Design, synthesis and antibacterial activity studies of thiazole derivatives as potent ecKAS III inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.05.006

Two series of thiazole derivatives containing amide skeleton were synthesized and developed as potent Escherichia coli β-ketoacyl-(acyl- carrier-protein) synthase III (ecKAS III) inhibitors. All the 24 new synthesized compounds were assayed for antibacterial activity against the respective Gram-negative and Gram-positive bacterial strains, including E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. In which, 10 compounds with broad-spectrum antibacterial activities were further tested for their ecKAS III inhibitory activity. Last, we have successfully found that compound 4e showed both the promising broad antibacterial activity with MIC of 1.56-6.25 μg/mL against the representative bacterial stains, and also processed the most potent ecKAS III inhibitory activity with IC₅₀ of 5.3 μM. In addition, docking simulation also carried out in this study to give a potent prediction binding mode between the small molecule and ecKAS III (PDB code: 1hnj) protein.

Full text of DOI:10.1016/j.bmcl.2013.05.006

Accepted Manuscript
Design, synthesis and antibacterial activity studies of thiazole derivatives as
potent ecKAS III inhibitors
Kui Cheng, Jia-Yu Xue, Hai-Liang Zhu
PII:
S0960-894X(13)00589-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.05.006
BMCL 20464
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
26 March 2013
27 April 2013
2 May 2013
Please cite this article as: Cheng, K., Xue, J-Y., Zhu, H-L., Design, synthesis and antibacterial activity studies of
thiazole derivatives as potent ecKAS III inhibitors, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://
dx.doi.org/10.1016/j.bmcl.2013.05.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis and antibacterial activity studies of thiazole
derivatives as potent ecKAS III inhibitors
Kui Cheng†, Jia-Yu Xue†, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China.
Abstract: Two series of thiazole derivatives containing amide skeleton were
synthesized and developed as potent Escherichia coli β-ketoacyl-(acyl-carrier-protein)
synthase III (ecKAS III) inhibitors. All the 24 new synthesized compounds were
assayed for antibacterial activity against the respective Gram-negative and
Gram-positive bacterial strains, including Escherichia coli, Pseudomonas aeruginosa,
Bacillus subtilis and Staphylococcus aureus. In which, 10 compounds with
broad-spectrum antibacterial activities were further tested for their ecKAS III
inhibitory activity. Last, we have successfully found that compound 4e showed both
the promising broad antibacterial activity with MIC of 1.56-6.25 µg/mL against the
representative bacterial stains, and also processed the most potent ecKAS III
inhibitory activity with IC50 of 5.3 µM. In addition, docking simulation also carried
out in this study to give a potent prediction binding mode between the small molecule
and ecKAS III (PDB code: 1hnj) protein.
Keywords: Antibacterial; Thiazole derivatives; Amide; ecKAS III inhibitors;
Computer simulation
* Corresponding authors: Tel.: +86-25-8359 2672; Fax: +86-25-8359 2572; E-mail address:
zhuhl@nju.edu.cn
†These two authors equally contributed to this paper
1

The increasing appearance and prevalence of multidrug-resistant pathogenic
bacteria have heightened the importance of the elaboration of new types of
antibacterial agents or the expansion of bioactivity of the previous drugs.1 Therefore,
in recent years research has been focused toward development of new antibacterial
agents, which may act through novel target, surpassing the problem of acquired
resistance.
Saturated fatty acid biosynthesis has emerged as a prime candidate for development
of such important and novel antibacterials.2 The ubiquitous type II fatty acid synthesis
system (FAS) in bacteria is not only essential to cell survival but also exhibits
significant differences between bacterial and human fatty acid synthesis systems
including the organization and structure of enzymes and the specific roles played by
fatty acids, which make this system an attractive target for antibacterial drug
discovery.3,4 Escherichia coli β-ketoacyl-(acyl-carrier-protein) synthase III, also
known as ecKAS III or FabH, plays an essential and regulatory role in bacterial FAS.5
The enzyme initiates the fatty acid elongation cycles (Figure. 1), and is involved in
the feedback regulation of the biosynthetic pathway via product inhibition.6 In
addition, ecKAS III proteins from both Gram-positive and -negative bacteria are
highly conserved at the sequence and structural level, while there are no significantly
homologous proteins in humans. Moreover, the residues that comprise the active site
are essentially invariant among Gram-positive and -negative organisms.7 These
attributes suggest that ecKAS III could be a promising target for the design of novel
antimicrobial drugs with selectivity, nontoxicity, and broad-spectrum antibacteria
activity.
Thiazoles and their derivatives have attracted continuing interest over the years
because of their varied biological activities,8 recently found application in drug
development for the treatment of allergies,9 hypertension,10 inflammation,11
schizophrenia,12 bacterial,13 HIV infections,14 hypnotics15 and more recently for the
treatment of pain.16 Besides, Kitagawa et al. find that thiazole derivatives show strong
FabI and FabK inhibitory activities with potent antibacterial activity.17
In view of the above mentioned findings, we described the synthesis of thiazole
2

derivatives containing amide skeleton. This combination was suggested in an attempt
to investigate the inhibitory activity against ecKAS III. The antimicrobial activity
against two Gram-negative bacterial strains (Escherichia coli and Pseudomonas
aeruginosa), and two Gram-positive bacterial strains (Bacillus subtilis and
Staphylococcu saureus) of these thiazole derivatives were also determined. Docking
simulation was performed using the X-ray crystallographic structure of ecKAS III
with the small molecule inhibitor 4e to explore the binding mode and the activity
relationship.
The synthesis of compounds 3a-3l and 4a-4l embarked on the preparation of two
key intermediates 2a and 2b followed by the general synthetic procedure shown in
Scheme 1. That is, treatment of bromides 1a and 1b with thiourea in refluxing EtOH
afforded 4-phenylthiazol-2-amine (2a) and 4-(4-bromophenyl)thiazol-2-amine (2b) in
92 % and 80 % yields, respectively. The methods for acylation of 2a and 2b were well
documented.18 Here, acylation of amines 2a and 2b with a series of phenylacetic acid
and naphthoic acid provided the corresponding final compounds 3a-3h, 3j-3k, 4a-4h,
4j-4k in the presence of EDC·HCl and HOBt in CH2Cl2. In addition, treatment of
amines 2a and 2b with benzoyl chloride or 4-morpholinecarbonyl chloride in pyridine
led to compounds 3i, 3l, 4i, 4l. All of the synthetic compounds (Table 1) gave
satisfactory analytical and spectroscopic data, which were in full accordance with
their depicted structures.
All the synthesized compounds (3a-l and 4a-l) were screened for antibacterial
activity against two Gram-negative bacterial strains: E.coli and P.aeruginosa and two
Gram-positive bacterial strains: B.subtilis and S.aureus by MTT method. The MICs
(minimum inhibitory concentrations) of the compounds against these bacteria were
presented in Table 2. Standard antibacterial agent kanamycin B and Penicillin G were
also screened under identical conditions for comparison. The results revealed that
most of the synthesized compounds exhibited significant antibacterial activity. It was
observed that many compounds exhibited interesting antibacterial activity displaying
MIC values between 1.56 and 50.0 µg/mL.
Out
of
the
24
compounds,
compound
4e,
3

N-(4-(4-bromophenyl)thiazol-2-yl)-2-(3-chlorophenyl)acetamide, exhibited the most
potent antibacterial activity with MIC of 1.56, 3.13, 6.25 and 3.13 µg/mL against
E.coli, P.aeruginosa, B.Subtilis and S.aureus, respectively, which was similar to the
broad-spectrum antibiotic kanamycin B with corresponding MIC of 1.56, 3.13, 0.39,
3.13 µg/mL and Penicillin G with corresponding MIC of 3.13, 6.25, 1.56, 6.25 µg/mL.
Modification of the parent compounds with various substituents such as halogen,
methoxyl and ethoxy were performed to explore the structure-activity relationships
(SAR) of theses thiazole derivatives containing amide skeleton. The parent
compounds can be divided into two rings: A ring and B ring. As shown in Table 2, we
found that compounds 4a-4l showed better antibacterial activity than compounds
(3a-3l). The slight structure difference between these two series was the substituent of
A ring: for 3a-3l, there was no substituent on A ring while a chloro group on A ring
for 4a-4l. These results demonstrated that chloro group on para- position of A ring
was favorable to the antibacterial activity of synthetic compounds. Compared to 3a,
compounds 3b-3f showed more potent antibacterial activity. The MIC values of
compound 3a and 3b-3f were 25-100 µg/mL, 3.13-50 µg/mL, respectively. This result
suggested that the introduction of halogen substituent on meta- or para- position of B
ring increased the hydrophobicity of compounds and lead to the increase of
antibacterial activity. In addition, the antibacterial activity of these compounds
enhanced slightly in the order of substituent on para- position of B ring component:
Br < Cl < F. The comparison between 4a and 4b-4f was similar to the example of 3a
and 3b-3f. Furthermore, compounds with substituent at para- position of B ring had
less potent activity than those at meta- position, which can be seen from the
comparison of 3b and 3c, 4b and 4c, respectively. This indicated that the location of
substituent also had an influence on the activity.
Compounds 3g, 3h, 4g and 4h showed similar moderate antibacterial activity with
MIC of 25-100 µg/mL against all the tested bacterial strains. This indicated that the
introduction of electron-donating group could not enhance the inhibitory activity.
Compounds 3i-3l and 4i-4l were found to be inactive against all the tested bacterial
strains. This indicated that the introduction of morpholine or naphthalene lead to the
4

decrease of antibacterial activity.
The ecKAS III inhibitory potency of the synthetic thiazole derivatives containing
amide skeleton with potent antibacterial activities (3b-3c, 3e-3f and 4b-4g) was
examined and the results were summarized in Table 3. Most of the tested compounds
displayed potent ecKAS III inhibitory. The more detailed inhibitory assay procedure
is described in the Supplementary data. Among them, compound 4e showed the most
potent inhibitory with IC50 of 5.3 µM. This result supported the potent antibacterial
activities of 4e. Compounds 4b-4g with substituent on A ring showed better ecKAS
III inhibitory activity than compounds 3b-3c and 3e-3f with no substituent on A ring.
Besides, a comparison of 4c and 4e, 4d and 4f, respectively, also indicated that
compounds with m-substituted chloride and bromine group on B ring exhibited more
potent inhibitory activity than those compounds with p-substituent on B ring. These
results of ecKAS III inhibitory activity of the test compounds were corresponding to
the structure relationships (SAR) of their antibacterial activities. This demonstrated
that the potent antibacterial activities of the synthetic compounds were probably
correlated to their ecKAS III inhibitory activities.
Molecular docking of compound 4e and ecKAS III was performed on the binding
model based on the ecKAS III-CoA complex structure (1HNJ.pdb).19 The ecKAS III
active site generally contains a catalytic triad tunnel consisting of Cys-His-Asn, which
is conserved in various bacteria. This catalytic triad plays an important role in the
regulation of chain elongation and substrate binding. Since the alkyl chain of CoA is
broken by Cys of the catalytic triad of ecKAS III, interactions between Cys and
substrate appear to play an important role in substrate binding. Qiu, X. et al. have
refined three-dimensional structure of ecKAS III in the presence and absence of
malonyl-CoA by X-ray spectroscopy. Since malonyl moiety was degraded by ecKAS
III, molecular docking studies for ecKAS III and malonyl-CoA were carried out to
identify a plausible malonyl-binding mode.19 They found that in one of the binding
modes appeared in the lower scored conformations, the malonyl carboxylate formed
hydrogen bonds to the backbone nitrogen of Phe304.
The binding mode of compound 4e and ecKAS III is depicted in Figure. 2. In the
5

binding mode, compound 4e is projected into the active site with a quite smooth
conformation. There is a π-π interaction between the benzyl ring of compound 4e
(C11, C12, C13, C14, C15, C16) and the benzyl ring of Phe304. The NH group of
compound 4e is also much close to the critical amino residue Phe304 in the active site.
Besides, compound 4e may form a hydrophobic interaction with Leu142, Phe157 and
Leu189. Though there is no hydrogen bond found in the binding model, the dominant
conformation and low binding energy (-8.56 kcal/mol) are still able to explain the
potent ecKAS III inhibitory activity of compound 4e.
In summary, a series of novel thiazole derivatives containing amide skeleton were
synthesized and assayed for their antibacterial activities against E.coli, P.aeruginosa,
B.Subtilis and S.aureus. Most compounds showed potent antibacterial and ecKAS III
inhibitory
activities.
In
particular,
compound
4e,
N-(4-(4-bromophenyl)thiazol-2-yl)-2-(3-chlorophenyl)acetamide showed both the
promising broad antibacterial activity with MIC of 1.56-6.25 µg/mL against the
representative bacterial stains, which was compared with the positive control
Kanamycin B and Penicillin G, and also processed the most potent ecKAS III
inhibitory activity with IC50 of 5.3 µM. Preliminary structure-activity relationships
and molecular modeling study provided further insight into interactions between the
enzyme and this small molecule ligand.
Acknowledgements
This work was supported by National Fundamental Fund of Science Personnel
Training (No. J1103512).
Supplementary data
Supplementary data associated with this article can be found, in the online version,
at http://dx.doi .org/xxxx.
6

References
1 Leeb, M. Nature. 2004, 431, 892.
2 Price, A. C.; Choi, K. H.; Heath, R. J.; Li, Z.; White, S. W.; Rock. C. O. J. Biol.
Chem. 2000, 276, 6551.
3 Khandekar, S. S.; Gentry, D. R.; VanAller, G. S.; Warren, P.; Xiang, H.; Silverman,
C.; Doyle, M. L.; Konstantinidis, A. K.; Brandt, M.; Daines, R. A.; Lonsdale, J. T. J.
Biol. Chem. 2001, 274, 30025.
4 Qiu, X.; Janson, C. A.; Smith, W. W.; Head, M.; Lonsdale, J.; Konstantinidis, A. K.
J. Mol. Biol. 2001, 307, 343.
5 Khandekar, S. S.; Daines, R. A.; Lonsdale, J. T. Curr. Protein Pept. Sci. 2003, 4, 21.
6 Heath, R. J.; Rock, C. O. J. Biol. Chem. 1996, 271,1833.
7 Nie, Z.; Perretta, C.; Lu, J.; Su, Y.; Margosiak, S.; Gajiwala, K. S.; Cortez, J.;
Nikulin,V.; Yager, K.M.; Appelt, K.; Chu, S. J. Med. Chem. 2005, 48, 1596.
8 Hutchinson, I.; Jennings, S. A.; Vishnuvajjala, B. R.; Westwell, A. D.; Stevens, M. F.
G. J. Med. Chem. 2002, 45, 744.
9 Hargrave, K. D.; Hess, F. K.; Oliver, J. T. J. Med. Chem. 1983, 26, 1158.
10 Patt, W. C.; Hamilton, H. W.; Taylor, M. D.; Ryan, M. J.; Taylor, D. G., Jr.;
Connolly, C. J. C.; Doherty, A. M.; Klutchko, S. R.; Sircar, I.; Steinbaugh, B. A.;
Batley, B. L.; Painchaud, C. A.; Rapundalo, S. T.; Michniewicz, B. M.; Olson, S. C. J.
J. Med. Chem. 1992, 35, 2562.
11 Sharma, P. K.; Sawnhney, S. N.; Gupta, A.; Singh, G. B.; Bani, S. Indian J. Chem.
1998, 37B, 376.
12 Jaen, J. C.; Wise, L. D.; Caprathe, B. W.; Tecle, H.; Bergmeier, S.; Humblet, C. C.;
Heffner, T. G.; Meltzner, L. T.; Pugsley, T. A. J. Med. Chem. 1990, 33, 311.
13 Tsuji, K.; Ishikawa, H. Bioorg. Med. Chem. Lett. 1994, 4, 1601.
14 Bell, F. W.; Cantrell, A. S.; Hogberg, M.; Jaskunas, S. R.; Johansson, N. G.; Jordon,
C. L.; Kinnick, M. D.; Lind, P.; Morin, J. M., Jr.; Noreen, R.; Oberg, B.; Palkowitz, J.
A.; Parrish, C. A.; Pranc, P.; Sahlberg, C.; Ternansky, R. J.; Vasileff, R. T.; Vrang, L.;
West, S. J.; Zhang, H.; Zhou, X. X. J. Med. Chem. 1995, 38, 4929.
15 Ergenc, N.; Capan, G.; Günay, N. S.; Özkirimli, S.; Güngor, M.; Özbey, S.; Kendi,
7

E. Arch. Pharm. Pharm. Med. Chem. 1999, 332, 343.
16 Carter, J. S.; Kramer, S.; Talley, J. J.; Penning, T.; Collins, P.; Graneto, M. J.;
Seibert, K.; Koboldt, C.; Masferrer, J.; Zweifel, B. Bioorg. Med. Chem. Lett. 1999, 9,
1171.
17 Kitagawa, H.; Ozawa, T.; Takahata, S.; Iida, M.; Saito, J.; Yamada, M. J. Med.
Chem. 2007, 50, 4710.
18 Qiu, X. L; Li, G. D; Wu, G. K; Zhu, J. W; Zhou, L; Chen, P. L; Chamberlin, A. R;
Lee, W. H. J. Med. Chem. 2009, 52, 1758.
19 Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.
Olson, A. J. J. Comput. Chem. 1998, 19, 1660.
Scheme 1. General Synthetic Procedure for the Preparation of Compounds 3a-3l and
4a-4l.
o
Reagents and conditions: (a) Thiourea, EtOH, rt; (b) benzoyl chloride, pyridine, 0 C
to rt; (c) substituted phenylacetic acid, EDC, HOBt, CH2Cl2, rt; (d)
4-Morpholinecarbonyl chloride, pyridine, 0 oC to rt; (e) 1-naphthoic acid, EDC, HOBt,
CH2Cl2, rt; (f) 2-naphthoic acid, EDC, HOBt, CH2Cl2, rt.
Figure 1. ecKAS III -catalyzed initiation reaction of fatty acid biosynthesis.
Figure 2. Molecular binding mode of compound 4e with ecKAS III.
8

R4
R1
R1
R3
O
O
N
N
R2
NH
NH
S
S
3i R1 = H
4i R1 = Br
3a-3h, 4a-4h
c
b
R1
R1
R1
a
d
O
N
N
S
N
O
Br
NH2
NH
S
O
2a R1 = H
2b R1 = Br
1a R1 = H
1b R1 = Br
3l R1 = H
4l R1 = Br
f
e
R1
R1
O
N
O
NH
S
N
NH
S
3k R1 = H
4k R1 = Br
3j R1 = H
4j R1 = Br
Scheme 1. General Synthetic Procedure for the Preparation of Compounds 3a-3l and
4a-4l.
o
Reagents and conditions: (a) Thiourea, EtOH, rt; (b) benzoyl chloride, pyridine, 0 C
to rt; (c) substituted phenylacetic acid, EDC, HOBt, CH2Cl2, rt; (d)
4-Morpholinecarbonyl chloride, pyridine, 0 oC to rt; (e) 1-naphthoic acid, EDC, HOBt,
CH2Cl2, rt; (f) 2-naphthoic acid, EDC, HOBt, CH2Cl2, rt.
9

Table 1. Structure of compounds 3a-3l and 4a-4l.
Compd
3a
R1
H
H
H
H
H
H
H
H
H
R2
H
H
H
H
H
H
H
H
R3
H
R4
H
Compd
4a
R1
Br
Br
Br
Br
Br
Br
Br
Br
Br
R2
H
H
H
H
H
H
H
H
R3
H
R4
H
3b
H
F
4b
H
F
3c
H
Cl
Br
H
4c
H
Cl
Br
H
3d
H
4d
H
3e
Cl
4e
Cl
3f
Br
H
4f
Br
H
3g
OMe
OEt
H
4g
OMe
OEt
H
3h
OEt
4h
OEt
3i
4i
R=
R=
3j
3k
3l
H
H
H
4j
4k
4l
Br
Br
Br
R=
R=
R=
R=
R=
R=
10

Table 2. MICs (minimum inhibitory concentrations) (µg/mL) of the synthetic
compounds.
Microorganisms
Compounds
Gram-negative
Gram-positive
E.coli
ATCC35218
100
P.aeruginosa
ATCC13525
100
B.subtilis
ATCC6633
50
S.aureus
ATCC6538
25
3a
12.5
25
50
25
25
50
12.5
12.5
25
12.5
25
50
3b
3c
3d
6.25
12.5
50
6.25
12.5
50
12.5
6.25
100
6.25
3.13
50
3e
3f
3g
50
50
>100
>100
>100
50
>100
25
>100
>100
>100
>100
>100
25
3h
3i
3j
3k
3l
4a
>100
>100
>100
>100
50
>100
>100
>100
>100
50
4b
4c
4d
12.5
12.5
25
12.5
25
50
6.25
12.5
25
12.5
12.5
25
1.56
6.25
25
3.13
12.5
50
6.25
6.25
25
3.13
6.25
50
4e
4f
4g
50
>100
50
>100
50
1.56
3.13
25
>100
100
>100
>100
3.13
6.25
50
>100
>100
>100
>100
>100
3.13
6.25
4h
4i
4j
4k
>100
>100
>100
100
0.39
1.56
4l
Kanamycin B
Penicillin G
11

Table 3. E.coli ecKAS III inhibitory activity of synthetic compounds.
Compounds
ecKAS III IC50 (µM)a
Hemolysis LC30 (mg/mL)
38.4±2.6
41.8±5.3
12.3±3.1
26.9±2.7
29.3±2.2
34.7±1.9
48.1±0.6
5.3±1.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3b
3c
3e
3f
4b
4c
4d
4e
4f
9.8±2.5
65.6±3.7
4g
a IC50 average values and corresponding SD values are determined from the results of
at least three independent repeats.
12

Figure 1. ecKAS III -catalyzed initiation reaction of fatty acid biosynthesis.
Figure 2. Molecular docking modeling of compound 4e with ecKAS III. Compund 4e
is displayed with bold bond.
13

Design, synthesis and antibacterial activity studies of thiazole
derivatives as potent ecKAS III inhibitors
Kui Cheng, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China.
Two series of thiazole derivatives containing amide skeleton were synthesized and
developed as potent Escherichia coli β-ketoacyl-(acyl-carrier-protein) synthase III
(ecKAS III) inhibitors. All the 24 new synthesized compounds were assayed for
antibacterial activity against the respective Gram-negative and Gram-positive
bacterial strains, including Escherichia coli, Pseudomonas aeruginosa, Bacillus
subtilis and Staphylococcus aureus. In which, 10 compounds with broad-spectrum
antibacterial activities were further tested for their ecKAS III inhibitory activity. Last,
we have successfully found that compound 4e showed both the promising broad
antibacterial activity with MIC of 1.56-6.25 µg/mL against the representative bacterial
stains, and also processed the most potent ecKAS III inhibitory activity with IC50 of
5.3 µM. In addition, docking simulation also carried out in this study to give a potent
prediction binding mode between the small molecule and ecKAS III (PDB code: 1hnj)
protein.
14