Structures and potential antitumor activity of sesterterpenes from the marine sponge hyrtios communis

Article abstract of DOI:10.1021/np400350k

The extract of marine sponge Hyrtios communis was found to inhibit activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) in T47D human breast tumor cells. Bioassay-guided isolation led to the identification of six new (1-6) and five previously reported (7-11) sesterterpene analogues and two unrelated sesterterpenes. Two new sesterterpenes, thorectidaeolide A (1) and 4-acetoxythorectidaeolide A (2), and luffariellolide (11) were among the most potent inhibitors of hypoxia (1% O ₂)-induced HIF-1 activation (IC₅₀ values of 3.2, 3.5, and 3.6 μM, respectively). Luffariellolide (11) exhibited a significant level of cytotoxicity that mirrored its HIF-1 inhibitory activity. Neither compound 1, compound 2, nor any of the other less active sesterterpenes suppressed breast tumor T47D or MDA-MB-231 cell viability.

Full text of DOI:10.1021/np400350k

Article
pubs.acs.org/jnp
Structures and Potential Antitumor Activity of Sesterterpenes from
the Marine Sponge Hyrtios communis
Jun Li,†,‡ Lin Du,†,§ Michelle Kelly,∥ Yu-Dong Zhou,*,† and Dale G. Nagle*,†,⊥
†Department of Pharmacognosy, and ⊥Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi,
University, Mississippi 38677, United States
∥Coasts and Oceans National Centre, National Institute of Water and Atmospheric Research Limited (NIWA), 41 Market Place,
Auckland Central, Auckland 1010, New Zealand
S
* Supporting Information
ABSTRACT: The extract of marine sponge Hyrtios communis was found to
inhibit activation of the transcription factor hypoxia-inducible factor-1 (HIF-
1) in T47D human breast tumor cells. Bioassay-guided isolation led to the
identification of six new (1−6) and five previously reported (7−11)
sesterterpene analogues and two unrelated sesterterpenes. Two new
sesterterpenes, thorectidaeolide A (1) and 4-acetoxythorectidaeolide A (2),
and luffariellolide (11) were among the most potent inhibitors of hypoxia
(1% O2)-induced HIF-1 activation (IC50 values of 3.2, 3.5, and 3.6 μM,
respectively). Luffariellolide (11) exhibited a significant level of cytotoxicity
that mirrored its HIF-1 inhibitory activity. Neither compound 1, compound
2, nor any of the other less active sesterterpenes suppressed breast tumor
T47D or MDA-MB-231 cell viability.
estertepenes represent an important group of active
RESULTS AND DISCUSSION
■
1
Ssecondary metabolites from marine sponges.
Marine
Compound 1 was obtained as a colorless oil. The molecular
formula C25H36O4 was deduced from high-resolution electro-
spray ionization mass spectrometry (HRESIMS) data (index of
hydrogen deficiency = 8). The infrared (IR) spectrum indicated
the presence of an α,β-unsaturated γ-butenolide group (1778
and 1745 cm−1), a ketone (1710 cm−1), and a hydroxy moiety
sesterterpenes that contain both a linear component and an
α,β-unsaturated γ-lactone ring moiety are found primarily in
sponges of the dictyoceratid families Thorectidae (Luffariella
Thiele, Hyrtios Duchassaing and Michelotti, Thorectandra
Lendenfeld, Cacospongia Schmidt, Fasciospongia Burton,
Aplysinopsis Lendenfeld, Fascaplysinopsis Bergquist, and Thor-
ecta Lendenfeld) and Irciniidae (Sarcotragus Schmidt), the
unrelated dendroceratid family Dictyodendrillidae (Acanthoden-
drilla Bergquist), and the astrophorid family Pachastrellidae
(Brachiaster Wilson).1−3 These relatively lipophilic sponge
metabolites possess a wide variety of biological activities,
including antibacterial, anti-inflammatory, Cdc25 phosphatase
inhibitory, cytotoxic, molluscicidal, nicotinic receptor antago-
nistic, and fish-deterrent activities (reviewed by Proksch and co-
workers1). As part of our molecular-targeted antitumor natural
product discovery program, an extract of the marine sponge
Hyrtios communis (Carter, 1885) (Order Dictyoceratida, Family
Thorectidae) [National Cancer Institute (NCI) Open
Repository collection number C030113] was found to inhibit
hypoxia-inducible factor-1 (HIF-1) activation in T47D cells.
Bioassay-guided isolation led to the identification of six new
(1−6) and five previously reported (7−11) sesterterpene
analogues and two unrelated sesterterpenes. The structures of
the previously reported sesterterpenes were confirmed by
comparison of the NMR spectra and specific rotation values to
published data. Herein, the chemical and biological character-
ization of these compounds is described.
1
(3422 cm−1). The H nuclear magnetic resonance (NMR)
spectrum displayed resonances corresponding to five methyl
groups [δH 1.72 (3H, s, CH3-20), 1.66 (3H, s, CH3-24), 1.65
(3H, s, CH3-23), and 1.17 (6H, s, CH3-21 and 22)], three
olefinic protons [δH 5.97 (1H, br s, H-2), 5.14 (1H, t, J = 7.2
Hz, H-6), and 5.11 (1H, t, J = 6.8 Hz, H-10)], one
oxymethylene [δH 4.87 (2H, br s, H-25)], one oxymethine
[δH 4.62 (1H, t, J = 6.4 Hz, H-4)], and seven methylene units
(δH 2.01−2.54). The 13C and distortionless enhancement by
polarization transfer (DEPT) NMR spectra revealed a total of
25 carbon resonances for five methyl, eight methylene, four
methine, and eight quaternary carbons. Six of the eight degrees
of hydrogen deficiency could be accounted for by the NMR
data (a ketone group, an ester carbonyl moiety, and four
carbon−carbon double bonds). Thus, it was apparent that two
rings were present in the structure. The presence of a
trimethylcyclohex-3-enone moiety was indicated by 13C
resonances [δC 136.4 (C-14), 48.1 (C-15), 215.9 (C-16),
36.1 (C-17), 31.7 (C-18), 127.4 (C-19), 19.6 (CH3-20), and
24.6 (CH3-21/22)] that were comparable to those reported for
Received: April 30, 2013
Published: August 14, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
1492
dx.doi.org/10.1021/np400350k | J. Nat. Prod. 2013, 76, 1492−1497

Journal of Natural Products
Article
olefinic methyl [δH 1.66 (H3-24) and 1.65 (H3-23) and δC 16.5
(C-24) and 16.1 (C-23)] and methylene [δC 40.1 (C-12), 39.7
(C-8), 35.4 (C-5), and 26.3 (C-9)] substituents in the NMR
spectra of compound 1 revealed the occurrence of a geranyl
moiety stretching from C5 to C12, which was further confirmed
by HMBC correlations, as shown in Figure 1. Correlations in
COSY and HMBC spectra succeeded in positioning a hydroxy
group at C-4, α to the γ-butenolide functionality and β to a
trisubstituted double bond (partial structure C1−C7) (Figure
1). Further comparison of 13C NMR chemical shifts to those
reported for a similarly C-4 hydroxy-substituted analogue,
luffarin R,7 supported assembly of the C1−C13 subunit. The
connectivity of the trimethylcyclohex-3-enone moiety with that
of the C1−C13 subunit was achieved by observation of HMBC
correlations between H2-13 and C-14, C-15, and C-19 as well
as between H2-12 and C-14. The configurations of both
trisubstituted double bonds were determined to be E because of
upfield 13C NMR chemical shifts for the olefinic methyl
substituents [δC 16.1 (C-23) and 16.5 (C-24)].8 Thus,
compound 1 could be envisioned to be an oxidized analogue
of the relatively simplified sesterterpene luffariellolide γ-lactone,
a compound Faulkner and co-workers originally prepared from
compound 11 by NaBH4 reduction of its γ-hydroxybutenolide.9
The absolute configuration at C-4 was determined by the
modified Mosher’s method. The (R)- and (S)-α-methoxy-α-
(trifluoromethyl)phenylacetyl (MTPA) esters (12 and 13) of
compound 1 were prepared in NMR tubes with deuterated
pyridine solutions of (S)- and (R)-MTPA chloride, respectively.
1
The H NMR spectra of compounds 12 and 13 were obtained
by recording data in the reaction NMR tubes. The Δδ (δS −
1
δR) values observed in the H NMR spectra were calculated.
2,2,4-trimethyl-3-(3-hydroxy-3-methylpent-4-enyl)cyclohex-3-
enone.4 Subsequent analysis of the heteronuclear multiple-bond
correlation (HMBC) spectrum identified the following long-
range correlations: gem-dimethyl system [δH 1.17 (H3-21 and
H3-22) coupled to δC 215.9 (C-16), 136.4 (C-14), and 48.1 (C-
15)]; methyl singlet [δH 1.72 (H3-20) coupled to δC 136.4 (C-
14), 127.4 (C-19), and 31.7 (C-18)]; methylene triplet [δH
2.52 (H2-17) coupled to δC 215.9 (C-16), 127.4 (C-19), and
31.7 (C-18) and a methylene triplet [δH 2.33 (H2-18) coupled
to δC 215.9 (C-16), 136.4 (C-14), 127.4 (C-19), and 36.1 (C-
17). These results, together with the observed correlation
spectroscopy (COSY) correlations between H2-17 and H2-18,
further confirmed the C14−C22 partial structure shown in
Figure 1. Analysis of the 1H and 13C NMR spectra of
The resulting negative Δδ values for H-2 and H2-25 and
positive Δδ values for H2-5, H-6, H2-8, H-10, and H3-24
(Figure 2) were consistent with the 4R configuration. On the
Figure 2. Δδ (δS − δR) values (in ppm, data obtained in pyridine-d5)
for the MTPA esters of compound 1.
basis of the family level classification of the sesquiterpene-rich
source sponge, H. communis, compound 1 was assigned the
name thorectidaeolide A.
The molecular formula C27H38O5 was deduced for
compound 2 from its HRESIMS data. As in compound 1,
the IR spectrum indicated the presence of an α,β-unsaturated γ-
butenolide moiety (1780 and 1745 cm−1) and a ketone moiety
1
(1710 cm−1). The H and 13C NMR spectra of compound 2
Figure 1. Selected COSY and HMBC correlations of compound 1
(arrows pointing protons to carbons).
(Tables 1 and 2) indicated that it was essentially identical to
compound 1, except for the presence of resonances for an
additional acetate ester substituent (δH 2.10 and δC 169.8 and
20.8). The downfield shift of C-4 (δC 69.6) and H-4 (δH 5.62, t,
J = 6.4 Hz) relative to the resonances observed in compound 1
indicated that the C-4 hydroxy group was acetylated in
compound 2. This was further confirmed by HMBC
correlations between H-4 (δH 5.62) and the carbonyl carbon
(δC 169.8) of acetate ester. Therefore, compound 2 was given
the trivial name 4-acetoxythorectidaeolide A.
compound 1 supported the presence of a β-substituted α,β-
unsaturated γ-butenolide moiety [δH 5.97 (1H, s, H-2) and 4.87
(2H, br s, H-25) and δC 173.7 (C-1), 114.9 (C-2), 171.9 (C-3),
and 71.2 (C-25)], as observed in the chemical structures of
luffariolide B,5 deoxymanoalide,6 and the luffarins P−W.7
Resonances for two trisubstituted double bonds [δH 5.14 (H-6)
and 5.11 (H-10) and δC 141.7 (C-7), 136.1 (C-11), 123.6 (C-
10), and 117.3 (C-6)] and their corresponding associated
1493
dx.doi.org/10.1021/np400350k | J. Nat. Prod. 2013, 76, 1492−1497

Journal of Natural Products
Article
a b
,
1
Table 1. H NMR Spectroscopic Data for Compounds 1−6 (Multiplicities, J in Hz)
number
1
2
3
4
5
6
2
5.97, br s
4.62, t (6.4)
2.45, t (6.8)
5.14, t (7.2)
2.10, m
5.97, br s
5.84, br s
5.85, br s
5.84, br s
2.45, t (6.0)
2.32, t (6.0)
5.09, m
5.98, br s
4.61, t (6.8)
2.45, t (6.8)
5.14, t (7.2)
1.99−2.06, m
2.08−2.16, m
5.09, t (6.4)
2.08−2.16, m
1.99−2.06, m
1.42, m
4
5.62, t (6.4)
2.52, t (6.8)
5.07, t (7.2)
2.02, m
2.51, 2.41 m
2.31, t (6.8)
5.12, t (6.8)
1.99−2.05, m
2.06−2.14, m
5.12, t (6.8)
2.06−2.14, m
1.99−2.05, m
2.49, m
5
2.34, m
6
5.18, t (6.8)
2.72, d (6.0)
8
2.01, t (6.4)
2.07, t (6.4)
5.08, m
9
2.10, m
2.04, m
5.62, dt (15.6, 6.0)
5.57, d (15.6)
1.61, m
10
12
13
16
17
18
20
21
22
23
24
25
5.11, t (6.8)
2.03, m
5.11, t (6.4)
2.00, m
1.90, 1.70, m
1.87, 1.57, m
1.65, 1.38, m
1.54 m
2.10, m
2.06, m
2.00, m
1.41, m
2.52, t (6.8)
2.33, t (6.8)
1.72, s
2.52, t (6.8)
2.32, t (6.8)
1.71, s
2.53, t (6.8)
2.34, t (6.8)
1.72, s
1.56, m
1.57, m
1.90, t (6.0)
1.58, s
2.30, 1.96, m
4.89, 4.83, s
0.96, s
1.91, t (6.0)
1.60, s
1.17, s
1.16, s
1.17, s
0.98, s
1.00, s
1.17, s
1.16, s
1.17, s
0.98, s
0.88, s
1.00, s
1.65, s
1.59, s
1.63, s
1.33, s
1.60, s
1.65, s
1.66, s
1.62, s
1.65, s
1.64, s
1.60, s
1.67, s
4.87, br s
4.83, dd (17.6, 1.6)
4.75, dd (17.6, 1.6)
2.10 s
5.99, br s
5.99, br s
5.98, br s
4.87, br s
OAc
a
b
Assignments based on heteronuclear single-quantum coherence (HSQC) and HMBC spectra. All spectra were recorded in CDCl3.
a b
,
Table 2. 13C NMR Spectroscopic Data for Compounds 1−6
position
1
2
3
4
5
6
1
173.7, C
172.9, C
171.2, C
171.0, C
171.5, C
173.6, C
2
114.9, CH
171.9, C
116.4, CH
167.4, C
117.6, CH
169.3, C
118.0, CH
169.1, C
117.7, CH
169.6, C
115.0, CH
171.7, C
3
4
68.3, CH
35.4, CH2
117.3, CH
141.7, C
69.6, CH
32.4, CH2
116.4, CH
140.7, C
27.7, CH2
25.1, CH2
122.2, CH
137.2, C
27.6, CH2
25.5, CH2
123.3, CH
136.2, C
27.6, CH2
25.5, CH2
122.3, CH
137.0, C
68.3, CH
35.4, CH2
117.2, CH
142.0, C
5
6
7
8
39.7, CH2
26.3, CH2
123.6, CH
136.1, C
39.7, CH2
26.5, CH2
123.6, CH
135.8, C
39.6, CH2
26.4, CH2
123.9, CH
135.5, C
42.2, CH2
126.0, CH
138.0, CH
73.4, C
39.4, CH2
26.0, CH2
123.9, CH
136.0, C
39.8, CH2
26.3, CH2
122.9, CH
136.9, C
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
4-OAc
40.1, CH2
28.2, CH2
136.4, C
40.1, CH2
28.3, CH2
136.5, C
40.1, CH2
28.3, CH2
136.5, C
42.8, CH2
22.9, CH2
136.6, C
33.6, CH2
30.9, CH2
80.6, C
40.2, CH2
27.9, CH2
137.0, C
48.1, C
48.1, C
48.2, C
35.1, C
39.7, C
35.0, C
215.9, C
215.8, C
216.2, C
39.8, CH2
19.5, CH2
32.8, CH2
127.1, C
38.0, CH2
22.7, CH2
34.0, CH2
150.4, C
39.8, CH2
19.5, CH2
32.7, CH2
127.0, C
36.1, CH2
31.7, CH2
127.4, C
36.1, CH2
31.6, CH2
127.3, C
36.1, CH2
31.6, CH2
127.3, C
19.6, CH3
24.6, CH3
24.6, CH3
16.1, CH3
16.5, CH3
71.2, CH2
19.6, CH3
24.6, CH3
24.6, CH3
16.0, CH3
16.4, CH3
71.1, CH2
169.8, C
19.6, CH3
24.6, CH3
24.6, CH3
16.0, CH3
16.2, CH3
99.0, CH
19.8, CH3
28.6, CH3
28.6, CH3
27.5, CH3
16.5, CH3
99.0, CH
108.3, CH2
24.1, CH3
22.1, CH3
16.0, CH3
16.3, CH3
99.3, CH
19.8, CH3
28.6, CH3
28.6, CH3
16.1, CH3
16.4, CH3
71.2, CH2
20.8, CH3
a
b
Assignments based on HSQC and HMBC spectra. Spectra were recorded in CDCl3.
Compound 3 was obtained as a colorless oil. The molecular
the presence of a hydroxy group. The 1H and 13C NMR spectra
(Tables 1 and 2) were comparable to those of compound 1,
with similar resonances for the region C8−C24. The 13C
resonances at δC 171.2 (C-1), 117.6 (C-2), 169.3 (C-3), and
99.0 (C-25) and their corresponding H resonances [δH 5.84
(1H, br s, H-2) and 5.99 (H, br s, H-25)] indicated that
formula C25H36O4 was deduced by HRESIMS (index of
hydrogen deficiency = 8). As in compound 1, the presence of
an ester carbonyl and ketone moiety was verified by the IR
(1759 and 1710 cm−1) and 13C NMR spectra (δC 171.2 and
216.2). A broad IR absorption centered at 3299 cm−1 suggested
1
1494
dx.doi.org/10.1021/np400350k | J. Nat. Prod. 2013, 76, 1492−1497

Journal of Natural Products
Article
compound 3 possessed a β-substituted α,β-unsaturated γ-
hydroxybutenolide moiety rather than the β-substituted α,β-
unsaturated γ-butenolide moiety in compound 1. A comparison
of the NMR spectra of the C1−C11 region to those reported for
the structural analogues acantholide B (8),10 acantholide C
(7),10 16-hydroxyluffariellolide (9),11 and luffariellolide (11)12
facilitated assembly of its C1−C11 component. The Δ6,7 and
cm−1). Similarities in the NMR spectra between compounds 5
(Tables 1 and 2) and 11 suggested that compound 5 is also a
luffariellolide-type sesterterpene. The main differences in the
1H NMR spectrum of compound 5 and that of compound 11
were the absence of one olefinic methyl group resonance in
compound 5 and the appearance of resonances attributable to
an exomethylene moiety [δH 4.89 and 4.83 (H2-20)].
Observation of a methylene resonance (δC 108.3) and a
corresponding quaternary carbon (δC 150.4) further supported
the presence of an exomethylene functionality in the structure
of compound 5. An oxygenated quaternary carbon [δC 80.6 (C-
14)] was observed by 13C NMR and DEPT experiments. The
assignment and placement of the C-14 hydroxy substituent and
the exomethylene at C-19 were deduced by observation of
Δ
10,11 double bonds were both assigned E configurations based
on the observation of the upfield 13C NMR chemical shifts of
the C-24 (δC 16.2) and C-23 (δC 16.0) methyl group
resonances, respectively. Compound 3 was assigned the trivial
name thorectidaeolide B.
The molecular formula of compound 4 (C25H38O4) was
deduced by HRESIMS (index of hydrogen deficiency = 7). The
13C NMR spectrum (Table 2) displayed resonances for a
disubstituted double bond (δC 126.0 and 138.0), two
trisubstitued double bonds (δC 118.0, 169.1, 123.3, and
136.2), a fully substituted double bond (δC 136.6 and 127.1),
and a carbonyl carbon (δC 171.0). According to the index of
hydrogen deficiency, the structure of compound 4 was deduced
to be bicyclic because of the absence of any other observable sp
or sp2 carbon resonances. These data suggested that compound
1
long-range H−13C HMBC correlations (H2-13 to C-14, H3-
21/22 to C-14, H2-18 to C-14, H2-13 to C-19, H2-18 to C-19,
H2-17 to C-19, H2-20 to C-19, H2-20 to C-14, and H2-20 to C-
18). The hydroxy and exomethylene-substituted cyclohexane
system in the structure was further supported by a comparison
of the relevant NMR spectra of compound 5 to those reported
for the similarly substituted marine sesterterpene metabolites
tasnemoxide C14 and epimuqubilin B.15 The 13C chemical shifts
of the C-23 and C-24 methyl groups (δC 16.0 and 16.3,
respectively) were indicative of E configurations for the Δ6,7
and Δ10,11 double bonds. Compound 5 was assigned the name
thorectidaeolide D.
1
4 is a luffariellolide-type sesterterpene. The H and 13C NMR
spectra (Tables 1 and 2) were comparable to those of
compound 11,8 indicating that both compounds possessed
identical cyclohexene and γ-hydroxybutenolide terminal ring
systems. The major differences between compounds 4 and 11
were found only in the C9−C11 region of the structure. The
C9−C11 segment of the structure was deduced from analysis of
the DEPT and HMBC spectra (Figure 3). The DEPT spectrum
Compound 6 was isolated as a colorless oil. The molecular
formula C25H38O3 was deduced from its HRESIMS data, 13C
NMR, and DEPT spectra. The IR spectrum indicated that the
structure contained an α,β-unsaturated γ-butenolide moiety
(1779 and 1742 cm−1) and a hydroxy group (3423 cm−1).
When the NMR spectra of compounds 6 and 1 were compared,
it was evident that they were structurally similar, except that
compound 6 lacked a ketone functionality. Examination of the
13C NMR spectrum of compound 6 suggested that the
resonances associated with the trimethylcyclohex-3-enone
moiety in compound 1 were replaced in this new compound
by a set of resonances [δC 137.0 (C-14), 35.0 (C-15), 39.8 (C-
16), 19.5 (C-17), 32.7 (C-18), 127.0 (C-19), 19.8 (CH3-20),
and 28.6 (CH3-21/22)] that was attributable to a trimethylcy-
clohexene moiety. The structure was further supported by
observation that the chemical shifts for the C13−C22 partial
structure in compound 6 matched those of the corresponding
unit in luffariellolide (11)12 and the luffariolides A−E.5 The
proton and carbon NMR resonances in the spectra of
compound 6 were readily assigned from the COSY, HSQC,
and HMBC spectra. As in compound 1, the upfield 13C NMR
chemical shifts for the olefinic methyl resonances (δC 16.1 and
16.4) indicated that both double bonds had E configurations.
As observed with similarly substituted sesterterpenes of the
series,2,6 compound 6 was deduced to have a 4R configuration
based on the observation that it exhibited a similar specific
rotation to that of compound 1 (+6.0 and +4.0, respectively).
Thus, compound 6 was given the name thorectidaeolide E.
The transcription factor HIF-1 regulates cellular oxygen
homeostasis, the ability of tumor cells to adapt and grow under
hypoxic conditions, and represents an important molecular
target for anticancer drug discovery.16 Concentration−response
studies were performed to determine the effects of compounds
1−10 and 11 on HIF-1 activation in a T47D cell-based reporter
assay. The three new sesterterpenes (1−3) that possess a
simple α,β-unsaturated γ-butenolide moiety inhibited hypoxia
(1% O2, 16 h)-induced HIF-1 activation with IC50 values of 3.2
Figure 3. Selected COSY (solid lines) and HMBC correlations of
compound 4 (arrows pointing from protons to carbons).
indicated the presence of an oxygenated quaternary carbon (δC
73.4), which was assigned to C-11. The HMBC correlations
between the methyl singlet at δH 1.33 (H3-23) and carbons at
δC 73.4 (C-11) and 138.0 (C-10) were observed. The HMBC
spectrum also showed long-range correlations between the H-9
(δH 5.62) and H-10 (δH 5.57) olefinic protons and the
oxygenated C-11 quaternary carbon (δC 73.4). The structure
was further supported by a comparison of the relevant NMR
data of compound 4 to those reported for two C-11 hydroxy-
substituted analogues luffariolide G13 and tasnemoxide A.14
The C9−C11 segment and both the C1−C8 and C12−C22 partial
structures were connected on the basis of observed COSY and
HMBC correlations between these three structural components
(Figure 3). The E configuration of the Δ9,10 double bond was
deduced from the large coupling constant (J = 15.6 Hz)
between H-9 and H-10. Compound 4 was assigned the trivial
name thorectidaeolide C. Insufficient material was available for
further experiments to determine the C-11 absolute config-
uration.
The C25H38O4 molecular formula of compound 5 was
deduced from its HRESIMS spectrum (index of hydrogen
deficiency = 7). The IR spectrum exhibited absorption bands
corresponding to a hydroxy group (3285 cm−1), an ester
carbonyl (1752 cm−1), and an exomethylene substituent (895
1495
dx.doi.org/10.1021/np400350k | J. Nat. Prod. 2013, 76, 1492−1497

Journal of Natural Products
Article
species is digitate, greenish brown and brick red, respectively, and in
the nature of the skeleton, which in the latter species forms a distinct
regular rounded reticulation of primary and secondary fibers packed
with sand. Voucher specimens of 0CDN9952 are held in the
collections of the Coral Reef Research Foundation, Palau, Michelle
Kelly, NIWA, Auckland, and in the Department of Invertebrate
Zoology, National Museum of Natural History, Smithsonian
Institution, Washington, D.C.
μM [95% confidence interval (CI) of 2.8−3.5 μM], 3.5 μM (CI
of 3.3−3.8 μM), and 6.2 μM (CI of 5.8−6.7 μM), respectively.
Among the other sesterterpenes that possess a γ-hydroxybute-
nolide moiety, only acantholide A (10) [IC50 of 7.1 μM (CI of
6.5−7.8 μM)] and compound 11 [IC50 of 3.6 μM (CI of 3.4−
3.9 μM)] significantly (i.e., IC50 < 10 μM) inhibited HIF-1
activation. A standard 48 h treatment study was performed in
T47D and MDA-MB-231 cells to determine the effects of test
compounds on cell proliferation/viability. None of the
compounds, except compound 11, significantly inhibited cell
proliferation (IC50 > 10 uM). Consistent with the previous
reports of cytotoxicity,17 compound 11 exhibited antiprolifer-
ative activity in both T47D and MDA-MB-231 cells [IC50
values of 5.1 μM (CI of 4.6−5.7) and 4.5 μM (CI of 4.1−4.8),
respectively] that mirrored its HIF-1 inhibitory activity. The
cytotoxic activity of compound 11 was previously attributed to
its ability to act as an agonist of retinoic acid receptors
(RARs).16 The Schiff-base-forming reactivity of the γ-
hydroxybutenolide ring system is believed to be responsible
for the anti-inflammatory and enzyme inhibitory (e.g.,
phosphatase cdc25A) properties of compound 11 and other
related marine natural products.9 Alternatively, compounds that
do not possess a hydroxylated γ-butenolide ring system (i.e.,
compounds 1, 2, and 6) exhibit pronounced HIF-1 inhibitory
activity. Further mechanistic studies are required to resolve the
precise means by which compounds such as these new
sestertepenes disrupt HIF-mediated hypoxic signaling in
tumor cells.
Extraction and Isolation. Ground material of H. communis was
extracted with H2O. The residual sample was then lyophilized and
extracted with 50% MeOH in CH2Cl2;18 residual solvents were
removed under vacuum; and the extract (number C030113) was
stored at −20 °C in the NCI repository at the Frederick Cancer
Research and Development Center. To reduce the loss of potential
HIF-1/antitumor activity,19 Si gel chromatography was avoided in the
bioassay-guided separation of the active constituents. The H. communis
extract (2.8 g) inhibited HIF-1 activation in T47D cells by 97% (5 μg
mL−1) and was passed over Sephadex LH-20 CC with 50% MeOH in
CH2Cl2 to yield four fractions. The active fourth fraction (1.81 g, 98%
HIF-1 inhibition at 5 μg mL−1) was further separated into eight
fractions by VLC (C18) and eluted with step gradients of MeOH in
H2O (30:70, 50:50, 60:40, 70:30, 80:20, 85:15, 90:10, and 100:0). The
active fourth fraction (71 mg, 95% HIF-1 inhibition at 5 μg mL−1) that
eluted with 70% MeOH was purified by semi-preparative HPLC [Luna
5 μm, C18(2) 100 Å, 250 × 10.0 mm, isocratic 82% MeOH in H2O, 4.0
mL min−1] to produce compounds 7 (acantholide C,10 1.1 mg, 0.04%
yield, tR of 8 min), 1 (2.1 mg, 0.08% yield, tR of 12 min), 8
(acantholide B,10 3.1 mg, 0.11% yield, tR of 15 min), 2 (4.0 mg, 0.14%
yield, tR of 17 min), a mixture of compounds 3 and 9 (5.1 mg, tR of 18
min), 4 (1.1 mg, 0.04% yield, tR of 20 min), 10 (acantholide A, 2.1 mg,
0.08% yield, tR of 21 min), and 5 (3.0 mg, 0.11% yield, tR of 23 min).
The mixture of compounds 3 and 9 was further purified by a semi-
preparative HPLC [Luna 5 μm, C18(2) 100 Å, 250 × 10.0 mm,
isocratic 75% MeCN in H2O, 4.0 mL min−1] to produce compounds 9
(16-hydroxyluffariellolide,11 1.4 mg, 0.05% yield, tR of 12 min), and 3
(2.4 mg, 0.09% yield, tR of 16 min). The active fifth fraction (1557 mg,
inhibited HIF-1 activation in T47D cells by 99% at 5 μg mL−1) that
eluted with 80% MeOH from the C18 VLC was purified by a semi-
preparative HPLC [Luna 5 μm, C18(2) 100 Å, 250 × 10.0 mm,
isocratic 90% MeOH in H2O, 4.0 mL min−1] to produce compounds 6
(1.8 mg, 0.06% yield, tR of 18 min), 11 (ircinolide B,
petrosaspongiolide B,20 2.5 mg, 0.09% yield, tR of 23 min) and 11
(luffariellolide,8 1000 mg, 35.71% yield, tR of 24 min). The active sixth
fraction (50 mg, inhibited HIF-1 activation in T47D cells by 86% at 5
μg mL−1) that eluted with 85% MeOH from the C18 VLC was purified
by recrystallization to produce ircinolide A (petrosaspongiolide A,20 20
mg, 0.7% yield).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Specific rotation values were
obtained on an AP IV/589-546 digital polarimeter. A Bruker Tensor
27 Genesis Series FTIR was used to obtain the IR spectra. The NMR
spectra were recorded in CDCl3 on AMX-NMR spectrometers
(Bruker) operating at 400 MHz for 1H and 100 MHz for 13C.
Residual solvent resonances (δ 7.26 for H and δ 77.16 for 13C) were
1
used as internal references for the NMR-spectra-recorded running
gradients. The HRESIMS spectra were determined on a Bruker
Daltonic micro TOF fitted with an Agilent 1100 series HPLC and an
electrospray ionization source. HPLC was performed on a Waters
system, equipped with a 600 controller and a 2998 photodiode array
detector. A semi-preparative HPLC column (Phenomenex Luna, RP-
18, 5 μm, 250 × 10.00 mm) was employed for isolation. TLC was
performed using Merck Si60F254 plates, sprayed with a 10% H2SO4
solution in EtOH, heated, and/or visualized under UV at 254 nm. The
purities of the compounds were judged on the percentage of the
integrated signal at UV of 220 nm. All purified compounds submitted
for bioassay were at least 95% pure as judged by HPLC and supported
Thorectidaeolide A (1): colorless oil; [α]2D5 +4.0 (c 0.20, MeOH);
IR (film) νmax 3422, 2928, 1778, 1745, 1710, 1443, 1131, 1025, 885,
859 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z
423.2516 [M + Na]+ (calcd for C25H36O4Na, 423.2511).
4-Acetoxythorectidaeolide A (2): colorless oil; [α]2D5 +3.8 (c 0.26,
MeOH); IR (film) νmax 2929, 1780, 1745, 1710, 1443, 1374, 1228,
1135, 1026, 885, 857 cm−1; 1H and 13C NMR data, see Tables 1 and 2;
HRESIMS m/z 465.2596 [M + Na]+ (calcd for C27H38O5Na,
465.2617).
1
by H NMR analysis.
Sponge Material. Sponge material was obtained from the U.S.
NCI’s Open Repository Program and collected from a depth of 18−21
m from the northern reefs region off the coast of Palau on June 22,
2009 (collection number 0CDN9952 and NPID number C030113).
Upon collection, material was frozen at −20 °C and later ground in a
meat grinder. The sponge was massive to lobate; the surface was
conulose and rough; and 5 mm diameter oscules are scattered on the
tops of lobes. The texture is cheese-like, compressible, and rubbery,
and the color in life is gray to black externally and beige to tan
internally. The skeleton consists of broad fascicles of sand grains that
extend vertically in the sponge toward the surface conules. There is no
obvious spongin surrounding the fascicles, and the cellular material
between the fascicules is dense, uniform, and very fleshy. The sponge
is most closely comparable to H. communis (Carter, 1885) (Order
Dictyoceratida, Family Thorectidae) and differs from other common
Indo-Pacific species Hyrtios erecta (Keller, 1899) and Hyrtios reticulatus
(Thiele, 1899) in gross morphology and coloration, which in the latter
Thorectidaeolide B (3): colorless oil; [α]2D5 +3.2 (c 0.13, MeOH);
IR (film) νmax 3299, 2929, 1759, 1710, 1443, 1357, 1277, 1126, 1024,
945 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z
399.2523 [M − H]− (calcd for C25H35O4, 399.2535).
Thorectidaeolide C (4): colorless oil; [α]2D5 +6.0 (c 0.07, MeOH);
1
IR (film) νmax 3340, 2925, 1741, 1439, 1360, 1128, 945 cm−1; H and
13C NMR data, see Tables 1 and 2; HRESIMS m/z 401.2691 [M −
H]− (calcd for C25H37O4, 401.2692).
Thorectidaeolide D (5): colorless oil; [α]2D5 −6.4 (c 0.19, MeOH);
IR (film) νmax 3285, 2930, 1752, 1444, 1383, 1137, 944, 895 cm−1; 1H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 401.2680 [M
− H]− (calcd for C25H37O4, 401.2692).
Thorectidaeolide E (6): colorless oil; [α]2D5 +6.0 (c 0.10, MeOH); IR
(film) νmax 3423, 2926, 1779, 1742, 1440, 1381, 1131, 1065, 1026, 886,
1496
dx.doi.org/10.1021/np400350k | J. Nat. Prod. 2013, 76, 1492−1497

Journal of Natural Products
Article
859 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z
409.2704 [M + Na]+ (calcd for C25H38O3Na, 409.2719).
Notes
The authors declare no competing financial interest.
Preparation of the (R)- and (S)-MTPA Ester Derivatives of
Compound 1. The (R)- and (S)-MTPA ester derivatives of
compound 1 were prepared as previously described.21 Briefly, two
portions of compound 1 (each 0.8 mg) were transferred into clean
NMR tubes and dried under vacuum. Pyridine-d5 [200 μL, 99.5%
deuterated, 0.05% tetramethylsilane (TMS)] was added under an
argon gas stream into each of two NMR tubes, and (R)-(−)- and (S)-
(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (3 μL each)
were immediately added into the NMR tubes, respectively. The NMR
tubes were shaken to mix the samples and MTPA chlorides evenly.
The reaction NMR tubes were allowed to stand in a desiccator at
ACKNOWLEDGMENTS
■
The authors thank the Natural Products Branch Repository
Program at the NCI for providing marine extracts from the
NCI Open Repository used in these studies, Dr. D. J. Newman
and E. C. Brown (NCI, Frederick, MD) for assistance with
sample logistics and collection information, and Dr. S. L.
McKnight (University of Texas Southwestern Medical Center
at Dallas, Dallas, TX) for providing the pTK-HRE3-luc
construct. This work was supported in part by the National
Institutes of Health NCI (Grant CA98787), and the National
Oceanic and Atmospheric Administration National Institute for
Undersea Science and Technology (Grant NA16RU1496).
This investigation was conducted in a facility constructed with
Research Facilities Improvement Grant C06 RR-14503-01 from
the National Institutes of Health.
1
room temperature and monitored hourly by H NMR. The reactions
1
were found to be complete after 5 h. The H NMR spectra were
obtained directly from the NMR reaction tubes and were assigned on
the basis of their respective COSY spectra.
Thorectidaeolide A, (R)-MTPA Ester (12): 1H NMR (pyridine-d5) δ
6.374 (1H, br s, H-2), 6.324 (1H, t, J = 6.4 Hz, H-4), 5.295 (1H, t, J =
6.4 Hz, H-10), 5.227 (1H, t, J = 6.8 Hz, H-6), 5.172 (1H, dd, J = 18.0
and 1.2 Hz, H-25a), 5.088 (1H, dd, J = 18.0 and 1.2 Hz, H-25b), 2.756
(2H, t, J = 6.4 Hz, H-5), 2.550 (2H, t, J = 6.8 Hz, H-17), 2.060 (2H, t,
J = 6.8 Hz, H-8), 1.719 (3H, s, H-20), 1.700 (3H, s, H-23), 1.615 (3H,
s, H-24), 1.273 (6H, s, H-21, H-22).
REFERENCES
■
(1) Ebada, S. S.; Lin, W.; Proksch, P. Mar. Drugs 2010, 8, 313−346.
(2) Ueoka, R.; Nakao, Y.; Fujii, S.; van Soest, R. W. M.; Matsunaga, S.
J. Nat. Prod. 2008, 71, 1089−1091.
(3) Wonganuchitmeta, S.; Yuenyongsawar, S.; Keawpradub, N.;
Plubrukarn, A. J. Nat. Prod. 2004, 67, 1767−1770.
(4) Marco, J. A.; Sanz, J. F.; Yuste, A.; Carda, M.; Jakupovic, J.
Phytochemistry 1991, 30, 3661−3668.
(5) Tsuda, M.; Shigemori, H.; Ishibashi, M.; Sasaki, T.; Kobayashi, J.
J. Org. Chem. 1992, 57, 3503−3507.
(6) Uddin, M. H.; Otsuka, M.; Muroi, T.; Ono, A.; Hanif, N.;
Matsuda, S.; Higa, T.; Tanaka, J. Chem. Pharm. Bull. 2009, 57, 885−
887.
(7) Butler, M. S.; Capon, R. J. Aust. J. Chem. 1992, 45, 1705−1743.
(8) Couperus, P. A.; Clague, A. D. H.; van Dongen, J. P. C. M. Org.
Magn. Reson. 1976, 8, 426−431.
(9) Potts, B. C. M.; Faulkner, D. J.; De Carvalho, M. S.; Jacobs, R. S.
J. Am. Chem. Soc. 1992, 114, 5093−5100.
Thorectidaeolide A, (S)-MTPA Ester (13): 1H NMR (pyridine-d5) δ
6.349 (1H, t, J = 6.4 Hz, H-4), 6.168 (1H, br s, H-2), 5.365 (1H, t, J =
6.8 Hz, H-6), 5.312 (1H, t, J = 6.8 Hz, H-10), 5.092 (1H, dd, J = 18.0
and 1.2 Hz, H-25a), 4.952 (1H, dd, J = 18.0 and 1.2 Hz, H-25b), 2.820
(2H, t, J = 6.4 Hz, H-5), 2.551 (2H, t, J = 6.8 Hz, H-17), 2.125 (2H, t,
J = 6.4 Hz, H-8), 1.722 (3H, s, H-20), 1.698 (3H, s, H-23), 1.684 (3H,
s, H-24), 1.273 (6H, s, H-21, H-22).
Cell-Based Reporter and Proliferation/Viability Assays.
Human breast tumor T47D and MDA-MB-231 cells were obtained
from American Type Culture Collection (ATCC). Cell maintenance,
experimental procedures, and data presentation for the cell-based
reporter and proliferation/viability assays (48 h) were as previously
described.22 Cycloheximide (protein synthesis inhibitor) and rotenone
(mitochondrial respiration inhibitor) were used as positive controls for
both the reporter and cell viability assays (results shown in S36 of the
Supporting Information). All extract, fraction, and compound samples
were prepared as stock solutions in dimethyl sulfoxide (DMSO) (final
solvent concentration less than 0.5% in all assays).
(10) Elkhayat, E.; Edrada, R.; Ebel, R.; Wray, V.; van Soest, R.;
Wiryowidagdo, S.; Mohamed, M. H.; Muller, W. E. G.; Proksch, P. J.
Nat. Prod. 2004, 67, 1809−1817.
Statistical Analysis. Data analyses were performed with GraphPad
Prism 4.
(11) Cao, S.; Foster, C.; Lazo, J. S.; Kingston, D. G. I. Bioorg. Med.
Chem. 2005, 13, 5094−5098.
(12) Albizati, K. F.; Holman, T.; Faulkner, D. J.; Glaser, K. B.; Jacobs,
R. S. Experientia 1987, 43, 949−950.
ASSOCIATED CONTENT
* Supporting Information
■
S
(13) Kobayashi, J.; Zeng, C.; Ishibashi, M.; Sasaki, T. J. Nat. Prod.
1993, 56, 436−439.
NMR spectra for compounds 1−6, a photo of H. communis
(collection number 0CDN9952 and NPID number C030113),
and additional positive control results from the reporter and
cell viability assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
(14) Youssef, D. T. J. Nat. Prod. 2004, 67, 112−114.
(15) Sperry, S.; Valeriote, F. A.; Corbett, T. H.; Crews, P. J. Nat.
Prod. 1998, 61, 241−247.
(16) Semenza, G. L. Oncogene 2010, 29, 625.
(17) Wang, S.; Wang, Z.; Lin, S.; Zheng, W.; Wang, R.; Jin, S.; Chen,
J.; Jin, L.; Li, Y. Biochem. J. 2012, 446, 79−87.
(18) McCloud, T. C. Molecules 2010, 15, 4526−4563.
(19) Datta, S.; Zhou, Y.-D.; Nagle, D. G. J. Nat. Prod. 2013, 76, 642−
647.
(20) Lal, A. R.; Cambie, R. C.; Rickard, C. E. F.; Bergquist, P. R.
Tetrahedron Lett. 1994, 35, 2603−2606.
(21) Su, B. N.; Park, E. J.; Mbwambo, Z. H.; Santarsiero, B. D.;
Mesecar, A. D.; Fong, H. H.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat.
Prod. 2002, 65, 1278−1282.
(22) Du, L.; Mahdi, F.; Jekabsons, M. B.; Nagle, D. G.; Zhou, Y.-D. J.
Nat. Prod. 2010, 73, 1868−1872.
AUTHOR INFORMATION
Corresponding Author
■
*Telephone: 662-915-7026 (Y.-D.Z.); 662-915-7026 (D.G.N.).
Fax: 662-915-6975 (Y.-D.Z.); 662-915-6975 (D.G.N.). E-mail:
ydzhou@olemiss.edu (Y.-D.Z.); dnagle@olemiss.edu (D.G.N.).
Present Addresses
‡Jun Li: Modern Research Center for Traditional Chinese
Medicine, Beijing University of Chinese Medicine, Beijing
100029, People’s Republic of China.
§Lin Du: Natural Products Discovery Group, Department of
Chemistry and Biochemistry, Stephenson Life Sciences
Research Center, University of Oklahoma, 101 Stephenson
Parkway, Room 1000, Norman, Oklahoma 73019-5251, United
States.
1497
dx.doi.org/10.1021/np400350k | J. Nat. Prod. 2013, 76, 1492−1497