Synthesis and antibacterial activity of a novel series of potent DNA gyrase inhibitors. Pyrazole derivatives

Article abstract of DOI:10.1021/jm030394f

We have previously found that a pyrazole derivative 1 possesses antibacterial activity and inhibitory activity against DNA gyrase and topoisomerase IV. Here, we synthesized new pyrazole derivatives and found that 5-[(E)-2-(5-chloroindol-3-yl)vinyl]pyrazol

Full text of DOI:10.1021/jm030394f

J . Med. Chem. 2004, 47, 3693-3696
3693
Syn th esis a n d An tiba cter ia l Activity of a Novel Ser ies of P oten t DNA Gyr a se
In h ibitor s. P yr a zole Der iva tives
Akihiko Tanitame,*,† Yoshihiro Oyamada,‡ Keiko Ofuji,† Mika Fujimoto,‡ Noritaka Iwai,§ Yoichi Hiyama,†
Kenji Suzuki,† Hideaki Ito,‡ Hideo Terauchi,† Motoji Kawasaki,† Kazuo Nagai,# Masaaki Wachi,§ and
J un-ichi Yamagishi‡
Chemistry Research Laboratories, Pharmacology and Microbiology Research Laboratories, Dainippon
Pharmaceutical Co., Ltd., 33-94, Enoki, Suita, Osaka 564-0053, J apan, Department of Bioengineering, Tokyo
Institute of Technology, 4259, Nagatsuda, Midori-ku, Yokohama 226-8501, J apan, and Department of Biological Chemistry,
Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501, J apan
Received August 14, 2003
We have previously found that a pyrazole derivative 1 possesses antibacterial activity and
inhibitory activity against DNA gyrase and topoisomerase IV. Here, we synthesized new
pyrazole derivatives and found that 5-[(E)-2-(5-chloroindol-3-yl)vinyl]pyrazole 16 possesses
potent antibacterial activity and selective inhibitory activity against bacterial topoisomerases.
Many of the synthesized pyrazole derivatives were potent against clinically isolated quinolone-
or coumarin-resistant Gram-positive strains and had minimal inhibitory concentration values
against these strains equivalent to those against susceptible strains.
In tr od u ction
Over the past decade, bacterial DNA gyrase has
drawn much attention as a selected target for finding
potent antibacterial agents. Accordingly, a number of
synthetic quinolone antibacterial agents have been
developed and are now widely used for treatment of
bacterial infectious diseases. Quinolones inhibit DNA
gyrase and topoisomerase IV and cause bacterial cell
death.1 In particular, fluoroquinolones, such as spar-
floxacin (SPFX, Figure 1), have been shown to be highly
successful inhibitors of bacterial DNA gyrase.2 Besides
the quinolones, naturally occurring bacterial DNA gy-
rase inhibitors such as the coumarins, which include
F igu r e 1.
novobiocin (NB, Figure 1), are also known as antibacte-
rial agents.3,4 The coumarins inhibit ATPase activity of
We have previously developed a new screening system
for specific inhibitors of chromosome partitioning in E.
coli.6 This assay system can detect the production of
bacterial anucleate cells (chromosomeless cells) caused
by inhibition of chromosome partitioning as develop-
ment of blue color around paper disks containing the
inhibitors. To generate a new class of DNA gyrase
inhibitors, we randomly screened our chemical library
on this assay system and found several new DNA gyrase
inhibitors. Among them, the pyrazole derivative 1
(Figure 1) was identified as an interesting lead candi-
date. Compound 1 showed the same minimal inhibitory
concentration value (MIC) against clinically isolated
multidrug resistant Gram-positive bacteria as against
susceptible strains. However, the antibacterial activity
of 1 was weak (MIC ) 64 µg/mL) because it only slightly
inhibited DNA gyrase and topoisomerase IV (IC50 ) 128
µg/mL). Our aim in this study was to optimize the lead
compound 1 and to find new, potent DNA gyrase
inhibitors with antibacterial activity against MRSA,
PRSP, and VRE. We report herein the synthesis and
structure-activity relationships of a series of pyrazole
derivatives.
DNA gyrase by competing with ATP for binding to the
subunit B of the enzyme. However, because of side
effects, no pharmaceutically useful drug has so far been
derived from the coumarins. Recently, multidrug-
resistant Gram-positive bacteria, such as methicillin-
resistant Staphylococcus aureus (MRSA), penicillin-
resistant Streptococcus pneumoniae (PRSP), and vanco-
mycin-resistant enterococci (VRE), have become a seri-
ous medical problem. Since most of these multidrug-
resistant bacteria are also resistant to treatment with
the quinolones, it is important to find a new class of
DNA gyrase inhibitors to solve this problem. Although
many efforts have been dedicated to finding potent
antibacterial agents that can overcome bacterial resis-
tance, promising lead structures of DNA gyrase inhibi-
tors with novel mechanisms of action have not been
found.5
* To whom correspondence should be addressed. Phone: +81-6-
6337-5906. Fax: +81-6-6338-7656. E-mail: akihiko-tanitame@
dainippon-pharm.co.jp.
† Chemistry Research Laboratories, Dainippon Pharmaceutical Co.,
Ltd.
‡ Pharmacology and Microbiology Research Laboratories, Dainippon
Pharmaceutical Co., Ltd.
§ Tokyo Institute of Technology.
#
Chubu University.
10.1021/jm030394f CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/05/2004

3694 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Brief Articles
Sch em e 1. General Synthesis of 3- and 5-(4-Piperidyl)pyrazole Derivativesa
a
Reagents: (a) (i) CDI/THF, (ii) R1COCH3/LDA or LHMDS; (b) NH2NH2‚H2O/EtOH; (c) R2 NHNH2/EtOH; (d) R2B(OH)2/Cu(OAc)2/
pyridine/CH2Cl2; (e) HCl/EtOH or TFA/CH2Cl2.
Ta ble 1. Antibacterial Activity of the Synthesized Pyrazole
Derivatives
Ch em istr y
The 3- and 5-(4-piperidyl)pyrazole derivatives 1 and
7-18 were synthesized as shown in Scheme 1. The
requisite intermediate 1,3-diketones 3a -g were pre-
pared by coupling reaction of the (1-tert-butyloxycarbo-
nyl)isonipecotic acid 2 with various methyl ketones in
THF using N,N′-carbonyldiimidazole and a base (e.g.,
lithium diisopropylamide or lithium hexamethydi-
silazide).7 Cyclization of the 1,3-diketones 3a -g with
hydrazine hydrate in EtOH gave the 1-unsubstituted
3-[4-(1-tert-butyloxycarbonyl)piperidyl]pyrazole 4, which
furnished the 1-unsubstituted 3-(4-piperidyl)pyrazoles
1 and 7-10 as acid salts by removal of the protecting
group under acidic conditions (method A). The 1-sub-
stituted 3- and 5-[4-(1-tert-butyloxycarbonyl)piperidyl]-
pyrazoles 5 and 6 were obtained as a mixture by two
different routes, i.e., either condensation of 3a -g with
phenyl- or alkylhydrazines (method B) or phenylation
of 4 using substituted phenylboronic acids with cupric
acetate and pyridine in CH2Cl2 (method C).8 Cleavage
of the protecting group in the mixture of 5 and 6 under
a
acidic conditions, followed by separation using CHP-20P
(reverse phase) column chromatography and/or recrys-
tallization gave the 1-substituted 3-(4-piperidyl)pyrazole
derivatives 11-16 and the 1-substituted 5-(4-piperidyl)-
pyrazole derivatives 17 and 18 as acid salts. The other
1-substituted 5-(4-piperidyl)pyrazole derivatives, except
for 17 and 18, were not isolated. The regiochemistry of
11-18 was determined on the basis of nuclear Over-
hauser effect (NOE) experiments.
S.a.1, S. aureus FDA 209P; S.a.2, S. aureus KMP9 (MRSA);
E.c.1, E. coli NIHJ J C-2; E.c.2, E. coli K901 (a multidrug efflux
pump mutant).
against S. aureus and was 16-fold more potent against
E. coli than 1. The MIC value of 10 against E. coli NIHJ
J C-2 was almost similar to that against E. coli K901.
From these results, it was considered that the (E)-2-
(3,4-dichlorophenyl)vinyl substituent on the R1 moiety
induces potent antibacterial activity against not only
Gram-positive bacteria but also Gram-negative bacteria.
Second, the effects of different substituents on the
nitrogen atom of the pyrazole ring were investigated
(11-14). The 1-methylpyrazole derivative 11 and the
1-unsubstituted pyrazole derivative 1 showed almost
similar antibacterial activity against the four strains
of bacteria studies. The 1-(3-chlorophenyl)pyrazole de-
rivatives 12 and 13 and the 1-benzyl derivative 14,
however, showed 16-fold more potent antibacterial
activity against S. aureus and 2- to 16-fold more potent
antibacterial activity against E. coli than the parent
compound 1. Thus, lipophilic substituents, such as a
phenyl (12, 13) or a benzyl (14) appended on the
nitrogen atom of the pyrazole ring, gave good antibacte-
rial activity, while small substituents, such as a hydro-
Resu lts a n d Discu ssion
To verify whether the lipophilicity of the synthesized
pyrazole derivatives affects their antibacterial activity,
we evaluated the in vitro antibacterial activity of the
3-(4-piperidyl)pyrazoles 7-16 and the 5-(4-piperidyl)-
pyrazoles 17 and 18 against two Gram-positive and two
Gram-negative bacteria (Table 1). First, transformation
of the 4-benzyloxyphenyl moiety of the lead compound
1 was examined (7-10). The 4-methoxyphenyl deriva-
tive 7 did not show any antibacterial activity. However,
the 4-phenoxyphenyl derivative 8 and the 2-naphthyl
derivative 9 showed the same antibacterial activity as
1. The (E)-2-(3,4-dichlorophenyl)vinyl derivative 10
revealed 16-fold more potent antibacterial activity

Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3695
Ta ble 2. Antibacterial Activity of Selected Pyrazole
Ta ble 3. Inhibitory Effects of Selected Pyrazole Derivatives
Derivatives against Susceptible and Resistant Strains
against DNA Gyrase and Topoisomerase IV
MIC (µg/mL)
IC50 (µg/mL)
organisma
1
12 16
SPFX
NB
1
12
16
SPFX
NB
0.25
28
0.25
3.5
>400
S. aureus FDA 209Pb
S. aureus KMP9 (MRSA)c
S. aureus RN4220b
64
64
64
64
64
4
4
8
8
8
8
8
8
1
1
2
2
2
2
2
2
0.125 0.25
128
0.125 0.125
0.125
0.125 0.5
4
0.25
32
S. aureus DNA gyrasea
S. aureus DNA Topo IVb
E. coli DNA gyrasea
128 32
128 32
>128 32
128 32
14
14
14
14
14
0.25
6.9
>400
0.25
S. aureus N175d
4
E. coli DNA Topo IVb
human DNA Topo IIc
8.0
S. pneumoniae ATCC49619b
200 50 >400
S. pneumoniae KT2524 (PRSP)c 64
0.5
2
2
a
b
DNA gyrase supercoiling activity. Topoisomerase IV decat-
enation activity. c Topoisomerase II relaxation activity.
E. faecalis ATCC29212b
64
64
E. faecalis KU1777 (VRE)e
a
(SPFX: sparfloxacin, CAM: clarithromycin, ABPC: ampicillin,
antibacterial activity against sensitive and resistant
Gram-positive bacteria with across-the-board MIC val-
ues of 1-2 µg/mL. Thus, it is suggested that the pyrazole
derivatives have potent antibacterial activity against
susceptible and quinolone- and coumarin-resistant Gram-
positive bacteria.
VCM: vancomycin). Susceptible strain. c SPFX-, CAM-, and
b
d
ABPC-resistant strain. S. aureus N175 (GyrB, R144I) was
derived from RN4220 with selection of novobiocin. e SPFX- and
VCM-resistant strain.
gen (1) or a methyl (11), ended in less potent antibac-
terial activity.
Next, the inhibitory activity of the selected compounds
1, 12, and 16 against DNA gyrase and topoisomerase
IV was examined. As shown in Table 3, 12 and 16
having potent antibacterial activity strongly inhibited
DNA gyrase and topoisomerase IV isolated from S.
aureus and E. coli. The SAR for this inhibition was
almost parallel to that for the antibacterial activity
mentioned above. In addition, MIC values for both
compounds correlated well with IC50 values especially
in Gram-positive microoraganisms (Tables 2 and 3),
indicating that inhibition of topoisomerases by pyrazole
derivatives causes bacterial cell growth inhibition.
Moreover, 16 did not inhibit human topoisomerase II
even at 400 µg/mL. This result indicates that 16, like
sparfloxacin and novobiocin, selectively inhibits bacte-
rial topoisomerase.
In summary, we have described the synthesis and
structure-activity relationships of new pyrazole ana-
logues. The 5-[(E)-2-(5-chloroindol-3-yl)vinyl]pyrazole
16, one of the most active compounds, showed potent
antibacterial activity against not only susceptible strains
but also multidrug-resistant strains. In addition, 16
showed a more potent antibacterial activity against
clinically isolated quinolone- and coumarin-resistant
Gram-positive bacteria than sparfloxacin and novobio-
cin, respectively. We are pursuing further modifications
of this novel pyrazole scaffold that can potently inhibit
DNA gyrase and topoisomerase IV.
As for the 5- and 3-vinyl derivatives (15, 16, 18), the
MIC values of the 5-[(E)-2-(2,6-dichlorophenyl)vinyl]-
pyrazole 15 against the four strains of bacteria studied
were almost similar to those of 13 or 14. However, the
5-[(E)-2-(5-chloroindol-3-yl)vinyl]pyrazole 16, having a
3-chlorophenyl moiety, showed more potent antibacte-
rial activity than 15. Although 18 had 8-fold more potent
antibacterial activity against S. aureus and E. coli K901
than the parent compound 1, its antibacterial activity
against E. coli NIHJ J C-2 was similar to that of 1. From
the MIC values of 8 and 13, and those of 10 and 15
against E. coli NIHJ J C-2 and E. coli K901, it seems
that the 1-(3-chlorophenyl)pyrazoles 13 and 15 were
pumped out of the bacteria by a bacterial outer mem-
brane pump. However, because 16 showed the most
potent antibacterial activity against the four strains of
bacteria, it is believed that this compound was not
affected by the bacterial outer membrane pump and that
it penetrated well through the bacterial membrane. The
5-[(E)-2-(2,6-dichlorophenyl)vinyl]pyrazole 15 showed
slightly more potent antibacterial activity than the
3-[(E)-2-(2,6-dichlorophenyl)vinyl]pyrazole 18. Although
most of the synthesized pyrazole derivatives revealed
potent antibacterial activity against not only S. aureus
FDA 209P but also S. aureus KMP9, 16 exhibited the
most potent antibacterial activity among all the com-
pounds synthesized in this study.
To examine whether the pyrazole derivatives are
effective against multidrug-resistant Gram-positive bac-
teria, MIC values of the selected pyrazole derivatives
1, 12, and 16 were determined against quinolone-
resistant clinical isolates and coumarin-resistant labo-
ratory isolates of Gram-positive organisms and were
compared with those of sparfloxacin and novobiocin.9
As shown in Table 2, MIC values of sparfloxacin against
MSSA, PSSP, and VSE were 0.125, 0.125, and 0.25 µg/
mL, respectively, and those against MRSA, PRSP, and
VRE were 128, 4, and 32 µg/mL, respectively. On the
other hand, 1, 12, and 16 revealed the same antibacte-
rial activity against sensitive and multidrug resistant
Gram-positive bacteria. MIC values of novobiocin against
S. aureus RN4220 and S. aureus N175 derived from
RN4220 were 0.125 and 4 µg/mL, respectively. On the
other hand, 1, 12, and 16 showed the same antibacterial
activity against sensitive and coumarin-resistant S.
aureus. Compound 16 demonstrated the most potent
Exp er im en ta l Section
Gen er a l P r oced u r es for Syn th esis of th e 3- a n d 5-(4-
P ip er id yl)p yr a zole Der iva tives. Meth od s A a n d B. (a) To
an EtOH (30 mL) solution of 1,3-diketones 3 (10 mmol) was
added hydrazine hydrate (20 mmol) or monosubstituted hy-
drazine (20 mmol), and the reaction mixture was refluxed for
12 h. To this reaction mixture was added 10% citric acid (30
mL), and the resulting solution was taken up in AcOEt (100
mL), washed with saturated aqueous NaHCO3 and brine, dried
over MgSO4, and filtered. The residue was purified by column
chromatography (silica gel, CHCl3-MeOH) to give 1-unsub-
stituted 3-[4-(1-tert-butyloxycarbonyl)piperidyl]pyrazoles 4 or
a mixture of 1-substituted 3-[4-(1-tert-butyloxycarbonyl)pip-
eridyl]pyrazoles 5 and 1-substituted 5-[4-(1-tert-butyloxycar-
bonyl)piperidyl]pyrazoles 6.
(b) A solution of 4 or a mixture of 5 and 6 (10 mmol) in CH2-
Cl2 (30 mL) containing CF3COOH (0.5 mL) was stirred for 4 h
at room temperature. The reaction mixture was evaporated,
and the resulting residue was separated using CHP-20P
(reverse phase) column chromatography and/or recrystalliza-

3696 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Brief Articles
Relationships of 5-Substituted 6,8-Difluoroquinolones, Including
Sparfloxacin, a New Quinolone Antibacterial Agent with Im-
proved Potency. J . Med. Chem. 1990, 33, 1645-1656.
(3) Kim, O. K.; Ohemeng, K. A. Patents on DNA gyrase inhibitors:
J anuary 1995 to March 1998. Expert Opin. Ther. Pat. 1998, 8,
959-969.
tion from suitable solvents to give the 3-(4-piperidyl)pyrazoles
or the 5-(4-piperidyl)pyrazoles, respectively.
Ack n ow led gm en t. We are grateful to Drs. K.
Chiba, Y. Furutani, and Y. Nishikawa for their encour-
agement throughout this work. Thanks are also due to
members of the pharmacology section for their phar-
macological support and to members of the analytical
chemistry section for their elements analysis and spec-
tral measurements. We also thank Keith Poole, Depart-
ment of Microbiology and Immunology, Queen’s Uni-
versity, for supplying E. coli K901.
(4) Maxwell, A. The Interaction between Coumarin Drugs and DNA
Gyrase. Mol. Microbiol. 1993, 9, 681-686.
(5) Boehm, H.; Boehringer, M.; Bur, D.; Gmuender, H.; Hunber, W.;
Klaus, W.; Kostrewa, D.; Kuehne, H.; Luebbers, T.; Meunier-
Keller, N.; Mueller, F. Novel Inhibitors of DNA Gyrase: 3D
Structure Based Biased Needle Screening, Hit Validation by
Biophysical Methods, and 3D Guided Optimization. A Promising
Alternative to Random Screening. J . Med. Chem. 2000, 43,
2664-2674.
(6) Wachi, M.; Iwai, N.; Kunihisa, A.; Nagai, K. Irregular Nuclear
Localization and Anucleate Cell Production in Escherichia coli
Induced by a Ca2+ Chelator, EGTA. Biochimie 1999, 81, 909-
913.
(7) Rowley, M.; Broughton, H. B.; Collins, I.; Baker, R.; Emms, F.;
Marwood, R.; Patel, S.; Patel, S.; Ragan, C. I.; Freedman, S. B.;
Leeson, P. D. 5-(4-Chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)-
piperidin-4-yl)isoxazole: A Potent, Selective Antagonist at Hu-
man Cloned Dopamine D4 Receptors. J . Med. Chem. 1996, 39,
1943-1945.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and characterization data of novel synthetic com-
pounds, combustion analysis data, a table of antibacterial
activity of selected pyrazole derivatives against different
strains including Gram-negative bacteria, and procedures of
biological assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
(8) Lam, P. Y. S.; Clark, C. G.; Saubern, S.; Adams, J .; Winters, M.
D.; Chan, D. M. T.; Combs, A. New Aryl/Heteroaryl C-N Bond
Cross-Coupling Reactions via Arylboronic Acid/Cupric Acetate
Arylation. Tetrahedron Lett. 1998, 39, 2941-2944.
(9) Stieger, M.; Angehrn, P.; Wohlgensinger, B.; Gmunder, H. GyrB
Mutations in Staphylococcus aureus Strains Resistant to Cy-
clothialidine, Coumermycin, and Novobiocin. Antimicrob. Agents
Chemother. 1996, 40, 1060-1062.
Refer en ces
(1) Ferrero, L.; Cameron, B.; Manse, B.; Lagneaux, D.; Crouzet, J .;
Famechon, A.; Blanche, F. Cloning and Primary Structure of
Staphylococcus aureus DNA Topoisomerase IV: A Primary
Target of Fluoroquinolones. Mol. Microbiol. 1994, 13, 641-653.
(2) Miyamoto, T.; Matsumoto, J .; Chiba, K.; Egawa, H.; Shibamori,
K.; Minamida, A.; Nishimura, Y.; Okada, H.; Kataoka, M.; Fujita,
M.; Hirose, T.; Nakano, J . Synthesis and Structure-Activity
J M030394F