Novel chalcone-conjugated, multi-flexible end-group coumarin thiazole hybrids as potential antibacterial repressors against methicillin-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.ejmech.2021.113628

The increasing resistance of methicillin-resistant Staphylococcus aureus (MRSA) to antibiotics has led to a growing effort to design and synthesize novel structural candidates of chalcone-conjugated, multi-flexible end-group coumarin thiazole hybrids with

Full text of DOI:10.1016/j.ejmech.2021.113628

European Journal of Medicinal Chemistry 222 (2021) 113628
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel chalcone-conjugated, multi-flexible end-group coumarin
thiazole hybrids as potential antibacterial repressors against
methicillin-resistant Staphylococcus aureus
,
,
, b
Yuanyuan Hu a b, Chunfang Hu c, Guangxing Pan a b, Congwei Yu a
,
Mohammad Fawad Ansari d, Rammohan R. Yadav Bheemanaboina e, Yu Cheng f,
Chenghe Zhou c **, Jiaheng Zhang a
,
, b, *
a Sauvage Laboratory for Smart Materials, Harbin Institute of Technology (Shenzhen), Shenzhen, 518055, China
b Shenzhen Key Laboratory of Flexible Printed Electronics Technology, School of Materials Science and Engineering, Harbin Institute of Technology,
Shenzhen, 518055, China
c Dongguan School Affiliated to South China Normal University, Dongguan, 523755, China
d School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, China
e Sokol Institute for Pharmaceutical Life Sciences, Department of Chemistry and Biochemistry, Montclair State University, New Jersey, 07043, USA
f School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The increasing resistance of methicillin-resistant Staphylococcus aureus (MRSA) to antibiotics has led to a
growing effort to design and synthesize novel structural candidates of chalcone-conjugated, multi-
flexible end-group coumarin thiazole hybrids with outstanding bacteriostatic potential. Bioactivity
screening showed that hybrid 5i, which was modified with methoxybenzene, exerted a significant
inhibitory activity against MRSA (MIC ¼ 0.004 mM), which was 6 times better than the anti-MRSA ac-
tivity of the reference drug norfloxacin (MIC ¼ 0.025 mM). Compound 5i neither conferred apparent
resistance onto MRSA strains even after multiple passages nor triggered evident toxicity to human he-
patocyte LO2 cells and normal mammalian cells (RAW 264.7). Molecular docking showed that highly
active molecule 5i might bind to DNA gyrase by forming stable hydrogen bonds. In addition, molecular
electrostatic potential surfaces were developed to explain the high antibacterial activity of the target
compounds. Furthermore, preliminary mechanism studies suggested that hybrid 5i could disrupt the
bacterial membrane of MRSA and insert itself into MRSA DNA to impede its replication, thus possibly
becoming a potential antibacterial repressor against MRSA.
Received 8 April 2021
Received in revised form
3 June 2021
Accepted 3 June 2021
Available online 9 June 2021
Keywords:
Coumarin
Thiazole
Chalcone
Antibacterial
MRSA
DNA
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
difficult to control [2]. Critically, MRSA exhibits varying degrees of
resistance against numerous antibiotics, such as b-lactams, fluo-
The high frequency and rapid transmission of microbial in-
fections, have inevitably become a significant challenge in modern
medicine, and pose a serious threat to human health [1]. Even more
concerning is the emergence of powerful drug-resistant strains
from common strains, particularly, the lethal methicillin-resistant
Staphylococcus aureus (MRSA), which makes infections more
roquinolones, macrolides and vancomycin, which have been clini-
cally approved to treat MRSA infections, thereby making it
extremely difficult to curb its spread [3,4]. All these concerns have
laid the foundation for the exploration of new therapeutic strate-
gies, and there is an urgent need to develop novel anti-MRSA agents
with high efficiency and low cytotoxicity to treat the infections
caused by such resistant strains [5].
Natural products have been invaluable in the discovery and
development of medicines, and have been used to treat diseases for
thousands of years. Coumarins are natural products that universally
exist among plant secondary metabolites [6]. To date, more than
1800 different natural and synthetic coumarins with important
* Corresponding author. Sauvage Laboratory for Smart Materials, Harbin Institute
of Technology (Shenzhen), Shenzhen, 518055, China.
** Corresponding author.
E-mail addresses: zhouch@swu.edu.cn (C. Zhou), zhangjiaheng@hit.edu.cn
(J. Zhang).
https://doi.org/10.1016/j.ejmech.2021.113628
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
biological activities, such as insecticidal, antimicrobial, anticancer,
and anti-HIV activities, have been documented [7]. Notably, cou-
marins have been widely used as antibiotics owing to their active
membrane penetration, e.g., chlorobiocin, novobiocin, and cou-
mermycin A1 are frequently used in clinical practice [8]. In addition,
coumarin agents can effectively inhibit the ATPase activity of DNA
gyrase, to compete with the B subunit of the ATP-binding enzyme
and exert their antimicrobial effects [9]. So far, more and more ef-
forts have been devoted to the structural reconstruction of the
coumarin skeleton, and the modified coumarin derivatives have
been found to have satisfactory antibacterial potential [10].
Therefore, a series of novel coumarin molecules with high anti-
microbial potential have been developed to reduce cytotoxicity and
drug resistance by splicing and recombining clinical antimicrobial
active fragments on coumarin scaffolds [11,12].
developed, and the thiazole ring and two hydroxyethyl fragments
have been incorporated into the reconstituted hybrids. These de-
rivatives, which were chalcone conjugates containing different
phenyl and indolyl groups, were obtained by condensing different
aldehyde molecules, namely indole-aldehydes and aromatic alde-
hydes, and the effects of different substituents on the antimicrobial
activity of these hybrids were measured (Fig. 1). The selection of
different substituents for the target molecules was based on the
following ideas: (1) Indole alkaloids are bacterial intercellular
signaling molecules with unique characteristics of mediating a
variety of biological processes. In addition, indole scaffolds may also
exert potential biological activity through groove binding or
insertion of DNA, especially the benign promotion of antimicrobial
activity has attracted our great attention [28]; (2) Various benzyl
groups can accelerate the regulation of cell membrane permeability
and lipid solubility, and facilitate the absorption and mobility of
novel heterozygous structures [20,25]; (3) Nitrogen and oxygen
atoms can react well on multiple active sites in biological systems
and may bind to diverse targets simultaneously [29].
Chalcones are typically composed of two aromatic rings bridged
by an
a,b-unsaturated system of three carbons, which are
commonly found in many medicinal plants, and have outstanding
medicinal value owing to their antibacterial, antifungal, anticancer
and anti-inflammatory properties [13]. Chalcones have large con-
jugated systems, which promote their photophysical properties
and facilitate their binding ability with bioactive molecules (en-
zymes or DNA), and further stimulate a wave of the design and
development of new drugs using chalcones as active fragments
[14]. Remarkably, the strong potential of chalcones as antimicrobial
agents is reflected in natural products. For example, licochalcone A,
a natural chalcone product, shows significant antimicrobial ability
against Staphylococcus aureus and Micrococcus luteus [15]. Addi-
tionally, in a large number of recent studies, many chalcone de-
rivatives have been developed by introducing heterocyclic
compounds, mainly azole compounds, into the chalcone skeleton,
or by using the large conjugated systems of chalcones to splice the
active ingredients of various antibacterial drugs to generate newly
conjugated drug molecules [16,17]. Given this, there is a large space
for the chalcone skeleton to be used in molecular modification,
which is brought up to date for the construction of novel multi-
component chalcone hybrids that can be used as multi-target
antimicrobial agents [18].
2. Results and discussion
2.1. Chemistry
A series of novel chalcone-conjugated coumarin thiazole hy-
brids was designed and synthesized using the synthetic routes
outlined in Schemes 1 and 2. Intermediate 2 was prepared from
commercial resorcinol 1 and ethyl 4-chloroacetoacetate by stirring
in concentrated sulfuric acid at 0 ꢀC for 24 h with a yield of 71%.
Thereafter, intermediate 2 and compound 7, which had been pre-
pared based on the literature, were reacted in acetonitrile in the
presence of potassium carbonate at 60 ꢀC for 8 h to obtain inter-
mediate 3 with a yield of 47%. Intermediate 3 was then treated with
diethanolamine in acetonitrile at 80 ꢀC to obtain key intermediate 4
with a yield of 70%. Furthermore, compound 4 was condensed with
different benzaldehydes (available in the market) and 3-
formylindoles 11 (obtained using a synthesis route from the liter-
ature) in ethanol in the case of piperidine at 80 ꢀC for 8 h to produce
target chalcone-conjugated coumarin thiazoles 5 and 8 in yields
ranging from 25% to 50%.
The frequent occurrence of azole heterocyclic compounds in
various antimicrobial drugs has made azoles, which are alternative
active fragments of drug recombination, attract much attention
[19]. Significantly, thiazoles are aromatic heterocyclic compounds
with a variety of covalent interactions, such as hydrogen bonds, van
2.2. Antibacterial activity
der Waals interactions, and
p
-
p
stacking. Therefore, the introduc-
In accordance with Clinical and Laboratory Standards Institute
(CLSI) guidelines, the two-fold serial dilution method was used to
evaluate the antibacterial activity of newly synthesized coumarin
thiazole derivatives in vitro. Norfloxacin, a widely used, clinically
effective antimicrobial agent, was employed to compare the anti-
bacterial activity for the series of coumarin thiazole hybrids. The
antibacterial activity results were summarized in Table 1, in which
the minimum inhibitory concentration (MIC) was the minimum
concentration (mM) at which the target compounds could
completely inhibit bacterial growth [30,31].
To further have a good knowledge of the structure-activity
relationship (SAR) of these chalcone-conjugated coumarin thia-
zole hybrids, the effects of different active fragments and their
various substituents on the antimicrobial activity of these com-
pounds was studied. For the series of phenyl-derived coumarin
thiazole hybrids, it could be observed that this series of derivatives
exhibited obvious selectivity toward MRSA strains. Remarkably,
methoxybenzene-modified hybrid 5i exhibited the strongest inhi-
bition against MRSA compared with the other products, with a MIC
value of 0.004 mM, which was 6 times better than that of the
reference drug norfloxacin (0.025 mM). In addition, the inhibitory
activity of compound 5i against E. coli was also noteworthy, with a
tion of thiazole fragment may effectively improve the physico-
chemical properties of drug molecules and enhance their
interactions with biological enzymes, as well as enhance the
binding ability of functional targets [20]. To date, the clinical use of
thiazole compounds has been considerable, especially in quino-
lones (cephalosporin, cefixime) and sulfonamides (sulfathiazole),
wherein this pharmacophore is widely observed [21]. Hence, thi-
azoles have strong antibacterial potential and promising biological
activity, which may provide a broad prospect for their further
development and utilization as active fragments [22].
Hydroxyethyl fragments are frequently used in lots of clinical
antimicrobial drugs such as antibacterial metronidazole and anti-
fungal fluconazole, demonstrating that hydroxyethyl group is
indeed beneficial for the improvement of antimicrobial activity
[23e25]. Therefore, the excellent contribution of hydroxyethyl
fragments in antimicrobial drug molecules and the strong vision of
using them as flexible end groups support a more profound idea for
exploring novel antimicrobial structures [26,27].
Based on all of these considerations, a series of new promising
antimicrobial chalcone-conjugated structural splice recombinants
with coumarin as the structural skeleton has been successfully
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Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
Fig. 1. Design of novel chalcone-conjugated coumarin thiazole hybrids.
Scheme 1. Synthetic route of phenyl-derived chalcone-conjugated coumarin thiazole hybrids.
Reagents and conditions: (i) ethyl 4-chloroacetoacetate, sulfuric acid, 0 ꢀC, 24 h, 71%; (ii) 2-bromoacetylthiazole, potassium carbonate, acetonitrile, 60 ꢀC, 8 h, 48%; (iii) dieth-
anolamine, acetonitrile, 80 ꢀC, 8 h, 70%; (iv) aryl aldehyde, piperidine, ethanol, 80 ꢀC, 8 h, 25e50%; (v) bromine, acetic acid, 50 ꢀC, 2 h, 78%.
3

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
Scheme 2. Synthetic route of indole-derived chalcone-conjugated coumarin thiazole hybrids.
Reagents and conditions: (iv) 3-formylindole, piperidine, ethanol, 80 ꢀC, 8 h, 40e45%; (v) dimethylformamide, phosphorus oxychloride, 0 ꢀC, 5 h, 81%; (vi) bromoethane, potassium
carbonate, acetonitrile, 60 ꢀC, 5 h, 78%.
Table 1
In vitro antibacterial data as MIC (mM) for coumarin thiazole hybrids 4, 5 and 8.a,b
.
Compds
Structure
Gram-positive bacteria
Gram-negative bacteria
R
MRSA
E. f.
S. a.
S. a. 25923
S. a. 29213
K. p.
E. c.
P. a.
A. b.
P. a. 27853
E. c. 25922
4
5a
e
0.020
0.130
0.316
0.130
0.316
0.260
0.633
1.040
1.266
1.040
0.316
1.040
0.316
1.040
0.633
0.520
0.633
1.040
1.266
0.520
0.633
0.260
5b
0.061
0.121
0.121
0.486
0.486
0.972
0.972
0.486
0.486
0.243
0.243
5c
0.121
0.007
0.243
0.228
0.243
0.228
0.243
0.456
0.243
0.456
0.486
0.228
0.486
0.228
0.061
0.114
0.243
0.114
0.243
0.228
0.486
0.456
5d
5e
5f
0.125
0.136
0.007
0.063
0.004
0.015
0.125
0.136
0.119
0.063
0.061
0.060
0.125
0.068
0.119
0.126
0.061
0.120
0.251
0.272
0.060
0.063
0.122
0.120
0.251
0.272
0.119
0.126
0.122
0.120
0.251
1.086
0.238
0.063
0.061
0.060
1.003
1.086
0.119
0.016
0.015
0.241
1.003
0.272
0.476
0.253
0.122
0.241
0.251
0.543
0.238
0.253
0.244
0.482
0.251
1.086
0.238
0.505
0.244
0.482
0.251
0.543
0.119
0.126
0.244
0.241
5g
5h
5i
8a
8b
8c
8d
0.057
0.117
0.114
0.057
0.015
0.014
0.113
0.029
0.029
0.057
0.007
0.007
0.057
0.117
0.057
0.057
0.235
0.229
0.226
0.235
0.229
0.226
0.117
0.114
0.113
0.117
0.057
0.113
0.059
0.057
0.226
0.235
0.229
Norfloxacin
e
0.025
0.025
0.006
0.003
0.006
0.013
0.050
0.025
0.025
0.003
0.025
a
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
MRSA, methicillin-resistant Staphylococcus aureus N315; E. f., Enterococcus faecalis; S. a., Staphylococcus aureus; S. a. 25923, Staphylococcus aureus ATCC 25923; S. a. 29213,
b
Staphylococcus aureus ATCC 29213; K. p., Klebsiella pneumonia; E. c., Escherichia coli; P. a., Pseudomonas aeruginosa; A. b., Acinetobacter baumanii; P. a. 27853, Pseudomonas
aeruginosa ATCC 27853; E. c. 25922, Escherichia coli ATCC 25922.
MIC value of 0.015 mM, which was more effective than the refer-
ence drug norfloxacin (MIC ¼ 0.05 mM). Furthermore, compounds
5d and 5g, modified with dichlorobenzene and nitrobenzene,
respectively, showed satisfactory inhibitory effect on MRSA, and
their MIC values were 0.007 mM. Otherwise, we also found that
derivative 5h, modified with methylbenzene, had slightly better
antibacterial activity (MIC ¼ 0.015 mM) against E. coli than
norfloxacin. Therefore, based on the antibacterial activity results of
the series of phenyl-derived coumarin thiazoles, it could be seen
that the inhibitory effect of the target hybrids was diversified due to
the different substituents on the phenyl group. Numerous studies
have pointed out that the introduction of nitro group as an active
functional group was beneficial for the antibacterial activity of the
target structures, and our activity analysis results of the series of
4

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
derivatives were consistent with this [29]. However, a few reports
stated that the activity of target molecules modified by methoxy
group and di-halogen atoms significantly improved, and it is pre-
cisely because of this that these fragments were of unique value as
functional group candidates for further research and development
of antibacterial agents with novel structural frameworks.
Subsequently, a series of indole-derived coumarin thiazole hy-
brids was derived by replacing the introduced phenyl ring with an
indole ring. However, in this series of derivatives, only compound
8a showed slightly better anti-MRSA activity (MIC ¼ 0.015 mM)
than the reference drug norfloxacin. Notably, although these
structural modifications did not induce the expected anti-MRSA
activity, they did show unforeseen sensitivity to E. faecalis. Specif-
ically, indole derivatives 8c and 8d, modified with methyl and ethyl
groups respectively, showed significantly better antibacterial ac-
tivity against E. faecalis than the reference drug (MIC ¼ 0.025 mM),
with MIC values of 0.015 and 0.014 mM, respectively. These results
indicated that the introduction of indole rings with different sub-
stituents contributed to the improvement of the antibacterial
properties of the target molecules. In addition to the positive effect
of the indole ring itself on the inhibitory activity against bacteria,
the introduction of alkyl chains also broadened the antibacterial
spectrum of indole-derived coumarin thiazoles to some extent.
In general, various phenyl- and indole-modified chalcone-
bridged coumarin thiazole hybrids showed effectively improved
inhibitory effects against bacteria, especially their selective inhi-
bition advantage of MRSA should not be underestimated. Besides,
some of the modified structures were also sensitive to E. coli and
E. faecalis and displayed relatively low MIC values. In particular,
highly active compound 5i, derived from the introduction of a
methoxybenzene group, had great potential as a new repressor
against MRSA, and provided a reliable case for the subsequent
screening of antimicrobial drug candidates [32].
Fig. 2. The calculated partition coefficients of coumarin thiazole hybrids 5 and 8 vs
antibacterial effect against MRSA.
reference drug norfloxacin (MIC ¼ 0.021 mM), with a Clog P value of
3.03. Furthermore, MRSA was relatively sensitive to derivatives 5g
(MIC ¼ 0.007 mM) and 8a (MIC ¼ 0.015 mM), and their Clog P values
were 2.86 and 3.10, respectively. However, no clear correlation was
observed between the Clog P values and MIC values. These results
indicated that when the Clog P values of the target compounds were
distributed between 2.86 and 4.54, it might be beneficial to inhibit
the growth of MRSA strains, and could promote the transport of the
target coumarin thiazoles and their release at the active site in the
organism [34].
2.4. Bactericidal kinetics
2.3. Analysis of Clog P values
In order to further verify whether highly active coumarin thia-
zole derivative 5i had an effective bactericidal ability against MRSA,
a time-kill kinetic experiment was carried out (Fig. 3). Notably, at a
reference concentration (4 ꢁ MIC), the viable cells number was less
than 103 CFU/mL after 2 h. These results showed that hybrid 5i had
a strong killing ability against MRSA and could quickly inactivate
the bacteria in a short time [35,36].
The lipophilic/hydrophilic properties of target active com-
pounds are generally reflected indirectly by the theoretical calcu-
lated value of the partition coefficient (Clog P). In general, the
appropriate range of Clog P values is conducive to the distribution
and transportation of target molecules in biological systems. The
Clog P values of all target coumarin thiazole derivatives were
summarized in Table 2 [33]. Obviously, the Clog P values of
coumarin thiazole hybrids 5 and 8 varied with the type and the
number of substituents on the benzene and indole rings, ranging
from 2.86 to 4.54.
To analyze the correlation between the Clog P value and the
antibacterial efficacy against MRSA, their relationship was estab-
lished, as shown in Fig. 2. It could be observed that dichlorobenzene-
modified hybrid 5d had the highest lipophilicity (Clog P ¼ 4.54) of all
target molecules, but did not show the best expected anti-MRSA
activity (MIC
¼
0.007 mM). Surprisingly, methoxybenzene-
modified compound 5i showed the most significant antimicrobial
activity (MIC ¼ 0.004 mM), which was better than that of the
Table 2
Clog P values of coumarin thiazole hybrids (Mean SD, n ¼ 3).a
Compds
Clog P
Compds
Clog P
Compds
Clog P
5a
5b
5c
5d
5e
3.11
3.83
3.83
4.54
3.26
5f
3.98
2.86
3.61
3.03
3.10
8b
8c
8d
-
3.99
3.60
4.10
-
5g
5h
5i
8a
-
-
a
Clog P values were calculated by ChemDraw Ultra 14.0.
Fig. 3. Bactericidal kinetics of compound 5i (4 ꢁ MIC) against MRSA.
5

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
2.5. Drug resistance development
The continuous rise in bacterial resistance was a huge obstacle
to drug development, hence, it was important to evaluate whether
newly synthesized coumarin thiazole derivatives could induce
bacterial resistance. In this study, highly active compound 5i was
selected to determine its ability to confer resistance onto MRSA,
and norfloxacin was used as a positive control. The results in Fig. 4
showed that MRSA remained almost unchanged for 7 consecutive
passages and produced slow resistance to molecule 5i compared
with the reference drug norfloxacin. These results indicated that it
was more difficult for the hybrid 5i to induce the development of
MRSA resistance than the control drug norfloxacin. Therefore, this
kind of modification of coumarin thiazole hybrids might yield a
promising low-resistance candidate structure against MRSA
[37,38].
2.6. Cytotoxicity
Cytotoxicity has long been used as an indicator to determine
whether novel and potentially modified drug molecules can be
employed as candidates for antimicrobial agents. The cytotoxic
effects of bioactive compound 5i on human hepatocyte LO2 cells
and normal mammalian cells (RAW 264.7) were assessed in vitro
using the MTT assay. The cytotoxicity results (Fig. 5) showed that no
less than 75% of living cells were present even when the hybrid
concentration was increased to 500 mg/mL. This result indicated
that the selected drug molecule 5i had low cytotoxicity, which was
very beneficial for its therapeutic potential, and its harmfulless
characteristic was also consistent with the screening criteria for
active drug candidate molecules [39,40].
2.7. Bacterial membrane permeabilization
Membrane-active drug molecules can penetrate bacterial
membranes to increase drug delivery rates, which is expected to
reduce bacterial resistance, due to their strong destructive power.
In addition, propidium iodide (PI) can successfully cross the cell
membrane of damaged bacteria and fluoresce when it binds to
DNA. Therefore, PI was used as a probe to determine the destructive
ability of hybrid 5i toward the MRSA membrane. As shown in Fig. 6,
the fluorescence intensity of PI with compound 5i showed a trend
Fig. 5. Cytotoxic assay of target compound 5i on human hepatocyte LO2 cells and
normal mammalian cells (RAW 264.7) tested by MTT methodology.
Fig. 4. Drug resistance development of compound 5i against MRSA.
Fig. 6. Membrane permeabilization of MRSA toward compound 5i (12 ꢁ MIC).
6

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
of rapid increase, and then gradually became flat after 60 min,
whereas the relative change in the control group was not obvious.
These results indicated that at the reference concentration
(12 ꢁ MIC), molecule 5i could destroy the membrane of MRSA and
form a PI-DNA complex, which might further penetrate the MRSA
membrane to achieve its effective antibacterial purpose [41,42].
bonds was beneficial to maintaining the stability of the coumarin
thiazole hybrid 5i-DNA complex, which might help explain why
compound 5i had a good inhibitory ability against MRSA.
2.9. Electronic properties
Molecular electrostatic potential (MEP) surfaces can effectively
highlight the distribution trend of electrons carried by the target
structures, and enable further analysis of the region formed by
hydrogen bonds, which have a positive impact on the interpreta-
tion of the electrostatic interactions between the target molecules
and the active sites in an organism. Herein, MEP simulation images
of target compounds 5d, 5g and 5i were recorded and summarized
in Table 3 Through a comparison of the following three maps, we
might roughly observe such a phenomenon. In the three selected
hybrids, the positively charged regions (in blue) were mainly
located on the hydroxyl group of the diethanolamine fragment and
the negatively charged regions (in red) were mainly concentrated
on the oxygen atom of the coumarin skeleton. This result precisely
confirmed that hydrogen bonds were formed because of such
electron sharing, and the formed hydrogen bonds were conducive
to the hydrophilic ability of the coumarin thiazole hybrids modified
2.8. Molecular docking
Flexible ligand-receptor docking of highly active compound 5i
was successfully conducted to rationalize antibacterial activity as
much as possible and to understand the possible mechanisms of
this type of coumarin thiazole derivatives. Crystal structure data
(gyrase-DNA complex) were obtained from the protein data bank
(PDB code: 2XCS), which was a potential target for antimicrobial
research as previously reported [8,43]. The molecular docking re-
sults (Fig. 7) indicated that the oxygen atom on the hydroxyl group
in hybrid 5i was adjacent to DC-11, forming a hydrogen bond at a
distance of 2.1 Å. Furthermore, hybrid 5i might also form hydrogen
bonds with the ARG-1122 residue via the nitrogen atom of thiazole
fragment and the oxygen atom on the carbonyl group, with dis-
tances of 2.0 Å and 2.7 Å. These stable existence of these hydrogen
Fig. 7. (A and B) Stereoview and three-dimensional conformations of hybrid 5i docked in gyrase DNA complex (PDB code: 2XCS); (C) Visualization of hybrid 5i interacting with the
hydrophobic residues at the active sites of gyrase DNA complex; (D) Two-dimensional conformation of hybrid 5i docked in gyrase DNA complex.
7

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
Table 3
MEP surfaces of coumarin thiazole hybrids 5d, 5g and 5i.
Compds Structures
MEPs
5d
5g
5i
with hydroxyethyl fragments, which thus made it easy to interact
with diverse enzymes and receptors in biological systems. This
conclusion, obtained from the study of electronic properties, was
consistent with the favorable mode of action indicated by the
aforementioned molecular docking research [34,44].
2.10. Interactions of compound 5i with MRSA DNA
DNA, the deliverer of genetic instructions, has played an
important role in the target construction of many antimicrobial
drugs, which provides a significant direction for the exploration of
novel potential DNA-targeting antimicrobial molecules [45]. Hence,
coumarin thiazole molecule 5i was selected as a sample for its
highly selective inhibition of MRSA strains, and its antibacterial
mechanism was preliminarily explored by reacting it with MRSA
DNA in vitro, and the interaction was monitored via UVevis spec-
troscopy. Herein, the tested DNA was isolated from the MRSA
strains, and neutral red (NR) dye as the spectroscopic probe was
used, and two spectral characteristics, hyperchromism and hypo-
chromism, were used to distinguish the variations in the DNA
double-helix structure. The UVevis spectra (Fig. 8) showed that the
maximum absorption peak (260 nm) of MRSA DNA increased along
with the increasing content of active compound 5i. In addition, the
absorption of the 5i-DNA complex was significantly lower than that
of the single sum of free hybrid 5i and MRSA DNA. This hypo-
chromic phenomenon might be related to the strong interaction
caused by the embedding of the aromatic chromophore of com-
pound 5i into the DNA helix structure [46]. The corresponding
equation and plot of the binding constant were shown in Fig. S2
(Supporting information).
Fig. 8. Different concentrations of coumarin thiazole hybrid 5i and DNA (pH ¼ 7.4,
T ¼ 298 K). Inset: comparison of absorption of the 5i-DNA complex at 260 nm and the
sum of free DNA and hybrid 5i.
c
(DNA)
¼
1.86
ꢁ
10ꢂ4 mol/L, and
c (hybrid
5i) ¼ 0e1.60 ꢁ 10ꢂ5 mol/L for curves aei respectively, at increment 0.20 ꢁ 10ꢂ5
.
absorbance was opposite to an increase in DNA content. These
obvious spectral changes suggested that highly active hybrid 5i
might compete with NR and effectively embed into the DNA
double-helix structure to block DNA replication and further exert
its highly efficient antibacterial activity [47].
2.10.1. Interactions of compound 5i, NR and MRSA DNA
2.11. Drug combination
As could be seen from Fig. 9, the maximum absorption peak
(530 nm) of the DNA-NR complex decreased with a gradual in-
crease in compound concentration, whereas the trend of free NR
Drug combination is an important means to improve the cure
rate of multiple diseases by combining potential drug candidates
8

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
norfloxacin
against
some
tested
strains.
Remarkably,
methoxybenzene-modified coumarin thiazole hybrid 5i showed
strong selectivity and significant inhibitory activity against MRSA
(MIC ¼ 0.004 mM), which was 6 times superior than that of nor-
floxacin (MIC ¼ 0.025 mM). When the Clog P values of the target
compounds were in the range of 2.63e4.54, it was beneficial to the
development of their physicochemical and pharmacokinetic
properties. In addition, compound 5i did not produce apparent
cytotoxicity or drug resistance. Molecular docking and MEP surface
studies have shown that highly active molecule 5i could facilitate
molecular interactions with various enzymes and receptors in
biological systems by forming stable hydrogen bonds. Moreover,
further research into the antibacterial mechanism revealed that
hybrid 5i might damage the MRSA cell membrane and could also be
inserted into MRSA DNA to inhibit its replication and growth. Based
on these research facts, the modification of this type of coumarin
thiazole derivatives could offer enormous possibilities for devel-
oping more novel antibacterial repressors and might also provide
an alternative for the synthesis of biologically active hybrids, in
addition to revealing their therapeutic targets and potential anti-
bacterial mechanism.
Fig. 9. Competitive reaction of coumarin thiazole hybrid 5i and neutral red with DNA.
c
(DNA)
¼
1.86
ꢁ
10ꢂ4 mol/L,
c
(NR)
¼
2
ꢁ
10ꢂ5 mol/L, and
c (compound
5i) ¼ 0e4.8 ꢁ 10ꢂ5 mol/L for curves aei respectively, at increment 0.6 ꢁ 10ꢂ5. Inset: in
the wavelength range of 380e620 nm, with the increase of compound 5i content, the
absorption spectra of the system reacted competitively with compound 5i, NR and
DNA.
4. Experimental protocols
4.1. General methods
with clinical drugs. It is believed to minimize the occurrence of side
effects and the development of bacterial resistance [20,23]. In this
study, highly active molecule 5i was combined with clinical nor-
floxacin against the test bacteria in vitro, and the effect of the
combination was verified by calculating the fractional inhibitory
concentration (FIC) index.
Unless otherwise stated, all materials are commercially available
without any additional processing. The mass was measured on a
micro-balance. Thin layer UV chromatography (254 nm) (TLC) was
employed. Silica gel (500e600 mesh) was used for column chro-
matography. Melting point (mp) was determined with an X-6
melting point meter. 1H NMR and 13C NMR spectra of coumarin
thiazole hybrids were recorded by Bruker AVANCE III spectrometer
(600 MHz). High resolution mass spectra (HRMS) was tested by
mass spectrometer (FT-ICR). The following abbreviations were used
to express the signal of NMR spectra: s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quadruplet, m ¼ multiplet. The coupling constant (J)
was applied to Hertz unit (Hz). Abbreviations for the following
were used to denote structural fragments: Coumarin ¼ Cou;
Ph ¼ Phenyl.
The results in Table 4 showed that the combination uses had a
significantly better inhibitory effect on bacteria than their single
use of either component, which was reflected by their final MIC
values. It was worth noting that compound 5i exhibited a signifi-
cantly improved inhibitory effect against MRSA after being com-
bined with norfloxacin, showing an expected synergistic effect
(FIC ¼ 0.45). In addition, hybrid 5i combined with norfloxacin
showed different degrees of additive effects against K. pneumonia
and E. coli, with FIC values of 0.58 and 0.51, respectively. However,
contrary to expectations, the combination of compound 5i and
norfloxacin had an indifferent effect on E. faecalis. These results
showed that the combination of coumarin thiazole molecule 5i and
clinical drugs could maximize the antibacterial efficiency, and
might also provide a feasible clue for broadening the antibacterial
spectrum and reducing drug resistance [25].
4.1.1. Synthesis of intermediates 2 and 7
The intermediates 2 and 7 were prepared according to the
literature procedures [8,48].
4.1.2. Synthesis of intermediates 10 and 11
The intermediates 10 and 11 were prepared according to the
literature procedures [28].
3. Conclusion
4.1.3. Synthesis of 4-(chloromethyl)-7-(2-oxo-2-(thiazol-2-yl)
ethoxy)-2H-chromen-2-one (3)
To a solution of intermediate 2 (2.10 g, 10.0 mmol), potassium
carbonate (1.70 g, 12.0 mmol) in acetonitrile (50 mL) was added
intermediate 7 (2.47 g, 12.0 mmol), the mixture was stirred at 60 ꢀC
for 8 h. After completion of the reaction, solvent was evaporated
A series of novel chalcone-conjugated, multi-flexible end-group
coumarin thiazole derivatives was successfully developed through
efficient and productive synthetic methods. In vitro antibacterial
evaluation displayed that the bacteriostatic ability of the prepared
hybrids was better than or equal to that of the reference drug
Table 4
Drug combinations of compound 5i with antibacterial drugs Norfloxacin.a
Bacteria
MRSA
E. faecalis
K. pneumonia
E. coli
Compound 5i MIC/10ꢂ3 mM
Compound N MIC/10ꢂ3 mM
FIC index
0.125
0.781
0.45
1.906
0.781
1.34
0.953
0.203
0.58
0.234
0.781
0.51
Effect
synergistic
indifferent
additivism
additivism
a
N, Norfloxacin; MRSA, Methicillin-Resistant Staphylococcus aureus N315; E. faecalis, Enterococcus faecalis; K. pneumonia, Klebsiella pneumonia; E. coli, Escherichia coli.
9

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
under reduced pressure and further purified by silica gel column
chromatography (eluent, dichloromethane/petroleum ether (10/1,
V/V)) to afford the desired compound 3 as white solid. Yield: 48%;
160.6,159.1,155.1,154.7,145.9,145.3,134.9,132.6,131.3,130.9,130.5,
130.4, 129.3, 128.3, 127.5, 114.4, 112.3, 112.2, 103.5, 59.7, 57.3,
56.2 ppm; HRMS (ESI) calcd. for C26H23ClN2O6S [MþH]þ, 527.1038,
found, 527.1043.
mp: 190e192 ꢀC; 1H NMR (600 MHz, DMSO‑d6)
d: 8.32 (d,
J ¼ 3.0 Hz, 1H, thiazole-5-H), 8.24 (d, J ¼ 3.0 Hz, 1H, thiazole-4-H),
7.78 (d, J ¼ 8.8 Hz, 1H, Cou-5-H), 7.12 (m, J ¼ 9.0, 3.2 Hz, 2H, Cou-6,
8-H), 6.52 (s, 1H, Cou-3-H), 5.82 (s, 2H, OCH2), 5.00 (s, 2H, CH2Cl)
4.1.7. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(4-chlorophenyl)-3-oxo-3-(thiazol-2-yl) prop-1-en-2-yl)oxy)-
2H-chromen-2-one (5c)
ppm; 13C NMR (150 MHz, DMSO‑d6)
d: 187.6, 164.1, 161.6, 160.4,
155.5, 151.2, 145.8, 128.7, 127.0, 126.9, 113.0, 112.8, 111.5, 102.6, 70.5,
55.4 ppm.
Compound 5c was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), p-chlorobenzaldehyde (140 mg, 1.00 mmol) and
piperidine (17 mg, 0.20 mmol). The pure product 5c was obtained
4.1.4. Synthesis of 4-((bis(2-hydroxyethyl)amino)methyl)-7-(2-oxo-
2-(thiazol-2-yl)ethoxy)-2H-chromen-2-one (4)
as yellow liquid. Yield: 42%; 1H NMR (600 MHz, DMSO‑d6)
d: 8.71 (s,
To a solution of intermediate 3 (336 mg, 1.00 mmol) in aceto-
nitrile (25 mL) was added diethanol amine (212 mg, 2.00 mmol),
the mixture was stirred at 80 ꢀC for 8 h. After completion of the
reaction, solvent was evaporated under reduced pressure and
further purified by silica gel column chromatography (eluent,
dichloromethane/methanol (3/1, V/V)) to afford the desired com-
pound 4 as brown liquid. Yield: 70%; 1H NMR (600 MHz, DMSO‑d6)
1H, C]CH), 8.32 (d, J ¼ 3.1 Hz, 1H, thiazole-5-H), 8.30 (d, J ¼ 3.1 Hz,
1H, thiazole-4-H), 7.93 (d, J ¼ 8.8 Hz, 1H, Cou-5-H), 7.83 (d,
J ¼ 8.7 Hz, 2H, Ph-2,6-H), 7.53 (d, J ¼ 8.6 Hz, 2H, Ph-3,5-H), 7.10 (d,
J ¼ 2.5 Hz, 1H, Cou-8-H), 7.07 (dd, J ¼ 8.8, 2.5 Hz, 1H, Cou-6-H), 6.58
(s, 1H, Cou-3-H), 4.51 (m, 2H, 2OH), 3.89 (s, 2H, Cou-4-CH2), 3.52 (d,
J ¼ 5.2 Hz, 4H, 2CH2CH2OH), 2.65 (t, J ¼ 6.1 Hz, 4H, 2CH2CH2OH)
ppm; 13C NMR (150 MHz, DMSO‑d6)
d: 178.6, 166.1, 160.6, 158.9,
d
: 8.33 (d, J ¼ 2.9 Hz, 1H, thiazole-5-H), 8.24 (d, J ¼ 2.9 Hz, 1H,
thiazole-4-H), 7.87 (d, J ¼ 8.9 Hz, 1H, Cou-5-H), 7.07 (d, J ¼ 2.3 Hz,
1H, Cou-8-H), 7.01 (dd, J ¼ 8.9, 2.4 Hz, 1H, Cou-6-H), 6.51 (s, 1H,
Cou-3-H), 5.80 (s, 2H, CH2CO), 4.49 (m, 2H, 2OH), 3.89 (s, 2H, Cou-4-
CH2), 3.51e3.48 (m, 4H, 2CH2CH2OH), 2.64 (t, J ¼ 5.8 Hz, 4H,
155.2, 154.7, 145.8, 144.5, 136.0, 134.4, 133.0, 131.3, 129.7, 129.0,
127.5, 114.4, 112.3, 112.1, 103.4, 59.7, 57.3, 56.2 ppm; HRMS (ESI)
calcd. for C26H23ClN2O6S [MþH]þ, 527.1038, found, 527.1043.
2CH2CH2OH) ppm; 13C NMR (150 MHz, DMSO‑d6)
d: 187.7, 164.1,
161.1, 160.9, 155.3, 155.0, 145.8, 128.7, 126.9, 112.9, 112.6, 111.4, 102.2,
70.4, 59.7, 57.4, 56.3, 55.4 ppm.
4.1.8. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(2,4-dichlorophenyl) -3-oxo-3-(thiazol-2-yl)prop-1-en-2-yl)
oxy)-2H-chromen-2-one (5d)
4.1.5. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((3-oxo-1-phenyl-3-(thiazol-2-yl)prop-1-en-2-yl) oxy)-2H-
chromen-2-one (5a)
To a solution of intermediate 4 (404 mg, 1.00 mmol), benzal-
dehyde (107 mg, 1.00 mmol) in ethanol (25 mL) was added piper-
idine (17 mg, 0.20 mmol), the mixture was stirred at 80 ꢀC for 8 h.
After completion of the reaction, solvent was evaporated under
reduced pressure and further purified by silica gel column chro-
matography (eluent, dichloromethane/petroleum ether (1/1, V/V))
to afford the desired compound 5a as yellow liquid. Yield: 25%; 1H
Compound 5d was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), 2, 4-dichlorobenzaldehyde (175 mg, 1.00 mmol) and
piperidine (17 mg, 0.20 mmol). The pure product 5d was obtained
as yellow liquid. Yield: 40%; 1H NMR (600 MHz, DMSO‑d6)
d: 9.11 (s,
1H, C]CH), 8.34 (d, J ¼ 3.1 Hz, 1H, thiazole-5-H), 8.31 (d, J ¼ 3.1 Hz,
1H, thiazole-4-H), 7.98 (d, J ¼ 8.7 Hz, 1H, Ph-6-H), 7.91 (d, J ¼ 8.9 Hz,
1H, Cou-5-H), 7.81 (d, J ¼ 2.1 Hz, 1H, Ph-5-H), 7.50 (dd, J ¼ 8.6,
2.1 Hz,1H, Ph-3-H), 7.10 (d, J ¼ 2.5 Hz,1H, Cou-8-H), 7.04 (dd, J ¼ 8.8,
2.5 Hz, 1H, Cou-6-H), 6.56 (s, 1H, Cou-3-H), 4.50 (m, 2H, 2OH), 3.88
(s, 2H, Cou-4-CH2), 3.50 (s, 4H, 2CH2CH2OH), 2.64 (t, J ¼ 6.0 Hz, 4H,
NMR (600 MHz, DMSO‑d6) d: 8.79 (s, 1H, C]CH), 8.27 (s, 2H,
thiazole-4,5-H), 7.92 (d, J ¼ 8.6 Hz, 1H, Cou-5-H), 7.79 (d, J ¼ 8.8 Hz,
2H, Ph-2,6-H), 7.06 (d, J ¼ 1.7 Hz, 2H, Ph-3,5-H), 7.03 (m, J ¼ 6.2,
2.7 Hz, 3H, Ph-4-H, Cou-6,8-H), 6.55 (s, 1H, Cou-3-H), 4.47 (m, 2H,
2OH), 3.88 (s, 2H, Cou-4-CH2), 3.51 (d, J ¼ 5.4 Hz, 4H, 2CH2CH2OH),
2.65 (d, J ¼ 6.3 Hz, 4H, 2CH2CH2OH) ppm; 13C NMR (150 MHz,
2CH2CH2OH) ppm; 13C NMR (150 MHz, DMSO‑d6)
d: 178.2, 165.9,
160.5, 158.9, 155.1, 154.7, 145.9, 145.6, 136.3, 135.8, 132.4, 130.1,
129.7, 129.4, 128.6, 127.5, 114.5, 112.4, 112.2, 103.6, 59.6, 57.3,
55.3 ppm; HRMS (ESI) calcd. for C26H22Cl2N2O6S [MþH]þ, 561.0648,
found, 561.0651.
DMSO‑d6) d: 178.2, 170.8, 166.6, 162.1, 160.6, 159.3, 155.2, 145.6,
142.6, 136.46, 133.8, 132.1, 129.1, 128.5, 127.4, 124.9, 115.3, 114.2,
112.1, 103.1, 102.2, 59.7, 57.5, 55.9 ppm; HRMS (ESI) calcd. for
C
26H24N2O6S [MþH]þ, 493.1428, found, 493.1426.
4.1.9. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(4-fluorophenyl)-3-oxo-3-(thiazol-2-yl) prop-1-en-2-yl)oxy)-
2H-chromen-2-one (5e)
4.1.6. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(2-chlorophenyl)-3-oxo-3-(thiazol-2-yl) prop-1-en-2-yl)oxy)-
2H-chromen-2-one (5b)
Compound 5e was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), p-fluorobenzaldehyde (124 mg, 1.00 mmol) and
piperidine (17 mg, 0.20 mmol). The pure product 5e was obtained
Compound 5b was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), o-chlorobenzaldehyde (140 mg, 1.00 mmol) and
piperidine (17 mg, 0.20 mmol). The pure product 5b was obtained
as yellow liquid. Yield: 50%; 1H NMR (600 MHz, DMSO‑d6)
d: 8.74 (s,
1H, C]CH), 8.31 (dd, J ¼ 7.1, 3.1 Hz, 2H, thiazole-4,5-H), 7.92e7.87
(m, 3H, Ph-2,6-H, Cou-5-H), 7.32 (s, 2H, Ph-3,5-H), 7.11 (d, J ¼ 2.5 Hz,
1H, Cou-8-H), 7.07 (dd, J ¼ 8.8, 2.6 Hz, 1H, Cou-6-H), 6.57 (s, 1H,
Cou-3-H), 4.52 (m, 2H, 2OH), 3.89 (s, 2H, Cou-4-CH2), 3.51 (t,
J ¼ 5.4 Hz, 4H, 2CH2CH2OH), 2.65 (t, J ¼ 6.0 Hz, 4H, 2CH2CH2OH)
as yellow liquid. Yield: 40%; 1H NMR (600 MHz, DMSO‑d6)
d: 9.19 (s,
1H, C]CH), 8.33 (d, J ¼ 11.0 Hz, 2H, thiazole-4,5-H), 7.98 (d,
J ¼ 7.7 Hz, 1H, Ph-6-H), 7.91 (d, J ¼ 8.8 Hz, 1H, Cou-5-H), 7.63 (d,
J ¼ 7.9 Hz, 1H, Ph-2-H), 7.47 (t, J ¼ 7.4 Hz, 1H, Ph-3-H), 7.39 (t,
J ¼ 7.4 Hz, 1H, Ph-5-H), 7.11 (s, 1H, Cou-8-H), 7.05 (d, J ¼ 8.6 Hz, 1H,
Cou-6-H), 6.56 (s, 1H, Cou-3-H), 4.49 (m, 2H, 2OH), 3.88 (s, 2H, Cou-
ppm; 13C NMR (150 MHz, DMSO‑d6)
d: 178.6, 166.2, 164.9, 162.5,
160.6, 159.0, 155.2, 154.8, 145.8, 143.8, 134.8, 134.08, 128.98, 127.58,
116.98, 116.88, 114.4, 112.1, 103.3, 59.6, 57.3, 56.14 ppm; HRMS (ESI)
calcd. for C26H23FN2O6S [MþH]þ, 511.1334, found, 511.1339.
4-CH2), 3.50 (s, 4H, 2CH2CH2OH), 2.63 (t,
J
¼
5.3 Hz, 4H,
: 178.3, 166.1,
2CH2CH2OH) ppm; 13C NMR (150 MHz, DMSO‑d6)
d
10

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
4.1.10. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(4-bromophenyl)-3-oxo-3-(thiazol-2-yl) prop-1-en-2-yl)oxy)-
2H-chromen-2-one (5f)
CH), 8.30 (q, J ¼ 3.1 Hz, 2H, thiazole-4,5-H), 7.92 (d, J ¼ 8.8 Hz, 1H,
Cou-5-H), 7.81 (dd, J ¼ 6.6, 3.0 Hz, 2H, Ph-2,6-H), 7.46 (d, J ¼ 2.8 Hz,
2H, Ph-3,5-H), 7.09 (d, J ¼ 2.5 Hz, 1H, Cou-8-H), 7.07 (dd, J ¼ 4.4,
1.7 Hz, 1H, Cou-6-H), 6.55 (s, 1H, Cou-3-H), 4.48 (m, 2H, 2OH), 4.04
(s, 2H, Cou-4-CH2), 3.89 (d, J ¼ 9.3 Hz, 3H, OCH3), 3.50 (d, J ¼ 5.5 Hz,
4H, 2CH2CH2OH), 2.65e2.64 (m, 4H, 2NCH2CH2OH); 13C NMR
Compound 5f was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), p-bromobenzaldehyde (184 mg, 1.00 mmol) and
piperidine (17 mg, 0.20 mmol). The pure product 5f was obtained as
(150 MHz, DMSO‑d6) d: 187.7, 178.7, 170.8, 166.2, 164.1, 160.6, 159.2,
yellow liquid. Yield: 40%; 1H NMR (600 MHz, DMSO‑d6)
d: 8.68 (s,
155.2,145.7,144.2,135.9,131.5,129.6,128.9,127.5,114.3,112.6,112.2,
112.1, 103.2, 102.2, 59.7, 57.5, 56.2, 53.5 ppm; HRMS (ESI) calcd. for
1H, C]CH), 8.32 (d, J ¼ 3.1 Hz, 1H, thiazole-5-H), 8.30 (d, J ¼ 3.1 Hz,
1H, thiazole-4-H), 7.92 (d, J ¼ 8.9 Hz, 1H, Cou-5-H), 7.74 (d,
J ¼ 8.6 Hz, 2H, Ph-2,6-H), 7.66 (d, J ¼ 8.6 Hz, 2H, Ph-3,5-H), 7.09 (d,
J ¼ 2.5 Hz, 1H, Cou-8-H), 7.06 (dd, J ¼ 8.8, 2.5 Hz, 1H, Cou-6-H), 6.56
(s, 1H, Cou-3-H), 4.49 (m, 2H, 2OH), 3.89 (s, 2H, Cou-4-CH2), 3.51 (d,
J ¼ 5.2 Hz, 4H, 2CH2CH2OH), 2.64 (t, J ¼ 6.1 Hz, 4H, 2CH2CH2OH)
C
27H26N2O7S [MþH]þ, 523.1533, found, 523.1532.
4.1.14. Synthesis of (Z)-7-((1-(1H-inden-3-yl)-3-oxo-3-(thiazol-2-
yl)prop-1-en-2-yl)oxy)-4-((bis(2-hydroxyethyl) amino)methyl)-2H-
chromen-2-one (8a)
ppm; 13C NMR (150 MHz, DMSO‑d6)
d
: 178.6, 166.1, 160.5, 158.9,
Compound 8a was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), compound 11a (145 mg, 1.00 mmol) and piperidine
(17 mg, 0.20 mmol). The pure product 8a was obtained as yellow
155.2, 154.7, 145.8, 144.6, 134.5, 133.2, 132.7, 131.6, 129.0, 127.5,
124.9, 114. 4, 112.3, 112.2, 103.4, 59.7, 57.3, 56.2 ppm; HRMS (ESI)
calcd. for C26H23BrN2O6S [MþH]þ, 571.0533, found, 571.0538.
liquid. Yield: 40%; 1H NMR (600 MHz, DMSO‑d6)
d: 12.10 (s, 1H, NH),
4.1.11. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(4-nitrophenyl)-3-oxo-3-(thiazol-2-yl) prop-1-en-2-yl)oxy)-
2H-chromen-2-one (5g)
9.47 (s, 1H, C]CH), 8.32 (d, J ¼ 3.0 Hz, 1H, thiazole-5-H), 8.25 (d,
J ¼ 3.1 Hz, 1H, thiazole-4-H), 7.94 (d, J ¼ 2.8 Hz, 1H, Cou-5-H), 7.90
(d, J ¼ 8.9 Hz, 1H, indole-2-H), 7.84 (dd, J ¼ 5.8, 3.0 Hz, 1H, indole-4-
H), 7.51 (dd, J ¼ 6.0, 2.8 Hz, 1H, indole-5-H), 7.26 (dd, J ¼ 5.9, 3.0 Hz,
2H, indole-6,7-H), 7.11 (d, J ¼ 2.3 Hz, 1H, Cou-8-H), 7.07 (dd, J ¼ 8.9,
2.3 Hz, 1H, Cou-6-H), 6.53 (s, 1H, Cou-3-H), 4.48 (m, 2H, 2OH), 3.88
(s, 2H, Cou-4-CH2), 3.49 (dd, J ¼ 10.5, 5.1 Hz, 4H, 2CH2CH2OH), 2.63
(t, J ¼ 5.4 Hz, 4H, 2NCH2CH2OH) ppm; 13C NMR (150 MHz,
Compound 5g was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), p-nitrobenzaldehyde (151 mg, 1.00 mmol) and piper-
idine (17 mg, 0.20 mmol). The pure product 5g was obtained as
yellow liquid. Yield: 45%; 1H NMR (600 MHz, DMSO‑d6)
d: 8.71 (s,
1H, C]CH), 8.32 (d, J ¼ 3.1 Hz, 1H, thiazole-5-H), 8.30 (d, J ¼ 3.1 Hz,
1H, thiazole-4-H), 7.93 (d, J ¼ 8.8 Hz, 1H, Cou-5-H), 7.83 (d,
J ¼ 8.7 Hz, 2H, Ph-2,6-H), 7.53 (d, J ¼ 8.6 Hz, 2H, Ph-3,5-H), 7.10 (d,
J ¼ 2.5 Hz, 1H, Cou-8-H), 7.07 (dd, J ¼ 8.8, 2.5 Hz, 1H, Cou-6-H), 6.58
(s, 1H, Cou-3-H), 4.51 (m, 2H, 2OH), 3.89 (s, 2H, Cou-4-CH2), 3.52 (d,
J ¼ 5.2 Hz, 4H, 2CH2CH2OH), 2.65 (t, J ¼ 6.1 Hz, 4H, 2CH2CH2OH)
DMSO‑d6) d: 176.3, 167.7, 160.7, 159.4, 155.3, 154.9, 145.5, 140.6,
136.6, 132. 5, 130.7, 127.8, 127.5, 127.3, 123.5, 121.9, 118.7, 113.9, 113.1,
111.9, 111.8, 108.9, 102.9, 59.7, 57.3, 56.2 ppm; HRMS (ESI) calcd. for
C
28H25N3O6S [MþH]þ, 532.1537, found, 532.1538.
4.1.15. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(6-chloro-1H-indol-3-yl) -3-oxo-3-(thiazol-2-yl)prop-1-en-2-
yl)oxy)-2H-chromen-2-one (8b)
ppm; 13C NMR (150 MHz, DMSO‑d6)
d: 178.6, 166.1, 160.6, 158.9,
155.2, 154.7, 145.8, 144.5, 136.0, 134.4, 133.0, 131.3, 129.7, 129.0,
127.5, 114.4, 112.3, 112.1, 103.4, 59.7, 57.3, 56.2 ppm; HRMS (ESI)
calcd. for C27H26N2O7S [MþH]þ, 538.1279, found, 538.1285.
Compound 8b was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), compound 11b (179 mg, 1.00 mmol) and piperidine
(17 mg, 0.20 mmol). The pure product 8b was obtained as yellow
4.1.12. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((3-oxo-3-(thiazol-2-yl)-1-(p-tolyl)prop-1-en-2-yl) oxy)-2H-
chromen-2-one (5h)
Compound 5h was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), p-tolualdehyde (120 mg, 1.00 mmol) and piperidine
(17 mg, 0.20 mmol). The pure product 5h was obtained as yellow
liquid. Yield: 40%; 1H NMR (600 MHz, DMSO‑d6) 1H NMR (600 MHz,
liquid. Yield: 43%; 1H NMR (600 MHz, DMSO‑d6)
d: 12.13 (s, 1H, NH),
9.37 (s, 1H, C]CH), 8.31 (d, J ¼ 2.7 Hz, 1H, thiazole-5-H), 8.26 (d,
J ¼ 2.8 Hz, 1H, thiazole-4-H), 7.97 (s, 1H, Cou-5-H), 7.90 (d,
J ¼ 8.9 Hz, 1H, indole-2-H), 7.83 (d, J ¼ 8.5 Hz, 1H, indole-4-H), 7.56
(s, 1H, indole-5-H), 7.27 (d, J ¼ 8.4 Hz, 1H, indole-7-H), 7.10 (s, 1H,
Cou-8-H), 7.06 (d, J ¼ 8.8 Hz, 1H, Cou-6-H), 6.53 (s, 1H, Cou-3-H),
4.46 (m, 2H, 2OH), 3.88 (s, 2H, Cou-4-CH2), 3.49 (d, J ¼ 4.8 Hz, 4H,
2CH2CH2OH), 2.64 (d, J ¼ 12.7 Hz, 4H, 2NCH2CH2OH) ppm; 13C NMR
DMSO‑d6) d: 8.75 (s, 1H, C]CH), 8.29 (s, 2H, thiazole-4,5-H), 7.91 (d,
J ¼ 8.7 Hz, 1H, Cou-5-H), 7.71 (d, J ¼ 8.1 Hz, 2H, Ph-2,6-H), 7.27 (d,
J ¼ 8.1 Hz, 2H, Ph-3,5-H), 7.08e7.04 (m, 2H, Cou-6,8-H), 6.55 (s, 1H,
Cou-3-H), 4.48 (m, 2H, 2OH), 3.88 (s, 2H, Cou-4-CH2), 3.51 (dd,
J ¼ 11.1, 5.6 Hz, 4H, 2CH2CH2OH), 2.64 (t, J ¼ 5.9 Hz, 4H,
2NCH2CH2OH), 2.32 (s, 3H, Ph-CH3) ppm; 13C NMR (150 MHz,
(150 MHz, DMSO‑d6) d: 176.5, 167.5, 160.7, 159.3, 155.3, 154.8, 145.5,
141.1, 137.0, 133.2, 130. 0, 128.1, 127.9, 127.4, 126.2, 122.1, 120.3, 114.0,
112.8, 111.9, 111.8, 109.0, 102.9, 59.7, 57.3, 56.2 ppm; HRMS (ESI)
calcd. for C28H24ClN3O6S [MþH]þ, 566.1142, found, 566.1148.
DMSO‑d6)
d
: 178.5, 166.4, 160.6, 159.2, 155.2, 154.8, 145. 7, 143.6,
4.1.16. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(6-methyl-1H-indol-3-yl)-3-oxo-3- (thiazol-2-yl)prop-1-en-2-
yl)oxy)-2H-chromen-2-one (8c)
141.9, 136.3, 131.6, 130.3, 129.6, 128.7, 127. 5, 114.2, 112.3, 112.1, 103.1,
59.7, 57.3, 56.2, 21.6 ppm; HRMS (ESI) calcd. for C27H26N2O6S
[MþH]þ, 507.1584, found, 507.1585.
Compound 8c was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), compound 11c (159 mg, 1.00 mmol) and piperidine
(17 mg, 0.20 mmol). The pure product 8c was obtained as yellow
4.1.13. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(4-methoxyphenyl)-3-oxo-3-(thiazol-2-yl) prop-1-en-2-yl)
oxy)-2H-chromen-2-one (5i)
liquid. Yield: 45%; 1H NMR (600 MHz, DMSO‑d6)
d: 11.96 (s, 1H, NH),
Compound 5i was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), p-anisaldehyde (136 mg, 1.00 mmol) and piperidine
(17 mg, 0.20 mmol). The pure product 5i was obtained as yellow
9.42 (s, 1H, C]CH), 8.31 (d, J ¼ 2.9 Hz, 1H, thiazole-5-H), 8.24 (d,
J ¼ 2.9 Hz, 1H, thiazole-4-H), 7.90 (d, J ¼ 8.9 Hz, 1H, Cou-5-H), 7.86
(s, 1H, indole-2-H), 7.71 (d, J ¼ 8.1 Hz, 1H, indole-4-H), 7.29 (s, 1H,
indole-5-H), 7.08 (d, J ¼ 6.7 Hz, 2H, indole-7-H, Cou-8-H), 7.06 (d,
J ¼ 8.8 Hz, 1H, Cou-6-H), 6.52 (s, 1H, Cou-3-H), 4.46 (m, 2H, 2OH),
liquid. Yield: 50%; 1H NMR (600 MHz, DMSO‑d6)
d: 8.74 (s, 1H, C]
11

Y. Hu, C. Hu, G. Pan et al.
European Journal of Medicinal Chemistry 222 (2021) 113628
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3.88 (s, 2H, Cou-4-CH2), 3.50 (d, J ¼ 5.2 Hz, 4H, 2CH2CH2OH), 2.64
(dd, J ¼ 11.6, 6.2 Hz, 4H, 2NCH2CH2OH), 2.43 (s, 3H, CH3) ppm; 13
C
NMR (150 MHz, DMSO‑d6) d: 176.2, 167.8, 160.7, 159.4, 155.3, 154.9,
145.4,140.5,137.0, 132.9,132.1,130.9,127.7,127.3, 125.4,123.5,118.5,
113.9, 112.8, 111.9, 111.7, 109.0, 102.9, 59.7, 57.4, 56.2, 21.7 ppm;
HRMS (ESI) calcd. for C29H27N3O6S [MþH]þ, 546.1693, found,
546.1695.
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4.1.17. Synthesis of (Z)-4-((bis(2-hydroxyethyl)amino)methyl)-7-
((1-(1-ethyl-1H-indol-3-yl)-3-oxo- 3-(thiazol-2-yl)prop-1-en-2-yl)
oxy)-2H-chromen-2-one (8d)
Compound 8d was prepared according to the procedure
described for compound 5a, starting from intermediate 4 (404 mg,
1.00 mmol), compound 11d (173 mg, 1.00 mmol) and piperidine
(17 mg, 0.20 mmol). The pure product 8d was obtained as yellow
liquid. Yield: 45%; 1H NMR (600 MHz, DMSO‑d6)
d: 9.43 (s, 1H, C]
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CH), 8.32 (d, J ¼ 3.1 Hz, 1H, thiazole-5-H), 8.25 (d, J ¼ 3.1 Hz, 1H,
thiazole-4-H), 8.05 (s,1H, Cou-5-H), 7.90 (d, J ¼ 8.9 Hz,1H, indole-2-
H), 7.86 (d, J ¼ 7.1 Hz, 1H, indole-5-H), 7.61 (d, J ¼ 7.2 Hz, 1H, indole-
5-H), 7.33e7.28 (m, 2H, indole-6,7-H), 7.12 (d, J ¼ 2.5 Hz, 1H, Cou-8-
H), 7.08 (dd, J ¼ 8.9, 2.5 Hz, 1H, Cou-6-H), 6.54 (s, 1H, Cou-3-H), 4.48
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3.52e3.49 (m, 4H, 2CH2CH2OH), 2.64 (t,
J
¼
6.1 Hz, 4H,
2NCH2CH2OH), 1.33 (t, J ¼ 7.2 Hz, 3H, NCH2CH3) ppm; 13C NMR
(150 MHz, DMSO‑d6) d: 176.2, 167.7, 160.7, 159.4, 155.2, 154.9, 145.4,
140.7, 136.3, 134.5, 130.1, 128.1, 127.8, 127.2, 123.5, 122.1, 119.1, 113.9,
112.1, 111.7, 111.5, 108.3, 103.1, 59.7, 57.4, 56.2, 41.8, 15.5 ppm; HRMS
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgments
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This work was supported by the National Natural Science Foun-
dation of China (21905069), the Shenzhen Science and Technology
Innovation Committee (JCYJ20180507183907224, KQTD20170
809110344233), Economic, Trade and Information Commission of
Shenzhen Municipality through the Graphene Manufacture Innova-
tion Center (201901161514), and Guangdong Province Covid-19
Pandemic Control Research Fund (2020KZDZX1220). The authors
would like to thank Zhou Qian from Shiyanjia Lab (www.shiyanjia.
com) for Molecular docking, He Daijia from Ceshigo Research Ser-
vice (www.ceshigo.com) for NMR analysis, Geng Huanran from the
testing technology center of materials and devices, Tsinghua Shenz-
hen international graduate school (https://mdtc.sz.tsinghua.edu.cn)
for the NMR analysis.
Appendix A. Supplementary data
(b) L. Zhang, X.M. Peng, G.L.V. Damu, R.X. Geng, C.H. Zhou, Comprehensive
review in current developments of imidazole-based medicinal chemistry,
Med. Res. Rev. 34 (2014) 340e437.
Supplementary data to this article can be found online at
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