Isolation of streptonigrin and its novel derivative from Micromonospora as inducing agents of p53-dependent cell apoptosis

Article abstract of DOI:10.1021/np0104572

Streptonigrin (1) and its novel natural derivative 7-(1-methyl-2-oxopropyl)streptonigrin (2) were isolated from an actinomycete strain, Micromonospora sp. IM 2670. The inductions for 1 and 2 are more potent in the human neuroblastoma SH-SY5Y cells that contain wild-type p53 than in SH-SY5Y-5.6 cells that overexpress a dominant negative mutant of p53, thus suggesting that they induce apoptosis through a p53-dependent pathway.

Full text of DOI:10.1021/np0104572

J . Nat. Prod. 2002, 65, 721-724
721
Isola tion of Str ep ton igr in a n d Its Novel Der iva tive fr om Micr om on ospor a a s
In d u cin g Agen ts of p 53-Dep en d en t Cell Ap op tosis
,#
Haishan Wang,†,# Su Ling Yeo,‡, J in Xu,† Xiaoli Xu,§ Hong He,‡ Francesca Ronca,| Anthony E. Ting,
Yue Wang,§ Victor C. Yu,| and Mui Mui Sim*,†
Medicinal and Combinatorial Chemistry Laboratory, Lead Discovery Group, Microbial Collection and Screening Laboratory,
Screening for Novel Inhibitors Group and Apoptosis Signaling Laboratory, Institute of Molecular and Cell Biology,
30 Medical Drive, Singapore 117609, Singapore
Received September 19, 2001
Streptonigrin (1) and its novel natural derivative 7-(1-methyl-2-oxopropyl)streptonigrin (2) were isolated
from an actinomycete strain, Micromonospora sp. IM 2670. The inductions for 1 and 2 are more potent
in the human neuroblastoma SH-SY5Y cells that contain wild-type p53 than in SH-SY5Y-5.6 cells that
overexpress a dominant negative mutant of p53, thus suggesting that they induce apoptosis through a
p53-dependent pathway.
The tumor suppressor protein p53 functions as a key
component of a cellular emergency response mechanism.
Activation of p53 can occur in response to a variety of stress
signals including DNA damage, hypoxia, and activated
oncogenes. Such activation then leads to the induction of
a number of genes whose products trigger growth arrest
or programmed cell death (apoptosis), thus eliminating
damaged and potentially dangerous cells from the organ-
ism.1 The importance of p53 has attracted considerable
attention in the scientific field, as over half of all human
cancers are linked with the loss of wild-type p53 function
due to mutation of the p53 gene. However, a large number
of cancers, such as human neuroblastoma, were found to
carry the wild-type p53 gene. Neuroblastoma is one of the
most common malignancies in childhood, and the cell lines
have been widely employed in studies involving the func-
tion and regulation of wild-type p53 in tumor cells. Yu and
colleagues reported that 1-(5-isoquinolinesulfonyl)-2-
methylpiperazine (H-7), at concentrations ranging from
20 to 100 µM, induced apoptosis in human neuroblastoma
SH-SY5Y cells.2 The cytotoxic effect of H-7 was correlated
with its ability to induce nuclear accumulation of p53 in
the cells. Furthermore, the apoptotic effect of H-7, but not
other apoptotic insults, was effectively diminished in SH-
SY5Y-5.6 cells that overproduced a dominant negative
mutant of p53, thus suggesting that H-7 mediates its
apoptotic effect through a p53-dependent mechanism.
Encouraged by the findings, a high-throughput screen was
established to identify novel natural products that can
trigger wild-type p53 function using human neuroblastoma
SH-SY5Y cells as the model system. Here we report the
isolation and characterization of a known aminoquinone
antibiotic streptonigrin (1) and its novel derivative (2) from
a Micromonospora strain as inducing agents of p53-
dependent apoptosis.
Six out of 12 000 actinomycete extracts that killed >70%
of the SH-SY5Y cells and <35% of the SH-SY5Y-5.6 cells
were identified from microtiter plate-based screening. One
extract, 2670-1, exhibited reproducible selective cytotoxicity
and caused an accumulation of p53 in the nucleus of SH-
SY5Y cells (data not shown) and was selected for further
purification.
The fermentation broth of Micromonospora sp. IM 2670
was freeze-dried and extracted with MeOH. The bioassay-
active extracts were dissolved in MeOH-H2O (1:1) and
extracted sequentially with hexane, CH2Cl2, EtOAc, and
n-butanol. The bioassay-active CH2Cl2 extract was frac-
tionated by reversed-phase HPLC (C18, MeOH-H2O +
0.04% TFA, method A). The bioassay-active HPLC fraction
was further purified by normal-phase preparative TLC
(15% MeOH in CHCl3) and reversed-phase HPLC (C18
,
MeOH-H2O + 0.04% TFA, method B) to give compounds
1 and 2. Compounds 3 and 4 were also isolated respectively
from the fractions collected just before and after the
bioactive HPLC fraction.
#
These authors contributed equally to this work.
* To whom correspondence should be addressed. Tel: (65) 6874-1443.
Fax: (65) 6779-1117. E-mail: mcbsimmm@imcb.nus.edu.sg.
† Medicinal and Combinatorial Chemistry Laboratory.
‡ Lead Discovery Group.
§ Microbial Collection and Screening Laboratory.
Screening for Novel Inhibitors Group.
Compound 1, dark red or black solid, had retention times
(tR) of 27.3 and 15.1 min as determined by HPLC methods
| Apoptosis Signaling Laboratory.
10.1021/np0104572 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/03/2002

722 J ournal of Natural Products, 2002, Vol. 65, No. 5
Notes
Ta ble 1. 1H and 13C NMR Data for Compounds 1 and 2 (δ in ppm, J in Hz) at 295 K
1
2
1H NMR
13C NMR
1H NMR
13C NMRd
dioxane-d8 lit.5
DMSO-d6
dioxane-d8 lit.5
DMSO-d6
CDCl3
CDCl3
a
no.
CDCl3
1
2
3
4
145.3
126.2
134.2
127.7
177.2
137.4
60.2
144.1
125.9
133.4
126.7
175.9
135.7
59.7
143.6
125.4
134.4
130.1
177.8
136.0
61.7
8.48 (d, 8.4)
8.69 (d, 8.4)
8.93 (d, 9)
8.38 (d, 9)
9.01 (d)
8.36 (d)
8.47 (8.4)
8.70 (8.4)
4a
5
6
6-OMe
7
7-NH2 or 7-NH
8
8a
4.104 (s)
3.96 (s)
3.81 (s)
4.001 (s)
141.0
(141.6)b
δN 122.4c
180.3
159.8
e
5.12 (br s)
6.00 (br s)
6.93 (br s)
5.97 (d, 6.9)
181.0
160.8
e
e
1′
2′
133.6
165.5
138.9
17.4
130.3
147.5
135.0
115.7
149.0
136.2
167.1
134.8
17.0
133.9
(145.7)b
129.5
114.8
148.1
139.5
e
131.2
17.5
134.0
e
2′-CO2H
3′
3′-Me
4′
5′
6′
7′
8′
12.39 (br s)
2.50 (s)
11.00 (br s)
2.34 (s)
12.22 (br s)
2.17 (s)
2.50 (s)
e
e
146.3
8′-OH
9′
9′-OMe
10′
10′-OMe
11′
12′
5.95 (br s)
3.956 (s)
7.83 (br s)
3.84 (s)
8.94 (br s)
3.76 (s)
5.94 (br s)
3.945 (s)
137.9
60.7
154.1
55.9
105.2
125.6
136.9
60.3
153.1
55.7
104.4
124.6
137.0
61.8
153.5
55.7
105.2
125.6
58.7
3.998 (s)
6.68 (d, 8.6)
6.80 (d, 8.5)
3.88 (s)
6.70 (d, 8.5)
6.77 (d, 8.5)
3.85 (s)
6.70 (d)
6.73 (d)
3.960 (s)
6.69 (d, 8.6)
6.81 (d, 8.6)
4.74 (pent, 7.1)
1′′
2′′
3′′
205.8
26.1
2.18 (s)
4′′
1.49 (d, 7.2)
18.9
a
b
The signals were assigned by COSY, 1H-13C HMQC, and HMBC (optimal J ) 10, 8, 6, 4, and 2 Hz). Not confirmed. c Obtained from
1H-15N HSQC. The chemical shifts obtained and assigned from COSY, H-13C HMQC, and HMBC (optimal J ) 8 and 4 Hz). e Unable
to determine or assign.
d
1
A and B, respectively. Its UV (MeOH-H2O + 0.04% TFA,
λmax 245, 292-302 (sh), and 374 nm) suggested that it
contained a highly conjugated system, such as benzo-
protons at δH 2.18 and δH 1.49 correlate with carbons at
δC 58.7 and δC 205.8 (CdO or imine CdN-). The formula
of 2 was deduced from its HRESIMS data (m/z 577.1935,
[M + H]+) as C29H28N4O9 (calcd for C29H28N4O9 + H,
577.1968). The difference between the formula of 1 and 2
was C4H6O; that is, compound 2 was most probably a
derivative of 1 with structure resembling either I or II
(Figure 2) and was derivatized at either 7-NH2 or 5′-NH2.
Since derivatization could cause a change in the chemical
shifts of the neighboring protons, a thorough study of the
chemical shift changes between 1 and 2 (∆δH ) -0.10 (6-
OMe), -0.04 (10′-OMe), -0.01 (for 9′-OMe, 11′-H, and 12′-
H) and +0.01 (8′-OH)) was carried out. The biggest change
occurred at the 6-CH3O group, thus suggesting that 7-NH2
had been altered. In addition, the disappearance of the
broad singlet of 7-NH2 at 5.12 ppm (2H) and the appear-
ance of a new doublet at 5.97 ppm (1H) in the 1H NMR
spectrum of 2 provided an important piece of evidence that
derivatization has indeed occurred at 7-NH2.
To further support that the structure was I and not II,
syntheses of imines 6, 8, and 9 were carried out (Scheme
1). The chemical shifts (δC and δN, in CDCl3) of imine 6
were deduced from the NMR data of the reaction mixture
of 5 and acetone. Reaction of 5 with acetoin 7 provided the
imine 9 (31%) and the acetamide 10 (51%) but not imine
8. As expected, the chemical shifts of the four-carbon unit
of compound 2 are very similar to that of 9, thus confirming
that compound 2 is a derivative of compound 1 at 7-NH2
and possesses a structure containing I.
1
quinone. The H NMR spectrum of 1 was relatively simple
(Table 1), although its 13C NMR indicated that it had 25
1
1
carbons. In addition, H-1H COSY, H-13C HMQC, and
HMBC experiments showed the presence of three CH3O,
one CH3, and four protons (in two coupled systems)
attached to aromatic rings. From its HRMS (electrospray,
m/z 507.1499, [M + H]+) and NMR data, the formula of 1
was deduced as C25H22N4O8 (calcd for C25H22N4O8 + H,
507.1516), which best matched that of the known amino-
quinone antibiotic streptonigrin.3 The 1H NMR (CDCl3)
data of the isolated 1 were identical to those reported for
14 and to those obtained from an authentic sample (Sigma,
S 1014). The 13C NMR (DMSO-d6) data were also in accord
with those recorded in dioxane-d8 (Table 1).5 The 7-NH2
was assigned by 1H-13C HMBC (3J to C-6 and C-8) and
1H-5N HSQC (δN 122.4). The isolated 1 and streptonigrin
(Sigma) had identical HPLC and UV profiles. Both com-
pounds induced apoptosis in SH-SY5Y cells through a p53-
dependent pathway.
Compound 2, a red solid, is less polar than 1 (tR ) 28.1
and 21.1 min as determined by HPLC methods A and B,
respectively) but has a similar UV spectrum: λmax 250,
1
295-305 (sh), 377 nm. Although the H NMR spectrum of
2 is similar to that of 1, it contains two additional CH3 at
δH 1.49 (d, J ) 7.2 Hz) and 2.18 (s) and two additional
protons at δH 4.74 (pent, J ) 7.1 Hz) and 5.97 (d, J ) 6.9
Hz). The 2D NMR experiments (1H-1H COSY, 1H-13C
HMQC, and HMBC) revealed that the proton at δH 4.74
(δC 58.7) is coupled to the protons at δH 1.49 and δH 5.97,
with the latter connected to an electronegative atom. Both
Compound 3 was obtained as a white solid (HPLC tR
)
1
26.4 min by method A, UV λmax 233, 318 nm). H and 13C
NMR (including DEPT) show that it has 15 protons and
15 carbons. The molecular formula of 3 was determined

Notes
J ournal of Natural Products, 2002, Vol. 65, No. 5 723
by 1D and 2D NMR experiments. Its 1H NMR data were
identical to those reported.8
Compound 11, a red compound isolated from the HPLC
fraction (16.5-17.0 min, a peak with tR ) 16.8 min, method
A), shows broad UV absorption from 380 to 470 nm (λmax
) 422 nm) and only very broad lines in the 1H NMR
spectrum, thus indicating that it is not an analogue of 1.
The formula of 11 was deduced as C27H46N6O9Fe by
HRESIMS, suggesting that it is the ferric salt of desferri-
oxamine E or norcardamine (12).9 The presence of iron is
consistent with the line broadening effect seen in the 1H
NMR spectrum. Furthermore, the presence of chelating
compound desferrioxamine E in Micromonospora is not
surprising, as it was found to protect isolated DNA and
bacterial cells against streptonigrin-induced toxic effects.10
When the MeOH solution of 11 was treated with Dowex
50W (H+) resin, a new peak showing M + H+ at m/z
601.403 (C27H48N4O9 + H) appeared in the MS. The ratio
of peak intensity of m/z 654/601 was less than 3% after
three treatments, thus indicating that the ferric cation has
undergone almost complete exchange with the proton.
Bioassay demonstrated that both 1 and 2 exhibit very
potent and selective cytotoxicity. Streptonigrin 1 is 92×
more toxic toward the SH-SY5Y (IC50 ) 0.05 µM) than
toward the SH-SY5Y-5.6 (IC50 ) 4.6 µM) cell lines, sug-
gesting that apoptosis occurs through a p53-dependent
pathway. Compound 2 is ca. 18× less potent (IC50 ) 0.9
and 75 µM for SH-SY5Y and SH-SY5Y-5.6 cell lines,
respectively) than its parent 1. This agrees with previous
reports indicating that cell toxicity of streptonigrin is
mediated by its aminoquinone domain since the antitumor
actions of streptonigrin are lost when the aminoquinone
moiety is blocked.11,12 Compounds 3 and 4 are not active
at up to 100 µM concentration. Compound 11 (tested as
crude) is also inactive. 1-(5-Isoquinolinesulfonyl)-2-meth-
ylpiperazine (H-7) is less potent and less selective when
compared with compounds 1 and 2 (IC50 ) 40 and 80 µM
for SH-SY5Y and SH-SY5Y-5.6 cell lines, respectively).
Many anticancer agents, such as cisplatin,13 bis-Pt(III)
complex,14 mitomycin C, and actinomycin D,15 which cause
DNA damage, are strong inducers of apoptosis. Many of
them have also been observed to induce nuclear accumula-
tion of p53 in cells expressing wild-type p53 protein.16
Streptonigrin has long been known to exert its genotoxic
effects by causing DNA and chromosome damage through
generation of free radicals. This, however, is the first report
demonstrating that streptonigrin, as well as its novel
derivative 2, can induce apoptosis through a p53-dependent
pathway in human neuroblastoma cells. Compound 1 also
caused nuclear accumulation of p53 and induced DNA
ladders in SH-SY5Y cells. Further studies on the effect of
streptonigrin in mediating p53-dependent apoptosis will
be published elsewhere.
F igu r e 1. 1H-13C HMBC correlations (arrows) for 1 (optimal J ) 10,
8, 6, 4, and 2 Hz, in DMSO-d6) and 2 (optimal J ) 8 and 4 Hz, CDCl3).
F igu r e 2.
Sch em e 1a
a
Reagents and conditions: (a) 5 (0.35 mmol) in acetone (ca. 10-15 mL)
stirred at room temperature for 23 h; (b) 5 (0.49 mmol) and 7 (2.3 mmol) in
CH2ClCH2Cl, stirred at room temperature for 73 h.
as C15H16N2O2 by HRESIMS. The structure of 3 was
determined by 2D NMR experiments (COSY, HMQC, and
HMBC). Compound 3 was confirmed to be the known
diketopiperazine alkaloid, albonoursin, with a 3Z,6Z-
configuration.6 The 1H NMR data of 3 were identical to
those reported.7
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es.17 All the 1D and 2D
Compound 4 has a UV spectrum similar to that of 3, but
is less polar (tR ) 28.9 min by HPLC method A). The
molecular formula of 4 was determined as C16H18N2O2 by
HRESIMS. It was found to be the known alkaloid 1-N-
methylalbonoursin with 3Z,6E-configuration as confirmed
1
NMR experiments for H (400.13 MHz), 13C (100.61 MHz), and
15N (40.55 MHz) nuclei were obtained on a Bruker AVANCE-
1
400 digital NMR spectrometer. 1H-13C and H-15N 2D experi-
ments (HMQC, HSQC, and HMBC) were run with Z-gradient

724 J ournal of Natural Products, 2002, Vol. 65, No. 5
Notes
selection. HRMS spectra were determined using a PerSeptive
Biosystems Mariner TOF spectrometer.
(dark red or black solid, 1.0 mg, tR ) 15.1 min) and compound
2 (red solid, 1.0 mg, tR ) 21.1 min) were obtained.
Isola tion a n d Ta xon om y of th e Actin om ycete Str a in
IM 2670. The procedures for the isolation and taxonomic
characterization of the strain IM 2670 were as described by
Wang et al.18 The actinomycete strain IM 2670, from which
streptonigrin and its derivative were purified, was isolated
from a soil sample collected in the Singapore Botanic Garden.
Its colony exhibits properties characteristic of Micromonospora
on an ISP 4 medium plate. The colonies do not grow aerial
mycelium. Dark-colored spore accumulation forms on the
colony surface. Single spores are born on short sporephores
on the substrate mycelium. The color of the colony is orange.
No diffusible pigment was produced on ISP 2, ISP 3, ISP 4,
and Bennett medium plates. The cell wall peptidoglycans
contained meso-diaminopimelic acid. The complete nucleotide
sequence of the 16S rRNA gene of IM 2670 was determined
for phylogenetic analysis. IM 2670 was placed within the
Micromonospora clade on the phylogenetic tree, and the 16S
rRNA gene sequences are higher than 97% identical between
IM 2670 and other Micromonospora species. On the basis of
morphological, chemotaxonomic, and phylogenetic evidences,
the actinomycete strain IM 2670 was assigned to the genus
Micromonospora.
Two HPLC fractions (method A, 25.5-27.0 min, major peak
tR ) 26.4 min and 28.5-29.5 min, major peak tR ) 28.9 min)
were purified by preparative TLC (5% MeOH in CHCl3) to give
a white solid 3 (2.5 mg) and a pale yellow solid 4 (1.7 mg),
respectively. In a scaled-up fermentation (10 L), the CH2Cl2
extract was first purified by Si flash chromatography (gradient
CH2Cl2-MeOH) and then followed by HPLC purification.
Compound 1 (8.4 mg) and a red solid 11 (20 mg, HPLC
fraction, 16.5-17.0 min, major peak tR ) 16.8 min at 450 nm,
method A) were obtained.
Str ep ton igr in (1): red or black solid; UV (65% MeOH in
H2O with 0.04% TFA) λmax (relative absorption) 245 (1.00),
292-302 (sh, 0.46), 374 (0.44) nm; NMR data, see Table 1;
HRESIMS m/z 507.1499 (calcd for C25H23N4O8 + H, 507.1516).
5-Am in o-4-(2-h ydr oxy-3,4-dim eth oxyph en yl)-6-[6-m eth -
oxy-7-(1-m et h yl-2-oxop r op yla m in o)-5,8-d ioxo-5,8-d ih y-
d r oqu in olin -2-yl]-3-m eth ylp yr id in e-2-ca r boxylic a cid or
7-(1-m eth yl-2-oxop r op yl)str ep ton igr in (2): red solid; UV
(65% MeOH in H2O with 0.04% TFA) λmax (relative absorption)
250 (1.00), 295-305 (sh, 0.40), 377 (0.42) nm; NMR data, see
Table 1; HRESIMS m/z 577.1935 (calcd for C29H28N4O9 + H,
577.1968).
High -Th r ou gh p u t Scr een s (HTS). HTS was performed
in transparent 96-well plates (Nunclon). Neuroblastoma cell
lines SH-SY5Y and SH-SY5Y-5.6 containing HA-tagged 91
amino acid mini p53 protein were employed in the primary
screens.2 Cell cytotoxicity was measured by using the Kit
CellTiter 96 AQueous Non-Radioactive Proliferation Assay
(Promega, http://www.promega.com). A fixed number of cells
(105 cells/mL) in 100 µL of RPMI media containing hygromycin
(0.1 mg/mL) was plated out the day before. The cells were
incubated with 1 µL of crude extracts for 24 h. At the 20th
hour, MTS (Owen’s reagent, or 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner slat), prepared according to manufacturer’s direction,
was added to a final concentration of 0.04 mg/well and
incubated for 4 h at 37 °C. The plates were read at OD490 in a
multilabel counter (Wallac 1420 Victor). Reference control
wells containing cells and blank wells containing media only
were treated with the appropriate amount of methanol.
Extracts, which were found to kill at least 70% of SH-SY5Y
cells but less than 35% of SH-5Y-5.6, were repeated in
duplicates in 96-well plates. Samples that exhibited reproduc-
ible inhibition were subjected to dose-response experiments.
Those with distinctive dose-response curves were selected for
western blotting.
Ack n ow led gm en t . This work was supported by grants
from the Agency for Science, Technology and Research (A*Star)
of Singapore. Dr. Victor C. Yu, Dr. Yue Wang, and Dr. Mui
Mui Sim are also an Adjunct Staff Member (Department of
Pharmacology), Adjunct Associate Professor (Department of
Microbiology), and Adjunct Senior Fellow (Department of
Biochemistry), respectively, at the National University of
Singapore.
Su p p or tin g In for m a tion Ava ila ble: NMR, UV, and MS data for
compounds
3 and 4; preparative procedures and NMR data for
compounds 6, 9, and 10. This material is available free of charge via
the Internet at http://pubs.acs.org.
Refer en ces a n d Notes
(1) (a) May, P.; May, E. Oncogene 1999, 18, 7621-7636. (b) Prives, C.;
Hall, P. A. J . Pathol. 1999, 187, 112-126.
(2) Ronca, F.; Chan, S.-L.; Yu, V. C. J . Biol. Chem. 1997, 272, 4252-
4260.
(3) The Combined Chemical Dictionary on CD-ROM (version 4:2); Chap-
man & Hall/CRC Press: New York, 2001.
(4) Herlt, A. J .; Rickards, R. W.; Wu, J .-P. J . Antibiot. 1985, 38, 516-
Isola tion a n d P u r ifica tion P r oced u r es. The bioassay-
active fermentation broth (4 L) of Micromonospora sp. IM 2670
was freeze-dried. The solid residue was extracted with MeOH
(3.5 L) and 10% H2O in MeOH (2 L × 2) at room temperature.
The bioassay-active extracts were combined and concentrated
under reduced pressure below 35 °C. The wet residue was
diluted with MeOH (250 mL) and H2O (250 mL), and then the
solution was extracted with hexane (300 mL × 3), CH2Cl2 (300
mL × 3), ethyl acetate (300 mL × 3), and n-butanol (300 mL
× 3). The bioassay data showed that the CH2Cl2 extract (11
g) was most active. This extract was purified (separately in
two equal portions) by a preparative HPLC system (Waters
Delta Prep 4000) equipped with a 996 photodiode array
detector, using Prep Nova-Pak HRC18 column segments (6 µm,
40 mm × 210 mm; flow rate, 30 mL/min; solvent A ) H2O +
0.04% TFA, B ) MeOH + 0.04% TFA; 5% to 100% B in 30
min, linear gradient, then 100% B for additional 5 min, method
A). Fractions corresponding to different peaks were collected.
The most active fraction (27.0-28.5 min, containing two major
peaks at tR ) 27.3 and 28.1 min, 370 nm) was further purified
by preparative TLC (silica, 15% MeOH in CHCl3) to give two
bands. They were separately subjected to another HPLC
purification (Novapak, C18, 19 × 300 mm, flow 10 mL/min, 65%
B for 30 min, then 65% to 100% B in 5 min, and maintained
at 100% B for an additional 5 min, method B). Compound 1
518.
(5) Isshiki, K.; Sawa, T.; Miura, K.; Li, B.; Naganawa, H.; Hamada, M.;
Takeuchi, T.; Umezawa, H. J . Antibiot. 1986, 39, 1013-1015.
(6) Shin, C.-G.; Hayakawa, M.; Mikami, K.; Yoshimura, J . Tetrahedron
Lett. 1977, 863-866.
(7) Kanzaki, H.; Yanagisawa, S.; Nitoda, T. J . Antibiot. 2000, 53, 1257-
1264.
(8) Gurney, K. A.; Mantle, P. G. J . Nat. Prod. 1993, 56, 1194-1198.
(9) (a) Meyer, J .-M.; Aballah, M. A. J . Gen. Microbiol. 1980, 118, 125-
129, and references therein. (b) Bergeron, R. J .; McManis, J . S.
Tetrahedron 1990, 46, 5881-5888.
(10) (a) Krishna, M. C.; Halevy, R. F.; Zhang, R.; Gutierrez, P. L.; Samuni,
A. Free Rad. Biol. Med. 1994, 17, 379-388. (b) White, J . R.; Yeowell,
H. N. Biochem. Biophys. Res. Commun. 1982, 106, 407-411.
(11) Bolza´n, A. D.; Bianchi, M. S. Mutat. Res. 2001, 488, 25-37.
(12) )Rao, K. V.; Rock, C. P. J . Heterocycl. Chem. 1996, 33, 447-458.
(13) Eastman, A. Cancer Cells 1990, 2, 275-280.
(14) Gonza´lez, V. M.; Fuertes, M. A.; Pe´rez-Alvarez, M. J .; Gervantes, G.;
Moreno, V.; Alonso, C.; Pe´rez, J . M. Biochem. Pharmacol. 2000, 60,
371-379.
(15) Fritche, M.; Haessler, C.; Brandner, G. Oncogene 1993, 8, 307-318.
(16) Chikayama, S.; Sugano, T.; Takahashi, Y.; Ikeda, M.; Kimura, S.;
Kobayashi, Y.; Kondo, M. Int. J . Oncol. 2000, 16, 1117-1124.
(17) )Wang, H.; Lim, K. L.; Yeo, S. L.; Xu, X.; Sim, M. M.; Ting, A. E.;
Wang, Y.; Yee, S.; Tan, Y. H.; Pallen, C. J . J . Nat. Prod. 2000, 63,
1641-1646.
(18) ) Wang, Y.; Zhang, Z.; Ruan, J .; Wang, Y.; Ali, S. M. J . Ind. Microbiol.
Biotech. 1999, 23, 178-187.
NP0104572