Phorone A and isophorbasone A, sesterterpenoids isolated from the marine sponge Phorbas sp.

Article abstract of DOI:10.1021/ol3019874

A chemical investigation of a Korean marine sponge, Phorbas sp., yielded unprecedented sesterterpenoids phorone A (1) and isophorbasone A (2) along with ansellone B (3) and phorbasone A acetate (4). Their complete structures were elucidated by the combina

Full text of DOI:10.1021/ol3019874

ORGANIC
LETTERS
2012
Vol. 14, No. 17
4486–4489
Phorone A and Isophorbasone A,
Sesterterpenoids Isolated from the
Marine Sponge Phorbas sp.
Weihong Wang,† Yehee Lee,† Tae Gu Lee,† Bora Mun,† Awadut G. Giri,† Jihye Lee,†
Hiyoung Kim,† Dongyup Hahn,† Inho Yang,† Jungwook Chin,†,‡ Hyukjae Choi,†
Sang-Jip Nam,*,§ and Heonjoong Kang*,†,‡
Center for Marine Natural Products and Drug Discovery, School of Earth and
Environmental Sciences, and Research Institute of Oceanography, Seoul National
University, NS-80, Seoul, 151-747, Korea, and College of Pharmacy and Research
Institute of Life and Pharmaceutical Sciences, Sunchon National University,
Suncheon 540-950, Korea
sjnam@sunchon.ac.kr; hjkang@snu.ac.kr
Received July 17, 2012
ABSTRACT
A chemical investigation of a Korean marine sponge, Phorbas sp., yielded unprecedented sesterterpenoids phorone A (1) and isophorbasone A (2)
along with ansellone B (3) and phorbasone A acetate (4). Their complete structures were elucidated by the combination of spectroscopic data and
chemical manipulation. Phorone A (1) and isophorbasone A (2) have the new “phorane”(5) and “isophorbasane”(6) sesterterpenoid carbon
skeletons, respectively. Ansellone B (3) and phorbasone A acetate (4) exhibited potent inhibitory activity on nitric oxide production in RAW 264.7
LPS-activated mouse macrophage cells with IC50 values of 4.5 and 2.8 μM, respectively.
Marine sponges of the genus Phorbas have been
proven to be an abundant source of structurally novel
secondary metabolites exhibiting diverse biological
activities. Compounds that represent a variety of dif-
ferent classes have been reported including alkaloids,1
macrolides,2 diterpenoids,3 steroids,4 sesterterpenoids,5
and tetraterpenoids6 in the order of their discovery. These
natural products displayed a wide range of biological
activities such as antifungal activity,2a cytostatic activity,2a
cytotoxicity,2c,4a,5b,6,7 inhibition of isocitrate lyase,7b
† SEES, Seoul National University.
‡ Research Institute of Oceanography, Seoul National University.
§ Sunchon National University.
(1) Rudi, A.; Stein, Z.; Green, S.; Goldberg, I.; Kashman, Y.;
Benayahu, Y.; Schleyer, M. Tetrahedron Lett. 1994, 35, 2589–2592.
(2) (a) Searle, P. A.; Molinski, T. F. J. Am. Chem. Soc. 1995, 117,
8126–8131. (b) Skepper, C. K.; MacMillan, J. B.; Zhou, G.-X.; Masuno,
M. N.; Molinski, T. F. J. Am. Chem. Soc. 2007, 129, 4150–4151. (c)
MacMillan, J. B.; Guang, X.-Z.; Skepper, C. K.; Molinski, T. F. J. Org.
Chem. 2008, 73, 3699–3706. (d) Dalisay, D. S.; Molinski, T. F. Org. Lett.
2009, 11, 1967–1970. (e) Dalisay, D. S.; Morinaka, B. I.; Skepper, C. K.;
Molinski, T. F. J. Am. Chem. Soc. 2009, 131, 7552–7553. (f) Dalisay,
D. S.; Molinski, T. F. J. Nat. Prod. 2010, 73, 679–682.
(3) (a) Vuong, D.; Capon, R. J. J. Nat. Prod. 2000, 63, 1684–1685. (b)
McNally, M.; Capon, R. J. J. Nat. Prod. 2001, 64, 645–647. (c) Rho,
J.-R.; Lee, H.-S.; Sim, C. J.; Shin, J. Tetrahedron 2002, 58, 9585–9591. (d)
Zhang, H.; Major, J. M.; Lewis, R. J.; Capon, R. J. Org. Biomol. Chem.
2008, 6, 3811–3815. (e) Zhang, H.; Capon, R. J. Org. Lett. 2008, 10,
1959–1962. (f) Jang, K. H.; Jeon, J. E.; Ryu, S.; Lee, H. S.; Oh, K. B.;
Shin, J. J. Nat. Prod. 2008, 71, 1701–1707. (g) Lee, H.-S.; Park, S. Y.;
Sim, C. J.; Rho, J.-R. Chem. Pharm. Bull. 2008, 56, 1198–1200.
(4) (a) Masuno, M. N.; Pawlik, J. R.; Molinski, T. F. J. Nat. Prod.
2004, 67, 731–733. (b) Morinaka, B. I.; Masuno, M. N.; Pawlik, J. R.;
Molinski, T. F. Org. Lett. 2007, 9, 5219–5222. (c) Morinaka, B. I.;
Pawlik, J. R.; Molinski, T. F. J. Nat. Prod. 2009, 72, 259–264.
(5) (a) Forestieri, R.; Merchant, C. E.; de Voogd, N. J.; Matainaho,
T.; Kieffer, T. J.; Andersen, R. J. Org. Lett. 2009, 11, 5166–5169. (b)
Rho, J.-R.; Hwang, B. S.; Sim, C. J.; Joung, S.; Lee, H.-Y.; Kim, H.-J.
Org. Lett. 2009, 11, 5590–5593. (c) Daoust, J.; Fontana, A.; Merchant,
C. E.; de, V. N. J.; Patrick, B. O.; Kieffer, T. J.; Andersen, R. J. Org. Lett.
2010, 12, 3208–3211. (d) Rho, J.-R.; Hwang, B. S.; Joung, S.; Byun,
M. R.; Hong, J.-H.; Lee, H.-Y. Org. Lett. 2011, 13, 884–887.
(6) Park, S. Y.; Choi, H.; Hwang, H.; Kang, H.; Rho, J.-R. J. Nat.
Prod. 2010, 73, 734–737.
(7) (a) Zhang, H.; Major, J. M.; Lewis, R. J.; Capon, R. J. Org.
Biomol. Chem. 2008, 6, 3811–3815. (b) Jang, K. H.; Jeon, J.-E.; Ryu, S.;
Lee, H.-S.; Oh, K.-B.; Shin, J. J. Nat. Prod. 2008, 71, 1701–1707.
r
10.1021/ol3019874
Published on Web 08/24/2012
2012 American Chemical Society

activation of the cAMP pathway,5a,c and induction of
osteoblast differentiation.5d
starting with a doublet at δ 1.13 (H-5) continuing to a
oxymethine at δ 4.03 (H-6) and ending with a diastereo-
topic methylene at δ 2.77 and 1.52 (H-7) (Figure 1). Each
methyl singlet at δ 0.48 (H3-23), 0.87 (H3-21), 1.15 (H3-20),
and 1.17 (H3-22) showed HMBC correlations to four
neighboring carbons. These observations allowed the as-
signment of a partial structure of 3,4a,8,8-tetramethylde-
cahydronaphthalen-1-ol.
Figure 1. COSY and key HMBC correlations of 1 and 2.
During the course of our search for bioactive metabo-
lites from Korean marine organisms, we encountered one
species of sponge of the genus Phorbas. Chemical investi-
gation of the extract uncovered a series of sesterterpenoids
representing different carbon skeletons including phorone
A (1), isophorbasone A (2), ansellone B (3), and phorba-
sone A acetate (4). Phorone A (1) and isophorbasone A (2)
possess unprecedented tetracyclic “phorane” and tricyclic
“isophorbasane” carbon skeletons, respectively. They
are presumably biogenetically related to the previously
reported phorbaketals, phorbasones, and ansellone.
Details of the structural elucidation, plausible biogenic
pathway, and biological activity of the compounds are
presented below.
Phorone A (1) has a molecular formula of C25H34O3
as established by high-resolution FABMS data of the
[M þ H]þ ion at m/z 383.2583 (Δ ꢀ0.3 mmu). A combina-
tion the 13C NMR data and edited HSQC data revealed
the presence of six methyl groups, four methylenes, two
methines, one oxymethine, three aliphatic quaternary
carbons, one R,β-unsaturated ketone group, and six
aromatic carbon resonances (Table 1). The aromatic
ring and the R,β-unsaturated ketone group account
for six of the nine sites of unsaturation required by
the molecular formula and indicated that phorone A
(1) contained three additional rings. The molecular
formula of phorone A (1) contains three oxygen atoms
according to the HRFABMS data. Therefore, the oxy-
methine carbon and the phenolic carbon must not be
linked by an ether linkage but be substituted by free
hydroxyl groups.
A tetrasubstituted phenyl ring was established by the
carbon chemical shifts and the HMBC correlations. The
methyl singlet at δ 2.28 (H3-25) showed HMBC correla-
tions to the aromatic carbons at δ 155.6 (C-16), 129.3
(C-17), and 130.5 (C-18). HMBC correlations from the
aromatic resonance at δ 7.10 (H-15) to the aromatic
carbons C-17 and C-19 (δ 138.5) and from the aromatic
resonance at δ 7.39 (H-18) to the aromatic carbons C-14
(δ 136.0) and C-16 were observed. The methyl singlet
at δ 2.35 (H3-24) showed HMBC correlations to a pair
of trisubstituted olefinic carbons at δ 132.3 (C-12) and
152.0 (C-13) and the aromatic carbon at δ 136.0 (C-14),
demonstrating the connection of C-13 to the phenyl ring.
HMBC correlation from H3-22 to the aliphatic quaternary
carbon C-8 and the aromatic carbon C-19 permitted
the connection of C-8 to C-19. Connection between the
R,β-unsaturated carbonyl carbon (C-11) and the aliphatic
methine carbon (C-9) was secured by the observation
of HMBC correlations from the methine proton resonance
at δ 2.32 (H-9) to the carbonyl carbon resonance at δ 204.0
(C-11) and the olefinic carbon C-12, which revealed a
seven-membered ring adjacent to the aromatic ring and
decahydronaphthalene ring.
The relative configuration for 1 was determined by
analysis of the coupling patterns and ROESY data. Owing
to the overlap of H-1, 2, and 3 in the 1H NMR spectrum,
the coupling patterns were deduced using homonuclear
decoupling experiments. Irradiation of the proton reso-
nance at δ 1.52 (H-2a and H-7b) induced the change in
coupling patterns of H-3b from a doublet of doublets
of doublets (J = 13.4, 13.4, 4.1 Hz) to a doublet ofdoublets
(J = 13.4, 4.1 Hz), which indicated that H-2a coupled to
H-3b in 13.4 Hz and, accordingly, both H-2a and H-3b
must be in the axial position. The appearance of H-7a
changed from a doublet of doublets (J = 13.7, 3.1 Hz) to
a doublet (J = 3.1 Hz); thus, one of the large coupling
The major portion of the backbone of the tetracyclic
terpene skeleton was established using COSY and HMBC
correlations. COSY correlations permitted a linear con-
nectivity of three contiguous diastereotopic methylenes in
the range of 1.00ꢀ2.00 ppm and also a linear spin system
Org. Lett., Vol. 14, No. 17, 2012
4487

Table 1. 1H and 13C NMR Data of Phorone A (1) in CD3OD
no.
δH
mult (J in Hz)
δC
1a
1b
2a
2b
3a
3b
4
1.31
1.03
1.52
1.33
1.28
1.22
m
41.0
CH2
CH2
CH2
br d (13.9)
m
19.5
45.0
m
m
Figure 2. δ values (ΔδSꢀR) in ppm for (S)- and (R)-MTPA esters
of 16-O-methyl ether of phorone A (1a) in CD3OD.
ddd (13.4, 13.4, 4.1)
35.1
63.7
68.2
53.4
C
5
1.13
4.03
2.77
1.52
d (10.8)
CH
CH
CH2
6
td (10.8, 3.1)
dd (13.7, 3.1)
m
7a
7b
8
40.0
76.0
C
9
2.32
6.30
7.10
7.39
s
s
s
s
CH
C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
41.7
204.0
132.3
152.0
136.0
118.3
155.6
129.3
130.5
138.5
37.6
C
CH
C
C
CH
C
C
CH
C
1.15
0.87
1.17
0.48
2.35
2.28
s
s
s
s
s
s
CH3
CH3
CH3
CH3
CH3
CH3
22.4
30.5
18.5
28.5
16.6
constants of H-6 (td, J = 10.8, 3.2 Hz) must be due to the
coupling with H-7b, suggesting the axial orientation of
H-7b. The strong 2D ROESY correlations between Me-23
and the three proton resonances of H-2a, H-6, and Me-21
revealed that the C-1 to C-10 decalin ring system was trans
fused with Me-21, Me-23, and H-6 in the axial orientation.
The same trans-fused decalin ring system was also ob-
served for phorbasones,5d ansellone,5c and isophorbasone
A (2), which is consistent with the proposal that they may
share the same biosynthetic pathway. The 2D ROESY
correlations between Me-22 and both H-7a and H-9
suggested that the rings B and C were cis fused.
In order to determine the absolute configuration of 1
by application of the modified Mosher’s method, its
16-O-methyl derivative (1a) was first prepared by treat-
ment with diazomethane. Esterification of1awith (R)-(ꢀ)-
and (S)-(þ)-R-methoxy-R-(trifluoromethyl) phenylacetyl
chloride yielded (S)- and (R)-MTPA esters, respectively.
The ΔδSꢀR value distribution pattern suggested the S con-
figuration at C-6 (Figure 2).
Isophorbasone A (2) gave a [M þ Na]þ ion at m/z
403.2244 in the HRFABMS, appropriate for a molecular
formula of C25H32O3Na. A detailed comparison of its NMR
data with those of phorbasone A5d indicated that 2 con-
tained the same ring system 5,5,8a-trimethyl-4a,5,6,7,8,8a-
hexahydronaphthalen-2(1H)-one, which was substituted by
an isopropene group as in phorbasone A. In addition to the
Figure 3. Proposed biosynthetic pathway to phorone and iso-
phorbasone skeletons.
4488
Org. Lett., Vol. 14, No. 17, 2012

signals assigned to the bicyclic ring, an olefinic methyl
carbon (δ 16.6), six aromatic carbons (δ 157.1, 137.4,
132.4, 131.9, 121.1, 114.7), a ketone carbonyl carbon (δ
199.7), and a methylene carbon (δ 40.4) were observed in the
13C NMR spectrum. The former three groups of carbon
signals and their HMBC correlations revealed the presence
of a trisubstituted phenyl ring instead of the cyclohexenone
moiety in phorbasone A and the attachment of C-12
(δ 199.7) on the phenyl ring. A pair of 1H signals at δ 3.91
(H-11a) and 3.86 (H-11b) showed HMBC correlations to the
carbons at δ 202.1 (C-8), 136.1 (C-7), and 151.9 (C-6) in the
bicyclic unit and the benzoyl carbonyl carbon C-12, which
assembled the two partial moieties of 2. The relative config-
uration of 2 was assigned by ROESY analysis and the values
of Hꢀ H vicinal coupling constants. The large coupling
constants between the protons H-1a (δ 1.47) and H-2a
(δ 1.78) and between the protons H-2a and H-3b (δ 1.27)
indicated that they were located in the axial position. The
configuration of the bicyclic ring was established as trans
ring fusion on the basis of the strong ROESY correlations
among H-21 (δ 1.15), H-20 (δ 0.98), and H-2a and among
H-5 (δ 2.61), H-1a, and H-3b. The ROESY correlations
between H-21 and H-9 (δ 2.58) and between H-5 and
H-24b (δ 4.83) indicated the trans relationship of Me-21
with the isopropene unit. The absolute configuration was
establishedby comparison of the CD data with those of the
ring C-aromatized derivative of phorbasone A (4a), which
was produced by hydrolysis of 4 followed by aromatizaion
with DBU (1,8-diazabicycloundec-7-ene). The CD spec-
trum of 2 revealed πfπ* Cotton effects (a positive Cotton
effect around 202 nm and a negative Cotton effect around
236 nm) having the same sign as those of 4a.
by the four classes of sesterterpenoids. B first undergoes
dehydroxylation and ketoꢀenol tautomerization of its
cyclohexenone ring before being subjected to further cycli-
zation to yield the phorone carbon skeleton. The latter step
could reasonably proceed via nucleophilic attack of the
para carbon of the phenol ring on the C-8 carbocation.
These proposals are supported by the key discovery
of ansellone B. As an offshoot of the biosynthetic path-
way, the stabilization of the C-8 carbocation through
rearrangements as reported by Rho would generate
the tricyclic ring of phorbasone A,5d which would be
hydroxylated to give phorbasone B. The formation of
isophorbasone A from phorbasone B could involve
an oxidation of C-11, a dehydration and carbon shift
of C-18 to C-7, and a dehydration and ketoꢀenol
tautomerization of the cyclohexenone ring to attain a
more stable structure.
It was reported that effective suppression of NO produc-
tion, via inhibition of inducible NOS (iNOS) expression,
has become an alternative strategy for developing new
compounds in the treatment of inflammatorydisease.8 The
iNOS inhibitory assay was performed for compounds 1ꢀ4
by measuring NO production in RAW 264.7 LPS-induced
mouse macrophage cells. Compounds 1 and 2 were not
active up to 100 μM, while compounds 3 and 4 were found
to exhibit potent inhibitory activity on nitric oxide produc-
tion with IC50 values of 4.5 and 2.8 μM, respectively, which
were better than those of prenylated flavonoids (IC50
values: 6.4ꢀ18.8 μM) from Artocarpus communis.8b In
particular, compound 3 showed a favorable selectivity
index (SI) of 3.8, which is indicative of its iNOS inhibitory
activity without cytotoxicity.
1
1
The surprising variety of sesterterpenoid skeletons iso-
lated from this specimen has prompted us to report
biosynthetic speculations in detail. The most realistic
hypothesis suggests that all these sesterterpenoids derive
from geranylfarnesyl pyrophosphate through cationic cy-
clizations and rearrangements. The early steps of the
biosynthetic process were proposed to involve the initial
cyclization of the geranyl unit and hydroxylation after
removal of the pyrophosphate moiety to yield the inter-
mediate A (phorbaketal carbon skeleton) (Figure 3). The
farnesyl part of A undergoes cationic cyclization to give B
(ansellone carbon skeleton), the last shared intermediate
of ansellone, phorbasone, phorone, and isophorbasone.
From a stereochemical point of view, this is a crucial step
for formation of the trans-fused bicyclic structure shared
Acknowledgment. This research was supported by the
Marine Biotechnology Program funded by the Ministry of
Land, Transport and Maritime Affairs of Korea.
Supporting Information Available. Structural elucida-
tion for compounds 3 and 4, experimental section, spec-
troscopic data of 1a, 1ꢀ4, and 4a, and biological activity
data of compounds 1ꢀ4. This material is available free of
charge via the Internet at http://pubs.acs.org.
(8) (a) Hobbs, A. J.; Higgs, A.; Moncada, S. Annu. Rev. Pharmacol.
Toxicol. 1999, 39, 191–220. (b) Han, A.-R.; Kang, Y.-J.; Windono, T.;
Lee, S. K.; Seo, E.-K. J. Nat. Prod. 2006, 69, 719–721.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 17, 2012
4489