Arch Pharm Res Vol 30, No 5, 552-555, 2007
A New Cytotoxic Phenazine Derivative from a Deep Sea
Bacterium Bacillus sp.
Dehai Li, Fengping Wang1, Xiang Xiao1, Xiang Zeng1, Qian-Qun Gu, and Weiming Zhu
Key Laboratory of Marine Drugs, Chinese Ministry of Education, Institute of Marine Drugs and Food, Ocean Uni-
versity of China, Qingdao 266003, PR China and 1Key Laboratory of Marine Biogenetic Resources, Third Institute
of Oceanography, The State Oceanic Administration, Xiamen, P.R. China
(Received November 20, 2006)
A novel phenazine derivative (1) together with six known compounds (2-7) were isolated by
bioassay-guided fractionation from the culture broth of a bacterium, Bacillus sp., collected from
a Pacific deep sea sediment sample (depth 5059 m). The structures of these compounds were
determined using spectroscopic methods. Their cytotoxic effects on P388 and K562 cell lines
were preliminarily examined using the sulforhodamine-B (SRB) assay.
Key words: Deep sea bacteria, Bacillus sp, Metabolite, Antitumor activities
Marine microorganisms are widely recognized as an
emerging source of secondary metabolites (Fenical, 1993).
These organisms, which flourish in diverse marine environ-
ments, have produced a wide variety of structurally unique
and biologically active compounds that have attracted
considerable attention for biomedical research (Fenical,
1993; Faulkner, 2002). The deep sea is a unique environ-
ment characterized by extreme factors such as hydrostatic
pressure, low temperature and darkness. Deep sea
microorganisms are diverse in their secondary metabolites
and functions. There is growing evidence that deep sea
microorganisms can be a source of unique natural products
(Faulkner, 2002; Chen et al., 2004).
In an effort to search for new antitumor compounds from
deep sea microorganisms, 260 bacterial strains isolated
from the sediment of the Pacific Ocean, have been
screened using the K562 cell line and strain 39, identified
as Bacillus sp., exhibited cytotoxic activity. The active
components of this strain were investigated using a bio-
assay-guided isolation procedure that led to the isolation
of a novel phenazine derivative, compound 1, together
with six known compounds (2-7). In this paper, we describe
the isolation and structural elucidation of compound 1 as
well as the cytotoxic activities of compounds 1-7 against
the P388 and K562 cell lines using the sulforhodamine-B
MATERIALS AND METHODS
Bacterial strain 39 was isolated using a marine 2216E
agar plate (0.5% tryptone, 0.1% yeast extract, 0.01%
FePO4, 3.4% NaCl and 1.5% agar) from an underwater
sediment sample (depth 5059 m) collected at site 39 from
W2003-03 (W154o57.04'; N8o20.30'), during an expedition
of Haiyang No. 4 in the west Pacific Ocean in 2003. The
sediment cores were transferred to sterile polypropylene
tubes on a clean bench, kept at 4oC for shipping to the
laboratory, and then stored at -20oC.
DNA samples were extracted from the strains and used
as templates for PCR amplification of the 16S rDNA
fragments according to the methods described previously
(Delong, 1992). Primers Eubac27F and Eubac1492R were
used to amplify the 1.5 kb fragment of the bacterial 16S
The 16S rRNA gene sequences were analyzed with the
Ribosomal Database Project (RDP) database, and manually
aligned to 16S rDNA sequences from the RDP and
GeneBank using the PHYLIP package (Felsenstein, J.,
PHYLIP, version 3.5c) and DNAMAN (version 5.1, Lynnon
Biosoft). A phylogenetic tree was constructed by the
Correspondence to: Qian-Qun Gu, Wei-Ming Zhu, Institute of
Marine Drugs and Food, Ocean University of China, Qingdao
266003, PR China
Tel: 86-532-82032065, Fax: 86-532-82033054
E-mail: email@example.com and firstname.lastname@example.org
A New Cytotoxic Phenazine Derivative from a Deep Sea Bacterium Bacillus sp.553
neighbor-joining method using the PHYLIP package and
DNAMAN programs (Saitou and Nei, 1992). Bootstrapping
analysis was used to evaluate the tree topology of the
neighbor-joining data and 1000 trials were performed.
The inhibition of cancer cell proliferation and the cyto-
toxicity of compounds 1-7 were measured using the
SRB assay (Skehan et al., 1990). Cell suspensions (200
µL) were plated in 96-cell plates at a density of 5×104
cell mL-1. 2 µL of the test compound solutions (in DMSO)
at various concentrations (10-4, 10-5, 10-6, 10-7and 10-8
µM) were added to each well and the culture was
incubated for 24 h at 37oC. After this time, the cells were
fixed with 12% trichloroacetic acid and the cell layer was
stained with 0.4% SRB. The absorbance of SRB solution
was measured at 515 nm. Dose response curves were
generated and the concentration of each compound
required to inhibit cell proliferation by 50% (IC50) was
calculated from the linear portion of the log dose
Fermentation and extraction
The deep sea sediment bacteria isolated using the
above procedure were cultured in 300 mL marine broth
(0.5 g peptone, 0.1 g yeast extract, and 0.1 g FePO4
dissolved in 1 L seawater, pH 7.2-7.6) for the production
of secondary metabolites in 500 mL Erlenmeyer flasks.
Flasks with a total of 100 L liquid medium were incubated
for 3 days on a rotary shaker at 100 rev/min at 20oC. The
broth was centrifuged at 5000 g for 30 min to remove the
cells and the supernatant was extracted three times with
100 mL ethyl acetate. The combined ethyl acetate extracts
were concentrated under reduced pressure to give a
crude extract (25 g).
The crude extract (25 g), which was highly cytotoxic to
K562 cells at 100 µg/mL, was separated into six fractions
on a silica gel column using a step gradient elution of
petroleum ether:acetone. Isolation of compounds 1-7 was
achieved through a bioassay-guided fractionation pro-
cedure using the bioassay method described above. The
bioactive fraction 2 (120 mg), eluted with petroleum ether:
acetone (v/v, 50:1), was recrystallized from MeOH, yielding
compound 1 (1.8 mg). Fraction 5 (356 mg) was chroma-
tographed by RP-18 to yield five fractions. Of these, frac-
tion 3 was chromotagraphed by preparative HPLC (column
YMC-pack ODS A, 10×250 mm, 5 µm, 4 mL/min) with
40% aqueous MeOH to give compounds 2 (8 mg, tR =
11.4 min), 3 (11 mg, tR = 11.9 min), 5 (3 mg, tR = 8.9 min), 6
(7 mg, tR = 5.4 min), and 7 (6 mg, tR = 10.0 min). Fraction
5 was similarly subjected to preparative HPLC, but with
20% aqueous MeOH, to give compound 4 (15 mg, tR =
Compound 1: obtained as yellow needles (MeOH): mp
λmax (log ε) 240 (3.72), 281 (3.46), 289 (3.41), 348 (3.39),
364 (3.48), 384 (3.31) nm; IR (KBr) νmax 3438, 2923, 2364,
1633, 1564, 1460, 1261, 1183, 798 cm-1; HRESIMS m/z
291.1143 [M+H]+ (calcd. for C18H15N2O2, 291.1134); 1H-
and 13C-NMR (see Table I).
25 -19.8o (c 0.050, CHCl3); UV (MeOH)
RESULTS AND DISCUSSION
The bioactive ethyl acetate extract of Bacillus sp. was
chromatographed on silica gel columns and then with
Sephadex LH-20 followed by recrystallization with chloro-
form/methanol to yield 1.8 mg compound 1 as yellow
The molecular formula of compound 1 was assigned
as C18H14N2O2 based on the HRESIMS measurement of
the [M+H]+ peak at m/z 291.1143 (calcd. 291.1134). The
molecular formula indicated thirteen degrees of unsatu-
ration within the molecule. The IR spectrum suggested
the presence of a conjugated carbonyl (1633 cm-1) and
an aromatic moiety (1564, 1460, 1261 cm-1). The UV
absorption profile [λmax (log ε) 240 (3.72), 281 (3.46), 289
(3.41), 348 (3.39), 364 (3.48), 384 (3.31) nm] indicated
Table I. 1H (600 MHz) and 13C (150 MHz) NMR Data for Compound
1 in CDCl3
δH (J in Hz)
1, 67.56, 2H br. s 120.4
1a, 6a 142.4
2, 7 134.9
3, 87.23, 2H dd (8.3, 1.8)127.7
4, 97.46, 2H d (8.3) 110.1
11, 122.50, 6H s121.5
2', 3'7.64, 2H s 123.5
Fig. 1. Structure of compounds 1-7
554 D. Li et al.
the presence of a moiety conjugated to the benzene
The 1H-NMR signals at δ 7.56 (1H, s), 7.23 (1H, dd, J =
8.3, 1.8) and 7.46 (1H, d, J = 8.3) and the 13C-NMR
signals at δ 148.9 (s), 142.4 (s), 134.9 (s), 120.4 (d), 127.7
(d) and 110.1 (d) implied a moiety of 1, 2, 4-trisubstituted
benzene ring in the molecule. Furthermore, the 13C-NMR
(DEPT) spectrum of 1 disclosed only nine signals for the
eighteen carbons required by the formula, attributed to
eight sp2 carbons [four quaternary carbons (δ 160.9,
148.9, 142.4, 134.9) and four methines (δ 127.7, 123.5,
120.4, 110.1)] and one methyl (δ 21.5), this indicated a
symmetrical dimeric structure for 1.
Routine analysis of the COSY and HMQC data
indicated the presence of a spin system as shown in Fig.
2. The partial structure a was also deduced by the HMBC
correlations of H-1 with C-3, C-11 and C-4a, H-11 with C-
1, C-2 and C-3, H-3 with C-1, C-11 and C-4a, and H-4
with C-2 and C-1a. As the structure of 1 was a symmetric
dimer, another partial structure should be present in the
molecule. Furthermore, partial structure b was also
confirmed by the HMBC correlations of H-2' with C-3' and
Eleven of the thirteen unsaturations were accounted for,
implying that 1 should also contain two additional rings.
Additionally, the downfield shift of the carbon signals at
δ 142.4 (C-1a) and 148.9 (C-4a) indicated that these two
carbons may be connected to nitrogen (Eberhard and
Ulrich, 1976). The carbonyl signal at δ 160.9 showed the
presence of two amide carbonyl groups and this was also
consistent with the IRCO signal at 1633 cm-1 (Carter et al.,
1961). Thus, fragment a must be connected to b through
two nitrogens as shown in Fig. 1.
All compounds were preliminarily evaluated for their
cytotoxicity against P388 and K562 cells by the SRB
assay. CDDP (cis-Diaminedichloroplatinum) was used as
a positive control (IC50 values of 0.039 and 0.078 µM
against P388 and K562 respectively). Compound 1 was
only tested at one concentration (50 µM) due to the small
quantity of this compound available. At this concentration
compound 1 was cytotoxic to P388 cells, inhibiting their
proliferation by 78.3%. Compound 2 was weakly cytotoxic
with an IC50 value 74 µM on P388 cells. Compound 1 was
not cytotoxic to K562 cells, while compound 2 had an IC50
value 87 µM on these cells. Other compounds showed no
activities against these two cells.
Phenazine derivatives have been isolated from terrestrial
Pseudomonas sp. (Ge et al., 2004) and Streptomyces sp.
(Chatterjee et al., 1995; Gebhardt et al., 2002) as well as
marine Streptomycin sp. (Pusecker et al., 1997) as bacterial
pigments. Some of them have symmetrical structures and
activities against bacteria and fungi (Chatterjee et al.,
1995; Gebhardt et al., 2002). The biosynthesis of the
phenazine pigments may involve the shikimic acid pathway
or the oxidative dimerization of anthranilic acid with the
loss of the carboxyl carbon where appropriate (Carter and
Richards, 1961; Ge et al., 2004). To the best of our
knowledge, compound 1 is the only phenazine derivative
with a [4, 2, 2] ring structure.
This work was founded by the Chinese Ocean Mineral
Resource R & D Association (DY105-2-04). We thank S.
Miao for reviewing the manuscript.
Carter R. E. and Richards J. H., The biogenesis of phenazine
pigments. J. Am. Chem. Soc., 83, 495-496 (1961).
Chatterjee, S., Vijayakumar, E. K. S., Franco, C. M. M., Maurya,
R., Blumbach, J., and Ganguli, B., Phencomycin, a new
antibiotic from a Streptomyces species HIL Y-9031725. J.
Antibio., 48, 1353-1354 (1995).
Chen, X. L., Zhang, Y. Zh., and Gao, P., Progress in deep-sea
microbiology. J. Marine Sci., 28, 61-66 (2004).
Delong, E. F., Archaea in coastal marine environments. Proc.
Natl. Acad. Sci. U.S.A., 89, 5685-5689 (1992).
Eberhard, B. and Ulrich H., Carbon-13 nuclear magnetic
resonance chemical shifts of substituted phenazines. J. Org.
Chem., 41, 2104-2108 (1976).
Ge Y., Huang X., Wang S., Zhang X., and Xu Y., Phenazine-1-
carboxylic acid is negatively regulated and pyoluteorin
positively regulated by gacA in Pseudomonas sp. M18.
FEMS Microbio. Lett., 237, 41-47 (2004).
Gebhardt K., Schimana J., Krastel P., Dettner K., Rheinheimer
J., Zeeck A., and Fiedler H., Endophenazines A-D, New
Phenazine Antibiotics from the Arthropod Associated Endo-
symbiont Streptomyces anulatus.I.Taxonomy, Fermentation,
Isolation and Biological Activities. J. Antibio., 55, 794-800
Faulkner, D. J., Marine natural products. J. Nat. Prod. Rep., 19,
Fenical, W., Chemical studies of marine bacteria: developing a
new resource. Chem. Rev., 93, 1673-1683 (1993).
Pusecker, K., Laatsch, H., Helmke, E., and Weyland, H., Dihy-
Fig. 2. Partial structures a and b in 1 and its selected 1H-13C HMBC
and 1H-1H COSY NMR correlations
A New Cytotoxic Phenazine Derivative from a Deep Sea Bacterium Bacillus sp. 555
drophencomycin methyl ester, a new phenazine derivative
from a marine Streptomycete. J. Antibio., 50, 479-83
Saitou, N. and Nei M., The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol. Biol.
Evol., 4, 406-425 (1987).
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon,
J., Vistica, D Warren, J. T., Bokesch, H., Kenney, S., and
Boyd, M. R., New colorimetric cytotoxicity assay for
anticancer drug screening. J. Natl Cancer Inst., 82, 1107-