Discovery and development of novel rhodanine derivatives targeting enoyl-acyl carrier protein reductase

Article abstract of DOI:10.1016/j.bmc.2019.02.043

A series of rhodanine derivatives RB1–RB23 were synthesized through a two-round screening. Their Mycobacterial tuberculosis (Mtb) InhA inhibitory activity and Mtb growth blocking capability were evaluated. The most potent hit compound RB23 indicated compa

Full text of DOI:10.1016/j.bmc.2019.02.043

Accepted Manuscript
Discovery and development of novel rhodanine derivatives targeting enoyl-acyl
carrier protein reductase
Jian-Fei Xua, Tian-Tian Wang, Qing Yuan, Yong-Tao Duan, Yun-Jie Xu, Peng-
Cheng Lv, Xiao-Ming Wang, Yu-Shun Yang, Hai-Liang Zhu
PII:
S0968-0896(19)30059-8
https://doi.org/10.1016/j.bmc.2019.02.043
BMC 14779
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
11 January 2019
18 February 2019
20 February 2019
Please cite this article as: Xua, J-F., Wang, T-T., Yuan, Q., Duan, Y-T., Xu, Y-J., Lv, P-C., Wang, X-M., Yang, Y-
S., Zhu, H-L., Discovery and development of novel rhodanine derivatives targeting enoyl-acyl carrier protein
reductase, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.043
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Discovery and development of novel rhodanine derivatives targeting enoyl-acyl
carrier protein reductase
Jian-Fei Xuaa,†, Tian-Tian Wanga,†, Qing Yuana, Yong-Tao Duanb, Yun-Jie Xua,
Peng-Cheng Lva, Xiao-Ming Wanga,*, Yu-Shun Yanga,*, Hai-Liang Zhua,*
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210023, China
bHenan Provincial Key Laboratory of Children's Genetics and Metabolic Diseases,
Children's Hospital Affiliated to Zhengzhou University, Zhengzhou Children's
Hospital, Zhengzhou, 450018, China
†Both authors contributed equally to the work
*Corresponding author. Tel. & Fax: +86-25-89682572; E-mail: zhuhl@nju.edu.cn;
ys_yang@nju.edu.cn; wangxm07@nju.edu.cn.
Abstract:
A series of rhodanine derivatives RB1-RB23 were synthesized through a
two-round screening. Their Mycobacterial tuberculosis (Mtb) InhA inhibitory activity
and Mtb growth blocking capability were evaluated. The most potent hit compound
RB23 indicated comparable InhA inhibiton (IC50 = 2.55 μM) with the positive control
Triclosan (IC50 = 6.14 μM) and Isoniazid (IC50 = 8.29 μM). Its improved
growth-blocking effect on Mtb and low toxicity were attractive for further
development. The docking simulation revealed the possible binding pattern of this
series and picked the key interacted residues as Ser20, Phe149, Lys165 and Thr196.
The 3D-QSAR model visualized the SAR discussion and hinted new information.
Modifying the surroundings near rhodanine moiety might be promising attempts in
later investigations.
Keywords:
Bacterial Infection; InhA Inhibition; Computer Assistant Drug Design; Rhodanine;
Molecular Docking; 3D-QSAR
1

1. Introduction
Bacterial infection is a nonnegligible risk worldwide and it has become even
more dreadful because of drug resistance and clinical complication.1-3 With increasing
knowledge of antibacterial targets and corresponding interactions, the development of
potent drug candidates gradually infers some traces to follow.4-6 One available
strategy is blocking the fatty acids biosynthesis of the pathogens.7-9 All through this
pathway, enoyl-acyl carrier protein reductase (InhA) is a convinced target for treating
tuberculosis by the first-line drug isoniazid.10-12 Being highly conserved and
distinguished from the mammalian fatty acid biosynthesis enzymes, InhA is suitable
as such a target.13-15 When computer assistant drug design (CADD) seems more
popular recently in medicinal chemistry, a variety of InhA inhibitory series have been
produced, such as triclosan16,17, isoniazid-NAD analogues18, pyrrolidine carboxamides
19,20, triazoles21,22 and pyridomycin23,24. Glancing at them, a typical purpose to inhibit
InhA was curing tuberculosis19,22,24. Since drug discovery for tuberculosis upon
common routes such as protein synthesis, cell wall, DNA gyrase and ATP synthesis
faces problems including resistance and poor selectivity25,26, blocking fatty acids
biosynthesis at InhA seemed a promising choice27,28. Though designed for InhA, the
in vitro inhibition of present inhibitors on Mycobacterial tuberculosis (Mtb) still need
to be improved.29 Therefore, exploiting new hit compounds for InhA to treat
tuberculosis is in urgent emergency.30,31
In this work, we started our series from the rhodanine moiety because of its
reported biological activities. Its bacterial-relating targets include penicillin-binding
proteins32, RNA polymerase33,34, oxyreductase enzymes35, and especially, the fatty
acid biosynthesis related ones such as Plasmodium falciparum FabI 36 and then InhA
37. By employing the similarities of previous work, we modified the detail of the
linker to introduce stretched group as shown in Figure 1. Seeking the new possibilities
of group B from amino acid residues and alkyl chains, we set it as a aryl one to further
balance the requirements of both group A and B. This strategy with a two-round
screening might raise new information during the comparisons between both the
bioactivities and molecular docking results. Through the improving procedure round
by round, this work might infer hints for future development of InhA inhibitors.
2

Figure 1. The designing concept of the novel series as InhA inhibitors.
2. Results and discussion
2. 1. Chemistry
The general synthesis route and the structures of the rhodanine derivatives
RB1-RB23 were shown in Scheme 1. All of them except (RB738 and RB939) were
synthesized for the first time. A series of substituted benzaldehyde, rhodanine and
urea were mixed and stirred in ethanol to acquire intermediate B. Then the following
reaction with different bromoacetophenones and sodium ethylate resulted in the
corresponding target compounds. Compounds RB16-RB23 were derived according to
the preliminary Structure-Activity Relationship (SAR) of RB1-RB15.
Recrystallisation was performed to guarantee the refined compounds gave satisfactory
analytical and spectroscopic data. Since the spectroscopic data of the synthesized
compounds did not show obvious isomerism, we carefully checked them and made
comparison with reported ones (including RB738 and RB939) to confirm that the
compounds in this work were all trans-forms.
Code
RB1
R1
R2 Code
R1
R2
Phenyl
H
2-Methoxyphenyl
H
RB13
3

4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
p-Tolyl
H
H
H
H
H
H
H
H
H
H
H
3-Methoxyphenyl
3,4,5-Trimethoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
p-Tolyl
H
RB2
RB3
RB4
RB5
RB6
RB7
RB8
RB9
RB10
RB11
RB12
RB14
RB15
RB16
RB17
RB18
RB19
RB20
RB21
RB22
RB23
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
4-Methoxyphenyl
4-Nitrophenyl
4-Hydroxyl
2-Chlorophenyl
3-Chlorophenyl
2-Methoxyphenyl
3,4,5-Trimethoxyphenyl
4-Dimethylaminophenyl
4-Acetamidephenyl
2-Chlorophenyl
3-Chlorophenyl
Scheme 1. General synthesis of RB1-RB23 and their structures.
2. 2. Biological activity
The Mtb InhA inhibitory effect and Mtb growth blocking of the synthesized
compounds were tested consulting the protocols in references22,37. We took three
controls. One was a typical InhA inhibitor Triclosan; another was a rhodanine-derived
drug Epalrestat; the other was a first-line tuberculosis drug Isoniazid. In this work,
two major concerns were enhancing the InhA inhibitory activity and improve the
performance on Mtb. Seen in Table 1, in the first round, most of the compounds
indicated potential for both of the demands.
Table 1. The Mtb InhA inhibitory and Mtb growth blocking activities of
RB1-RB15.
Code
R1
IC50 (μM)
MIC (μM)
Code
R1
IC50 (μM)
MIC (μM)
Mtb InhA
40.2  3.25
55.0  5.01
50.1  4.06
23.9  1.98
17.5  1.32
8.24  0.69
Mtb
Mtb InhA
Mtb
17.4  1.42
12.6  1.08
-H
4-F
4-N(Me)2
4-NHAc
2-Cl
RB1
RB2
RB3
RB4
RB5
RB6
32.3  2.89
57.4  5.32
48.2  4.61
21.1  1.98
15.1  1.26
2.95  0.27
RB9
RB10
RB11
RB12
RB13
RB14
11.6  1.01
25.8  2.23
47.3  4.28
28.1  2.54
22.6  2.03
3.18  0.29
25.5  2.24
40.9  3.87
30.4  2.53
20.5  1.89
4-Cl
4-Br
4-Me
4-OMe
3-Cl
2-OMe
3-OMe
4

3.12  0.28
0.38  0.035
4-NO2
4-OH
3,4,5-OMe
RB7
RB8
43.5  3.78
35.3  3.02
6.14  0.55
36.5  3.31
33.1  3.05
8.65  0.79
RB15
Triclosan
Isoniazid
Epalrestat
> 200
> 200
8.29  0.76 0.58  0.050
Preliminary SAR studies on Mtb InhA was conducted according to the data in
Table 1. Though the scale was not quite large, several clues could still be found. The
initial one was that electron-donating groups were basically preferred at the
para-position. The corresponding order was -OMe (RB6, IC50 = 8.24 μM) > -NHAc
(RB10, IC50 = 11.6 μM) > -N(Me)2 (RB9, IC50 = 17.4 μM) ≥ -Me (RB5, IC50 = 17.5
μM) > -Br (RB4, IC50 = 23.9 μM) > -OH (RB8, IC50 = 35.3 μM) > -H (RB1, IC50 =
40.2 μM) ≥ -NO2 (RB7, IC50 = 43.5 μM) > -Cl (RB3, IC50 = 50.1 μM) > -F (RB2,
IC50 = 55.0 μM). The second one came when we fixed the substitute but changed its
position. For the electron-donating group (here was -OMe), the tendency was para-
(RB6, IC50 = 8.24 μM) > meta- (RB14, IC50 = 22.6 μM) > ortho- (RB13, IC50 = 28.1
μM); whereas for the electron-withdrawing one (here was -Cl), the tendency was a
opposite ortho- (RB11, IC50 = 25.8 μM) > meta- (RB12, IC50 = 47.3 μM) ≥ para-
(RB3, IC50 = 50.1 μM). The last clue was for multi-substituted situation. Since
electron-donating seemed better for this series, we picked the 3,4,5-trimethoxyl one. It
suggested better potency (RB15, IC50 = 3.12 μM) than single-substituted ones.
Table 2. The Mtb InhA inhibitory and Mtb growth blocking activities of
RB16-RB23.
Code
R1
IC50 (μM)
MIC (μM)
Code
R1
IC50 (μM)
MIC (μM)
Mtb InhA
20.1  1.97
16.5  1.32
12.1  1.08
Mtb
Mtb InhA
Mtb
15.3  1.22
7.83  0.72
4-F
4-Cl
4-Br
4-Me
2-Cl
3-Cl
RB16
RB17
17.8  1.52
8.62  0.76
4.04  0.36
RB20
RB21
7.84  0.65
16.8  1.41
2.55  0.20
2.02  0.19
9.27  0.87
0.29  0.025
2-OMe
3,4,5-OMe
RB18
RB22
RB19
5.02  0.39 0.49  0.042
RB23
Triclosan
Isoniazid
Epalrestat
> 200
> 200
6.14  0.55
8.65  0.79
8.29  0.76 0.58  0.050
5

A second round was linked up by introducing the preferred electron-donating
group from Group A to Group B and giving a brief discussion on the reset Group A
(Table 2). This optimization was consulting both the above mentioned biological
activities and the later elaborated molecular docking results. An obvious progress in
potency could be indicated when the substitutes of Group A were the same. The
requirement for electron-donating substitute still existed with the observed tendency
of -Me (RB19, IC50 = 5.02 μM) > -Br (RB18, IC50 = 12.1 μM) > -Cl (RB17, IC50 =
16.5 μM) > -F (RB16, IC50 = 20.1 μM). The preference for substituent position
seemed interfered because the ortho- or meta-substituted compounds (RB20, RB21,
RB22) all suggested considerable potency. The top choice was still the
3,4,5-trimethoxyl one with potential InhA inhibitory activity (RB23, IC50 = 2.55 μM).
Moreover, it was cheerful that the growth-blocking effect on Mtb was improved. It
was still a slight pity that the top hits were subjected to antibacterial activity, which is
a common phenomenon mainly due to the complex compensatory mechanism of
bacteria to resist the drug action.
Top five compounds (RB23, RB15, RB19, RB21 and RB6) were chosen for the
evaluation of broad-spectrum antibacterial potency and cytotoxicity. The
Gram-positive MSSA (methicillin-sensitive Staphylococcus aureus ATCC 25923),
MRSA (methicillin-resistant Staphylococcus aureus ATCC 29213) and
Gram-negative (Pseudomonas aeruginosa ATCC 27583) pathogenic bacteria were
employed for the former while 293T (human embryonic kidney cell line) was
recruited for the latter. As presented in Table 3, the representatives indicated potent
broad-spectrum antibacterial ability and low toxicity.
Table 3. Broad-spectrum antibacterial and cytotoxic evaluation of top hits.
Compounds
MIC (μM)
MSSA
MIC (μM)
MRSA
MIC (μM)
IC50 (μM)
293T
P. aeruginosa
0.95  0.09
> 200
RB23
1.24  0.10
1.54  0.13
6

> 200
RB15
4.61  0.45
6.15  0.51
2.16  0.18
4.28  0.36
0.39  0.03
1.26  0.11
7.20  0.69
10.2  0.85
3.88  0.28
4.15  0.40
0.68  0.06
1.42  0.12
5.41  0.44
8.12  0.75
5.05  0.35
4.83  0.43
1.25  0.11
2.85  0.23
RB19
195  18.5
> 200
RB21
RB6
167  15.0
20.5  2.00
120  10.5
Triclosan
Isoniazid
For further determine the molecular target, two more reported antibacterial
targets were included. One was DNA gyrase40, the most common target for antibiotics,
while the other was FabH41, the fatty acid biosynthesis related enzyme. Seen in Table
4, the representatives did not indicated obvious inhibition against these two targets,
thus inferred the selectivity for InhA to some degree.
Table 3. The inhibitory capability against DNA gyrase and FabH of top hits.
Code
IC50 (μM)
IC50 (μM)
Code
IC50 (μM)
DNA gyrase
> 1000
IC50 (μM)
FabH
DNA gyrase FabH
> 1000
RB23
900  80
850  70
RB15
780  70
> 1000
Triclosan > 1000
Isoniazid
> 1000
2. 3. In silico study
The preliminary evaluation for the basic druggability was performed by
examining the ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity)
properties (Figure 2A). The AlogP (the partition coefficient of drug in
octanol/aqueous solution calculated by ACD/PhysChem Suite Software) and PSA_2D
(the fast calculated polar surface area from the 2D structure) were used to predict the
Absorption (human intestinal absorption) and BBB (blood-brain barrier penetration)
including 95% and 99% confidence ellipses as referenced.42 All the compounds
showed attractive pharmacokinetics potential which could lead to further applications.
7

Figure 2. (A) The ADMET properties predicted for RB1-RB23 indicated potential
druggability by locating inside the innermost oval (except RB7); (B) The consistency
between the Mtb InhA inhibitory activity and the negative values of calculated
interaction energy.
Molecular docking simulation was performed to reveal the probable binding
patterns of the synthesized compounds. The active site of Mtb InhA (PDB code: 4R9S)
was defined as the binding site. The Mtb InhA inhibitory activity and the negative
values of calculated interaction energy presented basically consistency (Figure 2B).
This parameter together with the binding patterns and bioactivities, helped us to
optimize from the first round to the second. For the first round, the 2D maps of RB6,
RB15 and Isoniazid within the binding position were displayed in Figure 3. We used a
more recent reported crystal structure of InhA than that in the related work (PDB code:
1P45)37. Compared with the previous work, the residues to form the key interactions
were similar but not exactly the same. They include Ser20, Phe149, Lys165 and
Thr196. Meanwhile, the positions of the key interactions were not exactly fixed. This
might infer the rotation of the two groups around rhodanine. In detail, RB6 interacted
with InhA via a potential hydrogen bond with Lys165 from the linker of Group B
(O…H-N: 1.81 Å, 130.884°) and π-π interactions with Phe149 from rhodanine
(distance: 5.14 Å). However, for RB15, the hydrogen bond with Lys165 moved to the
meta-methoxyl on Group A (O…H-N: 1.89 Å, 153.584°) and the linker of Group B
inferred another potential hydrogen bond with Thr196 (O…H-O: 1.95 Å, 160.955°).
8

Before confirming possible adduct with NAD+, Isoniazid also presented a similar
hydrogen bond with Lys165 (O…H-N: 2.03 Å, 122.105°) and another potential one
with ASP148 (N-H…O: 2.05 Å, 138.026°). The binding position, as reported, was
close to Tyr158 and NAD+. For the second round, the top hit RB23 indicated the
probable hydrogen bonds with Lys165 from the linker of Group B (O…H-N: 1.73 Å,
167.410°) and with Thr196 from the meta-methoxyl on Group A (O…H-O: 2.14 Å,
154.123°). It also retained the π-π interactions with Phe149 from rhodanine (distance:
5.63 Å) and the close steric distance to the reported residue Ser20.
Figure 3. The 2D docking patterns of the first round representatives (A) RB6, (B)
RB15 and (C) Isoniazid into Mtb InhA. Significant interactions and residues were
exhibited. The H-bonds were displayed as dotted lines. The π-π interactions were
shown as orange line. (D) 3D pattern of Isoniazid with NAD+ within InhA.
9

Figure 4. (A) The 2D and (B) 3D docking patterns the second round representative
RB23 into Mtb InhA. Significant interactions and residues were exhibited. The
H-bonds were displayed as dotted lines. The π-π interactions were shown as orange
line.
Moreover, the receptor surface model was generated in Figure 5A to visualize the
surrounding electronic conditions of the binding pocket. Then the two referenced
compounds in Figure 1 were put together with our series to participate a molecular
overlay. The result in Figure 5B suggested a reliable consistency to guarantee the
feasibility of the following QSAR model.
Figure 5. (A) The receptor surface model of RB23 into Mtb InhA; (B) The molecular
overlay of reference and our compounds indicated reliable consistency.
The 3D QSAR model was built according to the docked structures and the pIC50
scale (-log IC50) of their InhA inhibitory activities. The correlation coefficient r2
10

(0.996) ensured that the model was reliable. The electrostatic and Van der Waals
maps were presented in Figure 6. According to the electron-withdrawing (red),
electron-donating (blue), steric bulky (green) or small (yellow) requirements, the
modification of this series could be induced as the following points. Since the rotation
around the rhodanine moiety might exist, here Group A and B suggested the similar
requirements. Seen in a more brief view Figure 6C, they both preferred an
electron-donating and smaller substitute to stretch further but electron-withdrawing
and larger ones to fill in the near surrounding. This result basically agreed with the
above mentioned SAR discussion. Thus, a series of modification near the rhodanine
moiety might be a promising orientation to perform deeper design.
Figure 6. 3D-QSAR models of our series together with the referenced compounds (A
& B) and a brief view of the modification requirements (C). The maps of electronic
potential (A) and Van der Waals grids (B) visualized the requirements of each factor.
3. Conclusions
In summary, a two-round screening on rhodanine based series RB1-RB23 was
conducted to seek potential InhA inhibitory hit compounds. They were all novel
11

structures with their Mtb InhA inhibitory effect and Mtb growth blocking evaluated
for the first time. Among them, the most potent candidate RB23 provided comparable
activities against InhA and Mtb with the positive control Triclosan and Isoniazid. It
was also low toxic upon human embryonic cells. The ADMET analysis indicated the
practical druggability of the synthesized series. The docking simulation revealed the
possible binding patterns with the key residues picked as Ser20, Phe149, Lys165 and
Thr196. Then the 3D-QSAR model visualized the connection between activity data
and binding conformations. It would be a promising attempt to modify the
surroundings near rhodanine moiety to raise future designs.
4. Experimental Section
4. 1. Chemistry
4. 1. 1. General
All the commercially available chemicals were used directly as received without
further purification. All the melting points were determined on a WRS-1C
1
13
digital melting point apparatus and were uncorrected. H NMR and C NMR spectra
were recorded on a Bruker AM 600 (Rhenistetten-Forchheim, Germany) spectrometer.
1
Shifts are reported in parts per million based on residual solvent peaks (for H or
13C/CDCl3). NMR data were resolved with MestreNova software. Mass spectra were
obtained from an Agilent 6540 UHD Accurate Mass Q-TOF LC/MS.
4. 1. 2. Compounds
General method of synthesis of Intermediates B
Various substituted benzaldehydes (1 mmol) were added to the rhodanine (1
mmol) alcohol solution (10 mL), respectively. Then urea (1.5 mmol) was added and
the reaction continued in reflux for 5 h. After confirming the completion of the
reaction by thin layer chromatography, the sediment was filtered, washed with ethanol
and dried to obtain intermediates B.
The synthesis of (E)-5-arylidene-3-(2-aryl-2-oxoethyl)-2-thioxothiazolidin-4-one
(RB1-RB23)
12

Initially, the intermediates B (1 mmol) were dissolved in heated ethanol (8 mL),
respectively. The ethanol solution (2 mL) of different bromoacetophenones (1 mmol)
and sodium ethylate (1.5 mmol) was added dropwise. The mixture was kept reacting
overnight at 55 ℃. Finally the target compounds RB1-RB23 were achieved via
column chromatography and refined by recrystallization. Detailed NMR spectra were
organized in "Supporting Information".
(E)-5-benzylidene-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one (RB1)
1
Yellow solid; m.p. 135-137 ℃; yield: 87%; H NMR (600 MHz, DMSO-d6) δ
8.09-8.08 (m, 2H), 7.88 (s, 1H), 7.74 (t, J = 7.4 Hz, 1H), 7.68 (d, J = 7.3 Hz, 2H),
7.61 (t, J = 7.9 Hz, 2H), 7.57 (t, J = 7.0 Hz, 2H), 7.54-7.51 (m, 1H), 5.29 (s, 2H); 13C
NMR (151 MHz, DMSO) δ 192.08, 192.04, 136.03, 135.46, 134.58, 133.60, 131.52,
130.96, 129.92, 129.43, 129.02, 126.55, 42.28; HRMS (ESI-TOF) m/z: [M+H]+ Calcd.
for C18H14NO2S2 340.4450, Found 340.4454.
(E)-5-(4-fluorobenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB2)
1
Yellow solid; m.p. 112-114 ℃; yield: 81%; H NMR (600 MHz, DMSO-d6) δ
8.10-8.08 (m, 2H), 7.90 (s, 1H), 7.77-7.72 (m, 3H), 7.61 (t, J = 5.8 Hz, 2H), 7.44-7.40
(m, 2H), 5.29 (s, 2H); 13C NMR (151 MHz, DMSO) δ 192.06, 191.91, 179.19, 164.57,
162.90, 135.45, 134.95, 134.59, 133.51, 133.45, 130.33, 130.31, 129.44, 129.01,
126.32, 126.31, 117.21, 117.06, 58.14, 42.28; HRMS (ESI-TOF) m/z: [M+H]+ Calcd.
for C18H13FNO2S2 358.4354, Found 357.4356.
(E)-5-(4-chlorobenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB3)
1
Yellow solid; m.p. 171-172 ℃; yield: 88%; H NMR (600 MHz, CDCl3) δ
8.07-8.06 (m, 2H), 7.85 (s, 1H), 7.69-7.67 (m, 1H), 7.55 (t, J = 7.9 Hz, 2H), 7.50-7.47
(m, 4H), 5.15 (s, 2H); 13C NMR (151 MHz, DMSO) δ 192.04, 191.99, 179.15, 136.11,
135.44, 134.64, 134.60, 132.56, 130.00, 129.44, 129.01, 127.26, 42.34; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd. for C18H13ClNO2S2 374.8980, Found 373.8975.
(E)-5-(4-bromobenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB4)
13

1
Yellow solid; m.p. 139-141 ℃; yield: 82%; H NMR (600 MHz, DMSO-d6) δ
8.09-8.08 (m, 2H), 7.85 (s, 1H), 7.78-7.76 (m, 2H), 7.75-7.73 (m, 1H), 7.63-7.60 (m,
4H), 5.29 (s, 2H); 13C NMR (151 MHz, DMSO) δ 192.04, 191.94, 179.16, 135.45,
134.75, 134.60, 132.93, 132.86, 132.69, 129.44, 129.02, 127.32, 125.10, 42.35.;
HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C18H13BrNO2S2 419.3405, Found
419.3401.
(E)-5-(4-methylbenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB5)
1
Yellow solid; m.p. 125-127 ℃; yield: 85%; H NMR (600 MHz, DMSO-d6) δ
8.10-8.08 (m, 2H), 7.84 (s, 1H), 7.74 (t, J = 7.4 Hz, 1H), 7.61 (t, J = 8.0 Hz, 2H), 7.57
13
(d, J = 8.1 Hz, 2H), 7.38 (d, J = 9.0 Hz, 2H), 5.28 (s, 2H), 2.38 (s, 3H); C NMR
(151 MHz, DMSO) δ 192.11, 191.65, 179.32, 142.03, 136.17, 135.48, 134.58, 131.06,
130.86, 130.58, 129.43, 129.01, 125.36, 42.20, 21.62; HRMS (ESI-TOF) m/z:
[M+H]+ Calcd. for C19H16NO2S2 354.4720, Found 354.4723.
(E)-5-(4-methoxybenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB6)
1
Brown solid; m.p. 63-65 ℃; yield: 79%; H NMR (600 MHz, DMSO-d6) δ
8.10-8.08 (m, 2H), 7.83 (s, 1H), 7.74 (t, J = 7.4 Hz, 1H), 7.66-7.64 (m, 2H) ,7.61 (t, J
13
= 7.9 Hz, 2H), 7.14-7.12 (m, 2H), 5.28 (s, 2H), 3.85 (s, 3H); C NMR (151 MHz,
CDCl3) δ 191.63, 190.47, 180.10, 161.88, 136.65, 134.84, 134.36, 132.68, 129.00,
128.64, 126.12, 123.42, 114.91, 55.56, 42.53; HRMS (ESI-TOF) m/z: [M+H]+ Calcd.
for C19H16NO3S2 370.4713, Found 370.4710.
(E)-5-(4-nitrobenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB7)
Brown solid; m.p. 66-68 ℃; yield: 72%; 1H NMR (600 MHz, CDCl3) δ 8.27-8.25
(m, 2H), 7.98-7.97 (m, 2H), 7.82 (s, 1H), 7.62-7.58 (m, 3H), 7.47 (t, J = 8.0 Hz, 2H),
5.08 (s, 2H); 13C NMR (151 MHz, DMSO) δ 195.07, 192.53, 191.95, 178.89, 148.26,
139.90, 134.62, 133.16, 131.80, 129.44, 129.25, 129.02, 124.82, 42.56; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd. for C18H13N2O4S2 385.4423 Found 385.4425.
14

(E)-5-(4-hydroxybenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB8)
1
Yellow solid; m.p. 133-135 ℃; yield: 83%; H NMR (600 MHz, DMSO-d6) δ
10.46 (s, 1H), 8.10-8.08 (m, 2H), 7.78 (s, 1H), 7.74 (t, J = 7.4 Hz, 1H), 7.61 (t, J = 7.7
13
Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 5.27 (s, 2H); C NMR
(151 MHz, DMSO) δ 192.18, 190.66, 179.52, 161.10, 136.72, 135.51, 134.55, 133.56,
129.42, 129.00, 124.50, 122.21, 116.99, 42.06; HRMS (ESI-TOF) m/z: [M+H]+ Calcd.
for C18H14NO3S2 356.4443, Found 356.4446.
(E)-5-(4-(dimethylamino)benzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidi
n-4-one (RB9)
1
Yellow solid; m.p. 126-128 ℃; yield: 87%; H NMR (600 MHz, DMSO-d6) δ
8.10-8.08 (m, 2H), 7.75-7.72 (m, 2H), 7.62-7.60 (m, 2H), 7.50 (d, J = 9.0 Hz, 2H),
6.85-6.83 (m, 2H), 5.25 (s, 2H), 3.05 (s, 6H); 13C NMR (151 MHz, DMSO) δ 192.27,
188.89, 179.63, 152.46, 137.42, 135.55, 134.53, 133.39, 129.42, 129.00, 120.30,
118.88, 112.65, 41.88; HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C20H19N2O2S2
383.5136, Found 383.5141.
(E)-N-(4-((4-oxo-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-5-ylidene)methyl)ph
enyl)acetamide (RB10)
Brown solid; m.p. 75-77 ℃; yield: 86%; 1H NMR (600 MHz, DMSO-d6) δ 10.32
(s, 1H), 8.10-8.08 (m, 2H), 7.78 (t, J = 6.4 Hz, 1H), 7.74 (t, J = 7.4 Hz, 1H), 7.63-7.60
13
(m, 2H), 5.28 (s, 2H), 2.10 (s, 3H); C NMR (151 MHz, DMSO) δ 192.14, 191.21,
179.43, 169.42, 142.40, 135.93, 135.49, 134.57, 132.27, 129.43, 129.01, 127.91,
124.16, 119.64, 42.18, 24.65; HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for
C20H16N2O3S2 396.4890, Found 396.4885.
(E)-5-(2-chlorobenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB11)
1
White solid; m.p. 116-118 ℃; yield: 85%; H NMR (600 MHz, DMSO-d6) δ
8.09-8.08 (m, 2H), 7.98 (s, 1H), 7.75-7.72 (m, 1H), 7.67-7.63 (m, 2H), 7.60 (t, J = 8.0
15

Hz, 2H), 7.56-7.52 (m, 2H), 5.30 (s, 2H); 13C NMR (151 MHz, DMSO) δ 192.77,
191.99, 178.71, 135.42, 135.36, 134.60, 132.83, 131.77, 130.90, 130.63, 130.21,
129.84, 129.43, 129.01, 128.74, 42.48; HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for
C18H13ClNO2S2 374.8980, Found 374.8984.
(E)-5-(3-chlorobenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB12)
1
Yellow solid; m.p. 113-115 ℃; yield: 80%; H NMR (600 MHz, DMSO-d6) δ
8.09-8.08 (m, 2H), 7.86 (s, 1H), 7.75-7.72 (m, 2H), 7.62-7.57 (m, 5H), 5.29 (s, 2H);
13C NMR (151 MHz, DMSO) δ 192.18, 192.02, 178.99, 135.84, 135.44, 134.58,
134.52, 134.26, 131.69, 131.00, 130.67, 129.42, 129.00, 128.76, 128.26, 42.37;
HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C18H13ClNO2S2 374.8980, Found
374.8985.
(E)-5-(2-methoxybenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB13)
1
Yellow solid; m.p. 150-152 ℃; yield: 80%; H NMR (600 MHz, DMSO-d6) δ
8.09-8.08 (m, 2H), 8.03 (s, 1H), 7.75-7.72 (m, 1H), 7.61 (t, J = 8.0 Hz, 2H), 7.54-7.51
(m, 1H), 7.49-7.48 (m, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 5.27 (s,
2H), 3.90 (s, 3H); 13C NMR (151 MHz, DMSO) δ 192.12, 191.99, 179.26, 158.87,
135.48, 134.57, 133.67, 130.65, 129.86, 129.42, 129.00, 126.52, 122.03, 121.64,
112.50, 56.29, 42.12; HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C19H16NO3S2
370.4713, Found 370.4714.
(E)-5-(3-methoxybenzylidene)-3-(2-oxo-2-phenylethyl)-2-thioxothiazolidin-4-one
(RB14)
1
Yellow solid; m.p. 182-184 ℃; yield: 92%; H NMR (600 MHz, DMSO-d6) δ
8.10-8.08 (m, 2H), 7.84 (s, 1H), 7.75-7.72 (m, 1H), 7.62-7.60 (m, 2H), 7.48 (t, J = 8.0
13
Hz, 1H), 7.25-7.23 (m, 2H), 7.11-7.09 (m, 1H), 5.29 (s, 2H), 3.81 (s, 3H); C NMR
(151 MHz, DMSO) δ 192.09, 192.07, 179.16, 160.19, 135.98, 135.47, 135.00, 134.58,
16

131.02, 129.43, 129.01, 126.90, 122.87, 117.43, 116.12, 55.79, 42.23; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd. for C19H15NO3S2 370.4713, Found 370.4710.
(E)-3-(2-oxo-2-phenylethyl)-2-thioxo-5-(3,4,5-trimethoxybenzylidene)thiazolidin-
4-one (RB15)
1
Yellow solid; m.p. 136-138 ℃; yield: 90%; H NMR (600 MHz, DMSO-d6) δ
8.10-8.08 (m, 2H), 7.81 (s, 1H), 7.74 (t, J = 7.4Hz, 1H), 7.61 (t, J = 7.9 Hz, 2H), 6.98
13
(d, J = 1.4 Hz, 2H), 5.27 (s, 2H), 3.83 (s, 6H), 3.74 (s, 3H); C NMR (151 MHz,
DMSO) δ 192.09, 191.57, 179.18, 153.73, 140.43, 136.37, 135.52, 134.57, 129.43,
129.11, 128.98, 125.57, 108.46, 60.68, 56.50, 42.11; HRMS (ESI-TOF) m/z: [M+H]+
Calcd. for C21H20NO5S2 430.5239, Found 430.5243.
(E)-5-(4-fluorobenzylidene)-3-(2-(4-methoxyphenyl)-2-oxoethyl)-2-thioxothiazoli
din-4-one (RB16)
1
Yellow solid; m.p. 168-170 ℃; yield: 80%; H NMR (600 MHz, DMSO-d6) δ
8.07 (t, J = 2.8 Hz, 1H), 8.06 (t, J = 1.9 Hz, 1H), 7.89 (s, 1H), 7.76-7.74 (m, 2H),
7.43-7.40 (m, 2H), 7.13-7.10 (m, 2H), 5.24 (s, 2H), 3.88 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ 191.39, 189.90, 179.84, 164.75, 164.54, 163.06, 135.09, 132.65, 132.59,
131.07, 129.85, 129.83, 127.77, 126.06, 126.04, 116.74, 116.59, 114.20, 55.66, 42.61;
HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C19H15FNO3S2 388.4617, Found
388.4618.
(E)-5-(4-chlorobenzylidene)-3-(2-(4-methoxyphenyl)-2-oxoethyl)-2-thioxothiazoli
din-4-one (RB17)
1
Yellow solid; m.p. 111-113 ℃; yield: 77%; H NMR (600 MHz, DMSO-d6) δ
8.07 (t, J = 2.9 Hz, 1H), 8.06 (t, J = 2.0 Hz, 1H), 7.87 (s, 1H), 7.70-7.69 (m, 2H),
7.65-7.62 (m, 2H), 7.14-7.11 (m, 2H), 5.24 (s, 2H), 3.88 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ 191.41, 189.84, 179.77, 164.55, 136.98, 134.81, 132.00, 131.60, 131.07,
129.64, 127.75, 126.90, 114.20, 55.65, 42.65; HRMS (ESI-TOF) m/z: [M+H]+ Calcd.
for C19H15ClNO3S2 404.9243, Found 404.9239.
(E)-5-(4-bromobenzylidene)-3-(2-(4-methoxyphenyl)-2-oxoethyl)-2-thioxothiazoli
din-4-one (RB18)
17

1
Brown solid; m.p. 65-67 ℃; yield: 75%; H NMR (600 MHz, DMSO-d6) δ
8.06-8.03 (m, 2H), 7.83 (s, 1H), 7.65-7.63 (m, 2H), 7.42-7.41 (m, 2H), 5.12 (s, 2H),
3.93 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 191.43, 189.84, 179.78, 164.55, 134.87,
132.60, 132.41, 131.73, 131.07, 127.75, 127.03, 125.44, 114.20, 58.51, 55.65, 42.65,
18.46; HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for C19H15BrNO3S2 449.3669, Found
449.3666.
(E)-3-(2-(4-methoxyphenyl)-2-oxoethyl)-5-(4-methylbenzylidene)-2-thioxothiazoli
din-4-one (RB19)
1
Yellow solid; m.p. 145-147 ℃; yield: 81%; H NMR (600 MHz, DMSO-d6) δ
8.08-8.06 (m, 2H), 7.83 (s, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H),
13
7.13-7.11 (m, 2H), 5.23 (s, 2H), 3.86 (s, 3H), 2.38 (s, 3H) ; C NMR (151 MHz,
DMSO) δ 191.77, 190.38, 179.39, 164.30, 142.00, 136.08, 131.46, 131.06, 130.87,
130.58, 128.32, 125.39, 114.64, 56.18, 41.99, 21.62; HRMS (ESI-TOF) m/z: [M+H]+
Calcd. for C20H18NO3S2 384.4983, Found 384.4979.
(E)-5-(2-chlorobenzylidene)-3-(2-(4-methoxyphenyl)-2-oxoethyl)-2-thioxothiazoli
din-4-one (RB20)
1
Brown solid; m.p. 55-57 ℃; yield: 80%; H NMR (600 MHz, DMSO-d6) δ
8.07 (t, J = 1.8 Hz, 1H), 8.05 (t, J = 2.1 Hz, 1H), 7.99 (s, 1H), 7.67-7.64 (m, 2H),
7.55-7.53 (m, 2H), 7.12-7.10 (m, 2H), 5.24 (s, 2H), 3.88 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ 191.88, 189.88, 179.17, 164.55, 136.41, 132.30, 132.22, 131.54, 131.08,
130.51, 129.42, 129.24, 127.74, 127.37, 114.20, 55.66, 42.65; HRMS (ESI-TOF) m/z:
[M+H]+ Calcd. for C19H15ClNO3S2 404.9243, Found 404.9238.
(E)-5-(3-chlorobenzylidene)-3-(2-(4-methoxyphenyl)-2-oxoethyl)-2-thioxothiazoli
din-4-one (RB21)
Brown solid; m.p. 55-57 ℃; yield: 80%; 1H NMR (600 MHz, CDCl3) δ 8.05-8.03
(m, 2H), 7.81 (s, 1H), 7.51 (s, 1H), 7.44-7.42(m, 3H), 7.01-6.99 (m, 2H), 5.11 (s, 2H),
3.92 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 191.71, 189.80, 179.60, 164.54, 135.38,
135.33, 134.45, 131.06, 130.65, 130.50, 130.06, 128.41, 127.89, 127.74, 114.20,
18

58.50, 55.65, 42.68, 18.46; HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for
C19H15ClNO3S2 404.9243, Found 404.9240.
(E)-5-(2-methoxybenzylidene)-3-(2-(4-methoxyphenyl)-2-oxoethyl)-2-thioxothiaz
olidin-4-one (RB22)
1
Brown solid; m.p. 55-57 ℃; yield: 80%; H NMR (600 MHz, DMSO-d6) δ 8.06
(d, J = 7.4 Hz, 2H), 8.03 (s, 1H), 7.53-7.49 (m, 2H), 7.18 (d, J = 7.7 Hz, 1H), 7.11 (d,
J = 7.5 Hz, 3H), 5.22 (s, 2H), 3.91 (s, 3H), 3.88 (s, 3H); 13C NMR (151 MHz, CDCl3)
δ 191.42, 190.14, 179.96, 164.48, 158.91, 132.65, 132.02, 131.07, 129.77, 127.83,
126.24, 122.77, 120.98, 114.15, 111.20, 55.64, 55.55, 42.36; HRMS (ESI-TOF) m/z:
[M+H]+ Calcd. for C20H18NO4S2 400.4976, Found 400.4975.
(E)-3-(2-(4-methoxyphenyl)-2-oxoethyl)-2-thioxo-5-(3,4,5-trimethoxybenzylidene)
thiazolidin-4-one (RB23)
1
Brown solid; m.p. 55-57 ℃; yield: 80%; H NMR (600 MHz, DMSO-d6) δ
8.08-8.06 (m, 2H), 7.81 (s, 1H), 7.13-7.10 (m, 2H), 6.99 (s, 2H), 5.22 (s, 2H), 3.89 (s,
3H), 3.83 (s, 6H), 3.74 (s, 3H); 13C NMR (151 MHz, DMSO) δ 191.72, 190.35,
179.24, 164.30, 153.74, 140.42, 136.28, 131.43, 129.14, 128.37, 125.62, 114.64,
108.45, 60.73, 56.50, 56.18, 41.87; HRMS (ESI-TOF) m/z: [M+H]+ Calcd. for
C22H22NO6S2 460.5502, Found 460.5498.
4. 2. Biological assay
4. 2. 1. Minimum inhibitory concentration
The minimun inhibitory concentrations were determined according to the
references.22,37 Mtb was grown in a 7H9 medium with OADC for 24 h at 37 ℃ under
microaerobi conditions (5% O2, 10% CO2, and 85% N2). The other strains
Methicillin-sensitive Staphylococcus aureus ATCC 25923, methicillin-resistant
Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27583
were grown in Brucella Broth supplemented with 10% heat-inactivated horse serum.
Inoculums containing 1 × 105 bacteria were seeded in 96 well plates with different
concentrations of test compounds and incubated at 37 ℃ for 48 h. Absorbance was
19

measured at 590 nm. Wells without test compounds and with media only served as
control and blank, respectively. The percentages of growth inhibition were calculated,
and the concentrations of compounds producing 80% inhibition were considered as
MIC values.
4. 2. 2. Mtb InhA inhibition assay
The standard method in references was used.22,37 After the expression,
purification, ultrasonication and SDS-PAGE procedure, Mtb InhA was collected.
Then Mtb InhA inhibition was measured by monitoring the initial velocities (120 s) of
the decrease in the absorbance of NADH at 340 nm, 25 ℃. The standard reaction
mixture of 1.0 mL included buffer (30 mM PIPES, 150 mM NaCl, 1 mM EDTA, pH
6.8), 200 μM trans-2-decenoyl-N-acetyl cysteamine, 100 μM NADH and 1 μg
recombinant Mtb InhA. The data of positive control was close to literature value. All
assays were performed in triplicates.
4. 2. 3. DNA gyrase and FabH inhibition assay
The standard protocols of DNA gyrase40 and FabH41 inhibition assays in
references were used respectively.
4. 3. Cytotoxicity
293T (human embryonic kidney cell line) was used for cytotoxicity evaluation
via MTT method according to reference.43
4. 4. In silico assay
The Chem. 3D ultra 14.0 software [Chemical Structure Drawing Standard;
Cambridge Soft corporation, USA (2014)] was employed to construct the
three-dimensional structures of the aforementioned compounds. The CHARMm based
force field was recruited for minimization and molecular docking simulation. The
ADMET study was conducted by Calculate Molecular Properties in Small Molecules
module of Discovery Studio 3.5 (Accelrys, Inc. San Diego, CA). The ADMET
properties map and data were provided by the ADMET Descriptors tool. Molecular
docking was carried out by CDOCKER protocol as described.43 In this work, the
structures of Mtb InhA (PDB code: 4R9S) was used as the receptor. 3D-QSAR model
was built consulting the reference.44 After selecting the training set and the test set
20

(20%, RB5, RB8, RB10, RB13 and RB15), the Create 3D QSAR Model protocol was
used to provide a reliable 3D-QSAR model with visualized electrostatic and Van der
Waals potential maps.
Acknowledgment
This work was supported by the Major Science and Technology Program for Water
Pollution Control and Treatment, China (No. 2017ZX07602-002), the Public Science
and Technology Research Funds Projects of Ocean (No. 201505023), and the National
Natural Science Foundation of China (No. 21602103).
Conflict of Interest The authors declare no conflict of interest.
Supporting Information The NMR data of synthesized compounds.
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> 23 rhodanine derivatives were synthesized through a two-round screening.
> Their anti-bacterial activity was evaluated for the first time.
> The top hit showed improved growth-blocking effect on Mtb and low toxicity.
> Molecular docking indicated possible binding pattern and key interacted residues.
25

A series of rhodanine derivatives RB1-RB23 were synthesized through a
two-round screening with their Mycobacterial tuberculosis (Mtb) InhA inhibitory
activity and Mtb growth blocking capability evaluated. The improved
growth-blocking effect on Mtb and low toxicity were attractive for further
development. The docking simulation the key interacted residues as Ser20, Phe149,
Lys165 and Thr196 in the binding pattern.
26