Siladenoserinols A-L: New sulfonated serinol derivatives from a tunicate as inhibitors of p53-Hdm2 interaction

Article abstract of DOI:10.1021/ol3032363

Siladenoserinols A-L were isolated from a tunicate as inhibitors of p53-Hdm2 interaction, a promising target for cancer chemotherapy. Their structures including the absolute configurations were elucidated to be new sulfonated serinol derivatives, each of which contains a 6,8-dioxabicyclo[3.2.1] octane unit and either glycerophosphocholine or glycerophosphoethanolamine moiety. They inhibited p53-Hdm2 interaction with IC₅₀ values of 2.0-55 μM. Among them, siladenoserinol A and B exhibited the strongest inhibition with an IC₅₀ value of 2.0 μM.

Full text of DOI:10.1021/ol3032363

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Siladenoserinols AꢀL: New Sulfonated
Serinol Derivatives from a Tunicate as
Inhibitors of p53ꢀHdm2 Interaction
Yuichi Nakamura,† Hikaru Kato,† Tadateru Nishikawa,‡ Noriyuki Iwasaki,§ Yoshiaki Suwa,†
Henki Rotinsulu, Fije Losung,^ Wilmar Maarisit,# Remy E. P. Mangindaan,^
Hiroshi Morioka,† Hideyoshi Yokosawa,z and Sachiko Tsukamoto*,†
Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi,
Kumamoto 862-0973, Japan, Bruker BioSpin K.K., 3-9 Moriya, Kanagawa-ku,
Yokohama 221-0022, Japan, Bruker Daltonics K.K., 3-9 Moriya, Kanagawa-ku,
Yokohama 221-0022, Japan, Faculty of Agriculture, Universitas Pembangunan Indonesia,
Manado 95361, Indonesia, Faculty of Fisheries and Marine Science, Sam Ratulangi University,
Manado 95115, Indonesia, Faculty of Mathematics and Natural Sciences,
Christian University of Indonesia, Tomohon 95362, Indonesia, and School of Pharmacy,
Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan
sachiko@kumamoto-u.ac.jp.
Received November 24, 2012
ABSTRACT
Siladenoserinols AꢀL were isolated from a tunicate as inhibitors of p53ꢀHdm2 interaction, a promising target for cancer chemotherapy.
Their structures including the absolute configurations were elucidated to be new sulfonated serinol derivatives, each of which contains a
6,8-dioxabicyclo[3.2.1]octane unit and either glycerophosphocholine or glycerophosphoethanolamine moiety. They inhibited p53ꢀHdm2
interaction with IC50 values of 2.0ꢀ55 μM. Among them, siladenoserinol A and B exhibited the strongest inhibition with an IC50 value of 2.0 μM.
The ubiquitinꢀproteasome pathway consists of two
systems, the ubiquitin system and the protein degradation
system, i.e., the 26S proteasome. The former contains the
ubiquitin-activating enzyme (E1), ubiquitin-conjugating
enzyme (E2), and ubiquitin ligase (E3) and catalyzes the
ubiquitination of client proteins. In addition to inhibitors
targeting the proteasome, various inhibitors of the ubiq-
uitin system have been developed. Among them, E3s rec-
ognize huge numbers of client proteins for degrada-
tion.1 As E3 definitively determines which client pro-
teins are ubiquitinated, a specific inhibitor against an E3
recognizinga key client proteincould be a good leadfor the
treatment of diseases associated with degradation of the
key client protein. Among many E3s, Mdm2 or Hdm2
(human Mdm2 homologue), an E3 for tumor suppressor
p53,2 is frequently used as a target for inhibitor devel-
opment.3 The tumor suppressor p53 induces growth arrest
and apoptosis upon activation by various stimuli such as
DNA damage.4 The crystal structure of the complex com-
posed of the 109-residue amino-terminal domain of Mdm2
and a 15-residue transactivation domain peptide of p53
revealed that Mdm2 has a deep hydrophobic cleft, to which
the p53 peptide binds.5 Therefore, targeting Mdm2/Hdm2
is a promising way to reactivate p53, inducing apoptosis in
human cancer cells.
† Kumamoto University.
‡ Bruker BioSpin K.K.
§ Bruker Daltonics K.K.
Universitas Pembangunan Indonesia.
^ Sam Ratulangi University.
(2) Honda, R.; Tanaka, H.; Yasuda, H. FEBS Lett. 1997, 420, 25–27.
(3) Ashcroft, M.; Vousden, K. H. Oncogene 1999, 18, 7637–7643.
(4) Vogelstein, B.; Lane, D.; Levine, A. J. Nature 2000, 408, 307–310.
(5) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.;
Levine, A. J.; Pavletich, N. P. Science 1996, 274, 948–953.
# Christian University of Indonesia.
z Aichi Gakuin University.
(1) (a) Pickart, C. M. Annu. Rev. Biochem. 2001, 70, 503–533. (b)
Hershko, A.; Ciechanover, A. Annu. Rev. Biochem. 1998, 67, 425–479.
r
10.1021/ol3032363
XXXX American Chemical Society

Siladenoserinol A (1) has the molecular formula
C43H79N2O17PS, which was determined by HRESIMS
and 13C NMR (Table S1, Supporting Information) spec-
trometries. The 1H NMR spectrum (Table S1, Supporting
Information) showed two olefin signals at δ 7.02 (dt, J =
15.6, 7.0 Hz) and 5.86 (d, J = 15.6 Hz), 18 heteroatom-
bearing signals at δ5.27(m),4.67(brs),4.27(2H,m),4.38(dd,
J = 12.0, 3.7 Hz), 4.22 (dd, J = 12.0, 6.5 Hz), 4.16 (br s), 4.03
(2H, t, J = 5.8 Hz), 3.99 (br t, J = 4.6 Hz), 3.65 (2H, m), 3.68
(dd, J = 11.0, 5.5 Hz), 3.63 (dd, J = 11.0, 5.3 Hz), 3.58 (dd,
J = 9.4, 4.7 Hz), 3.51 (dd, J = 9.4, 6.6 Hz), 3.45 (2H, t, J =
6.6 Hz), and 3.43 (m), three singlet signals at δ 3.23 (9H), 2.07
(3H), and 2.03 (3H), a methylene signal at δ 2.25 (2H, br q,
J = 7.0 Hz), and methylene/methine signals at δ 1.3ꢀ2.1.
Analysis of 2D spectra, including COSY, HSQC, and HMBC
spectra, showed three partial structures a-c(Figure1).Inaunit
a, the COSY spectrum showed a linear connection from C-2 to
C-5 and from C-8 to C-13. HMBC correlations from δ 2.25
(H-4), 1.49 (H-5), and 1.36 (H-7) to δ 30.1 (C-6) and from δ
3.99 (H-9) to δ 26.3 (C-7) secured a whole connection from
C-2 to C-13. Two olefin hydrogens, H-2 and H-3, showed a
coupling constant, J2,3 = 15.6 Hz, which indicated a 2E con-
figuration. HMBC correlations from δ 7.02 (H-3) and 5.86
(H-2) to δ 167.3 (C-1) and from δ 2.07 (11-OCOMe) and 4.67
(H-11) to δ 172.4 (11- OCOMe) implied the presence of an
R,β-unsaturated carbonyl group and an acetoxy group on
In the course of our search for new classes of inhibitors
against the ubiquitin system from natural sources, we already
succeeded in isolating himeic acid A6 and hirtioreticulin A7 as
E1 inhibitors, leucettamol A8 and manadosterol A9 as inhibi-
tors of Ubc13 (E2)-Uev1A interaction, and (ꢀ)-hexylitaconic
acid10 as an inhibitor of p53ꢀHdm2 interaction. In the sub-
sequent search for more potent inhibitors of p53ꢀHdm2
interaction, we screened the extracts of marine invertebrates
by ELISA with recombinant p53 and Hdm2 proteins11 and
encountered the extract of a tunicate of the family Didemnidae
collected in Indonesia, which showed inhibitory activity
against p53ꢀHdm2 interaction. Here, we report the isolation
and structural determination of 12 compounds, designated
siladenoseriols AꢀL (1ꢀ12), as inhibitors of p53ꢀHdm2
interaction.
A tunicate was immediately extracted with EtOH after
collection. The extract was evaporated, and the aqueous resi-
due was successively extracted with EtOAc and n-BuOH. The
n-BuOH fraction, which showed inhibitory activity against
p53ꢀHdm2 interaction, was purified by ODS column chro-
matography and ODS HPLC to give 1ꢀ12.
Figure 1. Partial structures aꢀc of 1.
(6) Tsukamoto, S.; Hirota, H.; Imachi, M.; Fujimuro, M.; Onuki, H.;
Ohta, T.; Yokosawa, H. Bioorg. Med. Chem. Lett. 2005, 15, 191–194.
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Fujimuro, M.; Saeki, Y.; Yoshinaga, S.; Terasawa, H.; Iwasaki, N.; Rotinsulu,
H.; Losung, F.; Mangindaan, R. E. P.; Namikoshi, M.; de Voogd, N. J.;
Yokosawa, H.; Tsukamoto, S. Bioorg. Med. Chem. 2012, 20, 4437–4442.
(8) Tsukamoto, S.; Takeuchi, T.; Rotinsulu, H.; Mangindaan,
R. E. P.; van Soest, R. W.; Ukai, K.; Kobayashi, H.; Namikoshi, M.;
Ohta, T.; Yokosawa, H. Bioorg. Med. Chem. Lett. 2008, 18, 6319–6320.
(9) Ushiyama, S.; Umaoka, H.; Kato, H.; Suwa, Y.; Morioka, H.;
Rotinsulu, H.; Losung, F.; Mangindaan, R. E. P.; de Voogd, N. J.;
Yokosawa, H.; Tsukamoto, S. J. Nat. Prod. 2012, 75, 1495–1499.
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Figure 2. ESI-MS/MS analysis of 1.
C-11, respectively. HMBC correlations from δ 3.99 (H-9),
4.16 (H-10), and 1.72 (H-12) to δ 110.5 (C-14) indicated that
ꢀ
ꢀ
(11) (a) Gonzalez, N.; Rodrıguez, J.; Jimenez, C. J. Org. Chem. 1999,
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64, 5705–5707. (b) Kiyota, H.; Dixon, D. J.; Luscombe, C. K.; Hettstedt,
S.; Ley, S. V. Org. Lett. 2002, 4, 3223–3226.
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B
Org. Lett., Vol. XX, No. XX, XXXX

the a unit contained a 6,8-dioxabicyclo[3.2.1]octane skeleton
(Figure1), which is contained in known serinolipids, didemni-
serinolipids AꢀC11 and cyclodidemniserinol trisulfate.12
Further, the COSY spectrum and HMBC cross peaks from δ
1.52/1.74 (H2-13) to δ 38.3 (C-15) suggested that another
linear carbon chain was connected at C-14, but the COSY
correlation did not define the length of the carbon chain. From
these data, the a unit possesses the 6,8-dioxabicyclo[3.2.1]-
octane core with two long carbon chains, one of which has an
ester linkage. In a second unit b, the COSY spectrum implied
the presence of 1,2,3-trisubstituted propyl [δ 4.22 (dd, J =
12.0, 6.5 Hz, H-10), 4.38 (dd, J = 12.0, 3.7 Hz, H-10), 5.27 (m,
H-20),and4.03(2H,t,J= 5.8 Hz, H2-30)] and 1,2-disubstitued
ethyl [δ4.27 (2H, m, H2-100) and 3.65 (2H, m, H2-200)] moieties,
and the 13C chemical shifts clearly suggested that the five car-
bons were bonded to hetero atoms. A singlet signal at δ 3.23
matched nine hydrogens, which defined the presence of three
equivalent methyl groups. The signal (δ 3.23) had an HSQC
cross peak with a carbon at δ 54.6, which indicated that the
singlet signal was a methyl signal accommodated in a tri-
methylamino group. The HMBC spectrum showed a correla-
(δ 70.9) in 1 clearly showed that the linear aliphatic chain
attached to C-14 in the a unit was connected to the c unit
through an ether linkage. In addition, ESI MS2 and MS3
spectra of 1 were measured to confirm the connection among
the a, b, and c units and their observed peaks established a
planar structure, which was analyzed by NMR experiments
(Figures 2 and S10, Supporting Information). The lack of
coupling between H-9 and H-10 and between H-10 and H-11
revealed that the dihedral angles, H-9/H-10 and H-10/H-11,
were approximately 90ꢀ. Therefore, the relative configurations
were determined to be 9R*,10S*,11R*,14S*, which was sup-
ported by the NOE correlation between H-9 and H-11. In
order to determine the absolute configuration, a modified
Mosher’s method13 was applied to acidic methanolysis pro-
duct 13 prepared from 1 (Scheme 1). The differences in
chemical shifts (Δδ = δS ꢀ δR) between (R)- and (S)-MTPA
esters (14a and 14b, respectively) clearly indicated 9R,10S,
11R,14S,30S for 1. In order to determine the absolute config-
uration of C-20, the alkaline hydrolysis product 16 prepared
from 1 was subjected to esterification to give dibenzoate 17
(Scheme 2). The CD spectrum of 17 was identical to that
of a dibenzoate 19 derived from a commercially available
3-sn-phosphatidylcholine (Figure 3). Thus, the structure of 1,
14
tion from NMe3 to C-200 and a 1Hꢀ N HSQC (long-range)
spectrum showed correlations from H2-100 and NMe3 to NMe3
(Figures 1 and S8, Supporting Information), which showed
that the trimethylamino group was attached to C-200. The
presence of an acetoxy group at C-10 was revealed by HMBC
cross peaks from δ 4.22/4.38 (H2-10) and 2.03 (10-OCOMe) to
δ 172.3 (10-OCOMe). It was noted that the carbon signals
at δ 60.5 (C-100), 64.8 (C-30), 67.3 (C-200), and 71.7 (C-20)
appeared as doublet signals, which indicated that these car-
bons were coupled with a phosphorus atom. This was con-
firmed by detection of a 31P signal at δ ꢀ0.19, which showed
Scheme 1. Distribution of δΔSꢀR Values for the MTPA Deri-
vatives 14a and14b
31
1Hꢀ P HMBC cross peaks with δ 3.65 (H-200), 4.03 (H-30),
and 4.27 (H-100) (Figures 1 and S9, Supporting Information).
These data established the structure of a 20-O-substituted
glycerophosphocholine moiety. HMBC correlations from δ
5.27 (H-20) to δ 167.3 (C-1) clearly showed that the a and b
units were connected through an ester linkage at C-20. A third
unit cwas suggested to be a substituted propyl group (δH 3.51/
3.58 (H2-29), 3.43 (H-30), and 3.63/3.68 (H2-31); δC 70.9
(C-29), 56.0 (C-30), and 63.0 (C-31)), in which heteroatoms
were attached to each carbon judged by their chemical shifts.
Based on the molecular formula of 1, the c unit was implied to
consist of C3H8NO5S and the linear aliphatic chain linked to
C-14 in the a unit was composed of the remaining residue
C13H26, which thereby indicated that the c unit was a sulfo-
nated serinol derivative (Figure 1). Siladenoserinol G (7) has
the same molecular formula as 1, and the NMR spectra clearly
indicated that a structural difference between 1 and 7 was only
the sulfonated position (δH 3.62/3.66 (H2-29), 3.64 (H-30),
and 4.12/4.22 (H2-31); δC 68.6 (C-29), 52.3 (C-30), and 66.1
(C-31) in 7). In order to determine the sulfonated positions
in 1 and 7, the 13C NMR spectra in CD3OD and CD3OH
were measured. The differences [ΔδC = δC(CD3OD) ꢀ δC-
(CD3OH)] in the 13C chemical shift of C-31 were ꢀ0.24 and
0.00 in 1 and 7, respectively, which definitively showed that 1
had a hydroxy group at C-31 and a sulfamate group at C-30.
In contrast, 7 had amino and sulfate groups at C-30 and C-31,
respectively. The HMBC correlation from H2-28 to C-29
including the absolute configuration, was determined. The
structure determination of other siladenoserinols 2ꢀ6 and
8ꢀ12 is described in the Supporting Information.
The inhibitory effects of siladenoserinols AꢀL (1ꢀ12) on
p53ꢀHdm2 interaction were tested by ELISA10 (Table 1). In
spite of their structural similarities, they differed in their IC50
values from 2.0 to 55 μM. Compound 1 and 2 exhibited the
most significant inhibition, 8 was the next most potent
inhibitor, and 7 and 12 were the weakest inhibitors. Compar-
ing the sulfamate derivatives (R2 = SO3H, R3 = H) and the
corresponding sulfate derivatives (R2 = H, R3 = SO3H), the
sulfamate derivatives were more potent than the sulfate de-
rivatives (IC50 values: 2.0/53 μM for 1/7; 2.5/9.3 μM for 8/9;
(13) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092–4096.
Org. Lett., Vol. XX, No. XX, XXXX
C

Table 1. IC50 Values of 1-12 against p53-Hdm2 Interaction
Scheme 2. Preparation of 17 and 19 from 1 and
3-sn-Phosphatidylcholine, Respectively
compd
IC50 (μM)
compd
IC50 (μM)
1
2
3
4
5
6
7
2.0
2.0
4.0
7.7
18
8
9
2.5
9.3
11
10
11
13
12
nutlin-3a
55
29
0.1
53
a Positive control.
group (IC50 values: 2.0/7.7 μM for 1/4). Compound 8 with an
ester bond at the C-10 position of its glycerol moiety was more
potent than 4 with an ester bond at the C-20 position of its
glycerol moiety (IC50 values: 2.5/7.7 μM for 8/4); 1 with an
ester bond at the C-20 position and an acetoxy group at the
C-10 position was a little more potent than 8with an ester bond
at the C-10 position (IC50 values: 2.0/2.5 μM for 1/8). Thus, it
can be inferred that the sulfamate derivatives with an ester
bond at the C-20 position and acetoxy groups at the C-10 and
C-11 positions exhibit the strongest inhibitory activity against
p53ꢀHdm2 interaction.
After approval of a proteasome inhibitor, Velcade, for
cancer treatment,14 inhibitors targeting the ubiquitin system
including E1, E2, and E3 enzymes have also been developed,
and several compounds are now undergoing preclinical and
clinical trials for cancers.15 Among them, E3 inhibitors, such as
Nutlin-3,16 an Mdm2 antagonist, are expected to be excellent
leads for anticancer drugs, as the respective E3s recognize
specific target proteins functioning in the protection against
cancer progression. In this study, siladenoserinols 1ꢀ12 were
found to be a new class of inhibitors against p53ꢀHdm2
interaction, and the first sulfonated serinolipids containing
glycerophosphocholine or glycerophosphoethanolamine moi-
eties. With respect to cytotoxic activity of 1, it should be noted
that cell viability in A549 cells was reduced to approximately
80% at a concentration of 10 μM. A study on the optimization
of the inhibitory effect of siladenoserinol on the p53ꢀHdm2
interaction is now underway in our laboratory.
Figure 3. CD spectra of 17 (a) and 19 (b).
Acknowledgment. We thank Dr. H. Kobayashi of the
University of Tokyo and Prof. T. Nishikawa of Toho
University for collection and identification of the tunicate,
respectively. This work was supported by Grants-in-Aid
for Scientific Research (Nos. 22310138 and 22406001)
from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan and also by grants from the
Astellas Foundation for Research on Metabolic Disorders
and the Uehara Memorial Foundation.
13/55 μM for 11/12). In addition, in a comparison among the
acetyl (R1 = Ac) and corresponding hydroxy (R1 = H) de-
rivatives, the acetyl derivatives were more potent than the
hydroxy derivatives (IC50 values: 2.0/29 μMfor1/6;2.5/13μM
for 8/11;9.3/55μMfor9/12). On the other hand, a comparison
between the sulfamate derivatives, 1and 4, led to the following
result that 1, containing the acetoxy group at a C-10 position,
was more potent than 4, lacking the corresponding acetoxy
Supporting Information Available. Structure determi-
nation of 2ꢀ6 and 7ꢀ12, preparation of 14a,b, 17, and 19,
NMR spectral data tables, and 1D and 2D NMR spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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J. E. Nat. Rev. Drug Disc. 2011, 10, 29–46. (b) Cohen, P.; Tcherpakov,
M. Cell 2010, 143, 686–693. (c) Ande, S. R.; Chen, J.; Maddika, S. Eur.
J. Pharmacol. 2009, 625, 199–205.
(16) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.;
Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi,
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The authors declare no competing financial interest.
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