596
Published on the web May 15, 2010
Dinohydrazides A and B, Novel Hydrazides from a Symbiotic Marine Dinoflagellate
Norihito Maru,1 Osamu Ohno,2 Kaoru Yamada,2 and Daisuke Uemura*2
1Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602
2Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522
(Received April 1, 2010; CL-100313; E-mail: uemura@bio.keio.ac.jp)
Dinohydrazides A (1) and B (2), novel naturally-occurring
3'
6'
3'
6'
2'
1'
2'
1'
4'
5'
4'
5'
O
O
dibenzoylhydrazines, were isolated from a symbiotic marine
dinoflagellate of Okinawan sponge. Their structures were
determined by spectroscopic analyses and synthetic methods.
They moderately inhibited the growth of mammalian cells.
H
N
H
2
1"
2
5
1
3
1
7'
7'
N
3
7
7
N
N
2"
2"
H
H
4
O
4
O
6
6
5
1"
dinohydrazide A (1)
dinohydrazide B (2)
Marine organisms produce various molecules with remark-
able physiological activities. It has been suggested that most of
those metabolites are biosynthesized by marine microorganisms,
i.e., bacteria, blue-green algae, and dinoflagellates that are in the
food chain of, or in a symbiotic relationship with, their host
animals. Among them, dinoflagellates are widely known to be
a rich source of biologically active and structurally unique
secondary metabolites.1 Therefore, dinoflagellates that coexist
with marine invertebrates have been isolated and cultured to
search for useful compounds. In our recent studies, various
biologically active metabolites have been isolated from sym-
biotic marine dinoflagellates, such as symbioimines,2 symbio-
dinolide,3 symbiospirols,4 karatungiols,5 and durinskiols.6 In our
continuing search for biologically active compounds, unique
novel dibenzoylhydrazines, named dinohydrazides A (1) and
B (2), were isolated from a symbiotic dinoflagellate of the
Okinawan sponge. We describe here their isolation, structure
elucidation, and biological activities.
H
H
N
N
O
O
3
Scheme 1.
H2¤/C1¤ and H2¤/C7¤ indicated connectivity between the amide
group and one aromatic group. However, the connection around
two amide bonds could not be confirmed by 2D NMR analyses.
Dinohydrazide B (2)9 has the same molecular formula
(C16H16N2O2) as 1, as suggested by HRESIMS at m/z 291.1117
[M + Na]+ (calcd for C16H16N2O2Na, 291.1109). The H and
1
13C NMR data for 2 are summarized in Table 1. On the basis of
1H and 13C NMR spectra, the structure of 2 was elucidated to be
the same as that of 1 except for the ethyl residue. Therefore, we
sought to establish the final structures of dinohydrazides by
syntheses.
An unidentified dinoflagellate, isolated from the marine
sponge Xeospongia sp. which was collected at Bise, Okinawa
Prefecture, Japan, was cultured for 60 days in 20 L of seawater
medium enriched with 2% ES supplement.7 After cultivation,
the cells were harvested by centrifugation and extracted with
80% aqueous ethanol. The concentrated extract was partitioned
with ethyl acetate and water, and the aqueous layer was
chromatographed on TSK G-3000S polystyrene gel (water
to EtOH), ODS gel (50% aqueous MeOH to MeOH) and
reversed-phase HPLC (Develosil ODS-HG-5, 70% aqueous
MeOH to MeOH), monitoring the growth inhibitory effect
against HUVEC cells. Final purification was achieved by HPLC
(Develosil RPAQUEOUS, 50% aqueous MeOH to 80% aqueous
MeOH) to give dinohydrazides A (1) (1.1 mg) and B (2) (0.4
mg) as a white powder (Scheme 1).
1, 2, and their derivative 3 were synthesized according to
a previous report.10 Synthesis of compound 1 started with
3-bromobenzoyl chloride, which was stirred in THF at 0 °C,
followed by addition of benzoylhydrazine and sodium carbonate
in aqueous THF. After the mixture was stirred for 3 h at 0 °C, the
resulting white precipitate of 1-benzoyl-2-(3-bromobenzoyl)hy-
drazine in the reaction mixture was filtered. An ethyl group was
then introduced to the 3-bromobenzoyl moiety by the Suzuki-
Miyaura cross-coupling. The hydrazide, triethylborane, cesium
carbonate, and Pd(dppf)Cl2 catalyst were stirred in THF under
argon atmosphere at reflux for 3 h to give 1 as a white
amorphous powder (83% yield in 2 steps). Synthesis of
compound 2 was carried out following the same protocol as
that of the first step in the preparation of 1, which utilized 4-
ethylbenzoyl chloride as substrate (91% yield). The synthesis of
compound 3 began with benzoyl isothiocyanate, which was
dissolved in acetonitrile and treated with 3-ethylaniline for 3 h
at 40 °C to give a coupled product of thiourea as a white
precipitate. The thiocarbonyl unit was then oxidized to a
carbonyl by sodium metaperiodate in DMF/water for 15 min to
give 3 as a white amorphous powder (31% yield in 2 steps). The
NMR spectra of natural dinohydrazide A was not superimpos-
able with that of 3.11 Meanwhile, the NMR spectrum of
synthetic 1 and 2 were identical to those of natural dinohy-
drazides A and B. Thus, the structures of dinohydrazides A and
B were confirmed to be as proposed.
Dinohydrazide
A a molecular formula of
(1)8 has
C16H16N2O2, as suggested by HRESIMS at m/z 291.1129
1
[M + Na]+ (calcd for C16H16N2O2Na, 291.1109). The H and
13C NMR data for 1 are summarized in Table 1. Further 1H,
13C NMR and HMQC spectra in CD3OD revealed the presence
of one sp3-methyl, one methylene, two amide groups, and two
aromatic groups. A detailed analysis of the 1H NMR and COSY
spectra of 1 allowed us to elucidate three partial structures, C4-
C6, C2¤-C6¤, and C1¤¤-C2¤¤. The HMBC correlations at H2/C1,
H6/C1, H1¤¤/C2, H1¤¤/C3, H1¤¤/C4, and H2¤¤/C3 revealed the
structure of a disubstituted benzene. The HMBC correlations at
Chem. Lett. 2010, 39, 596-597
© 2010 The Chemical Society of Japan
Maru, Norihito
