Amphidinins C-F, amphidinolide Q analogues from marine dinoflagellate Amphidinium sp.

Article abstract of DOI:10.1021/ol502685z

Four new polyketides, amphidinins C-F (1-4), have been isolated from the culture broth of symbiotic dinoflagellate Amphidinium sp. The analysis of their spectral data revealed that amphidinins C-F (1-4) were 4,5-seco-analogues of amphidinolide Q (5). The absolute configurations of the new compounds were elucidated by the combination of J-based configuration analysis, modified Mosher's method, and chemical derivatization. Amphidinins D (2) and F (4) are the first glycosides related to amphidinolides. Amphidinins C-F (1-4) showed antimicrobial activity against bacteria and/or fungi.

Full text of DOI:10.1021/ol502685z

Letter
pubs.acs.org/OrgLett
Amphidinins C−F, Amphidinolide Q Analogues from Marine
Dinoflagellate Amphidinium sp.
Takaaki Kubota, Takahiro Iwai, Kanae Sakai, Tohru Gonoi, and Jun’ichi Kobayashi*
†
†
‡
‡
,†
†Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
‡Medical Mycology Research Center, Chiba University, Chiba 260-0856, Japan
*
S Supporting Information
ABSTRACT: Four new polyketides, amphidinins C−F (1−4), have been isolated
from the culture broth of symbiotic dinoflagellate Amphidinium sp. The analysis of their
spectral data revealed that amphidinins C−F (1−4) were 4,5-seco-analogues of
amphidinolide Q (5). The absolute configurations of the new compounds were
elucidated by the combination of J-based configuration analysis, modified Mosher’s
method, and chemical derivatization. Amphidinins D (2) and F (4) are the first
glycosides related to amphidinolides. Amphidinins C−F (1−4) showed antimicrobial
activity against bacteria and/or fungi.
arine dinoflagellates have been recognized as a source of
M
novel secondary metabolites with interesting structures
1
and bioactivities. In particular, carbon skeletons of dinoflagellate
polyketides, which might be synthesized by unexplained one-
carbon extension machinery, are unique and unavailable from
2
other organisms. In our continuing search for bioactive
metabolites from marine dinoflagellates, we have isolated a
series of macrolides, amphidinolides, and long-chain polyketide3s
from the cells of cultured dinoflagellates Amphidinium spp.
Amphidinolide Q (5), a cytotoxic 12-membered macrolide, was
one of them, the absolute structure of which was elucidated on
the basis of spectroscopic analyses and asymmetric total
4
synthesis. Recently, we have investigated the culture medium
of newly obtained dinoflagellates Amphidinium sp. and isolated
four new amphidinolide Q analogues, amphidinins C−F (1−4)
(Figure 1). Here, we describe the isolation, structure elucidation,
and biological activities of 1−4.
Figure 1. Structures of amphidinins C−F (1−4) and amphidinolide Q
The dinoflagellates Amphidinium sp. (2012-7-4A strain) were
isolated from the inside of a marine acoel flatworm Amphiscolops
sp. collected at Ishigaki Island, Okinawa, Japan. The dino-
flagellates were cultured at 25 °C for 3 weeks under 16 h light/8 h
dark schedule in seawater medium, and the supernatant was
subjected to a porous polymer gel column. The column was
(
5).
3
2
2
that 1 consists of six methyls, five sp methylenes, an sp
3
2
methylene, four sp methines, an sp methine, and four sp
quaternary carbons including a keto carbonyl carbon and an ester
carbonyl carbon (Table S1, Supporting Information). The planar
structure of 1 was elucidated from 2D NMR data (Figure 2).
washed with H O, and the adsorbed material was eluted with
2
MeOH, which was concentrated in vacuo and partitioned
between n-hexane and H O to afford n-hexane-soluble materials.
2
The n-hexane-soluble materials were separated by a silica gel
column, a C column, and C HPLC to afford amphidinins C−
1
8
18
5
4
6
F (1−4) with amphidinolides P and Q (5) and amphidinin A.
Amphidinin C (1) was obtained as an optically active colorless
amorphous solid [[α] −17.1 (c 0.50, MeOH)]. The molecular
formula of 1 was established as C H O by HRESIMS data (m/
z 375.25028 [M + Na] , Δ −0.30 mmu). IR absorptions implied
the existences of hydroxy (3361 cm ), ester carbonyl (1721
cm ), and keto carbonyl (1705 cm ) functionalities. Inspection
of the HMQC spectrum with H and C NMR data disclosed
2
2
D
2
1
36
4
+
Figure 2. Selected 2D NMR correlations for amphidinin C (1).
Received: September 11, 2014
−
1
−1
−1
1
13
©
XXXX American Chemical Society
A
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Organic Letters
Letter
1
1
8
H− H COSY and TOCSY spectra of 1 revealed connectivities
measured from hetero halcf-filtered TOCSY (HETLOC) and J-
9
13
of C-4 to 4-OH, C-7 to C-13, C-7 to C-18, C-9 to C-19, C-13 to
C-20, and C-15 to C-16. HMBC correlations between allylic
methyl protons H -17 (δ 2.08) and three carbons (C-2, C-3,
and C-4) indicated that the sp methine (C-2, δC 114.0),
oxymethylene (C-4, δ 67.1), and allylic methyl (C-17, δ 15.6)
carbons were linked at the sp quaternary carbon (C-3, δ 156.9).
An attachment of an acetate group (C-5 and C-6, δ 27.9 and
2
correlations from H -5 (δ 2.12) and H -18 (δ 1.06) to C-6. A
linkage of the sp methine carbon (C-13, δ 37.0), sp methylene
(
the sp quaternary carbon (C-14, δ 155.1) was suggested by the
HMBC correlations from H -21 (δ 4.73 and 4.72) to C-13 and
C-15 and from H -16 (δ 1.02) and H -20 (δ 1.03) to C-14.
Considering the molecular formula of 1 and the chemical shifts of
an oxymethine CH-11 (δ 4.99, δ 70.2), C-2 and C-11 were
resolved HMBC-2 spectra of the C-enriched sample. Relative
magnitudes of coupling constants assigned from J and JC,H
values (Table S2, Supporting Information) and NOESY
correlations implied that C-7−C-8, C-8−C-9, C-9−C-10, C-
0−C-11, and C-12−C-13 bonds existed in two major rotamers
and C-11−C-12 existed in a single major rotamer as shown in
Figure 3. Thus, the relative configurations at C-7, C-9, C-11, and
C-13 of 1 were assigned as R*, S*, R*, and R*, respectively.
The absolute configuration of 1 was established by the
3
2,3
H,H
3
H
2
1
C
C
2
C
C
3
12.9) to the sp methine (C-7, δ 44.7) was implied by HMBC
C
3
H
3
H
10
3
3
modified Mosher’s method. Amphidinin C (1) was treated
with K CO in MeOH, and the resulting mixture was separated
C
2
2 3
C-15, δ 26.0), and sp methylene (C-21, δ 107.2) carbons to
C
C
by HPLC to obtain the C-5−C-16 segment of 1, which was
successively treated with (R)- or (S)-2-methoxy-2-(trifluoro-
methyl)-2-phenylacetyl chloride (MTPACl) to obtain the (S)-
and (R)-MTPA esters (6a and 6b, respectively). Δδ values
2
C
2
H
3
H
3
H
1
obtained from H NMR data of 6a and 6b suggested that the
H
C
absolute configuration at C-11 of 1 was R (Scheme 1). Thus, the
absolute configuration of 1 was elucidated to be 7R,9S,11R,13R.
Amphidinin C (1) corresponds to 4,5-secoamphidinolide Q.
found to be connected through a remaining ester carbonyl
carbon C-1 (δ 166.4). The geometry of a double bond between
C-2 and C-3 was assigned as E by a NOESY correlation between
C
H-2 and H-4.
Scheme 1. Preparation of (S)- and (R)-MTPA Esters (6a and
a
The relative configuration of 1 was elucidated on the basis of J-
based configuration analysis (Figure 3). The JC,H values were
6b, Respectively) of the C-5−C-16 Part of Amphidinin C (1)
7
2,3
a
Δδ values (δ of 6a − δ of 6b) were indicated in italics.
H
H
H
Amphidinin D (2) was obtained as an optically active colorless
amorphous solid [[α]20 +76.0 (c 0.35, MeOH)]. The molecular
D
formula of 2 was defined as C H O by HRESIMS data (m/z
2
6
44
8
+
5
07.29276 [M + Na] , Δ-0.08 mmu). IR absorptions suggested
the presences of hydroxy (3445 cm ), ester carbonyl (1716
cm ), and keto carbonyl (1715 cm ) functionalities. Analyses
−1
−1
−1
of 2D NMR data revealed that 2 was the 4-O-pentofuranoside of
1
(Figure 4). Comparison of NMR data of 2 between methyl
pentofuranosides indicated that the sugar was ribofuranose
linked via an α-glycosidic bond.
11
Figure 4. Selected 2D NMR correlations for amphidinin D (2).
+
Amphidinin D (2) was treated with Dowex (H ) in MeOH,
and the resulting mixture was esterified with (R)-MTPACl to
obtain the tris-(S)-MTPA ester (7a) of a sugar moiety and the
1
(
S)-MTPA ester (8a) of an aglycon of 2 (Scheme 2). The H
Figure 3. Rotation models for (a) C-7−C-8, (b) C-8−C-9, (c) C-9−C-
NMR spectra of 7a and 8a were coincident with those of the tris-
S)-MTPA ester of methyl D-ribofuranoside and the (S)-MTPA
ester of 1, respectively. Thus, the absolute configuration of 2 was
elucidated to be 7R,9S,11R,13R,1′S,2′R,3′S,4′R. Amphidinin D
(2) corresponds to 4-O-α-D-ribofuranosyl-4,5-secoamphidino-
lide Q.
1
0, (d) C-10−C-11, (e) C-11−C-12, and (f) C-12−C-13 bonds of
(
amphidinin C (1). Protons with “a” and “b” are the germinal protons
whose signals were observed in lower and higher fields, respectively.
“nd” means that the magnitude was not determined. Blue dashed arrows
indicate NOESY correlations. The arrows pointing “C” indicate NOESY
correlations of protons attached to “C”.
B
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Organic Letters
Letter
Scheme 2. Preparation of Tris-(S)-MTPA Ester (7a) of a
Sugar Moiety and (S)-MTPA Ester (8a) of an Aglycon of
Amphidinin D (2)
Scheme 4. Preparation of Bis-(S)- and Bis-(R)-MTPA Esters
(10a and 10b, Respectively) of the C-5−C-16 Part of
a
Amphidinin E (3)
Amphidinin E (3) was obtained as an optically active colorless
a
Δδ values (δ of 10a − δ of 10b) are indicated in italics.
2
2
H
H
H
amorphous solid [[α] −9.6 (c 0.50, MeOH)]. The molecular
D
formula of 3, established as C H O by HRESIMS data (m/z
2
1
38
4
+
3
77.26570 [M + Na] , Δ −0.53 mmu), was bigger than that of 1
with two protons. Comparison of NMR data between 1 and 3
with consideration of their molecular formula concluded that the
planar structure of 3 was the 6-deoxo-6-hydroxy analogue of 1
(Figure 5).
Figure 6. Selected 2D NMR correlations for amphidinin F (4).
Scheme 5. Preparation of Tris-(S)-MTPA Ester (7a) of a
Sugar Moiety and Bis-(S)-MTPA Ester (11a) of an Aglycone
of Amphidinin F (4)
Figure 5. Selected 2D NMR correlations for amphidinin E (3).
Amphidinins C (1) and E (3) were independently oxidized by
1
2
1
AZADO (Scheme 3). The H NMR spectrum of oxidized
Scheme 3. Oxidation of Amphidinins C (1) and E (3)
Amphidinin F (4) corresponds to 4-O-α-D-ribofuranosyl-6-
deoxo-6β-hydroxy-4,5-secoamphidinolide Q.
Amphidinins C−F (1−4) and amphidinolide Q (5) showed
antimicrobial activity against several bacteria and fungi (Table 1).
Table 1. Antimicrobial Activities of Amphidinins C−F (1−4)
and Amphidinolide Q (5)
1
2
3
4
5
Escherichia coli
>32
32
>32
>32
>32
>32
>32
16
>32
32
>32
>32
>32
>32
>32
32
32
32
derivative (9) of 3 was coincident with that of the oxidized
derivative of 1. Thus, the relative configurations at C-7, C-9, C-
Staphylococcus aureus
Bacillus subtilis
32
32
16
11, and C-13 of 3 were assigned as R*, S*, R*, and R*,
Micrococcus luteus
Aspergillis niger
>32
32
>32
16
>32
>32
32
respectively.
The absolute configurations at C-6 and C-11 of 3 were
assigned as S and R, respectively, by applying modified Mosher’s
method for C-5-C-16 segment of 3, which was obtained by
alkaline methanolysis of 3 (Scheme 4). Thus, the absolute
configuration of 3 was elucidated to be 6S,7R,9S,11R,13R.
Amphidinin E (3) corresponds to 6-deoxo-6β-hydroxy-4,5-
secoamphidinolide Q.
Trichophyton mentagrophytes
Candida albicans
16
16
>32
>32
>32
>32
>32
>32
>32
>32
32
Cryptococcus neoformans
>32
The units for values indicating activities against bacteria and fungi are
MIC (μg/mL) and IC50 (μg/mL), respectively.
Amphidinin F (4) was obtained as an optically active colorless
All compounds were active against Trichophyton mentagrophytes.
In addition, 1 and 3 were active against Staphylococcus aureus,
Bacillus subtilis, and Aspergillis niger, while 5 was active against
Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and
Candida albicans. Amphidinin E (3) showed modest cytotoxicity
(IC 5.8 μg/mL) against murine lymphoma P388 cells in vitro,
2
0
amorphous solid [[α] +21.8 (c 0.50, MeOH)]. The molecular
D
formula of 4 was defined as C H O by HRESIMS data (m/z
2
6
46
8
+
5
09.30845 [M + Na] , Δ −0.04 mmu). Inspection of NMR data
disclosed that 4 was the 4-O-α-ribofuranoside of 3 (Figure 6).
The H NMR data of the tris-(S)-MTPA ester (7a) of a sugar
1
50
moiety and the bis-(S)-MTPA ester (11a) of an aglycone of 4,
obtained by acid methanolysis of 4 and subsequent esterification
with (R)-MTPACl (Scheme 5), were coincident with that of the
tris-(S)-MTPA ester of methyl-D-ribofuranoside and the bis-(S)-
MTPA ester of 3, respectively. Thus, the absolute configuration
of 4 was elucidated to be 6S,7R,9S,11R,13R,1′S,2′R,3′S,4′R.
while it did not show activity (IC50 > 10.0 μg/mL) against
murine lymphoma L1210 cells and human epidermoid
carcinoma KB cells in vitro. Amphidinins C (1), D (2), and F
(3) were not cytotoxic (IC50 > 10.0 μg/mL) against murine
lymphoma P388 and L1210 cells and human epidermoid
carcinoma KB cells in vitro.
C
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Organic Letters
Letter
Amphidinins C−F (1−4) were all 4,5-seco-analogues of
amphidinolide Q (5), of which 2 and 4 were the first glycosides
related to amphidinolides. Although the common biosynthetic
pathway might be involved with production of amphidinins C−F
(
1−4) and amphidinolide Q (5), it is unknown which comes
first. To identify the origin of each carbon composing
amphidinins C−F (1−4) and amphidinolide Q (5), feeding
experiments with C-labeled acetates are currently in progress.
13
ASSOCIATED CONTENT
Supporting Information
■
*
S
Experimental procedures, tabulated NMR data, and NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: jkobay@pharm.hokudai.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Y. Iwabuchi, Graduate School of Pharmaceutical
Sciences, Tohoku University, for supplying AZADO, Dr. E.
Fukushi, Graduate School of Agriculture, Hokkaido University,
for measurements of HETLOC and J-resolved HMBC-2 spectra,
and Ms. S. Oka and Ms. A. Tokumitsu, Center for Instrumental
Analysis, Hokkaido University, for measurements of ESIMS.
This work was partly supported by The Naito Foundation,
Cooperative Research Program of Medical Mycology Research
Center, Chiba University, and Grant-in-Aid for Sports, Science
and Technology of Japan.
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