Novel pyrazole derivatives as potent inhibitors of type II topoisomerases. Part 1: Synthesis and preliminary SAR analysis

Article abstract of DOI:10.1016/j.bmcl.2007.03.003

In an attempt to search for a new class of antibacterial agents, we have discovered a series of pyrazole analogs that possess good antibacterial activity for Gram-positive and Gram-negative organisms via inhibition of type II bacterial topoisomerases. We have investigated the structure-activity relationships of this series, with an emphasis on the length and conformation of the linker. This work led to the identification of tetrahydroindazole analogs, such as compound 1, as the most potent class of compounds.

Full text of DOI:10.1016/j.bmcl.2007.03.003

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2723–2727
Novel pyrazole derivatives as potent inhibitors of type II
topoisomerases. Part 1: Synthesis and preliminary SAR analysis
Laurent Gomez,* Michael D. Hack, Jiejun Wu, John J. M. Wiener, Hari Venkatesan,
´
Alejandro Santillan, Jr., Daniel J. Pippel, Neelakandha Mani, Brian J. Morrow,
S. Timothy Motley, Karen Joy Shaw, Ronald Wolin, Cheryl A. Grice and Todd K. Jones
Johnson & Johnson Pharmaceutical Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA
Received 14 December 2006; revised 28 February 2007; accepted 1 March 2007
Available online 6 March 2007
Abstract—In an attempt to search for a new class of antibacterial agents, we have discovered a series of pyrazole analogs that pos-
sess good antibacterial activity for Gram-positive and Gram-negative organisms via inhibition of type II bacterial topoisomerases.
We have investigated the structure–activity relationships of this series, with an emphasis on the length and conformation of the lin-
ker. This work led to the identification of tetrahydroindazole analogs, such as compound 1, as the most potent class of compounds.
Ó 2007 Elsevier Ltd. All rights reserved.
Over the past decade, type II topoisomerases have
drawn much attention as selected targets for the discov-
ery of potent antibacterial agents. Among the type II
topoisomerases are DNA gyrase and topoisomerase
IV. DNA gyrase is an essential prokaryotic type II topo-
isomerase with no direct mammalian counterpart.1 It is
involved in the vital processes of DNA replication, tran-
scription, and recombination. DNA gyrase catalyzes the
ATP-dependent introduction of negative supercoils into
bacterial DNA as well as the decatenation and unknot-
ting of DNA. Topoisomerase IV has a primary role in
decatenation of daughter chromosomes following
DNA replications. Those attributes make DNA gyrase
an attractive target for drug discovery. DNA gyrase is
mainly inhibited by quinolones and coumarins. Numer-
ous quinolone antibacterial agents have been developed
and are now widely used for the treatment of bacterial
infectious diseases (e.g., ciprofloxacin, Fig. 1).2,3 This
class of antibacterial agents inhibits the DNA break-
age-reunion cycle by binding to the A subunit and stabi-
lizing the gyrase–DNA complex. Unfortunately,
resistance to quinolones has emerged.4,5 In addition to
some quinolones, naturally occurring bacterial DNA
gyrase inhibitors such as the coumarin (e.g., novobiocin,
Fig. 1) have also shown some antibacterial activities.6,7
The coumarins inhibit ATPase activity of DNA gyrase
by competing with ATP for binding to the B subunit
of the enzyme.8,9 However, the coumarins suffer from
a developing resistance profile and toxicity concerns.3,10
Recently, multidrug-resistant Gram-positive bacteria,
such as methicillin-resistant Staphylococcus aureus
(MRSA), penicillin-resistant Streptococcus pneumoniae
(PRSA), and vancomycin-resistant enterococci (VRE),
have become a serious medical problem. To overcome
the limitations of the known DNA gyrase inhibitors, it
is important to identify new classes of compounds.
As part of our antibacterial program, we have identified
a series of pyrazole derivatives as potent inhibitors of
bacterial growth.11 These compounds target the parC
subunit of topoisomerase IV in S. pneumoniae. A series
of resistant mutants have been identified which map to
the parC subunit (unpublished results). We report herein
the racemic synthesis and the preliminary structure–
activity relationship of this series. Our initial efforts were
aimed at evaluating the influence of the nature of the
pyrazole on the antibacterial activity.
The synthesis of the initial pyrazole analogs is described
in Scheme 1. Treatment of CBz-protected 4-piperidi-
none with tert-butoxybis(dimethylamino) methane in
THF followed by hydrolysis (1 N HCl) of the corre-
sponding enamine afforded the desired intermediate 3
in quantitative yield. Pyrazole ring formation was
accomplished with (6-methoxy-quinolin-4-yl)-hydrazine
Keywords: Gyrase; Topoisomerase; Pyrazoles; SAR; Tetrahydroindazole.
*
Corresponding author. Tel.: +1 858 320 3383; fax: +1 858 450
2089; e-mail: lgomez4@prdus.jnj.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.003

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L. Gomez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2723–2727
NH2
O
OH
O
O
OH
OH
O
F
COOH
H
N
MeO
Me
Me
N
N
O
O
O
HN
Me
novobiocin
OMe
ciprofloxacin
S
O
H
N
N
F
COOH
N
H
O
N
N
N
N
N
N
O
Me
left-hand
side
linker
right-hand
side
levofloxacin
1
Figure 1. Selected inhibitors of type II topoisomerases.
OH
N
N
OMe
+
N
MeO
a
X
MeO
X
MeO
X
O
O
b
c
O
O
N
N
N
N
H
O
a, b
N
NH
2
c
OH
OMe
O
N
CBz
N
CBz
N
CBz
N
H N
2
CBz
H N
2
Br
NH
N
NH
3
4
5
d
e
MeO
X
MeO
X
MeO
X
N
OMe
+
N
N
HCl
N
2a X = CH
2b X = N
O
R1
=
d, e
N
N
N
N
O
OMe
Scheme 2. Reagents and conditions: (a) Meldrum’s acid, CH(OEt)3;
(b) Dowtherm A, 260 °C; (c) PBr3, DMF; (d) NH2NH2, NMP, 140 °C;
(e) basic extraction.
N
N
R1
R1
6
7
Scheme 1. Reagents and conditions: (a) [(CH3)2N]2CH(OtBu), THF,
65 °C; (b) 1 N HCl; (c) hydrazine 2a, THF then p-TsOH; (d) 10% Pd/C,
HCO2NH4, EtOH; (e) R1CHO, NaBH3CN, CH3CN.
It is worth mentioning that the regiochemical outcome
of the pyrazole ring formation resulted in a greater
selectivity (ratio 8:1, after optimization) favoring
isomer 9 when the intermediate 1,3-dicarbonyl 8 was used
shortly after preparation. 1H NMR analysis of this
material showed various isomerizations over time.
in THF. Complete dehydration of the intermediate is
accomplished with addition of p-TsOH to afford the
desired products, 4 and 5, as a 3:2 mixture of regioisom-
ers in 85% yield. A subsequent hydrogenolysis of the
CBz protective group with 10% Pd/C and ammonium
formate provided the unprotected secondary amine in
quantitative yield. A final derivatization step was
accomplished using various aldehydes in the presence of
sodium cyanoborohydride in CH3CN. Fortunately, the
regioisomers, 6 and 7, were readily separated by reversed
phase chromatography. The structure was fully assigned
using 2D NMR spectroscopy and NOE studies.
N
OMe
+
N
X
X
N
O
O
N
a, b
N
N
c
OH
OMe
NHBoc
NHBoc
NHBoc
NHBoc
9
13
8
X = CH
X = N
10
The synthesis of both quinoline and naphthyridine
hydrazines is described in Scheme 2.12 Treatment of
substituted aniline with Meldrum’s acid and triethyl
orthoformate afforded the corresponding enamines in
89% (X = N) and 85% (X = CH) yield, respectively.
The ring formation was accomplished in Dowtherm A
at 260 °C to afford the desired products in 86%
(X = N) and 78% (X = CH) yield, respectively. Subse-
quent bromination reaction using PBr3 in DMF fol-
lowed by hydrazine formation afforded the desired
products 2a and 2b in 52% and 62% overall yields.
N
OMe
N
X
H
N
X
N
O
d, e
R2
=
N
N
N
S
+
OMe
Y = H2 or O
1 or R2 NH
Y
X = CH
X = N
HN
R1 or R2
R
Y
11
14
12
Scheme 3. Reagents and condition: (a) [(CH3)2N]2CH(OtBu), THF,
65 °C; (b) 1 N HCl; (c) hydrazine 2a or 2b, THF then p-TsOH; (d) 4M
HCl, dioxane; (e) R1CHO or R2CHO, NaBH3CN, CH3CN or
R2COOH, EDC, HOBt, DMF.
Analogs bearing an exocyclic nitrogen were obtained via a
synthesis similar to the one described above (Scheme 3).

L. Gomez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2723–2727
2725
Interestingly, the same reaction was found to be regio-
specific when (6-methoxy-[1,5]naphthyridin-4-yl)-hydra-
zine 2b was used in place of (6-methoxy-quinolin-4-yl)-
hydrazine 2a. Indeed, the pyrazole ring formation led
exclusively to isomer 13 (Scheme 3).
an exocyclic position resulted in a potent inhibitor
against Gram-positive organisms as seen with com-
pound 21. The corresponding regioisomer 22 was found
to be about 32- and 64-fold less potent against S. aureus
and S. pneumoniae, respectively. Interestingly, the lack
of antibacterial activity of compound 21 against
Gram-negative organisms was overcome by modifying
the right-hand side. Analog 23, bearing 4H-
benzo[1,4]thiazin-3-one moiety, showed a 32-fold in-
crease in potency against E. coli and similar activity
against S. aureus and S. pneumoniae. In addition, com-
pound 25, bearing an amide functionality, displayed
comparable antibacterial activity to its amine counter-
part 23. However, the regioisomeric compound 24 was
found to have no antibacterial activity against both
Gram-positive and Gram-negative organisms. Replac-
ing the quinoline moiety on the pyrazole with naphthyri-
dine ring did not affect the antibacterial activity; since
both compounds 23 and 1 have similar potency against
all three strains of bacteria. This result is interesting
since we have shown that these two compounds display
different orientations of the bicyclic system with respect
to the pyrazole core structure. This would suggest that
only the relative orientation of both the right and
left-hand side of the molecule affects the antibacterial
activity of the compounds and not the interaction of
the linker itself (i.e., pyrazole) with the enzyme. An over-
lay of analogs 23 and 1 is depicted in Figure 3.
In addition, 2D NMR spectroscopy and NOE studies of
both compounds 9 and 13 revealed a different orienta-
tion of the bicyclic system with respect to the pyrazole
ring (Fig. 2). No additional correlations (H1a–H3a or
H1b–H2b) were detected by the NOE study suggesting
that the structures depicted in Figure 2 are energetically
favored conformations.
Investigation into the importance of the ring size was
conducted using a route similar to the one described
above. Condensation of N-Boc-hexahydro-1H-azepin-
4-one with tert-butoxybis(dimethylamino)methane yielded
a 3:1 mixture of the two regioisomers 15 and 16 in quan-
titative yield. Subsequent condensation with hydrazine
2b led to two single regioisomers in 65% yield, which
were further derivatized to afford compounds 17 and
18 using the conditions as shown in Scheme 4.
The antibacterial activity of the compounds was evalu-
ated in vitro against Gram-negative (Escherichia coli)
and Gram-positive (S. aureus and S. pneumoniae) organ-
isms and the results are summarized in Table 1. Explo-
ration of the linker length revealed some interesting
trends. Analogs bearing a nitrogen at the endocyclic po-
sition, compounds 19 and 20, did not show any antibac-
terial activity. Extending the position of the nitrogen to
This was further demonstrated with compound 27,
where the exocyclic nitrogen was attached at the adja-
cent carbon. This compound showed similar inhibitory
activity against topoisomerase IV (IC50 = 0.5 lM) with
the corresponding regioisomer 25 (IC50 = 0.25 lM).
Finally, expanding the size of the ring attached to the
pyrazole as in analog 28 resulted in a compound with
similar antibacterial activity to the parent analog 25.
N
O
N
H2b
N
Me
H3a
H2a
H1a
N
N
N
N
In summary, we have described the synthesis and struc-
ture–activity relationships of a series of racemic pyrazole
analogs as potent antibacterial agents. The present
investigation suggests the importance of the linker
length and the orientation of both right and left-hand
side moieties in the antibacterial activity. This work
led to the identification of compound 1, a tetrahydroin-
dazole core structure, as one of the most active com-
pounds, with potent antibacterial activity against both
Gram-positive and Gram-negative organisms. Further
development of the SAR on this compound will be pre-
sented in future publications.
O
Me
H1b
nOe
nOe
NHBoc
NHBoc
9
13
H1: 9.11 ppm, broad signal
H1: 7.63 ppm
Figure 2. NMR studies and assignment of conformations.
O
O
O
a, b
OH
HO
+
NBoc
NBoc
16
NBoc
15
N
N
N
c, d, e
+
N
N
N
N
N
N
MeO
2
MeO
17
18
N
O
2
R
R
O
Scheme 4. Reagents and condition: (a) [(CH3)2N]2CH(OtBu), THF,
65 °C; (b) 1 N HCl; (c) hydrazine 2b, THF then p-TsOH; (d) 4 M HCl,
dioxane; (e) R2COOH, EDC, HOBt, DMF.
Figure 3. Superposition of 23 (orange) and 1 (blue).

2726
L. Gomez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2723–2727
Table 1. Antibacterial activity of selected set of pyrazole analogs
Compound
Structure
DNA gyrase/Topo IV IC50 (lg/mL)
E.c./S.a./S.p MIC (lg/mL)
OMe
19
>8/ND
>128/>128/>128
O
O
N
N
N
N
O
O
N
N
MeO
N
20
21
22
23
24
25
1
>8/ND
2/0.06
>128/>128/>128
64/0.5/0.5
>128/16/32
2/0.25/0.5
>128/>128/>128
4/4/2
N
OMe
N
N
N
N
O
N
H
O
N
MeO
>8/ND
0.25/0.03
2/ND
N
O
O
N
H
OMe
N
N
H
N
N
O
N
H
N
S
N
MeO
H
N
N
O
N
H
OMe
N
S
N
N
1/0.125
0.25/0.016
0.06/0.25
>8/0.5
O
H
N
N
N
O
O
N
H
S
H
N
N
N
N
H
1/0.25/2
S
OMe
N
O
H
N
N
N
N
O
N
H
26
2713
28
128/0.125/4
S
OMe
OMe
S
H
N
8/8/4
N
N
N
H
O
N
N
N
O
O
N
N
N
0.25/ND
2/1/2
NH
O
OMe
S
E.c, Escherichia coli KL-16 (GSC strain 4245); S.a., Staphylococcus aureus ATCC13709; S.p, Streptococcus pneumoniae ATCC49619; ND, not
determined.

L. Gomez et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2723–2727
2727
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