Design, synthesis and evaluation of second generation MurF inhibitors based on a cyanothiophene scaffold

Article abstract of DOI:10.1016/j.ejmech.2013.11.031

MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target compounds had increased polarity compared to the first generation of inhibitors, with demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best inhibitors displayed promising antibacterial activities against selected Gram-positive and Gram-negative strains. These results represent an important step towards the development of new antibacterial agents targeting peptidoglycan biosynthesis.

Full text of DOI:10.1016/j.ejmech.2013.11.031

Accepted Manuscript
Design, synthesis and evaluation of second generation MurF inhibitors based on a
cyanothiophene scaffold
Martina Hrast, Marko Anderluh, Damijan Knez, Christopher P. Randall, Hélène
Barreteau, Alex J. O’Neill, Didier Blanot, Stanislav Gobec
PII:
S0223-5234(13)00773-3
10.1016/j.ejmech.2013.11.031
EJMECH 6577
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 July 2013
Revised Date: 15 November 2013
Accepted Date: 24 November 2013
Please cite this article as: M. Hrast, M. Anderluh, D. Knez, C.P. Randall, H. Barreteau, A.J. O’Neill,
D. Blanot, S. Gobec, Design, synthesis and evaluation of second generation MurF inhibitors based
on a cyanothiophene scaffold, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2013.11.031.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of second generation MurF inhibitors
based on a cyanothiophene scaffold
1
1
1
2
Martina Hrast , Marko Anderluh , Damijan Knez , Christopher P. Randall , Hélène
3
2
3
1
Barreteau , Alex J. O’Neill , Didier Blanot and Stanislav Gobec *
1
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
2
School of Molecular and Cellular Biology and Antimicrobial Research Centre, University of
Leeds, Leeds LS 9JT, U.K.
3
Univ Paris-Sud, Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS,
9
1405 Orsay, France
*
Corresponding author: Stanislav Gobec
University of Ljubljana
Faculty of Pharmacy, Aškerčeva 7
1
000 Ljubljana, Slovenia
Tel: +386-1-4769500
Fax: +386-1-4258031
E-mail: stanislav.gobec@ffa.uni-lj.si
1

ACCEPTED MANUSCRIPT
ABSTRACT
MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial
peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug
discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based
inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target
compounds had increased polarity compared to the first generation of inhibitors, with
demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best
inhibitors displayed promising antibacterial activities against selected gram-positive and
gram-negative strains. These results represent an important step towards the development of
new antibacterial agents targeting peptidoglycan biosynthesis.
2

ACCEPTED MANUSCRIPT
Abbreviations:
DAP, 2,6-diaminopimelic acid; GlcNAc, N-acetyl glucosamine; MurC, UDP-N-
acetylmuramate:L-Ala ligase; MurD, UDP-N-acetylmuramoyl-L-Ala:D-Glu ligase; MurE,
UDP-N-acetylmuramoyl-L-Ala-D-Glu:meso-DAP ligase; MurF, UDP-N-acetylmuramoyl-L-
Ala-γ-D-Glu-meso-DAP (or L-Lys):D-Ala-D-Ala ligase; MurNAc, N-acetyl muramic acid;
UDP, uridine 5’-diphosphate; UMtri-L-Lys, UDP-N-acetylmuramoyl-L-Ala-D-Glu-L-Lys;
UMtri-mDAP, UDP-N-acetylmuramoyl-L-Ala-γ-D-Glu-meso-DAP.
3

ACCEPTED MANUSCRIPT
1
. INTRODUCTION
The increasing emergence of pathogenic bacteria resistant to all known antibiotics has created
an urgent need for development of new antibacterial agents [1-3]. Cell wall biosynthesis in
bacteria is well characterised and thus represents an attractive target for the development of
novel antibacterial drugs [4-6]. The biosynthetic pathway of peptidoglycan encompasses
cytoplasmic, membrane and extracellular stages [4, 6, 7].
Mur ligases (MurC-F) are cytoplasmic ATP-dependent enzymes which catalyse the sequential
addition of L-Ala, D-Glu and meso-DAP in Gram-negative or L-Lys in Gram-positive bacteria,
and the dipeptide D-Ala-D-Ala to UDP-MurNAc, to form UDP-MurNAc-pentapeptide [7, 8].
All Mur ligases have similar mechanisms of action, and share the same three-domain
topology [9]. They are essential for the survival of bacteria and lack human counterparts;
consequently, they represent interesting targets for the discovery of new antibacterials with
selective toxicity [8]. There are only a few known classes of MurF inhibitors. The first classes
were pseudo-tripeptide and pseudo-tetrapeptide aminoalkylphosphinic acids [10], followed by
sulphonamides developed by Abbott Laboratories [11, 12]. Thiazolylaminopyrimidines [13],
8
-hydroxyquinolines [14] and diarylquinolines [15] were discovered by Johnson & Johnson.
Recently, a triazine derivative was identified through structure-based virtual screening [16].
Additionally, ligand-based virtual screening was successfully employed by our group and one
new compound with micromolar inhibitory activities against S. pneumoniae MurF (MurFSp
and E. coli MurF (MurFEc) was identified [17].
)
In our previous research, we also reported a series of cyanothiophene-based inhibitors of
MurF enzymes from different pathogenic bacteria, where systematic structural modifications
of the lead compound resulted in new potent nanomolar inhibitors of MurFSp and micromolar
inhibitors of MurFEc and S. aureus MurF (MurFSa). The crystal structures of MurFSp in
complex with two new inhibitors (PDB codes 3zm5 and 3zm6) revealed the binding modes of
compounds, which allowed us to undertake structure-based design of novel, improved
analogues with optimized physicochemical properties. Although some of the compounds from
the first series showed good MurF inhibitory potency and moderate antibacterial activity
against S. pneumoniae, the solubility of the majority of assayed compounds in water was low.
We postulated that the large planar lipophilic portion of the molecules associated with rather
4

ACCEPTED MANUSCRIPT
high molecular mass led to their high tendency for precipitation, making them inadequate
drug candidates [18].
In order to present further insight into the structure-activity relationship of cyanothiophene-
based MurF inhibitors, we report the design, synthesis and biological evaluation of a new
series of inhibitors. Different structural modifications of the parent compounds resulted in a
focused library of 37 new inhibitors, providing low micromolar inhibitors of MurF from E.
coli and S. pneumoniae.
2
2
. RESULTS AND DISCUSSION
.1. Design. According to the results from Abbott Laboratories [11, 12] and our previous
research [18], 2-aminothiophene-3-carbonitrile and 5-sulfamoyl-2-chlorobenzoic acid linked
together via an amide bond, are essential for good MurF inhibitory potency (i.e., compound I,
Figure 1.). Additionally, 3D similarity search revealed micromolar inhibitor II, where the
cyclohexene ring in compound I was replaced by cyclopentene ring, sulfamoyl substituent
was replaced by the 1-pyrazolylmethyloxy moiety and the chloro substituents of benzoic acid
part were replaced by hydrogens [17].
Due to promising potencies of compounds I and II, the structural features of both inhibitors
were combined. Overlapping both compounds yielded the key scaffold (in red, Fig. 1.), which
was used as a starting point in the design of novel MurF inhibitors. The main problem
observed with compounds I and II was their low aqueous solubility due to quite high
lipophilicity (ClogP(I) = 2.82, and ClogP(II) = 3.40). It is well known that antibacterial
agents have slightly different physiochemical properties than the rest of the drugs [19, 20].
Average molecular weights are usually higher for antibacterials, with a defined cut-off at 600
Da. In addition, the lipophilicity of the Gram-positive and especially of Gram-negative
antibacterials compared to “normal” drugs is reduced. These unique properties were taken
into account during the design of the second generation of MurF inhibitors, where more polar
compounds were targeted.
Two steps were performed in designing second-generation MurF inhibitors, as depicted in
Figure 1. In the first step, various alkyl or aryl substituents bearing mostly polar functional
1
groups (R ) were attached to the main scaffold in order to examine the inhibitory activities of
compounds with different replacements of the 4-chloropyrazole moiety. In the second step,
2
various phenyl and benzyl substituents were appended to position 6 (R substituent) of the
5

ACCEPTED MANUSCRIPT
4
,5,6,7-tetrahydrobenzo[b]thiophene
and
4,5,6,7-tetrahydrothieno[2,3-c]pyridine,
respectively. It is known from our previous study that extension of these ring systems
significantly improves the potency of compounds against S. pneumoniae MurF and gains the
potency against other MurF orthologes [18]. Although this extension increases lipophilicity,
the presence of the protonated amine or guanidine moiety outweighs the contribution of these
lipophilic residues so that the overall ClogP does not exceed the value of 2.8. (Table S1)
To predict the binding affinity of the designed compounds to MurF, a docking study was
performed using the crystal structure of MurF enzyme from S. pneumoniae (PDB code: 3zm5)
and OEDocking software (Release 3.0.1, OpenEye Scientific Software, Inc.). This revealed
1
the compounds with simple amine or guanidine at position R as the ones with the highest
predicted affinities (Fig. 2a-d). Furthermore, the replacement of H with Cl on the benzoic acid
moiety was proposed to be beneficial for potent inhibition of MurF.
As all predicted binding poses were plausible and with good docking scores, we decided to
incorporate both amino and guanidine groups into the structures of potential inhibitors. The
design presented herein proved to be successful, as majority of amines/guanidines
demonstrated low micromolar inhibiton of MurF from E. coli and S. pneumoniae.
2
.2. Chemistry. Compound 4 was synthesized as outlined in Scheme 1. In the first step, the
chlorosulfonated 2,4-dichlorobenzoic acid (1) reacted with the tert-butyl (2-
aminoethyl)carbamate to yield compound 2. Then, the free acid was coupled with 2-
aminothiophene through a two-stage process, including the conversion of the acid into acyl
chloride in the presence of oxalyl chloride and catalytic amount of DMF, followed by the
coupling in the presence of pyridine as a base, to obtain 3. Compound 4 was prepared by the
3
cleavage of the Boc protective group in the presence of CF COOH, and was subsequently
transformed into hydrochloride salt with HCl in ethyl acetate.
The synthesis of target compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a and 27b is presented
in Scheme 2. First, the hydroxy group of compounds 5 and 6 was acetylated with acetic
anhydride in pyridine. Next, compounds 9 and 10 were obtained using the same synthetic
strategy as described for 3. The acetoxy protective groups of compounds 9 and 10 were
cleaved in the presence of K CO in methanol. The diverse alkyl or aryl substituents were
2
3
attached to the hydroxyl group of compounds 11 and 12 with two different approaches. In the
first method, compounds 14, 17-19, 20a, 20b, 21a and 21b were obtained using K CO or
Cs CO and different benzyl or alkyl halides. An alternative method (the Mitsunobu reaction
2
3
2
3
6

ACCEPTED MANUSCRIPT
using PPh
3
, diisopropyl azodicarboxylate (DIAD) and the corresponding alcohol) was
employed to give compounds 15 and 16. As (1H-pyrazol-1-yl)methanol (13) was not
commercially available, we prepared it by the already reported procedure [21]. Compounds
2
2a, 22b, 23a and 23b were obtained after the cleavage of the Boc protective group using
CF COOH. Amine groups of compounds 22a, 23a and 23b were converted into di-Boc
protected guanidines in the presence of mercuric chloride and triethylamine. Finally, the
cleavage of both Boc protective groups with CF COOH, followed by direct transformation to
3
3
hydrochloride salts with HCl in ethyl acetate, gave target compounds 26a, 27a and 27b.
The synthesis of target compounds 33-36, 37a-f, 38a-f, 39c, 42a-e and 43 is outlined in
Scheme 3. 2-Chloro-5-hydroxybenzoic (6) acid was first protected as methyl ester and then
alkylated using tert-butyl (2-bromoethyl)carbamate and K
was obtained after alkaline hydrolysis of the methyl ester with aqueous sodium hydroxide,
followed by cleavage of Boc protective group using CF COOH. The amino group was further
protected as trifluoroacetamide with trifluoroacetic anhydride and pyridine as a base to give
2. Compounds 33a-b were obtained from 32 and the corresponding 2-aminothiophenes using
the same procedure as described for compound 3. Compound 34 was obtained after the
cleavage of the Boc group of 33a with the CF COOH. Final compounds 35 and 36 were
2 3
CO to yield 28. Compound 31
3
3
3
obtained by alkaline hydrolysis of the trifluoroacetamide group of 33b and 34, respectively.
The variously substituted benzyl fragments were attached to compound 34 via two different
strategies. The first method was a nucleophilic substitution, where K
benzyl bromides were used to yield compounds 37a-c; in the second method, compounds
7d-f were synthesized by sodium triacetoxyborohydride-promoted reductive amination of 34
with various benzaldehydes. In the next step, the removal of the trifluoroacetamide group of
7a-f was achieved through alkaline hydrolysis, which led to target compounds 38a-f. Finally,
target compounds 42a-e and 43 were obtained using the same strategy as described for 26a.
2 3
CO and different
3
3
2
.3. Inhibition of MurF. All target compounds were assayed for inhibition of MurF from E.
coli and S. pneumoniae, using the Malachite green assay [22], which detects the
orthophosphate generated during the enzymatic reaction. To avoid possible non-specific
inhibition, all of the compounds were tested in the presence of detergent (0.005% Triton X-
1
14) [23]. Results are presented as residual activities (RAs) of the respective enzyme in the
presence of 250 or 100 µM of each compound. For the most active compounds, IC50 values
were also determined.
7

ACCEPTED MANUSCRIPT
First, a small series of compounds (Table 1, compounds 14-19) was synthesized where the 4-
chloropyrazole moiety was replaced with different aryl and alkyl fragments. None of these
compounds significantly inhibited MurF. However, compounds with aminoethyl (23a) and
aminopropyl substituents (23b) showed modest inhibitory activities against both MurF
enzymes. As expected, the introduction of chlorine ortho to the amide bond was essential for
the activity while the length of alkyl chain had little influence on the potency. Furthermore,
compounds with the guanidino moiety (26a, 27a and 27b) were prepared and their potencies
were 2- to 3-fold higher than potencies for the corresponding amine.
If we compare the compound I with the compounds 23a and 27a, we can notice that the
morpholinosulfonyl fragment and chlorine next to sulfonamide linker are rather important for
good inhibition of MurFSp. This can be seen also in the co-crystal structure, where one of the
sulfonyl oxygens of compound I makes two hydrogen bonds with the protein and the chlorine
forms hydrophobic interactions [18]. These interactions are not present in our new inhibitors,
resulting in their higher IC50 values against MurFSp. However, this interaction pattern is not
necessary for inhibition of MurF , as compound I showed no inhibition of this ortholog.
Ec
Furthermore, to examine the importance of the sulfonamido group, compound 4 was
synthesized. By the comparison of compounds 4 and 23a, we can see that the inhibition is in
the same range as far as MurFSp is concerned (4, IC50 = 219 µM; 23a, IC50 = 170 µM).
However, in the case of MurFEc compound 4 was 2-fold less potent than 23b, which makes
sulfonamido group less favorable for inhibition of this enzyme.
In the next step, compounds with 4-hydroxyphenyl and different substituted benzyl
substituents attached to position 6 of the 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile
or 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile moiety were synthesized (Table 2),
as it is known that substitution of this ring leads to greater potency [11, 12, 18]. First,
compounds with the 4-hydroxyphenyl fragment appended to the saturated ring (33b and 35)
were prepared. The protected compound (33b) showed no inhibition against MurF while the
compound with free amino and hydroxyl groups (35) inhibited both MurF enzymes in the low
micromolar range (IC50 = 58 µM and 81 µM for MurFSp and MurFEc, respectively).
Compounds with different benzyl substituents and trifluoracetamide group (37a-f) showed
modest inhibitory activities against MurF. The most potent inhibitor of MurFSp and MurFEc
within this set was 37f with the p-tetrazole group attached to the benzyl fragment, with IC50
values of 103 and 152 µM, respectively. Furthermore, inhibitors with a free amino group
(38a-f and 49c) were synthesized and most of them showed improved activity against both
8

ACCEPTED MANUSCRIPT
MurF enzymes. Exceptions were 38c and 38f with carboxylic acid and tetrazole on para
position, respectively, which showed significantly lower inhibition of MurFSp activity. The
introduction of substituents to either para or meta position on the benzyl ring led to 2-fold
lower potency against MurFSp compared to 38a (IC50 = 51 µM), while the p-substituents on
the benzyl ring did not influence the activity against MurFEc within this series. Finally,
compounds with the guanidine substituent (42a-e and 43) were prepared. The most potent
inhibitor within this set was compound 43, with the p-hydroxyphenyl group attached to the
saturated ring: IC50 values were 20 and 25 µM against MurFSp and MurFEc, respectively.
Compounds 42a and 42d had similar inhibitory activities against both MurF orthologs as the
corresponding amine precursors, while inhibitors 42b, 42c and 42e showed about 2-fold better
activity against MurFSp than their corresponding amines (38b, 38c and 38e, respectively).
Unfortunately, the introduction of the guanidine moiety does not improve the activity of
inhibitors (42a-e) against MurFEc compared to their respective amine precursors (38a-e and
3
9c).
2
.4. Antibacterial activity. For antibacterial evaluation of selected compounds, typical
representatives of Gram-positive (S. aureus) and Gram-negative bacteria (E. coli) were
chosen (Table 3). In the case of E. coli, compounds were further evaluated against efflux-
deficient strains (E. coli SM1411 and ES100), and those in which the outer membrane had
been artificially permeabilized with polymyxin B (PMBN). Compounds 23a-b, 26a and 27a-
b showed modest antibacterial activities against S. aureus and E. coli, which increased against
the latter species upon deletion of the AcrAB-TolC efflux transporter. Compounds 33a, 34, 36
and 37a-f showed no activity against E. coli and S. aureus; this was expected since none of
these compounds showed potent inhibition of MurFEc and MurFSp. Compound 35 was inactive
against S. aureus and E. coli, although antibacterial activity was observed against PMBN-
permeabilized and/or efflux-deficient strains of E. coli. Compounds 38a-f and 39c showed
modest antibacterial activities against both bacterial species. 38c showed limited inhibition of
both MurF enzymes (IC50 ~ 500 ꢀM), and no antibacterial activity was observed. On the other
hand, compounds 38b, 38d, 38e and 39c demonstrated antibacterial activities against S.
aureus and E. coli strains, and inhibited isolated enzymes more potently (IC50 around or below
1
00 ꢀM). The most potent inhibitor of MurF within this series was 38a, which has 2-fold
lower MIC values against all strains compared to its analogues. Finally, compounds with the
guanidine moiety 42a-e and 43 showed promising antibacterial activities against S. aureus
with MICs ranging between 4 and 32 µg/mL. The latter compounds also demonstrated a
9

ACCEPTED MANUSCRIPT
modest antibacterial activity against E. coli. All compounds with antibacterial activity against
E. coli exhibited increased activity against efflux-deficient strains and strains with increased
membrane permeability.
Additional studies were performed to determine whether the antibacterial activities of the
compounds are the consequence of peptidoglycan biosynthesis inhibition or non-specific
TM
effect. The BacLight assay was used to assess the membrane integrity of S. aureus SH1000
at 4 × MIC values of inhibitors [24]. Unfortunately, all of the tested compounds (38b, 42a,
4
2c, 42e and 43) significantly decreased the membrane integrity thus showing non-specific
mechanism of the antibacterial activity.
3
. CONCLUSION
We have designed, synthesized and evaluated a second generation of 37 new MurF inhibitors
with cyanothiophene scaffold. The major improvement of the present compounds over the
first-generation inhibitors is their reduced lipophilicity and increased solubility, with
conserved enzyme inhibitory potency in micromolar range. The most potent inhibitors have
well balanced inhibitory properties against both MurF from S. pneumonie and MurF from E.
coli (the best inhibitor 42 inhibits MurFSp and MurFEc with IC50 values of 20 µM and 25 µM,
respectively). Furthermore, the most potent compounds within this series demonstrate
antibacterial activities. As these compounds achieve part of their antimicrobial action by
damaging the bacterial cytoplasmic membrane, they are unlikely to be developed as
antibacterial drug candidates until this nonspecific effect is diminished, without
compromising their inhibitory effect on MurF.
4
4
. EXPERIMENTAL SECTION
.1. Inhibition assay. The inhibition of MurF ligases was determined using the Malachite
green assay with slight modifications. The mixture with final volume of 50 µL contained:
S. pneumoniae MurF: 50 mM Hepes, pH 8.0, 50 mM MgCl , 0.005% Triton X-114, 100 µM
2
D-Ala-D-Ala, 50 µM UMtri-L-Lys, 250 µM ATP, purified MurFSp [17], and 250 or 100 µM of
each tested compound dissolved in DMSO.
2
E. coli MurF: 50 mM Hepes, pH 8.0, 50 mM MgCl , 0.005% Triton X-114, 600 µM D-Ala-
D-Ala, 100 µM UMtri-mDAP, 500 µM ATP, purified MurFEc [25], and 250 or 100 µM of
each tested compound dissolved in DMSO.
1
0

ACCEPTED MANUSCRIPT
In both cases, the final concentration of DMSO was 5% (v/v). After incubation for 15 min at
®
3
7 °C, the enzyme reaction was stopped by adding Biomol reagent and the absorbance was
measured at 650 nm. All of the experiments were run in duplicate. 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 concentration for which the RA was 50%.
4
.2. Microbiological evaluation. Minimum inhibitory concentrations (MICs) for selected
compounds were determined by broth microdilution in Mueller-Hinton broth (MHB), against
S. aureus SH1000 [26], and E. coli strains 1411, SM1411 (acrAB derivative of 1411) [27],
AB734 and ES100 (tolC derivative of AB734) [28], according to CLSI guidelines [29]. 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 µg/mL
TM
polymyxin B nonapeptide (PMBN) [30]. The BacLight
assay was used to measure
membrane damage in S. aureus SH1000 induced by compounds at 4 × MIC, compared to a
drug free control [24].
4
.3. Chemistry. All of the chemicals used were obtained from commercial sources (Acros,
Sigma-Aldrich, Alfa Aeser, Apollo Scientific, Fluka and Merck), and were used without
further purification. Solvents were used without purification or drying, unless otherwise
stated. Reactions were monitored using analytical thin-layer chromatography plates (Merck,
silica gel 60 F254, 0.25 mm), and compounds were visualized with ultraviolet light and
ninhydrin or ferric chloride. Silica gel grade 60 (particle size 0.040–0.063 mm, Merck,
1
13
Germany) was used for flash column chromatography. H and C NMR spectra were
recorded on a Bruker AVANCE III 400 MHz spectrometer in acetone-d , CDCl , DMSO-d
CD OD or pyridine-d , with TMS as the internal standard. Mass spectra were obtained with a
6
3
6
,
3
5
VG-Analytical Autospec Q mass spectrometer (Centre for Mass Spectrometry, Institute Jožef
Stefan, Ljubljana). Infrared spectra were recorded on a Perkin-Elmer FTIR 1600
spectrometer. Melting points were determined using a Reichert hot-stage microscope and are
uncorrected. Microanalyses were performed on a 240 C Perkin Elmer elemental analyzer.
Analyses indicated by the symbols of the elements were within 0.4% of the theoretical values.
HPLC analyses were performed on an Agilent Technologies HP 1100 instrument with a
G1365B UV-VIS detector (220 and 254 nm), using a Luna C18 column (4.6 × 250 mm) at a
1
1

ACCEPTED MANUSCRIPT
flow rate of 1 mL/min. The eluent was a mixture of 0.1% CF
acetonitrile (B). The gradient was 10% to 90% B in 19 min.
3
COOH in water (A) and
4
.3.1. 5-(N-(2-((tert-butoxycarbonyl)amino)ethyl)sulfamoyl)-2,4-dichlorobenzoic acid (2). To
a solution of tert-butyl (2-aminoethyl)carbamate (0.450 g, 2.81 mmol) in DCM (15 mL), Et
3
N
(
0.59 mL, 4.21 mmol) was added. The reaction mixture was cooled on an ice bath and 1
0.813 g, 2.81 mmol) was added portion wise. The reaction mixture was stirred for 16 h,
(
diluted with 30 mL of DCM, and washed with 1 M HCl (2 × 40 mL) and brine (1 × 50 mL),
and dried with Na SO . The solvent was evaporated under reduced pressure and the crude
product was purified by flash chromatography.
2
4
1
Yield = 15%; white needles, mp = 192-193 °C; H NMR (400 MHz, CDCl
CH ), 2.90 (q, J = 5.6 Hz, 2H, SO -NH-CH ), 2.96 (q, J = 6.4 Hz,2H, CO-NH-CH
J = 5.6 Hz, 1H, SO -NH), 8.01 (s, 1H, Ar-H), 8.17 (t, J = 6.4 Hz, 1H, O-CO-NH), 8.34 (s, 1H,
3
): δ 1.34 (s, 9H, 3
×
3
2
2
2
), 6.72 (t,
2
13
Ar-H) ppm. C NMR (100 MHz, CD
3
OD): δ 28.77, 28.83, 40.74, 41.08, 41.23, 43.57, 80.26,
8
0.35, 132.19, 133.93, 134.67, 136.52, 137.01, 138.40, 158.46, 158.60, 167.88 ppm. ESI
+
HRMS calcd. for [M+(H)] 555.1447, found 555.1448. IR (KBr) νmax: 3377, 2980, 2935,
-1
1
694, 1586, 1526, 1454, 1385, 1367, 1336, 1277, 1253, 1163, 1086 cm .
4
.3.2. Tert-butyl (2-(2,4-dichloro-5-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)carbamoyl) phenylsulfonamido)ethyl)carbamate (3). To a suspension of 2 (0.305 g, 0.74
mmol) in anhydrous DCM (15 mL), catalytic amounts of DMF and (COCl) (0.190 mL, 2.21
2
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 DCM (20 mL), the
mixture of 2-aminothiophene (0.125 g, 0.70 mmol) and pyridine (0.180 mL, 2.21 mmol) in
DCM (15 mL) was added drop-wise, and the reaction was stirred overnight at room
temperature. The reaction mixture was washed with 1 M HCl (2 × 40 mL), saturated aqueous
3 2 4
NaHCO (1 × 40 mL) and brine (1 × 40 mL), and dried (Na SO ). The solvent was evaporated
under reduced pressure. The crude product was purified by flash chromatography.
1
Yield = 24%; white crystals, mp = 196–198 °C; H NMR (400 MHz, CDCl
3
): δ 1.43 (s, 9H, 3
-CH -CH -CH ),
×
CH
3
), 1.85–1.90 (m, 4H, CH
2
-CH
2
-CH
2
-CH
2
), 2.63 (t, J = 5.6 Hz, 2H, CH
2
2
2
2
2
.70 (t, J = 5.6 Hz, 2H, CH -CH -CH -CH ), 3.06 (q, J = 4.8 Hz, 2H, SO -NH-CH ), 3.23 (q,
2
2
2
2
2
2
J = 4.8 Hz, 2H, SO
2
-NH-CH
2
2 2
-CH ), 4.95 (t, J = 4.8 Hz, 1H, SO -NH), 6.21 (t, J = 4.8 Hz, 1H,
SO
2 2 2
-NH-CH -CH -NH), 7.61 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 10.04 (bs, 1H, NH-CO-Ar)
13
ppm. C NMR (100 MHz, CDCl ): δ 22.10, 23.05, 24.01, 24.06, 28.35, 40.24, 43.44, 79.99,
3
1
2

ACCEPTED MANUSCRIPT
9
1
4.88, 114.59, 129.62, 131.57, 132.12, 132.61, 132.90, 134.42, 136.12, 136.57, 145.83,
-
56.34, 161.00 ppm. ESI HRMS calcd. for [M-(H)] 571.0643, found 571.0644. IR (KBr)
ν
max: 3414, 3077, 2974, 2227, 1683, 1637, 1616, 1579, 1481, 1447, 1400, 1383, 1369, 1347,
-1
1
327, 1286, 1259, 1167, 1105, 1078 cm .
4
.3.3. 5-[(2-aminoethyl)sulfamoyl]-2,4-dichloro-N-(3-cyano-4,5,6,7-tetrahydro-1-
benzothiophen-2-yl)benzamide hydrochloride (4). To a solution of 3 (0.095 g, 0.166 mmol) in
DCM (10 mL), CF COOH (0.6 mL, 8.0 mmol) was added. The reaction mixture was stirred at
3
room temperature for 1 h. The solvent was evaporated under reduced pressure and the product
was dissolved in 1 M HCl in EtOAc (3 mL). EtOAc was removed and the product was dried
in a desiccator overnight.
1
Yield = 100%; pale brown crystals, mp = 204–206 °C; H NMR (400 MHz, MeOD): δ 1.85–
1
5
2
.93 (m, 4H, CH -CH -CH -CH ), 2.62 (t, J = 5.6 Hz, 2H, CH -CH -CH -CH ), 2.72 (t, J =
2
2
2
2
2
2
2
2
.6 Hz, 2H, CH
2
-CH
2
-CH
2
-CH
2
), 3.10 (t, J = 6.0 Hz, 2H, SO
2
-NH-CH
2
), 3.22 (t, J = 6.0 Hz,
+
3
H, SO
2
-NH-CH
2
-CH
2
), 7.91 (s, 1H, Ar-H), 8.33 (s, 1H, Ar-H) ppm, NH
and CONH are
13
exchanged. C NMR (100 MHz, MeOD): δ 23.31, 24.23, 24.91, 25.09, 40.77, 41.13, 97.21,
1
14.61, 131.01, 133.05, 133.15, 134.25, 134.82, 135.66, 137.55, 137.99, 146.59, 164.12 ppm.
+
ESI HRMS calcd. for [M+(H)] 473.0276, found 473.0268. IR (KBr) νmax: 3437, 2928, 2361,
-1
2
2
228, 1671, 1582, 1448, 1334, 1295, 1174, 1084, 1034 cm . HPLC t = 11.977 min (100% at
R
20 nm, 100% at 254 nm).
4
.3.4. General procedure for synthesis of compounds 7 and 8.
To a solution of 5-hydroxybenzoic acid (5) (17.4 mmol) in pyridine (10 mL), acetic anhydride
was added dropwise (6.6 mL, 70.0 mmol). The reaction mixture was stirred at room
temperature for 1 h and was subsequently poured into water (50 mL). The water phase was
acidified to pH 2 and extracted with EtOAc (3 × 40 mL). The combined organic phases were
2 4
washed with 0.1 M HCl (1 × 100 mL), brine (1 × 100 mL), and dried with Na SO . The
solvent was evaporated under reduced pressure and the crude product was used without
further purification.
4
1
8
7
.3.4.1. 3-acetoxybenzoic acid (7). Yield = 89%; white crystals, mp = 127–129 °C (lit. [31]
1
28.7–131.3 °C); H NMR (400 MHz, DMSO-d ): δ 2.28 (s, 3H, CO-CH ), 7.39 (ddd, J =
6
3
1
.1, J
2 3
= 2.4, J = 1.1 Hz, 1H, Ar-H), 7.52–7.56 (m, 1H, Ar-H), 7.65–7.69 (m, 1H, Ar-H),
.80–7.86 (m, 1H, Ar-H), 13.18 (bs, 1H, COOH) ppm.
1
3

ACCEPTED MANUSCRIPT
4
.3.5. General procedure for synthesis of 9 and 10.
To a solution of 7 (14.0 mmol) in anhydrous DCM (30 mL), DMF (catalytic amount) and
COCl) (3.60 mL, 42 mmol) were added drop-wise, and the mixture was stirred for 0.5 h at
(
2
room temperature. The solvent was evaporated to dryness and the residue was dissolved in
DCM (20 mL). A mixture of 2-aminothiophene (2.245 g, 12.6 mmol) and pyridine (3.30 mL,
4
2 mmol) in DCM (30 mL) was added drop-wise, and the reaction was stirred overnight at
room temperature. The reaction mixture was washed with 1 M HCl (40 mL), saturated
aqueous NaHCO (40 mL) and brine (40 mL), and dried (Na SO ). The solvent was
3
2
4
evaporated under reduced pressure. The crude product was purified by flash chromatography
and recrystallized from ethanol.
4
.3.5.1. 3-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamoyl)phenyl acetate (9).
1
Yield = 65%; white crystals, mp = 161–162 °C; H NMR (400 MHz, CDCl ): δ 1.82–1.88 (m,
3
4
7
H, CH
.35 (ddd, J
2.0 Hz, 1H, Ar-H), 7.78 (ddd, J = 7.8, J = 1.7, J = 1.0 Hz, 1H, Ar-H), 8.99 (s, 1H, CO-
2
-CH
2
-CH
2
-CH
2
), 2.34 (s, 3H, OCO-CH
3 2 2 2 2
), 2.59–2.71 (m, 4H, CH -CH -CH -CH ),
1
= 8.1, J
2
= 2.3, J
3
= 1,0 Hz, 1H, Ar-H), 7.54 (t, J = 8.0 Hz, 1H, Ar-H), 7.65 (t, J
=
1
2
3
13
NH) ppm. C NMR (100 MHz, CDCl
3
): δ 21.11, 22.12, 23.09, 23.98, 94.26, 114.54, 121.12,
1
24.93, 126.26, 128.94, 130.07, 131.25, 133.41, 146.53, 150.95, 162.87, 169.30 ppm. ESI
+
HRMS calcd. for [M+(H)] 341.0960, found 341.0966. IR (KBr) νmax: 3237, 3202, 3074,
2
1
8
936, 2842, 2365, 2345, 2223, 2212, 1765, 1671, 1572, 1545, 1458, 1369, 1325, 1291, 1272,
-1
206, 1146, 1094,1010 cm . Anal. Calcd. for C18
H
16
N
2
O
3
S × 0.1 H
2
O: C, 63.18; H, 4.77; N,
.19. Found C, 62.90; H, 4.45; N, 8.05.
4
.3.6. General procedure for synthesis of 11 and 12.
To a suspension of 9 (3.0 g, 8.0 mmol) in methanol (20 mL), K
2
CO
3
(1.69 g, 12.0 mmol) was
added. The reaction mixture was stirred at room temperature for 1 h, and the solvent was
evaporated under reduced pressure. The residue was dissolved in 1 M HCl (40 mL) and
extracted with EtOAc (3 × 40 mL). The combined organic phases were washed with brine,
2 4
dried with Na SO , and the solvent was removed under reduced pressure. The product was
used without further purification.
4
.3.6.1. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-hydroxybenzamide (11).
1
Yield = 87%; small white crystals, mp = 249–250°C; H NMR (400 MHz, acetone-d
6
): δ
), 7.07–7.13
m, 1H, Ar–H), 7.35–7.42 (m, 1H, Ar–H), 7.45–7,54 (m, 2H, 2 × Ar–H), 8.85 (s, 1H, CO–
1
2 2 2 2 2 2 2 2
.80–1.90 (m, 4H, CH -CH -CH -CH ), 2.53–2.71 (m, 4H, CH -CH -CH -CH
(
1
4

ACCEPTED MANUSCRIPT
13
NH), 10.52 (s, 1H, Ar–OH) ppm. C NMR (100 MHz, acetone-d
6
): δ 22.92, 23.84, 24.46,
2
1
3
1
4.67, 96.20, 114.52, 115.62, 119.87, 120.44, 129.47, 130.70, 132.17, 134.96, 147.33, 158.47,
+
65,45 ppm. ESI HRMS calcd. for [M+(H)] 299.0854, found 299.0856. IR (KBr) νmax: 3380,
244, 3077, 2992, 2943, 2922, 2840, 2345, 2222, 1660, 1616, 1587, 1576, 1559, 1490, 1458,
-1
14 2 2
399, 1327, 1306, 1283, 1260, 1216, 1147, 1078 , 1029 cm . Anal. Calcd. for C16H N O S:
C, 64.41; H, 4.73; N, 9.39. Found C, 64.19; H,4.56; N, 9.27.
4
.3.7. General procedure for synthesis of 14-29, 20a-b and 21a-b.
Method A: To a solution of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-
hydroxybenzamide (12) (0.270 g, 0.90 mmol) in DMF (5 mL), K CO or Cs CO (1.35 mmol)
2
3
2
3
was added. After 15 min, the corresponding bromide or chloride (1.35 mmol) was added and
the reaction mixture was stirred for 16 h at 50 °C. The solvent was evaporated under reduced
pressure and the residue was dissolved in EtOAc (40 mL). The organic phase was washed
2 4
with water (2 × 30 mL), 1 M HCl (1 × 30 mL), brine (1 × 30 mL), and dried with Na SO .
The solvent was removed under reduced pressure and the crude product was purified by flash
chromatography.
4
.3.7.1.
3-((4-chlorobenzyl)oxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
1
yl)benzamide (14). Yield = 44%; white crystals, mp = 195–196 °C; H NMR (400 MHz,
CDCl ): δ 1.79–1.90 (m, 4H, CH -CH -CH -CH ), 2.58–2.70 (m, 4H, CH -CH -CH -CH ),
3
2
2
2
2
2
2
2
2
5
2
.11 (s, 2H, O-CH -Ar), 7.17–7.21 (m, 1H, Ar-H), 7.34–7.42 (m, 4H, 4 × Ar-H), 7.42–7.48
13
(
m, 2H, 2 × Ar-H), 7.51–7.55 (m, 1H, Ar-H), 8.85 (s, 1H, CO-NH) ppm. C NMR (100
MHz, CDCl ): δ 22.12, 23.10, 24.00, 69.46, 94.08, 113.64, 114.49, 119.42, 120.07, 128.87,
28.92, 130.26, 131.12, 133.29, 134.06, 134.74, 146.57, 159.04, 163.27 ppm. ESI HRMS
3
1
+
calcd. for [M+(H)] 429.0934, found 423.0939. IR (KBr) νmax: 3446, 3252, 3200, 3076, 2932,
2
1
855, 2366, 2217, 1889, 1670, 1602, 1595, 1573, 1558, 1490, 1470, 1456, 1411, 1399, 1378,
-1
327, 1296, 1279, 1229, 1164, 1153, 1116, 1095, 1082, 1054, 1032, 1014 cm . Anal. Calcd.
for C H ClN O S × 0.3 H O: C, 64.49; H, 4.61; N, 6.54. Found C, 64.29; H, 4.32; N, 6.44.
23
19
2
2
2
Method B: To a solution of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-
hydroxybenzamide (12) (0.250 g, 0.838 mmol) in anhydrous THF (5 mL), the corresponding
alcohol (0.838 mmol) and PPh (0.330 g, 1.25 mmol) were added. The reaction mixture was
3
cooled on ice bath and DIAD (0.25 mL, 1.25 mmol) was added. The reaction was stirred
overnight at room temperature. The solvent was evaporated under reduced pressure and the
crude product was purified by flash chromatography.
1
5

ACCEPTED MANUSCRIPT
4
.3.7.2.
3-((1H-pyrazol-1-yl)methoxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
1
yl)benzamide (16). Yield = 26%; pale yellow crystals, mp = 86–88 °C; H NMR (400 MHz,
CDCl
CH ), 2.56 (t, J = 4.8 Hz, 2H, CH
H, CH-CH-CH-pyrazol), 6.84 (ddd, J
3
): δ 1.69–1.79 (m, 4H, CH
2
-CH
2
-CH
-CH
= 8.0, J
2
-CH
-CH
= 2.8, J
2
), 2.44 (t, J = 5.2 Hz, 2H, CH
), 6.09 (s, 2H, CH -O), 6.37 (t, J = 2.4 Hz,
1
= 0.8 Hz, 1H, Ar-H), 6.92 (dt, J =
2 2 2
-CH -CH -
2
2
-CH
2
2
2
2
1
7
1
2
3
.6, J = 1.6 Hz, 1H, Ar-H), 6.98 (dd, J = 2.4, J = 1.6 Hz, 1H, Ar-H), 7.10 (t, J = 8.0 Hz, 1H,
2
1
2
Ar-H), 7.54 (dd, J
1
= 1.6, J
2
= 0.4 Hz, 1H, CH
A
-CH-CH
B
-pyrazole), 7.62 (bs, 1H, CO-NH),
1
3
7
2
1
3
1
.84 (d, J = 2.4 Hz, 1H, CH
A
-CH-CH
B
-pyrazole) ppm. C NMR (100 MHz, CDCl
3
): δ 21.62,
2.62, 24.12, 24.62, 64.25, 107.30, 110.01, 112.66, 115.25, 118.54, 119.74, 129.46, 131.28,
+
34.42, 134.79, 136.96, 140.30, 148.79, 156.04, 170.75 ppm. ESI HRMS calcd. for [M+(H)]
79.1229, found 379.1220. IR (KBr) νmax: 3418, 2937, 2225, 1668, 1599, 1449, 1376, 1291,
-1
248, 1194, 1128, 1086, 1054 cm . HPLC t = 12.696 min (100% at 220 nm, 100% at 254
R
nm).
4
.3.8. General procedure for synthesis of 22a-b and 23a-b.
To a solution of Boc-protected amine (20a-b and 21a-b) (0.1 mmol) in DCM, CF COOH (3–
3
5
mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 h.
Solvent was evaporated and some products were converted into hydrochloride salts with HCl
in EtOAc.
4
.3.8.1. 3-(2-aminoethoxy)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide
1
(
22a). Yield = 76%; white crystals, mp = 158–160 °C; H NMR (400 MHz, DMSO-d
6
): δ
), 3.25–3.31
2
-O), 7.25–7.31 (m, 1H, Ar-H),
1
.73–1.84 (m, 4H, CH
-CH -O), 4.22–4.30 (m, 2H, NH-CH
.50-7.57 (m, 2H, 2 × Ar–H), 7.58–7.64 (m, 1H, Ar-H), 7.93–8.14 (bs, 3H, NH
): δ 21.67, 22.57, 23.45, 23.59, 38.32, 64.63, 96.29, 114.18,
14.30, 118.81, 121.07, 128.84, 129.82, 131.45, 133.80, 146.16, 157.74, 164.79 ppm. ESI
2
-CH
2
-CH
2
-CH
2
), 2.54–2.67 (m, 4H, CH
2 2 2 2
-CH -CH -CH
(m, 2H, NH-CH
2
2
2
-CH
+
13
7
3
) ppm. C
NMR (100 MHz, DMSO-d
6
1
+
HRMS calcd. for [M+(H)] 342.1276, found 342.1277. IR (KBr) νmax: 3434, 3235, 3195,
3
1
9
080, 2936, 2220, 1669, 1576, 1559, 1511, 467, 1450, 1326, 1297, 1282, 1230, 1203, 1183,
1
128, 1076, 1025 cm- . Anal. Calcd. for C18
.23. Found C, 52.56; H, 4.22; N, 9.13.
19 3 2 3
H N O S × CF COOH: C, 52.74; H, 4.43; N,
4
.3.9. General procedure for synthesis of compounds 24a, 25a-b, 40a-e and 41.
To a solution of amine (22a, 23a-b, 35, 38a-e or 39c) (0.150 mmol) in anhydrous DMF (5
mL), di-Boc-S-methylisothiourea (0.150 mmol), Et N (0.300 mmol) and HgCl (0.150 mmol)
3
2
1
6

ACCEPTED MANUSCRIPT
were added. The reaction mixture was stirred for 2 h at room temperature. The formed
precipitate was filtered off and the solvent was evaporated under reduced pressure. The
residue was dissolved in EtOAc (30 mL), washed with water (3 × 30 mL), saturated NaHCO
30 mL), brine (30 mL), and dried with Na SO . The crude product was purified by flash
chromatography.
.3.9.1. Tert-butyl(tert-butoxycarbonylamino)(2-(3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]
3
(
2
4
4
thiophen-2-ylcarbamoyl)phenoxy)ethylamino)methylencarbamate (24a). Yield = 51%; white
1
crystals, mp = 165–166 °C; H NMR (400 MHz, CDCl
3
): δ 1.53 (dd, J
1
= 6.8, J
2
= 1.2 Hz,
1
6H, 2 × COO-(CH ) ), 1.81–1.94 (m, 4H, CH -CH -CH -CH ), 2.61–2.74 (m, 4H, CH -CH -
3
3
2
2
2
2
2
2
CH
2
-CH
2
), 3.90 (q, J = 5.3 Hz, 2H, NH-CH
2
2
-CH -O), 4.20 (t, J = 5.2 Hz, 2H, NH-CH
2
-CH
2
-
O), 7.18–7.27 (m, 1H, Ar-H), 7.41–7.50 (m, 2H, 2 × Ar-H), 7.50–7.56 (m, 1H, Ar-H), 8.78 (t,
13
J = 5.3 Hz, 1H, C-NH-CH ), 8.90 (s, 1H, CO-NH-C), 11.20 (s, 1H, C-NH-Boc) ppm.
C
2
NMR (100 MHz, acetone-d
6
): δ 14.37, 22.92, 23.30, 23.83, 24.46, 24.68, 28.14, 28.47, 32.32,
4
1
0.70, 67.21, 79.02, 83.84, 96.29, 114.50, 114.93, 119.77, 121.46, 129.56, 130.78, 132.27,
+
35.00, 159.79 ppm. ESI HRMS calcd. for [M+(H)] 584.2543, found 584.2545. IR (KBr)
max: 3341, 3285, 3080, 2976, 2934, 2362, 2344, 2219, 1741, 1656, 1623, 1576, 1546, 1493,
ν
-1
1
459, 1434, 1415, 1397, 1362, 1330, 1300, 1276, 1223, 1146, 1086, 1062, 1046, 1027 cm .
S × 0.9 H O: C, 58.06; H, 6.52; N, 11.67. Found C, 58.33; H,
Anal. Calcd. for C29
37
H N
5
O
6
2
6
.44; N, 11.27.
4
.3.10. General procedure for synthesis of compounds 26a, 27a-b, 42a-e and 43.
To a solution of Boc-protected guanidine (24a, 25a-b, 40a-e and 41) (0.095 mmol) in DCM
5 mL), CF COOH (0.210 mL, 2.84 mmol) was added, and the mixture was stirred at room
(
3
temperature for 2 h. The solvent was evaporated under reduced pressure and 1 M HCl in
EtOAc (3 ml) was added to the residue. The formed precipitate was collected by filtration.
4
.3.10.1.
N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(2-guanidinoethoxy)
1
benzamide (26a). Yield = 86%; white crystals, mp = 179–181 °C; H NMR (400 MHz,
acetone-d
CH ), 2.68–2.72 (m, 2H, CH
5.2 Hz, 2H, NH-CH -CH -O), 7.25–7,29 (m, 1H, Ar-H), 7.48–7.53 (m, 1H, Ar-H), 7.62–
6
): δ 1.84–1.89 (m, 4H, CH
2
-CH
2
-CH
2
-CH
2 2 2 2
), 2.57–2.61 (m, 2H, CH -CH -CH -
2
2
-CH -CH
2
2
-CH
2
), 3.78–3.83 (m, 2H, NH-CH
2
-CH -O), 4.33 (t, J
2
=
2
2
+
7
3
.69 (m, 2H, 2 × Ar-H), 7.79 (bs, 3H, C–NH ), 8.68 (bs, 1H, NH), 10.72 (bs, 1H, NH) ppm.
1
3
C NMR (100 MHz, MeOD): δ 22.91, 23.82, 24.47, 24.68, 41.68, 41.79, 67.71, 96.50,
1
14.50, 114.94, 119.87, 121.62, 129.64, 130.74, 132.31, 135.02, 147.20, 159.52, 165.34 ppm.
+
ESI HRMS calcd. for [M+(H)] 384.1494, found 384.1493. IR (KBr) νmax: 3425, 3366, 3179,
1
7

ACCEPTED MANUSCRIPT
2
1
936, 2855, 2363, 2344, 2225, 1674, 1579, 1558, 1454, 1397, 1326, 1304, 1272, 1206, 1186,
-
1
134, 1063, 1028 cm . HPLC t
R
= 11,836 min, (98.2% at 220 nm, 100% at 254 nm).
4
.3.11. Methyl 2-chloro-5-hydroxybenzoate (28). To a cooled (0°C) solution of 2-chloro-5-
hydroxybenzoic acid (6) (4 g, 23.6 mmol) in methanol (50 mL), thionyl chloride (2.5 mL, 35
mmol) was added drop-wise. The reaction mixture was stirred at room temperature for 3 h.
The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc
3
(100 ml). The organic phase was washed with saturated NaHCO (3 × 50 mL), water (50 mL)
and brine (50 mL), dried with Na
2
SO
4
and evaporated under reduced pressure.
1
Yield = 99%; white crystals, mp = 96–97 °C; H NMR (400 MHz, CDCl ): δ 3.91 (s, 3H, O-
3
CH
3
1 2
), 6.91 (dd, J = 8.7, J = 3.1 Hz, 1H, Ar-H), 7.27 (d, J = 8.7 Hz, 1H, Ar-H), 7.31 (d, J =
1
3
3
1
.1 Hz, 1H, Ar-H). C NMR (100 MHz, CDCl
3
): δ 52.79, 118.15, 120.33, 124.67, 130.25,
+
32.14, 154.51, 166.77 ppm. ESI HRMS calcd. for [M+(H)] 187.0162, found 187.0155.
R
HPLC t = 10.618 min (100% at 220 nm, 100% at 254 nm).
4
.3.12. Methyl 5-(2-((tert-butoxycarbonyl)amino)ethoxy)-2-chlorobenzoate (29). To a solution
of 28 (1.00 g, 5.36 mmol) in DMF (15 mL), K CO (1.11 g, 8.04 mmol) was added. After 10
2
3
min at room temperature, tert-butyl 2-bromoethylcarbamate (1.56 g, 6.97 mmol) was added
and the reaction mixture was stirred at 80 °C for 16 h. DMF was evaporated, the crude residue
was dissolved in EtOAc (50 mL), washed with water (2 × 30 mL), 1 M NaOH (2 × 30 mL),
water (1 × 50 mL), brine (10 mL), and dried with Na
flash chromatography.
2 4
SO . The crude residue was purified by
1
Yield = 73%; colourless oil; H NMR (400 MHz, CDCl
3
): δ 1.47 (s, 9H, 3 × CH
), 4.05 (t, J = 4.8 Hz, 2H, O-CH
= 2.8 Hz, 1H, Ar-H), 7.36 (s, 1H, Ar-H),
.37 (d, J = 6.0 Hz, 1H, Ar-H) ppm. C NMR (100 MHz, CDCl ): δ 28.38, 39.95, 52.53,
3
), 3.56 (q, J
=
5.6 Hz, 2H, O-CH
2
-CH
2
-NH), 3.95 (s, 3H, O-CH
= 8.8, J
3
2
-CH -
2
NH), 4.98 (bs, 1H, NH-CO), 6.98 (dd, J
1
2
1
3
7
6
3
7.71, 79.72, 116.72, 119.22, 125.39, 130.56, 131.95, 155.83, 156.88, 165.87 ppm. ESI
+
HRMS calcd. for [M+(H)] 330.1108, found 330.1103.
4
.3.13. 5-(2-(tert-butoxycarbonylamino)ethoxy)-2-chlorobenzoic acid (30). To a solution of
9 (1.00 g, 3.03 mmol) in a mixture of dioxane and water (1/1, 30 mL), NaOH (0.24 g, 6.00
2
mmol) was added. The reaction mixture was stirred at room temperature for 4 h. Dioxane was
evaporated under reduced pressure and the remaining water was acidified with 1 M HCl to pH
3
. The water phase was extracted with EtOAc (3 × 30 mL). The combined organic phases
1
8

ACCEPTED MANUSCRIPT
were washed with brine (1 × 50 mL), dried with Na
pressure.
2 4
SO and evaporated under reduced
1
Yield = 98%; white crystals, mp = 117–119 °C; H NMR (400 MHz, CDCl ): δ 1.48 (s, 9H, 3
3
×
CH
.03 (bs, 1H, NH-CO), 7.03 (dd, J
H), 7.52 (d, J = 2.8 Hz, 1H, Ar-H), 7.64 (bs, 1H, COOH) ppm. C NMR (100 MHz, acetone-
3
), 3.58 (q, J = 5.6 Hz, 2H, O-CH
2
-CH
2 2 2
-NH), 4.07 (t, J = 4.8 Hz, 2H, O-CH -CH -NH),
5
1
= 8.0, J
2
= 2.4 Hz, 1H, Ar-H), 7.40 (d, J = 8.8 Hz, 1H, Ar-
13
d
1
3
1
6
): δ 28.63, 40.51, 68.35, 78.98, 117.93, 119.83, 124.93, 132.34, 132.64, 156.77, 158.37,
+
66.56 ppm. ESI HRMS m/z calcd. for [M-(H)] 314.0795, found 314.0791. IR (KBr) νmax
:
403, 2981, 2576, 1717, 1669, 1605, 1572, 1527, 1478, 1464, 1430, 1394, 1366, 1275, 1252,
-1
207, 1162, 1110, 1077, 1053, 1037 cm .
4
.3.14. 2-(3-carboxy-4-chlorophenoxy)ethan aminium 2,2,2-trifluoroacetate (31). To a
solution of 30 (1.00 g, 3.17 mmol) in DCM, CF COOH (7.0 mL, 91.4 mmol) was added and
3
stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and
the crude product was used without further purification.
1
Yield = 100%; mp = 147–149 °C; H NMR (400 MHz, acetone-d ): δ 4.29 (t, J = 5.6 Hz, 2H,
6
O-CH
2
-CH
2 2 2 2 2 1 2
-NH ), 4.51 (t, J = 4.8 Hz, 2H, O-CH -CH -NH ), 7.17 (dd, J = 8.8, J = 3.2 Hz,
1
H, Ar-H), 7.42 (d, J = 3.2 Hz, 1H, Ar-H), 7.43 (d , J = 8.8 Hz, 1H, Ar-H) ppm.
4
.3.15. 2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzoic acid (32). To a suspension of
1 (1.00 g, 4.64 mmol) in anhydrous DCM (15 mL), anhydrous pyridine (1.12 mL, 13.9
3
3 2
mmol) was added and the reaction mixture was cooled to 0°C. (CF CO) O (0.72 mL, 5.10
mmol) was added drop-wise and the mixture was stirred for 2 h at 0 °C then allowed to warm
to room temperature. The reaction mixture was diluted with DCM (30 mL), washed with 1 M
HCl (2 × 30 mL), water (30 mL), and saturated NaHCO
water phases were acidified with concentrated HCl to pH = 1 and extracted with EtOAc (3 ×
0 mL). The combined organic phases were washed with water (50 mL), brine (50 mL) and
dried with Na SO . The solvent was evaporated under reduced pressure.
3
(3 × 30 ml). The combined alkaline
5
2
4
1
Yield = 76%; white crystals, mp = 141–143°C; H NMR (400 MHz, acetone-d
5.6 Hz, 2H, O-CH -CH -NH), 4.28 (t, J = 5.2 Hz, 2H, O-CH -CH -NH), 7.15 (dd, J
J2 = 3.2 Hz, 1H, Ar-H), 7.43 (d, J = 2.8 Hz, 1H, Ar-H), 7.45 (d, J = 8.4 Hz, 1H, Ar-H), 8.79
6
): δ 3.79 (q, J
=
2
2
2
2
1
= 8.8,
1
3
4
(
6 2
bs, 1H, NH-CO) ppm. C NMR (100 MHz, acetone-d ): δ 39.98 (d, JF,C = 12 Hz, CH -NH),
1
6
7.01, 117.08 (q, JF,C = 285.8 Hz, CF
3
), 117.89, 119.86, 125.21, 132.46, 132.70, 157.93 (qd,
1
9

ACCEPTED MANUSCRIPT
2
2
J
3
F,C = 36.5 Hz, JF,C = 8 Hz, CO-CF ), 158.07, 166.52 ppm. ESI HRMS m/z calcd. for [M-
+
(
H)] 310.0094, found 310.0088. IR (KBr) νmax: 3414, 3316, 3110, 2927, 2633, 1710, 1597,
1
1
560, 1486, 1469, 1432, 1414, 1384, 1374, 1349, 1321, 1277, 1260, 1235, 1206, 1182, 1116,
-
1
061, 1052, 1039 cm . HPLC t
R
= 10.996 min (100% at 220 nm, 100% at 254 nm).
4
.3.16. General procedure for synthesis of compounds 33a and 33b.
To a solution of 32 (2.15 g, 6.88 mmol) in anhydrous DCM (30 mL), DMF (catalytic amount)
and (COCl) (1.77 mL, 20.6 mmol) were added drop-wise, and the mixture was stirred for 0.5
2
h at room temperature. The solvent was evaporated to dryness and the residue was dissolved
in DCM (20 mL). A mixture of the corresponding 2-aminothiophene (1.73 g, 6.19 mmol) and
pyridine (1.63 mL, 20.6 mmol) in DCM (20 mL) was added drop-wise, and the reaction was
stirred overnight at room temperature. The reaction mixture was washed with 1 M HCl (30
mL), saturated aqueous NaHCO (30 mL) and brine (30 mL), and dried (Na SO ). The solvent
3
2
4
was evaporated under reduced pressure. The crude product was purified by flash
chromatography.
4
4
.3.16.1. Tert-butyl 2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano-
,5-dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (33a). Yield = 79%; orange crystals, mp
1
=
89–91 °C; H NMR (400 MHz, CDCl
3
): δ 1.46 (s, 9H, 3 × CH
-CH -N), 3.78 (q, J = 5.4 Hz, 2H, O-CH
-NH), 4.51 (s, 2H, CH -N-CO), 6.98 (bs, 1H, NH-
2
= 3.1 Hz, 1H, Ar-H), 7.37 (d, J = 8.7 Hz, 1H, Ar-H), 7.48 (d, J
3
), 2.67 (t, J = 5.6 Hz,
2
H,CH
NH), 4.13 (t, J = 5.1 Hz, 2H, O-CH
CO-CF ), 7.01 (dd, J = 8.9, J
3
3.1 Hz, 1H, Ar-H), 9.84 (s, 1H, NH-CO-thiophene) ppm. C NMR (100 MHz, CDCl ): δ
2
-CH
2
-N), 3.68 (t, J = 5.6 Hz, 2H, CH
2
2
2
2
-CH -
2
-CH
2
2
3
1
13
=
2
1
1
3
1
4.07, 28.39, 31.45, 36.56, 39.18, 66.27, 80.65, 94.18, 113.65, 114.32, 116.95, 117.18,
2
19.95, 122.99, 131.64, 131.98, 147.04, 154.54, 157.21, 157.57 (q, JC,F = 37.4 Hz), 161.82,
+
62.62 ppm. ESI HRMS m/z calcd. for [M+(H)] 573.1186, found 573.1201. IR (KBr) νmax
261, 2982, 2935, 2217, 1666, 1545, 1459, 1416, 1367, 1287, 1237, 1210, 1153, 1118, 1063,
-1
038, 1005 cm . HPLC t = 16.299 min (100% at 220 nm, 100% at 254 nm).
R
4
.3.17. 2-(2-chloro-5-(2-(2,2,2-trifluoroacetamido)ethoxy)benzamido)-3-cyano-4,5,6,7-tetra-
hydrothieno[2,3-c]pyridin-6-ium trifluoroactetate (34). To a solution of 33a (2.13 g, 3.72
mmol) in DCM (30 mL), CF COOH (8.60 mL, 112 mmol) was added and stirred at room
3
temperature for 2 h. The solvent was evaporated under reduced pressure and the residue was
dried in a desiccator overnight.
2
0

ACCEPTED MANUSCRIPT
1
Yield = 96%; orange crystals, mp = 90–92 °C; H NMR (400 MHz, CDCl
3
): δ 3.07 (t, J = 6.2
Hz, 2H, CH
CH -NH), 4.16 (t, J = 5.1 Hz, 2H, O-CH
Hz, 1H, NH-CO-CF ), 7.07 (dd, J = 8.8, J
2
-CH
2
-N), 3.60 (t, J = 6.2 Hz, 2H, CH
2
-CH
2
2
-N), 3.81 (q, J = 5.1 Hz, 2H, O-CH -
+
2
2
-CH
2
-NH), 4.39 (s, 2H, CH
2
-NH
2
), 6.71 (t, J = 5.7
3
1
2
= 3.1 Hz, 1H, Ar-H), 7.44 (d, J = 8.8 Hz, 1H, Ar-
13
H), 7.56 (d, J = 3.1 Hz, 1H, Ar-H), 10.00 (s, 1H, NH-CO-thiophene) ppm. C NMR (100
MHz, MeOD): δ 22.26, 27.78, 40.29, 42.35, 42.76, 67.46, 95.33, 113.83, 116.08, 116.57,
1
18.94, 119.78, 121.38, 124.03, 130.43, 132.32, 135.79, 149.83, 158.83, 161.78 (q, J = 36.8
+
Hz), 166.53 ppm. ESI HRMS m/z calcd. for [M+(H)] 473.0662, found 473.0668. IR (KBr)
-1
νmax 2992, 2842, 2221, 1711, 1667, 1547, 1462, 1419, 1292, 1135, 1067, 1039 cm . HPLC t
R
=
9.914 min (96.6% at 220 nm, 97.1% at 254 nm).
4
.3.18. General procedure for synthesis of compounds 35, 36 and 38a-f.
To a solution of 33b, 34 or 37a-f (0.17 mmol) in methanol (2 mL), 1 M NaOH (2 mL) was
added. The reaction mixture was stirred overnight. The methanol was evaporated and the
residue was diluted with water (20 mL). The water phase was extracted with EtOAc (3 × 20
mL) and the combined organic phases were washed with water (30 mL), brine (30 mL) and
dried with Na
2 4
SO . The solvent was evaporated under reduced pressure and the residue was
purified by flash chromatography.
4
.3.18.1.
5-(2-aminoethoxy)-2-chloro-N-(3-cyano-6-(4-hydroxyphenyl)-4,5,6,7-tetrahydro
1
benzo[b]thiophen-2-yl)benzamide (35). Yield = 100%; white crystals, mp = 162–164 °C; H
NMR (400 MHz, DMSO-d
CH -CH ), 2.61–2.68 (m, 3H, CH
4.8 Hz, 2H, O-CH -CH -NH ), 4.22 (t, J = 4.8 Hz, 2H, O-CH
Hz, 2H, Ar-H), 7.09–7.13 (m, , 3H, 3 × Ar-H), 7.24 (d, J = 3.2 Hz, 1H, Ar-H), 7.45 (d, J = 8.8
6
): δ 1.81–1.90 (m, 1H, CH-CH
-CH-CH -CH ), 2.80–2.92 (m, 2H, CH
-CH -NH ), 6.72 (d, J = 8.4
A
H
B
-CH
2
), 1.97–2.02 (m, 1H, CH-
A
H
B
2
2
2
2
2
-CH), 3.22 (t, J
=
2
2
2
2
2
2
1
3
Hz, 1H, Ar-H), 9.25 (bs, 1H, NH-CO) ppm. C NMR (100 MHz, pyridine-d
6
): δ 26.76,
3
1
2.06, 34.27, 41.92, 42.18, 69.31, 97.49, 116.97, 117.96, 118.38, 120.29, 125.17, 130.44,
30.72, 133.03, 133.68, 138.26, 138.39, 149.53, 159.40, 159.57, 167.05 ppm. ESI HRMS
+
calcd. for [M+(H)] 468.1149, found 468.1143. IR (KBr) νmax: 3549, 3475, 3413, 3237, 3084,
3
1
012, 2922, 2224, 2032, 1677, 1656, 1638, 1617, 1574, 1556, 1514, 1460, 1408, 1384, 1317,
-
1
296, 1234, 1201, 1178, 1156, 1139, 1074, 1017 cm . HPLC t = 11.363 min (100% at 220
R
nm, 100% at 254 nm).
4
.3.19. General procedure for synthesis of compounds 37a-f.
Method A: To a solution of 34 (0.30 g, 0.511 mmol) in DMF (3 mL), Et N (1.5 mmol) was
3
added. After 10 min of stirring at room temperature, the corresponding benzyl bromide (0.766
2
1

ACCEPTED MANUSCRIPT
mmol) was added. The reaction mixture was stirred overnight at room temperature. DMF was
then evaporated, the crude residue was dissolved in EtOAc (20 mL) and washed with water (3
×
10 mL), brine (10 mL) and dried with Na
2 4
SO . The crude product was purified by flash
chromatography.
Method B: To a solution of 34 (0.50 g, 0.852 mmol) in THF (5 mL), the corresponding
benzaldehyde (1.28 mmol) and Na(OAc) BH (0.45 g, 2.13 mmol) were added, and the
3
reaction mixture was stirred overnight at room temperature. To quench the reaction, saturated
3
aqueous NaHCO (10 mL) was used, and the water phase was extracted with EtOAc (3 × 20
mL). The combined organic phases were washed with brine (30 mL) and dried with Na SO .
2
4
The crude residue was purified by flash chromatography.
.3.19.1. N-(6-benzyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-2-chloro-5-(2-
4
(2,2,2-trifluoroacetamido)ethoxy)benzamide (37a). Yield = 35%; pale brown crystals, mp =
1
7
1–73 °C; H NMR (400 MHz, acetone-d
6
): δ 2.68 (t, J = 5.6 Hz, 2H, CH
-N), 3.61 (s, 2H, CH -N), 3.76–3.79 (m, 4H, N-CH
NH ), 4.26 (t, J = 5.1 Hz, 2H, O-CH -CH -NH), 7.11 (dd, J = 8.9, J = 3.1 Hz, 1H, Ar-H),
2
-CH
2
-N), 2.85 (t, J
=
5.6 Hz, 2H, CH
2
-CH
2
2
2
-Ar+ O-CH
2
-CH
2
-
2
2
1
2
7
8
.25–7.29 (m, 2H, 2 × Ar-H), 7.33–7.37 (m, 2H, 2 × Ar-H), 7.39-7.44 (m, 3H, 3 × Ar-H),
13
.79 (bs, 1H, NH-CO-CF
): δ 24.98, 40.04, 50.15, 51.41, 62.04, 67.18, 95.27, 114.08, 115.69, 116.38,
18.54, 119.28, 132.30, 127.55, 128.05, 129.21, 129.77, 131.14, 131.85, 135.97, 139.53,
3
), 11.01 (s, 1H, NH-CO-thiophene) ppm. C NMR (100 MHz,
acetone-d
6
1
1
+
47.26, 158.29, 164.51 ppm. ESI HRMS m/z calcd. for [M+(H)] 563.1132, found 563.1137.
IR νmax 3319, 3081, 2923, 2807, 2214, 1714, 1667, 1545, 1456, 1405, 1366, 1288, 1209, 1154,
-1
1
R
057, 1036, 1007 cm . HPLC t = 11.907 min (100% at 220 nm, 100% at 254 nm).
4
.4. Molecular docking studies
A docking study was performed using the crystal structure of MurF enzyme from S.
pneumoniae (PDB code: 3zm5) and OEDocking software (Release 3.0.1, OpenEye Scientific
Software, Inc.).
4
.4.1. Ligand preparation
The molecules were built with ChemBioDraw Ultra 12.0 (CambridgeSoft). The ligand
geometries were optimized with ChemBio 3D Ultra 12.0 (CambridgeSoft) using MM2 force
field until a minimum 0.100 root mean square (RMS) gradient was reached. The optimized
structure was refined with GAMESS interface using the semi-empirical AM1 method, QA
2
2

ACCEPTED MANUSCRIPT
optimization algorithm and Gasteiger Hückel charges for all atoms for 100 steps. Amines and
guanidines were kept in their ionized state, corresponding to pH 7.4. FRED requires a set of
input conformers for each ligand, which were generated with OMEGA 2.4.6, with maximum
number of conformations set to 1000, and using the RMS cut-off value (Root Mean Square
(RMS) cartesian distance below which two conformers are duplicates) of 0.3 Å. All the other
options were left as default values.
4
.4.2. Receptor preparation & docking protocol
The crystal structure of MurF enzyme from S. pneumoniae in complex with the ligand (PDB
code: 3zm5) was taken as a starting-point. The ligand (2,4-dichloro-N-[3-cyano-6-[(4-
hydroxyphenyl)methyl]-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium-2-yl]-5-
morpholinosulfonyl-benzamide) was taken as a reference structure and a grid box including
all active-site atoms (including hydrogens) with a volume of 32562 Å, dimensions of 36.22 Å
×
36.33 Å × 24.67 Å, and outer contour of 2953 Å, was created around it as a “docking
receptor” using Make Receptor 3.0.1.
The docking software FRED (OEDocking 3.0.1) was used for docking studies with the default
settings, except for dock resolution, which was set to “high”, and number of poses, which was
set to 50. The proposed 10 binding modes with the highest rank of the docked inhibitors were
evaluated using final score and root mean square deviation (RMSD) as a tool to explore
relative structural differences between proposed binding modes. The graphical representations
of the proposed binding positions of all the molecules were obtained using Accelrys
Discovery Studio 2.5 (Accelrys Software Inc.).
4
.4.3. Validation of the docking protocol
The docking procedure should be able to correctly predict the binding poses of the molecules
in the PDB database i.e., the crystal structures of ligands in complex with proteins. We have
attempted to reproduce the pose of the ligand as seen in its X-ray complex with the target
MurF enzyme from S. pneumoniae (PDB code: 3zm5). The top 10 docking poses of the
docked ligand were within 1.5 Å RMSD of the ligand crystal structure. The predicted top
score pose was the one that best fitted the crystal structure of the ligand, overlapping it almost
completely, which offers the proof of the protocol validation (Fig. 3).
ACKNOWLEDGEMENTS
2
3

ACCEPTED MANUSCRIPT
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
an L1-4039 grant to SG, both from the Slovenian Research Agency.
2
4

ACCEPTED MANUSCRIPT
REFERENCES
[
[
[
[
[
[
[
[
[
[
[
1] A.J. Alanis, Resistance to antibiotics: are we in the post-antibiotic era?, Arch. Med. Res.
6 (2005) 697–705.
3
2] P.C. Appelbaum, 2012 and beyond: potential for the start of a second pre-antibiotic era?,
J. Antimicrob. Chemoth. 67 (2012) 2062–2068.
3] I. Chopra, The 2012 Garrod Lecture: Discovery of antibacterial drugs in the 21st century,
J. Antimicrob. Chemoth., (2012).
4] T.D.H. Bugg, D. Braddick, C.G. Dowson, D.I. Roper, Bacterial cell wall assembly: still an
attractive antibacterial target, Trends Biotechnol. 29 (2011) 167–173.
5] A. Gautam, R. Vyas, R. Tewari, Peptidoglycan biosynthesis machinery: A rich source of
drug targets, Cr. Rev. in Biotechn. 31 (2011) 295–336.
6] D.W. Green, The bacterial cell wall as a source of antibacterial targets, Expert Opin. Ther.
Tar. 6 (2002) 1–20.
7] H. Barreteau, A. Kovač, A. Boniface, M. Sova, S. Gobec, D. Blanot, Cytoplasmic steps of
peptidoglycan biosynthesis, FEMS Microbiol. Rev. 32 (2008) 168–207.
8] A. El Zoeiby, F. Sanschagrin, R.C. Levesque, Structure and function of the Mur enzymes:
development of novel inhibitors, Mol. Microbiol. 47 (2003) 1–12.
9] C.A. Smith, Structure, function and dynamics in the mur family of bacterial cell wall
ligases, J. Mol. Biol. 362 (2006) 640–655.
10] D. J. Miller, S. M. Hammond, D. Anderluzzi, T. D. H. Bugg, Aminoalkylphosphinate
inhibitors of D-Ala-D-Ala adding enzyme, J. Chem. Soc., Perk. T. 1. (1998) 131–142.
11] Y.G. Gu, A.S. Florjancic, R.F. Clark, T. Zhang, C.S. Cooper, D.D. Anderson, C.G.
Lerner, J.O. McCall, Y. Cai, C.L. Black-Schaefer, G.F. Stamper, P.J. Hajduk, B.A.
Beutel, Structure–activity relationships of novel potent MurF inhibitors, Bioorg. Med.
Chem. Lett. 14 (2004) 267–270.
[
[
12] G.F. Stamper, K.L. Longenecker, E.H. Fry, C.G. Jakob, A.S. Florjancic, Y.G. Gu, D.D.
Anderson, C.S. Cooper, T. Zhang, R.F. Clark, Y. Cia, C.L. Black-Schaefer, J. Owen
McCall, C.G. Lerner, P.J. Hajduk, B.A. Beutel, V.S. Stoll, Structure-based optimization
of MurF inhibitors, Chem. Biol. Drug Des. 67 (2006) 58–65.
13] E.Z. Baum, S.M. Crespo-Carbone, D. Abbanat, B. Foleno, A. Maden, R. Goldschmidt,
K. Bush, Utility of muropeptide ligase for identification of inhibitors of the cell wall
biosynthesis enzyme MurF, Antimicrob. Agents Ch. 50 (2006) 230–236.
2
5

ACCEPTED MANUSCRIPT
[
[
[
14] E.Z. Baum, S.M. Crespo-Carbone, A. Klinger, B.D. Foleno, I. Turchi, M. Macielag, K.
Bush, A MurF inhibitor that disrupts cell wall biosynthesis in Escherichia coli,
Antimicrob. Agents Ch. 51 (2007) 4420–4426.
15] E.Z. Baum, S.M. Crespo-Carbone, B.D. Foleno, L.D. Simon, J. Guillemont, M.
Macielag, K. Bush, MurF inhibitors with antibacterial activity: Effect on muropeptide
levels, Antimicrob. Agents Ch. 53 (2009) 3240–3247.
16] S. Turk, A. Kovač, A. Boniface, J.M. Bostock, I. Chopra, D. Blanot, S. Gobec,
Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD
and MurF by structure-based virtual screening, Bioorg. Med. Chem. 17 (2009) 1884–
1
889.
[
[
17] S. Turk, M. Hrast, I. Sosič, H. Barreteau, D. Mengin-Lecreulx, D. Blanot, S. Gobec,
Biochemical characterization of MurF from Streptococcus pneumoniae and the
identification of a new MurF inhibitor through ligand-based virtual screening, Acta
Chim. Slov. 60 (2013) 294–299.
18] M. Hrast, S. Turk, I. Sosič, D. Knez, C.P. Randall, H. Barreteau, C. Contreras-Martel, A.
Dessen, A.J. O’Neill, D. Mengin-Lecreulx, D. Blanot, S. Gobec, Structure-activity
relationships of new cyanothiophene inhibitors of the essential peptidoglycan
biosynthesis enzyme MurF, Eur. J. Med. Chem. 66 (2013) 32–45.
[
[
[
[
[
[
19] P.D. Leeson, A.M. Davis, Time-related differences in the physical property profiles of
oral drugs. J. Med. Chem. 47 (2004) 6338–6348.
20] R. O’Shea, H.E. Moser, Physicochemical Properties of Antibacterial Compounds:
Implication for Drug Discovery, J. Med. Chem. 51 (2008) 2871–2878.
21] I. Dvoretzky, G.H. Richter, Formaldehyde condensation in the pyrazole series, J. Org.
Chem. 15 (1950) 1285–1288.
22] P.A. Lanzetta, L.J. Alvarez, P.S. Reinach, O.A. Candia, An improved assay for nanomole
amounts of inorganic phosphate, Anal. Biochem. 100 (1979) 95–97.
23] S.L. McGovern, B.T. Helfand, B. Feng, B.K. Shoichet, A specific mechanism of
nonspecific inhibition, J. Med. Chem. 46 (2003) 4265–4272.
24] J.K. Hobbs, K. Miller, A.J. O’Neill et al. Consequences of daptomycin-mediated
membrane damage in Staphylococcus aureus. J. Antimicrob. Chemother. 62 (2008)
1
003–1008.
[
25] S. Dementin, A. Bouhss, G. Auger, C. Parquet, D. Mengin-Lecreulx, O. Dideberg, J. van
Heijenoort, D. Blanot, Evidence of a functional requirement for a carbamoylated lysine
2
6

ACCEPTED MANUSCRIPT
residue in MurD, MurE and MurF synthetases as established by chemical rescue
experiments, Eur. J. Biochem. 268 (2001) 5800–5807.
[
[
[
[
[
26] A.J. O'Neill, Staphylococcus aureus SH1000 and 8325-4: comparative genome
sequences of key laboratory strains in staphylococcal research. Lett. Appl. Microbiol. 51
(2010) 358–361.
27] A.J. O'Neill, J.M. Bostock, A.M. Moita, I. Chopra, Antimicrobial activity and
mechanisms of resistance to cephalosporin P1, an antibiotic related to fusidic acid. J.
Antimicrob. Chemother. 50 (2002) 839–848.
28] E. Shapiro, F. Baneyx, Stress-based identification and classification of antibacterial
agents: second-generation Escherichia coli reporter strains and optimization of
detection, Antimicrob. Agents Chemother. 46 (2002) 2490–2497.
29] P.A. Wayne, Methods for dilution antimicrobial susceptibility tests for bacteria that grow
aerobically; approved standard-Ninth Edition. Clinical and Laboratory Standards
Institute (2012) CLSI Document M07–A9.
30] R.A. Dixon, I. Chopra, Leakage of periplasmic proteins from Escherichia coli mediated
by polymyxin B nonapeptide, Antimicrob. Agents Chemother. 29 (1986) 781–788.
[
31] E.R. Marshall, J.A. Kuck, R.C. Elderfield, Studies on lactones related to the cardiac
α,β
aglycones. X. Synthesis of simple hydroxylated β-substituted ꢁ -butenolides, J. Org.
Chem. 7 (1942) 444–456.
2
7

ACCEPTED MANUSCRIPT
List of table and figure captions:
Table 1. Inhibitory activities of compounds 14-19, 22a, 22b, 23a, 23b, 26a, 27a and 27b
against MurF from S. pneumoniae and E. coli.
Table 2. Inhibitory activities of compounds 33-38f, 39c, 42a-e and 43 against MurF from S.
pneumoniae and E. coli.
Table 3. Antibacterial activities of compounds 23a-b, 26a, 27a-b, 33a, 34-38f, 39c, 42a-e
and 43 against S. aureus and E. coli bacterial strains.
Figure 1. Two-step design of new cyanothiophene MurF inhibitors. Overlapping scaffold
essential for good inhibitory potency is shown in red.
Figure 2. New cyanothiophene MurF inhibitors (sticks rendering, coloured by atom) docked
to MurFSp (PDB code: 3zm5) with a co-crystallized inhibitor (sticks rendering, in green). The
best ranked predicted binding mode can be seen for a) amine, and b) guanidine of truncated
analogue of II; guanidine is predicted to fit into the “morpholine” pocket (binds morpholine
moiety from the co-crystallized inhibitor) by forming 4 H-bonds with Asn134, Asn137 and
Asn326 (in green, demonstrated by the distances between guanidine protons and aminoacid
carbonyl oxygens), while the rest of the molecule overlaps the structure of a co-crystallized
cyanothiophene scaffold. Virtually the same binding mode is predicted for c) amine with
benzyl substituent attached to position 6 of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine (c), while
its guanidine analogue (d) moves towards the interior of the binding site to form close contact
with Asp323 (in green, ionic bond and two H-bonds).
Figure
3. Crystal
structure
of the
ligand
(2,4-dichloro-N-[3-cyano-6-[(4-
hydroxyphenyl)methyl]-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-ium-2-yl]-5-morpholino
sulfonyl-benzamide, shown as green sticks) and top docked pose of the ligand, as predicted by
FRED (shown as stick colored by atom type). The top docked ligand pose had an RMSD of
1
.15 Å.
2
8