Sacchathridine A, a prostaglandin release inhibitor from Saccharothrix sp.

Article abstract of DOI:10.1021/np3006327

Sacchathridine A (1) was isolated from the fermentation broth of strain Saccharothrix sp. MI559-46F5. The structure was determined as a new naphthoquinone derivative with an acetylhydrazino moiety by a combination of NMR, MS spectral analyses, and chemical degradation. Compound 1 showed inhibitory activity of prostaglandin E₂ release in a concentration-dependent manner from human synovial sarcoma cells, SW982, with an IC₅₀ value of 1.0 μM, but had no effect on cell growth up to 30 μM.

Full text of DOI:10.1021/np3006327

Note
pubs.acs.org/jnp
Sacchathridine A, a Prostaglandin Release Inhibitor from
Saccharothrix sp.
Koichi Nakae,* Ikuko Kurata, Fukiko Kojima, Masayuki Igarashi, Masaki Hatano, Ryuichi Sawa,
Yumiko Kubota, Hayamitsu Adachi, and Akio Nomoto
Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
*
S Supporting Information
ABSTRACT: Sacchathridine A (1) was isolated from the fermentation
broth of strain Saccharothrix sp. MI559-46F5. The structure was
determined as a new naphthoquinone derivative with an acetylhydrazino
moiety by a combination of NMR, MS spectral analyses, and chemical
degradation. Compound 1 showed inhibitory activity of prostaglandin E2
release in a concentration-dependent manner from human synovial
sarcoma cells, SW982, with an IC50 value of 1.0 μM, but had no effect
on cell growth up to 30 μM.
rostanoids are bioactive lipid mediators composed of
unsaturated twenty-carbon fatty acids. The biosynthesis of
prostanoids involves oxidation and subsequent isomerization of
P
arachidonic acid, which is released from the cell membrane by
1
phospholipase A enzymes. Arachidonic acid is enzymatically
2
converted to various kinds of prostanoid via cyclooxygenase
2
and prostanoid synthase. The released prostanoid binds to its
specific G protein-coupled receptor and activates second
3
messengers.
Prostaglandin E (PGE ) is known to play a prominent role
in several diseases such as inflammatory disorders, sensory
2
2
and two exchangeable protons from these spectra. The analysis
1
1
of the H− H COSY spectrum of 1 revealed the connectivity
neuron related disorders, and cancer, as well as in maintaining
3
4
−7
from an anomeric proton of a sugar at δ 5.41 (H-1′) to an sp
physiological homeostasis.
To discover effective molecules
methine proton at δ 3.94 (H-2′), 3.75 (H-3′), 3.30 (H-4′), and
that could be beneficial against these disorders, we conducted a
2
3
.37 (H-5′) and a methyl at δ 1.10 (H-6′) and from sp
screening program for inhibitors of PGE release from natural
2
methine protons at δ 7.50 (H-5) to 7.37 (H-6) (Figure 1). The
connectivities from H-5′ to δ 99.0 (C-1′), from H-1′ to δ 69.9
sources and identified an inhibitory sample from the culture
medium of a Saccharothrix sp. In this study, we report the
isolation, physicochemical properties, structural determination,
and biological activities of sacchathridine A found from this
microorganism.
The strain Saccharothrix sp. MI559-46F5 was cultured in a
00 mL Erlenmeyer flask containing 110 mL of a seed medium.
5
The inoculated medium was incubated at 27 °C for 48 h on a
rotary shaker. This seed culture was used to inoculate into 110
mL of the same medium in a 500 mL Erlenmeyer flask, which
was then incubated at 27 °C for 5 days on a rotary shaker.
From multiple flasks, 15 L of culture broth was loaded onto
DIAION HP-20 and eluted with MeOH. The extract was
concentrated, and the resulting aqueous solution was extracted
with EtOAc. The organic phase was evaporated to give a solid,
which was subjected to silica gel column chromatography
followed by gel filtration and reverse phase HPLC to give the
red powder of 1.
Figure 1. Key correlations observed in the 2D NMR spectra of
sacchathridine A by COSY, HMBC, and SIMBA.
1
13
1
13
H and C signals for 1 were assigned by H, C, DEPT135,
and HMQC spectral analysis. Compound 1 has four methyls,
Received: September 13, 2012
Published: April 12, 2013
3
2
five sp methines, two sp methines, nine quartnary carbons,
©
2013 American Chemical Society and
American Society of Pharmacognosy
720
dx.doi.org/10.1021/np3006327 | J. Nat. Prod. 2013, 76, 720−722

Journal of Natural Products
Note
2
(
C-5′), and from an sp carbon at δ 151.0 (C-7) revealed the
identical to those of standard methyl-α-L-rhamnoside (Wako
Pure Chemical Industries, Osaka, Japan) (Figures S3 and S4).
Finally, the structure of 2 was determined to be methyl-α-L-
presence of a sugar moiety that was attached to an olefinic or an
1
13
aromatic moiety. In addition to H− C long-range correlations
from H-5 to δ 184.1 (C-4), C-7, and δ 115.8 (C-8a), from H-6
to δ 125.6 (C-4a) and 142.0 (C-8), from methyl protons at δ
.94 (H-13) to δ 158.5 (C-2), δ 130.3 (C-3), and C-4, and
from a methoxy group at δ 3.93 (H-14) to C-2, the long-range
correlation at δ 182.8 (C-1) was obtained by the analysis of a
2
1
rhamnoside by comparison of the optical rotation (2, [α]
=
D
21
−57.9 (c 0.15 H O); standard, [α] = −59.1 (c 0.15 H O)).
2
D
2
1
1
Combined with this result and the J (C-1′) values (171.6
CH
Hz) of compound 1, the sugar moiety was proven to be α-L-
rhamnose. On the basis of all of these results, structure 1 was
proposed for sacchathridine A as N′-(7-methoxy-6-methyl-5,8-
dioxo-2-(α-L-rhamnosyloxy)-5,8-dihydronaphthalen-1-yl)-
acetohydrazide.
8
selective inverse multiple-bond analysis (SIMBA) experiment,
which revealed four-bond correlations. In particular, irradiation
at the C-1 carbon by SIMBA revealed the correlation at the
methyl proton of H-13 and the aromatic proton of H-5. These
observations indicated that 1 has a 2-methoxy-3-methylnaph-
thalene-1,4-dione skeleton. Remaining exchangeable protons (δ
.89 and 10.03) were assigned to be from the two NH groups
by H− N-HMQC. Finally, long-range coupling from the
methyl proton at δ 1.83 (H-12) to the carbonyl carbon at δ
The effect of compound 1 on PGE
release from human
2
synovial sarcoma SW982 cells was investigated. SW982 cells are
9
,10
known to produce prostaglandins and cytokines.
monitored bradykinin-induced PGE release from SW982
cells in a concentration-dependent manner (Figure 2a). As
shown in Figure 2b, compound 1 inhibited PGE release in a
We
9
2
1
15
2
1
68.2 (C-11), from NH-10 (δ 9.89) to C-8 and C-11, and from
concentration-dependent manner from SW982 cells with an
IC50 value of 1.0 μM. We also examined the effect of cell
growth on 1, and the results indicated that there was no cell
growth inhibition up to 30 μM (Figure 2c).
In conclusion, we isolated sacchathridine A (1) from the
culture broth of Saccharothrix sp. MI559-46F5. The structure of
NH-9 (δ 10.03) to C-7 and C-8a established that the
acetylhydrazino moiety was attached to C-8 and the sugar
moiety was attached to C-7 of the 2-methoxy-3-methylnaph-
thalene-1,4-dione skeleton. The planar structure of 1 was
elucidated as shown in Figure 1.
1
was elucidated to be a new naphthoquinone derivative with
Table 1. H and 13C NMR Spectroscopic Data for
Sacchathridine A (DMSO-d6)
1
an acetylhydrazino moiety. Sacchathridine A inhibited PGE2
release from SW982 cells without affecting cell growth and
would be a useful tool for the analysis of the mechanism of
multiplicity,
PGE release as a bioprobe.
δ
δ
2
position
C
type
H
J Hz
HMBC
SIMBA
5, 13
1
2
3
4
182.8
158.5
130.3
184.1
125.6
120.1
118.6
151.0
142.0
115.8
C
EXPERIMENTAL SECTION
C
■
C
General Experimental Procedures. The UV spectrum was
obtained on a U-2800 spectrophotometer (Hitachi High-Technolo-
gies, Tokyo, Japan). The IR spectrum was obtained on an FT-210
Fourier transform infrared spectrometer (Horiba, Kyoto, Japan).
Optical rotations were measured on a P-1030 polarimeter (JASCO,
Tokyo, Japan). NMR spectra were recorded using a JNM-ECA 600
C
4
5
6
7
8
8
9
1
1
1
1
1
1
2
3
4
5
6
a
C
CH
CH
C
7.50
7.37
d (8.5)
4, 6, 7, 8a
4a, 5, 8, 7
d (8.5)
(JEOL, Tokyo, Japan) with TMS as an internal standard. The
C
HRESIMS spectrum was measured using an LTQ Orbitrap mass
spectrometer (Thermo Fischer Scientific, San Jose, CA, USA).
Microorganism. The strain MI559-46F5 was isolated from a soil
sample collected at Higashikagawa, Kagawa Prefecture, Japan. The
substrate hyphae were branched and were pale yellow to pale brown.
The aerial mycelia were straight or flexuous and were fragmented into
cylindrical and ellipsoidal spores (0.5 to 0.7 by 0.9 to 1.3 μm). The
surface of the spore was smooth and was white to brownish-white.
These characteristics were observed on oatmeal agar (ISP medium
No.3) and inorganic salt-starch agar (ISP medium No.4). Whole-cell
hydrolysates contained meso-A2 pm, rhamnose, and galactose as the
characteristic whole-cell sugars. A polar lipid analysis showed
phosphatidylethanolamine as the diagnostic phospholipid. The
predominant menaquinone was MK-9(H4), and a trace amount of
MK-10(H4) was contained. The cell-wall acyl type was acetyl type.
The partial 16S rRNA gene sequence (1446 bp) of the strain showed
high identity with those of the genus Saccharothrix such as
a
C
NH
NH
C
10.03
9.89
br
br
7, 8a
8, 11
0
1
2
3
4
′
′
′
′
′
168.2
20.5
9.0
CH3
CH3
CH3
CH
CH
CH
CH
CH
CH3
1.83
1.94
3.93
5.41
3.94
3.75
3.30
3.37
1.10
(s)
11
(s)
2, 3, 4
2
60.7
99.0
69.7
70.3
71.8
69.9
17.9
(s)
brd (1.4)
m
5′, 7
3′, 4′
4′
2′, 3′, 6′
1′, 3′, 4′
4′, 5′
dd (3.4, 9.3)
t (9.3)
dq (6.1, 9.3)
d (6.1)
′
T
In regard to the analysis of the sugar constituent of 1, the
Saccharothrix texasensis (NRRL B-16107 , T: type strain, 1429/1444
3
3
3
T
vicinal coupling constant J values ( J = 1.4 Hz, J = 3.4
Hz, J3 = 9.3 Hz, J = 9.3 Hz, and J5′,6′ = 6.1 Hz) suggested
bp, 98.9%) and S. xinjiangensis (NBRC 101911 , 1418/1441 bp,
HH
1′,2′
2′,3′
3
3
98.4%). The GenBank/EMBL/DDBJ accession number for the 16S
rRNA gene sequence of MI559-46F5 is AB795585. These phenotypic
and genotypic analyses suggested that the strain MI559-46F5 belonged
to the genus Saccharothrix. Therefore, the strain was tentatively
designated as Saccharothrix sp. MI559-46F5.
′,4′
4′,5′
that the sugar moiety was rhamnose. The absolute config-
uration of rhamnose was determined by chemical degradation.
Sacchathridine A (15.4 mg) was dissolved in 1 mL of 5−10%
HCl−methanol and stirred at 60 °C for 24 h. The reaction
mixture was diluted by EtOAc, and the aqueous phase was
evaporated. The residue was purified by preparative TLC
Fermentation and Isolation of Sacchathridine A. A slant
culture of MI559-46F5 was used for inoculation in 500 mL Erlenmeyer
flasks. Each flask contained 110 mL of seed medium consisting of
galactose 2%, dextrin 2%, glycerol 1%, soy peptone 1%, corn steep
liquor 0.5%, (NH ) SO 0.2% and CaCO 0.2% in deionized water
(
CHCl −MeOH−H O, 10:5:1) to give pure methylrhamno-
3
2
1 13
side (2) (2.6 mg). The H and C NMR data of 2 were
4
2
4
3
7
21
dx.doi.org/10.1021/np3006327 | J. Nat. Prod. 2013, 76, 720−722

Journal of Natural Products
Note
Figure 2. Biological activity of 1. (a) Concentration−response of PGE release from SW982 cells induced by bradykinin. (b) Effect of sacchathridine
2
A on PGE release from SW982 cells. (c) Effect of sacchathridine A on cell growth of SW982 cells. Data represent mean ± SD of 3 or 4 independent
2
experiments. 1 was initially dissolved in DMSO and diluted by assay buffer. The final concentration of DMSO was 0.1%.
and adjusted to pH 7.0 before sterilization. The flasks were shaken on
a rotary shaker at 200 rpm at 37 °C for 2 days. An aliquot of 2 mL of
the seed culture was inoculated into 110 mL of the culture medium
consisting of glycerol 2%, dextrin 2%, soy peptone 1%, yeast extract
ASSOCIATED CONTENT
Supporting Information
■
*
S
1
13
1
15
H NMR, C NMR, and H− N-HMQC spectra in DMSO-d
6
1
13
for sacchathridine A (1) and H NMR and C NMR spectra of
0
.3%, (NH ) SO 0.2% and CaCO 0.2% in deionized water (pH 7.4)
4 2 4 3
methylrhamnoside (2) and methyl-α-L-rhamnoside in CD OD
are available free of charge via the Internet at http://pubs.acs.
org.
3
in a 500 mL Erlenmeyer flask and incubated at 27 °C for 5 days on a
rotary shaker (180 rpm). The cultured broth (15 L) was centrifuged to
separate the supernatant and a mycelial cake. The supernatant was
loaded on DIAION HP-20 (1.5 L) (Mitsubishi Chemical Co., Tokyo,
Japan), washed with 50% MeOH, and then eluted with MeOH. The
MeOH fraction was concentrated, and the resulting aqueous solution
was extracted with EtOAc (4.5 L). The extract was dried over
anhydrous Na SO and evaporated to give a red oil (1.13 g). The oil
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-3-3441-4173. Fax: +81-3-3441-7589. E-mail:
nakaek@bikaken.or.jp.
■
2
4
was subjected to silica gel column chromatography (60 g) and eluted
Notes
with 900 mL of CHCl −MeOH (10:1) followed by 1200 mL of
3
The authors declare no competing financial interest.
CHCl −MeOH (50:1). The active fractions (127 mg) were combined
3
and evaporated to give a solid, which was further subjected to column
chromatography using TOYOPEARL HW-40F (Tosoh Co., Tokyo,
Japan) eluted with MeOH to yield a red residue (44 mg). Finally,
active fractions were separated via preparative HPLC (CAPCELL PAK
C18 UG column, 5 μM 20 i.d. × 250 mm (Shiseido, Tokyo, Japan)
with photodiode array detector) with CH CN−H O (20:80)
ACKNOWLEDGMENTS
■
This work was supported in part by a grant from JSPS
KAKENHI (grant no. 22710227). The authors would like to
thank M. Kawada, S. Ohba, and T. Masuda for providing
technical assistance and helpful discussions.
3
2
containing 0.01% TFA at a flow rate of 8 mL/min. The fraction
with a retention time of 12.9 min yielded compound 1 (31.8 mg).
Physicochemical Properties of 1. The physicochemical proper-
REFERENCES
■
(1) Murakami, M.; Kudo, I. J. Biochem. 2002, 131, 285−292.
2) Bos, C. L.; Richel, D. J.; Ritsema, T.; Peppelenbosch, M. P.;
ties of 1 are as follows: red powder; [α]23 −358 (c 0.0036 in MeOH);
(
D
Versteeg, H. H. Int. J. Biochem. Cell Biol. 2004, 36, 1187−1205.
(3) Breyer, R. M.; Bagdassarian, C. K.; Myers, S. A.; Breyer, M. D.
Annu. Rev. Pharmacol. Toxicol. 2001, 41, 661−690.
UV λmax (MeOH) 228, 263, 470 nm (ε 17 254, 9908, 2739); IR
−1
spectrum νmax (KBr) 3400, 1681, 1643, 1139, 953 cm . The
molecular formula of sacchathridine A was determined to be
C H N O by HRESIMS (positive ion mode, m/z 459.1371 (M +
(
4) Funk, C. D. Science 2001, 294, 1871−1875.
2
0
24
2
9
+
(5) Furuyashiki, T.; Narumiya, S. Curr. Opin. Pharmacol. 2009, 9,
Na) Δ −0.3 mDa as C H N O +Na).
20
24
2
9
3
(
1−38.
Measurement of PGE . SW982 cells were incubated for 24 h in a
2
6) Ushikubi, F.; Segi, E.; Sugimoto, Y.; Murata, T.; Matsuoka, T.;
96-well plate (10 000 cells/well) at 37 °C and then washed with assay
Kobayashi, T.; Hizaki, H.; Tuboi, K.; Katsuyama, M.; Ichikawa, A.;
Tanaka, T.; Yoshida, N.; Narumiya, S. Nature 1998, 395, 281−284.
7) Cathcart, M. C.; O’Byrne, K. J.; Reynolds, J. V.; O’Sullivan, J.;
buffer (Hank’s balanced salt solution, 17 mM HEPES pH 7.4, 0.1%
BSA). Cells were treated with 1 nM bradykinin (Sigma) at 37 °C. To
detect PGE , cells were incubated for 30 min. The release of PGE into
(
2
2
Pidgeon, G. P. Biochim. Biophys. Acta 2012, 1825, 49−63.
the assay buffer was quantified using a fluorescence resonance energy
(
(
8) Crouch, R.; Martin, G. J. Magn. Reson. 1991, 92, 189−194.
11
transfer-based assay kit (Cisbio, Codolet, France) by an Envision
microplate reader (PerkinElmer Inc., Waltham, MA, USA). Under this
condition, the IC50 value of SC-58125, selective cyclooxygenase-2
inhibitor, was 33 nM.
9) Yamazaki, T.; Yokoyama, T.; Akatsu, H.; Tukiyama, T.; Tokiwa,
T. In Vitro Cell. Dev. Biol. Anim. 2003, 39, 337−339.
(10) Wada, Y.; Shimada, K.; Kimura, T.; Ushiyama, S. Immunol. Lett.
2005, 101, 50−59.
(11) Goedken, E. R.; Gagnon, A. I.; Overmeyer, G. T.; Liu, J.;
Petrillo, R. A.; Burchat, A. F.; Tomlinson, M. J. J. Biomol. Screen. 2008,
13, 619−625.
Cell Proliferation Assay. SW982 cells were grown in DMEM/
F12 medium supplemented with 10% fetal bovine serum, penicillin,
streptomycin, MEM-nonessential amino acids, and 1 mM sodium
pyruvate at 37 °C. SW982 cells were seeded in a 96-well plate (3000
cells/well). Sacchathridine A was added, and the plates were incubated
for 48 h. Cell counting kit-8 solution was added, and the cells were
incubated for 90 min before measuring the absorbance at 450 nm
using an ARVO microplate reader (PerkinElmer Inc.).
7
22
dx.doi.org/10.1021/np3006327 | J. Nat. Prod. 2013, 76, 720−722