A novel ketone derivative of artemisinin biotransformed by Streptomyces griseus ATCC 13273

Article abstract of DOI:10.1016/j.bmcl.2005.12.076

A novel ketone derivative of artemisinin, artemisitone-9, was produced by the biotransformation of cultured Streptomyces griseus ATCC 13273. The structure of the ketone product was fully elucidated by various spectroscopic techniques, and the mechanism of generating such novel metabolite is also discussed.

Full text of DOI:10.1016/j.bmcl.2005.12.076

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1909–1912
A novel ketone derivative of artemisinin biotransformed by
Streptomyces griseus ATCC 13273
Ji-Hua Liu, You-Gen Chen, Bo-Yang Yu and Yi-Jun Chen*
*
Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing 210038, People’s Republic of China
Received 17 October 2005; revised 5 December 2005; accepted 24 December 2005
Available online 24 January 2006
Abstract—A novel ketone derivative of artemisinin, artemisitone-9, was produced by the biotransformation of cultured Streptomy-
ces griseus ATCC 13273. The structure of the ketone product was fully elucidated by various spectroscopic techniques, and the
mechanism of generating such novel metabolite is also discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
Artemisinin (1, Qinghaosu) is the active component of
the antimalarial herb Qinghao (Artemisia annua L,
Asteraceae) . Because of its high therapeutic values in
analogues, such as artemether, arteether, artemisitene,
and 12-deoxoartemisinin, have been reported to produce
6–10
oxidative products by different microorganisms.
1
treating malaria, especially chloroquine-resistant
Plasmodium falciparum and the cerebral infections, tre-
mendous efforts have been made toward structure mod-
ification and analogue synthesis with the aim of
developing more potent antimalarial agents with im-
proved in vivo stability since it was discovered in 1970s.
In order to obtain novel analogues for structural modi-
fication of artemisinin, we initiated a screening of differ-
ent microorganisms with a two-stage fermentation
1
1
protocol, and metabolite generation was monitored
by analyzing the fermentation broth by HPLC over
the time. A number of microorganisms were able to pro-
duce polar metabolites in the screening. However, unlike
other microorganisms, Streptomyces griseus ATCC
13273 produced a major metabolite that was eluted be-
tween artemisinin and other three polar metabolites
from the HPLC analyses. This novel phenomenon led
us to pursue isolation and identification of these
metabolites.
Thus far, most of the structural modifications took place
at the lactone moiety of artemisinin, modifications and
structure–activity relationship on the two saturated
rings remain unexplored due mainly to the difficulty of
introducing functionalities on the ring systems by con-
ventional chemical methods. Therefore, microbial trans-
formation comes to play an important role to overcome
the inaccessibility by chemical reactions and to serve as a
valuable tool to introduce hydroxy group(s) on the sat-
urated rings. To date, a number of oxidation products of
artemisinin at different positions have been reported.
These transformations include conversion to 3a-hy-
A preparative fermentation at 1.2 L scale was conducted
with S. griseus ATCC 13273 by transformation of
720 mg artemisinin substrate to isolate and identify the
products. At 84 h of the second stage of fermentation,
the fermentation broth was pooled and filtered, and
the filtrate (1.2 L) was extracted three times with ethyl
acetate. The extracts were dried over anhydrous Na SO
2
,3
droxy-deoxyartemisinin
and
deoxyartemisinin;
conversion to 9b-hydroxy-artemisinin and 3a-hydroxy-
artemisinin , and conversion to 10-hydroxy-artemisi-
3
–5
2
4
5
nin and 9b-hydroxy-11a-artemisinin. In addition,
microbial transformations on some artemisinin
and evaporated to dryness under reduced pressure to af-
ford 0.84 g of dark brown residue. Then, the residue was
purified by column chromatography over an 80 g silica
gel column eluting with a chloroform–methanol gradi-
ent to afford artemisitone-9 (2) (90.1 mg, 12.5% yield),
9a-hydroxy-artemisinin (3) (118.7 mg, 16.5%), 9b-hy-
droxy-artemisinin (4) (115.8 mg, 16.1%), and 3a-hy-
droxy-deoxyartemisinin (5) (68.7 mg, 9.5%) as shown
Keywords: Biotransformation; Streptomyces griseus ATCC 13273;
Artemisinin; regioselectivity; Artemisitone-9.
*
Corresponding authors. Tel.: +86 25 83313080; fax: +86 25
5302355; e-mail addresses: boyangyu59@163.com; boyangyu@
yahoo.com.cn
8
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.076

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J.-H. Liu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1909–1912
in Scheme 1. The structures of 2, 3, 4, and 5 were fully
characterized by mass spectrometry and NMR
spectroscopy.
In the present work, a novel ketone derivative of 1 has
been isolated and identified from microbial transforma-
tion. The regioselective introduction of a carbonyl group
on the saturated ring is not possible to achieve by con-
ventional chemical methods, and this type of transfor-
mation by an actinomycete like S. griseus ATCC
13273 has not been reported as well. We speculated that
the formation of 2 from 1 underwent two consecutive
steps by this actinomycete. First, compound 1 was
hydroxylated at C-9 position by a hydroxylase to gener-
ate the monohydroxy compounds 3 and 4 with a- and
b-configuration, respectively. Upon the formation of
monohydroxylated compounds, an alcohol dehydroge-
nase in the organism catalyzed a dehydrogenation reac-
HREIMS analysis of 2 indicated its molecular formu-
la of C H O . Peaks of IR spectra at 822, 878,
1
5
20
6
À1
suggested the existence of the
1
113, and 1739 cm
endoperoxide bridge and lactone skeleton.
NMR signal at d205.8 and the inverted peaks at d
6.1, 35.8, and 38.3 of DEPT spectra recorded with
1
3
C
2
h = 135° confirmed that one of the secondary carbons
on the 6-membered ring of 1 was converted to a car-
bonyl group, and this carbonyl group is located at
C-9 position according to the HMQC data. Based
on the spectroscopic data compared with established
+
+
tion, in the presence of NAD or NADP as a cofactor,
to form the ketone product 2 (Scheme 2). To confirm
such sequence, we used 3 and 4 as a substrate, respec-
tively, with same biotransformation procedures. After
1
13
12
H and
C NMR assignments,
this metabolite
1
3
was identified as artemisitone-9 (2), a novel ketone
derivative of 1.
4
8 h of incubation, both compounds were indeed con-
All data from IR, CIMS, NMR spectra for metabolite 3
indicated as 9a-hydroxy-artemisinin, a mammalian
metabolite previously reported by Chi et al.
verted to 2 at similar rates, whereas no changes of 3
and 4 were observed in the controls without the actino-
mycete. This result indicated that the second step was
catalyzed by a non-stereospecific alcohol dehydroge-
nase. Meanwhile, we measured the concentrations of
products 2, 3, and 4 while prolonging the reaction time.
Compounds 3 and 4 were produced to a peak at day 4,
then gradually decreased over the period of a week,
whereas product 2 was formed at day 3 and reached
the peak at day 7 with conversion yield greater than
50%. Such product profiles provided additional evidence
that 3, and 4 are the intermediate metabolites for 2.
1
4
All data from IR, CIMS, NMR spectra for metabolite 4
were in excellent agreement with 9b-hydroxy-artemisi-
nin, an epimer of metabolite 3 and previously reported
3
–5
in the literature.
All data from IR, CIMS, NMR spectra for metabolite 5
were confirmed to be 3a-hydroxy-deoxyartemisinin, a
compound previously reported in the literature.
2
,3
CH3
OH
H
O
H C
O
3
O
H
H
O
CH3
O
3
CH3
14
CH3
HO
H C
H
CH3
H
2
H
1
3
O
O
10
7
9
O
3
4
O
O
H C
3
H C
O
5
O
3
8
H
O
H
CH3
15
6
H
O
H
H
O
H
O
12
O
CH3
1
1 CH
3
O
5
O
2
1
3
O
1
CH3
H
OH
O
H C
O
3
O
H
CH3
H
O
O
4
Scheme 1. Biotransformation of artimisinin by Streptomyces griseus ATCC 13273.

J.-H. Liu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1909–1912
1911
CH3
H
OH
O
H C
3
O
O
O
H
CH3
H
CH3
H
H
CH
H
3
O
O
3
O
O
H
H C
3
O
O
H C
O
O
3
Hydroxylase
O
H
+
Dehydrogenase
H
CH3
O
CH3
O
1
CH3
O
2
H
O
O
H C
3
O
O
O
H
CH3
H
O
4
Scheme 2. Proposed mechanism of producing 2 from 1 by Streptomyces griseus ATCC 13273.
The antimalarial activity of the metabolites 2, 3, 4 and 5
was compared with 1 by a 48-h in vitro assay against
and grants for Outstanding Young Scholars at Universi-
ties in Jiangsu Province, PR China, awarded to B-Y.
Yu.
1
5
P. falciparum FCC-1/HN strain. Not surprisingly,
the metabolite without the endoperoxide bridge 5 was
inactive, whereas metabolites 2, 3, and 4 retained the
activity with IC50 values of 25.7, 34.6, and 29.3 ng/ml,
respectively, even though they were less active than arte-
misinin (IC50 = 3.1 ng/ml). These results were similar to
previous reports on the comparisons of artemisitene,
arteether, anhydrodihydroartemisinin, and their micro-
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.12.076.
9
,16,17
bial metabolites.
The decrease of activity by oxida-
tion of 1 was expected because oxidative modifications
generally are the metabolic reactions of detoxification
of drugs in mammals, plants and microorganisms.
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This work was supported by grants from National
Natural Science Foundation of China (No. 2997256)

1912
J.-H. Liu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1909–1912
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3