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Mar. Drugs 2012, 10(2), 349-357; doi:10.3390/md10020349
Published: 10 February 2012
Abstract: Two new tryptamine-derived alkaloids, named as leptoclinidamide (1) and (-)-leptoclinidamine B (2), were isolated from an Indonesian ascidian Leptoclinides dubius together with C2-α-D-mannosylpyranosyl-L-tryptophan (3). The structure of 1 was assigned on the basis of spectroscopic data for 1 and its N-acetyl derivative (4). Compound 1 was an amide of tryptamine with two β-alanine units. Although the planar structure of 2 is identical to that of the known compound (+)-leptoclinidamine B (5), compound 2 was determined to be the enantiomer of 5 based on amino acid analysis using HPLC methods. Compounds 1 to 4 were evaluated for cytotoxicity against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma) cells, but none of the compounds showed activity.
Ascidians are a rich source of biologically-active nitrogenous substances with high chemical diversity [1,2]. More than 80% of new compounds from ascidians contained nitrogen, and about 70% of nitrogenous compounds are alkaloids .
In the course of our studies on the bioactive components from marine invertebrates, we found that the EtOH extract of an Indonesian ascidian Leptoclinides dubius inhibited the growth of Escherichia coli. Chemical study on the EtOH extract led to the isolation of two tryptamine-derived alkaloids, a C-glycosylated tryptophan and an antibacterial compound. Two alkaloids were revealed to be new compounds and named as leptoclinidamide (1) and (-)-leptoclinidamine B (2, Figure 1), and a tryptophan derivative was assigned as C2-α-D-mannosylpyranosyl-L-tryptophan (3) [4,5,6,7,8]. The major bioactive constituent could not be identified because the amount obtained from the ascidian was not enough to measure 2D NMR spectra.
We report herein the isolation and structures of two new tryptamine-derived alkaloids leptoclinidamide (1) and (-)-leptoclinidamine B (2), which had unique amide moiety with two β-alanine units and D-arginine moiety, respectively.
2. Results and Discussion
The EtOH extract of an Indonesian ascidian L. dubius showed antimicrobial activity in the screening bioassay against Escherichia coli and was separated into nine fractions (fraction 1–fraction 9) by octadecylsilyl (ODS) column chromatography. Leptoclinidamide (1) was isolated from fraction 6 (50% MeOH eluate) by HPLC (ODS) and compounds 2 and 3 were obtained from fraction 3 (30% MeOH eluate) and fraction 2 (water eluate), respectively. The antibacterial activity against E. coli was detected in fraction 5 (50% MeOH eluate, 10 mm inhibition zone at 250 μg/disk), and HPLC separation of fraction 5 gave an antibacterial compound as a single HPLC peak.
Unfortunately, the antibacterial component did not give an informative 1H NMR spectrum because of the small amounts obtained, and, therefore, 2D NMR experiments could not be recorded. The structure of compound 3 was assigned on the basis of its spectral data and comparison with that of the reported values for C2-α-D-mannosylpyranosyl-L-tryptophan [6,7]. Compound 3 was first identified as a novel post-translationally modified tryptophan in human RNases  and isolated thereafter from the ascidian L. dubius in 2000 , but the role of compound 3 have not yet been elucidated.
Leptoclinidamide (1) was obtained as a TFA salt. The FAB-MS spectrum of 1 showed a peak at m/z 303.1821, and 16 13C signals were detected in the 13C NMR spectrum (DMSO-d6). However, the 1H signals due to amine moiety were observed as broad peak in the NMR spectrum of 1. Therefore, the molecular weight and formula of 1 were confirmed by the spectroscopic data for an N-acetyl derivative (4) of 1. Compound 4 gave an [M + H]+ ion at m/z 345.1930 in the HRFAB-MS, and the molecular formula of 4 was determined as C18H25N4O3. Accordingly, the molecular formula of 1, which revealed an [M + H]+ ion at m/z 303.1821 in the HRFAB-MS, were deduced as C16H22N4O2 (8 degrees of unsaturation).
The IR spectrum of 1 showed absorption bands at 1681 and 1584 cm−1, which were ascribable to two amide carbonyl groups. The 13C signals of 1 were classified into six methylene, four sp2 methine, one nitrogenated sp2 methine, two sp2 quaternary, one nitrogenated sp2 quaternary and two amide carbonyl carbons by the analysis of 13C NMR, DEPT and HMQC spectra of 1. The 1H NMR spectrum (DMSO-d6) of 1 displayed 22 signals including N-H protons due to a primary amine (δH 7.66, 2H), two amides (δH 7.93 and 8.07), and an indole ring (δH 10.8). Table 1 shows 1H and 13C NMR data for 1 assigned by the analysis of 1H-1H COSY, HMQC and HMBC spectra. The 1H-1H COSY spectrum of 1 revealed the partial structures I through V (Figure 2). The presence of an indole ring was suggested by the 1H NMR signals at δ 7.09 (H-2), 7.48 (H-5), 6.93 (H-6), 7.02 (H-7), 7.29 (H-8), and 10.8 (1-NH) and HMBC correlations from these signals to the expected 13C NMR signals. HMBC correlations from H2-10 (δH 2.77) to C-2 (δC 122.5), C-3 (δC 111.8), and C-4 (δC 127.2) and from H2-11 (δH 3.29) to C-3 revealed a tryptamine unit in the molecule of 1. The 1H NMR signals at δ 3.29 (H2-11) showed an HMBC correlation to one of two amide carbonyl carbons at δC 170.0 (C-13). The other amide linkage was determined by HMBC correlations to C-17 (δC 169.2) from 16-NH (δH 8.07), H2-18 (δH 2.38), and H2-19 (δH 2.93). These data suggested the structure of 1 as shown in Figure 1. The structure of 1 was further confirmed by the analysis of the N-acetyl derivative (4). The 1H NMR spectrum of 4 showed new signals corresponding to a methyl proton signal at δ 1.75 and a new amide proton signal at δH7.78 and a corresponding loss of two primary amine proton signals detected in the 1H NMR spectrum of 1 (δH7.66). These data led to the conclusion that leptoclinidamide has the structure 1 as shown in Figure 1.
|Table 1. 13C (100 MHz) and 1H (400 MHz) NMR data for leptoclinidamide (1) in DMSO-d6.|
|No.||δC||δH ( J in Hz)||HMBC|
|1-NH||-||10.8 brs||3, 4, 9|
|2||122.5||7.09 d (2.0)||3, 4, 9|
|5||118.1||7.48 d (8.0)||9|
|6||118.1||6.93 t (8.0)||4|
|7||120.8||7.02 t (8.0)||9|
|8||111.3||7.29 d (8.0)||4|
|10||25.1||2.77 brt (7.4)||2, 3, 4|
|11||39.2||3.29 brt (7.4)||3, 13|
|12-NH||-||7.93 brt (5.8)|
|14||35.2||2.24 brt (7.4)||13|
|15||35.3||3.23 brt (7.4)||13|
|16-NH||-||8.07 brt (5.8)||17|
|18||31.9||2.38 brt (7.4)||17|
|19||35.3||2.93 brt (7.4)||17|
Compound 2 showed a molecular ion peak at m/z 362 [M + H]+ in the FAB-MS, and the molecular formula C16H20N5O5 was deduced from HRFAB-MS. The NMR spectrum of 2 revealed the presence of a disubstituted indole ring and an arginine moiety. A literature to search suggested the structure of (+)-leptoclinidamine B (5) as a candidate, and this structure was confirmed by comparing 1H and 13C NMR data for 2 with those for the reported values . Compound 5 was originally isolated from an ascidian Leptoclinides durus and showed no apparent antiprotozoal or cytotoxic activity . Although 1H and 13C NMR spectra of 2 were identical to those of 5, the sign of the specific rotation of 2 was negative ([α]D −20.6 in MeOH), suggesting that compound 2 was the enantiomer of 5 ([α]D +27.0 in MeOH) . To determine the absolute configulation of 2, amino acid analysis was carried out by the methods described in the Experimental Section. The acid hydrolysate of 2 was analyzed by using chiral HPLC. In the analysis, an amino acid in the hydrolysate of 2 was detected at 3.7 min, which corresponded to D-Arg (L-Arg eluted at 11.3 min). This result was confirmed by Marfey’s method [10,11]. The hydrolysate of 2 was treated with Marfey’s reagent, 5-fluoro-2,4-dinitrophenyl-L-leucine amide (L-FDLA), and the resulting derivative was analyzed by HPLC using an ODS column. An L-FDLA derivative of an amino acid in the hydrolysate of 2 showed the same retention time as that of the derivative from D-Arg (13.4 min), whereas the L-FDLA derivative of L-Arg eluted at 15.9 min. Consequently, the absolute stereochemistry of 2 was determined to be 13R, as shown in Figure 1. Very recently, herdmanines A-D containing a D-Arg unit were isolated from a solitary ascidian Herdmania momus .
No biological activities have been reported for compound 3. Compounds 1–4 were tested for their cytotoxicity against two human cancer cell lines (colon adenocarcinoma HCT-15 and T-cell leukemia Jurkat cells). However, none of the four compounds displayed activity against these cell lines at 30 μM. Since fractions 2, 3, and 6, from which respective compounds 3, 2, and 1 were isolated, did not show growth inhibitory activity against four microorganisms (see Experimental Section), compounds 1–3 will not have antimicrobial activity. Further study on biological activity of 1 is now in progress. More L. dubius will be collected from the same site to provide sufficient amounts of the antibacterial component for structural characterization.
3. Experimental Section
FAB-MS spectra were obtained using a JEOL JMS-MS 700 mass spectrometer (Tokyo, Japan). 1H and 13C NMR spectra were recorded on a JEOL JNM-AL-400 NMR spectrometer (400 MHz for 1H and 100 MHz for 13C) in DMSO-d6 (δH 2.46, δC 39.5) or CD3OD (δH 3.31, δC 49.0). Optical rotations were measured with a JASCO P-2300 digital polarimeter (Tokyo, Japan). UV spectra were recorded on a Hitachi U-3310 UV-Visible spectrophotometer (Tokyo, Japan) and IR spectra on a PerkinElmer Spectrum One Fourier transform infrared spectrometer (Waltham, MA, USA). Preparative HPLC was carried out with a Hitachi L-6200 system.
Fetal bovine serum (FBS) and other culture materials were purchased from Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and organic solvents were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).
The ascidian was collected by scuba diving at the coral reef in the Lembeh Strait, North Sulawesi, Indonesia in October 2009 and identified as Leptoclinides dubius by T. N. The voucher specimen is deposited at the National Museum of Nature and Science, Tokyo as NSMT Pc-1123.
3.4. Extraction and Isolation
The ascidian (250 g, wet weight) was cut into small pieces and soaked in EtOH on a boat immediately after collection. The organism was further extracted twice with EtOH. The EtOH extract (3.57 g) was suspended in H2O and adsorbed on an ODS column (100 g). The ODS column was eluted stepwise with 0, 30, 50, 70, and 100% MeOH in 0.10% TFA aqueous solution into nine fractions (fraction 1–fraction 9). Fraction 2, eluted with H2O, was concentrated to yield a red brown oil (108.0 mg), and 20.0 mg of the fraction was purified by preparative HPLC column, PEGASIL ODS (10 mm × 250 mm); solvent, 35% MeOH containing 0.10% TFA; flow rate, 2.0 mL/min; detection, UV at 210 nm] to give compound 3 (eluted at 9.9 min) as a colorless solid (6.0 mg). Fraction 3, eluted with 30% MeOH, was concentrated to yield a red brown oil (399.5 mg), and 20.0 mg was fractionated by preparative HPLC (same conditions as Fraction 2) to yield compound 2 (eluted at 11.0 min) as a pale yellow oil (4.0 mg). Fraction 5 (95. 0 mg), eluted with 50% MeOH, was active against E. coli and subjected to ODS HPLC with 40% MeOH containing 0.10% TFA (the other conditions were the same as above) into six fractions (Fraction 5-1–Fraction 5-6). Fraction 5-6 showed the growth inhibition against E. coli, but the amounts were not enough to obtain a good 1H NMR spectrum. Fraction 6, eluted with 50% MeOH, was concentrated to dryness (50.5 mg), and 20.0 mg was separated by preparative HPLC with the same conditions as Fraction 5 and afforded compound 1 (eluted at 15.0 min) as a colorless oil (2.0 mg).
Leptoclinidamide (1): obtained as TFA salt; UV λmax (MeOH) nm (log ε): 202 (4.47), 221 (4.31); IR νmax (KBr) cm−1: 3418, 2938, 1680, 1584, 1556, 1457; HRFAB-MS (m/z) found: 303.1821, calcd: 303.1821 [M + H]+ for C16H23N4O2; 1H and 13C NMR data, see Table 1.
(-)-Leptoclinidamine B (2): obtained as TFA salt; [α]20D −20.6 (c = 0.033, MeOH); UV λmax (MeOH) nm (log ε): 203 (4.38), 247 (3.78), 284 (3.78), 334 (3.22); IR νmax (KBr) cm−1: 3384, 1684, 1638, 1450, 1275, 1131; HRFAB-MS (m/z) found: 362.1465, calcd: 362.1464 [M + H]+ for C16H20N5O5; 1H-NMR (DMSO-d6) δH 1.53 (2H, m, H-15), 1.77, 1.86 (2H, m, H-14), 3.14 (2H, m, H-16), 4.28 (1H, brs, H-13), 6.75 (1H, d, J = 8.0 Hz, H-6), 6.87 (1H, s, H-8), 7.12 (4H, brs, 18-N2H4), 7.62 (1H, brs, 17-NH), 7.97 (1H, d, J = 8.0 Hz, H-5), 8.52 (1H, s, 12-NH), 8.72 (1H, brd, H-2), 9.33 (1H, brs, 7-OH), 11.9 (1H, brs, 1-NH); 13C-NMR (DMSO-d6) δC25.2 (C-15), 27.9 (C-14), 40.3 (C-16), 51.9 (C-13), 97.2 (C-8), 112.4 (C-6), 112.5 (C-3), 118.8 (C-4), 121.7 (C-5), 137.3 (C-2), 137.5 (C-9), 154.6 (C-7), 156.7 (C-18), 163.5 (C-11), 173.0 (C-20), 181.3 (C-10).
C2-α-D-mannosylpyranosyl-L-tryptophan (3): obtained as TFA salt; [α]20D +15.5 (c = 0.52, MeOH); FAB-MS (m/z): 367 [M + H]+; 1H-NMR (CD3OD) δH 3.31, 3.65 (2H, m), 3.65 (1H, m), 3.65, 4.29 (2H, m), 3.89 (1H, m), 3.97 (1H, m), 4.05 (1H, brd), 4.25 (1H, brdd), 5.04 (1H, brd), 7.07 (1H, t, J = 7.8 Hz), 7.11 (1H, t, J = 7.8 Hz), 7.38 (1H, d, J = 7.8 Hz), 7.63 (1H, d, J = 7.8 Hz).
3.5. Synthesis of N-Acetyl Derivative (4) of Compound 1
Acetic anhydride (120 μL) was added to a solution of fraction 6 (20 mg) in MeOH (340 μL) at room temperature. The mixture was stirred for 12 h and evaporated. The residue was purified by preparative HPLC (40% MeOH containing 0.10% TFA) using ODS column (PEGASIL ODS) to give 1.0 mg of 4: HRFAB-MS (m/z) found: 345.1930, calcd: 345.1927 [M + H]+ for C18H25N4O3; 1H-NMR (DMSO-d6) δH 1.75 (3H, s), 2.17 (2H, t, J = 7.6 Hz), 2.21 (2H, t, J = 7.6 Hz), 2.79 (2H, t, J = 7.6 Hz), 3.18 (2H, t, J = 7.6 Hz), 3.22 (2H, t, J = 7.6 Hz), 3.31 (2H, t, J = 7.6 Hz), 6.95 (1H, t, J = 7.4 Hz), 7.04 (1H, t, J = 7.4 Hz), 7.11 (1H, d, J = 8.3 Hz), 7.31 (1H, d, J = 8.3 Hz), 7.50 (1H, d, J = 2.0 Hz), 7.78 (NH, 1H, brt, J = 5.4 Hz), 7.85 (NH, 1H, brt, J = 5.8 Hz), 7.93 (NH, 1H, brt, J = 5.8 Hz), 10.8 (NH, 1H, brs).
3.6. Acid Hydrolysis of Compound 2
Compound 2 (0.5 mg) was suspended in 6.0 N HCl (1.0 mL) and heated at 100 °C for 8 h. The mixture was cooled to room temperature and evaporated to dryness.
3.7. Chiral HPLC of Peptide Hydrolysate
Hydrolysate of 2 was dissolved in H2O (0.4 mL) and 5.0 μL was analyzed by HPLC using the following condition (A): L-6200 system; column, SUMICHIRAL OA-6000 (Sumika Chemical Analysis Service, Ltd., Tokyo, Japan), 4.6 × 150 mm; flow rate, 1.0 mL/min; detection, UV 254 nm; mobile phase, 2 mM CuSO4 in 2.0% CH3CN. Authentic L- and D-Arg were eluted at 11.3 and 3.7 min, respectively. An amino acid in the hydrolysate of 2 was detected at 3.7 min.
Hydrolysate of 2 was dissolved in 1 M NaHCO3 (200 μL) and reacted with 1% L-FDLA (100 μL in acetone) at 40 °C for 2 h. After cooling, the sample was quenched with 1 N HCl and dried under vacuum. The solid residue was dissolved in 50% aq CH3CN (400 μL) and analyzed under the following condition (B): L-6200 system; column, Pegasil ODS SP100 (Senshu Scientific), 4.6 × 250 mm; flow rate, 0.8 mL/min; detection, UV 340 nm; mobile phase, a liner gradient from 30% to 60% CH3CN containing 0.05% TFA. L-FDLA derivatives of authentic L- and D-Arg eluted at 15.9 and 13.4 min, respectively, and the L-FDLA derivative in the hydrolysate of 2 was detected at 13.4 min.
3.9. Antimicrobial Assay
The growth inhibitory activity was examined by the paper disk method against Mucor hiemalis IAM 6088 (fungus), Saccharomyces cerevisiae IAM 1438T (yeast), Staphylococcus aureus IAM 12544T (Gram-positive bacterium), and Escherichia coli IAM 12119T (Gram-negative bacterium) as test microorganisms. A paper disk containing 250 μg of the test sample was placed on a test plate.
3.10. Cytotoxicity Assay
HCT-15 and Jurkat cells were obtained from the Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University (Miyagi, Japan). Two cell lines were cultured in RPMI-1640 medium. The medium was supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Exponentially growing cells cultured in a humidified chamber at 37 °C containing 5.0% CO2 were used for the experiments.
Cytotoxic activity was evaluated using the colorimetric MTT assay . HCT-15 cells (1.0 × 104 cells in 100 μL) or Jurkat cells (2.0 × 104 cells in 100 μL) were added to each well of a 96-well plastic plate (Corning Inc., Corning, NY, USA). A sample (1.0 μL in MeOH) was added to each well to make the final concentration from 0 to 27 μM and the cells were incubated for 48 hours at 37 °C. MTT (10 μL of 5.5 mg/mL stock solution) and a cell lysate solution (90 μL, 40% N,N-dimethylformamide, 20% sodium dodecyl sulfate, 2.0% CH3COOH and 0.030% HCl) were added to each well, and the plate was shaken thoroughly by agitation at room temperature for overnight. The optical density of each well was measured at 570 nm using an MTP-500 microplate reader (Corona Electric Co., LTD., Ibaraki, Japan).
Two new tryptamine derivatives, leptoclinidamide (1) and (-)-leptoclinidamine B (2), were isolated from the EtOH extract of an Indonesian ascidian Leptoclinides dubius together with a known compound, C2-α-D-mannosylpyranosyl-L-tryptophan (3). Biological activity of compound 3 has not been clarified, and, in this study, we found that compounds 1–4 were not active against two human cancer cell lines (HCT-15 and Jurkat) and four microorganisms (Gram positive and negative bacteria, yeast, and fungus). An antibacterial component against E. coli was also obtained from the EtOH extract, but the structure has not been determined because the amounts were not enough to measure 2D NMR spectra.
This work was supported in part by a Grant-in-aid for Scientific Research (21603012) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan to MN and by a Colab. Res. & Int. Pub. Project No. 492/SP 2H/PL/2011 from DGHE, Ministry of National Education of Indonesia to REPM. We are grateful to the Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University for kindly providing human cancer cell lines.
- Faulkner, D.J. Marine natural products. Nat. Prod. Rep. 2002, 19, 1–48. and previous reports in this series. 11902436
- Blunt, J.W.; Copp, B.R.; Munro, M.H.G.; Northcote, P.T.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2011, 28, 196–268. and previous reports in this series, doi:10.1039/c005001f.
- Wang, W.F.; Namikoshi, M. Bioactive nitrogenous metabolites from ascidians. Heterocycles 2007, 74, 53–88, doi:10.3987/REV-07-SR(W)2.
- De Beer, T.; Vliegenthart, J.F.G.; Loffler, A.; Hofsteenge, J. The hexopyranosyl residue that is C-glycosidically linked to the side chain of tryptophan-7 in human RNase Us is α-mannopyranose. Biochemistry 1995, 34, 11785–11789, doi:10.1021/bi00037a016. 7547911
- Gutsche, B.; Grun, C.; Scheutzow, D.; Herderich, M. Tryptophan glycoconjugates in food and human urine. Biochem. J. 1999, 343, 11–19, doi:10.1042/0264-6021:3430011. 10493906
- Manabe, S.; Ito, Y. Total synthesis of novel subclass of glyco-amino acid structure motif: C2-α-L-C-mannosylpyranosyl-L-tryptophan. J. Am. Chem. Soc. 1999, 121, 9754–9755, doi:10.1021/ja990926a.
- Nishikawa, T.; Ishikawa, M.; Isobe, M. Synthesis of a α-C-mannosyltryptophan derivative, naturally occurring C-glycosyl amino acid found in human ribonuclease. Synlett 1999, 123–125.
- Garcia, A.; Lenis, L.A.; Jimenez, C.; Debitus, C.; Quinoa, E.; Riguera, R. The occurrence of the human glycoconjugate C2-α-D-mannosylpyranosyl-L-tryptophan in marine ascidians. Org. Lett. 2000, 2, 2765–2767, doi:10.1021/ol0061384. 10964360
- Carroll, A.R.; Avery, V.M. Leptoclinidamines A-C, indole alkaloids from the Australian ascidian Leptoclinides durus. J. Nat. Prod. 2009, 72, 696–699, doi:10.1021/np800831z. 19236031
- Marfey, P. Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 1984, 49, 591–596, doi:10.1007/BF02908688.
- Kuo, H.K.; Kai, K.; Akiyama, K.; Hayashi, H. Novel bioactive peptides, PF1171F and PF1171G, from unidentified ascomycete OK-128. Tetrahedron Lett. 2012, 53, 429–431, doi:10.1016/j.tetlet.2011.11.068.
- Li, J.L.; Han, S.C.; Yoo, E.S.; Shin, S.; Hong, J.; Cui, Z.; Li, H.; Jung, J.H. Anti-inflammatory amino acid derivatives from the ascidian Herdmania momus. J. Nat. Prod. 2011, 74, 1792–1797, doi:10.1021/np200397g. 21770369
- Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63, doi:10.1016/0022-1759(83)90303-4. 6606682
- Samples Availability: Not available.
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