Synthesis and Evaluation of N-Phenylpyrrolamides as DNA Gyrase B Inhibitors

Article abstract of DOI:10.1002/cmdc.201700549

ATP-competitive inhibitors of DNA gyrase and topoisomerase IV are among the most interesting classes of antibacterial drugs that are unrepresented in the antibacterial pipeline. We developed 32 new N-phenylpyrrolamides and evaluated them against DNA gyrase and topoisomerase IV from E. coli and Staphylococcus aureus. Antibacterial activities were studied against Gram-positive and Gram-negative bacterial strains. The most potent compound displayed an IC₅₀ of 47 nm against E. coli DNA gyrase, and a minimum inhibitory concentration (MIC) of 12.5 μm against the Gram-positive Enterococcus faecalis. Some compounds displayed good antibacterial activities against an efflux-pump-deficient E. coli strain (MIC=6.25 μm) and against wild-type E. coli in the presence of efflux pump inhibitor PAβN (MIC=3.13 μm). Here we describe new findings regarding the structure–activity relationships of N-phenylpyrrolamide DNA gyrase B inhibitors and investigate the factors that are important for the antibacterial activity of this class of compounds.

Full text of DOI:10.1002/cmdc.201700549

Accepted Article
Title: Synthesis and Evaluation of N-Phenylpyrrolamides as DNA
Gyrase B Inhibitors
Authors: Martina Durcik, Päivi Tammela, Michaela Barančoková,
Tihomir Tomašič, Janez Ilaš, Danijel Kikelj, and Nace Zidar
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201700549
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Synthesis and Evaluation of N-Phenylpyrrolamides as DNA
Gyrase B Inhibitors
Martina Durcik,[a] Päivi Tammela,[b] Michaela Barančoková,[a] Tihomir Tomašič,[a] Janez Ilaš,[a] Danijel
Kikelj,[a] and Nace Zidar*[a]
[a]
M. Durcik (0000-0002-9218-1771), M. Barančoková, Assist. Prof. T. Tomašič (0000-0001-5534-209X), Assoc. Prof. J. Ilaš (0000-0002-0124-0474), Prof. D.
Kikelj (0000-0002-2067-7604), Assist. Prof. N. Zidar (0000-0003-1905-0158)
Department of Pharmaceutical Chemistry
Faculty of Pharmacy, University of Ljubljana
Aškerčeva cesta 7, 1000 Ljubljana (Slovenia)
E-mail: nace.zidar@ffa.uni-lj.si
[b]
Prof. P. Tammela (0000-0003-4697-8066)
Drug Research Program, Division of Pharmaceutical Biosciences
Faculty of Pharmacy, University of Helsinki
P.O. Box 56 (Viikinkaari 5 E), Helsinki FI-00014 (Finland)
Supporting information for this article is given via a link at the end of the document.
Abstract: ATP-competitive inhibitors of DNA gyrase and
topoisomerase IV (topo IV) are among the most interesting classes of
antibacterial drugs that do not have any representative in the
antibacterial pipeline. We have developed thirty-two new N-
phenylpyrrolamides and evaluated them against DNA gyrase and
topoisomerase IV from Escherichia coli and Staphylococcus aureus.
Antibacterial activities were studied against Gram-positive and Gram-
negative bacterial strains. The most potent compound displayed an
IC50 of 47 nM against E. coli DNA gyrase, and a minimum inhibitory
concentration (MIC) of 12.5 µM against Gram-positive Enterococcus
faecalis. Some compounds displayed good antibacterial activities
against the efflux pump deficient E. coli strain (MICs = 6.25 µM) and
against wild type E. coli in the presence of efflux pump inhibitor PAβN
(MIC = 3.13 µM). We describe here new findings regarding structure-
activity relationship of N-phenylpyrrolamide DNA gyrase B inhibitors
and explore factors that are important for antibacterial activity of this
class of compounds.
antibacterial drug discovery. DNA gyrase is involved primarily in
the negative supercoiling of DNA, while topoisomerase IV is
responsible mainly for the decatenation of the two daughter
chromosomes after replication. DNA gyrase and topoisomerase
IV both belong to type IIA topoisomerases. They are
heterotetrameric enzymes composed of two pairs of subunits: two
GyrA and two GyrB subunits in DNA gyrase, and two ParC and
two ParE subunits in topoisomerase IV. The GyrA and GyrB
subunits of DNA gyrase are similar in amino acid sequence to the
ParC and ParE subunits of topoisomerase IV, respectively. The
main function of GyrA/ParC is cleavage and reunion of DNA,
whereas GyrB/ParE binds ATP, which is crucial for providing the
energy for the conformational movements of the enzyme,
required for the ligation process.[4]
There are two main mechanisms of action of drugs targeting DNA
gyrase and topoisomerase IV. The first is stabilisation of the
covalent enzyme–DNA complex, which is typical for the
fluoroquinolone class of antibacterials.[5] The presence of some
serious side effects together with the growing bacterial resistance
to fluoroquinolones has stimulated the search for inhibitors
targeting various binding sites on DNA gyrase.[6] The second
possible mechanism of action of drugs targeting DNA gyrase and
topoisomerase IV involves inhibition of ATP binding on the
Introduction
Infections are among the major causes of human morbidity and
mortality and the pharmaceutical industry is not able to keep up
with the growing needs for effective novel antibacterials. The main
reason for this situation is the rapid adaptation of bacteria to
antibiotics, which results in the development of resistance soon
after antibacterial drugs are introduced to therapy.[1] Another
problem is the low number of new approved antibacterial drugs in
the last decades, which is associated with the strict regulatory
requirements and relatively low cost-to-benefit ratio for
antibacterial R&D compared to other pharmacological fields.[2][3]
Bacterial topoisomerases are enzymes that catalyse changes in
DNA topology during DNA replication, transcription and
recombination. They are essential in all bacteria but absent in
higher eukaryotes, which makes them attractive targets for
GyrB/ParE subunit. Novobiocin,
a natural aminocoumarin
antibiotic that was withdrawn from the clinic because of toxicity or
lack of effectiveness,[7] is a classic example of ATP-competitive
GyrB inhibitors. In recent decades, several new scaffolds have
been identified as GyrB and/or ParE inhibitors, such as
pyridylureas,[8]
benzimidazole
pyrimidinoindoles,[9]
ureas,[11]
pyrrolopyrimidines,[10]
and
pyrrolamides,[12]
pyrazolopyridones[13] (Figure 1). Even though some of the new
inhibitors have advanced to Phase I, none of them has so far
reached clinical practice.[14] In the present study, we describe a
new series of N-phenylpyrrolamide DNA gyrase (GyrB) and
topoisomerase IV (ParE) inhibitors, resulting in optimised on-
target and antibacterial activities.
1
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Figure 1. Representative inhibitors of DNA gyrase (GyrB) and topoisomerase IV (ParE), and their IC50, Ki and MIC (S. aureus) values.
in kibdelomycin, a complex antibiotic isolated from extracts of the
soil bacterium Kibdelosporangium sp.,[18] and in some
Results and Discussion
pyrrolamide DNA gyrase
AstraZeneca.[12d]
B
inhibitors developed by
Design. The design of the present series of compounds was
based on the crystal structure of N-phenylpyrrolamide inhibitor A
in complex with E. coli DNA gyrase B (Figure 2, PDB code:
4ZVI),[15] and was aimed at improving its GyrB/ParE inhibitory
potency and antibacterial activity. Three structural types of
compounds (type I-III, Figure 2) were prepared, in which we
introduced modifications proposed by the detailed study of the
binding mode of inhibitor A in the GyrB active site, and by
molecular docking (Figure 3). In all structural types (I-III) we have
retained the adjacent hydrogen bond donor (pyrrole NH) and
hydrogen bond acceptor (pyrrolamide C=O) groups that are
necessary for establishing the H-bonding network with Asp73 (E.
coli numbering) and the structurally conserved water molecule
(Figure 2). The amide NH of the pyrrolamide moiety was left
unsubstituted because it is important for indirect contacts with
Asn46 through a crystal water molecule. In some derivatives, we
have replaced the 4,5-dibromo-1H-pyrrole group on the left-hand
side of the molecules with 3,4-dichloro-5-methyl-1H-pyrrole. Thus
far, replacing the 4,5-dibromo-1H-pyrrole moiety by 4-bromo-1H-
pyrrole, 1H-indole, or by 4,5-dichloro-1H-pyrrole resulted in
weaker activity.[15-16] The 3,4-dichloro-5-methyl-1H-pyrrole was
selected because the chlorine atoms are slightly smaller than the
bromine atoms and are thus proposed to bind more strongly, not
only to DNA gyrase, but also to the slightly smaller binding
pockets of S. aureus DNA gyrase and topoisomerase IV.[4b,17]
Additionally, the 3,4-dichloro-5-methylpyrrole moiety is found e.g.
The right-hand side of the molecules was designed to contain
groups able to form ionic interactions with Arg136 (e.g. carboxylic
acid groups) or π-stacking interactions with the Glu50-Arg76 salt
bridge (e.g. 4-amino-2-hydroxybenzoic acid or 1,3,4-oxadiazol-2-
one ring). Whereas type I compounds were all glycine derivatives,
in some type II compounds, the glycine group (n = 1, Figure 2)
was modified to beta-alanine group (n = 2, Figure 2). In order to
improve transport across the bacterial cell membranes, the
ionizable terminal carboxylic acid functionality was replaced in
some type I and type II compounds by its less acidic bioisostere
1,3,4-oxadiazol-2-one. The 1,3,4-oxadiazol-2-one ring was
proposed to form similar interactions with Arg136 as does the
terminal carboxylic group, but its lower acidity promises better
permeability across bacterial membranes and, consequently,
better antibacterial activity.[8] Additionally, some type I and type II
compounds with terminal hydrazide groups were analyzed.
With the type II series, we have introduced additional alkoxy
substituents to the 3-position of the central benzene ring. The
preliminary docking studies have indicated the possibility of
additional hydrophobic contacts between these groups and the
lipophilic floor of the enzyme formed by the amino acid residues
Ile94, Ile78, Val120 and Val167 (Figure 3). The size of
substituents was increased from methoxy, through isopropoxy, to
the benzyloxy group.
2
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Figure 2. Design of three structural types (I-III) of N-phenylpyrrolamides as DNA gyrase B inhibitors, based on the co-crystal structure of the inhibitor A – GyrB (E.
coli) (PDB code: 4ZVI[15]).
relationships (SAR) of all the prepared compounds were studied,
enabling the essential structural elements for GyrB/ParE inhibition
and for antibacterial activity to be determined.
Chemistry. The synthesis of type I compounds (7-11) is
presented in Scheme 1. First, 4-nitrobenzoyl chloride (1) was
reacted with glycine methyl ester hydrochloride (2) in the
presence of potassium carbonate to give compound 3. The nitro
group of 3 was reduced by catalytic hydrogenation and the
obtained amine 4 was coupled with 4,5-dibromopyrrole-2-
carboxylic acid using N,N,N',N'-tetramethyl-O-(benzotriazol-1-
yl)uronium tetrafluoroborate (TBTU), to yield pyrrolamide 5, or
with 3,4-dichloro-5-methylpyrrole-2-carboxylic acid chloride
prepared in situ from the carboxylic acid using oxalyl chloride, to
Figure 3. The docking binding mode of inhibitor 19f (in magenta) in the ATP
give pyrrolamide 6. Compound 7 was prepared by alkaline
hydrolysis of methyl ester 6 while hydrazides 8 and 9 were
binding site of E. coli DNA gyrase B (in grey, PDB code: 4DUH[19]). The ligand
and the neighbouring protein side-chains are shown as stick models, coloured
prepared by heating 5 or 6 with hydrazine at 78 °C. To obtain the
target 1,3,4-oxadiazol-2-ones 10 and 11, compounds 8 and 9
were reacted with 1,1'-carbonyldiimidazole (CDI).
according to the chemical atom type (blue, N; red, O; orange, S; green, Cl).
Hydrogen bonds are indicated by black dotted lines.
Type II compounds (19a-g, 20 and 21) were synthesized
according to Scheme 2. First, methyl ester 13 was prepared from
3-hydroxy-4-nitrobenzoic acid (12) and thionyl chloride in
methanol. The phenolic group of 13 was then alkylated with
methyl (compound 14a), isopropyl (compound 14b) or benzyl
(compound 14c) substituents. Compounds 14a and 14c were
prepared using methyl iodide or benzyl bromide and potassium
carbonate as base, while compound 14b was prepared using
isopropanol under Mitsunobu conditions. Methyl esters 14a-c
were hydrolyzed with 1 M sodium hydroxide to give carboxylic
acids 15a-c, which were subsequently coupled with glycine
methyl ester hydrochloride to give compounds 16a-c, or with
methyl 3-aminopropanoate to give compounds 16d-f. The nitro
groups of 16a-f were reduced to amino groups by catalytic
hydrogenation (for products 17a-b, 17d-e) or with tin(II) chloride
(for products 17c and 17f). Pyrrolamides 18a-c were prepared by
Based on the relatively high activity of one of our previously
reported inhibitors with only a short COCH3 substituent on the
para position of the central aminobenzene ring (i.e. N-(4-
acetylphenyl)-4,5-dibromo-1H-pyrrole-2-carboxamide, IC50 = 1.6
µM),[16b] we decided to further explore the chemical space around
this molecule by preparing a small library of its analogues (type III
compounds, Figure 2). Analogues were prepared with carboxylic
groups attached directly to the central phenyl ring (X = OH, Y = H,
Figure 2), compounds with an additional hydroxyl group on the
phenyl ring (X = OH, Y = OH, Figure 2), and compounds with an
acetophenone functionality (X = CH3, Y = H, Figure 2). To
determine the influence of increased length of the molecules on
gyrase B and topoisomerase IV inhibition, we prepared a
compound with an additional aromatic ring (X = 4-NH2-2-OH-
(C6H3)-COOH,
Y
= H, Figure 2). The structure-activity
3
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coupling 17a-b or 17d with 4,5-dibromopyrrole-2-carboxylic acid
using TBTU methodology, while pyrrolamides 18d-h were
prepared from 17a-c or 17e-f and the corresponding pyrrole-2-
carboxylic acid chloride prepared in situ using oxalyl chloride as
reagent. Alkaline hydrolysis of compounds 18a-g gave the final
products 19a-g, while hydrolysis of 18h was unsuccessful.
Compound 18b was additionally reacted with hydrazine
monohydrate to give hydrazide 20, and, after heating 20 with CDI,
the final product 21 was obtained.
Scheme 1. Reagents and conditions: a) K2CO3, CH3CN, 0 °C  rt, 15 h; b) H2, Pd-C, THF, 3 h; c) 4,5-dibromopyrrole-2-carboxylic acid, TBTU, NMM, CH2Cl2, 50 °C,
15 h (for the synthesis of 5), i) 3,4-dichloro-5-methylpyrrole-2-carboxylic acid, oxalyl chloride, CH2Cl2, rt, 15 h, then ii) 4, pyridine, CH2Cl2, rt, 15 h (for the synthesis
of 6); d) 1 M NaOH, THF, rt, 15 h; e) hydrazine monohydrate, EtOH, reflux, 15 h; f) CDI, 1,4-dioxane, 101 °C, 15 h (for the synthesis of 10), CDI, DMF, 50 °C, 15 h
(for the synthesis of 11).
Compounds 25a-c and 29 were prepared according to Scheme 3.
In the first step, the carboxylic acids 22a and 22b were converted
to methyl esters using thionyl chloride and methanol. The
obtained esters 23a and 23b were then coupled with 4,5-
dibromopyrrole-2-carboxylic acid or with 3,4-dichloro-5-
methylpyrrole-2-carboxylic acid, as described above, to give
compounds 24a-d. The final products 25a-c were obtained after
alkaline hydrolysis of methyl esters 24a-c. For the synthesis of
compound 29, first, 4-nitrobenzoyl chloride (1) was reacted with
methyl 4-aminosalicylate (23b) in the presence of pyridine to give
the amide 26. After reduction of the nitro group of compound 26
by catalytic hydrogenation, the obtained amine 27 was coupled
with 4,5-dibromopyrrole-2-carboxylic acid to give compound 28
which was submitted to alkaline hydrolysis to give the final
compound 29.
showed inhibitory activities in the low nanomolar range (IC50 < 0.1
µM) and their enzymatic inhibition was thus better than that of
novobiocin (IC50 = 0.17 µM). Generally, compounds containing
3,4-dichloro-5-methylpyrrole groups on the left-hand side of the
molecules displayed higher activities against E. coli DNA gyrase
than compounds containing 4,5-dibromopyrrole moieties. For
example, in the type I series, 3,4-dichloro-5-methylpyrrolamide 11
(IC50 = 0.080 µM) displayed a four-fold higher activity than its 4,5-
dibromopyrrolamide analog 10 (IC50 = 0.30 µM). Similarly,
compound 9 (IC50 = 0.43 µM) was five-times more active than its
4,5-dibromopyrrolamide analog 8 (IC50
= 2.4 µM). Similar
differences in activity could be observed between 4,5-
dibromopyrrolamides and 3,4-dichloro-5-methylpyrrolamides of
the type II (Table 2) and type III (Table 3) series. These results
indicate that both pyrroles can occupy the hydrophobic binding
pocket of the enzyme and form hydrophobic interactions with
Val71, Val167, Val43 and Ala47, but the smaller methyl and
chlorine atoms fit better into the enzyme’s binding site. A further
observation from Tables 1-3 is that compounds containing methyl
ester groups on the right-hand side show weaker activities than
their carboxylic acid counterparts, probably because they cannot
form ionic interactions with Arg136. This can be seen by
comparing the esters 6 (IC50 = 0.41 µM), 18a (IC50 = 3.7 µM) and
24c (IC50 = 1.6 µM) on the one hand, with the corresponding
carboxylic acids 7 (IC50 = 0.28 µM), 19a (IC50 = 0.31 µM) and with
25c (IC50 = 0.79 µM) on the other. When comparing the activities
of compounds with carboxylic acid groups with their hydrazide
Inhibitory Activities against DNA Gyrase and Topoisomerase
IV. Thirty-two new compounds were prepared and evaluated
against E. coli DNA gyrase in a supercoiling inhibition assay. The
results are presented in Tables 1-3 as residual activities (RA) of
the enzyme in the presence of 10 µM of compounds or as
concentrations of compounds that inhibit the enzyme activity by
50% (IC50 values). The most active compounds were additionally
tested against topoisomerase IV from E. coli, and against DNA
gyrase and topoisomerase IV from S. aureus.
In total, twelve compounds displayed IC50 values lower than 0.5
µM against E. coli DNA gyrase, three of which (11, 19f and 19g)
4
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analogs, the former were generally more active. For example,
carboxylic acid 7 (IC50 = 0.28 µM) displayed greater activity than
acid 19b (IC50 = 0.16 µM) was more potent than the hydrazide 20
(IC50 = 1.6 µM). These differences are, most probably, due to the
loss of ionic interactions with Arg136 in the case of hydrazide
groups.
its hydrazide analogue 9 (IC50 = 0.43 µM), compound A (IC50
=
0.45 µM, reported previously)[16b] was more active than the
corresponding hydrazide 8 (IC50 = 2.4 µM), and the carboxylic
Scheme 2. Reagents and conditions: a) thionyl chloride, MeOH, 0 °C  rt, 15 h; b) MeI, K2CO3, DMF, rt, 15 h (for the synthesis of 14a), isopropanol,
triphenylphosphine, DIAD, THF, rt, 15 h (for the synthesis of 14b), K2CO3, benzyl bromide, CH3CN, rt  60 °C, 3 h (for the synthesis of 14c); c) 1 M NaOH, MeOH,
rt, 15 h; d) glycine methyl ester hydrochloride (for the synthesis of 16a-c) or methyl 3-aminopropanoate (for the synthesis of 16d-f), TBTU, NMM, CH2Cl2, rt, 2-15
h; e) H2, Pd-C, MeOH, rt, 1-4 h (for the synthesis of 17a-b and 17d-e), SnCl2, EtOAc, EtOH, 55 °C, 24 h (for the synthesis of 17c and 17f); f) i) 4,5-dibromopyrrole-
2-carboxylic acid, TBTU, NMM, CH2Cl2, rt, 30 min, then ii) 17a or 17b or 17d, DMAP, 60 °C, 15 h (for the synthesis of 18a-c), i) 4,5-dibromopyrrole-2-carboxylic
acid, oxalyl chloride, CH2Cl2, rt, 15 h, then ii) 17c or 17e or 17f, pyridine, CH2Cl2, rt, 15 h (for the synthesis of 18d and 18g-h), i) 3,4-dichloro-5-methylpyrrole-2-
carboxylic acid, oxalyl chloride, CH2Cl2, rt, 15 h, then ii) 17a or 17b, pyridine, CH2Cl2, rt, 15 h (for the synthesis of 18e-f); g) 1-2 M NaOH, MeOH/THF, rt, 15 h; h)
hydrazine monohydrate, EtOH, reflux, 2 d; i) CDI, 1,4-dioxane, 101 °C, 15 h.
There were no significant differences in activity between
compounds that contain the glycine (n = 1, Table 2) and those
containing the longer beta-alanine substituent (n = 2, Table 2) on
the right-hand side of the molecules. Although in the carboxylic
acid subset (compounds 19a-g, Table 2), the glycine derivative
19b (IC50 = 0.16 µM) was slightly more active than the beta-
alanine derivative 19d (IC50 = 0.36 µM), compounds 19a (IC50 =
0.31 µM) and 19c (IC50 = 0.35 µM) showed almost no difference
in activities. Also, by observing the methyl ester subset
compounds (18a-h, Table 2) the optimal length of the molecules
could not be determined with certainty. These results can be due
to the high flexibility of this side chain in solution, as recently
5
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proposed by molecular dynamics simulation.[15] Probably both, the
glycine and the beta-alanine groups can adopt conformations in
which the terminal carboxylate group is either in contact with
Arg136 or with the solvent.
Scheme 3. Reagents and conditions: a) thionyl chloride, MeOH, 0 °C  70 °C, 15 h; b) i) 4,5-dibromopyrrole-2-carboxylic acid (for the synthesis of 24a-b and 28)
or 3,4-dichloro-5-methylpyrrole-2-carboxylic acid (for the synthesis of 24c-d), oxalyl chloride, CH2Cl2, rt, 15 h, then ii) 23a or 23b or 23c or 27, pyridine, CH2Cl2, rt,
15 h; c) 1 M NaOH, MeOH/THF, rt  60 °C, 30 h (for the synthesis of 25a-b and 29), 1 M NaOH, THF, 45 °C, 15 h (for the synthesis of 25c); d) 1, CH2Cl2, pyridine,
rt 15 h; e) H2, Pd-C, MeOH/THF, rt, 3 h.
Table 1. Inhibitory activities of type I compounds 6-11 against DNA gyrase and topoisomerase IV.
IC50 (µM)[a] or RA (%)[b]
S. aureus
gyrase
E. coli
topo IV
S. aureus topo
Compd
R1
R2
E. coli gyrase
IV
6
7
8
9
3,4-diCl-5-Me
3,4-diCl-5-Me
4,5-diBr
COOMe
0.41±0.20 µM
0.28±0.21 µM
2.4±1.5 µM
n.d.[c]
11±0 µM
93%
n.d.
100%
79%
n.d.
100%
95%
COOH
CONHNH2
CONHNH2
3,4-diCl-5-Me
0.43±0.26 µM
60%
100%
100%
10
4,5-diBr
0.30±0.09 µM
100%
98%
100%
11
3,4-diCl-5-Me
0.080±0.027 µM
0.17 µM
23±5 µM
0.041 µM
100%
62%
novobiocin
11 µM
27 µM
[a] Concentration of compound that inhibits the enzyme activity by 50%. [b] Residual activity of the enzyme at 10 µM of the compound.
[c] Not determined.
6
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Table 2. Inhibitory activities of type II compounds 18a-h, 19a-g, 20 and 21 against DNA gyrase and topoisomerase IV.
IC50 (µM)[a] or RA (%)[b]
S. aureus
gyrase
n.d.[c]
n.d.
n.d.
n.d.
99%
n.d.
n.d.
E. coli
topo IV
n.d.
n.d.
n.d.
n.d.
100%
n.d.
n.d.
n.d.
S. aureus topo
IV
Compd
R1
R2
R3
n
E. coli gyrase
18a
18b
18c
18d
18e
18f
18g
18h
19a
19b
19c
19d
19e
19f
4,5-diBr
4,5-diBr
4,5-diBr
4,5-diBr
3,4-diCl-5-Me
3,4-diCl-5-Me
4,5-diBr
4,5-diBr
4,5-diBr
4,5-diBr
4,5-diBr
4,5-diBr
3,4-diCl-5-Me
3,4-diCl-5-Me
4,5-diBr
Me
iPr
Me
iPr
Me
iPr
Bzl
Bzl
Me
iPr
Me
iPr
Me
iPr
Bzl
iPr
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOMe
COOH
COOH
COOH
COOH
COOH
1
1
2
2
1
1
2
1
1
1
2
2
1
1
2
1
3.7±2.1 µM
97%
n.d.
n.d.
n.d.
n.d.
100%
n.d.
n.d.
n.d.
96%
13±10 µM
98%
90%
65%
2.3±0.7 µM
98%
4.2±0.3 µM
3.9±0.8 µM
0.54±0.36 µM
2.3±0.1 µM
21%
77%
n.d.
0.31±0.11 µM
0.16±0.06 µM
0.35±0.10 µM
0.36±0.29 µM
0.12±0.10 µM
0.047±0.021 µM
0.087±0.052 µM
1.6±0.0 µM
17±2 µM
22±4 µM
67%
28±10 µM
4.1±2.9 µM
2.3±0.5 µM
20±5 µM
n.d.
85%
5.0±5.2 µM
100%
76%
99%
COOH
COOH
CONHNH2
1.4±0.5 µM
100%
n.d.
19g
20
4,5-diBr
n.d.
21
4,5-diBr
iPr
1
1.7±0.0 µM
65%
100%
99%
novobiocin
0.17 µM
0.041 µM
11 µM
27 µM
[a] Concentration of compound that inhibits the enzyme activity by 50%. [b] Residual activity of the enzyme at 10 µM of the compound. [c] Not
determined.
Table 3. Inhibitory activities of type III compounds 24a-d, 25a-c, 28 and 29 against DNA gyrase and topoisomerase IV.
IC50 (µM)[a] or RA (%)[b]
S. aureus topo
Compd
R1
R2
R3
E. coli gyrase
S. aureus gyrase
E. coli topo IV
IV
n.d.
n.d.
24a
24b
24c
4,5-diBr
4,5-diBr
3,4-diCl-5-Me
H
OH
H
OMe
OMe
OMe
5.8±1.7 µM
94%
1.6±0.5 µM
n.d.[c]
n.d.
n.d.
n.d.
100%
94%
100%
24d
3,4-diCl-5-Me
H
Me
0.72±0.09 µM
100%
100%
100%
25a
25b
25c
4,5-diBr
4,5-diBr
3,4-diCl-5-Me
H
OH
H
OH
OH
OH
2.9±0.4 µM
2.7±0.1 µM
0.79±0.02 µM
n.d.
n.d.
62%
n.d.
n.d.
11±2 µM
n.d.
n.d.
45±4 µM
4-NH-2-OH-(C6H3)-
COOCH3
4-NH-2-OH-(C6H3)-
COOH
28
4,5-diBr
4,5-diBr
H
H
80%
n.d.
n.d.
n.d.
29
0.60±0.37 µM
0.17 µM
n.d.
n.d.
n.d.
novobiocin
0.041 µM
11 µM
27 µM
[a] Concentration of compound that inhibits the enzyme activity by 50%. [b] Residual activity of the enzyme at 10 µM of the compound. [c] Not
determined.
With the introduction of methoxy (compounds 18a, 18e, 19a and
19e, Table 2), isopropoxy (compounds 18b, 18d, 18f, 19b, 19d,
19f, 20 and 21, Table 2) or benzyloxy substituents (compounds
18g, 18h and 19g, Table 2) at the 3-position of the central
benzene ring, we intended to make additional contacts with the
lipophilic floor of the enzyme. Among the 3,4-dichloro-5-
methylpyrrolamides, the most active was the isopropoxy
compound 19f (IC50 = 0.047 µM), followed by the methoxy
compound 19e (IC50 = 0.12 µM) and the unsubstituted compound
7 (IC50 = 0.28 µM). In the 4,5-dibromopyrrolamide series the
= 21%) did not display significant activity, its carboxylic acid
derivative 19g (IC50 = 0.087 µM) was among the most potent of
the series. Overall, the results indicate that lipophilic substituents
at the 3-position of the benzene ring are favorable for enhancing
interactions with the enzyme.
With the type III series of compounds, we studied the activities of
analogues without the glycine or beta-alanine groups on the right-
hand side of the molecules (compounds 24a-d and 25a-c, Table
3). Some prepared compounds lacking the glycine or beta-alanine
groups showed good inhibitory activities, e.g. compound 24c had
an IC50 value of 1.6 µM and its carboxylic acid analogue 25c an
IC50 of 0.79 µM against E. coli DNA gyrase. These results are
particularly interesting when taking into account the simple,
fragment-like structures of these molecules that could still be
relationship was similar, as the isopropoxy compound 19b (IC50
=
0.16 µM) and the methoxy compound 19a (IC50 = 0.31 µM) were
both more potent than the unsubstituted analog A (IC50 = 0.45
µM). Even though the benzyloxy-substituted compound 18g (RA
7
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10.1002/cmdc.201700549
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optimized. Furthermore, two longer molecules, 28 and 29 (Table
3) have been prepared with an additional 4-aminobenzoyl group,
but their activities were weaker, probably because the terminal
carboxylic acid group in these molecules is too far from Arg136 to
form interactions.
125 µM (Table 4). The highest antibacterial activity of compound
11 is in line with our design strategy, that the bioisosteric
replacement of the terminal carboxylic acid group with the 1,3,4-
oxadiazol-2-one ring might lead to easier entry of inhibitors into
the bacterial cell due to their lower acidity and polarity.
Overall, the activities against S. aureus DNA gyrase, E. coli
topoisomerase IV, and S. aureus topoisomerase IV were lower
than those against E. coli DNA gyrase (Tables 1-3). The two
compounds with the greatest activity against S. aureus DNA
gyrase were 19f with an IC50 of 2.3 µM and 19e with an IC50 of 4.1
µM. Six other compounds (7, 11, 19a, 19b, 19d and 19g)
displayed S. aureus DNA gyrase inhibitory activities in the low
micromolar range, but none of the compounds have reached a
comparable inhibition to that of novobiocin with an IC50 value of
41 nM. Seven of these compounds contained a free carboxylic
acid group, which thus appears crucial for the activity. A possible
reason for the weaker activities of compounds against S. aureus
gyrase is that the adenine-binding pocket of this enzyme is slightly
smaller than that of E. coli gyrase, which would indicate that
smaller groups are more effective in binding to the hydrophobic
binding site, as discussed recently.[16b] In line with this, the two
most active compounds against S. aureus gyrase, 19e (IC50 = 4.1
µM) and 19f (IC50 = 2.3 µM), contain smaller 3,4-dichloro-5-
methylpyrrole moieties, while the activities of their 4,5-
Fifteen compounds (6, 7, 9, 11, 18f, 19f, 24a-d, 25a, 25b, 25c, 28
and 29) were additionally tested against two E. coli mutant strains,
JD17464 and JW5503. E. coli JD17464 is an lpxC deletion mutant
with impaired outer membrane, while E. coli JW5503 is a tolC
deletion mutant with a defective efflux pump. Inhibition of growth
was measured at 50 µM concentration of compounds after 24 h
incubation (Table 2S). MIC values for six selected compounds (6,
9, 11, 19f, 24a and 25c) against E. coli JW5503 are presented in
Table 5. The activities against the mutant with impaired outer
membrane (JD17464) were comparable with those against wild
type E. coli. On the other hand, activities against the mutant with
a defective efflux pump (JW5503) were greater for the majority of
the tested compounds. The most active compound was
oxadiazolone 11 with an MIC value of 6.25 µM, followed by a type
III compound 25c with 25 µM MIC. Compounds 6, 9, 19f and 24a
had an MIC of 50 µM. These results indicate that the efflux of
inhibitors is probably the major factor causing the lower activities
of these compounds against the Gram-negative bacteria. To
confirm these findings, compounds 6, 9, 11, 19f and 25c were
additionally evaluated against wild type E. coli in the presence of
100 µg/mL concentration of efflux inhibitor phenylalanine-arginine
β-naphthylamide (PAβN)[20] (Table 5). The most active compound
was again compound 11, with an MIC of 3.13 µM, followed by 25c
with an MIC of 12.5 µM, and compounds 6 (MIC = 25 µM), 19f
(MIC = 25 µM) and 9 (MIC = 50 µM). Observing the inhibitory
activity of compound 11 against wild type E. coli with and without
PAβN at different time-points (4, 8 and 24 h) clearly demonstrates
that this compound is able to affect the growth in the logarithmic
growth phase also without PAβN, but that at the 24 h end-point
the inhibitory activity no longer reaches the MIC (Figure 1S). In
the presence of PAβN, stronger inhibition is seen at all time
points. These results confirm that, in Gram-negative bacteria, the
prepared compounds are most probably substrates for efflux
pumps, which should be taken into account in the design of all
future series of N-phenylpyrrolamide inhibitors. As has been
demonstrated for the pyrrolopyrimidine series of DNA gyrase
inhibitors,[10] despite the susceptibility to efflux, it is nevertheless
dibromopyrrolamide analogs 19a (IC50 =17 µM) and 19b (IC50
=
22 µM) were slightly lower. The most active compound against
topoisomerase IV enzyme was again compound 19f with an IC50
values of 1.8 µM against E. coli topo IV and 2.7 µM against S.
aureus topo IV. The weaker activities on E. coli topoisomerase IV
than on E. coli DNA gyrase are probably due to the previously
discussed differences in the binding sites of these enzymes.[16b]
Antibacterial Activity. Compounds were evaluated against two
Gram-positive (S. aureus ATCC 25923 and Enterococcus faecalis
ATCC 29212) and two Gram-negative (E. coli ATCC 25922 and
Pseudomonas aeruginosa ATCC 27853) wild type bacterial
strains at 50 µM concentration. Results are presented in Table 1S
(Supporting information) as percent inhibition of growth after 24 h
incubation. In general, the activities against Gram-positive
bacteria were greater than those against Gram-negative bacteria.
This could be due to difficulties in permeation of compounds
through the Gram-negative bacterial cell wall, or to efflux of
inhibitors from the bacteria. In total, nine compounds (6, 9, 11, 18f, possible to achieve good MICs against Gram-negative bacteria.
19f, 19g, 21, 25c and 29) inhibited the growth of Gram-positive E.
faecalis by more than 50% at 50 µM concentration, four of which
(6, 11, 19f and 25c) displayed more than 70% inhibition of growth.
Compound 11 also showed 64% inhibition of growth of S. aureus
and 51% inhibition of growth of Gram-negative E. coli (Table 1S).
The results of the minimum inhibitory concentration (MIC)
experiments against S. aureus, E. faecalis and E. coli for the most
interesting compounds are presented in Table 4. The MIC values
for compounds 11 and 19f against E. faecalis were 12.5 µM and
50 µM (MIC for ciprofloxacin = 3.02 µM). The MIC value for
compound 11 against S. aureus was 75 µM (MIC for ciprofloxacin
= 1.51 µM). With other tested compounds, the MIC level (≥ 90%
inhibition) was not reached even at the highest concentration of
It is likely, that weaker antibacterial activity of the presented N-
phenylpyrrolamides with respect to some other published DNA
gyrase B inhibitors is due to their lower on-target activity. For
example, the representative pyrrolamide developed at
AstraZeneca (Figure 1) with S. aureus MIC of 0.06 µg/mL
displayed an IC50 of less than 10 nM against S. aureus DNA
gyrase, and the representative pyrazolopyridones from Cubist
Pharmaceuticals (Figure 1) with S. aureus MIC of 0.06 µg/mL
displayed an IC50 of 6 nM against S. aureus DNA gyrase.
Therefore, to obtain higher antibacterial potency, DNA gyrase
activity of N-phenylpyrrolamides will likely need to be improved to
low nanomolar range.
8
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10.1002/cmdc.201700549
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FULL PAPER
Table 4. Minimum inhibitory concentrations (MICs) of compounds 6, 11, 19f, 19g, 21, 25c and 29 against S. aureus (ATCC 25923), E.
faecalis (ATCC 29212) and E. coli (ATCC 25922).
MIC (µM)[a]
E. faecalis (ATCC 29212)
Compd
S. aureus (ATCC 25923)
E. coli (ATCC 25922)
6
11
n.d.[b]
75
>125
12.5
50
>125
>125
>125
>125
n.d.
>125
n.d.
n.d.
n.d.
n.d.
n.d.
19f
19g
21
25c
29
n.d.
>125
>125
n.d.
n.d.
[a] MIC = minimum inhibitory concentration (µM) that inhibited the growth of bacteria by ≥ 90%. MIC values of the positive control
ciprofloxacin against S. aureus, E. faecalis and E. coli were 1.51, 3.02 and 0.05 µM. [b] Not determined.
Table 5. Minimum inhibitory concentrations (MIC) of compounds 6, 9, 11,
mM KCl, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5 % w/v glycerol,
19f and 25c against the efflux pump deficient strain E. coli JW5503, and
against wild type E. coli in the presence of efflux inhibitor PAβN.
MIC (µM)[a]
0.1 mg/mL albumin) contained 1.5 U of DNA gyrase from E. coli or S.
aureus, 0.75 μg of relaxed pNO1 plasmid, and 3 μL solution of the inhibitor
in 10% DMSO and 0.008% Tween 20. Reaction solutions were incubated
at 37 °C for 30 min. After that, the TF buffer (50 mM NaOAc with pH 5.0,
50 mM NaCl and 50 mM MgCl2) was added to terminate the enzymatic
reaction. After additional incubation for 30 min at rt, during which biotin-
oligonucleotide-plasmid triplex was formed, the unbound plasmid was
washed off using TF buffer and SybrGOLD in T10 buffer (10 mM Tris HCl
with pH 8.0 and 1 mM EDTA) was added. The fluorescence was measured
with a microplate reader (BioTek Synergy H4, excitation: 485 nm,
emission: 535 nm). Initial screening was done at 100 or 10 μM
concentration of inhibitors. For the most active inhibitors IC50 was
determined using seven concentrations of tested compounds. GraphPad
Prism software was used to calculate the IC50 values. The result is given
as the average value of three independent measurements. As the internal
standard novobiocin (IC50 = 0.168 µM (lit.[21] 0.08 µM) for E. coli gyrase
and IC50 = 0.041 µM (lit.[22] 0.01 µM) for S. aureus gyrase) was used.
Compd
E. coli (ATCC 25922) with
E. coli (JW5503)[b]
PaβN[c]
25
6
9
50
50
50
11
19f
24a
25c
6.25
50
50
3.13
25
n.d.[d]
12.5
25
[a] MIC = minimum inhibitory concentration that inhibited the growth of
bacteria by ≥ 90%. Ciprofloxacin was used as a positive control. [b] E. coli
tolC deletion mutant with defective efflux pump. [c] Phenylalanine-arginine
β-naphthylamide; an efflux pump inhibitor. The tests were performed in the
presence of 100 µg/mL concentration of PAβN. [d] Not determined.
Conclusions
Despite the huge amount of research work, there are currently no
inhibitors of the ATPase activities of DNA gyrase and/or
topoisomerase IV in clinical use. To optimise and further develop
the promising class of N-phenylpyrrolamide DNA gyrase B
inhibitors, we have designed and prepared thirty-two new
compounds of this class and evaluated their activities against
DNA gyrase and topoisomerase IV from E. coli and S. aureus.
The most potent compounds, 11, 19f and 19g, had IC50 values
Determination of Inhibitory Activities on E. coli and S. aureus
Topoisomerase IV. IC50 values were determined in an assay from
Inspiralis on streptavidin-coated 96-well microtiter plates from Thermo
scientific Pierce. First, the plates were rehydrated with buffer (20 mM Tris-
HCl with pH 7.6, 0.01% w/v BSA, 0.05% v/v Tween 20, 137 mM NaCl) and
biotinylated oligonucleotide was then immobilized. After washing off the
unbound oligonucleotide, the enzyme test was performed. The reaction
volume of 30 μL in buffer (40 mM HEPES KOH with pH 7.6, 100 mM
potassium glutamate, 10 mM magnesium acetate, 10 mM DTT, 1 mM ATP,
0.05 mg/mL albumin) contained 1.5 U of topoisomerase IV from E. coli or
S. aureus, 0.75 μg of pNO1 supercoiled plasmid, and 3 μL solution of the
inhibitor in DMSO (10%) and Tween 20 (0.008%). Reaction mixtures were
incubated at 37 °C for 30 min and after that, the TF buffer (50 mM NaOAc
with pH 5.0, 50 mM NaCl and 50 mM MgCl2) was added to terminate the
enzymatic reaction. After additional incubation for 30 min at rt, during
which triplex (biotin-oligonucleotide-plasmid) was formed, the unbound
plasmid was washed off using TF buffer and SybrGOLD in T10 buffer (10
mM Tris HCl with pH 8.0 and 1 mM EDTA) was added. The fluorescence
was measured with a microplate reader (BioTek Synergy H4, excitation:
485 nm, emission: 535 nm). Initial screening was done at 100 or 10 μM
concentration of inhibitors. For the most active inhibitors IC50 was
determined using seven concentrations of tested compounds. GraphPad
Prism software was used to calculate the IC50 values. The result is given
as the average value of three independent measurements. As the internal
against E. coli DNA gyrase in the low nanomolar range (IC50
<
100 nM). The activities against DNA gyrase from S. aureus and
against topoisomerase IV were lower. The most potent compound,
11, had an MIC value of 12.5 µM against Gram-positive E. faecalis.
Even though the activities against wild type Gram-negative E. coli
were lower, interesting results were obtained against the efflux
pump deficient and against the wild type E. coli strains in the
presence of efflux pump inhibitor PAβN, where the MICs of
compound 11 were 6.25 µM and 3.13 µM, respectively. These
results indicate that major factor responsible for the lower activity
of this type of compounds against the Gram-negative bacteria is
their efflux from the cells. These results should be taken into
account in the design of future series of this class of GyrB
inhibitors.
standard novobiocin (IC50
topoisomerase IV and IC50 = 26.7 µM (lit.[21] 20 µM) for S. aureus
=
11.1 µM (lit.[21] 10 µM) for E. coli
topoisomerase IV) was used.
Experimental Section
Determination of Antibacterial Activity. Clinical microbiology control
strains of Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC
25922), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus
aureus (ATCC 25923) were obtained from Microbiologics Inc. (St. Cloud,
Minnesota, USA). Single-gene knock-out mutant strains of E. coli, JW5503
(tolC knock-out)[22] and JD17464 (lpxC knock-out), were obtained from the
NBRP-E.coli collection at the National Institute of Genetics (NIG, Japan).
To determine antibacterial activity, broth microdilution assays in 96-well
Determination of Inhibitory Activities on E. coli and S. aureus DNA
Gyrase. Inhibitory activities were determined in an assay from Inspiralis
on streptavidin-coated 96-well microtiter plates from Thermo scientific
Pierce. First, the plates were rehydrated with buffer (20 mM Tris-HCl with
pH 7.6, 0.01% w/v BSA, 0.05% v/v Tween 20, 137 mM NaCl) and the
biotinylated oligonucleotide was then immobilized. After washing off the
unbound oligonucleotide, the enzyme test was performed. The reaction
volume of 30 μL in buffer (35 mM Tris × HCl with pH 7.5, 4 mM MgCl2, 24
9
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10.1002/cmdc.201700549
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plates were carried out by following the CLSI guidelines.[23] For selected
compounds minimum inhibitory concentrations (MIC) were determined by
dose-response experiments (reported values are from at least two
independent experiments, each with three replicates per concentration). In
the experiments with the efflux inhibitor, the assay media was
supplemented with 100 µg/mL of Phe-Arg-β-naphthylamide (PAβN;
Sigma-Aldrich, Switzerland).
General procedure A. Synthesis of Compounds 4, 17a-b, 17d-e and
27 (with 4 as an Example). Compound 3 (0.920 g, 3.86 mmol) was
dissolved in THF (50 mL), Pd/C (200 mg) was added and the reaction
mixture was stirred under hydrogen atmosphere for 3 h. The catalyst was
filtered off and the solvent removed under reduced pressure to obtain 4
(0.796 g) as white crystals.
Methyl (4-aminobenzoyl)glycinate (4). White crystals; yield 99% (0.796
g); mp: 129-131 °C (130-131 °C, lit.[29]).
General procedure B. Synthesis of Compounds 6, 18d-h and 24b-d
(with 6 as an Example). To a suspension of 3,4-dichloro-5-methylpyrrole-
2-carboxylic acid (280 mg, 1.44 mmol) in anhydrous dichloromethane (16
mL) oxalyl chloride (2 M solution in dichloromethane, 2.20 mL, 4.32 mmol)
was added and the mixture stirred at rt for 15 h. The solvent was removed
under reduced pressure, fresh anhydrous dichloromethane (4 mL),
pyridine (2 mL) and compound 4 (300 mg, 1.44 mmol) were added and the
reaction mixture was stirred at rt for 15 h. The solvent was removed under
reduced pressure, the residue dissolved in ethyl acetate (20 mL) and
washed with 1 M HCl (2 × 20 mL) and brine (2 × 20 mL). The organic phase
was dried over Na2SO4, filtered and the solvent removed under reduced
pressure. To the residue ether (15 mL) was added, the obtained
suspension was sonicated, filtered off, washed with ether (2 × 5 mL) and
dried to afford 6 (245 mg) as a brown solid.
Molecular Modeling.
Protein and Ligand Preparation. 3D compound models were built using
ChemBio3D Ultra 16.0.[24] MMFF94 force field[25] was used for the
optimization of geometries and partial atomic charges were added. Energy
was minimized to less than 0.001 kcal/(mol Å) gradient value. The
structure was refined with GAMESS interface using PM3 method, QA
optimization algorithm and Gasteiger Hückel charges for all atoms for 100
steps.[24] GOLD Suite v5.4[26-27] was used for molecular docking
calculations. GOLD graphical user interface was used for receptor
preparation. To the protein hydrogen atoms were added and correct
tautomers and protonation states were assigned. Except for HOH614, all
water molecules and ligands were deleted from the crystal structure.
Amino acid residues within 7 Å around the ligand (PDB entry: 4DUH[19]
were selected as the binding site.
)
Methyl
(4-(3,4-dichloro-5-methyl-1H-pyrrole-2-
carboxamido)benzoyl)glycinate (6). Brown solid; yield 42% (245 mg);
mp: >300 °C.
Ligand Docking. Compounds were docked to the defined binding site in
25 independent genetic algorithm (GA) runs by applying different GA
parameters (population size = 100, selection pressure = 1.1, number of
operations = 100,000, niche size = 2, number of islands = 5, mutation
frequency = 95, crossover frequency = 95, migration frequency = 10) and
scoring functions (GoldScore, ChemScore, CHEMPLP). The most
representative results were obtained using GoldScore as a scoring
function. Ligands with RMSD value less than 1.5 Å were joined in clusters
and early termination was allowed if the top 3 solutions were within 1.0 Å
of the RMSD value. Proposed binding modes of the top 5 highest scored
docking poses were evaluated for each ligand and used for graphical
representation in PyMOL.[28]
General procedure C. Synthesis of Compounds 7, 15a-c, 19e-f and
25c (with 7 as an Example). To a stirred solution of compound 6 (48 mg,
0.12 mmol) in THF (15 mL) 1 M NaOH (240 µL, 0.24 mmol) was added.
The mixture was stirred at rt for 15 h, concentrated under reduced pressure
and the residue was acidified with 1 M HCl to pH 1. The product was
extracted with ethyl acetate (2 × 20 mL), the combined organic phases
washed with water (2 × 20 mL) and brine (20 mL), dried over Na2SO4,
filtered and the solvent evaporated under reduced pressure. To the residue
ether (5 mL) was added, the obtained suspension was sonicated, filtered
off, washed with ether (2 × 3 mL) and dried to give 7 (25 mg) as a brown
solid.
(4-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzoyl)glycine
(7). Brown solid; yield 52% (25 mg); mp: >250 °C.
Chemistry. Chemicals were obtained from Apollo Scientific (Stockport,
UK), Sigma-Aldrich (St. Louis, MO, USA) and Acros Organics (Geel,
Belgium). TLC analyses were performed on Merck 60 F254 plates (0.25
mm). Flash column chromatography was performed on silica gel 60
(particle size 240–400 mesh). For HPLC analyses an Agilent Technologies
General procedure D. Synthesis of Compounds 8, 9 and 20 (with 8 as
an Example). To the solution of compound 5 (96 mg, 0.209 mmol) in
absolute ethanol (10 mL) hydrazine monohydrate (101 µL, 2.09 mmol) was
added and the mixture was stirred at reflux for 15 h. The solvent was
evaporated under reduced pressure, to the residue absolute ethanol (10
mL) was added, the obtained precipitate was filtered off and dried to give
8 (25 mg) as a white solid.
4,5-Dibromo-N-(4-((2-hydrazineyl-2-oxoethyl)carbamoyl)phenyl)-1H-
pyrrole-2-carboxamide (8). White solid; yield 26% (25 mg); mp: 265-266
°C.
General procedure E. Synthesis of Compounds 10 and 21 (with 10 as
an Example). The suspension of compound 8 (108 mg, 0.235 mmol) and
1,1'-carbonyldiimidazole (CDI, 57 mg, 0.353 mmol) in 1,4-dioxane (15 mL)
was stirred at 101 °C for 15 h upon which a clear solution formed. The
solvent was removed under reduced pressure and the oily residue was
1100 instrument (Agilent Technologies, CA, USA) with
a G1313A
autosampler, a G1316A thermostat and a G1365B UV-Vis detector was
used. An Agilent Eclipse Plus C18 column was used (5 µm, 4.6 × 150 mm)
with a flow rate of 1.0 mL/min. Melting points were determined on a hot-
stage microscope from Reichert and are uncorrected. 1H and 13C NMR
spectra were recorded at 400 and 100 MHz on a Bruker AVANCE III 400
spectrometer (Bruker Corporation, MA, USA) as [D6]DMSO or CDCl3
solutions. For the IR measurements, a Thermo Nicolet Nexus 470 ESP
FT-IR spectrometer (Thermo Fisher Scientific, MA, USA) was used. Mass
spectra were recorded on an Advion CMS spectrometer (Advion Inc.,
Ithaca, USA) or VG Analytical Autospec Q mass spectrometer (Fisons, VG
Analytical, Manchester, UK). All tested compounds were more than 95%
pure as established by HPLC.
purified
with
flash
column
chromatography
using
dichloromethane/methanol (20/1) as an eluent to afford 10 (25 mg) as a
white solid.
4,5-Dibromo-N-(4-(((5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-
yl)methyl)carbamoyl)phenyl)-1H-pyrrole-2-carboxamide (10). White
solid, yield 22% (25 mg); mp: 261-263 °C.
General procedure F. Synthesis of Compounds 13 and 23a-b (with 13
as an Example). To the solution of 3-hydroxy-4-nitrobenzoic acid (12)
(10.00 g, 55 mmol) in methanol (500 mL) cooled on ice bath thionyl
chloride (32.20 mL, 165 mmol) was added dropwise. The mixture was
stirred at rt for 15 h. The solvent was evaporated under reduced pressure
to obtain 13 (10.35 g) as a yellow solid.
Methyl 3-hydroxy-4-nitrobenzoate (13). Yellow solid, yield 97% (10.53
g); mp: 87-91 °C (86-88 °C, lit.[30]).
Methyl 3-methoxy-4-nitrobenzoate (14a).[31] To a stirred suspension of
compound 13 (3.43 g, 17.4 mmol) and potassium carbonate (3.61 g, 26.1
Synthetic Procedures.
Methyl (4-nitrobenzoyl)glycinate (3).[16b] To a suspension of glycine
methyl ester hydrochloride (2) (0.677 g, 5.39 mmol) and potassium
carbonate (2.23 g, 16.17 mmol) in acetonitrile (50 mL) cooled to 0 °C, a
solution of 4-nitrobenzoil chloride (1) (1.00 g, 5.39 mmol) in acetonitrile (10
mL) was added dropwise. After 15 h of stirring at rt, the solvent was
evaporated under reduced pressure, the residue dissolved in ethyl acetate
(100 mL) and washed with water (2 × 20 mL) and brine (20 mL). The
organic phase was dried over Na2SO4, filtered and the solvent evaporated
under reduced pressure to afford 3 (929 mg) as yellow crystals. Yield 72%
(0.929 g).
10
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10.1002/cmdc.201700549
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mmol) in N,N-dimethylformamide (30 mL) methyl iodide (2.20 mL, 34.8
mmol) was added dropwise. The mixture was stirred at rt for 15 h,
concentrated under reduced pressure, the residue was dissolved in ethyl
acetate (20 mL) and the organic phase was washed with water (2 × 30 mL)
and brine (30 mL). The organic phase was dried over Na2SO4, filtered and
the solvent removed under reduced pressure to obtain 14a (3.01 g) as a
pale yellow solid. Yield 82% (3.01 g); mp: 90-93 °C.
4,5-Dibromo-N-(4-((2-hydrazineyl-2-oxoethyl)carbamoyl)-2-
isopropoxyphenyl)-1H-pyrrole-2-carboxamide (20). Synthesized
according to General procedure D with the addition of further 10 eq. of
hydrazine monohydrate after 15 h (34 µL, 0.700 mmol) and stirring at reflux
for additional 48 h. The product was isolated without evaporating the
solvent but with filtering off the white precipitate (20, 34 mg) formed during
reaction. White solid; yield 92% (34 mg); mp: 253-257 °C.
3-Methoxy-4-nitrobenzoic acid (15a). Synthesized according to General
procedure C with methanol (50 mL) as solvent. Pale yellow solid; yield 86
% (2.38 g); mp: 229-233 °C (230-233 °C, lit.[32]).
4,5-Dibromo-N-(2-isopropoxy-4-(((5-oxo-4,5-dihydro-1,3,4-oxadiazol-
2-yl)methyl)carbamoyl)phenyl)-1H-pyrrole-2-carboxamide
(21).
Synthesized according to General procedure E and additionally purifying
the crude product by adding ether (10 mL) to the crude product, sonicating
the obtained suspension, filtering off the solid, washing with ether (2 × 5
mL) and drying to give 21 (12 mg) as a white solid. Yield 39% (12 mg); mp:
160-162 °C.
Methyl 4-aminobenzoate (23a). Synthesized according to General
procedure F with heaing the reaction mixture at 70 °C. After the completion
of the reaction, the solvent was evaporated under reduced pressure, the
residue was dissolved in ethyl acetate (120 mL) and washed with aqueous
NaHCO3 solution (3 × 40 mL) and brine (3 × 40 mL). The organic phase
was dried over Na2SO4, filtered and the solvent removed under reduced
pressure to obtain 23a (4.88g) as off-white crystals. Yield 89% (4.88 g);
mp: 106-107 °C (107-110 °C, lit).
General procedure G. Synthesis of Compounds 16a-f (with 16a as an
Example). To a solution of compound 15a (2.32 g, 11.8 mmol) and TBTU
(4.90 g, 15.3 mmol) in dichloromethane (80 mL) N-methylmorpholine (3.9
mL, 35.2 mmol) was added and the mixture was stirred at rt for 15 min.
Glycine methyl ester hydrochloride (2) (1.62 g, 12.9 mmol) was added and
the mixture was stirred at rt for 2 h. The solvent was removed under
reduced pressure, the residue was dissolved in ethyl acetate (20 mL) and
the organic phase was washed with water (2 × 20 mL), saturated aqueous
NaHCO3 solution (2 × 20 mL), 1 M HCl (2 × 20 mL) and brine (2 × 20 mL).
The organic phase was dried over Na2SO4, filtered and the solvent
removed under reduced pressure to afford 16a (2.27 g) as a yellow solid.
Methyl (3-methoxy-4-nitrobenzoyl)glycinate (16a). Yellow solid; yield
72% (2.27 g); mp: 102-105 °C.
Methyl 4-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzoate (24a). To
a suspension of 4,5-dibromopyrrole-2-carboxylic acid (268 mg, 0.997
mmol) in anhydrous dichloromethane (9 mL) oxalyl chloride (392 µL, 4.48
mmol) was added and the mixture stirred at rt for 15 h. The solvent was
removed under reduced pressure, fresh anhydrous dichloromethane (9
mL), pyridine (4.5 mL) and compound 23a (126 mg, 0.833 mmol) were
added and the reaction mixture stirred at rt for 15 h. The solvent was
removed under reduced pressure, to the residue ethyl acetate (15 mL) and
water (10 mL) were added. The undissolved solid was filtered off, phases
were separated and the organic phase was washed with aqueous NaHCO3
solution (3 × 10 mL) and brine (2 × 10 mL), dried over Na2SO4, filtered and
the solvent removed under reduced pressure. To the crude product
methanol (10 mL) was added, the obtained suspension was sonicated,
heated, filtered off, washed with methanol (2 × 5 mL) and dried to give 24a
(96 mg) as a pale brown solid. Yield 29% (96 mg); mp: 230-233 °C.
General procedure K. Synthesis of Compounds 25a-b and 29 (with
25a as an example). To a stirred solution of compound 24a (70 mg, 0.174
mmol) in a mixture of methanol and THF (3.5:1, 9 mL) 1 M NaOH (696 µL,
0.696 mmol) was added. The mixture was stirred at rt for 15 h. Additional
4 eq. of 1 M NaOH (696 µL, 0.696 mmol) were added and the mixture was
stirred at 60 °C for 5 h, concentrated under reduced pressure, the basic
water phase was washed with ethyl acetate (10 mL) and then acidified with
1 M HCl to pH 1. The product was extracted with ethyl acetate (3 × 15 mL),
the combined organic phases washed with water (3 × 10 mL) and brine
(10 mL), dried over Na2SO4, filtered and the solvent evaporated under
reduced pressure. To the residue ether (15 mL) was added, the obtained
suspension was sonicated, filtered, washed with ether (5 mL) and dried to
give 25a (42 mg) as a pale brown solid.
Methyl (4-amino-3-methoxybenzoyl)glycinate (17a). Synthesized
according to General procedure A with methanol (20 mL) as solvent.
Brown oil; yield 99% (1.845 g).
General procedure H. Synthesis of Compounds 17c and 17f (with 17c
as an Example). A mixture of compound 16c (143 mg, 0.42 mmol) and
SnCl2 (394 mg, 2.08 mmol) in ethyl acetate (2 mL) and ethanol (2 mL) was
stirred at 55 °C for 24 h. The mixture was poured slowly into an aqueous
NaHCO3 solution (20 mL) and stirred vigoroursly for 2 h, then extracted
with EtOAc (20 mL) and washed with brine (20 mL). The organic layer was
dried over Na2SO4, filtered and the solvent was evaporated under reduced
pressure. The residue was purified with flash column chromatography
using dichloromethane/methanol (30/1  20/1) as an eluent to give
compound 17c (58 mg) as a brown oil.
Methyl (4-amino-3-(benzyloxy)benzoyl)glycinate (17c). Brown oil; yield
44% (58 mg).
General procedure I. Synthesis of Compounds 18a-c (with 18a as an
Example). To a suspension of 4,5-dibromopyrrole-2-carboxylic acid (296
mg, 1.10 mmol) and TBTU (405 mg, 1.26 mmol) in dichloromethane (10
mL) N-methylmorpholine (350 µL, 3.15 mmol) was added and the
suspension was stirred at rt for 30 min upon which a clear solution formed.
Compound 17a (145 mg, 1.05 mmol) and DMAP (14 mg, 0.115 mmol)
were added and the mixture was stirred at 60 °C for 15 h. The solvent was
removed under reduced pressue, to the residue ethyl acetate (10 ml) and
water (10 mL) were added and the organic phase was washed with
aqueous NaHCO3 solution (10 mL), 1 M HCl (10 mL) and brine (2 × 10
mL), dried over Na2SO4, filtered and the solvent removed under reduced
pressure. The crude product was purified with flash column
chromatography using ethyl acetate/petroleum ether (1/1) as an eluent to
obtain a white solid which was further purified by sonicating with ether (25
mL), filtering off the precipitate and drying to give 18a (40 mg) as a white
solid.
4-(4,5-Dibromo-1H-pyrrole-2-carboxamido)benzoic
acid
(25a).
Synthesized according to General procedure K. Pale brown solid; yield
62% (42mg); mp: >300 °C.
Methyl 2-hydroxy-4-(4-nitrobenzamido)benzoate (26). The solution of
4-nitrobenzoyl chloride (1) (0.550 g, 2.96 mmol) and methyl 4-amino-3-
hydroxybenzoate (23b) (495 mg, 2.96 mmol) in anhydrous
dichloromethane (20 mL) and pyridine (10 mL) was stirred at rt for 15 h.
The solvent was removed under reduced pressure, the residue was
dissolved in ethyl acetate (40 mL) and washed with water (2 × 15 mL),
aqueous NaHCO3 solution (3 × 15 mL) and brine (2 × 10 mL). The organic
phase was dried over Na2SO4, filtered and the solvent evaporated under
reduced pressure. To the crude product ether (15 mL) was added, the
obtained suspension sonicated, filtered off, washed with ether (2 × 5 mL)
and dried to obtain 26 (641 mg) as a white solid. Yield 68% (641 mg), mp:
200-202 °C.
Methyl
(4-(4,5-dibromo-1H-pyrrole-2-carboxamido)-3-
methoxybenzoyl)glycinate (18a). White solid; yield 10% (40 mg); mp:
230-233 °C.
General procedure J. Synthesis of Compounds 19a-d and 19g (with
19a as an Example). To a stirred solution of compound 18a (34 mg, 0.070
mmol) in methanol (10 mL) 2 M NaOH (110 µL, 0.210 mmol) was added.
The mixture was stirred at rt for 15 h, concentrated under reduced pressure
and the residue was acidified with 1 M HCl to pH 1. The product was
extracted with ethyl acetate (2 × 10 mL), the combined organic phases
washed with water (2 × 10 mL) and brine (10 mL), dried over Na2SO4,
filtered and the solvent evaporated under reduced pressure to afford 19a
(18 mg) as a white solid.
Methyl 4-(4-aminobenzamido)-2-hydroxybenzoate (27). Synthesized
according to General procedure A. After evaporation of the solvent, ether
(20 mL) was added to the residue, the obtained suspension sonicated,
(4-(4,5-Dibromo-1H-pyrrole-2-carboxamido)-3-
methoxybenzoyl)glycine (19a). White solid; yield 94% (18 mg); mp: 272-
275 °C.
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700549
ChemMedChem
FULL PAPER
filtered, washed with ether (2 × 5 mL) and dried to afford 27 (458 mg) as
white solid. Yield 83% (458 mg); mp: 207-210 °C.
References:
Methyl 4-(4-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzamido)-2-
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a suspension of 4,5-dibromopyrrole-2-
carboxylic acid (338 mg, 1.26 mmol) in anhydrous dichloromethane (10
mL) oxalyl chloride (2 M solution in dichloromethane, 3.8 mL, 7.55 mmol)
was added and the mixture was stirred at rt for 15 h. The solvent was
removed under reduced pressure, fresh anhydrous dichloromethane (10
mL), pyridine (5 mL) and compound 27 (290 mg, 1.01 mmol) were added
and the reaction mixture was stirred at rt for 15 h. The solvent was removed
under reduced pressure, to the residue ethyl acetate (30 mL) and water
(15 mL) were added. The undissolved solid was filtered off, phases were
separated and the organic phase was washed with water (2 × 10 mL),
aqueous NaHCO3 solution (3 × 10 mL) and brine (2 × 10 mL), dried over
Na2SO4, filtered and the solvent was removed under reduced pressure.
The solid residue after extraction was combined with the filtered solid and
acetonitrile (20 mL) was added, the obtained suspension was sonicated,
heated, filtered off, washed with acetonitrile (2 × 5 mL) and dried to give
28 (326 mg) as an off-white solid. Yield 60% (326 mg); mp: >300 °C.
4-(4-(4,5-Dibromo-1H-pyrrole-2-carboxamido)benzamido)-2-
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Acknowledgements
This work was supported by the Slovenian Research Agency
(Grant No. P1-0208), EU FP7 Integrated Project MAREX (Project
No. FP7-KBBE-2009-3-245137), EU H2020 ITN-ETN Project
INTEGRATE (Project Reference: 642620), and Academy of
Finland (Grant No. 277001 and 284477). We thank Dr. Dušan
Žigon (Mass Spectrometry Center, Jožef Stefan Institute,
Ljubljana, Slovenia) for recording mass spectra, Helena Macut,
Nataša Šijanec and Francesca Magari for their help with chemical
syntheses, and Heidi Mäkkylä and Cristina Carbonell Duacastella
for their technical assistance in the antibacterial assays. The
authors thank Prof. Roger Pain for proofreading the manuscript.
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Abbreviations
ATCC, American type culture collection; ATR, attenuated total
reflectance; CDI, 1,1'-carbonyldiimidazole; CFU, colony-forming
unit; CLSI, Clinical and Laboratory Standards Institute; DIAD,
diisopropyl azodicarboxylate; DMAP, 4-dimethylaminopyridine;
DTT, dithiothreitol; GyrA, DNA gyrase A; GyrB, DNA gyrase B;
MH, Mueller Hinton; NMM, N-methylmorpholine; ParC,
topoisomerase IV subunit A; ParE, topoisomerase IV subunit B;
[13] a) J. B. Cross, J. Zhang, Q. Y. Yang, M. F. Mesleh, J. A. C. Romero, B.
Wang, D. Bevan, K. M. Poutsiaka, F. Epie, T. Moy, A. Daniel, J. Shotwell,
B. Chamberlain, N. Carter, O. Andersen, J. Barker, M. D. Ryan, C. A.
Metcalf, J. Silverman, K. Nguyen, B. Lippa, R. E. Dolle, ACS Med. Chem.
Lett. 2016, 7, 374-378; b) M. F. Mesleh, J. B. Cross, J. Zhang, J.
Kahmann, O. A. Andersen, J. Barker, R. K. Cheng, B. Felicetti, M.
Woodc, A. T. Hadfield, C. Scheich, T. I. Moya, Q. Y. Yang, J. Shotwell,
K. Nguyen, B. Lippa, R. Dolle, M. D. Ryan, Bioorg. Med. Chem. Lett.
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RA,
residual
activity;
TBTU,
tetrafluoroborate;
N,N,N′,N′-tetramethyl-O-
topo IV,
(benzotriazol-1-yl)uronium
topoisomerase IV
Conflict of interest
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Solmajer, L. P. Mašič, J. Ilaš, D. Kikelj, J. Med. Chem. 2015, 58, 6179-
6194.
The authors declare no conflict of interest.
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Mašič, D. Kikelj, J. Med. Chem. 2015, 58, 5501-5521; b) N. Zidar, T.
Keywords: antibacterials • DNA gyrase • GyrB • inhibitors •
pyrrolamide
12
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Table of Contents
New N-phenylpyrrolamides were prepared as DNA gyrase and topoisomerase IV inhibitors and their structure-activity relationships
were studied. Most potent compounds displayed IC50 values lower than 100 nM against E. coli gyrase. Minimum inhibitory
concentration of 12.5 µM was determined for 11 against Gram-positive E. faecalis. MICs against efflux pump deficient and against
the E. coli strains in the presence of efflux pump inhibitor were studied.
14
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