DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: Synthesis and antibacterial activity

Article abstract of DOI:10.1016/j.bmcl.2008.11.102

The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.

Full text of DOI:10.1016/j.bmcl.2008.11.102

Bioorganic & Medicinal Chemistry Letters 19 (2009) 894–899
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: Synthesis
and antibacterial activity
a
a
b
b
b
Stephen P. East a, , Clara Bantry White , Oliver Barker , Stephanie Barker , James Bennett , David Brown ,
*
E. Andrew Boyd a, Christopher Brennan a, Chandana Chowdhury a, Ian Collins b,
Emmanuelle Convers-Reignier a, Brian W. Dymock a, Rowena Fletcher b, David J. Haydon b,
Mihaly Gardiner a, Stuart Hatcher a, Peter Ingram a, Paul Lancett b, Paul Mortenson a,
Konstantinos Papadopoulos a, Carol Smee b, Helena B. Thomaides-Brears b, Heather Tye a,
James Workman a, Lloyd G. Czaplewski b,
*
a Evotec (UK) Ltd, Discovery Services, 114 Milton Park, Abingdon, Oxfordshire OX14 4SA, UK
b Prolysis Ltd, Oxford University Begbroke Science Park, Sandy Lane, Yarnton, Oxfordshire OX5 1PF, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on
imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of
DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for opti-
mization leading to a series with potent Gram-positive antibacterial activity and a low resistance
frequency.
Received 10 October 2008
Revised 24 November 2008
Accepted 26 November 2008
Available online 3 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Antibacterial
Gyrase
Imidazopyridine
Triazolopyridine
The type II bacterial topoisomerases DNA gyrase and topoiso-
merase IV, which are responsible for the control of DNA topology
and chromosome function, have been validated as a therapeutic
drug target by members of the quinolone/fluoroquinolone class
of antibacterial agents (e.g. gemifloxacin).1 This particular class of
compounds interact with the catalytic subunits of DNA gyrase
(GyrA) and topoisomerase IV (ParC). The ATPase subunits of these
enzymes GyrB and ParE have also been identified as the target for
small molecule inhibitors (e.g. novobiocin), however, to date, is-
sues surrounding resistance, toxicity and permeability of these
inhibitors have limited their therapeutic use (Fig. 1).2
Recently, interest in identifying small molecule inhibitors of
GyrB to overcome the liabilities of the existing compounds has
intensified.3,4 Elucidation of protein–ligand structures of GyrB by
X-ray crystallography5,6 has aided the search for new inhibitors
and Tanitame et al.7 (e.g. pyrazole 1), Mani et al.,8 Grossman
et al.9 and Charifson et al. (e.g. benzimidazole 2)10 have provided
examples of potent dual-targeting GyrB/ParE inhibitors which
demonstrate good antibacterial activity (Fig. 2).
Encouraged by these disclosures, we explored a number of pos-
sible replacements for the benzimidazole core of 2. We reasoned
that it might be possible to find other 5,6-fused heterocyclic scaf-
folds that could provide different SAR and optimization opportuni-
ties as well as alternative ADME, pharmacology, toxicology and
efficacy profiles. Our requirements for each new scaffold were to
fix the 2-amino substituent as the ethylurea and one of the core
substituents as the 3-pyridyl group. These proposals were docked
into the ATP binding site of a GyrB model generated from the struc-
ture (PDB 1EI1) deposited by Brino et al.5 We scrutinized our pro-
O
CO2H
F
O
O
O
O
O
O
H2N
N
N
N
N
H
OH
H2N
O
OH
OH
N
O
* Corresponding authors. Tel.: +44 (0) 1235 441735 (S.P. East), +44 (0) 1865
854700 (L.G. Czaplewski).
OMe
gemifloxacin
novobiocin
E-mail addresses: stephen.east@evotec.com (S.P. East), lloyd.czaplewski@proly-
sis.com (L.G. Czaplewski).
Figure 1. Gyrase inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.102

S. P. East et al. / Bioorg. Med. Chem. Lett. 19 (2009) 894–899
895
Table 1
Cl
N
GyrB and ParE inhibitory activity and MICs
N
Compound
GyrB IC50
(
lM)
ParE IC50
(lM)
MIC (lg/ml)
Cl
N
N
S. aureus
E. faecalis
E. coli
H
N
H
Novobiocin
0.03
0.009
<0.004
0.39
1.32
3.5
0.035
nd
0.125
0.25
0.063
32
128
>256
8
8
1
nd
32
32
nd
N
H
N
HN
2
0.125
nd
32
32
>256
O
R
2a
3
N
1
2 R = CO2Me
4
5
2.68
9.64
145
nd
Figure 2. Reported GyrB/ParE inhibitors.
nd, not determined.
a
Data (Ki) from Charifson et al.10
posals according to the interactions that they made with the en-
zyme and the three scaffolds that were the most appealing as pre-
dicted by our model were the imidazo[1,2-a]pyridine and the two
isomeric [1,2,4]triazolo[1,5-a]pyridines (Fig. 3). The key binding
interactions according to our docking studies are illustrated for
triazolopyridine 5. The scaffold and urea interactions have been
previously described by Charifson et al.,10 but the interaction be-
tween the 3-pyridyl substituent and Arg136, which they described
strated modest MICs against all three species in the antibacterial
assay. The triazolopyridine 4 was ꢀ7-fold less potent against GyrB
compared with 3, however, 4 also showed modest antibacterial
activity. Triazolopyridine 5 was approximately 25-fold less active
than 3 against the target and did not show any antibacterial activ-
ity. The ParE enzyme used in the ATPase assay has a lower specific
activity than GyrB, requires more enzyme and therefore more
inhibitor to reach an IC50. The ParE inhibitory activity of triazolo-
pyridine 4 was confirmed in this assay. Compared to benzimid-
azole 2 and novobiocin, compounds 3, 4 and 5 were significantly
less potent but they do lack the additional substituent present in
2. With the exception of the ParE assay our results for compound
2 were in agreement with data reported by Charifson et al.10
as a H-bond, appears more likely to be a
our model.
p-stacking interaction in
To test our modeling hypothesis we prepared 3, 4 and 5. Com-
pounds 3 and 4 were synthesised from a common bipyridyl amine
intermediate 7, which was accessed via a modified Suzuki reaction
using 2-amino-4-chloropyridine 611 and 3-pyridyl boronic acid.
The imidazopyridine 3 was synthesized in six steps from interme-
diate 7. First, 7, was converted to the corresponding tosylate using
p-toluenesulfonyl chloride and this was followed by alkylation
with iodoacetamide to give 8. Cyclisation to provide the imidazo-
pyridine scaffold12 was effected by treatment of 8 with trifluoro-
acetic anhydride. After removal of the trifluoroacetate group
using potassium carbonate and methanol, compound 9 was ob-
tained. Attempts to form the urea by reaction of 9 with ethyl isocy-
anate were unsuccessful. Instead we resorted to a two-step
protocol with phenyl chloroformate to provide the phenyl carba-
mate followed by reaction with ethylamine to give 3, albeit in very
low yield.13,14
To obtain the triazolopyridine 4, compound 7 was treated with
ethoxycarbonylisothiocyanate to provide thiourea 10 which was
reacted with hydroxylamine according to the procedure described
by Nettekoven et al.15 to give the triazolopyridine (not shown)
which was then treated with ethyl isocyanate to give compound
4 in moderate overall yield. Triazolopyridine 5 was prepared via
an analogous route to 4 but using 2-amino-5-iodopyridine as the
starting material.
Although the IC50s for all of the scaffolds were below 10 lM, we
reasoned that 3 and 4 represented the best opportunities for opti-
mization because they were antibacterial. The lack of antibacterial
activity for compound 5 might be related to the higher GyrB IC50
observed for this compound. The higher IC50 for 4 (compared with
3) might be attributed to the N3 atom in 4 being a weaker H-bond
acceptor than the N1 atom in 3 and hence the H-bonding network
in the active site of GyrB is weaker.
Further insights from molecular modeling and SAR from Mani
et al.8 suggested that the 5-position of the new scaffolds would
be a good area for SAR exploration to improve the potency. Exam-
ination of the chemistry required to access 5-substituted triazolo-
pyridines and imidazopyridines indicated that compound 4 would
be more amenable to parallel synthesis.19 We therefore elected to
focus our efforts on the synthesis of 5-substituted triazolopyridines
and for this study we continued to fix the 7-substituent as the 3-
pyridyl moiety.
To incorporate functionality into the 5-position of the triazolo-
pyridine scaffold, we modified the original route outlined in
Scheme 1 so that the 5-substituent could be introduced during
the latter stages of the synthetic sequence. Key to the success of
this route was the preparation of 11 on a multi-gram scale. This
was achieved in three steps from 6 using an adaptation of the con-
ditions described by Nettekoven et al.15 as shown in Scheme 2.
From compound 11, we introduced iodine regioselectively into
the 5-position using a modification of a procedure reported by Fin-
kelstein.20,21 This provided the iodo intermediate 12 which allowed
us to incorporate aromatic (Ar) and heteroaromatic (HetAr) sub-
stituents into the 5-position using palladium cross-coupling reac-
tions with boronic acids and trialkyl stannanes. These reactions
provided intermediates that were subjected to a second palladium
cross-coupling reaction to install the 3-pyridyl moiety in the 7-po-
sition to give compounds 13–29. Alternatively, intermediate 12
was shown to be a substrate for SNAr or copper-promoted reactions
to access N-linked analogues 30–32, again after a palladium cross-
coupling step to introduce the 3-pyridyl group.
The compounds were screened in GyrB and ParE ATPase assays
(Escherichia coli)16 and minimum inhibitory concentrations
(MICs)17 were determined against Gram-positive (Staphylococcus
aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) and
Gram-negative (E. coli N43 efflux pump mutant) bacterial strains
(Table 1).18
The imidazopyridine 3 was the most active of the compounds
tested with a sub-micromolar IC50 against GyrB and it demon-
Arg136
HN
NH+3
HN
π
Asp73
O
O
H
N
N
H
O
N
N
N
R
H
H
N
N
N
H
H
N
N
N
O
O
3 R = CH
4 R = N
Regioselective deprotonation of intermediate 11 also facilitated
the introduction of carboxylic acid derivatives in the 5-position of
the triazolopyridine ring related to benzimidazole 2 (Scheme 3).
5
Figure 3. Proposed GyrB/ParE inhibitors.

896
S. P. East et al. / Bioorg. Med. Chem. Lett. 19 (2009) 894–899
N
N
N
Cl
NH2
(i)
(ii), (iii)
(iv), (v)
NTs
O
NH2
N
N
NH2
N
N
N
NH2
6
7
8
9
(vi)
N
(vii)
N
N
N
H
H
(viii), (ix)
N
N
O
H
H
N
N
N
H
N
H
N
N
N
N
S
O
N
O
O
10
4
3
Scheme 1. Reagents and conditions: (i) 3-pyridyl boronic acid, Pd(dppf)Cl2, Na2CO3, MeCN, lW, 170 °C, 95%; (ii) TsCl, pyridine, 85 °C, 40%; (iii) ICH2CONH2, DIPEA, DMF, rt,
50%; (iv) (CF3CO)2O, CH2Cl2, 30 °C; (v) K2CO3, MeOH, reflux, 20% (2 steps); (vi) a—PhOCOCl, THF, rt; b—EtNH2, THF, rt, 2% (2 steps); (vii) EtOC(O)NCS, 1,4-dioxane, rt; (viii)
NH2OHÁHCl, DIPEA, MeOH:EtOH (1:1), 60 °C; (ix) EtNCO, 1,4-dioxane, 80 °C, 23% (3 steps).
Cl
vided compounds with improved IC50s against GyrB compared
with the parent unsubstituted compound 4. The 5-phenyl com-
pound 13 is the most potent (GyrB IC50 150 nM), although substi-
tutions in the ortho-, meta- and para-positions of the phenyl ring
are inhibitors of GyrB. Electron-withdrawing and electron-donat-
ing substituents in the phenyl ring provided potent GyrB inhibitors
however none of the compounds exhibit antibacterial activity.
Compounds 23–26, containing a six-membered heteroaromatic
N
N
H
N
Cl
NH2
(i)-(iii)
H
N
N
N
O
R
6
11 R = H
12 R = I
(iv)
(vii), (vi)
(v), (vi)
N
N
N
ring in the 5-position are also active against GyrB (IC50 ꢀ 1
lM).
With the exception of the 2-pyridyl analogue 23, which shows
modest activity against E. faecalis, these compounds are inactive
in the antimicrobial screen.
N
N
H
N
H
N
H
H
N
N
N
N
N
O
O
NR1R2
Ar/HetAr
Some highly potent GyrB inhibitors were identified when the
substituent in the 5-position of the triazolopyridine scaffold was
a five-membered heteroaromatic ring. In particular, thiazole 27
and pyrazole 31 demonstrate IC50s of 42 nM and 54 nM, respec-
tively. Compound 27 shows good antibacterial activity against
30-32
13-29
Scheme 2. Reagents and conditions: (i) EtOC(O)NCS, 1,4-dioxane, rt, 95%; (ii)
NH2OHÁHCl, DIPEA, MeOH:EtOH (1:1), 60 °C, 72%; (iii) EtNCO, 1,4-dioxane, 80 °C,
84%; (iv) n-BuLi (3 equiv), THF, À78 °C, then I2, 52%; (v) ArB(OH)2 or HetArB(OH)2,
Pd(dppf)Cl2, K3PO4, MeOH:1,4-dioxane (1:4), 80 °C, ꢀ60%; or RSnBu3, Pd(PPh3)2,
CuI, THF, 80 °C, ꢀ35%; (vi) 3-pyridyl boronic acid, Pd(dppf)Cl2, K3PO4, MeOH:1,4-
dioxane (1:4), 100 °C, 7–25%; (vii) a—HNR1R2, DMF, 100 °C, ꢀ25%; or b—HNR1R2,
CuI, Cs2CO3, NMP, 80 °C, ꢀ30%.
the Gram-positive organisms with MICs of 1 lg/ml. The MICs of
compound 31 suggests that there is potential for broad-spectrum
inhibitors from this class. Of the carboxylic acid derivatives 33–
38, the ethylamide 37 is the most potent GyrB inhibitor with an
IC50 of 41 nM. The most potent compounds 27, 31 and 37 were also
screened in the ParE assay and they were all found to be dual
inhibitors.
N
N
N
(i)-(iii)
Comparison of substituted triazolopyridines 23, 24, 31, 33 and
36 with the analogous benzimidazoles described by Charifson
et al.10 shows that the benzimidazole scaffold (2, 39–42) leads to
more potent enzyme inhibitors (Table 3). One key difference be-
tween the scaffolds is the potential for intramolecular H-bonding
in the benzimidazole series involving the NH and the R substituent
and this might explain some of the observed differences with the
enzyme potency SAR, for example, compound 23 versus 39. Indeed
Charifson et al.10 suggest that coplanarity between the scaffold and
the R substituent is particularly important for ParE activity (com-
pound 39 compared with compound 40). There is clear evidence
of divergent antimicrobial SAR between the two scaffolds. Com-
pounds 23, 24 and 33 have little or no antimicrobial activity but
the corresponding benzimidazole analogues 39, 40 and 2 are anti-
bacterial indicating that the triazolopyridine scaffold is more con-
servative. The compounds containing a 1-pyrazolyl substituent, 31
and 41 are antibacterial on both scaffolds, although the benzimid-
azole compound 41 is more potent. Compound 36 is equipotent
compared to 42 despite being a less effective enzyme inhibitor
on the triazolopyridine scaffold. Further SAR evaluation is neces-
sary in order to optimize the enzyme potency and antibacterial
activity on the triazolopyridine scaffold.
H
N
11
H
N
N
O
R
33
34
R = CO2Me
R = CO2H
35-38 R = CONR1R2
(v)
(iv)
(see Table 2)
Scheme 3. Reagents and conditions: (i) n-BuLi (3 equiv), THF, À78 °C, then CO2; (ii)
SOCl2, MeOH, rt to 75 °C, 18% (over 2 steps); (iii) 3-pyridyl boronic acid, Pd(dppf)Cl2,
K3PO4, MeOH:1,4-dioxane (1:4), 100 °C, 39%; (iv) LiOH, 1,4-dioxane, H2O, rt, 88%;
(v) HNR1R2, AlMe3, 1,4-dioxane, toluene, 45 °C, ꢀ90%.
Thus, treatment of 11 with n-butyllithium was followed by a car-
bon dioxide quench to afford a carboxylic acid (not shown). Ester-
ification of this acid by activation with thionyl chloride and
subsequent reaction with methanol was followed by a palladium
cross-coupling reaction using 3-pyridyl boronic acid to provide es-
ter 33. Further functional group manipulation gave access to the
parent carboxylic acid 34 or amides, for example, 35–38.
Biological data for selected compounds synthesized via the
routes shown in Schemes 2 and 3 are shown in Table 2.
As indicated by the entries 13–22 in Table 2 a six-membered
aromatic ring in the 5-position of the triazolopyridine scaffold pro-
The three most potent triazolopyridines, compounds 27, 31 and
37, were selected for further characterization. Dual-targeting

S. P. East et al. / Bioorg. Med. Chem. Lett. 19 (2009) 894–899
897
Table 2
GyrB/ParE inhibitory activity and MICs for 5-substituted triazolopyridines
N
N
N
H
N
H
N
N
O
R
Compound
R
GyrB IC50
(l
M)
ParE IC50
(
lM)
MIC (lg/ml)
SA
128
EF
SP
EC
4
H
Ph
2-F Ph
2.69
0.15
1.52
0.69
0.51
0.37
1.08
1.01
1.01
0.35
0.84
0.89
145
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
32
32
nd
32
32
13
14
15
16
17
18
19
20
21
22
23
>256
>256
>256
>128
>128
>256
>256
>256
>256
>256
>256
128
>256
>256
>128
>128
>256
>256
>256
>256
>256
>256
>256
>256
>128
>128
>256
>256
>256
>256
>256
32
>256
>256
>128
>128
>256
>256
>256
>128
>256
>256
2-OH Ph
3-OMe Ph
4-OMe Ph
3-NHCOMe Ph
3-COMe Ph
3-CONH2 Ph
4-OH Ph
3-NMe2Ph
N
24
25
26
0.61
1.5
nd
nd
nd
>256
>256
>128
>256
>256
>128
>256
>256
>128
>256
>256
>128
N
N
N
N
0.98
N
S
27
28
0.042
0.2
11
nd
1
1
8
1
4
>128
>256
N
>256
N
N
H
29
30
0.34
1.15
nd
nd
32
32
8
32
32
>128
32
N
NMe
N
32
N
31
32
0.054
1.43
25
nd
2
2
2
4
N
N
N
N
16
16
32
32
N
33
34
35
36
37
38
–CO2Me
–CO2H
–CO2NH2
–CONHMe
–CONHEt
–CONMe2
1.3
2.8
0.64
0.64
0.041
3.2
nd
nd
nd
nd
40
nd
>256
>256
32
8
8
256
>256
>256
16
4
4
256
>256
>256
16
8
8
256
>256
>256
8
8
8
256
SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, Streptococcus pyogenes ATCC 51339; EC, E. coli ATCC N43; nd, not determined.

898
S. P. East et al. / Bioorg. Med. Chem. Lett. 19 (2009) 894–899
Table 3
Head-to-head comparison of the triazolopyridine and benzimidazole scaffolds
a
a
Compound
R
GyrB IC50
(l
M)
ParE IC50
(l
M)
SA MIC (lg/ml)
23
24
31
33
36
2-Pyridyl
3-Pyridyl
1-Pyrazolyl
–CO2Me
0.89
0.61
0.054
1.3
nd
nd
25
nd
nd
>256
>256
2
>256
8
N
N
N
H
N
H
N
–CONHMe
0.64
N
O
R
39
40
41
2
2-Pyridyl
3-Pyridyl
1-Pyrazolyl
–CO2Me
<0.004
0.006
<0.004
<0.004
0.005
0.014
1.3
0.046
0.035
0.15
0.031
4
0.063
0.063
16
N
N
H
N
H
42
–CONHMe
N
H
N
O
R
a
Ki data for compounds 39–42.
Table 4
In conclusion, we have identified an imidazopyridine and a triaz-
olopyridine scaffold which can be functionalized to provide com-
pounds that demonstrate antibacterial activity through inhibition
of GyrB/ParE. The triazolopyridine scaffold was selected for further
SAR evaluation and compounds with good antibacterial activity,
particularly against Gram-positive organisms, have been identified.
From our preliminary evaluation it appears that the GyrB IC50s need
to be ꢀ500 nM or lower in order to see single digit MICs in the anti-
microbial screen. The evaluation of the pharmacokinetics and effi-
cacies of these compounds will be presented in due course.
Antibacterial activities for compounds 27, 31 and 37
Strain
MIC (
l
g/ml)
27
31
37
E. coli ATCC 25922
>128
>128
0.5
>128
1
1
1
0.5
1
1
>128
16
1
>128
4
4
4
4
4
2
>128
16
2
>128
4
4
4
2
4
4
H. influenzae ATCC 49247
M. catarrhalis ATCC 25240
P. aeruginosa 101021
E. faecalis 1.5604 (VRE)
S. aureus 601055 (MSSA)
S. aureus 43300 (MRSA)
S. aureus ATCC 700698 (MRSA)
S. aureus Smith ATCC 19636 (MSSA)
S. pneumoniae ATCC 49619
Acknowledgments
The authors extend their thanks to Dr. Steve Ruston, Dr. Mark
Whittaker and Dr. Geoff Lawton for useful discussions during
course of this research programme. This work was funded by
investments from East Hill Management (Boston, USA). The Proly-
sis authors declare financial interests.
antibacterials should demonstrate low spontaneous resistance fre-
quencies. No bona fide resistant strains of S. aureus at 2Â, 4Â or 8Â
the MICs of the compounds were isolated despite multiple at-
tempts. The resistance frequency was estimated to be
<1.8 Â 10À9, consistent with a dual-targeting inhibitor series. The
three compounds demonstrate good activity against a range of
pathogenic bacteria including drug-resistant clinical isolates (Table
4) with MICs comparable with the oxazolidinone antibacterial
agent linezolid.22 It is interesting to note that all three compounds
were ineffective against the E. coli strain used in this study. This
data, coupled with the data on the efflux pump mutant (shown
in Table 2), suggests that compounds in the triazolopyridine series
may have reduced permeability as well as some susceptibility to
efflux, at least in E. coli, however, this has not been investigated
further. Overall, the microbiology suggests that this series of com-
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Grillot, A.-L.; Stamos, D.; Charifson, P. S.; Grossman, T. H. Antimicrob. Agents
Chemother. 2006, 50, 1228.
9. Grossman, T. H.; Bartels, D. J.; Mullin, S.; Gross, C. H.; Parsons, J. D.; Liao, Y.;
Grillot, A.-L.; Stamos, D.; Olson, E. R.; Charifson, P. S.; Mani, N. Antimicrob.
Agents Chemother. 2007, 51, 657.
pounds would be more suited to
indication.
a Gram-positive clinical
The mammalian cytotoxicity of 27, 31 and 37 was evaluated in a
HepG223 cell assay to test the effect of the compounds on mito-
chondrial metabolism after 24 h exposure. The IC50s for the com-
pounds were all >64
selectivity for antimicrobial versus cytotoxic activity and indicates
that the compounds are not general ATPase inhibitors.
During the course of this work, a patent application describing
some of the triazolopyridines described here as antibacterial
agents was published.24 In this patent activity against a strain of
Neisseria gonorrhoeae was reported (e.g. compound 37 MIC of
1 lg/ml) but to our knowledge no additional data on the antimi-
crobial activity of these compounds has appeared in the literature
and the compounds 27 and 31 have not been reported previously.
l
g/ml providing a significant window of
10. Charifson, P. S.; Grillot, A.-L.; Grossman, T. H.; Parsons, J. D.; Badia, M.; Bellon,
S.; Deininger, D. D.; Drumm, J. E.; Gross, C. H.; LeTiran, A.; Liao, Y.; Mani, N.;
Nicolau, D. P.; Perola, E.; Ronkin, S.; Shannon, D.; Swenson, L. L.; Tang, Q.;
Tessier, P. R.; Tian, S. K.; Trudeau, M.; Wang, T.; Wei, Y.; Zhang, H.; Stamos, D. J.
Med. Chem. 2008, 51, 5243.
11. Meigh, J.-P.; Álvarez, M.; Joule, J. A. J. Chem. Soc., Perkin Trans. 1 2001, 2012.
12. Hamdouchi, C.; de Blas, J.; del Prado, M.; Gruber, J.; Heinz, B. A.; Vance, L. J. Med.
Chem. 1999, 42, 50.
13. All compounds described in this manuscript were characterized by LC–MS and
1H NMR.
14. The yield for step (vi) of the reaction sequence illustrated in Scheme
represents the yield from one unoptimised reaction.
1
15. Nettekoven, M.; Püllmann, B.; Schmitt, S. Synthesis 2003, 1649.

S. P. East et al. / Bioorg. Med. Chem. Lett. 19 (2009) 894–899
899
16. Gyrase B and ParE convert ATP into ADP and inorganic phosphate. The released
phosphate can be detected by the addition of malachite green solution and
measured by monitoring the increase in absorbance at 600 nm. The GyrB
ATPase assay is carried out in a buffer containing 12.8 nM Gyrase enzyme (A2B2
complex from E. coli), 0.08 mg/mL ssDNA, 35 mM Tris, pH 7.5, 24 mM KCl,
2 mM MgCl2, 6.5% glycerol, 2 mM DDT, 1.8 mM spermidine, 0.1 mg/mL BSA,
19. During the course of our optimization programme on the triazolopyridine
series we became aware of patent that reported imidazopyridines and
a
imidazopyrimidines as antibacterial agents: Sciotti, R. J.; Starr, J. T.; Richardson,
C.; Rewcastle, G. W.; Palmer, B. D.; Sutherland, H. S.; Spicer, J. A.; Chen, H. Int.
Pat. Appl. WO 2005089763, 2005.
20. Finkelstein, B. J. Org. Chem. 1992, 57, 5538.
and 5% DMSO solution containing the inhibitor in a total volume of 25
reaction is started by adding ATP to a final concentration of 1 mM and allowed
to incubate at 30 °C for 60 min. The reaction is stopped by adding 200 l of
malachite green solution (0.034% malachite green, 10 mM ammonium
molybdate, 1 M HCl, 3.4% ethanol, 0.01% Tween 20). Colour is allowed to
develop for 5 min and the absorbance at 600 nm is measured
spectrophotometrically. The IC50 values are determined from the absorbance
readings using no compound and no enzyme controls. The ParE assay is
l
l. The
21. Three equivalents of n-butyllithium were critical to the success of this reaction.
Experimental procedure: To
a solution of 1-(7-chloro-[1,2,4]triazolo[1,5-
l
a]pyridin-2-yl)-3-ethyl-urea 11 (200 mg, 0.834 mmol) in THF (8 mL) at
À78 °C was added n-BuLi (1.5 mL of a 1.67 M solution, 2.5 mmol) dropwise.
The resulting mixture was stirred at À78 °C for 5 min and then warmed to 0 °C
where it was maintained for 1 h. After this time, the solution was re-cooled to
À78 °C and iodine (648 mg, 2.5 mmol) as
a solution in THF (4 mL) was
introduced dropwise. The reaction mixture was stirred for 2 h at À78 °C before
sat. NH4Cl (4 mL) was added followed by sat. sodium thiosulfate (1 mL). The
solution was extracted with dichloromethane (3Â) and the combined organic
extracts were dried (Na2SO4), filtered and concentrated in vacuo to reveal a
yellow solid. This solid was purified by chromatography on silica, using 1–2%
MeOH/dichloromethane as the eluent, to provide 1-(7-chloro-5-iodo-
[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-ethyl-urea 12 as a yellow solid (160 mg,
52%). MS: m/z 366 [M+H]+. 1H NMR (DMSO): d = 10.16 (s, 1H), 8.16 (t, J = 5 Hz,
1H), 7.97 (d, J = 2 Hz, 1H), 7.86 (d, J = 2 Hz, 1H), 3.34 (m, J = 7, 5 Hz, 2H), 1.24 (t,
J = 7 Hz, 3H).
performed in a buffer containing 43 nM TopoIV enzyme from E. coli, 10 lg/mL
ssDNA, 100 mM Tris, pH 7.5, 20 mM KCl, 6 mM MgCl2, 10 mM DDT, 0.1 mg/mL
BSA, and 5% DMSO solution containing the inhibitor with 0.5 mM ATP added to
start the reaction.
17. Compounds were tested for antimicrobial activity by susceptibility testing in
liquid or on solid media. MICs for compounds against each strain were
determined by the broth microdilution or agar dilution method according to
the guidelines of the Clinical Laboratories and Standards Institute described in
CLSI Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria
Approved Standard, 6th ed., Document M11-A6, Vol. 24(2), January 2004 and
CLSI Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That
Grow Aerobically; Approved Standard, 7th ed.: M7-A7E, Vol. 26(2), January
2006.
22. Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010.
23. HepG2 cytotoxicity assays were performed by Precos Ltd, University of
Nottingham, UK. Briefly, HepG2 cells were incubated with compound for
48 hours. Mitochondrial dehydrogenase activity was measured.
18. MICs estimated in triplicate once. Activity of compounds confirmed. For
compounds 27, 31 and 37 n > 10.
24. Butler, D. C. D.; Chen, H.; Hegde, V. R.; Limberakis, C.; Rasne, R. M.; Sciotti, R. J.;
Starr, J. T. Int. Pat. Appl. WO2006038116, 2006