Bromotyrosine-derived alkaloids from the Caribbean sponge Aplysina lacunosa.

Three new bromotyrosine-derived alkaloids 14-debromo-11-deoxyfistularin-3 (1), aplysinin A (2), and aplysinin B (3), together with 15 known compounds (4-18) were isolated from the sponge Aplysina lacunosa collected from Stirrup Cay, Bahamas. The structures of the isolated compounds were identified on the basis of MS and NMR data analysis. The (13)C NMR assignment of spirocyclohexadienylisoxazoline moieties of 1 and 2 were confirmed by an 1,1-ADEQUATE experiment. Compounds 1 and 2 showed a mild to moderate cytotoxic activities against KB-31 and FS4-LTM cell lines. Only aplysinin A (2) exhibited cytotoxicity against MCF-7 cells.

Introduction

Bromotyrosine-derived alkaloids are unique brominated metabolites which were isolated mainly from marine sponges of the order Verongida. For more than 50 years, bromotyrosine alkaloids raised the interests of synthetic and natural products chemists due to their high chemical diversity (besides the common spirocyclohexadienylisoxazoline moiety) and interesting biological activities. Since the first derivative 2,6-dibromo-4-acetamido-4-hydroxycyclohexadienone was isolated in 1967 [1], a series of bromotyrosine alkaloids were discovered with various biological activities, including antiviral [2], antibiotic [3-5], Na+/K+ ATPase inhibition [6-8], anti-HIV [9-10], antifungal [11], histidine-H3 antagonist [12], cytotoxic [13-14], and antimalarial activities [15-17]. During our investigation of the chemical constituents of Aplysina lacunosa (Aplysinidae, Verongida), three new bromotyrosine-derived alkaloids: 14-debromo-11-deoxyfistularin-3 (1), aplysinin A (2), and aplysinin B (3) (Figure 1), together with 15 known compounds were obtained. In this report we describe the structure elucidation of 1 to 3 and the biological activities of all the isolated compounds.

Figure 1

Three new bromotyrosine derivatives isolated from sponge Aplysina lacunosa: 14-debromo-11-deoxyfistularin-3 (1), aplysinin A (2), and aplysinin B (3).

Results and Discussion

The freeze-dried sponge was extracted three times with CH2Cl2/MeOH (1:1, v/v). The resulting crude extract was partitioned between n-hexane and MeOH. The MeOH extract was further partitioned between ethyl acetate and H2O. The resulting ethyl acetate phase was purified by vacuum liquid chromatography using silica gel with a stepwise gradient eluent from 100:0 to 80:20 (CH2Cl2/MeOH, v/v). The two fractions eluting with 97:3 and 94:6 (CH2Cl2/MeOH, v/v) were further purified by HPLC and yielded the three new bromotyrosine derivatives 14-debromo-11-deoxyfistularin-3 (1), aplysinins A (2) and B (3), as well as the 15 known compounds: 14-debromoaraplysillin I (4) [18], fistularin-3 (5) [19], 11,19-dideoxyfistularin-3 (6) [20], 19-deoxyfistularin-3 (7) [21], 11-deoxyfistularin-3 (8) [22], 11-ketofistularin-3 (9) [2], hexadellin B (10) [23], aerothionin (11) [13,24-25], 11-hydroxyaerothionin (12) [20], 11-oxoaerothionin (13) [13], 11-oxo-12-hydroxyaerothionin (14) [26], N-methyl-aerophobin-2 (15) [27], aeroplysinin-2 (16) [28], subereaphenol B (17) [29], and the unnamed bromotyrosine 18 [30]. Compounds 4 to 18 were identified by comparison of their MS data as well as 1H and 13C NMR chemical shifts with those reported in the literature (Figure 2).

Figure 2

Bromotyrosine alkaloids and brominated compounds isolated from the sponge Aplysina lacunosa.

Compound 1 was obtained as a white solid. The MS–ESI(+) showed a characteristic pentabrominated ion peak cluster at m/z 1037/1039/1041/1043/1045/1047 [M + Na]+ (1:5:10:10:5:1). The molecular formula of C31H31Br5N4O10 was deduced from HRMS–ESI(+) at m/z 1036.7844 [M + Na]+ (calcd for C31H3179Br5N4O10Na, 1036.7855) which required 16 double bond equivalents (DBEs). The 13C NMR spectrum of 1 indicated two amide groups at δ 158.9 (C-9´) and 159.5 (C-9), 14 olefinic carbons, two hetero-olefinic carbons at δ 154.5 (C-8´) and 155.0 (C-8), and 5 ring systems to fulfill the DBEs. The comparison of the 1H and 13C NMR data from positions C-1, C-1´ to C-9, C-9´(Table 1) of 1 with those of aerothionin (11) [13,24-25] allowed the assignment of the two dibromospirocyclohexadienylisoxazole carbonyl groups which was further confirmed by 1H,13C-HMBC and 1,1-ADEQUATE experiments. The 13C NMR assignment of C-2 and C-4 were reversed before Ciminiello´s revision in 1994 [26]. Nevertheless, the wrong assignment has still continued to be used as reference in the literature [29,31]. We therefore applied an 1,1-ADEQUATE experiment which allows the selective observation of two-bond H,C correlations [32]. The signals in the 1,1-ADEQUATE spectrum from δH 3.92 (H-1, H-1´) to δC 113.6 (C-2, C-2´) and from δH 6.57 (H-5, H-5´) to δC 120.9 and 120.8 (C-4 and C-4´, respectively) confirmed the assignments of C-2, C-2´ and C-4, C-4´ from 1994 (Figure 3). 1H,1H-COSY correlations were observed among 9-NH (δ 8.60)/H-10 (δ 3.36, 2H)/H-11 (δ 1.95, 2H)/H-12 (δ 4.06, 2H) establishing an propanamine moiety. In a similar manner, 1H,1H-COSY correlations among 9´-NH (δ 8.35)/H-20 (δ 3.35)/H-19 (δ 4.65, 2H) revealed the presence of a hydroxylethylamine moiety. The remaining signals at δH 7.05 (d, J = 8.6 Hz, H-14), 7.28 (dd, J = 1.5, 8.6 Hz, H-15), and 7.52 (d, J = 1.5 Hz, H-17) together with 1H,13C-HMBC correlations indicated the presence of a 1,2,4-trisubstituted phenoxy group. The phenoxy group was connected to the propanamine and the hydroxyethylamine substructures according to 1H,13C-HMBC correlations from H-12 to δC 153.8 (C-13) and from H-19 to δC 137.2 (C-16). Both sides of the linear fragment were connected to dibromospirocyclohexadiene moieties through amide bonds according to 1H,13C-HMBC correlations from H-7 (δ 3.21, 3.63) and 9-NH to C-9 (δ 159.1) as well as from H-7´ (δ 3.19, 3.62) and 9´-NH to C-9´ (δ 159.0). The structure of 1 is closely related to 11-deoxyfistularin-3 (8) which was originally isolated from the Caribbean sponge Aplysina fistularis insularis [22]. The only difference between 1 and 8 is the lack of one bromine atom in the central benzene ring of compound 1 at C-14. Therefore, compound 1 was named 14-debromo-11-deoxyfistularin-3. The 13C chemical shifts assignment of 1 according to the 1,1-ADEQUATE suggested a revision of the chemical shifts of C-2, C-2´, C-6, and C-6´ of the two related compounds 11-deoxyfistularin-3 (8) and 14-debromoaraplysilin I (4) [18] (Table 1 and Table 2, respectively).

Table 1

NMR data (600 MHz, DMSO-d6) of 14-debromo-11-deoxyfistularin-3 (1) and 11-deoxyfistularin-3 (8).

position	1				8			8a
	δC	δH	1,1-ADEQ		δC	δH		δC
1, 1´	73.6, CH; 73.5, CH	3.92, 2H s	2, 2´, 6, 6´		74.1; 74.0	3.93, d (7.9)		74.67; 74.60
2, 2´	113.6, 2C	–	–		113.6	–		121.66b
3, 3´	147.1, 2C	–	–		147.6	–		147.92
4, 4´	120.9, C; 120.8, C	–	–		121.4; 121.3	–		115.16b
5, 5´	131.2, CH; 131.1, CH	6.57, 2H s	6, 6´		131.7; 131.6	6.57, s;6.59, s		132.31; 132.15
6, 6´	90.3, C; 90.2, C	–	–		90.8; 90.7	–		91.78; 91.72
7	40.0c, CH2	3.21, d (18.2)	6, 8		39.9	3.22, d (18.2)		40.27
		3.63, d (18.2)				3.63, d (18.2)
7´	39.9c, CH2	3.19, d (18.2)3.62, d (18.2)	6´, 8´		39.6	3.18, d (18.1)3.62, d (18.1)
8, 8´	154.5, C; 154.6, C	–	–		155.0; 154.9	–		155.23; 155.10
9, 9´	159.1, C; 159.0, C	–	–		159.5; 159.4	–		160.44; 160.05
10	36.1, CH2	3.36c, 2H, overlapped	11		36.7	–		37.13
11	28.6, CH2	1.95, 2H qui (6.4)	10, 12		29.9	2.01, qui (7.2)		30.37
12	66.5, CH2	4.06, 2H t (6.2)	11		71.7	3.98, t (6.4)		71.51
13	153.8, C	–	–		151.8	–		152.27
14	113.3, CH	7.05, d (8.6)	13, 15		117.8	–		118.35
15	126.7, CH	7.28, dd (1.5, 8.6)	–		130.9	7.58, s		130.90
16	137.2, C	–	–		143.1	–-		143.35
17	130.5, CH	7.52, d (1.5)	–		130.9	7.58, s		130.90
18	110.8, C	–	–		117.8	–		118.35
19	69.9, CH	4.65, dt (4.5, 7.2)	16, 20		69.9	4.69, q (5.3)		70.70
20	46.8, CH2	3.35c, 2H overlapped	–		46.8	3.29, m3.34c		47.99
3, 3´-OMe	59.7, 2CH3	3.64, 6H s	–		60.1	3.66, s		59.75
9-NH	–	8.60, t (5.8)	–		–	8.57, t (5.7)		–
9´-NH	–	8.35, t (5.8)	–		–	8.39, t (5.7)		–
1-OH	–	6.36d	–		–	6.36, d (7.9)		–
1´-OH	–	6.37d	–		–	6.37, d (7.9)		–
19-OH	–	5.54, d (4.5)	–		–	5.73, d (5.3)		–

aThe 13C NMR data were obtained in pyridine-d5 at 67.5 MHz [22]. bAssignments should be reversed. cSignal obscured by the H2O residual signal in DMSO-d6; chemical shift was obtained from 2D NMR spectra. dChemical shifts were obtained from the sample before purification. Peaks were not observed in the purified sample.

Figure 3

1,1-ADEQUATE spectrum of 14-debromo-11-deoxyfistularin-3 (1).

Table 2

13C NMR data (600 MHz, DMSO-d6) of the central benzene ring of the isolated compounds 1 and 4.

position	1	4	4a
13	153.8	153.3	153.4
14	113.3	111.3	134.4b
15	126.7	133.8	133.4
16	137.2	133.4	112.2b
17	130.5	129.6	128.8
18	110.8	113.6	113.3

aThe 13C NMR data were obtained in CDCl3 [18]. bAssignments should be reversed.

The relative configuration of the spiroisoxazoline rings of 1 was investigated using a NOESY experiment. NOEs were observed between δH 6.36 (1-OH) and 3.60 (H-7); 6.37 (1´-OH) and 3.63 (H-7´); 3.57 (2H, H-5-and H-5´) and 3.21 (2H, H-7 and H-7´) indicated a trans-hydroxyspiroisoxazoline ring which was supported by a W-coupling between the olefinic proton H-5 and the methine proton H-1 (4J ~ 0.7 Hz) [28,33]. An NOE was also observed between δH 5.54 (19-OH) and δH 6.37 (1´-OH) suggested that both hydroxy groups are on the same side of the molecule. The absolute configuration of compound 1 was investigated by CD spectroscopy. The CD spectrum of 1 showed positive Cotton effects (λmax 248, Δε +5.16, λmax 285, Δε +4.58) with the same sign and magnitude as observed for (+)-aerothionin (11) [25,34-35]. Thus, the absolute configuration of spiroisoxazoline moieties were assigned as 1,1´-(R),6,6´-(S) (Figure 1). The absolute configuration of C-19 was assigned as 19-(R) according to NOE data which is in agreement with the proposed configuration by Molinski and co-workers [34,36]. The configuration of C-19 of fistularin-3 (5) was proposed to be the same as of 19, chemical fragmentation of 5 releasing from the sponge Aplysina spp. after induction by tissue damage (Figure 4). However, the conversion from 5 to 19 has not been confirmed. A single data set in the 13C NMR spectrum supported the presence of one diastereomer of 1.

Figure 4

The tissue damage induced chemical conversion from fistularin-3 (5) to 19 by an undefined enzyme in the sponge Aplysina spp. [36].

Compound 2 was isolated as a white solid. MS–ESI(+) data of 2 showed a pseudomolecular ion cluster at m/z 793/795/797/799/801 [M + Na]+ (1:4:6:4:1) indicating a tetrabrominated compound. HRMS–ESI(+) of 2 at m/z 793.8320 [M + Na]+ suggested a molecular formula of C23H25Br4N3O7 (calcd for C23H2579Br4N3O7Na, 793.8324). The 1H and 13C NMR spectra of 2 resemble to 1 except that the spectrum of 2 showed one extra methyl group (δH 1.80 s; δC 22.6) and one aromatic proton less (C-14, δH 7.05 for 1). According to one set of δH 3.17 (H-7), 3.61 (H-7), 3.93 (H-1), 6.56 (H-5), δC 39.7 (C-7), 73.5 (C-1), 90.3 (C-6), 113.1 (C-2), 120.9 (C-4), 131.1 (C-5), 147.1 (C-3), 154.3 (C-8), and 159.0 (C-9) together with 1H,13C-HMBC correlations revealed that 2 consist of only one spirocyclohexadienylisoxazoline moiety in comparison with 1. The structure determination of 2 was accomplished based on 1H,1H-COSY and 1H,13C-HMBC correlations in the same manner as for 1. Once again, 1H,1H-COSY revealed propanamine [H-18 (δ 3.96, 2H)/H-19 (δ 1.91, 2H)/H-20 (δ 3.25)/20-NH (δ 7.86)] and hydroxyethylamine [9-NH (δ 8.39)/H-10 (δ 3.33, 2H)/H-11 (δ 4.67)/11-OH (δ 5.72)] substructures. The signals at δH 7.57 (2H, s, H-13,17), δC 117.3 (C-14, 16), 130.4 (C-13, 17), 142.5 (C-12), and 151.4 (C-15) and 1H,13C-HMBC correlations among those signals showed a 1,2,4,6-dibromophenyl moiety. The substructures were assembled by 1H,13C-HMBC correlations from H-10 to C-9 and C-12, from H-11 to C-12 and C-13 as well as from δH H-18 to C-15. The terminal of side chain was connected to an acetamide moiety according to 1H,13C-HMBC correlations from H-20, 20-NH, and δH 1.80 (3H; H-22) to δC 169.1 (C-21). Compound 2 was named aplysinin A. The structure of 2 is similar to right-side portions of 1 and 8 (start at C-10). However, compound 2 contained an acetamide in the left-side portion instead of a ring system in comparison with 8 and showed great similarity with hexadellin B (10) isolating from the same organism. Hexadellin B (10) was originally isolated from the sponge Hexadella sp. The spectroscopic data of 10 was coincidently obtained from diacetylhexadellin B (20, Figure 5 and Table 3) [23] which supported the assignment of the acetamide moiety. The relative configuration of the spiroisoxazoline moiety (C-1 and C-6) was determined by comparison of the 1H and 13C NMR data with 10 and 20. The NOESY spectrum showed correlations between δH 6.36 (1-OH) and 3.61 (C-7) as well as between 6.56 (H-6) and 3.17 (H-7) suggesting a trans-hydroxyspiroisoxazoline ring similar to compound 1. An NOE was also observed between δH 5.72 (11-OH) and 1-OH indicating the same planar alignment of both hydroxy groups. The absolute configuration of spiroisoxazoline moiety was confirmed as 1-(R),6-(S) by positive Cotton effects (λmax 252, ∆ε +4.77, λmax 283, ∆ε +3.34) comparing to (+)-aerothionin (11) in the same manner of 1 [25,37]. The arrangement of 1-OH and 19-OH on the same side of the structure allowed assigning the configuration of C-19. The presence of one diastereomer of 2 was confirmed by a single data set in the 13C NMR spectrum.

Figure 5

Diacetylhexadellin B (20) isolated from sponge Hexadella sp.

Table 3

NMR data (600 MHz, DMSO-d6) of aplysinin A (2), hexadellin B (10), and diacetylhexadellin B (20).

position	2			10			20a
	δC	δH		δC	δH		δC
1	73.5, CH	3.93, d (8.2)		74.0	3.92, d (7.3)		73.1
2	113.1, C	–		113.5	–		122.1b
3	147.1, C	–		147.6	–		149.7
4	120.9, C	–		121.4	–		107.8b
5	131.1, CH	6.56, s		131.6	6.58, d (0.8)		130.2
6	90.3, C	–		90.7	–		89.9
7	39.7, CH2	3.17, d (18.4);		39.6	3.19, d (18.1);		39.9
		3.61, d (18.4)			3.60, d (18.1)
8	154.3, C	–		154.9	–		153.5
9	159.0, C	–		159.4	–		158.6
10	46.3, CH2	3.33, 2H, overlapped		40.4	3.38, m		40.4
11	69.3, CH	4.67, t (6.1)		33.6	2.77, t (7.1)		34.4
12	142.5, C	–		139.5	–		137.2
13,17	130.4, 2CH	7.57, 2H, s		133.5	7.54, s		132.8
14,16	117.3, 2C	–		117.7	–		118.2
15	151.4, C	–		150.9	–		151.5
18	71.3, CH2	3.96, 2H, t (6.2)		70.8	4.00, t (6.1)		72.1
19	29.8, CH2	1.91, 2H, q (6.9)		28.2	2.08, m		29.4
20	35.7, CH2	3.25, 2H, q (6.7)		37.0	3.08, br s		37.7
21	169.1, C	–		–	–		170.0
22	22.6, CH3	1.80, 3H, s		–	–		23.6
3-OMe	59.6, CH3	3.65, 3H, s		60.1	3.65, s		60.3
9-NH	–	8.39, t (5.9)		–	8.59, t (5.9)		–
1-OH	–	6.36, d (8.2)		–	6.37, d (7.8)		–
11-OH	–	5.72, d (4.4)		–	–		–
20-NH	–	7.86, t (5.3)		–	–		–

aThe 13C NMR data were obtained in CDCl3 [23]. bAssignments should be reversed.

Compound 3 was isolated together with N-methylaerophobin-2 (15) as a mixture (approximate ratio 1:5). The ESIMS spectrum exhibited a 1:2:1 ion cluster at m/z 457/459/461, indicating the presence of two bromine atoms. The HRMS–ESI(+) spectrum revealed a pseudomolecular ion [M + H]+ at m/z 456.9892, which indicated a molecular formula of C16H18Br2N4O2 (calcd for C16H1979Br2N4O2, 456.9875), containing nine DBEs. Two singlet aromatic protons δH 7.88 (δC 131.7) suggested a tetrasubstituted benzene pattern which was confirmed by 1H,13C-HMBC correlations; from δH 7.88 (2H, H-2, H-6) to δC 118.5 (C-3, C-5), δC 154.4 (C-4), δC 131.7 (C-2, C-6), and δC 135.7 (C-7), from δH 3.82 (4-OMe) to C-4. The connection of the benzene fragment to the E-vinyl moiety was confirmed by HMBC correlations from two olefinic protons δH 7.33 (d, J = 15.8 Hz, H-7) and δH 6.66 (d, J = 15.8 Hz, H-8) to δC 134.9 (C-1). 1H,1H-COSY correlations in between 9-NH (δ 8.17)/H-10 (δ 3.20, 2H)/H-11 (δ 1.71, 2H)/H-12 (δ 2.45, 2H) indicated a propanamine fragment which was connected to a 2-aminoimidazole moiety according to 1H,13C-HMBC correlations from H-12 to δC 126.8 (C-13) and 109.2 (C-14) (Table 4). The two substructures are connected through an amide bond according to the 1H,13C-HMBC correlations from H-7, 9-NH, and H-10 to δC 165.0 (C-9). The structure of 3 is very similar to compound 21 (Figure 6) which was isolated from the Caribbean sponge Verongula sp. [38]. In comparison with 21, compound 3 showed one extra aliphatic carbon and was named aplysinin B.

Table 4

NMR data of aplysinin B (3) (600 MHz, DMSO-d6) and compound 21.

position	3			21a
	δC	δH		δC
1	134.9, C	–		136.36
2, 6	131.7, CH	7.88, 2H, s		132.98
3, 5	118.5, C	–		119.48
4	154.4, C	–		154.46
7	135.7, CH	7.33, d (15.8)		138.42
8	124.9, CH	6.66, d (15.8)		123.95
9	165.0, C	–		167.50
10	38.4, CH2	3.20, 2H, m		39.27
11	28.1, CH2	1.71, 2H, m		25.92
12	22.0, CH2	2.45, 2H, m		125.89
13	126.8, C	–		110.87
14	109.2, CH	6.60, s		146.43
15	147.4, C	–		–
4-OMe	61.0, CH3	3.82, s		61.26
9-NH	–	8.17, t (5.58)		–

aThe 13C NMR data were obtained in CD3OD [38].

Figure 6

Bromotyrosine alkaloid (21) isolated from the sponge Verongula sp.

The new compounds 14-debromo-11-deoxyfistularin-3 (1) and aplysinin A (2) were tested for their antimicrobial activity against different Gram-positive and Gram-negative bacteria, fungi, and for their antiproliferative activity. Aplysinin B (3) was not subjected to any biological activity test due to the minute amount and its existence as the minor compound of a mixture. The results showed that 1 and 2 exhibited mild cytotoxic activity against KB-31 epidermoid carcinoma cells (IC50 = 69 and 26 µM, respectively). Only 2 showed mild toxicity against the breast cancer cell line MCF-7 and to FS4-LTM conditional immortalization human fibroblasts (IC50 = 78 and 32 µM, respectively). The cytotoxicities of the known compounds (4–17) are also listed in Table 5. None of the isolated compounds showed any antimicrobial activity.

Table 5

Cytotoxicity of the isolated compounds (IC50).a

compound	IC50 [µM]
	L929	KB-31	MCF-7	FS4-LTM
1	–	68.8	–	–
2	–	25.8	77.5	32.2
4	94.3	–	78.6	–
5	–	–	206.9	–
6	117.6	88.2	–	–
7	–	–	60.0	–
8	–	–	47.2	87.3
10	–	–	90.6	73.4
15	55.9	48.9	–	–
16	–	–	96.3	–
17	–	–	64.8	–

aCompounds with no activity are not listed in the table.

Experimental

UV spectra were recorded during HPLC separation with a DAD detector (JASCO MD-2010 Plus). CD spectra were recorded on a JASCO J-810 spectropolarimeter. Low and high resolution ESIMS was performed with a Bruker micrOTOF mass spectrometer. Mass calibration was performed using sodium formate cluster ions prior each measurement. 1H and 13C spectra were recorded on a Bruker Avance 600 NMR spectrometer equipped with a cryo platform (1H at 600 MHz, 13C at 150 MHz) and a Bruker Avance NMR spectrometer (1H at 400 MHz, 13C at 100 MHz). All NMR experiments were measured at a temperature of 303 K using DMSO-d6 (δH 2.50, δC 39.5) as internal standard. HPLC separation was achieved by Jasco PU-1580 using a Kromasil RP18 column (16 mm × 250 mm, 5 µm) and a Kromasil RP18 column (1 mm × 50 mm, 5 µm) and was eluted with gradient H2O (0.1% TFA) and MeCN (0.1% TFA).

The sponge Aplysina lacunosa was collected by SCUBA diving at a depth of 8 m from Stirrup Cay in the Bahamas in June 2008. The sample was immediately frozen and kept at −20 °C until extraction. A voucher specimen of this species is deposited in AG Köck, Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung (voucher number: Aplysina lacunosa 08/21). The freeze-dried sponge (200 g) was extracted three times with CH2Cl2/MeOH (1:1, v/v) at room temperature. The filtrates were pooled and evaporated to yield 24.3 g of crude extract which was further partitioned between n-hexane and MeOH. The MeOH extract was then partitioned between EtOAc and H2O. The EtOAc fraction was further purified by vacuum liquid chromatography using silica gel eluting with stepwise gradient from 100:0 to 80:20 (CH2Cl2/MeOH, v/v). The fraction eluted with 97:3 (CH2Cl2/MeOH, v/v) was concentrated and further purified by HPLC using an RP C18 column (stepwise gradient 60:40, 40:60, and 20:80 H2O/MeCN, v/v) to yield 5 (198.0 mg), 11 (263.0 mg), 12 (445.0 mg), 13 (55.0 mg), 14 (7.9 mg), 16 (15.0 mg), 17 (9.1 mg), and two other fractions. The first fraction was purified using reversed-phase HPLC [52:48 H2O (0.1% TFA)/MeCN (0.1% TFA), v/v] to obtain 7 (3.2 mg), 8 (4.5 mg), 9 (9.6 mg) and following with analytical RP18 column [gradient 60:40 to 20:80 H2O (0.1% TFA)/MeCN (0.1% TFA), v/v] yielding 1 (0.8 mg). The other fraction was purified using RP18 HPLC [60:40 H2O (0.1% TFA)/MeCN (0.1% TFA), v/v] and follow with an analytical RP18 column [65:35 H2O (0.1% TFA)/MeCN (0.1% TFA), v/v] yielding 2 (0.8 mg) and 18 (5.3 mg). The 94:6 (CH2Cl2/MeOH, v/v) fraction was purified by HPLC using a RP C18 column (gradient 80:20 to 40:60 H2O/MeCN, v/v) yielding 6 (10.7 mg), 10 (25.5 mg), 15 (5.0 mg), and one fraction which was purified using reversed-phase HPLC [70:30 H2O (0.1% TFA)/MeCN (0.1% TFA), v/v] to get a mixture of 3 and 15 (3.5 mg) as well as 4 (3.0 mg).

Biological activity test

The antimicrobial activities of isolated compounds were evaluated against five microorganisms [Gram-positive: Straptococcus aureus (MRSA and MSSA) and Micrococcus luteus; Gram-negative: Peumonia aruginosa and Klebsiella pneumonia] and antifungal Candida albicans using microdilution technique. The MIC was defined as lowest concentration that shows 50% growth inhibition after 24 hour incubation.

Cytotoxicity assay

The cytotoxicity was determined using WST-1 cell proliferation assays. Targeting cell lines are L929 mouse fibroblasts, KB-31 epidermoid carcinoma, and MCF-7 breast cancer cell lines which were incubated for 5 days with the test substances. The acute toxicity was determined using the FS4-LTM conditional immortalization human fibroblasts cell line which was incubated for 24 hours with the test compounds.

Experimental data

14-Debromo-11-deoxyfistularin-3 (1): white solid; UV (DAD) λmax 226 nm; CD (MeOH) λmax 248 nm (∆ε +5.16), 288 nm (∆ε +4.55); 1H NMR and 13C NMR see Table 1; HRMS–ESI(+) m/z = 1036.7844 [M + Na]+ (calcd for C31H3179Br5N4O10Na, 1036.7855, Δm = 1.1 ppm).

Aplysinin A (2): white solid; UV (DAD) λmax 225 nm; CD (MeOH) λmax 252 nm (∆ε +4.77), 283 nm (∆ε +3.34); 1H NMR and 13C NMR see Table 3; HRMS–ESI(+) m/z = 793.8320 [M + Na]+ (calcd for C23H2579Br4N3O7Na, 793.8324, Δm = 0.4 ppm).

Aplysinin B (3): white solid; UV (DAD) λmax 227 nm; 1H NMR and 13C NMR see Table 4; HRMS–ESI(+) m/z = 456.9892 [M + H]+ (calcd for C16H1979Br2N4O2, 456.9875, Δm = 1.7 ppm).

1D, 2D NMR, and CD spectra of three new compounds. 1D NMR, mass and CD spetra of all known isolated compounds.