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Tetrahedron 68 (2012) 8545e8550

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Tetrahedron

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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,*aGraduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, JapanbMedicinal Mycology Research Center, Chiba University, Chiba 260-0856, JapancWestern Australian Museum, Locked Bag 49, Weishpool DC, WA 6986, Australia

a r t i c l e i n f o

Article history:Received 13 July 2012Received in revised form 6 August 2012Accepted 7 August 2012Available online 15 August 2012

Keywords:Heteroaromatic alkaloidsMarine spongeSuberites sp.Nakijinamines

* Correspondingauthor. E-mail address: jkobay@phar

0040-4020/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.tet.2012.08.018

a b s t r a c t

The structures of seven new secondary metabolites isolated from an Okinawan marine sponge Suberitessp., nakijinamines A (1), B (2), and FeI (3e6) and 6-bromoconicamin (7), have been elucidated on thebasis of spectroscopic analysis, chemical conversion, and conformational analysis. These analyses dis-closed that 1e6 were heteroaromatic alkaloids having the hybrid structures of an aaptamine-type al-kaloid and an indole alkaloid, while 7 was a bromoindole alkaloid. Nakijinamine I (6) is the first exampleof an aaptamine-type alkaloid possessing a 1,4-dioxane ring. Antimicrobial activities of 1e7 wereevaluated.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Marine sponges have been recognized as a rich source of in-teresting bioactive metabolites with fascinating chemical struc-tures.1 Among them, aaptamine is a representative heteroaromaticalkaloid with a 1H-benzo[de][1,6]naphthyridine ring system.2

Various aaptamine-type alkaloids have been isolated from marinesponges belonging to the genus Aaptos, Suberites, Luffariella,Hymeniacidon, Suberea, and Xestospongia so far.3 A lot of syntheticand structureeactivity relationship studies for these alkaloids havebeen carried out,3 because of their various biological activities, suchas cytotoxic, antimicrobial, antiviral, antioxidant, and enzymaticinhibitory activities.3,4

During our continuing search for secondary metabolites pos-sessing interesting chemical structures from marine sponges,5 weexamined 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 reportedthe structure elucidation of nakijinamines CeE (8e10),6 which areheteroaromatic alkaloids possessing the hybrid structures of anaaptamine-type alkaloid and a bromoindole alkaloid. Further in-vestigation for the chemical structures of nakijinamines resulted inthe structure elucidation of six new heteroaromatic alkaloids,nakijinamines A (1), B (2), and FeI (3e6). The structure of one new

m.hokudai.ac.jp (J. Kobayashi).

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bromoindole alkaloid, 6-bromoconicamin (7), which was purifiedin the isolation process of 1e6, was also assigned. In this paper, wedescribe the isolation and structure elucidation of 1e7.

2. Results and discussion

The sponge Suberites sp. (SS-1084, 0.4 kg, wet weight) collectedoff Unten Port, Nakijin, Okinawa was extracted with MeOH. Theextract was partitioned between EtOAc and H2O, and subsequentlythe aqueous layer was extracted with n-BuOH. The n-BuOH-solublematerials were subjected to a C18 column and a Sephadex LH-20column followed by reversed-phase HPLC to afford nakijinaminesA (1, 1.7�10�3%, wet weight), B (2, 9.6�10�5%), F (3, 9.6�10�5%), G(4, 4.2�10�5%), H (5, 2.1�10�5%), and I (6, 7.8�10�5%) and 6-bromoconicamin (7, 1.3�10�4%) together with nakijinamines CeE(8e10)6 and two known aaptamine-type alkaloids, aaptamine2 andbisdemethylaaptamine.7

Nakijinamine A (1) was obtained as a yellow amorphous solid.Themolecular formula of 1was elucidated to be C24H24BrN4O2 {m/z239.04965 ([M�H]2þ, D �0.02 mmu) and 477.09206 ([M�2H]þ, D�0.01 mmu)} by the HRESIMS. IR absorptions at 3396 and3242 cm�1 implied the existence of OH and/or NH functionalities.The UV spectrum suggested the occurrence of a conjugated aro-matic chromophore. Inspection of the 1H and 13C NMR and theHMQC spectra disclosed the presence of 11 sp2 quaternary carbons,eight sp2 methines, one sp3 methine, one sp3 methylene, and threesp3 methyls (Table 1). The 13C NMR spectrum of 1 was similar to

HN

NH

OHHO

NMe3

NH

Br1

4

79 3'

1'

1''

2''

6

33a

9a9b

3'a

7'a

4'

7'

5

8

unit A

unit B

6'

2

5'

2'6a

NOESY

1H-1H COSY

HMBC& TOCSY

HN

NH

NH

OHHO

NMe3

HN

NH

NH

Br

NMe3ON

R

HN

NH

NH

Br

NMe3OHHN

OR

7

3: R =

4: R =

5: R =

9: R =

6

SO3

1: R = Br2: R = H

8: R =

10: R =

SO3

ON

N

R

HN

NH

OO

Me3N

HN

Br

NH

Br

98

7

6

54

32

9a1

3a

9b 6a

1"

2"

7'

6'

5'4'

3'2' 1'

3'a

7'a

1'

2'

7

6

54

3

2 1

3a

7a

1"2"

7' 6'

5'

4'3'

2'

1'

3'a

7'a

1"'

2"'3"'

4"'

5"'6"'

7"'8"'

2"' 3"'

4"'

5"'

2"' 3"' 4"'

5"'

2"'

Me3N

Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e85508546

that of nakijinamine C (8)6 except for the resonances of the sidechain at C-8 and C-9 in 8, implying that 1 has the hybrid structure ofan aaptamine-type alkaloid and a bromoindole alkaloid. The com-parison of 13C NMR data for unit A (N-1eC-9b) in 1 with that forbisdemethylaaptamine7 indicated that 1 has the same 8,9-dihydroxy-1H-benzo[de][1,6]naphthyridine ring system as bisde-methylaaptamine. This was supported by a 1He1H COSY cross-peakof H-2/H-3 and HMBC correlations for H-2/C-9a, H-2/C-3a, andH-3/C-9b (Fig. 1). A large 1JCH value of H-5 (182.4 Hz) implied thatC-5 is an sp2 methine.6,8 On the other hand, the gross structure ofunit B (N-10, C-20eC-70a, C-100, and C-200) was confirmed by analysisof the 2D NMR spectra (Fig. 1). Although the connectivity of C-200 inunit B to C-6 or C-7 in unit Awas assumed by themolecular formula

Table 11H (600 MHz) and 13C (150 MHz) NMR data for nakijinamines A (1) and B (2) inCD3OD

Position 1 2

dCa dH

a (J in Hz) dCa dH

b (J in Hz)

2 142.5 7.74, d (7.0) 142.6 7.78, d (7.0)3 100.1 6.32, d (7.0) 100.0 6.34, d (7.0)3a 152.6 d 152.6 d

5 130.2 7.20, br 130.2 7.20, br6 111.3 7.22, br 111.5 7.20, br6a 129.7 d 129.9 d

7 118.1 d 119.0 d

8 153.6 d 153.5 d

9 130.7 d 130.5 d

9a 134.3 d 134.6 d

9b 119.0 d 118.6 d

20 125.9 7.65, s 125.0 7.61, s30 115.4 d 115.0 d

30a 127.1 d 128.2 d

40 121.7 7.35, br 120.2 7.47, br50 124.1 7.02, br d (8.1) 121.0 6.96, br t (7.7)60 117.1 d 123.8 7.10, t (7.7)70 116.2 7.51, d (1.3) 113.4 7.37, d (7.7)70a 139.7 d 138.9 d

100 71.1 4.58, br 71.2 4.62, br4.09, br 4.40, br

200 31.8 5.58, br 31.8 5.61, brNeMe 55.2 3.25 (9H), s 55.0 3.26 (9H), s

a Recorded at 300 K.b Recorded at 315 K.

Fig. 1. Selected 2D NMR correlations for nakijinamine A (1).

of 1 and the chemical shift of CH-200 (dH 5.58 and dC 31.8), no cor-relation that might provide an information on the connectivity wasobserved in the 2D NMR spectra because of its broadening protonsignals (Table 1).

To elucidate the gross structure of nakijinamine A (1), chemicalconversion was carried out as follows. Treatment of 1 with methyliodide in acetone under basic condition gave two separable de-rivatives (1a and 1b) (Scheme 1), whose 1H NMR spectra showedthe sharp signals. Detailed analysis of the 2D NMR spectra of 1a and1b suggested that they are stereoisomers of the methyl and acetoneadduct of 1 (Supplementary data). Though the relative configura-tions of C-9 and C-200 in 1a and 1b were not elucidated, the grossstructures of them revealed the connectivity of C-7 to C-200 in 1.Accordingly, the gross structure of nakijinamine Awas elucidated tobe 1.

Nakijinamine A (1) gave the broadening signals especially in H-5, 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 theinterference by three bulky substituents attached to C-200.6 The 1HNMR spectrum of 1 measured at 223 K provided two sets of sharpsignals with ca. 5:3 proportions (Table 2). Thesewere assigned to berotamers (1c and 1d, respectively) by analysis of the ROESY spec-trum. Therefore, 1 presumed to exist as an equilibrium mixture oftwo rotamers in CD3OD at 300 K.

Nakijinamine B (2) was yielded as a yellowamorphous solid, andthe molecular formula, C24H25N4O2, was revealed by the HRESIMS

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 wereshown.

Table 21H (500 MHz) and 13C (125 MHz) NMR dataa for the major (1c) and minor (1d) ro-tamers of nakijinamine A (1) in CD3OD

Position 1c 1d

dC dH (J in Hz) dC dH (J in Hz)

2 142.2 7.80, mb 142.5 7.80, mb

3 99.6 6.30, d (7.1) 99.6 6.36, d (7.1)3a 152.2 d 152.6 d

5 130.3 7.13, d (7.4) 131.1 7.44, d (7.5)6 111.3 6.88, d (7.4) 110.9 7.74, mb

6a 129.5c d 128.9f d

7 118.1d d 118.2g d

8 153.2 d 155.3 d

9 130.4c d 129.9 d

9a 134.9 d 135.1f d

9b 119.0d d 118.7g d

20 125.2 7.77, s 125.2 7.75, s30 115.7e d 115.8h d

30a 127.1 d 127.2 d

40 122.1 7.14, d (8.5) 121.9 7.62, d (8.7)50 123.7 6.96, dd (8.5, 1.5) 123.8 7.12, mb

60 117.1 d 126.5 d

70 115.9e 7.49, d (1.5) 115.9h 7.50, d (1.5)70a 139.7 d 139.0 d

100a 69.6 4.76, dd (13.5, 4.5) 71.6 4.71, dd (13.6, 6.9)100b 4.23, dd (13.5, 6.0) 4.36, dd (13.6, 3.9)200 30.2 5.84, br s 32.1 5.23, br sNeMe 54.3 3.28 (9H), s 54.3 3.22 (9H), s

a Recorded at 223 K.b Coupling constants were not determined due to overlapping of signals.c Interchangeable.d Interchangeable.e Interchangeable.f Interchangeable.g Interchangeable.h Interchangeable.

Table 31H (600 MHz) and 13C (150 MHz) NMR dataa for nakijinamines FeH (3e5) in CD3OD

Position 3 4 5

dC dH (J in Hz) dC dH (J in Hz) dC dH (J in Hz)

2 142.5 7.86,d (6.9)

142.7 7.92,d (7.0)

142.3 7.89,d (7.0)

3 101.4 6.53,d (6.9)

101.5 6.56,d (7.0)

101.2 6.55,d (7.0)

3a 152.3 d 152.4 d 152.1 d

5 132.4 7.40, br 132.6 7.40, br 132.4 7.39, br6 111.2 7.40, br 111.3 7.40, br 111.1 7.45, br6a 130.2 d 129.9 d 130.2 d

7 118.3 d 118.8 d 118.2 d

8 154.2b d 154.6b d 153.4b d

9 124.2 d 124.2 d 124.2 d

9a 140.2 d 139.7 d 140.3 d

9b 118.8 d 118.8 d 120.0 d

20 126.1 7.64, s 126.2 7.64, s 125.8 7.62, s30 115.3 d 115.3 d 115.3 d

30a 127.1 d 127.1 d 127.1 d

40 121.8 7.40, br 121.8 7.40, br 121.8 7.45, br50 124.2 7.05,

br d (8.3)124.2 7.06,

br d (8.4)124.2 7.05,

br d (8.9)60 117.2 d 117.2 d 117.1 d

70 116.2 7.52, s 116.2 7.53,d (1.6)

116.2 7.53,d (1.7)

70a 139.7 d 139.0 d 139.7 d

100 70.8 4.58, br 70.9 4.60, br 70.7 4.56, br4.41, br 4.42, br 4.40, br

200 31.8 5.60, br 31.5 5.60, br 31.6 5.60, brNeMe 55.0 3.26

(9H), s55.1 3.29

(9H), s55.0 3.24

(9H), s1000 177.4 d 181.8 d 175.8 d

2000 46.0 2.60 (2H),d (7.0)

44.2 2.74, m 43.6 4.05(2H), m

3000 27.0 2.29, m 28.8 1.96, m 136.7 d

1.64, m4000 23.9 1.11 (3H),

d (6.8)12.9 1.09 (3H),

t (7.4)132.0 7.47,

d (7.5)5000 23.9 1.11 (3H),

d (6.8)28.8 1.37 (3H),

d (7.0)130.1 7.39,

t (7.5)6000 127.8 7.31,

t (7.5)7000 130.1 7.39,

t (7.5)8000 132.0 7.47,

d (7.5)

a Recorded at 315 K.b Not observed clearly.

Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550 8547

{m/z 201.10237 ([MþH]2þ, Dþ0.13 mmu) and 399.18181 ([M�2H]þ,D þ0.26 mmu)}. Inspection of the 1H and 13C NMR spectra sug-gested that 2 was structurally related to 1 (Table 1). In addition, 2showed the signal of one sp2 methine (CH-60) in place of the bro-minated sp2 quaternary carbon in 1. This proton signal was corre-lated to H-50 and H-70 in the 1He1H COSY spectrum. From thesedata, the gross structure of nakijinamine B (2) was concluded to bea debromo analog of 1.

Nakijinamine F (3) was isolated as a yellow amorphous solid.The molecular formula was assigned to be C29H33BrN5O2 based onthe 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 for3 (Table 3) were reminiscent of those for nakijinamine C (8),6 andthe signals for a 3-methylbutanamide (C-1000eC-5000) in 3 were dis-cerned in place of the resonances of the substituent at C-8 and C-9in 8. The presence of the 3-methylbutanamide was confirmed bythe 2D NMR analysis (Fig. 2). The positions of the 3-methylbutanamide at C-9 and a hydroxy group at C-8 were

assigned based on the chemical shifts for C-8 (dC 154.2) and C-9 (dC124.2).6 Thus, the gross structure of nakijinamine F was elucidatedto be 3.

Nakijinamines G (4) and H (5) were isolated as yellow amor-phous solids. The molecular formula of 4, C29H33BrN5O2 {m/z281.59436 ([MþH]2þ, D þ0.11 mmu) and 562.18147 ([M]þ, Dþ0.20 mmu)}, and of 5, C32H31BrN5O2 {m/z 298.58649 ([MþH]2þ, D

HN

NH

OO

Me3N

HN

Br

1"2"

7'6'

5'4'

3'

2'1'

3'a

7'a

9

87

6

54

32

9a1

3a

9b 6a

ROESY

1H-1H COSYHMBC

Fig. 3. Selected 2D NMR correlations of nakijinamine I (6).

HN

NH

OHHN

NMe3

NH

2"

Br

O

1

4

79

6

33a

9a

9b

5

8

2

3'

1'

3'a

7'a

4'

7'

6'

5'1''

2'

1'''2'''

3'''

4''' 5'''

NOESY

1H-1H COSY

HMBC& TOCSY

Fig. 2. Selected 2D NMR correlations of nakijinamine F (3).

Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e85508548

þ0.07 mmu) and 596.16577 ([M]þ, D þ0.01 mmu)}, were assignedby 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 wereelucidated to be a 2-methylbutanamide (C-1000eC-5000) and a phe-nylethanamide (C-1000eC-8000), respectively, by analysis of the 2DNMR spectra (Supplementary data). Thus, the gross structures ofnakijinamines 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 theHRESIMS analysis {m/z 239.04990 ([MþH]2þ, D þ0.23 mmu) and477.09248 ([M]þ, D þ0.41 mmu)}. The UV spectrum suggested theexistence of a conjugated aromatic chromophore. The 1H and 13CNMR spectra (Table 4) exhibited the signals of three D2O-ex-changeable protons, 19 aromatic carbons including nine sp2

methines, two sp3 methines, and one N,N,N-trimethylammoniogroup. Interpretation of the 2D NMR data for 6 indicated thepresence of a 8,9-dihydroxy-1H-benzo[de][1,6]naphthyridine ringsystem (N-1eC-9b) and a 6-bromoindole moiety (N-10eC-70a)(Fig. 3). In addition, a 1He1H COSY cross-peak of H-100/H-200 andHMBC correlations for H-200/C-30 and 100-NMe3/C-100 revealed theconnectivities of C-200 to C-100 and C-30, and of C-100 to the N,N,N-trimethylammonio group. The chemical shifts of CH-100 (dH 6.41; dC91.2) and CH-200 (dH 6.82; dC 64.6) implied that these sp3 methineswere oxygenated. HMBC cross-peaks of H-100/C-9 and H-200/C-8

Table 41H (600 MHz) and 13C (150 MHz) NMR dataa for nakijinamine I (6) in DMSO-d6

Position dC dH (J in Hz)

1 d 13.11, br s2 141.5 8.07, d (7.3)3 99.5 6.55, d (7.3)3a 149.8 d

4 d 12.77, br s5 129.4 7.34, d (7.4)6 112.1 6.74, d (7.4)6a 131.0 d

7 105.1 6.87, s8 144.8 d

9 124.4 d

9a 129.5 d

9b 117.2 d

10 d 11.60, d (2.5)20 125.9 7.39, d (2.5)30 108.1 d

30a 124.5 d

40 120.7 7.83, dd (8.5, 1.7)50 122.8 7.28, d (8.5)60 115.0 d

70 114.6 7.61, d (1.7)70a 136.8 d

100 91.2 6.41, s200 64.6 6.82, sNeMe 49.9 3.34 (9H), s

a Recorded at 300 K.

revealed the connectivity of C-100 to C-9 and C-200 to C-8 via etherbonds, forming a 2,3-dihydro-1,4-dioxane ring. Thus, the grossstructure of nakijinamine I (6) was assigned as shown in Fig. 3.

The relative stereochemistry of nakijinamine I (6) was assignedby the conformational search on MacroModel program9 and theROESY analysis. The conformational search for anti- and syn-naki-jinamine 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 6aand 6bwere 79� and 58�, respectively. In the 1HNMR spectrum of 6,the signals for H-100 and H-200 were observed as both singlet (Table4), 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. ROESYcorrelations for H-40/H-100 and 100-NMe3/H-200 also implied the antirelationship for H-100/H-200. Therefore, the relative stereochemistryof nakijinamine I (6) was concluded to be 6a (Fig. 4).

Fig. 4. The most stable conformers for anti- (6a) and syn- (6b) nakijinamine I (protonsof methyl groups were omitted).

The molecular formula of 6-bromoconicamin (7), C13H16BrN2,was established by the HRESIMS (m/z 279.04975 [M]þ, Dþ0.61 mmu). Analysis of the 1He1H COSY, HMBC, and NOESYspectra (Fig. 5) indicated the existence of a 3,6-disubstituted indolering, a 1,2-disubstituted olefin, and an N,N,N-trimethylammonio

Fig. 5. Selected 2D NMR correlations of 6-bromoconicamin (7).

Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e8550 8549

group. HMBC correlations for 10-NMe3 to C-10 and H-20 to C-2 andC-3a suggested that an N,N,N-trimethylammonio group was con-nected to C-3 via the 1,2-disubstituted olefin, and therefore a bro-mine atom was assigned to locate at C-6. The coupling constant ofH-10/H-20 (J¼14.3 Hz) disclosed the geometry of the olefin to be E.Thus, the structure of 6-bromoconicamin (7) was assigned asshown 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 bychiral HPLC resulted in the separation of two enantiomers, the ratioof which was approximately 1:1. Therefore, nakijinamine A (1) wasconcluded to be a racemate. Similarly, the enantiomers of nakijin-amines B (2), F (3), and I (6) were separated by chiral HPLC, in-dicating that they are racemates. Although optical resolution ofnakijinamines G (4) and H (5) was not successful under any con-ditions, these compounds were also presumed as being racematessince the biogenetic pathway of 4 and 5 might be similar to that of1e3.

Recently, we reported the isolation and structure elucidation ofheteroaromatic alkaloids, nakijinamines CeE (8e10), from the ex-tract of an Okinawan marine sponge Suberites sp. (SS-1084).6 Fur-ther investigation for the chemical structure of alkaloids from theextract resulted in the structure elucidation of six new hetero-aromatic alkaloids, nakijinamines A (1), B (2), and FeH (3e6), andone new bromoindole alkaloid, 6-bromoconicamin (7). Nakijin-amines A (1) and B (2) possess the hybrid structure of bisdeme-thylaaptamine and 6-bromoconicamin or conicamin.9

Nakijinamines FeH (3e5) are similar hybrid compounds to 1 witha 3-methylbutanamide, a 2-methylbutanamide, and a phenyl-ethanamide moieties, respectively, which seem to be derived fromleucine, isoleucine, and phenylalanine. Nakijinamine I (6) is the firstexample of an aaptamine-type alkaloid having a 1,4-dioxane ring.

Nakijinamines A (1), B (2), and FeI (3e6) and 6-bromoconicamin (7) were evaluated for their antimicrobial activi-ties. Among the evaluated compounds, 1 exhibited antimicrobialactivity against Candida albicans (IC50 0.25 mg/mL), Cryptococcusneoformans (IC50 0.5 mg/mL), Trichophyton mentagrophytes (IC500.25 mg/mL), Staphylococcus aureus (MIC 16 mg/mL), Bacillus subtilis(MIC 16 mg/mL), and Micrococcus luteus (MIC 2 mg/mL), while 2 and3 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 epider-moid carcinoma KB cells in vitro.

3. Experimental section

3.1. General procedures

IR and UV spectra were recorded on a JASCO FT/IR-5300 anda Shimadzu UV-1600PC spectrophotometers, respectively. NMRspectra were measured on a Bruker AMX-600 and a Bruker AMX-500 spectrometers. The 2.49 and 3.35 ppm resonances of residualCHD2SOCD3 and CHD2OD, and 39.5 and 49.8 ppm resonances ofDMSO-d6 and CD3OD were used as internal references for 1H and

13C chemical shifts, respectively. ESIMS spectra were obtained ona 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

The sponge Suberites sp. (SS-1084, family Suberitidae) was col-lected off Unten Port, Nakijin, Okinawa, and kept frozen until used.This specimen is a dark brown flattened mound. The skeleton isa dense mass of styles of various sizes: 1320�15 mm, 970�12 mm,480�10 mm. Some styles occur in tracts in the mesohyl. There areno microscleres. The voucher specimen is deposited at the Gradu-ate School of Pharmaceutical Sciences, Hokkaido University.

3.3. Extraction and isolation

The sponge (SS-1084, 0.4 kg, wet weight) was extracted withMeOH. The extracts (18.5 g) were partitioned between EtOAc(3�500 mL) and H2O (500 mL), and then the aqueous layer wasextracted with n-BuOH (3�500 mL). A part (1.2 g) of the n-BuOH-soluble materials (5.0 g) was separated by a C18 column chroma-tography (MeOH/H2O/CF3CO2H) to give four fractions IeIV. FractionIwas applied to gel filtration on Sephadex LH-20 (MeOH), and thensubjected to Phenyl-Hexyl HPLC (250�10 mm ID; eluent MeCN/H2O/CF3CO2H, 17:83:0.1 to 50:50:0.1; flow rate 2.0 mL/min; UVdetection at 255 nm) to yield 6-bromoconicamin (7, 0.5 mg,1.3�10�4%, wet weight) and aaptamine. Fraction II was purified bygel filtration on Sephadex LH-20 (MeOH/CF3CO2H, 100:0.1) fol-lowed by Phenyl-Hexyl HPLC (250�10 mm ID; MeCN/H2O/CF3CO2H, 20:80:0.2 to 25:75:0.2; 2.0 mL/min; 280 nm) to affordnakijinamines A (1, 28.5 mg, 1.7�10�3%) and B (2, 1.6 mg,9.6�10�5%) together with bisdemethylaaptamine. Fraction III wassubjected to Phenyl-Hexyl HPLC (250�10 mm ID; MeCN/H2O/CF3CO2H, 25:75:0.1 to 35:65:0.1; 2.0 mL/min; 280 nm) to give threefractions. Each fraction was purified by C18 HPLC {250�4.6 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,9.6�10�5%), G (4, 0.7 mg, 4.2�10�5%), and H (5, 0.4 mg, 2.1�10�5%),respectively. Fraction IV was purified by Phenyl-Hexyl HPLC(250�10 mm ID; MeOH/H2O/CF3CO2H, 55:45:0.1; 3.0 mL/min;300 nm) and then C18 HPLC (250�4.6 mm ID; MeCN/H2O/CF3CO2H,20:80:0.1; 1.0 mL/min; 265 nm) to yield nakijinamines I (6, 1.3 mg,7.8�10�5%) and CeE (8e10).6

3.3.1. Nakijinamine A (1). Yellow amorphous solid; [a]D20 �0 (c 0.15,MeOH); UV (MeOH) lmax 227 (log 34.4), 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 cm�1; 1H and 13CNMR data (Table 1); ESIMS (pos.) m/z 239/240 (1:1, [M�H]2þ) and477/479 (1:1, [M�2H]þ); HRESIMS (pos.)m/z 239.04965 ([M�H]2þ,calcd for C24H23

79BrN4O2, 239.04967) and 477.09206 ([M�2H]þ,calcd for C24H22

79BrN4O2, 477.09207).

3.3.2. Nakijinamine B (2). Yellow amorphous solid; [a]D20�0 (c 0.15,MeOH); UV (MeOH) lmax 217 (log 34.2), 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 cm�1; 1H and 13C NMR data (Table1); ESIMS (pos.) m/z 201 [MþH]2þ and 399 [M�2H]þ; HRESIMS(pos.) m/z 201.10237 ([MþH]2þ, calcd for C24H26N4O2, 201.10224)and 399.18181 ([M�2H]þ, calcd for C24H23N4O2, 399.18155).

3.3.3. Nakijinamine F (3). Yellow amorphous solid; [a]D20 �0 (c 0.15,MeOH); UV (0.1 M HCl aq, pH 1) lmax 225 (log 34.4), 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

Y. Takahashi et al. / Tetrahedron 68 (2012) 8545e85508550

sh) nm; IR (film) nmax 3393, 3242, 2960,1650,1618,1543,1478,1232,and 807 cm�1; 1H and 13CNMRdata (Table 3); ESIMS (pos.)m/z 282/283 (1:1, [MþH]2þ) and 562/564 (1:1, [M]þ); HRESIMS (pos.) m/z281.59420 ([MþH]2þ, calcd for C29H34

79BrN5O2, 281.59425) and562.18118 ([M]þ, calcd for C29H33

79BrN5O2, 562.18121).

3.3.4. Nakijinamine G (4). Yellowamorphous solid; [a]D20�0 (c 0.15,MeOH); UV (0.1 M HCl aq, pH 1) lmax 225 (log 34.3), 263 (4.1), 295(3.6 sh), 316 (3.5), 350 (3.4 sh), 376 (3.5), 391 (3.5 sh), and 412 (3.1sh) nm; IR (film) nmax 3242, 2969, 2865,1649,1618,1543,1478,1235,and 804 cm�1; 1H and 13CNMRdata (Table 3); ESIMS (pos.)m/z 282/283 (1:1, [MþH]2þ) and 562/564 (1:1, [M]þ); HRESIMS (pos.) m/z281.59436 ([MþH]2þ, calcd for C29H34

79BrN5O2, 281.59425) and562.18147 ([M]þ, calcd for C29H33

79BrN5O2, 562.18121).

3.3.5. Nakijinamine H (5). Yellowamorphous solid; [a]D20�0 (c 0.15,MeOH); UV (0.1 M HCl aq, pH 1) lmax 223 (log 34.4), 264 (4.2), 294(3.8 sh), 316 (3.6), 352 (3.5 sh), 375 (3.6), 392 (3.5 sh), and 414 (3.2sh) nm; IR (film) nmax 3245, 2926, 1650, 1618, 1545, 1479, 1232, and805 cm�1; 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/z298.58649 ([MþH]2þ, calcd for C32H32

79BrN5O2, 298.58642) and596.16577 ([M]þ, calcd for C32H31

79BrN5O2, 596.16556).

3.3.6. Nakijinamine I (6). Yellow amorphous solid; [a]D20 �0 (c 0.15,MeOH); UV (0.1 MHCl aq, pH 1) lmax 220 (log 34.2), 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, and819 cm�1; 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/z239.04990 ([MþH]2þ, calcd for C24H23

79BrN4O2, 239.04967) and477.09248 ([M]þ, calcd for C24H22

79BrN4O2, 477.09207).

3.3.7. 6-Bromoconicamin (7). Colorless amorphous solid; UV(MeOH) lmax 225 (log 34.0), 267 (3.8), and 289 (3.6 sh) nm; IR (film)nmax 3402, 3242, 3028, 2946, 1717, 1660, 1611, 1528, 1453, 1334, 1117,954, and810 cm�1; 1HNMR (DMSO-d6, 300 K) dH11.70 (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,H-7), 7.30 (1H, d, J¼14.3 Hz,H-20), 7.27 (1H, brd, 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 (C-3a), 123.0 (C-5), 121.6 (C-4), 115.0 (C-7), 114.9 (C-6), 107.8 (C-3), and54.8�3 (NeMe3); ESIMS (pos.) m/z 279/281 (1:1, [M]þ); HRESIMS(pos.) m/z 279.04975 ([M]þ, calcd for C13H16

79BrN2, 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 hin the presence of K2CO3 (23 mg). After filtration, the solvent wasevaporated to dryness in vacuo, and the residue was purified by C18HPLC (250�4.6 mmID;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 (Supplementarydata); ESIMS (pos.) m/z 549/551 (1:1, [M]þ); HRESIMS (pos.) m/z549.15051 ([M]þ, calcd for C28H30

79BrN4O3, 549.14958). Compound1b: orange-red amorphous solid; 1H and 13C NMR (Supplementarydata); ESIMS (pos.) m/z 549/551 (1:1, [M]þ); HRESIMS (pos.) m/z549.15029 ([M]þ, calcd for C28H30

79BrN4O3, 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

(250�4.6 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; 20:80:0.1for 6) to separate each enantiomer (tR 17.6 and 19.2 min in 1; tR 14.4and 16.4 min in 2; tR 37.6 and 40.4 min in 3; tR 23.2 and 25.2 min in6), respectively.

3.3.10. Conformational search for anti- (6a) and syn-nakijinamine I(6b). Conformational searches were carried out using mixedMCMM/Low-Mode conformational search methods10 on Macro-Model 9.8 (Schr€odinger, LLC). Each generated conformers wereminimized with MMFF94s force field.11

Acknowledgements

We thank Ms. S. Oka, Ms. A. Tokumitsu, and Ms. T. Komiya, In-strumental Analysis Division, Equipment Management Center,Creative Research Institution, Hokkaido University, for measure-ments of ESIMS; Dr. Eri Fukushi, Graduate School of Agriculture,Hokkaido University and Mr. T. Hirose, Instrumental Analysis Di-vision, Equipment Management Center, Creative Research In-stitution, Hokkaido University for measurements of a part of NMRspectra; Mr. Z. Nagahama and K. Uehara for their help with col-lection of the sponge. This work was partly supported by a researchfellowship for young scientists from the Japan Society for the Pro-motion of Science (to Y.T.) and a Grant-in-Aid for Science Researchfrom the Ministry of Education, Culture, Sports, Science, andTechnology of Japan.

Supplementary data

Supplementary data associated with this article can be found inthe online version, at http://dx.doi.org/10.1016/j.tet.2012.08.018.

References and notes

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