Synthesis and biological evaluation of 2-arylimino-3-pyridin-thiazolineone derivatives as antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2016.03.089

With an intention to find more potent antibacterial agents, four halogen disubstituted thiazolineone derivatives (2a-d), five halogen monosubstituted thiazolineone derivatives (2e-i), and eleven 2-arylimino-3-pyridin-thiazolineone derivatives (2j-t) were

Full text of DOI:10.1016/j.bmcl.2016.03.089

Accepted Manuscript
Synthesis and biological evaluation of 2-arylimino-3-pyridin-thiazolineone de-
rivatives as antibacterial agents
Ming-Guang Cai, Yang Wu, Jun Chang
PII:
S0960-894X(16)30321-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.089
BMCL 23732
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
7 January 2016
6 March 2016
25 March 2016
Please cite this article as: Cai, M-G., Wu, Y., Chang, J., Synthesis and biological evaluation of 2-arylimino-3-pyridin-
thiazolineone derivatives as antibacterial agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.03.089
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Synthesis and biological evaluation of 2-arylimino-3-pyridin-thiazolineone
derivatives as antibacterial agents
Ming-Guang Caia, Yang Wuc,, Jun Changb,
a College of Chemistry and Molecular Engineering, Qingdao University of Science and
Technology, 53 Zhengzhou Road, Qingdao 266042, China
b Department of Natural Products Chemistry, School of Pharmacy, Fudan University,
826 Zhangheng Road, Shanghai 201203, China
c
Key Laboratory of Medical Molecular Virology of Ministries of Education and
Health, School of Basic Medical Science and Institutes of Biomedical Sciences,
Shanghai Medical College of Fudan University, Shanghai 200032, China
 Corresponding author. Tel./fax: +86 21 51980102.
E-mail address: jchang@fudan.edu.cn (J. Chang), yangwu@fudan.edu.cn (Y. Wu).
1

Abstract
With an intention to find more potent antibacterial agents, four halogen
disubstituted thiazolineone derivatives (2a–d), five halogen monosubstituted
thiazolineone derivatives (2e–i), and eleven 2-arylimino-3-pyridin-thiazolineone
derivatives (2j–t) were synthesized and screened for their antibacterial activity,
bactericidal activity, cytotoxicity, and erythrocyte hemolysis. Most of the synthesized
derivatives showed antibacterial activity in inhibiting the growth of S. epidermidis and
MRSA, and exhibited safety in the cytotoxicity study on the Vero cells and hemolytic
activities test on healthy human erythrocytes. 2-arylimino-3-pyridin-thiazolineone
derivatives not only improved the clog P, but also showed potent antibacterial activity
in inhibiting the growth of S. epidermidis and MRSA. In particularly, several
compounds (2f, 2i, 2r and 2t) showed bactericidal activity, in which compound 2r
displayed the best inhibitory capacity among the synthesized compounds, and further
druggability research is on going.
Keywords
Synthesis; biological evaluation; 2-arylimino-3-pyridin-thiazolineone derivatives;
antibacterial agents
2

Infections caused by drug-resistant Gram-positive bacteria such as
methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant
Streptococcus pneumoniae (PRSP), and vancomycin-resistant Enterococcus faecalis
(VRE) are still the most serious threat to global health.1–2 NIH findings indicated that
more than 80% of bacterial infections were related with biofilm.3 Bacterial biofilm
can resist not only the host immune destruction, but also most of the antibiotics,
moreover, the resistance was up to a thousand times more than planktonic bacteria.4
Most of the existing antibiotics fail to adequately penetrate the biofilm or have limited
activity against surface-attached cells and cells with low metabolic activity.5
Daptomycin and linezolid are now available for biofilm-associated infections caused
by staphylococci, but neither was found to be bactericidal against biofilm-embedded
bacteria.6 Vancomycin is regarded as an antibiotic of last resort against MRSA, and
other multiple antibiotic-resistant infections caused by Gram-positive bacteria, but it
has no significant effect on the bacteria in the biofilm.7
Bacterial two-component systems (TCSs) were proposed as attractive targets more
than 20 years ago because they are absent in mammals and essential or conditionally
essential for viability in several important bacterial pathogens. WalKR system (a.k.a.,
YycGF, VicKR, MicAB) is an obligate essential regulatory system in Firmicutes
including MRSA, VRE, and some other notorious pathogens.8-9 It plays important
roles in the growth, cell wall metabolism, and biofilm formation of pathogenic
staphylococcal species.10 We have previously described two series of YycG inhibitors
that target the HK domain of S. epidermidis YycG and show bactericidal and
antibiofilm activities against S. epidermidis and S. aureus.7, 11-12 One of the two lead
compounds
is
{4-[3-(2-ethylphenyl)-2-(2-ethylphenylimino)-4-oxothiazolidin-5-ylidenemethyl]-2-m
ethoxyphenoxy}-acetic acid (1). In this study, we reported our further research results
on the structure-activity relationships (SAR) of compound 1.
3

According to the previous SAR, it was found that the introduction of halogen
substituent on phenyl rings displayed more potent antibacterial activity. Considering a
significant number of antibacterial agents in clinical development are chlorinated
structures,13 and the introduction of the strongly electron-withdrawing fluorine group
into drugs or drug candidates can substantially enhanced binding interactions,
metabolic stability, changes in physical properties, and selective reactivities,14 we paid
more attention to chlorine or fluorine substitution. Therefore, halogen disubstituted
thiazolineone derivatives (2a–d) and halogen monosubstituted thiazolineone
derivatives (2e–i) were synthesized. Log P is an important parameter in the
druggability research, and clog P of the good lead compound should be between 0 and
3.0. However, clog P of most of the derivatives (2a–i) was so high that heterocyclic
rings derivatives (2j–t) were designed and synthesized to increase the hydrophility by
applying the principle of bioisosterism. (Figure 1)
2
R
2
R
1
R
1
R
Optimization
Bioisosterism
N
S
N
S
N
N
S
N
N
N
COOH
O
COOH
O
X
O
O
X
O
COOH
2j-t
1
2a-i
Figure 1. Design strategy
The synthesis of halogen disubstituted thiazolineone derivatives (2a–d)
commenced from corresponding halogen substituted aniline according to the previous
report (Scheme 1),15 while halogen monosubstituted thiazolineone derivatives (2e-i)
were synthesized from asymmetric thioureas (4c-d). The substituted anilines were
converted to asymmetric thioureas (4c-d) by using phenyl isothiocyanate in CH2Cl2
under reflux for 2 h in 70% yields. 4c was transformed into iminothiazolidinones 5c
and 5d (1:1) via the reaction of ethyl bromoacetate in absolute EtOH and NaOAc,
4

while 4d was selectively transformed into iminothiazolidinones 5e in 88% yield. 2e-i
were achieved by the Knoevenagel condensation of substituted iminothiazolidinones
(5c-e) with equivalent amount of aromatic aldehydes (6a/6b) catalyzed by -alanine
in acetic acid under reflux for 2 h in good yields.16
1
R
1
H
H
R
N
N
N
S
CS
2
S
N
c
a
1
1
R
R
O
NH
2
1
1
4a: R = Cl
5a: R = Cl
C
N
1
1
S
4b: R = F
2
5b: R = F
R
H
H
1
1
R
R
N
N
N
2
R
3
S
N
S
2
1
R
c
b
R
O
1
2
1
2
4c: R = Cl, R = H
5c: R = Cl, R = H
1
2
1
2
4d: R = F, R = H
5d: R = H, R = Cl
1
2
5e: R = F, R = H
1
R
HO
O
1
R
N
S
1
O
2a: R = Cl, X = O
N
X
1
2b: R = Cl, X = S
COOH
H
1
O
2c: R = F, X = O
X
1
2d: R = F, X = S
6a: X = O
6b: X = S
2
R
d
1
R
1
2
N
2e: R = Cl, R = H, X = O
1
2
2f: R = Cl, R = H, X = S
N
S
1
2
COOH
2g: R = H, R = Cl, X = O
O
1
2
X
2h: R = F, R = H, X = O
1
2
2i: R = F, R = H, X = S
o
Scheme 1. Reagents and conditions: a) 10% NaOH solution, 60 C; b) CH2Cl2, reflux;
c) ethyl bromoacetate, NaOAc, absolute alcohol, reflux; d) β-alanine, acetic acid, reflux.
To avoid of the two possible regioisomeric iminothiazolidinone products from the
asymmetric thioureas, many reagents were tried, including ethyl bromoacetate, ethyl
chloroacetate, chloroacetic acid, bromoacetic acid. However, unfortunately they all
didn’t work. At last, we found a different route to selectively synthesize 5f-n, as
illustrated in Scheme 2.17 The reaction of aminopyridine or substituted aminopyridine
5

(7a-g) with choloro acetylcholoride afforded the corresponding amides 8a-g. Then the
compounds 8a-g reacted smoothly with isothiocyanates in the presence of a weak
base such as K2CO3 in CH3CN to produce iminothiazolidinones (5f-n). The final step
of the Knoevenagel condensation of iminothiazolidinones (5f-n) with equivalent
amount of substituted aromatic aldehydes (6a/6b) was catalyzed by -alanine to give
the target compounds 2j-t in good yields.18 The structures of a total of twenty
synthesized compounds were confirmed by 1H NMR, 13C NMR and mass spectra.
1
1
2
2
R
5f: R = H,
R
= 4-F
O
1
1
R
1
R
2
N
C
5g: R = H,
R
= 4-Cl
R
Cl
N
S
N
b
N
N
1
2
N
NH
2
5h: R = 3-F, R = 4-Cl
H
N
1
2
O
a
1
1
S
5i: R = 3-F, R = H
7a: R = H
8a: R = H
2
R
1
2
Cl
1
1
5j: R = 5-F, R = H
O
7b: R = 3-F
8b: R = 3-F
Cl
1
2
1
1
5k: R = 6-F, R = H
7c: R = 5-F
8c: R = 5-F
1
1
7d: R = 6-F
8d: R = 6-F
H
N
N
NH
2
2
Cl
R
N
1
1
2
1
R
5l: R = 2-Cl,
R
= H
= H
R
O
N
1
1
2
2
R
N
5m: R = 2-Me,
R
N
S
1
1
5n: R = 2-NO , R = H
1
2
8e: R = 2-Cl
7e: R = 2-Cl
1
O
1
8f: R = 2-Me
7f:
R = 2-Me
1
1
8g: R = 2-NO
7g: R = 2-NO
2
2
1
R
HO
O
1
1
2
2j:
R
= H, R = 4-F, X = O
2
N
2
N
R
2k: R = H,
1
R = 4-Cl, X = O
O
2
2l:
R
= 3-F, R = 4-Cl, X = O
2
N
X
S
1
COOH
2m: R = 3-F, R = H, X = O
H
O
1
2
X
2n: R = 5-F, R = H, X = O
1
2
6a: X = O
6b: X = S
2o: R = 5-F, R = H, X = S
1
2
2p: R = 6-F, R = H, X = O
c
N
2
R
N
1
2 =
2
2q: R = 2-Cl,
R
H, X = O
1
1
R
N
2r: R = 2-Cl,
R
R
= H, X = S
S
1
2 =
COOH
2s: R = 2-Me,
H, X = S
O
1
2 =
X
2t:
R
= 2-NO , R H, X = S
2
Scheme 2. Reagents and conditions: a) Et3N, CH2Cl2, rt; b) K2CO3, CH3CN, rt; c) β-alanine,
acetic acid, reflux.
The in vitro antibacterial activity (MIC) was evaluated with Gram-positive bacterial
strains (S. epidermidis ATCC35984 and clinical bacterial strain MRSA 8282) and
Gram-negative bacterial strain (E. Coli ATCC25922) according to the broth
micro-dilution (in tubes) methods of the clinical and Laboratory Standards Institute
(CLSI) of America. Most of the synthesized compounds showed good potency in
6

inhibiting the growth of S. epidermidis ATCC35984 and MRSA 8282, while none of
them was able to inhibit the growth of E. Coli.
Table 1. Biological activity of the compounds 2a-t
Compounds S. epidermidis
ATCC 35984
MRSA
8282
S. epidermidis
E. Coli
CC50
Hemolysis d
ATCC 35984
ATCC 25922 (M) c
MIC (M) a
MIC (M) a
MIC (M) a MBC (M) b
1
25
25
100
>200
>200
>200
100
50
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
50






















2a
25
25
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
2b
2c
12.5
100
6.25
6.25
1.56
6.25
25
25
100
6.25
6.25
3.13
6.25
25
2d
2e
2f
25
2g
100
50
2h
2i
6.25
12.5
6.25
12.5
12.5
12.5
3.13
6.25
3.13
1.56
12.5
1.56
3.13
12.5
12.5
6.25
12.5
12.5
12.5
3.13
6.25
6.25
3.13
12.5
3.13
100
25
2j
>200
50
2k
2l
>200
>200
>200
50
2m
2n
2o
2p
2q
2r
100
50
6.3
2s
100
12.6
>200
2t
methicillin
a MIC represented the minimal inhibitory concentration of the compounds against S. epidermidi
ATCC 35984, MRSA 8282, and E. Coli ATCC 25922. b MBC represented minimal bactericidal
concentration of the compounds against S. epidermidi ATCC 35984. c CC50 represented the
d
concentration of the compounds which produces 50% cytotoxicity effect on Vero cells.
Hemolytic activities of the compounds on healthy human erythrocytes were tested at 200 M,
“”: no hemolytic activity at 200 M.
7

The results showed that halogen monosubstituted series (2e-i) exhibited more
potent antibacterial activity than the corresponding disubstituted series (2a-d) (Table
1). The replacement of furan moiety with thiophene moiety based on the bioisosterism
improved the inhibitory activity compared with the corresponding furan derivatives.
2e showed same inhibitory activity as 2g, which suggested that the site of halogen
monosubstituted on the phenyl ring connected with amide or imino didn’t influence
the inhibitory activity. Heterocyclic rings derivatives (2j–t) still maintain the
antibacterial activity. Similar as the above SAR, compound 2j with the halogen
substituted on the phenyl ring exhibited comparable antibacterial activity to
compounds 2m and 2n, with the halogen substituted on the pyridine ring. It was
noteworthy that although the positions of fluorine and nitrogen on the pyridine didn’t
influence the activity significantly, chlorine substitution derivatives showed more
potent antibacterial activity than fluorine substitution derivatives. Compound 2s with
the methyl substituted on the pyridine ring significantly reduced the antibacterial
activity, compared with compound 2t with the nitro group substituted. Moreover,
compounds 2a-2t exhibited similar antibacterial activity against clinical bacterial
strain MRSA 8282. It was worth mentioning that compounds 2f, 2i, 2r and 2t also
showed bactericidal activity. In the cytotoxicity study on the Vero cells, the CC50
values of all the derivatives were greater than 200 M. At the MIC concentrations, all
the derivatives lysed healthy human erythrocytes by less than 1%, and showed no
obvious hemolysis even at the highest concentration 200 M.
In conclusion, four halogen disubstituted thiazolineone derivatives (2a–d), five
halogen
monosubstituted
thiazolineone
derivatives
(2e–i),
and
nine
2-arylimino-3-pyridin-thiazolineone derivatives (2j–t) were synthesized and screened
for their antibacterial activity, bactericidal activity, cytotoxicity, and erythrocyte
hemolysis. Most of the synthesized derivatives showed antibacterial activity in
inhibiting the growth of S. epidermidis and clinical bacterial strain MRSA 8282, and
8

exhibited safety in the cytotoxicity study on the Vero cells and hemolytic activities
test on healthy human erythrocytes. 2-arylimino-3-pyridin-thiazolineone derivatives
not only improved the clog P, but also maintained the antibacterial activity in
inhibiting the growth of S. epidermidis and MRSA. In particularly, several compounds
(2f, 2i, 2r and 2t) showed bactericidal activity, in which compound 2r displayed the
best inhibitory capacity among the synthesized compounds, and further druggability
research is on going.
Acknowledgments
Financial support by the National Natural Science Foundation of China (No:
81573268), and the Shanghai Municipal Committee of Science and Technology (No:
14431900300, 15431900400) are acknowledged.
References and notes
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Antimicrob. Chemother. 2012, 67, 2682.
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Danchin, A.; Jiang, H.; Qu, D. BMC Microbiol. 2006, 6, 96.
8. Bem, A. E.; Velikova, N.; Pellicer, M. T.; van Baarlen, P.; Marina, A.; Wells, J. M.
ACS Chem. Biol. 2015, 10, 213.
9. Velikova, N.; Bem, A. E.; van Baarlen, P.; Wells, J. M.; Marina, A. ACS Med.
Chem. Lett. 2013, 4, 891.
9

10. Winkler, M. E.; Hoch, J. A. J. Bacteriol. 2008, 190, 2645.
11. Huang, R-Z.; Zheng, L-K.; Liu, H-Y.; Pan, B.; Hu, J.; Zhu, T.; Wang, W.; Jiang,
D-B.; Wu, Y.; Wu, Y-C.; Han, S-Q.; Qu, D. Acta Pharmacol. Sin. 2012, 33, 418.
12. Liu, H.; Zhao, D.; Chang, J.; Yan, L.; Zhao, F.; Wu, Y.; Xu, T.; Gong, T.; Chen,
L.; He, N.; Wu, Y.; Han, S.; Qu, D. Appl. Microbiol. Biotechnol. 2014, 98, 6003.
13. McArthur, K. A.; Mitchell, S. S.; Tsueng, G.; Rheingold, A.; White, D. J.;
Grodberg, J.; Lam, K. S.; Potts, B. C. M. J. Nat. Prod. 2008, 71, 1732.
14. Hagmann, W. K. J. Med. Chem. 2008, 51, 4359.
15. Zhang, J.; Zheng, L.; Liu, H.; Zhao, D.; Qu, D.; Han, S. Lett. Drug Des. Disc.
2013, 10, 928.
16. Luo,Y.; Ma, L.; Zheng, H.; Chen, L.; Li, R.; He,C.; Yang, S.; Ye, X.; Chen, Z.; Li,
Z.; Gao, Y.; Han, J.; He,G.; Yang L.; Wei, Y. J. Med. Chem. 2010, 53, 273.
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18.
Representative
experiment
for
the
preparation
of
3-{5-{[2-phenylimino-3-(2-chloropyridin-3-yl)-4-oxothiazolidin-5-ylidene]methyl}thi
ophene-2-yl}-benzoic acid (2r): choloro acetylcholoride (24 mmol) and Et3N (24
mmol) was added to a solution of 2-chloro-3-aminopyridine 7e (20 mmol) in CH2Cl2
(20 mL) at room temperature. The mixture was stirred for 5 hrs, and the solvent was
evaporated under vacuum. The residue was purified by column chromatography
(CH2Cl2:CH3OH: 30:1) on silica gel to obtain pure compound 8e as a white powder in
72% yield. To a solution of amide derivative 8e (5 mmol) and potassium carbonate
(7.5 mmol) in acetonitrile (20 ml) was added isothiocyanate (6 mmol) during about 5
minutes. The reaction mixture was stirred at room temperature overnight, and the
solvent was evaporated under vacuum. The residue was extracted with ethyl acetate
(20 mL × 3). The combined organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The obtained residue was purified by silica gel
flash chromatography column (CH2Cl2:CH3OH: 30:1) to afford 5l as a white solid in
10

82% yield. To a solution of 5l (1 mmol) in glacial acetic acid (5 mL) were added
aldehyde 6b (1 mmol) and β-alanine (1 mmol). The resulting mixture was stirred
under reflux for 2 h. Upon completion of the reaction, the mixture was cooled, the
reaction was quenched with water, and the precipitate was filtered off, then the
residue was purified by column chromatography (CH2Cl2:CH3OH: 15:1) on silica gel
to obtain pure compound 2r as a faint yellow powder in 80% yield. Mp 230-232 °C;
1H NMR (400 MHz, DMSO-d6) δ 13.23(s, 1H), 8.59 (s, 1H), 8.17 (m, 2H), 7.97 (d, J
= 7.5 Hz, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.80 (m, 1H), 7.75 (m, 1H), 7.56 (m, 4H),
7.41 (d, J = 7.5 Hz, 1H), 6.97 (d, J = 7.5 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ
165.86, 163.42, 154.79, 149.70, 148.83, 148.54, 148.07, 146.17, 140.02, 130.99,
128.95, 128.64 (3), 128.54, 128.26, 127.12, 124.33, 123.61, 123.50, 120.03 (3),
116.83, 116.73, 109.71. ESI-MS m/z 518.0 [M + H]+; HR-ESIMS m/z calcd. for
C26H17ClN3O3S2 [M + H]+: 518.0400, found 518.0402.
11

Graphic Abstract
2
R
2
R
1
R
1
R
Optimization
Bioisosterism
N
S
N
S
N
N
N
N
N
COOH
O
S
COOH
O
X
O
O
X
O
COOH
2j-t
1
2a-i
-6
-6
2r MIC: 1.56×10
MBC: 6.3×10
M
M
2-arylimino-3-pyridin-thiazolineone derivatives (2j-t) not only improved the clog P,
but also showed potent antibacterial activity in inhibiting the growth of S. epidermidis
and MRSA. In particularly, several compounds (2f, 2i, 2r and 2t) showed bactericidal
activity, in which compound 2r displayed the best inhibitory capacity among the
synthesized compounds, and further druggability research is on going.
12