Sinulasulfoxide and sinulasulfone, sulfur-containing alkaloids from the Indonesian soft coral Sinularia sp.

Article abstract of DOI:10.1016/j.tetlet.2012.05.095

Chemical analysis of the Indonesian soft coral Sinularia sp. (order Alcyonacea, family Alcyoniidae) afforded two new alkaloids, named sinulasulfoxide (1) and sinulasulfone (2), characterized by an amide linkage between a phytanic acid moiety and an uncomm

Full text of DOI:10.1016/j.tetlet.2012.05.095

Tetrahedron Letters 53 (2012) 3937–3939
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Sinulasulfoxide and sinulasulfone, sulfur-containing alkaloids
from the Indonesian soft coral Sinularia sp.
Masteria Yunovilsa Putra a,b, Angela Ianaro c, Elisabetta Panza c, Giorgio Bavestrello d,
Carlo Cerrano b, Ernesto Fattorusso a, Orazio Taglialatela-Scafati a,
⇑
a Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli ‘Federico II’, Via D. Montesano 49, 80131 Napoli, Italy
b Dipartimento di Scienze del Mare, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy
c Dipartimento di Farmacologia Sperimentale, Università di Napoli ‘Federico II’, Via D. Montesano 49, 80131 Napoli, Italy
d Dipartimento per lo Studio del Territorio e delle sue Risorse, Università di Genova, Corso Europa 26, 16132 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical analysis of the Indonesian soft coral Sinularia sp. (order Alcyonacea, family Alcyoniidae) affor-
ded two new alkaloids, named sinulasulfoxide (1) and sinulasulfone (2), characterized by an amide link-
Received 2 March 2012
Revised 16 May 2012
Accepted 21 May 2012
Available online 26 May 2012
age between
a phytanic acid moiety and an uncommon sulfur-containing unit. Their complete
stereostructures were elucidated by interpretation of MS and NMR data along with CD analysis and
chemical modifications. Sinulasulfoxide (1) proved to moderately inhibit LPS-induced NO release.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Sinularia
Soft coral
Sulfur-containing compounds
Alkaloids
iNOS inhibition
Soft corals belonging to the family Alcyoniidae are by far the
dominant reef dwelling octocorals in the Indo-West Pacific area.
These organisms, characterized by a great variety in colors, shapes,
and sizes, have been recognized as prolific producers of a wide ar-
ray of secondary metabolites, particularly cembrane diterpenoids
and steroids, often characterized by uncommon structural features
and potent bioactivities.1–3 Since some of these soft corals host
large populations of zooxanthellae symbionts, a biosynthetic role
for these microorganisms has been postulated.4
We have recently started a research project aimed at the evalu-
ation of the chemical composition and of the biomedical potential
of marine octocorals from the Indonesian coasts,5–7 one of the rich-
est biodiversity hot spots of the oceans. As part of this investiga-
tion, we have been analyzing a specimen of Sinularia sp. (order
Alcyonacea, family Alcyoniidae), collected along the island of Sila-
den, in the Bunaken Marine Park of Manado (North Sulawesi, Indo-
nesia). From the organic extract of this organism, we have recently
reported the isolation of chloroscabrolides,8 a class of chlorinated
norcembranoids, and of polyhydroxylated steroids endowed with
antagonistic activity against the farnesoid X-activated receptor.9
Inspection of more polar fractions obtained from the organic ex-
tract of Sinularia sp. has now resulted in the isolation of two new
sulfur-containing alkaloids, named sinulasulfoxide (1) and sinula-
sulfone (2), along with the known furanosesquiterpenoid 310
(Fig. 1). In this Letter we will provide details about the stereostruc-
tural elucidation of these metabolites and the results of the
evaluation of their inducible NO-synthase (iNOS) protein inhibitory
activity.
Colonies of Sinularia sp. (580 g wet weight) were homogenized
and repeatedly extracted with MeOH and CHCl3 at room tempera-
ture. The obtained organic extract (6.8 g) was chromatographed by
MPLC over silica gel eluting with a gradient system of increasing
polarity from n-hexane to EtOAc to MeOH. Fractions eluted with
EtOAc were further purified by reversed-phase HPLC (eluent
MeOH/H2O 96:4) to afford sinulasulfone (2, 1.0 mg), fractions
eluted with EtOAc/MeOH 9:1, purified by HPLC (eluent MeOH/
H2O 9:1), gave sinulasulfoxide (1, 3.9 mg), while fractions eluted
with EtOAc/n-hexane 9:1 were purified by HPLC (eluent EtOAc/n-
hexane 85:15) to give the known furanosesquiterpene (E)-5-(2,
6-dimethylocta-5,7-dienyl)furan-3-carboxylic acid (3, 3.2 mg)
identified by comparison of its spectral data with those reported
in the literature.10
Sinulasulfoxide (1)11 was obtained as an optically active amor-
phous solid and showed pseudomolecular ion peak at m/z 424
[M+Na]+ in the ESI-MS (positive ions). The molecular formula
C
23H47NO2S was assigned to sinulasulfoxide on the basis of HR-
⇑
Corresponding author. Tel.: +39 081 678509; fax: +39 081 678552.
E-mail address: scatagli@unina.it (O. Taglialatela-Scafati).
ESIMS measurements.11 The 1H NMR spectrum of 1 (CD3OD)
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.095

3938
M. Y. Putra et al. / Tetrahedron Letters 53 (2012) 3937–3939
18
negative sign (
assignment of the R configuration at the sulfur atom in 1.
D
e
= ꢀ0.8), according to Mislow’s rule, allowed the
19
20
3
17
O-
S
O
1'
Phytanic acid is a branched polyisoprenoid of considerable bio-
logical interest which, in the ocean, should be produced by sedi-
ment bacteria through oxidation and hydrogenation of the phytol
unit present in the chlorophyll side chain.13 Since two diastereo-
mers of phytanic acid have been found in nature (3S,7R,11R and
3R,7R,11R), an evaluation of the configurational features of the phy-
tanic acid unit present in sinulasulfoxide was needed. To this aim,
sinulasulfoxide (1.2 mg) was treated with 70% acetic acid at 80 °C
for 2 days, and the obtained mixture was partitioned between
EtOAc and water. The organic phase was finally purified by HPLC
(n-hexane/EtOAc 95:5) to obtain pure phytanic acid, whose 1H
N
H
1
2'
3'
16
1
O
O
O
S
N
H
2
HOOC
NMR spectrum and [
a
]
(ꢀ3.9, c 0.1) value were compared with
D
those reported in the literature for the two diastereoisomers.14 This
comparison suggested its assignment as the 3S,7R,11R isomer.
Sinulasulfoxide is a unique secondary metabolite constituted by
a phytanic acid linked to a sulfoxide-containing unit through an
amide bond. The 2-methylsulfinyl ethanamine unit has been very
rarely found in the structures of natural compounds and some Pi-
per alkaloids15 or psammaplin N from the sponge Aplysinella rhax16
are among the few examples. On the other hand, its homolog 3-
methylsulfinyl propanamine, the decarboxylated methionine sulf-
oxide, has been much more frequently reported.
O
3
Figure 1. Chemical structures of sinulasulfoxide (1), sinulasulfone (2) and of the
known furanosesquiterpenoid 3.
showed six methyl resonances (five doublets at d 0.94, 0.90, 0.89,
0.88, and 0.88, in addition to a deshielded methyl singlet at dH
2.70), a broad cluster of signals between dH 1.10–1.50, three
methine multiplets at dH 1.54, 1.99, and 2.20, and four relatively
deshielded methine signals at 2.94, 3.07, 3.58, and 3.64. All these
proton signals were associated to those of the directly attached
carbon atoms by means of the 2D NMR HSQC experiment, and, in
particular, the multiplets resonating in the midfield region could
be sorted out as two methylene groups (dC 43.5 and 53.9, respec-
tively). The 13C NMR spectrum of 1 (CD3OD) showed the signal of
a single unprotonated carbon, a carbonyl resonating at dC 174.9.
The COSY spectrum of 1 evidenced the mutual coupling of the
two deshielded methylenes, which constituted an isolated spin
system (Fig. 2), while the remaining multiplets could be organized
in a single saturated polyisoprenoid spin system starting from the
relatively deshielded methylene H2-2 (dH 2.20 and 1.99) and termi-
nating with the two methyl groups H3-16 and H3-17 to build up a
phytanic acid moiety. The presence of an amide linkage between
this moiety and 2-methylsulfinyl ethanamine fully accounted for
the molecular formula and the correlations coming from the g-
HMBC experiment (Fig. 1). In particular, the key g-HMBC cross-
peaks of both H2-2 and H2-10 with C-1 and of H3-30 with C-20 con-
firmed the planar structure of sinulasulfoxide (1).
Sinulasulfoxide includes four stereogenic centers, three carbon
atoms (C-3, C-7, C-11), and the sulfur atom. The assignment of
the absolute configuration at this latter center was achieved
through analysis of the CD spectrum of 1. Indeed, Mislow et al.12
have demonstrated the existence of a direct correlation between
the absolute configuration of methyl alkyl sulfoxides and their opti-
cal activity in terms of the signs and rotational strengths of selected
Cotton effects. In the absence of strongly perturbing groups, as in
the case of monoallyl(aryl) or diallyl(aryl) sulfoxides, a negative
Cotton effect, centered at the absorption band in the region 220–
240 nm in acetonitrile, was found to be indicative of the R configu-
ration. This correlation proved to be not significantly influenced by
the presence of stereogenic centers on the alkyl group itself; since
in sinulasulfoxide (1) the first asymmetric carbon is six-atoms far
from the sulfur center, this rule could be confidently applied. The
CD spectrum of 1 showed an absorption band at k = 236 nm, whose
Sinulasulfone (2)17 is a secondary metabolite strictly related to
sinulasulfoxide (1), from which it differs only for oxidation at the
sulfur carbon. Compound 2 showed pseudomolecular ion peak at
m/z 440 [M+Na]+ in the ESI-MS (positive ions) and its molecular
formula C23H47NO3S, assigned on the basis of HR-ESIMS measure-
ments, presented an additional oxygen atom compared to that of
sinulasulfoxide. The 1H and 13C NMR spectra of 217 closely paral-
leled those of 1 and strongly suggested the attachment of the addi-
tional oxygen atom at the sulfur atom. In particular, the 1H NMR
spectrum showed that, while the signals of the phytanic moiety
were practically superimposable to those detected for 1, consistent
differences could be evidenced in the signals of the sulfur contain-
ing moiety. In particular, the methyl singlet was downfield shifted
to dH 2.95, while the two methylenes of this moiety resonated as
mutually coupled triplets at dH 3.22 (the S-linking) and 3.77 (the
N-linking). The multiplicity of these protons was in perfect agree-
ment with the lack of chirality of the sulfone group. All the proton
and the carbon resonances of 2 were assigned on the basis of de-
tailed 2D NMR (COSY, HSQC, and g-HMBC) analysis. Oxidation of
sinulasulfoxide (1, 1.5 mg) with meta-chloroperoxybenzoic acid
(rt, 3 h) afforded 2 in nearly quantitative yields, thus finally sup-
porting the stereostructure assignment of sinulasulfone as re-
ported in 2. To our knowledge, sinulasulfone (2) is only the
second sulfone-containing derivative from a soft coral, the first
being austrasulfone, recently isolated as a neuroprotective agent
from Cladiella australis.18 Sinulasulfoxide (1) was left in MeOH/
H2O, EtOAc, and CHCl3 solutions for one week at room temperature
but no detectable changes were observed in any of the three sol-
vents. Although this plays in favor of a genuine origin, we cannot
exclude that sinulasulfone (2) could be an artifact obtained by oxi-
dation of sinulasulfoxide during the isolation procedure.
It is interesting to notice that one of the taxonomic markers of
Sinularia soft corals is the presence of spermidine amides of long
chain fatty acids, such as the strongly cytotoxic compound 419 or
the geranylneroic derivative sinulamide (5)20 ( Fig. 3), showing
H,K ATPase inhibitory activity. Sinulasulfoxide and sinulasulfone
innovate this class of amphipathic compounds by showing, for
the first time, a sulfur-containing polar moiety. To our knowledge,
the role of this class of compounds in the producing organism has
never been investigated.
17
O-
O
1
COSY
1'
S
g-HMBC
N
H
2'
3'
16
Sinulasulfoxide (1) and furanosesquiterpene 3 were evaluated
for inducible NO-synthase (iNOS) protein inhibitory activity. iNOS
Figure 2. COSY (bold) and key g-HMBC correlations evidenced for sinulasulfoxide
(1).

M. Y. Putra et al. / Tetrahedron Letters 53 (2012) 3937–3939
3939
2. Lindel, T.; Jensen, P. R.; Fenical, W.; Long, B. H.; Casazza, A. M.; Carboni, J.;
Fairchild, C. R. J. Am. Chem. Soc. 1997, 119, 8744–8745.
3. El Sayed, K. A.; Hamann, M. T.; Waddling, C. A.; Jensen, C.; Lee, S. K.; Danstan, C.
A.; Pezzuto, J. M. J. Org. Chem. 1998, 63, 7449–7455.
O
N
N
H
N
4. Khalesi, M. K.; Beeftink, H. H.; Wjffels, R. H. Mar. Biotechnol. 2009, 11, 488–494.
5. Fattorusso, E.; Romano, A.; Taglialatela-Scafati, O.; Bavestrello, G.; Bonelli, P.;
Calcinai, B. Tetrahedron Lett. 2006, 47, 2197–2200.
4
6. Fattorusso, E.; Romano, A.; Taglialatela-Scafati, O.; Achmad, M. J.; Bavestrello,
G.; Cerrano, C. Tetrahedron 2008, 64, 3141–3146.
7. Fattorusso, E.; Romano, A.; Taglialatela-Scafati, O.; Irace, C.; Maffettone, C.;
Bavestrello, G.; Cerrano, C. Tetrahedron 2009, 65, 2898–2904.
N
NH3
O
N
H
N
8. Fattorusso, E.; Luciano, P.; Putra, M. Y.; Taglialatela-Scafati, O.; Ianaro, A.;
Panza, E.; Bavestrello, G.; Cerrano, C. Tetrahedron 2011, 67, 7983–7988.
9. Putra, M. Y.; Bavestrello, G.; Cerrano, C.; Renga, B.; D’Amore, C.; Fiorucci, S.;
Fattorusso, E.; Taglialatela-Scafati, O. Steroids 2012, 77, 433–440.
10. Bowden, B. F.; Coll, J. C.; de Silva, E. D.; de Costa, M. S. L.; Djiura, P. J.;
Mahendran, M.; Tapiolas, D. M. Aust. J. Chem. 1983, 36, 371–376.
5
Figure 3. The structures of some spermidine derivatives obtained from Sinularia
species.
11. Sinulasulfoxide (1). Amorphous colorless solid; ½a D25
ꢁ
+11.7 (c 0.2, MeOH); (+)ESI-
MS (positive ions) m/z 424 [M+Na]+. IR (KBr) mmax 3288, 1662, 1630, 1609,
is regulated by inflammatory mediators (LPS, cytokines), and the
excessive production of NO by iNOS has been implicated in the path-
ogenesis of the inflammatory response.21 In this assay, the produc-
1255, 1050, 1014, 990 cmꢀ1; CD (CH3CN): k = 236 nm (
De = ꢀ0.8); HR-ESIMS:
m/z 424.3229 (calcd for C23H47NaNO2S 424.3225); 1H NMR (CD3OD, 500 MHz, J
in Hz): 3.64 (1H, m, H-10a), 3.59 (1H, m, H-10b), 3.07 (1H, m, H-20a), 2.94 (1H,
m, H-20b), 2.70 (3H, s, H3-30), 2.20 (1H, dd, J = 11.5, 6.5, H-2a), 1.99 (1H, t,
J = 11.5, H-2b), 1.95 (1H, m, H-3), 1.54 (1H, non, J = 6.5, H-15), 1.41 (2H, m, H-7
and H-11), 1.25–1.30 (14H, m, H2-5, H2-6, H2-8, H2-9, H2-10, H2-13, H2-14),
1.20 (2H, m, H-4a, H-13a), 1.10 (2H, m, H-4b, H-13b), 0.94 (3H, d, J = 6.5 Hz, H3-
20), 0.90 (3H, d, J = 6.5 Hz, H3-16), 0.89 (3H, d, J = 6.5 Hz, H3-17), 0.88 (6H, d,
J = 6.5 Hz, H3-18 and H3-19); 13C NMR (CD3OD, 125 MHz): 174.9 (C-1), 53.9 (C-
20), 43.7 (C-2), 39.3 (C-14), 37.5 (C-30), 37.4 (C-6, C-8, C-10, C-12), 37.0 (C-4),
35.5 (C-10), 32.7 (C-7, C-11), 30.9 (C-3), 28.1 (C-15), 24.8 (C-5), 24.6 (C-9), 24.3
(C-13), 21.9 (C-16, C-17), 21.0 (C-18, C-19), 20.2 (C-20).
ꢀ
tion of NO2 (stable metabolite of NO) was evaluated as a
parameter of macrophage activation and iNOS induction. Unstimu-
lated J774 cells generated undetectable (<5 nmol/mL) amounts of
NO2ꢀ, while stimulation of the cells with LPS (1
lg/mL) for 24 h pro-
duced a dose-dependent release of NO2ꢀ (15.6 0.1 nmol/mL). After
incubation of the cells with a concentration 30 lM of sinulasulfox-
ide (1) or of compound 3, a moderate inhibition of NO2ꢀ production
12. Mislow, K.; Green, M. M.; Laur, P.; Melillo, J. T.; Simmons, T.; Ternay, A. L., Jr. J.
Am. Chem. Soc. 1965, 87, 1958–1976.
(22.6% and 25.7%, respectively), was observed.
13. Gillan, F. T.; Nichols, P. D.; Johns, R. B.; Bavor, H. J. Appl. Environ. Microbiol. 1983,
45, 1423–1428.
Acknowledgments
14. Sita, L. R. J. Org. Chem. 1993, 58, 5285–5287.
15. Tang, G.-H.; Chen, D.-M.; Qiu, B.-Y.; Sheng, L.; Wang, Y.-H.; Hu, G.-W.; Zhao, F.-
W.; Ma, L.-J.; Wang, H.; Huang, Q.-Q.; Xu, J.-J.; Long, C. L.; Li, J. J. Nat. Prod. 2011,
74, 45–49.
This research was partially conduced during the Master Course
‘Tropical Marine Biodiversity and Natural Products’ of Università
Politecnica delle Marche and was partially financed by 7th Frame-
work Programme of the European Community (REGPOT-2008-1-
NatPharma contract). Mass and NMR spectra were recorded at
‘Centro di Servizio Interdipartimentale di Analisi Strumentale’,
Università di Napoli ‘Federico II’.
16. Graham, S. K.; Lambert, L. K.; Pierens, G. K.; Hooper, J. N. A.; Garson, M. J. Aust. J.
Chem. 2010, 63, 867–872.
17. Sinulasulfone (2). Amorphous colorless solid; ½a D25
ꢁ
+3.7 (c 0.1, MeOH); (+)ESI-
MS (positive ions) m/z 440 [M+Na]+. HR-ESIMS: m/z 440.3179 (calcd for
C
23H47NaNO3S 440.3174); 1H NMR (CD3OD, 500 MHz, J in Hz): 3.77 (2H, t,
J = 7.0, H2-10), 3.22 (2H, t, J = 7.0, H2-20), 2.95 (3H, s, H3-30), 2.20 (1H, dd,
J = 11.5, 6.5, H-2a), 1.95 (1H, t, J = 11.5, H-2b), 1.94 (1H, m, H-3), 1.54 (1H, non,
J = 6.5, H-15), 1.41 (2H, m, H-7 and H-11), 1.25–1.30 (14H, m, H2-5, H2-6, H2-8,
H2-9, H2-10, H2-13, H2-14), 1.20 (2H, m, H-4a, H-13a), 1.10 (2H, m, H-4b, H-
13b), 0.94 (3H, d, J = 6.5 Hz, H3-20), 0.90 (3H, d, J = 6.5 Hz, H3-16), 0.89 (3H, d,
J = 6.5 Hz, H3-17), 0.88 (6H, d, J = 6.5 Hz, H3-18 and H3-19); 13C NMR (CD3OD,
125 MHz): 173.2 (C-1), 53.0 (C-20), 43.7 (C-2), 39.3 (C-14), 40.6 (C-30), 37.4 (C-
6, C-8, C-10, C-12), 37.0 (C-4), 35.1 (C-10), 32.7 (C-7, C-11), 30.9 (C-3), 28.1 (C-
15), 24.8 (C-5), 24.6 (C-9), 24.3 (C-13), 21.9 (C-16, C-17), 21.0 (C-18, C-19), 20.2
(C-20).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.05.
095. These data include MOL files and InChiKeys of the most
important compounds described in this article.
18. Wen, Z.-H.; Chao, C.-H.; Wu, M.-H.; Sheu, J.-H. Eur. J. Med. Chem. 2010, 45,
5998–6004.
19. Choi, Y.-H.; Schmitz, F. J. J. Nat. Prod. 1997, 60, 495–496.
20. Sata, N. U.; Sugano, M.; Matsunaga, S.; Fusetani, N. Tetrahedron Lett. 1999, 40,
719–722.
21. Ianaro, A.; O’Donnell, C. A.; Di Rosa, M.; Liew, F. Y. Immunology 1994, 82, 370–
375.
References and notes
1. Coval, S. J.; Patton, R. W.; Petrin, J. M.; James, L.; Rothofsky, M. L.; Lin, S. L.; Patel,
M.; Reed, J. K.; Mc Phail, A. T.; Bishop, W. R. Bioorg. Med. Chem. Lett. 1996, 6,
909–912.