Biological evaluation of halogenated thiazolo[3,2-a]pyrimidin-3-one carboxylic acid derivatives targeting the YycG histidine kinase

Article abstract of DOI:10.1016/j.ejmech.2014.09.096

With an intention to potent inhibitors of YycG histidine kinase, a series of halogenated thiazolo[3,2-a]pyrimidin-3-one carboxylic acid derivatives were synthesized and evaluated for their antibacterial, antibiofilm and hemolytic activities. The majority of the compounds showed good activity against Staphylococcus epidermidis and Staphylococcus aureus, with MIC values of 1.56-6.25 μM, simultaneously presented promising antiobifilm activity against S. epidermidis ATCC35984 at 50 μM. The test of inhibitory activity on YycG kinase suggested the antibacterial activities of these derivatives are based on inhibiting the enzyme activity of the YycG HK domain. The hemolytic activity test suggested these compounds exhibited in vitro antibacterial activity at non-hemolytic concentrations.

Full text of DOI:10.1016/j.ejmech.2014.09.096

European Journal of Medicinal Chemistry 87 (2014) 500e507
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Biological evaluation of halogenated thiazolo[3,2-a]pyrimidin-3-one
carboxylic acid derivatives targeting the YycG histidine kinase
,
,
,
, *
Dan Zhao a 1, Chen Chen a 1, Huayong Liu b 1, Likang Zheng a, Yao Tong a, Di Qu b
,
Shiqing Han a
,
*
a College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, China
b Key Laboratory of Medical Molecular Virology of Ministry of Education and Ministry of Public Health, Institute of Medical Microbiology and Institutes of
Biomedical Sciences, Shanghai Medical School of Fudan University Box 228, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
With an intention to potent inhibitors of YycG histidine kinase, a series of halogenated thiazolo[3,2-a]
pyrimidin-3-one carboxylic acid derivatives were synthesized and evaluated for their antibacterial,
antibiofilm and hemolytic activities. The majority of the compounds showed good activity against
Received 20 May 2014
Received in revised form
18 September 2014
Accepted 30 September 2014
Available online
Staphylococcus epidermidis and Staphylococcus aureus, with MIC values of 1.56e6.25
mM, simultaneously
presented promising antiobifilm activity against S. epidermidis ATCC35984 at 50 M. The test of inhibi-
m
tory activity on YycG kinase suggested the antibacterial activities of these derivatives are based on
inhibiting the enzyme activity of the YycG HK domain. The hemolytic activity test suggested these
compounds exhibited in vitro antibacterial activity at non-hemolytic concentrations.
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Halogenated thiazolo[3,2-a]pyrimidin-3-
one
Antibacterial activity
Antibiofilm activity
YycG histidine kinase inhibitor
Staphylococcus epidermidis
Staphylococcus aureus
1. Introduction
these inhibitors, a typical compound A (Fig. 1), which had a thia-
zolopyrimidinone core substituted by aromatic ring, showed potent
Antibiotic-resistant bacteria have become a major concern for
public health professionals since bacterial resistance has dramati-
cally increased during the last two decades [1]. Indeed, bacteria
growing as an adherent biofilm on catheters, orthopedic implants,
and bones are inherently resistant to antibiotics and are poorly
recognized by the host immune defenses [2]. Biofilm associated
bacteria exhibit enhanced resistance to many conventional antibi-
otics [3]. Staphylococcus epidermidis has become a significant
pathogen causing infections due to biofilm formation on surfaces of
indwelling medical devices [4]. Consequently, it has an urgent need
to design novel antibiotics against staphylococci infections, espe-
cially those can kill cells embedded in biofilm.
antibacterial activity, moderate antibiofim activity, low cytotoxicity
on mammalian cells and was not hemolytic. Our previous work on
analogs of thiazolo[3,2-a]pyrimidin-3-ones A suggests that car-
boxylic acid fragment at C-2 position of A-ring was indispensable
and B-ring opening analogs of thiazolo[3,2-a]pyrimidin-3-ones A
maintained the antibiofilm potency [6,7]. However, improvement
of antibacterial and antibiofilm potency of the compound A is still
needed and its structure modification is a challenging task.
Herein, we describe further modifications that focused on pre-
serving A-ring and carboxylic acid moieties at C-2 position of A-ring
while simultaneously investigating the necessity of B-ring and
variations to the B-ring substituents. Considering a significant
number of antimycobacterial agents in clinical development are
chlorinated structures [8], and the introduction of the strongly
electron-withdrawing fluorine group into drugs or drug candidates
can substantially enhanced binding interactions, metabolic stabil-
ity, changes in physical properties, and selective reactivities [9], we
paid attention to chlorine and fluorine substitution. A series of
thiazolo[3, 2-a]pyrimidine-3-one derivatives were designed,
accordingly (Fig. 1).
In recent years, some novel inhibitors of YycG histidine kinase
had been reported which are of potential value as leads for devel-
oping new antibiotics against infecting staphylococci [5]. Among
* Corresponding authors.
E-mail addresses: dqu@shmu.edu.cn (D. Qu), hanshiqing@njut.edu.cn, ssqqhan@
yahoo.com (S. Han).
1
Those authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.09.096
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

D. Zhao et al. / European Journal of Medicinal Chemistry 87 (2014) 500e507
501
Fig. 1. Leading compound and structure-based design of the target compounds.
2. Chemistry
4k, MIC ¼ 3.125
m
M), it revealed that
p
e
p
stacking effect of phenyl
group might led thiazolo[3,2-a]pyrimidine-3-ones bind the target
protein more easily. Moreover, in order to check out whether the
ester moiety on 6-position of B-ring is essential for activity or not,
compounds 8aec without 6-ester moiety were also synthesized.
The biological results revealed that removing the ester moiety did
not decrease the antibacterial activity.
The synthetic route to 4aeo is depicted in Scheme 1. A multiple-
componentchemical reaction known as Biginelli reaction of aryl
aldehyde or substituted aryl aldehyde, ethyl benzoylacetate and
thiourea gave different dihydropyrimidine scaffolds (DHPMs,
2aeh). Cyclization of 2aeh with bromoacetic acid afforded 3aeh
[10], an aldol condensation of 3aeh with equivalent amount
substituted aromatic aldehydes [11] of was catalyzed by b-alanine
to give 4aeo in good yields [12]. As shown in Scheme 2, an aldol
reaction of acetophenone and substituted phenylaldehyde gave
5aec, 5aec reacted with thiourea under base condition gave 6aec
[13], cyclization of 6aec with bromoacetic acid afforded 7aec. 8aec
were prepared via aldol condensation between 7aec and aromatic
aldehydes. The identities of all of the synthesized compounds were
confirmed by IR, 1H NMR, 13C NMR and mass spectral.
3.2. Antibiofilm and hemolysis activity of the derivatives
The newly synthesized compounds were further screened for
their antibiofilm activities against S. epidermidis ATCC35984 and
hemolytic activities of the derivatives on healthy human
erythrocytes.
As indicated in Table 2, the compounds 4b, 4e, 4f, 4i, 4j, 8b and
8c displayed antibiofilm activity at 50
hemolytic activity in human erythrocytes at 50
m
M. No compound showed
3. Results and discussion
m
M. Interestingly, in
compare with the compound A, the antibiofilm activity of 8a dis-
appeared, although its antibacterial activity was improved (Ta-
ble 1). While 4-Cl or 4-F substituted compounds 8b and 8c regained
3.1. Evaluation of antibacterial activity
The in vitro antibacterial activity was evaluated with Gram-
positive (S. epidermidis ATCC35984, S. epidermidis ATCC12228,
Staphylococcus aureus ATCC25923) and Gram-negative (Escherichia
coli and Shigella flexneri 301) bacterial strains, using the minimum
inhibitory concentration (MIC). Cefazolin was used as positive
control for bacteria.
As it can be concluded from the data in Table 1, most of the
compounds were highly active against S. epidermidis ATCC35984,
S. epidermidis ATCC12228 and S. aureus ATCC25923. Unlike the
good activity of compounds against Gram positive bacterial strains,
this class of compounds did not show any antibacterial activity in
vitro against the Gram-negative (E. coli ATCC25922 and S. flexneri
promising antibiofilm activity at 50
Cl or 4-F substituted 4e, 4f, 4i, 4j also exhibited promising anti-
biofilm activity at 50 M. All the results showed that an ester
mM. In addition, compounds 4-
m
moiety at C-2 position of B-ring and the substituent phenyl moi-
eties at C-5 position of B-ring might be two decisive factors influ-
enced the antibiofilm activity.
3.3. Inhibition of YycG enzyme activity by the derivatives
The newly synthesized compounds are derivatives of lead
compound A which is the inhibitor of YycG histidine kinase. To
detect whether the derivatives still have the inhibitory activities on
YycG kinase, the recombinant HATPase_c and HisKA domains of
YycG were expressed in E. coli BL21 (DE3) and purified by the Ni-
NTA column, then ATP-Luminescence assay kit (Promega) were
used to measure the inhibitory activities of the compounds on YycG
autophosphorylation activity. With good antibacterial activities as
above mentioned, compounds 4e, 8a and 8b were selected for the
enzyme inhibitor assay. As it was envisaged, all of them displayed
inhibition of phosphorylation of the recombinant YycG. The IC50
301) strains at 200
In the compounds containing substituents on the phenyl ring,
compounds 4e (MIC ¼ 1.56e6.25 M), 4i (MIC ¼ 3.125e6.25 M)
and 4k (MIC ¼ 3.125 M) with 4-Cl substituent, compounds 4f
(MIC ¼ 3.125e6.25 M) with 4-F substituent presented much more
potent antibacterial activity, relative to the leading compound A
and cefazolin. Compounds (4bef, MIC ¼ 1.56e50 M) containing
substituents on the phenyl ring showed higher levels of activity
than non-substituted one (4a, MIC > 200 M), Based on this fact, we
mM.
m
m
m
m
m
m
inferred that the presence of 4-Cl or 4-F may have a good contri-
bution to the activity. The derivatives bearing methyl group (4g,
values were 12.65 M, respectively, and
m
M, 49.92
m
M and 55.61
m
MIC ¼ 6.25
m
M and 4m, MIC ¼ 200
m
M) on 6-position of B-ring
the IC50 of prototype compound A was 14 mM (Table 3). This sug-
gested that the bactericidal activity of these derivatives is based on
inhibiting the enzyme activity of the YycG HK domain.
showed lower levels of activity against S. epidermidis ATCC35984
than the corresponding derivatives bearing phenyl group (4b and

502
D. Zhao et al. / European Journal of Medicinal Chemistry 87 (2014) 500e507
Scheme 1. Reagents and conditions: (i) CeCl3$7H2O, EtOH, reflux; (ii) bromoacetic acid, NaOAc, AcOH/Ac2O, reflux; (iii) b-alanine, acetic acid, reflux.
4. Conclusion
valuable leads for development of novel inhibitors of YycG histidine
kinase. More modifications on compounds 4e, 4f, 8b and 8c and
biological evaluation are on going in our laboratories.
A series of thiazolo[3,2-a]pyrimidin-3-one carboxylic acid de-
rivatives were synthesized and evaluated for antibacterial, anti-
biofilm and hemolytic activities. Most of the compounds showed
good antibacterial activity and antibiofilm activity. In particular,
compounds 4e, 4f, 8b and 8c exhibited stronger activity against
S. epidermidis and S. aureus than the standard drug cefazolin.
Compound 4e bearing 4-Cl substituent exhibited higher level of
inhibitory activity on YycG kinase. This suggested that chlorinated
or fluorinated thiazolo[3,2-a]pyrimidin-3-ones might provide
5. Experimental protocols
5.1. Chemistry
All reagents and solvents were commercially available and used
without further purification. Melting points were determined on an
electrothermal digital apparatus model WRS-1B (Shanghai, China),
Scheme 2. Reagents and conditions: (i) methanol, 2 N NaOH (aq); (ii) KOH, ethanol; (iii) BrCH2COOH, NaOAc, AcOH/Ac2O; (iv) b-alanine, acetic acid, reflux.

D. Zhao et al. / European Journal of Medicinal Chemistry 87 (2014) 500e507
503
Table 1
Antibacterial activities of the compounds 4aeo and 8aec.
Compound
MIC (m
M)a
S. epidermidis ATCC35984
S. epidermidis ATCC12228
S. aureus ATCC25923
E. coli ATCC25922
Shigella flexneri 301
A
4a
4b
4c
4d
4e
6.25
200
3.125
50
1.56
1.56
6.25
6.25
6.25
6.25
6.25
3.125
50
200
200
50
1.56
1.56
6.25
17.6
6.25
200
3.125
50
3.125
3.125
3.125
nd
6.25
3.125
6.25
3.125
50
200
200
50
6.25
6.25
3.125
17.6
6.25
nd
nd
nd
25
6.25
6.25
nd
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
e
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
8a
8b
8c
nd
6.25
6.25
nd
nd
nd
nd
nd
6.25
3.125
6.25
17.6
Cefazolin
nd ¼ no detected.
a
MIC assay was performed following the broth micro-dilution method (in tubes) of the CLSI of America. In this assay, the highest concentration of each compound was
200 mM.
without correction. All 1H NMR (300 or 400 MHz) and 13C NMR
(100 MHz) spectra were recorded on a Bruker AM-300 or 400 MHz
spectrometer (Bruker) as solutions in DMSO-d6 or CDCl3 with TMS
as an internal standard. Mass spectra (MS) were recorded on an HP-
5973 instrument (HP). IR spectra were recorded (in KBr) on an FTIR
1730. The purity of the final compounds was determined by HPLC
on a DIONEX-P680 instrument (DIONEX) at 254 nm and all final
compounds have a purity of above 95%.
Table 2
Antibiofilm and hemolysis activities of the compounds 4aeo and 8aec.
Compound
Antibiofilma
Hemolysisb
A
þ
e
þ
e
e
þ
þ
e
e
þ
þ
e
e
e
e
e
e
þ
þ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
nd
nd
e
e
e
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
8a
8b
8c
5.2. General procedure for the preparation of compounds 2aeh
To a solution of phenylaldehyde or substituted phenylaldehyde
1aef (3 mmol), ethyl benzoylacetate (3 mmol), thiourea (9 mmol)
and CeCl3$7H2O (0.75 mmol) in absolute ethanol (10 ml) was
refluxed for 5 h. Then the mixture was poured into 200 ml ice-
water and solid was precipitated. The crude products were filtered
and recrystallized in methanol to obtain pure compound as a white
solid.
5.3. General procedure for the preparation of compounds 3aeh
nd ¼ no detected.
a
Antibiofilm activities of the derivatives against S. epidermidis ATCC35984 were
To a solution of 2aeh (3 mmol), bromoacetic acid (3 mmol),
NaOAc (3 mmol) in 20 ml HAc/Ac2O (3:1 v/v) was refluxed for 6 h.
Then the mixture was cooled to room temperature and poured into
200 ml water and solid was precipitated. The crude products were
filtered and recrystallized in methanol to obtain pure compound as
a golden crystal 3aeh.
tested at 50
m
M; þ: active at 50
m
M; ꢁ: no inhibition at 50
mM.
b
Hemolytic activities of the derivatives on healthy human erythrocytes were
tested at 50
m
M; ꢁ: no hemolytic activity at 50
mM.
Table 3
Inhibition of YycG enzyme activities of the compound
4e and 8a.
a
Compound
IC50
14
(mM)
5.4. General procedure for the preparation of compounds 4aeo
A
4e
8a
8b
12.65
49.92
55.61
The corresponding aldehyde (1 equiv), b-alanine (1 equiv), and
3aeh (1 equiv) were heated at 100 ꢀC for 1 h in glacial acetic acid.
Upon completion of the reaction, the mixture was cooled, the re-
action was quenched with water, and the precipitate was filtered
off. The solid products were filtered and recrystallized in methanol
to obtain pure compound.
a
IC50 represents half maximal inhibitory concen-
tration of the derivatives, which inhibit half of the
autophosphorylation of recombinant YycG.

504
D. Zhao et al. / European Journal of Medicinal Chemistry 87 (2014) 500e507
5.4.1. 3-(5-((6-(Ethoxycarbonyl)-3-oxo-5,7-diphenyl-3,5-dihydro-
2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)furan-2-yl)benzoic
acid (4a) [6]
5.4.6. 3-(5-((6-(Ethoxycarbonyl)-5-(4-fluorophenyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4f)
Yellow solid: yield 93%; mp: 284e285 ꢀC; IR (KBr) cmꢁ1: 3387
Yellow solid: yield 92.1%; mp: 232.1e233.5 ꢀC; IR (KBr): v
(COeOH), 1698 (C]O), 1549 (C]C); 1H NMR (DMSO-d6, 400 MHz,
(cmꢁ1): 3436 (COeOH), 1712 (C]O), 1546 (C]C); 1H NMR (DMSO-
ppm):
d
13.07 (s, 1H, CH), 8.40 (s, 1H, CH), 8.13e8.05 (d, J ¼ 6 Hz, 1H,
d6, 400 MHz, ppm): d 13.06 (s, 1H, COOH), 8.38 (s, 1H, CH),
furan-H), 7.93e7.91 (d, J ¼ 6 Hz, 1H, furan-H), 7.69 (d, J ¼ 7.9 Hz, 2H,
AreH), 7.48e7.40 (m,10H, AreH), 7.29 (d, J ¼ 3.8 Hz, 2H, AreH), 6.16
(s, 1H, CH), 3.86e3.81 (q, J ¼ 7.0 Hz, 2H, CH2), 0.82e0.80 (t,
J ¼ 7.0 Hz, 3H, CH3); ESI-MS m/z 575.2 [MꢁH]ꢁ.
8.12e8.04 (d, J ¼ 8.4 Hz, 1H, furan-H), 7.97 (d, J ¼ 8.4 Hz, 1H, furan-
H), 7.67e7.27 (m, 13H, AreH), 6.16 (s, 1H, CH), 3.84e3.80 (q,
J ¼ 7.2 Hz, 2H, CH2), 0.81e0.78 (t, J ¼ 7.2 Hz, 3H, CH3); 13C NMR
(DMSO-d6, 100 MHz, ppm): d 166.68, 165.33, 163.94, 156.04, 155.86,
150.17, 149.77, 149.15, 138.69, 138.52, 131.84, 129.53, 128.97, 128.69,
128.56, 128.25, 128.19, 127.64, 127.57, 124.72, 121.70, 118.71, 116.61,
110.65, 109.70, 108.47, 60.14, 56.00, 13.25; ESI-MS m/z 595.2
[MþH]þ.
5.4.2. 3-(5-((6-(Ethoxycarbonyl)-5-(4-methoxyphenyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4b) [6]
Orange solid: yield 91.4%; mp: 252.3e253.4 ꢀC; IR (KBr) cmꢁ1
:
3371 (COeOH), 1608 (C]O), 1547 (C]C); 1H NMR (DMSO-d6,
300 MHz, ppm):
5.4.7. 3-(5-((6-(Ethoxycarbonyl)-5-(4-methoxyphenyl)-7-methyl-
3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4g)
d
8.34 (s, 1H, CH), 8.05e8.03 (d, J ¼ 6 Hz, 1H, furan-
H), 7.93e7.91 (d, J ¼ 6 Hz, 1H, furan-H), 7.64e6.92 (m, 13H, AreH),
6.06 (s, 1H, CH), 3.85e3.78 (q, J ¼ 6 Hz, 2H, CH2), 3.69 (s, 3H, OCH3),
0.79e0.75 (t, J ¼ 6 Hz, 3H, CH3); ESI-MS m/z 607.2 [MþH]þ.
Orange solid: yield 91.3%; mp: 276.5e277.4 ꢀC; IR (KBr) cmꢁ1
:
3441 (COeOH), 1709 (C]O), 1633 (C]N), 1533 (C]C); 1H NMR
(DMSO-d6, 400 MHz, ppm): 13.21 (s, 1H, COOH), 8.37 (s, 1H, CH),
d
8.07e8.05 (d, J ¼ 7.8 Hz, 1H, furan-H), 7.97e7.95 (d, J ¼ 7.8 Hz 1H,
furan-H), 7.69e6.88 (m, 8H, AreH), 5.96 (s, 1H, CH), 4.08e4.01 (q,
J ¼ 7.2 Hz, 2H, CH2), 3.71 (s, 3H, OCH3), 2.39 (s, 3H, CH3),1.17e1.12 (t,
5.4.3. 3-(5-((6-(Ethoxycarbonyl)-5-(4-hydroxyphenyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4c)
J ¼ 7.2 Hz 3H, CH3); 13C NMR (DMSO-d6, 100 MHz, ppm):
d 166.69,
Orange solid: yield 91.6%; mp: 273.3e274.6 ꢀC; IR (KBr) cmꢁ1
:
164.88, 163.95, 159.20, 155.93, 155.90, 150.99, 149.14, 132.56, 131.81,
129.64, 129.50, 128.96, 128.69, 128.25, 124.62, 121.55, 118.53, 116.67,
113.93, 110.60, 108.65, 60.06, 55.06, 54.24, 22.33, 13.88; ESI-MS m/z
545.1 [MþH]þ.
3431 (COeOH), 1608 (C]O), 1547 (C]C); 1H NMR (DMSO-d6,
400 MHz, ppm): 13.05 (s, 1H, CH), 8.38 (s, 1H, CH), 8.11e8.03 (d,
d
J ¼ 6 Hz, 1H, furan-H), 7.96 (d, J ¼ 6 Hz, 1H, furan-H), 7.66e7.27 (m,
13H, AreH), 6.16 (s, 1H, CH), 4.17 (s, 1H, OH); 3.83e3.79 (q, J ¼ 7 Hz,
2H, CH2), 0.80e0.77 (t, J ¼ 7 Hz, 3H, CH3); 13C NMR (DMSO-
5.4.8. 4-(5-((6-(Ethoxycarbonyl)-5-(4-methoxyphenyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4h)
d6,100 MHz, ppm):
d 169.04, 166.67, 165.37, 163.93, 156.02, 155.91,
150.53, 149.83, 149.14, 138.55, 137.32, 131.82, 129.65, 129.52, 128.95,
128.70, 128.27, 127.64, 124.72, 122.22, 121.69, 118.76, 116.57, 110.63,
109.67, 60.16, 55.10, 13.24; ESI-MS m/z 593.1 [MþH]þ.
Orange solid: yield 88.2%; mp: 252.3e253.4 ꢀC; IR (KBr) cmꢁ1
:
3452 (COeOH), 1710 (C]O), 1536 (C]C); 1H NMR (DMSO-d6,
400 MHz, ppm):
d
13.05 (s,1H, COOH), 8.06 (d, J ¼ 6 Hz, 2H, furan-H,
5.4.4. 3-(5-((6-(Ethoxycarbonyl)-5-(4-nitrophenyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4d)
AreH), 7.92 (d, J ¼ 6 Hz, 1H, furan-H, AreH), 7.65 (s, 1H, CH), 7.42
(m, 6H, AreH), 7.36e7.29 (m, 2H, AreH), 7.26 (d, J ¼ 3.8 Hz, 1H,
AreH), 6.95 (d, J ¼ 8.8 Hz, 2H, AreH), 6.09 (s, 1H, CH), 3.85e3.78 (q,
J ¼ 7.1 Hz, 2H, CH2), 3.73 (s, 3H, OCH3), 0.80e0.76 (t, J ¼ 7.1 Hz, 3H,
Orange solid: yield 93.6%; mp: 232.3e233.4 ꢀC; IR (KBr) cmꢁ1
:
3492 (COeOH), 1709 (C]O), 1630 (C]N), 1528 (C]C); 1H NMR
(DMSO-d6, 400 MHz, ppm): 13.15 (s, 1H, COOH), 8.39 (s, 1H, CH),
CH3); 13C NMR (DMSO-d6, 100 MHz, ppm):
d 166.71, 165.42, 163.93,
d
159.40, 155.82, 155.62, 149.68, 149.41, 138.59, 132.20, 131.90, 130.54,
130.22, 128.84, 128.59, 128.22, 127.62, 124.12, 121.49, 118.37, 117.37,
114.13, 111.76, 110.18, 60.09, 55.09, 13.28; ESI-MS m/z 607.2 [MþH]þ.
8.08 (d, J ¼ 7.8 Hz, 1H, furan-H), 8.00e7.93 (d, J ¼ 7.8 Hz, 1H, furan-
H), 7.79e7.43 (m, 12H, AreH), 7.29 (d, J ¼ 3.8 Hz, 1H), 6.27 (s, 1H,
CH), 3.83 (q, J ¼ 6.9 Hz, 2H, CH2), 0.80 (t, J ¼ 6.9 Hz, 3H, CH3); 13C
NMR (DMSO-d6, 100 MHz, ppm):
d 166.67, 165.13, 163.89, 156.29,
5.4.9. 4-(5-((5-(4-Chlorophenyl)-6-(ethoxycarbonyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4i)
156.15, 150.55, 149.12, 147.47, 146.62, 138.46, 131.84, 129.69, 129.58,
129.07, 128.94, 128.83, 128.29, 127.66, 124.74, 124.04, 123.87, 121.94,
118.98, 116.33, 110.71, 108.64, 60.26, 56.00, 13.22; ESI-MS m/z 622.1
[MþH]þ.
Orange solid: yield 92.5%; mp: 256.2e257.1 ꢀC; IR (KBr) cmꢁ1
:
3441 (COeOH), 1706 (C]O), 1556 (C]C); 1H NMR (DMSO-d6,
400 MHz, ppm): 13.05 (s, 1H, COOH), 8.38 (s, 1H, CH), 8.10e8.07
d
d
5.4.5. 3-(5-((5-(4-Chlorophenyl)-6-(ethoxycarbonyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4e)
(d, J ¼ 7.8 Hz, 1H, furan-H), 7.97e7.95 (d, J ¼ 7.8 Hz, 1H, furan-H),
7.70e7.29 (m, 13H, AreH), 6.16 (s, 1H, CH), 3.87e3.80 (q, J ¼ 7.2 Hz,
2H, CH2), 0.82e0.78 (t, J ¼ 7.2 Hz, 3H, CH3); 13C NMR (DMSO-d6,
Orange solid: yield 92.5%; mp: 256.2e257.1 ꢀC; IR (KBr) cmꢁ1
:
100 MHz, ppm) d 166.70, 165.25, 163.87, 155.93, 155.91, 149.92,
3431 (COeOH), 1710 (C]O), 1547 (C]C); 1H NMR (DMSO-d6,
300 MHz, ppm) 13.08 (s, 1H, COOH), 8.38 (s, 1H, CH), 8.10e8.07 (d,
149.64, 138.77, 138.48, 133.32, 132.16, 130.58, 130.22, 129.45, 128.85,
128.72, 128.25, 127.62, 124.13, 121.68, 118.61, 117.09, 111.79, 109.40,
60.18, 55.12, 13.24; ESI-MS m/z 611.1 [MþH]þ.
d
J ¼ 7.8 Hz, 1H, furan-H), 7.97e7.95 (d, J ¼ 7.8 Hz, 1H, furan-H),
7.70e7.67 (d, J ¼ 9 Hz, 2H, AreH), 7.46e7.41 (m, 10H, AreH),
7.31e7.29 (d, J ¼ 3.9 Hz 1H, AreH), 6.16 (s, 1H, CH), 3.86e3.79 (q,
J ¼ 6.9 Hz, 2H, CH2), 0.82e0.78 (t, J ¼ 6.9 Hz 3H, CH3); 13C NMR
5.4.10. 4-(5-((6-(Ethoxycarbonyl)-5-(4-fluorophenyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
furan-2-yl)benzoic acid (4j)
(DMSO-d6, 100 MHz, ppm):
d 166.77, 165.42, 164.03, 156.13, 155.96,
149.87, 149.25, 138.79, 138.62, 131.94, 129.76, 129.06, 128.78, 128.65,
128.34, 128.28, 127.74, 127.66, 124.82, 121.79, 118.81, 116.71, 115.87,
115.66,110.74,109.80, 60.23, 56.10,13.34; ESI-MS m/z 611.1 [MþH]þ.
Yellow solid: yield 92.1%; mp: 237.1e238.5 ꢀC; IR (KBr) cmꢁ1
:
3444 (COeOH), 1706 (C]O), 1637 (C]N), 1533 (C]C); 1H NMR
(DMSO-d6, 400 MHz, ppm): 13.08 (s, 1H, COOH), 8.07 (d,
d

D. Zhao et al. / European Journal of Medicinal Chemistry 87 (2014) 500e507
505
J ¼ 8.5 Hz, 2H, furan-H, AreH), 7.93 (d, J ¼ 8.5 Hz, 2H, furan-H,
5.4.15. 2-(4-((6-(Ethoxycarbonyl)-5-(4-methoxyphenyl)-7-methyl-
AreH), 7.67 (s, 1H, CH), 7.53e7.38 (m, 8H, AreH), 7.32e7.17 (m, 3H,
AreH), 6.15 (s, 1H, CH), 3.83 (q, J ¼ 7.0 Hz, 2H, CH2), 0.80 (t,
3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)-
2-methoxyphenoxy)acetic acid (4o)
J ¼ 7.0 Hz, 3H, CH3); 13C NMR (DMSO-d6, 100 MHz, ppm):
d
166.71,
Yellow solid: yield 90.2%; mp:167.3e168.4 ꢀC; IR (KBr) cmꢁ1
:
165.30, 163.90, 160.78, 155.88, 155.83, 149.74, 149.66, 138.49, 132.18,
130.58, 130.23, 129.81, 129.73, 128.69, 128.24, 127.63, 124.14, 121.63,
117.18, 115.79, 115.57, 111.79, 109.72, 60.15, 55.03, 13.25; ESI-MS m/z
595.2 [MþH]þ.
3444 (COeOH), 1709 (C]O), 1595 (C]C); 1H NMR (DMSO-d6,
400 MHz, ppm):
d
13.10 (s, 1H, COOH), 7.72 (d, J ¼ 8.3 Hz, 1H),
7.31e7.18 (m, 3H, AreH), 7.14 (d, J ¼ 8.5 Hz, 1H, AreH), 7.01 (d,
J ¼ 8.5 Hz, 1H, AreH), 6.89 (d, J ¼ 8.7 Hz, 2H, AreH), 5.99 (s, 1H),
4.77 (s, 2H, CH), 4.04 (q, J ¼ 7.0 Hz, 2H, CH2), 3.83 (s, 3H, OCH3), 3.71
(s, 3H, OCH3), 2.39 (s, 3H, CH3), 1.13 (t, J ¼ 7.1 Hz, 3H, CH3); 13C NMR
5.4.11. 2-(4-((5-(4-Chlorophenyl)-6-(ethoxycarbonyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
phenoxy)acetic acid (4k)
(DMSO-d6, 100 MHz, ppm): d 169.73, 164.90, 164.35, 159.23, 155.56,
151.10, 149.51, 148.95, 133.19, 132.50, 128.72, 125.95, 123.39, 117.02,
113.94, 113.66, 113.17, 108.65, 64.98, 60.08, 55.59, 55.06, 54.22,
22.34, 13.87; ESI-MS m/z 539.1 [MþH]þ.
Yellow solid: yield 85.6%; mp:156.8e157.9 ꢀC; IR (KBr) cmꢁ1
:
3445 (COeOH), 1715 (C]O), 1690 (C]N), 1598 (C]C); 1H NMR
(DMSO-d6, 400 MHz, ppm): 13.11 (s, 1H, COOH), 7.78 (s, 1H, CH),
d
7.59 (d, J ¼ 8.9 Hz, 2H, AreH), 7.50e7.36 (m, 9H, AreH), 7.09 (d,
J ¼ 8.9 Hz, 2H, AreH), 6.16 (s, 1H, CH), 4.78 (s, 2H, CH2), 3.81 (q,
J ¼ 7.0 Hz, 2H, CH2), 0.79 (t, J ¼ 7.0 Hz, 3H, CH3); 13C NMR (DMSO-d6,
5.5. General procedure for the preparation of compounds 5aec
Chalcones 5aec were prepared by condensing substituted
benzaldehydes 1aec with acetophenone in the presence of sodium
hydroxide under the ClaseineSchmidt reaction conditions.
100 MHz, ppm):
d 169.67, 165.35, 164.35, 159.73, 155.52, 149.93,
138.75, 138.44, 133.05, 132.11, 129.49, 128.85, 128.74,128.23,127.66,
125.70, 116.65, 115.50, 109.46, 64.54, 60.19, 55.11, 13.24; ESI-MS m/z
575.1 [MþH]þ.
5.6. General procedure for the preparation of compounds 6aec
5.4.12. 2-(4-((6-(Ethoxycarbonyl)-5-(4-methoxyphenyl)-7-methyl-
3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
phenoxy)acetic acid (4l)
A mixture of chalcones 5 (1 mol) and thiourea (1.5 mmol) in
ethanolic potassium hydroxide (60 g in 50 ml) was heated under
reflux for 4 h. The volume of the reaction mixture was reduced to
half of its original volume, diluted with ice cold water, then acidi-
fied with dilute acetic acid and kept overnight. The crude products
were filtered and recrystallized to obtain pure compound 6aec.
Yellow solid: yield 85.6%; mp: 189.8e191.7 ꢀC; IR (KBr) cmꢁ1
:
3445 (COeOH), 1710 (C]O), 1596 (C]C); 1H NMR (DMSO-d6,
400 MHz, ppm):
d
13.13 (s, 1H, COOH), 7.74 (d, J ¼ 6.6 Hz, 1H, CH),
7.56 (t, J ¼ 7.3 Hz, 2H, ArH), 7.22 (d, J ¼ 8.7 Hz, 2H, ArH), 7.13e7.02
(m, 2H, ArH), 6.89 (t, J ¼ 5.7 Hz, 2H, ArH), 5.99 (s, 1H, CH), 4.78 (s,
2H, CH2), 4.09e3.98 (q, J ¼ 7.1 Hz, 2H, CH2), 3.71 (d, J ¼ 1.9 Hz, 3H,
CH3), 2.39 (s, 3H, CH3), 1.13 (t, J ¼ 7.1 Hz, 3H, CH3); 13C NMR (DMSO-
5.7. General procedure for the preparation of compounds 7aec
d6, 100 MHz, ppm)
d 169.67, 164.89, 164.41, 159.65, 159.23, 155.49,
A mixture of thione 6 (3 mmol), bromoacetic acid (3 mmol),
anhydrous sodium acetate (3 mmol), glacial acetic acid (15 ml) and
acetic anhydride (3 ml) was heated under reflux for 3 h. Then the
reaction mixture was cooled and poured onto crushed ice with
vigorous stirring. The separated solid was filtered and washed with
water, recrystallized from methanol to give 7aec.
151.11, 132.79, 132.49, 132.01, 128.76, 125.71, 116.80, 115.44, 113.92,
108.64, 64.53, 60.07, 55.05, 54.25, 22.35, 13.86; ESI-MS m/z 509.1
[MþH]þ.
5.4.13. 2-(4-((5-(4-Chlorophenyl)-6-(ethoxycarbonyl)-7-methyl-3-
oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)
phenoxy)acetic acid (4m)
Yellow solid: yield 87.6%; mp:164.3e166.4 ꢀC; IR (KBr) cmꢁ1
:
5.8. General procedure for the preparation of compounds 8aec
3453 (COeOH), 1712 (C]O), 1587 (C]C); 1H NMR (DMSO-d6,
400 MHz, ppm):
d
13.09 (s, 1H, COOH), 7.76 (d, J ¼ 6.6 Hz, 1H, CH),
The corresponding aldehyde (1 mmol), b-alanine (1 mmol) and
7.61 (m, 2H, ArH), 7.22e7.02 (m, 4H, ArH), 6.89 (m, 2H, ArH), 6.08 (s,
1H, CH), 4.78 (s, 2H, CH2), 4.11e3.97 (q, J ¼ 7.0 Hz, 2H, CH2), 2.41 (s,
3H, CH3), 1.09 (t, J ¼ 7.0 Hz, 3H, CH3); 13C NMR (DMSO-d6, 100 MHz,
7 (1 mmol) were heated at 100 ꢀC for 1 h in glacial acetic acid. Upon
completion of the reaction, the mixture was cooled, the reaction
was quenched with water, and the precipitate was filtered off. The
solid products were filtered and recrystallized in methanol to
obtain 8aec.
ppm):
d 169.42, 164.64, 164.16, 159.40, 158.98, 155.24, 150.86,
132.54, 132.24, 131.76, 128.51, 125.46, 116.55, 115.19, 113.67, 108.39,
64.28, 59.82, 54.00, 22.10, 13.61; ESI-MS m/z 513.1 [MþH]þ.
5.4.14. 2-(4-((5-(4-Chlorophenyl)-6-(ethoxycarbonyl)-3-oxo-7-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-2-ylidene)
methyl)-2-methoxyphenoxy)acetic acid (4n)
5.8.1. 3-(5-((5-(4-Methoxyphenyl)-3-oxo-7-phenyl-3,5-dihydro-
2H-thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)furan-2-yl)benzoic
acid (8a)
Yellow solid: yield 87.2%; mp:160.8e161.9 ꢀC; IR (KBr) cmꢁ1
:
Yellow solid: yield 90.1%; mp: 267.3e268.4 ꢀC; IR (KBr) cmꢁ1
:
3454 (COeOH), 1720 (C]O), 1667 (C]N), 1589 (C]C); 1H NMR
(DMSO-d6, 400 MHz, ppm): 7.76 (s, 1H, CH), 7.52e7.31 (m, 9H,
3434(COeOH), 1706 (C]O), 1595 (C]C); 1H NMR (DMSO-d6,
300 MHz, ppm): 13.08 (s,1H, COOH), 8.41 (s,1H, CH), 8.11e8.08 (d,
d
d
ArH), 7.22 (d, J ¼ 1.9 Hz, 1H, ArH), 7.17 (dd, J ¼ 8.5, 1.9 Hz, 1H, ArH),
7.02 (d, J ¼ 8.6 Hz, 1H, ArH), 6.16 (s, 1H, CH), 4.79 (s, 2H, CH2),
3.89e3.74 (m, 5H, CH2, CH3), 0.80 (t, J ¼ 7.1 Hz, 3H, CH3); 13C NMR
J ¼ 7.8 Hz, 1H, furan-H), 7.99e7.96 (d, J ¼ 7.8 Hz, 1H, furan-H),
7.84e6.93 (m, 13H, AreH), 6.13e6.11 (d, J ¼ 4.5 Hz, 1H), 5.93e5.92
(d, J ¼ 4.5 Hz, 1H), 3.73 (s, 3H, OCH3); 13C NMR (DMSO-d6, 100 MHz,
ppm): 166.75, 164.01, 160.86, 156.11, 150.25, 149.23, 138.77, 138.60,
136.25, 131.92, 129.89, 129.80, 129.74, 129.61, 129.04, 128.32, 128.26,
127.72, 127.64, 121.77, 118.79, 116.69, 115.86, 110.72, 108.55, 60.21,
56.08; ESI-MS m/z 535.1 [MþH]þ.
(DMSO-d6, 100 MHz, ppm): d 169.66, 165.32, 164.28, 155.53, 149.91,
149.46,148.98,138.75,138.39,133.40,133.35,129.48,128.85,128.77,
128.26, 127.64, 126.01, 123.53, 116.89, 113.63, 113.20, 109.41, 64.77,
60.20, 55.58, 55.10, 13.24; ESI-MS m/z 605.1 [MþH]þ.

506
D. Zhao et al. / European Journal of Medicinal Chemistry 87 (2014) 500e507
5.8.2. 3-(5-((5-(4-Chlorophenyl)-3-oxo-7-phenyl-3,5-dihydro-2H-
thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)furan-2-yl)benzoic acid
(8b)
OD570 of the negative control, the ODc was calculated as 0.28 in
this paper. Each assay was performed in triplicate.
Yellow solid: yield 91.3%; mp: 257.1e258.4 ꢀC; IR (KBr) cmꢁ1
:
5.9.3. Erythrocyte hemolysis
To determine the hemolytic activities of the derivatives on
erythrocytes, 5% (v/v) healthy human erythrocytes resuspended in
3441 (COeOH), 1710 (C]O), 1546 (C]C); 1H NMR (DMSO-d6,
300 MHz, ppm):
d 13.00 (s, 1H, COOH), 8.42 (s, 1H, CH), 8.12e8.09
(d, J ¼ 7.8 Hz, 1H, furan-H), 7.99e7.96 (d, J ¼ 7.8 Hz, 1H, furan-H),
NS were co-incubated with the derivatives at 100 mm for 1 h at
37 ꢀC in 96-well microtiter plates. After the incubation, the sus-
pensions were centrifuged at 350 ꢂ g for 10 min, and the level of
hemolysis was determined by measuring the absorbance of the
supernatant at 570 nm. Cells treated with DMSO (0.1%) and Triton-
X100 (1%) were used as negative and positive controls, respectively
[16].
7.83e7.24 (m, 13H, AreH), 6.15e6.13 (d, J ¼ 4.5 Hz, 1H), 6.02e6.01
(d, J ¼ 4.5 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz, ppm):
d 167.22,
164.84,160.62,156.00,153.35,149.88,138.90,137.52,136.59,132.30,
130.08,129.79,129.62,129.47,129.36,128.79,128.54,125.66,125.14,
120.97,117.41, 116.27,115.98,110.88,106.77, 55.81; ESI-MS m/z 539.1
[MþH]þ.
5.8.3. 3-(5-((5-(4-Fluorophenyl)-3-oxo-7-phenyl-3,5-dihydro-2H-
thiazolo[3,2-a]pyrimidin-2-ylidene)methyl)furan-2-yl)benzoic acid
(8c)
5.9.4. Inhibition assay for YycG autophosphorylation activity
To detect whether the compound still have the inhibitory ac-
tivities on YycG kinase, the recombinant HATPase_c and HisKA
domains of YycG were expressed in E. coli BL21 (DE3) and purified
by the Ni-NTA column, then ATP-Luminescence assay kit (Promega,
Madison, WI, USA) were used to measure the inhibitory activities of
the compounds on YycG autophosphorylation activity [5]. Briefly,
Orange solid: yield 89.6%; mp: 267.1e268.1 ꢀC; IR (KBr) cmꢁ1
:
3429 (COeOH), 1710 (C]O), 1528 (C]C); 1H NMR (DMSO-d6,
300 MHz, ppm):
d
13.08, (s, 1H, COOH), 8.12e8.09 (d, J ¼ 7.8 Hz, 1H,
furan-H), 7.99e7.96 (d, J ¼ 7.8 Hz, 1H, furan-H), 7.87e7.81 (m, 4H,
AreH), 7.70 (s, 1H, CH), 7.38e7.24 (m, 9H, AreH), 6.14e6.13 (d,
J ¼ 4.5 Hz, 1H), 6.02e6.01 (d, J ¼ 4.5 Hz, 1H); 13C NMR (DMSO-d6,
0.13 mmol/L recombinant YycG was pre-incubated with serial di-
lutions of the derivatives in reaction buffer (40 mmol/L Tris Ph 8.0,
20 mmol/L MgCl2, and 0.1 mg/mL BSA) at 25 ꢀC for 30 min. Then,
75 MHz, ppm):
d 166.60, 164.22, 160.00, 155.37, 152.73, 149.26,
138.28, 136.90, 135.97, 131.68, 129.46, 129.17, 129.00, 128.85, 128.74,
128.17, 127.92, 125.04, 124.52, 120.35, 116.79, 115.65, 115.36, 110.25,
106.15, 55.19; ESI-MS m/z 523.1 [MþH]þ.
4
mmol/L ATP was added and the plates were incubated for
30 min at 25 ꢀC, and Kinase-Glo™ Reagent was added to detect the
remaining ATP, recorded by luminescence measurement (RLU). The
rate of protein phosphorylation (Rp) inhibition by the derivatives
was calculated using the equation [5]. The half maximal inhibitory
concentration (IC50, the concentration of the derivatives required to
inhibit half of the autophosphorylation of the recombinant YycG)
was determined by Origin 8.0 software (OriginLab, Northampton,
USA) [5].
5.9. Evaluation of biological activity assay
5.9.1. Minimal inhibitory assay
MIC assays for the antibacterial activities of the derivatives were
performed according to the broth micro-dilution (in tubes) method
of the Clinical and Laboratory Standards Institute (CLSI) of America
[14]. In brief, a serial twofold dilution of derivatives was added to
eight tubes containing 4 ml Mueller-Hinton Broth (OXOID, En-
Acknowledgments
gland), making the final concentration from 200 mM to 0.78 mM. The
This work was supported by National Natural Science Founda-
tion of China (501100001809) Grant No. 21072095 (S-Q. Han), Grant
No. 30800036 (D. Qu).
turbidity of 6 h strain cultures is adjusted to match that of a 0.5
McFarland standard (approximately 108,142 CFU/ml), and then
0.02 ml of the bacterial inoculum was added to each tube. An
inoculated broth containing no antibiotic was included as a bac-
terial growth control and a tube of un-inoculated broth was used as
a sterility control. These bacteria were incubated at 37 ꢀC for
12e16 h, the lowest concentration that completely inhibits visible
growth of the organism as detected by the unaided eye is recorded
as the MIC.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.096.
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