Design and synthesis of thiazole derivatives as potent FabH inhibitors with antibacterial activity

Article abstract of DOI:10.1016/j.ejmech.2013.11.020

Components of fatty acid biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. Compounds of series A (4a-4g) and series B (5a-5g) were synthesized by the formation of an amine bond between aromati

Full text of DOI:10.1016/j.ejmech.2013.11.020

European Journal of Medicinal Chemistry 75 (2014) 438e447
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of thiazole derivatives as potent FabH inhibitors
with antibacterial activity
,
b
,
,
b
,
,
,
,b
Jing-Ran Li a 1, Dong-Dong Li a 1, Rong-Rong Wang a b, Jian Sun a b, Jing-Jun Dong a
,
Qian-Ru Du a b, Fei Fang a b, Wei-Ming Zhang a , Hai-Liang Zhu
,
,
,
b,
a,b,
*
*
a Nanjing Institute for the Comprehensive Utilization of Wild Plant, Nanjing 210042, People’s Republic of China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Components of fatty acid biosynthetic pathway have been identified as attractive targets for the
development of new antibacterial agents. Compounds of series A (4ae4g) and series B (5ae5g) were
synthesized by the formation of an amine bond between aromatic acid and 4-phenylthiazol-2-amine or
4-(4-bromophenyl)thiazol-2-amine. These thiazole derivatives have evaluated as potent FabH inhibitors.
Nineteen compounds (4be4h, 4k, 4l, 5ae5h, 5k, 5l) are reported for the first time. Most of the syn-
thesized compounds exhibited antibacterial activity in the MTT assay. The MIC value of these compounds
Received 19 June 2013
Received in revised form
2 November 2013
Accepted 14 November 2013
Available online 4 December 2013
ranged from 1.56
ability with IC50 value ranging from 5.8
pound 5f has exhibited the best antibacterial and Escherichia coli FabH inhibitory activity. Docking
simulation and the QSAR study was conducted for learning about binding mode and the relationship
between structure and activity.
m
g/mL to 100
m
g/mL. Moreover, the tested compounds also showed FabH inhibition
M to 48.1 M. The IC50 values are near the MIC values. Com-
Keywords:
m
m
Thiazole derivatives
Antibacterial
FabH
Molecular docking
3D-QSAR
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Gram-positive and negative organisms [7]. These attributes suggest
that FabH could be a promising target for designing novel antimi-
Saturated fatty acid biosynthesis pathway has emerged as a
prime candidate for developing an important and novel antibac-
terial [1]. The ubiquitous type II fatty acid synthesis system (FAS) in
bacteria is not only essential to cell survival, but also exhibits sig-
nificant differences between bacterial and human fatty acid syn-
thesis, including the organization, the structure of enzymes and
their specific roles. As a result, the differences mentioned above
make this system be an attractive target for antibacterial drug
crobial drugs with selectivity, low-toxicity, and broad-spectrum
antibacterial activity [8,9].
At the moment, the rise and prevalence of multidrug-resistant
pathogenic bacteria have brought us challenges which increase
the need to create new types of antibacterial agents and expand the
bio-utility of old drugs [2]. Thiazoles and their derivatives have
attracted continuing interest over the years for their various bio-
logical activities [10]. In past years, they have been used for the
treatment of allergies [11], hypertension [12], inflammation [13],
schizophrenia [14], bacterial [15], HIV infections [16], hypnotics
[17], and more recently for the treatment of pain [18,20]. Further-
more, Kitagawa et al. found that thiazole derivatives not only
showed potent FabI and FabK inhibitory activity, but also had an
antibacterial effect [19].
In view of these findings, we describe here the synthesis of
thiazole derivatives containing an amide skeleton as antibacterial
agents [21,22]. This thiazole and amide skeleton combination was
suggested in an attempt to investigate the inhibitory activity
against Escherichia coli FabH. The antimicrobial activity against two
Gram-negative bacterial strains (E. coli and Pseudomonas aerugi-
nosa) and two Gram-positive bacterial strains (Bacillus subtilis and
Staphylococcus aureus) of these thiazole derivatives was deter-
mined by applying the standard MTT colorimetric method. The
discovery [3,4]. b-Ketoacyl-acyl carrier protein (ACP) synthase III,
also known as FabH or KAS III, plays an important and regulatory
role in bacterial FAS [5]. As is well-known, FabH initiates the fatty
acid elongation cycles (Fig. 1), and is involved in feedback regula-
tion of biosynthetic pathway via product inhibition [6]. In addition,
FabH proteins from both Gram-positive and Gram-negative bacte-
ria are highly conserved at the sequence and structural level, while
there are no significantly homologous proteins in humans. More-
over, the residues composing the active site are almost invariant in
* Corresponding authors. State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210093, People's Republic of China. Tel./fax: þ86 25
83592672.
E-mail addresses: botanyzh@163.com (W.-M. Zhang), zhuhl@nju.edu.cn
(H.-L. Zhu).
1
These two authors equally contribute to this paper.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.020

J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
439
Fig. 1. FabH-catalyzed initiation reaction of fatty acid biosynthesis.
antimicrobial activity was shown by the MIC (minimum inhibitory
G
(corresponding MIC: 3.13, 6.25, 1.56 and 6.25
mg/mL),
concentrations) values. The enzyme assay was also performed to
explore the anti-FabH activity with the selected compounds.
Docking simulations were performed using the X-ray crystallo-
graphic structure of the FabH of E. coli (PDB code: 3IL9) in complex
with an inhibitor to explore the binding modes of these compounds
at the active site. The structureeactivity relationship (SAR) study
was also conducted for a better understanding of their activity and
their structure relationship using DS 3.5 (Discovery Studio 3.5,
Accelrys, Co. Ltd).
respectively.
The modification of parent compounds with various sub-
stituents such as halogen, methoxyl and ethoxy groups was per-
formed to explore the structureeactivity relationships of theses
thiazole derivatives. Moreover, we measured the MIC values for
each derivative and compared each series (Table 1e3). From Table 2
we found that series B (compounds 5ae5g) showed better anti-
bacterial activity than series A (compounds 4ae4g). The mean
values of series B in antibacterial activity against four bacterial
strains: E. coli, P. aeruginosa, B. subtilis, and S. aureus, were all lower
than series A, respectively. What’s more, the maximums and min-
imums of series B in antibacterial activity were also lower than that
of series A. This result corresponded directly with their mean
values. As shown in Table 3, the total mean value of series A was
2. Results and discussion
2.1. Chemistry
29.93 mg/mL and the series B was 14.85 mg/mL, which indicated that
The synthesis of key intermediates, 3a and 3b, is outlined in
series B had better antibacterial activity than series A. The struc-
tural difference between these two series confirmed that Br at the
para-position of the benzene ring was beneficial to the antibacterial
activity of the compounds. Still, series A and B both exhibited better
activity against Gram-positive bacterial strains compared to Gram-
negative bacterial strains. The mean values of series A and B against
Gram-positive and Gram-negative bacterial strains were
23.96 < 32.29 and 11.98 < 17.97, respectively (Table 3).
Scheme 1 [24]. The different substituted a-bromo-acetophenones
(2a, 2b) were synthesized by adding bromine (Br2) in the presence
of aluminum chloride (AlCl3) as catalyst in THF liquid containing
acetophenone or 1-(4-bromophenyl)ethanone. Then, treatment of
freshly prepared 2a or 2b with thiourea in EtOH produced the 4-
phenyl-1,3-thiazol-2-amine (3a) and 4-(4-bromo-phenyl)-1,3-
thiazol-2-amine (3b) in 52% and 47% yields, respectively. Accord-
ingly, the acetyl chloride was synthesized according to Scheme 2.
Treatment of 3a and 3b with acetyl chloride in acetone in an ice-
bath yielded the desired compounds 4ae4h and 5ae5h with
moderate to high overall yields. Moreover, as shown in Scheme 3,
treatment of the amines, 3a and 3b, with benzoic, naphthoic acid or
4-morpholine formic acid in the presence of EDC$HCl and HOBt in
CH2Cl2 yielded the desired compound series 4ie4l and 5ie5l.
Although the compounds of 4he4l; 5he5l had ꢀ100
mg/mL MIC
values, we still could conclude that the shorter chain amide group
and larger volume acid substituent group at the amide binding sites
may be related to their antimicrobial activities. Specifically, com-
pounds 4g, 4h and 5h showed similar moderate antibacterial ac-
tivity (MIC value: 25e100 mg/mL) against all tested bacterial strains.
This indicated that the introduction of a methyl or ethyl (electron-
donating group) ether could not enhance the inhibitory activity.
Compounds 4ie4l and 5ie5l were found to be inactive against all
tested bacterial strains, which indicated that the introduction of
morpholine or naphthalene led to the decrease of antibacterial
activity. Compound 5g showed better antibacterial activity than 4g,
which may be due to the Br at the para-position of the benzene ring
attached to the thiazole ring.
2.2. Biological activity and molecular modeling
2.2.1. Antibacterial activity and structureeactivity relationships
analysis
All compounds (4ae4l and 5ae5l) were screened for antibac-
terial activity against two Gram-negative bacterial strains: E. coli,
P. aeruginosa and two Gram-positive bacterial strains: B. subtilis,
S. aureus by applying the MTT colorimetric method [26]. The MIC
(minimum inhibitory concentrations) values of these compounds
were presented in Table 1. Antibacterial agents Kanamycin B and
Penicillin G were also screened under identical conditions as pos-
itive controls. The results revealed that most synthesized com-
pounds exhibited antibacterial activity with MIC values ranging
Compared to 4a, compounds 4be4f showed more potent anti-
bacterial activity. The MIC values of compound 4a (25e100
mg/mL)
and 4be4f (3.13e50 g/mL) suggested that the introduction of
m
halogen substituent at meta or para position of benzene ring
binding with amide could increase antibacterial activity. In addi-
tion, for substituent, at the para position of the benzene ring of the
from 1.56 mg/mL to 100 mg/mL. Among of all the tested twenty-four
compounds, fourteen compounds with completed activity infor-
mation were marked in Table 1. Overall, compounds 4b, 4e, 4f, 5b,
5e and 5f exhibited the greatest antibacterial activity. Particularly,
compound 5f which had MIC values of 1.56, 3.13, 1.56 and 6.25 mg/
mL against E. coli, P. aeruginosa, B. Subtilis and S. aureus, respectively,
Scheme 1. Synthesis of 3a and 3b. Reagents and conditions: (i) Br2, AlCl3, THF, 0 ꢁC; (ii)
thiourea, EtOH, reflux.
and was comparable to the broad-spectrum antibiotic kanamycin B
(corresponding MIC: 1.56, 3.13, 0.39 and 3.13 mg/mL) and Penicillin

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J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
Compounds
R
H
R1
Compounds
R
R1
4a
5a
Br
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
5b
5c
5d
5e
5f
Br
Br
Br
Br
Br
Br
Br
4g
4h
5g
5h
Scheme 2. Synthesis of 4ae4h; 5ae5h. Reagents and conditions: (i) SOCl2, 70e80 ꢁC, reflux, 3 h; (ii) acetone/K2CO3, ice-bath, reflux for 5 h.
Compounds
R
H
R2
Compounds
R
R2
4i
5i
Br
4j
4k
4l
H
H
H
5j
5k
5l
Br
Br
Br
Scheme 3. Synthesis of 4ie4l; 5ie5l. Reagents and conditions: (i) acid, EDC, HOBt, CH2Cl2, RT.
amide, the antibacterial activity of these compounds was slightly
enhanced in the following order: Br < Cl < F. The comparison be-
tween 5a and 5be5f was similar to the example of 4a and 4be4f.
More interestingly, compounds with substituents at the para
position of the benzene ring of the amide had less antibacterial
activity than those at meta-position, which could be seen from the
comparison: 4b > 4e, 4c > 4f; 5b > 5e, 5c > 5f. This indicated that
the location of the substituent also had an influence on the

J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
441
Table 1
antibacterial activity. So, the compounds of series B with halogen
substituent could be the potential antibacterials.
MIC (minimum inhibitory concentrations)
compounds.
(
m
g/mL) values of the synthesized
Compounds
Microorganismsa
Gram-negative
2.2.2. E. coli FabH inhibitory activity and QSAR study
The E. coli FabH inhibitory activity of some thiazole derivatives
containing an amide skeleton (4b, 4c, 4e, 4f, 4i and 5be5g, 5i) are
examined and the results were summarized in Table 4. The
CDOCKER_INTERACTION_ENERGY values of these compounds
calculated with DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd) are
shown in Fig. 2 [25,27]. The lower value indicates better stability of
the interaction FabH protein complex (PDB code: 3IL9). The colored
dots represent the tested compounds in E. coli FabH inhibitory ac-
tivity assay in Fig. 2. Most of the tested compounds displayed E. coli
FabH inhibitory activity. Compounds 5be5g with substituent on
the benzene ring located at the C4 position of the thiazole ring
showed better E. coli FabH inhibitory activity than compounds 4b,
4c, 4e and 4f which had no substituent on this ring. Another
comparison of 5c and 5e, 5d and 5f, respectively, also indicated that
compounds with m-substituted Cl or Br located at the benzene ring
attached to the amide exhibited better inhibitory activity than
those compounds with p-substituents. In addition, inactive com-
pounds 4i and 5i had no detectable FabH inhibitory activity
Gram-positive
E. coli
ATCC35218
P. aeruginosa
ATCC13525
B. subtilis
ATCC6633
S. aureus
ATCC6538
4ab
100
12.5
25
100
25
25
50
12.5
12.5
25
12.5
6.25
100
>100
>100
>100
50
>100
25
6.25
12.5
25
6.25
1.56
6.25
50
>100
>100
>100
100
25
12.5
25
4bb
4cb
4db
50
50
50
4eb
6.25
12.5
50
6.25
12.5
25
6.25
3.13
50
>100
>100
>100
>100
>100
25
4fb
4gb
4h
4i
4j
4k
50
50
>100
>100
>100
>100
50
12.5
12.5
25
6.25
1.56
12.5
50
>100
50
>100
>100
>100
>100
50
12.5
25
50
12.5
3.13
3.13
25
>100
100
>100
>100
3.13
6.25
4l
5ac
5bc
12.5
12.5
25
5cc
5dc
5ec
3.13
6.25
3.13
>100
>100
>100
>100
>100
3.13
6.25
5fc
5gc
(IC50 > 100 mM). However, in Fig. 2, we found that the CDOCK-
ER_INTERACTION_ENERGY values of compounds 4i (ꢂ49.56) and 5i
(ꢂ57.08) were at a tolerated level. Thus the lack of inhibitory ac-
tivity displayed by these two compounds may be caused by some
other reasons, such as poor permeability and solubility.
5h
5i
5j
5k
>100
50
5l
Kanamycin Bd
Penicillin Gd
1.56
3.13
0.39
1.56
These IC50 values with E. coli FabH were correlated with their
antibacterial activities. Just as discussed in Tables 1 and 2, series B
showed better antibacterial activity than series A. Compounds 4b,
4f, 5b, 5e and 5f exhibited the greatest antibacterial and FabH
inhibitory activity. This demonstrated that the antibacterial activ-
ities of the synthesized compounds were likely due to their FabH
inhibitory activities. Besides, the CDOCKER_INTERACTION_ENERGY
values of these compounds were also at a lower level. Based on
above analysis, some compounds could be the potential antibac-
terials, especially 5f, which exhibited the greatest antibacterial and
FabH inhibitory activity.
As a supplementary analysis, a 3D-QSAR study was performed
by means of the DS 3.5 software (Discovery Studio 3.5, Accelrys, Co.
Ltd), aiming for further exploration of structure and activity re-
lationships of these compounds. The 3D-QSAR model was created
with the corresponding pIC50 values which obtained from the IC50
a
Bacterial strains were kindly supplied by State Key Laboratory of Pharmaceutical
Biotechnology, Nanjing University.
b
Compounds 4ae4g were belonged to series A.
Compounds 5ae5g were belonged to series B.
Used as a positive control.
c
d
Table 2
The mean MIC (minimum inhibitory concentrations) (
four bacterial strains.
mg/mL) values of the tested
Series and value
E. coli
ATCC35218
P. aeruginosa
ATCC13525
B. subtilis
ATCC6633
S. aureus
ATCC6538
Series Aa
Series Bb
Averc
28.75
12.5
100
11.876
1.56
50
26.25
12.5
100
20.626
3.13
50
22.5
6.25
100
11.25
1.56
25
23.75
3.13
50
11.876
3.13
25
Minic
Maxc
Aver
Mini
Max
(mM) values of FabH protein inhibition. The pIC50 values were
derived from an online calculator developed from an Indian me-
dicinal chemistry lab (http://www.sanjeevslab.org/tools-IC50.
html) [23]. The observed and predicted values with correspond-
ing residual values in 3D-QSAR model of the training set and test set
molecules were listed in Table 5. For a further step, a graphical
relationship of the observed pIC50 values versus the predicted
values is shown in Fig. 3. The linear R2 value was 0.751.
a
Series A comprise compounds 4ae4g.
Series B comprise compounds 5ae5g.
Aver represent average value, mini represent minimum value, max represent
b
c
maximum value. The minimum value and maximum value are excluded from the
average value calculation.
As shown in Fig. 4, the potent compounds containing an
electron-withdrawing atom and small substituents not only
circumvent the red (in the web version) subregion but also get
more close to the favorable yellow spaces. This reference figure
displayed that these small molecules could have favorable elec-
trostatic interactions with the protein with minimal steric clashes.
This QSAR study could predict that electron-withdrawing atom and
small substituents located at the benzene ring may enhance bind-
ing affinity of the thiazole derivatives.
Table 3
The mean MIC (minimum inhibitory concentrations) (
negative and Gram-positive bacterial strains with the total mean values.
mg/mL) values of Gram-
Seriesa Gram-negative
Gram-positive Total
Averb Minib Maxb Aver
Mini Max Aver
Mini Max
29.93 3.13 100
A
B
32.29 12.5
17.97 1.56
100
50
23.96 3.13
11.98 1.56
100
25
14.85 1.56
50
2.2.3. Binding model of 5f and Penicillin G with FabH protein
complex
In order to gain better understanding of the correlations be-
tween binding model and biological activity, a docking study was
a
Series A comprise compounds 4ae4g; Series B comprise compounds 5ae5g.
Aver represent average value, mini represent minimum value, max represent
b
maximum value. The minimum value and maximum value are excluded from the
average value calculation.

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J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
Table 4
E. coli FabH inhibitory activity, CDOCKER_INTERACTION_ENERGY and Macrophage
CC50 values.
Compounds E. coli FabH CDOCKER_INTERACTION_ENERGY Macrophage
IC50
(
m
M)
(kcal/mol)
CC50 SD (mM)
4b
4c
4e
4f
38.4 ꢃ 0.41 ꢂ42.7372
41.8 ꢃ 0.38 ꢂ47.8179
12.3 ꢃ 0.15 ꢂ43.5665
26.9 ꢃ 0.33 ꢂ41.9839
318.4 ꢃ 27.44
357.1 ꢃ 33.71
264.2 ꢃ 21.43
256.8 ꢃ 24.37
aNA
264.3 ꢃ 27.82
285.7 ꢃ 26.58
314.9 ꢃ 28.78
193.6 ꢃ 27.44
219.5 ꢃ 21.43
192.3 ꢃ 19.01
NA
4i
>100
ꢂ49.5635
5b
5c
5d
5e
5f
29.3 ꢃ 0.31 ꢂ43.5641
34.7 ꢃ 0.39 ꢂ49.6976
48.1 ꢃ 0.44 ꢂ50.3613
9.3 ꢃ 0.17
5.8 ꢃ 0.24
6.6 ꢃ 0.11
>100
ꢂ46.7522
ꢂ52.532
ꢂ53.435
ꢂ57.0833
ꢂ61.7367
5g
5i
Penicillin G 4.9 ꢃ 0.13
228.9 ꢃ 20.43
Note: the experiment for E. coli FabH inhibitory activity repeats 3 times.
a
NA: not assayed.
Fig. 3. Linear fit curve of the observed pIC50 values versus the predicted values of the
training set part and the test set part.
the binding site well. The binding model of 5f in Fig. 6 was the
active site of the E. coli FabH protein had two Pi-cation and one
hydrogen bond. The thiazole ring and benzene ring formed two Pi-
ꢀ
ꢀ
cation bond (PieN: 5.9 A; PieN: 5.4 A) with the nitrogen atom of
Arg36 and the sulfur-atom locating at the thiazole ring formed a
ꢀ
hydrogen bond (S/HeN: 2.2 A) with the hydrogen atom of Asn247.
The two amino acid residues Arg36 and Asn247 located in the
binding pocket could be indispensable for the active conformation
of the compounds. Though 5f had a different binding mode with
the E. coli FabH protein than Penicillin G, it exhibited stabilizing
binding interactions with the receptor.
Fig. 2. The CDOCKER_INTERACTION_ENERGY (kcal/mol) obtained from the docking
study (Discovery Studio 3.5, Accelrys, Co. Ltd).
performed by fitting compound 5f or Penicillin G into the active site
of E. coli FabH (PDB code: 3IL9). The binding model of compound 5f
and Penicillin G with FabH protein are depicted in Figs. 5 and 6. In
Fig. 5, the 2D projection drawing of compound 5f or Penicillin G
with FabH shows that the two small molecular ligands both fit into
Table 5
Experimental, predicted inhibitory activity of compounds 4b, 4c, 4e, 4f and 5be5g
against E. coli FabH protein by 3D-QSAR model.
Compounds
E. coli FabH protein
Residue value
Actual pIC50
Predicted pIC50
4b
4c
4e
4f
5b
5c
5d
5e
5f
4.42
4.38
4.91
4.57
4.53
4.46
4.32
5.27
5.01
5.25
4.50086
4.36322
4.96114
4.53882
4.48553
4.51869
4.75605
5.26691
4.9519
ꢂ0.08086
0.016781
ꢂ0.05114
0.031179
0.044468
ꢂ0.05869
ꢂ0.43605
0.003093
0.058096
ꢂ0.01929
5g
5.26929
Fig. 4. Isosurface of the 3D-QSAR model coefficients.

J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
443
Fig. 5. The 2D map of Penicillin G and compound 5f.
2.2.4. Cytotoxic
Gel 60 F254 (Merck). Melting points (uncorrected) were determined
on a XT4MP apparatus (Taike Corp., Beijing, China). ESI mass
spectra were obtained on a Mariner System 5304 mass spectrom-
eter, and 1H NMR spectra were recorded on a Bruker DPX300
spectrometer at 25 ꢁC with TMS and solvent signals allotted as
internal standards. Chemical shifts were reported in ppm (d).
Elemental analyses were performed on a CHNeO-Rapid instrument
and were within ꢃ0.4% of the theoretical values.
Compounds were evaluated for their toxicity against human
macrophage with the median cytotoxic concentration (CC50) data
of tested compounds by the MTT assay. As showed in Table 4, these
compounds were tested at multiple doses to study the viability of
macrophage. The results showed that they almost did not exhibit
cytotoxic activity.
3. Conclusion
A series of novel thiazole derivatives containing an amide
skeleton were synthesized and their antibacterial activities against
E. coli, P. aeruginosa, B. Subtilis and S. aureus were also assayed by
applying standard MTT colorimetric method. Some compounds
showed moderate antibacterial activities that were generally
consistent with their potencies as FabH inhibitors. In particular,
compound 5f was the most potent one with an MIC of 1.56e
6.25 mg/mL against the tested bacterial strains, which was compa-
rable to the positive control Kanamycin B and Penicillin G. It also
exhibited the best E. coli FabH inhibitory activity with IC50 of
5.8 mM. The enzyme assay data and the molecular docking results
suggest that compound 5f as a potential inhibitor of E. coli FabH
protein. This study reports compounds with thiazole frameworks
containing an amide skeleton may serve as model compounds for
the design and development of antibacterial agents. Preliminary
structureeactivity relationships (3D-QSAR) and molecular
modeling study provide further insight into the interactions be-
tween FabH and these compounds.
4. Experimental
4.1. General
All commercially available reagents were obtained from various
producers and used without further purification. The reactions
were monitored and the purity of the compounds was checked by
thin layer chromatography (TLC) using aluminum sheets with Silica
Fig. 6. The binding modes of the complexes.

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J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
4.2. Chemistry synthesis procedure
312.1 (C17H13FN2OS, [M þ H]þ). Anal. Calcd for C17H12FN2OS: C,
65.37; H, 4.19; N, 8.97. Found: C, 65.73; H, 4.48; N, 8.61%.
4.2.1. General procedure for the synthesis of 4-phenylthiazol-2-
amine derivatives 3a, 3b
4.2.6. 2-(4-Chlorophenyl)-N-(4-phenylthiazol-2-yl)acetamide (4c)
Pink crystal. Yield, 73%. Mp 199e200 ꢁC. 1H NMR (300 MHz,
Just as outlined in Scheme 1, the compound 1a (20 mmol) or 1b
(20 mmol) and catalytic amount of AlCl3 was added to a three-
necked flask containing THF (40 mL). Then the mixture was
cooled to 0 ꢁC and Br2 (20 mmol) stored in the constant pressure
funnel was slowly and cautiously added to the mixture in 20 min.
After the dropwise add completed, the mixture was evaporated
under reduced pressure immediately. Then, precipitation crystal-
lization was washed with mixed solvent of water and petroleum
ether to get 2a, 2b. Freshly prepared 2a (20 mmol) or 2b (20 mmol)
and thiourea (22 mmol) in anhydrous EtOH (30 mL) was refluxed
for 30 min. After that, the solvent was evaporated under reduced
pressure and then the saturated aqueous NaHCO3 was added to
make the mixture basic (pH 8e9). Then the mixture was extracted
with CH2Cl2 (3 ꢄ 30 mL). The combined organic phases were dried
with anhydrous Na2SO4. After removal of all the solvent, the residue
was purified by recrystallization from anhydrous EtOH to afford
product 3a or 3b, 91% and 93%, respectively.
CDCl3):
d
3.68 (s, 2H, CH2), 7.16 (t, J ¼ 6.03 Hz, 3H), 7.32e7.42 (m,
5H), 7.8 (d, J ¼ 7.14 Hz, 2H), 9.29 (s, 1H). MS (ESI): 328.1
(C17H13ClN2OS, [M þ H]þ). Anal. Calcd for C17H12ClN2OS: C, 62.10; H,
3.98; N, 8.52. Found: C, 62.43; H, 3.59; N, 8.75%.
4.2.7. 2-(4-Bromophenyl)-N-(4-phenylthiazol-2-yl)acetamide (4d)
Pink crystal. Yield, 68%. Mp 200e201 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
3.64 (s, 2H, CH2), 7.1 (d, J ¼ 8.4 Hz, 2H), 7.14 (s, 1H), 7.30e
7.35 (m, 1H), 7.40 (t, J ¼ 7.5 Hz, 2H), 7.49 (d, J ¼ 6.42 Hz, 2H), 7.78 (d,
J ¼ 6.96 Hz, 2H), 9.37 (s, 1H). MS (ESI): 372.0 (C17H13BrN2OS,
[M þ H]þ). Anal. Calcd for C17H12BrN2OS: C, 54.70; H, 3.51; N, 7.50.
Found: C, 54.91; H, 3.22; N, 7.22%.
4.2.8. 2-(3-Chlorophenyl)-N-(4-phenylthiazol-2-yl)acetamide (4e)
Colorless crystal. Yield, 75%. Mp 180e181 ꢁC. 1H NMR (300 MHz,
CDCl3):
7.40 (t, J ¼ 7.68 Hz, 2H), 7.77 (d, J ¼ 7.14 Hz, 2H), 9.14 (s, 1H). MS
(ESI): 328.0 (C17H13ClN2OS, [M
H]þ). Anal. Calcd for
17H12ClN2OS: C, 62.10; H, 3.98; N, 8.52. Found: C, 62.31; H, 3.69; N,
8.74%.
d 3.73 (s, 2H, CH2), 7.14e7.18 (m, 2H), 7.28e7.35 (m, 4H),
4.2.2. General procedure for the synthesis of acetamide derivatives
4ae4h and 5ae5h
þ
C
For the synthesis of compounds 4ae4h and 5ae5h was depicted
in Scheme 2. Firstly, 2-phenylacetic acid (1 mmol) and SOCl2 (4e
6 mL) were refluxed at 80 ꢁC for 3 h. The reaction liquid was cooled
to room temperature and then evaporated to give reactive acyl
chloride. The product was obtained as an oil matter, which would
be dissolved in acetone (5e6 mL) in the next step. Treatment of 3a
(5-phenylthiazol-2-amine) with 2-phenylacetyl chloride in acetone
for 5 h at ice-bath afforded the aimed amine. Meanwhile, K2CO3
(0.8 g) was added to the mixture. Then the mixture was evaporated
under reduced pressure and the resulting solid was washed with
diluted NaOH liquid. The aimed amide was extracted from the
NaOH liquid with ethyl acetate for column chromatography. Col-
umn chromatography was performed using silica gel (200e
300 mesh) eluting with ethyl acetate and petroleum ether to give
the aimed amine (4a). Compounds of 4be4h and 5ae5h could be
got with corresponding acids by the procedures above.
4.2.9. 2-(3-Bromophenyl)-N-(4-phenylthiazol-2-yl)acetamide (4f)
Colorless crystal. Yield, 68%. Mp 183e184 ꢁC. 1H NMR (300 MHz,
CDCl3): d 3.73 (s, 2H, CH2), 7.14 (s, 1H), 7.18e7.24 (m, 2H), 7.31e7.48
(m, 5H), 7.77 (d, J ¼ 6.93 Hz, 2H), 9.57 (s, 1H). MS (ESI): 372.0
(C17H13BrN2OS, [M þ H]þ). Anal. Calcd for C17H12BrN2OS: C, 54.70;
H, 3.51; N, 7.50. Found: C, 54.99; H, 3.82; N, 7.81%.
4.2.10. 2-(3-Methoxyphenyl)-N-(4-phenylthiazol-2-yl)acetamide
(4g)
Pink crystal. Yield, 56%. Mp 99e101 ꢁC. 1H NMR (300 MHz,
CDCl3):
d 3.76 (s, 2H, CH2), 3.84 (s, 3H), 6.59 (s, 1H), 6.81e6.89 (m,
3H), 7.11 (s, 1H), 7.42e7.47 (m, 3H), 7.72 (d, J ¼ 8.04 Hz, 2H), 8.60 (s,
1H). MS (ESI): 324.1 (C18H16N2O2S, [M þ H]þ). Anal. Calcd for
C18H15N2O2S: C, 66.64; H, 4.97; N, 8.64. Found: C, 66.99; H, 4.66; N,
8.37%.
4.2.3. General procedure for the synthesis of acetamide derivatives
4ie4l and 5ie5l
4.2.11. 2-(3,4-Diethoxyphenyl)-N-(4-phenylthiazol-2-yl)acetamide
(4h)
The key intermediates 3a or 3b (1 mmol), corresponding acids
(1 mmol) and carbodiimide hydrochloride (EDC$HCl, 1.5 mmol) and
N-hydroxybenzotriazole (HOBt, 0.05 mmol) were stirred in CH2Cl2
(20e30 mL) at room temperature for 24 h. After that, the solvent
was evaporated under reduced pressure to afford a residue, which
was extracted with EtOAc (3 ꢄ 20 mL). The combined organic
phases were dried with anhydrous Na2SO4. Removal of all the
solvent under reduced pressure distillation to get the residue,
which was purified with recrystallization to give target products
4ie4l and 5ie5l.
White crystal. Yield, 58%. Mp 116e118 ꢁC. 1H NMR (300 MHz,
CDCl3):
d 1.43e1.49 (m, 6H), 3.72 (s, 2H, CH2), 4.05e4.15 (m, 4H),
6.80 (d, J ¼ 6.96 Hz, 2H), 6.89 (d, J ¼ 8.76 Hz, 1H), 7.12 (s, 1H), 7.28e
7.31 (m, 1H), 7.32e7.42 (m, 2H), 7.76 (d, J ¼ 7.14 Hz, 2H), 8.93 (s, 1H).
MS (ESI): 382.1 (C21H22N2O3S, [M
þ
H]þ). Anal. Calcd for
C21H21N2O3S: C, 65.95; H, 5.80; N, 7.32. Found: C, 65.59; H, 5.51; N,
7.65%.
4.2.12. N-(4-Phenylthiazol-2-yl)benzamide (4i)
Yellow crystal. Yield, 84%. Mp 85e86 ꢁC. 1H NMR (300 MHz,
4.2.4. 2-Phenyl-N-(4-phenylthiazol-2-yl)acetamide (4a)
CDCl3):
d
7.20 (s, 1H), 7.31e7.36 (m, 1H), 7.42 (t, J ¼ 7.14 Hz, 2H), 7.53
White crystal. Yield, 78%. Mp 160e161 ꢁC. 1H NMR (300 MHz,
(t, J ¼ 6.78 Hz, 2H), 7.60e7.64 (m, 1H), 7.83 (d, J ¼ 6.96 Hz, 2H), 7.97
(d, J ¼ 8.43 Hz, 2H), 9.89 (s, 1H). MS (ESI): 280.1 (C16H12N2OS,
[M þ H]þ). Anal. Calcd for C16H11N2OS: C, 68.55; H, 4.31; N, 9.99.
Found: C, 68.23; H, 4.62; N, 9.66%.
CDCl3): d 3.81 (s, 2H, CH2), 7.12 (s, 1H), 7.28e7.33 (m, 3H), 7.36e7.44
(m, 5H), 7.76 (d, J ¼ 8.04 Hz, 2H), 8.85 (s, 1H). MS (ESI): 294.1
(C17H14N2OS, [M þ H]þ). Anal. Calcd for C17H13N2OS: C, 69.36; H,
4.79; N, 9.52. Found: C, 69.03; H, 4.48; N, 9.85%.
4.2.13. N-(4-Phenylthiazol-2-yl)-1-naphthamide (4j)
4.2.5. 2-(4-Fluorophenyl)-N-(4-phenylthiazol-2-yl)acetamide (4b)
Colorless crystal. Yield, 70%. Mp 189e190 ꢁC. 1H NMR (300 MHz,
Colorless crystal. Yield, 62%. Mp 178e180 ꢁC. 1H NMR (300 MHz,
CDCl3):
d 7.22 (s, 1H), 7.32e7.37 (m, 1H), 7.41e7.46 (m, 2H), 7.83 (d,
CDCl3):
d
3.77 (s, 2H, CH2), 7.06e7.13 (m, 3H), 7.30e7.34 (m, 3H),
J ¼ 6.96 Hz, 2H), 7.89e7.97 (m, 3H), 8.06 (d, J ¼ 8.22 Hz, 2H), 8.34 (d,
7.39 (t, J ¼ 7.5 Hz, 2H), 7.71 (d, J ¼ 6.96 Hz, 2H), 8.89 (s,1H). MS (ESI):
J ¼ 7.32 Hz, 1H), 8.55 (d, J ¼ 8.25 Hz, 1H), 8.89 (d, J ¼ 8.58 Hz, 1H).

J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
445
MS (ESI): 330.1 (C20H14N2OS, [M
20H13N2OS: C, 72.70; H, 4.27; N, 8.48. Found: C, 72.96; H, 4.48; N,
8.79%.
þ
H]þ). Anal. Calcd for
J ¼ 8.61 Hz, 3H), 9.19 (s, 1H). MS (ESI): 450.0 (C17H12Br2N2OS,
[M þ H]þ). Anal. Calcd for C17H11Br2N2OS: C, 45.16; H, 2.67; N, 6.20.
Found: C, 45.48; H, 2.88; N, 6.01%.
C
4.2.14. N-(4-Phenylthiazol-2-yl)-2-naphthamide (3k)
4.2.22. N-(4-(4-Bromophenyl)thiazol-2-yl)-2-(3-methoxyphenyl)
acetamide (5g)
Colorless crystal. Yield, 64%. Mp 168e169 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
7.22 (s, 1H), 7.30e7.35 (m, 1H), 7.40e7.44 (m, 2H), 7.58e
Pink crystal. Yield, 55%. Mp 112e113 ꢁC. 1H NMR (300 MHz,
7.67 (m, 2H), 7.85 (d, J ¼ 6.96 Hz, 2H), 7.91e7.97 (m, 2H), 7.99 (s, 2H),
CDCl3): d 3.71 (s, 2H, CH2), 3.83 (s, 3H), 6.85e6.93 (m, 3H), 7.12 (s,
8.50 (s, 1H). MS (ESI): 330.1 (C20H14N2OS, [M þ H]þ). Anal. Calcd for
1H), 7.34 (t, J ¼ 7.86 Hz, 1H), 7.49 (d, J ¼ 6.39 Hz, 2H), 7.63 (d,
J ¼ 6.75 Hz, 2H), 8.78 (s, 1H). MS (ESI): 402.0 (C18H15BrN2O2S,
[M þ H]þ). Anal. Calcd for C18H14BrN2O2S: C, 53.61; H, 3.75; N, 6.95.
Found: C, 53.89; H, 3.45; N, 7.16%.
C
20H13N2OS: C, 72.70; H, 4.27; N, 8.48. Found: C, 72.39; H, 4.47; N,
8.60%.
4.2.15. N-(4-Phenylthiazol-2-yl)morpholine-4-carboxamide (4l)
Yellow crystal. Yield, 88%. Mp 155e156 ꢁC. 1H NMR (300 MHz,
CDCl3): d 3.76e3.78 (m, 8H), 7.02 (s, 1H), 7.47e7.54 (m, 3H), 7.75 (d,
J ¼ 6.48 Hz, 2H), 9.44 (s, 1H). MS (ESI): 389.1 (C14H15N3O2S,
[M þ H]þ). Anal. Calcd for C14H14N3O2S: C, 58.11; H, 5.23; N, 14.52.
Found: C, 58.49; H, 5.42; N, 14.25%.
4.2.23. N-(4-(4-Bromophenyl)thiazol-2-yl)-2-(3,4-diethoxyphenyl)
acetamide (5h)
. White crystal. Yield, 56%. Mp 149e150 ꢁC. 1H NMR (300 MHz,
CDCl3):
d 1.44e1.50 (m, 6H), 3.76 (s, 2H, CH2), 4.06e4.16 (m, 4H),
6.83 (d, J ¼ 8.97 Hz, 2H), 6.91 (d, J ¼ 7.86 Hz, 1H), 7.11 (s, 1H), 7.50 (d,
J ¼ 8.79 Hz, 2H), 7.64 (d, J ¼ 8.58 Hz, 2H), 8.66 (s, 1H). MS (ESI):
460.1 (C21H21BrN2O3S, [M þ H]þ). Anal. Calcd for C21H20BrN2O3S: C,
54.67; H, 4.59; N, 6.07. Found: C, 54.95; H, 4.70; N, 6.29%.
4.2.16. N-(4-(4-Bromophenyl)thiazol-2-yl)-2-phenylacetamide
(5a)
Pink crystal. Yield, 80%. Mp 210e211 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
3.84 (s, 2H, CH2), 7.12 (s, 1H), 7.32e7.43 (m, 5H), 7.50 (d,
4.2.24. N-(4-(4-Bromophenyl)thiazol-2-yl)benzamide (5i)
J ¼ 8.76 Hz, 2H), 7.64 (d, J ¼ 8.76 Hz, 2H), 8.67 (s,1H). MS (ESI): 372.0
(C17H13BrN2OS, [M þ H]þ). Anal. Calcd for C17H12BrN2OS: C, 54.70;
H, 3.51; N, 7.50. Found: C, 54.96; H, 3.72; N, 7.89%.
Colorless crystal. Yield, 88%. Mp 120e122 ꢁC. 1H NMR (300 MHz,
DMSO-d6): d 7.17 (s, 2H), 7.60e7.62 (m, 4H), 7.71e7.78 (m, 4H), 9.67
(s, 1H). MS (ESI): 358.0 (C16H11BrN2OS, [M þ H]þ). Anal. Calcd for
C16H10BrN2OS: C, 53.49; H, 3.09; N, 7.80. Found: C, 53.76; H, 3.40; N,
4.2.17. N-(4-(4-Bromophenyl)thiazol-2-yl)-2-(4-fluorophenyl)
acetamide (5b)
7.52%.
Pink crystal. Yield, 68%. Mp 218e219 ꢁC. 1H NMR (300 MHz,
4.2.25. N-(4-(4-Bromophenyl)thiazol-2-yl)-1-naphthamide (5j)
Colorless crystal. Yield, 64%. Mp 150e151 ꢁC. 1H NMR (300 MHz,
CDCl3): d 3.81 (s, 2H, CH2), 7.11e7.14 (m, 3H), 7.28e7.33 (m, 2H), 7.51
(d, J ¼ 8.61 Hz, 2H), 7.60e7.66 (m, 2H), 8.80 (s, 1H). MS (ESI): 390.0
(C17H12BrFN2OS, [M þ H]þ). Anal. Calcd for C17H11BrFN2OS: C, 52.19;
H, 3.09; N, 7.16. Found: C, 52.56; H, 3.40; N, 6.98%.
DMSO-d6): d 7.29 (s, 1H), 7.69e7.74 (m, 4H), 7.79e7.87 (m, 3H), 7.95
(d, J ¼ 8.04 Hz, 2H), 8.11 (d, J ¼ 7.32 Hz, 1H), 8.31 (d, J ¼ 9.69 Hz, 1H),
8.87 (d, J ¼ 8.4 Hz, 1H). MS (ESI): 408.0 (C20H13BrN2OS, [M þ H]þ).
Anal. Calcd for C20H12BrN2OS: C, 58.69; H, 3.20; N, 6.84. Found: C,
58.96; H, 3.49; N, 6.99%.
4.2.18. N-(4-(4-Bromophenyl)thiazol-2-yl)-2-(4-chlorophenyl)
acetamide (5c)
Yellow crystal. Yield, 70%. Mp 218e219 ꢁC. 1H NMR (300 MHz,
4.2.26. N-(4-(4-Bromophenyl)thiazol-2-yl)-2-naphthamide (5k)
Colorless crystal. Yield, 67%. Mp 123e125 ꢁC. 1H NMR (300 MHz,
CDCl3):
d 3.74 (s, 2H, CH2), 7.13 (s, 1H), 7.30 (s, 2H), 7.39 (d,
J ¼ 8.4 Hz, 2H), 7.54 (d, J ¼ 8.4 Hz, 2H), 7.64 (d, J ¼ 8.4 Hz, 2H), 9.55
CDCl3):
d
7.22 (s, 1H), 7.53 (d, J ¼ 8.58 Hz, 2H), 7.59e7.70 (m, 2H),
(s, 1H). MS (ESI): 406.0 (C17H12BrClN2OS, [M þ H]þ). Anal. Calcd for
7.92e8.04 (m, 4H), 8.20 (d, J ¼ 8.58 Hz, 1H), 8.50 (s, 1H), 8.79 (s, 1H).
C
17H11BrClN2OS: C, 50.08; H, 2.97; N, 6.87. Found: C, 50.36; H, 2.68;
MS (ESI): 408.0 (C20H13BrN2OS, [M þ H]þ). Anal. Calcd for
N, 7.08%.
C20H12BrN2OS: C, 58.69; H, 3.20; N, 6.84. Found: C, 58.99; H, 3.49;
N, 6.53%.
4.2.19. 2-(4-Bromophenyl)-N-(4-(4-bromophenyl)thiazol-2-yl)
acetamide (5d)
4.2.27. N-(4-(4-Bromophenyl)thiazol-2-yl)morpholine-4-
carboxamide (5l)
Pink crystal. Yield, 71%. Mp 211e212 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
3.78 (s, 2H, CH2), 7.13 (s, 1H), 7.21 (d, J ¼ 8.43 Hz, 2H), 7.53
White crystal. Yield, 83%. Mp 143e144 ꢁC. 1H NMR (300 MHz,
(t, J ¼ 8.43 Hz, 4H), 7.64 (d, J ¼ 8.61 Hz, 2H), 8.99 (s, 1H). MS (ESI):
450.0 (C17H12Br2N2OS, [M þ H]þ). Anal. Calcd for C17H11Br2N2OS: C,
45.16; H, 2.67; N, 6.20. Found: C, 45.46; H, 2.88; N, 6.02%.
DMSO-d6): d 3.48e3.53 (m, 4H), 3.58e3.60 (m, 4H), 7.03 (s, 1H),
7.42e7.45 (m, 2H), 7.69 (d, J ¼ 8.61 Hz, 2H), 9.58 (s, 1H). MS (ESI):
367.0 (C14H14BrN3O2S, [M þ H]þ). Anal. Calcd for C14H13BrN3O2S: C,
45.66; H, 3.83; N, 11.41. Found: C, 45.89; H, 3.62; N, 11.63%.
4.2.20. N-(4-(4-Bromophenyl)thiazol-2-yl)-2-(3-chlorophenyl)
acetamide (5e)
4.3. Antibacterial activity
Yellow crystal. Yield, 78%. Mp 170e172 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
3.76 (s, 2H, CH2), 7.14 (s, 1H), 7.17e7.20 (m, 1H), 7.31e7.34
The antibacterial activity of the synthesized compounds was
tested against E. coli, P. aeruginosa, B. subtilis and S. aureus using MH
medium (MuellereeHinton medium: casein hydrolyzate 17.5 g,
soluble starch 1.5 g, beef extract 1000 mL). The MICs (minimum
inhibitory concentrations) of the test compounds were determined
(m, 3H), 7.52 (d, J ¼ 8.61 Hz, 2H), 7.64 (d, J ¼ 8.61 Hz, 2H), 9.23 (s,
1H). MS (ESI): 406.0 (C17H12BrClN2OS, [M þ H]þ). Anal. Calcd for
C
17H11BrClN2OS: C, 50.08; H, 2.97; N, 6.87. Found: C, 50.38; H, 2.68;
N, 7.09%.
by
a
colorimetric method using the dye MTT (3-(4,5-
4.2.21. 2-(3-Bromophenyl)-N-(4-(4-bromophenyl)thiazol-2-yl)
acetamide (5f)
dimethylthiazol-2-yl)-2,5-diphenyl-trtrazolium bromide). A stock
solution of the synthesized compound (1 mg/mL) in DMSO was
prepared and graded quantities of the test compounds were
incorporated in specified quantity of sterilized liquid MH medium.
Pink crystal. Yield, 65%. Mp 182e183 ꢁC. 1H NMR (300 MHz,
CDCl3):
d 3.74 (s, 2H, CH2), 7.13 (s, 1H), 7.48e7.54 (m, 5H), 7.64 (d,

446
J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
A specified quantity of the medium containing the compound was
poured into microtitration plates. Suspension of the microorganism
was prepared to contain approximately 105 cfu/mL and applied to
microtitration plates with serially diluted compounds in DMSO to
be tested and incubated at 37 ꢁC for 24 h. After the MICs were
4.5. Cytotoxicity on human macrophage
The cytotoxicity test of compounds was measured by the
colorimetric MTT assay. Human macrophage suspension in
DMEM medium supplemented with 10% FBS and 1ꢄ antimycotic
was added in 96-well microplates at 1.8 ꢄ 104 cells/well. Then,
various concentrations of the test compounds (160, 40, 10, 2.5,
visually determined on each of the microtitration plates, 50 mL of
PBS (Phosphate Buffered Saline 0.01 mol/L, pH 7.4: Na2HPO4$12H2O
2.9 g, KH2PO4 0.2 g, NaCl l8.0 g, KCl 0.2 g, distilled water 1000 mL)
containing 2 mg of MTT/mL was added to each well. Incubation was
continued at room temperature for 4e5 h. The content of each well
0.25
each well. After incubation for 24 h at 37 ꢁC under 5% CO2,
2.5 mg/mL of MTT solution ( L) was added to each well. The plate
was incubated for a further 4 h. Then, the incubation medium
was aspirated, and DMSO (100 L) was added to solubilize the
mg/mL) dissolved in distilled 10% DMSO (10 mL) was added to
m
was removed, and 100 mL of isopropanol containing 5% 1 mol/L HCl
was added to extract the dye. After 12 h of incubation at room
temperature, the opticaldensity (OD) was measured with a micro-
plate reader at 550 nm. The observed MICs were presented in
Table 1.
m
MTT formazan product. After mixing, the absorbances at 570 and
655 nm were measured. The difference between absorbance at
570 and 630 nm was used as an index of the cell viability. The
morphology of the cells was observed using Giemsa stain under
Phase contrast microscope. Metronidazoleethiazoles derivatives
were used as a positive control, whereas untreated cells were
used as negative controls. The concentration of the compound
that killed 50% of the cells (CC50) was calculated by GraphPad
Prism 4 software. All determinations were performed in tripli-
cate. The results were summarized in Table 4.
4.4. E. coli FabH purification and activity assay
Full-length E. coli acyl carrier protein (ACP), acyl carrier protein
synthase (ACPS), and
b-ketoacyl-ACP synthase III (FabH) were
individually cloned into pET expression vectors with an N-terminal
His-tag (ACP, ACPS in pET 19; FabH in pET 28).
All proteins were expressed in E. coli strain BL21 (DE3). Trans-
formed cells were grown on LuriaeBertani (LB) agar plates sup-
plemented with kanamycin (30 mg/mL). Sodium dodecyl sulfate-
4.6. Virtual calculation
polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used
to screen colonies for overexpression of proteins. One such positive
4.6.1. 3D-QSAR study
Ligand-based 3D-QSAR approach was performed by QSAR
software of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). The
training sets were composed of 6 small molecular while test sets
comprised 4 compounds. The corresponding pIC50 values were
converted from the obtained IC50 (nM) (http://www.sanjeevslab.
org/tools-IC50.html).2 All the definition of the descriptors can be
seen in the help of DS 3.5 software and they were calculated by
QSAR protocol of DS 3.5. The alignment conformation of each
molecule was the one with lowest interaction energy in the docked
results of CDOCKER.
colony was used to inoculate 10 mL of LB medium with 30
kanamycin and grown overnight at 37 ꢁC,1 mL of which was used to
inoculate 100 mL LB medium supplemented with 30 g/mL of
kanamycin. The culture was shaken for 4 h at 37 ꢁC, and then
induced with 0.5 mM isopropyl b- -thio galacto pyranoside (IPTG).
mg/mL of
m
D
The culture was grown for 4 h, and harvested by centrifugation
(30 min at 15,000 rpm).
Harvested cells containing His-tagged ACP, ACPS, and FabHs
were lysed by sonication in 20 mM Tris, pH 7.6, 5 mM imidazole,
0.5 M NaCl and centrifuged at 20, 000 rpm for 30 min. The super-
natant was applied to a NieNTA agarose column, washed, and
eluted using a 5e500 mM imidazole gradient over 20 column
volumes. Eluted protein was dialyzed against 20 mM Tris, pH 7.6,
1 mM DTT, and 100 mM NaCl. Purified FabHs were concentrated up
to 2 mg/mL and stored at ꢂ80 ꢁC in 20 mM Tris, pH 7.6, 100 mM
NaCl, 1 mM DTT, and 20% glycerol for enzymatic assays.
4.6.2. Molecular docking modeling
The crystal structures of the proteins complex were retrieved
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do). The three-dimensional structures of the aforementioned
compounds were constructed using Chem. 3D ultra 12.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2010)], then they were energetically minimized by using
MMFF94 with 5000 iterations and minimum RMS gradient of 0.10.
Molecular docking of compound 5f into the three-
dimensional E. coli FabH protein complex structure (3IL9.pdb,
downloaded from the PDB) was carried out using the Discovery
Studio (version 3.5) as implemented through the graphical user
interface DS-CDOCKER protocol, all bound waters and ligands
were eliminated from the protein and the polar hydrogen was
added to the proteins.
Purified ACP contains the apo-form that needs to be converted
into the holo-form. The conversion reaction is catalyzed by ACP
synthase (ACPS). In the final volume of 50 mL, 50 mg ACP, 50 mM
Tris, 2 mM DTT, 10 mM MgCl2, 600 mM CoA, and 0.2 mM ACPS was
incubated for 1 h at 37 ꢁC. The pH of the reaction was then adjusted
to approximately 7.0 using 1 M potassium phosphate. Holo-ACP
was purified by fractionation of the reaction mixture by Source
Q-15 ion exchange chromatography using a 0e500 mM NaCl
gradient over 25 column volumes.
In a final 20
0.5 mM DTT, 0.25 mM MgCl2, and 2.5 mM holo-ACP were mixed
with 1 nM FabH, and H2O was added to 15 L. After1 min incu-
bation, a 2 L mixture of 25 M acetyl-CoA and 0.75
Ci [3H]
acetyl-CoA was added for FabH reaction for 25 min. The reaction
was stopped by adding 20 L of ice-cold 50% TCA, incubating for
5 min on ice, and centrifuging to pellet the protein. The pellet was
mL reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0,
Acknowledgments
m
m
m
m
We thank Miss Zhu Shen in Nanjing Foreign Languages School
for the synthesis of compounds 3a and 3b of this paper. This work
was supported by National Science & Technology Pillar Program
during the Twelfth Five-year Plan Period (No. 2012BAD36B01).
m
washed with 10% ice-cold TCA and resuspended with 5
mL of
0.5 M NaOH. The incorporation of the 3H signal in the final
product was read by liquid scintillation. When determining the
inhibition constant (IC50), inhibitors were added from a concen-
trated DMSO stock such that the final concentration of DMSO did
not exceed 2%.
2
The pIC50 value calculator is a web tool which is developed by using JavaScript
and HTML, to convert IC50 to pIC50 values. It is available online. To enter this
website, the IC50 values were input into the calculator surface one by one, and then
we got the corresponding pIC50 values.

J.-R. Li et al. / European Journal of Medicinal Chemistry 75 (2014) 438e447
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