 45.9, 58.9), four methine carbons (δ_C 50.1, 46.0, 25.6, 26.6), and three quaternary carbons (δ_C 19.6, 44.0, 79.1). These NMR data (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– (δ_H 3.16, s) with δ_C 79.1 indicates the methoxygenation of C-10. The HMBC correlations of H-16 [δ_H 2.72 (d, J = 12 Hz), 2.94 (d, J = 12 Hz)] with C-3 (δ_C 37.0)/C-4 (δ_C 44.0)/C-5 (δ_C 46.0)/Me-14 (δ_C 17.6) indicate that there is a side chain at C-4 instead of a –OH group. The HMBC spectrum also shows correlations of H-16 with C-17 (δ_C 45.8) and H-17 [δ_H 3.54 (t, J = 5.0 Hz)] with C-16 (δ_C 45.8)/C-18 (δ_C 45.8). In the ¹H–¹H COSY spectrum (Figure 4), correlation of H-17 with H-18 [δ_H 3.31 (t, J = 5.0 Hz)] is observed. The above HMBC and ¹H–¹H COSY spectral data, combined with the molecular formula of C₁₉H₃₅NSO₄, suggest the presence of a –CH₂NHCH₂CH₂SO₃H group. The suggestion was supported by the ¹H and ¹³C NMR data comparison of the –CH₂NHCH₂CH₂SO₃H group in 6 with literature data [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 –NHCH₂CH₂SO₃H 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 [7-11], which suggests the cis-orientation of the cyclopropane ring [7,8]. The coupling constants (J = 10.0 Hz) between H-5 and H-1/H-6 indicate the trans-relationship of H-5 with H-1 and H-6. By comparison with the known chemical-shift data and shift parameters [7-11], the β-orientation of H-1, H-6, H-7 and α-orientation of H-5 were assigned. In the seven-numbered ring, the methyl group C-15 (δ_C 17.8) appears at a high field, reflecting that Me-15 is not in the same orientation as H-6 and H-7 [7]. In the NOESY spectrum, correlations of OMe-10 with H-1, H-6, H-7 are observed (Figure 5), which suggest that OMe-10, H-1, H-6, H-7 are in β-orientation, meanwhile, the presence of correlations between H-5 and Me-14/Me-13 indicates that H-5, Me-14, Me-13 are in α-orientation. Thus, the structure of 6 was elucidated to be as shown and named as (+)-4β-N-methenetauryl-10β-methoxy-1β,5α,6β,7β-aromadendrane.

Compound 7 showed the same molecular formula of C₁₉H₃₅NSO₄ as 6 on the basis of NMR spectra and HRMS–ESI (m/z 372.2172 [M − H]⁻). The ¹H and ¹³C NMR spectral data of 7 (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 ¹H–¹H 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 IC₅₀ > 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

Optical rotations were measured with a Horiba SEAP-300 spectropolarimeter. UV spectra were measured with a Shimadzu double-beam 210A spectrophotometer in MeOH solution. ¹H, ¹³C NMR and 2D NMR spectra were recorded on a Bruker AV-500 MHz NMR spectrometer with TMS as internal standard. MS spectral data were obtained on an LCQDECA XP HPLC/MSn spectrometer for ESIMS. Silica gel (200–300 mesh) for column chromatography and GF₂₅₄ for TLC were obtained from the Qindao Marine Chemical Factory, Qindao, People’s Republic of China.

Animal material

The South China Sea gorgonian coral Melitodes squamata Nutting was collected in Sanya, Hainan province, China in July 2008 and identified by Prof. Huang H., the South China Sea Institute of Oceanology, Academia Sinica. A voucher specimen (No. 0802) was deposited in the South China Sea Institute of Oceanology, Academia Sinica, Guangzhou, China.

Extraction and isolation

The frozen specimen of the South China Sea gorgonian coral M. squamata (20 kg, wet weight) was extracted with EtOH/CH₂Cl₂ (2:1) three times at room temperature and the solvent was evaporated in vacuo. The residue was partitioned in H₂O and extracted with EtOAc and n-BuOH three times each. The extracts of each respective solvent were combined. The EtOAc and n-BuOH extracts were concentrated in vacuo to afford 35.17 g and 16.85 g of residue, respectively. The EtOAc extract was subjected to silica-gel column chromatography (column A), eluted with CHCl₃/MeOH (from 1:10 to 0:10). By combining the fractions with TLC (GF₂₅₄) monitoring, 17 fractions were obtained. Fraction A-5 was applied to a Sephadex LH-20 column (column B), eluted with CHCl₃/MeOH (1:1), to obtain four fractions. Fraction B-4 was further purified by HPLC (MeOH/H₂O 45:100) to yield 1 (1.8 mg, t_R = 23.5 min), 2 (1.8 mg, t_R = 41.2 min), 3 (2.8 mg, t_R = 38.2 min), 4 (2.7 mg, t_R = 13.8 min) and 5 (1.8 mg, t_R = 9.0 min), separately. Fraction B-3 was further purified by RP-18 reverse-phase silica-gel column chromatography (column C), eluted with MeOH/H₂O (30:100), to yield 8 (14.8 mg) and 9 (9.5 mg). Fraction A-11 was separated by a Sephadex LH-20 column (column D), eluted with CHCl₃/MeOH (1:1) to give five fractions. Fraction D-2 was subjected to silica-gel column chromatography (column E), eluted with CHCl₃/MeOH (10:1), to yield 6 (4.2 mg) and three other fractions. Fraction E-2 was furthered purified by a Sephadex LH-20 column (MeOH/H₂O 95:5) to yield 7 (4.8 mg).

Obtucarbamate C (1): White powder; UV (MeOH): 258, 216 nm; ¹H and ¹³C NMR data, see Table 1; (+)-ESIMS (m/z): 409.0 [M + Na]⁺, 795.0 [2M + Na]⁺; HRMS–ESI (m/z): [M + Na]⁺ calcd for C₁₉H₂₃N₄O₅Na, 409.1590; found, 409.1568.

Obtucarbamate D (2): White powder; UV (MeOH): 258, 216 nm; ¹H and ¹³C NMR data, see Table 1; (+)-ESIMS (m/z): 409.1 [M + Na]⁺, 795.3 [2M + Na]⁺; HRMS–ESI (m/z): [M + Na]⁺ calcd for C₁₉H₂₃N₄O₅Na, 409.1590; found, 409.1576.

(+)-4β-N-methenetauryl-10β-methoxy-1β,5α,6β,7β-aromadendrane (6): White powder; [α]_D²⁰ +26.4° (c 0.28, MeOH); ¹H and ¹³C NMR data, see Table 2; (−)-ESIMS (m/z): 372.1 [M − H]⁻, 745.4 [2M − H]⁻; HRMS–ESI (m/z): [M − H]⁻ calcd for C₁₉H₃₄NSO₄, 372.2209; found, 372.2170.

(−)-4β-N-methenetauryl-10β-methoxy-1β,5β,6α,7α-aromadendrane (7): White powder; [α]_D²⁰ −7.5° (c 0.48, MeOH); ¹H and ¹³C NMR data, see Table 2; (−)-ESIMS (m/z): 372.2 [M − H]⁻, 745.4 [2M − H]⁻; HRMS–ESI (m/z): [M − H]⁻ calcd for C₁₉H₃₄NSO₄, 372.2209; found, 372.2172.