Merobatzelladines A and B, anti-infective tricyclic guanidines from a marine sponge monanchora sp.

Article abstract of DOI:10.1021/ol9006794

Merobatzelladines A (1) and B (2) have been isolated from a marine sponge Monanchora sp. as antibacterial constituents. Their structures including relative stereochemistry were determined by interpretation of spectral data. The absolute stereochemistry of

Full text of DOI:10.1021/ol9006794

ORGANIC
LETTERS
2009
Vol. 11, No. 12
2655-2658
Merobatzelladines A and B,
Anti-Infective Tricyclic Guanidines from
a Marine Sponge Monanchora sp.
Shunsuke Takishima,† Aki Ishiyama,‡ Masato Iwatsuki,‡ Kazuhiko Otoguro,‡
‡
‡
†
§
˜
Haruki Yamada, Satoshi Omura, Hirotsugu Kobayashi, Rob W. M. van Soest,
and Shigeki Matsunaga*,†
Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural
and Life Sciences, The UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan,
Research Center for Tropical Diseases, Kitasato Institute for Life Sciences, Kitasato
UniVersity, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan,
and Zoological Museum, UniVersity of Amsterdam,
1090 GT Amsterdam, The Netherlands
assmats@mail.ecc.u-tokyo.ac.jp
Received April 3, 2009
ABSTRACT
Merobatzelladines A (1) and B (2) have been isolated from a marine sponge Monanchora sp. as antibacterial constituents. Their structures
including relative stereochemistry were determined by interpretation of spectral data. The absolute stereochemistry of merobatzelladine B (2)
was elucidated after introduction of the fourth ring system preinstalled with a secondary hydroxyl group to which the modified Mosher method
was applied. Merobatzelladines exhibit moderate anti-infective activity against a bacterium and protozoa.
Even though a variety of secondary metabolites with
cytotoxic activity have been discovered from marine inverte-
brates, those with antibacterial activity are less pronounced.1
In the course of our search for antibacterial agents against fish
pathogenic bacteria,2 the extract of a marine sponge Monan-
chora sp. (collected off Amami-Oshima, ZMAPOR 19862)
exhibited potent activity against Vibrio anguillarum. Bio-
assay-guided fractionation afforded two tricyclic guanidine
derivatives merobatzelladines A (1) and B (2).3,4
The MeOH and CHCl3/MeOH (1:1) extract of the
sponge Monanchora sp. was partitioned between water
and CHCl3, and the organic layer was subjected to a
modification of the Kupchan’s solvent partitioning
scheme5 to give the antibacterial CHCl3 fraction. This
material was subjected to ODS flash chromatography
followed by reversed-phase HPLC permitting us to isolate
merobatzelladines A (1) and B (2) from a complex mixture
of closely related compounds.
† The University of Tokyo.
‡ Kitasato University.
§ University of Amsterdam.
(1) Donia, M.; Hamann, M. T. Lancet Infect. Dis. 2003, 3, 338–348.
(2) Kobayashi, H.; Miyata, Y.; Okada, K.; Fujita, T.; Iwashita, T.; Nakao,
Y.; Fusetani, N.; Matsunaga, S. Tetrahedron 2007, 63, 6748–6754.
(3) The major cytotoxic constituent of this sponge was identified as
azaspiracid-2.4 A reexamination of the sponge in ref 4 indicated that the
taxonomy of the sponge should be corrected from Echinoclathria sp. to
Merobatzelladine A (1) was isolated as the TFA salt and
the molecular formula of C23H42N3 was assigned for the free
base by HRESIMS. Interpretation of the 1H NMR, 13C NMR,
Monanchora sp
.
10.1021/ol9006794 CCC: $40.75
Published on Web 05/26/2009
 2009 American Chemical Society

and HSQC data (Table 1) showed the presence of 2 terminal
methyls, 14 methylenes, 4 methines, 1 disubstituted olefin,
and 1 nonprotonated sp2 carbon.
linkage between units a and b was not assigned by the COSY
data due to degenerate H signals for H2-1′ and H2-2′, a
1
TOCSY cross peak between H2-3′ and H-1 demonstrated the
connection between C-1′ and C-2′. In the same way, the
linkage between units b and c was displayed on the basis of
1
a TOCSY cross peak between H-3 and H-5a. The H and
1
Table 1. H and 13C NMR Data of Merobatzelladines A (1) and
13C shifts for the methine carbons (C-1, C-3, C-6, and C-8)
suggested that they were all substituted by a nitrogen atom.7
Considering the molecular formula, which showed the
absence of oxygen atom, and the chemical shift of the
remaining sp2 carbon (δ 150.6), the four methine carbons
were linked to a guanidyl group to form the hexahydro-
5,6,6a-triazaacenaphthalene ring system as observed in
ptilomycalin and batzelladine class of sponge metabolites.8
The NMR data within the ring system of 1 agreed well with
those in the literature. To satisfy the molecular formula the
remaining portion was an n-hexyl group (unit d), which
should be attached to C-1′′ (Figure 1).
B (2) in CD3ODa
1
2
no.
δC (typeb)
δH
HMBC #H δC (type)
δH
J (Hz)
1
51.5, CH
3.43, m
2a,2′
51.5, CH
3.43, m
2a
2b
3
34.8, CH2 1.25, m
2.25, m
9,7b
34.8, CH2 1.26, m
2.27, m
57.5, CH
3.76, m
2a,4a,4b
57.5, CH
3.76, m
4a
4b
5a
5b
6
30.8, CH2 1.70, m
2.23, m
30.8, CH2 1.68, m
2.23, m
31.3, CH2 1.64, m
2.24, m
31.2, CH2 1.66, m
2.24, m
53.5, CH
3.73, m
4a
53.5, CH
3.74, m
7a
7b
8
31.9, CH2 1.57, m
2.16, m
31.9, CH2 1.58, m
2.17, m
The relative stereochemistry of the tricyclic portion was
assigned by analysis of NMR data. The ROESY cross peak
between H-1 and H-3 showed that they were syn, whereas
ROESY cross peaks between H-6 and H2-1′′ suggested that H-6
and H-8 were anti. The assignment of the relationship between
the angular hydrogen atoms (H-3 and H-6) was not possible
50.1, CH
150.6, C
3.49, m
50.1, CH
150.6, C
3.49, m
9
8
3′
1′a
1′b
2′
35.5, CH2 1.54, m
1.59, m
26.2, CH2 1.45, m
27.7, CH2 2.09, m
35.9, CH2 1.54, m
1.59, m
25.8, CH2 1.40, m
32.9, CH2 1.35, m
23.6, CH2 1.35, m
14.3, CH3 0.92, m
3′
4′,5′
2′,3′,6′
3′,6′,7′
3′
4′
129.3, CH
133.3, CH
5.32, m
5.38, m
2.04,
by analysis of NMR data due to the degeneracy of 1H and 13
C
5′
signals. Then we turned our attention to compare carbon
chemical shifts of 1 with those of synthetic compounds with
defined stereochemistry: the data for the isomer with 1,3-syn,
3,6-syn, 6,8-syn-stereochemistry (plane-symmetric isomer) and
those with 1,3- syn, 3,6-anti, 6,8-syn-stereochemistry (C2-
symmetric isomer) had been reported (Figure 2).9 Carbon
6′
7′
21.5, CH2
quint (7)
4′,5′,7′
5′,6′
14.7, CH3 0.95, t (7)
36.2, CH2 1.48, m
1.56, m
27.1, CH2 1.36, m
30.3, CH2 1.30, m
30.4, CH2 1.30, m
33.0, CH2 1.29, m
23.7, CH2 1.30, m
14.4, CH3 0.89, t (7)
1′′a
1′′b
2′′
3′′
4′′
5′′
6′′
7′′
36.2, CH2 1.49, m
1.57, m
26.8, CH2 1.40, m
32.9, CH2 1.35, m
23.6, CH2 1.35, m
14.3, CH3 0.92, m
7′′
5′′,7′′
6′′
a 600 MHz for 1H and 150 MHz for 13C. b Multiplicity assigned by the
HSQC data.
Interpretation of the COSY spectrum, in conjunction with
the HSQC data, starting from the lower field methyl triplet
(δ 0.95; H3-7′) indicated unit “a” (Figure 1) in which ∆4′-
Figure 2. Carbon chemical shifts of the tricyclic portion of 1 and
model compounds.9
Figure 1. Partial structures in merobatzelladine A (1).
chemical shifts of C-1, C-2, and C-3 in 1 coincided well with
those of the plane-symmetric isomer within errors less than 0.1
ppm, but differed from those of the C2-symmetric isomer by
between 0.4 and 1.6 ppm.9a As expected the chemical shifts
for the C-6 to C-8 portion in 1 differed from both of the model
olefin was assigned as E on the basis of the carbon chemical
shifts of an allylic carbon (δ 27.7; C-3′).6 Analysis of the
COSY data also suggested units b and c. Although the
(5) Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Sigel, C. W. J. Org.
Chem. 1973, 38, 178–179.
(4) Ueoka, R.; Ito, A.; Izumikawa, M.; Maeda, S.; Takagi, M.; Shin-ya,
K.; Yoshida, M.; van Soest, R. W. M.; Matsunaga, S. Toxicon 2009, 53,
680–684.
(6) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; VCH:
Weinheim, 1990; pp 192-194.
2656
Org. Lett., Vol. 11, No. 12, 2009

compounds, because 1 had 6,8-anti stereochemistry. In related
compounds with the same tricyclic skeleton 3,6-syn- and 3,6-
anti-isomers gave diagnostic carbon chemical shift values
corresponding to each skeleton and long-distance substituent
effects were hardly observed.10 Therefore, we assigned 3,6-
syn stereochemistry for 1.
In 6 H-10a and H-12a were coupled to H-11 by 8.5 Hz
indicating that H-11 was axial, suggesting that 6 could be
represented by either 6a or 6b (Figure 3). Structure 6a
Merobatzelladine B (2) had a molecular formula of
C19H36N3 as the free base, which was analyzed by HRESIMS.
The 1H and 13C NMR data of 2 coincided well with those of
1 except for the absence of the olefinic carbons. Interpretation
of the COSY and HSQC data and comparison of the NMR
data with those of 1 demonstrated that 2 had the tricyclic
portion identical with that of 1: the identity of the relative
stereochemistry was implied by the superimposable NMR
data and confirmed by the ROESY data. The molecular
formula and the NMR data suggested that 2 had two saturated
linear side chains composed of 10 carbons in total. The
lengths of the side chains were determined by interpretation
of 13C NMR data. 13C chemical shifts of three carbons from
the terminus of the side chains were assigned by the HMBC
data as δ 14.3 (2C; C-5′ and C-5′′), 23.6 (2C; C-4′ and C-4′′),
and 32.8/32.9 (C-3′ and C-3′′), whereas HSQC spectrum
allowed the assignment of C-1′/C-1′′ and C-2′/C-2′’ signals,
leaving no unassigned carbon signals. Therefore, both side
chains are n-pentyl group.
Then, we sought to determine the absolute stereochemistry
of merobatzelladine B (2). It was reported that related cyclic
guanidines could be converted to the triamines by reduction of
the guanidyl group followed by acidic hydrolysis.11 The
resulting triamine would be a good substrate for the modified
Mosher analysis. However, our attempts to reduce 1 or 2 with
NaBH4 were unsuccessful. Then we turned our attention to use
two of the nitrogen atoms as a scaffold to introduce a fused
six-membered ring in which a secondary hydroxyl group was
preinstalled (Scheme 1).11 Even though 1 decomposed under
Figure 3. Possible structures of 5 (5a and 5b) and 6 (6a and 6b)
with NOESY correlations shown by solid arrows.
accounted for all the observed NOESY cross peaks. How-
ever, NOESY data were not consistent with 6b: the distances
between H2-1′ and H-10b are shorter than those between H2-
1′ and H-10b, but cross peaks not between the former pairs
but between the latter pairs were observed; the same was
true of the NOESY cross peaks and the distances between
protons on C-1′′ and C-12. Therefore, 6 should be represented
by structure 6a. With the structure of 6 established, 5 should
be the diastereomer of 6 differing in the stereochemistry at
C-11. H-11 in 5 was in the equatorial position because all
the C-10 and C-12 methylene protons appeared as a broad
doublet. Therefore, compound 5 was represented by structure
5a, because structure 5b was a conformational isomer of 6a.
The absolute stereochemistry of 6 was determined by
application of the modified Mosher’s method.12 Compound
6 was converted to the (S)- and (R)-MTPA esters (7 and
8, respectively). In both 7 and 8, the OMTPA group
Scheme 1
.
Introduction of the Fourth Ring System in
Merobatzelladine B (2)
1
occupied the axial position as demonstrated by the H
NMR data. Even though the distribution of the signs of
chemical shift differences in axial esters are not completely
uniform, previous studies suggested that this methodology
could be applicable to the axial esters.13,14 Analysis of the
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the reported reaction condition to conjugate with 1,3-ditosyloxy-
2-benxyloxypropane, 2 was stable enough to be converted to a
mixture of diastereomeric tetracyclic derivatives 3 and 4. They
were separated by HPLC and the benzyl group was removed
by catalytic hydrogenation to afford 5 and 6 whose relative
stereochemistry was assigned by interpretation of the NMR data
in DMSO-d6 as described below.
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Org. Lett., Vol. 11, No. 12, 2009
2657

∆δ values allowed us to assign the 11S-stereochemistry for
6 (Figure 4). From this result the absolute stereochemistry
of merobatzelladine B (2) was assigned as 1R,3S,6R,8R.
in the syn-direction to the angular hydrogen within the six-
membered ring system: in all previously isolated congeners
with this ring system, the relevant portion has anti-relation-
ship.17 This deviation in the stereochemistry distinguishes
merobatzelladines from a fragment of batzelladines such as
batzelladine K.19 This feature is interesting in light of the
recently proposed biogenesis of sponge-derived cyclic
guanidines.20 The assignment of the stereochemistry of the
unfunctionalized hexahydro-5,6,6a-triazaacenaphthalene ring
system had only been accomplished by total synthesis.21 We
have overcome the difficulty in the structure elucidation by
devising a method using a derivatization reaction for this
ring system followed by the modified Mosher analysis. Even
though the assignment of the relative stereochemistry of the
newly introduced hydroxyl group was not simple, we were
able to assign the absolute stereochemistry of merobatzel-
ladine B (2).
Figure 4. Modified Mosher analysis of 6.
Merobatzelladines exhibit moderate antimicrobial activity
against Vibrio anguillarum: inhibitory zones of 9-10 mm
on application of 50 µg of a sample to a paper disk of 6 mm
diameter. Merobatzelladines A and B also inhibit Tripano-
soma brucei brucei (GUT at 3.1) with IC50 value of 0.24
µg/mL each.15 They display moderate inhibitory activity
against the K1 strain of Plasmodium falciparum with IC50
values of 0.48 µg/mL and 0.97 µg/mL, respectively.16
Merobatzelladines are new members of the batzelladines/
ptilomycalin class of metabolites which contain one hexahy-
dro-5,6,6a-triazaacenaphthalene ring system or two. A wide
range of biological activities have been reported for this class
of metabolites.17 It is interesting to speculate whether
merobatzelladines inhibit protein-protein interactions rel-
evant to the infection of AIDS virus.18 Although merobat-
zelladine A or B may be considered as a fragment of
batzelladines, one side-chain in merobatzelladines emanates
Acknowledgment. We thank the officers and crew of T/S
Toyoshio-maru of Hiroshima University for their assistance
in collecting the sponge specimen. We acknowledge K. Hori,
Hiroshima University, for the arrangement of the cruise. We
thank K. Ogawa, The University of Tokyo, for a gift of
bacterial strains. We are indebted to S. Murayama, Labora-
tory of Aquatic Natural Products Chemistry, for HRESIMS
measurements. This work was partly supported by Grant-
in-Aids for Scientific Research 16380142 from Japan Society
for Promotion of Science and on Priority Area 16073207
from Ministry of Education, Culture, Sports, Science, and
Technology and Funds from the Drugs for Neglected
Diseases initiative and a Grant from All Kitasato Project
Study.
Supporting Information Available: Experimental details,
1H and 13C NMR assignments of 5-8, and NMR spectra of
1, 2, and 5-8. This material is available free of charge via
the Internet at http://pubs.acs.org.
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de, C.; Cuevas, C.; Garcia-Fernandez, L. F.; Kelly, M.; Hamann, M. T.
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