Potent DNA gyrase inhibitors; novel 5-vinylpyrazole analogues with Gram-positive antibacterial activity

Article abstract of DOI:10.1016/j.bmcl.2004.03.045

In this study, we designed and synthesized novel 5-vinylpyrazole analogues by decreasing the lipophilicity of the parent compounds 1a,b; 3-[(3-methoxycarbonyl)cyclohexylaminomethyl]indazoles while keeping the van der Waals interaction with the lipophilic area of DNA gyrase B. The selected compound 8bb exhibited good antibacterial activity against staphylococci and enterococci, including multi-drug resistant strains.

Full text of DOI:10.1016/j.bmcl.2004.03.045

Bioorganic& Medicinal Chemistry Letters 14 (2004) 2863–2866
Potent DNA gyrase inhibitors; novel 5-vinylpyrazole analogues with
Gram-positive antibacterial activity
Akihiko Tanitame,a,* Yoshihiro Oyamada,b Keiko Ofuji,a Kenji Suzuki,a Hideaki Ito,b
Motoji Kawasaki,a Masaaki Wachic and Jun-ichi Yamagishib
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
cDepartment of Bioengineering, Tokyo Institute of Technology, 4259, Nagatsuda, Midori-ku, Yokohama 226-8501, Japan
Received 18 February 2004; accepted 15 March 2004
Abstract—In this study, we designed and synthesized novel 5-vinylpyrazole analogues by decreasing the lipophilicity of the parent
compounds 1a,b; 3-[(3-methoxycarbonyl)cyclohexylaminomethyl]indazoles while keeping the van der Waals interaction with the
lipophilic area of DNA gyrase B. The selected compound 8bb exhibited good antibacterial activity against staphylococci and
enterococci, including multi-drug resistant strains.
Ó 2004 Elsevier Ltd. All rights reserved.
The increasing use of antibacterial agents such as
N
O
NH
Cl
H
N
b-lactams, macrolides, vancomycin or quinolones has
resulted in the emergence of multi-drug resistant
pathogens, especially Gram-positive bacteria.1;2 Over
the past decade, DNA gyrase has drawn much attention
as selected target for finding potent antibacterial agents
against multi-drug resistant strains.3
MeOOC
N
N
Cl
+
HN
N
R1
2a; R2 = H
1a; R1 = 4-tBu
N
2b; R2 = Me
1b; R1 = 3,4-diCl
R2
We have previously found compounds 1a,b (Fig. 1) as
potent and selective inhibitors of DNA gyrase with
moderate antibacterial activity against multi-drug
resistant strains as well as susceptible strains (Table 1).4
Docking studies of 1a with a 43 kDa fragment of DNA
gyrase subunit B from Escherichia (E.) coli revealed that
(1) the indazole scaffold forms postulated H-bond net-
work with Asp73 and H2O, (2) the methyl ester moiety is
H-bonded to Arg136, (3) the benzyloxy side chain
interacts––van der Waals interaction––with the lipo-
philicarea around Ile94, (4) an additional H-bond is
formed between Gly77 and the nitrogen atom on the
secondary amine of 1a (Fig. 2).
NH
H
MeOOC
N
R3
3
Figure 1.
In addition to our studies on compounds 1a,b, we have
previously reported a new class of antibacterial agents
with potent inhibitory activity against bacterial type II
topoisomerase using a new screening system for specific
inhibitors of chromosome partitioning in E. coli.5;6
Among these compounds, the pyrazole analogues 2a,b
(Fig. 1) showed potent Gram-positive antibacterial
activity (Table 1).7
Keywords: DNA gyrase inhibitor; 5-Vinylpyrazole analogues; Multi-
drug resistant strains.
Although compounds 1a,b and 2a,b were active in vitro,
they had apparently no in vivo antibacterial activity and
* Corresponding author. Tel.: +81-6-6337-5906; fax: +81-6-6338-7656;
e-mail: akihiko-tanitame@dainippon-pharm.co.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.045

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A. Tanitame et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2863–2866
Table 1. Inhibitory activity against E. coli DNA gyrase and topoisomerase IV (IC50) and minimum inhibitory concentration (MIC) of compounds
1a,b and 2a,b
No.
IC50 (lg/mL)
MIC (lg/mL)
Gyrasea
Topo IVb
Topo IIc
S. aureus
KMP9e
E. faecalis
ATCC 29212d KU 1777f
FDA 209Pd
1a
1b
2a
2b
4
1
>128
>128
8.0
>400
>400
>400
>400
4
4
1
1
4
4
1
1
8
4
2
2
4
4
2
2
14
14
8.0
(CAM: clarithromycin, ABPC: ampicillin, SPFX: sparfloxacin, VCM: vancomycin).
a DNA gyrase supercoiling activity.
b Topoisomerase IV decatenation activity.
c Human topoisomerase II relaxation activity.
d Susceptible strain.
e SPFX-, CAM-, ABPC-resistant strain.
f SPFX-, VCM-resistant strain.
The novel 5-vinylpyrazole analogues were prepared as
shown in Scheme 1. Condensation of the aldehydes 4a,b
with acetone in the presence of aq NaOH in EtOH,8
followed by condensation reaction with (COOEt)2 and
NaOEt gave the 1,3-diketones 5a,b. Cyclization of 5a,b
with NH2NH2 in EtOH, and reduction with LiAlH4
afforded the 3-hydroxymethylpyrazoles 6a,b. Chlorina-
tion of 6a,b with (COCl)2, followed by reaction with
excess 3-(methoxycarbonyl)cyclohexylamine gave the
desired products 7a,b. Next, separation of the diaste-
reomericmixture of 7b using CHP-20P (reverse phase
column) furnished the less polar diastereoisomer 7ba
and the more polar diastereoisomer 7bb, respectively.9
Moreover, we found out that the (E)-forms 7a, 7ba and
7bb were easily isomerized to the (Z)-forms 8a, 8ba and
8bb, respectively, by irradiation with visible light in
highly diluted MeOH. The stereochemistry of each iso-
mer of 7 and 8 was determined on the basis of NOE
experiments. For example, in compound 8a, an
enhancement was observed between the two protons of
the vinyl moiety. On the other hand, in compound 7a,
no enhancement was observed between the two protons
of the vinyl moiety.
H2O
H
Asp73
Gly77
O
H
N
-O
H
O
H
O
NH
NH2
Arg136
Me
O
N
N
H
N
O
O
Ile94
Figure 2. Schematic representation of the indazole analogue 1a bound
to the 43 kDa fragment of DNA gyrase B from E. coli.
showed weak antibacterial activity in the presence of
10% horse blood, indicating that these compounds, due
to their lipophilicity, bind to serum proteins.
From the findings above and in order to improve the
bioavailability and antibacterial activity of 1a,b, we
generated the novel 5-vinylpyrazoles 3 from the 3-[(3-
methoxycarbonyl)cyclohexylaminomethyl]indazole deriva-
tives 1a,b and the cited 5-vinylpyrazole analogues 2a,b
(Fig. 1). The idea behind our strategy was to decrease
the lipophilicity of compounds 1a,b while keeping the
van der Waals interaction with the lipophilic area
around Ile94 of the DNA gyrase B.
Next, compounds 7a, 7ba, 7bb, 8a, 8ba and 8bb were
tested for inhibition of DNA gyrase and topoisomerase
IV,10;11 and their MIC values against Staphylococcus (S.)
aureus and Enterococcus (E.) faecalis including suscep-
tible and multi-drug resistant strains were determined by
a microdilution method (Table 2).
N
NH
O
O
N
NH
N
NH
R3
H
N
H
N
HO
g
a,b
c,d
e,f
MeOOC
MeOOC
R3 CHO
EtOOC
R3
R3
R3
5a,b
6a,b
7a,b
8a,b
4a,b
Cl
Cl
Cl
b; R3
=
a; R3
=
N
Me
Scheme 1. Synthesis of the novel 5-vinylpyrazole analogues. Reagents and conditions: (a) acetone, aq NaOH, EtOH; (b) (COOEt)2, NaOEt, Et2O;
(c) NH2NH2, EtOH; (d) LiAlH4, THF (e) (COCl)2, CH2Cl2; (f) 3-methoxycarbonyl-cyclohexylamine (mixture of two stereoisomers), CHCl3–MeOH;
(g) hm, MeOH.

A. Tanitame et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2863–2866
2865
Table 2. Inhibitory activity against E. coli DNA gyrase (IC50) and minimum inhibitory concentration (MIC) of the novel 5-vinylpyrazole analogues
N
NH
H
N
MeOOC
R3
No.
R3
IC50 (lg/mL)
Gyrase
MIC (lg/mL)
S. aureus
E. faecalis
ATCC 29212 KU 1777
FDA 209P
KMP9
Cl
Cl
Cl
7a
8a
E
Z
16
16
4
4
8
8
16
16
16
16
7ba
7bb
8ba
8bb
E
E
Z
Z
1
8
16
4
8
16
8
32
64
8
32
32
8
1
1
0.5
N
Me
8
16
64
32
Compounds 7a and 8a showed less potent DNA gyrase
inhibitory activity and slightly less potent antibacterial
activity against four Gram-positive strains than the
parent compound 1b (Table 2). These results suggest
that the 2-[3,4-(dichlorophenyl)]vinyl moiety renders
good permeability through bacterial cell membrane.
Since 7a and 8a inhibited DNA gyrase with the same
IC50 value, it was assumed that both compounds bind to
DNA gyrase in a similar manner. In addition, both
compounds 7a and 8a showed the same MIC value
against four Gram-positive strains.
In summary, we have designed and synthesized novel
5-vinylpyrazole analogues by decreasing the lipophilicity
of the parent compounds 1a,b while keeping the van der
Waals interaction with the lipophilic area around Ile94
of the DNA gyrase B. The selected compound 8bb
(clogP ¼ 4.630) was less lipophilicthan 1a (clogP ¼
6.445) or 1b (clogP ¼ 5.925),13 and showed more potent
inhibitory activity against DNA gyrase than the parent
compounds. Although 8bb showed weaker antibacterial
activity against four Gram-positive strains than the
parent compounds 1a,b, it exhibited relatively good
antibacterial activity against staphylococci and entero-
cocci, including multi-drug resistant strains. These
results provide a good strategy for the development
of Gram-positive antibacterial agents targeting DNA
gyrase.
Compounds 7ba, 7bb, 8ba and 8bb exhibited the same or
2-fold more potent inhibitory activity against DNA
gyrase compared with the parent compound 1b,
although their antibacterial activity was less potent than
that of 1b. Among these compounds, 8bb showed the
most potent inhibitory activity against DNA gyrase. In
addition, 8ba and 8bb showed slightly more potent
antibacterial activity against four Gram-positive strains
than 7ba and 7bb, respectively. Finally, the less polar
diastereoisomers 7ba and 8ba exhibited slightly more
potent antibacterial activity than the more polar dia-
stereoisomers 7bb and 8bb, respectively (Table 2).
Acknowledgements
We are grateful to Dr. K. Nagai, Dr. K. Chiba, Dr. S.
Kato, Dr. H. Yoshida and Dr. H. Terauchi for their
encouragement throughout this work.
In this study, all the novel 5-vinylpyrazole analogues
had moderate to strong inhibitory activity against DNA
gyrase, but not against topoisomerase IV11 (IC50s >
128 lg/mL) and human topoisomerase II12 inhibitory
activity (IC50s > 400 lg/mL). In addition, all compounds
exhibited antibacterial activity against both susceptible
and multi-drug resistant strains.
References and notes
1. Witte, W. J. Antimicrob. Agents Chemother. 1999, 14, 1.
2. Marchese, A.; Schito, G. C.; Debbia, E. A. J. Chemother.
2000, 12, 12.
3. Ferrero, L.; Cameron, B.; Manse, B.; Lagneaux, D.;
Crouzet, J.; Famechon, A.; Blanche, F. Mol. Microbiol.
1994, 13, 641.
4. 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, preced-
ing paper in this issue. doi:10.1016/j.bmcl.2004.03.044.
5. Wachi, M.; Iwai, N.; Kunihisa, A.; Nagai, K. Biochimie.
1999, 81, 909.
6. Hiraga, S.; Niki, H.; Ogura, T.; Ichinose, C.; Mori, H.;
Ezaki, B.; Jaffe, A. J. Bacteriol. 1989, 171, 1496.
7. J. Med. Chem., in press.
Compound 8bb exhibited 8-fold more potent inhibitory
activity against DNA gyrase than 1a. On the other hand,
we previously reported that the 3-[(3-methoxycar-
bonyl)cyclohexylaminomethyl]indazole portion of 1a
has three H-bond interactions with the 43 kDa fragment
of DNA gyrase B on ATP binding site (Fig. 2).4 Taken
together, these results suggest that 8bb interacts with the
43 kDa fragment of DNA gyrase B on ATP binding site
as does 1a.

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A. Tanitame et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2863–2866
8. Edwards, M. L.; Ritter, H. W.; Stemerich, D. M.; Stewart,
K. T. J. Med. Chem. 1983, 26, 431.
9. Compounds 7a and 8a were an inseparable mixture of
diastereomers.
10. Sato, K.; Inoue, Y.; Fujii, T.; Aoyama, H.; Inoue, M.;
Mitsuhashi, S. Antimicrob. Agents Chemother. 1986, 30, 777.
11. Peng, H.; Marians, K. J. J. Biol. Chem. 1993, 268, 24481.
12. Spitzner, J. R.; Chung, I. K.; Muller, M. T. Nucleic Acids
Res. 1990, 18, 1.
13. The clogPs of these compounds were calculated by cLogP
for Windows Version 4.0 (Biobyte Corp., Claremont, CA,
U.S.A).