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Cite This: J. Nat. Prod. 2018, 81, 1295−1299
Poecillastrin H, a Chondropsin-Type Macrolide with a Conjugated Pentaene Moiety, from a Characella sp. Marine Sponge Rei Suo,† Kentaro Takada,*,† Raku Irie,† Ryuichi Watanabe,§ Toshiyuki Suzuki,§ Yuji Ise,‡ Susumu Ohtsuka,⊥ Shigeru Okada,† and Shigeki Matsunaga*,† †
Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan § National Research Institute of Fisheries Science, 2-12-4 Fukuura, Kanazawa, Yokohama 236-8648, Japan ‡ Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Toba, Mie 517-0004, Japan ⊥ Takehara Marine Science Station, Hiroshima University, Takehara, Hiroshima 725-0024, Japan S Supporting Information *
ABSTRACT: Poecillastrin H (1), a chondropsin-type macrolide with a conjugated pentaene moiety, was isolated from the Characella sp. marine sponge. The planar structure of 1 was elucidated by analysis of spectroscopic data. The absolute configuration of the β-hydroxyaspartic acid residue (βOHAsp) was determined to be D-threo by Marfey’s analysis, and the mode of lactone ring formation through the OHAsp residue was determined by chemical degradation. Poecillastrin H was extremely sensitive toward light and showed potent cytotoxic activity against 3Y1 cells with an IC50 value of 4.1 nM.
M
poecillastrin H (1, 0.6 mg). All procedures were carried out in the dark due to the sensitivity of 1 toward light.
arine invertebrates, e.g., sponges, ascidians, and bryozoans, constitute about 60% of marine animal diversity and are rich sources of structurally unique and biologically active secondary metabolites.1 So far, metabolites derived from shallow-water marine invertebrates have been the main targets for drug discovery; a variety of invertebrates living in deep sea are largely untapped.2 Because deep-sea organisms inhabit environments that are under extreme conditions such as high pressure, low levels of oxygen, and absence of light, their primary and secondary metabolisms are likely to be adapted to such conditions,3 thereby affording structurally unique natural products with promising biological activities. We have conducted cell-morphology-based screenings against extracts of deep-sea sponges4 and found that the extract of a Characella sp. deep-sea sponge induced characteristic morphological changes in rat embryonic fibroblast 3Y1 cells. Similar morphological changes were observed by treatment with bafilomycin, a V-ATPase inhibitor.5 From this sponge, we isolated a new member of the chondropsin family of metabolites, poecillastrin H (1), which was extremely sensitive to light. The isolation, structure elucidation, and cytotoxic activity of 1 are the subjects of this paper. The extract of the Characella sp. marine sponge was partitioned between H2O and CHCl3, and the organic layer was further partitioned between n-hexane and MeOH−H2O (9:1). The aqueous MeOH fraction was separated by Sephadex LH-20 chromatography followed by reversed-phase HPLC under guidance with the cell-morphological changes to afford © 2018 American Chemical Society and American Society of Pharmacognosy
The molecular formula of 1 was determined to be C78H127N3O19 by HRESIMS, implying the unsaturation degree of 17. Preliminary analysis of 2D NMR data showed the presence of 54 sp3-hybridized carbons (CH3 × 19, CH2 × 8, CH × 25, and C × 2) and 24 sp2-hybridized carbons (CH × 15, C × 3, and CO × 6), from which 113 protons were shown to Received: February 27, 2018 Published: April 27, 2018 1295
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Figure 1. Partial structures A−G of poecillastrin H (1).
Table 1. 1H (800 MHz) and 13C NMR Data (200 MHz) of Poecillastrin H (1) in CD3OD position
δC, type
1 2 3
174.7, C 73.2, CH 57.5, CH
4
176.4,b C
5 6
168.4, C 125.1, CH
7
142.3, CH
8
131.4, CH
9
141.4, CH
10
133.3, CH
11
138.4, CH
12
131.5, CH
13
137.4, CH
14
133.1, CH
15
136.3, CH
16a
35.8, CH2
16b 17 18a 18b 19
73.0, CH 38.1, CH2 27.2, CH
δH, mult (J in Hz)
position
4.88, brsa 4.88, brsa
34 34-Me 35
41.1, CH 9.6, CH3 78.1, CH
36
55.0, CH
37 38
70.1, CH 21.5, CH3
39
179.0, C
40
48.3, CH
2.56, m
40-Me
15.7, CH3
41
74.7, CH
1.20, d (7.2) 3.57, m
42a
33.5, CH2
1.57, m
6.16, d (14.9)
δC, type
δH, mult (J in Hz)
44-Me
15.9, CH3
2.15, m
45
85.3, CH
4.09, 1.52, 1.35, 1.88,
46 46-Me 47 48
136.2, C 13.7, CH3 1.70, brs 136.3, CH 5.43, s 41.6, C
m m m m
42b 43a
19-Me
22.9, CH3
0.92c
20a
41.8, CH2
1.49, m
20b
1.48, m 30.6, CH2
43b 44
δC, type
1.78, m 0.93c 5.18, d (10.1) 4.14, d (5.1) 3.73, m 1.17, d (6.0)
7.22, dd (14,9 11.1) 6.45, dd (14.4, 11.1) 6.70, dd (14.4, 11.6) 6.41, dd (14.2, 11.6) 6.60, dd (14.2, 11.7) 6.32, dd (15.1, 11.7) 6.49, dd (15.1, 10.6) 6.27, dd (15.8, 10.6) 5.85, ddd (15.8, 10.7, 3.9) 2.84, m
1.44, m 1.28, m
37.1, CH2
δH, mult (J in Hz)
position
1.62, m 0.96, d (6.2) 3.59, brd (8.3)
66.9, CH 43.3, CH2
position
0.87, m
48-Me (a) 48-Me (b) 49
3.64, 1.41, 1.13, 4.10, 1.34, 0.51, 3.49,
50 51 52 53 54 55 56
1.13, s
26.7, CH3
1.22, s
82.3, CH
3.49, d (5.3) 4.19, m 1.49, m 1.53, m 0.90c 0.91c
66.3, CH 42.5, CH 8.8, CH3 81.0, CH
26
137.3, C
26-Me 27
10.6, CH3 135.3, CH
1.55, brs 4.89, d (9.5)
28
36.0, CH
2.49, m
28-Me 29
17.6, CH3 84.3, CH
0.56, d (6.6) 3.44, brdc
58 58-Me (a) 58-Me (b) 59 60
30 30-Me 31 32a
137.2, C 10.9, CH3 129.2, CH 35.0, CH2
1.58, brs 5.26, m 2.33, m
60-Me 61 62 63
15.8, 78.8, 30.3, 14.5,
1.88, m
64
20.6, CH3
33
69.8, CH
57
δH, mult (J in Hz)
26.1, CH3
21 22a 22b 23 24 24-Me 25
32b
m m m m m d (7.0) d (9.0)
δC, type
51.1, CH 41.1, CH2 26.1, CH 22.1, CH3 24.5, CH3 167.1, C 124.3, CH 6.07, d (15.2) 148.6, CH 6.93, d (15.2) 52.2, C 23.9, CH3 1.27, s 24.1, CH3 217.4, C 45.8, CH CH3 CH CH CH3
1.30, s
3.15, dq (9.1, 6.7) 0.89c 3.55, m 1.77, m 0.84, d (6.9) 0.95, d (6.7)
3.44, brdc
a
Overlapped with HDO. bAssigned by HMBC data. cCoupling constant was not determined due to signal overlap.
with the oxymethine proton (H-29). The connections from C30 to C-33 and C-34 to C-37 in partial structure C were similarly assigned on the basis of the COSY data. Although COSY cross-peak was not observed between H-33 and H-34, an HMBC correlation between the C-34 methyl proton signal to C-33 allowed us to link C-33 and C-34. The 13C chemical shift values indicated that C-33, C-35, and C-37 were substituted by an oxygen atom and that C-36 was substituted by a nitrogen atom. The 1H chemical shift of H-35 suggested that the oxygen atom on C-35 was esterified. The 1H NMR spin system in partial structure D was assigned by analysis of
be attached to a carbon atom, implying the presence of two rings and 14 exchangeable protons. Further analysis of the 2D NMR data showed partial structures A−G (Figure 1). One end of partial structure A was a carbonyl conjugated with a pentaene system whose terminus was linked to a C10aliphatic chain including two methyl-substituted methines (C19 and C-24) and four oxygenated methines (C-17, C-21, C-23, and C-25). The ether linkage between C-17 and C-21 was suggested by a NOESY correlation between H-16a (δH 2.84) and H-21. In partial structure B, H-27 was coupled with the C26 methyl proton signal and H-28, which was in turn coupled 1296
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Figure 2. Interconversions of poecillastrin H (1) and its isomers. HPLC chart of the following preparations with UV detection at 365 nm. (a) Freshly purified compound 1; (b) compound 1 after exposure to ambient light for 48 h; (c) freshly purified peak C; (d) peak C after exposure to ambient light for 48 h; (e) freshly purified peak D; (f) peak D after exposure to ambient light for 48 h.
the COSY and TOCSY data, whereas the 1H chemical shift of H-40 suggested that C-40 was adjacent to a carbonyl carbon. The C-46 to C-47 portion and the C-49 to C-54 portion in partial structure E were assigned by interpretation of the COSY data, and the linkage of these portions through a quaternary carbon with a gem-dimethyl group (C-48) was established on the basis of the HMBC correlations between the gem-dimethyl protons and both C-47 and C-49. Substitution of C-50 by a nitrogen atom was suggested by the 13C chemical shift. Partial structure F contained an isolated disubstituted double bond conjugated with a carbonyl carbon as assigned by HMBC crosspeaks from H-56 and H-57 to C-55. Interpretation of the COSY spectrum allowed us to assign the 2-substituted 3hydroxy-4-methylpentane moiety (C-60−C-64). Substitution of C-60 by a keto-carbonyl carbon (C-59) which was connected to C-57 through a quaternary carbon with a gem-dimethyl group (C-58) was indicated by HMBC cross-peaks from the C58 methyl proton signals to C-57, C-58, and C-59 as well as the C-60 methyl proton signal to C-59. After these assignments, two methine carbons and two carbonyl carbons were left unassigned. Their 1H and 13C chemical shifts suggested the presence of a β-hydroxyaspartic acid (β-OHAsp) residue (partial structure G). Partial structures A and B were connected between C-25 and C-26 on the basis of an HMBC cross-peak between the C-26 methyl proton signal and C-25. Partial structures C and D were connected between the C-36 nitrogen atom and C-39 on the basis of HMBC cross-peaks between H-36 and C-39 and between the C-40 methyl proton signal and C-39. Partial structures D and E were connected between C-45 and C-46 on the basis of an HMBC cross-peak between the C-46 methyl proton signal and C-45. HMBC correlations between H-35 and a carbonyl carbon at δC 174.7 (C-1) and between H-3 and C-5 suggested the connection of partial structure G to partial structure C through an ester bond and the connection of partial structure G to partial structure A through an amide bond. The precise mode of the linkage formation involving the β-OHAsp residue was determined through chemical degradation as described below. The last bond was formed between the nitrogen atom at C-50 and C-55 by a process of elimination. All E-configurations were assigned to the conjugated pentaene moiety based on large coupling constant values (Table 1), whereas the E-configurations were assigned to the three trisubstituted double bonds based on NOESY correla-
tions (H-25 to H-27 and 26-Me to H-28; H-29 to H-31 and 30Me to H-32; and H-45 to H-47) and the 13C chemical shift values of the vinylic methyl groups. An E-configuration was assigned to the Δ56-olefin on the basis of a large coupling constant value between H-56 and H-57. Therefore, poecillastrin H (1) was assigned as a new member of the chondropsin family of metabolites.6−16 The mode of ester formation of the β-OHAsp residue in 1 was determined by a chemical method as previously described.16 First, the absolute configuration of the β-OHAsp was determined to be D-threo by Marfey’s method (Figure S8).17 Then, 2-amino-3,4-dihydroxybutanoic acid was obtained by reduction of 1 with NaBH4 followed by acid hydrolysis. The HPLC analysis of the Marfey’s derivative of the aminodihydroxybutanoic acid showed the (2R,3R)-configuration (Figure S9). In our initial 1H NMR experiments of 1, we measured the spectrum of the sample, which was prepared by using glassware covered by aluminum foil throughout the isolation procedures. The 1H NMR spectrum of the sample measured immediately after the HPLC purification contained many small signals in addition to the main signals (Figure S10). We suspected the possibility that 1 underwent light-catalyzed isomerization. Then, we conducted the final HPLC purification and evaporation of the solvent at night with the room light off. The 1H NMR spectrum of the resulting sample was improved significantly. Then, we examined the changes of 1 by LC-MS. Poecillastrin H (1) gave a single sharp peak immediately after purification in the dark and gave one peak after 3 days of storage in the dark. By exposing to ambient light for 30 min, the compound started to give multiple HPLC peaks. These peaks exhibited the same molecular weight and UV spectrum as the starting material (Figure S11). We purified peak C and peak D and exposed these compounds and 1 to ambient light for 48 h. The three compounds stayed unchanged in the dark, but were converted to the same sets of isomers (Figure 2). This experiment demonstrated that compound 1 and its isomers were interconverted by exposure to light. Even though we did not characterize the newly formed peaks, it is likely that each peak arose from cis−trans isomerization within the pentaene moiety. Poecillastrin H (1) showed potent cytotoxicity against 3Y1 cells with an IC50 value of 4.1 nM. The morphological changes of elongated cells as induced by poecillastrin H were similar to 1297
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have not been reported from C. poecillastroides. The specimen used for the identification (SMBL-Po051) is deposited at the Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University. Extraction and Isolation. The sponge (800 g) was homogenized in EtOH and extract with EtOH and MeOH−CHCl3 (1:1). The extracts were combined, concentrated in vacuo, and partitioned between H2O and CHCl3. The CHCl3 layer was further partitioned between n-hexane and MeOH−H2O (9:1). The aqueous MeOHsoluble material (2.5 g) was purified by gel permeation chromatography (Sephadex LH-20, MeOH) to afford seven fractions (A−G). Fraction B was purified by RP-HPLC on a COSMOSIL 5C18-AR-II column with an isocratic elution with 52% MeCN in the presence of 1% AcOH to give poecillastrin H (1, 0.6 mg). Poecillastrin H (1): yellowish solid; [α]D +4.3 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 378 (4.2), 353 (4.4), 208 (4.4); 1H and 13C NMR data, see Table 1; HRESIMS m/z 1432.8945 [M + Na]+ (calcd for C46H64N4O13Na,1432.8961). Marfey’s Analysis of 1. The sample was hydrolyzed in 6 N HCl at 110 °C for 5 h. The solution was concentrated and redissolved in H2O (100 μL). To the solution were added 1% L-FDAA in acetone (100 μL) and 1 M NaHCO3 in H2O (20 μL). The resulting solution was kept at 55 °C for 30 min. After neutralization with 2 N HCl (10 μL), the reaction mixture was analyzed by LC-MS on a COSMOSIL 2.5C18-MS-II column with an isocratic elution (7% MeCN−H2O + 0.5% AcOH). Retention time (min): D-threo β-OHAsp (9.7), L-threo β-OHAsp (10.5), D-erythro β-OHAsp (24.4), L-erythro β-OHAsp (32.0), and the hydrolysate from poecillastrin H (9.7). Reduction of 1. To a solution of 1 (100 μg) in MeOH (150 μL) was added NaBH4 (0.5 mg), and the mixture was left at rt for 4 h and dried by a stream of N2 to afford a crude diol, ESIMS [M + H]+ 1417. The reaction product was hydrolyzed and subjected to Marfey’s analysis as described above. Retention time (min): (2S,3S)-2-amino3,4-dihydroxybutanoic acid (13.6), (2R,3S)-3-amino-2,4-dihydroxybutanoic acid (14.3), (2R,3R)-2-amino-3,4-dihydroxybutanoic acid (20.2), (2S,3R)-3-amino-2,4-dihydroxybutanoic acid (32.3): acid hydrolysate of the reduction product of poecillastrin H (20.1). Cytotoxicity Assay. The cytotoxicity of 1 against 3Y1 cells was evaluated by an MTT assay. The 3Y1 cells were cultured in Dulbecco’s modified Eagle’s medium containing penicillin, streptomycin, and 10% fetal bovine serum at 37 °C under as atmosphere of 5% CO2. After overnight preincubation, 1 was added to each well of a 96-well microplate containing 200 μL of 3Y1 cell suspension and further incubated for 72 h. Then, MTT saline solution was added to each well, and the plate was further incubated for 3 h. The medium was excluded, and formazan dye was dissolved in 150 μL of DMSO to be quantitated.
those observed by treatment with bafilomycin. Poecillastrins and chondropsins are known to inhibit V-ATPase,18 demonstrating that the morphology-guided cell-based assay is an efficient means to detect biologically active compounds with specific modes of action.4 Poecillastrin H (1) exhibited extreme sensitivity to light. Apart from the presence of an extra methyl group at C-48, poecillastrin H is different from poecillastrin C (2) by incorporation of an additional unsaturation at C-10 and the absence of the methoxy group at C-16. When we handled poecillastrins C (2), E (3), and F (4),13 it was possible to isolate significantly pure samples by covering the containers with aluminum foil during the isolation procedures. Poecillastrin E has a conjugated pentaene moiety and the C-16 methoxy group, whereas poecillastrin F has an interrupted polyene system with the C-16 methoxy group lacking. Both congeners were more stable than poecillastrin H, showing that the combination of the pentaene system and the lack of the C-16 methoxy group bestowed poecillastrin H with the extreme sensitivity toward light. Such light sensitivity may be the reason that poecillastrins have been isolated from sponges inhabiting the deep sea. Although this class of compounds exhibits potent cytotoxicity with a specific mode of action, the absolute configuration has been assigned only for the β-hydroxyaspartic acid residue. The elucidation of the full relative and absolute configurations of these molecules will promote the endeavor toward their total synthesis, culminating in the evaluation as drug leads.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a Jasco DIP-1000 polarimeter. UV spectra were measured on a Shimadzu BioSpec-1600 spectrophotometer. NMR spectra were measured on a JEOL alpha 600 NMR spectrometer and referenced to the solvent peak: δH 3.30 and δC 49.0 for methanol-d4. ESI mass spectra were recorded on a JEOL JMS-T100LC mass spectrometer. LC-MS experiments were performed on a Shimadzu LC-20AD solvent delivery system and interfaced to a Bruker amaZon SL mass spectrometer. The results of the MTT assay were recorded with a Molecular Devices SPECTRA Max M2. Time-lapse imaging of cells was conducted on the Essen Bioscience IncuCyteZoom. Animal Material. The Characella sp. sponge was collected by dredging at a depth of 191 m at seamount Yaku-Shinsone (29°49.073′ N, 130°22.594′ E), southern Japan, during a cruise of the T/S Toyoshio-maru, on October 21, 016. The specimen was a broken part of the sponge, and thus the whole external morphology was not precisely observed. However, it was either a plate-like or fan-shaped sponge and ca. 2 cm in thickness. The surface of the sponge is hispid because of the protruding large oxeas. Spicules are composed of oxeas, orthotriaenes, and microxeas in two size classes and one type of amphiasters. Oxeas are very large, straight, or slightly curved at midpoint, smooth and fushiform with sharp tips, mainly 2−3 mm in length and 70−80 μm in thickness. Orthotriaenes are rare, with smooth shafts. The length/thickness of the cladomes and rhabdome are almost uniform, 320−400 μm in length and 13−15 μm in thickness. Microxeas I are very abundant, straight, fushiform and have a rough surface, 130−175 μm in length and 1.2−1.6 μm in thickness. Microxeas II are also straight, fushiform, with a rough surface and sometimes centrotylote, 25−38 μm in length, 2−4 μm in thickness. Style-like derivatives are frequently observed for this spicule type. Amphiasters have a straight central axis, 20−30 μm in total length. Up to now, 14 species have been reported as valid for the genus Characella Sollas, 1888 according to the World Porifera Database. Of these, Characella poecillastroides once collected from Bonaire, Caribbean Netherlands, is comparable to our specimen for its spicule composition. However, the size of each spicule type is different from those of our specimen except amphiaster, and style-like derivatives
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00180. 1D and 2D NMR spectra of 1; LC-MS chromatograms of Marfey’s derivatives (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail (K. Takada):
[email protected]. *Tel (S. Matsunaga): 81-3-5841-5297. Fax: 81-3-5841-8166. Email:
[email protected]. ORCID
Shigeki Matsunaga: 0000-0002-8360-2386 Notes
The authors declare no competing financial interest. 1298
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Note
ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grants Numbers 25252037, 16H04980, and 17H06403 and JSPS Research Fellowship for Young Researchers 16J07837 and 17J08775. We thank the captain and crew of the T/S Toyoshio-maru for sample collection.
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REFERENCES
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