Design, synthesis and structure-activity relationship studies of novel indazole analogues as DNA gyrase inhibitors with Gram-positive antibacterial activity

Article abstract of DOI:10.1016/j.bmcl.2004.03.044

In this study, we report the design, synthesis and structure-activity relationships of novel indazole derivatives as DNA gyrase inhibitors with Gram-positive antibacterial activity. Our results show that selected compounds from this series exhibit potent antibacterial activity against Gram-positive bacteria including multi-drug resistant strains that is methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE).

Full text of DOI:10.1016/j.bmcl.2004.03.044

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2857–2862
Design, synthesis and structure–activity relationship studies of novel
indazole analogues as DNA gyrase inhibitors with Gram-positive
antibacterial activity
Akihiko Tanitame,a,* Yoshihiro Oyamada,b Keiko Ofuji,a Yoko Kyoya,a Kenji Suzuki,a
Hideaki Ito,b Motoji Kawasaki,a Kazuo Nagai,c Masaaki Wachid 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 Biological Chemistry, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501, Japan
dDepartment 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 report the design, synthesis and structure–activity relationships of novel indazole derivatives as DNA
gyrase inhibitors with Gram-positive antibacterial activity. Our results show that selected compounds from this series exhibit potent
antibacterial activity against Gram-positive bacteria including multi-drug resistant strains that is methicillin-resistant Staphylo-
coccus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE).
Ó 2004 Elsevier Ltd. All rights reserved.
NH2
O
In recent years, the emergence and spread of multi-drug
resistant microorganisms have become a serious prob-
lem in the treatment of infectious diseases.1 To over-
come this problem, new and effective antibacterial
agents are needed.
OH
O
O
OH
O
H
N
O
OH
O
O
O
Novobiocin
Over the past decade, bacterial DNA gyrase has drawn
much attention as a selected target for finding potent
antibacterial agents.2 Accordingly, a number of syn-
thetic quinolone antibacterial agents have been devel-
oped and are now widely used for treatment of bacterial
infectious diseases.3 Quinolones inhibit DNA gyrase and
topoisomerase IV, and cause bacterial cell death.2
Besides the quinolones, naturally occurringantibacterial
agents, such as novobiocin (NB; Fig. 1) have also been
identified as bacterial DNA gyrase inhibitors.4;5 Novo-
biocin inhibits ATPase activity of DNA gyrase by
competingwith ATP for bindingto the gyrase subunit
B. Usingdetailed 3D structural information of ATP
bindingsite located on the subunit B of DNA gyrase,
Hoffmann–La Roche’s group has recently developed a
N
O
NH
N
NH
O
O
S
HN
O
2
1
Figure 1.
new lead DNA gyrase inhibitor (1; Fig. 1).6;7 X-ray
studies of indazole analogues with a 24 kDa fragment of
DNA gyrase B from Staphylococcus (S.) aureus revealed
that (1) the indazole scaffold forms postulated H-bond
network with Asp73 and H2O, (2) the ‘X’ moiety stacks
on the Glu50-Arg76 salt bridge and the ‘Y’ moiety is H-
bonded to Arg136 and (3) the benzyloxy side chain
interacts––van der Waals interaction––with the lipo-
philic area around Ile94 (Fig. 2).6 Although compound 1
Keywords: DNA gyrase inhibitor; Indazole analogues; Multi-drug
resistant strains; ATP bindingsite.
* Correspondingauthor. 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.2004.03.044

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A. Tanitame et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2857–2862
etc.11), followed by protection of the amino function, if
Asp73
O
necessary, and deprotection of the tert-butyldiphenylsi-
lyl (TBDPS) group afforded the intermediates 5, 7 and
9a,b. Benzylation of these phenols with 4-tert-butylb-
enzyl bromide and NaH in DMF, followed by cleavage
of the protecting groups gave the desired products 6, 8
and 10a,b, respectively. Reduction of 10a,b with NaBH4
afforded the correspondinghydroxymethyl derivatives
11a,b.
H2O
-O
H
O
Arg76
Glu50
H
H
N
N
NH
NH2
Y
X
Arg136
O
Z
Compounds 6, 8, 10a,b and 11a,b were then tested for
inhibition of DNA gyrase and topoisomerase IV,12;13
and their MIC values against S. aureus and E. faecalis
includingsusceptible and multi-drugresistant strains
were determined by a microdilution method (Table 2).
While all compounds showed some antibacterial activ-
ity, compounds 6, 8 and 11a,b exhibited only weak
DNA gyrase inhibitory activity and no inhibition of
topoisomerase IV (IC50s > 128 lg/mL). On the other
hand, the methyl ester derivatives 10a,b showed strong
DNA gyrase inhibitory activity with the same IC50 value
(8 lg/mL). From these findings, we assumed that
the methyl ester group of 10a,b could form a specific
H-bond with Arg136 of ATP biding site in the DNA
gyrase B (Fig. 2).
Ile94
Figure 2. Schematic representation of an indazole analogue bound to
the 24 kDa fragment of DNA gyrase B.
has strong inhibitory activity against DNA gyrase, its
antibacterial activity is weak, suggesting that 1 cannot
penetrate bacterial cells (Table 1).
Independently, we have previously discovered a new
class of bacterial DNA gyrase inhibitors that have po-
tent activity against S. aureus and Enterococcus (E.)
faecalis.8 Amongthese inhibitors, compound 2 havinga
piperidine ringwas identified as a hit compound usinga
new screeningsystem for specific inhibitors of chromo-
some partitioningin Escherichia (E.) coli.9;10 As com-
pound 2 exhibited both weak inhibitory activity against
bacterial type II topoisomerase and moderate antibac-
terial activity against Gram-positive pathogens (Table
1), it was assumed that this compound could penetrate
bacterial cells.
With the results of 10a,b in hand, we attempted to
modify the position of ‘Y’ moiety, which forms a H-
bond with Arg136 of the DNA gyrase B while keeping
‘X’ moiety as an amine group (Fig. 2). Compounds 13–
19 with an ester group were synthesized using the cor-
respondingamines instead of the methylamine or N-Boc
piperazine in a similar manner to that described in
Scheme 1.
Usingthe results obtained with compound 2 to generate
more potent DNA gyrase inhibitors with strong anti-
bacterial activity, we attempted in this study to improve
compound 1 ability to penetrate bacterial cells by
replacingits coumarin moiety with a variety of basic
amines such as the 4-piperidyl moiety of compound 2.
Although compound 16 had stronginhibitory activity
against DNA gyrase, it did not show any antibacterial
activity, presumably because of low permeability
through bacterial membrane. Compounds 13, 14 and 15
had potent DNA gyrase inhibitory activity and showed
moderate antibacterial activity (Table 2).
The six compounds 6, 8, 10a,b and 11a,b were prepared
as the first set of analogues (Scheme 1). Reaction of the
3-(bromomethyl)indazole 46 with an excess or equiva-
lent amount of the appropriate amine (12a or 12b,
When the secondary amine derivatives 17–19 were tes-
ted, 17 and 18 had moderate inhibitory activity against
DNA gyrase with IC50 values of 16 and 32 lg/mL,
Table 1. Inhibitory activity against E. coli DNA gyrase and topoisomerase IV (IC50) and minimum inhibitory concentration (MIC) of compounds 1
and 2
IC50 (lg/mL)
MIC (lg/mL)
No.
Gyrasea
Topo IVb
Topo IIc
S. aureus
KMP9e
E. faecalis
ATCC 29212d KU 1777f
FDA 209Pd
1
0.25
128
0.25
>128
128
25
>400
200
>400
>128
64
>128
64
>128
64
>128
64
2
NB
0.25
0.25
2
2
(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.

A. Tanitame et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2857–2862
2859
N
O
NH
H
N
Boc
Boc
N
N
N
Me
f,g
Me
NH2
MeOOC
HO
5
N
d,b,e
6
Boc
12a (2R, 4R)
N
O
NH
Boc
Boc
HN
N
N
NH
N
O
N
N
N
f,g
a,b,c
h,e
Br
Boc
N
N
NH2
Me
MeOOC
N
Boc
HO
HO
TBDPS
7
8
3
4
12b (2R, 4S)
i,b,e
N
N
O
NH
N
O
NH
Boc
H
N
H
N
Boc
N
HO
f,g
*
*
j
N
MeOOC
*
N
H
MeOOC
Boc
HN
N
HO
11a,b
9a,b
10a,b
Scheme 1. Synthesis of 6, 8, 10a,b and 11a,b. Reagents and conditions: (a) TBDPSCl, imidazole, DMF; (b) Boc2O, Et3N, DMAP, CH3CN; (c) NBS,
(PhCO2)2, CCl4; (d) aq CH3NH2 (excess), THF; (e) TBAF, THF; (f) tert-butylbenzylbromide, NaH, DMF; (g) TFA, CH2Cl2; (h) N-Boc-piperazine,
Et3N, THF; (i) 12a or 12b (excess), CHCl3–CH3OH; (j) NaBH4, CH3OH.
respectively. However, the carboxylic acid derivative 18
did not show any antibacterial activity, presumably
because of low permeability. Compound 19, on the
other hand, had potent inhibitory activity against DNA
Table 2. Inhibitory activity against E. coli DNA gyrase (IC50) and minimum inhibitory concentration (MIC) of the synthesized indazole analogues
N
NH
R1
O
No.
R1
IC50 (lg/mL)
Gyrase
MIC (lg/mL)
S. aureus
KMP9
E. faecalis
ATCC 29212 KU 1777
FDA 209P
32
H
N
6
8
64
64
64
32
32
32
16
16
Me
HN
N
16
64
H
N
MeOOC
10a
10b
8
8
128
64
128
64
64
64
N
H
H
N
64
32
MeOOC
HO
N
H
H
N
11a
32
64
64
32
N
H
(continued on next page)

2860
A. Tanitame et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2857–2862
Table 2 (continued)
R1
IC50 (lg/mL)
Gyrase
MIC (lg/mL)
No.
S. aureus
KMP9
E. faecalis
ATCC 29212
FDA 209P
KU 1777
H
N
HO
11b
64
64
64
64
64
N
H
13
14
MeOOC
N
16
16
32
32
32
64
32
64
32
32
N
MeOOC
EtOOC
15
16
8
4
16
32
16
16
N
>128
>128
>128
>128
O
N
MeOOC
H
N
17
18
16
32
16
16
16
16
EtOOC
HOOC
H
>128
>128
>128
>128
N
H
N
MeOOC
19
4
4
4
8
4
gyrase (IC50 ¼ 4 lg/mL) and good antibacterial activity
against Gram-positive bacteria with MIC values of 4–
8 lg/mL (Table 2).14 Compound 19 revealed 16-fold less
potent inhibitory activity against DNA gyrase and >32-
fold more potent antibacterial activity against S. aureus
than compound 1. These results suggests that the
3-(methoxycarbonyl)cyclohexylamine moiety allows
good permeability through bacterial membrane com-
pared with other moieties.
the amine function and deprotection of TBDPS group
afforded the intermediate 20. Benzylation of 20 with
R2X and NaH in DMF or R2OH and Mitsunobu
reagents, followed by cleavage of the protecting groups
gave the desired products 21a–e.14
Amongthe non-, mono- or di-haloegnated benzyl
derivatives 21a–d, the 3,4-dichlorobenzyl derivative 21d
revealed more potent DNA gyrase inhibitory activity
than the parent compound 19 and moderate antibacte-
rial activity against four Gram-positive strains (Table 3).
As the lipophilicity of R2 group increased, DNA gyrase
inhibitory activity and antibacterial activity of the
indazole analogues became stronger. However, the
3-pyridylmethyl derivative 21e ended in less potent
Next, transformation of the 4-tert-butylbenzyl moiety of
19 was examined. The substituted benzyloxy derivatives
were prepared as shown in Scheme 2. Reaction of the 3-
(bromomethyl)indazole 4 with an excess of 3-(meth-
oxycarbonyl)cyclohexylamine, followed by protection of
Boc
Boc
N
NH
N
O
N
Boc
N
N
N
H
N
a,b,c
d or e, then f
MeOOC
Br
MeOOC
O
HO
R2
TBDPS
21a-e
4
20
Scheme 2. Synthesis of 21a–e. Reagents and conditions: (a) 3-(methoxycarbonyl)cyclohexylamine (excess), THF; (b) Boc2O, Et3N, DMAP, CH3CN;
(c) TBAF, THF; (d) R2X, NaH, DMF; (e) R2OH, DIAD, Ph3P, DMF; (f) TFA, CH2Cl2.

A. Tanitame et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2857–2862
Table 3. Inhibitory activity against E. coli DNA gyrase (IC50) and
2861
minimum inhibitory concentration (MIC) of 3-[(3-methoxycar-
bonyl)cyclohexylaminomethyl]indazoles
N
NH
H
N
MeOOC
O
R2
No.
R2
IC50
(lg/mL)
Gyrase
MIC (lg/mL)
S. aureus E. faecalis
FDA KMP9 ATCC KU
209P
29212 1777
21a
21b
8
64
64
64
64
F
F
16
128
128
128
128
21c
16
32
4
32
4
64
4
32
4
Cl
Cl
Cl
21d
21e
1
Cl
Cl
64
>128 >128 >128 >128
Figure 3. Docking study of the 43 kDa fragment of DNA gyrase B
from E. coli with compound 19 (1S,3R). Dockingstudies of the four
isomers of 19 and the crystal structure of DNA gyrase B (PDB code;
1EI1) were performed usingthe dockingsoftware FlexX (Tripos, Inc.,
St. Louis MO, U.S.A.).16 The models of 19 (1S,3R) and (1S,3S) iso-
mers had the same bindingform as that shown in Figure 2. Molecular
dynamic (MD) simulations were carried out on these two models for
1 ns at 298 K usingthe molecular modellingsoftware InsightII/Di-
cover95.0 (Accerlys, Inc., San Diego, CA, U.S.A.) with cvff force
field17 with each structure alongthe trajectory path beingeneryg-
minimized.
N
DNA gyrase inhibitory activity and less antibacterial
activity (Table 3).
All compounds tested in this study had selective DNA
gyrase inhibitory activity, but had no topoisomerase
IV13 (IC50s > 128 lg/mL) and no human topoisomerase
II15 inhibitory activity (IC50s > 400 lg/mL). Compounds
19 and 21d showed good antibacterial activity
against not only susceptible but also multi-drug resistant
strains.
Acknowledgements
We are grateful to Dr. K. Chiba, Dr. S. Kato, Dr. H.
Yoshida and Dr. H. Terauchi for their encouragement
throughout this work.
Finally, dockingstudies of compound 19 with a 43 kDa
fragment of the DNA gyrase B from E. coli (Fig. 3)
revealed the followinginteractions: (1) specific H-bonds
with both Asp73 and a water molecule, (2) ionic inter-
action with Arg136 and (3) lipophilic interaction with
Ile94. Moreover, another H-bond could be formed
between Gly77 and the nitrogen atom on the secondary
amine of 19.
References and notes
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novel indazole derivatives as DNA gyrase inhibitors.
Amongthese compounds, we have identified the 3-[(3-
methoxycarbonyl)cyclohexylaminomethyl]indazole deriva-
tive 21d as havingpotent inhibitory activity aaginst
DNA gyrase with exceptionally good antibacterial
activity against staphylococci and enterococci, including
multi-drugresistant strains.

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