Synthesis and antibacterial activity of novel and potent DNA gyrase inhibitors with azole ring

Article abstract of DOI:10.1016/j.bmc.2004.08.010

The pyrazole, oxazole and imidazole derivatives synthesized in this study exhibited potent antibacterial activity against multidrug resistant Gram-positive bacteria with minimal inhibitory concentration values equivalent to those against susceptible strains. The 4-piperidyl moiety and the pyrazole ring in 1-(3-chlorophenyl)-5-(4-phenoxyphenyl)-3-(4-piperidyl)pyrazole 2, which has previously shown improved DNA gyrase inhibition and target-related antibacterial activity, were transformed to other groups and the in vitro antibacterial activity of the synthesized compounds was evaluated. The selected pyrazole, oxazole and imidazole derivatives showed moderate inhibition against DNA gyrase and topoisomerase IV with similar IC₅₀ values (IC ₅₀ = 9.4-25 μg/mL). In addition, many of the pyrazole, oxazole a1nd imidazole derivatives synthesized in this study exhibited potent antibacterial activity against quinolone-resistant clinical isolates and coumarin-resistant laboratory isolates of Gram-positive bacteria with minimal inhibitory concentration values equivalent to those against susceptible strains.

Full text of DOI:10.1016/j.bmc.2004.08.010

Bioorganic & Medicinal Chemistry 12 (2004) 5515–5524
Synthesis and antibacterial activity of novel and potent DNA
gyrase inhibitors with azole ring
Akihiko Tanitame,a,* Yoshihiro Oyamada,b Keiko Ofuji,a Mika Fujimoto,b Kenji Suzuki,a
Tomohiko Ueda,a Hideo Terauchi,a,c Motoji Kawasaki,a Kazuo Nagai,d Masaaki Wachie
and Jun-ichi Yamagishib,c
aChemistry Research Laboratories, Dainippon Pharmaceutical Co., Ltd, 33-94, Enoki, Suita, Osaka 564-0053, Japan
bPharmacology and Microbiology Research Laboratories, Dainippon Pharmaceutical Co., Ltd, 33-94, Enoki, Suita,
Osaka 564-0053, Japan
cPharmaceutical Research and Technology Center, Dainippon Pharmaceutical Co., Ltd, 33-94, Ebie 1-5-51, Fukushima,
Osaka 553-0001, Japan
dDepartment of Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501, Japan
eDepartment of Bioengineering, Tokyo Institute of Technology, 4259, Nagatsuda, Midori-ku, Yokohama 226-8501, Japan
Received 14 July 2004; accepted 10 August 2004
Available online 11 September 2004
Abstract—The 4-piperidyl moiety and the pyrazole ring in 1-(3-chlorophenyl)-5-(4-phenoxyphenyl)-3-(4-piperidyl)pyrazole 2, which
has previously shown improved DNA gyrase inhibition and target-related antibacterial activity, were transformed to other groups
and the in vitro antibacterial activity of the synthesized compounds was evaluated. The selected pyrazole, oxazole and imidazole
derivatives showed moderate inhibition against DNA gyrase and topoisomerase IV with similar IC50 values (IC50 = 9.4–25lg/
mL). In addition, many of the pyrazole, oxazole and imidazole derivatives synthesized in this study exhibited potent antibacterial
activity against quinolone-resistant clinical isolates and coumarin-resistant laboratory isolates of Gram-positive bacteria with min-
imal inhibitory concentration values equivalent to those against susceptible strains.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Bacterial DNA gyrase is a proven target for antibacte-
rial chemotherapy.2 Quinolones, such as sparfloxacin3
(SPFX, Fig. 1), inhibit bacterial DNA gyrase and topo-
isomerase IV and cause bacterial cell death. Besides the
quinolones, other naturally occurring bacterial DNA
gyrase inhibitors, such as the coumarins, which include
novobiocin (NB, Fig. 1), clorobiocin and coumermycin
The emergence and spread of multidrug resistant Gram-
positive bacteria, such as methicillin-resistant Staphylo-
coccus aureus (MRSA), penicillin-resistant Streptococcus
pneumoniae (PRSP) and vancomycin-resistant entero-
cocci (VRE), have made treatment of infectious diseases
difficult and have over the last decades become a serious
medical problem. As pathogenic bacteria continuously
evolve mechanisms of resistance to currently used anti-
bacterial agents, the discovery of novel and potent bac-
tericides is the best way to overcome bacterial resistance
and develop effective therapies. Unfortunately, finding
novel antibacterial agents has been difficult with the
oxazolidinones, identified in 1980, being the last exam-
ple to successfully reach the clinic.1
NH2
O
NH2
O
N
OH
F
COOH
O
O
OH
O
H
N
O
OH
N
O
HN
F
O
O
NB
SPFX
Cl
N
NH
N
N
HN
Keywords: DNA gyrase inhibitor; Pyrazole, oxazole and imidazole
derivatives; Multidrug resistant strains.
O
HN
1
2
O
*
Corresponding author. Tel.: +81 6 6337 5906; fax: +81 6 6338
7656; e-mail: akihiko-tanitame@dainippon-pharm.co.jp
Figure 1.
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.010

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A. Tanitame et al. / Bioorg. Med. Chem. 12 (2004) 5515–5524
A1 have also been known as antibacterial agents.4 The
coumarins inhibit ATPase activity of DNA gyrase by
competing with ATP for binding to the B subunit of
the enzyme. However, no pharmaceutically useful drug
has so far been derived from the coumarins. Although
many efforts have been dedicated to finding potent anti-
bacterial agents that can overcome bacterial resistance,
promising lead structures of DNA gyrase inhibitors
have not been found.5–10
conditions, followed by separation using CHP-20P
(reverse phase) column chromatography and/or recrys-
tallization gave the 3-substituted 1-(3-chloro-
phenyl)pyrazoles 8, 10–12, 14, 15 and the regioisomers
9, 13, 16 as acid salts. Compound 7 having a cyclohexyl
group at the 3-position instead of the 4-piperidyl group
of 2 was synthesized from cyclohexanecarboxylic acid
and 4-phenoxyacetophenone in a similar manner to that
described above. The corresponding 5-substituted
isomers of 1-(3-chlorophenyl)pyrazole derivatives 7,
10, 11 and 14 were not isolated.
Using a newscreening system for specific inhibitors of
chromosome partitioning in Escherichia (E.) coli11,12
we have previously reported that one of our hit com-
pounds (compound 1, Fig. 1) having a piperidine ring
represents a newclass of bacterial DNA gyrase inhibi-
tors that have potent antibacterial activity against
Staphylococcus (S.) aureus and Enterococcus (E.)
faecalis.13 We have also demonstrated that compound
2 (Fig. 1), a derivative of 1, shows improved DNA gyr-
ase inhibition and target-related antibacterial activity.11
Moreover compound 2 exhibits the same minimal inhib-
itory concentration (MIC) values against clinically iso-
lated multidrug resistant Gram-positive bacteria as
against drug susceptible strains.
The position of 3-chlorophenyl group in 7–16 was deter-
mined on the basis of nuclear Overhauser effects (NOE)
experiments. For example, in compound 8, a correlation
of NOE was observed between the protons of the phenyl
moiety on R1-Ph and the ortho protons of the 3-chloro-
phenyl moiety. On the other hand, in compound 9, a
correlation of NOE was observed between the 2-H pro-
tons of the 3-piperidyl moiety on R2 and the ortho pro-
tons of the 3-chlorophenyl moiety.
The novel oxazole derivatives 21 and 22 and the imidaz-
ole derivatives 24 and 25 were synthesized as shown in
Scheme 2. The 2-trimethylsilyloxyacetonitrile 18a was
prepared by condensation of 4-phenoxybenzalde-
hyde (R3 = 4-phenoxy) with trimethylsilylcyanide and
zinc iodide in CH2Cl2.16 Coupling reaction of 18a
with 3-chlorobenzaldehyde (R4 = 3-chlorophenyl) using
LHMDS in THF, followed by treatment with aqueous
H2SO4 afforded the 2-hydroxyethane-1-one 19a. Reac-
tion of 19a with 1-(tert-butoxycarbonyl)piperidine-4-
carbxylic acid in the presence of 1-ethyl-3-[3-(dimethyl-
amino)propyl]carbodiimide hydrochloride (EDC) and
4-dimethylaminopyridine (DMAP) gave the ester 20a,
which was treated with NH4OAc in AcOH to afford
the desired 21.17 In a similar manner to that described
above, 22 was obtained from 3-chlorobenzaldehyde
(R3 = 3-chloro) as starting material via 18b–20b
(R4 = 4-benzyloxy).
Our aim in this study was to optimize the piperidine
moiety and pyrazole ring of compound 2 and find new
potent DNA gyrase inhibitors with antibacterial activity
against MRSA, PRSP and VRE. Here we report the
synthesis and structure–activity relationships (SARs)
of a series of pyrazole and other five-membered hetero-
cyclic derivatives.
2. Chemistry
The novel pyrazole derivatives 8–16 were synthesized as
shown in Scheme 1. The requisite intermediate 1,3-dike-
tones 4a–f were prepared by coupling reaction of the
N-(tert-butoxycarbonyl) amino acids 3 with various
benzophenone derivatives in THF using 1,10-carbonyldi-
1H-imidazole (CDI) and lithium hexamethyldisilazide
(LHMDS).14,15 Condensation of 4a–f with 3-chloro-
phenylhydrazine hydrochloride with aqueous NaOH
gave a mixture of the 1-(3-chlorophenyl)pyrazoles 5
and 6. Cleavage of the protecting group under acidic
The imidazole analogues 24 and 25 were obtained by
condensation of 23a,b, which were prepared by oxida-
tion of 19a,b using CuSO4 in aqueous pyridine, with
1-(tert-butoxycarbonyl)-4-formylpiperidine in the pres-
ence of NH4OAc in AcOH.18
Cl
Cl
+
N
N
N
N
O
O
O
tBuOOC
tBuOOC
tBuOOC
tBuOOC
COOH
R2
R2
R2
a
R2
R1
R1
b
R1
3
4a-f
5
6
or
or
c
O
Cl
Cl
COOH
+
R1
N
N
N N
4g
R1
H
R2
H
R2
R1
7, 8, 10-12, 14, 15
9, 13, 16
Scheme 1. General synthesis of the 1-(3-chlorophenyl)pyrazole derivatives. Reagents: (a) i. CDI, THF, ii. R1-PhCOCH3, LHMDS, THF; (b) 3-
chlorophenylhydrazineÆHCl, aq NaOH, EtOH; (c) HCl, EtOH or TFA, CH2Cl2.

A. Tanitame et al. / Bioorg. Med. Chem. 12 (2004) 5515–5524
5517
R4
O
TMS
R4
O
O
R4
O
CHO
a
b
d
c
R3
R3
CN
R3
R3
O
N
R3
N
COOtBu
OH
HN
O
17a,b
18a,b
19a,b
20a,b
21, 22
e
R4
O
R4
f
HN
R3
N
O
R3
HN
23a,b
24, 25
Scheme 2. Synthesis of the oxazole and imidazole derivatives. Reagents: (a) TMSCN, ZnI, CH2Cl2; (b) i. R4-CHO, LHMDS, THF, ii. aq H2SO4,
MeOH; (c) 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid, EDC, DMAP, CH2Cl2; (d) NH4OAc, AcOH; (e) CuSO4, pyridine, H2O; (f) 1-(tert-
butoxycarbonyl)-4-formylpiperidine, NH4OAc, AcOH.
3. Results and discussion
3.1. Antibacterial activity
As shown in Table 1, the cyclohexyl derivative 7 having
no basic amine did not exhibit any antibacterial activity.
This result suggests that basic amines in the R2 moiety
appended on the pyrazoles are essential for antibacterial
activity. The 3-piperidyl analogue 8 showed 2-fold more
potent antibacterial activity against the four strains of
bacteria than the corresponding 4-piperidyl derivative
2. However, the 2-piperidyl analogue 10 showed four-
to eightfold less potent antibacterial activity against S.
aureus than the 4-piperidyl derivative 2 and the 3-piper-
idyl derivative 8. These results suggest that the position
of NH group in the R2 moiety is important for antibac-
terial activity.
To investigate the effect of basic amine appended on the
pyrazole ring on the antibacterial activity, the 4-piper-
idyl moiety in 2 was transformed to other groups
(7–14) and the in vitro antibacterial activity of the com-
pounds obtained was evaluated against two Gram-posi-
tive bacteria, that is a drug susceptible strain, S. aureus
FDA 209P and a multidrug resistant strain, S. aureus
KMP9 (MRSA), and against two Gram-negative bacte-
ria that is a susceptible strain, E. coli NIHJ JC-2 and a
multidrug efflux pump mutant, E. coli W3110 DacrA.
For comparison, the in vitro antibacterial activity of
sparfloxacin and novobiocin has also been evaluated.
Compound 11 with an acyclic substituent, N-methylami-
nopropyl, exhibited twofold and fourfold more potent
Table 1. Antibacterial activity of the pyrazole derivatives
Cl
Cl
N
N
N
N
R2
R1
H
R2
H
R1
9, 13, 16
2, 7, 8, 10-12, 14, 15
Compd
R1
H-R2
MIC (lg/mL)
S. aureus
E. coli
W3110 DacrAb
FDA 209P
KMP9a
NIHJ JC-2
2
4-PhO
4-PhO
4-PhO
4-PhO
4-PhO
4-PhO
4-PhO
4-PhO
4-PhO
3-PhCH2O
3-PhCH2O
––
4-Piperidyl
c-Hexyl
4
>128
4
32
4
>128
7
>128
2
>128
16
32
8
3-Piperidyl
3-Piperidyl
2-Piperidyl
2
2
16
9
4
4
10
11
12
13
14
15
16
1
16
2
16
2
>128
8
16
8
CH3NHCH2CH2CH2
CH3NHCH2
CH3NHCH2
PhCH2NHCH2
4-Piperidyl
4-Piperidyl
––
2
4
4
2
8
8
>128
>128
32
4
64
2
128
2
>128
1
4
4
32
2
64
0.125
0.25
64
128
0.25
128
0.032
64
64
0.004
0.5
SPFX
NB
––
––
––
––
a Multidrug resistant S. aureus.
b A multidrug efflux pump mutant.

5518
A. Tanitame et al. / Bioorg. Med. Chem. 12 (2004) 5515–5524
antibacterial activity against S. aureus and E. coli NIHJ
JC-2, respectively, than the lead compound 2. Com-
pound 12 having another acyclic substituent, N-methyl-
aminomethyl, showed the same or twofold more potent
antibacterial activity against the four strains of bacteria
studied compared to compound 2. However the N-benz-
ylaminomethyl derivative 14 showed 16–32-fold less
potent antibacterial activity against S. aureus than com-
pound 2. The SARs of these acyclic substituent suggest
that contrary to cyclic amines, the position of NH group
in the R2 moiety is not important for antibacterial activ-
ity. It is also noted that a small group, such as a methyl
on NH retains strong potency whereas a bulky group,
such as benzyl weaken the potency.
is assumed that the imidazole ring induces potent anti-
bacterial activity against not only Gram-positive bacte-
ria but against Gram-negative bacteria. Since the
imidazole derivatives showed nearly the same antibacte-
rial activity against E. coli NIHJ JC-2 and W3110
DacrA, it is presumed that these derivatives are not
affected by bacterial outer membrane pump.
The SARs of the pyrazole derivatives described in this
study can be summarized as follows: a basic amine
structure on the R2 moiety of the pyrazoles is essential
for antibacterial activity. In addition, the position of
the nitrogen atom in the cyclic amine and the size of
the substituents on the nitrogen atom of the acyclic
amine are important for potent antibacterial activity.
Although no significant difference in the antibacterial
activity against S. aureus among the three types of azole,
the imidazole derivatives had more potent antibacterial
activity against E. coli NIHJ JC-2 than the correspond-
ing pyrazoles. On the whole, the order of antibacterial
activity for these five-membered ring derivatives can be
summarized as follows: imidazole > pyrazole > oxazole.
The 3-benzyloxyphenyl pyrazole 15 revealed twofold
more potent antibacterial activity against S. aureus
and the same potency against E. coli NIHJ JC-2 as
compound 2. When regioisomers of the synthesized
compounds were compared, the 3-aminoalkyl-1-(3-
chlorophenyl)pyrazoles 8, 12 and 15 showed slightly
more potent antibacterial activity than the correspond-
ing 5-aminoalkyl-1-(3-chlorophenyl)pyrazoles 9, 13
and 16. Among all pyrazole analogues synthesized in
this study, compounds 8, 11 and 15 had comparatively
strong antibacterial activity against the four strains of
bacteria studied.
3.2. Inhibitory effects against DNA gyrase and topo-
isomerase IV
To elucidate the mechanism by which the pyrazole,
oxazole and imidazole derivatives induce their antibac-
terial activity, the inhibitory activity of selected com-
pounds (15, 22 and 25) against DNA gyrase and
topoisomerase IV isolated from E. coli was exam-
ined.19,20 As shown in Table 3, the IC50 values of the ini-
tial lead compound 1 against DNA gyrase and
topoisomerase IV were >128 and 128lg/mL, respec-
tively. Compounds 15, 22 and 25 having the same three
substituents (i.e., 4-piperidyl, 3-chlorophenyl and 3-benz-
yloxyphenyl) showed moderate inhibition against the
two enzymes (IC50 = 9.4–25lg/mL). The oxazole deriva-
tive 22 and the imidazole derivative 25 showed twofold
more potent inhibitory activity against DNA gyrase
than the pyrazole derivative 15. On the other hand, com-
pounds 22 and 25 showed an inhibitory activity against
topoisomerase IV almost similar to that of the pyrazole
derivative 15.
Next, the effects of a transformation of the pyrazole ring
of compound 2 on the antibacterial activity were exam-
ined (Table 2). The oxazole and imidazole derivatives
configured with three substituents in the structure of
the pyrazole derivatives 2 and 15 (i.e., 4-piperidyl, 3-
chlorophenyl and a substituted phenyl) were synthesized.
The oxazole derivative 21 and 22 showed the same anti-
bacterial activity against S. aureus and less potent anti-
bacterial activity against E. coli NIHJ JC-2 compared
to the corresponding pyrazole derivatives 2 and 15.
On the other hand, the imidazole derivatives 24 and 25
showed similar antibacterial activity against S. aureus
and four- to eightfold more potent antibacterial activity
against E. coli NIHJ JC-2 compared to the correspond-
ing pyrazole derivatives 2 and 15. From these results, it
Table 2. Antibacterial activity of the oxazole and imidazole derivatives
R4
X
N
R3
HN
Compd
X
R3
R4
MIC (lg/mL)
S. aureus
E. coli
FDA 209P
KMP9a
NIHJ JC-2
W3110 DacrAb
21
22
24
25
O
4-PhO
3-Cl
3-Cl
4
2
2
2
4
2
4
2
128
64
8
4
4
2
2
O
3-PhCH2O
3-Cl
3-PhCH2O
NH
NH
4-PhO
3-Cl
4
a Multidrug resistant S. aureus.
b A multidrug efflux pump mutant.

A. Tanitame et al. / Bioorg. Med. Chem. 12 (2004) 5515–5524
5519
Table 3. Inhibitory effects of selected compounds against DNA gyrase
and topoisomerase IV
As shown in Table 4, MIC values of sparfloxacin against
the susceptible strains (MSSA, PSSP and VSE) were
0.125, 0.125 and 0.25lg/mL, respectively, and those
against MRSA, PRSP and VRE were 128, 4 and
64lg/mL, respectively. MIC values of novobiocin
against S. aureus RN4220 and S. aureus N175 derived
from RN4220 were 0.125 and 4lg/mL, respectively.
On the other hand, the pyrazole derivatives 8, 11 and
15, the oxazole derivative 22 and the imidazole deriva-
tive 25 revealed almost the same antibacterial activity
against both susceptible and resistant Gram-positive
bacteria. Although sparfloxacin and novobiocin ratio
of MIC values against susceptible and resistant strains
was more than 32, that of the pyrazole, oxazole and imi-
dazole derivatives ranged between 1 and 2.
Compd
IC50 (lg/mL)a
Gyraseb
Topo IVc
1
15
>128
18.8
9.4
128
25
22
25
25
9.4
18.8
6.9
3.5
SPFX
NB
0.25
0.25
a Isolated from E. coli.
b DNA gyrase supercoiling activity.
c Topoisomerase IV decatenation activity.
There was a good correlation between the MICs and the
IC50s (Tables 1 and 2), suggesting that inhibition of the
DNA gyrase and topoisomerase IV by the pyrazole,
oxazole and imidazole derivatives suppresses bacterial
cell growth. While sparfloxacin ratio of inhibition of
DNA gyrase and topoisomerase IV was more than 20,
that of the pyrazole, oxazole and imidazole derivatives
ranged between 1.3 and 2.7. These results suggest that
the pyrazole, oxazole and imidazole derivatives would
not be easily tolerated by bacteria.
The pyrazole, oxazole and imidazole derivatives were
also effective against quinolone- and coumarin-resistant
Gram-positive bacteria with compounds 8, 11, 15, 22
and 25 showing a more potent antibacterial activity
against clinically isolated quinolone-resistant Gram-pos-
itive bacteria than sparfloxacin, and compounds 22 and
25 revealing the same antibacterial activity against labo-
ratory isolated coumarin-resistant Gram-positive as
novobiocin. Compounds 8 and 15 demonstrated the
most potent antibacterial activity against susceptible
and resistant Gram-positive bacteria with across-the-
board MIC values of 1–4lg/mL.
3.3. Effect against multidrug resistant Gram-positive
bacteria
To examine whether the pyrazole, oxazole and imidaz-
ole derivatives are effective against multidrug resistant
Gram-positive bacteria, MIC values of selected com-
pounds against quinolone-resistant clinical isolates and
coumarin-resistant laboratory isolates of Gram-positive
bacteria were determined and compared with those of
sparfloxacin and novobiocin. The strains of bacteria
used for MIC values determination were: S. aureus
KMP9 (MRSA; a sparfloxacin-, clarithromycin- and
ampicillin-resistant strain), S. aureus N175 (a novobio-
cin-resistant strain) derived from RN4220,21 Streptococ-
cus (S.) pneumoniae KT2524 (PRSP; a sparfloxacin-,
clarithromycin- and ampicillin-resistant strain) and
Enterococcus (E.) faecium KU1778 (VRE; a sparfloxa-
cin-, clarithromycin-, ampicillin- and vancomycin-resist-
ant strain).
It is therefore clear that the pyrazole, oxazole and imi-
dazole derivatives have potent antibacterial activity
against both susceptible and quinolone- and coumarin-
resistant Gram-positive bacteria. Additionally, the
results above suggest that the mode of action of these
analogues against DNA gyrase and topoisomerase IV
is different from that of the quinolones or the coumarins.
4. Conclusions
In this study, we have described the synthesis and SARs
of newpyrazole, oxazole and imidazole analogues.
Among the pyrazole analogues, compounds 8 and 11
had comparatively strong antibacterial activity, whereas
Table 4. Antibacterial activity of selected pyrazole derivatives against susceptible and resistant Gram-positive bacteria
Organisma
MIC (lg/mL)
8
11
15
22
25
SPFX
0.125
128
0.125
NB
S. aureus FDA 209P (MSSA)b
S. aureus KMP9 (MRSA)c
S. aureus RN4220b
2
2
2
2
2
2
2
4
4
4
4
4
2
0.25
0.25
0.125
4
2
2
2
––
––
1
––
––
4
––
––
2
4
S. aureus N175d
4
0.125
0.125
4
S. pneumoniae ATCC49619 (PSSP)b
S. pneumoniae KT2524 (PRSP)e
E. faecium ATCC19434 (VSE)b
E. faecium KU1778 (VRE)f
4
0.5
0.5
2
2
4
2
4
4
8
4
16
8
0.25
2
4
4
64
2
a SPFX: sparfloxacin, CAM: clarithromycin, ABPC: ampicillin, VCM: vancomycin.
b Susceptible strain.
c SPFX-, CAM-, ABPC-resistant strain.
d S. aureus N175 (GyrB; R144I) was derived from RN4220 with selection of novobiocin.
e SPFX-, CAM-, ABPC-resistant strain.
f SPFX-, CAM-, ABPC-, VCM-resistant strain.

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A. Tanitame et al. / Bioorg. Med. Chem. 12 (2004) 5515–5524
among the oxazole and imidazole analogues, com-
pounds 22 and 25 showed comparatively strong antibac-
terial activity. These selected compounds 8, 11, 22 and
25 possessed potent antibacterial activity against not
only susceptible strains, but also against multidrug
resistant strains. In addition, 8, 11, 22 and 25 showed
a more potent antibacterial activity against clinically iso-
lated quinolone-resistant Gram-positive bacteria than
sparfloxacin. Compounds 22 and 25 revealed the same
antibacterial activity against laboratory isolated couma-
rin-resistant Gram-positive bacteria compared to
novobiocin.
5.3. General procedure for 1,3-diketones 4a–g
To a solution of lithium hexamethyldisilazide (11mmol)
in THF (30mL) was added benzophenone derivatives
(10mmol) in THF (10mL) at ꢀ78°C under argon
atmosphere, and the resulting yellowsuspension was
stirred for 30min at the same temperature (solution
A). A solution of 1,10-carbonyldi-1H-imidazole (CDI,
16.2g, 10mmol) and an N-(tert-butoxycarbonyl) amino
acid derivatives 3 [e.g., 1-(tert-butoxycarbonyl)piperi-
dine-4-carboxylic acid] (10mmol) in THF (30mL) was
stirred for 45min at room temperature (solution B).
The resulting solution B was added dropwise to solution
A over 30min at ꢀ78°C. After removal of the cold bath,
the reaction mixture was warmed to room temperature
and stirred for 12h. To this reaction mixture was added
10% aqueous citric acid (30mL), and the resulting solu-
tion was taken up in AcOEt (100mL), washed with
brine, dried over MgSO4, and concentrated to dryness.
The residue was purified by silica gel column chroma-
tography (MeOH/CHCl3) to give 4 as an oil.
Though the inhibitory activity of sparfloxacin against
DNA gyrase was more than 20-fold that against topo-
isomerase IV, the pyrazole, oxazole and imidazole deriv-
atives synthesized in this study showed almost the same
inhibitory activity against both enzymes. These results
suggest that the pyrazole, oxazole and imidazole deriva-
tives would not be easily resisted by bacteria. We are
pursuing further modifications of the pyrazole, oxazole
and imidazole scaffold to obtain more potent inhibitors
of both DNA gyrase and topoisomerase IV.
5.3.1. tert-Butyl 3-[1,3-dioxo-3-(4-phenoxyphenyl)-1-pro-
pyl]piperidine-1-carboxylate (4a). 56% Yield. H NMR
1
(300MHz, CDCl3): d 1.48 (s, 9H), 1.61–1.67 (m, 3H),
2.31–2.36 (m, 2H), 2.94–2.98 (m, 2H), 4.09 (br, 2H),
6.46 (s, 1H), 6.99 (d, 2H, J = 8.7Hz), 7.04–7.06 (m,
2H), 7.15–7.20 (m, 1H), 7.35–7.40 (m, 2H), 7.83 (d,
2H, J = 8.7Hz), 16.02 (br, 1H); HRMS calcd for
424.2124, found 424.2127.
5. Experimental
5.1. Spectroscopic measurements
All melting points were determined on a Yanaco micro-
melting point apparatus and are uncorrected. H NMR
1
spectra were taken at 200MHz on a Varian Gemini
spectrometer, at 300MHz on a JEOL spectrometer, or
at 400MHz on a Varian Mercury-400 spectrometer in
DMSO-d6 or CDCl3. All chemical shift values are
reported as ppm (d) values with tetramethylsilane as
internal standard. The abbreviations used are as follows:
s; singlet, d; doublet, t; triplet, q; quartet, m; multiplet,
and br; broad. Mass spectra were obtained on a JEOL
JMS-SX 102A QQ for fast atom bombardment mass
spectra (FABMS) or a Hitachi M-1000 LC APCI
mass spectrometer for atmospheric pressure chemical
ionization mass spectra (APCIMS). For elements
analysis, C, H and N were analyzed using a CE instru-
ments EA1110 CHN elements analyzer, and F, Cl and
Br were analyzed using a Yokogawa IC-7000 ion
chromatograph. All analytical results were within
0.4% of the theoretical values obtained by a given
formula.
5.3.2. tert-Butyl 2-[1,3-dioxo-3-(4-phenoxyphenyl)pro-
pyl]piperidine-1-carboxylate (4b). 29% Yield. H NMR
1
(300MHz, CDCl3): d 1.44 (s, 9H), 1.65–1.69 (m, 3H),
1.93–1.97 (m, 1H), 2.43–2.51 (m, 1H), 2.82–3.07
(m, 2H), 3.90–4.13 (m, 2H), 6.17 (s, 1H), 6.96 (d,
2H, J = 8.8Hz), 7.01–7.04 (m, 2H), 7.12–7.17 (m,
1H), 7.27–7.37 (m, 2H), 7.85 (d, 2H, J = 8.8Hz),
15.95 (br, 1H); HRMS calcd for 424.2124, found
424.2119.
5.3.3.
1-[(tert-Butoxycarbonyl)-N-methyl]amino-6-(4-
phenoxyphenyl)hexane-4,6-dione (4c). 29% Yield. 1H
NMR (300MHz, CDCl3): d 1.45 (s, 9H), 1.81–1.95 (m,
2H), 2.42 (t, 2H, J = 7.3Hz), 2.86 (s, 3H), 3.29 (t, 2H,
J = 7.0Hz), 6.13 (s, 1H), 6.99 (d, 2H, J = 8.8Hz),
7.02–7.08 (m, 2H), 7.16–7.21 (m, 1H), 7.36–7.41 (m,
2H), 7.86 (d, 2H, J = 8.8Hz), 16.08 (br, 1H); HRMS
calcd for 412.2124, found 412.2119.
5.2. Starting material
5.3.4.
1-[(tert-Butoxycarbonyl)-N-methyl]amino-4-(4-
phenoxyphenyl)butane-2,4-dione (4d). 49% Yield. 1H
NMR (400MHz, CDCl3): d 1.45 (s, 9H), 2.99 (s, 3H),
4.03 (s, 2H), 6.12 (s, 1H), 7.01 (d, 2H, J = 8.7Hz),
7.04–7.23 (m, 3H), 7.39–7.42 (m, 2H), 7.83 (d, 2H,
J = 8.7Hz), 15.98 (br, 1H); HRMS calcd for 383.1732,
found 383.1735.
The following compounds 3 were commercially availa-
ble from Aldrich or Watanabe Chemical Industries, Ltd,
Japan:
1-(tert-butoxycarbonyl)piperidine-4-carboxy-
lic acid, 1-(tert-butoxycarbonyl)piperidine-3-carboxy-
lic acid, 1-(tert-butoxycarbonyl)piperidine-2-carboxylic
acid, N-(tert-butoxycarbonyl)-N-methylglycine and N-
(tert-butoxycarbonyl)-N-benzylglycine. The following
compounds were synthesized according to published
procedures: N-(tert-butoxycarbonyl)-N-methyl-c-ami-
5.3.5.
1-[N-Benzyl-(tert-butoxycarbonyl)]amino-4-(4-
phenoxyphenyl)butane-2,4-dione (4e). 33% Yield. 1H
NMR (300MHz, CDCl3): d 1.46 (s, 9H), 3.93 (s, 2H),
4.58 (s, 2H), 6.03 (s, 1H), 6.99 (d, 2H, J = 8.6Hz),
7.05–7.08 (m, 2H), 7.17–7.42 (m, 8H), 7.79 (d, 2H,
nobutylic
acid22
and
1-(tert-butoxycarbonyl)-4-
formylpiperidine.23

A. Tanitame et al. / Bioorg. Med. Chem. 12 (2004) 5515–5524
5521
J = 8.6Hz), 16.16 (br, 1H); HRMS calcd for 460.2124,
found 460.2129.
3.17–3.51 (m, 3H), 6.96 (s, 1H), 7.04–7.22 (m, 5H),
7.39–7.78 (m, 6H), 7.85 (d, 2H, J = 8.3Hz), 8.76–9.16
(m, 2H); MS (APCI+), m/z 430 (MH+). Anal. Calcd
for C26H24ClN3OÆ1.2HClÆ2H2O: C, 61.55; H, 5.77; N,
8.24; Cl, 15.30. Found: C, 61.55; H, 5.60; N, 8.17; Cl,
15.12.
5.3.6. tert-Butyl 4-[3-(3-benzyloxyphenyl)-1,3-dioxo-1-
propyl]piperidine-1-carboxylate (4f). 68% Yield. 1H
NMR (300MHz, CDCl3): d 1.39 (s, 9H), 1.45–1.60 (m,
2H), 1.73–1.76 (m, 2H), 2.28–2.38 (m, 1H), 2.62–2.70
(m, 2H), 3.98–4.08 (m, 2H), 4.95 (s, 2H), 6.05 (s, 1H),
6.98 (m, 1H), 7.19–7.42 (m, 8H), 16.13 (br, 1H); HRMS
calcd for 437.2202, found 437.2204.
5.4.3. 1-(3-Chlorophenyl)-5-(4-phenoxyphenyl)-3-(2-pip-
eridyl)pyrazole hydrochloride (10). 8% Yield from 4b,
mp 245–248°C (acetone–Et2O). 1H NMR (400MHz,
DMSO-d6): d 1.56 (br, 1H), 1.76–1.94 (m, 3H), 2.11–
2.15 (m, 2H), 3.08 (br, 2H), 4.27 (br, 1H), 7.07–7.09
(m, 4H), 7.16–7.26 (m, 2H), 7.38–7.48 (m, 2H), 7.55–
7.69 (m, 4H), 7.79–7.82 (m, 2H), 9.31 (br, 1H), 9.37
(br, 1H); MS (APCI+), m/z 430 (MH+). Anal. Calcd
for C26H24ClN3OÆ1.25HClÆ0.75H2O: C, 63.86; H, 5.51;
N, 8.59; Cl, 16.31. Found: C, 63.96; H, 5.29; N, 8.58;
Cl, 16.02.
5.3.7.
1-Cyclohexyl-3-(4-phenoxyphenyl)propane-1,3-
1
dione (4g). 69% Yield. H NMR (300MHz, CDCl3): d
1.20–1.51 (m, 5H), 1.69–1.95 (m, 5H), 2.30 (tt, 1H,
J = 11.4Hz, J = 3.1Hz), 6.12 (s, 1H), 7.00 (d, 2H,
J = 8.9Hz), 7.05–7.08 (m, 2H), 7.15–7.20 (m, 1H),
7.35–7.42 (m, 2H), 7.86 (d, 2H, J = 8.9Hz), 15.95 (br,
1H); HRMS calcd for 323.1647, found 323.1645.
5.4. General procedure for 1-(3-chlorophenyl)pyrazole
derivatives 8–16
5.4.4. 1-(3-Chlorophenyl)-3-[3-(N-methyl)aminopropyl]-5-
(4-phenoxyphenyl)pyrazole hydrochloride (11). 53% Yield
from 4c, prepared by lyophilization. 1H NMR
(300MHz, DMSO-d6): d 1.99–2.09 (m, 2H), 2.52–2.56
(m, 3H), 2.73 (t, 2H, J = 7.3Hz), 2.98 (br, 2H), 6.54 (s,
1H), 6.98–7.01 (m, 2H), 7.04 (d, 2H, J = 8.4Hz), 7.16–
7.21 (m, 2H), 7.26 (d, 2H, J = 8.4Hz), 7.35–7.45 (m,
5H), 9.03 (br, 2H); MS (APCI+), m/z 418 (MH+). Anal.
Calcd for C25H24ClN3OÆ1.25HClÆ0.75H2O: C, 63.19; H,
5.66; N, 8.84; Cl, 16.41. Found: C, 63.13; H, 5.80; N,
8.76; Cl, 16.23.
(a) A solution of 1,3-diketone derivatives 4 (10mmol), 3-
chlorophenylhydrazine hydrochloride (3.58g, 20mmol)
and 2mol/L NaOH (19mmol) in EtOH (30mL) was
heated to reflux for 12h and cooled to 0°C. To this reac-
tion mixture was added 10% aqueous citric acid (30mL)
and the resulting solution was taken up in AcOEt
(100mL), washed successively with saturated aqueous
NaHCO3 and brine, and dried over MgSO4. The residue
was purified by silica gel column chromatography
(MeOH/CHCl3) to give a mixture of the 1-(3-chlorophe-
5.4.5. 1-(3-Chlorophenyl)-3-(N-methylaminomethyl)-5-(4-
phenoxyphenyl)pyrazole fumarate (12). 14% Yield from
4d, mp 145–148°C (i-PrOH–Et2O). 1H NMR
(200MHz, DMSO-d6): d 2.48–2.54 (m, 3H), 4.05 (br,
2H), 6.49 (s, 2H), 6.72 (s, 1H), 6.98–7.08 (m, 4H),
7.15–7.28 (m, 4H), 7.38–7.46 (m, 5H), 9.87 (br, 3H);
MS (APCI+), m/z 390 (MH+). Anal. Calcd for
C23H20ClN3OÆC4H4O4: C, 64.10; H, 4.78; N, 8.31; Cl,
7.01. Found: C, 64.11; H, 4.76; N, 8.31; Cl, 6.91.
nyl)-3-[N-(tert-butoxycarbonyl)]aminoalkylpyrazoles
and the 1-(3-chlorophenyl)-5-[N-(tert-butoxycarbo-
nyl)]aminoalkylpyrazoles 6.
5
(b) A solution of the mixture of 5 and 6 (10mmol) and
CF3COOH (0.5mL) in CH2Cl2 (30mL) was stirred for
4h at room temperature. The reaction mixture was con-
centrated to dryness, and the resulting residue was sep-
arated using CHP-20P (reverse phase, Mitsubishi
Chemical Co., Ltd, Japan; CH3CN/H2O) column chro-
matography and/or recrystallization from suitable
solvents to give the 3-aminoalkyl-1-(3-chlorophenyl)pyr-
azole derivatives and the 5-aminoalkyl-1-(3-chlorophe-
nyl)pyrazole derivatives.
5.4.6. 1-(3-Chlorophenyl)-5-(N-methylaminomethyl)-3-(4-
phenoxyphenyl)pyrazole hydrochloride (13). 57% Yield
from 4d, mp 184–187°C (CH3CN). 1H NMR (200MHz,
DMSO-d6): d 2.48–2.53 (m, 3H), 4.25 (m, 2H), 7.05–
7.31 (m, 6H), 7.42 (d, 2H, J = 8.4Hz), 7.47–7.76 (m,
4H), 7.83 (d, 2H, J = 8.4Hz), 9.93 (br, 2H); MS (APCI+),
m/z 390 (MH+). Anal. Calcd for C23H20ClN3OÆ0.95-
HClÆ0.10H2O: C, 64.80; H, 5.00; N, 9.86; Cl, 16.22.
Found: C, 64.53; H, 4.91; N, 10.00; Cl, 16.07.
5.4.1. 1-(3-Chlorophenyl)-5-(4-phenoxyphenyl)-3-(3-pip-
eridyl)pyrazole hydrochloride (8). 38% Yield from 4a,
prepared by lyophilization. 1H NMR (400MHz,
DMSO-d6): d 1.68–1.86 (m, 3H), 2.11–2.14 (m, 1H),
2.85–2.92 (m, 1H), 3.04–3.10 (m, 1H), 3.17–3.29 (m,
2H), 3.49–3.51 (m, 1H), 6.63 (s, 1H), 6.99 (d, 2H,
J = 8.8Hz), 7.04 (d, 2H, J = 7.6Hz), 7.15–7.20 (m,
2H), 7.24 (d, 2H, J = 8.8Hz), 7.38–7.43 (m, 5H), 8.81–
9.19 (m, 2H); MS (APCI+), m/z 430 (MH+). Anal. Calcd
for C26H24ClN3OÆHClÆ2H2O: C, 62.15; H, 5.82; N, 8.36;
Cl, 14.11. Found: C, 62.38; H, 5.70; N, 8.32; Cl, 14.12.
5.4.7. 3-(N-Benzylaminomethyl)-1-(3-chlorophenyl)-5-(4-
phenoxyphenyl)pyrazole hydrochloride (14). 25% Yield
1
from 4e as an oil. H NMR (400MHz, DMSO-d6): d
4.21 (s, 2H), 4.24 (s, 2H), 6.91 (s, 1H), 7.00–7.10 (m,
4H), 7.16–7.26 (m, 4H), 7.37–7.49 (m, 8H), 7.54–7.60
(m, 2H), 9.99 (br, 2H); HRMS calcd for 466.1686, found
466.1693.
5.4.2. 1-(3-Chlorophenyl)-3-(4-phenoxyphenyl)-5-(3-pip-
eridyl)pyrazole hydrochloride (9). 3% Yield from 4a,
prepared by lyophilization. 1H NMR (200MHz,
DMSO-d6): d 1.64–2.04 (m, 4H), 2.83–3.09 (m, 2H),
5.4.8. 5-(3-Benzyloxyphenyl)-1-(3-chlorophenyl)-3-(4-pip-
eridyl)pyrazole hydrochloride (15). 19% Yield from 4f,
mp 90–93°C (acetone–Et2O). 1H NMR (200MHz,
DMSO-d6): d 1.84–2.03 (m, 2H), 2.13–2.21 (m, 2H),

5522
A. Tanitame et al. / Bioorg. Med. Chem. 12 (2004) 5515–5524
2.96–3.10 (m, 3H), 3.31–3.34 (m, 2H), 5.05 (s, 2H), 6.59
(s, 1H), 6.76–6.82 (m, 1H), 6.94–7.18 (m, 3H), 7.25–7.44
(m, 9H), 9.02 (br, 1H), 9.18 (br, 1H); MS (APCI+), m/z
444 (MH+). Anal. Calcd for C27H26ClN3OÆHClÆ0.50-
H2O: C, 66.26; H, 5.77; N, 8.59; Cl, 14.49. Found: C,
66.37; H, 5.84; N, 8.55; Cl, 14.79.
up in Et2O (50mL), washed with 0.5mol/L NaOH
(15mL), dried over MgSO4, and concentrated to dry-
ness. The residue was purified by silica gel column chro-
matography (CHCl3) to give 2.01g (19% from 17a) of
1
19a as an oil. H NMR (200MHz, DMSO-d6): d 4.61
(d, 1H, J = 6.0Hz) 5.84 (d, 1H, J = 6.0Hz), 6.94 (d,
2H, J = 8.6Hz), 7.01–7,42 (m, 9H), 7.88 (d, 2H,
J = 8.6Hz); MS (APCI+), m/z 339 (MH+).
5.4.9. 3-(3-Benzyloxyphenyl)-1-(3-chlorophenyl)-5-(4-pip-
eridyl)pyrazole hydrochloride (16). 41% Yield from 4f,
mp 135–138°C (CH3CN). 1H NMR (200MHz,
DMSO-d6): d 1.75–1.98 (m, 4H), 2.85–2.99 (m, 2H),
3.07–3.32 (m, 3H), 5.17 (s, 2H), 6.88 (s, 1H), 6.97–7.04
(m, 1H), 7.31–7.70 (m, 12H), 9.11 (br, 2H); MS (APCI+),
m/z 444 (MH+). Anal. Calcd for C27H26ClN3OÆ1.40-
HClÆ0.25H2O: C, 64.92; H, 5.63; N, 8.41; Cl, 17.03.
Found: C, 65.16; H, 5.45; N, 8.57; Cl, 16.90.
5.5.2.
2-(3-Benzyloxyphenyl)-1-(3-chlorophenyl)-2-
hydroxyethane-1-one (19b). In a similar manner to that
described above, 19b was prepared from 17b via 18b in
1
78% overall yield. H NMR (300MHz, CDCl3): d 4.40
(d, 1H, J = 6.1Hz), 5.05 (s, 2H), 5.85 (d, 1H,
J = 6.1Hz), 6.89–6.94 (m, 2H), 7.23–7.51 (m, 9H),
7.70–7.90 (m, 2H); MS (APCI+), m/z 353 (MH+).
5.4.10. 1-(3-Chlorophenyl)-3-(cyclohexyl)-5-(4-phenoxy-
phenyl)pyrazole (7). A solution of 1-cyclohexyl-3-(4-
phenoxyphenyl)propane-1,3-dione (2.11g, 6.54mmol)
and 3-chlorophenylhydrazine hydrochloride (2.34g,
13.1mmol) in EtOH (20mL) was heated to reflux for
12h and cooled to 0°C. To this reaction mixture was
added 10% aqueous citric acid (20mL) and the resulting
solution was taken up in AcOEt (70mL), washed succes-
sively with saturated aqueous NaHCO3 and brine, dried
over MgSO4 and evaporated to dryness. The residue was
then purified by silica gel column chromatography
(AcOEt/n-hexane) to give 1.15g (41%) of 7 as an oil.
1H NMR (300MHz, CDCl3): d 1.21–1.48 (m, 5H),
1.70–1.91 (m, 6H), 6.48 (s, 1H), 7.00–7.18 (m, 5H),
7.28–7.53 (m, 6H), 7.81 (d, 2H, J = 8.8Hz); HRMS
calcd for 429.1734, found 429.1730.
5.6. Synthesis of oxazole derivatives 21, 22
5.6.1. 5-(3-Chlorophenyl)-4-(4-phenoxyphenyl)-2-(4-pip-
eridyl)oxazole fumarate (21). A solution of 19a (1.60g,
4.72mmol), 1-(tert-butoxycarbonyl)piperidine-4-carb-
oxylic acid (1.19g, 5.18mmol), 1-ethyl-3[3-(dimethyl-
amino)propyl]carbodiimide
hydrochloride
(EDC,
0.92g, 5.18mmol) and 4-dimethylaminopyridine
(DMAP, 0.58g, 4.72mmol) in CH2Cl2 (40mL) was stir-
red for 6h at room temperature. To this reaction mix-
ture was added 10% aqueous citric acid (30mL) and
the resulting solution was taken up in CHCl3 (50mL),
dried over MgSO4 and concentrated to dryness. The res-
idue was dissolved in AcOH (40mL) and was added
NH4OAc (0.91g, 11.8mmol). The reaction mixture
was heated to reflux for 6h and cooled to room temper-
ature. The solvent was evaporated, and the residue was
purified by silica gel column chromatography (2:1
CHCl3/MeOH) and recrystallization from EtOH–Et2O
to give 1.16g (51% from 19a) of 21, mp 145–148°C.
1H NMR (400MHz, DMSO-d6): d 1.62–1.74 (m, 1H),
1.91–2.02 (m, 2H), 2.22–2.26 (m, 1H), 2.83–2.92 (m,
1H), 2.99–3.07 (m, 1H), 3.18–3.32 (m, 3H), 6.48 (s,
2H), 6.63 (br, 2H), 7.04–7.10 (m, 4H), 7.16–7.21 (m,
1H), 7.42–7.51 (m, 5H), 7.54–7.61 (m, 3H), 8.12 (br,
1H); HRMS calcd for 430.1448, found 430.1454.
5.5. Synthesis of 2-hydroxyethane-1-one 19a,b
5.5.1. 2-(3-Chlorophenyl)-1-(4-phenoxyphenyl)-2-hydro-
xyethane-1-one (19a). (a) To a solution of 17a (6.33g,
31.3mmol) in CH2Cl2 (30mL) was added dropwise tri-
methylsilylcyanide (3.11g, 31.3mmol) at 0°C and added
zinc iodide (15mg) at the same temperature. The mix-
ture was stirred for 48h at room temperature. The sol-
vent was evaporated and the residue was purified by
silica gel column chromatography (CHCl3) to give 18a
as an oil, which was used in the next step without further
purification.
5.6.2. 5-(3-Benzyloxyphenyl)-4-(3-chlorophenyl)-2-(4-pip-
eridyl)oxazole fumarate (22). In a similar manner to that
described above, 22 was prepared from 19b via 20b in
44% overall yield, mp 123–126°C (CHCl3). H NMR
(300MHz, CDCl3): d 1.67 (br, 3H), 1.81–1.93 (m, 2H),
2.10–2.14 (m, 2H), 2.74–2.81 (m, 2H), 2.97–3.05 (m,
1H), 3.19–3.23 (m, 2H), 5.00 (s, 2H), 6.95–6.97 (m,
1H), 7.15–7.18 (m, 2H), 7.23–7.39 (m, 10H), 7.50–7.70
(m, 2H); MS (APCI+), m/z 445 (MH+). Anal. Calcd
for C27H25ClN2O2ClÆC4H4O4: C, 66.37; H, 5.21; N,
4.99; Cl, 6.32. Found: C, 66.06; H, 4.86; N, 4.93; Cl,
6.43.
1
(b) To a solution of 1mol/L lithium hexamethyldisilaz-
ide (25mmol) in THF (50mL) was added dropwise
18a (7.44g, 25mmol) in THF (15mL) at ꢀ78°C under
argon atmosphere. The yellowsuspension was stirred
for 30min at the same temperature. To the resulting
solution was added dropwise 3-chlorobenzaldehyde
(3.51g, 25mmol) in THF (20mL) at the same tempera-
ture. After removal of the cold bath, the reaction mix-
ture was raised to room temperature and stirred for
12h. To this reaction mixture was added saturated aque-
ous NH4Cl (60mL), and the solution was taken up in
AcOEt (100mL), washed with brine, dried over MgSO4,
and concentrated to dryness. The residue was dissolved
in MeOH (30mL) and was added 5% aqueous H2SO4
(15mL). The reaction mixture was stirred for 30min at
room temperature. The resulting solution was taken
5.7. Synthesis of imidazole derivatives 24, 25
5.7.1. 5-(3-Chlorophenyl)-4-(4-phenoxyphenyl)-2-(4-pip-
eridyl)imidazole difumarate (24). (a) A solution of
CuSO4 (1.35g, 8.50mmol) in pyridine (5mL) and H2O
(1mL) was heated to reflux for 10min and cooled to

A. Tanitame et al. / Bioorg. Med. Chem. 12 (2004) 5515–5524
5523
room temperature. To the resulting solution was added
References and notes
19a (1.44g, 4.25mmol) and reheated to reflux for 1h and
cooled to room temperature. The solvent was evapo-
rated and the residue was taken up Et2O (20mL) and
H2O (10mL). The organic layer was separated and dried
over MgSO4, and concentrated to dryness. The residue
was purified by silica gel column chromatography (3:1
CHCl3/n-hexane) to give 23a as an oil, which was used
in the next step without further purification.
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(b) A solution of 23a (0.65g, 1.93mmol), 1-(tert-butoxy-
carbonyl)-4-formylpiperidine (0.41g, 1.93mmol) and
NH4OAc (1.19g, 1.54mmol) in AcOH (15mL) was stir-
red for 4h at 90°C. The solvent was evaporated, and the
residue was taken up in CHCl3 (20mL), washed with
saturated aqueous NaHCO3, and dried over MgSO4.
The solvent was evaporated. The residue was purified
by CHP-20P (3:1 CH3CN/H2O) column chromatogra-
phy to give 0.22g (12% from 19a) of 24, mp 93–95°C
(EtOH–Et2O). 1H NMR (300MHz, DMSO-d6): d
1.89–2.02 (m, 2H), 2.10–2.16 (m, 2H), 2.95–3.06 (m,
3H), 3.30 (br, 5H), 3.32–3.36 (m, 2H), 6.51 (s, 4H),
7.00–7.06 (m, 4H), 7.13–7.18 (m, 1H), 7.24–7.49 (m,
9H); HRMS calcd for 429.1608, found 429.1599.
7. Boehm, H.; Boehringer, M.; Bur, D.; Gmuender, H.;
Hunber, W.; Klaus, W.; Kostrewa, D.; Kuehne, H.;
Luebbers, T.; Meunier-Keller, N.; Mueller, F. J. Med.
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9. Galm, U.; Heller, S.; Shapiro, S.; Page, M.; Li, S.; Heide,
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10. (a) Tanitame, A.; Oyamada, Y.; Ofuji, K.; Kyoya, Y.;
Suzuki, K.; Ito, H.; Kawasaki, M.; Nagai, K.; Wachi, M.;
Yamagishi, J. Bioorg. Med. Chem. Lett. 2004, 14, 2857–
2862; (b) Tanitame, A.; Oyamada, Y.; Ofuji, K.; Suzuki,
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5.7.2. 5-(3-Benzyloxyphenyl)-4-(3-chlorophenyl)-2-(4-pip-
eridyl)imidazole dihydrochloride (25). In a similar man-
ner to that described above, 25 was prepared from 19b
via 23b in 24% overall yield, mp 140–141°C (CH3CN).
1H NMR (200MHz, DMSO-d6): d 2.06–2.34 (m, 4H),
2.97–3.16 (m, 2H), 3.39–3.53 (m, 3H), 5.11 (s, 2H),
7.03 (d, J = 8.0Hz, 1H), 7.09–7.21 (m, 2H), 7.35–7.64
(m, 11H), 9.24 (br, 3H); MS (APCI+), m/z 444 (MH+).
Anal. Calcd for C27H26ClN3OClÆ2HClÆ1.30H2O: C,
60.02; H, 5.71; N, 7.78; Cl, 19.68. Found: C, 59.65; H,
5.33; N, 7.75; Cl, 19.71.
5.8. Microbiology
5.8.1. In vitro antibacterial activity. The minimal inhibi-
tory concentration (MIC) of each test-compound was
determined by a microdilution method according to
guidelines of the National Committee for Clinical Lab-
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5.8.2. Enzyme inhibition. DNA gyrase supercoiling and
topoisomerase IV decatenation assays using E. coli en-
zymes were carried out by the methods of Sato et al.19
and Peng and Marians,20 respectively.
Acknowledgements
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We are grateful to Drs. K. Chiba, Y. Furutani, S. Kato
and H. Yoshida for their encouragement throughout
this work and to Dr. M. Fujita for his helpful discus-
sions. Thanks are also due to members of the pharma-
cology section for their pharmacological support and
members of the analytical chemistry section for their ele-
ments analysis and spectral measurements. We also
thank Dr. S. Matsuyama, for supplying the E. coli
W3110 DacrA.

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