The Halicylindramides, Farnesoid X Receptor Antagonizing Depsipeptides from a Petrosia sp. Marine Sponge Collected in Korea

Article abstract of DOI:10.1021/acs.jnatprod.5b00871

Three new structurally related depsipeptides, halicylindramides F-H (1-3), and two known halicylindramides were isolated from a Petrosia sp. marine sponge collected off the shore of Youngdeok-Gun, East Sea, Republic of Korea. Their planar structures were elucidated by extensive spectroscopic data analyses including 1D and 2D NMR data as well as MS data. The absolute configurations of halicylindramides F-H (1-3) were determined by Marfey's method in combination with Edman degradation. The absolute configurations at C-4 of the dioxyindolyl alanine (Dioia) residues of halicylindramides G (2) and H (3) were determined as 4S and 4R, respectively, based on ECD spectroscopy. The C-2 configurations of Dioia in 2 and 3 were speculated to both be 2R based on the shared biogenesis of the halicylindramides. Halicylindramides F (1), A (4), and C (5) showed human farnesoid X receptor (hFXR) antagonistic activities, but did not bind directly to hFXR.

Full text of DOI:10.1021/acs.jnatprod.5b00871

Article
pubs.acs.org/jnp
The Halicylindramides, Farnesoid X Receptor Antagonizing
Depsipeptides from a Petrosia sp. Marine Sponge Collected in Korea
Dongyup Hahn,† Hiyoung Kim,‡ Inho Yang,‡ Jungwook Chin,† Hoosang Hwang,‡ Dong Hwan Won,‡
Byoungchan Lee,‡ Sang-Jip Nam,§ Merrick Ekins,⊥ Hyukjae Choi,*,∥ and Heonjoong Kang*,‡
†New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, 41061, Republic of Korea
‡School of Earth and Environmental Sciences, Seoul National University, NS-80, Seoul, 08826, Republic of Korea
§Department of Chemistry and Nano Science, Global Top 5 Program, Ewha Womans University, Seoul, 03760, Republic of Korea
⊥Queensland Museum, P.O. Box 3300, South Brisbane, Queensland 4101, Australia
∥College of Pharmacy, Yeungnam University, Gyeongsan, 38541, Republic of Korea
S
* Supporting Information
ABSTRACT: Three new structurally related depsipeptides,
halicylindramides F−H (1−3), and two known halicylindra-
mides were isolated from a Petrosia sp. marine sponge
collected off the shore of Youngdeok-Gun, East Sea, Republic
of Korea. Their planar structures were elucidated by extensive
spectroscopic data analyses including 1D and 2D NMR data as
well as MS data. The absolute configurations of halicylin-
dramides F−H (1−3) were determined by Marfey’s method in
combination with Edman degradation. The absolute config-
urations at C-4 of the dioxyindolyl alanine (Dioia) residues of
halicylindramides G (2) and H (3) were determined as 4S and
4R, respectively, based on ECD spectroscopy. The C-2
configurations of Dioia in 2 and 3 were speculated to both
be 2R based on the shared biogenesis of the halicylindramides. Halicylindramides F (1), A (4), and C (5) showed human
farnesoid X receptor (hFXR) antagonistic activities, but did not bind directly to hFXR.
dependent transcription regulates cholesterol metabolism at
multiple stages, such as synthesis, transport, elimination, and
reabsorption of BAs.13,14 Therefore, FXR has attracted
attention as a therapeutic target for the treatment of
dyslipidemia and cholestasis. In an animal model of non-
obstructive cholestasis, FXR activation improved BA clearance
from hepatocytes and protected against cholestatic liver
damage.15 However, in an animal model of obstructive
cholestasis, FXR activation aggravated hepatocyte injury and
parenchymal necrosis.16 Although potential risks of FXR
activation have been raised by studies based on preclinical
models of obstructive cholestasis, FXR antagonists are
considered to offer a potential means of treating obstructive
and/or severe cholestasis.17
arine sponges are one of the richest sources of bioactive
Mnatural products, including peptides with complex
structures and molecular weight exceeding 1000 Da. These
peptides, isolated from marine sponges, have intriguing
structural features, such as halogen atoms, macrocyclic rings,
and uncommon amino acids with high levels of modification,
such as N-methylation, epimerization (D-amino acids), and
unusual branches.1 Peptides found in marine sponges have also
been reported to be potent cytotoxins (e.g., arenastatin A, IC50
value of 8.3 pM against KB cells;2 hemiasterlin, ED50 value of
0.5 nM for antiproliferative activity against MCF-7 cells3), anti-
inflammatory agents (halipeptin A, 60% inhibition of paw
edema in mice at 0.3 mg/kg ip),4 antibacterial agents
(discodermins),5 antifungal agents (theonellamide F, MIC of
1.8−7.3 μM against pathogenic fungi;6 the discobahamins
against Candida albicans;7 the halicylindramides against
Mortierella ramanniana;8 jaspamide9 and the theopapuamides10
against amphotericin B-resistant C. albicans), and antiviral
agents (papuamide A, EC50 of 2.6 nM for inhibition of HIV-1RF
infection;11 homophymine A, cytoprotective activity against
HIV-1 infection with an IC50 of 75 nM).12
During our search for nonsteroidal human FXR (hFXR)
antagonists in marine organisms, we isolated three new and two
known halicylindramides, which are a type of depsipeptide,8
from a Petrosia sp. collected in Korean waters. Herein, the
Special Issue: Special Issue in Honor of John Blunt and Murray
Munro
The farnesoid X receptor (FXR) is expressed in the liver and
in the intestine and is a nuclear hormone receptor of bile acids
(BAs; e.g., chenodeoxycholic acid). In addition, its ligand-
Received: October 1, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00871
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Key TOCSY, HMBC, and NOESY correlations in halicylidramides F (1) and G (2).
isolation and structure elucidation of the three new compounds
and their hFXR antagonistic activities are described.
assigned on the basis of COSY, TOCSY, and HMBC spectra
analyses (Figure 1 and Table 1).
Connections between neighboring amino acids were
determined by HMBC and NOESY data analyses (Figure 1).
The deshielded H NMR signal of Hβ in Thr-1 at 5.14 ppm
and the strong HMBC correlation from the Hβ of Thr-1 to the
carbonyl carbon of Sar supported the presence of an ester
linkage between Thr-1 and Sar to form a macrocyclic lactone
ring.
RESULTS AND DISCUSSION
■
1
Halicylindramide F (1) was isolated as a pale yellow oil, and its
molecular formula was determined as C79H113N20O22S by
HRESIMS. The IR spectrum of 1 displayed absorption bands at
1655 and 1745 cm−1, indicating the presence of amide and ester
carbonyls, respectively. Amide carbonyl signals (δC 167.6−
173.6), amide NH signals (δH 6.78−8.45), and α proton signals
(δH 3.46−4.98) suggested that compound 1 was a peptide. A
formyl group signal (δH 7.89, δC 160.5) in the 1D NMR spectra
and a negative ninhydrin reaction suggested that the N-
terminus of 1 was blocked with a formyl group.
The conformation of Pro of 1 was deduced to be trans
because the value of Δδβγ (differential value between 13C
chemical shift carbon β and carbon γ of proline) is smaller than
9 ppm.18 The NOESY correlation between Hδ of Pro and Hβ
of Phe-1 also supported the trans conformation (Figure 1).
The absolute configurations of the constituent amino acids of
halicylindramide F (1) were determined using Marfey’s method
and the advanced Marfey’s method (Table S2).19,20 As a result,
D-Ala, L-Pro, D-Val, L-t-Leu, D-Trp, L-Arg, D-Cys(SO3H), L-Thr,
L-NMeGln, D-Asn, D-Phe, and L-Phe were found to be the
amino acids constituting 1. The absolute configurations of both
Thr-1 and Thr-2 residues were identified as L-Thr. The exact
positions of D- and L-Phe were assigned by coupled Edman
degradation21 followed by advanced Marfey’s analysis. Com-
pound 1 was deformylated and subjected to two Edman
reaction cycles to remove two amino acids (Ala and Phe-1)
from the N-terminus of halicylindramide F. The resulting
dodecapeptide was analyzed by the advanced Marfey’s method
and found to contain D-Phe without L-Phe, and thus the
configuration of Phe-1 was assigned as L.
Spectroscopic analyses of 1D and 2D NMR data, including
COSY, HMQC, HMBC, and TOCSY data, allowed us to assign
the constitutive amino acids of 1 (Figure 1 and Table S1,
1
Supporting Information). The H and 13C NMR peaks of N-
formylated Ala, Pro, Val, Arg, Cys(SO3H), Thr, and Asn were
assigned using COSY, TOCSY, and HMBC spectra (Figure 1).
The assignment of tert-Leu (t-Leu) was established by the
COSY correlation between amide NH and the α proton and
the HMBC correlation from singlet methyl protons (9H, δH
0.70) to the α and β carbons (δC 60.4 and 33.7, respectively,
Table 1).
NMeGln and NMeGly (Sar) were also assigned by COSY,
TOCSY, and HMBC spectroscopic analysis (Figure 1). The 1H
and 13C NMR chemical shifts of two singlet methyl signals (δH
2.93/δC 30.6 and δH 2.73/δC 35.6) and the HMBC correlations
from these two methyl protons to α carbons (δC 54.9 and 48.6,
respectively) indicated the presence of NMeGln and Sar
(Figure 1 and Table 1).
The molecular formula of 2 was determined as
C79H11279BrN20O24S based on the protonated molecule peak
at m/z 1835.7062 [M + H]+ as determined by HRESIMS. The
presence of a bromine atom in compound 2 was confirmed by
the pattern of the ion cluster of the protonated molecule at m/z
1835.7062:1836.6591:1837.6467:1838.6130 with an intensity
ratio of 6:7:9:10, and this isotope pattern is similar to that
calculated for a compound with the assigned molecular formula
The COSY correlation between the most deshielded
exchangeable proton signal (δH 10.6) and one singlet aromatic
proton signal (δH 7.15) and the COSY correlations between
four aromatic protons (δH 7.60, 6.96, 7.03, and 7.31) suggested
the presence of an indole of Trp. Two sets of Phe were also
B
DOI: 10.1021/acs.jnatprod.5b00871
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Journal of Natural Products
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Table 1. NMR Data of Halicylindramides F−H (1−3) in DMSO-d6 at 600 MHz (1H) and 150 MHz (13C)
1
2
3
a
a
a
position
NCHO
δC, mult.
δH, (J in Hz)
7.89, s
position
NCHO
δC, mult.
δH, (J in Hz)
7.88, s
δC, mult.
δH, (J in Hz)
160.5, C
160.5, C
161.4, C
7.90, s
b
Ala α
β
46.4, CH
19.4, CH3
4.33, q (7.3)
0.89, d (7.2)
8.14, brd (7.8)
Ala α
β
46.4, CH
18.6, CH3
4.31, q (7.3)
0.93, d (7.0)
8.18, brs
47.6, CH
19.5, CH3
4.31, m
0.92, d (7.0)
8.18, brs
NH
CO
Phe-1 α
β
NH
CO
BrPhe α
β
171.4, C
51.7, CH
34.7, CH2
171.4, C
51.9, CH
36.2, CH2
172.3, C
52.6, CH
37.1, CH2
b
b
4.72, m
4.71, m
4.72, m
2.74, d (13.8)
3.01, m
2.69−2.72 m
2.71, m
b
b
3.02, m
3.01, m
C1
137.7, C
C1
137.1, C
138.2, C
b
b
C2/C6
C3/C5
C4
129.3, CH
128.0, CH
126.7, CH
7.25, m
C2/C6
C3/C5
C4
131.8, CH
130.8, CH
119.5, C
7.26, m
132.6, CH
131.7, CH
120.3, C
7.26, m
7.22, m
7.42, m
7.43, d (8.1)
7.16, m
b
NH
8.45, brd (8.4)
NH
8.44, brd (8.3)
8.42, m
CO
169.4, C
59.8, CH
29.8, CH2
CO
171.0, C
59.5, CH
29.5, CH2
171.4, C
59.8, C
b
Pro α
β
4.46, m
1.88, m
2.07 m
1.89, m
3.67, m
Pro α
β
4.46, m
4.40, m
b
1.89, m
30.5, CH2
1.80, m
b
2.10, m
2.02, m
b
γ
24.4, CH2
47.0, CH2
γ
24.6, CH2
46.9, CH2
1.90, m
25.2, CH2
47.8, CH2
1.83, m
δ
δ
3.70−3.62 m
3.60, m
3.71, m
CO
Val α
β
171.1, C
CO
Val α
β
169.8, C
168.7, C
b
b
b
57.2, CH
31.2, CH
17.3, CH3
18.7, CH3
4.38, m
57.2, CH
30.7, CH
17.4, CH3
19.4, CH3
4.38, m
58.1, CH
31.8, CH
18.2, CH3
20.3, CH3
4.37, m
b
2.07, m
2.08, m
2.09, m
γ
0.78, d (6.7)
0.83, d (7.2)
7.81, m
γ
0.76, d (6.7)
0.81, d (6.7)
0.77, d (6.7)
0.82, d (6.5)
γ′
γ′
b
b
NH
CO
t-Leu α
β
NH
CO
t-Leu α
β
7.80, m
7.78, m
171.0, C
60.4, CH
33.7, C
171.9, C
60.2, CH
34.1, C
171.4, C
61.3, CH
34.9, C
b
b
4.18, d (8.7)
4.13, m
4.13, m
γ
26.5, CH3
0.70, s
γ
26.6, CH3
0.86, s
27.6, CH3
0.89, s
b
b
NH
CO
Trp α
β
7.76, m
NH
CO
Dioia α
β
7.82, m
7.91, m
169.9, C
54.0, CH
26.8, CH2
170.0, C
48.5, CH
39.0, CH2
170.9, C
50.8, CH
39.9, CH2
b
b
4.57, m
3.16, m
2.96, m
7.15, s
4.46, m
4.42, m
2.30, m
1.89, m
2.18, m
1.96, m
C2
124.1, CH
109.8, C
C2
178.2, C
74.0, C
179.8, C
75.2, C
C3
C3
C3a
C7a
C4
136.6, C
C3a
131.7, C
141.7, C
124.0, CH
122.0, CH
128.0, CH
109.8, CH
132.0, C
142.1, C
125.0, CH
122.6, CH
129.3, CH
110.8, CH
119.5, C
C7a
b
118.1, CH
120.8, CH
111.2, CH
127.2, CH
7.60, d (7.9)
C4
7.35, d (7.6)
7.23, m
C5
6.96, dd (7.5, 7.1)
7.03, dd (7.4, 7.6)
7.31, d (8.0)
C5
6.90, dd (7.6, 7.3)
6.94, brt (7.3)
b
b
C6
C6
7.18, m
7.19, m
C7
C7
6.78, d (8.0)
10.24, brs
6.80, brd (7.5)
10.32, brs
6.18, brs
1-NH
10.6, brs
1-NH
3-OH
6.16, brs
NH
8.29, brd (6.5)
NH
8.21, brd (7.9)
8.33, brs
CO
170.0, C
CO
169.8, C
169.1, C
b
b
Arg α
51.5, CH
29.5, CH2
23.6, CH2
39.7, CH2
156.7, C
4.39, m
1.61, m
1.28, m
2.94, m
Arg α
52.0, CH
29.1, CH2
23.2, CH2
40.2, CH2
156.8, C
4.22, m
53.0, CH
29.5, CH2
24.8, CH2
41.2, CH2
157.7, C
4.27, m
b
β
β
1.65, m
1.40, m
1.68, m
γ
γ
1.45, m
b
b
δ
δ
3.01, m
3.01, m
guanidine
guanidine
b
b
NH
7.99, brs
NH
7.81, brs
7.82, brs
CO
171.4, C
50.9, CH
52.4, CH2
CO
171.0, C
50.9, CH
52.5, CH2
172.3, C
51.8, CH
53.4, CH2
Cys(SO3H) α
4.60, m
Cys(SO3H) α
4.59, m
4.59, m
b
β
2.93, m
β
2.90, m
2.96, m
NH
CO
8.35, brd (6.5)
NH
CO
8.30, brd (8.3)
8.30, brd (7.2)
170.8, C
51.8, CH
169.4, C
51.9, CH
169.0, C
52.8, CH
Thr-1 α
4.83, d (8.5)
Thr-1 α
4.84, d (8.5)
4.83, d (8.6)
C
DOI: 10.1021/acs.jnatprod.5b00871
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Journal of Natural Products
Article
Table 1. continued
1
2
3
a
a
a
position
δC, mult.
δH, (J in Hz)
position
δC, mult.
δH, (J in Hz)
δC, mult.
δH, (J in Hz)
β
γ
69.5, CH
17.3, CH3
5.14, d (6.7)
1.16, d (6.5)
7.98, m
β
γ
69.2, CH
17.4, CH3
5.14, d (6.7)
1.17, d (6.5)
7.96, m
69.8, CH
18.2, CH3
5.13, brq (6.6)
1.16, d (6.5)
8.01, brs
NH
NH
CO
167.9, C
54.9, CH
24.7, CH2
CO
169.2, C
54.5, CH
24.7, CH2
170.5, C
55.2, CH
26.7, CH2
b
b
NMeGln α
4.97, m
1.70, m
1.89, m
1.87, m
NMeGln α
4.98, m
4.96, m
b
β
β
1.70, m
1.70, m
b
1.89, m
b
γ
31.6, CH2
γ
31.5, CH2
1.89, m
32.4, CH2
1.87, m
b
1.92, m
NMe
30.6, CH3
173.6, C
2.93, s
NMe
30.4, CH3
173.5, C
2.94, s
31.3, CH3
174.4, C
2.94, s
CONH2
6.68, brs
7.17, brs
CONH2
6.64, brs
6.67, brs
b
b
7.17, brs
7.16, brs
CO
168.8, C
54.2, CH
38.0, CH2
CO
Phe α
β
167.5, C
54.0, CH
37.9, CH2
168.6, C
55.0, CH
39.2, CH2
b
Phe-2 α
β
4.67, d (7.2)
3.00, m
4.69, d (7.2)
2.98, m
4.69, m
b
2.96, m
b
3.02, m
C1
137.3, C
C1
137.1, C
138.0, C
b
b
C2/C6
C3/C5
C4
129.4, CH
128.0, CH
126.3, CH
7.22, m
C2/C6
C3/C5
C4
129.3, CH
129.0, CH
126.2, CH
7.18, m
130.2, CH
128.9, CH
127.2, CH
7.18, m
b
b
7.14, m
7.23, m
7.23, m
b
b
7.12, m
7.15, m
7.17, m
NH
CO
7.55, brd (6.6)
NH
CO
7.56, brd (6.6)
7.56, brs
171.1, C
60.9, CH
65.6, CH
20.2, CH3
170.7, C
60.7, CH
65.5, CH
20.1, CH3
171.9, C
61.9, CH
66.3, CH
21.0, CH3
Thr-2 α
β
3.85, m
Thr-2 α
β
3.85, m
3.86, m
3.96, m
3.96, m
3.96, brs
b
γ
0.89, d (4.8)
4.87, d (5.1)
7.92, brd (7.9)
γ
0.89, d (6.3)
4.88, d (4.9)
7.95, brs
0.85, m
OH
NH
CO
OH
NH
CO
4.90, brs
7.96, brd (6.8)
169.8, C
45.9, CH
36.9, CH2
169.6, C
46.0, CH
36.9, CH2
170.1, C
46.8, CH
38.1, CH2
b
b
Asn α
β
4.98, m
Asn α
β
4.98, m
4.99, m
b
2.08, m
2.09, m
2.08, m
b
2.64, m
2.30, m
2.66, m
NH
7.50, brd (9.6)
6.78, brs
7.32, brs
NH
7.51, brd (7.6)
6.72, brs
7.33, brs
7.50, brd (9.1)
6.77, brs
CONH2
171.2, C
CONH2
172.1, C
173.0, C
7.32, brs
CO
172.1, C
CO
169.4, C
170.2, C
b
Sar α
48.6, CH2
3.46, d (15.7)
4.40, m
Sar α
49.5, CH2
3.49, d (17.3)
50.6, CH2
3.48, m
b
b
4.42, m
4.42, m
NMe
35.6, CH3
167.6, C
2.73, s
NMe
35.4, CH3
167.5, C
2.74, s
36.4, CH3
168.5, C
2.73, s
CO
CO
a
b
Derived from the combination of 13C NMR, HSQC, and HMBC data. Signals overlapped.
(S28, Supporting Information). Like compound 1, the NMR
data of 2 also showed characteristic features of a peptide, such
as amide carbonyl signals (δC 167.5−173.5), amide NH signals
(δH 6.64−8.44), and α proton signals (δH 3.49−4.98). The 1D
and 2D NMR spectra of 2 were closely related to those of
compound 1, indicating that compound 2 was also a
halicylindramide (Tables 1 and S3). However, aromatic proton
and carbon signals (δH 7.15, δC 124.1, C-2 of Trp) and the
nonprotonated carbon signal (δC 109.8, C-3 of Trp) of 1 were
not found in the 1H and 13C NMR spectra of 2, and a
deshielded carbonyl carbon at 178.2 ppm and an oxygenated
carbon at 74.0 ppm were additionally observed in the 13C NMR
spectrum of 2, which suggested the Trp residue of 1 was
modified in 2. Furthermore, the methylene protons at 2.30 and
1.89 ppm of 2 showed HMBC correlations to the carbons at
178.2 and 74.0 ppm. In addition, a deshielded exchangeable
proton (δH 10.24) exhibited an HMBC correlation with the
carbon at 74.0 ppm. These spectroscopic features indicated the
presence of a dioxindolyalanine (Dioia) group in compound 2
rather than the indole of Trp. In addition, the 1H and 13C NMR
spectra of compound 2 showed a 1,4-disubstituted benzene ring
and a monosubstituted benzene ring rather than the two
monosubstituted benzene rings in the spectra of 1. The 13C
NMR chemical shift of C-4 of the 1,4-disubstituted benzene of
2 was 119.5 ppm, which corresponded to bromination at the C-
4 position. Furthermore, spectroscopic data analyses enabled us
to assign N-formylated Ala, Pro, Val, t-Leu, Arg, Cys(SO3H),
Thr, NMeGln, Asn, and Sar. Their assignments were secured
based on HMBC and NOESY correlations, and the structure of
halicylindramide G (2) was determined (Figure 1).
The molecular formula of 3 was also determined to be
C79H11279BrN20O24S based on its protonated molecule peak at
m/z 1835.7119 in [M + H]+ and its ion cluster by HRESIMS.
1
The H NMR and 13C NMR spectra of compound 3 were
similar to those of compound 2 (Table 1). Spectroscopic data
analysis including 1D and 2D NMR data analysis indicated that
D
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Chart 1
the planar structure of compound 3 was identical to the planar
structure of compound 2 (Table S4).
The conformation of Pro in compounds 2 and 3 was
assigned to be trans based on the small Δδβγ values and on the
NOESY correlations between Hδ of Pro and Hα of BrPhe
(Figure 1).18
The absolute configurations of the constitutive amino acids,
except for the Dioia unit of halicylindramides G (2) and H (3),
were determined by Marfey’s method and the advanced
Marfey’s method. They were found to possess identical
absolute configurations of the amino acids except for the
Dioia unit (Table S5). The Dioia unit decomposed during the
acid hydrolysis, and attempts to prepare Marfey’s derivatives
from the Dioia standards also failed.22,23 The absolute
configurations of its stereogenic centers were investigated by
comparing the ECD absorption of the parent compounds with
four authentic Dioia standards (Figure 2).24,25 The ECD
spectra of 2 at 240 and 265 nm showed positive and negative
Cotton effects, respectively, indicating a Dioia unit with the 4S
configuration. On the other hand, the ECD absorption spectra
of 3 showed negative and positive Cotton effects at 240 and
265 nm, respectively, and thus, the absolute configuration of C-
4 of Dioia in 3 was assigned as R. The Trp residue of previously
reported halicylindramides A−E and halicylindramide F (1)
was found to be a D-type amino acid, and thus, the absolute
configurations of the α position of Dioia in 2 and 3 were
speculated to be R based on a shared biogenesis of the
halicylindramides.
Along with compounds 1−3, compounds 4 and 5 were also
obtained, and their spectroscopic data including LRESIMS data
and 1D/2D NMR data were identical to those of previously
reported data of halicylindramides A and C.8
Due to the structural similarity, compounds 2 and 3 were
assumed to be oxidation artifacts derived from compound 5. A
portion of compound 5 in a dried vial and a portion of 5
dissolved in MeOH (1 mg/mL) were stored at room
temperature and −20 °C under dark conditions for 8 weeks,
and no changes were obversed in this examination. Therefore, it
could be speculated that the oxidation from Trp to Dioia occurs
under the highly oxidative conditions in living organisms such
Figure 2. ECD spectra of halicylindramides G (2) and H (3) and four
authentic standards of Dioia.
as cyanobacteria or marine sponges or the Dioia unit is
prepared before the halicylindramides’ biosynthesis.
The antagonistic activities of compounds 1−5 against hFXR
were evaluated using a co-transfection assay. Compounds 1, 4,
and 5 showed potent inhibitory effects of hFXR transactivation
with IC50 values of 6.0, 0.5, and 5.0 μM, respectively, while 2
and 3 were inactive up to 100 μM. Compounds 1−5 are not
cytotoxic against HepG2 cells (IC50 values of 100, 100, 100, 20,
and 100 μM, respectively; IC50 value of doxorubicin = 2.1 μM,
cytotoxicity = IC50 values less than 10 μM). In particular,
halicylindramide A (4) most potently inhibited hFXR trans-
activation with an IC50 value of 0.5 μM, which is much lower
than that of the well-known FXR antagonist E-guggulsterone
(IC50 41.0 μM,).26,27 Interestingly, halicylindramides G (2) and
E
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Article
further partitioned between EtOAc and H2O. Finally, the H2O-soluble
fraction was extracted with 1-BuOH. Fractions were assayed for their
hFXR antagonizing activities. The 1-BuOH fraction exhibited the most
potent bioactivity on both activity screening platforms. 1-BuOH
solubles were fractionated by size exclusion chromatography through
Sephadex LH-20 (1000 × 50 mm) using 70% MeOH in CH2Cl2. The
bioactive compounds were finally purified by RP-HPLC [Phenomenex
HydroRP, 250 × 10 mm, flow rate 2.0 mL/min, UV detection at 210
nm] with 30% CH3CN in H2O. Finally, three new depsipeptides,
halicylindramides F (1, 10 mg), G (2, 8 mg), and H (3, 7 mg) and two
known analogue, halicylindramides A (4, 30 mg)8 and C (5, 260 mg),8
were obtained.
H (3) did not antagonize ligand-dependent hFXR trans-
activation, and the presence of Trp in the halicylindramides is
speculated to be crucial for their hFXR antagonistic activity. In
direct binding experiments using the BIAcore system, 1, 4, and
5 did not show any significant inhibition of the recruitment of
cofactor SRC-1 peptide to hFXR at concentrations higher than
their IC50 values determined in a cell-based co-transfection
assay. Therefore, it seems that the hFXR antagonism of the
halicylindramides F, A, and C is achieved not by their direct
binding to LBD of the receptor but rather by an indirect
manner such as through a membrane-bound receptor or a
kinase/phosphatase pathway. Halicylindramides A−E have
been previously reported as antifungal agents against Mortierella
ramanniana and cytotoxic agents against P388 murine leukemia
cells.8
All of the previously reported halicylindramides A−E were
discovered from Halichondria cylindrata, while the two known
halicylindramides A and C along with three new analogues (1−
3) in this study were isolated from Petrosia sp. 4921. The
structures of the halicylindramides show high degrees of
modification such as N-methylation, epimerization to D-amino
acids, and halogenation, which is reminiscent of cyanobacterial
peptides.28 Due to the structural features and the occurrence of
the halicylindramides in two different genera of marine
sponges, the producers of the halicylindramides may be
sponge-associated cyanobacteria.
Halicylindramide F (1): pale yellow oil; [α]25D −8.0 (c 0.1, MeOH);
UV (MeOH) λmax 207 nm (log ε 4.32), 287 nm (log ε 3.06); IR (KBr)
νmax 1745, 1655 cm−1; 1H, 13C, and 2D NMR data, Table 1; HRESIMS
m/z 1725.7976 [M + H]+ (calcd for C79H113N20O22S 1725.8059).
Halicylindramide G (2): pale yellow oil; [α]25 −10 (c 0.1, MeOH);
D
UV (MeOH) λmax 207 nm (log ε 4.40), 261 nm (log ε 3.32); ECD
(1.0 mg/mL, CH3CN), λmax (Δε) 322 (0.59), 265 (−3.65), and 231
1
(9.39) nm; IR (KBr) νmax 1735, 1640 cm−1; H, 13C, and 2D NMR
data, Table 1; HRESIMS m/z 1835.7062 [M + H]+ (calcd for
C79H11279BrN20O24S 1835.7063).
Halicylindramide H (3): pale yellow oil; [α]25D −10 (c 0.1, MeOH);
UV (MeOH) λmax 207 nm (log ε 4.41), 260 nm (log ε 3.31); ECD
(1.0 mg/mL, CH3CN), λmax (Δε) 319 (0.88), 266 (2.99), and 239
(−10.54) nm; IR (KBr) νmax 1735, 1640 cm−1; 1H, 13C, and 2D NMR
data, Table 1; HRESIMS m/z 1835.7119 [M + H]+ (calcd for
C79H11279BrN20O24S 1835.7063).
Halicylindramide A (4): [α]25 −1.5 (c 0.1, MeOH) (reported
D
[α]23 −1.4).8
In conclusion, three new and two previously reported
halicylindramides were isolated from the extract of a Korean
Petrosia sp. marine sponge. The structures of compounds 1−3
are closely related to halicylindramide C (5). The L-BrPhe of 5
is substituted with L-Phe in 1. The Dioia unit in compounds 2
and 3, oxidation products of Trp, have not been reported in the
previously reported halicylindramides. Halicylindramides F (1),
A (4), and C (5) were found to be potent antagonists of hFXR,
with IC50 values as low as 0.5 μM. Currently, target
identification of the halicylindramides is under way.
D
Halicylindramide C (5): [α]25 −6.0 (c 0.1, MeOH); (reported
D
[α]23 −6.1).8
D
Advanced Marfey’s Method.19,20 A peptide (200 μg) was
treated with 100 μL of 6 N HCl at 110 °C for 30 min, and the
resulting acid hydrolysates were lyophilized. Dried products were
dissolved in 100 μL of 1 M NaHCO3, and samples were halved and
transferred to two separate vials. A 25 μL amount of 1% L-FDLA (1-
fluoro-2,4-dinitrophenyl-5-L-leucine amide) in acetone was added to
one, and 25 μL of a 1% D- and L-FDLA mixture in acetone was added
to the other. Solutions were vortexed and incubated at 40 °C for 60
min, reactions were quenched by adding 25 μL of 2 N HCl, reaction
mixtures were diluted with 100 μL of MeOH, and 10 μL of each
solution was injected into a HPLC-ESIMS.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Rudolph Research Autopol III using a 5 cm cell. UV
spectra were acquired on a Scinco UVS-2100 using a path length of 1
cm. ECD spectra were collected in the wavelength range 200 to 400
nm using a JASCO J-715 (path length: 0.01 dm; cell volume: 400 μL;
scan speed: 50 nm/min; resolution: 0.5 nm). IR spectra were obtained
using a Nicolet ThermoElectron Nicolet 5700 FT-IR spectrometer
using KBr plates. NMR spectra were recorded in DMSO-d6 (δC 39.5,
δH 2.50) using solvent peaks as internal standards on a Bruker Avance
The reaction products from advanced Marfey’s method were
separated on an RP-HPLC column (Capcell Pak C-18 MGII column,
20 × 50 mm, 5.0 μm, Shiseido, Japan) by step gradient elution [0.1%
TFA in H2O (A) and 100% CH3CN (B); 0−10 min, B, 30%; 10−50
min, B, 30−70%; 50−60 min, B, 70%; 60−70 min, B, 100%; flow rate,
100 μL/min; column temperature, 30 °C] and detected by ESIMS
(sheath gas flow rate, 20 arbitrary units; auxiliary gas flow rate, 1.40
arbitrary units; sweep gas flow rate, 1.5 arbitrary units; capillary
voltage, 36 V; capillary temperature, 277 °C; tube lens, 120 V).
Retention times of individual Marfey’s derivatives are included in the
Supporting Information (Table S2).
Preparation of Dodecapeptide from 1 by Modified Edman
Degradation.21 Halicylindramide F (1, 300 μg) was deformylated by
treating it with 100 μL of 1.5 N HCl at rt for 24 h, and reaction
products were lyophilized. To couple peptide N-termini with the
Edman reagent, the products were dissolved in 60 μL of Edman
coupling solution (phenylisothiocyanate/H2O/EtOH/triethylamine =
1:1:7:1), vortexed, and heated at 50 °C for 8 min. Mixtures were then
desolvated in a centrifugal evaporator at 50 °C for 5 min, and the
residue were resuspended in 100 μL of deionized H2O and washed
three times with 100 μL of heptane/EtOAc (7:1, v/v). The resulting
aqueous layers were dried in a centrifugal evaporator at 50 °C for 15
min. To perform cyclization and cleavage, 50 μL of 8 mM BF3/Et2O in
CH3CN was added to the residue, and the reaction mixture was
vortexed and heated at 50 °C for 5 min. The product mixture was
dried under a N2 stream, resuspended in 100 μL of H2O, and washed
three times with 100 μL of heptane/EtOAc (1:5, v/v). The aqueous
DPX-600 spectrometer (600 and 150 MHz for H and 13C NMR,
1
respectively). LR- and HRFABMS were obtained using a JEOL JMS-
AX505WA mass spectrometer. HPLC was carried out using a
Younglin SDV 30 Plus HPLC system equipped with a Younglin M
720 UV detector.
Animal Material. The Petrosia sp. 4921 specimen (HK11) was
collected by scuba at a depth of 10−20 m off the shore of Youngdeok-
Gun, East Sea, Republic of Korea. The identification of the specimen
was done by an Australian sponge taxonomist, Merrick Ekins (S84,
Supporting Information). A voucher specimen (G331811) is deposited
at the Queensland Museum, Australia, and at the Center for Marine
Natural Products and Drug Discovery, Seoul National University,
Korea (registered as HK11).
Isolation of Halicylindramides F−H, A, and C (1−5). Collected
sponges (18 kg, wet weight) were lyophilized, and the dried materials
(2.1 kg) were powdered and extracted three times with 50% MeOH in
CH2Cl2 at room temperature (rt) for 3 days. The extract (570 g) was
partitioned between hexanes and MeOH, and MeOH solubles were
F
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layers were concentrated in a centrifugal evaporator to obtain the
dealanyl peptide, which was subjected to a second Edman reaction to
prepare the dodecapeptide that lacks 2-Phe.
with test compounds. Serially diluted samples were added to wells, and
doxorubicin was used as a positive control. Plates were incubated for
approximately 24 h at 37 °C in humidified air containing 5% CO2
before staining with MTT. MTT assessment of cytotoxicity was
performed as a modification of Mosmann’s method,30 replacing the
overlying medium with 60 μL of a 1:6 dilution of MTT stock (5 mg/
mL PBS, pH = 7.4) in complete culture medium. Mitochondrial
dehydrogenases in live cells converted the MTT to an insoluble purple
formazan crystal. Following the incubation (30 min at 37 °C) with the
MTT media mixture, the remaining solution was removed from the
wells and the crystals were solubilized by the addition of 50 μL of
DMSO. After incubating the plate at rt in the dark for 15 min,
absorbance was read at 550 nm with a VICTOR2 (PerkinElmer).
Preparation of Authentic Dioia Standards from D- and L-
Trp.23 DMSO (450 μL) was slowly added to a vial of 2.5 mL of 12 N
HCl at rt. Phenol (50 mg) was then added followed by D-Trp (500
mg) in 15 mL of acetic acid. The reaction mixture was stirred at rt for
5 h, and the acetic acid, HCl, and DMSO were then removed in vacuo.
The resulting syrup was resuspended in 750 μL of acetic acid, stored at
4 °C for 12 h, and dried under vacuum, and the oxyindolyl-D-alanine
C-4 epimers (D-Oia, 243 mg, 45%) produced were obtained by RP-
HPLC (Phenomenex Polar RP, 10 × 250 mm, 4 μm, 100% H2O, 3.0
mL/min, 250 nm, retention time 24 min).
D-Oia diastereomers: C11H12N2O3, ESIMS m/z [M + H]+ 221; UV
1
(H2O) 252 nm; H NMR (DMSO-d6) 11.01 (1H, brs), 10.65 (1H,
ASSOCIATED CONTENT
■
brs), 9.18 (2H, brs), 8.50 (2H, brs), 7.64−6.83 (8H, m), 4.68 (1H, m),
4.45 (1H, m), 3.10−2.90 (2H, m), 2.35 (2H, m), 2.15 (2H, m).
D-Oia diastereomers (100 mg) were dissolved in 10 mL of 0.1 N
NaOH, and the solution was aerated with O2 for 12 h at rt. Conversion
was confirmed by the formation of two products, which were both
more polar than D-Oia on NP-TLC (1-BuOH/EtOAc/H2O/AcOH =
1:1:1:1) and produced spots of almost equal intensity (ninhydrin).
The reaction mixture was adjusted to pH 4 with acetic acid and
refrigerated overnight, and the residual syrup was dried in vacuo. The
two diastereomers were separated by RP-HPLC (YMC AQ S-5, 10 ×
250 mm, 5 μm, 280 nm) by step gradient elution [100% H2O (eluent
A) and 100% CH3CN (eluent B); 0−18 min, A, 100%, 2.5 mL/min;
18−28 min, B: 25%, 3.5 mL/min]. D-Dioia-1 and -2 were eluted with
retention times of 22 and 26 min, respectively. In addition, L-Dioia-1
and -2 were also prepared using the same procedure from L-Trp via L-
Oia.
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00871.
One-dimensional (1H and 13C) and two-dimensional
(HMQC, COSY, TOCSY, NOESY, and HMBC) NMR
spectra of 1−3 and surface plasmon resonance spectros-
copy data of 1, 4, and 5 (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*Tel (H. Choi): (82) 53-810-2824. Fax: (82) 53-810-4654. E-
mail: h5choi@yu.ac.kr.
*Tel (H. Kang): (82) 2-880-5730. Fax: (82) 2-883-9289. E-
mail: hjkang@snu.ac.kr.
D-Dioia-1: [α]25 +20 (c 0.1, H2O); ECD (0.5 mg/mL, CH3CN),
D
λmax (Δε) 318 (−0.24), 265 (21.49), and 239 (−50.77) nm; LRESIMS
m/z [M + H]+ 237.
D-Dioia-2: [α]25 −39 (c 0.1, H2O); ECD (0.5 mg/mL, CH3CN),
Notes
D
λmax (Δε) 315 (0.42), 263 (−20.07), and 240 (52.64) nm; LRESIMS
The authors declare no competing financial interest.
m/z [M + H]+ 237.
L-Dioia-1: [α]25 −20 (c 0.1, H2O); ECD (0.5 mg/mL, CH3CN),
D
ACKNOWLEDGMENTS
λmax (Δε) 318 (0.57), 266 (−20.53), and 238 (51.54) nm; LRESIMS
■
m/z [M + H]+ 237.
This work was supported by the 2013 Yeungnam University
research grant.
L-Dioia-2: [α]25 +39 (c 0.1, H2O); ECD (0.5 mg/mL, CH3CN),
D
λmax (Δε) 315 (−0.42), 263 (20.07), and 240 (−52.64) nm; LRESIMS
m/z [M + H]+ 237.
REFERENCES
■
Cell-Based Co-transfection Assay. CV-1 cells were seeded in 96-
well plates in Dulbecco’s modified Eagle’s medium (GIBCO)
supplemented with 10% resin-charcoal-stripped fetal bovine serum in
humidified air containing 5% CO2 at 37 °C for 24 h. Transient co-
transfections with pCMX-hFXR, CMX-β-GAL, and Tk-(EcRE)6-LUC
were carried out using SuperFect (Qiagen), according to the
manufacturer’s instructions. After incubation for 24 h, co-transfected
cells were treated with a control vehicle (DMSO) or indicated
compounds for the hFXR agonist test. Ligands were treated with 50
μM chenodeoxycholic acid (CDCA) to test for antagonism on hFXR.
Cells were harvested at 24 h, and luciferase activities were assayed as
described previously.13,14 Luciferase activities were normalized versus
β-galactosidase activity expressed by the control plasmid CMX-β-GAL.
Cotransfections were performed in triplicate.
Surface Plasmon Resonance (SPR) Spectroscopy. Direct
bindings of 1, 4, and 5 to the ligand binding domain (LBD) of
hFXR were monitored by SPR spectroscopy using a BIAcore system.29
hFXR LBD (4 μM), preincubated for 1 h with E-guggulsterone or 1−5
in the presence of 50 μM CDCA, was injected over a sensor chip
surface coated with immobilized coactivator peptide (SRC-1).
Changes in resonance units (RUs) were used to monitor the ligand-
induced association of the hFXR LBD and SRC-1 peptide.
(1) Ebada, S. S.; Proksch, P. In Handbook of Marine Natural Products;
Fattorusso, E.; Gerwick, W. H.; Taglialatela-Scafati, O., Eds.; Springer:
New York, 2012; Vol. 1, Chapter 4, pp 191−293.
(2) Kobayashi, M.; Aoki, S.; Ohyabu, N.; Kurosu, M.; Wang, W.;
Kitagawa, I. Tetrahedron Lett. 1994, 35, 7969−7972.
(3) Anderson, H. J.; Coleman, J. E.; Andersen, R. J.; Roberge, M.
Cancer Chemother. Pharmacol. 1997, 39, 223−226.
(4) Randazzo, A.; Bifulco, G.; Giannini, C.; Bucci, M.; Debitus, C.;
Cirino, G.; Gomez-Paloma, L. J. Am. Chem. Soc. 2001, 123, 10870−
10876.
(5) Ryu, G.; Matsunaga, S.; Fusetani, N. Tetrahedron Lett. 1994, 35,
8251−8254.
(6) Matsunaga, S.; Fusetani, N.; Hashimoto, K.; Waelchli, M. J. Am.
Chem. Soc. 1989, 111, 2582−2588.
(7) Gunasekera, S. P.; Pomponi, S. A.; McCarthy, P. J. J. Nat. Prod.
1994, 57, 79−83.
(8) Li, H.; Matsunaga, S.; Fusetani, N. J. Med. Chem. 1995, 38, 338−
343.
(9) Scott, V. R.; Boehme, R.; Matthews, T. R. Antimicrob. Agents
Chemother. 1998, 32, 1154−1157.
(10) Plaza, A.; Bifulco, G.; Keffer, J. L.; Lloyd, J. R.; Baker, H. L.;
Bewley, C. A. J. Org. Chem. 2009, 74, 504−512.
(11) Ford, P. W.; Gustafson, K. R.; McKee, M. C.; Shigematsu, N.;
Maurizi, L. K.; Pannell, L. K.; Williams, D. E.; de Silva, D.; Lassota, P.;
Allen, T. M.; van Soest, R.; Andersen, R. J.; Boyd, M. R. J. Am. Chem.
Soc. 1999, 121, 5899−5909.
Cytotoxicity Assay. HepG2 human liver carcinoma cells (5.0 ×
103 cells/mL) were plated to 96-well plates containing 0.1 mL of
Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with
10% resin-charcoal-stripped fetal bovine serum. Cells were incubated
overnight (37 °C, 5% CO2) to allow recovery before being treated
G
DOI: 10.1021/acs.jnatprod.5b00871
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(12) Zampella, A.; Sepe, V.; Luciano, P.; Bellotta, F.; Monti, M. C.;
́
D’Auria, M. V.; Jepsen, T.; Petek, S.; Adeline, M.; Laprevote, O.;
Aubertin, A.; Debitus, C.; Poupat, C.; Ahond, A. J. Org. Chem. 2008,
73, 5319−5327.
(13) Makishima, M.; Okamoto, A. Y.; Repa, J. J.; Tu, H.; Learned, R.
M.; Luk, A.; Hull, M. V.; Lustig, K. D.; Mangelsdorf, D. J.; Shan, B.
Science 1999, 284, 1362−1365.
(14) Parks, D. J.; Blanchard, S. G.; Bledsoe, R. K.; Chandra, G.;
Consler, T. G.; Kliewer, S. A.; Stimmel, J. B.; Willson, T. M.; Zavacki,
A. M.; Moore, D. D.; Lehmann, J. M. Science 1999, 284, 1365−1368.
(15) Liu, Y.; Binz, J.; Numerick, M. J.; Dennis, S.; Luo, G.; Desai, B.;
MacKenzie, K. I.; Mansfield, T. A.; Kliewer, S. A.; Goodwin, B.; Jones,
S. A. J. Clin. Invest. 2003, 112, 1678−1687.
(16) Fiorucci, S.; Antonelli, E.; Rizzo, G.; Renga, B.; Mencarelli, A.;
Riccardi, L.; Orlandi, S.; Pellicciari, R.; Morelli, A. Gastroenterology
2004, 127, 1497−1512.
(17) Fiorucci, S.; Distrutti, E.; Ricci, P.; Giuliano, V.; Donini, A.;
Baldelli, F. Expert Opin. Ther. Targets 2014, 18, 1449−1459.
(18) Siemion, I. Z.; Wieland, T.; Pook, K. Angew. Chem., Int. Ed. Engl.
1975, 14, 702−703.
(19) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
(20) Fuji, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem.
1997, 69, 5146−5151.
(21) Edman, P. Acta Chem. Scand. 1950, 4, 283−293.
(22) Simat, T. J.; Steinhart, H. J. Agric. Food Chem. 1998, 46, 490−
498.
(23) Labroo, R. B.; Cohen, L. A. J. Org. Chem. 1990, 55, 4901−4904.
(24) Takayama, H.; Shimizu, T.; Sada, H.; Harada, Y.; Kitajima, M.;
Aimi, N. Tetrahedron 1999, 55, 6841−6846.
(25) Robinson, S. J.; Morinaka, B. I.; Amagata, T.; Tenney, K.; Bray,
W. M.; Gassner, N. C.; Lokey, R. S.; Crews, P. J. Med. Chem. 2010, 53,
1651−1661.
(26) Urizar, N. L.; Liverman, A. B.; Dodds, D. T.; Silva, F. V.;
Ordentlich, P.; Yan, Y.; Gonzalez, F. J.; Heyman, R. A.; Mangelsdorf,
D. J.; Moore, D. D. Science 2002, 296, 1703−1706.
(27) Burris, T. P.; Montrose, C.; Houck, K. A.; Osborne, H. E.;
Bocchinfuso, W. P.; Yaden, B. C.; Cheng, C. C.; Zink, R. W.; Barr, R.
J.; Hepler, C. D.; Krishnan, V.; Bullock, H. A.; Burris, L. L.; Galvin, R.
J.; Bramlett, K.; Stayrook, K. R. Mol. Pharmacol. 2005, 67, 948−954.
(28) Choi, H.; Pereira, A. R.; Gerwick, W. H. In Handbook of Marine
Natural Products; Fattorusso, E.; Gerwick, W. H.; Taglialatela-Scafati,
O., Eds.; Springer: New York, 2012; Vol. 1, Chapter 2, pp 55−152.
(29) Fujino, T.; Sato, Y.; Une, M.; Kanayasu-Toyoda, T.; Yamaguchi,
T.; Shudo, K.; Inoue, K.; Nishimaki-Mogami, T. J. Steroid Biochem.
Mol. Biol. 2003, 87, 247−252.
(30) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.
H
DOI: 10.1021/acs.jnatprod.5b00871
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