Synthesis, antimicrobial activity and possible mechanism of action of 9-bromo-substituted indolizinoquinoline-5,12-dione derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.07.042

A series of 9-bromo-substituted indolizinoquinoline-5,12-dione derivatives was synthesized. Antimicrobial activity assessment indicates that compounds 1, 26, 27 and 28 exhibit strong activity against gram-positive bacterial strains, including Beta-hemolyt

Full text of DOI:10.1016/j.ejmech.2011.07.042

European Journal of Medicinal Chemistry 46 (2011) 4625e4633
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antimicrobial activity and possible mechanism of action
of 9-bromo-substituted indolizinoquinoline-5,12-dione derivatives
,
,
Xi-Wei Wu a 1, Zu-Ping Wu a 1, Lu-Xia Wang b, Hong-Bin Zhang b, Jian-Wen Chen a, Wei Zhang a,
Lian-Quan Gu a, Zhi-Shu Huang a, Lin-Kun An a
,
*
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b Department of Clinical Laboratory, Guangzhou Liuhuaqiao Hospital, Guangzhou 510010, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 9-bromo-substituted indolizinoquinoline-5,12-dione derivatives was synthesized. Antimi-
crobial activity assessment indicates that compounds 1, 26, 27 and 28 exhibit strong activity against
gram-positive bacterial strains, including Beta-hemolytic streptococcus CMCC32210, Staphylococcus aureus
ATCC25923, Staphylococcus epidermidis ATCC12228, Enterococcus faecalis ATCC29212 and methicillin-
resistant S. aureus ATCC43300 (MRSA). Compound 27 shows the best anti-MRSA activity with an MIC
Received 8 March 2011
Received in revised form
22 July 2011
Accepted 22 July 2011
Available online 28 July 2011
value of 0.031 mg/ml. To assess the mechanism of action, the inhibitory activities of compound 1 against
DNA gyrase and DNA topoisomerase IV were also measured. The results indicate that compound 1 has
strong inhibitory effects on the Escherichia coli DNA gyrase supercoiling activity and S. aureus Topo IV
relaxing activity.
Keywords:
9-bromo-substituted indolizinoquinoline-
5,12-dione derivatives
Antimicrobial activity
Ó 2011 Elsevier Masson SAS. All rights reserved.
Methicillin-resistant Staphylococcus aureus
DNA gyrase
DNA topoisomerase IV
1. Introduction
2. Results and discussion
Pathogen infections have threatened human’s health for
thousands years [1]. During past decades, with the abuse of
antimicrobial agents, more and more drug-resistant pathogens
were found. Among them, methicillin-resistant Staphylococcus
aureus (MRSA) is a prominent pathogen, which causes a public
health concern worldwide and associates with a high mortality
[2e4]. Novel antimicrobial agents against MRSA have been
introduced recently. However, the emergency of resistance and
side effects for those agents raise the need for novel antimicro-
bial agents [5,6]. In this work, a series of 9-bromo-substituted
indolizinoquinoline-5,12-dione derivatives was synthesized, and
their antimicrobial activity was screened. The inhibitory activi-
ties against DNA gyrase and topoisomerase IV were also
investigated.
2.1. Chemistry
In our previous study of novel anticancer agents, the reaction of
6,7-dichloroquinoline-5,8-dione with active methylene agent and
pyridine gave two main products, N,N-syn and N,N-anti indolizi-
noquinoline-5,12-dione derivatives [7]. According to the mecha-
nism of heterocyclization proposed by Defant et al [8], four isomers,
9/7-bromo N,N-syn isomers and 9/7-bromo N,N-anti isomers,
should be obtained theoretically if using 3-bromopyridine as
material. In fact, only two main products, 7-bromo N,N-syn isomer
and 7-bromo N,N-anti isomer, were obtained in our previous
experiment [7]. In order to obtain the corresponding 9-bromo
isomers, we examined the reaction conditions and have found
that an increase of 3-bromopyridine amount and the reaction
concentration improved the yield of 9-bromo products. When 12
equivalents of 3-bromopyridine was used, the reaction gave 9-
bromo isomers as the main products, 9-bromo N,N-syn isomer (1)
in 28% yield and 9-bromo N,N-anti isomer (2) in 16% yield, as well as
7-bromo N,N-syn isomer (3) in 20% yield and 7-bromo N,N-anti
isomer (4) in 4% yield (Scheme 1). The NMR, MS spectra and
melting point of compounds 3 and 4 are similar to our reported
* Corresponding author. Tel./fax: þ86 20 39943052.
E-mail address: lssalk@mail.sysu.edu.cn (L.-K. An).
1
These authors contributed equally to this paper.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.042

4626
X.-W. Wu et al. / European Journal of Medicinal Chemistry 46 (2011) 4625e4633
Scheme 1. Synthesis of compounds 1e31.
results and confirm their structures [7]. HRMS spectrum of
compound and shows they have the same formula,
and 83160) is up to 0.016
vancomycin.
mg/ml, 64-fold superior to that of
1
2
C18H11N2O4Br, as that of 3 and 4. The HMBC spectrum of 1 shows
a long-range hetero-correlation of C-5 at 178.8 ppm with 4-H at
8.57 ppm (dd, J ¼ 7.8, 1.8 Hz). In the 1H NMR spectrum, the signal at
10.10 ppm (dd, J ¼ 1.6, 0.4 Hz) can be assigned to 10-H. As
a consequence, the signals at 8.27 ppm and 7.56 ppm can be
assigned to 7-H and 8-H, respectively. HRMS, 1D NMR and 2D NMR
spectra of compound 1 prove its ethyl 9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate structure, also
confirmed by X-ray single crystal analysis (Fig. 1). Similarly,
compound 2 can be assigned as the 9-bromo N,N-anti isomer.
An antimicrobial screening indicates that compound 1 exhibits
a strong activity against gram-positive bacterial strains, especially
MRSA, but only a low solubility in water and organic solvent, such
as dimethyl sulfoxide. This drawback pushed us to prepare its
analogs to find more potent agents with higher antimicrobial
activity and better solubility. The synthesis of these analogs,
compounds 6e31, is shown in Scheme 1. At first, we tried to convert
compound 1 to its amide analogs according to Defant’s method [9].
Unfortunately, it did not react with the corresponding amines
under those conditions. Therefore, we hydrolyzed compound 1 in
isopropanol with 15% K2CO3 aqueous solution to give a corre-
The antimicrobial activity of amide and ester derivatives 6e31 is
summarized in Table 2. The results indicate that all products exhibit
a low activity against fungal strain. Compared to compound 1, its
amide analogs 6e25 show a lower antimicrobial activity against
bacterial and fungal strains, whereas ester analogs 26e31 exhibit
a substantially higher activity against five gram-positive bacterial
strains than its amide analogs. It is clear that alkoxycarbonyl group
plays an important role in the antimicrobial activity of ester
products. It is noteworthy that compound 27, whose solubility in
dimethyl sulfoxide is three times better than that of compound 1,
exhibits stronger anti-MRSA activity than compound 1.
2.3. Inhibitory activities against DNA gyrase and DNA
topoisomerase IV
To assess the mechanism of antimicrobial activity, DNA gyrase
supercoiling assay was performed with compound 1 using relaxed
pHOT1 DNA as a substrate. Ciprofloxacin (CFX) was used as
sponding carboxylic acid
5 [10]. Following chlorination of
compound 5 with thionyl chloride excess gave a corresponding acid
chloride intermediate that was aminated or esterified to give the
target products 6e31. Their structures were elucidated by MS and
NMR spectra.
2.2. Antimicrobial activity
The antimicrobial activity of compound 1 was firstly assessed by
a standard two-fold microdilution assay in Mueller-Hinton broth
against three gram-negative bacterial strains, one fungal strain, and
sixteen gram-positive bacterial strains including twelve MRSA
strains [11]. The minimum inhibition concentration (MIC) is
summarized in Table 1. The results indicate that compound 1
exhibits a low activity against gram-negative bacterial strains and
fungal strain. The MIC values are more than 64
mg/ml against E. coli
(ATCC25922), P. aeruginosa (ATCC27853), S. paratyphi
B
(CMCC50094) and C. Albicans (ATCC10231). However, compound 1
exhibits a strong activity against gram-positive bacterial strains,
especially MRSA (ATCC43300) with a MIC value of 0.063 mg/ml, 16-
fold superior to that of vancomycin. The activity of compound 1
against five clinical MRSA strains (72754, 071843, 82731, 071429
Fig. 1. Perspective ORTEP drawing of compound 1.

X.-W. Wu et al. / European Journal of Medicinal Chemistry 46 (2011) 4625e4633
4627
Table 1
4. Methods and materials
In vitro antimicrobial activity of compound 1.
Microorganism
MIC (
1
m
g/ml)a
4.1. General experiments
Vancomycin
The major chemical reagents for synthesis were purchased from
Alfa Aesar or Sigma Aldrich Co. 6,7-Dichloroquinoline-5,8-dinone
was prepared according to our reported method [7]. The common
solvents were obtained from local commercial suppliers and used
without further purification. Ciprofloxacin and vancomycin, used as
positive control for biological assay, were purchased from National
Institute for the Control of Pharmaceutical and Biological Products
(Beijing, China). Chemical reaction courses were monitored by silica
gel GF254 thin layer chromatography. Melting points were deter-
mined in open capillary tubes on a MPA100 Optimelt Automated
Melting Point System without being corrected. Nuclear magnetic
resonance spectra were recorded on a Bruker AVANCE III 400 MHz
spectrometer using tetramethylsilane as an internal reference.
Mass spectra were analyzed on an Agilent 6120 (Quadrupole LC-
MS) mass spectrometer. The high-resolution mass spectra were
analyzed on a SHIMADZU LCMS-IT-TOF mass spectrometer. UV
spectra were recorded on a SHIMADZU UV-2501 PC spectropho-
tometer. All compounds tested for biological activity were analyzed
by HPLC and their contents were more than 95%.
E. coli ATCC25922
P. aeruginosa ATCC27853
S. paratyphi B CMCC50094
H. streptococcus B CMCC32210
S. aureus ATCC25923
S. epidermidis ATCC12228
E. faecalis ATCC29212
C. albicans ATCC10231
MRSA
>64.00
64.00
>64.00
0.031
0.063
0.063
ndb
nd
nd
nd
nd
nd
nd
nd
0.016
>64.00
ATCC43300
0.063
0.063
0.063
0.032
0.032
0.016
0.016
0.016
0.016
0.016
0.032
0.032
1.00
0.50
2.00
1.00
0.50
1.00
1.00
1.00
1.00
1.00
1.00
1.00
80754c
7365
0441
071410
72754
071843
82731
071429
83160
82896
82924
a
The antimicrobial activity was expressed by the minimum inhibitory concen-
tration, which was obtained in at least two independent experiments.
b
“nd” means “not determined”.
These MRSA strains were clinically isolated from Guangzhou Liuhuaqiao
4.2. Synthesis of compounds 1e4
c
Hospital, China.
To
a yellow suspension of 6,7-dichloroquinoline-5,8-dione
(1.14 g, 5.00 mmol) in absolute ethanol (25 ml), ethyl acetoace-
tate (1.20 ml, 10.00 mmol) and 3-bromopyridine (6.00 ml,
60.00 mmol) were added. The resultant solution was stirred and
refluxed for 5 h. The reaction solution gradually became a dark red
suspension. After completion of reaction, the reaction solution was
cooled to room temperature. The precipitate was collected by
filtration and purified by silica gel column chromatography with
eluent CH2Cl2:AcOEt ¼ 30:1. Four products, 1 (0.56 g, 28%), 2
(0.32 g, 16%), 3 (0.40 g, 20%) and 4 (0.08 g, 4%), were obtained. The
structures of products 3 and 4 were identified by mass and NMR
spectra, which were similar to those reported in our previous
reference [7].
a positive control. As shown in Fig. 2A, compound 1 shows a strong
inhibition of the supercoiling activity of E. coli DNA gyrase but
lesser than that of CFX. DNA gyrase supercoiling activity is inhibited
over 78% and 98% at 1
compound 1 does not exhibit a supercoiling inhibitory activity
against S. aureus DNA gyrase at 125 M (data not shown) [12].
mM and 100 mM, respectively. Likely to CFX,
m
DNA topoisomerase IV (Topo IV) relaxation assay was performed
with compound 1 using supercoiled pBR322 DNA as a substrate.
CFX was used as a positive control. As shown in Fig. 2B, likely to
CFX, compound 1 exhibits a slight inhibitory activity against
S. aureus Topo IV at 1
activity at 25 M. On the contrary, compound 1 is not effective
against E. coli Topo IV at 125 M (data not shown).
mM, and completely inhibits the relaxing
m
m
4.2.1. Ethyl 9-bromo-5,12-dioxo-5,12-dihydroindolizino[2,3-g]
quinoline-6-carboxylate (1)
Topo IV decatenation assay was performed with compound 1
using catenated kDNA as a substrate. CFX was used as a positive
control. As shown in Fig. 2C, the decatenation activity of S. aureus
Topo IV is almost completely inhibited by CFX at 25 mM. Compound
1 has a lower inhibitory activity than CFX. There is 84% monomer
circle DNA remained after being treated with compound 1 at
Orange solid, mp ¼ 279.8e281.8 ꢀC. 1H NMR (CDCl3)
d 10.10 (dd,
J ¼ 1.6, 0.4 Hz, 1H, 10-H), 9.05 (dd, J ¼ 4.6, 1.8 Hz, 1H, 2-H), 8.57 (dd,
J ¼ 7.8, 1.8 Hz, 1H, 4-H), 8.27 (dd, J ¼ 9.6, 0.4 Hz, 1H, 7-H), 7.67 (dd,
J ¼ 8.0, 4.8 Hz, 1H, 3-H), 7.56 (dd, J ¼ 9.6, 1.6 Hz, 1H, 8-H), 4.52 (q,
J ¼ 7.1 Hz, 2H, eOCH2e), 1.50 (t, J ¼ 7.1 Hz, 3H, eCH3). 13C NMR
125 mM concentration.
(CDCl3) d 178.8 (C-5),173.3 (C-12),162.7 (eCOOe),154.2 (C-2),149.2
(C-12a), 138.1 (C-6a), 135.5 (C-4), 131.9 (C-8), 131.0 (C-4a), 128.4 (C-
10), 127.8 (C-5a), 127.2 (C-3), 122.8 (C-11a), 121.6 (C-7), 113.5 (C-6),
107.1 (C-9), 61.4 (eCH2e), 14.3 (eCH3). ESI-MS m/z: 401.0 (100%),
399.0 (88%) [MþH]þ. HRMS (ESI) m/z: 398.9988 [MþH]þ, calcd for
3. Conclusion
In conclusion, we have synthesized a series of 9-bromo-
substituted indolizinoquinoline-5,12-dione derivatives. The ester
analogs exhibit strong antibacterial activity against gram-positive
strains, especially MRSA. The MIC values of compounds 1, 26 and
C18H12N2O4Br 398.9980. The structure of compound
1 was
confirmed with 2D NMR spectra and single crystal analysis.
Compound 1 was crystallized from CHCl3/AcOEt as orange block
28 against MRSA are up to 0.063
vancomycin. Compound 27 shows the best activity with a MIC value
of 0.031 g/ml, 32-fold superior to that of vancomycin. DNA gyrase
supercoiling assay and Topo IV inhibition assays indicate that
compound 1 shows the mechanism of action similar to cipro-
floxacin [12]. It effectively inhibits the supercoiling activity of E. coli
DNA gyrase and the relaxation activity of S. aureus Topo IV, but has
only a low decatenation inhibitory activity against S. aureus Topo IV.
Our findings indicate that DNA gyrase and Topo IV might be the
biological target for compounds under study.
m
g/ml, 16-fold superior to that of
crystals. Molecular formula
¼
C18H11N2O4Br, molecular
mass ¼ 399.20, monoclinic, a ¼ 7.1719(3) Å, b ¼ 12.1221(5) Å,
m
c ¼ 17.0506(8) Å,
b
¼ 90.658(4)ꢀ, U ¼ 1482.25(12) Å3, T ¼ 153, space
group P21/c (no. 14), Z ¼ 4,
m
(Cu K
a
) ¼ 4.044, 5836 reflections
measured, 2698 unique (Rint ¼ 0.0240) which were used in all
calculations. The final (reflections) 0.0391, wR2
R
¼
(reflections) ¼ 0.1075. Crystallographic data for compound 1 has
been deposited with the Cambridge Crystallographic Data Center as
supplementary publication number CCDC 828381. Copies of the
data can be obtained, free of charge, on application to CCDC, 12

4628
X.-W. Wu et al. / European Journal of Medicinal Chemistry 46 (2011) 4625e4633
Table 2
Structures and in vitro antimicrobial activity of compounds 6e31.
Comp
R
MIC (
m
g/ml)a
E. coli
P. aeruginosa S. paratyphi B H. streptococcus B S. aureus
S. epidermidis E. faecalis
MRSA
C. albicans
ATCC25922 ATCC27853
CMCC50094
CMCC32210
ATCC25923 ATCC12228 ATCC29212 ATCC43300 ATCC10231
6
7
8
9
10
11
12
13
14
eNH(CH2)2CH3
eNHCH(CH3)2
eNH(CH2)3CH3
eNEt2
eb
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
>64.00
e
>64.00
e
>64.00
e
8.00
e
8.00
16.00
16.00
16.00
>64.00
e
eNHCH2Ph
e
e
e
e
eNH(CH2)2NMe2
eNH(CH2)3NMe2
eNH(CH2)2NEt2
eNH(CH2)3NEt2
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
0.50
1.00
0.50
1.00
4.00
4.00
4.00
8.00
2.00
8.00
2.00
4.00
1.00
4.00
4.00
4.00
4.00
4.00
4.00
8.00
>64.00
>64.00
>64.00
>64.00
15
64.00
64.00
>64.00
0.50
1.00
4.00
4.00
8.00
>64.00
16
17
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
1.00
0.50
8.00
4.00
4.00
1.00
4.00
2.00
4.00
4.00
>64.00
>64.00
18
19
>64.00
>64.00
>64.00
1.00
0.50
8.00
1.00
4.00
1.00
4.00
2.00
4.00
2.00
>64.00
>64.00
64.00
64.00
64.00
20
21
22
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
1.00
16.00
8.00
2.00
8.00
8.00
32.00
32.00
2.00
4.00
64.00
32.00
>64.00
>64.00
>64.00
>64.00
>64.00
16.00
23
24
>64.00
>64.00
>64.00
>64.00
>64.00
>64.00
16.00
64.00
16.00
32.00
>64.00
>64.00
64.00
64.00
>64.00
>64.00
>64.00
>64.00
>64.00
25
>64.00
>64.00
>64.00
16.00
64.00
>64.00
>64.00
>64.00
26
27
64.00
>64.00
>64.00
>64.00
>64.00
0.031
0.031
0.031
0.031
0.016
0.016
0.031
0.016
0.063
0.031
>64.00
>64.00
>64.00
28
29
>64.00
>64.00
>64.00
>64.00
0.031
0.031
0.031
0.13
0.016
0.13
0.016
0.13
0.063
0.13
>64.00
>64.00
2.00
1.00
30
4.00
>64.00
16.00
0.031
0.063
0.063
0.031
0.50
>64.00

X.-W. Wu et al. / European Journal of Medicinal Chemistry 46 (2011) 4625e4633
4629
Table 2 (continued )
Comp
R
MIC (
m
g/ml)a
E. coli
P. aeruginosa S. paratyphi B H. streptococcus B S. aureus
S. epidermidis E. faecalis
MRSA
C. albicans
ATCC25922 ATCC27853
CMCC50094
CMCC32210
ATCC25923 ATCC12228 ATCC29212 ATCC43300 ATCC10231
31
32.00
>64.00
>64.00
0.063
0.25
0.25
0.13
2.00
0.25
>64.00
Penc
64.00
>64.00
2.00
<0.031
<0.031
16.00
>64.00
ndd
a
The MIC values represent the results obtained in at least two independent experiments.
“-” means “not determined” because of poor solubility of the compound.
“Pen” means the standard penicillin.
b
c
d
“nd” means “not determined”.
Union Road, Cambridge CB2 1EZ, UK [fax: þ44 (0) 1223-336033 or
7.54 (dd, J ¼ 9.6,1.6 Hz,1H), 4.50 (q, J ¼ 7.2 Hz, 2H),1.52 (t, J ¼ 7.2 Hz,
3H). 13C NMR (CDCl3)
177.9, 174.0, 163.1, 154.0, 149.6, 138.3, 134.4,
e-mail: deposit@ccdc.cam.ac.uk].
d
131.7, 130.3, 128.1, 127.3, 121.6, 113.4, 107.6, 61.5, 14.1. ESI-MS m/z:
399.0 (100%), 401.0 (98%) [M þ H]þ. HRMS (ESI) m/z: 420.9794
[M þ Na]þ, calcd for C18H11N2O4BrNa 420.9800.
4.2.2. Ethyl 8-bromo-5,12-dioxo-5,12-dihydroindolizino[3,2-g]
quinoline-11-carboxylate (2)
Orange solid, mp ¼ 266.1e268.9 ꢀC. 1H NMR (CDCl3)
d 9.98 (dd,
J ¼ 1.6, 0.8 Hz, 1H), 9.03 (dd, J ¼ 4.4, 1.6 Hz, 1H), 8.57 (dd, J ¼ 8.0,
1.6 Hz,1H), 8.30 (dd, J ¼ 9.6, 0.8 Hz,1H), 7.69 (dd, J ¼ 8.0, 4.4 Hz,1H),
4.3. Synthesis of 9-bromo-5,12-dioxo-5,12-dihydroindolizino[2,3-g]
quinoline-6-carboxylic acid (5)
To a yellow suspension of compound 1 (1.60 g, 4.00 mmol) in
isopropanol (250 ml), an aqueous solution of K2CO3 (15%, 30 ml)
was added. The yellow suspension was stirred at room temperature
for 15 min, and then refluxed for 24 h to give a red suspension. The
suspension was cooled to room temperature. The solvent was
removed in vacuo. The residue was dissolved in water (2 L), filtered
to remove the undissolved precipitate. The filtrate was adjusted to
pH 3 with 2 M aqueous HCl to give abundant aubergine precipitate.
The resultant precipitate was collected by filtration and washed
with water, and then dried in vacuo to give an aubergine solid 5
(1.40 g), yield 94%, mp > 300 ꢀC. 1H NMR (CDCl3)
d 9.93 (br.s, 1H),
8.95 (d, J ¼ 4.4 Hz, 1H), 8.46 (d, J ¼ 7.6 Hz, 1H), 8.21 (d, J ¼ 9.6 Hz,
1H), 7.54 (dd, J ¼ 7.6, 4.4 Hz,1H), 7.34 (d, J ¼ 9.6 Hz,1H). ESI-MS m/z:
370.9 [MþH]þ.
4.4. General procedure for the synthesis of compounds 6-31
At room temperature, to a red solution of compound 5 (0.19 g,
0.50 mmol) and triethylamine (0.14 ml, 1.00 mmol) in chloroform
(40 ml), thionyl chloride (2.5 ml) was added dropwise. The
mixture was stirred and refluxed for 5 h. The mixture gradually
became a red solution. The reaction solution was then cooled to
room temperature. The solvent was evaporated under reduced
pressure. The residue was contained under reduced pressure for
a period to get rid of most of the residual SOCl2 to give an orange
solid residue. 4-(Dimethylamino)pyridine (0.07 g, 0.6 mmol) and
different amine or alcohol derivative (1.80 mmol) in CHCl3
(30 ml) were added dropwise to the resultant residue. The
reaction mixture instantaneously became a red solution. The
reaction mixture was refluxed for 5 h, and cooled to room
temperature. The solvent was evaporated under reduced pres-
sure. The crude product was purified by silica gel column
chromatography.
Fig. 2. (A) Inhibition of the supercoiling activity of E. coli gyrase by compound 1 (Upper
panel) or ciprofloxacin (Bottom panel). Lane 1, relaxed pHOT1 DNA only. Lane 2,
relaxed pHOT1 DNA and enzyme. Lane 3e9, relaxed pHOT1 DNA, enzyme and
compound at various concentrations. (B) Inhibition of S. aureus Topo IV-catalyzed DNA
relaxation by compound 1 (Left panel) or ciprofloxacin (Right panel). Lane 1, pBR322
DNA only. Lane 2, pBR322 DNA and enzyme. Lane 3e6, pBR322 DNA, enzyme and
compound at various concentrations. (C) Inhibition of S. aureus Topo IV-catalyzed
4.4.1. N-Propyl 9-bromo-5,12-dioxo-5,12-dihydroindolizino[2,3-g]
quinoline-6-carboxamide (6)
The product was purified by silica gel column chromatography
with eluent CHCl3:AcOEt ¼ 11:1 to give an aubergine solid 6
(0.20 g), yield 96%, mp ¼ 280.2e283.1 ꢀC. 1H NMR (CDCl3)
d 10.19 (t,
decatenation by compound
1 (Left panel) or ciprofloxacin (Right panel). Lane 1,
J ¼ 5.6 Hz, 1H), 10.10 (br.s, 1H), 9.06 (dd, J ¼ 4.8, 1.2 Hz, 1H), 9.04 (d,
J ¼ 9.6 Hz, 1H), 8.58 (dd, J ¼ 7.8, 1.2 Hz, 1H), 7.69 (dd, J ¼ 7.8, 4.6 Hz,
1H), 7.53 (dd, J ¼ 9.6, 1.5 Hz, 1H), 3.50e3.45 (m, 2H), 1.76 (sext,
kDNA only. Lane 2, kDNA and enzyme. Lane 3e6, kDNA, enzyme and compound at
various concentrations. Key: R, relaxed DNA; SC, supercoiled DNA; C, catenated kDNA;
M, monomer circle DNA.

4630
X.-W. Wu et al. / European Journal of Medicinal Chemistry 46 (2011) 4625e4633
J ¼ 7.4 Hz, 2H),1.09 (t, J ¼ 7.4 Hz, 3H). 13C NMR (CDCl3)
d
183.0,172.5,
4.4.6. N-(2-(Dimethylamino)ethyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (11)
162.2, 155.0, 149.5, 139.2, 135.7, 131.7, 130.2, 127.7, 127.0, 124.3, 124.1,
121.9, 114.8, 111.3, 41.4, 22.7, 11.7. UV/vis lmax (CHCl3, ε): 259
(26,100), 334 (8500), 346 (9100), 367 (5300), 503 (4400) nm. ESI-
MS m/z: 414.1 (100%), 412.1 (97%) [M þ H]þ. HRMS (ESI) m/z:
412.0289 [M þ H]þ, calcd for C19H15N3O3Br 412.0297.
The product was purified by silica gel column chromatography
with eluent CHCl3:MeOH ¼ 12:1 to give a red solid 11 (0.20 g), yield
93%, mp ¼ 234.8e236.4 ꢀC. 1H NMR (CDCl3)
d
10.25 (t, J ¼ 5.2 Hz,
1H), 10.12 (br.s, 1H), 9.07 (dd, J ¼ 4.8, 1.6 Hz, 1H), 9.03 (d, J ¼ 10.0 Hz,
1H), 8.59 (dd, J ¼ 7.6, 1.6 Hz, 1H), 7.68 (dd, J ¼ 7.6, 4.8 Hz, 1H), 7.54
(dd, J ¼ 9.6, 1.6 Hz, 1H), 3.66 (q, J ¼ 6.1 Hz, 2H), 2.69 (t, J ¼ 6.6 Hz,
4.4.2. N-Isopropyl 9-bromo-5,12-dioxo-5,12-dihydroindolizino[2,3-
g]quinoline-6-carboxamide (7)
2H), 2.40 (s, 6H). 13C NMR (CDCl3)
d 182.9, 172.6, 162.4, 155.0, 149.5,
The product was purified by silica gel column chromatography
with eluent CHCl3:AcOEt ¼ 8:1 to give a bright red solid 7 (0.19 g),
139.2, 135.7, 131.7, 130.2, 127.8, 127.0, 124.5, 124.0, 121.9, 114.7, 111.1,
58.3, 45.5, 37.7. UV/vis lmax (MeCN, ε): 259 (34,500), 332 (11,100),
364 (6,300), 491 (5,000) nm. ESI-MS m/z: 441.0 (100%), 443.0 (98%)
[M þ H]þ. HRMS (ESI) m/z: 441.0557 [M þ H]þ, calcd for
C20H18N4O3Br 441.0562.
yield 92%; mp ¼ 258.4e260.2 ꢀC. 1H NMR (CDCl3)
d 10.16e10.08 (m,
2H), 9.09e9.02 (m, 2H), 8.59 (d, J ¼ 8.0 Hz, 1H), 7.69 (dd, J ¼ 7.8,
4.6 Hz, 1H), 7.54 (dd, J ¼ 9.6, 1.6 Hz, 1H), 4.33e4.25 (m, 1H), 1.39 (d,
J ¼ 6.6 Hz, 6H). 13C NMR (CDCl3)
d 183.0, 172.5, 161.4, 155.0, 149.5,
139.3, 135.7, 131.7, 130.2, 127.7, 127.0, 124.3, 124.1, 121.9, 114.8, 111.7,
41.6, 22.7. UV/vis lmax (CHCl3, ε): 260 (16,400), 345 (5700), 504
(2800) nm. ESI-MS m/z: 412.0 (100%), 414.0 (97%) [M þ H]þ. HRMS
(ESI) m/z: 412.0310 [M þ H]þ, calcd for C19H15N3O3Br 412.0297.
4.4.7. N-(3-(Dimethylamino)propyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (12)
The product was purified by silica gel column chromatography
with eluent CHCl3:MeOH ¼ 14:1 to give a red solid 12 (0.20 g), yield
87%, mp ¼ 165.6e169.1 ꢀC. 1H NMR (CDCl3)
d
10.21 (t, J ¼ 5.2 Hz,
4.4.3. N-Butyl 9-bromo-5,12-dioxo-5,12-dihydroindolizino[2,3-g]
quinoline-6-carboxamide (8)
1H), 10.13 (dd, J ¼ 1.6, 0.8 Hz, 1H), 9.07 (dd, J ¼ 4.8, 1.8 Hz, 1H), 9.04
(dd, J ¼ 9.6, 0.8 Hz, 1H), 8.58 (dd, J ¼ 7.8, 1.8 Hz,1H), 7.68 (dd, J ¼ 8.0,
4.4 Hz, 1H), 7.54 (dd, J ¼ 9.6, 1.6 Hz, 1H), 3.58 (q, J ¼ 6.4 Hz, 2H), 2.54
(t, J ¼ 7.4 Hz, 2H), 2.36 (s, 6H), 1.96 (quint, J ¼ 7.2 Hz, 2H). 13C NMR
The product was purified by silica gel column chromatography
with eluent CHCl3:AcOEt ¼ 11:1 to give a red solid 8 (0.19 g), yield
87%, mp ¼ 264.8e267.1 ꢀC. 1H NMR (CDCl3)
d
10.16 (t, J ¼ 4.6 Hz,
(CDCl3) d 183.1, 172.6, 162.4, 155.1, 149.6, 139.3, 135.7, 131.8, 130.3,
1H), 10.07 (br.s, 1H), 9.06 (d, J ¼ 4.4 Hz, 1H), 9.01 (d, J ¼ 9.6 Hz, 1H),
8.57 (d, J ¼ 7.6 Hz, 1H), 7.68 (dd, J ¼ 7.6, 4.4 Hz, 1H), 7.52 (dd, J ¼ 9.6,
1.4 Hz,1H), 3.52e3.47 (m, 2H),1.72 (quint, J ¼ 7.3 Hz, 2H),1.52 (sext,
127.8, 127.1, 124.4, 124.1, 122.0, 114.8, 111.2, 57.2, 45.3, 37.6, 27.3. UV/
vis lmax (MeCN, ε): 259 (50,000), 339 (15,500), 489 (6600) nm. ESI-
MS m/z: 457.1 (100%), 455.1 (98%) [M þ H]þ. HRMS (ESI) m/z:
455.0698 [M þ H]þ, calcd for C21H20N4O3Br 455.0719.
J ¼ 7.4 Hz, 2H),1.02 (t, J ¼ 7.4 Hz, 3H). 13C NMR (CDCl3)
d 182.9,172.5,
162.1, 155.0, 149.4, 139.2, 135.7, 131.7, 130.2, 127.7, 127.0, 124.2, 124.1,
121.9, 114.8, 111.3, 39.4, 31.5, 20.4, 13.9. UV/vis lmax (CHCl3, ε): 259
(17,500), 346 (6000), 366 (3500), 504 (2900) nm. ESI-MS m/z: 426.0
(100%), 428.0 (98%) [M þ H]þ. HRMS (ESI) m/z: 426.0473 [M þ H]þ,
calcd for C20H17N3O3Br 426.0453.
4.4.8. N-(2-(Diethylamino)ethyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (13)
The product was purified by silica gel column chromatography
with eluent CHCl3:MeOH ¼ 15:1 to give a red solid 13 (0.21 g), yield
91%, mp ¼ 200.0e202.6 ꢀC. 1H NMR (CDCl3)
d
10.28 (t, J ¼ 5.2 Hz,
4.4.4. N,N-Diethyl 9-bromo-5,12-dioxo-5,12-dihydroindolizino[2,3-
g]quinoline-6-carboxamide (9)
1H), 10.10 (s, 1H), 9.07 (d, J ¼ 4.0 Hz, 1H), 9.00 (d, J ¼ 10.0 Hz, 1H),
8.57 (d, J ¼ 8.0 Hz, 1H), 7.69 (dd, J ¼ 7.6, 4.8 Hz, 1H), 7.54 (d,
J ¼ 9.6 Hz, 1H), 3.70 (q, J ¼ 6.3 Hz, 2H), 2.91 (t, J ¼ 6.8 Hz, 2H), 2.79
The product was purified by silica gel column chromatography
with eluent CH2Cl2:AcOEt:MeOH ¼ 60:3:1 to give a red solid 9
(q, J ¼ 7.1 Hz, 4H), 1.18 (t, J ¼ 7.2 Hz, 6H). 13C NMR (CDCl3)
d 182.6,
(0.20 g), yield 93%, mp ¼ 281.9e284.3 ꢀC. 1H NMR (CDCl3)
d
9.92
172.6,162.5, 155.0, 149.5,139.2,135.7,131.8,130.2,127.8,127.1,124.5,
124.0, 121.9, 114.7, 110.9, 51.5, 47.1, 37.2, 11.2. UV/vis lmax (MeCN, ε):
259 (43,200), 332 (13,700), 364 (7,700), 494 (6,300) nm. ESI-MS m/
z: 469.1 (100%), 471.1 (88%) [M þ H]þ. HRMS (ESI) m/z: 469.0881
[M þ H]þ, calcd for C22H22N4O3Br 469.0875.
(dd, J ¼ 1.6, 0.8 Hz, 1H), 9.04 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.51 (dd,
J ¼ 7.8, 1.6 Hz, 1H), 7.64 (dd, J ¼ 7.8, 4.8 Hz, 1H), 7.59 (dd, J ¼ 9.2,
0.8 Hz, 1H), 7.41 (dd, J ¼ 9.2, 1.6 Hz, 1H), 3.95e3.86 (m, 1H),
3.59e3.51 (m, 1H), 3.40e3.21 (m, 2H), 1.42 (t, J ¼ 7.0 Hz, 3H), 1.02
(t, J ¼ 7.2 Hz, 3H). 13C NMR (CDCl3)
d 180.1, 172.3, 163.2, 154.4,
150.5, 135.0, 134.9, 130.3, 130.2, 128.2, 126.8, 125.1, 121.0, 120.1,
113.4, 113.0, 43.2, 39.6, 14.5, 12.8. UV/vis lmax (MeCN, ε): 257
(32,900), 334 (9500), 479 (6400) nm. ESI-MS m/z: 426.1 (100%),
428.0 (92%) [M þ H]þ. HRMS (ESI) m/z: 426.0473 [M þ H]þ, calcd
for C20H17N3O3Br 426.0453.
4.4.9. N-(3-(Diethylamino)propyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (14)
The product was purified by silica gel column chromatography
with eluent CHCl3:MeOH ¼ 15:1 to give a red solid 14 (0.23 g), yield
94%, mp ¼ 243.3e244.9 ꢀC. 1H NMR (CDCl3)
d
10.24 (t, J ¼ 5.5 Hz,
1H), 10.05 (d, J ¼ 1.2 Hz, 1H), 9.02 (dd, J ¼ 4.6, 1.8 Hz, 1H), 8.88 (d,
J ¼ 10.0 Hz,1H), 8.50 (dd, J ¼ 8.0, 2.0 Hz,1H), 7.64 (dd, J ¼ 8.0, 4.8 Hz,
1H), 7.48 (dd, J ¼ 10.0, 1.8 Hz, 1H), 3.56 (q, J ¼ 6.0 Hz, 2H), 3.10e2.96
(m, 6H), 2.23e2.12 (m, 2H), 1.31 (t, J ¼ 7.2 Hz, 6H). 13C NMR (CDCl3)
4.4.5. N-Benzyl 9-bromo-5,12-dioxo-5,12-dihydroindolizino[2,3-g]
quinoline-6-carboxamide (10)
The product was purified by silica gel column chromatography
with eluent CHCl3:AcOEt ¼ 8:1 to give an aubergine solid 10
d
182.2, 171.7, 161.8, 154.2, 148.4, 138.2, 134.8, 131.0, 129.2, 126.8,
(0.19 g), yield 81%, mp ¼ 264.6e266.2 ꢀC. 1H NMR (CDCl3)
d
10.62 (t,
126.3, 123.4, 122.7, 121.0, 113.8, 109.4, 48.5, 45.6, 36.0, 23.4, 8.1. UV/
vis lmax (MeCN, ε): 260 (30,800), 332 (10,000), 340 (9800), 489
(4400) nm. ESI-MS m/z: 485.2 (100%), 483.2 (93%) [M þ H]þ. HRMS
(ESI) m/z: 483.1051 [M þ H]þ, calcd for C23H24N4O3Br 483.1032.
J ¼ 5.2 Hz, 1H), 10.10 (br.s, 1H), 9.07e9.04 (m, 2H), 8.53 (dd, J ¼ 8.0,
1.6 Hz, 1H), 7.65 (dd, J ¼ 8.0, 4.6 Hz, 1H), 7.54 (dd, J ¼ 9.8, 1.6 Hz, 1H),
7.47e7.45 (m, 2H), 7.40e7.36 (m, 2H), 7.32e7.27 (m, 1H), 4.72 (d,
J ¼ 5.6 Hz, 2H). 13C NMR (CDCl3)
d 183.0, 172.6, 162.3, 155.1, 149.4,
139.4, 138.5, 135.8, 131.9, 130.2, 128.7, 127.8, 127.7, 127.3, 127.1, 124.4,
124.1, 122.0, 114.8, 110.9, 43.6. UV/vis lmax (CHCl3, ε): 260 (52,500),
334 (16,600), 344 (17,800), 366 (10,400), 501 (8400) nm. ESI-MS m/
z: 460.1 (100%), 462.1 (96%) [M þ H]þ. HRMS (ESI) m/z: 460.0297
[M þ H]þ, calcd for C23H15N3O3Br 460.0297.
4.4.10. N-(2-Morpholinoethyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (15)
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH:Et3N ¼ 30:1:0.04 to give a red
solid 15 (0.20 g) as, yield 81%, mp ¼ 227.5e228.4 ꢀC. 1H NMR

X.-W. Wu et al. / European Journal of Medicinal Chemistry 46 (2011) 4625e4633
4631
(CDCl3)
d
10.32 (t, J ¼ 5.2 Hz, 1H), 10.14 (d, J ¼ 1.6 Hz 1H), 9.08 (dd,
7.55 (dd, J ¼ 9.8, 1.8 Hz, 1H), 3.68 (q, J ¼ 6.2 Hz, 2H), 2.69 (t,
J ¼ 6.6 Hz, 2H), 2.56 (br.s, 4H), 1.70e1.65 (m, 4H), 1.53e1.45 (m, 2H).
J ¼ 4.8, 1.6 Hz, 1H), 9.04 (d, J ¼ 9.6 Hz, 1H), 8.58 (dd, J ¼ 8.0, 1.6 Hz,
1H), 7.70 (dd, J ¼ 8.0, 4.4 Hz,1H), 7.55 (dd, J ¼ 9.6,1.6 Hz,1H), 3.82 (t,
J ¼ 4.4 Hz, 4H), 3.67 (q, J ¼ 6.0 Hz, 2H), 2.71 (t, J ¼ 6.2 Hz, 2H), 2.62
13C NMR (CDCl3)
d 182.7, 172.6, 162.3, 155.0, 149.5, 139.3, 135.6,
131.7, 130.3, 127.8, 127.1, 124.5, 124.1, 121.9, 114.7, 111.2, 57.7, 54.6,
37.1, 26.0, 24.5. UV/vis lmax (MeCN, ε): 259 (30,900), 333 (9900),
491 (4400) nm. ESI-MS m/z: 483.1 (100%), 481.1 (73%) [M þ H]þ.
HRMS (ESI) m/z: 481.0877 [M þ H]þ, calcd for C23H22N4O3Br
481.0875.
(s, br. 4H). 13C NMR (CDCl3)
d 182.7, 172.6, 162.3, 155.0, 149.5, 139.2,
135.6, 131.7, 130.2, 127.8, 127.1, 124.4, 124.0, 121.9, 114.7, 111.0, 67.0,
57.2, 53.5, 36.6. UV/vis lmax (MeCN, ε): 259 (34,400), 331 (11,000),
365 (6200), 492 (4900) nm. ESI-MS m/z: 483.1 (90%), 485.1 (100%)
[M þ H]þ. HRMS (ESI) m/z: 483.0647 [M þ H]þ, calcd for
C22H20N4O4Br 483.0668.
4.4.15. N-(3-(Piperidin-1-yl)propyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (20)
4.4.11. N-(3-Morpholinopropyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (16)
The crude product was purified by silica gel column chroma-
tography with eluent CHCl3:MeOH ¼ 20:1 to give a red solid 20
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH ¼ 25:1 to give a bright red solid
(0.21 g), yield 85%, mp ¼ 213.5e214.5 ꢀC. 1H NMR (CDCl3)
d 10.20 (t,
J ¼ 5.2 Hz, 1H), 10.08 (dd, J ¼ 1.8, 0.6 Hz, 1H), 9.06 (dd, J ¼ 4.8, 1.6 Hz,
1H), 9.01 (dd, J ¼ 9.8, 0.6 Hz, 1H), 8.56 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.69
(dd, J ¼ 7.8, 4.6 Hz, 1H), 7.53 (dd, J ¼ 9.8, 1.8 Hz, 1H), 3.54 (q,
J ¼ 6.4 Hz, 2H), 2.56e2.40 (m, 6H), 1.95 (quint, J ¼ 7.3 Hz, 2H),
16 (0.23 g), yield 92%, mp ¼ 230.1e232.8 ꢀC. 1H NMR (CDCl3)
d 10.22
(t, J ¼ 5.2 Hz, 1H), 10.11 (dd, J ¼ 1.8, 0.8 Hz, 1H), 9.07 (dd, J ¼ 4.8,
1.6 Hz,1H), 9.04 (dd, J ¼ 9.6, 0.8 Hz,1H), 8.56 (dd, J ¼ 8.0,1.6 Hz,1H),
7.69 (dd, J ¼ 8.0, 4.4 Hz, 1H), 7.54 (dd, J ¼ 9.6, 2.0 Hz, 1H), 3.74 (t,
J ¼ 4.6 Hz, 4H), 3.60e3.55 (m, 2H), 2.58e2.46 (m, 6H), 1.93 (quint,
1.63e1.60 (m, 4H), 1.50e1.40 (m, 2H). 13C NMR (CDCl3)
d 182.9,
172.5, 162.2, 155.0, 149.4, 139.2, 135.7, 131.7, 130.2, 127.7, 127.1, 124.3,
124.0, 121.9, 114.8, 111.2, 56.8, 54.6, 37.9, 26.7, 25.9, 24.4. UV/vis lmax
(MeCN, ε): 260 (44,900), 337 (12,700), 360 (7400), 489 (5300) nm.
ESI-MS m/z: 495.1 (100%), 497.1 (87%) [M þ H]þ. HRMS (ESI) m/z:
495.1018 [M þ H]þ, calcd for C24H24N4O3Br 495.1032.
J ¼ 7.1 Hz, 2H). 13C NMR (CDCl3)
d 183.1, 172.6, 162.3, 155.1, 149.5,
139.3, 135.6, 131.8, 130.2, 127.8, 127.1, 124.3, 124.1, 122.0, 114.8, 111.2,
67.1, 56.4, 53.8, 37.7, 26.4. UV/vis lmax (MeCN, ε): 258 (39,400), 332
(12,600), 365 (7100), 496 (5500) nm. ESI-MS m/z: 499.1 (100%),
497.1 (92%) [M þ H]þ. HRMS (ESI) m/z: 497.0827 [M þ H]þ, calcd for
C23H22N4O4Br 497.0824.
4.4.16. 9-Bromo-6-(pyrrolidine-1-carbonyl)indolizino[2,3-g]
quinoline-5,12-dione (21)
4.4.12. N-(2-(Pyrrolidin-1-yl)ethyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (17)
The crude product was purified by silica gel column chroma-
tography with eluent CHCl3:MeOH ¼ 15:1 to give a red solid 21
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH:Et3N ¼ 25:1:0.25 to give a red
solid 17 (0.20 g), yield 85%, mp ¼ 225.1e226.3 ꢀC. 1H NMR (CDCl3)
(0.20 g), yield 96%, mp > 300 ꢀC. 1H NMR (CDCl3)
d
9.95 (dd, J ¼ 1.6,
0.8 Hz,1H), 9.06 (dd, J ¼ 4.6,1.8 Hz,1H), 8.51 (dd, J ¼ 8.0, 2.0 Hz,1H),
7.73 (dd, J ¼ 9.6, 0.8 Hz, 1H), 7.65 (dd, J ¼ 8.0, 4.8 Hz, 1H), 7.43 (dd,
J ¼ 9.4, 1.8 Hz, 1H), 3.93e3.82 (m, 1H), 3.81e3.71 (m, 1H), 3.50e3.37
(m, 1H), 3.23e3.14 (m, 1H), 2.12e1.80 (m, 4H). 13C NMR (CDCl3)
d
10.31 (t, J ¼ 4.6 Hz,1H), 10.16 (d, J ¼ 1.8 Hz, 1H), 9.09e9.04 (m, 2H),
8.60 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.69 (dd, J ¼ 8.0, 4.4 Hz, 1H), 7.55 (dd,
J ¼ 9.8, 1.8 Hz, 1H), 3.72 (q, J ¼ 6.4 Hz, 2H), 2.88 (t, J ¼ 6.2 Hz, 2H),
d
180.3, 172.3, 162.1, 154.4, 150.3, 135.4, 135.1, 130.31, 130.29, 128.1,
2.72 (br.s, 4H), 1.87 (br.s, 4H). 13C NMR (CDCl3)
d
182.8, 172.6, 162.4,
126.9, 125.0, 121.1, 120.7, 113.5, 113.4, 47.5, 46.1, 25.9, 24.5. UV/vis
lmax (MeCN, ε): 256 (32,200), 334 (9800), 479 (6100) nm. ESI-MS m/
z: 426.1 (100%), 424.1 (92%) [M þ H]þ. HRMS (ESI) m/z: 424.0287
[M þ H]þ, calcd for C20H15N3O3Br 424.0297.
155.0, 149.5, 139.2, 135.7, 131.7, 130.2, 127.7, 127.0, 124.5,124.0, 121.9,
114.7, 111.1, 55.0, 54.2, 38.8, 23.6. UV/vis lmax (MeCN, ε): 259
(36,400), 331 (11,500), 492 (5100) nm. ESI-MS m/z: 469.1 (100%),
467.2 (68%) [M þ H]þ. HRMS (ESI) m/z: 467.0708 [M þ H]þ, calcd for
C22H20N4O3Br 467.0719.
4.4.17. 9-Bromo-6-(4-methylpiperazine-1-carbonyl)indolizino[2,3-
g]quinoline-5,12-dione (22)
4.4.13. N-(3-(Pyrrolidin-1-yl)propyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (18)
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH:Et3N ¼ 12:1:0.25 to give a red
solid 18 (0.22 g), yield 90%, mp ¼ 195.7e196.9 ꢀC. 1H NMR (CDCl3)
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH:Et3N ¼ 25:1:0.05 to give a red
solid 22 (0.18 g) as, yield 81%, mp ¼ 248.3e250.1 ꢀC. 1H NMR
(CDCl3)
d
9.93 (dd, J ¼ 1.6, 0.8 Hz, 1H), 9.05 (dd, J ¼ 4.8, 1.6 Hz, 1H),
8.52 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.70e7.62 (m, 2H), 7.44 (dd, J ¼ 9.6,
1.6 Hz, 1H), 4.13e4.04 (m, 1H), 3.95e3.84 (m, 1H), 3.52e3.42 (m,
1H), 3.41e3.34 (m, 1H), 2.79e2.71 (m, 1H), 2.53e2.42 (m, 2H), 2.35
d
10.26 (t, J ¼ 5.2 Hz, 1H), 10.13 (dd, J ¼ 1.8, 0.8 Hz, 1H), 9.08 (dd,
J ¼ 4.8, 1.6 Hz, 1H), 9.05 (dd, J ¼ 9.8, 0.8 Hz, 1H), 8.59 (dd, J ¼ 8.0,
1.6 Hz, 1H), 7.70 (dd, J ¼ 8.0, 4.8 Hz, 1H), 7.55 (dd, J ¼ 9.8, 1.8 Hz, 1H),
3.59 (q, J ¼ 6.2 Hz, 2H), 2.68 (t, J ¼ 7.6 Hz, 2H), 2.60 (br.s, 4H), 1.98
(s, 3H), 2.22e2.12 (m, 1H). 13C NMR (CDCl3)
d 180.0, 172.4, 162.3,
154.5, 150.3, 135.4, 135.1, 130.5, 130.3, 128.2, 126.9, 125.2, 121.1,
120.3, 113.5, 111.5, 55.2, 54.6, 46.8, 46.0, 42.1. UV/vis lmax (MeCN, ε):
256 (32,700), 330 (10,200), 477 (5700) nm. ESI-MS m/z: 455.1
(100%), 453.1 (90%) [M þ H]þ. HRMS (ESI) m/z: 453.0582 [M þ H]þ,
calcd for C21H18N4O3Br 453.0562.
(quint, J ¼ 7.2 Hz, 2H), 1.88e1.75 (m, 4H). 13C NMR (CDCl3)
d 183.0,
172.6, 162.3, 155.0, 149.6, 139.3, 135.7, 131.7, 130.3, 127.8, 127.1, 124.4,
124.1, 122.0, 114.8, 111.3, 54.3, 54.1, 37.9, 28.8, 23.5. UV/vis lmax
(MeCN, ε): 259 (40,600), 340 (12,600), 361 (7400), 491 (5400) nm.
ESI-MS m/z: 481.1 (100%), 483.1 (98%) [M þ H]þ. HRMS (ESI) m/z:
481.0872 [M þ H]þ, calcd for C23H22N4O3Br 481.0875.
4.4.18. 9-Bromo-6-(morpholine-4-carbonyl)indolizino[2,3-g]
quinoline-5,12-dione (23)
4.4.14. N-(2-(Piperidin-1-yl)ethyl)-9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxamide (19)
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH:Et3N ¼ 30:1:0.3 to give a red
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH:Et3N ¼ 20:1:0.05 to give a red
solid 19 (0.23 g), yield 96%, mp ¼ 237.8e239.6 ꢀC. 1H NMR (CDCl3)
solid 23 (0.19 g), yield 86%, mp > 300 ꢀC. 1H NMR (CDCl3)
d 9.95 (dd,
J ¼ 1.6, 0.8 Hz, 1H), 9.06 (dd, J ¼ 4.6, 1.8 Hz, 1H), 8.52 (dd, J ¼ 7.8,
1.8 Hz, 1H), 7.73e7.64 (m, 2H), 7.45 (dd, J ¼ 9.2, 1.6 Hz, 1H),
4.06e3.89 (m, 3H), 3.84e3.72 (m, 2H), 3.54e3.45 (m, 2H),
d
10.28 (t, J ¼ 4.6 Hz, 1H), 10.15 (dd, J ¼ 1.8, 0.8 Hz, 1H), 9.09e9.05
(m, 2H), 8.59 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.70 (dd, J ¼ 8.0, 4.4 Hz, 1H),
3.38e3.26 (m, 1H). 13C NMR (CDCl3)
d 180.1, 172.3, 162.5, 154.5,

4632
X.-W. Wu et al. / European Journal of Medicinal Chemistry 46 (2011) 4625e4633
150.2, 135.6,135.1,130.6,130.2,128.2,127.0,125.2,121.1,120.4,113.6,
110.9, 66.8, 47.3, 42.6. UV/vis lmax (MeCN, ε): 257 (32 500), 334
(9600), 478 (5900) nm. ESI-MS m/z: 440.0 (100%), 442.0 (99%)
[M þ H]þ. HRMS (ESI) m/z: 462.0057 [M þ Na]þ, calcd for
C20H14N3O4BrNa 462.0065.
4.4.23. 2-Fluoroethyl 9-bromo-5,12-dioxo-5,12-dihydroindolizino
[2,3-g]quinoline-6-carboxylate (28)
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:AcOEt:MeOH ¼ 60:3:1 to give an
orange solid 28 (0.12 g), yield 61%, mp ¼ 265.4e267.3 ꢀC. 1H NMR
(CDCl3)
d
10.11 (dd, J ¼ 1.6, 0.8 Hz, 1H), 9.05 (dd, J ¼ 4.4, 1.6 Hz, 1H),
4.4.19. 9-Bromo-6-(thiomorpholine-4-carbonyl)indolizino[2,3-g]
quinoline-5,12-dione (24)
The crude product was purified by silica gel column chroma-
tography with eluent CHCl3:MeOH:Et3N ¼ 130:7:0.2 to give an
orange solid 24 (0.22 g), yield 96%, mp > 300 ꢀC. 1H NMR (CDCl3)
8.57 (dd, J ¼ 8.0, 1.6 Hz, 1H), 8.28 (dd, J ¼ 9.6, 0.8 Hz, 1H), 7.68 (dd,
J ¼ 8.0, 4.8 Hz, 1H), 7.58 (dd, J ¼ 9.6, 1.6 Hz, 1H), 4.92e4.88 (m, 1H),
4.80e4.77 (m, 1H), 4.75e4.72 (m, 1H), 4.68e4.65 (m, 1H). 13C NMR
(CDCl3)
d 178.7, 173.4, 162.2, 154.3, 149.2, 138.3, 135.6, 132.3, 131.0,
128.4, 128.1, 127.2, 123.1, 121.6, 113.6, 106.0, 81.3 (d, JCeF ¼ 169.5 Hz),
64.0 (d, JC-F ¼ 19.8 Hz). UV/vis lmax (MeCN, ε): 260 (43,300), 326
(11,300), 358 (6100), 462 (6200) nm. ESI-MS m/z: 417.0 (100%),
419.0 (96%) [M þ H]þ. HRMS (ESI) m/z: 416.9900 [M þ H]þ, calcd for
C18H11N2O4FBr 416.9886.
d
9.94 (dd, J ¼ 1.6, 0.8 Hz, 1H), 9.06 (dd, J ¼ 4.6, 1.8 Hz, 1H), 8.52 (dd,
J ¼ 7.8, 1.8 Hz, 1H), 7.67 (dd, J ¼ 7.8, 4.6 Hz, 1H), 7.64 (dd, J ¼ 9.2,
0.8 Hz, 1H), 7.44 (dd, J ¼ 9.2, 1.6 Hz, 1H), 4.22 (t, J ¼ 4.9 Hz, 2H),
3.70e3.65 (m, 2H), 2.96e2.90 (m, 1H), 2.84e2.75 (m, 1H),
2.69e2.61 (m, 1H), 2.45e2.36 (m, 1H). 13C NMR (CDCl3)
d 180.1,
172.3, 162.8, 154.5, 150.3, 135.2, 135.1, 130.6, 130.3, 128.6, 127.0,
125.1, 121.2, 120.1, 113.6, 111.3, 49.7, 44.7, 28.1, 27.5. UV/vis lmax
(MeCN, ε): 257 (33,600), 334 (9900), 481 (6100) nm. ESI-MS m/z:
456.1 (100%), 458.1 (92%) [M þ H]þ. HRMS (ESI) m/z: 477.9851
[M þ Na]þ, calcd for C20H14N3O3SBrNa 477.9837.
4.4.24. 2-(Dimethylamino)ethyl 9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (29)
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH ¼ 20:1 to give an orange solid
29 (0.16 g), yield 73%, mp ¼ 182.2e184.7 ꢀC. 1H NMR (CDCl3)
d 10.09
(br.s, 1H), 9.05 (dd, J ¼ 4.4, 1.6 Hz, 1H), 8.56 (dd, J ¼ 7.6, 1.4 Hz, 1H),
8.36 (d, J ¼ 9.6 Hz, 1H), 7.67 (dd, J ¼ 7.8, 4.6 Hz, 1H), 7.55 (dd, J ¼ 9.6,
1.6 Hz,1H), 4.56 (t, J ¼ 5.8 Hz, 2H), 2.83 (t, J ¼ 5.8 Hz, 2H), 2.38 (s, 6H).
4.4.20. 6-(4-Acetylpiperazine-1-carbonyl)-9-bromoindolizino[2,3-
g]quinoline-5,12-dione (25)
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH ¼ 30:1 to give a isomers
mixture 25 (0.21 g, red solid), yield 89%, mp > 300 ꢀC. 1H NMR
13C NMR (CDCl3)
d 178.8,173.3,162.5,154.3,149.2,138.2,135.5,131.9,
131.0,128.3,127.9,127.2,122.8,121.8,113.5,107.0, 62.8, 57.7, 45.7. UV/
vis lmax (MeCN, ε): 259 (54,400), 326 (11,500), 358 (6300), 463
(6100) nm. ESI-MS m/z: 442.0 (100%), 444.0 (98%) [M þ H]þ. HRMS
(ESI) m/z: 442.0404 [M þ H]þ, calcd for C20H17N3O4Br 442.0402.
(CDCl3)
d
9.95 (dd, J ¼ 1.2, 0.8 Hz, 1H), 9.07 (dd, J ¼ 4.8, 1.6 Hz, 1H),
8.50 (d, J ¼ 7.2 Hz, 1H), 7.67 (m, 2H), 7.46 (dd, J ¼ 9.2, 1.2 Hz, 1H),
4.07e3.86 (m, 3H), 3.80e3.70 (m, 2H), 3.65e3.58 (m, 1H),
3.49e3.43 (m, 1H), 3.36e3.29 (m, 1H), 1.65 (s, 3H). 13C NMR (CDCl3)
4.4.25. 2-(Piperidin-1-yl)ethyl 9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (30)
d
180.2, 172.34, 172.31, 169.3, 169.1, 163.0, 162.6, 154.6, 150.2, 135.8,
135.6, 130.7, 130.2, 128.2, 128.1, 127.0, 125.2, 125.1, 121.2, 121.1, 120.4,
120.3, 113.8, 113.7, 110.8, 110.6, 46.9, 46.6, 46.3, 45.9, 42.3, 42.1, 41.5,
41.1, 21.4. UV/vis lmax (MeCN, ε): 257 (35 200), 334 (10 500), 478
(6400) nm. ESI-MS m/z: 481.1 (100%), 483.1 (86%) [M þ H]þ. HRMS
(ESI) m/z: 503.0317 [M þ Na]þ, calcd for C22H17N4O4BrNa 503.0331.
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH ¼ 25:1 to give an orange solid
30 (0.18 g), yield 76%, mp ¼ 188.1e189.2 ꢀC. 1H NMR (CDCl3)
d 10.10
(dd, J ¼ 2.0, 0.8 Hz,1H), 9.04 (dd, J ¼ 4.8,1.6 Hz,1H), 8.57 (dd, J ¼ 8.0,
1.6 Hz,1H), 8.52 (dd, J ¼ 9.6, 0.8 Hz,1H), 7.67 (dd, J ¼ 7.8, 4.6 Hz,1H),
7.54 (dd, J ¼ 9.6, 2.0 Hz, 1H), 4.57 (t, J ¼ 5.8 Hz, 2H), 2.82 (t,
J ¼ 5.8 Hz, 2H), 2.54 (br.s, 4H), 1.68e1.58 (m, 4H), 1.52e1.44 (m, 2H).
4.4.21. Methyl 9-bromo-5,12-dioxo-5,12-dihydroindolizino[2,3-g]
quinoline-6-carboxylate (26)
13C NMR (CDCl3)
d 178.8, 173.2, 162.2, 154.2, 149.2, 138.2, 135.5,
The crude product was purified by silica gel column chroma-
tography with eluent CHCl3:AcOEt ¼ 5:1 to give an orange solid 26
131.8, 130.9, 128.2, 127.9, 127.2, 122.9, 122.2, 113.5, 107.2, 62.2, 57.1,
54.7, 26.0, 24.2. UV/vis lmax (MeCN, ε): 260 (44,700), 327 (12,700),
358 (7400), 463 (7400) nm. ESI-MS m/z: 482.1 (100%), 484.1 (96%)
[M þ H]þ. HRMS (ESI) m/z: 482.0692 [M þ H]þ, calcd for
C23H21N3O4Br 482.0715.
(0.12 g), yield 53%, mp ¼ 285.7e286.9 ꢀC. 1H NMR (CDCl3)
d 10.08
(br.s, 1H), 9.04 (dd, J ¼ 4.4, 1.6 Hz, 1H), 8.55 (dd, J ¼ 8.0, 1.6 Hz, 1H),
8.26 (dd, J ¼ 9.6, 0.8 Hz, 1H), 7.68 (dd, J ¼ 8.0, 4.4 Hz, 1H), 7.55 (dd,
J ¼ 9.6,1.6 Hz,1H), 4.04 (s, 3H). 13C NMR (CDCl3)
d 178.8, 173.3,163.1,
154.3, 149.2, 138.2, 135.5, 132.0, 130.9, 128.3, 127.8, 127.2, 122.9,
121.6, 113.6, 106.5, 52.3. UV/vis lmax (MeCN, ε): 259 (34,200), 326
(9200), 459 (4900) nm. ESI-MS m/z: 384.9 (100%), 386.9 (96%)
[M þ H]þ. HRMS (ESI) m/z: 384.9812 [M þ H]þ, calcd for
C17H10N2O4Br 384.9824.
4.4.26. 2-Morpholinoethyl 9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (31)
The crude product was purified by silica gel column chroma-
tography with eluent CH2Cl2:MeOH ¼ 25:1 to give an orange solid
31 (0.17 g), yield 69%, mp ¼ 138.4e139.7 ꢀC. 1H NMR (CDCl3)
d 10.10
(dd, J ¼ 1.6, 0.8 Hz, 1H), 9.05 (dd, J ¼ 4.6, 1.8 Hz,1H), 8.56 (dd, J ¼ 7.8,
1.8 Hz,1H), 8.43 (dd, J ¼ 9.6, 0.8 Hz,1H), 7.68 (dd, J ¼ 7.8, 4.6 Hz,1H),
7.55 (dd, J ¼ 9.6, 1.6 Hz, 1H), 4.58 (t, J ¼ 5.8 Hz, 2H), 3.80e3.70 (m,
4H), 2.87 (t, J ¼ 5.8 Hz, 2H), 2.67e2.57 (m, 4H). 13C NMR (CDCl3)
4.4.22. 2,2,2-Trifluoroethyl 9-bromo-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (27)
The crude product was purified by silica gel column chroma-
tography with eluent CHCl3:AcOEt ¼ 5:1 to give an orange solid 27
d
178.8, 173.3, 162.3, 154.3, 149.2, 138.2, 135.5, 131.9, 130.9, 128.3,
(0.15 g), yield 67%, mp ¼ 262.1e264.9 ꢀC. 1H NMR (CDCl3)
d
10.15
127.9, 127.2, 122.9, 121.9, 113.5, 106.9, 67.0, 61.8, 56.9, 53.7. UV/vis
lmax (MeCN, ε): 260 (30,000), 326 (9500), 461 (5400) nm. ESI-MS
m/z: 486.1 (100%), 484.1 (92%) [M þ H]þ. HRMS (ESI) m/z:
484.0519 [M þ H]þ, calcd for C22H19N3O5Br 484.0508.
(br.s Hz, 1H), 9.07 (d, J ¼ 3.2 Hz, 1H), 8.60(dd, J ¼ 7.6, 0.8 Hz, 1H),
8.26 (dd, J ¼ 9.6, 0.4 Hz, 1H), 7.71 (dd, J ¼ 7.8, 4.6 Hz, 1H), 7.64 (dd,
J ¼ 9.6, 1.6 Hz, 1H), 4.84 (q, J ¼ 8.4 Hz, 2H). 13C NMR (CDCl3)
d 178.5,
173.5, 160.7, 154.4, 149.0, 138.5, 135.7, 132.9, 130.9, 128.6, 128.5,
127.4, 123.4, 121.3, 113.7, 104.1, 60.8 (q, JCeF ¼ 36.6 Hz), 29.7. UV/vis
lmax (MeCN, ε): 261 (40,400), 325 (10,900), 357 (6300), 453 (5500)
nm. ESI-MS m/z: 453.0 (100%), 455.0 (94%) [M þ H]þ. HRMS (ESI) m/
z: 452.9708 [M þ H]þ, calcd for C18H9N2O4F3Br 452.9698.
4.5. In vitro antimicrobial activity evaluation by MIC assay
The MIC values were determined using a broth dilution method
[11]. The starting concentrations of tested compounds were 64 mg/

X.-W. Wu et al. / European Journal of Medicinal Chemistry 46 (2011) 4625e4633
4633
ml. The solution of compound in DMSO (15
m
l) was added to 285
m
l
reaction buffer (20 mM HEPESeKOH, pH 7.6, 50 mM potassium
glutamate, 5 mM magnesium acetate, 5 mM dithiothreitol, 1 mM
of bacterial culture (5 ꢁ 105 cells/ml) at the first well of flat-
bottomed 96-well tissue culture plates (JET BIOFILÒ, JET BIO-
CHEMICALS Intl., Inc, CANADA). The solution was then double-
diluted. Bacterial culture solution containing appropriate
ATP and 25
stopped by the addition of 30
(24:1). 5 l of the reaction solution was mixed with 1
loading buffer, and then analyzed by electrophoresis on 1% agarose
gel at 5 V/cm (for relaxation assay) or 10 V/cm (for decatenation
assay) in 1 ꢁ TAE buffer. The gel was stained with 1 ꢁ Gel Red,
visualized with a UV transilluminator and analyzed by AlphaEaseFC
software.
m
g/ml BSA) at 37 ꢀC for 30 min. The reaction was
ml of chloroform/iso-amyl alcohol
m
ml of 6 ꢁ DNA
compound (150
ml) was discarded at the last well in order to ensure
150 l volume of bacterial culture in every well. The plate was
m
incubated at 37 ꢀC overnight in electro-heating standing-temper-
ature cultivator before the measurement of the absorbance value.
The optical density values at 600 nm were measured using
a multifunction microplate reader (PowerWaveÔ XS2, BioTekÒ
Instruments Inc, USA).
Acknowledgments
4.6. DNA gyrase supercoiling assay
This work was supported by the National Natural Science
Foundation of China (No. 30801425), National S & T Major Project
(No. 2009ZX09103-042), Ph.D. Programs Foundation of Ministry of
Education of China (No. 2009171110050) and Guangdong Natural
Science Fund (No. 10151008901000022). We thank the reviewers’
helpful suggestions on the manuscript’s revision.
DNA gyrase supercoiling assay was performed with relaxed
pHOT1 DNA (TopoGEN, USA) as a substrate according to the
manufacturer’s protocol. Briefly, 1 U of DNA gyrase (TopoGEN, USA)
from E. coli or S. aureus and 0.5 mg of relaxed pHOT1 DNA in 30 ml
supercoiling assay buffer (35 mM TriseHCl, pH 7.5, 24 mM KCl,
4 mM MgCl2, 2 mM DTT, 1.8 mM Spermidine, 1 mM ATP, 6.5%
glycerol and 0.1 mg/ml BSA) were incubated with or without the
compound at 37 ꢀC for 60 min. After incubation, the reactions were
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the reaction solution was mixed with 1
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4.7. Topoisomerase IV relaxation assay and decatenation assay
Topo IV relaxation assay or decatenation assay was performed
according to a slightly modified method [13]. 1 U of Topo IV
(TopoGEN, USA) was incubated with 0.5
DNA (for relaxation assay, Takara Biotechnology Co., Ltd.) or 200 ng
of catenated kDNA (for decatenation assay, TopoGEN, USA) in 30
mg of supercoiled pBR322
ml