Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF

Article abstract of DOI:10.1016/j.ejmech.2013.05.013

Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials.

Full text of DOI:10.1016/j.ejmech.2013.05.013

European Journal of Medicinal Chemistry 66 (2013) 32e45
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationships of new cyanothiophene inhibitors
of the essential peptidoglycan biosynthesis enzyme MurF
a
a
a
b
ꢀ a
Martina Hrast , Samo Turk , Izidor Sosic , Damijan Knez , Christopher P. Randall ,
Hélène Barreteau c, Carlos Contreras-Martel d, Andréa Dessen d, Alex J. O’Neill b,
Dominique Mengin-Lecreulxc, Didier Blanot c, Stanislav Gobec a
,
*
a
ꢀ
ꢀ
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
b Institute of Molecular and Cellular Biology and Antimicrobial Research Centre, University of Leeds, Leeds LS 9JT, UK
c Univ Paris-Sud, Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS, 91405 Orsay, France
d Institut de Biologie Structurale, Bacterial Pathogenesis Group (CEA, CNRS, Université Grenoble I), 41 rue Jules Horowitz, 38027 Grenoble, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosyn-
Received 15 February 2013
Received in revised form
10 May 2013
Accepted 13 May 2013
Available online 21 May 2013
thesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular
peptidoglycan biosynthesis step: the addition of
D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-
Ala- -Glu-meso-DAP (or -Lys). As MurF has no human counterpart, it represents an attractive target
g
-D
L
for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of
MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of
structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial
species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar
inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and
Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6.
These findings, together with two new co-crystal structures, represent an excellent starting point for
further optimization toward effective novel antibacterials.
Keywords:
MurF inhibitors
Antibacterial agents
Crystal structures
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
drug targets [5,6]. Peptidoglycan is an essential component of the
bacterial cell wall, and its main function is the preservation of cell
Infectious diseases represent the second major cause of death
globally [1]. Although numerous antibacterial agents are currently
available, their extensive and sometimes inappropriate use has
resulted in the development of bacterial resistance [2,3]. Therefore,
intensive research toward the development of new antibacterial
agents with novel mechanisms of action has become an urgent
need [4].
integrity by withstanding the internal osmotic pressure. It also
helps to retain the characteristic shape of the bacterial cell, and
protects the cell content from environmental factors. Peptidoglycan
is composed of repeating disaccharide units of N-acetyl muramic
acid (MurNAc) and N-acetyl glucosamine (GlcNAc) that are cross-
linked by peptide chains [5,7e9]. Although the extracellular
stages of cell wall biosynthesis are well exploited as drug targets
and are inhibited by several therapeutically used drugs, such as the
The enzymes involved in peptidoglycan biosynthesis rank
among the best known and most extensively validated antibacterial
b-lactams and glycopeptide antibiotics [5], the intracellular stages
remain underexploited. Only two currently used drugs act at the
stage of cytoplasmic precursor formation: fosfomycin, which acts as
an inhibitor of MurA, and
D
-cycloserine, which inhibits both alanine
Abbreviations: DAP, 2,6-diaminopimelic acid; GlcNAc, N-acetyl glucosamine;
racemase and -alanine:
D
D-alanine ligase [10,11].
MurC, UDP-N-acetylmuramate:
Glu ligase; MurE, UDP-N-acetylmuramoyl-
MurF, UDP-N-acetylmuramoyl- -Ala- -Glu-meso-DAP (or
ligase; MurNAc, N-acetyl muramic acid; UDP, uridine 50-diphosphate; UMtri-
UDP-N-acetylmuramoyl- -Ala- -Glu-
Ala- -Glu-meso-DAP.
L
-Ala ligase; MurD, UDP-N-acetylmuramoyl-
-Ala- -Glu:meso-DAP ligase or
-Lys): -Ala-
L
-Ala:
D-
The Mur ligases (MurC, MurD, MurE and MurF) are intracellular
ATP-dependent enzymes that share similar reaction mechanisms
and three-dimensional structures [12,13]. They catalyze the
L
D
L-Lys;
L
g-
D
L
D
D
-Ala
L
-Lys,
L
D
L-Lys; UMtri-mDAP, UDP-N-acetylmuramoyl-L-
sequential addition of
dipeptide -Ala- -Ala to UDP-MurNAc, to form UDP-MurNAc-
pentapeptide. In most Gram-negative bacteria, mycobacteria and
L-Ala, D-Glu, meso-DAP or L-Lys, and the
g-D
D
D
* Corresponding author. Tel.: þ386 1 4769500; fax: þ386 1 4258031.
E-mail address: stanislav.gobec@ffa.uni-lj.si (S. Gobec).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.013

M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
33
bacilli, MurF catalyzes the addition of
precursor UDP-MurNAc- -Ala-
(UMtri-mDAP). In Gram-positive bacteria, the nucleotide precursor
contains -Lys instead of meso-DAP [7,14,15]. MurF has been
D
-Ala-
D
-Ala to the nucleotide
2. Results and discussion
L
g-D-Glu-meso-diaminopimelate
2.1. Design
L
recognized as essential for the growth of bacteria [16e18], and its
active site is highly conserved in all medically important pathogens
[19]. As MurF has no human counterpart, it represents an attractive
target for the development of new antibacterial drugs.
The first reported MurF inhibitors were pseudo-tripeptide and
pseudo-tetrapeptide aminoalkylphosphinate mimetics of the
It is known from the Abbott Laboratories series of compounds
that 2-aminothiophene-3-carbonitrile and 5-sulfamoyl-2-chloro
benzoic acid linked together via an amide bond are essential for
good inhibitory activity (Fig. 2). The introduction of 4-chlorine to
the benzoic acid resulted in even greater potency [16,22]. However,
the greatest increase in potency was achieved when an aryl group
was attached to position 6 of the 4,5,6,7-tetrahydrobenzo[b]thio-
phene ring. These structural modifications led to compounds with
14-fold to 45-fold improved potencies compared to 1 [23].
Due to its good MurF inhibitory activity and appropriate mo-
lecular mass, we initiated systematic structural modifications of
lead compound 1, as illustrated in Fig. 2. First, various sulfonamides
were synthesized to examine the inhibitory potency of compounds
with different replacements of the morpholino group (Fig. 2: var-
iations of substituent R1). Furthermore, different substituents were
attached to position 6 of the 4,5,6,7-tetrahydrobenzo[b]thiophene
core (Fig. 2: variations of substituent R2), as it is known that
extension of this ring system at position 6 leads to greater potency
[23]. Given that the fused 4,5,6,7-tetrahydrobenzo ring is not
essential for biological activity and can be replaced by other rings
[22,23], 4,5,6,7-tetrahydrothieno[2,3-c]pyridine was introduced,
which allows for rapid introduction of N-phenyl, -benzyl and -alkyl
substituents at position 6 and enabled the synthesis of a compre-
hensive series of compounds to study the structureeactivity re-
lationships (SARs).
transition state, with Ki values ranging from 200 mM to 700 mM [20].
The sulfonamide inhibitors (e.g., Fig. 1: 1) were discovered by
Abbott Laboratories, using affinity selection screening technology
[21], and they were co-crystallized with the enzyme [16]. The
subsequent structure-based optimization led to highly potent
nanomolar inhibitors (e.g. 2) [22,23]. A muropeptide-ligase-based
assay was used to identify the thiazolylaminopyrimidine series of
MurF inhibitors with IC50 values as low as 2.5 mM (e.g. 3) [19]. The
crystal structure of Streptococcus pneumoniae MurF (PDB code
2AM1) [16] was also used for structure-based virtual screening,
which identified a novel MurF inhibitor 4 [24]. Furthermore, a se-
ries of 8-hydroxyquinolines was found to inhibit Escherichia coli
MurF; this series was later used as the basis for pharmacophore
modeling, to identify additional inhibitors without chelating
properties [25]. Two structurally different classes of inhibitors were
discovered as well; namely, the 4-phenylpiperidine derivatives and
the diarylquinolines (5) [26]. However, most of these inhibitors lack
good antibacterial activities, most likely due to the poor cell
permeability, with the exception of the 4-phenylpiperidine de-
rivatives and diarylquinolines that show modest antibacterial ac-
tivity against E. coli (MIC values 8e16 mg/mL) [26].
2.2. Chemistry
We present here the design, synthesis, biological evaluation and
co-crystallization of a new series of cyanothiophene-based in-
hibitors of MurF enzymes from different pathogenic bacteria. The
design of these compounds was based on previously published
inhibitors (1 and 2) [22,23]. Systematic structural modifications of
these lead compounds resulted in a comprehensive series of new
inhibitors, providing nanomolar inhibitors of S. pneumoniae MurF
(MurFSp) and micromolar inhibitors of MurF of E. coli (MurFEc) and
Staphylococcus aureus (MurFSa). These structural optimizations
represent an important development of MurF inhibitors toward
potential antibacterial drugs.
The synthesis of the fused 2-aminothiophenes 9aeg (Table 1),
the crucial building blocks for all of the target compounds, is pre-
sented in Scheme 1. The synthesis was performed via different
cyclic ketones (Scheme 1: 8aec, 10, 12eef, 14) that were either
commercially available (such as cyclohexanone (10)) or were syn-
thesized, as follows (Scheme 1):
(i) The synthesis of 1-phenylpiperidin-4-ones (8aec) involved
the displacement of fluorine from activated aryl fluorides with
4-piperidone ethylene acetal (6) in the presence of sodium
Fig. 1. Structural formulas of the known MurF inhibitors, with their corresponding inhibitory and antimicrobial activities.

34
M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
Fig. 2. A typical MurFSp inhibitor. Groups essential for inhibitory activity are shown in red. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
carbonate in DMSO, yielding compounds 7aec. This was fol-
lowed by mild acidic hydrolysis (10% H2SO4 in THF) of the
acetal protective group.
Compounds 21, 22aew and 23e25 were synthesized as outlined
in Scheme 3. The Boc protective group of compound 18i was
cleaved under anhydrous conditions in the presence of CF3COOH or
HCl in ethanol, to obtain 21. The variously substituted benzyl or
alkyl fragments were then attached to the main scaffold via two
different strategies. The first method was a classical nucleophilic
substitution reaction, where K2CO3 and different benzyl or alkyl
bromides were used to obtain compounds 22aeh; in the second
method, compounds 22iew were synthesized by reductive ami-
nation of 21 with a variety of benzaldehydes, using sodium tri-
acetoxyborohydride as the reducing agent and CH3COOH as the
catalyst. The formation of the amide 23 was achieved with the
coupling of benzoyl chloride and 21 in the presence of TEA. Com-
pound 24 was obtained by the cleavage of the Boc protective group
of 22a, whereas the removal of the acetyl groups of 22q with so-
dium methoxide yielded compound 25.
(ii) The hydroxyl group of 11 was converted into the methoxy
moiety using iodomethane, via Williamson synthesis of
ethers, or acetylated with acetic anhydride in pyridine,
yielding compounds 12eef.
(iii) 4-Oxopiperidine hydrochloride (13) was reacted with di-tert-
butyl dicarbonate ((Boc)2O) under aqueous conditions in the
presence of TEA, to give the Boc-protected compound 14.
Finally, 2-aminothiophenes (9aeg) were obtained via three
component condensation between sulfur, cyclic ketone and
malononitrile in the presence of morpholine as a base, using
the Gewald reaction.
The synthesis of target compounds 18aen, 19dee and 20 is
presented in Scheme 2. In the first step, 2,4-dichlorobenzoic (15)
acid was chlorosulfonated, to yield the sulfonyl chloride (16) that
was subsequently reacted with different secondary amines to give
the various sulfonamides (17aeh). These were coupled with 2-
aminothiophenes (9aeg) via a two-stage process, involving the
conversion of the acid into acyl chloride in the presence of oxalyl
chloride and catalytic amounts of DMF, followed by the coupling in
the presence of pyridine as a base, to give 18aen. As far as
carboxylate esters 18dee are concerned, their hydrolysis with KOH
provided 19dee. Finally, compound 20 was obtained by the
reduction of the keto group of 18f with sodium borohydride.
2.3. Biological activity
Target compounds 18aen, 19dee, 20, 21, 22bew and 23e25
(Schemes 2 and 3) were tested for their inhibitory activities on
MurFSp, MurFEc and MurFSa using the Malachite green assay [27]. To
exclude possible non-specific inhibition, all of the compounds were
tested in the presence of detergent (0.005% Triton X-114) [28]. The
results are presented as residual activities (RAs) of the respective
MurF in the presence of 100 mM of each compound. For the com-
pounds with RAs lower than 50%, the IC50 values were also deter-
mined. To confirm the suitability of the MurFSp inhibition assay, and
for a better comparison of newly synthesized compounds with
known inhibitors, Abbott inhibitor 1 (Figs. 1 and 2) was re-
synthesized. Compound 1 was 4-fold less potent in our assay as
Table 1
Different substituents applied to the saturated ring (Fig. 2: R2).
Compound
X
R2
compared with the published activity (IC50 ¼ 4
mM versus 1 mM),
which can be attributed to the different assay conditions [22].
9a
N
2.4. Structureeactivity relationships: the influence of different R1
substituents
With the intention of further exploring the SARs of these com-
pounds, a focused library of compound 1 analogs was synthesized
where the morpholine moiety was replaced by different saturated
heterocycles (Scheme 2; Table 2: compounds 18aeh, 19dee, 20).
The thiomorpholine (18a), thiazolidine (18b) and 3-oxopiperazine
(18c) derivates showed significantly lower inhibition of MurFSp
than parent compound 1, most likely due to the introduction of the
bulky sulfur atom in 18a and 18b, or to the increased rigidity of the
ring in 18c. Most of the compounds with the piperidine moiety
(18dee, 18h,19dee, 20) were inactive or less active than compound
1. The introduction of a carboxyethyl group in the meta (18d) or
para (18e) position led to a decrease in potency; however, the
carboxylic derivates (19dee) showed modest inhibitory activities.
The p-methylpiperidine derivate (18h) completely lost the inhibi-
tion, although compound 18g with a methyl group in the ortho
position on the piperidine ring showed very promising activity
9b
N
9c
9d
9e
N
CH2
CH
/
9f
CH
N
9g
Boc

M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
35
Scheme 1. Reagents and conditions: (a) Na2CO3, aryl fluoride, DMSO, 120 ꢀC, 16 h; (b) H2SO4, THF, rt, 48 h; (c) 8 or 10 or 12 or 14, S, CNCH2CN, morpholine, EtOH, reflux, 2 h; (d)
Cs2CO3, MeI, acetone, reflux, 1 h; (e) Ac2O, pyridine, rt, 18 h; (f) (Boc)2O, TEA, rt, 16 h.
(IC50 ¼ 3.75
m
M), as comparable to compound 1. Compound 20,
position 6 of the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine ring (18le
n) showed no inhibition of any of these MurF enzymes, most likely
due to differences in the conformation between the 4,5,6,7-
tetrahydrobenzo[b]thiophene and 4,5,6,7-tetrahydrothieno[2,3-c]
pyridine rings. Compound 23, with a benzoyl group, showed
with a p-hydroxyl group, was only 2.5-fold less potent than 1;
however compound 18f with a carbonyl function at the para posi-
tion again showed a comparable activity (IC50 ¼ 3.37
mM) to 1. All of
these sulfonamide analogs were also assayed for inhibition of
MurFEc and MurFSa, and they were inactive against both of these
enzymes.
moderate inhibitory activity of MurFSp (IC50 ¼ 27
mM), but was
devoid of MurFEc and MurFSa inhibition. Inhibitor 24 showed good
inhibitory activity against MurFSp, with an IC50 of 0.60
also inactive against the other two MurF enzymes.
mM, but was
2.5. Structureeactivity relationships: the influence of different R2
Finally, a series of inhibitors containing different benzyl sub-
stituents at position 6 were synthesized, and all of these showed
promising inhibitory activities against all of these MurF enzymes.
The 6-benzyl derivative 22c and its saturated derivative 22b with a
6-cyclohexylmethyl substituent were both potent inhibitors of
substituents
A number of compounds (Table 3) were prepared to examine
the influence on MurF inhibition of different 6-substituents
attached to either the 4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carbonitrile or the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carbonitrile core. The most potent inhibitors of MurFSp were com-
pounds 18jek with p-methoxy and p-acetoxyphenyl rings attached
to the fused 4,5,6,7-tetrahydrobenzene ring at position 6. They
were 33-fold and 23-fold more potent than the parent compound 1,
MurFSp (IC50 values of 0.64 mM and 0.46 mM, respectively), most
likely due to the same hydrophobic interaction pattern between the
inhibitor and the enzyme; 22b was, however, less potent on MurFEc
and MurFSa. A general conclusion can be made for the benzyl-
substituted derivatives 22cew: electron withdrawing groups
substituted at the para or meta positions of the benzyl ring (com-
pounds 22deh and 22jel) only slightly reduced the inhibitory ac-
tivity against MurFSp compared to 22c, while electron donating
with IC50 values of 0.12 mM and 0.17 mM, respectively. However,
neither of these two compounds showed inhibitory activity against
MurFEc and MurFSa. The compounds with phenyl substituents in
Scheme 2. Reagents and conditions: (a) HSO3Cl, 140 ꢀC, 16 h; (b) corresponding secondary amine, TEA, CH2Cl2, 0 ꢀC to rt, 16 h; (c) 1. (COCl)2, DMF, CH2Cl2, 0 ꢀC to rt, 1 h; 2. 9aeg,
pyridine, CH2Cl2, 0 ꢀC to rt, 16 h; (d) 18d or 18e, KOH, dioxane/water, rt, 2 h; (e) 18f, NaBH4, ethanol/CH2Cl2, rt, 3 h.

36
M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
Scheme 3. Reagents and conditions: (a) HCl, EtOH, rt, 18 h or CF3COOH, CH2Cl2, rt, 2 h; (b) A: K2CO3, corresponding benzyl bromide, DMF, rt, 16 h; B: corresponding benzaldehyde,
Na(OAc)3BH, AcOH, THF, rt, 16 h; (c) benzoyl chloride, TEA, CH2Cl2, rt, 16 h; (d) 22a, TFA, CH2Cl2, rt, 2 h; (e) 22q, 30% NaOMe, THF/MeOH, rt, 0.5 h.
groups (compounds 22i, 22n and 22o) improved the inhibition by
more than 3-fold. The only exception to this rule was compound
22m, which was 2-fold more potent than the parent compound
22c. In addition, if we compare compound 22k with 22l and
compound 22n with 22o, it appears that para substitution was
more favorable than the meta substitution. The bioisoteric
replacement of the carboxylic group of 22k with a more bulky and
less acidic tetrazole group (22m) led to a 2.5-fold improvement in
(Supporting information e Figure S1). Because of the specific
MurFSa and MurFEc variations in the active site, several important
interactions with cyanothiophene inhibitors are lost, leading to
lower activity on both enzymes. It thus appears most probable that
this type of cyanothiophene inhibitors of MurF have the potential to
be developed into species-specific antibacterial agents for the
treatment of infections with S. pneumoniae.
the inhibitory activity (IC50 ¼ 0.32
m
M). The compounds with two
2.6. Crystal structures of MurFSp in complex with compounds 22m
and 22n
or more substituents on the benzyl ring were mostly less potent
than compound 22c. The exception was compound 25, with two OH
groups positioned at the para and meta sites of the benzyl ring,
which showed a comparable inhibitory activity to 22c with an IC50
The crystal structures of MurFSp in complex with compounds
ꢁ
22m and 22n were solved to 1.9 and 3 A resolution (PDB codes
of 0.43
methoxy moiety led to the similarly active compound 22s
M). The substitution of the meta hydroxyl with a
carboxy (22w) or methoxy (22r) moiety resulted in 3-fold and 10-
fold lower activities, respectively. The replacement of both OH
groups with acetoxy (22q) and methoxy (22r) moieties resulted in
significantly lower inhibitory activities. The most promising in-
hibitor within this series was compound 22n, with an IC50 of
m
M. Here, the replacement of the para OH group with the
3zm6 and 3zm5), respectively, to determine the binding mode of
the inhibitors within the active site of the enzyme. The data were
collected at the European Synchotron Radiation Facility (Grenoble,
France), and the structures of the complexes were solved by mo-
lecular replacement using the known structure of complex of
MurFSp (PDB code 2AM1) [16].
Considering the similarity of our compound with Abbott in-
hibitors [22], there is no surprise that the co-crystals are in closed
conformation and that the interaction pattern is similar to the one
described before [16]. However, our crystal structures revealed
new conformations of cyanothiophene inhibitors and disclosed
some new interactions (Fig. 3). We found that the morpholino
ring could occupy either chair (22m) or boat (22n) conformation.
One of the sulfonyl oxygen atoms from the sulfonamide linker is
in a position that allows it to make two hydrogen bonds, one with
Nd from the side chain of Asn326, and one with Nd from the side
(IC50 ¼ 0.60
m
0.18 mM.
Most of the compounds from this series demonstrated moderate
inhibitory activities for both MurFEc and MurFSa (IC50 values ca.
100
mM). The most potent inhibitor of MurFEc and MurFSa was
compound 22r, with IC50 values of 37
m
M and 67 M, respectively.
m
Interestingly, compound 22n, with the 6-(p-hydroxybenzyl) sub-
stituent, and which was the best inhibitor of MurFSp within this
series, showed very low inhibitory activity against the orthologs
from E. coli and S. aureus. However, inhibitor 22o, with the 6-(m-
hydroxybenzyl) substituent, and which possessed good inhibitory
chain of Asn328. The central benzene ring is held in place by pep
interactions with Phe31. The chlorine next to sulfonamide linker
forms hydrophobic interactions with Ile139 and the chlorine
ortho to the amide linker forms the same type of interaction with
Leu45. The amine group from amide is hydrogen bonded to the
hydroxyl group of the side chain of Thr330. The nitrile group from
the 3-cyanothiophene ring forms hydrogen bonds with the
backbone of Arg49 and Ala48. Furthermore, the piperidine ring
condensed with the cyanothiophene moiety makes hydrophobic
interaction with side chains of residues Pro329, Leu360, Leu367
and Phe54. Finally, the new fragment in the molecules, the
p-substituted benzyl ring, contributes to the recognition in the
activity against MurFSp (IC50 ¼ 0.42
mM), showed promising inhi-
bition of MurFEc and MurFSa (IC50 values of 81
respectively).
mM and 91 mM,
The differences in the inhibitory activities of these compounds
on these different MurF ligases from different species were not
surprising given that MurFSp shows only 33% and 27% sequence
identity with MurFSa and MurFEc, respectively. MurFSa and MurFEc
themselves share 33% amino-acid sequence identity. These differ-
ences among the orthologs are further underlined by their different
substrate selectivities. A closer look at the active sites of all
three MurF enzymes reveals important differences among them
active site by forming the
pep interactions with Phe54 and

M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
37
Table 2
Inhibitory activities of compounds 1, 18aeh, 19dee and 20 against MurF, and their antibacterial activities against S. pneumoniae.
R1
RAa (%) or IC50
(
mM) MurFSp
RAa (%) MurFEc
RAa (%) MurFSa
MIC (mg/mL)
b
Compound
S. pneumoniae
1
4
m
M
100
91
107
95
64
18a
18b
42%
66%
>128
>128
104
85
18c
18d
89%
131
91
94
95
93
n.d.
n.d.
m
M
18e
18f
100%
3.37
95
100
103
n.d.
m
M
M
102
>128
18g
3.75
90%
m
107
103
>128
18h
19d
87
80
n.d.
50
m
M
100
102
>128
19e
20
17.9
m
M
102
104
96
64
64
10.45
m
M
101
n.d., antibacterial activity not determined.
a
Residual activity (%) of the enzyme at 100
m
M of the tested compound. Data are means of two independent experiments. Standard deviations were within ꢁ10% of the
mean.
b
Concentration of the inhibitor for which the residual activity of the enzyme is 50%.
making the hydrophobic interaction with Arg34 and Leu360. The
tetrazole moiety from compound 22m is additionally hydrogen-
bonded to carboxyl group of Asp362, while the hydroxyl group
of compound 22n forms the hydrogen bonds with the side chain
carboxyl group of Asp362 and Od of Gln363.
inhibition of MurFEc and MurFSa in vitro. However, some of the
compounds showed moderate antibacterial activity against
S. pneumoniae (Tables 2 and 3). The compounds within the first
series of inhibitors with the morpholino group (1) and their analogs
(19e, 20) prevented the growth of S. pneumoniae at a concentration
of 64 mg/mL, while compounds 22p, 22o and 22v showed the best
activities, with MICs of 16 mg/mL to 32 mg/mL.
2.7. Antibacterial activity
The antibacterial activities of selected compounds were deter-
mined against E. coli, S. aureus and S. pneumoniae. None of the
compounds tested demonstrated useful activity against E. coli
(including efflux-deficient strains, or those in which the outer
membrane had been artificially permeabilised) or S. aureus; this
was as expected, as none of these compounds showed potent
3. Conclusions
We have designed, synthesized and evaluated a comprehensive
series of cyanothiophene-based inhibitors of the MurF enzymes
from important pathogenic species: S. pneumoniae, E. coli and
S. aureus. Systematic structural modifications of the lead compound

38
M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
Table 3
Inhibitory activities of compounds 18ien, 21, 22bew and 23e25 against the MurF enzymes, and their antibacterial activity against S. pneumoniae.
RAa (%) or IC50
M) MurFSp
RA (%) or IC50
M) MurFEc
RA (%) or IC50
M) MurFSa
MIC (mg/mL)
(S. pneumoniae)
b
Compound
X
R
(
m
(
m
(
m
1
CH
H
4
m
M
100
107
64
18i
18j
N
C
19
m
M
88%
78%
128
0.12
0.17
80%
63%
m
M
M
91%
90%
98%
95%
103%
85%
79%
85%
>128
18k
18l
C
m
>128
>128
>128
N
N
18m
18n
21
N
N
80%
74%
94%
83%
81%
>128
Hþ2 Clꢂ
47
m
M
128
22a
N
n.d.
n.d.
n.d.
64%
n.d.
22b
22c
N
N
0.46
0.64
m
M
63%
109
>128
>128
mM
m
m
M
M
80
79
m
M
M
22d
22e
N
N
0.98
1.92
m
m
M
100
58%
m
>128
>128
M
92
90
m
M
M
22f
N
1.17
mM
99
62
m
M
M
m
>128
22g
22h
22i
N
N
N
1.36
1.09
0.60
mM
mM
mM
m
80
94
95
m
m
m
M
M
M
>128
128
101
m
M
82
m
M
>128

M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
39
Table 3 (continued )
b
Compound
X
N
N
R
RAa (%) or IC50
M) MurFSp
RA (%) or IC50
M) MurFEc
RA (%) or IC50
M) MurFSa
MIC (mg/mL)
(S. pneumoniae)
(
m
(
m
(m
22j
0.72
0.86
m
m
M
M
59
mM
88 mM
>128
22k
118
m
M
110
118
m
m
M
>128
>128
22l
N
2.55
m
M
67
55
m
M
M
M
22m
22n
22o
N
N
N
0.30
0.18
0.42
m
m
m
M
M
M
mM
120
84%
m
>128
>128
16
68%
81
mM
91 mM
22p
N
2.75
m
M
81%
79%
32
22q
22r
N
N
3.03
10.4
m
m
M
M
88
37
m
M
89
m
M
>128
>128
mM
66.5 mM
22s
22t
N
N
0.60
4.15
m
m
M
M
77
98
m
M
M
115
64%
m
M
>128
>128
m
22u
22v
N
N
0.99
m
M
73
78
m
M
M
109
m
M
>128
48
m
M
m
84
m
M
16
22w
23
N
N
1.41
m
M
106
93%
m
M
132
m
M
>128
27
m
M
107%
>128
(continued on next page)

40
M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
Table 3 (continued )
RAa (%) or IC50
M) MurFSp
RA (%) or IC50
M) MurFEc
RA (%) or IC50
M) MurFSa
MIC (mg/mL)
(S. pneumoniae)
b
Compound
X
R
(
m
(
m
(
m
24
25
N
N
0.60
0.43
m
M
M
93%
78%
84%
64%
>128
m
>128
n.d., antibacterial activity not determined.
a
Residual activity (%) of the enzyme at 100
m
M of the tested compound. Data are means of two independent experiments. Standard deviations were within ꢁ10% of the
mean.
b
Concentration of the inhibitor for which residual activity of the enzyme is 50%.
resulted in new potent nanomolar inhibitors of MurFSp and
micromolar inhibitors of MurFEc and MurFSa. Two new co-crystal
structures were obtained and they provide important information
for future structure-based design of improved inhibitors. Some of
the compounds showed moderate antibacterial activity against
S. pneumoniae, and thus represent a good basis for further opti-
mization toward an effective novel antibacterial drug.
4.1.2. E. coli MurF
50 mM Hepes, pH 8.0, 50 mM MgCl2, 0.005% Triton X-114,
600
mM
D-Ala-
D-Ala, 100
m
M UMtri-mDAP, 500
mM ATP, purified
MurFEc [29] and 100
DMSO.
mM of each tested compound dissolved in
4.1.3. S. aureus MurF
50 mM Hepes, pH 8.0, 20 mM MgCl2, 0.005% Triton X-114,
600 -Ala- -Ala, 50 M UMtri- -Lys, 500 M ATP, purified
M of each tested compound dissolved in
m
M
D
D
m
L
m
4. Experimental section
MurFSa [14] and 100
DMSO.
m
4.1. Inhibition assay
In all cases, the final concentration of DMSO was 5% (v/v). The
mixtures were incubated at 37 ꢀC for 15 min, and then quenched
The compounds were tested for inhibition of the addition of
D-
with 100
m
L BiomolÒ reagent. After 5 min, the absorbance at
Ala- -Ala to either UMtri- -Lys or UMtri-mDAP catalyzed by
D
L
650 nm was measured. All of the experiments were run in dupli-
cate. Residual activities (RAs) were calculated with respect to
similar assays without the tested compounds and with 5% DMSO.
The IC50 values, which were determined by measuring the residual
activities at seven different compound concentrations, represented
the concentrations for which the RA was 50%.
MurFSp, MurFEc or MurFSa. This assay was also based on the Mala-
chite green method described above, with slight modifications,
using a mixture (final volume, 50 mL) containing:
4.1.1. S. pneumoniae MurF
50 mM Hepes, pH 8.0, 50 mM MgCl2, 0.005% Triton X-114,
100 -Ala- -Ala, 50 M UMtri- -Lys, 250 M ATP, purified
MurFSp and 100 M of each tested compound dissolved in DMSO.
m
M
D
D
m
L
m
m
4.2. Microbiological evaluation
Minimum inhibitory concentrations (MICs) for selected com-
pounds were determined by broth microdilution in MuellereHin-
ton broth (MHB), against S. aureus SH1000 [30], S. pneumoniae R6
[31], and E. coli strains 1411, SM1411 (acrAB derivative of 1411) [32],
AB734 and ES100 (tolC derivative of AB734) [33], according to CLSI
guidelines [34]. In the case of S. pneumoniae, MHB was supple-
mented with 5% (v/v) lysed horse blood. To assess the impact of
outer-membrane permeability on antimicrobial activity against
E. coli, MICs were also determined against strains 1411 and AB734 in
the presence of 4 mg/mL polymyxin B nonapeptide (PMBN) [35].
4.3. Co-crystallization, diffraction data collection, structure solution
and model refinement
Crystals of conformationally closed MurFSp ligase were obtained
by co-crystallizing MurF at 288 K with inhibitors 22m and 22n. The
crystals were obtained using the vapor diffusion method and the
hanging drop system in Linbro plates, by mixing drops of 2
MurF solution (15 mg/mL purified enzyme, 20 mM HEPES pH 7.4,
200 mM NaCl, 5 mM dithiothreitol and 1 mM 22m or 22n) and 2
of reservoir solution (0.1 M tris sodium citrate pH 5.6, 0.2 M K/Na-
tartrate and 2.2 M (NH4)2SO4). The crystals were grown over 7 days.
Crystals were flash cooled in liquid nitrogen using Paratone oil as a
cryoprotectant. Diffraction data were collected at ID29 and BM14
mL of
Fig. 3. Superposition of co-crystal structures of inhibitors 22m (cyan) and 22n
(magenta) with MurFSp. MurF active site is colored green and only relevant amino acid
residues are shown as lines and colored green if they form hydrogen bond or yellow if
they form hydrophobic interactions with the inhibitors. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article.)
m
L

M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
41
Table 5
beamlines of the European Synchrotron Radiation Facility (ESRF,
Data collection, molecular replacement and structure refinement statistics 22n.
Grenoble, France).
Statistics on diffraction data collection and refinement are
summarized in Tables 4 and 5. Datasets were indexed and scaled
with XDS program suite [36]. The structures of MurF-22m and
MurF-22n were solved by molecular replacement using MurF
(2AM1) as a search model with PHASER 2.3.0 [37]. The models were
rebuilt de novo to reduce bias using the AutoBuild protocol as
implemented in PHENIX 1.8-1069 suite [38] and in ARP/wARP 7.2
[39]. Water molecules were added using the ARP/wARP Solvent
protocol. COOT 0.6.2 was used for manual corrections of the model
[40], and 22m and 22n geometry description was performed with
JLigand [41]. Cycles of restrained refinement employing TLS were
performed by REFMAC 5.5 [42] as implemented in the CCP4-6.2.0
suite of programs [43]. The stereo chemical quality of the refined
models was verified with PROCHECK [44]. A detailed representa-
tion of the binding mode of 22m and 22n in MurF active site was
made by Ligplot [45] and is presented in Fig. 3. Solved structures
displayed 99.5 and 99.8% of the non glycine residues in the most
favored and allowed regions of the Ramachandran plot.
Figures were generated with PyMol (http://www.pymol.org).
Data collection
X-ray source
Detector
Wavelength (A)
Scan-range (deg)
Oscillation (deg)
Space group
ID29
Pilatus 6M
0.979
200
0.25
P6122
119.2, 119.2, 160.7
0.502
2.94 (2.94e3.11)
87,684/13,459
89.8 (85.3)
3.9 (48.9)
ꢁ
ꢁ
Cell dimensions a, b, c (A)
Mosaicity (ꢀ)
ꢁ
Resolution (A) (last shell)
No. observed/unique reflections
Completeness (%) (last shell)
Rsym (last shell)
I/s
(I) (last shell)
46.0 (9.0)
76.16
2
ꢁ
Wilson plot B factor (A )
Molecular replacement
Mol/ASU
Phaser LLG
1
2265
Refinement
Rwork/Rfree (%)
22.48/30.30
3527/46
95.75
86.79
0.009
No. of atoms (protein/water)
2
ꢁ
Mean B factor (A )
2
ꢁ
Compound 22n mean B factor (A )
4.4. Chemistry
ꢁ
Rmsd bonds (A)
Rmsd angles (deg)
1.353
All of the chemicals used were obtained from commercial
sources (Acros, Aldrich, Alfa Aeser, Fluka and Merck), and were used
without further purification. Solvents were used without purifica-
tion or drying, unless otherwise stated. Reactions were monitored
using analytical thin-layer chromatography plates (Merck, silica gel
60 F254, 0.25 mm), and the compounds were visualized with ul-
traviolet light and ninhydrin or bromocresol green. Silica gel grade
60 (particle size 0.040e0.063 mm; Merck, Germany) was used for
flash column chromatography. 1H and 13C NMR spectra were
recorded on a Bruker AVANCE III 400 MHz spectrometer in CDCl3,
DMSO-d6, acetone-d6 or pyridine-d5, with TMS as the internal
Residues in most favored/allowed
region of Ramachandran plot (%)
Protein Data Bank entry
99.8
3zm5
standard. Mass spectra were obtained with a VG-Analytical Auto-
spec Q mass spectrometer (Centre for Mass Spectrometry, Institute
ꢀ
Jozef Stefan, Ljubljana). Infrared spectra were recorded on a Per-
kineElmer FTIR 1600 spectrometer. Melting points were deter-
mined using a Reichert hot-stage microscope, and are uncorrected.
Microanalyses were performed on a 240 C, PerkineElmer elemental
analyzer. Analyses indicated by the symbols of the elements were
within 0.4% of the theoretical values. HPLC analyses were per-
formed on an Agilent Technologies HP 1100 instrument with a
G1365B UVeVIS detector (220 and 254 nm), using a Luna C18
column (4.6 ꢃ 250 mm) at a flow rate of 1 mL/min. In method A the
eluant was a mixture of 0.1% TFA in water (A) and acetonitrile (B).
The gradient was 40% B to 90% B in 20 min. In method B the eluent
was a mixture of water (A) and acetonitrile (B). The gradient was
10% B to 90% B in 20 min. The purity of the tested compounds was
established to be ꢄ95%.
Table 4
Data collection, molecular replacement and structure refinement statistics 22m.
Data collection
X-ray source
Detector
Wavelength (A)
Scan-range (deg)
Oscillation (deg)
Space group
BM14
MARCCD 225
0.978
90
1.0
P6122
116.2, 116.6, 160.3
0.110
1.84 (1.84e1.95)
290,140/52,244
93.3 (83.1)
4.3 (52.1)
37.6 (3.5)
32.28
ꢁ
ꢁ
Cell dimensions a, b, c (A)
Mosaicity (ꢀ)
4.5. General procedure for synthesis of compounds 7aec
ꢁ
Resolution (A) (last shell)
No. observed/unique reflections
Completeness (%) (last shell)
Rsym (last shell)
I/s(I) (last shell)
Wilson plot B factor (A )
To a solution of 6 (2 g,14.2 mmol) in DMSO (10 mL), K2CO3 (2.49 g,
18.05 mmol) and the corresponding aryl fluoride (12.9 mmol) were
added, and the reaction mixture was stirred at 120 ꢀC for 16 h. The
mixture was cooled to room temperature, poured into water (50 mL),
and the yellow precipitate was filtered off.
2
ꢁ
Molecular replacement
Mol/ASU
1
4.5.1. 4-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)benzonitrile (7a)
Yield ¼ 98%; pale yellow solid; mp 129e131 ꢀC (lit. [46], mp
Phaser LLG
2308
132e133 ꢀC): 1H NMR (400 MHz, DMSO-d6):
d
1.65 (t, J ¼ 5.8 Hz, 4H,
Refinement
Rwork/Rfree (%)
No. of atoms (protein/water)
Mean B factor (A )
22.41/25.52
3577/299
37.49
29.86
0.012
2ꢃ CeCH2eCH2eN), 3.46 (t, J ¼ 5.6 Hz, 4H, 2ꢃ CeCH2eCH2eN), 3.9
(s, 4H, OeCH2eCH2eO), 7.03 (d, J ¼ 9.0 Hz, 2H, 2ꢃ AreH), 7.55 (d,
J ¼ 9.0 Hz, 2H, 2ꢃ AreH) ppm.
2
ꢁ
2
ꢁ
Compound 22m mean B factor (A )
ꢁ
Rmsd bonds (A)
Rmsd angles (deg)
1.667
99.5
4.6. General procedure for synthesis of compounds 8aec
Residues in most favored/allowed
region of Ramachandran plot (%)
Protein Data Bank entry
To a solution of 7aec (3 g) in THF (50 mL), 10% H2SO4 was added
(50 mL), and the reaction mixture was stirred at room temperature
3zm6

42
M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
for 48 h. THF was evaporated, and the water phase was treated with
saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 ꢃ 30 mL).
The combined organic phases were washed with water (40 mL) and
then brine (40 mL), and dried. CH2Cl2 was evaporated to obtain
products 8aec.
149.18, 169.67, 211.00 ppm. ESI HRMS m/z calcd. for [M þ (H)]þ
233.1178, found 233.1169. IR (KBr) nmax: 3396, 3051, 2963, 2931,
2865,1749, 1710, 1509, 1465, 1432, 1419,1370,1348, 1331, 1219,1197,
1165, 1109, 1042, 1015 cmꢂ1. Anal. calcd. for C14H16O3 ꢃ 0.1H2O: C,
71.84; H, 6.98. Found C, 71.92; H, 6.95.
4.6.1. 4-(4-Oxopiperidin-1-yl)benzonitrile (8a)
4.7.4. Synthesis of tert-butyl 4-oxopiperidine-1-carboxylate (14)
To a solution of 13 (15 g, 97.6 mmol) in a mixture of dioxane and
water (4/1, 200 mL), TEA (33.8 mL, 0.244 mol) was added. To this
mixture Boc2O (21.3 g, 146 mmol) was added portion-wise. The
mixture was stirred overnight at room temperature. The solvent
was evaporated, and the residue dissolved in 5% citric acid (100 mL)
and extracted with CH2Cl2 (3 ꢃ 75 mL). The combined organic
phases were washed with brine (100 mL) and dried with Na2SO4.
The solvent was evaporated and the crude product was recrystal-
lized from cyclohexane to obtain pure compound 14. Yield: 89%;
white needles; mp 73e75 ꢀC (lit. [48] 72 ꢀC); 1H NMR (400 MHz,
Yield ¼ 78%; pale yellow solid; mp 92e94 ꢀC (lit. [46], mp 98e
100 ꢀC); 1H NMR (400 MHz, CDCl3)
d
2.56 (t, J ¼ 6.2 Hz, 4H, 2ꢃ CH2e
CO), 3.72 (t, J ¼ 6.1 Hz, 4H, 2ꢃ NeCH2), 6.88 (d, J ¼ 9.0 Hz, 2H, 2ꢃ
AreH), 7.52 (d, J ¼ 9.0 Hz, 2H, 2ꢃ AreH) ppm.
4.7. General procedure for synthesis of compounds 9aeg
To a solution of ketone (8aec, 10, 12eef or 14) (25 mmol) and
malononitrile (1.65 g, 25 mmol) in EtOH (50 mL), sulfur (0.88 g,
27.5 mmol) and morpholine (4.35 mL, 50 mmol) were added, and
the mixture was stirred at reflux for 2 h. The reaction mixture was
cooled to room temperature and the orange solid was collected by
filtration and washed with cold EtOH.
CDCl3):
d
1.50 (s, 9H, 3ꢃ CH3), 2.44 (t, J ¼ 6.5 Hz, 4H, 2ꢃ CH2-N), 3.72
(t, J ¼ 6.1 Hz, 4H, 2ꢃ CH2-CO) ppm.
4.7.5. 2,4-Dichloro-5-(chlorosulfonyl)benzoic acid (16)
4.7.1. 2-Amino-6-(4-cyanophenyl)-4,5,6,7-tetrahydrothieno[2,3-c]
pyridine-3-carbonitrile (9a)
To a cooled (0 ꢀC) chlorosulfonic acid (10.5 mL, 157 mmol),
compound 15 (5 g, 26.2 mmol) was added portion-wise. The re-
action mixture was subsequently heated to 140 ꢀC and stirred for
16 h. The reaction mixture was cooled to room temperature, poured
into crushed ice, and the white solid formed was collected by
filtration. Yield ¼ 77%; white solid; mp 182e184 ꢀC (lit. [49] 183e
Yield ¼ 82%, orange crystals; mp 224e226 ꢀC; 1H NMR
(400 MHz, CDCl3):
d 2.69e2.73 (m, 2H, CH2eCH2eN), 3.70 (t,
J ¼ 5.7 Hz, 2H, CH2eCH2eN), 4.28 (t, J ¼ 1.7 Hz, NeCH2eAr), 4.70
(bs, 2H, NH2), 6.86 (d, J ¼ 9.0 Hz, 2H, 2ꢃ AreH), 7.50 (d, J ¼ 9.0 Hz,
2H, 2ꢃ AreH) ppm. 13C NMR (DMSO-d6, 400 MHz):
d
23.41, 43.97,
185 ꢀC); 1H NMR (400 MHz, DMSO-d6):
d 7.66 (s, 1H, AreH), 8.29 (s,
45.31, 82.60, 98.18, 113.84, 114.16, 115.81, 120.03, 130.40, 133.43,
152.31, 163.75 ppm. ESI-HRMS m/z calcd. for [M þ (H)]þ 281.0852,
found 281.0861. IR (KBr) nmax 3725, 3406, 3317, 3223, 2838, 2439,
2211, 1899, 1644, 1606, 1533, 1514, 1464, 1419, 1390, 1353, 1300,
1H, AreH), 13.93 (s, 1H, COOH) ppm.
4.8. General procedure for synthesis of compounds 17aeh
1246, 1224, 1179, 1132, 1038 cmꢂ1
.
Anal. calcd. for
28H28Cl2N4O7S2: C, 50.38; H, 4.23; N, 8.39. Found C, 50.14; H,
4.02; N, 8.17.
To a solution of 16 (3 g, 10.4 mmol) in CH2Cl2 (20 mL), TEA was
added (3.4 mL, 26 mmol) and the mixture was cooled to 0 ꢀC. The
corresponding secondary amine (11.4 mmol) was added slowly and
the mixture was stirred overnight at room temperature. Then 1 M
HCl was added (20 mL) and it was extracted with CH2Cl2
(3 ꢃ 30 mL). The combined organic phases were washed with brine
(50 mL), dried (Na2SO4), and evaporated under reduced pressure.
The crude product was purified by flash chromatography.
C
4.7.2. 4-(4-Methoxyphenyl)cyclohexanone (12e)
To a mixture of 11 (1.7 g, 8.94 mmol) and Cs2CO3 (4.37 g,
13.4 mmol) in acetone (20 mL), MeI was added (7.84 mL,126 mmol)
dropwise. The reaction mixture was refluxed for 1 h. The solvent
was evaporated, and water (30 mL) was added to the crude product.
The water phase was extracted with CH2Cl2 (3 ꢃ 30 mL), the
combined organic phases were dried with Na2SO4, and the solvent
evaporated under reduced pressure. The solid obtained was puri-
fied by chromatography. Yield ¼ 93%; white needles; mp ¼ 78e
4.8.1. 2,4-Dichloro-5-(thiomorpholin-4-ylsulfonyl)benzoic acid (17a)
Yield ¼ 8%; white crystals; mp 176e178 ꢀC: 1H NMR (400 MHz,
CDCl3):
d 2.68 (bs, 4H, CH2eSeCH2), 3.58 (bs, 4H, CH2eNeCH2),
7.62 (s, 1H, AreH), 8.59 (s, 1H, AreH) ppm, COOH exchanged. 13C
NMR (400 MHz, Py-d5): 29.69, 50.00, 136.01, 136.42, 136.73,
79 ꢀC (lit [47] 74 ꢀC); 1H NMR (400 MHz, CDCl3):
d
1.88e1.99 (m, 2H,
d
CHeCH2eCH2eCO), 2.20e2.24 (m, 2H, CHeCH2eCH2eCO), 2.51e
2.55 (m, 4H, CH2eCOeCH2), 3.01 (tt, J1 ¼ 11.9, J2 ¼ 2.9 Hz, 1H, CH),
3.82 (s, 3H, OeCH3), 6.89 (d, J ¼ 8.6 Hz, 2H, 2ꢃ AreH), 7.19 (d,
J ¼ 8.6 Hz, 2H, 2ꢃ AreH) ppm.
137.37, 137.59, 140.43, 169.72 ppm. ESI HRMS m/z calcd. for
[M þ (H)]þ 355.9585, found 355.9576. IR (KBr) nmax: 3444, 2903,
1694, 1580, 1544, 1398, 1353, 1289, 1251, 1166, 1127, 1085, 1068,
1021 cmꢂ1. Anal. calcd. for C11H11Cl2NO4S2: C, 37.09; H, 3.11; N, 3.93.
Found C, 37.00; H, 2.95; N, 3.82.
4.7.3. 4-(4-Oxocyclohexyl)phenyl acetate (12f)
To a solution of 11 (5 g, 26.3 mmol) in pyridine (15 mL), Ac2O
(10 mL) was added dropwise. The reaction mixture was stirred at
room temperature for 18 h. Then water (100 mL) was added and the
pH was adjusted to 1 with HCl (37%). The water was extracted with
EtOAc (4 ꢃ 50 mL), the organic phase was washed with brine
(100 mL), dried (Na2SO4), and evaporated under reduced pressure.
The crude product was purified by flash chromatography.
Yield ¼ 58%; white needles; mp 108e111 ꢀC; 1H NMR (400 MHz,
4.9. General procedure for synthesis of compounds 18aen
To a solution of 17aeh (2.78 g, 8.17 mmol) in anhydrous CH2Cl2
(30 mL), catalytic amounts of DMF and (COCl)2 (2.1 mL, 24.5 mmol)
were added drop-wise, and the mixture was stirred for 0.5 h at
room temperature. The solvent was evaporated to dryness and the
residue was dissolved in CH2Cl2 (20 mL), 9aeg (2.05 g, 7.35 mmol)
and pyridine (2 mL, 24.5 mmol) were added drop-wise, and the
mixture was stirred overnight at room temperature. The reaction
mixture was washed with 1 M HCl (30 mL), saturated aqueous
NaHCO3 (30 mL) and brine (30 mL), and dried (Na2SO4), and the
solvent evaporated under reduced pressure. The crude product was
purified by flash chromatography.
CDCl3):
d 1.87e1.97 (m, 2H, CHeCH2eCH2eCO), 2.19e2.25 (m, 2H,
CHeCH2eCH2eCO), 2.30 (s, 3H, CH3eCO), 2.49e2.53 (m, 4H, CH2e
COeCH2), 3.03 (tt, J1 ¼ 12.4, J2 ¼ 3.4 Hz, 1H, CH), 7.04 (d, J ¼ 8.4 Hz,
2H, 2ꢃ AreH), 7.25 (d, J ¼ 8.4 Hz, 2H, 2ꢃ AreH) ppm. 13C NMR
(400 MHz, CDCl3): d 21.16, 34.01, 41.32, 42.21, 121.64, 127.67, 142.32,

M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
43
4.9.1. 2,4-Dichloro-N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-
2-yl)-5-(thiomorpholin-4-ylsulfonyl)benzamide (18a)
[M þ (H)]þ 514.0429, found 514.0416. IR (KBr) nmax: 3260, 3085, 2931,
2215, 1678, 1572, 1447, 1406, 1329, 1286, 1259, 1157, 1077, 1039 cmꢂ1
.
Yield ¼ 25%; pale orange crystals; mp 252e254 ꢀC; 1H NMR
Anal. calcd. for C21H21Cl2N3O4S2: C, 49.03; H, 4.11; N, 8.17. Found: C,
48.79; H, 4.14; N, 7.78.
(400 MHz, CDCl3):
d 1.84e1.89 (m, 4H, CeCH2eCH2eCH2eCH2eC),
2.61 (t, J ¼ 4.8 Hz, 2H, CeCH2eCH2eCH2eCH2eC), 2.69e2.71 (m,
6H, CeCH2eCH2eCH2eCH2eC þ CH2eSeCH2), 3.62 (t, J ¼ 4.8 Hz,
4H, CH2eNeCH2), 7.69 (s, 1H, AreH), 8.54 (s, 1H, AreH), 9.58 (s, 1H,
4.10.3. 3-Cyano-2-(2,4-dichloro-5-(morpholinosulfonyl)benzamido)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium 2,2,2-trifluoroacetate
(21)
NH) ppm. 13C NMR (400 MHz, CDCl3):
d 22.04, 23.01, 23.98, 24.03,
27.54, 47.78, 95.07, 114.05, 129.79, 130.98, 131.56, 133.74, 134.26,
135.64, 135.91, 136.56, 145.50, 160.09 ppm. ESI HRMS m/z calcd. for
[M þ (H)]þ 516.0044, found 516.0050. IR (KBr) nmax: 3448, 3303,
3094, 2944, 2914, 2860, 2214, 1694, 1573, 1549, 1449, 1357, 1342,
1329, 1292, 1281, 1264, 1153, 1118, 1083, 1068, 1024 cmꢂ1. Anal.
calcd. for C20H19Cl2N3O3S3: C, 46.51; H, 3.71; N, 8.14. Found: C,
46.71; H, 3.58; N, 8.23.
To a solution of 18i (1 g, 1.66 mmol) in CH2Cl2 (20 mL), CF3COOH
(5 mL) or HCl in EtOH (2 M, 10 mL) was added and stirred at room
temperature for 2 h. The solvent was evaporated under reduced
pressure and the residue was recrystallized from EtOH to obtain 21.
Yield ¼ 94%; white powder; mp 268e270 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d
2.75 (t, J ¼ 5.1 Hz, 2H, CH2), 3.18 (t, J ¼ 4.5 Hz, 4H, 2ꢃ
CH2eN-morpholine), 3.63 (t, J ¼ 4.4 Hz, 4H, 2ꢃ CH2eO-morpho-
line), 4.14 (s, 2H, CH2eN), 7.88 (s, 1H, AreH), 8.30 (s,1H, AreH), 8.94
(bs, 1H, NH) ppm, CH2eCH2eN covered with water from DMSO,
4.10. Synthesis of compounds 19dee
NH2 exchanged. 13C NMR (400 MHz, DMSO-d6):
d 21.13, 40.91,
To a solution of 18dee (0.150 g, 0.263 mmol) in a mixture of
dioxane and water (1/1, 5 mL), KOH (0.06 g, 1.05 mmol) was added.
The reaction mixture was stirred at room temperature for 2 h.
Dioxane was then evaporated under reduced pressure and the
remaining water was acidified to pH 1. The precipitate obtained was
collected by filtration.
41.72, 45.53, 65.68, 90.63, 105.17, 114.89, 117.24, 126.65, 130.75,
132.93, 132.96, 133.14, 136.50, 138.61, 165.97 ppm. ESI-HRMS m/z
calcd. for [M þ (H)]þ 501.0225, found 501.0223. IR (KBr) nmax 3448,
3064, 2970, 2921, 2867, 2738, 2617, 2481, 2223, 1698, 1678, 1583,
1566, 1471, 1454, 1434, 1390, 1367, 1341, 1322, 1293, 1262,
1246, 1193, 1157, 1134, 1120, 1086, 1018 cmꢂ1. Anal. calcd. for
C
19H18Cl2N4O4S2 ꢃ CF3COOH: C, 40.98; H, 3.11; N, 9.10. Found: C,
4.10.1. 1-(2,4-Dichloro-5-(3-cyano-4,5,6,7-tetrahydrobenzo[b]
thiophen-2-ylcarbamoyl)phenylsulfonyl) piperidine-3-carboxylic
acid (19d)
41.04; H, 3.13; N, 8.83.
4.11. General procedures for synthesis of compounds 22aew
Yield ¼ 84%; white solid; mp ¼ 278e280 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d
1.47e1.56 (m, 2H, NeCH2eCH2eCH2eCH), 1.71e1.78
Method A: To a solution of 21 (0.100 g, 0.186 mmol) in DMF
(3 mL), K2CO3 (0.100 g, 0.740 mmol) was added. After 10 min of
stirring at room temperature, the corresponding benzyl bromide
was added (0.195 mmol). The reaction mixture was stirred over-
night at room temperature. DMF was then evaporated, the crude
residue was dissolved in EtOAc (20 mL) and washed with water
(3 ꢃ 10 mL), brine (10 mL) and dried with Na2SO4. The yellow solid
was purified by flash chromatography.
(m, 5H, CeCH2eCH2eCH2eCH2eC þ CHAHBeCH), 1.90e1.94 (m,1H,
CHAHBeCH), 2.65 (bs, 2H, CeCH2eCH2eCH2eCH2eC), 2.94 (t,
J ¼ 9.7 Hz, NeCHAHBeCH2eCH2eCH), 3.04 (t, J ¼ 11.0 Hz, Ne
CHAHBeCH2eCH2eCH), 3.52 (d, J ¼ 12.6 Hz, NeCHACHBeCH), 3.72
(dd, J1 ¼ 12.6, J2 ¼ 3.7 Hz, NeCHACHBeCH), 8.11 (s, 1H, AreH), 8.16
(s, 1H, AreH), 12.35 (s, 1H, NH), 12.53 (bs, 1H, COOH) ppm. 13C NMR
(400 MHz, DMSO-d6):
d 21.70, 22.60, 23.45, 23.57, 23.90, 25.75,
40.43. 45.47, 46.84, 94.43, 114.04, 128.36, 131.30, 131.74, 132.78,
133.37, 133.87, 134.83, 135.79, 145.64, 162.32, 173.72 ppm. ESI-
HRMS m/z calcd. for [M þ (H)]þ 542.0378, found 542.0395. IR
(KBr) nmax 3854, 3317, 3097, 2941, 2860, 2361, 2215, 1833, 1702,
1672, 1570, 1552, 1448, 1337, 1286, 1255, 1218, 1171, 1157, 1136, 1081,
1052, 1030 cmꢂ1. Anal. calcd. for C22H21Cl2N3O5S2 ꢃ 0.6H2O: C,
47.76; H, 4.04; N, 7.59. Found: C, 47.50; H, 3.65; N, 7.32.
Method B: To a solution of 21 (0.150 g, 0.244 mmol) in THF
(10 mL), the corresponding benzaldehyde (0.268 mmol), CH3COOH
(0.014 mL, 0.244 mmol) and Na(OAc)3BH (0.103 g, 4.88 mmol) were
added, and the reaction mixture was stirred overnight at room
temperature. To quench the reaction, saturated aqueous NaHCO3
(10 mL) was used, and the water phase was extracted with EtOAc
(3 ꢃ 10 mL), washed with brine (20 mL) and dried with Na2SO4. The
crude residue was purified by flash chromatography.
4.10.2. 2,4-Dichloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]
thiophen-2-yl)-5-((4-hydroxypiperidin-1-yl)sulfonyl)benzamide
(20)
4.11.1. N-(6-Benzyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]
pyridin-2-yl)-2,4-dichloro-5-(morpholino sulfonyl)benzamide (22c)
To a solution of 18f (0.180 g, 0.351 mmol) in a mixture of EtOH and
CH2Cl2 (1/1,10 mL), NaBH4 (0.027 g, 0.703 mmol) was added and the
reaction mixture was stirred at roomtemperature for 3 h. The solvent
was evaporated to dryness, the crude residue was dissolved in EtOAc
(20 mL), washed with saturated aqueous NaHCO3 (20 mL) and brine
(20 mL), dried with Na2SO4, and evaporated under reduced pressure.
The solid was purified by flash chromatography. Yield ¼ 67%; pale
Yield ¼ 45%; mp 126e128 ꢀC; 1H NMR (400 MHz, CDCl3):
d 2.74
(t, J ¼ 5.1 Hz, 2H, CH2eCH2eN), 2.84 (t, J ¼ 5.6 Hz, 2H, CH2eCH2eN),
3.31 (t, J ¼ 4.7 Hz, 4H, 2ꢃ CH2eN-morpholine), 3.58 (s, 2H, NeCH2),
3.70e3.73 (m, 6H, 2ꢃ CH2eO-morpholine þ NeCH2-Bz), 7.28e7.53
(m, 5H, 5ꢃ AreH), 7.70 (s, 1H, AreH), 8.57 (s, 1H, AreH), 9.57 (bs,
1H, NH) ppm. 13C NMR (400 MHz, CDCl3):
d 24.26, 46.08, 49.51,
50.83, 61.81, 66.72, 94.86, 113.89, 116.45, 123.76, 127.37, 127.74,
128.74, 129.33, 130.38, 130.94, 134.12, 134.68, 135.89,136.25, 146.40,
160.30 ppm. ESI-HRMS m/z calcd. for [M þ (H)]þ 591.0694, found
591.0699. IR (KBr) nmax 3774, 3320, 2858, 2457, 2212, 1912, 1669,
1582, 1550, 1451, 1358, 1329, 1262, 1172, 1113, 1072 cmꢂ1. HPLC
(method A) tR ¼ 6.181 min (99.42% at 220 nm, 97.60% at 254 nm).
yellow crystals; mp 217e219 ꢀC; 1H NMR (400 MHz, CDCl3):
d 1.54 (d,
J ¼ 3.42 Hz,1H, CHeOH),1.60e1.66 (m, 2H, CH2eCHeCH2),1.83e1.88
(m, 4H, CeCH2eCH2eCH2eCH2eC), 1.90e1.97 (m, 2H, CH2eCHe
CH2), 2.60e2.62 (t, J ¼ 4.8 Hz, 2H, CeCH2 CH2eCH2eCH2eC), 2.68e
2.70 (t, J ¼ 4.8 Hz, 2H, CeCH2eCH2eCH2eCH2eC), 3.16e3.22 (m, 2H,
CH2eNeCH2), 3.56e3.61 (m, 2H, CH2eNeCH2), 3.88e3.92 (m, 1H,
CH), 7.68 (s, 1H, AreH), 8.52 (s, 1H, AreH), 9.62 (s, 1H, NHeCO) ppm.
4.11.2. N-(6-Benzoyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]
pyridin-2-yl)-2,4-dichloro-5-(morpholinosulfonyl)benzamide (23)
To a solution of 21 (0.100 g, 0.16 mmol) in CH2Cl2 (5 mL), TEA
(0.055 mL, 0.40 mmol) was added. After 10 min benzoyl chloride
13C NMR (400 MHz, CDCl3):
d 22.04, 23.02, 23.99, 24.04, 33.64, 42.88,
66.95, 95.16, 114.08, 129.79, 130.69, 131.56, 133.80, 134.32, 135.55,
136.03, 136.64, 145.41, 160.04 ppm. ESI HRMS m/z calcd. for

44
M. Hrast et al. / European Journal of Medicinal Chemistry 66 (2013) 32e45
(0.028 mL, 0.25 mmol) was added drop-wise. The reaction mixture
was stirred for 16 h at room temperature. The mixture was diluted
with CH2Cl2 (20 mL) and washed with water (10 mL), 1 M HCl
(10 mL), saturated aqueous NaHCO3 (10 mL) and brine (15 mL). The
solvent was dried over Na2SO4 and evaporated under reduced
pressure. The crude product was purified by flash chromatography.
Yield ¼ 62%; white solid; mp 258e260 ꢀC; 1H NMR (400 MHz,
3376, 3089, 2856, 2363, 2223, 1832, 1700, 1579, 1560, 1448, 1336,
1285, 1164, 1112, 1084, 1067, 1000 cmꢂ1
HPLC (method A)
tR ¼ 3.534 min (97.25% at 220 nm, 98.15% at 254 nm).
.
Acknowledgments
We thank OpenEye Scientific Software, Inc. for free academic
licenses of their software, and the Ministry of Higher Education,
Science and Technology of the Republic of Slovenia for financial
support. This study was partially supported by a Young Researcher
grant to MH, and a L1-4039 grant to ST and SG, both from the
Slovenian Research Agency. We also thank Institute Charles Nodier
and Proteus PHC program for supporting our project.
CDCl3):
d
2.73 (t, J ¼ 5.5 Hz, 2H, NeCH2eCH2), 3.23 (t, J ¼ 4.4 Hz,
4H, 2ꢃ CH2eN-morpholine), 3.63e3.65 (m, 6H, 2ꢃ CH2eO-
morpholine þ NeCH2eCH2), 4.77 (s, 2H, COeNeCH2), 7.47e7.52
(m, 5H, 5ꢃ AreH), 8.16 (s, 1H AreH), 8.19 (s, 1H, AreH), 12.53 (bs,
1H, NH) ppm. 13C NMR (400 MHz, DMSO-d6):
d 22.11, 40.82, 44.13,
45.52, 65.73, 93.95, 113.60, 124.74, 126.85, 128.57, 129.27, 129.88,
130.73, 132.03, 132.91, 133.66, 133.98, 135.76, 136.03, 146.84, 162.38,
169.77 ppm. ESI-HRMS m/z calcd. for [M þ (H)]þ 605.0487, found
605.0496. IR (KBr) nmax 3437, 3252, 3088, 2919, 2860, 2216, 1690,
1641,1576,1556,1493,1433,1352,1331,1288,1262,1156,1116,1086,
1072, 1050, 1027, 1007 cmꢂ1. HPLC (method B) tR ¼ 12.908 min
(98.65% at 220 nm, 100% at 254 nm).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.05.013.
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benzamido)-4,5-dihydrothieno[2,3-c]pyridin-6(7H)-yl)ethyl)
piperidinium trifluoroacetate (24)
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CF3COOH was added (1 mL), and the reaction mixture was stirred at
room temperature for 3 h. The solvent was evaporated and the
product was dried in a desiccator. Yield ¼ 80%; pale yellow solid;
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