P

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THE JOURNAL OF ANTIBIOTICS

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1997

NOTES

Isolation and Structure Elucidation of Novelin 500-ml Erlenmeyer flasks containing 100ml (totallyliters)

Neuronal Cell Protecting Substances,
Carbazomadurins A and B Producedby Actinomadura madurae

7.4 beforeand Haruo Seto*

Institute of Molecular and Cellular Biosciences,
The University of Tokyo,
Tokyo

1 13, Japan

1 Division of Agriculture and Agricultural Life Sciences,
The University of Tokyo,
Bunkyo-ku,(ReceivedTokyofor publication

It was recently reported
centration of L-glutamate,

1 13, Japan

June 9, 1997)

that the extracellularconan excitatory amino acid,acting as a major neurotransmitter in the brain, increases

after brain ischemic attack1>2). In addition, neuronal cell
death is suggested to be related to excessive excitementof glutamate receptors and inhibition of the uptake ofcystine, a precursor of glutathione which is the major
reducing substance in the cells3'4). Therefore, antagonists
for glutamate receptors and radical scavengers may be
useful candidates

to cure brain ischemia

medium consisting

Pharmamedia 1.5%, L-glutamate

NOBUHIRO KOTODA, KAZUO SfflN-YA,
KAZUO FURIHATAf,YOICHI HAYAKAWA

Bunkyo-ku,of theinjury.

In thesterilizationgelcolumnpackedwith

The physico-chemical

UV and visible
spectra, which resembled those ofcarbazole compounds such as epocarbazolins12)
andcarbazomycin B13). The molecular formulae of 1 and2 were establishedbyofcarbazomadurins

A (1) and B (2).
BUVX<£á"nm(£)

UV /lMeOH+NaOH nm (£)

739692)0,0),10),062
7(,4200
1,6393(,008,5055
4223( ,),0)00,1,4),h26s(+)3).cM
OedN(5(701210H
12-.238250C
1 1991.353
3rw
edow
lleeolyap,)000
63
9,0
788
563
2(,002,7(,)hs

IR v^ CKBrJ cm"1
Pale yellow
powder108-110(dec.)
C23H29NO3
367.2158(M)+367.2148+4° (c 0.05,MeOH)
237 (35,700),
249 (29,100,sh),289 (7,400,299 (10,400),
347 (6,500),
357 (7,300)
236 (33,600),
255 (27,300),
304 (8,100),
363 (6,600)
3440 (br),1620, 1580, 1440,1380sh),

3440(br);

1630,1585,1440,1380

high-resolution

Fig. 1. Structure ofcarbazomadurins

The producing organism of carbazomadurins, iden-MP(°C)
Molecular formula
HRFAB-MS found:
calcd:of 1 and 2 aresummarized in Table 1. Both 1 and 2 showed identicaltified as Actinomadura madurae 2808-SV1 , was cultivatedAppearance

w-hexane-EtOAc-MeOH

properties1).

properties

0.5%, NaCl 0.3%, pH(20 : 5 : 1), and eluted with the same solvent system in thedark. Further purification of the active eluate by HPLC
using PEGASIL ODS (Senshu-Pak 20 i.d. x250mm)
developed with 60% CH3CNgave pure 1 and 2 (1.6mg
and 2.0 mg, respectively).

investigation has resulted in the isolation of novel compounds designated carbazomadurins A and B (1 and 2,Table 1. Physico-chemical

3shaker. The mycelial acetone extract was concentrated
to a small volumeand the aqueousresidue wasextractedwith EtOAc. The solvent layer was dried over Na2SO4
and concentrated to dryness in the dark to give an oilybrownish residue. This material was subjected to a silica

hybridoma N18-RE-105 cells as an in vitro ischemia
model, we isolated
several compounds5~11}. FurtherFig.

1.5%,

at 27°C for 5 days on a rotary

course of our screening for substances protecting centralnervous system from glutamate toxicity using neuronalrespectively,of glycerol

FAB-MS as

A (1) and B (2).

VOL.50

NO.9

7

THE JOURNAL OF ANTIBIOTICS

771

A (1) and B (2).

B

A

B

A

Table 2. 13C and XHchemical shifts ofcarbazomadurins
No.

5

<5H

<__1

12
131415
16
1718

122.1

122.4

122.1

150.0

149.7

32122.4

1920

10

121.1

123.6

123.2

128.4

128.1

1

7.59

0

121.5

118.5

6.72

109.3

05

6.91

109.7

6.92
6.72

8

118.9

133.7
120.4

143.0

142.7

130.6

130.2

17.7

1.56

37.1

2.35

35.31.70,

34.7

1.50

ll.4
13.2

0.99
2.26

63.4
29.7
19.1

5.01
1.48,
0.94
8.89

1.46

92

1.26'OH
6.43

133.3
120.1!OH

6.43

C22H27NO3and C23H29NO3, respectively.
The *H and13C NMRspectral data are shown in the Table 2. Theirstructures were elucidated as follows.
The DQF-COSYand HMBCspectra of2 revealed the1,2,3,4-tetrasubstituted
benzene ring with an alkyl side
chain as shown in Fig. 2. In the DQF-COSYspectrumof 2, an allylic methylene proton 13-H (2.35ppm) was
coupled to adjacent methylene protons 14-H (1.70ppm,1.46ppm), while a methyl proton 16-H (0.99ppm) was
coupled to a methine proton 15-H (1.50ppm). Anothermethyl proton 20-H (0.94 ppm) was coupled to methylene

protons 19-H (1.48ppm and 1.26ppm). In the HMBC
spectrum of2, the methylene proton 13-H was long-rage
coupled to two sp2 carbons C-10 (120.1 ppm) and C-ll(142.5ppm) together with a methyl carbon C-12 (17.7ppm).

22.9

24a4b56788a106.6

142.5

9-NH

47.59

107.0

142.8
18.0
37.8
38.0
28.622.9
13.6
63.8

1ll

2

<5H<5c1No

In addition,an allylmethylproton

12-H (1.56ppm) was long-range coupled to C-10, C-ll and C-13
(37.1 ppm). Thus, the sequence from C-ll to C-13 wasrevealed as shown in Fig. 2. The connectivities between

3.90

Fig. 2. NMRanalyses ofcarbazomadurin

17-H (2.26ppm)

to aromaticcarbons

B (2).

C-l (122.1ppm),

C-14, C-15 and C-19 were established by observing^-^C long-range couplings from 16-H to C-14 (35.3C-2 and C-3, and from an olefinic proton 10-H (6.43 ppm)
to C-l, C-2 and C-9a established the positions of themethylated heptenyl side chain and the aromatic methyl
residue on the benzene ring as shown in Fig. 2. Appm), C-15 (34.7 ppm) and C-19 (29.7 ppm). These resultshydroxymethyl proton 18-H (5.01 ppm) was long-range

together with other long-range couplings established theexistence of the 2,5-dimethylheptene side chain moiety
as shown in Fig. 2.

coupled to C-4b, C-6 (118.5ppm) and C-5 (128.1 ppm)
which in turn long-range coupled to an aromatic proton
7-H (6.72ppm). Thus, the hydroxymethyl moiety is de-Since the UVspectrum of 2 suggested the presence ofduced to be substituted

a carbazole nucleus, the remaining six sp2 carbons except

paring the 13C chemical shifts of aromatic carbons withthose of epocarbazolins and by analyzing the long-range
couplings from an exchangeable proton 9-NH (8.89 ppm)
to C-4a (121.1 ppm), C-4b and C-9a, it was concluded
that the nitrogen atom was connected to both C-8afor C-17 (13.2ppm) and C-18 (63.4ppm) are considered
to consist the carbazole skeleton. In the HMBC
spectrumof 2, an aromatic proton 4-H (7.59 ppm) was long-range
coupledto aromaticcarbons

C-2 (122.1ppm),

C-3(149.7ppm), C-4b (123.3ppm) and C-9a (133.3ppm). In
addition, long-range couplings from a methyl proton(130.2ppm)

at the C-5 position.

and C-9a. The remaining

residues were substituted

By com-two hydroxyl

at the C-3 and C-8 (142.7ppm)

772

SEPT.

THE JOURNAL OF ANTIBIOTICS

References

positions. These data revealed the structure of2 as shownin Fig. 2.

The stereochemistry of the C10=Cn double bond in
the side chain of2 was determined to be E by the NOE
between 10-H and 13-H as well as by 13C chemical shiftof the methyl carbon C-12 (17.7ppm) as shown in Fig. 2.
The structure of 1 was elucidated by comparing NMR
spectral data with those of 2. The NMRdata of 1 wasidentical with those of 2 except for the signals due to thesidechain.

Detailed

analysisof the DQF-COSYandHMBCspectrum of 1 proved the presence of an isoamyl
residue in 1. The stereochemistry of the C10=C1JLdoublebond in 1 was also determined to be E according to the13C chemical shift of methyl carbon C-12 (18.0ppm).
Thus, the structure of 1 was determined to be that shownin Fig. 1.

In the evaluation system we employed, 1 and 2 suppressed effectively the toxicity of L-glutamate in N18RE-105

cells

with

EC50

values97nM

and 84nM,

re-spectively. Since the carbazole nucleus, which constitutes
the chromophore of 1 and 2, was reported to exhibit
potent antioxidative
activity,
we also examined theantioxidative
activity of 1 and 2 by observing the inhibitory effect against buthionine sulfoximine (BSO14),
an inhibitorof glutathionesynthesis)
toxicity. It wasreported that L-glutamate toxicity in N18-RE-105 cells
was mediated by inhibiting the uptake of cystine, which
led to the loss of intracellular reducing agent glutathione
and the accumulationof intracellularperoxides.

As

expected, 1 and 2 also showed a protective effect fromthe BSOtoxicity with identical EC50 values to those in
the glutamate

toxicity.

These data suggested

that themodeof action of carbazomadurins was based on theantioxidativeactivity.
biological activities

Further studies on the detailed
are now under way.

Choi, D. W.: Glutamate neurotoxicity

nervous system. Neuron 1: 623-634,
Coyle,J. T. & P. Puttfarcken:

This work was supported in part by a Grant-in Aid for
Scientific Research (C) to K.S. and by Research for the Future,
Japan Society for the Promotion of Science to H. S.

and diseases of the1988
Oxidative

stress,glutamate,and1993neurodegenerative disorders. Science 262:
689-695,
Murphy, T. H.; M. Miyamoto, A. Sastre, R. L. Schnaar& J. T. Coyle: Glutamate toxicity in a neuronal cell line

involves inhibitionof cystine transportleadingtooxidative stress. Neuron 2: 1547- 1558, 1989
Murphy, T. H.; R. L. Schnaar & J. T. Coyle: Immatureneurons are unique sensitive to glutamate toxicity byinhibitionof cystine uptake. FASEB J. 4: 1624- 1633,
1990

Shin-ya, K.; M. Tanaka, K. Furihata, Y. Hayakawa&H. Seto: Structure ofcarquinostatin A, a new neuronalcell protecting substance produced by Streptomyces
exfoliatus. Tetrahedron Lett. 34: 4943 -4944, 1993
Shin-ya, K.; S. Shimizu, T. Kunigami, K. Furihata, K.
Furihata & H. Seto: A new neuronal cell protecting
substance, lavanduquinocin, produced by Streptomyces
viridochromogenes.
J. Antibiotics 48: 574- 578, 1995
Shin-ya, K.; S. Shimizu, T. Kunigami, K. Furihata,Y.

Hayakawa & H. Seto: Novel neuronal cell protecting
substances,aestivophoenins

Streptomycespurpeofuscus.
1995

A and B, produced

J. Antibiotics

by48: 1378 - 138 1,Kunigami, T.; K. Shin-ya, K. Furihata, K. Furihata,
Y. Hayakawa & H. Seto: A neuronal cell protecting
substance, 4-demethoxymichigazone, produced by Strep-

tomyces halstedi.
J. Antibiotics
49: 312-313, 1996
Murakami, Y.; S. Kato, M. Nakajima, M. Matsuoka,
H. Kawai, K. Shin-ya & H. Seto: Formobactin, a novel
free radical scavenging and neuronal cell protecting
substance from Nocardia sp. J. Antibiotics 49: 839- 845,
1996

Kim, J.-S.;

K. Shin-ya,

J. Eishima,

K. Furihata& H.

Seto:producedA novel byneuronalprotecting substance, espicufolin,49:
Streptomyces sp. cu39. J. Antibiotics
947-948,

1996

Kim, J.-S.;
K. Shin-ya, J. Eishima, K. furihata& H.
Seto: A novel neuronal cell protecting
substance

naphthomycinol, produced by Streptomyces sp. PF7. J.
Antibiotics

Acknowledgments

1997

49:

1172-1174,

1996

Nihei, Y.; H. Yamamoto, M. Hasegawa, M. Haneda,
Y. Fukagawa & T. Oki: Epocarbazolins
A and B, novel
5-lipoxygenase inhibitors.
J. Antibiotics 46: 25 - 33, 1993
Sakano, K. & S. Nakamura: New antibiotics,
carbazomycins A and B. J. Antibiotics
33: 961-966, 1980Griffith, O. W.: Mechsnism of action, metabolism
and toxicity of buthionine sulfoximine and its higher

homologs,
potent inhibitors of glutathione
sysnthesis.
Biol.
Chem. 257: 13704-13712,
1982

J.