Identification, biological activity, and mechanism of the anti-ischemic quinolone analog

Article abstract of DOI:10.1016/j.bmc.2007.07.009

The quinolone analog SQ-4004 has been identified as a potentially excellent anti-ischemic agent, which exhibited highly potent efficacy in reducing infarct volume size in vivo rat MCAO model (32.1% at 0.01 mg/kg) and potent cardioprotective effect at myoc

Full text of DOI:10.1016/j.bmc.2007.07.009

Bioorganic & Medicinal Chemistry 15 (2007) 6517–6526
Identification, biological activity, and mechanism
of the anti-ischemic quinolone analog
Chan-Hee Park,a Jongwon Lee,b Hwi Young Jung,b Min Ji Kim,b Sun Ha Lim,b
Hyung Tae Yeo,c Eung Chil Choi,a Eun Jeong Yoon,a Kyu Won Kim,a Jong Ho Cha,a
Seok-Ho Kim,a Dong-Jo Chang,a Do-Yeon Kwon,a Funan Lia and Young-Ger Suha,*
aCollege of Pharmacy, Seoul National University, San 56-1 Shinrim-Dong, Kwanak-Gu, Seoul 151-742, Republic of Korea
bDepartment of Biochemistry, School of Medicine, Catholic University of Daegu, Daegu, Republic of Korea
cDepartment of Neurosurgery, School of Medicine, Catholic University of Daegu, Daegu, Republic of Korea
Received 6 June 2007; revised 6 July 2007; accepted 10 July 2007
Available online 26 July 2007
Abstract—The quinolone analog SQ-4004 has been identified as a potentially excellent anti-ischemic agent, which exhibited highly
potent efficacy in reducing infarct volume size in vivo rat MCAO model (32.1% at 0.01 mg/kg) and potent cardioprotective effect at
myocardial infarction in vivo model (26.6% at 0.01 mg/kg) while it exhibited highly reduced anti-bacterial activity. The mechanistic
study revealed that the anti-ischemic activity might exert via an anti-apoptotic pathway, which implies its therapeutic uses against
the ischemic cell injuries including ischemic stroke and ischemic heart disease.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Current studies on the new anti-ischemic agents include
glutamate antagonists, anti-inflammatory agents, anti-
apoptotic agents, anti-oxidant agents, and ion-channel
modulators.2–4 In particular, the recent efforts focused
on the evaluation of the currently available antibiotics
on the basis of their potential role in reducing infarct
size via an inhibition of inflammation and/or infection
accompanied by ischemic injury. The tetracycline ana-
logs,5 the representative example, showed reasonable
efficacy in in vivo animal model in reducing the infarct
size during the ischemic insult. Several research groups
also recently reported that the anti-ischemic effect by
pretreatment of antibiotics is likely due to their anti-
inflammatory or anti-apoptotic effect.6–11
Ischemic cell injury under hypoxic conditions such as
ischemic stroke and/or myocardial infarction, in many
cases, leads to fatal situation or causes a long-lasting dis-
ability.1 To the best of our knowledge, the currently ap-
proved treatment for the patients is only an
administration of the tissue-type plasminogen activator
(t-PA) as a thrombolytic therapy for the restoration of
blood flow to the ischemic region. However, the throm-
bolytic therapy has some limitation to be treated within
3 h after a stroke and is applicable to a limited number
of patients because of the risk of symptomatic intracra-
nial hemorrhage. Moreover, during the reperfusion, cells
are often endangered in a circumstance of oxidative
stress, which results in further tissue injury. In this re-
gard, antithrombolytic agents seem not effective enough
to stop or retard the on-going tissue injury at a reperfu-
sion phase. Therefore, development of the new anti-
ischemic agents with different modes of action as well
as with wider therapeutic window is in urgent need.
In the course of our search for new anti-ischemic agents,
we have also discovered that several antibiotics provide
significant cell survivals under in vitro hypoxic condi-
tion.12 In particular, among the fluoroquinolones tested,
ciprofloxacin (CPFX) exhibited in vitro cell viability and
furthermore considerably reduced infarct volume size in
in vivo rat focal cerebral ischemic animal model
(MCAO) at low concentration. During our extensive
studies on anti-ischemic agent, Christian Meisel et al.
also reported the moderate reduction of infarct volume
size by moxifloxacin pretreatment in mouse MCAO
Keywords: Anti-ischemic agents; Quinolone analog; Structure–activity
relationship; MCAO; MI; Anti-apoptotic activity.
*
Corresponding author. Tel.: +82 2 880 7875; fax: +82 2 888 0649;
e-mail: ygsuh@snu.ac.kr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.009

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C.-H. Park et al. / Bioorg. Med. Chem. 15 (2007) 6517–6526
model (about 14% reduction in infarct size after six
times intravenous administration of 100 mg/kg) and its
anti-infection mechanism for the anti-ischemic
activity.13
2. Results and discussion
2.1. Structural modification of CPFX
According to the structure–activity relationship for the
anti-bacterial activity of fluoroquinolones, N1, C2–H,
C3-Carboxylic acid, C4-carbonyl, C6-F, and C7-pipera-
zine are essential or beneficial for the anti-bacterial
activity.14,15 Thus, modification or elimination of these
groups would give us the valuable structural informa-
tion for separation from the anti-bacterial activity as
well as further improvement of anti-ischemic effect of
CPFX. The structural modification of CPFX and the
rationale for the modification are summarized in Figure 1.
The CPFX analogs prepared for the preliminary
structure–activity relationship of CPFX appeared in a
few literatures except SQ-4007 and SQ-4008 as synthetic
intermediates without biological property (SQ-4001,16
SQ-4004,17 and SQ-400518) or with anti-bacterial activ-
ity (SQ-4002,19,20 SQ-4003,21 and SQ-400622). All the
analogs were synthesized by modification (method A
or method B) of the established procedure as shown in
Scheme 1. In method A, the cis-isomer of 2 was directly
subjected to acylation right after purification by column
chromatography on NH–silica gel to afford the desired
product 3 with a minimum isomerization to the corre-
sponding trans-isomer. Cyclization of 3 under basic con-
dition and further hydrolysis gave the quinolone
intermediate 4. The analogs, SQ-4003 and SQ-4004,
On the basis of the excellent anti-ischemic activity of
CPFX at both in vitro and in vivo experiments, our
initial efforts for new anti-ischemic agents included
the systematic structural modification of CPFX in an
anticipation of minimization of the anti-bacterial activ-
ity for the separation of anti-ischemic activity from the
anti-bacterial activity of CPFX as well as enhancement
of its anti-ischemic activity. As our preliminary work
to understand structural requirements for the anti-
ischemic effect of CPFX, we manipulated the func-
tional groups of CPFX associated with the anti-bacte-
rial effect. Consequently, we were able to identify
SQ-4004, which exhibited potent cell viability with highly
reduced anti-bacterial efficacy among the CPFX ana-
logs. Moreover, SQ-4004 showed high anti-ischemic
efficacy in further in vivo animal model studies. Thus,
we herein communicate our initial success to identify
the quinolone-based anti-ischemic agent, SQ-4004,
which envisions separation of the potent neuroprotec-
tive and cardioprotective activity from the anti-bacte-
rial effect. In addition, we describe the preliminary
structure–activity relationship of CPFX and the mech-
anism of the anti-ischemic activity associated with the
anti-apoptotic activity.
O
O
F
OH
N
O
O
N
O
F
F
SQ-4001
OH
Elimination of C7-piperazine
N
N
N
HN
HN
SQ-4002
Elimination of C3-CO2H
SQ-4008
Block of C2
O
4
O
O
O
S
O
F
6
F
OH
F
3
OH
OH
N
7
N
1
N
N
N
N
HN
HN
CPFX
SQ-4003
SQ-4007
Role of piperazine-N'4
Role of C4-carbonyl
O
O
O
O
F
OH
OH
N
N
N
O
N
O
HN
HN
OH
SQ-4006
Role of piperazine-N'1
SQ-4004
Elimination of C6-F
SQ-4005
Role of C7-piperazine
and aromatic ring
Figure 1. Structure of CPFX and its structural modification.

C.-H. Park et al. / Bioorg. Med. Chem. 15 (2007) 6517–6526
6519
Method A
O
X
1
O
O
Cl
O
O
O
X1
X2
O
d, e
X
2
X
3
a
X1
X2
OR
OR
N
H
OR
c
OR
N
X3
N
H
cis;trans = ca.1:3
1 (R=Et or Me)
b
3
4
2, cis only
X1 = H or F
X2, X3 =H or Cl or F
SQ-4001 (X1=F, X2=H, R=H)
SQ-4005 (X1, X2, R=H)
f
Method B
B
O
O
O
O
X1
N
O
OR
X1
X1
N
k or k, l
or k, m
g
h, i, j
X1
OR
N
N
F
F
N
H
A
X1
F
A
A
5
8
6
7
SQ-4003 (X1=F, R=H, A=C, B=O)
SQ-4004 (X1=H, R=H, A=NH, B=O)
CPFX (X1=F, R=H, A=NH, B=O)
B
N
F
R'
n or o ~ r
or q
N
R''
HN
9
SQ-4002(R', R''=H, B=O)
SQ-4007(R'=CO2H, R"=H, B=S)
SQ-4008(R'=CO2H, R''=CH3, B=O)
Scheme 1. General synthetic routes for SQ-analogs. Reagents and conditions, Method A: (a) cyclopropylamine, CH3CN, 5–10 ꢁC, 5 h, 100%; (b)
column chromatography on NH–silica gel, 20%; (c) Et3N, dioxane, reflux, 12 h, 60–96%; (d) K2CO3, DMF, 110 ꢁC, 12 h, 70–88%; (e) 6 N HCl,
EtOH, reflux, 6 h, 78–96%; (f) piperazine or piperidine, DMSO, 130–140 ꢁC, 5 h, 40–67%. Method B: (g) piperidine or N-Boc-piperazine, pyridine,
Et3N, rt, 48 h, 95–100%; (h) diethylcarbonate, NaH, rt ! 80 ꢁC, 3 h, 60–79%; (i) triethylorthoformate, Ac2O, 110 ꢁC, 2 h; (j) cyclopropylamine,
CH2Cl2, rt, 3 h, 79–87% for two steps; (k) NaH, THF, reflux, 2 h, 90–98%; (l) 6 N HCl, EtOH, reflux, 6 h, 96%; (m) P2S10, pyridine, reflux, 2 h, 33%;
(n) NaCN, DMSO, 170–180ꢁC, 2 h, 30%; (o) CH3MgBr, CuI, THF, ꢀ78 ꢁC, 3 h, 62%; (p) NaH, PhSeCl, THF, 0 ꢁC, 10 min then H2O2, CH2Cl2, rt,
30 min, 70% for two steps; (q) concd HCl, EtOH/CH2Cl2, rt, 2 h, 77%; (r)1 N NaOH, THF/EtOH, rt, 1 day, 54%.
were synthesized by aromatic nucleophilic substitution
of the corresponding C7-fluorobenzenes with piperazine
or piperidine. The analogs SQ-4003 and SQ-4004 could
be also synthesized by method B under the much milder
reaction condition. The nucleophilic substitution took
place even at room temperature with an excellent isola-
tion yield and then facile alkoxycarbonylation of the
resulting substitution product, followed by aminometh-
ylenation, gave the intermediate 7 with good yields.
Finally, sequential cyclization, Grignard reaction or
conversion of carbonyl to thiocarbonyl, and then hydro-
lysis provided SQ-4007 and SQ-4008 with moderate
yields. The analogs, SQ-4002 and SQ-4006, were synthe-
sized by the reported procedure.20,22
Table 1. Cell viability for SQ-analogs by MTS assay in SH-SY5Y cell
line
Analogs
Cell viability (%) (mean SD)
Positive control
CPFX
43.3 5.4
46.5 5.3
45.1 7.4
57.4 4.3*
44.1 7.9
61.7 4.6**
39.0 1.2
42.4 0.9
27.1 2.2
49.9 3.9
SQ-4001
SQ-4002
SQ-4003
SQ-4004
SQ-4005
SQ-4006
SQ-4007
SQ-4008
* P < 0.1.
** P < 0.05 as compared to the control.
2.2. Evaluation of the pharmacophoric parts of CPFX
(in vitro cell viability and anti-bacterial effect)
cell viability, respectively, compared to the control.
However, SQ-4007 seems to show cell cytotoxicity even
at low concentration (Table 1). The anti-bacterial activ-
ity for both SQ-4002 and SQ-4004, which exhibited
excellent cell viability, was also tested against four rep-
resentative gram-positive and three gram-negative
strains. As anticipated, SQ-4002 and SQ-4004 appar-
ently exhibited the highly reduced anti-bacterial activi-
ties and especially, SQ-4004 exhibited 8- to 32-fold
Anti-ischemic effects of CPFX-analogs were assessed
preliminarly at 0.1 lM concentration in vitro in SH-
SY5Y, neuroblastoma cell line. Ischemic insult was in-
duced by 200 lM of H2O2 and the level of cell viability
was determined using MTS assay and was expressed as
a percentage compared to that of the control. As shown
in Table 1, among the tested CPFX-analogs, SQ-4002
and SQ-4004 exhibited 33% and 42% enhancement of

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C.-H. Park et al. / Bioorg. Med. Chem. 15 (2007) 6517–6526
Table 2. Anti-bacterial activity of SQ-4002, SQ-4004, and CPFX against Gram-positive and Gram-negative strains
Analogs
MIC (lg/mL)
S. aureus
S. epidermidis
E. faecalis
B. subtilis
E. coli
E. clocaes
S. marcesens
CPFX
SQ-4002
SQ-4004
2
16
32
1
8
2
8
1
8
8
0.031
<0.12
0.5
0.063
0.5
1
0.125
1
4
16
64
reduced anti-bacterial activities compared to CPFX
against all the tested strains (Table 2). Obviously, elim-
ination of carboxyl and fluoro groups of CPFX, which
are essential or beneficial for the anti-bacterial activity,
respectively, seems to enhance the anti-ischemic activ-
ity. On the other hand, modification of C7-piperazine
group (SQ-4001, 4003, 4005, and 4006), which is bene-
ficial for the anti-bacterial activity of CPFX, slightly re-
duced the anti-ischemic activity. Thiocarbonyl
substitution of C4-carbonyl group of the enone moiety,
which is essential for the anti-bacterial effect signifi-
cantly dropped the anti-ischemic activity. However,
methyl introduction at C2 slightly increased the anti-
ischemic activity.
2.3. In vivo MCAO model
To assess the anti-ischemic activity of SQ-4004 in vivo,
SQ-4004 was evaluated in rat MCAO model under both
pre- and post-ischemic conditions (ischemia/reperfu-
sion = 2 h/22 h) and CPFX was used as a control drug.
Both compounds were tested by intraperitoneal admin-
istration three times (pre-ischemic condition; 3 h, 1 h
prior, and 1 h after occlusion of MCA) at the doses of
0.001, 0.01 and 0.1 mg/kg, respectively. As a result, the
significant reduction of the infarct size of brain was ob-
served as shown in Table 3 and Figure 2. The infarct
volumes for the group of rats treated with SQ-4004 were
54.6 5.0 mm3
(0.001 mg/kg,
N = 4;
P < 0.05),
Table 3. Anti-ischemic effect of CPFX and SQ-4004 in the rat MCAO model
Condition
Drugs
Dose (mg/kg, ip)
Infarct volume (mm3 SD)
Infarct size reduction (%)
Pre-ischemia
Vehicle
CPFX
(n = 28)
60.4 7.6
52.0 8.0
49.0 8.3*
58.8 8.5
54.6 5.0*
41.0 7.3**
57.2 8.7
0.001 (n = 4)
0.01 (n = 8)
0.1 (n = 5)
0.001 (n = 4)
0.01 (n = 9)
0.1 (n = 7)
13.9
18.8
2.7
SQ-4004
9.6
32.1
5.3
Post-ischemia
SQ-4004
0.01 (n = 5)
55.1 5.6
8.9
* P < 0.05.
** P = 0 as compared to vehicle treated.
Figure 2. Effect of SQ-4004 under the condition of ischemia/reperfusion (2/22 h) in the rat MCAO model. Brains were dissected into seven slices,
stained with triphenyltetrazolium chloride, and assessed for infarct size.

C.-H. Park et al. / Bioorg. Med. Chem. 15 (2007) 6517–6526
6521
41.0 7.3 mm3 (0.01 mg/kg, N = 9; P = 0) and
57.2 8.7 mm3 (0.1 mg/kg, N = 7), respectively, com-
pared to 60.4 7.6 mm3 (N = 28) for the vehicle group.
trations of SQ-4004 1 h prior to the local ischemia at
the doses of 0.001, 0.01 and 0.1 mg/kg, respectively.
After transient ischemia/reperfusion (30 min/3 h) insult,
the infarct area of the hearts was assessed and the highly
potent cardioprotective effect at 0.01 mg/kg was also ob-
served as shown in Table 4. Again, no reduction of the
infarct area at 0.1 mg/kg was observed in MI model.
The analog SQ-4004 exhibited a highly potent anti-
ischemic activity of 32.1% infarct reduction compared
to the vehicle at 0.01 mg/kg and 1.7-fold potency com-
pared to CPFX. Based on the high efficacy at pre-ische-
mic condition, SQ-4004 were also tested by
intraperitoneal administration three times under the
post-ischemic condition (right after, 2 h after occlusion
of MCA and 2 h after reperfusion) at the dose of
0.01 mg/kg. However, only 8.9% reduction of the infarct
size was observed under this condition. No reduction of
the infarct area at 0.1 mg/kg of SQ-4004 impinged on
dose-dependent manner seems to imply the possible
rebounding aspect of SQ-4004.
2.4. Anti-apoptotic activity of SQ-4004
On the basis of potent anti-ischemic activities of SQ-
4004 at both MCAO and MI models, we investigated
its modes of action associated with the inhibitory activ-
ity of programmed cell death during hypoxic injury. His-
topathological examination of the heart tissue was
carried out after a transient ischemic/reperfusion insult
followed by assessing the degree of apoptotic cell death
via TUNEL (terminal deoxynucleotidyl transferase-
mediated uridine 50-triphosphate-biotin nick end-label-
ing) staining. As a result, the significant apoptosis reduc-
tion compared to that of the vehicle was observed as
shown in Figure 3. These results strongly implicate the
possible anti-apoptotic activity for the anti-ischemic
activity of SQ-4004.
Considering the highly potent anti-ischemic effect of SQ-
4004, its in vivo evaluation was extended to myocardial
infarction animal model. The reduction in myocardial
infarction was examined after intraperitoneal adminis-
Table 4. Cardioprotective efficacy of SQ-4004 in the myocardial
infarction (MI) model in rats
3. Conclusion
Condition Drugs
Dose
Infarct area Infarct size
(mg/kg, ip) (% of area reduction (%)
at risk)
In conclusion, starting from an observation of the highly
potent anti-ischemic activity of CPFX at both in vitro
and in vivo assays, we have established the preliminary
structure–activity relationship for anti-ischemic effect
of CPFX. In addition, we have identified two potent
anti-ischemic quinolones. In particular, SQ-4004 exhib-
ited highly enhanced in vitro anti-ischemic activity with
Myocardial Vehicle (n = 37)
infarction
72.4 9.8
SQ-4004 0.001 (n = 5) 62.1 6.0* 14.2
0.01 (n = 10) 53.1 14.3* 26.6
0.1 (n = 6)
78.3 13.1
—
* P < 0.05 as compared to the vehicle treated.
Figure 3. Histological examination on the apoptotic pathway of SQ-4004, TUNEL staining and DAPI staining: (a) vehicle, (b) SQ-4004 (0.01 mg/
kg).

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C.-H. Park et al. / Bioorg. Med. Chem. 15 (2007) 6517–6526
significantly reduced anti-bacterial activity compared to
that of CPFX. Moreover, SQ-4004 showed a highly po-
tent neuroprotective effect as well as an excellent cardio-
protective effect. The anti-apoptotic mechanism for the
anti-ischemic activity was also elucidated, which pro-
vides the valuable information for further studies on
the novel anti-ischemic quinolones. Currently, the inten-
sive studies on SQ-4004 including the detailed structure–
activity relationship and the anti-ischemic mechanism
are in good progress. The successful results will be re-
ported in due course.
5-fluorobenzoyl chloride (666 mg, 1.69 mmol) in
5.00 mL dioxane was heated at reflux for 1 h and then
cooled down to room temperature. The reaction mixture
was diluted with CH2Cl2 (10.0 mL) and washed with
water and brine. After drying over anhydrous MgSO4,
removal of the solvent in vacuo gave a residue, which
was chromatographed over SiO2 using EtOAc/n-hexane
(1:8) to give the title compound, 3 (434 mg, 44%). Pale
1
yellowish solid; H NMR (CDCl3, 300 MHz) d 11.10
(br s, 1H), 8.20 (d, 1H, J = 13.8 Hz), 7.36 (s, 1H), 7.25
(d, 1H, J = 7.7 Hz), 7.11 (d, 1H, J = 7.9 Hz), 3.55 (s,
3H), 3.00 (m, 1H), 0.80–1.00 (m, 4H). ESI-MS: m/z
314 [M+H]+.
4. Experimental
4.1. Materials
4.2.1.4. 7-Chloro-1-cyclopropyl-4-oxo-1,4-dihydroquin-
oline-3-carboxylic acid (4). A mixture of 3 (355 mg,
1.13 mmol), K2CO3 (171 mg, 1.24 mmol) in 5.00 mL
dimethylformamide was heated at 110–120 ꢁC for 1 h
and then cooled down to room temperature. The reac-
tion mixture was diluted with cold water (10.0 mL)
and stirred for 1 h at room temperature. The resulting
solid was filtered, washed with cold water, and dried un-
der vacuum to give 7-chloro-1-cyclopropyl-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid methyl ester
All reagents including starting materials and solvents
were purchased from Aldrich Chemical Co. or TCI
and used without further purification. Reaction was
routinely checked with thin-layer chromatography
(Kiesegel 60F254, Merck) and column chromatography
was performed with Kiesegel 60, 230–400 mesh (Merck)
or NH–silica gel, Chromatorex, 100–200 mesh (Fuji
Silysia), respectively. NMR spectra were recorded on a
JEOL LNM-LA 300 (300 MHz), Bruker ARX 300 and
TMS (tetramethylsilane) was used as an internal stan-
dard. Chemical shifts (d) were recorded in ppm and cou-
pling constants (J) in hertz (Hz). IR (infrared) spectra
were recorded on a Jasco FT/IR-300E and Perkin-Elmer
1710 FT spectrometer. High resolution mass spectra
were obtained on a JEOL JMS-AX 505wA and JEOL
JMS-HX/HX 110A spectrometer.
1
(245 mg, 78%). Pale yellowish solid; H NMR (CDCl3,
300 MHz) d 8.58 (s, 1H), 8.40 (d, 1H, J = 8.3 Hz), 7.90
(d, 1H, J = 1.9 Hz), 7.40 (dd, 1H, J = 8.4, 1.9 Hz), 3.92
(s, 3H), 3.47 (m, 1H), 1.33 (m, 2H), 1.17 (m, 2H). ESI-
MS: m/z 278 [M+H]+.
A mixture of 7-chloro-1-cyclopropyl-4-oxo-1,4-dihydro-
quinoline-3-carboxylic acid methyl ester (37 mg,
0.13 mmol) in 6 N HCl / EtOH (8.00 mL, 1 : 1 v/v)
was heated at reflux for 4 h and then cooled down to
room temperature. The reaction mixture was diluted
with cold water (5.00 mL) and stirred for 1 h at room
temperature. The resulting solid was filtered, washed
with cold water, and dried under vacuum to give the title
4.2. Chemistry
The representative synthetic procedure was selected for
the preparation of SQ-4004, SQ-4007 and SQ-4008.
1
compound, 4 (31 mg, 91%). Yellowish solid; H NMR
4.2.1. Method A
(CDCl3, 300 MHz) d 15.00 (br s, 1H), 9.00 (s, 1H),
8.57(d, 1H, J = 8.9 Hz), 8.51 (d, 1H, J = 1.6 Hz), 7.95
(dd, 1H, J = 8.4, 1.9 Hz), 4.10 (m, 1H), 1.38–1.56 (com-
plex m, 4H).
4.2.1.1. Synthesis of cis-3-cyclopropylaminoacrylic
acid methyl ester (2). To a stirred solution of c-propyl-
amine, 1 (1.14 mL, 16.5 mmol) in 10.0 mL CH3CN
was added dropwise ethylpropiolate (1.06 g, 12.6 mmol)
at 5–10 ꢁC and stirred for 3 h. The reaction mixture was
concentrated in vacuo, and then the residue was purified
by NH–silica column chromatography using EtOAc/n-
hexane (1:5) as an eluent to give the title compound, 2
(600 mg, 22%): colorless oil; 1H NMR (CDCl3,
300 MHz) d 7.91 (br s, 1H), 7.44 (dd, 1H, J = 13.0,
8.2 Hz), 4.50 (d, 1H, J = 8.4 Hz), 3.63 (s, 3H), 2.69 (m,
1H), 0.50–0.75 (complex m, 4H). Bp 65–68 ꢁC/
0.09 mm Hg.
4.2.1.5. 1-Cyclopropyl-4-oxo-7-piperazin-1-yl-1,4-dihy-
droquinoline-3-carboxylic acid (8a, SQ-4004). A mixture
of 4 (233 mg, 0.88 mmol) and piperazine (227 mg,
3.00 mmol) in anhydrous dimethylsulfoxide (2.00 mL)
was heated at 130–140 ꢁC for 5 h. The reaction mixture
was diluted with water and treated conc. HCl to adjust
pH 4–5 and stirred for 1 h under steam bath. The result-
ing solid was filtered, washed with hot water several
times and dried under vacuum to give the title com-
pound, 8a (SQ-4004, 100 mg, 40%) (Table 5).
4.2.1.2. For trans-3-cyclopropylaminoacrylic acid methyl
ester (2). Colorless crystal; 1H NMR (CDCl3, 300 MHz)
d 7.44 (dd, 1H, J = 13.2, 7.6 Hz), 5.04 (d, 1H,
J = 13.3 Hz), 4.74 (br s, 1H), 3.67 (s, 3H), 2.43 (m,
1H), 0.50–0.75 (complex m, 4H).
4.2.2. Method B
4.2.2.1. Synthesis of 4-(4-acetyl-2, 5-difluorophe-
nyl)piperazine-1-carboxylic acid tert-butyl ester (6). To
a stirred solution of commercially available 2,4,6-diflu-
oro acetophenone (1.00 g, 5.74 mmol) and Et3N
(1.10 mL, 8.04 mmol) in pyridine (6.00 mL) was added
N-Boc-piperazine (1.18 g, 6.34 mmol) at room tempera-
ture, and the reaction mixture was stirred for 2 days.
4.2.1.3. 3-Cyclopropylamino-2-(2,4-dichlorobenzoyl)
acrylic acid methyl ester (3). A mixture of 2 (464 mg,
3.29 mmol), Et3N (0.24 mL, 3.39 mmol), and 2-chloro-

Table 5. Physicochemical and spectral properties of the CPFX analogs
SQ-analogs Empirical formula 1H NMR (DMSO-d6, 300 MHz) d (ppm), J (Hz)
IR (KBr, cmꢀ1
)
HR-MS (FAB+)
Appearance
SQ-4001
SQ-4002
SQ-4003
C
C
13H10FNO3
16H18FN3O
14.90 (br s, 1H), 8.77 (s, 1H), 8.38 (dd, 1H, J = 9.6, 4.3 Hz), 8.04 2918, 1731, 1617, 1548, 1459, 1079,
936, 829**
Calcd for C13H11FNO3 248.0723, White solid
found 248.0719
(dd, 1H, J = 8.8, 3.1 Hz), 7.94 (td, 1H, J = 7.9, 3.0 Hz), 3.95 (m,
1H), 1.34 (m, 2H), 1.20 (m, 2H)
8.00 (d, 1H, J = 13.3 Hz), 7.60 (d, 1H, J = 8.1 Hz), 7.26 (d, 1H,
J = 7.2 Hz), 6.15 (d, 1H, J = 7.8 Hz), 3.35 (m, 1H), 3.25 (m, 4H),
3.13 (m, 4H), 1.30 (m, 2H), 1.05 (m, 2H)*
3436, 1623, 1484, 1382, 1295, 1261,
1129, 823, 737, 620
Calcd for C16H19N3OF 288.1512, White solid
found 288.1515
C18H19FN2O3
15.10 (s, 1H), 8.76 (s, 1H), 8.00 (d, 1H, J = 13.4 Hz), 7.35 (d, 1H, 3441, 2935, 1725, 1626, 1505, 1469,
Calcd for C18H20FN2O3
White solid
J = 7.3 Hz), 3.60 (m, 1H), 3.35 (m, 4H), 1.60–1.95 (complex m,
6H), 1.40 (m, 2H), 1.20 (m, 2H)
8.61 (S, 1H), 8.14 (d, 1H, J = 8.9 Hz), 7.35 (m, 2H), 3.60 (m, 1H), 2819, 1681, 1619, 1515, 1463, 1271,
1383, 1338, 1252, 1101, 957, 887, 858, 331.1458, found 331.1458
827
SQ-4004
SQ-4005
C
C
17H19N3O3
13H11NO3
Calcd for C17H20N3O3 314.1505, Yellowish solid
found 314.1496
3.50 (m, 4H), 3.10 (m, 4H), 1.35 (m, 2H), 1.14 (m, 2H)
1156, 1044, 944, 839, 800. 625
14.90 (s, 1H), 8.91 (s, 1H), 8.53 (dd, 1H, J = 8.3, 1.5 Hz), 8.10 (d, 2918, 1722, 1616, 1542, 1451, 1336,
1H, J = 8.8 Hz), 7.90 (m, 1H), 7.60 (m, 1H), 3.60 (m, 1H), 1.45 (m, 1235, 1171, 1050, 908, 810, 757**
2H), 1.20 (m, 2H)*
Calcd for C13H12NO3 230.0817,
found 230.0818
White solid
White solid
Yellowish solid
White solid
SQ-4006
SQ-4007
SQ-4008
C18H19FN2O3
8.93 (s, 1H), 8.38 (d, 1H, J = 6.1 Hz), 8.20 (d, 1H, J = 10.2 Hz),
4.10 (m, 1H), 3.50–3.65 (m, 3H), 3.21–3.49 (m, 2H), 2.15–2.42 (m, 1543, 1509, 1468, 1394, 1338, 1261,
4H), 1.57 (m, 2H), 1.39 (m, 2H) 808, 555
9.29 (br s, 1H), 9.00 (s, 1H), 8.58 (d, 1H, J = 14.7 Hz), 7.73 (d, 1H, 3426, 2496, 1704, 1623, 1494, 1455,
3439, 2935, 2712, 2499, 1730, 1615,
Calcd for C18H20FN2O3
331.1458, found 331.1461
C
C
17H18FN3O2S
18H20FN3O3
Calcd for C17H19FN3O2S
J = 7.8 Hz), 4.11 (m, 1H), 3.69 (m, 4H), 3.48 (m, 4H), 1.50 (m, 2H), 1319, 1268, 1138, 1027, 938, 775, 536 348.1182, found 348.1178
1.30 (m, 2H)
7.85 (d, 1H, J = 13.2 Hz), 7.53 (d, 1H, J = 7.5 Hz), 3.65 (m, 1H), 3443, 2677, 1602, 1478, 1404, 1293,
3.33 (m, 4H), 3.09 (s, 3H), 3.01 (m, 4H), 1.48 (m, 2H), 1.04 (m, 2H) 1262, 1142, 848, 728, 609
Calcd for C18H21FN3O3
346.1567, found 346.1562
* 1H NMR (300 MHz, CDCl3).
** IR (CHCl3, neat).

6524
C.-H. Park et al. / Bioorg. Med. Chem. 15 (2007) 6517–6526
The resulting solution was concentrated and the crude
residue was partitioned between CH2Cl2 (20.0 mL) and
water (2· 20.0 mL). The combined organic layers were
dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was purified
by recrystallization (EtOAc/n-hexane, 1:5) to give the ti-
was partitioned between CH2Cl2 (10.0 mL) and water
(2· 10.0 mL). The combined organic layers were dried
over anhydrous Na2SO4, filtered, and concentrated un-
der reduced pressure. The residue was purified by col-
umn chromatography on silica gel (CH2Cl2/MeOH,
40:1) to give title compound, 8b (220 mg, 84%). Yellow-
1
1
tle compound, 6 (1.96 g, 100%). White solid; H NMR
ish solid; H NMR (CDCl3, 300 MHz) d 8.53 (s, 1H),
(CDCl3, 300 MHz) d 7.58 (dd, 1H, J = 13.6, 6.8 Hz),
6.58 (dd, 1H, J = 12.7, 6.9 Hz), 3.60 (m, 4H), 3.17 (m,
4H), 2.59 and 2.57 (s, 3H), 1.49 (s, 9H). ESI-MS: m/z
341 [M+H]+, 363 [M+Na]+.
8.04(d, 1H, J = 13.2 Hz), 7.26 (br s, 1H), 4.40(q, 2H,
J = 7.2 Hz), 3.65 (m, 4H), 3.45 (m, 4H), 3.20 (m, 1H),
1.49 (s, 9H), 1.40 (t, 3H, J = 7.1 Hz), 1.35 (m, 2H),
1.25 (m, 2H). ESI-MS: m/z 460 [M+H]+, 482 [M+Na]+.
4.2.2.2. 4-[4-(3-Cyclopropylamino-2-ethoxycarbonyl-
acryloyl)-2,5-difluorophenyl]piperazine-1-carboxylic acid
tert-butyl ester (7). To a stirred solution of 6 (653 mg,
1.92 mmol) in diethylcarbonate (10.0 mL) was added
NaH (60% in dispersion oil, 307 mg, 7.68 mmol) at
room temperature, and the reaction mixture was stirred
at 80 ꢁC for 2 h. The resulting solution was concentrated
by evaporation of diethylcarbonate and the crude mix-
ture was partitioned between Et2O (20.0 mL) and water
(2· 20.0 mL). The combined organic layers were dried
over anhydrous Na2SO4, filtered, and concentrated un-
der reduced pressure. The residue was purified by col-
umn chromatography on silica gel (EtOAc/n-hexane,
1:7) to give 4-[4-(2-ethoxycarbonylacetyl)-2,5-difluor-
ophenyl]piperazine-1-carboxylic acid tert-butyl ester
(394 mg, 50%). Beige solid; 1H NMR (CDCl3,
300 MHz) d 12.72 (s, 1/3H), 7.60 and 7.58 (dd, 1H,
J = 13.6, 6.8 Hz), 6.59 and 6.58 (dd, 1H, J = 12.7,
6.9 Hz), 5.81 (s, 1/3H), 4.24 (q, 2H, J = 7.1 Hz), 3.90
(d, 1 and 2/3H, J = 3.8 Hz), 3.58 (m, 4H), 3.12 and
3.20 (m, 4H), 1.43 (s, 9H), 1.23 (m, 3H). ESI-MS: m/z
413 [M+H]+, 435 [M+Na]+.
4.2.2.4.
7-(4-tert-Butoxycarbonylpiperazin-1-yl)-1-
cyclopropyl-6-fluoro-4-thioxo-1,4-dihydroquinoline-3-car-
boxylic acid ethyl ester (8c). A mixture of 8b (163 mg,
0.35 mmol) and P4S10 (101 mg, 0.23 mmol) in pyridine
(1.20 mL) was heated at reflux for 2 h. The resulting
solution was concentrated by evaporation of pyridine
and the crude mixture was partitioned between CHCl3
(20.0 mL) and water (2· 20.0 mL). The combined organ-
ic layers were dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel
(EtOAc/n-hexane, 3:1) to give title compound, 8c
(54 mg, 33%). Orange yellowish solid; 1H NMR (CDCl3,
300 MHz) d 8.63 (d, 1H, J = 11.1 Hz), 7.97 (s, 1H), 4.38
(q, 2H, J = 5.4 Hz), 3.64 (m, 4H), 3.50 (m, 1H), 3.23 (m,
4H), 1.39 (s, 9H), 1.36 (m+t, 5H), 1.11 (m, 2H).
4.2.2.5. 1-Cyclopropyl-6-fluoro-2-methyl-4-oxo-7-pip-
erazin-1-yl-1,4-dihydroquinoline-3-carboxylic acid (9a,
SQ-4008). To a stirred solution of 8b (615 mg,
1.34 mmol) and CuI (76.6 mg, 0.40 mmol) in anhydrous
THF (7.00 mL) was added CH3MgBr (3 M in ether,
0.67 mL, 2.00 mmol) at ꢀ78 ꢁC dropwise and then stir-
red for 3 h. The reaction mixture was quenched with
satd NaHCO3 (aq) and concentrated under reduced
pressure. The crude mixture was partitioned between
CHCl3 (10.0 mL) and water (2· 20.0 mL). The com-
bined organic layers were dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography on
silica gel (EtOAc/n-hexane, 1:5) to give 7-(4-tert-but-
oxycarbonylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-2-
methyl-4- oxo 1,2,3,4-tetrahydroquinoline-3-carboxylic
To a stirred solution of 4-[4-(2-Ethoxycarbonylacetyl)-
2,5-difluorophenyl]piperazine-1-carboxylic acid tert-bu-
tyl ester (394 mg, 0.96 mmol) in acetic anhydride
(1.00 mL) was added triethylorthoformate (0.32 mL,
1.92 mmol) at room temperature and the reaction mix-
ture was stirred at 110 ꢁC for 2 days. The solution was
concentrated by evaporation of acetic anhydride and tri-
ethylorthoformate under vacuum and the resulting
crude mixture was dissolved in anhydrous CH2Cl2
(1.00 mL) and then cyclopropylamine (80.0 lL) was
added. The mixture was concentrated under reduced
pressure and the resulting residue was purified by col-
umn chromatography on silica gel (EtOAc/n-hexane,
1:4) to give the title compound, 7 (398 mg, 87%). Yel-
lowish amorphous; 1H NMR (CDCl3, 300 MHz) d
10.76 (br d, 2/3H, J = 13.1 Hz), 9.21 (br d, 1/3H,
J = 13.2 Hz), 8.14 and 8.05 (d, 1H, J = 13.7 Hz), 7.10
(m, 1H), 6.50 (m, 1H), 4.02 (m, 2H), 3.60 (m, 4H),
3.05 (m, 4H), 2.98 (m, 1H), 1.50 (s, 9H), 0.80–1.20 (com-
plex m, 7H). ESI-MS: m/z 480 [M+H]+, 502 [M+Na]+.
1
acid ethyl ester (274 mg, 70%). White solid; H NMR
(CDCl3, 300 MHz) d 7.54 (d, 1H, J = 13.4 Hz), 6.54
(d, 1H, J = 7.1 Hz), 4.26 (m, 1H), 4.10 (qd, 2H,
J = 7.1, 0.9 Hz), 3.59 (m, 4H), 3.19 (m, 5H), 2.40 (m,
1H), 1.60 (s, 9H), 0.92–1.35 (m, 6H), 0.79–1.12 (m,
4H). ESI-MS: m/z 476 [M+H]+.
To a stirred solution of 7-(4-tert-butoxycarbonylpipera-
zin-1-yl)-1-cyclopropyl-6-fluoro-2-methyl-4-oxo 1,2,3,4-
tetrahydroquinoline-3-carboxylic acid ethyl ester
(393 mg, 0.83 mmol) in anhydrous THF (4.00 mL) was
added NaH (60% dispersion in oil, 40.0 mg, 0.99 mmol)
portionwise and then added PhSeCl (175 mg,
0.91 mmol) in anhydrous THF (0.50 mL) rapidly at
room temperature. The crude mixture was partitioned
between pentane/ether (6.00 mL, 1/1 v/v) and satd NaH-
CO3 (aq) (3.00 mL), then the organic layer was sepa-
rated. The organic layer was treated with H2O2 (30%
4.2.2.3.
7-(4-tert-Butoxycarbonylpiperazin-1-yl)-1-
cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-car-
boxylic acid ethyl ester (8b). To a stirred solution of
compound 7 (275 mg, 0.57 mmol) in THF (2.00 mL)
was added NaH (60% in dispersion oil, 27.4 mg,
0.68 mmol) at room temperature and the mixture was
heated at reflux for 2 h. The resulting crude mixture

C.-H. Park et al. / Bioorg. Med. Chem. 15 (2007) 6517–6526
6525
w/w, 0.20 mL) in CH2Cl2 (11.0 mL). After stirring for
1 h at room temperature, the reaction mixture was di-
luted with CH2Cl2 (10.0 mL) and washed with 10% w/w
NaHCO3 (aq), water, and brine. The combined
organic layers were dried over anhydrous Na2SO4, fil-
tered, and concentrated under reduced pressure. The
residue was purified by column chromatography on
silica gel (EtOAc/n-hexane, 2:1) to give 7-(4-tert-
butoxycarbonylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-
2-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
ethyl ester (274 mg, 70%). White solid; 1H NMR
(CDCl3, 300 MHz) d 7.90 (d, 1H, J = 13.0 Hz), 7.22
(d, 1H, J = 7.0 Hz), 4.39 (q, 2H, J = 7.1 Hz), 3.66 (m,
4H), 3.20 (m, 4H), 2.61 (s, 3H), 1.49 (s, 9H), 1.35
(m+t, 5H), 0.88 (m, 2H). ESI-MS: m/z 474 [M+H]+,
496 [M+Na]+.
per well in a 96-well plate in a 1:1 mixture of FBS-free
EMEM and F12K medium at a humidified CO2 incuba-
tor. After one day, cells were pre-treated with 0.1 lM of
the test compounds for 2 h and then treated with
200 lM of hydrogen peroxide (Sigma Aldrich Co.) fol-
lowed by incubation for 24 h. To assess the cell viability,
cells were incubated with 20 lL of MTS solution (CelT-
iter 96 Aqueous one solution cell proliferation assay,
Promega) for 3 h at 37.0ꢁC, and OD was determined
at 490 nm using a spectrophotometer. All values are
averages of at least two independent experiments, each
done in triplicate.
4.3.2. In vitro anti-bacterial activity. Minimal Inhibitory
Concentrations (MICs) were determined by a standard-
ized agar dilution method according to CLSI guidelines.
Staphylococcus aureus ATCC 25923, Staphylococcus epi-
dermidis ATCC 12228, Bacillus subtilis ATCC 6633,
Escherichia coli ATCC 25922, Enterobacter clocae
ATCC 13047, and Serratia marcescens ATCC 27117
were tested on Mueller Hinton agar and Enterococcus
faecalis ATCC 29212 was on Brain–Heart Infusion agar.
The stock solutions of test compounds were diluted in
dimethylsulfoxide (DMSO) or 0.1 M NaOH solution
to give a serial and twofold series yielding final drug
concentrations in a range of 0.06–128 lg/mL. A micro-
inoculator (Sakuma Co. Ltd, Tokyo, Japan) was used
to inoculate the bacterial suspensions (104 cfu/spot).
The inoculated plates were incubated in ambient air at
36 ꢁC for 16–18 h. The MIC of each compound was de-
fined as the lowest concentration that inhibited visible
growth of the organism.23
A mixture of 7-(4-tert-butoxycarbonylpiperazin-1-yl)-1-
cyclopropyl-6-fluoro-2-methyl-4-oxo-1,4-dihydroquino-
line-3-carboxylic acid ethyl ester (274 mg, 0.58 mmol) in
EtOH/CH2Cl2/concd HCl (2.00/2.00/0.20 mL) was stir-
red for 3 h at room temperature. The reaction mixture
was evaporated under reduced pressure. The residue
was diluted with CH2Cl2 (10.0 mL) and washed with
water and brine. The combined organic layers were
dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was purified
by column chromatography on silica gel (CH2Cl2/
MeOH, 15:1) to give 1-cyclopropyl-6-fluoro-2-methyl-
4-oxo-7-piperazin-1-yl-1,4-dihydroquinoline-3-carbox-
ylic acid ethyl ester (167 mg, 77%). White solid; 1H
NMR (CDCl3, 300 MHz) d 7.95(d, 1H, J = 12.9 Hz),
7.23 (d, 1H, J = 7.2 Hz), 4.41 (q, 2H, J = 6.9 Hz), 3.23
(m, 4H), 3.18 (m, 4H), 2.62 (s, 3H), 1.43 (m+t, 5H),
1.00 (m, 2H). ESI-MS: m/z 374 [M+H]+.
4.3.3. In vivo MCAO animal model. Transient focal
ischemic stroke was modeled in rats using the intralumi-
nal thread procedure according to Longa’s protocol
with a minor modification.24 The male Sprague–Dawley
rats (240–270 g, Charles River, USA) were anesthetized
with 5% enflurane. The rectal temperature was main-
tained in the range of 37 0.5 ꢁC with a heating pad.
A 4–0 nylon suture was inserted through the internal
carotid artery into the origin of the middle cerebral ar-
tery (MCA). The suture was carefully removed after
2 h of MCA occlusion. Drugs were administered total
three times divided into two groups under pre-ischemic
condition and post-ischemic condition as mentioned in
the main text. Then, brains were isolated at 24 h after
ischemia, sliced, and processed for TTC (2,3,5-tripenyl-
tetrazolium chloride) histochemistry. The infarcts were
measured using the computer-assisted planimetry, Gel
Documentation System, Bio-Rad. The effect of drug
treatment is expressed as the percent reduction in infarct
volume compared to the vehicle controls. Statistical sig-
nificance was determined using the Student’s t-test with
a confidence interval accepted when P < 0.05.
A mixture of 1-cyclopropyl-6-fluoro-2-methyl-4-oxo-
7-piperazin-1-yl-1,4-dihydroquinoline-3-carboxylic acid
ethyl ester (100 mg, 0.27 mmol) in EtOH/THF/1 N
NaOH (aq) (0.50/0.50/0.20 mL) was stirred for 12 h at
room temperature. The reaction mixture was acidified
with concd HCl adjust to pH 5–6. The resulting solid
was filtered, washed with cold water, and dried under
vacuum to give the title compound 9a, SQ-4008
(50 mg, 54%) (Table 5).
4.2.2.6. 1-Cyclopropyl-6-fluoro-7-piperazin-1-yl-4-thi-
oxo-1,4-dihydroquinoline-3-carboxylic acid (9b, SQ-
4007). A mixture of 8c (55.0 mg, 0.11 mmol) in 6 N
HCl/EtOH (1.00 mL/1.00 mL) was heated at reflux for
12 h. The reaction mixture was cooled down to room
temperature and the resulting solid was filtered, washed
with cold EtOH, and dried under vacuum to give the ti-
tle compound 9b, SQ-4007 (19 mg, 50%) (Table 5).
4.3. Biology
4.3.4. In vivo MI animal model. In vivo rat MI model was
performed according to Ju’s protocol with a slight mod-
ification.25 The male Sprague–Dawley rats (260–290 g,
Charles River, USA) were anesthetized with ketamine
(10 mg/kg) and xylazine (5 mg/kg) and then SQ-4004
was intraperitoneally administered 1 h before regional
ischemia. The heart was exposed by a median sternot-
4.3.1. In vitro MTS assay. Human neuroblastoma cell
line, SH-SY5Y (ATCC, CRL-2262), was cultured under
1:1 mixture of EMEM (Eagle’s minimum essential med-
ium) and F12K (Ham’s F12 medium) supplemented by
10% heat-inactivated FBS (fetal bovine serum) at
37.0 ꢁC in 5% CO2. Cells were plated at 5 · 104 cells

6526
C.-H. Park et al. / Bioorg. Med. Chem. 15 (2007) 6517–6526
5. Yrja¨nheikki, J.; Tikka, T.; Keina¨nen, R.; Goldstein, G. P.;
Chan, H.; Koistinaho, J. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 13496.
6. Chen, M.; Ona, V. O.; Li, M.; Ferrante, R. J.; Fink, K. B.;
Zhu, S.; Bian, J.; Guo, L.; Farrell, L. A.; Hersch, S. M.;
Hobbs, W.; Vonsattel, J.; Cha, J. J.; Friedlander, R. M.
Nat. Med. 2000, 6, 797.
7. Szymanska, A.; Biernaskie, J.; Laidley, D.; Granter-
Button, S.; Corbett, D. Exp. Neurol. 2006, 197,
189.
omy and the left anterior descending artery was snare-
occluded for 30 min and then reperfused for 3 h. After
injection of 1% Evans blue, rats were sacrificed and
the hearts were separated. The left ventricle was cut into
two slices and then processed for TTC histochemistry.
The infarct size was calculated as a percentage of the
area at risk. Statistical significance was determined using
the Student’s t-test with a confidence interval accepted
when P < 0.05.
8. Weng, Y. C.; Kriz, J. Exp. Neurol. 2007, 204, 433.
9. Meier, C. R. J. Infect. Dis. 2000, 181, S558.
10. Khan, S. A.; Salloum, F.; Das, A.; Xi, L.; Vetrovec, G.
W.; Kukreja, R. C. J. Mol. Cell. Cardiol. 2006, 41, 256.
11. Granville, D. J.; Tashakkor, B.; Takeuchi, C.; Gustafsson,
4.3.5. TUNEL staining. Cardiomyocyte apoptosis was
analyzed by the terminal deoxynucleotidyl transferase
mediated nick end labeling (TUNEL) staining using
Apop-Tag (POD Roche Mannheim, Germany). After
3 h reperfusion, the heart was separated and subjected
to the general process (dehydration, clearing, impregna-
tion, and embedding) for the tissue preparation. The tis-
sue was washed with 0.02 M phosphate buffered saline,
at pH 7.4 and treated with proteinase K. The prepared
tissue slides were treated with a mixture of 1:9 of enzyme
solution of Apop-Tag kit and label solution and then
left for 1 h at dark room, followed by DAPI (4,6-diami-
no-2-phenylindole) staining. Cells, which lay in the de-
fined rectangular field area (20· objective) and
exhibited positive staining for apoptosis, were examined
and photographed using a fluorescent microscope.
˚
A. B.; Huang, C.; Sayen, M. R.; Wentworth, P.; Yeager,
M.; Gottlieb, R. A. Proc. Natl. Acad. Sci. U.S.A. 2004,
101, 1321.
12. Lee, J. WO 0151615 A1, 2001.
13. Meisel, C.; Prass, K.; Braun, J.; Victorov, I.; Wolf, T.;
Megow, D.; Halle, E.; Volk, H. D.; Dirnagl, U.; Meisel, A.
Stroke 2004, 35, 2.
14. Walsh, C. Antibiotics, Actions, Origins, Resistance; Har-
vard Medical School, Boston: ASM Press, 2003.
15. Ronald, A. R.; Low, D. E. Fluoroquinolones Antibiotics;
Birkha¨user Verlag: Basel, 2003.
16. Ohta, M.; Koga, H. J. Med. Chem. 1991, 34, 131.
17. Peterson, U.; Grohe, K.; Kuhle, E.; Zeiler, H.; Metzger,
¨
K. G. US 4559341, 1984.
18. Grohe, K.; Heitzer, H.; Helmut, W. Liebigs Ann. Der
Chemie 1987, 1, 29.
19. Kerns, R. J.; Rybak, M. J.; Kaatz, G. W.; Vaka, F.; Cha,
R.; Grucz, R. G.; Diwadkard, V. U. Bioorg. Med. Chem.
Lett. 2003, 13, 2109.
20. Reuman, M.; Eissenstat, M. A.; Weaver, J. D. Tetrahe-
dron Lett. 1994, 35, 8303.
21. Cecchetti, V.; Fravolini, A.; Palumbo, M.; Sissi, C.;
Tabarrini, O.; Terni, P.; Xin, T. J. Med. Chem. 1996, 39,
4952.
Acknowledgments
This work was supported by a grant from the Seoul R&
BD Program and the Research Institute of Medical Sci-
ence, Catholic University of Daegu.
References and notes
22. Laborde, E.; Kiely, J. S.; Culbertson, T. P.; Lesheski, L. E.
J. Med. Chem. 1993, 36, 1964.
1. Wolf, P. A., Cobb, J. L., D’Agostino, R. B. Eds.; Stroke,
Pathophysiology, Diagnosis and Management; Churchill
Livingstone: New York, 1997, pp. 3–27.
2. Habeck, M. Drug Discovery Today 2002, 7, 157.
3. Boxer, P. A.; Bigge, C. F. Drug Discovery Today 1997, 2,
219.
23. Clinical and Laboratory Standards Institute. Performance
standards for antimicrobial susceptibility testing, fifteenth
informational supplement. Approved standard MS100-
S15, 2005. CLSI, Wayne, PA.
24. Longa, E. Z.; Weinstein, P. R.; Carlson, S.; Cummins, R.
Stroke 1989, 20, 84.
4. Green, A. R.; Shuaib, A. Drug Discovery Today 2006, 11,
681.
25. Ju, H.; Zhao, S.; Tappia, P. S.; Panagia, V.; Dixon, I. M.
C. Circulation 1998, 97, 892.