Synthesis, molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential antibacterial agents

Article abstract of DOI:10.1016/j.bmc.2014.09.048

A series of 4-hydroxycoumarin derivatives were designed and synthesized in order to find some more potent antibacterial drugs. Their antibacterial activities against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus were tested. These compounds showed good antibacterial activities against Gram-positive strains. Compound 4g represented the most potent antibacterial activity against Bacillus subtilis and S. aureus with MIC of 0.236, 0.355 μg/mL, respectively. What's more, it showed the most potent activity against SaFabI with IC₅₀ of 0.57 μM. Molecular docking of 4g into S. aureus Enoyl-ACP-reductase active site were performed to determine the probable binding mode, while the QSAR model was built to check the previous work as well as to introduce new directions.

Full text of DOI:10.1016/j.bmc.2014.09.048

Accepted Manuscript
Synthesis, molecular docking and biological evaluation of coumarin derivatives
containing piperazine skeleton as potential antibacterial agents
She-Feng Wang, Yong Yin, Xun Wu, Fang Qiao, Shao Sha, Peng-Cheng Lv,
Jing Zhao, Hai-Liang Zhu
PII:
S0968-0896(14)00702-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.09.048
BMC 11835
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 July 2014
6 September 2014
23 September 2014
Please cite this article as: Wang, S-F., Yin, Y., Wu, X., Qiao, F., Sha, S., Lv, P-C., Zhao, J., Zhu, H-L., Synthesis,
molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential
antibacterial agents, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.09.048
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Synthesis, molecular docking and biological evaluation of coumarin derivatives
containing piperazine skeleton as potential antibacterial agents
She-Feng Wang+, Yong Yin+, Xun Wu, Fang Qiao, Shao Sha, Peng-Cheng Lv*,
Jing Zhao*, Hai-Liang Zhu*.
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China
+ These authors contributed equally to this work.
* Corresponding authors. Tel.: +86 25 83592572; Fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
1

Abstract
A series of 4-hydroxycoumarin derivatives were designed and synthesized in
order to find some more potent antibacterial drugs. Their antibacterial activities
against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and
Staphylococcus aureus were tested. These compounds showed good antibacterial
activities against Gram-positive strains. Compound 4g represented the most potent
antibacterial activity against Bacillus subtilis and S.aureus with MIC of 0.236, 0.355
µg/mL, respectively. What’s more, it showed the most potent activity against SaFabI
with IC50 of 0.57 µM. Molecular docking of 4g into S.aureus Enoyl-ACP-reductase
active site were performed to determine the probable binding mode, while the QSAR
model was built to check the previous work as well as to introduce new directions.
Keywords
Coumarin derivatives
Antibacterial activities
SaFabI
Molecular docking
3D-QSAR
2

1. Introduction
The emergence of bacterial resistance to most of the antibacterials is becoming a
1
serious global healthcare problem.
Particularly, the spread of multiresistant
Gram-positive bacteria, such as methicillin resistant Staphylococcus aureus (MRSA),
2
was fast. It was only two years after the introduction of the antibiotic methicillin in
1959 that resistant strains of the gram-positive human pathogen Staphylococcus
aureus emerged. Since then, methicillin-resistant S. aureus (MRSA) infections have
become an increasing problem in hospitals worldwide. However, antibiotic resistance
has drastically outpaced new antibiotic discovery, which flourished between the
1940s and 1960s. Only a few novel scaffolds were approved between 1962 and 2000,
such as the carbapenems. It is an urgent to elaborate new, highly potent antibiotics
with alternative modes of actions. In this regard, bacterial fatty acid biosynthesis
(FAS) is an attractive target. 3
Fatty acid synthesis (FAS) pathways are divided into two distinct forms: FAS-I
4
and FAS-II. Enzymes of FAS-II pathway have been selectively targets for the
5
development of novel antibacterial agents. The FAS-II pathway is consist of some
distinct and highly conserved enzymes: β-hydroxacyl-ACP-dehydratase (FabZ),
β-ketoacyl-ACP-reductase (FabG), and enoyl-ACP-reductase (FabI, also known as
ENR). For most bacterias, FabI, the unique ENR, is responsible for the concluding
reduction step of each elongation cycle. It represents a key physiological regulator of
fatty acid biosynthesis, and it has been carried out to be an important drug target for
the development of novel antibacterials and antimalarials. 6-8 Furthermore, it has been
proved that the sequences of human FAS have many similarities with some other
creatures.9 This has led to the pursuit of specific FabI inhibitors as novel antibacterial
agents which would not be expected to interfere with comparable human biochemical
processes. 10 The antibacterial target of isoniazid (Scheme 1), 11 used in the treatment
12
of tuberculosis for 50 years, and triclosan (Scheme 1), the broad spectrum biocide
in a wide range of consumer goods, have been determined to be the enoyl-ACP
reductase (FabI). Here we describe a series of novel FabI inhibitors.
3

Coumarins are a class of compounds found widely in nature, they show a broad
13
spectrum of activities and are frequently associated with low toxicity, and they can
be considered as a privileged scaffold and an ideal framework for the design of
compounds that can interact with different targets as their inherent affinity for several
14
biological targets.
Like ensaculine, one kind of AChE inhibitors, a coumarin
derivative containing piperazine ring (Scheme 1). There have been performed a lot of
efforts which aimed at designing functionalized synthetic coumarins as effective
15–24
agents that can affect some cellular functions potently and selectively.
Since
piperazines and its derivatives are important pharmacophores, they are effective
ingredients in many marketed drugs like olaparib and Piperazinyl-linked ciprofloxacin
25-26
dimers which was reported as potent antibacterial agents (Scheme 1).
These
results promoted us to synthesize new derivatives of coumarins with piperazine
skeleton for the sake of finding new effectively antibacterial agents. Based on these
things mentioned above, we synthesized two series of novel coumarins derivatives
owning piperazine skeleton (4a-4n) and (5a-5n) (Table 1), while compounds 5a, 5i, 5l
27
and 5m were reported before.
We studied their antibacterial activities against
Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus), Escherichia coli
(E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus
Enoyl-ACP-reductase (SaFabI) (PDB code: 4CV0) inhibitory activities. Docking
simulation was performed using the X-ray crystallographic structure of SaFabI in
complex with the most potent inhibitor to explore the binding mode of the compound
at the active site. Additionally, we also used the results of molecular orbital
calculations (3D-QSAR model) to study the structure–activity relationship and guide
the further study.
2. Results and discussion
2.1. Chemistry
Compounds 4a-4n and 5a-5n were synthesized by the routes outlined in Scheme
4

2. They were first designed and synthesized from coumarin and piperazine derivatives
except componds 5a, 5i, 5l and 5m. The chemical structures of these coumarin
derivatives were summarized in Table 1. All of the synthetic compounds were
1
characterized by H NMR, elemental analysis and mass spectrum, and they gave
satisfactory analytical and spectroscopic data, which were in full accordance with
their depicted structures.
2.2. Biological activity
2.2.1. Antibacterial activity
The synthesized compounds were tested for their antibacterial activities against
two Gram-negative bacterial strains: E. coli and P. aeruginosa and two Gram-positive
bacterial strains: B. subtilis and S. aureus by MTT method with MH medium
(Muellere-Hinton medium: casein hydrolysate 17.5 g, soluble starch 1.5 g, beef
extract 1000 mL). The MICs (minimum inhibitory concentrations) of the compounds
against these bacteria were reported in Table 2, the activity of reference drug
Penicillin G was also included. The results revealed that those compounds showed
good activity against Gram-positive bacteria but inactive against Gram-negative
bacteria.
Most of the new compounds exhibited good inhibitory activities against both B.
Subtilis and S. Aureus. As shown in Table 2. Compound 4g showed the most potent
activities with MIC values of 0.236 and 0.355 µg/mL against B. subtilis and S. aureus,
respectively, which were superior to the positive control Penicillin G with MIC values
of 0.752 and 1.685 µg/mL. Compound 5g, with MIC values of 0.861, 0.327 µg/mL
against B. subtilis and S. aureus, respectively, also displayed better activities than the
positive control Penicillin G. 4f (MIC=0.612 µg/mL) and 4i (MIC=0.339 µg/mL)
showed better inhibitory activities against B. subtilis than Penicillin G; 5i
(MIC=1.857 µg/mL) and 5n (MIC=0.916 µg/mL) exhibited moderate inhibitory
against B. subtilis; 4c (MIC=0.612 µg/mL), 4n (MIC=0.612 µg/mL), 5f (MIC=0.612
5

µg/mL) and 5n (MIC=0.612 µg/mL) displayed better inhibitory activities against S.
aureus than Penicillin G (MIC=1.685 µg/mL).
According to these data, in 4a-4n, we found that compounds which have a
methoxy group on the position of benzene ring showed higher antibacterial activity
than other compounds. Especially compound 4g, which had a methoxy group on the
4-position of benzene ring, showed higher antibacterial activity against B. Subtilis and
S. aureus with MIC values of 0.236 µg/mL and 0.355 µg/mL than compound 4e
which had a methoxy group on the 2-position of benzene ring with MIC values of
3.883 µg/mL and 1.832 µg/mL and compound 4f which had a methoxy group on the
3-position of benzene ring with MIC values of 0.612 µg/mL and 2.523 µg/mL, the
same trend was also found in compounds 5e-5g. Compounds 4i-4h and 5i-5h, which
introduced two chlorine atoms at 2,3-position or 3,4-positon in benzene ring, showed
that compounds with substitutes at 3,4-position displayed better inhibitory activity
than substitutes at 2,3-postion. These results indicated that compounds with
substitution at the para- position exhibited more potent antibacterial activities than
compounds with substitution at the meta- and ortho-position.
Compounds 4l-4m and 5l-5m which introduced fluorine atom at 2-position or
4-positon in benzene ring, displayed a dramatic loss in activity compared to
compounds 4n and 5n with a trifluoromethyl at 3-position. Among compounds 4a-4d
and 5a-5d the order of inhibitory activities showed the potency of 4c > 4d > 4a> 4b
and 5d>5b>5c>5a which indicated the activity of compounds with substitutes were
superior to that of without sbustitutes. Varieties of substitutes of phenyl like
trifluoromethyl, methyl and halogen can lead to different antibacterial activities. For
example, the derivatives which have electron-withdrawing substituents (like halogen
and trifluoromethyl) displayed weaker activities against B. subtilis and S. aureus than
those having electron-donating substituents (such as methyl and methoxyl), which
suggested that substituent group can affect the inhibitory activities, and compounds
with electron-donating substituents exhibited better inhibitory activities than those
with electron-withdrawing substituents.
6

2.2.2. S. aureus FabI inhibitory activity
The ability of 4a-4n and 5a-5n to inhibit the recombinant SaFabI enzyme were
examined. All data were shown in Table 3. Among compounds 4a-4n, 4d and 4f
exhibited moderate SaFabI inhibition with IC50 values of 3.82 µM and 2.73 µM,
respectively. While 4g and 4n showed the highest inhibitory activity with IC50 values
of 0.57 µM and 0.97 µM, respectively. With compounds 5a-5n, all compounds in this
subset displayed moderate activity toward SaFabI. With the exception of 5g featuring
a p- methoxylsubunit, which turned out to be the most active compound within series
II (IC50 = 0.83 µM). 5f and 5n inhibited SaFabI with IC50 values of 54.62 and 1.82 µM,
respectively. As showed in Table 3, compounds with substituents on the position of
benzene ring displayed better inhibitory activity than that with no substituents on the
position of benzene ring (4a). Compounds 4g (IC50 =0.57 µM) and 5g (IC50 =0.83 µM)
with p-methoxyl on the position of benzene ring showed the highest inhibitory
activity. Compounds 4e-4g and 5e-5g, which introduced a methoxy group at
2-position, 3-positon or 4-positon in benzene ring, showed the SaFabI inhibitory
activity trend: 4e (IC50 =5.54 µM)<4f (IC50 =2.73 µM)<4g (IC50 =0.57 µM), 5e (IC50
=7.15 µM)<5f (IC50 =4.62 µM)<5g (IC50 =0.83 µM), these results indicated that
compounds with substitution at the para- position exhibited more potent inhibitory
activities than compounds with substitution at the meta- and ortho-position. What’s
more, the inhibitory activity of series I (4a-4n) were generally better than Series II
(5a-5n), indicating that compounds with hydroxyl showed better SaFabI inhibitory
activity than compounds with aldehyde.
The results of SaFabI inhibitory activity of the test compounds were
corresponding to the structure-activity relationships (SAR) of their antibacterial
activities. It demonstrated that the potent antibacterial activities of the synthetic
compounds were probably correlated to their FabI inhibitory activities.
2.2.3. Cytotoxicity test
7

In order to find potent antibiotics, it is important to measure cytotoxicity. One of
the major hindrances to the clinical use of many compounds with effective
antibacterial activities, however, is their ability to damage mammalian cell that leads
to high hemolytic and cytotoxic activity dangerous to the host organism. Hemolytic
activity is conventionally used as a measure of cytotoxicity and model for mammalian
cells because red blood cells are, in general, extremely fragile. However, sometimes
compounds which show low hemolytic activities have the severe cytotoxicity against
mammalian cells. So the hemolysis and cytotoxicity assays were both tested.
The pharmacological results of these selected compounds were summarized in
Table 4. It can be seen from Table 4 that the selected compounds displayed low
hemolytic activities. Besides, based on the dates, it showed that the compounds with
potent inhibitory activity were low toxic.
2.2.4. Molecular docking
In order to better understanding of the structure-activity relationships observed,
molecular docking of the most potent inhibitor 4g into the active site cavity of
Staphylococcus aureus Enoyl-ACP-reductase (SaFabI) was performed on the binding
model based on the Staphylococcus aureus Enoyl-ACP-reductase complex structure
(PDB ID: 4CV0). The binding model of compound 4g and SaFabI was described in
Fig.1A, and the enzyme surface model was shown in Fig. 1B, which revealed that the
molecule is well filled in the active pocket.
As illustrated in Figure 1, 4g located in the substrate binding site of
Staphylococcus aureus Enoyl-ACP-reductase. The docking results revealed that
amino acids Lys164 and Ile20 located in the binding pocket of protein played
important roles in the combination with compound 4g, which were stabilized two
hydrogen bonds. As showed in Figure 1, one of the two hydrogen bonds was formed
between the hydrogen atom of Lys164 and the oxygen atom of compound 4g (bond
length: C-O…H = 2.4 Å); The other one was formed between the hydrogen atom of
8

Ile20 and the oxygen atom of compound 4g (bond length: C=O…H = 2.0 Å). All
above, this molecular docking results, along with the biological assay data, suggested
that compound 4g was a potential inhibitor of SaFabI.
2.2.5. 3D-QSAR
For further exploration of structure and activity relationships of these compounds,
the 3D-QSAR study was performed by means of the DS 3.5 software (Discovery
Studio 3.5, Accelrys, Co. Ltd). The 3D-QSAR model was created with corresponding
pIC50 values (-logIC50) which were converted from the obtained IC50 (µM) values of
SaFabI inhibition. The observed and predicted pIC50 values with corresponding
residual errors of 28 compounds by this QSAR model had been given in Table 5. The
graphical relationship of the observed pIC50 values versus the predicted values had
been illustrated in Figure 2. The linear R2 value was 0.88, which meant the
dependability of linear fit.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR model
coefficients on Van der Waals grids (Fig. 3C) and electrostatic potential grids (Fig.
3D) were listed. Electrostatic map indicated red contours around regions where high
electron density (negative charge) was expected to increase activity, and blue contours
represent areas where low electron density (partial positive charge) was expected to
increase activity. Similarly, steric map indicated areas where steric bulk was predicted
to increase (green) or decrease (yellow) activity. It was widely acceptable that a better
inhibitor based on the 3D-QSAR model should have strong Van der Waals attraction
in the green areas and a polar group in the blue electrostatic potential areas (which
were dominant close to the skeleton).
As shown in Figure 3, compounds with electron-donating atom not only
circumvented the red subregion but also got more close to the favorable green spaces.
This figure displayed that these bulkier group substituted in the aromatic ring could
have good interaction with the protein for the nice molecule electrostatic and steric
features. So, this QSAR study could predict that electron-donating atom at the
9

benzene ring might enhance binding affinity of coumarin derivatives. A high negative
charged R group would help obtain good activity, validating the methoxy substituent
could be more effective. As a result, data summarized above demonstrated that
compounds 4g, the most potent SaFabI inhibitor (IC50=0.57 µM) with suitable
substituent possessed an outstanding activity.
The 3D QSAR models fitted the inhibitory activity well thus provide us the
direction of further modification. It could be predicted that electron-donating
substituent (OCH3) for the R group could increase the SaFabI inhibitory activity.
3. Conclusion
In summary, a series of novel 4-hydroxycoumarin derivatives have been
designed and synthesized, and their biological activities were also evaluated as potent
SaFabI inhibitors. Compound 4g demonstrated the most potent antibacterial activity
(MIC = 0.236 µg/mL for B. subtilis and MIC = 0.355 µg/mL for S.aureus) and SaFabI
inhibitory activity(IC50=0.57 µM), which was compared with the positive control
Penicillin G. Docking simulation was performed to position compound 4g into the
S.aureus Enoyl-ACP-reductase active site to determine the potential binding model.
Analysis of the binding conformation of compound 4g displayed that compound 4g
was stabilized by hydrogen bonding interactions with Lys164 and Ile20. Antibacterial
assay results also showed that these 4-hydroxycoumarin derivatives had the potential
to be developed for antibacterial agents. Particularly, compound 4g has demonstrated
significant S.aureus Enoyl-ACP-reductase inhibitory activity as a potential
antibacterial agent.
4. Experiments
4.1 Materials and measurements
10

All chemicals and reagents used in current study were of analytical grade. TLC
was performed on the glass-backed silica gel sheets (silica GF254) and visualized in
UV light (254 nm). Column chromatography was performed using silica gel (200–300
mesh) eluting with ethyl acetate and petroleum ether. Melting points (uncorrected)
1
were determined on a XT4MP apparatus (Taike Corp., Beijing, China). All the H
NMR spectra were recorded on a Bruker DPX 400model Spectrometer in CDCl3 and
chemical shifts were reported in ppm (d). ESI-MS spectra were recorded on a Mariner
System 5304 Mass spectrometer. Elemental analyses were performed on a
CHN-O-Rapid instrument.
4.2. General method of synthesis of coumarin derivatives
4.2.1 General procedure for synthesis of compounds 4a-4n and 5a-5n
Epoxy chloropropane (2 mmol) and anhydrous K2CO3 (1 mmol) were dissolved
in 50 acetone (50 mL), then compound 1 was added. The reaction mixture was
refluxed at 50℃ for 10-12 h, the reaction mixture was vacuum distillated, then some
water was added, the aqueous layer was extracted with ethyl acetate (2×50 mL). The
organic layer was combined and dried with anhydrous Na2SO4 for 0.5 h, removal of
the solvent to give compound 2. Compounds 4a-4n were synthesized by the following
procedure. Compound 2 (1mmol) and anhydrous K2CO3 (1mmol) were dissolved in
DMF (10 mL), and then one of those substituted piperazines (1mmol) was added. The
reaction mixture was allowed to stand at 110 ℃ for 20-24 h. The reaction mixture
was poured in 200 mL water and extracted with ethyl acetate（3×50 mL). The organic
layer was combined and dried with anhydrous Na2SO4 for 0.5 h.After the reaction
mixture was vacuum distillated, the target product 4a-4n was separated by column
chromatography.
Compounds 5a-5n were synthesized by the following procedure. Bromoacetic
acid (1.2 mmol) and anhydrous Cs2CO3 (1 mmol) were dissolved in DMF (10 mL),
and then one of those substituted piperazines (1 mmol) was added. The reaction
mixture was allowed to stand at 90 ℃ for 8-10 h. The reaction mixture was poured
11

in water and extracted with ethyl acetate（3×50 mL). The organic layer was combined
and dried with anhydrous Na2SO4 for 0.5 h. After the reaction mixture was vacuum
distillated, the intermediate products 6a-6n was separated by column chromatography.
Compounds 5a-5n were synthesized by the following procedure. To a solution of
compound 1 (1 mmol) and 6a-6n (1 mmol) in dichloromethane (25 mL),
N-hydroxybenzotriazole
(HOBt)
(0.5
mmol)
and
1-[3-(dimethyamino)-propyl]-3-ethylcarbodiimide hydrochloride (EDC) (1.5 mmol)
were added. The mixture was refluxed at 50 ℃ for 8-12 h. After completion of the
reaction, the solution was cooled, and was washed successively with 5% HCl,
saturated NaHCO3 and brine, respectively, then dried over anhydrous Na2SO4, and
concentrated in vacuum. The residue was subjected to silica gel chromatography
orcrystallization if necessary to afford the compounds 5a-5n.
4.2.1. 4-(2-hydroxy-3-(4-phenylpiperazin-1-yl)propoxy)-2H-chromen-2-one (4a)
1
White solid, yield: 67%, m.p. 80-82 ℃. H NMR (400 MHz, CDCl3) δ:
7.59-7.86 (t, J=3.98 Hz, 1H, ArH), 7.54-7.58 (m, 1H,ArH), 7.26-7.34 (m, 4H, ArH),
6.91-6.96 (t, J=9.58 Hz, 2H, ArH), 6.87-6.89 (d, J=7.32 Hz, 1H, ArH), 5.72 (s, 1H),
4.26-4.30 (m, 1H, CH), 4.13-4.20 (m, 2H, CH2), 3.71 (s, 1H, -OH), 3.21-3.30 (br, 4H,
-CH2CH2-), 2.73-2.94 (m, 2H, CH2), 2.63-2.73 (br, 4H, -CH2CH2-). ESI-MS: 381.21
(C22H25N2O4, [M+H]+), Anal.Calcd for C22H24N2O4: C, 69.46; H, 6.36; N, 7.36%;
Found: C, 69.48; H, 6.34; N, 7.40%.
4.2.2. 4-(2-hydroxy 3-(4-benzylpiperazin-1-yl)propoxy)-2H-chromen-2-one (4b)
Yellow oil, yield: 48%. 1H NMR (400 MHz, CDCl3) δ: 7.86-7.88 (m, 1H, ArH),
7.56-7.60 (m, 1H, ArH), 7.36-7.37 (d, J=4.4 Hz, 5H, ArH),7.32-7.33 (t, J=2.1 Hz, 1H,
ArH), 7.31 (s, 1H, ArH), 7.28-7.30 (m, 1H, CH), 5.73 (s, 1H), 4.21-4.26 (m, 1H, OH),
4.12-4.20 (m, 2H, CH2), 3.57 (s, 2H, CH2), 2.78 (s, 2H, CH2), 2.64-2.67 (t, J=6.16 Hz,
4H, -CH2CH2-), 2.47-2.61 (m, 4H, -CH2CH2-). ESI-MS: 395.15 (C23H27N2O4,
[M+H]+), Anal.Calcd for C23H26N2O4: C, 70.03; H, 6.64; N, 7.10%; Found: C, 70.05;
H, 6.62; N, 7.09%.
12

4.3.3.
4-(2-hydroxy-3-(4-(2,4-dimethylphenyl)piperazin-1-yl)propoxy)-2H-chromen-2-o
ne (4c)
1
Brown oil, yield: 52%. H NMR (400 MHz, CDCl3) δ: 7.85-7.87 (t, J=3.96 Hz,
1H, ArH ), 7.54-7.58 (m, 1H, ArH), 7.28-7.34 (m, 1H, ArH), 6.93-7.01 (m, 3H, ArH),
5.72 (s, 1H), 4.24-4.29 (m, 1H, CH), 4.14-4.17 (m, 2H, CH2), 2.93-2.97 (m, 4H,
-CH2CH2-), 2.89-2.91 (d, J=9.32 Hz, 2H, CH2), 2.62-2.66 (m, 4H, -CH2CH2-), 2.78 (s,
6H, 2CH3). ESI-MS: 409.26 (C24H29N2O4, [M+H]+). Anal.Calcd for C24H28N2O4: C,
70.57; H, 6.91; N, 6.86%; Found: C, 70.57; H, 6.92; N, 6.88%.
4.2.4.
4-(2-hydroxy-3-(4-(4-nitrophenyl)piperazin-1-yl)propoxy)-2H-chromen-2-one
(4d)
1
Yellow solid, yield: 58%, m.p. 170-172 ℃. H NMR (400 MHz, CDCl3) δ:
8.19-8.21 (d, J=9.32 Hz, 2H, ArH), 7.87-7.89 (d, J=8.08 Hz, 1H, ArH), 7.39 (s, 1H,
ArH), 7.36-7.37 (d, J=4.92 Hz, 2H, ArH), 6.90-6.92 (d, J=9.24 Hz, 2H, ArH), 5.78(s,
1H); 4.41-4.43 (d, J=9.2 Hz, 1H, CH), 4.23-4.24 (d, J=4.48 Hz, 2H, CH2), 3.60 (s, 4H,
-CH2CH2-), 3.02 (s; 2H, CH2), 2.85 (s, 4H, -CH2CH2-). ESI-MS: 426.22 (C22H24N3O6,
[M+H]+). Anal.Calcd for C22H23N3O6: C, 62.11; H, 5.45; N, 9.88%; Found: C, 62.12;
H, 5.43; N, 9.89%.
4.2.5.
4-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propoxy)-2H-chromen-2-one
(4e)
1
Brown oil, yield: 43%. H NMR (400 MHz, CDCl3) δ: 7.89（s, 1H, ArH),
7.56-7.89 (m, 1H, ArH), 7.29-7.35 (m, 3H, ArH), 7.20-7.24 (t, J=8.16 Hz, 1H, ArH),
6.58-6.60 (m, 1H, CH), 6.46-6.51 (m, 2H, ArH), 5.75 (s, 1H), 4.23-4.33 (m, 1H, OH),
4.13-4.23 (m, 2H, CH2), 3.83 (s, 3H, CH3), 3.22-3.31 (m, 4H, -CH2CH2-), 2.88-2.93
(m, 2H, CH2), 2.62-2.74 (m, 4H, -CH2CH2-). ESI-MS: 411.12 (C23H27N2O5, [M+H]+).
13

Anal.Calcd for C23H26N2O5: C, 67.30; H, 6.38; N, 6.82%; Found: C, 67.33; H, 6.35; N,
6.84%.
4.2.6.
4-(2-hydroxy-3-(4-(3-methoxyphenyl)piperazin-1-yl)propoxy)-2H-chromen-2-one
(4f)
1
White solid, yield: 61%, m.p. 208-210 ℃. H NMR (400 MHz, CDCl3) δ:
7.87-7.89 (d, J=7.6 Hz, 1H, ArH), 7.55-7.59 (t, J=7.44 Hz, 1H, ArH), 7.19-7.34 (m,
3H, ArH), 6.46-6.59 (m, 3H, ArH), 5,75 (s, 1H), 4.29-4.30 (d, J=3.96 Hz,1H,CH),
4.15-4.19 (t, J=7.98 Hz, 2H,CH2), 3.82 (s, 3H, CH3),3.26 (s, 4H, -CH2CH2-,),
2.88-2.89 (d, J=4.28 Hz, 2H, CH2), 2.66-2.70 (t, J=8.28 Hz, 4H, -CH2CH2-). ESI-MS:
411.12 (C23H27N2O5, [M+H]+). Anal.Calcd for C23H26N2O5: C, 67.30; H, 6.38; N,
6.82%; Found: C, 67.32; H, 6.40; N, 6.80%.
4.2.7.
4-(2-hydroxy-3-(4-(4-methoxyphenyl)piperazin-1-yl)propoxy)-2H-chromen-2-one
(4g)
1
Brown solid, yield: 43%, m.p. 120-122 ℃. H NMR (400 MHz, CDCl3) δ:
7.89-7.91 (m, 1H, ArH); 7.58-7.62 (m, 1H, ArH); 7.33-7.38 (m, 2H, ArH); 6.94-6.97
(m, 2H, ArH); 6.90-6.91 (d, J=2.36 Hz, 2H, ArH); 6.89 (s, 1H, CH); 5.76 (s, 1H);
4.35-4.38 (m, 1H, OH); 4.20-4.24 (m, 2H, CH2); 3.81-3.82 (d, J=3.84 Hz, 4H,
-CH2CH2-); 3.20-3.24 (m, 4H, -CH2CH2-); 2.99-3.02 (m, 2H, CH2); 2.73-2.82 (m, 3H,
CH3). ESI-MS: 411.12 (C23H27N2O5, [M+H]+). Anal.Calcd for C23H26N2O5: C, 67.30;
H, 6.38; N, 6.82%; Found: C, 67.30; H, 6.37; N, 6.84%.
4.2.8.
4-(2-hydroxy-3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propoxy)-2H-chromen-2-o
ne (4h)
1
Yellow solid, yield: 55%, m.p. 176-178 ℃. H NMR (400 MHz, CDCl3) δ:
7.89-7.91 (d, J=7.84 Hz, 1H, ArH), 7.59-7.63 (t, J=7.78 Hz, 1H, ArH), 7.33-7.39 (m,
14

2H, ArH), 7.21-7.23 (d, J=6.88 Hz, 2H, ArH), 7.01-7.03 (t, J=3.4 Hz, 1H, ArH), 5.77
(s, 1H), 4.31-4.34 (m, 1H, CH), 4.21-4.22 (d, J=4.96 Hz, 2H, CH2), 3.78 (s, 1H, OH),
3.17-3.22 (t, J=2.32 Hz, 4H, -CH2CH2-); 2.99 (s, 2H, CH2), 2.73-2.78 (m, 4H,
-CH2CH2-). ESI-MS: 449.15 (C22H23Cl2N2O4, [M+H]+). Anal.Calcd for
C22H22Cl2N2O4: C, 58.81; H, 4.94; N, 6.23%; Found: C, 58.80; H, 4.95; N, 6.22%.
4.2.9.
4-(2-hydroxy-3-(4-(3,4-dichlorophenyl)piperazin-1-yl)propoxy)-2H-chromen-2-o
ne (4i)
1
Brown solid, yield: 62%, m.p. 82-85 ℃. H NMR (400 MHz, CDCl3) δ:
7.88-7.90 (d, J=7.84 Hz, 1H), 7.59-7.63 (m, 1H, ArH), 7.32-7.38 (m, 3H, ArH), 7.01
(d, J=2.72 Hz, 1H, ArH), 6.78-6.81 (m, 1H, ArH), 5.76 (s, 1H), 4.30-4.34 (m, 1H,
CH), 4.17-4.25 (m, 2H, CH2), 3.68-3.75 (m, 1H, OH), 3.25-3.29 (m, 4H, -CH2CH2-),
2.90-2.95 (m, 2H, CH2), 2.66-2.77 (m, 4H, -CH2CH2-). ESI-MS: 449.15
(C22H23Cl2N2O4, [M+H]+). Anal.Calcd for C22H22Cl2N2O4: C, 58.81; H, 4.94; N,
6.23%; Found: C, 58.83; H, 4.91; N, 6.20%.
4.2.10.
4-(2-hydroxy-3-(4-(2-chlorophenyl)piperazin-1-yl)propoxy)-2H-chromen-2-one
(4j)
Yellow oil, yield: 47%. 1H NMR (400 MHz, CDCl3) δ: 7.89-7.91（m, 1H, ArH）,
7.57-7.62 (m, 1H, ArH); 7.40-7.42 (m, 1H, ArH), 7.28-7.34 (m, 3H, ArH), 7.09-7.11
(m, 1H, ArH), 7.01-7.05 (m, 1H, ArH), 5.76 (s, 1H), 4.29-4.36 (m, 1H, CH), 4.17-4.25
(m, 2H, CH2), 3.75-3.78 (t, J=4.90 Hz, 1H, OH), 3.17 (s, 4H, -CH2CH2-), 2.96-2.98 (t,
J=4.98 Hz, 2H, CH2), 2.68-2.77 (m, 4H, -CH2CH2-). ESI-MS: 415.21 (C22H24ClN2O4,
[M+H]+). Anal.Calcd for C22H23ClN2O4: C, 63.69; H, 5.59; N, 6.75%; Found, C,
63.66; H, 5.61; N, 6.74%.
4.2.11
4-(2-hydroxy-3-(4-(3-chlorophenyl)piperazin-1-yl)propoxy)-2H-chromen-2-one
15

(4k)
1
Brown oil, yield: 51%. H NMR (400 MHz, CDCl3) δ: 7.88-7.90 (m, 1H, ArH),
7.58-7.62 (m, 1H, ArH), 7.30-7.36 (m, 2H, ArH), 7.20-7.24 (t, J=8.1 Hz, 1H, ArH),
6.92-6.93 (t, J=2.06 Hz, 1H, ArH), 6.78-6.89 (m, 2H, ArH), 5.75-5.78 (d, J=9.48 Hz,
1H), 4.29-4.35 (m, 1H, CH), 4.16-4.24 (m, 2H, CH2), 3.67-3.68 (d, J=4.2 Hz, 1H,
OH), 3.25-3.33 (m, 4H, -CH2CH2-), 2.89-2.94 (m, 2H, CH2), 2.65-2.77 (m, 4H,
-CH2CH2-). ESI-MS: 415.21 (C22H24ClN2O4, [M+H]+). Anal.Calcd for C22H23ClN2O4:
C, 63.69; H, 5.59; N, 6.75%; Found: C, 63.69; H, 5.57; N, 6.78%.
4.2.12.
4-(2-hydroxy-3-(4-(2-fluorophenyl)piperazin-1-yl)propoxy)-2H-chromen-2-one
(4l)
Yellow oil, yield: 47%. 1H NMR (400 MHz, CDCl3) δ: 7.88-7.89 (d, J=7.84 Hz,
1H, ArH), 7.57-7.61 (t, J=7.72 Hz, 1H, ArH), 7.34-7.36 (d, J=9.6 Hz; 2H, ArH),
7.08-7.13 (m, 2H, ArH), 6.98-7.01 (d, J=14.24 Hz, 2H, ArH), 5.76 (s, 1H), 4.30-4.31
(d, J=3.72 Hz, 1H, CH), 4.20-4.21 (d, J=5.52 Hz, 2H, CH2), 3.75 (s, 1H, OH),
3.19-3.25 (t, J=10.32 Hz, 4H, -CH2CH2-), 2.93-2.94 (d, J=4.84 Hz, 2H, CH2),
2.70-2.75 (t, J=9.32 Hz, 4H, -CH2CH2-). ESI-MS: 399.07 (C22H24FN2O4, [M+H]+).
Anal.Calcd for C22H23FN2O4: C, 66.32; H, 5.82; F, N, 7.03%; Found: C, 66.31; H,
5.82; N, 7.05%.
4.2.13.
4-(2-hydroxy-3-(4-(4-fluorophenyl)piperazin-1-yl)propoxy)-2H-chromen-2-one
(4m)
Yellow oil, yield: 50%. 1H NMR (400 MHz, CDCl3) δ: 7.88-7.90 (m, 1H, ArH),
7.57-7.61 (m, 1H, ArH), 7.31-7.36 (m, 2H, ArH), 7.05-7.13 (m, 2H, ArH), 6.97-7.02
(m, 2H, ArH), 5.76 (s, 1H), 4.28-4.34 (m, 1H, CH), 4.16-4.24 (m, 2H, CH2), 3.74-3.78
(m, 1H, OH), 3.15-3.24 (m, 4H, -CH2CH2-), 2.92-2.97 (m, 2H, CH2), 2.65-2.75 (m,
4H, -CH2CH2-). ESI-MS: 399.07 (C22H24FN2O4, [M+H]+). Anal.Calcd for
C22H23FN2O4: C, 66.32; H, 5.82; N, 7.03%; Found: C, 66.34; H, 5.79; N, 7.02%.
16

4.2.14.
4-(2-hydroxy-3-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)propoxy)-2H-chrom
en-2-one (4n)
1
Yellow solid, yield: 52%, m.p. 93-96 ℃. H NMR (400 MHz, CDCl3) δ:
7.84-7.86 (m, 1H, ArH), 7.54-7.58 (m, 1H, ArH), 7.29-7.38 (m, 3H, ArH), 7.07-7.12 (t,
J=10.44 HZ, 3H, ArH), 5.72 (s, 1H), 4.25-4.30 (m, 1H, CH), 4.13-4.4.21 (m, 2H,
CH2), 3.57-3.66 (m, 1H, OH), 3.25-3.34(m, 4H, -CH2CH2-), 2.87-2.92 (m, 2H, CH2),
2.61-2.70 (m, 4H, -CH2CH2-). ESI-MS: 449.09 (C23H24F3N2O4, [M+H]+). Anal.Calcd
for C23H23F3N2O4: C, 61.60; H, 5.17; N, 6.25%; Found: C, 61.58; H, 5.18; N, 6.27%.
4.2.15.4-(2-(4-(4-chlorophenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one
(5b)
1
White solid, yield: 57%, m.p. 173-175 ℃. HNMR (400 MHz, DMSO)δ:
7.88-7.90 (t, J=7.84 Hz, 1H, ArH), 7.68-7.72 (t, J=7.84 Hz, 1H, ArH), 7.40-7.46 (m,
3H, ArH), 7.31-7.33 (d, J=7.4 Hz, 1H, ArH), 7.16-7.18 (d, J=7.12 Hz, 1H, ArH),
7.09-7.11 (d, J=7.72 Hz, 1H, ArH), 5.98 (s, 1H), 5.25 (s, 2H, CH2), 3.62-3.66 (m, 4H,
-CH2CH2-), 2.99-3.66 (t, J=11.32 Hz, 4H, -CH2CH2-).ESI-MS: 399.13 (C21H20ClN2O4,
[M+H]+). Anal.Calcd for C21H19ClN2O4:C, 63.24; H, 4.80; N, 7.02%; Found: C, 63.22;
H, 4.81; N, 7.04%.
4.2.16.
4-(2-(4-(2,4-dimethylphenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one (5c)
1
White solid, yield: 66%, m.p. 185-188 ℃. HNMR (400 MHz, DMSO) δ:
7.88-7.90 (d, J=7.8 Hz, 1H, ArH), 7.67-7.69 (d, J=7.4 Hz, 1H, ArH), 7.39-7.45 (m,
2H, ArH), 6.90-7.00 (m, 3H, ArH), 5.97 (s, 1H); 5.24 (s, 2H, CH2), 3.59-3.63 (d,
J=17.88 Hz, 4H, -CH2CH2- ), 2.79-2.85 (d, J=23.56 Hz, 4H, -CH2CH2-), 2.22-2.25 (d,
J=11.92 Hz, 6H, 2CH3).ESI-MS: 393.15(C23H24N2O4, [M+H]+). Anal.Calcd for
C23H24N2O4:C, 70.39; H, 6.16; N, 7.14%; Found: C, 70.40; H, 6.18; N, 7.12%.
17

4.2.17.
(5d)
4-(2-(4-(4-nitrophenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one
1
Yellow solid, yield: 52%, m.p. 202-204 ℃. HNMR (400 MHz, DMSO)δ:
8.05-8.07 (d, J=8.36 Hz, 1H, ArH); 7.91-7.93 (d, J=6.88 Hz, 1H); 7.63-7.78 (d,
J=8.32 Hz, 1H, ArH); 7.52-7.63 (m, 1H, ArH); 7.35-7.45 (m, 1H, ArH); 7.23-7.28 (m,
1H, ArH); 6.94-7.00 (m, 2H, ArH); 6.82-6.85 (m, 1H, ArH), 5.91 (s, 1H); 3.77 (s, 2H,
ArH); 3.60-3.62 (t, J=4.84 Hz, 4H, -CH2CH2-); 3.22-3.25 (t, J=5.08 Hz, 4H,
-CH2CH2-). ESI-MS: 410.21(C21H20N3O6, [M+H]+). Anal.Calcd for C21H19N3O6:C,
61.61; H, 4.68; N, 10.26%; Found: C, 61.58; H, 4.65; N, 10.28%.
4.2.18.
4-(2-(4-(2-methoxyphenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one (5e)
Pink solid, yield: 63%, m.p. 145-147 ℃. 1HNMR (400 MHz, DMSO) δ: 7.89 (s,
1H, ArH), 7.69 (s, 1H, ArH), 7.43 (s, 2H, ArH), 6.91-6.98 (d, J=29.96 Hz, ArH), 5.96
(s, 1H), 5.23 (s, 2H, CH2), 3.81 (s, 3H, CH3), 3.62 (s, 4H, -CH2CH2-), 2.97-3.02 (d,
J=20.56 Hz, 4H, -CH2CH2-).ESI-MS: 395.17(C22H23N2O5, [M+H]+). Anal.Calcd for
C22H22N2O5:C, 66.99; H, 5.62; N, 7.10%; Found: C, 66.97; H, 5.64; N, 7.11%.
4.2.19.
4-(2-(4-(3-methoxyphenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one (5f)
1
White solid, yield: 67%, m.p. 220-222 ℃. HNMR (400 MHz, DMSO) δ:
7.87-7.89 (d, J=7.84 Hz, 1H, ArH), 7.67-7.71 (t, J=7.8 Hz, 1H, ArH), 7.39-7.45 (m,
2H, ArH), 7.12-7.16 (t, J=8.18 Hz, 1H, ArH), 6.56-6.58 (m, 1H, ArH), 6.50 (s, 1H,
ArH), 6.40-6.43 (m, 1H, ArH), 5.97 (s, 1H), 5.25 (s, 2H, CH2), 3.73 (s, 3H, CH3),
3.59-3.62 (m, 4H, -CH2CH2-), 3.16-3.23 (t, J=14.06 Hz, 4H, -CH2CH2-).ESI-MS:
395.17(C22H23N2O5, [M+H]+). Anal.Calcd for C22H22N2O5:C, 66.99; H, 5.62; N,
7.10%; Found: C, 66.70; H, 5.60, N, 7.08%.
4.2.20.
4-(2-(4-(4-methoxyphenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one (5g)
18

1
White solid, yield: 66%, m.p. 205-207 ℃. HNMR (400 MHz, CDCl3) δ:
7.93-7.95 (m, 1H, ArH), 7.60-7.64 (m, 1H, ArH), 7.35-7.39 (m, 2H, ArH), 6.98 (s, 2H,
ArH), 6.90-6.92 (d, J=8.84 Hz, 2H, ArH), 5.73 (s, 1H), 4.96 (s, 2H, CH2), 3.89 (s, 2H,
CH2), 3.83 (s, 3H, CH3), 3.72 (s, 2H, CH2), 3.16 (s, 4H, -CH2CH2-).ESI-MS:
395.17(C22H23N2O5, [M+H]+). Anal.Calcd for C22H22N2O5:C, 66.99; H, 5.62; N,
7.10%; Found: C, 66.97; H, 5.63; N, 7.11%.
4.2.21.
4-(2-(4-(2,3-dichlorophenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one (5h)
1
White solid, yield: 57%, m.p. 174-176 ℃. HNMR (400 MHz, DMSO) δ:
7.88-7.90 (m, 1H, ArH), 7.67-7.72 (m, 1H, ArH), 7.40-7.45 (m, 2H, ArH), 7.35-7.36 (t,
J=2.62 Hz, 2H, ArH), 7.16-7.19 (m, 1H, ArH), 5.98 (s, 1H), 5.25 (s, 2H, CH2),
3.63-3.66 (m, 4H, -CH2CH2-), 3.00-3.06 (t, J=13.2 Hz, 4H, -CH2CH2-).ESI-MS:
433.09(C21H19Cl2N2O4, [M+H]+). Anal.Calcd for C21H18Cl2N2O4:C, 58.21; H, 4.19; N,
6.47%; Found: C, 58.20; H, 4.17; N, 6.48%.
4.2.22. 4-(2-(4-(2-chlorophenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one
(5j)
1
White solid, yield: 59%, m.p. 172-175 ℃. HNMR (400 MHz, DMSO) δ:
7.88-7.90 (m, 1H, ArH), 7.67-7.71 (m, 1H, ArH), 7.41-7.46 (m, 3H, ArH), 7.31-7.40
(m, 1H, ArH), 7.16-7.18 (m, 1H, ArH), 7.07-7.11 (m, 1H, ArH), 5.98 (s, 1H), 5.25 (s,
2H, CH2), 3.63-3.66 (m, 4H, -CH2CH2-), 3.00-3.05 (t, J=11.12 Hz, 4H,
-CH2CH2-).ESI-MS:
399.13(C21H20ClN2O4,
[M+H]+).
Anal.Calcd
for
C21H19ClN2O4:C, 63.24; H, 4.80; N, 7.02%; Found: C, 63.26; H, 4.76; N, 7.01%.
4.2.23. 4-(2-(4-(3-chlorophenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one
(5k)
1
White solid, yield: 63%, m.p. 197-199 ℃. HNMR (400 MHz, DMSO) δ: 7.87
(d, J=1.44 Hz, 1H, ArH), 7.67-7.72(m, 1H, ArH), 7.40-7.45 (m, 2H, ArH), 7.07-7.11
(t, J=8.86 Hz, 2H, ArH), 6.99-7.02 (m, 2H, ArH), 5.97 (s, 1H), 5.24 (s, 2H, CH2),
19

3.60-3.64 (m, 4H, -CH2CH2-); 3.10-3.17 (t, J=14.36 Hz, 4H, -CH2CH2-). ESI-MS:
399.13(C21H20ClN2O4, [M+H]+). Anal.Calcd for C21H19ClN2O4:C, 63.24; H, 4.80; N,
7.02%; Found: C, 63.22, H, 4.76, N, 7.05%.
4.2.24
4-(2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-o
ne (5n)
1
White solid, yield: 67%, m.p. 159-161 ℃. HNMR (400 MHz, DMSO) δ:
7.87-7.90 (m, 1H, ArH), 7.67-7.71 (m, 1H, ArH), 7.45-7.48 (t, J=5.54 Hz, 1H, ArH),
7.39-7.43 (t, J=7.16 Hz, 2H, ArH), 7.23-7.28 (t, J=11.02 Hz, 2H, ArH), 7.11-7.13 (d,
J=7.16 Hz, 1H, ArH), 5.98 (s, 1H), 5.26 (s, 2H, CH2), 3.63-3.64 (t, J=5.24 Hz, 4H,
-CH2CH2-), 3.28-3.29 (d, J=4.6 Hz, 4H, -CH2CH2-).ESI-MS: 433.15(C22H20F3N2O4,
[M+H]+). Anal.Calcd for C22H19F3N2O4:C, 61.11; H, 4.43; N, 6.48%; Found: C, 61.12;
H, 4.40; N, 6.45%.
4.3. Antibacterial activity
The antibacterial activities of the synthetic compounds were tested against two
Gram-negative bacterial strains: E. coli ATCC 25922 and P. aeruginosa ATCC 27853,
two Gram-positive bacterial strains: B. subtilis ATCC 530 and S. aureus ATCC 25923,
using method recommended by National Committee for Clinical Laboratory
Standards (NCCLS). 28
In vitro activities of the compounds were tested in Nutrient broth (NB) for
bacteria by the two-fold serial dilution method. Seeded broth (broth containing
microbial spores) was prepared in NB from 24 h old bacterial cultures on nutrient agar
(Hi-media) at 37 1 ℃. The bacterial suspension was adjusted with sterile saline to a
concentration of 1 ×104-105 CFU/mL. The tested compounds and reference drugs
were prepared by two-fold serial dilution to obtain the required concentrations of 25,
12.5, 6.25, 3.13, 1.57, 0.79 µg/mL and even lower concentration. The tubes were
20

incubated in BOD incubators at 37 1 ℃ for bacteria. The MICs were recorded by
visual observations after 24h (for bacteria) of incubation. Penicillin G and
chloramphenicol were used as standards for bacteria. The observed MICs are
presented in Table 2.
4.4. S. aureus FabI inhibitory activity
All measurements were performed on a Uvikon 941 Plus spectrophotometer
(Kontron Instruments) in 1 mL of 20 mM Tris/HCl PH 7.4 and 150 mM NaCl. The
compounds were dissolved in MeOH with a series of concentration as 60, 20, 6.67,
2.22, 0.74, 0.25 and 0.082 µg/mL, respectively, then tested in the presence of 1µg (20
nM) enzyme and 200 µM NADH. The reaction was started by addition of 50 µM
crotonoyl-CoA. The reaction mixture was read spectrophotometrically for 1 min by
follow the oxidation of NADH to NAD+ at 340 nm. IC50 values were estimated from
graphically plotted dose-response curves.
4.5. Hemolysis test
Hemolytic activity was assayed using fresh capillary human blood. Erythrocytes
were collected by centrifuging the blood three times in chilled phosphate buffered
saline (PBS at 4 ℃) at 1000× g for 10 min. The final pellet was resuspended in PBS
to give a 2% w/v solution. Using a microtitre plate, 100 mL of the erythrocyte
solution was added to dextran, PLL, stearyl-PLL or stearyl-PLL t LDL
(1-1000/mg/mL) in a volume of 100 mL. Samples were then incubated for 3 h and the
microtitre plate was centrifuged then at 1000× g for 10 min and the supernatants (100
mL) transferred into a new microtitre plate. Hemoglobin release was determined
spectrophotometrically using a microtitre plate reader (absorbance at 550 nm). Results
were expressed as the amount of released hemoglobin induced by the compounds as a
percentage of the total.
21

4.6. Cytotoxicity test
The cytotoxic activity in vitro was measured against mammalian cells, human
macrophage using the MTT assay. The cell was grown in DMEM medium
supplemented with 10% PBS and 1× antimycotic and antibacterial solution (sigma
USA) at 37 ℃, in humidified atmosphere having 5% CO2. 100 µL of the confluent
fibroblast stock suspension was dispensed in 96-well tissue culture plate. The original
medium from the wells was replaced with 100 µL serum free DMEM when the cells
reached 90% confluence after 5 h incubation in a CO2 incubator. Various
concentrations of the compounds were added to the growing cells and incubated for
24 h. The absorbance was measured at a wavelength of 570 nm (OD570 nm) on an
ELISA microplate reader. Three replicate wells were used for each concentration and
each assay was measured three times, after which the average of IC50 was calculated.
The cytotoxicity of each compound was expressed as the concentration of compound
that reduced cell viability to 50% (IC50). The results were summarized in Table 4.
4.7. Docking simulations
The crystal structures of SaFabI (PDB code: 4CV0) was obtained from the
Protein Data Bank (http://www.rcsb.org). Molecular docking of compound 4g into the
three-dimensional X-ray structure of FabI was carried out using CDOCKER Dock
protocol of Discovery Studio 3.5.
4.8. QSAR model
Among all the 28 compounds, 80% (that is 23) were utilized as a training set for
QSAR modeling. The remaining 20% (that is 5) were chosen as an external test subset
for validating the reliability of the QSAR model by the Diverse Molecules protocol in
Discovery Studio3.5. The selected test compounds were: 4b, 4n, 5a, 5d and 5h.
The inhibitory activity of the compounds in literatures [IC50 (µM)] was initially
22

changed into the minus logarithmic scale [pIC50 (µM)] and then used for subsequent
QSAR analysis as the response variable.
In Discovery Studio, the CHARMm force field is applied and the electrostatic
potential together with the Van der Waals potential are treated as separate terms. As
the electrostatic potential probe, A + le point change is used while distance-dependent
dielectric constant is used to mimic the solvent effect. As for the Van der Waals
potential, a carbon atom with a radius of 1.73 A is used as probe.
A Partial Least-Squares (PLS) model is built using energy grids as descriptors.
QSAR models were built by using the Create 3D-QSAR Model protocol in Discovery
Studio 3.5. The models were also validated by test sets, in which the compounds are
not included in the training sets. Usually, one can believe that the modeling is reliable,
when the R2 for test sets is larger than 0.6, respectively.
Acknowledgment
This work was supported by Natural Science Foundation of Jiangsu Province
(No. BK20130554) and supported by Major Projects on Control and Rectification of
Water Body Pollution (No. 2011ZX07204-001-2214 004).
23

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26

Figure Captions
Table 1. Structures of compounds 4a-4n and 5a-5n
Table 2. Antibacterial activities of synthetic compounds.
Table 3. S. aureus FabI inhibitory activity of synthetic compounds.
Table 4. Hemolytic activities and cytotoxicity of the selected compounds.
Table 5. Experimental, predicted inhibitory activity of all synthesized compounds
against SaFabI by 3D-QSAR models based on active conformations selected from the
preliminary molecular docking study.
Figure 1A. Binding mode of compound 4g with FabI from S. aureus. For clarity, only
interacting residues were labeled. Hydrogen bonding interactions are shown in green
dotted lines. This figure was made using DS.
Figure 1B. Binding mode of compound 4g with FabI. The enzyme is shown as surface;
while 4g docked structures are shown as sticks. This figure was made using DS.
Figure 2．Linear fit curve of the observed pIC50 values versus the predicted values of
the training set part and the test set part, R2= 0.88
Figure 3．3D-QSAR of coumarinderivatives containing piperazine for SaFabI (pdb:
4CV0). (C) Iso-surface of the 3D-QSAR model coefficients on electrostatic potential
grids. The blue triangle mesh represents positive electrostatic potential and the red
area represents negative electrostatic potential. (D) Iso-surface of the 3D-QSAR
model coefficients on Van der Waals grids. The green triangle mesh representation
indicates positive coefficients; the yellow triangle mesh indicates negative
coefficients.
Scheme 1. Chemical structure of coumarin derivatives.
Scheme 2. Reagents and conditions: (a) Acetone, K2CO3, 50 ℃, 10-12 h; (b) K2CO3,
DMF, 110 ℃, 20-24 h; (c) Cs2CO3, DMF,90 ℃, 8-10 h; (d) CH2Cl2, HOBt, EDC,
50℃, 8-12 h.
27

Table 1. Structures of compounds 4a-4n and 5a-5n.
4a-4n
compound
4a
R
compound
4h
R
4b
4c
4i
4j
4d
4e
4k
4l
4f
4m
4n
4g
28

5a-5n
compound
5a
R
compound
5h
R
5b
5c
5i
5j
5d
5e
5k
5l
5f
5m
5n
5g
29