Two New Tryptamine Derivatives, Leptoclinidamide and (-)-Leptoclinidamine B, from an Indonesian Ascidian Leptoclinides dubius

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.


Introduction
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 [3].
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 C 2 -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.

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 1 H 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 C 2 -α-D-mannosylpyranosyl-L-tryptophan [6,7]. Compound 3 was first identified as a novel post-translationally modified tryptophan in human RNases [4] and isolated thereafter from the ascidian L. dubius in 2000 [8], 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 13 C signals were detected in the 13 C NMR spectrum (DMSO-d 6 ). However, the 1 H 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. The IR spectrum of 1 showed absorption bands at 1681 and 1584 cm −1 , which were ascribable to two amide carbonyl groups. The 13 C signals of 1 were classified into six methylene, four sp 2 methine, one nitrogenated sp 2 methine, two sp 2 quaternary, one nitrogenated sp 2 quaternary and two amide carbonyl carbons by the analysis of 13 C NMR, DEPT and HMQC spectra of 1. The 1 H NMR spectrum (DMSO-d 6 ) of 1 displayed 22 signals including N-H protons due to a primary amine (δ H 7.66, 2H), two amides (δ H 7.93 and 8.07), and an indole ring (δ H 10.8). Table 1 shows 1 H and 13 C NMR data for 1 assigned by the analysis of 1 H-1 H COSY, HMQC and HMBC spectra. The 1 H-1 H COSY spectrum of 1 revealed the partial structures I through V ( Figure 2). The presence of an indole ring was suggested by the 1 H 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 13 C NMR signals. HMBC correlations from H 2 -10 (δ H 2.77) to C-2 (δ C 122.5), C-3 (δ C 111.8), and C-4 (δ C 127.2) and from H 2 -11 (δ H 3.29) to C-3 revealed a tryptamine unit in the molecule of 1. The 1 H NMR signals at δ 3.29 (H 2 -11) showed an HMBC correlation to one of two amide carbonyl carbons at δ C 170.0 (C-13). The other amide linkage was determined by HMBC correlations to C-17 (δ C 169.2) from 16-NH (δ H 8.07), H 2 -18 (δ H 2.38), and H 2 -19 (δ H 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 1 H NMR spectrum of 4 showed new signals corresponding to a methyl proton signal at δ 1.75 and a new amide proton signal at δ H 7.78 and a corresponding loss of two primary amine proton signals detected in the 1 H NMR spectrum of 1 (δ H 7.66). These data led to the conclusion that leptoclinidamide has the structure 1 as shown in Figure 1.   Compound 2 showed a molecular ion peak at m/z 362 [M + H] + in the FAB-MS, and the molecular formula C 16 H 20 N 5 O 5 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 1 H and 13 C NMR data for 2 with those for the reported values [9]. Compound 5 was originally isolated from an ascidian Leptoclinides durus and showed no apparent antiprotozoal or cytotoxic activity [9]. Although 1 H and 13 C 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) [4]. 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 [12].
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.

General
FAB-MS spectra were obtained using a JEOL JMS-MS 700 mass spectrometer (Tokyo, Japan). 1 H and 13 C NMR spectra were recorded on a JEOL JNM-AL-400 NMR spectrometer (400 MHz for 1 H and 100 MHz for 13 C) in DMSO-d 6 (δ H 2.46, δ C 39.5) or CD 3 OD (δ H 3.31, δ C 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.

Ascidian
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.

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 H 2 O 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 H 2 O, 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 1 H 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).

Conclusions
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, C 2 -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.