Note pubs.acs.org/jnp
Zamamidine D, a Manzamine Alkaloid from an Okinawan Amphimedon sp. Marine Sponge Takaaki Kubota,*,† Kenta Nakamura,‡ Shin-ichiro Kurimoto,† Kanae Sakai,§ Jane Fromont,⊥ Tohru Gonoi,§ and Jun’ichi Kobayashi*,‡ †
Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan § Medical Mycology Research Center, Chiba University, Chiba 260-0856, Japan ⊥ Western Australian Museum, Locked Bag 49, Welshpool DC, WA 6986, Australia ‡
S Supporting Information *
ABSTRACT: A new manzamine alkaloid, zamamidine D (1), was isolated from an Okinawan Amphimedon sp. marine sponge. The structure of zamamidine D (1) including the relative configuration was elucidated on the basis of spectroscopic data. Zamamidine D (1) is the first manzamine alkaloid possessing a 2,2′-methylenebistryptamine unit as the aromatic moiety instead of a β-carboline unit. Zamamidine D (1) showed antimicrobial activity against several bacteria and fungi.
T
he manzamine alkaloids have been reported from several marine sponge genera and are attractive as structurally unique and biosynthetically interesting bioactive natural products.1,2 In our continuing search for bioactive natural products from marine organisms, we have isolated many manzamine alkaloids from Okinawan marine sponges.3−5 More recently, we have investigated the extract of an Okinawan marine sponge Amphimedon sp. (SS-1231) and isolated a manzamine-related alkaloid, zamamiphidin A, from its EtOAcsoluble materials.5 Subsequently, we investigated BuOH-soluble materials of the extract and found a new manzamine alkaloid, zamamidine D (1). Here we describe the isolation and structure elucidation of 1. The sponge Amphimedon sp. (SS-1231) collected at Zamami, Okinawa, was extracted with MeOH. The extract was partitioned stepwise between organic solvents (EtOAc and then BuOH, successively) and H2O. BuOH-soluble materials were separated by a Sephadex LH-20 column, a C18 flash column, and C18 HPLC to obtain zamamidine D (1, 1.7 mg, 0.00025% wet weight). Zamamidine D (1) was obtained as an optically active pale yellow, amorphous solid. The UV absorptions [λmax 291 and 282 nm] were attributed to heteroaromatic rings, while the IR absorption (3436 cm−1) indicated the presence of hydroxy and/or amino functionalities. The molecular formula of 1 was established as C46H60N6O by HRESIMS data. The analysis of the HSQC spectrum with 1H and 13C NMR data of 1 (Table 1) disclosed the existence of 46 carbons consisting of nine sp2 © 2017 American Chemical Society and American Society of Pharmacognosy
nonprotonated carbons, 13 sp2 methines, two sp3 nonprotonated carbons, four sp3 methines, and 18 sp3 methylenes. The inspection of the COSY, TOCSY, and HSQC-TOCSY spectra of 1 revealed connectivities of C-3 to C-4, C-5 to C-8, C-13 to C-20, C-22 to C-24, C-28 to C-34, C-3′ to C-4′, and C5′ to C-8′. The detailed analyses of the HMBC spectrum of 1 revealed the existence of a 5/6/6/8/13 ring system (C-10−CReceived: December 1, 2016 Published: February 16, 2017 1196
DOI: 10.1021/acs.jnatprod.6b01110 J. Nat. Prod. 2017, 80, 1196−1199
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Table 1. 1H and 13C NMR Data of Zamamidine D (1) in CD3OD position 1 3 4 4a 4b 5 6 7 8 8a 9a 3′ 4′a 4′b 4a′ 4b′ 5′ 6′ 7′ 8′ 8a′ 9a′ 10 11 12 13a 13b 14a 14b 15 a
δC,a type 41.0, 42.4, 24.3, 110.5, 129.8, 119.6, 121.7, 124.1, 113.5, 138.4, 134.2, 42.0, 24.6,
CH CH2 CH2 C C CH CH CH CH C C CH2 CH2
108.1, 129.6, 119.4, 121.4, 123.6, 113.5, 138.3, 135.6, 144.7, 133.0, 68.5, 41.5,
C C CH CH CH CH C C C CH C CH2
22.6, CH2 128.5, CH
δH,b multi (J in Hz) 5.45, s 3.03, ndc 3.28, ndc
7.60, 7.13, 7.22, 7.54,
d (7.9) dd (7.9, 7.9) dd (7.9, 7.9) d (7.9)
3.19, ndc 3.34, ndc 3.20, ndc
7.58, 7.08, 7.12, 7.41,
d (7.9) dd (7.9, 7.9) dd (7.9, 7.9) d (7.9)
5.35, s 2.00, 1.45, 2.28, 1.96, 5.50,
ndc ndc ndc ndc ndc
position
δC,a type
16 17a 17b 18a 18b 19a 19b 20a 20b 22a 22b 23a 23b 24 25 26 28a 28b 29 30a 30b 31a 31b 32 33 34 35a 35b 36a 36b
135.1, CH 26.7, CH2 28.4, CH2 26.5, CH2 54.7, CH2 51.0, CH2 33.9, CH2 41.8, 48.8, 78.9, 55.3,
CH C CH CH2
28.6, CH2 26.1, CH2 29.8, CH2 143.8, 126.0, 59.6, 45.6,
CH CH CH CH2
72.0, CH2
δH,b multi (J in Hz) 5.55, 2.57, 1.61, 1.49, 1.22, 1.84, 1.60, 2.74, 2.34, 3.00, 1.75, 2.30, 1.61, 1.92,
ddd (11.0, 11.0, 4.2) m ndc ndc m br s ndc ddd (12.1, 12.1, 5.1) ndc ndc ddd (12.1, 12.1, 2.9) ndc ndc dd (12.0, 6.9)
3.71, 3.91, 3.45, 2.06, 2.01, 1.53, 2.44, 2.35, 6.33, 5.41, 4.82, 2.13, 1.66, 3.05, 2.26,
s m dd (11.9, 9.6) ndc ndc ndc m ndc ddd (11.1, 7.1, 7.1) dd (11.1, 8.4) ndc dd (14.1, 8.1) d (14.1) ndc d (11.6)
150 MHz. b600 MHz. cnd: J-values were not determined because of overlapping with other signals.
20, N-21, C-22−C-26, N-27, and C-28−C-36), which was characteristic for manzamine alkaloids such as manzamine A.6,7 Considering the molecular formula and 1H and 13C NMR chemical shifts, eight sp2 nonprotonated carbons, eight sp2 methine carbons, and four sp3 methylene carbons of remaining carbons were ascribed to two 2-substituted tryptamines (N-2, C-3−C-8a, N-9, and C-9a; N-2′, C-3′−C-8a′, N-9′, and C-9a′). The existence of two 2-substituted tryptamines was supported by HMBC correlations. Finally, the HMBC correlations for H1/C-4a, H-1/C-9a, H-1/C-10, and H-1/C-4a′ revealed that C9a and C-9a′ of two 2-substituted tryptamines and C-10 of the manzamine A-type 5/6/6/8/13 ring were connected through C-1. The configurations of the double bonds at C-15−C-16 and C-32−C-33 were assigned as both Z from the 3JH/H values of olefinic protons (3JH‑15/H‑16 = 11.0 Hz and 3JH‑32/H‑33 = 11.1 Hz). Thus, the gross structure of zamamidine D was elucidated to be 1 (Figure 1). The relative configuration of zamamidine D (1) was deduced by analysis of the NOESY spectrum (Figure 2). The proximity of H-24 to H-36b was implied by the NOESY correlation for H-24/H-36b, while the NOESY correlations for H-26/H-36a and H-33/H-36a suggested that H-26 and H-33 were located close to H-36a. The NOESY correlations for H-14a/H-26 and H-33/H-35b showed proximity of H-14a to H-26, and H-33 to H-35b, respectively. Thus, the relative configuration of 1 was assigned as 12S*, 24S*, 25R*, 26R*, and 34R*. Considering the shape of the whole molecule in addition to these NOESY correlations, the cyclohexene ring (C-10−C-12 and C-24−C-
Figure 1. Selected 2D NMR correlations for zamamidine D (1) in CD3OD.
26) and the piperidine ring (N-21, C-22−C-25, and C-36) in the cis-octahydroisoquinoline ring of 1 might adopt the pseudoboat form and the chair form, respectively. Nearly 100 manzamine alkaloids have been reported so far.1 Most of the manzamine alkaloids possess an aromatic moiety, typically a β-carboline unit or its derivative. Zamamidines A−C possess a bis-β-carboline unit as the aromatic moiety.3,4 Acanthomanzamines A and B are rare manzamine alkaloids 1197
DOI: 10.1021/acs.jnatprod.6b01110 J. Nat. Prod. 2017, 80, 1196−1199
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line,9−11 is shown in Scheme 1. Presumably manzamine A is formed by aromatization of 3,4-dihydromanzamine A, which is expected to be generated from a tryptamine and ircinal A by the Pictet−Spengler reaction.12,13 Zamamidine D (1) may be derived from 3,4-dihydromanzamine A through hypothetical intermediates X−Z. Cleavage of the C-1 and N-2′ bond of 3,4dihydromanzamine A might afford the intermediate X. Condensation of intermediate X and tryptamine and intermolecular cyclization of the resulting intermediate Y could give intermediate Z. Zamamidine D (1) is assumed to arise from intermediate Z as a result of reductive cleavage of its C-1 and N-2 bond. Zamamidine D (1) exhibited significant activity against a variety of bacterial and fungal microbes (Table 2).
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Figure 2. Selected NOESY correlations for zamamidine D (1) in CD3OD.
EXPERIMENTAL SECTION
General Experimental Procedures. The optical rotation was recorded on a JASCO P-2200 polarimeter. The UV spectrum was recorded on a JASCO Ubest-55 spectrophotometer. The ECD spectrum was recorded on a JASCO J-1500 spectropolarimeter. The IR spectrum was recorded on a Horiba FT710 spectrophotometer. 1H and 13C NMR spectra were recorded on a Bruker Avance II 600 MHz NMR spectrometer equipped with a cryoplatform using 3.0 mm micro cells (Shigemi Co., Ltd.) for CD3OD. The 3.35 ppm resonance of
possessing a single tetrahydroisoquinoline unit as the aromatic moiety.8 Zamamidine D (1) is the first manzamine alkaloid possessing a 2,2′-methylenebistryptamine unit as the aromatic moiety. A possible biosynthetic pathway for zamamidine D (1), inspired by the total synthesis of a marine alkaloid, bengacarbo-
Scheme 1. Possible Biosynthetic Pathway for Manzamine A and Zamamidine D (1)
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DOI: 10.1021/acs.jnatprod.6b01110 J. Nat. Prod. 2017, 80, 1196−1199
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value was determined after 72 h of incubation comparing with the drug-free control.
Table 2. Antimicrobial Activities of Zamamidine D (1) antibacterial activity strain Escherichia coli ATCC25922 Stapylococcus aureus 209P Bacillus subtilis PC1219 Micrococcus luteus IFM2066
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antifungal activity
MIC (μg/mL) 32 8 8 8
strain Aspergillus niger IFM62678 Trichophyton mentagrophytes IFM62679 Candida albicans IFM62680 Cryptococcus neoformans IFM62681
IC50 (μg/mL)
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01110. NMR, UV, IR, ECD, and MS spectra of 1 and photographs of the sponge Amphimedon sp. (SS-1231) (PDF)
16 8 16
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2
AUTHOR INFORMATION
Corresponding Authors
residual CD2HOD and 49.8 ppm resonance of CD3OD were used as internal references for 1H and 13C NMR spectra, respectively. MS spectra were recorded on a JMS-T100LP spectrometer. Flash column chromatography was performed with a Biotage Isolera flash purification system. Sponge Description. The sponge (SS-1231) Amphimedon sp. (order Haplosclerida, family Niphatidae) was collected at Zamami, Okinawa, on October 29, 1982, and kept frozen until used. The sponge was flattened, firm, compressible, and springy, and had large internal canals. The sponge was light to medium brown in color in EtOH, having a smooth but microscopically hispid surface. The reticulate fiber skeleton was fully cored by spicules. Primary fibers were multispicular and ∼50 μm wide. Secondary fibers forming either isodictyal or rectangular meshes had 1−3 spicules across a ∼35 μm width. The sponge had numerous oxeas throughout the skeleton. The spicules were small oxeas with short pencil points, ∼105 × 5 μm. This specimen is likely to be the same species as the sponges Amphimedon sp. (SS-264, SS-975, and SS-1251).3,4 The voucher specimen was deposited at Graduate School of Pharmaceutical Sciences, Hokkaido University. Extraction and Isolation. The sponge Amphimedon sp. (SS-1231, 0.68 kg, wet weight) was extracted with MeOH (500 mL × 3) to obtain the extract (54.49 g), which was partitioned between EtOAc (500 mL × 3) and H2O (500 mL) to give EtOAc-soluble materials (4.30 g). The aqueous layer was extracted with BuOH (500 mL × 3) to give BuOH-soluble materials (8.54 g). The BuOH-soluble materials (8.54 g) were fractionated by gel filtration (Sephadex LH-20, GE Healthcare; 70 × 850 mm; eluent, MeOH), and a fraction was separated by a flash C18 column chromatography (Isolera SNAP Ultra C18 30 g, Biotage; eluent, MeOH/H2O/TFA, 0:100:0.1 to 100:0:0.1) and C18 HPLC (Mightysil RP-18 GP, 20 × 250 mm, Kanto Chemical Co., Inc.; eluent, MeOH/H2O/TFA, 50:50:0.1; flow rate, 2.0 mL/min; UV detection at 220 nm) to yield zamamidine D (1, 1.7 mg, 0.00025% wet weight). Zamamidine D (1): pale yellow, amorphous solid; [α]25D +22 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 218 (4.16), 282 (3.61), and 291 (3.55) nm; IR (KBr plate) νmax 3436, 2923, 1685, 1432, 1351, 1205, 1137, and 1025 cm−1; ECD (MeOH) λmax (Δε) 204 (8.00), 221 (0.31), 230 (3.79), and 261 (−1.44), nm; 1H and 13C NMR data, Table 1; HRESIMS m/z 713.48951 [M + H]+ (calcd for C46H61N6O, 713.49068). Antimicrobial Assay. Zamamidine D (1, 0.1 mg) was dissolved in DMSO and diluted 100 times with RPMI 1640 medium to get a final concentration of 64 μg/mL. The sample solution (100 μL) was added to a well of a 96-well plate and serially diluted with the same medium. Bacterium or yeast-type fungus was cultured overnight in LB or PDB medium, respectively. The turbidity of the suspension was adjusted to a McFarland No. 0.5 using RPMI 1640 medium, and the suspension was further diluted 100 times with the same medium. Then, the diluted suspension (100 μL) was mixed with each sample solution in the 96-well plate. Aspergillus niger (5 days old) or Trichophyton mentagrophytes (7 days old) was collected from a PDA slant and suspended in RPMI 1640 medium to get a final concentration of 1 × 105 or 1 × 106 conidia/mL, respectively. The suspension (100 μL) was mixed with each sample solution in the 96-well plate. The MIC or IC50
*E-mail (T. Kubota):
[email protected]. *E-mail (J. Kobayashi):
[email protected]. ORCID
Takaaki Kubota: 0000-0002-3553-5844 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the late Mr. Z. Nagahama for his help with sponge collection and Mr. K. Chiba, the Instrument Analysis Equipment Research Center, Showa Pharmaceutical University, for measurements of MS. This work was supported by JSPS Kakenhi grant number 25460115.
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REFERENCES
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