Two New Indole Alkaloids from Toad Venom of Bufo bufo gargarizans

Two new indole alkaloids, Bufotenidine B (2) and Bufocarboline A (6), along with seven known indole alkaloids (1, 3–5, and 7–9) and three organic acids (10–12), were isolated from the water extract of toad venom. The structures of the new alkaloids were elucidated by extensive spectroscopic methods. The absolute configurations of 4, 6, and 8 were determined for the first time by electronic circular dichroism (ECD) calculations. The cytotoxic activity of all compounds was tested against human malignant melanoma cells A375 by the MTT method, and no antitumor activity was observed.


Results and Discussion
Compound 2 was afforded as a yellow powder. The molecular formula C 14   The DEPT (135 • ) spectrum disclosed the presence of a pair of coupled methylene groups at δ C 23.6 (C-10) and 69.2 (C-11), which correlated with 3.32 (m, 2H, H-10), 3.23 (m, 2H, H-11) in its HSQC spectrum. Combining with the HMBC correlations between 3.32 (m, 2H, H-10) and 69.2 (C-11), and between 3.23 (m, 2H, H-11) and 23.6 (C-10) indicated the two methylene groups were directly connected. In addition, the HMBC correlations between 7.15 (s, 1H, H-2) and 23.6 (C-10); between 3.32 (m, 2H, H-10) and 125.4 (C-9), 110.7(C-3), and 128.9 (C-2); and between 3.23 (m, 2H, H-11) and 110.7 (C-3) disclosed a pair of coupled methylene groups was attached to the 3, 4, 5-trisubstituted indole moiety at C-3, thus forming a 5-hydroxytryptamine moiety. The HSQC spectrum showed 2.98 (s, 16H, N-CH 3 ) correlated with 54.6 (C-13, 14, and 15). Meanwhile, the DEPT (135 • ) displayed 54.6 (C-13, 14, and 15) was a positive peak, and the peak height about three times higher than that of other carbon signals, suggesting that there were three identical methyl groups in 2. Finally, three identical methyl groups were connected to the 5-hydroxytryptamine moiety at N-2 by the HMBC correlations between 3.23 (m, 2H, H-11) and 54.6 (C- 13,14,15), and between 2.98 (s, 16H, N-CH 3 ) and 69.2 (C-11) ( Figure 2). 1 H-NMR spectrum showed a methine signal at δH 4.19 (d, J = 8.0Hz, 1H, H-1), which correspond to 51.9 (C-1) in HSQC spectrum. In addition, the HMBC spectrum showed two methylene groups 2.40-2.21 (m, 2H, H-11) and 1.90-2.10 (m, 2H, H-10) were both associated with 51.9 (C-1), indicating that the carboxyethyl is connect to C-1 (51.9). Further, the HMBC spectrum showed correlations between 4.19 (d, J = 8.0Hz, 1H, H-1), 1.90-2.10 (m, 2H, H-10) and 134.1 (C-9a), showing that 51.9 (C-1) is linked to 134.1 (C-9a) to form a tricyclic structure of β-carboline ( Figure 2). According to the aforementioned information, the plane structure of 6 was unambiguously assigned as 6-hydroxy-1-(2 ＇carboxyethyl)-1,2,3,4-tetrahydro-β-carboline, which is a new compound named bufocarboline A. Compounds 4 and 8 were isolated for the first time from toad venom, and the plane structures of them were confirmed respectively as 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid [8] and 6-Hydroxy-2,3,4,9-tetrahydro-1H-β-carboline-1-carboxylic acid [8] by the comparison with extensive spectroscopic data. The chemical synthesis and biological activities of them have been reported in the literature [9][10][11], but the absolute configurations have not been determined. Compounds 4, 6, and 8 are all derivatives of tetrahydro-β-carboline with one chiral carbon at C-1. The absolute configurations of 4, 6, and 8 were determined by comparison with their experimental CD spectra and calculated electronic circular dichroism (ECD) spectra, which were calculated by a quantum chemical method. Compound 4 has two possible enantiomers (1S)-4 (4a) and (1R)-4 (4b), and the calculated ECD curve of 4a was similar to the experimental CD spectrum, both showing Cotton effects at 205-220 (negative) and 220-240 nm (positive) ( Figure 3). Therefore, the absolute configuration 4 was assigned as 1S. The structure of 6 was similar to 4 except for the loss of methyl at C-1 instead of carboxyethyl. The calculated ECD spectrum of enantiomer (1S)-6 (6a) was in good agreement with the experimental spectrum, both showing negative Cotton effect at 210-230 and 260-300 nm and positive Cotton effect 230-260 nm ( Figure 4). Therefore, the absolute configuration of 6 was assigned as 1S. Compound 8 was a demethylated derivative of 4. Considering the biosynthesis of 4 and 8 in B. bufo gargarizans, it could be concluded that the stereoconfiguration of carboxyl group at C-1 of 8 should be the same as that of 4. The comparison of the CD and ECD spectra of enantiomers (1R)-8 (8a) and (1S)-8 (8b) showed that the calculated ECD spectra of 8b was matched better with that of CD than 8a ( Figure 5). Furthermore, the calculated specific rotation value of 8a was 103.7, and the calculated specific rotation value of 8b was corresponded to −103.7, which yielded a good match with the measured value [ ] −115.2 (c, 0.033, H2O) of 8. Consequently, the absolute configuration of 8 was assigned as 1S. The absolute configurations of 4, 6, and 8 were all identified as 1S, indicating they have similar biosynthesis pathways. According to the aforementioned information, a structural fragment of C 13 H 18 N 2 O which missed a [COO − ] fragment compared to the molecular formula C 14 H 18 N 2 O 3 of 2 was formed. The 13 C-NMR spectrum showed the presence of a carbonyl group at δ C 176.9, suggesting the [COO -] fragment was attached to C-4 to form an inner salt. The suggestion was further demonstrated by its UV spectrum, which showed that the maximum absorption wavelength of the indole ring shifted from 275 nm to 312 nm. This indicated that the C-4 substituent could prolong the conjugated system. Based on the above analysis, 2 was unambiguously assigned as 5-hydroxy-3-(2-(trimethylammonio) ethyl) -1H-indole-4-carboxylate, and named bufotenidine B, which is a new compound confirmed by Scifinder investigation.
Compounds 4 and 8 were isolated for the first time from toad venom, and the plane structures of them were confirmed respectively as 6-hydroxy-l-methyltetrahydro-β-carboline-1-carboxylic acid [8] and 6-Hydroxy-2,3,4,9-tetrahydro-1H-β-carboline-1-carboxylic acid [8] by the comparison with extensive spectroscopic data. The chemical synthesis and biological activities of them have been reported in the literature [9][10][11], but the absolute configurations have not been determined. Compounds 4, 6, and 8 are all derivatives of tetrahydro-β-carboline with one chiral carbon at C-1. The absolute configurations of 4, 6, and 8 were determined by comparison with their experimental CD spectra and calculated electronic circular dichroism (ECD) spectra, which were calculated by a quantum chemical method. Compound 4 has two possible enantiomers (1S)-4 (4a) and (1R)-4 (4b), and the calculated ECD curve of 4a was similar to the experimental CD spectrum, both showing Cotton effects at 205-220 (negative) and 220-240 nm (positive) (Figure 3). Therefore, the absolute configuration 4 was assigned as 1S. The structure of 6 was similar to 4 except for the loss of methyl at C-1 instead of carboxyethyl. The calculated ECD spectrum of enantiomer (1S)-6 (6a) was in good agreement with the experimental spectrum, both showing negative Cotton effect at 210-230 and 260-300 nm and positive Cotton effect 230-260 nm ( Figure 4). Therefore, the absolute configuration of 6 was assigned as 1S. Compound 8 was a demethylated derivative of 4. Considering the biosynthesis of 4 and 8 in B. bufo gargarizans, it could be concluded that the stereoconfiguration of carboxyl group at C-1 of 8 should be the same as that of 4. The comparison of the CD and ECD spectra of enantiomers (1R)-8 (8a) and (1S)-8 (8b) showed that the calculated ECD spectra of 8b was matched better with that of CD than 8a ( Figure 5). Furthermore, the calculated specific rotation value of 8a was 103.7, and the calculated specific rotation value of 8b was corresponded to −103.7, which yielded a good match with the measured value    The other six compounds 3, 7, 9-12 were identified as dehydrobufothionine [12], bufobutarginine [6], 6-Hydroxy-1-oxo-3,4-dihydro-β-carboline [13], 1,7-pimelic acid [14], suberic acid [15], azelaic acid [16] in comparison with their NMR data in the literature respectively.
The cytotoxic activities of 1-12 were evaluated against human malignant melanoma cells A375 using the MTT method. Unfortunately, the experimental results disclosed that the IC 50 values of them were greater than 100 µM, and no antitumor activity was observed. In our previous study, we isolated one organic acid and ten bufotenines from the water extract of the toad venom. Among them, only bufoserotonin C exhibited cytotoxic effects against human lung adenocarcinoma epithelial cells A549 with IC 50 of 34.3 µM than that of positive control 5-FU (IC 50 of 48.65 µM) [12]. There we speculated that the antitumor activity of water extract and water-soluble preparations of toad venom may be manifested by the trace bufadienolides which possessed powerful cytotoxicity despite their solubility being very low in water.

Materials
The crude drug ChanSu was collected from Linyi, Shandong Province, China, in March 2010 and authenticated by the Associate Professor Dong Wang from Shenyang Pharmaceutical University.
Human malignant melanoma cells A375 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Extraction and Isolation
The dried and roughly powdered ChanSu (250 g) was extracted with dichloromethane (7 × 2.5 L) under reflux. The solvent was removed in vacuo to afford dichloromethane extract (50 g). The residue (200 g) was extracted 6 times by distilled water (6 × 2 L) with an ultrasonator (200 W, 59 kHz, 30 min), and concentrated in vacuo to obtain crude water extract (120 g). The crude water extract was suspended with 1 L distilled water and partitioned 3 times with 1 L of n-butanol saturated with water. Collected water phase and concentrated under vacuum to afford water extract (100 g). The water extract was dispersed with 0.5 L distilled water, and added ethanol to a final concentration of 75% (v/v), then kept for 12 h at 4 • C. The filtrate was evaporated to give water soluble low molecular components (WSLM) (50 g). The WSLM was subjected to 101 macroporous resin, adsorbed overnight, and then eluted  Table 1.   Table 2.  Table 2.

ECD Calculations
For the computational details for ECD spectra of compounds 4, 6, and 8 based on their known relative configuration, all six possible stereoisomers were employed for the conformational random search using the MMFF94s force field by CONFLEX software package [17]. The conformational search results with an energy cut off of 3 kcal/mol were selected to further geometry optimization and ECD calculation. The initial conformers were optimized at B3LYP/6-31G(d) theoretical level by using Gaussian09 software [18]. They were then checked by frequency calculation and resulted in no imaginary frequencies. The ECD of the conformers were then calculated by the TDDFT method at the B3LYP/6-311++G(2d,p) level with the CPCM model in methanol solution. The calculated ECD curve was generated based on Boltzmann weighted average of the conformations search results using SpecDis 1.51 [19].