4-Hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-one as inhibitors of tyrosyl-tRNA synthase: Synthesis, molecular docking and antibacterial evaluation

Article abstract of DOI:10.1016/j.molstruc.2013.10.032

A series of novel 4-hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-ones as tyrosyl-tRNA synthetase inhibitors were synthesized. Of these compounds, 4-(naphthalen-1-ylmethyl)-5-oxo-2,5-dihydrothiophen-3-yl-2-(4-hydroxyphenyl) acetate (29) was the most potent. The binding model and structure-activity relationship indicate that replacement of phenyl acetate in the side chain of 29 with a substituent containing more hydrophilic groups would be more suitable for further modification. Antibacterial assay revealed that the synthetic compounds are effective against growth of Gram-positive organisms, and 29 is the most potent agent against Staphylococcus aureus ATCC 25923 with MIC ₅₀ value of 0.21 μg/mL.

Full text of DOI:10.1016/j.molstruc.2013.10.032

Journal of Molecular Structure 1056–1057 (2014) 104–109
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
4-Hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-one as inhibitors
of tyrosyl-tRNA synthase: Synthesis, molecular docking and antibacterial
evaluation
a
a
b
b
b
a,b,
⇑
Juan Sun , Jia-Jia Liu , Wei Zhou , Feng-Jiao Guo , Xin-Yi Wang , Hai-Liang Zhu
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
School of Life Sciences, Shandong University of Technology, Shandong 255049, People’s Republic of China
b
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
A series of 4-hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-ones were first synthesized.
Compound 29 is the most potent agent against Staphylococcus aureus ATCC 25923 with MIC50 value of 0.21
Their biological activities are also evaluated for tyrosyl-tRNA synthase inhibitory activity.
l
g/mL.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 August 2013
Received in revised form 11 October 2013
Accepted 11 October 2013
Available online 19 October 2013
A series of novel 4-hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-ones as tyrosyl-tRNA synthetase
inhibitors were synthesized. Of these compounds, 4-(naphthalen-1-ylmethyl)-5-oxo-2,5-dihydrothio-
phen-3-yl-2-(4-hydroxyphenyl)acetate (29) was the most potent. The binding model and structure–
activity relationship indicate that replacement of phenyl acetate in the side chain of 29 with a substituent
containing more hydrophilic groups would be more suitable for further modification. Antibacterial assay
revealed that the synthetic compounds are effective against growth of Gram-positive organisms, and 29
Keywords:
Thiophenone
Antibacterial
is the most potent agent against Staphylococcus aureus ATCC 25923 with MIC50 value of 0.21 lg/mL.
Ó 2013 Elsevier B.V. All rights reserved.
Tyrosyl-tRNA synthase
Structure–activity relationship
Molecular docking
1
. Introduction
the topical treatment of onychomycosis and icofungipen. These
enzymes, therefore, are attractive targets for antibacterial agents
[10–12].
The emergence of resistance to clinically used drugs has
promoted development of novel antimicrobial agents which
selectively inhibit novel bacterial targets [1–3]. In this regard, ami-
noacyl-tRNA synthetases (aaRSs) have attracted considerable
interest in antibacterial drug discovery [4–6]. It is well known that
aaRSs are the enzymes that catalyze the transfer of amino acids to
their cognate tRNAs [7,8] and their catalytic activities determine
the genetic code. Consequently, once these enzymes are inhibited,
cell growth is inhibited due to protein biosynthesis being halted.
This concept is proven by the success of the broad-spectrum anti-
bacterial drug mupirocin, which targets bacterial isoleucyl-tRNA
synthetases (IleRS) [9]. Additionally, AN-2690 (Fig. 1), an inhibitor
of leucyl-tRNA synthetases, is currently in clinical development for
Thiophenone framework is a part of many natural and synthetic
compounds, which possess useful biological activities such as
antitubercular and antibacterial activity [13–19]. Xiao et al. have
shown that 3-aryl-4-alkylaminofuran-2(5H)-ones are potent
inhibitors against tyrosyl-tRNA synthetase (TyrRS) [20,21], one of
the aminoacyl-tRNA synthetases (aaRSs). Structure–activity
relationships of the two classes of compounds disclosed that
bioisosteric replacement of NH group (a) with an O (b) was more
favorable for activity (Fig. 1). Recently, Benneche et al. reported
5-(chloromethylene)-and
5-(bromoalkylidene)thiophen-2(5H)-
ones showing better antibacterial activity than furanones [22].
Therefore, derivatives of thiophenone have been receiving signifi-
cant attention, which encourages us to synthesize and evaluate
the antibacterial activity of a series of substituted thiophenones.
To learn more about the structure-antibacterial activity
relationship of thiophenones, we herein report the synthesis of
⇑
Corresponding author at: School of Life Sciences, Shandong University of
Technology, Shandong 255049, People’s Republic of China. Tel./fax: +86 25 8359
2
672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
4
-hydroxy-3-(naphthalen-1-ylmethyl) thiophen-2(5H)-ones as
0
022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.10.032

J. Sun et al. / Journal of Molecular Structure 1056–1057 (2014) 104–109
OH
105
HO
B
O
OH
OH
H
O
HO
O
O
F
O
O
H
mupirocin
AN-2690
O
O
O
O
HN
O
N
O
N
O
Br
Br
a
b
Fig. 1. Structures of some compounds.
5
TyrRS inhibitors and antibacterial agents. The results show that
some of the synthesized compounds exhibit excellent antibacterial
activities.
the microorganism was prepared to contain approximate 10 cfu/
mL and applied to 96-well plates with serially diluted compounds
to be tested and incubated at 37 °C for 24 h. The optical density
(
OD) was measured with a microplate reader at 550 nm. The ob-
served MIC50s were presented in Table 2.
2
. Experimental
2.3. Protocol of docking study
2.1. Preparation of the TyrRS and enzyme assay
Automated docking studies were carried out using Discovery
Studio (version 3.1) as implemented through the graphical user
interface DS-CDocker protocol.
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 12.0 software
Chemical Structure Drawing Standard; Cambridge Soft corpora-
Staphylococcus aureus TyrRS was over-expressed in Escherichia
coli and purified to near homogeneity (ꢁ98% as judged by SDS–
PAGE) using standard purification procedures [7]. TyrRS activity
was measured by aminoacylation using modifications to previously
described methods [8]. The assays were performed at 37 °C in a
mixture containing (final concentrations) 100 mM Tris/Cl pH 7.9,
[
tion, USA (2010)], then they were energetically minimized by using
MOPAC with 100 iterations and minimum RMS gradient of 0.10.
The Gasteiger–Hückel charges of ligands were assigned. The crystal
structures of TyrRS (PDB code: 1jij.) complex were retrieved from
the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do). All bound waters and ligands were eliminated from
the protein and the polar hydrogens and the Kollman-united
charges were added to the proteins.
5
0 mM KC1, 16 mM MgCl
2
, 5 mM ATP, 3 mM DTT, 4 mg/mL E. coli
-tyrosine (0.3 M L-[ring-3,5-
H] tyrosine (PerkinElmer, Specific activity: 1.48–2.22 TBq/mmol),
M carrier). TyrRS (0.2 nM) was preincubated with a range of
MRE600 tRNA (Roche) and 10
3
1
inhibitor concentrations for 10 min at room temperature followed
by the addition of pre-warmed mixture at 37 °C. After specific
intervals, the reaction was terminated by adding aliquots of the
reaction mix into ice-cold 7% trichloroacetic acid and harvesting
onto 0.45 mm hydrophilic Durapore filters (Millipore Multiscreen
lM
L
l
0
l
2.4. Chemistry
9
6-well plates) and counted by liquid scintillation. The rate of reac-
tion in the experiments was linear with respect to protein and time
with less than 50% total tRNA acylation. IC50s correspond to the con-
centration at which half of the enzyme activity is inhibited by the
compound. The results are presented in Table 1.
All chemicals and reagents used in the current study were of
analytical grade. The reactions were monitored by thin layer chro-
matography (TLC) on Merck pre-coated silica GF254 plates. Melt-
ing points (uncorrected) were determined on a XT4MP apparatus
(
Taike Corp., Beijing, China). ESI mass spectra were obtained on a
2.2. Antimicrobial activity
1
Mariner System 5304 mass spectrometer, and H NMR spectra
were collected on a Bruker DPX300 spectrometer at room temper-
ature with TMS and solvent signals allotted as internal standards.
Chemical shifts are reported in ppm (d). Elemental analyses were
performed on a CHN-O-Rapid instrument, and were within ±0.4%
of the theoretical values.
The antibacterial activities of the synthesized compounds was
tested against two Gram-positive bacterial strains (Bacillus subtilis
ATCC 6633, S. aureus ATCC 25923, kanamycin as positive control)
and two Gram-negative bacterial strains (Pseudomonas aeruginosa
ATCC13525, E. coli ATCC 35218, penicillin G as positive control)
using LB medium. The MIC50s of the test compounds were deter-
mined by a colorimetric method using the dye MTT. A stock solu-
3. Results and discussion
tion of the synthesized compound (1000 lg/mL) in DMSO was
prepared and graded quantities of the test compounds were incor-
porated in specified quantity of sterilized liquid medium (50% (v/v)
of DMSO in PBS). A specified quantity of the medium containing
the test compound was poured into 96-well plates. Suspension of
3.1. Chemistry
Thirty 4-hydroxy-3-(naphthalen-1-ylmethyl) thiophen-2(5H)-
ones (5–34) were designed and synthesized by the routes outlined

1
06
J. Sun et al. / Journal of Molecular Structure 1056–1057 (2014) 104–109
Table 1
In vitro inhibitory activity data of the synthesized compounds against S. aureus TyrRS.
O
S
O
R
O
Compound
R
IC50
(l
M)
Compound
R
IC50 (lM)
5
F
>100
>100
>100
20
50.08
44.76
48.94
43.32
8.30
C
6
7
8
9
Cl
Br
21
22
23
24
25
O
N
CH
3
88.62
92.10
>100
OCH
3
1
0
NO
2
18.98
1
1
1
1
1
1
1
2
3
4
5
6
F
82.14
86.13
83.13
69.00
72.11
71.09
26
27
28
29
30
31
15.86
11.10
13.79
0.25
Cl
Br
C
O
>100
>100
N
N
N
Cl

J. Sun et al. / Journal of Molecular Structure 1056–1057 (2014) 104–109
107
Table 1 (continued)
Compound
R
IC50
(l
M)
Compound
R
IC50 (lM)
1
7
65.14
32
>100
N
F
O
1
8
55.23
33
34
Cl
>100
N
N
Cl
1
9
51.17
>100
Br
were obtained by treatment with different benzoic acids in the
presence of EDC.HCl and HoBt in anhydrous dichloromethane. All
Table 2
Inhibitory activity of the synthetic compounds against microbes.
4
-hydroxy-3-(naphthalen-1-ylmethyl) thiophen-2(5H)-ones are
reported here for the first time and were fully characterized by
spectroscopic methods and elemental analysis (see Supporting
Information).
Compound
MIC50
A
(lg/mL)
B
C
D
>50
2
2
2
2
2
2
4
5
6
7
8
9
23.50
>50
>50
42.12
29.81
3.66
0.48
–
10.08
33.12
35.78
21.99
33.12
0.21
1.20
–
>50
>50
>50
40.0
>50
>50
3.93
–
>50
>50
>50
>50
>50
>50
2.70
3.2. Inhibitory activities of 4-hydroxy-3-(naphthalene-1-ylmethyl)
thiophen-2(5H)-ones against TyrRS from S. aureus
All the synthesized compounds (5–34) were tested for inhibi-
tory activity against TyrRS from S. aureus. The IC50s of these com-
pounds are presented in Table 1. Compounds 5–23 were
prepared to study the utility of benzoic acid side chains and all
of the compounds showed poor activity, indicating that separation
of benzene moiety and thiophenone fragment with an carbon atom
spacer led to compounds (24–29) being more potent. Therefore,
this position seems to be more suitable for further modification
with a substituent containing more aliphatic chains.
Kanamycin
Penicillin
Note: (A) B. subtilis ATCC 6633; (B) S. aureus ATCC 25923; (C) P. aeruginosa
ATCC13525; (D) E. coli ATCC 35218.
in Scheme 1. In brief, compound 2 was formed in the presence of
thionyl chloride. Ester 3 was prepared using concentrated sulfuric
acid in methanol. The key compound thiophenone (4) was ob-
tained by alkalization of 3 with appropriate methyl thioglycolate
in THF in the presence of t-BuOK. The target compounds (5–34)
We then turned our attention toward exploring the SAR profile
of the phenylacetic acid group (24–29). The compound 29 with a
hydroxyl group at the para position of the benzene-ring fragment
O
O
HOOC
Cl
O
i
ii
2
1
3
O
O
S
S
O
HS
O
iv
OH
O
R
iii
O
4
5
-34
Scheme 1. General synthesis of compounds 5–34. Reagents and conditions: (i) Thionyl chloride, reflux, 1 h; (ii) Methanol, reflux, 3 h; (iii) Tetrahydrofuran, Potassium tert-
butoxide, reflux, overnight; (iv) Dichloromethane, EDC.HCl, HoBt. rt, 3–10 h.

1
08
J. Sun et al. / Journal of Molecular Structure 1056–1057 (2014) 104–109
exerted the highest inhibitory activity against S. aureus TyrRS with
an IC50 of 0.25 g/mL. Replacement of 4-hydroxyl group with
chloro or bromo group resulted in 100-fold loss of activity (27–
8). In the case of compounds (30–34) with pyridine-ring substit-
l
2
uents, introduction of electron-donating group (methoxy) and
electron-withdrawing group (chloro) led to complete loss in activ-
ity, indicating that these substituents may cause a steric clash with
the protein.
3.3. Antibacterial activity
Compounds with IC50 < 20 lM against S. aureus TyrRS were
tested against representative Gram-positive organisms (B. subtilis
ATCC 6633 and S. aureus ATCC 25923) and Gram-negative organ-
isms (P. aeruginosa ATCC13525 and E. coli ATCC 35218), and the re-
sults are presented in Table 2. The tested compounds showed good
activity against Gram-positive organisms, especially against S. aur-
eus, while all of them were inactive against Gram-negative bacte-
rium, P .aeruginosa and E. coli. Compound 29 displayed levels of
inhibition against S. aureus compared to Kanamycin. From compar-
ing the data in Tables 1 and 2, a positive correlation between the
IC50 values in the enzyme assay and the MIC50 values against whole
cell bacteria was found. This suggests that the antibacterial activity
of thiophenone, as that of 4-hydroxy-3-(naphthalen-1-ylmethyl)
thiophen-2(5H)-ones, may be due to their inhibition against TyrRS.
Fig. 3. The 3D model structure of compound 29 binding model with TyrRS.
TyrRS is depicted in Fig. 2 and the enzyme surface model is showed
in Fig. 3, which revealed that the molecular is well filled in the ac-
tive pocket.
Several hydrogen-bonding interactions together with some
hydrophobic interactions anchoring 29 to the active site tightly
may explain its excellent inhibitory activity. In brief, the ben-
zene-ring system in the side chain of 29 occupies the SB-239629
piperidyl-binding pocket, and is oriented towards the entrance
cavity (Fig. 3) surrounded by Ala39, Leu70, Gln174, Val191,
Gly192 and Ile200. The O atom of thiophenone-ring as a hydrogen
bond acceptor is hydrogen-bonded to the backbone nitrogen of
Gly193 with O. . .H bond. (Fig. 2). Obviously, the benzene-ring sys-
tem is located at a hydrophilic pocket of the active site. Thus,
replacement with a substituent containing stronger hydrophilic
3.4. Molecular docking
With the aim to explore the structural determinants responsible
for the activity of these new inhibitors of TyrRS, molecular docking
of the most potent inhibitor 29 into SB-239629 binding site of Tyr-
RS was performed on the binding model based on the TyrRS com-
plex structure (1jij.pdb). The binding model of compound 29 and
Fig. 2. Ligand interaction diagram of compound 29 with TyrRS using Discovery Studio program with the essential amino acid residues at the binding site are tagged in circles.
The purple circles show the amino acids which participate in hydrogen bonding, electrostatic or polar interactions and the green circles show the amino acids which
participate in the Van der Waals interactions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

J. Sun et al. / Journal of Molecular Structure 1056–1057 (2014) 104–109
109
group should increase the potency, and further investigations on
Appendix A. Supplementary material
the optimization of 29 as leading compounds are being carried
out in our laboratory.
The 4-hydroxy-3-(naphthalen-1-ylmethyl)-thiophenone moi-
ety is located at the bottom of the active site cavity (Fig. 3), making
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.2013.
10.032.
some
p-cation interactions with Lys84 and Arg88 side chains. In
addition to these interactions, the hydroxyl group forms one O–
H. . .O and one O. . .H–N hydrogen bonds with Lys84 and Asp195
residue, respectively (Fig. 2).
Our modeling results reveal that hydrophilic group substituted
at the side chain are important to the interactions of the protein–
ligand complex and are crucial to the potency of TyrRS inhibitory
activity. Therefore, removing the hydrophilic group or substituting
a hydrophobic group can obviously reduce the enzyme inhibitory
activity.
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Acknowledgements
This work was supported by the National Forestry Common-
weal Project (200904001) and by Major Projects on Control and
Rectification of Water Body Pollution (No. 2011ZX07204-001-
0
04).