Iotrochamides A and B, antitrypanosomal compounds from the Australian marine sponge Iotrochota sp.

Article abstract of DOI:10.1016/j.bmcl.2012.05.029

Bioassay-guided isolation of the CH₂Cl₂/MeOH extract from the Australian sponge Iotrochota sp. resulted in the purification of two new N-cinnamoyl-amino acids, iotrochamides A (1) and B (2). The chemical structures of 1 and 2 were determined by 1D/2D NMR and MS data analyses. Compounds 1 and 2 were shown to inhibit Trypanosoma brucei brucei with IC ₅₀ values of 3.4 and 4.7 μM, respectively.

Full text of DOI:10.1016/j.bmcl.2012.05.029

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4873–4876
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Iotrochamides A and B, antitrypanosomal compounds from the Australian
marine sponge Iotrochota sp.
⇑
Yunjiang Feng, Rohan A. Davis, Melissa L. Sykes, Vicky M. Avery, Ronald J. Quinn
Eskitis Institute, Griffith University, Brisbane, QLD 4111, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 February 2012
Revised 4 May 2012
Accepted 8 May 2012
Available online 17 May 2012
Bioassay-guided isolation of the CH Cl /MeOH extract from the Australian sponge Iotrochota sp. resulted
in the purification of two new N-cinnamoyl-amino acids, iotrochamides A (1) and B (2). The chemical
structures of 1 and 2 were determined by 1D/2D NMR and MS data analyses. Compounds 1 and 2 were
2
2
shown to inhibit Trypanosoma brucei brucei with IC50 values of 3.4 and 4.7 lM, respectively.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Iotrochamides A and B
Cinnamoyl-amino acids
Human African trypanosomiasis
Trypanocidal
Human African Trypanosomiasis (HAT) is a fatal disease caused
by two subspecies of a protozoan parasite Trypanosoma brucei rho-
desiense and Trypanosoma brucei gambiense. T. b. rhodesiense is the
agent of the acute form of the disease, prevailing in Eastern and
Southern Africa and T. b. gambiense causes the chronic form of
the disease in Western and Central Africa. The World Health
Organization (WHO) has estimated there are currently 30,000
ated natural product library consisting of 202,983 fractions.15 T. b.
brucei is routinely used in screening for antitrypanosomal com-
pounds with potential to be developed into HAT drugs. It is pos-
sible to develop a library of drug like natural products.17 However
to overcome the limited diversity and to identify drug-like natural
products following HTS campaigns, we recently reported a proce-
16
dure to capture natural products with LogP less than 5 in ex-
1
15
cases of the disease. The registered drugs suramin and pentami-
tracts.
The extracts of over 18,000 marine and terrestrial
dine are not effective against both stages of the disease. The second
stage effective drug melarsoprol has associated toxicity which has
extracts were then fractionated with 11 fractions collected per
sample. From the 11 fractions derived from the Australian sponge
Iotrochota sp., a single fraction was identified that showed activity
against T. b. brucei and desirable selectivity against a human
embryonic kidney cell line HEK293 (>10-fold). Bioassay-guided
2
been reported as lethal in up to 12% of cases. There have also been
reports of incidence of drug resistance in HAT cases.2 These fac-
tors highlight the need for the development of new, safer and more
effective drugs to fight HAT.
,3
Natural products have traditionally played a highly significant
14
4
HO
O
OH
role in drug discovery and development, particularly in the areas
O
of cancer and infectious diseases.4 The basis for this can be ex-
3
1
6
1
2
10
N
H
1
6
4
plained by the similarity of the biosynthetic imprint, the molecular
recognition between substrate and biosynthetic enzyme, and the
natural product binding motif, the molecular recognition between
a ligand and its therapeutic target that led to the concept of protein
OMe
Br
1
8
15
13
17
O
OH
5
O
fold topology (PFT). PFT describes cavity recognition points unre-
3
HN
lated to fold and sequence similarity, and defines a natural prod-
12
10
N
H
1
6
4
6
–8
19
uct’s ability to recognize biology space.
for new lead compounds for neglected diseases,9
fluorescence based trypanosomal high throughput screening
In our ongoing search
OMe
–13
a 384-well
Br
2
8
MeO
Br
1
4
O
(
HTS) assay against T. b. brucei was used to screen a pre-fraction-
3
1
6
N
H
4
OMe
⇑
Corresponding author.
E-mail address: R.Quinn@griffith.edu.au (R.J. Quinn).
OH
3
8
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.029

4
874
Y. Feng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4873–4876
fractionation of the CH
namoyl-amino acids, iotrochamides A (1) and B (2), which both dis-
played activity towards T. b. brucei.
The Australian sponge Iotrochota sp. (Order: Poecilosclerida,
Family: Iotrochotidae) was collected from the west side of Curacao
Island, Queensland, at a depth of 30 m by SCUBA diving in 1999. A
voucher sample (G314368) is stored at the Queensland Museum,
Brisbane, Australia. The freeze-dried and ground Iotrochota sp.
2
Cl
2
/MeOH extract afforded two new N-cin-
configuration of the amino acid residue in 1 was determined by
Marfey’s method. The acid hydrolysis of compound 1 followed
2
1
by derivatisation with Marfey’s reagent (FDAA, 1-fluoro-2,4-dini-
2
1
trophen-5-yl-
at 33.95 min in LC–MS analysis, in agreement with the standard
bis- -tyrosine FDAA derivative at 33.70 min, while a bis- -tyrosine
L-alanine amide) showed a bis-tyrosine FDAA peak
2
2
L
D
FDAA derivative gave a retention time of 36.38 min. This was con-
firmed by co-injections. The absolute configuration of the amino
(
(
10 g) was sequentially extracted with n-hexane, CH
4:1), and MeOH. The CH Cl /MeOH extracts were combined and
O/0.1%
TFA). Following biological evaluation, the active fractions were
combined and further purified by C18 HPLC (MeOH/H O/0.1%
2
Cl
2
/MeOH
acid residue in 1 was therefore
the D
L
-tyrosine. With the geometry of
double bond and the absolute configuration of the tyrosine
moiety determined, iotrochamide A was assigned as N-(Z)-2-meth-
oxycinnamoyl- -tyrosine.
Iotrochamide B (2) was also obtained as an optically active
2
,3
2
2
chromatographed using C18 bonded silica HPLC (MeOH/H
2
L
2
2
3
TFA) to yield two new N-cinnamoyl-amino acids, iotrochamides
A (1, 1.8 mg, 0.018% dry weight) and B (2, 1.4 mg, 0.014% dry
weight).
glassy solid. LRESIMS showed a cluster of two isotopic ions at
m/z 443 and 445 (1:1), suggesting the presence of one bromine
atom in the molecule. HRESIMS measurement on [M+Na] ion
(m/z 465.0416), in combination with H and C NMR spectroscopic
data (Table 1), supported the molecular formula of C21
with 13 double bond equiv. Comparison of the H and C NMR
spectroscopic data between 1 and 2 (Table 1) showed that 2 had
an identical N-cinnamoyl moiety as 1. The only difference between
1 and 2 was that the two aromatic doublets from the
idue in 1 were replaced by signals characteristic of a 3-(6-bromo)-
indole system in 2. These included three doublets (d 7.52, d,
+
1
13
Iotrochamide A (1) was obtained as an optically active glassy
1
8
+
solid. HRESIMS measurement on [M+Na] ion (m/z 364.1146),
H
19BrN
2 4
O
1
13
1
13
in combination with H and C NMR spectroscopic data (Table 1),
supported the molecular formula of C19 with 11 double
bond equiv. The H NMR spectrum of 1 displayed signals due to
two exchangeable protons (d 9.18 and 8.20), ten aromatic/olefinic
protons (d 6.59–7.64), one methine (d 4.49), one methylene (d
.96 and 3.05) and one methoxy group (d 3.49). Fifteen carbons
were observed in the C NMR spectrum, including two carbonyl
H19NO
5
1
L-tyrosine res-
H
H
H
H
2
H
H
1
3
J = 8.4 Hz; 7.50, d, J = 1.8 Hz; 7.20, d, J = 2.4 Hz), one doublet of dou-
blet (7.10, dd, J = 1.8 and 8.4 Hz;) and one broad indole-NH
2
3
(d
C
162.9 and 172.7), ten sp and three sp carbons.
The mono-substituted benzene ring was evident by the cou-
exchangeable proton (d
atom at the 6-position of the indole ring was determined by the
ROESY correlation between H-17 (d 7.50) and the indole-NH,
and confirmed by the detailed analysis of the HMBC correlations
(Fig. 1)
H
10.97). The placement of the bromine
pling patterns of five aromatic protons, including one doublet of
doublet of doublets (d 7.32, 1H, J = 7.8, 7.8, 1.2 Hz), and two dou-
blet of doublets (d 7.37, 2H, J = 7.8, 7.8 Hz; d 7.64, 2H, J = 7.8,
.2 Hz). An enamide group was established by the HMBC correla-
tions from an olefinic proton (d 6.59) and an exchangeable amide
proton to a conjugated carbonyl carbon (d 162.9) (Fig. 1). The
H
H
H
H
1
Comparison of the C-3 chemical shift between compound 1 and
H
2 (d
compound 2 also had a Z
confirmed by the ROESY correlation between H-5/9 (d
2-OMe (d 3.46) (Fig. 1). The Marfey’s method was not performed
on compound 2 as - and -6-bromotryptophan standards are not
C 6
117.4 and 117.3 respectively, in DMSO-d
) indicated that
double bond. This assignment was
7.62) and
C
2
,3
attachment of the enamide functionality to the benzene ring was
elucidated by the HMBC correlation from the same olefinic proton
D
H
to the aromatic carbon (d
the olefinic proton and methoxy protons (d
nary carbon (d 148.7) established the formation of an enol methyl
ether moiety. A para-substituted benzene ring was indicated by the
presence of two coupled aromatic doublets (d 7.05, 2H, J = 8.4 Hz
and d 6.65, 2H, J = 8.4 Hz). HMBC correlations from the aromatic
doublets and an exchangeable phenolic proton (d 9.18) to an aro-
matic oxygenated quaternary carbon (d 155.4) suggested that this
C
129.2). Further HMBC correlations from
H
2
3.49) to a sp quater-
L
D
H
available. However, compound 2 had the same sign of optical rota-
C
2
D
4
24
D
tion ½
c 0.2, MeOH) and compound 1 (½
ing a -6-bromotryptophan in 2. Iotrochamide B was therefore as-
signed as N-(Z)-2-methoxycinnamoyl- -6-bromotryptophan.
a
ꢀ
ꢁ12.5, c 0.1, MeOH) as that of
L
-tryptophan (½
a
ꢀ
= ꢁ54.3,
2
D
4
a
ꢀ
ꢁ13.6, c 0.1, MeOH), suggest-
H
L
H
L
H
Marine sponges belonging to the genus Iotrochota have been re-
C
benzene ring was a 4-hydroxyphenyl group. A 2-carboxy ethyl
moiety was established by the COSY correlation between the
ported to produce fatty acids, bromotyrosine derivatives, indole
2
4
and pyrrole alkaloids. Of the compounds isolated so far, iotrori-
2
5
26
methine (d
the ethyl group, and the HMBC correlations from the ethyl protons
to the carboxylic acid carbon (d 172.7) (Fig. 1). The 2-carboxy ethyl
H
4.49) and methylene (d
H
2.96 and 3.05) protons in
doside A and matemone displayed cytotoxic activity, while
2
7
purpurone
exhibited ATP-citrate lyase inhibition. Cinnamoyl
amides have been frequently isolated from plants of different gen-
C
2
4
unit was directly attached to the para-substituted benzene ring
since HMBC correlations were observed from the ethyl protons to
era. They have also been reported from marine organisms such as
1
9,28,29
ascidians belonging to the genus Botrylloides sp.
Iotrocha-
a quaternary aromatic carbon (d
C
127.6). Finally, the HMBC corre-
mides A (1) and B (2), however, are the first cinnamoyl amino acids
reported from the marine sponge Iotrochota sp. Biological study
from three experimental replicates (n = 3) showed that both com-
pounds exhibited moderate activity against T. b. brucei with IC50
values of 4.7 ± 2.5 and 3.4 ± 1.9 lM, respectively. A preliminary
cytotoxicity study using a human embryonic kidney cell line,
lation from the ethyl methine proton to the conjugated carbonyl
carbon established the connectivity of the 2-carboxy ethyl unit
and the enamide moiety to give the planar structure of 1.
2
,3
The geometry of the
D
double bond in 1 was established by
1
3
the characteristic C chemical shift of C-3 in comparison with
structurally similar botryllamides, such as botryllamide A (3). Pre-
HEK293, showed that compounds 1 and 2 had 85 ± 14% inhibition
at 50 lM and 100 ± 0% inhibition at 70 lM, respectively. These re-
sults indicated that both compounds 1 and 2 showed moderate
selectivity towards T. b. brucei.
In conclusion, two new cinnamoyl amino acids, iotrochamides A
(1) and B (2) were isolated from the marine sponge Iotrochota sp.
Compounds 1 and 2 were active against T. b. brucei with single digit
2,3
vious study suggested that botryllamides possess a
bond when the C-3 resonates at d >120 in CDCl
6
double bond when C-3 resonates at d <110 in acetone-d and
D
Z double
2
,3
C
3
, and a
D
E
C
1
9,20
DMSO-d
DMSO-d
6
.
The C-3 in compound 1 resonates at d
C
,3
117.4 in
Z double
2
6
and 120.9 in CDCl
3
, in agreement with a
D
bond. This assignment was supported by the ROESY correlation be-
tween H-5/9 (d 7.64) and 2-OMe (d 3.49) (Fig. 1). The absolute
lM IC50 values.
H
H

Y. Feng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4873–4876
4875
Table 1
a
NMR data for iotrochamides A (1) and B (2)
Position
1 in DMSO-d
6
1 in CDCl
3
2 in DMSO-d
6
d
C
d
H
mult. (J in Hz)
HMBC
ROESY
d
C
d
H
mult. (J in Hz)
d
C
d
H
mult. (J in Hz)
1
2
3
4
5
6
7
8
9
1
1
162.9
148.7
117.4
133.2
129.2
128.1
127.9
128.1
129.2
53.6
—
—
6.59 s
—
7.64 dd (7.8, 1.2)
7.37 dd (7.8, 7.8)
7.32 ddd (1.2, 7.8, 7.8)
7.37 dd (7.8, 7.8)
7.64 dd (7.8, 1.2)
4.49 ddd (4.8, 6.6, 9.6)
2.96 dd (9.6, 13.8)
—
—
—
—
H5, H9
—
H3, H6
H5
—
H9
H8, 2-OMe
H13
H10, H13
165.1
147.8
120.9
133.2
130.1
128.7
129.0
128.7
130.1
53.7
—
—
7.06 s
163.1
148.9
117.3
133.3
129.1
128.4
128.1
128.4
129.1
52.7
—
—
6.58 s
C1, C2, C5, C9
—
C3, C7, C9
C4, C8
C5, C9
C4, C6
C3, C5, C7
C1, C11, C12, 10-COOH
C10, C12, C13, 10-COOH
—
—
7.64 d (7.2)
7.37 dd (7.2, 7.2)
7.31 dd (7.2, 7.2)
7.37 dd (7.2, 7.2)
7.64 d (7.2)
4.86 ddd (6.0, 6.6, 6.6)
3.16 dd (6.6, 13.8)
3.26 dd (6.0, 13.8)
—
7.08 d (8.4)
6.77 d (8.4)
—
6.77 d (8.4)
7.08 d (8.4)
—
7.62 d (7.8)
7.37 dd (7.8, 7.8)
7.29 dd (7.8, 7.8)
7.37 dd (7.8, 7.8)
7.62 d (7.8)
4.59 ddd (4.8, 7.8, 9.0)
3.20 dd (9.0, 15.0)
3.26 dd (4.8, 15.0)
—
0
1
34.9
36.4
26.1
3.05 dd (4.8, 13.8)
1
1
1
1
1
1
1
1
2
1
1
2
3
4
5
6
7
8
9
127.6
129.5
114.7
155.4
114.7
129.5
—
—
—
—
H14
H13, 15-OH
—
H17, 15-OH
H16
—
—
H9
—
H14, H16
—
—
128.1
130.5
115.7
155.1
115.7
130.5
—
110.5
125.8
119.8
121.0
113.5
113.6
136.9
124.6
58.3
173.4
—
7.05 d (8.4)
6.65 d (8.4)
—
6.65 d (8.4)
7.05 d (8.4)
—
—
3.49 s
—
9.18 br s
8.20 br d (6.6)
—
C11, C15, C17
C12, C15, C16
—
C12, C14, C15
C11, C13, C15
—
—
—
7.52 d (8.4)
7.10 dd (1.8, 8.4)
—
7.50 d (1.8)
—
7.20 d (2.4)
3.46 s
—
—
—
—
3.49 s
-OMe
0-COOH
5-OH
58.6
172.7
—
—
—
C2
—
59.7
172.4
—
—
—
C14, C15, C16
C1
—
—
NH
indole-NH
7.01 br d (6.6)
—
—
—
8.19 br d (7.8)
10.97 br s
a
Spectra were recorded at 600 MHz for 1H and 150 MHz for 13C at 30 °C.
HEK293 cytotoxicity assay) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2012.05.029.
HO
O
O
OH
O
N
H
1
OMe
OMe
Br
References and notes
OH
O
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5.
6.
7.
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9
.
Feng, Y. J.; Davis, R. A.; Sykes, M.; Avery, V. M.; Camp, D.; Quinn, R. J. J. Nat. Prod.
2010, 73, 716.
Financial support from Drugs for Neglected Disease initiative
DNDi) and Deutsche Gesellschaft für Technische Zusammenarbeit
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(
GmbH on behalf of the Government of the Federal Republic of
Germany is acknowledged. We thank H. Vu from Griffith Univer-
sity for HRESIMS measurements. The authors thank D. Camp,
C. Lewis and K. Watts from Eskitis Institute, Griffith University
for their technical assistance. We are indebted to J. Hooper of the
Queensland Museum for collection and identification of the
sponge. The authors thank A. Schnaufer (University of Edinburgh)
who kindly supplied the trypanosome cell stock. We thank the
Australian Research Council (ARC) for support toward NMR and
MS equipment (LE0668477 and LE0237908).
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2
4
aꢀ
ꢁ13.6° (c 0.1, MeOH); UV
D
1 13
Supplementary data
e
C
C
19
H
5
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Table 1; (+)-HRESIMS m/z 465.0416 [M+Na] (calcd
65.0420).
4. In Dictionary of Natural Products on DVD; Chapman and Hall: 2009; Vol. 18.2.
a
ꢀ
ꢁ12.5° (c 0.1, MeOH); UV
D
1 13
(
e
+
21 2 4
C H19BrN NaO ,
4
2