Novel small molecule inhibitors targeting the "switch region" of bacterial RNAP: Structure-based optimization of a virtual screening hit

Article abstract of DOI:10.1016/j.ejmech.2013.04.060

Rising resistance against current antibiotics necessitates the development of antibacterial agents with alternative targets. The "switch region" of RNA polymerase (RNAP), addressed by the myxopyronins, could be such a novel target site. Based on a hit can

Full text of DOI:10.1016/j.ejmech.2013.04.060

European Journal of Medicinal Chemistry 65 (2013) 223e231
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel small molecule inhibitors targeting the “switch region” of
bacterial RNAP: Structure-based optimization of a virtual screening hit
*
J. Henning Sahner, Matthias Groh, Matthias Negri, Jörg Haupenthal, Rolf W. Hartmann
Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug
Design and Optimization, Campus C2 3, 66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Rising resistance against current antibiotics necessitates the development of antibacterial agents with
alternative targets. The “switch region” of RNA polymerase (RNAP), addressed by the myxopyronins,
could be such a novel target site. Based on a hit candidate discovered by virtual screening, a small library
of 5-phenyl-3-ureidothiophene-2-carboxylic acids was synthesized resulting in compounds with
Received 4 April 2013
Received in revised form
26 April 2013
Accepted 27 April 2013
Available online 7 May 2013
increased RNAP inhibition. Hansch analysis revealed
p (lipophilicity constant) and s (Hammet substit-
uent constant) of the substituents at the 5-phenyl moiety to be crucial for activity. The binding mode was
proven by the targeted introduction of a moiety mimicking the enecarbamate side chain of myxopyronin
into the hit compound, accompanied by enhanced RNAP inhibitory potency. The new compounds dis-
played good antibacterial activities against Gram positive bacteria and Gram negative Escherichia coli TolC
and a reduced resistance frequency compared to the established antibiotic rifampicin.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Bacterial RNAP inhibitor
Switch region
Myxopyronin
Ureidothiophene carboxylic acid
QSAR
Antibacterial agent
1. Introduction
discovered as an interesting new binding domain of bacterial RNAP
[2,4]. Myxopyronin B (1), a natural
a-pyrone antibiotic isolated
The bacterial RNA polymerase (RNAP) catalyzes the pivotal
transcription process and therefore is an attractive drug target for
antibacterial agents [1e3]. Due to its high conservation among a
wide range of bacteria, inhibitors of the RNAP are applicable against
a broad spectrum of bacterial pathogens [4]. The architecture of the
bacterial enzyme significantly differs from eukaryotic RNAP, facili-
tating a selective blockade of the essential bacterial cell function
[5,6]. This is exemplified by a wide range of compounds which
selectively effect the prokaryotic RNAP [2]. Several classes of in-
hibitors targeting bacterial RNAP are known [1,3]. But at the current
state the clinically used compounds are limited to lately approved
fidaxomicin and members of the rifamycin family [2,7]. For
example rifampicin is used in combination with other antibacte-
rials, in the first line therapy of tuberculosis [3,8]. In the meantime
some resistant strains, including multi-resistant Mycobacterium
tuberculosis, emerged [4,9,10]. This creates an urgent need for the
development of new classes of antibacterial agents [2,5,11].
from the myxobacterium Myxococcus fulvus [13], and its synthetic
derivative desmethyl myxopyronin B (2) [14], have been demon-
strated to target this region [4,15]. Due to the different binding
mode compared to the rifamycin antibiotics, compounds binding to
the “switch region” are expected to overcome existent resistance
[4]. Studies concerning the activity of myxopyronins towards rifa-
mycin-resistant strains of Staphylococcus aureus have confirmed,
that the “switch region” inhibitors do not exhibit cross-resistance
to the rifamycins [16,17]. Although the natural compound myx-
opyronin B (1) is highly active in vitro (IC50 ¼ 0.35
mM; minimal
g/mL for Escherichia coli TolC), its
inhibitory concentration ¼ 0.8
m
use as a drug is hampered by insufficient physicochemical prop-
erties [18,19]. Therefore small molecule inhibitors possessing a
higher in vivo efficacy should be developed.
Another project of our group is focused on the optimization of
novel RNAP inhibitors of the benzamidobenzoic acid type, identi-
fied by a virtual screening, based on the flexible alignment of
known inhibitors. Besides good in vitro activity these compounds
also possess antibacterial effects [20]. In a previous study Fishwick
et al. developed inhibitors of bacterial RNAP, using a structure-
based de novo design approach. These compounds, predicted to
bind to the “switch region”, possessed inhibitory activity against
E. coli RNAP, but displayed no antibacterial properties [21]. Very
A promising strategy to face resistance is the exploration of
novel target sites [6,12]. Recently, the “switch region” has been
* Corresponding author. Tel.: þ49 681 302 70300.
E-mail addresses: rolf.hartmann@helmholtz-hzi.de, rwh@mx.uni-saarland.de
(R.W. Hartmann).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.060

224
J.H. Sahner et al. / European Journal of Medicinal Chemistry 65 (2013) 223e231
Me
O
O
OH
O
Bu
H
N
OMe
1: R = Me
2: R = H
R
O
Cl
Ph
H
O
O
Ph
N
S
N
HN
HN
S
5: R = H
6: R = Et
7: R = tBu
COOR
COOR
3: R = H
4: R = Me
Chart 1. Myxopyronins 1 and 2, hit compound 3, its ester 4 and S. aureus RNAP in-
hibitors 5, 6 and 7.
were subjected to the virtual screening using our 3D-pharmaco-
phore model. None of them was recognized. This is in accordance
with the observation of Buurmann et al. that these ureido cyclo-
octathiophenes failed to dock to the “switch region” of the RNAP
[22]. In both crystal structures of Thermus thermophilus RNAP with
1 (PDB ID: 3DXJ) [4] and 2 (PDB ID: 3EQL) [15] the myxopyronins
adopt a U-shaped conformation filling the “switch region” binding
pocket. According to the docking pose (Fig. 1) hit compound 3 is
predicted to bind in a tilted conformation. The thiophene core is
placed on the top of the entrance to the switch-2 binding cavity,
anchored by atomic interactions (H-bond or ion-pair) of its car-
boxylic acid moiety with Lys334. Such an interaction is not possible
with the corresponding ester 4. The 4-chlorophenyl ring occupies
the lower part of the enecarbamate-binding pocket of myxopyr-
onins. Thereby, the chloro atom is fitted into a small negatively-
charged site delimited by Leu343, Gly344 and Lys345 (b0 subunit)
Fig. 1. Binding mode of desmethyl myxopyronin 2 (yellow) and compound 3 (green) as
front- (a) and top-view (b). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
recently Buurmann et al. reported on the first synthetic RNAP in-
hibitors which have been shown to target the “switch region”.
Although these compounds were highly active (IC50 as low as
at one side, and Phe1270, Gly1271, Val1275 and Leu1291 (b subunit)
on the other side (red area of the electrostatic potential surface in
the orange circle in Fig. 1a) The ureido moiety, stabilized by an
intramolecular hydrogen-bond (Chart 2) with the carboxylic acid
group, overlaps with the dienone side chain of myxopyronins,
pointing the lipophilic substituents ethyl and benzyl into the hy-
4.4
mM), they showed only weak antibacterial effects [22]. In a
further work compounds supposed to interact with the “switch
region” were identified by virtual screening, but the putative hits
were not experimentally validated [23].
Recently, we identified novel RNAP inhibitors also following a
virtual screening approach. A homology model of E. coli RNAP was
used to set up a 3D-pharmacophore model, which included ligand
features from 1 and 2, but also protein-derived characteristics,
matching the “switch region”. Finally, a pharmacophore-based
virtual screening of the Chemiotheque Nationale library
(w42,000 compounds) was performed resulting in a selection of 70
virtual hits. In vitro activity determination using an E. coli RNAP
inhibition assay led to the identification of hit compound 3 showing
drophobic pocket delimited by Leu1326 and Ile1337 (b subunit) and
Phe1319, Ile1320, Ala1323, Thr1328, Ile1352 (b0 subunit). Based on
this binding mode, 3 was divided into the carboxyethiophene core
and two variable fragments for structureeactivity exploration and
optimization: the substituted phenyl ring (A), and the ureido group
(B) as depicted in chart 2.
3. Chemistry
a moderate inhibitory activity with an IC50 value of 75
mM and MIC
The synthesis of the 5-aryl-3-ureidothiophene-2-carboxylic
acids (Scheme 1) started from readily available acetophenones (I)
which were converted to the 5-aryl thiophene anthranilic acid
value of 11 g/mL (E. coli TolC) [24]. Interestingly, the related ureido
m
cyclooctathiophenes reported by Arhin et al. for example the acid 5
and especially the esters 6 and 7 have been described to inhibit
S. aureus RNAP, but their binding site was not further investigated.
They showed antibacterial effects against several S. aureus strains
but not against other Gram positive or Gram negative bacteria [25].
As we expect our compounds to bind to the “switch region” of
RNAP, which is highly conserved among various bacterial strains
[4], we considered compound 3 to be a good starting point for the
development of novel antibacterials with broad spectrum activity.
variable
variable
R2
O
R1
N
B
A
R3
N
H
S
R1 = Me, OMe, Cl, CN, NO2
CF3, OCF3, di-Cl, etc.
O
OH
R2, R3 = H, Alkyl, Phenyl,
Benzyl, etc.
2. Molecular modeling and optimization strategy
To find out whether the SAR of the ureido cyclooctathiophenes
is reasonable to be considered in our study, compounds 5, 6 and 7
Chart 2. Optimization strategy of 5-aryl-3-ureidothiophene-2-carboxylic acids.

J.H. Sahner et al. / European Journal of Medicinal Chemistry 65 (2013) 223e231
225
R1
R1
O
H2N
(c)
H2N
(a), (b)
R1
S
S
MeOOC
HOOC
I
II
III
R1
H
N
R2
O
R1
O
(d)
(e)
N
R3
O
N
H
S
S
V
IV
O
COOH
Scheme 1. Synthesis of 5-aryl-3-ureidothiophene-2-carboxylic acids (V). Reagents and conditions: (a) POCl3, DMF, 50 ꢁC to rt, then NH2OH∙HCl, up to 150 ꢁC, 75e90%. (b) Methyl-
thioglycolate, NaOMe, MeOH, reflux, 65e85%. (c) KOH, MeOH, THF, H2O, reflux, 40e80%. (d) COCl2, THF, 50e70%. (e) Amine, H2O, 100 ꢁC then at 0 ꢁC conc. HCl, 40e80%.
methylesters (II) via an ArnoldeVilsmaiereHaack reaction fol-
lowed by a cyclization using methylmercaptoacetate [26]. The es-
ters (II) were then hydrolysed under basic conditions to afford the
thiophene anthranilic acids (III) which were converted into the
thiaisatoic anhydrides (IV) [27,28]. The anhydrides (IV) were reac-
ted with various primary and secondary amines giving rise to the 5-
aryl-3-ureidothiophene-2-carboxylic acids (V) [29]. Compounds 5,
6 and 7 were synthesized as previously described [25]. Compound
4 was obtained by methylation of compound 3.
a larger blue area on ring A the electrostatic potential was less
negative for the potent compound 18, followed by 13, 3 and the
inactive compound 9. As seen in the docking poses of compounds 3
and 18 ring A is sandwiched between Leu343, Val1351, Ile1352 and
the alkyl chain of Lys345, almost perpendicular to the
a-pyrone ring
of myxopyronin (Figs. 1 and 5). CHe and van der Waals in-
p
teractions are formed between ring A and these residues. The
introduction of inductive electron-withdrawing substituents on
ring A leads to a less polarized/negative potential as shown in the
MEP maps (progressively less negative potential e Fig. 4). The in-
crease in potency seen for electron-withdrawing groups might
depend on stronger London dispersion forces resulting from more
balanced electron redistribution at both sides of the ring. The fact
that lipophilic (e.g. Cl, CF3) substituents outreach hydrophilic ones
(e.g. CN) fits well with the presence of several apolar residues in the
surrounding, i.e. Phe1270, Val1275, Leu1291, Val1351, Ile1352,
Val1353 (Fig. 2).
Within the small negatively-charged subpocket (Fig. 1 (orange
circle) and Fig. 2) the chloro derivatives 3 and especially 18 are
favored due to a complementary electrostatic surface (blue at
chloro atom). The more negative potential (green at nitrogen or
oxygen) on the methoxy in 9 and cyano group in 13 probably causes
electrostatic repulsions and is consequently associated with a drop
in activity. Subsuming, the substitution pattern of ring A finetunes
charge density distribution of the phenyl system and the thiophene
core, enabling enhanced interactions with the target protein.
4. Results and discussion
As expected, the ester of 3, compound 4 did not show E. coli
RNAP inhibition. Interestingly, not only the esters 6 and 7, but also
the free acid 5 were inactive (Table S2 in Supporting information).
During the hit optimization process of the discovered 5-phenyl-3-
ureidothiophene-2-carboxylic acids, two aims were pursued:
Firstly, the putative binding mode of hit compound 3 was investi-
gated by chemical modifications of the substituents on ring A.
Thereby the Topliss’ logical was followed to optimize the hydro-
phobic and electronic effects [30]. Secondly, the hydrophobic
pocket of the “switch region”, occupied by the dienone side chain of
myxopyronins, was explored by varying the substitution pattern of
the ureido moiety B.
The removal of the chlorine (ring A) decreased activity as well as
its exchange by a methoxy or methyl group. The introduction of the
strongly electron-withdrawing and lipophilic trifluoromethyl
group was accompanied by a slight increase in activity. Whereas a
more hydrophilic, electron-withdrawing nitro group (12) was
tolerated, but did not enhance the activity, a cyano group at the
same position (13) led to a decreased inhibitory effect. An addi-
tional chloro substituent improved activity independent of the
position of both substituents. This correlates well with the distri-
bution of favorable interaction contour plots for chloro atoms
within the subpocket of the “switch region” as computed with
Molecular Operating Environment (MOE) [31] (Fig. 2) localized
close to positions 2, 3 and 4. The most active compounds in this
series were the 3,4-di-chloro compound 18 and 22 with a tri-
fluoromethyl and a chloro substituent present in positions 3 and 4.
Regarding quantitative structureeactivity relationship (QSAR) an
excellent correlation was observed when
p and s were used in a
multiparameter regression analysis. The obtained Hansch equation
(Equation (1): pIC50 ¼ 3.71 þ 0.34 ꢀ
p
þ 0.60 ꢀ
s
(n ¼ 16, R2 ¼ 0.95,
RMSE ¼ 0.071)) clearly indicates that highly lipophilic and strongly
electron-withdrawing groups result in the most potent compounds
(Fig. 3).
In order to elucidate the influence of the substitution pattern of
ring A on the inhibitory potency, ab initio geometry optimizations
for compounds 3, 9, 13 and 18 were carried out and molecular
electrostatic potentials (MEP) were visualized (Fig. 4). As shown by
Fig. 2. Small subpocket of the “switch region” with contour graphics (yellow grid)
indicating regions where chlorine has interaction energies within isovalues
of ꢂ4 kcal/mol. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

226
J.H. Sahner et al. / European Journal of Medicinal Chemistry 65 (2013) 223e231
more active than 3, despite these two negative effects, corroborates
the validity of the docking pose.
Turning our interest to the ureido motif B, we retained the 3,4-
di-chloro ring A. The unsubstituted ureido compound 24
(R2 ¼ R3 ¼ H) displayed low affinity compared to 18 containing two
lipophilic alkyl chains. This was concordant with the proposed
binding mode since B is located in a highly lipophilic cavity where
hydrophobic contacts, Van der Waals and CHep interactions, pre-
vail. As depicted in Fig. 5 the urea moiety does not form hydrogen-
bonds with surrounding residues, but acts as a planar linker for the
obligatory hydrophobic groups. Within the compound series
derived from primary alkyl amines an increased activity was
observed for chain elongation. For example the n-hexyl derivative
28 displayed the highest potency compared to smaller substituents.
Besides long aliphatic chains, aromatic residues (phenyl, benzyl or
phenethyl) were also suitable. The introduction of a second chain at
the nitrogen was in general accompanied by an increasing activity.
The increase in potency of compounds 35ꢂ40 compared to the
mono-substituted derivatives 29ꢂ31 appears to rely on additional
hydrophobic contacts within the lipophilic pocket. In fact these
compounds present a second sterically demanding, hydrophobic
substituent at the ureido motif (B). These moieties better fill the
dienone-binding cavity reaching into the pocket delimited by
Fig. 3. Comparison of experimentally determined and calculated activities (based on
Equation (1)) of compounds 3 and 8e22.
To proof the binding mode proposed in Fig. 1, compound 23 was
synthesized [40]. It was deduced from the docking pose, that po-
sition 3 at the phenyl ring A should be appropriate for an elongation
into the pocket occupied by the enecarbamate chain of myxopyr-
onins (red circle in Fig. 1). As a linker a C4 unit has been considered
to be suitable for the positioning of a carbamate group. For syn-
thetic reasons we introduced a saturated chain as the corre-
sponding myxopyronin analogue has been described to show a
similar biological activity as 1 [14]. The resulting compound 23 that
contains a mimic of the myxopyronin enecarbamate chain indeed
possessed increased activity compared to the parent compound 3
(Table 2). Nevertheless the putative new interactions (Figure S2 in
Supporting information) would suggest an even higher gain in
activity. We suppose that the entropic penalty for the binding of the
highly flexible chain has a negative effect on the affinity. Further-
more, it can be rationalized that the positive inductive effect of the
introduced alkylcarbamate chain negatively affects the electronic
properties of ring A according to equation (1). The fact that 23 is
Leu1326 and Ile1330 (
and Leu1332 (b0 subunit). For instance, the higher affinity of 40 can
be explained by additional CHe interactions between the second
b subunit), and Lys332, Ser1324, Thr1328,
p
aromatic ring and Lys334 (b0 subunit) as well as by non-bonded
interactions with Leu1332 and Thr1328, whereas compound 18
only interacts with Ile1320 and Leu1326 in the dienone pocket
(Fig. 5). Subsuming, the increase of activity by extension of the
lipophilic side chains in part B can be attributed to a better fit into
the binding cavity and therefore extension of the hydrophobic
contact area and displacement of water molecules.
In addition to the RNAP in vitro inhibition, the effect of 18 on
RNA synthesis was investigated in a whole-cell assay with E. coli
TolC using radiolabeled 3H uridine. At a concentration of 100
mM the
RNA level was reduced by 60% after 40 min (Figure S2).
Further biological evaluation was carried out by determination
of minimal inhibitory concentrations (MIC) in E. coli TolC (Table 1),
which is defective in the multidrug AcrABeTolC efflux system. Most
Fig. 4. Ab initio molecular electrostatic potential (MEP) of compounds 3, 9, 13 and 18.

J.H. Sahner et al. / European Journal of Medicinal Chemistry 65 (2013) 223e231
227
Fig. 5. Docking simulation of compounds 18 (a) and 40 (c) and two-dimensional illustration of the interactions between 18 (b) and 40 (d) and RNAP. The main interacting amino
acids and the key interactions are highlighted. Numbers of amino acid residues from
and b0 subunit are labeled in black and blue. Polar amino acids are illustrated in purple and
b
hydrophobic amino acids in green circles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1
Inhibitory activities against E. coli RNA polymerase in vitro and antibacterial activities.
Cpd
R1
Inhibition of E. coli RNAPa
(
mM)
MIC (
m
g/mL)b,c
Cpd
R2
R3
Inhibition of E. coli RNAPa
MIC (m
g/mL)b,c
3
8
9
4-Cl
H
4-OCH3
4-CH3
4-CF3
4-NO2
4-CN
4-OCF3
3-Cl
3-CF3
75
11
17
15
21
21
23
25
21
9
38
22
10
8
24
25
26
27
28
29
30
31
32
33
34
18
35
36
37
38
39
40
H
H
H
H
H
H
H
H
H
26%
26%
>25
15
13
10
>25
12
10
8
9
5
292
240
137
51
73
133
45
72
53
80
22
32
35
25
21
0.35
0.03
i-propyl
n-butyl
n-pentyl
n-hexyl
Phenyl
Benzyl
Phenethyl
Phenyl
Benzyl
Phenyl
Benzyl
Benzyl
Phenethyl
Phenyl
Benzyl
Phenethyl
Benzyl
91
35
19
25
45
26
45
m
m
m
m
m
m
m
M
M
M
M
M
M
M
10
11
12
13
14
15
16
17
18
19
20
21
22
1
Methyl
Methyl
Ethyl
Ethyl
n-propyl
n-propyl
n-butyl
n-butyl
n-butyl
Benzyl
12%
3-NO2
36
22
14
12
14
m
M
M
M
M
M
16
10
3,4-di-Cl
2,4-di-Cl
2,5-di-Cl
3,5-di-Cl
3-CF3e, 4-Cl
Myxopyronin B
Rifampicin
m
m
m
m
>25
>25
>25
>50
>25
>25
9
n.d.
12
0.8
5
9
7
6
m
m
m
M
M
M
a
IC50 values [
Minimal inhibitory concentration in E. coli TolC.
>: MIC-determination was limited due to insufficient solubility of the tested compounds. n.d. ¼ not determined.
m
M] or inhibition at 100
mM [%] of E. coli RNA polymerase.
b
c

228
J.H. Sahner et al. / European Journal of Medicinal Chemistry 65 (2013) 223e231
Table 2
Table 3
Structure and biological data of 23.
Antibacterial activities of selected 5-aryl-3-ureidothiophene-2-carboxylic acids.
Cpd
MICa
E. coli TolC E. coli K12
g/mL) g/mL)
MICa
MICa
P. aeruginosa B. subtilis S. aureus
g/mL) g/mL) g/mL)
MICa
MICa
(
m
(
m
(
m
(m
(m
IC50 E. coli RNAP: 20
MIC E. coli TolC: 25
mM
g/mL
14
15
18
19
22
27
29
31
21
9
10
8
12
10
12
8
>50
>25
0.8
10
>100
>100
>100
>25
>50
>50
>25
>50
>25
>50
>25
>25
13
20
12
11
10
20
8
9
21
8
12
5
>25
6
6
>50
3
0.5
0.02
0.5
m
>100
>25
>50
>50
>25
>50
>25
>50
>25
>25
7
12
7
of the compounds active against the bacterial enzyme displayed
growth inhibitory effects in the range of 5e25 g/mL. Their anti-
bacterial activity is comparable to that of the reference drug
rifampicin (5 g/mL E. coli TolC). The effect on the bacterial growth
38
40
1
>50
>25
0.9
4.8
0.9
m
m
Rifampicin
Myxopyronin B 0.8
reflects that the ureidothiophene carboxylic acids can diffuse across
the asymmetric bilayer to gain access to the cell interior, as
described for lipophilic antibacterials including macrolides and
rifamycins [32]. However, the most potent RNAP inhibitors 35ꢂ40
e the compounds with the highest lipophilicity in that class e
showed lower antibacterial potency against E. coli TolC. These re-
sults can be explained by the fact that the outer membrane of Gram
negative bacteria is well known to be a potent barrier, hindering too
lipophilic compounds from entering the cell [33,34].
To explore the spectrum of bacteria possessing susceptibility to
selected compounds, MIC values in two Gram positive and two
Gram negative strains were determined (Table 3). While the Gram
positive strains (Bacillus subtilis, S. aureus) were in general sensitive,
wild type Gram negative bacteria (E. coli K12, Pseudomonas aeru-
ginosa) were not affected. For B. subtilis and S. aureus similar
inhibitory effects compared to E. coli TolC were observed. Consid-
ering that the RNAP “switch region” is highly conserved among
various bacterial strains, we assume that the unequal antibacterial
potencies are due to differences in cell wall permeability between
Gram negative and Gram positive bacteria.
>25
>25
a
>: MIC-determination was limited due to insufficient solubility of the tested
compounds.
via virtual screening using a 3D-pharmacophore model [24], based
on the cocrystal structures of myxopyronins 1 and 2 in the “switch
region” [4,15]. In this paper the SAR of compound 3 was explored
and the inhibitory potency was optimized following a rational
approach (Hansch, Topliss) supported by computational methods
(docking studies, MEPs). Furthermore, the binding mode was
experimentally validated by mimicking the natural ligand myx-
opyronin via introduction of a carbamate side chain into compound
3. The new inhibitors show activity against Gram positive
(B. subtilis, S. aureus) and Gram negative (E. coli TolC) bacterial
strains accompanied by outstanding low resistance frequencies. In
contrast, other synthetic RNAP inhibitors possess weak bacterial
growth inhibitory activity [21,22] or have only narrow spectrum
activity among the S. aureus genus [25]. The lack of activity towards
Gram negative wild type strains, is most likely due to poor pene-
tration and/or drug efflux. Therefore further optimization is
required. Improving the physicochemical properties by introduc-
tion of hydrophilic or ionisable groups could enhance cellular
availability in Gram negative bacteria. Ring A seems to be inap-
propriate since Equation (1) shows the importance of lipophilic
substituents in this part. In our opinion, the ureido motif B is so far
underexploited and should be utilized. As it is known, that the
introduction of polar groups can have negative impact on the ac-
tivity unless they contribute to favorable enthalpic interactions
[36,37], this issue has to be taken into consideration. In the binding
area of B the polar amino acids (Ser1324, Thr1328) and some
backbone regions could be addressed for such interactions. Thus,
clinically applicable, broad spectrum antibacterial agents, that are
urgently needed to combat infectious diseases, could finally be
obtained. The present work lays the foundation for future
structure-based design and expansion of the chemical space in this
class.
The fact that most compounds were potent against the TolC
mutant of E. coli and almost ineffective against E. coli K12 suggests
that drug efflux, deactivated in E. coli TolC, is responsible for the lack
of antibacterial potency in Gram negative bacteria.
An essential requirement for an effective antibacterial agent is
that its potency is not reduced by the occurrence of resistant
strains. Spontaneous resistance towards myxopyronin B in S. aureus
occurs at a frequency of 8 ꢀ 10ꢂ8, similar to that of rifampicin [17].
The determination of the in vitro resistance frequencies in E. coli
TolC at 2 ꢀ MIC revealed a significant (>500-fold) lower value for 15
(<4.2 ꢀ 10ꢂ11) compared to rifampicin (8.3 ꢀ 10ꢂ8) and myxopyr-
onin B (7.1 ꢀ 10ꢂ8). This phenomenon can be explained by the fact
that 15 occupies only a part of the “switch region” whereas myx-
opyronin fills a larger area, including the narrow enecarbamate-
binding pocket. Especially mutations in this part, which lead to
resistance against myxopyronin [4,35], should not affect the anti-
bacterial activity of our compounds. Another mechanism that can
contribute to the low resistance frequency can be the effect on an
additional target. This will be investigated in further experiments.
Our finding indicates, that the probability of occurring resistant
strains, is reduced with our compounds compared to existing
drugs. This makes these compounds promising candidates for
further optimization as novel antibacterials.
6. Experimental procedures
6.1. Chemistry
6.1.1. Materials and methods
Starting materials were purchased from commercial suppliers
and used without further purification. Column flash chromatog-
5. Conclusions
raphy was performed on silica gel (40e63
mM), and reaction
With the aim to develop antibacterial substances with broad
spectrum activity, we have chosen the “switch region” of bacterial
RNAP as a target site, as it is conserved among various bacterial
strains [4]. In a previous work we had discovered hit compound 3
progress was monitored by TLC on TLC Silica Gel 60 F254 (Merck). All
moisture-sensitive reactions were performed under nitrogen at-
mosphere using oven-dried glassware and anhydrous solvents. 1H
and 13C NMR spectra were recorded on
a Bruker Fourier

J.H. Sahner et al. / European Journal of Medicinal Chemistry 65 (2013) 223e231
229
spectrometer (300 or 75 MHz) at ambient temperature with the
chemical shifts recorded as values in ppm units by reference to the
solution of NaHCO3 (30 mL) and H2O (50 mL). The resulting
mixture was extracted with EtOAc/THF (1:1, 3 ꢀ 100 mL). The
organic layer was washed with saturated aqueous NaCl (100 mL),
dried (MgSO4) and concentrated. The crude material was sus-
pended in a mixture of n-hexane/EtOAc (2:1, 50 mL) heated to
50 ꢁC and after cooling to room temperature separated via
filtration.
d
hydrogenated residues of deuteriated solvent as internal standard.
Coupling constants (J) are given in Hz and signal patterns are
indicated as follows: s, singlet; d, doublet; dd, doublet of doublets;
t, triplet; m, multiplet, br., broad signal. The melting points (m.p.)
were determined using a Stuart Scientific SMP3. The purity of the
final compounds was ꢃ95% as measured by HPLC. The Surveyor LC
system consisted of a pump, an autosampler, and a PDA detector.
Mass spectrometry was performed on an MSQ electrospray mass
spectrometer (ThermoFisher, Dreieich, Germany). The system was
operated by the standard software Xcalibur. An RP C18 NUCLEODUR
100-5 (125 mm ꢀ 3 mm) column (MachereyeNagel GmbH, Dühren,
Germany) was used as the stationary phase. All solvents were HPLC
grade. In a gradient run the percentage of acetonitrile (containing
0.1% trifluoroacetic acid) was increased from an initial concentra-
tion of 0% at 0 min to 100% at 15 min and kept at 100% for 5 min. The
6.1.2.4. General procedure for the synthesis of 5-aryl-3-
ureidothiophene-2-carboxylic acid (V). The 5-aryl-2-thiaisatoic-
anhydrid (IV) (0.46 mmol) was suspended in water (7.5 mL) and the
appropriate amine (4.60 mmol) was added. The reaction mixture
was stirred, heated to 100 ꢁC and then cooled to room temperature.
The reaction mixture was poured into a mixture of concentrated
HCl and ice (1:1) and extracted with EtOAc/THF (1:1, 60 mL). The
organic layer was washed with aqueous HCl (2 M), followed by
saturated aqueous NaCl (2 ꢀ 50 mL), dried (MgSO4) and concen-
trated. The crude material was suspended in a mixture of n-hexane/
EtOAc (2:1, 20 mL) heated to 50 ꢁC and after cooling to room
temperature separated via filtration.
injection volume was 10 mL, and flow rate was set to 800 mL/min. MS
analysis was carried out at a spray voltage of 3800 V and a capillary
temperature of 350 ꢁC and a source CID of 10 V. Spectra were ac-
quired in positive mode from 100 to 1000 m/z at 254 nm for the UV
trace.
6.1.3. Spectroscopic data of final compounds
Spectroscopic data of final compounds can be found in the
Supporting information. Compound 3 is presented as example.
6.1.2. General procedure for the synthesis of ureidothiophene-2-
carboxylic acids
6.1.2.1. General procedure for the synthesis of 5-aryl-3-amino-2-
carboxylic acid methyl ester (II). POCl3 (26.1 g, 0.17 mol) was
added dropwise to DMF (24.9 g, 0.34 mol) maintaining the
temperature beyond 25 ꢁC (cooling in ice bath) and stirred for
additional 15 min. The acetophenone I (85.0 mmol) was added
slowly and the temperature was kept between 40 and 60 ꢁC.
After complete addition, the mixture was stirred for 30 min at
room temperature. Hydroxylamine hydrochloride (23.6 g,
0.34 mol) was carefully added portionwise (exothermic reac-
tion!) and the reaction was stirred for additional 30 min without
heating. After cooling to room temperature, the mixture was
6.1.3.1. 5-(40-chlorophenyl)-3-[(N-ethylbenzylamino)carbon-
ylamino]-thiophene-2-carboxylic acid (3). Yellow powder, m.p.
173e174 ꢁC.
1H NMR (300 MHz, DMSO-d6):
d
¼ 1.16 (t, J ¼ 7.1 Hz, 3H), 3.39 (q,
J ¼ 7.1 Hz, 2H), 4.58 (s, 2H), 7.24e7.38 (m, 5H), 7.50 (d, J ¼ 8.5 Hz,
2H), 7.71 (d, J ¼ 8.5 Hz, 2H), 8.29 (s,1H),10.06 (s,1H),13.40 (br. s,1H,
COOH) ppm.
13C NMR (75 MHz, DMSO-d6):
d
¼ 13.1, 41.8, 49.3, 107.2, 117.8,
127.1, 127.1, 127.4, 128.5, 129.3, 131.5, 133.8, 138.1, 146.1, 146.7, 153.0,
165.6 ppm.
poured into ice water (300 mL). The precipitated
b
-chloro-cin-
6.2. Biology
namonitrile was collected by filtration, washed with H2O
(2 ꢀ 50 mL) and dried under reduced pressure over CaCl2. In the
next step sodium (1.93 g, 84.0 mmol) was dissolved in MeOH
(85 mL) and methylthioglycolate (6.97 g, 65.6 mmol) was added
6.2.1. Transcription assay
E. coli RNA polymerase holo enzyme was purchased from Epi-
centre Biotechnologies (Madison, WI). Final concentrations in a total
to the stirred solution. The
b
-chloro-cinnamonitrile (61.1 mmol)
volume of 30
were used along with 60 nCi of [5,6-3H]-UTP, 400
GTP as well as 100 M of UTP, 20 units of RNAse inhibitor (RiboLock,
m
L were one unit of RNA polymerase (0.5
mg) which
was added and the mixture was heated to reflux for 30 min. After
cooling to room temperature, the mixture was poured into ice
water (300 mL). The precipitated solid was collected by filtration,
washed with H2O (2 ꢀ 50 mL) and dried under reduced pressure
over CaCl2. If necessary, recrystallisation from EtOH was
performed.
mM of ATP, CTP and
m
Fermentas), 10 mM DTT, 40 mM TriseHCl (pH 7.5), 150 mM KCl,
10 mM MgCl2 and 0.1% CHAPS. As a DNA template 3500 ng of reli-
gated pcDNA3.1/V5-His-TOPO were used per reaction [38]. Prior to
starting the experiment, the compounds were dissolved in DMSO
(final concentration during experiments: 2%). Dilution series of
compounds were prepared using a liquid handling system (Janus,
Perkin Elmer, Waltham, MA). The components described above
(including the inhibitors) were preincubated in absence of NTPs and
DNA for 10 min at 25 ꢁC. Transcription reactions were started by the
addition of a mixture containing DNA template and NTPs and
incubated for 10 min at 37 ꢁC. The reaction was stopped by the
addition of 10% TCA, followed by a transfer of this mixture to a 96-
well Multiscreen GFB plate (Millipore, Billerica, MA) and incubation
for 45 min at 4 ꢁC. The plate underwent several centrifugation and
washing steps with 10% TCA and 95% ethanol to remove residual
unincorporated 3H UTP. After that the platewas dried (30 min, 50 ꢁC)
6.1.2.2. General procedure for the synthesis of 5-aryl-3-amino-2-
carboxylic acid (III). The 5-aryl-3-amino-2-carboxylic acid methyl
ester (16.6 mmol) was added to a solution of KOH (60 mL, 0.6 M in
H2O) and MeOH (60 mL). The mixture was heated to reflux for 3 h,
concentrated, and washed with EtOAc (2 ꢀ 50 mL). The aqueous
layer was cooled with ice and acidified by addition of a saturated
aqueous solution of KHSO4. The precipitated solid was collected by
filtration, washed with H2O (2 ꢀ 30 mL) and dried under reduced
pressure over CaCl2.
6.1.2.3. General procedure for the synthesis of 5-aryl-2-thiaisatoic-
anhydrid (IV). To a solution of the 5-aryl-3-amino-2-carboxylic
acid (III) (5.28 mmol) in THF (50 mL) a solution of phosgene
(6.10 mL, 20 wt% in toluene, 11.6 mmol) was added dropwise over
a period of 30 min. The reaction mixture was stirred for 2 h at
room temperature, followed by the addition of saturated aqueous
and 30 mL of scintillation fluid (Optiphase Supermix, Perkin Elmer)
was added to each well. After 10 min the wells were assayed for
presence of 3H RNA by counting using a Wallac MicroBeta TriLux
system (Perkin Elmer). To obtain inhibition values for each sample,
their counts were related to DMSO controls.

230
J.H. Sahner et al. / European Journal of Medicinal Chemistry 65 (2013) 223e231
6.2.2. RNA synthesis assay
6.3.3. QSAR model
The QSAR model (Equation (1)) was obtained using the software
Molecular Operating Environment (MOE) v. 2010.10.
E. coli TolC was cultured in lysogeny broth (LB) medium. 3H
uridine (1
and 3 min before the addition of compound 18 at 100
DMSO). 300 L of the cultured bacteria were harvested 40 min after
mCi/mL) was added during the logarithmic growth phase
mM (0.5%
m
Acknowledgments
addition of the inhibitors and supplemented with 2 volumes of 10%
TCA. After 45 min at 4 ꢁC the precipitates were collected and
washed using a 96-well glass fiber filter plate (Multiscreen GFB)
(Millipore, Billerica, MA). After adding Optiphase Supermix (Perkin
Elmer, Waltham, MA) the quantification of radioactivity was per-
formed using a Wallac MicroBeta TriLux system (Perkin Elmer).
The authors thank Jeannine Jung and Jannine Ludwig for per-
forming the in vitro tests. We also appreciate Ailke Gerstner for her
help in performing the synthesis. Furthermore we are grateful to K.
Gerth (Helmholtz Centre for Infection Research (HZI) in
Braunschweig) for kindly providing myxopyronin B. J. Henning
Sahner gratefully acknowledges a scholarship from the “Stiftung
der Deutschen Wirtschaft” (SDW).
6.2.3. Determination of IC50 values
For the determination of IC50 values three different concentra-
tions of a compound were chosen (two samples for each concen-
tration) including data points above and below the IC50 value. The
calculation of the IC50 value was performed by plotting the percent
inhibition vs. the concentration of inhibitor on a semi-log plot.
From this the molar concentration causing 50% inhibition was
calculated. At least three independent determinations were per-
formed for each compound.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.04.060.
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in DMSO (maximal DMSO concentration in the experiment: 1%). Final
compound concentrations prepared from serial dilutions ranged from
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