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Heteroaromatic alkaloids, nakijinamines, from a sponge Suberites sp.
Tetrahedron 68 (2012) 8545e8550
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Heteroaromatic alkaloids, nakijinamines, from a sponge Suberites sp. Yohei Takahashi a, Naonobu Tanaka a, Takaaki Kubota a, Haruaki Ishiyama a, Azusa Shibazaki b, Tohru Gonoi b, Jane Fromont c, Jun’ichi Kobayashi a, * a
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Medicinal Mycology Research Center, Chiba University, Chiba 260-0856, Japan c Western Australian Museum, Locked Bag 49, Weishpool DC, WA 6986, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2012 Received in revised form 6 August 2012 Accepted 7 August 2012 Available online 15 August 2012
The structures of seven new secondary metabolites isolated from an Okinawan marine sponge Suberites sp., nakijinamines A (1), B (2), and FeI (3e6) and 6-bromoconicamin (7), have been elucidated on the basis of spectroscopic analysis, chemical conversion, and conformational analysis. These analyses disclosed that 1e6 were heteroaromatic alkaloids having the hybrid structures of an aaptamine-type alkaloid and an indole alkaloid, while 7 was a bromoindole alkaloid. Nakijinamine I (6) is the first example of an aaptamine-type alkaloid possessing a 1,4-dioxane ring. Antimicrobial activities of 1e7 were evaluated. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Heteroaromatic alkaloids Marine sponge Suberites sp. Nakijinamines
1. Introduction Marine sponges have been recognized as a rich source of interesting bioactive metabolites with fascinating chemical structures.1 Among them, aaptamine is a representative heteroaromatic alkaloid with a 1H-benzo[de][1,6]naphthyridine ring system.2 Various aaptamine-type alkaloids have been isolated from marine sponges belonging to the genus Aaptos, Suberites, Luffariella, Hymeniacidon, Suberea, and Xestospongia so far.3 A lot of synthetic and structureeactivity relationship studies for these alkaloids have been carried out,3 because of their various biological activities, such as cytotoxic, antimicrobial, antiviral, antioxidant, and enzymatic inhibitory activities.3,4 During our continuing search for secondary metabolites possessing interesting chemical structures from marine sponges,5 we examined the extract of an Okinawan marine sponge Suberites sp. (SS-1084), and isolated a series of new alkaloids, nakijinamines A (1), B (2), CeE (8e10), and FeI (3e6). Recently, we have reported the structure elucidation of nakijinamines CeE (8e10),6 which are heteroaromatic alkaloids possessing the hybrid structures of an aaptamine-type alkaloid and a bromoindole alkaloid. Further investigation for the chemical structures of nakijinamines resulted in the structure elucidation of six new heteroaromatic alkaloids, nakijinamines A (1), B (2), and FeI (3e6). The structure of one new
* Corresponding author. E-mail address: [email protected] (J. Kobayashi). 0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2012.08.018
bromoindole alkaloid, 6-bromoconicamin (7), which was purified in the isolation process of 1e6, was also assigned. In this paper, we describe the isolation and structure elucidation of 1e7.
2. Results and discussion The sponge Suberites sp. (SS-1084, 0.4 kg, wet weight) collected off Unten Port, Nakijin, Okinawa was extracted with MeOH. The extract was partitioned between EtOAc and H2O, and subsequently the aqueous layer was extracted with n-BuOH. The n-BuOH-soluble materials were subjected to a C18 column and a Sephadex LH-20 column followed by reversed-phase HPLC to afford nakijinamines A (1, 1.7103%, wet weight), B (2, 9.6105%), F (3, 9.6105%), G (4, 4.2105%), H (5, 2.1105%), and I (6, 7.8105%) and 6bromoconicamin (7, 1.3104%) together with nakijinamines CeE (8e10)6 and two known aaptamine-type alkaloids, aaptamine2 and bisdemethylaaptamine.7 Nakijinamine A (1) was obtained as a yellow amorphous solid. The molecular formula of 1 was elucidated to be C24H24BrN4O2 {m/z 239.04965 ([MH]2þ, D 0.02 mmu) and 477.09206 ([M2H]þ, D 0.01 mmu)} by the HRESIMS. IR absorptions at 3396 and 3242 cm1 implied the existence of OH and/or NH functionalities. The UV spectrum suggested the occurrence of a conjugated aromatic chromophore. Inspection of the 1H and 13C NMR and the HMQC spectra disclosed the presence of 11 sp2 quaternary carbons, eight sp2 methines, one sp3 methine, one sp3 methylene, and three sp3 methyls (Table 1). The 13C NMR spectrum of 1 was similar to
8546
Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550
HO
9
1 9a
HN 2
3
8
NMe3
1"
OH
2"
7
2'
1'
4
5
N H
6' 7'a 7'
N H
6
3a
7'
1' 7'a
3'
Me3N
1"
2"
1"'
O
OH
6'
N H
4'
4: R =
2"'
2"' 3"'
4"'
5"'
5"'
2"' 3"'
HN 9: R =
2"'
5
N H
6 7a
Br
7
NMe3
O
N
4"'
4"'
5: R =
6
3"'
1
7
R
5"'
3: R =
N H
2
N H
Br
O
4 3a
3
Br
HN
O
2'
1'
R
5' 3'a
Me3N
NMe3
HN
1: R = Br 2: R = H
HN 2'
5'
3'a
3'
6a
9b
R
4'
Br
HN
N H
N H
6"'
SO3
8: R =
7"' 8"'
N
SO3 10: R =
N O
that of nakijinamine C (8)6 except for the resonances of the side chain at C-8 and C-9 in 8, implying that 1 has the hybrid structure of an aaptamine-type alkaloid and a bromoindole alkaloid. The comparison of 13C NMR data for unit A (N-1eC-9b) in 1 with that for bisdemethylaaptamine7 indicated that 1 has the same 8,9dihydroxy-1H-benzo[de][1,6]naphthyridine ring system as bisdemethylaaptamine. This was supported by a 1He1H COSY cross-peak of H-2/H-3 and HMBC correlations for H-2/C-9a, H-2/C-3a, and H-3/C-9b (Fig. 1). A large 1JCH value of H-5 (182.4 Hz) implied that C-5 is an sp2 methine.6,8 On the other hand, the gross structure of unit B (N-10, C-20 eC-70 a, C-100, and C-200 ) was confirmed by analysis of the 2D NMR spectra (Fig. 1). Although the connectivity of C-200 in unit B to C-6 or C-7 in unit A was assumed by the molecular formula
unit B
OH HO
9
1 9a
HN 2
3
8
7
3a
6 4
N H
unit A
4' 3' 3'a
2''
6a
9b
NMe3
1''
2' 5
1'
N H
5' 6'
7'a 7'
Br 1H-1H
COSY & TOCSY HMBC NOESY
Fig. 1. Selected 2D NMR correlations for nakijinamine A (1). Table 1 1 H (600 MHz) and CD3OD Position
2 3 3a 5 6 6a 7 8 9 9a 9b 20 30 30 a 40 50 60 70 70 a 100 200 NeMe a b
13
C (150 MHz) NMR data for nakijinamines A (1) and B (2) in
1
2
dCa
dHa (J in Hz)
dCa
dHb (J in Hz)
142.5 100.1 152.6 130.2 111.3 129.7 118.1 153.6 130.7 134.3 119.0 125.9 115.4 127.1 121.7 124.1 117.1 116.2 139.7 71.1
7.74, d (7.0) 6.32, d (7.0) d 7.20, br 7.22, br d d d d d d 7.65, s d d 7.35, br 7.02, br d (8.1) d 7.51, d (1.3) d 4.58, br 4.09, br 5.58, br 3.25 (9H), s
142.6 100.0 152.6 130.2 111.5 129.9 119.0 153.5 130.5 134.6 118.6 125.0 115.0 128.2 120.2 121.0 123.8 113.4 138.9 71.2
7.78, d (7.0) 6.34, d (7.0) d 7.20, br 7.20, br d d d d d d 7.61, s d d 7.47, br 6.96, br t (7.7) 7.10, t (7.7) 7.37, d (7.7) d 4.62, br 4.40, br 5.61, br 3.26 (9H), s
31.8 55.2
Recorded at 300 K. Recorded at 315 K.
31.8 55.0
of 1 and the chemical shift of CH-200 (dH 5.58 and dC 31.8), no correlation that might provide an information on the connectivity was observed in the 2D NMR spectra because of its broadening proton signals (Table 1). To elucidate the gross structure of nakijinamine A (1), chemical conversion was carried out as follows. Treatment of 1 with methyl iodide in acetone under basic condition gave two separable derivatives (1a and 1b) (Scheme 1), whose 1H NMR spectra showed the sharp signals. Detailed analysis of the 2D NMR spectra of 1a and 1b suggested that they are stereoisomers of the methyl and acetone adduct of 1 (Supplementary data). Though the relative configurations of C-9 and C-200 in 1a and 1b were not elucidated, the gross structures of them revealed the connectivity of C-7 to C-200 in 1. Accordingly, the gross structure of nakijinamine A was elucidated to be 1. Nakijinamine A (1) gave the broadening signals especially in H5, H-6, H-40 , H2-100, and H-200 in the 1H NMR spectrum (Table 1), suggesting the restriction of C-7eC-200 bond rotation due to the interference by three bulky substituents attached to C-200 .6 The 1H NMR spectrum of 1 measured at 223 K provided two sets of sharp signals with ca. 5:3 proportions (Table 2). These were assigned to be rotamers (1c and 1d, respectively) by analysis of the ROESY spectrum. Therefore, 1 presumed to exist as an equilibrium mixture of two rotamers in CD3OD at 300 K. Nakijinamine B (2) was yielded as a yellow amorphous solid, and the molecular formula, C24H25N4O2, was revealed by the HRESIMS
Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550
8547
Scheme 1. Chemical conversion of nakijinamine A (1) into two stereoisomers (1a and 1b) of the methyl and acetone adduct of 1. Selected 2D NMR correlations for 1a and 1b were shown.
Table 2 1 H (500 MHz) and 13C (125 MHz) NMR dataa for the major (1c) and minor (1d) rotamers of nakijinamine A (1) in CD3OD Position
2 3 3a 5 6 6a 7 8 9 9a 9b 20 30 30 a 40 50 60 70 70 a 100 a 100 b 200 NeMe a b c d e f g h
1c
Position
1d
dC
dH (J in Hz)
dC
dH (J in Hz)
142.2 99.6 152.2 130.3 111.3 129.5c 118.1d 153.2 130.4c 134.9 119.0d 125.2 115.7e 127.1 122.1 123.7 117.1 115.9e 139.7 69.6
7.80, mb 6.30, d (7.1) d 7.13, d (7.4) 6.88, d (7.4) d d d d d d 7.77, s d d 7.14, d (8.5) 6.96, dd (8.5, 1.5) d 7.49, d (1.5) d 4.76, dd (13.5, 4.5) 4.23, dd (13.5, 6.0) 5.84, br s 3.28 (9H), s
142.5 99.6 152.6 131.1 110.9 128.9f 118.2g 155.3 129.9 135.1f 118.7g 125.2 115.8h 127.2 121.9 123.8 126.5 115.9h 139.0 71.6
7.80, mb 6.36, d (7.1) d 7.44, d (7.5) 7.74, mb d d d d d d 7.75, s d d 7.62, d (8.7) 7.12, mb d 7.50, d (1.5) d 4.71, dd (13.6, 6.9) 4.36, dd (13.6, 3.9) 5.23, br s 3.22 (9H), s
30.2 54.3
Table 3 1 H (600 MHz) and 13C (150 MHz) NMR dataa for nakijinamines FeH (3e5) in CD3OD
32.1 54.3
Recorded at 223 K. Coupling constants were not determined due to overlapping of signals. Interchangeable. Interchangeable. Interchangeable. Interchangeable. Interchangeable. Interchangeable.
{m/z 201.10237 ([MþH]2þ, Dþ0.13 mmu) and 399.18181 ([M2H]þ, D þ0.26 mmu)}. Inspection of the 1H and 13C NMR spectra suggested that 2 was structurally related to 1 (Table 1). In addition, 2 showed the signal of one sp2 methine (CH-60 ) in place of the brominated sp2 quaternary carbon in 1. This proton signal was correlated to H-50 and H-70 in the 1He1H COSY spectrum. From these data, the gross structure of nakijinamine B (2) was concluded to be a debromo analog of 1. Nakijinamine F (3) was isolated as a yellow amorphous solid. The molecular formula was assigned to be C29H33BrN5O2 based on the HRESIMS analysis {m/z 281.59420 ([MþH]2þ, D 0.05 mmu) and 562.18118 ([M]þ, D 0.03 mmu)}. The 1H and 13C NMR data for 3 (Table 3) were reminiscent of those for nakijinamine C (8),6 and the signals for a 3-methylbutanamide (C-1000 eC-5000 ) in 3 were discerned in place of the resonances of the substituent at C-8 and C-9 in 8. The presence of the 3-methylbutanamide was confirmed by the 2D NMR analysis (Fig. 2). The positions of the 3methylbutanamide at C-9 and a hydroxy group at C-8 were
3
4
5
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
2
142.5
142.7
101.4
3a 5 6 6a 7 8 9 9a 9b 20 30 30 a 40 50
152.3 132.4 111.2 130.2 118.3 154.2b 124.2 140.2 118.8 126.1 115.3 127.1 121.8 124.2
60 70
117.2 116.2
70 a 100
139.7 70.8
7.92, d (7.0) 6.56, d (7.0) d 7.40, br 7.40, br d d d d d d 7.64, s d d 7.40, br 7.06, br d (8.4) d 7.53, d (1.6) d 4.60, br 4.42, br 5.60, br 3.29 (9H), s d 2.74, m
142.3
3
7.86, d (6.9) 6.53, d (6.9) d 7.40, br 7.40, br d d d d d d 7.64, s d d 7.40, br 7.05, br d (8.3) d 7.52, s
7.89, d (7.0) 6.55, d (7.0) d 7.39, br 7.45, br d d d d d d 7.62, s d d 7.45, br 7.05, br d (8.9) d 7.53, d (1.7) d 4.56, br 4.40, br 5.60, br 3.24 (9H), s d 4.05 (2H), m d
200 NeMe
31.8 55.0
1000 2000
177.4 46.0
3000
27.0
4000
23.9
5000
23.9
d 4.58, br 4.41, br 5.60, br 3.26 (9H), s d 2.60 (2H), d (7.0) 2.29, m 1.11 (3H), d (6.8) 1.11 (3H), d (6.8)
101.5 152.4 132.6 111.3 129.9 118.8 154.6b 124.2 139.7 118.8 126.2 115.3 127.1 121.8 124.2 117.2 116.2 139.0 70.9 31.5 55.1 181.8 44.2 28.8 12.9 28.8
1.96, m 1.64, m 1.09 (3H), t (7.4) 1.37 (3H), d (7.0)
101.2 152.1 132.4 111.1 130.2 118.2 153.4b 124.2 140.3 120.0 125.8 115.3 127.1 121.8 124.2 117.1 116.2 139.7 70.7 31.6 55.0 175.8 43.6 136.7 132.0 130.1
000
6
127.8
7000
130.1
8000
132.0
a b
7.47, d (7.5) 7.39, t (7.5) 7.31, t (7.5) 7.39, t (7.5) 7.47, d (7.5)
Recorded at 315 K. Not observed clearly.
assigned based on the chemical shifts for C-8 (dC 154.2) and C-9 (dC 124.2).6 Thus, the gross structure of nakijinamine F was elucidated to be 3. Nakijinamines G (4) and H (5) were isolated as yellow amorphous solids. The molecular formula of 4, C29H33BrN5O2 {m/z 281.59436 ([MþH]2þ, D þ0.11 mmu) and 562.18147 ([M]þ, D þ0.20 mmu)}, and of 5, C32H31BrN5O2 {m/z 298.58649 ([MþH]2þ, D
8548
Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550 4'''
5'''
2''' 1'''
O
HN
9
OH 8
1'' 2"
7
1 9a
HN
6
9b
2
3a
3
4
N H
2' 5
HN
NMe3
2'
4' 3' 3'a
1'
N H
7'a
Me3N
Br
7'
1H-1H
COSY & TOCSY HMBC NOESY
þ0.07 mmu) and 596.16577 ([M]þ, D þ0.01 mmu)}, were assigned by the HRESIMS analysis. The 1H and 13C NMR spectra (Table 3) suggested that 4 and 5 have similar structures to nakijinamine F (3) with different side chains at C-9. The side chains of 4 and 5 were elucidated to be a 2-methylbutanamide (C-1000 eC-5000 ) and a phenylethanamide (C-1000 eC-8000 ), respectively, by analysis of the 2D NMR spectra (Supplementary data). Thus, the gross structures of nakijinamines G (4) and H (5) were elucidated. Nakijinamine I (6) was obtained as a yellow amorphous solid, and the molecular formula, C24H22BrN4O2, was established by the HRESIMS analysis {m/z 239.04990 ([MþH]2þ, D þ0.23 mmu) and 477.09248 ([M]þ, D þ0.41 mmu)}. The UV spectrum suggested the existence of a conjugated aromatic chromophore. The 1H and 13C NMR spectra (Table 4) exhibited the signals of three D2O-exchangeable protons, 19 aromatic carbons including nine sp2 methines, two sp3 methines, and one N,N,N-trimethylammonio group. Interpretation of the 2D NMR data for 6 indicated the presence of a 8,9-dihydroxy-1H-benzo[de][1,6]naphthyridine ring system (N-1eC-9b) and a 6-bromoindole moiety (N-10 eC-70 a) (Fig. 3). In addition, a 1He1H COSY cross-peak of H-100 /H-200 and HMBC correlations for H-200 /C-30 and 100 -NMe3/C-100 revealed the connectivities of C-200 to C-100 and C-30 , and of C-100 to the N,N,Ntrimethylammonio group. The chemical shifts of CH-100 (dH 6.41; dC 91.2) and CH-200 (dH 6.82; dC 64.6) implied that these sp3 methines were oxygenated. HMBC cross-peaks of H-100 /C-9 and H-200 /C-8
13
1"
O
4'
3'a
2"
O 8 9
1 9a
HN
7
9b 6a 6
2 3
3a
4
N H
5
1H-1H
COSY HMBC ROESY
Fig. 3. Selected 2D NMR correlations of nakijinamine I (6).
revealed the connectivity of C-100 to C-9 and C-200 to C-8 via ether bonds, forming a 2,3-dihydro-1,4-dioxane ring. Thus, the gross structure of nakijinamine I (6) was assigned as shown in Fig. 3. The relative stereochemistry of nakijinamine I (6) was assigned by the conformational search on MacroModel program9 and the ROESY analysis. The conformational search for anti- and syn-nakijinamine I was carried out to give the most stable conformers 6a (anti) and 6b (syn) (Fig. 4). The dihedral angles of H-100 /H-200 in 6a and 6b were 79 and 58 , respectively. In the 1H NMR spectrum of 6, the signals for H-100 and H-200 were observed as both singlet (Table 4), suggesting that the dihedral angle of H-100 /H-200 was close to 90 . It was similar to calculated value of 6a rather than that of 6b. ROESY correlations for H-40 /H-100 and 100 -NMe3/H-200 also implied the anti relationship for H-100 /H-200 . Therefore, the relative stereochemistry of nakijinamine I (6) was concluded to be 6a (Fig. 4).
C (150 MHz) NMR dataa for nakijinamine I (6) in DMSO-d6
Position
dC
dH (J in Hz)
1 2 3 3a 4 5 6 6a 7 8 9 9a 9b 10 20 30 30 a 40 50 60 70 70 a 100 200 NeMe
d 141.5 99.5 149.8 d 129.4 112.1 131.0 105.1 144.8 124.4 129.5 117.2 d 125.9 108.1 124.5 120.7 122.8 115.0 114.6 136.8 91.2 64.6 49.9
13.11, br s 8.07, d (7.3) 6.55, d (7.3) d 12.77, br s 7.34, d (7.4) 6.74, d (7.4) d 6.87, s d d d d 11.60, d (2.5) 7.39, d (2.5) d d 7.83, dd (8.5, 1.7) 7.28, d (8.5) d 7.61, d (1.7) d 6.41, s 6.82, s 3.34 (9H), s
a
5' 3'
6'
6'
7'a
1'
5'
Fig. 2. Selected 2D NMR correlations of nakijinamine F (3).
Table 4 1 H (600 MHz) and
Br
7'
3'''
Recorded at 300 K.
Fig. 4. The most stable conformers for anti- (6a) and syn- (6b) nakijinamine I (protons of methyl groups were omitted).
The molecular formula of 6-bromoconicamin (7), C13H16BrN2, was established by the HRESIMS (m/z 279.04975 [M]þ, D þ0.61 mmu). Analysis of the 1He1H COSY, HMBC, and NOESY spectra (Fig. 5) indicated the existence of a 3,6-disubstituted indole ring, a 1,2-disubstituted olefin, and an N,N,N-trimethylammonio
Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550
8549
13
C chemical shifts, respectively. ESIMS spectra were obtained on a JEOL JMS-T100LC and a Thermo Scientific Exactive spectrometers. HPLC column: C18 (Cosmosil 5C18-PAQ, Nacalai Tesque, Inc.), Phenyl-Hexyl (Luna 5u Phenyl-Hexyl, Phenomenex, Inc.), chiral (CHIRALCEL OD-R, Daicel Chemical Ind., Ltd.). 3.2. Sponge description
Fig. 5. Selected 2D NMR correlations of 6-bromoconicamin (7).
group. HMBC correlations for 10 -NMe3 to C-10 and H-20 to C-2 and C-3a suggested that an N,N,N-trimethylammonio group was connected to C-3 via the 1,2-disubstituted olefin, and therefore a bromine atom was assigned to locate at C-6. The coupling constant of H-10 /H-20 (J¼14.3 Hz) disclosed the geometry of the olefin to be E. Thus, the structure of 6-bromoconicamin (7) was assigned as shown in Fig. 5. Nakijinamines A (1), B (2), and FeI (3e6) were optically inactive, though they have a chiral center (C-200 ). Optical resolution of 1 by chiral HPLC resulted in the separation of two enantiomers, the ratio of which was approximately 1:1. Therefore, nakijinamine A (1) was concluded to be a racemate. Similarly, the enantiomers of nakijinamines B (2), F (3), and I (6) were separated by chiral HPLC, indicating that they are racemates. Although optical resolution of nakijinamines G (4) and H (5) was not successful under any conditions, these compounds were also presumed as being racemates since the biogenetic pathway of 4 and 5 might be similar to that of 1e3. Recently, we reported the isolation and structure elucidation of heteroaromatic alkaloids, nakijinamines CeE (8e10), from the extract of an Okinawan marine sponge Suberites sp. (SS-1084).6 Further investigation for the chemical structure of alkaloids from the extract resulted in the structure elucidation of six new heteroaromatic alkaloids, nakijinamines A (1), B (2), and FeH (3e6), and one new bromoindole alkaloid, 6-bromoconicamin (7). Nakijinamines A (1) and B (2) possess the hybrid structure of bisdemethylaaptamine and 6-bromoconicamin or conicamin.9 Nakijinamines FeH (3e5) are similar hybrid compounds to 1 with a 3-methylbutanamide, a 2-methylbutanamide, and a phenylethanamide moieties, respectively, which seem to be derived from leucine, isoleucine, and phenylalanine. Nakijinamine I (6) is the first example of an aaptamine-type alkaloid having a 1,4-dioxane ring. Nakijinamines A (1), B (2), and FeI (3e6) and 6bromoconicamin (7) were evaluated for their antimicrobial activities. Among the evaluated compounds, 1 exhibited antimicrobial activity against Candida albicans (IC50 0.25 mg/mL), Cryptococcus neoformans (IC50 0.5 mg/mL), Trichophyton mentagrophytes (IC50 0.25 mg/mL), Staphylococcus aureus (MIC 16 mg/mL), Bacillus subtilis (MIC 16 mg/mL), and Micrococcus luteus (MIC 2 mg/mL), while 2 and 3 showed antimicrobial activity against C. albicans (IC50 8 mg/mL, each). On the other hand, 1e7 did not show cytotoxicity (IC50 >10 mg/mL) against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro. 3. Experimental section 3.1. General procedures IR and UV spectra were recorded on a JASCO FT/IR-5300 and a Shimadzu UV-1600PC spectrophotometers, respectively. NMR spectra were measured on a Bruker AMX-600 and a Bruker AMX500 spectrometers. The 2.49 and 3.35 ppm resonances of residual CHD2SOCD3 and CHD2OD, and 39.5 and 49.8 ppm resonances of DMSO-d6 and CD3OD were used as internal references for 1H and
The sponge Suberites sp. (SS-1084, family Suberitidae) was collected off Unten Port, Nakijin, Okinawa, and kept frozen until used. This specimen is a dark brown flattened mound. The skeleton is a dense mass of styles of various sizes: 132015 mm, 97012 mm, 48010 mm. Some styles occur in tracts in the mesohyl. There are no microscleres. The voucher specimen is deposited at the Graduate School of Pharmaceutical Sciences, Hokkaido University. 3.3. Extraction and isolation The sponge (SS-1084, 0.4 kg, wet weight) was extracted with MeOH. The extracts (18.5 g) were partitioned between EtOAc (3500 mL) and H2O (500 mL), and then the aqueous layer was extracted with n-BuOH (3500 mL). A part (1.2 g) of the n-BuOHsoluble materials (5.0 g) was separated by a C18 column chromatography (MeOH/H2O/CF3CO2H) to give four fractions IeIV. Fraction I was applied to gel filtration on Sephadex LH-20 (MeOH), and then subjected to Phenyl-Hexyl HPLC (25010 mm ID; eluent MeCN/ H2O/CF3CO2H, 17:83:0.1 to 50:50:0.1; flow rate 2.0 mL/min; UV detection at 255 nm) to yield 6-bromoconicamin (7, 0.5 mg, 1.3104%, wet weight) and aaptamine. Fraction II was purified by gel filtration on Sephadex LH-20 (MeOH/CF3CO2H, 100:0.1) followed by Phenyl-Hexyl HPLC (25010 mm ID; MeCN/H2O/ CF3CO2H, 20:80:0.2 to 25:75:0.2; 2.0 mL/min; 280 nm) to afford nakijinamines A (1, 28.5 mg, 1.7103%) and B (2, 1.6 mg, 9.6105%) together with bisdemethylaaptamine. Fraction III was subjected to Phenyl-Hexyl HPLC (25010 mm ID; MeCN/H2O/ CF3CO2H, 25:75:0.1 to 35:65:0.1; 2.0 mL/min; 280 nm) to give three fractions. Each fraction was purified by C18 HPLC {2504.6 mm ID; MeCN/H2O/CF3CO2H, 21:79:0.1 (3 and 4) and 22:78:0.1 (5); 1.0 mL/min; 280 nm} to afford nakijinamines F (3, 1.6 mg, 9.6105%), G (4, 0.7 mg, 4.2105%), and H (5, 0.4 mg, 2.1105%), respectively. Fraction IV was purified by Phenyl-Hexyl HPLC (25010 mm ID; MeOH/H2O/CF3CO2H, 55:45:0.1; 3.0 mL/min; 300 nm) and then C18 HPLC (2504.6 mm ID; MeCN/H2O/CF3CO2H, 20:80:0.1; 1.0 mL/min; 265 nm) to yield nakijinamines I (6, 1.3 mg, 7.8105%) and CeE (8e10).6 3.3.1. Nakijinamine A (1). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (MeOH) lmax 227 (log 3 4.4), 278 (4.3), 297 (3.9 sh), 352 (3.5), 371 (3.4), and 426 (3.2) nm; IR (film) nmax 3396, 3242, 3078, 3028, 1651, 1615, 1551, 1481, 1229, 813, and 778 cm1; 1H and 13C NMR data (Table 1); ESIMS (pos.) m/z 239/240 (1:1, [MH]2þ) and 477/479 (1:1, [M2H]þ); HRESIMS (pos.) m/z 239.04965 ([MH]2þ, calcd for C24H2379BrN4O2, 239.04967) and 477.09206 ([M2H]þ, calcd for C24H2279BrN4O2, 477.09207). 3.3.2. Nakijinamine B (2). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (MeOH) lmax 217 (log 3 4.2), 239 (3.9), 248 (3.9), 257 (3.9), 317 (3.4), 353 (3.3), and 367 (3.3) nm; IR (film) nmax 3464, 3018, 2906, 1629, 1481, 1230, and 749 cm1; 1H and 13C NMR data (Table 1); ESIMS (pos.) m/z 201 [MþH]2þ and 399 [M2H]þ; HRESIMS (pos.) m/z 201.10237 ([MþH]2þ, calcd for C24H26N4O2, 201.10224) and 399.18181 ([M2H]þ, calcd for C24H23N4O2, 399.18155). 3.3.3. Nakijinamine F (3). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (0.1 M HCl aq, pH 1) lmax 225 (log 3 4.4), 264 (4.2), 295 (3.8 sh), 315 (3.6), 352 (3.5 sh), 375 (3.6), 391 (3.5 sh), and 414 (3.2
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Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550
sh) nm; IR (film) nmax 3393, 3242, 2960, 1650, 1618, 1543, 1478, 1232, and 807 cm1; 1H and 13C NMR data (Table 3); ESIMS (pos.) m/z 282/ 283 (1:1, [MþH]2þ) and 562/564 (1:1, [M]þ); HRESIMS (pos.) m/z 281.59420 ([MþH]2þ, calcd for C29H3479BrN5O2, 281.59425) and 562.18118 ([M]þ, calcd for C29H3379BrN5O2, 562.18121).
(2504.6 mm ID; 1.0 mL/min; 265 nm) with eluent MeCN/H2O/ CF3CO2H (12:88:0.1 for 1; 8:92:0.1 for 2; 12:88:0.1 for 3; 20:80:0.1 for 6) to separate each enantiomer (tR 17.6 and 19.2 min in 1; tR 14.4 and 16.4 min in 2; tR 37.6 and 40.4 min in 3; tR 23.2 and 25.2 min in 6), respectively.
3.3.4. Nakijinamine G (4). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (0.1 M HCl aq, pH 1) lmax 225 (log 3 4.3), 263 (4.1), 295 (3.6 sh), 316 (3.5), 350 (3.4 sh), 376 (3.5), 391 (3.5 sh), and 412 (3.1 sh) nm; IR (film) nmax 3242, 2969, 2865, 1649, 1618, 1543, 1478, 1235, and 804 cm1; 1H and 13C NMR data (Table 3); ESIMS (pos.) m/z 282/ 283 (1:1, [MþH]2þ) and 562/564 (1:1, [M]þ); HRESIMS (pos.) m/z 281.59436 ([MþH]2þ, calcd for C29H3479BrN5O2, 281.59425) and 562.18147 ([M]þ, calcd for C29H3379BrN5O2, 562.18121).
3.3.10. Conformational search for anti- (6a) and syn-nakijinamine I (6b). Conformational searches were carried out using mixed MCMM/Low-Mode conformational search methods10 on Macro€dinger, LLC). Each generated conformers were Model 9.8 (Schro minimized with MMFF94s force field.11
3.3.5. Nakijinamine H (5). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (0.1 M HCl aq, pH 1) lmax 223 (log 3 4.4), 264 (4.2), 294 (3.8 sh), 316 (3.6), 352 (3.5 sh), 375 (3.6), 392 (3.5 sh), and 414 (3.2 sh) nm; IR (film) nmax 3245, 2926, 1650, 1618, 1545, 1479, 1232, and 805 cm1; 1H and 13C NMR (Table 3); ESIMS (pos.) m/z 299/300 (1:1, [MþH]2þ) and 596/598 (1:1, [M]þ); HRESIMS (pos.) m/z 298.58649 ([MþH]2þ, calcd for C32H3279BrN5O2, 298.58642) and 596.16577 ([M]þ, calcd for C32H3179BrN5O2, 596.16556).
We thank Ms. S. Oka, Ms. A. Tokumitsu, and Ms. T. Komiya, Instrumental Analysis Division, Equipment Management Center, Creative Research Institution, Hokkaido University, for measurements of ESIMS; Dr. Eri Fukushi, Graduate School of Agriculture, Hokkaido University and Mr. T. Hirose, Instrumental Analysis Division, Equipment Management Center, Creative Research Institution, Hokkaido University for measurements of a part of NMR spectra; Mr. Z. Nagahama and K. Uehara for their help with collection of the sponge. This work was partly supported by a research fellowship for young scientists from the Japan Society for the Promotion of Science (to Y.T.) and a Grant-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
3.3.6. Nakijinamine I (6). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (0.1 M HCl aq, pH 1) lmax 220 (log 3 4.2), 241 (4.0 sh), 252 (4.0 sh), 260 (4.0), 268 (4.0 sh), 284 (3.6 sh), 293 (3.5 sh), 313 (3.5), 355 (3.2 sh), 385 (3.2), 405 (3.3 sh), and 429 (3.0 sh) nm; IR (film) nmax 3378, 3240, 3151, 3019, 2936, 1658, 1619, 1569, 1437, 1234, and 819 cm1; 1H and 13C NMR data (Table 4); ESIMS (pos.) m/z 239/240 (1:1, [MþH]2þ) and 477/479 (1:1, [M]þ); HRESIMS (pos.) m/z 239.04990 ([MþH]2þ, calcd for C24H2379BrN4O2, 239.04967) and 477.09248 ([M]þ, calcd for C24H2279BrN4O2, 477.09207). 3.3.7. 6-Bromoconicamin (7). Colorless amorphous solid; UV (MeOH) lmax 225 (log 3 4.0), 267 (3.8), and 289 (3.6 sh) nm; IR (film) nmax 3402, 3242, 3028, 2946, 1717, 1660, 1611, 1528, 1453, 1334, 1117, 954, and 810 cm1; 1H NMR (DMSO-d6, 300 K) dH 11.70 (1H, br s, N-1), 7.87 (1H, d, J¼8.6 Hz, H-4), 7.71 (1H, d, J¼2.5 Hz, H-2), 7.66 (1H, br s, H7), 7.30 (1H, d, J¼14.3 Hz, H-20 ), 7.27 (1H, br d, J¼8.6 Hz, H-5), 7.01 (1H, d, J¼14.3 Hz, H-10 ), and 3.37 (9H, s, NeMe3); 13C NMR (DMSO-d6, 300 K) dC 138.0 (C-7a), 131.8 (C-10 ), 129.8 (C-2), 129.8 (C-20 ), 123.4 (C3a), 123.0 (C-5), 121.6 (C-4), 115.0 (C-7), 114.9 (C-6), 107.8 (C-3), and 54.83 (NeMe3); ESIMS (pos.) m/z 279/281 (1:1, [M]þ); HRESIMS (pos.) m/z 279.04975 ([M]þ, calcd for C13H1679BrN2, 279.04914). 3.3.8. Methyl and acetone adducts (1a and 1b) of nakijinamine A (1). A mixture of nakijinamine A (1, 1.7 mg) and methyl iodide (10 mL) in acetone (200 mL) was stirred at room temperature for 18 h in the presence of K2CO3 (23 mg). After filtration, the solvent was evaporated to dryness in vacuo, and the residue was purified by C18 HPLC (2504.6 mm ID; MeCN/H2O/CF3CO2H, 20:80:0.1; 1.0 mL/min; 265 nm) to afford 1a (0.4 mg) and 1b (0.3 mg). Compound 1a: orange-red amorphous solid; 1H and 13C NMR (Supplementary data); ESIMS (pos.) m/z 549/551 (1:1, [M]þ); HRESIMS (pos.) m/z 549.15051 ([M]þ, calcd for C28H3079BrN4O3, 549.14958). Compound 1b: orange-red amorphous solid; 1H and 13C NMR (Supplementary data); ESIMS (pos.) m/z 549/551 (1:1, [M]þ); HRESIMS (pos.) m/z 549.15029 ([M]þ, calcd for C28H3079BrN4O3, 549.14958). 3.3.9. Optical resolution of nakijinamines A (1), B (2), F (3), and I (6). Racemic forms of 1e3 and 6 were analyzed using chiral HPLC
Acknowledgements
Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2012.08.018.
References and notes 1. Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2012, 29, 144e222. 2. Nakamura, H.; Kobayashi, J.; Ohizumi, Y.; Hirata, Y. Tetrahedron Lett. 1982, 23, 5555e5558. 3. (a) Shubina, L. K.; Makarieva, T. N.; Dyshlovoy, S. A.; Fedorov, S. N.; Dmitrenok, P. S.; Stonik, V. A. Nat. Prod. Commun. 2010, 5, 1881e1884; (b) Larghi, E. L.; Bohn, M. L.; Kaufman, T. Tetrahedron 2009, 65, 4257e4282; (c) Utkina, N. K.; Denisenko, V. A.; Pushilin, M. A. Tetrahedron Lett. 2009, 50, 2580e2582; (d) Shubina, L. K.; Kalinovsky, A. I.; Federov, S. N.; Radchenko, O. S.; Denisenko, V. A.; Dmitrenok, P. S.; Dyshlovoy, S. A.; Krasokhin, V. B.; Stonik, V. A. Nat. Prod. Commun. 2009, 4, 1085e1088. 4. (a) Tsukamoto, S.; Yamanokuchi, R.; Yoshitomi, M.; Sato, K.; Ikeda, T.; Rotinsulu, H.; Mangindaan, R. E. P.; de Voogd, N. J.; van Soest, R. W. M.; Yokosawa, H. Bioorg. Med. Chem. Lett. 2010, 20, 3341e3343; (b) Utkina, N. K. Chem. Nat. Compd. 2009, 45, 849e853. 5. (a) Tanaka, N.; Suto, S.; Ishiyama, H.; Kubota, T.; Yamano, A.; Shiro, M.; Fromont, J.; Kobayashi, J. Org. Lett. 2012, 14, 3498e3501; (b) Takahashi, Y.; Iinuma, Y.; Kubota, T.; Tsuda, M.; Sekiguchi, M.; Mikami, Y.; Fromont, J.; Kobayashi, J. Org. Lett. 2011, 13, 628e631; (c) Takahashi, Y.; Kubota, T.; Fromont, J.; Kobayashi, J. Org. Lett. 2009, 11, 21e24. 6. Takahashi, Y.; Kubota, T.; Shibazaki, A.; Gonoi, T.; Fromont, J.; Kobayashi, J. Org. Lett 2011, 13, 3016e3019. 7. (a) Herlt, A.; Mander, L.; Rombang, W.; Rumampuk, R.; Soemitro, S.; Steglich, W.; Tarigan, P.; von Nussbaum, F. Tetrahedron 2004, 60, 6101e6104; (b) von Nussbaum, F.; Schumann, S.; Steglich, W. Tetrahedron 2001, 57, 2331e2335. 8. Kalinowski, H.-O.; Berger, S.; Braun, S. Carbon-13 NMR Spectroscopy; John Wiley & Sons: Chichester, UK, 1988, pp 495e512. 9. Aiello, A.; Borrelli, F.; Capasso, R.; Fattorusso, E.; Luciano, P.; Menna, M. Bioorg. Med. Chem. Lett. 2003, 13, 4481e4483. 10. Kolossv ary, I.; Guida, W. C. J. Comput. Chem. 1999, 20, 1671e1684. 11. Halgren, T. A. J. Comput. Chem. 1999, 20, 720e729.
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Heteroaromatic alkaloids, nakijinamines, from a sponge Suberites sp. Yohei Takahashi a, Naonobu Tanaka a, Takaaki Kubota a, Haruaki Ishiyama a, Azusa Shibazaki b, Tohru Gonoi b, Jane Fromont c, Jun’ichi Kobayashi a, * a
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Medicinal Mycology Research Center, Chiba University, Chiba 260-0856, Japan c Western Australian Museum, Locked Bag 49, Weishpool DC, WA 6986, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2012 Received in revised form 6 August 2012 Accepted 7 August 2012 Available online 15 August 2012
The structures of seven new secondary metabolites isolated from an Okinawan marine sponge Suberites sp., nakijinamines A (1), B (2), and FeI (3e6) and 6-bromoconicamin (7), have been elucidated on the basis of spectroscopic analysis, chemical conversion, and conformational analysis. These analyses disclosed that 1e6 were heteroaromatic alkaloids having the hybrid structures of an aaptamine-type alkaloid and an indole alkaloid, while 7 was a bromoindole alkaloid. Nakijinamine I (6) is the first example of an aaptamine-type alkaloid possessing a 1,4-dioxane ring. Antimicrobial activities of 1e7 were evaluated. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Heteroaromatic alkaloids Marine sponge Suberites sp. Nakijinamines
1. Introduction Marine sponges have been recognized as a rich source of interesting bioactive metabolites with fascinating chemical structures.1 Among them, aaptamine is a representative heteroaromatic alkaloid with a 1H-benzo[de][1,6]naphthyridine ring system.2 Various aaptamine-type alkaloids have been isolated from marine sponges belonging to the genus Aaptos, Suberites, Luffariella, Hymeniacidon, Suberea, and Xestospongia so far.3 A lot of synthetic and structureeactivity relationship studies for these alkaloids have been carried out,3 because of their various biological activities, such as cytotoxic, antimicrobial, antiviral, antioxidant, and enzymatic inhibitory activities.3,4 During our continuing search for secondary metabolites possessing interesting chemical structures from marine sponges,5 we examined the extract of an Okinawan marine sponge Suberites sp. (SS-1084), and isolated a series of new alkaloids, nakijinamines A (1), B (2), CeE (8e10), and FeI (3e6). Recently, we have reported the structure elucidation of nakijinamines CeE (8e10),6 which are heteroaromatic alkaloids possessing the hybrid structures of an aaptamine-type alkaloid and a bromoindole alkaloid. Further investigation for the chemical structures of nakijinamines resulted in the structure elucidation of six new heteroaromatic alkaloids, nakijinamines A (1), B (2), and FeI (3e6). The structure of one new
* Corresponding author. E-mail address: [email protected] (J. Kobayashi). 0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2012.08.018
bromoindole alkaloid, 6-bromoconicamin (7), which was purified in the isolation process of 1e6, was also assigned. In this paper, we describe the isolation and structure elucidation of 1e7.
2. Results and discussion The sponge Suberites sp. (SS-1084, 0.4 kg, wet weight) collected off Unten Port, Nakijin, Okinawa was extracted with MeOH. The extract was partitioned between EtOAc and H2O, and subsequently the aqueous layer was extracted with n-BuOH. The n-BuOH-soluble materials were subjected to a C18 column and a Sephadex LH-20 column followed by reversed-phase HPLC to afford nakijinamines A (1, 1.7103%, wet weight), B (2, 9.6105%), F (3, 9.6105%), G (4, 4.2105%), H (5, 2.1105%), and I (6, 7.8105%) and 6bromoconicamin (7, 1.3104%) together with nakijinamines CeE (8e10)6 and two known aaptamine-type alkaloids, aaptamine2 and bisdemethylaaptamine.7 Nakijinamine A (1) was obtained as a yellow amorphous solid. The molecular formula of 1 was elucidated to be C24H24BrN4O2 {m/z 239.04965 ([MH]2þ, D 0.02 mmu) and 477.09206 ([M2H]þ, D 0.01 mmu)} by the HRESIMS. IR absorptions at 3396 and 3242 cm1 implied the existence of OH and/or NH functionalities. The UV spectrum suggested the occurrence of a conjugated aromatic chromophore. Inspection of the 1H and 13C NMR and the HMQC spectra disclosed the presence of 11 sp2 quaternary carbons, eight sp2 methines, one sp3 methine, one sp3 methylene, and three sp3 methyls (Table 1). The 13C NMR spectrum of 1 was similar to
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Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550
HO
9
1 9a
HN 2
3
8
NMe3
1"
OH
2"
7
2'
1'
4
5
N H
6' 7'a 7'
N H
6
3a
7'
1' 7'a
3'
Me3N
1"
2"
1"'
O
OH
6'
N H
4'
4: R =
2"'
2"' 3"'
4"'
5"'
5"'
2"' 3"'
HN 9: R =
2"'
5
N H
6 7a
Br
7
NMe3
O
N
4"'
4"'
5: R =
6
3"'
1
7
R
5"'
3: R =
N H
2
N H
Br
O
4 3a
3
Br
HN
O
2'
1'
R
5' 3'a
Me3N
NMe3
HN
1: R = Br 2: R = H
HN 2'
5'
3'a
3'
6a
9b
R
4'
Br
HN
N H
N H
6"'
SO3
8: R =
7"' 8"'
N
SO3 10: R =
N O
that of nakijinamine C (8)6 except for the resonances of the side chain at C-8 and C-9 in 8, implying that 1 has the hybrid structure of an aaptamine-type alkaloid and a bromoindole alkaloid. The comparison of 13C NMR data for unit A (N-1eC-9b) in 1 with that for bisdemethylaaptamine7 indicated that 1 has the same 8,9dihydroxy-1H-benzo[de][1,6]naphthyridine ring system as bisdemethylaaptamine. This was supported by a 1He1H COSY cross-peak of H-2/H-3 and HMBC correlations for H-2/C-9a, H-2/C-3a, and H-3/C-9b (Fig. 1). A large 1JCH value of H-5 (182.4 Hz) implied that C-5 is an sp2 methine.6,8 On the other hand, the gross structure of unit B (N-10, C-20 eC-70 a, C-100, and C-200 ) was confirmed by analysis of the 2D NMR spectra (Fig. 1). Although the connectivity of C-200 in unit B to C-6 or C-7 in unit A was assumed by the molecular formula
unit B
OH HO
9
1 9a
HN 2
3
8
7
3a
6 4
N H
unit A
4' 3' 3'a
2''
6a
9b
NMe3
1''
2' 5
1'
N H
5' 6'
7'a 7'
Br 1H-1H
COSY & TOCSY HMBC NOESY
Fig. 1. Selected 2D NMR correlations for nakijinamine A (1). Table 1 1 H (600 MHz) and CD3OD Position
2 3 3a 5 6 6a 7 8 9 9a 9b 20 30 30 a 40 50 60 70 70 a 100 200 NeMe a b
13
C (150 MHz) NMR data for nakijinamines A (1) and B (2) in
1
2
dCa
dHa (J in Hz)
dCa
dHb (J in Hz)
142.5 100.1 152.6 130.2 111.3 129.7 118.1 153.6 130.7 134.3 119.0 125.9 115.4 127.1 121.7 124.1 117.1 116.2 139.7 71.1
7.74, d (7.0) 6.32, d (7.0) d 7.20, br 7.22, br d d d d d d 7.65, s d d 7.35, br 7.02, br d (8.1) d 7.51, d (1.3) d 4.58, br 4.09, br 5.58, br 3.25 (9H), s
142.6 100.0 152.6 130.2 111.5 129.9 119.0 153.5 130.5 134.6 118.6 125.0 115.0 128.2 120.2 121.0 123.8 113.4 138.9 71.2
7.78, d (7.0) 6.34, d (7.0) d 7.20, br 7.20, br d d d d d d 7.61, s d d 7.47, br 6.96, br t (7.7) 7.10, t (7.7) 7.37, d (7.7) d 4.62, br 4.40, br 5.61, br 3.26 (9H), s
31.8 55.2
Recorded at 300 K. Recorded at 315 K.
31.8 55.0
of 1 and the chemical shift of CH-200 (dH 5.58 and dC 31.8), no correlation that might provide an information on the connectivity was observed in the 2D NMR spectra because of its broadening proton signals (Table 1). To elucidate the gross structure of nakijinamine A (1), chemical conversion was carried out as follows. Treatment of 1 with methyl iodide in acetone under basic condition gave two separable derivatives (1a and 1b) (Scheme 1), whose 1H NMR spectra showed the sharp signals. Detailed analysis of the 2D NMR spectra of 1a and 1b suggested that they are stereoisomers of the methyl and acetone adduct of 1 (Supplementary data). Though the relative configurations of C-9 and C-200 in 1a and 1b were not elucidated, the gross structures of them revealed the connectivity of C-7 to C-200 in 1. Accordingly, the gross structure of nakijinamine A was elucidated to be 1. Nakijinamine A (1) gave the broadening signals especially in H5, H-6, H-40 , H2-100, and H-200 in the 1H NMR spectrum (Table 1), suggesting the restriction of C-7eC-200 bond rotation due to the interference by three bulky substituents attached to C-200 .6 The 1H NMR spectrum of 1 measured at 223 K provided two sets of sharp signals with ca. 5:3 proportions (Table 2). These were assigned to be rotamers (1c and 1d, respectively) by analysis of the ROESY spectrum. Therefore, 1 presumed to exist as an equilibrium mixture of two rotamers in CD3OD at 300 K. Nakijinamine B (2) was yielded as a yellow amorphous solid, and the molecular formula, C24H25N4O2, was revealed by the HRESIMS
Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550
8547
Scheme 1. Chemical conversion of nakijinamine A (1) into two stereoisomers (1a and 1b) of the methyl and acetone adduct of 1. Selected 2D NMR correlations for 1a and 1b were shown.
Table 2 1 H (500 MHz) and 13C (125 MHz) NMR dataa for the major (1c) and minor (1d) rotamers of nakijinamine A (1) in CD3OD Position
2 3 3a 5 6 6a 7 8 9 9a 9b 20 30 30 a 40 50 60 70 70 a 100 a 100 b 200 NeMe a b c d e f g h
1c
Position
1d
dC
dH (J in Hz)
dC
dH (J in Hz)
142.2 99.6 152.2 130.3 111.3 129.5c 118.1d 153.2 130.4c 134.9 119.0d 125.2 115.7e 127.1 122.1 123.7 117.1 115.9e 139.7 69.6
7.80, mb 6.30, d (7.1) d 7.13, d (7.4) 6.88, d (7.4) d d d d d d 7.77, s d d 7.14, d (8.5) 6.96, dd (8.5, 1.5) d 7.49, d (1.5) d 4.76, dd (13.5, 4.5) 4.23, dd (13.5, 6.0) 5.84, br s 3.28 (9H), s
142.5 99.6 152.6 131.1 110.9 128.9f 118.2g 155.3 129.9 135.1f 118.7g 125.2 115.8h 127.2 121.9 123.8 126.5 115.9h 139.0 71.6
7.80, mb 6.36, d (7.1) d 7.44, d (7.5) 7.74, mb d d d d d d 7.75, s d d 7.62, d (8.7) 7.12, mb d 7.50, d (1.5) d 4.71, dd (13.6, 6.9) 4.36, dd (13.6, 3.9) 5.23, br s 3.22 (9H), s
30.2 54.3
Table 3 1 H (600 MHz) and 13C (150 MHz) NMR dataa for nakijinamines FeH (3e5) in CD3OD
32.1 54.3
Recorded at 223 K. Coupling constants were not determined due to overlapping of signals. Interchangeable. Interchangeable. Interchangeable. Interchangeable. Interchangeable. Interchangeable.
{m/z 201.10237 ([MþH]2þ, Dþ0.13 mmu) and 399.18181 ([M2H]þ, D þ0.26 mmu)}. Inspection of the 1H and 13C NMR spectra suggested that 2 was structurally related to 1 (Table 1). In addition, 2 showed the signal of one sp2 methine (CH-60 ) in place of the brominated sp2 quaternary carbon in 1. This proton signal was correlated to H-50 and H-70 in the 1He1H COSY spectrum. From these data, the gross structure of nakijinamine B (2) was concluded to be a debromo analog of 1. Nakijinamine F (3) was isolated as a yellow amorphous solid. The molecular formula was assigned to be C29H33BrN5O2 based on the HRESIMS analysis {m/z 281.59420 ([MþH]2þ, D 0.05 mmu) and 562.18118 ([M]þ, D 0.03 mmu)}. The 1H and 13C NMR data for 3 (Table 3) were reminiscent of those for nakijinamine C (8),6 and the signals for a 3-methylbutanamide (C-1000 eC-5000 ) in 3 were discerned in place of the resonances of the substituent at C-8 and C-9 in 8. The presence of the 3-methylbutanamide was confirmed by the 2D NMR analysis (Fig. 2). The positions of the 3methylbutanamide at C-9 and a hydroxy group at C-8 were
3
4
5
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
2
142.5
142.7
101.4
3a 5 6 6a 7 8 9 9a 9b 20 30 30 a 40 50
152.3 132.4 111.2 130.2 118.3 154.2b 124.2 140.2 118.8 126.1 115.3 127.1 121.8 124.2
60 70
117.2 116.2
70 a 100
139.7 70.8
7.92, d (7.0) 6.56, d (7.0) d 7.40, br 7.40, br d d d d d d 7.64, s d d 7.40, br 7.06, br d (8.4) d 7.53, d (1.6) d 4.60, br 4.42, br 5.60, br 3.29 (9H), s d 2.74, m
142.3
3
7.86, d (6.9) 6.53, d (6.9) d 7.40, br 7.40, br d d d d d d 7.64, s d d 7.40, br 7.05, br d (8.3) d 7.52, s
7.89, d (7.0) 6.55, d (7.0) d 7.39, br 7.45, br d d d d d d 7.62, s d d 7.45, br 7.05, br d (8.9) d 7.53, d (1.7) d 4.56, br 4.40, br 5.60, br 3.24 (9H), s d 4.05 (2H), m d
200 NeMe
31.8 55.0
1000 2000
177.4 46.0
3000
27.0
4000
23.9
5000
23.9
d 4.58, br 4.41, br 5.60, br 3.26 (9H), s d 2.60 (2H), d (7.0) 2.29, m 1.11 (3H), d (6.8) 1.11 (3H), d (6.8)
101.5 152.4 132.6 111.3 129.9 118.8 154.6b 124.2 139.7 118.8 126.2 115.3 127.1 121.8 124.2 117.2 116.2 139.0 70.9 31.5 55.1 181.8 44.2 28.8 12.9 28.8
1.96, m 1.64, m 1.09 (3H), t (7.4) 1.37 (3H), d (7.0)
101.2 152.1 132.4 111.1 130.2 118.2 153.4b 124.2 140.3 120.0 125.8 115.3 127.1 121.8 124.2 117.1 116.2 139.7 70.7 31.6 55.0 175.8 43.6 136.7 132.0 130.1
000
6
127.8
7000
130.1
8000
132.0
a b
7.47, d (7.5) 7.39, t (7.5) 7.31, t (7.5) 7.39, t (7.5) 7.47, d (7.5)
Recorded at 315 K. Not observed clearly.
assigned based on the chemical shifts for C-8 (dC 154.2) and C-9 (dC 124.2).6 Thus, the gross structure of nakijinamine F was elucidated to be 3. Nakijinamines G (4) and H (5) were isolated as yellow amorphous solids. The molecular formula of 4, C29H33BrN5O2 {m/z 281.59436 ([MþH]2þ, D þ0.11 mmu) and 562.18147 ([M]þ, D þ0.20 mmu)}, and of 5, C32H31BrN5O2 {m/z 298.58649 ([MþH]2þ, D
8548
Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550 4'''
5'''
2''' 1'''
O
HN
9
OH 8
1'' 2"
7
1 9a
HN
6
9b
2
3a
3
4
N H
2' 5
HN
NMe3
2'
4' 3' 3'a
1'
N H
7'a
Me3N
Br
7'
1H-1H
COSY & TOCSY HMBC NOESY
þ0.07 mmu) and 596.16577 ([M]þ, D þ0.01 mmu)}, were assigned by the HRESIMS analysis. The 1H and 13C NMR spectra (Table 3) suggested that 4 and 5 have similar structures to nakijinamine F (3) with different side chains at C-9. The side chains of 4 and 5 were elucidated to be a 2-methylbutanamide (C-1000 eC-5000 ) and a phenylethanamide (C-1000 eC-8000 ), respectively, by analysis of the 2D NMR spectra (Supplementary data). Thus, the gross structures of nakijinamines G (4) and H (5) were elucidated. Nakijinamine I (6) was obtained as a yellow amorphous solid, and the molecular formula, C24H22BrN4O2, was established by the HRESIMS analysis {m/z 239.04990 ([MþH]2þ, D þ0.23 mmu) and 477.09248 ([M]þ, D þ0.41 mmu)}. The UV spectrum suggested the existence of a conjugated aromatic chromophore. The 1H and 13C NMR spectra (Table 4) exhibited the signals of three D2O-exchangeable protons, 19 aromatic carbons including nine sp2 methines, two sp3 methines, and one N,N,N-trimethylammonio group. Interpretation of the 2D NMR data for 6 indicated the presence of a 8,9-dihydroxy-1H-benzo[de][1,6]naphthyridine ring system (N-1eC-9b) and a 6-bromoindole moiety (N-10 eC-70 a) (Fig. 3). In addition, a 1He1H COSY cross-peak of H-100 /H-200 and HMBC correlations for H-200 /C-30 and 100 -NMe3/C-100 revealed the connectivities of C-200 to C-100 and C-30 , and of C-100 to the N,N,Ntrimethylammonio group. The chemical shifts of CH-100 (dH 6.41; dC 91.2) and CH-200 (dH 6.82; dC 64.6) implied that these sp3 methines were oxygenated. HMBC cross-peaks of H-100 /C-9 and H-200 /C-8
13
1"
O
4'
3'a
2"
O 8 9
1 9a
HN
7
9b 6a 6
2 3
3a
4
N H
5
1H-1H
COSY HMBC ROESY
Fig. 3. Selected 2D NMR correlations of nakijinamine I (6).
revealed the connectivity of C-100 to C-9 and C-200 to C-8 via ether bonds, forming a 2,3-dihydro-1,4-dioxane ring. Thus, the gross structure of nakijinamine I (6) was assigned as shown in Fig. 3. The relative stereochemistry of nakijinamine I (6) was assigned by the conformational search on MacroModel program9 and the ROESY analysis. The conformational search for anti- and syn-nakijinamine I was carried out to give the most stable conformers 6a (anti) and 6b (syn) (Fig. 4). The dihedral angles of H-100 /H-200 in 6a and 6b were 79 and 58 , respectively. In the 1H NMR spectrum of 6, the signals for H-100 and H-200 were observed as both singlet (Table 4), suggesting that the dihedral angle of H-100 /H-200 was close to 90 . It was similar to calculated value of 6a rather than that of 6b. ROESY correlations for H-40 /H-100 and 100 -NMe3/H-200 also implied the anti relationship for H-100 /H-200 . Therefore, the relative stereochemistry of nakijinamine I (6) was concluded to be 6a (Fig. 4).
C (150 MHz) NMR dataa for nakijinamine I (6) in DMSO-d6
Position
dC
dH (J in Hz)
1 2 3 3a 4 5 6 6a 7 8 9 9a 9b 10 20 30 30 a 40 50 60 70 70 a 100 200 NeMe
d 141.5 99.5 149.8 d 129.4 112.1 131.0 105.1 144.8 124.4 129.5 117.2 d 125.9 108.1 124.5 120.7 122.8 115.0 114.6 136.8 91.2 64.6 49.9
13.11, br s 8.07, d (7.3) 6.55, d (7.3) d 12.77, br s 7.34, d (7.4) 6.74, d (7.4) d 6.87, s d d d d 11.60, d (2.5) 7.39, d (2.5) d d 7.83, dd (8.5, 1.7) 7.28, d (8.5) d 7.61, d (1.7) d 6.41, s 6.82, s 3.34 (9H), s
a
5' 3'
6'
6'
7'a
1'
5'
Fig. 2. Selected 2D NMR correlations of nakijinamine F (3).
Table 4 1 H (600 MHz) and
Br
7'
3'''
Recorded at 300 K.
Fig. 4. The most stable conformers for anti- (6a) and syn- (6b) nakijinamine I (protons of methyl groups were omitted).
The molecular formula of 6-bromoconicamin (7), C13H16BrN2, was established by the HRESIMS (m/z 279.04975 [M]þ, D þ0.61 mmu). Analysis of the 1He1H COSY, HMBC, and NOESY spectra (Fig. 5) indicated the existence of a 3,6-disubstituted indole ring, a 1,2-disubstituted olefin, and an N,N,N-trimethylammonio
Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550
8549
13
C chemical shifts, respectively. ESIMS spectra were obtained on a JEOL JMS-T100LC and a Thermo Scientific Exactive spectrometers. HPLC column: C18 (Cosmosil 5C18-PAQ, Nacalai Tesque, Inc.), Phenyl-Hexyl (Luna 5u Phenyl-Hexyl, Phenomenex, Inc.), chiral (CHIRALCEL OD-R, Daicel Chemical Ind., Ltd.). 3.2. Sponge description
Fig. 5. Selected 2D NMR correlations of 6-bromoconicamin (7).
group. HMBC correlations for 10 -NMe3 to C-10 and H-20 to C-2 and C-3a suggested that an N,N,N-trimethylammonio group was connected to C-3 via the 1,2-disubstituted olefin, and therefore a bromine atom was assigned to locate at C-6. The coupling constant of H-10 /H-20 (J¼14.3 Hz) disclosed the geometry of the olefin to be E. Thus, the structure of 6-bromoconicamin (7) was assigned as shown in Fig. 5. Nakijinamines A (1), B (2), and FeI (3e6) were optically inactive, though they have a chiral center (C-200 ). Optical resolution of 1 by chiral HPLC resulted in the separation of two enantiomers, the ratio of which was approximately 1:1. Therefore, nakijinamine A (1) was concluded to be a racemate. Similarly, the enantiomers of nakijinamines B (2), F (3), and I (6) were separated by chiral HPLC, indicating that they are racemates. Although optical resolution of nakijinamines G (4) and H (5) was not successful under any conditions, these compounds were also presumed as being racemates since the biogenetic pathway of 4 and 5 might be similar to that of 1e3. Recently, we reported the isolation and structure elucidation of heteroaromatic alkaloids, nakijinamines CeE (8e10), from the extract of an Okinawan marine sponge Suberites sp. (SS-1084).6 Further investigation for the chemical structure of alkaloids from the extract resulted in the structure elucidation of six new heteroaromatic alkaloids, nakijinamines A (1), B (2), and FeH (3e6), and one new bromoindole alkaloid, 6-bromoconicamin (7). Nakijinamines A (1) and B (2) possess the hybrid structure of bisdemethylaaptamine and 6-bromoconicamin or conicamin.9 Nakijinamines FeH (3e5) are similar hybrid compounds to 1 with a 3-methylbutanamide, a 2-methylbutanamide, and a phenylethanamide moieties, respectively, which seem to be derived from leucine, isoleucine, and phenylalanine. Nakijinamine I (6) is the first example of an aaptamine-type alkaloid having a 1,4-dioxane ring. Nakijinamines A (1), B (2), and FeI (3e6) and 6bromoconicamin (7) were evaluated for their antimicrobial activities. Among the evaluated compounds, 1 exhibited antimicrobial activity against Candida albicans (IC50 0.25 mg/mL), Cryptococcus neoformans (IC50 0.5 mg/mL), Trichophyton mentagrophytes (IC50 0.25 mg/mL), Staphylococcus aureus (MIC 16 mg/mL), Bacillus subtilis (MIC 16 mg/mL), and Micrococcus luteus (MIC 2 mg/mL), while 2 and 3 showed antimicrobial activity against C. albicans (IC50 8 mg/mL, each). On the other hand, 1e7 did not show cytotoxicity (IC50 >10 mg/mL) against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro. 3. Experimental section 3.1. General procedures IR and UV spectra were recorded on a JASCO FT/IR-5300 and a Shimadzu UV-1600PC spectrophotometers, respectively. NMR spectra were measured on a Bruker AMX-600 and a Bruker AMX500 spectrometers. The 2.49 and 3.35 ppm resonances of residual CHD2SOCD3 and CHD2OD, and 39.5 and 49.8 ppm resonances of DMSO-d6 and CD3OD were used as internal references for 1H and
The sponge Suberites sp. (SS-1084, family Suberitidae) was collected off Unten Port, Nakijin, Okinawa, and kept frozen until used. This specimen is a dark brown flattened mound. The skeleton is a dense mass of styles of various sizes: 132015 mm, 97012 mm, 48010 mm. Some styles occur in tracts in the mesohyl. There are no microscleres. The voucher specimen is deposited at the Graduate School of Pharmaceutical Sciences, Hokkaido University. 3.3. Extraction and isolation The sponge (SS-1084, 0.4 kg, wet weight) was extracted with MeOH. The extracts (18.5 g) were partitioned between EtOAc (3500 mL) and H2O (500 mL), and then the aqueous layer was extracted with n-BuOH (3500 mL). A part (1.2 g) of the n-BuOHsoluble materials (5.0 g) was separated by a C18 column chromatography (MeOH/H2O/CF3CO2H) to give four fractions IeIV. Fraction I was applied to gel filtration on Sephadex LH-20 (MeOH), and then subjected to Phenyl-Hexyl HPLC (25010 mm ID; eluent MeCN/ H2O/CF3CO2H, 17:83:0.1 to 50:50:0.1; flow rate 2.0 mL/min; UV detection at 255 nm) to yield 6-bromoconicamin (7, 0.5 mg, 1.3104%, wet weight) and aaptamine. Fraction II was purified by gel filtration on Sephadex LH-20 (MeOH/CF3CO2H, 100:0.1) followed by Phenyl-Hexyl HPLC (25010 mm ID; MeCN/H2O/ CF3CO2H, 20:80:0.2 to 25:75:0.2; 2.0 mL/min; 280 nm) to afford nakijinamines A (1, 28.5 mg, 1.7103%) and B (2, 1.6 mg, 9.6105%) together with bisdemethylaaptamine. Fraction III was subjected to Phenyl-Hexyl HPLC (25010 mm ID; MeCN/H2O/ CF3CO2H, 25:75:0.1 to 35:65:0.1; 2.0 mL/min; 280 nm) to give three fractions. Each fraction was purified by C18 HPLC {2504.6 mm ID; MeCN/H2O/CF3CO2H, 21:79:0.1 (3 and 4) and 22:78:0.1 (5); 1.0 mL/min; 280 nm} to afford nakijinamines F (3, 1.6 mg, 9.6105%), G (4, 0.7 mg, 4.2105%), and H (5, 0.4 mg, 2.1105%), respectively. Fraction IV was purified by Phenyl-Hexyl HPLC (25010 mm ID; MeOH/H2O/CF3CO2H, 55:45:0.1; 3.0 mL/min; 300 nm) and then C18 HPLC (2504.6 mm ID; MeCN/H2O/CF3CO2H, 20:80:0.1; 1.0 mL/min; 265 nm) to yield nakijinamines I (6, 1.3 mg, 7.8105%) and CeE (8e10).6 3.3.1. Nakijinamine A (1). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (MeOH) lmax 227 (log 3 4.4), 278 (4.3), 297 (3.9 sh), 352 (3.5), 371 (3.4), and 426 (3.2) nm; IR (film) nmax 3396, 3242, 3078, 3028, 1651, 1615, 1551, 1481, 1229, 813, and 778 cm1; 1H and 13C NMR data (Table 1); ESIMS (pos.) m/z 239/240 (1:1, [MH]2þ) and 477/479 (1:1, [M2H]þ); HRESIMS (pos.) m/z 239.04965 ([MH]2þ, calcd for C24H2379BrN4O2, 239.04967) and 477.09206 ([M2H]þ, calcd for C24H2279BrN4O2, 477.09207). 3.3.2. Nakijinamine B (2). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (MeOH) lmax 217 (log 3 4.2), 239 (3.9), 248 (3.9), 257 (3.9), 317 (3.4), 353 (3.3), and 367 (3.3) nm; IR (film) nmax 3464, 3018, 2906, 1629, 1481, 1230, and 749 cm1; 1H and 13C NMR data (Table 1); ESIMS (pos.) m/z 201 [MþH]2þ and 399 [M2H]þ; HRESIMS (pos.) m/z 201.10237 ([MþH]2þ, calcd for C24H26N4O2, 201.10224) and 399.18181 ([M2H]þ, calcd for C24H23N4O2, 399.18155). 3.3.3. Nakijinamine F (3). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (0.1 M HCl aq, pH 1) lmax 225 (log 3 4.4), 264 (4.2), 295 (3.8 sh), 315 (3.6), 352 (3.5 sh), 375 (3.6), 391 (3.5 sh), and 414 (3.2
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Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550
sh) nm; IR (film) nmax 3393, 3242, 2960, 1650, 1618, 1543, 1478, 1232, and 807 cm1; 1H and 13C NMR data (Table 3); ESIMS (pos.) m/z 282/ 283 (1:1, [MþH]2þ) and 562/564 (1:1, [M]þ); HRESIMS (pos.) m/z 281.59420 ([MþH]2þ, calcd for C29H3479BrN5O2, 281.59425) and 562.18118 ([M]þ, calcd for C29H3379BrN5O2, 562.18121).
(2504.6 mm ID; 1.0 mL/min; 265 nm) with eluent MeCN/H2O/ CF3CO2H (12:88:0.1 for 1; 8:92:0.1 for 2; 12:88:0.1 for 3; 20:80:0.1 for 6) to separate each enantiomer (tR 17.6 and 19.2 min in 1; tR 14.4 and 16.4 min in 2; tR 37.6 and 40.4 min in 3; tR 23.2 and 25.2 min in 6), respectively.
3.3.4. Nakijinamine G (4). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (0.1 M HCl aq, pH 1) lmax 225 (log 3 4.3), 263 (4.1), 295 (3.6 sh), 316 (3.5), 350 (3.4 sh), 376 (3.5), 391 (3.5 sh), and 412 (3.1 sh) nm; IR (film) nmax 3242, 2969, 2865, 1649, 1618, 1543, 1478, 1235, and 804 cm1; 1H and 13C NMR data (Table 3); ESIMS (pos.) m/z 282/ 283 (1:1, [MþH]2þ) and 562/564 (1:1, [M]þ); HRESIMS (pos.) m/z 281.59436 ([MþH]2þ, calcd for C29H3479BrN5O2, 281.59425) and 562.18147 ([M]þ, calcd for C29H3379BrN5O2, 562.18121).
3.3.10. Conformational search for anti- (6a) and syn-nakijinamine I (6b). Conformational searches were carried out using mixed MCMM/Low-Mode conformational search methods10 on Macro€dinger, LLC). Each generated conformers were Model 9.8 (Schro minimized with MMFF94s force field.11
3.3.5. Nakijinamine H (5). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (0.1 M HCl aq, pH 1) lmax 223 (log 3 4.4), 264 (4.2), 294 (3.8 sh), 316 (3.6), 352 (3.5 sh), 375 (3.6), 392 (3.5 sh), and 414 (3.2 sh) nm; IR (film) nmax 3245, 2926, 1650, 1618, 1545, 1479, 1232, and 805 cm1; 1H and 13C NMR (Table 3); ESIMS (pos.) m/z 299/300 (1:1, [MþH]2þ) and 596/598 (1:1, [M]þ); HRESIMS (pos.) m/z 298.58649 ([MþH]2þ, calcd for C32H3279BrN5O2, 298.58642) and 596.16577 ([M]þ, calcd for C32H3179BrN5O2, 596.16556).
We thank Ms. S. Oka, Ms. A. Tokumitsu, and Ms. T. Komiya, Instrumental Analysis Division, Equipment Management Center, Creative Research Institution, Hokkaido University, for measurements of ESIMS; Dr. Eri Fukushi, Graduate School of Agriculture, Hokkaido University and Mr. T. Hirose, Instrumental Analysis Division, Equipment Management Center, Creative Research Institution, Hokkaido University for measurements of a part of NMR spectra; Mr. Z. Nagahama and K. Uehara for their help with collection of the sponge. This work was partly supported by a research fellowship for young scientists from the Japan Society for the Promotion of Science (to Y.T.) and a Grant-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
3.3.6. Nakijinamine I (6). Yellow amorphous solid; [a]20 D 0 (c 0.15, MeOH); UV (0.1 M HCl aq, pH 1) lmax 220 (log 3 4.2), 241 (4.0 sh), 252 (4.0 sh), 260 (4.0), 268 (4.0 sh), 284 (3.6 sh), 293 (3.5 sh), 313 (3.5), 355 (3.2 sh), 385 (3.2), 405 (3.3 sh), and 429 (3.0 sh) nm; IR (film) nmax 3378, 3240, 3151, 3019, 2936, 1658, 1619, 1569, 1437, 1234, and 819 cm1; 1H and 13C NMR data (Table 4); ESIMS (pos.) m/z 239/240 (1:1, [MþH]2þ) and 477/479 (1:1, [M]þ); HRESIMS (pos.) m/z 239.04990 ([MþH]2þ, calcd for C24H2379BrN4O2, 239.04967) and 477.09248 ([M]þ, calcd for C24H2279BrN4O2, 477.09207). 3.3.7. 6-Bromoconicamin (7). Colorless amorphous solid; UV (MeOH) lmax 225 (log 3 4.0), 267 (3.8), and 289 (3.6 sh) nm; IR (film) nmax 3402, 3242, 3028, 2946, 1717, 1660, 1611, 1528, 1453, 1334, 1117, 954, and 810 cm1; 1H NMR (DMSO-d6, 300 K) dH 11.70 (1H, br s, N-1), 7.87 (1H, d, J¼8.6 Hz, H-4), 7.71 (1H, d, J¼2.5 Hz, H-2), 7.66 (1H, br s, H7), 7.30 (1H, d, J¼14.3 Hz, H-20 ), 7.27 (1H, br d, J¼8.6 Hz, H-5), 7.01 (1H, d, J¼14.3 Hz, H-10 ), and 3.37 (9H, s, NeMe3); 13C NMR (DMSO-d6, 300 K) dC 138.0 (C-7a), 131.8 (C-10 ), 129.8 (C-2), 129.8 (C-20 ), 123.4 (C3a), 123.0 (C-5), 121.6 (C-4), 115.0 (C-7), 114.9 (C-6), 107.8 (C-3), and 54.83 (NeMe3); ESIMS (pos.) m/z 279/281 (1:1, [M]þ); HRESIMS (pos.) m/z 279.04975 ([M]þ, calcd for C13H1679BrN2, 279.04914). 3.3.8. Methyl and acetone adducts (1a and 1b) of nakijinamine A (1). A mixture of nakijinamine A (1, 1.7 mg) and methyl iodide (10 mL) in acetone (200 mL) was stirred at room temperature for 18 h in the presence of K2CO3 (23 mg). After filtration, the solvent was evaporated to dryness in vacuo, and the residue was purified by C18 HPLC (2504.6 mm ID; MeCN/H2O/CF3CO2H, 20:80:0.1; 1.0 mL/min; 265 nm) to afford 1a (0.4 mg) and 1b (0.3 mg). Compound 1a: orange-red amorphous solid; 1H and 13C NMR (Supplementary data); ESIMS (pos.) m/z 549/551 (1:1, [M]þ); HRESIMS (pos.) m/z 549.15051 ([M]þ, calcd for C28H3079BrN4O3, 549.14958). Compound 1b: orange-red amorphous solid; 1H and 13C NMR (Supplementary data); ESIMS (pos.) m/z 549/551 (1:1, [M]þ); HRESIMS (pos.) m/z 549.15029 ([M]þ, calcd for C28H3079BrN4O3, 549.14958). 3.3.9. Optical resolution of nakijinamines A (1), B (2), F (3), and I (6). Racemic forms of 1e3 and 6 were analyzed using chiral HPLC
Acknowledgements
Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2012.08.018.
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