Pyridine-3-carboxamide-6-yl-ureas as novel inhibitors of bacterial DNA gyrase: Structure based design, synthesis, SAR and antimicrobial activity

Article abstract of DOI:10.1016/j.ejmech.2014.08.025

The development of antibacterial drugs based on novel chemotypes is essential to the future management of serious drug resistant infections. We herein report the design, synthesis and SAR of a novel series of N-ethylurea inhibitors based on a pyridine-3-carboxamide scaffold targeting the ATPase sub-unit of DNA gyrase. Consideration of structural aspects of the GyrB ATPase site has aided the development of this series resulting in derivatives that demonstrate excellent enzyme inhibitory activity coupled to potent Gram positive antibacterial efficacy.

Full text of DOI:10.1016/j.ejmech.2014.08.025

European Journal of Medicinal Chemistry 86 (2014) 31e38
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Pyridine-3-carboxamide-6-yl-ureas as novel inhibitors of bacterial
DNA gyrase: Structure based design, synthesis, SAR and antimicrobial
activity
Ian A. Yule a, Lloyd G. Czaplewski b, Stephanie Pommier b, David T. Davies b,
,
*
Sarah K. Narramore a, Colin W.G. Fishwick a
a School of Chemistry, University of Leeds, Leeds LS2 9JT, UK
b Biota Europe Ltd, Begbroke Science Park, Woodstock Road, Begbroke, 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 development of antibacterial drugs based on novel chemotypes is essential to the future manage-
ment of serious drug resistant infections. We herein report the design, synthesis and SAR of a novel series
of N-ethylurea inhibitors based on a pyridine-3-carboxamide scaffold targeting the ATPase sub-unit of
DNA gyrase. Consideration of structural aspects of the GyrB ATPase site has aided the development of this
series resulting in derivatives that demonstrate excellent enzyme inhibitory activity coupled to potent
Gram positive antibacterial efficacy.
Received 6 June 2014
Received in revised form
4 August 2014
Accepted 7 August 2014
Available online 7 August 2014
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Bacterial resistance
DNA gyrase
Pyridine-3-carboxamide
Molecular modelling
1. Introduction
DNA gyrase and the structurally homologous enzyme topo-
isomerase IV (topo IV) are highly conserved ATP dependant bac-
Due in part to disproportionate prescription of antibiotics dur-
ing the latter half of the 20th century, the past 2 decades have seen
the inevitable emergence of bacterial resistance to all currently
available therapeutic agents [1]. Of particular concern are the so-
called ‘ESKAPE’ pathogens (multi-drug resistant Enterococcus fae-
cium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa and Enterobacter species),
which cause infections associated with elevated rates of morbidity
and mortality [2,3]. The need for antibiotics based upon novel
chemotypes which act upon novel molecular targets has long been
recognised as critical to the future management of the problem. A
recent manifestation of this concept is the ‘ten-by-twenty’ initia-
tive, a gauntlet thrown down by the Infectious Diseases Society of
America (IDSA) to those involved in antimicrobial drug discovery,
urging a more concerted effort with the goal of delivering 10 new
antibacterial drugs by 2020 [4].
terial type IIa topoisomerase enzymes which play an essential role
within bacteria linked to the conservation of chromosomal integ-
rity during DNA replication and transcription [5]. These enzymes
have long been validated as therapeutic drug targets owing to the
commercial and clinical success of members of the fluoroquinolone
antibiotic class (e.g. ciprofloxacin, gemifloxacin) which target the
catalytic (GyrA/ParC) sub-units in a dual-inhibitory fashion [6].
More recently, the development of small molecule ATPase in-
hibitors of gyrase (GyrB) and topo IV (ParE), aided by the elucida-
tion of proteineligand structures of GyrB, has gained attention as a
means to inhibit these classical enzyme targets in the pursuit of
novel antibacterial compounds [7e10].
Encouraged by these reports, our interest in the discovery of
novel ATPase inhibitors of GyrB/ParE began with the observation
that a number of reports in the literature demonstrated that het-
erocyclic N-ethylurea derivatives to be conducive with potent dual-
inhibitory ATPase activity coupled to antimicrobial activity
[7,8,10,11]. Notably, benzimidazol-2-yl-ureas (1), benzathiazol-2-
yl-ureas (2), imidazopyridin-2-yl-ureas (3), triazolopyridine-5-
carboxamides (4), isoquinolin-3-yl-ureas (5) and pyridin-3-yl-
ureas (6) reported by groups at Vertex [7], Prolysis [11], Pfizer [10],
* Corresponding author.
E-mail address: C.W.G.Fishwick@leeds.ac.uk (C.W.G. Fishwick).
http://dx.doi.org/10.1016/j.ejmech.2014.08.025
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

32
I.A. Yule et al. / European Journal of Medicinal Chemistry 86 (2014) 31e38
Fig. 1. Reported GyrB/ParE inhibitors featuring the N-ethylurea moiety.
Evotec [8], Actelion [12] and AstraZeneca [13] respectively, exem-
plify the prevalence of this moiety amongst antibacterial GyrB/ParE
inhibitors (Fig. 1).
2. Molecular design of inhibitors
Employing an in house crystal structure depicting the ATP
binding domain of GyrB, we explored and modelled a number of
alternative heterocyclic core scaffolds which, when coupled to the
N-ethylurea side-chain, were predicted to have the potential for
potent GyrB inhibitory activity whilst offering distinct advantages
in terms of optimisation opportunities to compounds 1e6. Specif-
ically, the de novo design software SPROUT [14] aided the identifi-
cation of putative inhibitors with features complementary to the
ATP binding pocket.
Using SPROUT, key interactions within the GyrB ATP binding
pocket, specifically involving Asn-55, Asp-82, Gly-86, Lys-145 and
Arg-85 (Fig. 2), were selected and an ethylurea moiety was posi-
tioned so that it would form hydrogen bonding interactions to Asp-
82 and Asn-55 (Fig. 2). A variety of small structural fragments were
also positioned in the other selected target sites (for Arg-85 aryl
and heteroaryl fragments, for Lys-145 H-bond acceptors and
Fig. 3. Simple pyridine-3-carboxamid-6-yl-urea 7 derived using de novo ligand design.
carboxylates and for Gly-86 H-bond donors) and connected with
linker fragments (consisting of aryl, amide and single carbon
atoms) to give a selection of complete molecules. A number of such
design runs were conducted within SPROUT involving variations in
the specific fragments placed at each of the chosen residues and the
order in which these were connected. Following the SPROUT runs
the designed molecules were analysed based on their predicted
binding scores and ease of synthesis.
Fig. 4. Compound 7 docked within the E. faecalis GyrB ATP binding site. Compound 7 is
shown as orange sticks, key residues as grey sticks and polar interactions as dashed
black lines. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Fig. 2. The first stages of de novo design using SPROUT. The ATP binding site of
Enterococcus faecalis GyrB with several target sites selected and an ethylurea fragment
docked into the target sites for Asp-82 and Asn-55.

I.A. Yule et al. / European Journal of Medicinal Chemistry 86 (2014) 31e38
33
site within the protein using AutoDock [15] acted as an indepen-
dent check and confirmed that key binding interactions of known
importance to GyrB/ParE inhibition would be maintained. These
include hydrogen bonds to Asn-55/Asp-82 and an aryl
pep stack
interaction with Arg-85 (E. faecalis GyrB numbering, Fig. 4) [16].
Although treated as neutral by AutoDock during the docking pro-
cess, Arg-85 is likely to be positively charged under physiological
conditions, and therefore interaction of this residue with the pyr-
idine ring may be expected to have both
character.
pep and pecation
3. Synthesis of putative inhibitors
Scheme 1. Reagents and conditions: (a) EtNCO, 1,4-dioxane, 100 ꢀC, 18 h, 63%; (b) 2 M
NaOH (aq), RT, 16 h, 95%; (c) ArNH2, EDC$HCl, HOBT, DMF, RT-40 ꢀC, 16 h, 15e70%.
To test the modelling hypothesis, compound 7 was prepared
according to the procedure described in Scheme 1 from commer-
cially available 6-aminonicotinate 8.
Compound 7 was screened in vitro for GyrB inhibitory activity
(ATPase, Escherichia coli) and minimum inhibitory concentrations
(MICs) against S. aureus (ATCC 29213), E. faecalis (ATCC 29212),
Streptococcus pyogenes (ATCC 51339) and Haemophilus influenzae
(ATCC 49247) (Table 1).
As indicated from inspection of the data in Table 1, although
compound 7 synthesised as part of the present work was 1e2 or-
ders of magnitude less potent against GyrB than those previously
reported, this was encouraging given the lower molecular weight/
complexity of this inhibitor. Furthermore, the antimicrobial activity
found for this compound, albeit weak, indicated the potential for
optimisation. We therefore elected that our continuing investiga-
tion would focus on further developing these pyridine-3-
carboxamides.
Table 1
Comparison of compound 5 with reported GyrB inhibitors.
Compound
GyrB IC50
(m
M)
MIC (
m
g/mL)
EF
SA
SP
HI
1a
2b
3c
4d
7
0.004
<0.75
0.053
0.041
6.0
0.03
nd
0.5
8
nd
<0.25
nd
4
256
nd
nd
0.5
8
1
nd
nd
nd
>256
>256
32
E. coli GyrB; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes
ATCC 51339; HI, H. influenzae ATCC 49247; nd, not determined.
a
Data (Ki) from Charifson et al. [7].
Data from Haydon et al. [11].
Data from Starr et al. [10].
Data from East et al. [8].
b
c
d
4. C3 position SAR
From a synthetic viewpoint, the most attractive structure to
emerge from this process was based on a pyridine-3-carboxamide
(7) core (Fig. 3). Docking of this structure into the ATP binding
Our initial SAR investigation concerned variation of the aryl
moiety at the pyridyl C3 position of structure 7 and the target
Table 2
GyrB inhibitory activity and MICs for pyridine-3-carboxamides.
Cmpd
R
GyrB IC50
(m
M)
MIC (mg/mL)
SA
SA (T173N)
EF
256
>16
16
>64
>64
32
SP
SP (A53S)
HI
7
Pyridin-3-yl
Phenyl
6.0
7.7
1.6
2.1
3.3
3.0
3.4
3.8
24.5
40.4
6.2
7.5
3.5
>256
>64
16
>64
>64
32
nd
nd
>64
nd
nd
>64
>64
nd
nd
nd
>64
nd
nd
nd
nd
nd
nd
nd
nd
32
nd
nd
>64
nd
nd
>64
>64
nd
nd
nd
>64
nd
nd
nd
nd
nd
nd
nd
nd
>256
>16
>64
>64
>64
>64
>64
>64
>256
>256
>64
>64
>64
>64
>64
>64
>64
>64
>64
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
>16
16
>64
>64
32
3-Chlorophenyl
2-Chlorophenyl
4-Chlorophenyl
3-Flourophenyl
3-Methylphenyl
3-Cyanophenyl
3-Carbamoylphenyl
3-Ethylphenyl
3,4-Bismethoxyphenyl
5-Chloro, 2-methylphenyl
Pyridin-4-yl
2-Methyl pyridin-4-yl
4-CO2Me pyridin-2-yl
Thiazol-2-yl
5-CO2H thiazol-2-yl
Cyclohexyl
32
32
32
32
>64
>256
>256
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>256
>256
32
>64
64
>64
>64
>64
>64
>64
>64
>256
>256
32
>64
64
>64
>64
>64
>64
>64
>64
5.3
14.0
0.42
0.96
23.1
8.4
4-Methylcoumarin-7-yl
E. coli GyrB; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; HI, H. influenzae ATCC 49247; SA T173N, S. aureus GyrB mutant; SP A53S,
S. pyogenes ParE mutant; nd, not determined.

34
I.A. Yule et al. / European Journal of Medicinal Chemistry 86 (2014) 31e38
activity in the strains tested (Table 2). Compounds 12, 15, 16 and 20,
which possessed antibacterial activity against the wild type strains,
were further tested for MICs against a S. aureus GyrB mutant
(T173N) and a S. pyogenes ParE mutant (A53S) respectively. The
compounds showed diminished antibacterial activity relative to
that observed for the wild-type, consistent with a target based,
dual-inhibitory mode of action. None of the compounds tested
demonstrated antibacterial activity against the Gram negative or-
ganism H. influenzae, highlighting the challenges involved in tar-
geting Gram negative organisms.
5. Variation of the C4 position: synthesis and SAR
According to our earlier modelling (Fig. 3), a sizeable sub-pocket
was predicted such that substitution at the pyridyl C4 position in
structure 7 was highlighted as an option for structural optimisation.
Synthetic routes to these compounds are summarised in Schemes
2e5. Our initial route proceeded via 5-iodopyridine 30, which
was prepared following regioselective iodination of 2-amino-4-
chloropyridine. A modified Rosenmundevon Braun reaction [17]
installed the carbonitrile functionality as in 31, which was hydro-
lysed under aqueous acidic conditions to give tri-substituted
nicotinic acid 32. Coupling under standard conditions with an
appropriate aniline led, unexpectedly, to the 6-amino-3,4-
bisanilino intermediate which, when treated with ethyl isocya-
nate, gave pyridin-6-yl-urea 33 (Scheme 2).
Alternatively, C4 substituted compounds were prepared via
dichloronicotinate 36 [18] using a range of synthetic methodolo-
gies, as indicated in Schemes 3e5. Sequential SNAr at the C4 and C6
positions of pyridine 36, followed by facile benzyl deprotection
using TFA and triethylsilane, gave 6-amino-4-anilinonicotinate 39.
This intermediate was treated with ethyl isocyanate to give the N-
Scheme 2. Reagents and conditions: (a) NIS, DMF, rt, 18 h, 60%; (b) Zn(CN)2, Pd(PPh3)4,
NMP, 135 ꢀC, 2 h, 60%; (c) 1:2 c$H2SO4eH2O, 100 ꢀC, 18 h, 81%; (d) m-toluidine, HOBT,
EDC$MeI, DMF, rt, 5 h; (e) EtNCO, 1,4-dioxane, 80 ꢀC, 16 h, 17% (over 2 steps).
molecules were accessed conveniently through coupling of nico-
tinic acid 10 with commercially available aromatic amines (Scheme
1, Table 2). We reasoned that variation here could increase the
importance of the
pep stacking with Arg-85. Biological data is
given in Table 2. In general, substituted phenyl derivatives (entries
11e21) led to improvements in enzyme potency and some MICs
were also improved relative to compound 7. In particular the ortho-
chloro compound 12 (GyrB IC50 1.6 mM, MIC 16 mg/mL SA, EF, SP)
demonstrated a good balance between enzyme and cellular po-
tency. Potent GyrB inhibitors were also identified when the C3
substituent was a 5-membered heterocycle. Thiazole compounds
25 and 26 have IC50s of 420 nM and 960 nM respectively, though
these compounds did not demonstrate measurable antibacterial
Scheme 3. Reagents and conditions: (a) HC(OEt)3, Ac2O, NH3(aq), 120 ꢀC, 2 h, 55%; (b) POCl3, 110 ꢀC, 2.5 h, 79%; (c) m-toluidine, HCl, EtOH, 80 ꢀC, 3 h, 51%; (d) p-methox-
ybenzylamine, PhMe, reflux, 72 h, 62%; (e) TFA, Et3SiH, DCM, rt, 4 h, 98%; (f) EtNCO, 1,4-dioxane, 100 ꢀC, 48 h, 43%; (g) 2 M NaOH(aq), 75 ꢀC, 48 h, 90%; (h) EDC$HCl, HOBT, ArNH2,
DMF, 40 ꢀC, 18 h, 35%; (i) ImH, NaH, DMF, 0 ꢀC e RT, 34% or N-methylpiperazine, TEA, EtOH, 0 ꢀC, 3 h, 80%; (j) Pd(OAc)2, Xantphos, KOtBu, N-ethylurea, 1,4-dioxane, H2O, 100 ꢀC, 16 h,
79e92%; (k) 2 M NaOH (aq), rt or 40 ꢀC, 2 h, 56e97%; (l) ArNH2, EDC$HCl, HOBT, DMF, rt-40 ꢀC, 5e51%.

I.A. Yule et al. / European Journal of Medicinal Chemistry 86 (2014) 31e38
35
Scheme 4. Reagents and conditions: (a) Pd(OAc)2, Xantphos, KOt-Bu, N-ethylurea, 1,4-dioxane, H2O, 100 ꢀC, 16 h, 40%; (b) NaH, NuH, DMF, 0 ꢀCe70 ꢀC, 48 h, 21e32%, or RNH2, EtOH,
reflux, 68e71%, or ArNH2, HCl, EtOH, 60 ꢀC, 16 h, 93%; (c) Pd(PPh3)4, ArB(OH)2, 2 M Na2CO3(aq), 1,4-dioxane or THF, reflux, 2e16 h, 63%; (d) Pd(OAc)2, Xantphos, KOt-Bu, ArNH2, 1,4-
dioxane, H2O, 100 ꢀC, 3 h; (e) 2 M NaOH(aq), rt-70 ꢀC, 47%-quantitative or LiOH (aq), THF, EtOH, 60 ꢀC, 3 h, 95%; (f) ArNH2, EDC$HCl, HOBT, DMF, rt-40 ꢀC, 22e55%.
ethylurea 40 as per Scheme 3. Ester hydrolysis under more forcing
conditions than those used previously, preceded synthesis of the
target molecules under standard amide coupling conditions. In an
improvement to this synthetic sequence, we discovered that the N-
ethylurea side-chain could be installed directly to C4 functional-
ised-6-chloro-nicotinates (i.e. compounds 43 and 44) via a Buch-
waldeHartwig type coupling [19]. The target nicotinamides were
thus accessed following ester hydrolysis (Scheme 3). A key
improvement was realised upon direct reaction of dichloro com-
pound 36 with N-ethylurea under the same conditions to give the
4-chloro-pyridin-6-yl-urea intermediate 54 with C6 regiose-
lectivity. Variation at the C4 position was then readily achieved via
SNAr, BuchwaldeHartwig coupling or SuzukieMiyaura coupling
accordingly either following or prior to nicotinamide synthesis
using the previously described conditions (Scheme 4). Further C4-
substituted derivatives were synthesised using a variant synthetic
route. Ester hydrolysis of the 4-chloro-pyridin-6-yl urea interme-
diate 52 followed by amide coupling using T3P gave 4-chloro-
pyridine carboxamide 90 which was converted to C4 substituted
compounds 91e93 via SNAr reactions (Scheme 5).
suitability of these compounds for broad spectrum therapeutic use,
selected compounds were further tested for MICs against an
extended panel of Gram positive (Streptococcus epidermidis ATCC
12228, Streptococcus pneumonia ATCC 49616), Gram negative
(E. coli ATCC 25922, E. coli N43 efflux knock-out) and mutant
(S. aureus T173N GyrB, S. pyogenes A53S ParE, S. aureus T173N/A53S
GyrBeParE dual mutant) bacterial species. These additional data
are presented in Table 5.
As indicated from inspection of the data in Table 3, significant
improvements in enzyme potency and antimicrobial activity were
realised upon the addition of a substituted aniline to the pyridyl C4
position. In particular, compound 33 has an IC50 of 99 nM and MIC
of 0.5 mg/mL against E. faecalis. Thiazol-2-yl nicotinamide 42, being
structurally analogous, demonstrated comparable enzyme inhibi-
tion (GyrB IC50 98 nM) but was not antibacterial within the con-
centration ranges tested, illustrating the importance of structural
modification on bacterial membrane penetration. Structural de-
parture from an amino aryl substituent at C4 (compounds 49e76)
led to reduced enzyme potency and antibacterial activity, with the
exception of pyrazol-1-yl compound 73 which, despite having
Biological data for the C4 substituted pyridine-3-carboxamides
is given in Tables 3 and 4. Additionally, in order to probe the
moderate enzyme potency (GyrB IC50 2.96
tivity against Gram positive bacterial species. The 4-
mM), retained good ac-
Scheme 5. Reagents and conditions: (a) 2 M NaOH(aq), reflux, 80 ꢀC, 18 h, 83%; (b) ArNH2, T3P, EtOAc, rt, 18 h, 34e57%; (c) 3-aminopyridine, HCl, EtOH, 60 ꢀC, 16 h, 46e83%.

36
I.A. Yule et al. / European Journal of Medicinal Chemistry 86 (2014) 31e38
Table 3
(Table 2). It was evident that a single atom-linked aromatic ring at
the C4 position of the pyridine core was important to inhibitor
efficacy. The reduced potency demonstrated by C4-
cyclohexylamino derivative 74, further confirmed this apparently
strict requirement. In order to aid our understanding, compound 33
was modelled within the E. faecalis ATP binding domain (Fig. 5).
Again, important polar contacts were predicted to be maintained
and it appeared plausible that ligand ‘pre-organisation’ through an
internal hydrogen bond involving the carbonyl at C3 and the NH at
C4 may explain the improvement in potency.
GyrB inhibitory activity and MICs for C4 functionalised pyridine-3-carboxamides.
Cmpd R1
R2
GyrB IC50
(m
M) MIC (
m
g/mL)
SA
EF
SP
HI
A number of compounds were synthesised to further explore
this phenomenon through preservation of the C4 amino-aryl motif
(Table 4). In this instance, simple, unsubstituted phenyl/pyridine-3-
yl moieties were chosen and found to be adequate replacements for
the m-toluidyl group present in compound 33. Compounds
featuring the amino-aryl appendage were predominantly found to
have potent GyrB inhibitory activity (IC50s < 500 nM) and Gram
33
42
0.099
0.098
1
0.5
32 >64
>64 >64
>64 >64
positive antibacterial activity (MICs 0.125e2 mg/mL). Tellingly, C4
O-aryl compound 85, which lacks the ability to form an internal
hydrogen bond, is comparatively inactive and had no antibacterial
activity (GyrB IC50 4.9 mM). Para substitution at the C3 aromatic
49
50
5.14
5.66
>64 >64
>64 >64
>64 >64
>64 >64
group was found to be well tolerated, with compounds 81e83
designed to probe the solvent exposed area of the binding pocket
using morpholine, acetamide and triazole side-chains respectively.
In particular, compound 82 is a highly potent GyrB inhibitor (GyrB
IC50 39 nM), presumably forming additional contacts with Lys-145
(Arg-136 E. coli GyrB) through the acetamide carbonyl oxygen.
Incorporating a 3-chlorophenylamine moiety at C3 identified
compound 79 as the most potent anti Staphylococcal agent tested
51
52
53
18.0
33.2
0.55
>64 >64
>64 >64
>64 >64
>64 >64
64 >64
>64
>64
32
64
here (MIC S. aureus 0.5 mg/mL). Where MICs against S. aureus were
determined in the presence of plasma proteins (horse serum,
compounds 80e82, 86), MIC shifts of 4e8-fold were observed,
indicating that compounds of this class are not greatly impeded by
high plasma-protein binding (Table 4).
Table 5 details additional biological profiling of the most
promising C4 substituted pyridine-3-carboxamides. Firstly, excel-
lent antibacterial activity was demonstrated against S. epidermidis
68
69
70
71
72
0.56
1.13
3.02
7.27
0.81
64 >64
64 >64
>64
>64
>64
>64
8
64
64
64
4
64 >64
and S. pneumonia (MICs 0.125e1
mg/mL), confirming the com-
pounds possess a broad spectrum of Gram positive activity.
Furthermore, though none of the compounds tested showed ac-
tivity versus E. coli, it is significant that certain compounds (73, 77
and 80) were active against the efflux knock-out mutant indicating
that active efflux plays a role in the lack of Gram negative activity of
this compound class. The MICs for these three compounds were
higher against the efflux-knock out E. coli than against wild type
S. aureus suggesting that they are not able to permeate Gram
negative cells as effectively as Gram positive cells.
Finally, mutant data provided useful insights into the mode of
action, dual-inhibitory nature and resistance vulnerability of these
compounds. In all cases, MIC shifts were observed versus GyrB/
ParE mutants suggesting bacterial cell death to be target specific
rather than due to promiscuous cell damaging effects. Compounds
33 and 78, whilst potent against wild-type species, were ineffec-
tive against S. aureus and S. pyogenes single-step GyrB and ParE
mutants respectively. Compounds 81 and 82 lost activity against
the S. aureus GyrB mutant but maintained activity against the
S. pyogenes ParE mutant. This would suggest GyrB to be the pri-
mary target for these compounds. Pleasingly, compounds 73, 77,
79 and 80 were active against both GyrB and ParE single-step
mutants but lost all antibacterial activity versus the S. aureus
dual GyrB/ParE mutant. The indication is therefore that these
compounds are potent dual inhibitors of GyrB/ParE, are not
vulnerable to single-step resistance and would require a statisti-
cally unlikely spontaneous double mutation for resistance to be
conferred.
>64 >64
>64 >64
73
74
2.96
2.12
1
4
>64
>64 >64
>64 >64
75
76
0.66
4.81
>64
>64
8
2
8
8
>64
>64
E. coli GyrB; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes
ATCC 51339; HI, H. influenza ATCC 49247.
methylpiperazine moiety at C4 (compounds 49e52) is conducive
with both a reduction in enzyme inhibitory activity and a loss of
antibacterial activity, whilst directly linked aromatic groups at this
position (compounds 53e73) resulted in inhibitors with broadly
similar activity profiles to the parent unsubstituted compounds

I.A. Yule et al. / European Journal of Medicinal Chemistry 86 (2014) 31e38
37
Table 4
GyrB inhibitory activity and MICs for C4 amino-aryl pyridine-3-carboxamides.
Cmpd
R1
R2
GyrB IC50
(
m
M)
MIC (mg/mL)
SA
SA þ HS
EF
SP
HI
77
78
79
80
81
82
83
84
85a
86
87
Phenyl
3-Methylphenyl
3-Chlorophenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
0.040
0.140
0.150
0.170
0.110
0.039
0.150
2.0
1
1
0.5
1
2
2
nd
nd
nd
4
16
8
nd
nd
nd
2
0.25
2
2
4
1
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.125
0.125
0.125
0.25
0.125
0.25
Pyridin-3-yl
6-(Morpholin-1-yl)pyridin-3-yl
6-(Acetamid-1-yl)pyridin-3-yl
6-(1,2,4-Triazol-1-yl)pyridin-3-yl
3-Picolylamin-1-yl
Phenyl
1
0.5
>16
1
>16
1
>16
>16
>16
1
0.5
Phenyl
Pyridin-3-yl
Pyridin-3-yl
4.9
0.43
0.093
>16
0.25
0.5
Phenyl
Pyridin-3-yl
8
nd
1
E. coli GyrB; SA, S. aureus ATCC 29213; SA þ HS, S. aureus þ 50% horse serum; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; HI, H. influenzae ATCC 49247. Unless stated,
X ¼ NH.
nd, not determined.
a
X ¼ O.
Subsequently a small library of compounds was synthesised
with the goal of investigating alternative substitution at the C4
position (Schemes 4 and 5). Hydroxy- and methoxy-phenyl de-
rivatives were chosen, as docking indicated that the C4 group
would be oriented in a solvent exposed area, and more polar sub-
stituents would be predicted to experience stabilisation in the
water-exposed region. Enzyme inhibition was measured against
both GyrB and ParE enzymes from E. coli and S. aureus and MICs
were measured against both S. aureus and E. coli (Table 6). In
keeping with our previous observations, none of the compounds
synthesised showed meaningful activity against the Gram negative
species suggesting that simple variation at the C4 position is
insufficient to provide compounds with Gram negative activity. All
compounds showed good inhibitory activity against GyrB and
reasonable activity against ParE, although they were less active
against ParE from E. coli which may offer an additional barrier to
activity of these compounds in Gram negative bacteria.
Through structural optimisation we have developed the pyridine-
3-carboxamide-6-yl-urea core to offer derivatives with excellent
GyrB inhibitory activity and Gram positive antibacterial efficacy. A
key activity breakthrough via the introduction of amino-aryl i.e.
NH-aryl functionality at the pyridyl C4 position is reported.
Encouragingly, inhibitors were identified (e.g. compound 80) which
demonstrate broad spectrum Gram positive antibacterial activity,
activity against a Gram negative efflux pump mutant, and activity
versus single-step GyrB/ParE mutants. These observations may
prove important in the future therapeutic utility of this compound
class. Compounds reported in this paper have been patented [20].
Further optimisation of compounds in this series is ongoing, with a
focus on modification of physicochemical properties with the
hopes of broadening their spectrum of activity to include Gram
negative organisms.
6. Conclusion
In summary we have reported the discovery, synthesis and an
initial SAR study of a novel series of antibacterial GyrB inhibitors.
Table 5
MICs for selected compounds against GyrB/ParE mutants, additional Gram positive
species and E. coli.
Cmpd MIC (
mg/mL)
SA SA T173N SA T173N/T167N SP SP A53S SE
SPn
EC EC
(N43)
(GyrB)
(GyrB/ParE)
(ParE)
73
77
78
79
80
81
82
8
1
1
0.5
1
32
2
>64
1
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
4
2
2
4
1
1
0.5
16
16
>64
32
2
2
0.25
0.5 0.5
0.5
1
1
>64 32
>64 8
>64 >64
>64 >64
1
0.25 0.25 >64
0.5 >64 >64
0.25 0.125 >64 >64
8
2
2
4
2
1
SA, S. aureus ATCC 29213; SA T173N, S. aureus GyrB mutant; SA T173N/T167N,
S. aureus dual GyrB/ParE mutant; SP, S. pyogenes ATCC 51339; SP A53S, S. pyogenes
ParE mutant; SE, S. epidermidis ATCC 12228; SPn, S. pneumonia ATCC 49616; EC,
E. coli ATCC 25922; EC (N43), E. coli efflux pump mutant.
Fig. 5. Compound 33 docked within the E. faecalis GyrB ATP binding site. 33 is shown
as orange sticks, key residues (EF numbering) as grey sticks and predicted polar con-
tacts as dashed lines. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

38
I.A. Yule et al. / European Journal of Medicinal Chemistry 86 (2014) 31e38
[5] C. Levine, H. Hiasa, K.J. Marians, DNA gyrase and topoisomerase IV:
Table 6
biochemical activities, physiological roles during chromosome replication,
and drug sensitivities, Biochim. Biophys. Acta 1400 (1998) 29e43.
[6] L.A. Mitscher, Bacterial topolsomerase inhibitors: quinolone and pyridone
antibacterial agents, Chem. Rev. 105 (2005) 559e592.
GyrB and ParE inhibitory activity and MICs for C4 amino-aryl pyridine-3-
carboxamides.
[7] P.S. Charifson, A.L. Grillot, T.H. Grossman, J.D. Parsons, M. Badia, S. Bellon,
D.D. Deininger, J.E. Drumm, C.H. Gross, A. LeTiran, Y.S. Liao, N. Mani,
D.P. Nicolau, E. Perola, S. Ronkin, D. Shannon, L.L. Swenson, Q. Tang,
P.R. Tessier, S.K. Tian, M. Trudeau, T.S. Wang, Y.Y. Wei, H. Zhang, D. Stamos,
Novel dual-targeting benzimidazole urea inhibitors of DNA gyrase and topo-
isomerase IV possessing potent antibacterial activity: Intelligent design and
evolution through the judicious use of structure-guided design and structure-
activity relationships, J. Med. Chem. 51 (2008) 5243e5263.
[8] S.P. East, C.B. White, O. Barker, S. Barker, J. Bennett, D. Brown, E.A. Boyd,
C. Brennan, C. Chowdhury, I. Collins, E. Convers-Reignier, B.W. Dymock,
R. Fletcher, D.J. Haydon, M. Gardiner, S. Hatcher, P. Ingram, P. Lancett,
P. Mortenson, K. Papadopoulos, C. Smee, H.B. Thomaides-Brears, H. Tye,
J. Workman, L.G. Czaplewski, DNA gyrase (GyrB)/topoisomerase IV (ParE) in-
hibitors: synthesis and antibacterial activity, Bioorg. Med. Chem. Lett. 19
(2009) 894e899.
Compound R1
IC50s (
GyrB
SA
m
M)
MIC (
mL)
mg/
ParE
EC
SA
EC
0.94
SA EC
8 >64
88
91
92
4-Hydroxyphenyl
3-Hydroxyphenyl
2,4-Bismethoxyphenyl 0.041 0.046 0.80
0.024 0.028 0.086
nd 0.036 nd
0.78 nd >64
42 >64
1
[9] S.M. Ronkin, M. Badia, S. Bellon, A.L. Grillot, C.H. Gross, T.H. Grossman,
N. Mani, J.D. Parsons, D. Stamos, M. Trudeau, Y.Y. Wei, P.S. Charifson, Dis-
covery of pyrazolthiazoles as novel and potent inhibitors of bacterial gyrase,
Bioorg. Med. Chem. Lett. 20 (2010) 2828e2831.
S. aureus and E. coli GyrB; S. aureus and E. coli ParE; SA, S. aureus ATCC 29213; EC,
E. coli ATCC 25922; nd, not determined.
[10] J.T. Starr, R.J. Sciotti, D.L. Hanna, M.D. Huband, L.M. Mullins, H. Cai, J.W. Gage,
M. Lockard, M.R. Rauckhorst, R.M. Owen, M.S. Lall, M. Tomilo, H.F. Chen,
S.P. McCurdy, M.R. Barbachyn, 5-(2-Pyrimidinyl)-imidazo[1,2-a]pyridines are
antibacterial agents targeting the ATPase domains of DNA gyrase and topo-
isomerase IV, Bioorg. Med. Chem. Lett. 19 (2009) 5302e5306.
[11] D.R. Haydon, L.G. Czaplewski, N.J. Palmer, D.R. Mitchell, J.F. Atherall,
C.R. Steele, T. Ladduwahetty, Antibacterial Compositions, 2007.
[12] D. Bur, M. Gude, C. Hubschwerlen, P. Panchaud, Antibacterial Isoquinolin-3-
ylurea Derivatives, 2011.
Acknowledgements
The authors would like to thank Craig Avery for useful discus-
sion and Biota Europe Ltd. for their financial support and biological
evaluation during the course of this research.
[13] G.S. Basarab, J.I. Manchester, S. Bist, P.A. Boriack-Sjodin, B. Dangel,
R. Illingworth, B.A. Sherer, S. Sriram, M. Uria-Nickelsen, A.E. Eakin, Fragment-
to-hit-to-lead discovery of a novel pyridylurea scaffold of ATP competitive
dual targeting type II topoisomerase inhibiting antibacterial agents, J. Med.
Chem. 56 (2013) 8712e8735.
[14] V. Gillet, A.P. Johnson, P. Mata, S. Sike, P. Williams, Sprout e a program for
structure generation, J. Comput. Aided Mol. Des. 7 (1993) 127e153.
[15] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (2009) 2785e2791.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.025.
References
[16] S.P. East, L.G. Czaplewski, D.J. Haydon, Chapter 20 ethyl urea inhibitors of the
bacterial type II topoisomerases DNA gyrase (GyrB) and topoisomerase IV
(ParE), in: Designing Multi-target Drugs, The Royal Society of Chemistry, 2012,
pp. 335e352.
[17] A. Tsuruoka, T. Matsushima, K. Miyazaki, J. Kamata, Y. Fukuda, K. Takahashi, M.
Matsukura, Int. Pat. Appl. WO 200420434, 2005.
[18] E. Wallace, B. Hurley, H.W. Yang, J. Lyssikatos, J. Blake, Int. Pat. App.
WO200523759, 2005.
[19] A. Abad, C. Vilanova, Regioselective preparation of pyridin-2-yl ureas from 2-
chloropyridines catalyzed by Pd(0), Synthesis 6 (2005) 915e924.
[20] L.G. Czaplewski, C.W.G. Fishwick, I.A. Yule, J.P. Mitchell, K.H. Anderson, G.R.W.
Pitt, WO 2013/091011 A1, 2013.
[1] A.J. Alanis, Resistance to antibiotics: are we in the post-antibiotic era? Arch.
Med. Res. 36 (2005) 697e705.
[2] L.B. Rice, Federal funding for the study of antimicrobial resistance in noso-
comial pathogens: no ESKAPE, J. Infect. Dis. 197 (2008) 1079e1081.
[3] H.W. Boucher, G.H. Talbot, J.S. Bradley, J.E. Edwards, D. Gilbert, L.B. Rice,
M. Scheld, B. Spellberg, J. Bartlett, Bad Bugs, no drugs: no ESKAPE! An update
from the infectious diseases society of America, Clin. Infect. Dis. 48 (2009)
1e12.
[4] D.N. Gilbert, R.J. Guidos, H.W. Boucher, G.H. Talbot, B. Spellberg,
J.E. Edwards Jr., W.M. Scheld, J.S. Bradley, J.G. Bartlett, The 10 ꢁ '20 initiative:
pursuing a global commitment to develop 10 new antibacterial drugs by 2020,
Clin. Infect. Dis. 50 (2010) 1081e1083.