Synthesis and biological evaluation of coumarin derivatives containing imidazole skeleton as potential antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2017.11.100

Emergence of multidrug-resistant bacteria causes an urgent need for new generation of antibiotics, which may have a different mechanism of inhibition or killing action from the existing. Here, we report on the design, synthesis, and biological evaluation of thirty-nine coumarin derivatives in order to solve the antibacterial resistance by targeting at the inhibition of biosynthesis pathway of fatty acids. Their antibacterial activities against Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and Flavobacterium cloumnare are tested and action mechanism against the key enzyme in bacterial fatty acid synthesis pathway are studied. The results show that compounds 13 and 18 have potent and broad spectrum antimicrobial activity. In addition, 9, 14 and 19 show eminent antimicrobial efficacy toward S. aureus, S. agalactiae, and F. cloumnare. Mechanistically, coumarin derivatives display the antibacterial activity via the control of FabI and FabK function. The structure-activity relationship analysis indicate that the length of linker and imidazole substitute group could significantly influence the antimicrobial activity, as well as the inhibitory activity against FabI and FabK. The structural optimization analysis of coumarin suggest that derivatives 9, 13, 14, 18 and 19 could be a viable way of preventing and controlling bacteria and considered as promising lead compounds for the development of commercial drugs.

Full text of DOI:10.1016/j.ejmech.2017.11.100

Accepted Manuscript
Synthesis and biological evaluation of coumarin derivatives containing imidazole
skeleton as potential antibacterial agents
Yang Hu, Yufeng Shen, Xiao Tu, Xiaohu Wu, Gao-Xue Wang
PII:
S0223-5234(17)31003-6
10.1016/j.ejmech.2017.11.100
EJMECH 9975
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 June 2017
Revised Date: 16 November 2017
Accepted Date: 29 November 2017
Please cite this article as: Y. Hu, Y. Shen, X. Tu, X. Wu, G.-X. Wang, Synthesis and biological
evaluation of coumarin derivatives containing imidazole skeleton as potential antibacterial agents,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.11.100.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1
Synthesis and biological evaluation of coumarin derivatives
containing imidazole skeleton as potential antibacterial agents
Yang Hua, Yufeng Shena, Xiao Tua, Xiaohu Wua, Gao-Xue Wang a,*
2
3
4
5
aCollege of Animal Science and Technology, Northwest A&F University, Xinong Road 22nd, Yangling,
Shaanxi, 712100, China;
6
*Author to whom correspondence should be addressed; E-mail address: wanggaoxue@126.com；
Tel./Fax: +86-029-8709-2102.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21

ACCEPTED MANUSCRIPT
22
Abstract
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Emergence of multidrug-resistant bacteria causes an urgent need for new generation of antibiotics,
which may have a different mechanism of inhibition or killing action from the existing. Here, we report
on the design, synthesis, and biological evaluation of thirty-nine coumarin derivatives in order to solve
the antibacterial resistance by targeting at the inhibition of biosynthesis pathway of fatty acids. Their
antibacterial activities against Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and
Flavobacterium cloumnare are tested and action mechanism against the key enzyme in bacterial fatty
acid synthesis pathway are studied. The results show that compounds 13 and 18 have potent and broad
spectrum antimicrobial activity. In addition, 9, 14 and 19 show eminent antimicrobial efficacy toward S.
aureus, S. agalactiae, and F. cloumnare. Mechanistically, coumarin derivatives display the antibacterial
activity via the control of FabI and FabK function. The structure-activity relationship analysis indicate
that the length of linker and imidazole substitute group could significantly influence the antimicrobial
activity, as well as the inhibitory activity against FabI and FabK. The structural optimization analysis of
coumarin suggest that derivatives 9, 13, 14, 18 and 19 could be a viable way of preventing and
controlling bacteria and considered as promising lead compounds for the development of commercial
drugs.
Keywords: coumarin derivatives, antibacterial activity, FabI, FabK
40
41
42

ACCEPTED MANUSCRIPT
43
1. Introduction
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
Antibiotic resistance has been a global healthcare problem due to the indiscriminate use of
antibiotics and the subsequent creation of bacteria that can survive traditional treatment [1,2]. Namely,
antibiotics have dramatically reduced the incidence of many diseases infected by bacteria. In addition,
antibiotic resistance has drastically outpaced new antibiotic discovery. For instance,
methicillin-resistant Staphylococcus aureus is one of the main species of bacteria that cause
nosocomial infections in hospitals worldwide [3-5]. It was only two years after the introduction of the
methicillin in 1959 that resistant strains of the gram-positive human pathogen Staphylococcus aureus
emerged. Additionally, many problems remain unresolved due to occasional serious side effects and the
appearance of antibiotic-resistant mutant bacteria. Therefore, the search for new drugs effective for the
treatment of bacterial infections is of high priority. Focusing on known validated intracellular targets
remains a valid approach to identify new drug candidates with novel chemical structures [6].
Interestingly, use of natural products as a screening pool for novel antibiotics has several advantages
related to safety, availability, and minimizing the risk of side-effect and addiction [7,8].
Natural products have been used to treat disease for thousands of years and play an increasingly
important role in drug discovery and development. In fact, the majority of anti-infectious agents are of
natural origin [9,10]. Natural product coumarin, the simplest member of the group of oxygen
heterocyclics called benzo-2-pyrones, a secondary metabolite has been reported in many families of
plants such as Apiaceae, Asteraceae, Fabiaceae, Rosaceae, Rubiaceae, Rutaceae and Solanaceae [15].
Natural products with a coumarinic moiety have been reported to have multiple biological activities
including anticancer, antioxidant, antiinflammatory and antiviral [10-17]. In addition, some
investigators have focused on the antimicrobial activity of coumarins [18-20]. For example, Wang et al.

ACCEPTED MANUSCRIPT
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
has found that coumarin derivatives showed the antibacterial activity by inhibiting the key enzyme of
biosynthesis pathway of fatty acids (FAS) [21].
One of the strategies adopted to control bacterial pathogens is to target the FAS-II, in which the
highly conserved enoyl-acyl carrier protein reductase plays a key role [22]. Most eukaryotes contain
type I FAS, where all the catalytic domains reside on one polypeptide. Prokaryotes and plants contain
the type II or the dissociated FAS-II system, where each enzyme is found on a separate polypeptide.
Enzyme catalyzes the last step in the bacterial FAS-II cycle, the reduction of α, β-unsaturated fatty
acids esterified to the acyl carrier protein (ACP). The FAS-II pathway consists of some distinct and
highly conserved enzymes: β-hydroxacyl-ACP-dehydratase (FabA, FabZ), β-ketoacyl-ACP-reductase
(FabG), and enoyl-ACP-reductase (FabI, FabK, FabL) [23,24]. For most bacterias, FabI and FabK are
responsible for the concluding reduction step of each elongation cycle. They represent a key
physiological regulator of fatty acid biosynthesis, and they have been carried out to be the important
drug targets for the development of novel antibacterials [25,26]. This has led to the pursuit of specific
FabI and FabK inhibitors as novel antibacterial agents which would not be expected to interfere with
comparable human biochemical processes. However, the information about the relationships between
the chemical structures and activity of inhibiting FAS-II enzymes is currently limited.
The exploration of new heterocycles that can accommodate potency to multiple biological targets
remains an intriguing scientific endeavour. In recent years, many hybrid molecules with coumarin
based ring systems have been synthesized utilizing novel synthetic methodologies. Some coumarin
derivatives conjugated with nitrogen-containing heterocyclic moieties, such as triazole and pyridine,
were synthesized and proved to possess antibacterial bioactivity [27,28]. Thus, hybridizing the
coumarin nucleus with other moieties has afforded new molecules with improved antimicrobial activity

ACCEPTED MANUSCRIPT
87
88
89
90
91
92
93
94
95
96
97
98
99
100
profiles.
In this study, our work are directed toward the coumarin antibiotics and their structural
modification. According to the previous studies, we hypothesized 1). Coumarin derivatives showed the
antibacterial activity by targeting the FAS-II. 2). The chemical structures of coumarin derivatives
influenced the antimicrobial activity. The previous researches promoted us to synthesize new
derivatives of coumarins with imidazoles skeleton for the sake of finding new effectively antibacterial
agents. Based on these things mentioned above, we synthesized several series of novel coumarins
derivatives owning nitrogen-containing heterocyclic with different length of chemical bridges. We
studied their antibacterial activities against four strains of bacteria of Escherichia coli (E. coli),
Staphylococcus aureus (S. aureus), Streptococcus agalactiae (S. agalactiae), and Flavobacterium
cloumnare (F. cloumnare). Futhermore, we focused on the expression and purification of the
recombinant FabI and FabK protein to investigate the mechanism of the inhibitory activity of
coumarins derivatives. This study also helped rationalize activities at the investigated drugs targets,
pointing to potential strategies for designing more selective compounds.
101
102
2. Results and discussion
2.1 Chemistry
103
104
105
106
107
The synthesis of initial compounds 7-hydroxy coumarin and 4-methyl-7-hydroxycoumarin were
shown in Scheme 1. The 7-hydroxycoumarin was obtained by heating the solution of
+
2,4-dihydroxybenzaldehyde and ClCH2COOC2H5 together with PH3 -CH2COOC2H5 in ethanol. The
4-methyl-7-hydroxycoumarin was obtained by heating the solution of resorcinol and ethyl acetoacetate
together in sulfuric acid.

ACCEPTED MANUSCRIPT
Scheme 1
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
The syntheses of the thirty-nine derivatives are based on well described reactions in previous
study [29]. Synthesis of coumarin derivatives 1-33 followed the general pathway depicted in Scheme
2-4. Compounds 1-5 were synthesized from 7-hydroxy coumarin by reacting with corresponding α,
ω-dibromoalkanes and triethylamine in anhydrous acetone at reflux condition. Compounds 6-33 were
synthesized in 60-90% yield by treatment of 1-5 compounds with corresponding amines and anhydrous
potassium carbonate in acetonitrile at room temperature. In particularly, the derivative 29 was
synthesized in 95% yield by treatment of compound 4 using Gabriel reaction.
Scheme 2
Scheme 3
Scheme 4
Scheme 5 reported the synthetic pathway used to prepare compounds 34-39. To assess the effects
of the methyl at the para-position of oxygen atom, we changed the initial compound 7-hydroxy
coumarin to 4-methyl-7-hydroxy coumarin for synthesizing derivatives 34-39. The structures of a total
thirty-nine synthesized compounds were confirmed by ESI-MS, 1H and 13C NMR (see the
Supplementary data).
Scheme 5
125
126
Antibacterial activity
2.2 MICs
127
128
The synthesized compounds were tested for their antibacterial activities against two
Gram-negative bacteria strains: E. coli and F. cloumnare and two Gram-positive bacteria strains: S.

ACCEPTED MANUSCRIPT
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
aureus and S. agalactiae. The MICs (minimum inhibitory concentrations) of the compounds against
these bacteria were reported in Table 1 and 2, the activities of reference drugs enrofloxacin and
norfloxacin were also included. We sought to characterize the structure activity relationship (SAR) for
this class of compounds by introducing substituents on the aromatic ring of hydroxy and investigating
those compounds with different length of “C-linker”. We also considered the role of the imidazole
analogues in the other item of the linker. Finally, we studied a limited set of variants by exploring
substituents of methyl at the C-4 position.
In line with results reported for 14 were against F. cloumnare, S. agalactiae and S. aureus with 2,
4, and 16 µM MIC values, respectively (Table 1). Compound 14 was equal to the positive control
enrofloxacin with MIC values of 2 and 8 µM against F. cloumnare, S. agalactiae, respectively (Table 1).
Consistently and in line with the results obtained with the imidazole and 4-methylimidazole derivatives,
compounds 9 and 19, displayed the potent activities against F. cloumnare, S. agalactiae and S. aureus
with MIC values of 4, 8, 16 µM and 4, 4, 8 µM, respectively. Remarkably, derivative 28 which
introduced a benzimidazole group at the position of the end of linker which had the length of eight
atom, exhibited a dramatic loss in activities against F. cloumnare, S. agalactiae and S. aureus with MIC
values of 8, 256, 32 µM. Additionally, introducing other substituent group at the position of the item of
linker, as with bromine-bearing (1, 2, 3, 4, and 5) or 2-phenylimidazole bearing (22, 23, and 24)
derivatives, did not show activities at these three strains of bacteria.
Table 1
In light with these results, we proposed a rationale for the chemical features required for a
balanced and potent activities at F. cloumnare, S. agalactiae and S. aureus. It was found that the length
of the R1 substituent group (“linker”) played an important role in the antibacterial activities of

ACCEPTED MANUSCRIPT
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
compounds. We therefore turned our attention to derivatives bearing this moiety. Reducing or
increasing the length of the linker of 14 to six (13) or ten (15) methylene units were detrimental for
activities at the three targets. As anticipated, the antibacterial activities of compounds 13 and 15 against
F. cloumnare, S. agalactiae and S. aureus decreased quite substantially (13: 16, 128, 64 µM; 15: 8, 8,
64 µM;). This pattern was also observed in other two coumarin-imidazole hybrid derivatives. Among
compounds 6-10 and 16-20 the order of antibacterial activities showed the potency of 9 > 10 > 8 > 7 >
6 and 19 > 20 > 18 > 17 > 16 which indicated the activities of compounds against these three bacteria
with eight carbon atoms of linker were superior to that of with other length of linker.
Notably, activity at E. coli of compounds 1-28 behaved different rule from the other three strains.
According to the date, compounds with six carbon atoms of linker showed the significantly higher
activities against E. coli than other length of linker. For instance, in 13, the efficacy against E. coli was
8 µM MIC value. By contrast, the results for 11, 12, 14, and 15 suggested that extending or narrowing
the carbon chains would lead to a drop in activity against E. coli. Derivative 8 maintained the potent
activities with MIC values of 32 µM. In agreement with the behavior of 8, 13, and 18, introducing a six
carbon atoms of linker would be the best choice for activity against E. coli. On the other hand, when
the R2 position coupled with a 2-phenylimidazole or benzimidazole, the suitable length of linker would
be four methylene units. It was found that compounds 22 and 26 were 32, 8 µM MIC values
respectively with the highest activities among their own groups.
The effect of imidazole group on potency was apparent throughout our results, with its presence at
the R2 position increasing the potency in every example. According to the results, we suggested that the
types of substituent group of R2 could be crucial to the efficacy against bacteria. We synthesized five
compounds with the same linker but different types of heterocyclic nitrogen groups in R2 substituted

ACCEPTED MANUSCRIPT
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
position. Predictably, the analogues displayed huge difference of antibacterial activities. Among
compounds 9, 14, 19, 24, and 28, the order of antibacterial activities showed the potency of 14 > 19 >
9 > 28> 24 which indicated the activities of compounds with 2-methylimidazole substitutes were
superior to that of with the other four substitutes.
To assess the effects of the types of R2 substituents, we synthesized and tested compounds 29-33
which had same length of linker at R1 position but different substituent groups at R2 position against F.
cloumnare and S. agalactiae. As shown in Table 2, only derivative 30 coupled with a triazole
maintained the activities against F. cloumnare and S. agalactiae among these five compounds, with the
MIC values of 64 and 32 µM, respectively. The other four derivatives displayed no activities against
two bacteria at the concentration of 256 µM.
In addition, we further investigated the effect of methyl at the C-4 position. In light with the
results of compounds 34-39 against four strains of bacteria, we suggested that introducing a methyl
group in the distal ring of the O-biphenyl moiety led to a slight drop in activities. Namely, compound
35 showed anti-F. cloumnare, S. agalactiae and S. aureus activities with the MIC values of 4, 8, 64 µM
while 9 which lacked a methyl but had the same length of linker was 4, 8, 16 µM MIC values,
respectively. Similarly, derivatives 36 and 37 decreased the activities against F. cloumnare, S.
agalactiae and S. aureus to 4, 16, 64 µM and 8, 8, 32 µM, respectively. Interestingly, compound 38
increased the activity at S. agalactiae to 32 µM compared to 28 (256 µM). Derivative 39 was 16 µM
against F. cloumnare while its analogue 30 exhibited the activity with MIC value of 64 µM.
Consistently, all six derivatives didn’t display the activity against E. coli at the concentration of 256
µM.
Table 2

ACCEPTED MANUSCRIPT
195
2.3 MBCs
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
We further investigated the MBC values of all compounds against four bacterials. The results
confirmed that the four overarching trends what we had noted previously were also suitable for the date
of MBC values. First, the eight methylene chains was the best choice for the coumarin derivatives
against the three strains of F. cloumnare, S. agalactiae and S. aureus. Extending or narrowing the
length of alkyl chain between the corresponding amine and coumarin led to a significant reduction in
activities at the three bacterials. For example, the compounds 8, 9, and 10, with the same R2 substituent
group but different in the length of linker, exhibited a great difference in antibacterial activity.
Compound 9 which had a six carbon atoms of linker on the position of R1 showed activity against F.
cloumnare with the MBC value of 8 µM. Otherwise, the MBC values were increased to 16 or 128 µM,
if the length was ten or six carbon atoms.
Secondly, we suggested that compounds with six carbon atoms of linker would increase the anti-E.
coli activity notably. Derivatives 8 and 13 were the same MBC values of 64 µM. Most importantly,
when the same substitution was coupled with a longer or shorter linker, activity at E. coli was
intensively affected. As shown in Table 3, compounds 7 and 12 displayed the activities with the MBC
values of 256 µM. For another, the same trend noted previously was observed from MBC values of
21-28. Derivatives 22 and 26 which were coupled with the four methylene units exhibited the highest
activities with MBC values of 128 and 64 µM, respectively. In addition, there are several investigators
demonstrated that the bioactivities were influenced by the carbon linker, such as antibacterial, anti-HIV
and anti-parasites [30-32]. Indeed, the length of carbon chain was crecial to the antibacterial activities.
Table 3
Subsequently, the substituents at R2 position also occupied an important place in the efficacy of

ACCEPTED MANUSCRIPT
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
antibacterial. The coumarin derivatives which coupled with imidazole, 2-methylimidazole,
4-methylimidazole or triazole revealed superiority in against bacteria to the compounds coupled with
2-phenylimidazole, benzimidazole, and so on. In 30, coupled with a triazole group, activities at F.
cloumnare and S. agalactiae were 128 and 64 µM MBC values respectively while other four
derivatives (29, 31, 32, 33) did not show efficacy against the two strians of bacteria.
Moreover, imidazole analogues had reported other antibacterial activities which was consistent to
our result that it could increase the activity against bacteria [33,34]. Furthermore, its derivatives are of
great importance in medicinal chemistry and can be used for the synthesis of myriad heterocyclic
compounds with different biological activities such as anti-tumor, antithrombotic, antioxidative [35-38].
These studies indicated that synthesis of coumarin-imidazole hybrid derivatives is a right guidance for
further researches. The presence of 2-methyl was the structural feature exerting the more evident effect,
as suggested by activity data on 14.
Lastly, it was also confirmed that introducing a methyl group in the distal ring of the O-biphenyl
moiety led to a slight influence in activities by the results of 34-39. Similarly, compound 35 showed
anti-F. cloumnare, S. agalactiae and S. aureus activities with the MBC values of 16, 16, 256 µM while
9 which had the same carbon chain was 8, 16, 128 µM MBC values, respectively. Consistently,
compounds 36 and 37 decreased the activities against F. cloumnare, S. agalactiae and S. aureus to 8, 16,
256 µM and 16, 32, 256 µM, respectively. Conversely, compound 38 increased the activity at S.
agalactiae to 64 µM compared to 28 (512 µM). Derivative 39 was 64 µM against F. cloumnare while
its analogue 30 exhibited the activity with MIC value of 128 µM.
Table 4
2.4 Expression and characterization of recombinant protein

ACCEPTED MANUSCRIPT
239
240
241
242
243
244
245
246
247
248
249
250
The amplification of the FabI gene (expected size 760 bp) of E. coli isolate ATCC 35150 and the
FabK gene (expected size 960 bp) of S. agalactiae isolate WB1445 were shown in Fig. 1A and Fig. 2A,
respectively. The plasmids pET32a-FabI and pET32a-FabK were confirmed by restriction enzyme
digestion (Fig. 1B and Fig. 2B) and sequence analysis (data not show). Both results confirmed that
recombinant plasmids were successfully constructed. Expression analysis of abundant FabI protein and
FabK protein were performed after 4 h of induction with 1 mM IPTG. And SDS-PAGE analysis
showed that the molecular size of recombinant protein containing 6-histidine tag were estimated to be
approximately 43 kDa and 48 kDa, respectively. (Fig. 1C, Fig. 2C). Western blot analysis using
anti-His-tag monoclonal antibody confirmed the authenticity of the FabI and FabK as clear bands at the
expected position on polyvinylidene fluoride membrane (Fig. 1D, Fig. 2D).
Figure 1
Figure 2
251
2.5 Identification of (E)-2-octenoyl-acyl-N-acetylcysteamine (t-O-NAC)
252
253
254
255
By analyzing the data of 1H NMR (nuclear magnetic resonance) (500.23 MHz, MeOD), 13C NMR
(125.78 MHz, MeOD), t-O-NAC was identified and its chemical structure was shown in Scheme 6. In
addition, the date of general nuclear magnetic resonance spectrum was shown in supplementary date.
Scheme 6
256
2.6 E. coli FabI and S. agalactiae FabK inhibitory activity
257
258
259
In order to investigate the structural requirements involving the pyridoindole core, we have chose
10 derivatives with a substituent of diverse functionalities to rationalize the FabI and FabK inhibitory
activities of these compounds in terms of structural modifications. The IC50 values of these ten

ACCEPTED MANUSCRIPT
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
compounds which showed superior antibacterial activities to inhibit the recombinant FabI and FabK
enzyme were shown in Table 5. All derivatives showed the activities of inhibitory FabI and FabK to
varying degrees. In 13, activity at FabI was 1.20 µM IC50 value which was the highest inhibitory
efficacy. Similarly, in line with result reported for 14, exhibited the best inhibitory FabK activity with
IC50 value of 1.13 µM. Conversely, compounds 10 and 12, displayed the lowest activities inhibitory
FabI and FabK, respectively. In light with these results, we suggested a rationale for the chemical
features required for potent inhibitory FabI and FabK activities. When the coumarin derivatives
coupled with a six methylene units, it would show the balanced perferable inhibitory efficacy of the
two enzymes. Interestingly, extending the length of alkyl chain led a change in the balance of inhibitory
two proteins. When the length of linker was more than six carbon atoms, derivatives showed the better
inhibitory FabK activity than FabI. In comparison, narrowing the length of chain made a converse
result. Namely, when the length of alkyl chain was less than six methylene units, activity of derivatives
would be led a enhancement at FabI but a drop at FabK.
Table 5
The results of FabI and FabK inhibitory activity of the test compounds were corresponding to the
structure-activity relationships of their antibacterial activities. It demonstrated that the potent
antibacterial activities of the synthetic compounds were probably correlated to their FabI and FabK
inhibitory activities.
278
3. Conclusions
279
280
281
The coumarin derivatives were structurally designed by employing a molecular hybridization of
the coumarin group with the heterocyclic ring imidazole. Thirty-nine coumarin derivatives were
synthesized and their antibacterial activity was evaluated against E. coli, F. cloumnare, S. aureus and S.

ACCEPTED MANUSCRIPT
282
283
284
285
286
287
288
289
290
291
292
293
294
295
agalactiae. Moreover, the inhibitory activity was evaluated against FabI and FabK. The most
promising compounds showed both antibacterial and inhibitory activity and, in particular, the
compounds 9, 13, 14, 18 and 19, also exhibited remarkable activity against F. cloumnare, S. aureus and
S. agalactiae. The ensuing structure-activity relationships study predictably identified antimicrobial
activity represented by the R1 and R2 substituent group. Eight carbon atoms length of linker (R1
substitute position) and imidazole could significantly increase the antimicrobial activity. In addition,
six carbon atoms length of linker could be the suitable choice for the inhibitory activity against FabI
and FabK. Furthermore, derivative 14, the most potent compound, showed broad range activity in all in
vitro experiments and might represent a good starting point for optimizing the structure to develop
more active antibacterials.
In conclusion, the results obtained in this work highlight imidazole substituted coumarin as very
promising antibacterial scaffolds acting through enoyl-acyl carrier protein reductases inhibition. This
study suggests that the development of more potent enoyl-acyl carrier protein reductases inhibitors
could lead to novel broad-spectrum antibacterial drug candidates.
296
297
4. Experimental section
4.1 Bacterial strain and growth conditions
298
299
300
301
302
Bacterial reference strains of E. coli ATCC 35150 and S. aureus ATCC 29213 were kindly
provided by Prof. Jin-you Duan in college of Chemistry & Pharmacy, Northwest A&F University,
Yangling, Shaanxi, China. Bacterial reference strain of S. agalactiae WB1445 was kindly provided by
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou,
Guangdong, China. Bacterial reference strain of F. cloumnare FC-G1 was preserved in our laboratory

ACCEPTED MANUSCRIPT
303
304
305
with 20% glycerol at -80 °C. For experimental work, E. coli and S. aureus were cultured in Broth
medium (BM) at 28 °C and grown overnight with constant shaking. Brain heart infusion (BHI) broth
and Shieh broth were chosed for culturing S. agalactiae and F. cloumnare, respectively.
306
4.2 Minimal inhibitory concentration (MIC)
307
308
309
310
311
312
313
314
315
316
317
318
319
The minimal inhibitory concentration (MIC) of the synthetic compounds were tested against two
Gram-negative bacterial strains: E. coli and F. cloumnare, two Gram-positive bacterial strains: S.
aureus and S. agalactiae, using method recommended by Clinical and Laboratory Standards Institute
(CLSI) guidelines (CLSI, 2009, 2012). 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 corresponding broth at 28 ±
1 °C. The bacterial suspension was adjusted with sterile saline to a concentration of 1×107-2×107
cfu/mL and diluted with corresponding broth to a concentration of 1×105-2×105 cfu/mL. The tested
compounds and reference drugs were prepared by two-fold serial dilution to obtain the required
concentrations of 256.00, 128.00, 64.00, 32.00, 16.00, 8.00, 4.00, 2.00, 1.00, 0.50 µM and even lower
concentration. The tubes were incubated in BOD incubators at 28 ± 1 °C for bacteria. The MICs were
recorded by visual observations after 24 h (for bacteria) of incubation. Enrofloxacin and norfloxacin
were used as standards for bacteria.
320
4.3 Minimal bactericidal concentration (MBC)
321
322
323
The method used and described below is an amended version of the procedure described in the
BSAC Guide to Sensitivity Testing and can be adapted for determining the minimal bactericidal
concentration (MBC) of coumarin derivatives by substituting IsoSensitest broth (ISA;Oxoid) with

ACCEPTED MANUSCRIPT
324
325
326
327
328
corresponding broth. After MIC determination of the compounds tested, an aliquot of 100 µl from all
tubes in which no visible bacterial growth was observed were seeded in Nutrient Agar (NA) plates not
supplemented with coumarin derivatives. The plates were then incubated for overnight at 28 ± 1 °C.
The MBC endpoint is defined as the lowest concentration of antimicrobial agent that kills >99.9% of
the initial bacterial population where no visible growth of the bacteria was observed on the NA plates.
329
4.4 Expression and purification of the recombinant FabI and FabK protein
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
Primers were designed according to the FabI sequence of E. coli ATCC 35150 (Genebank
accession: YP_006119687). The FabI gene amplification was carried out using genomic DNA of E. coli
as
template
and
two
oligonucleotide
primers
(Forward:
5’-
AGGAGATATACCATGGGTTTTCTTTCCGGTAAGCGCATTCTG
-3’;
Reverse:
5’-CGCCGAGCTCGAATTCTTATTTCAGTTCGAGTTCGTTCATTGC -3’) which introduced BamH
I and Hind III restriction enzyme sites, respectively. The PCR products were ligated with the cloning
vector pMD19T (Takara, Dalian, China) to generate the pMD19T-FabI, which was confirmed by
sequence analysis (Sangon, Shanghai, China).
Similarly, primers were designed according to the FabK sequence of S. agalactiae WB1445
(Genbank accession: NC_004116.1). The FabK gene amplification was carried out using genomic
DNA of S. agalactiae as template and two oligonucleotide primers (Forward: 5’-
ATCGCCATGGTTGATCGGTACTCACTCTTCTTC
-3’;
Reverse:
5’-
ATCGGAATTCTTATAAATCTGACCAGCGGC-3’) which introduced BamH I and Hind III restriction
enzyme sites, respectively. The PCR products were ligated with the cloning vector pMD19T (Takara,
Dalian, China) to generate the pMD19T-FabK, which was confirmed by sequence analysis (Sangon,
Shanghai, China).

ACCEPTED MANUSCRIPT
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
The FabI (FabK) gene excised from pMD19T-FabI (pMD19T-FabK) by digestion with BamH
I/Hind III restriction enzyme (Takara, Dalian, China) was inserted into expression plasmid pET32a (+)
(Novagen, Madison, USA) to generate pET32a-FabI (pET32a-FabK). The resulting recombinant
plasmid was transformed into E. coli BL21 (DE3) (Novagen, Madison, USA), and the recombinant
constructs pET32a-FabI (pET32a-FabK) were sequenced by Sangon Biological Company (Shanghai,
China). The positive recombinant E. coli was grown in Luria bertani (LB) broth with ampicillin (100
mg/mL) at 37 °C with shaking, and the expression of recombinant FabI (Fabk) protein was induced by
1 mM isopropyl-b-D-thiogalacto-pyranoside (IPTG; SigmaeAldrich Trading Co., Ltd, Shanghai,
China). The obtained fusion proteins were determined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and visualized after staining with Coommassie brilliant blue R-250
(Sigma, USA). Then the recombinant proteins were assessed by western blotting analysis using diluted
anti-His-tag monoclonal antibody (1:2000, Abcam, Cambridge, MA, USA) and HRP-conjugated goat
anti-mouse IgG (1:2000, Beijing CoWin Biotech Corp., Beijing, China) as antibodies. The result was
visualized by staining with DAB (3,3-diamonobenzidine tetrahydrochloride) horseradish peroxidase
color development kit (Qiagen, Hilden, Germany).
Owning to the hexa-histidine tag at the N-terminus, the recombinant proteins from the constructs
were purified using nickelnitrilotriacetic acid (Ni-NTA) column (Qiagen, Shanghai, China) under
native conditions as recommended by the manufacturer. After affinity chromatography, the fractions
were pooled and dialysed using urea gradient dialysis method. The protein concentration was
determined by Micro BCA Protein Assay Kit (Beijing CoWin Biotech Corp., Beijing, China).
366
367
4.5 The procedure for the synthesis of (E)-2-octenoyl-acyl-N-acetylcysteamine (t-O-NAC)
To a solution of (E)-2-octenoic acid (569 mg, 4 mmol) in dichloromethane (20 mL) was added N,

ACCEPTED MANUSCRIPT
368
369
370
371
372
373
374
375
N-diisopropylcarbodiimide (528 mg, 4mmol) and 4-dimethylaminopyridine (200 mg, 2mmol) at room
temperature. After stirring at room temperature for 10 min, N-Acetylcysteamine (300 mg, 3 mmol) was
added to the reaction mixture, and whole was stirred under room temperature for 24 h. The precipitate
was filtered off and washed with dichloromethane (3×40 mL). The solvent was evaporated under
reduced pressure, and the residue was treated with water (30 mL) and extracted with ethyl acetate
(3×40 mL). The organic layer were combined, dried with anhydrous Na2SO4, and concentrated under
reduced pressure. The crude product was purified via silica gel column chromatography with mixed
petroleum ether and ethyl acetate (3:1, v/v) as eluent.
376
4.6 E. coli FabI and S. agalactiae FabK inhibitory activity
377
378
379
380
381
382
383
384
385
386
387
388
389
The measurements of E. coli FabI inhibitory activity were performed on an Infinit eM200 pro
plate reader (Tecan, Switzerland) in 100 µL of 0.1 M PBS-DTT PH 7.5. The compounds were
dissolved in 0.1 M PBS-DTT with a series of concentration as 128, 64, 32, 16, 8, 4, 2 and 1 µM,
respectively, then tested in the presence of 20 µL (100 µg/mL) FabI and 10 µL (4 mM) t-O-NAC. The
reaction was started by addition of 20 µL (2 mM) NADH. 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.
The measurements of S. agalactiae FabK inhibitory activity were performed on an Infinit eM200
pro plate reader (Tecan, Switzerland) in 100 µL of 0.1 M ADA-DTT PH 6.5. The compounds were
dissolved in 0.1 M ADA-DTT with a series of concentration as 128, 64, 32, 16, 8, 4, 2 and 1 µM,
respectively, then tested in the presence of 20 µL (150 µg/mL) FabK and 10 µL (4 mM) t-O-NAC. The
reaction was started by addition of 20 µL (2 mM) NADH. The reaction mixture was read
spectrophotometrically for 1 min by follow the oxidation of NADH to NAD+ at 340 nm. IC50 values

ACCEPTED MANUSCRIPT
390
391
392
393
394
395
were estimated from graphically plotted dose–response curves.
Notes
The authors declare no competing financial interest.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (No. 31372559).
References
396
397
[1] H. Yoneyama, R. Katsumata, Antibiotic resistance in bacteria and its future for novel antibiotic
development, Bioscience Biotechnology & Biochemistry, 70 (2006) 1060.
398
399
400
[2] J.S. Molton, P.A. Tambyah, B.S. Ang, M.L. Ling, D.A. Fisher, Apropos "The global spread of
healthcare-associated multidrug-resistant bacteria: a perspective from Asia", Clinical Infectious
Diseases, 56 (2013) 1310-1318.
401
402
403
[3] S. Ichiyama, [Genomic DNA fingerprinting by pulsed-field gel electrophoresis for study of
nosocomial infection caused by methicillin-resistant Staphylococcus aureus], Nippon Rinsho
Japanese Journal of Clinical Medicine, 50 (1992) 992-997.
404
405
[4] K. Yasunaka, K. Kono, Epidemiological study of methicillin-resistant Staphylococcus aureus at
Fukuoka University Hospital, Microbial Drug Resistance, 5 (1999) 207-213.
406
407
408
[5] S. Takeda, K. Yasunaka, K. Kono, K. Arakawa, Methicillin-resistant Staphylococcus aureus (MRSA)
isolated at Fukuoka University Hospital and hospitals and clinics in the Fukuoka city area,
International Journal of Antimicrobial Agents, 14 (2000) 39-43.

ACCEPTED MANUSCRIPT
[6] D.E. Goldberg, R.F. Siliciano, J.W. Jr, Outwitting evolution: fighting drug-resistant TB, malaria,
and HIV, Cell, 148 (2012) 1271.
409
410
411
412
413
[7] D.H. Hahm, M.J. Yeom, E.H. Lee, I. Shim, H.J. Lee, H.Y. Kim, Effect of Scutellariae radix as a
novel antibacterial herb on the ppk(Polyphosphate kinase) mutant of Salmonella typhimurium,
Biochemical Society Transactions, 28 (2001) 1061-1065.
414
415
[8] S.-H. Lee, C.-J. Kim, Antibacterial activity of antimycotic miconazole against methicillin-resistant
Staphylococcus aureus, Journal of microbiology and biotechnology, 9 (1999) 572-575.
416
417
418
[9] I. Weinmann, History of the development and applications of coumarin and coumarin-related
compounds, Coumarins: biology, applications and mode of action. John Wiley & Sons, Inc., New
York, NY, (1997).
419
420
421
[10] W. Li, Y.N. Sun, X.T. Yan, S.Y. Yang, E.J. Kim, H.K. Kang, Y.H. Kim, Coumarins and Lignans
from Zanthoxylum schinifolium and Their Anticancer Activities, Journal of Agricultural & Food
Chemistry, 61 (2013) 10730-10740.
422
423
424
[11] S. Maiti, N. Park, J.H. Han, H.M. Jeon, J.H. Lee, S. Bhuniya, C. Kang, J.S. Kim,
Gemcitabine-coumarin-biotin conjugates: a target specific theranostic anticancer prodrug, Journal
of the American Chemical Society, 135 (2013) 4567-4572.
425
426
[12] G.L. Xi, Z.Q. Liu, Coumarin-fused coumarin: antioxidant story from n,n-dimethylamino and
hydroxyl groups, Journal of Agricultural & Food Chemistry, 63 (2015) 3516-3523.
427
428
[13] G. Mazzone, A. Galano, J.R. Alvarezidaboy, N. Russo, Coumarin-Chalcone Hybrids as Peroxyl
Radical Scavengers: Kinetics and Mechanisms, Journal of Chemical Information & Modeling, 56

ACCEPTED MANUSCRIPT
429
(2016) 662.
430
431
[14] C.A. Kontogiorgis, D.J. Hadjipavlou-Litina, Synthesis and antiinflammatory activity of coumarin
derivatives, Journal of Medicinal Chemistry, 48 (2005) 6400-6408.
432
433
434
[15] T. Symeonidis, K.C. Fylaktakidou, D.J. Hadjipavloulitina, K.E. Litinas, Synthesis and
anti-inflammatory evaluation of novel angularly or linearly fused coumarins, European Journal of
Medicinal Chemistry, 44 (2009) 5012.
435
436
437
[16] J. Neyts, C.E. De, R. Singha, Y.H. Chang, A.R. Das, S.K. Chakraborty, S.C. Hong, S.C. Tsay, M.H.
Hsu, J.R. Hwu, Structure-activity relationship of new anti-hepatitis
C virus agents:
heterobicycle-coumarin conjugates, Journal of Medicinal Chemistry, 52 (2009) 1486-1490.
438
439
[17] M.Z. Hassan, H. Osman, M.A. Ali, M.J. Ahsan, Therapeutic potential of coumarins as antiviral
agents, European Journal of Medicinal Chemistry, 123 (2016) 236-255.
440
441
442
443
444
[18] K.B. Puttaraju, K. Shivashankar, Chandra, M. Mahendra, V.P. Rasal, P.N. Venkata Vivek, K. Rai,
M.B. Chanu, Microwave assisted synthesis of dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-ones;
synthesis, in vitro antimicrobial and anticancer activities of novel coumarin substituted
dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-ones, European Journal of Medicinal Chemistry, 69
(2013) 316-322.
445
446
447
[19] M. Basanagouda, K. Shivashankar, M.V. Kulkarni, V.P. Rasal, H. Patel, S.S. Mutha, A.A. Mohite,
Synthesis and antimicrobial studies on novel sulfonamides containing 4-azidomethyl coumarin,
European Journal of Medicinal Chemistry, 45 (2010) 1151-1157.
448
[20] L. Bonsignore, A.D. Logu, G. Loy, S.M. Lavagna, D. Secci, Synthesis and antimicrobial activity

ACCEPTED MANUSCRIPT
of coumarin and benzodioxazepine-, diazazepine- and benzoxazepine-substituted penicillins,
Cheminform, 25 (2010) no-no.
449
450
451
452
[21] Y.M. Zhang, Y.J. Lu, C.O. Rock, The reductase steps of the type II fatty acid synthase as
antimicrobial targets, Lipids, 39 (2004) 1055.
453
454
455
456
[22] C.Y. Lai, B.G. Trewyn, D.M. Jeftinija, K. Jeftinija, S. Xu, S. Jeftinija, V.S. Lin, A mesoporous
silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for
stimuli-responsive controlled release of neurotransmitters and drug molecules, Journal of the
American Chemical Society, 125 (2003) 4451.
457
458
459
[23] L. Shi, R.Q. Fang, Z.W. Zhu, Y. Yang, K. Cheng, W.Q. Zhong, H.L. Zhu, Design and synthesis of
potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III (FabH) as potential antibacterial
agents, European Journal of Medicinal Chemistry, 45 (2010) 4358-4364.
460
461
[24] A. Surolia, T.N. Ramya, V. Ramya, N. Surolia, 'FAS't inhibition of malaria, Biochemical Journal,
383 (2004) 401-412.
462
463
464
[25] R.F. Waller, S.A. Ralph, M.B. Reed, V. Su, J.D. Douglas, D.E. Minnikin, A.F. Cowman, G.S.
Besra, G.I. Mcfadden, A type II pathway for fatty acid biosynthesis presents drug targets in
Plasmodium falciparum, Antimicrobial Agents & Chemotherapy, 47 (2003) 297-301.
465
466
467
[26] S. F. Wang, Y. Yin, X. Wu, F. Qiao, S. Sha, P. C. Lv, J. Zhao, H. L. Zhu, Synthesis, molecular
docking and biological evaluation of coumarin derivatives containing piperazine skeleton as
potential antibacterial agents, Bioorganic & medicinal chemistry, 22 (2014) 5727-5737.
468
[27] T.G. Kraljević, A. Harej, M. Sedić, S.K. Pavelić, V. Stepanić, D. Drenjančević, J. Talapko, S.

ACCEPTED MANUSCRIPT
Raić-Malić, Synthesis, in vitro anticancer and antibacterial activities and in silico studies of new
4-substituted 1, 2, 3-triazole–coumarin hybrids, European journal of medicinal chemistry, 124
(2016) 794-808.
469
470
471
472
473
474
[28] R.M. Mohareb, E. Ezz EL-ARAB, K.A. El-Sharkawy, The reaction of cyanoacetic acid hydrazide
with 2-acetylfuran: Synthesis of coumarin, pyridine, thiophene and thiazole derivatives with
potential antimicrobial activities, Scientia Pharmaceutica, 77 (2009) 355-366.
475
476
[29] Y. Shi, C.-H. Zhou, Synthesis and evaluation of a class of new coumarin triazole derivatives as
potential antimicrobial agents, Bioorganic & medicinal chemistry letters, 21 (2011) 956-960.
477
478
479
[30] D. Tang, Y. Gai, A. Polemeropoulos, Z. Chen, Z. Wang, Y.S. Or, Design, synthesis, and
antibacterial activities of novel 3, 6-bicyclolide oximes: Length optimization and zero carbon
linker oximes, Bioorganic & medicinal chemistry letters, 18 (2008) 5078-5082.
480
481
482
[31] W.M. Golebiewski, R.F. Keyes, M. Cushman, Exploration of the effects of linker chain
modifications on anti-HIV activities in a series of cosalane analogues, Bioorganic & medicinal
chemistry, 4 (1996) 1637-1648.
483
484
485
[32] G.-L. Liu, Y. Hu, X.-H. Chen, G.-X. Wang, F. Ling, Synthesis and anthelmintic activity of
coumarin–imidazole hybrid derivatives against Dactylogyrus intermedius in goldfish, Bioorganic
& Medicinal Chemistry Letters, 26 (2016) 5039-5043.
486
487
488
[33] S. Sharma, S. Gangal, A. Rauf, Convenient one-pot synthesis of novel 2-substituted
benzimidazoles, tetrahydrobenzimidazoles and imidazoles and evaluation of their in vitro
antibacterial and antifungal activities, European Journal of Medicinal Chemistry, 44 (2009) 1751.

ACCEPTED MANUSCRIPT
489
490
491
[34] V. Padmavathi, K.C. Prema, B.C. Venkatesh, A. Padmaja, Synthesis and antimicrobial activity of
amido linked pyrrolyl and pyrazolyl-oxazoles, thiazoles and imidazoles, European Journal of
Medicinal Chemistry, 43 (2012) 5317-5326.
492
493
494
[35] L.X. Liu, X.Q. Wang, J.M. Yan, Y. Li, C.J. Sun, W. Chen, B. Zhou, H.B. Zhang, X.D. Yang,
Synthesis and antitumor activities of novel dibenzo[b,d]furan-imidazole hybrid compounds,
European Journal of Medicinal Chemistry, 66 (2013) 423.
495
496
497
498
[36] F. Bellina, S.S. Cauteruccio, R. Rossi, Novel Imidazole-Based Combretastatin A-4 Analogues:
Evaluation of Their in vitro Antitumor Activity and Molecular Modeling Study of Their Binding
to the Colchicine Site of Tubulin, Bioorganic & Medicinal Chemistry Letters, 16 (2006)
5757-5762.
499
500
501
502
[37] J.C. Barrow, P.G. Nantermet, S.R. Stauffer, P.L. Ngo, M.A. Steinbeiser, S.S. Mao, S.S. Carroll, C.
Bailey, D. Colussi, M. Bosserman, Synthesis and evaluation of imidazole acetic acid inhibitors of
activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics, Journal of
Medicinal Chemistry, 46 (2003) 5294.
503
504
505
[38] M. Alaiz, R. Zamora, F.J. Hidalgo, Antioxidative Activity of Pyrrole, Imidazole, Dihydropyridine,
and Pyridinium Salt Derivatives Produced in Oxidized Lipid/Amino Acid Browning Reactions,
Journal of Agricultural & Food Chemistry, 44 (1996) 686-691.
506
507
508

ACCEPTED MANUSCRIPT
509
Figure captions
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
Figure 1. Analysis of FabI expression. (A) PCR amplification of FabI: lane M, DNA marker; lane 1,
FabI. (B) Analysis of recombinant plasmid: lane M, DNA marker; lane 1, double enzymes digested
pET32a-FabI with BamH I and Hind III.(C) SDS-PAGE analysis of expressed pET32a-FabI: lane M,
Standard protein marker; lane 1, Soluble lysates from uninduced cells containing recombinant
pET32a-FabI; lane 2, Insoluble lysates from uninduced cells containing recombinant pET32a-FabI;
lane 3, Soluble lysates from induced cells containing recombinant pET32a-FabI; lane 4, Insoluble
lysates from induced cells containing recombinant pET32a-FabI. (D) Western blotting analysis of
purified FabI using anti-his-tag monoclonal antibody.
Figure 2. Analysis of FabK expression. (A) PCR amplification of FabK: lane M, DNA marker; lane 1,
FabK. (B) Analysis of recombinant plasmid: lane M, DNA marker; lane 1, double enzymes digested
pET32a-FabK with BamH I and Hind III.(C) SDS-PAGE analysis of expressed pET32a-FabI: lane M,
Standard protein marker; lane 1, Empty pET32a control (uninduced by IPTG); lane 2, Insoluble lysates
from induced cells containing recombinant pET32a-FabK; lane 3, Soluble lysates from induced cells
containing recombinant pET32a-FabK; lane 4, Insoluble lysates from uninduced cells containing
recombinant pET32a-FabK; lane 5, Soluble lysates from uninduced cells containing recombinant
pET32a-FabK. (D) Western blotting analysis of purified FabK using anti-his-tag monoclonal antibody.
Scheme 1. Synthetic route of initial compounds 7-hydroxycoumarin and 4-methyl-7-hydroxycoumarin.
+
Reagents and conditions: (a) ClCH2COOC2H5, Ph3 -CH2COOC2H5, CH3CH2OH, 80 °C, 2 h; (b) Ethyl
acetoacetate, H2SO4, ice bath, 16 h.

ACCEPTED MANUSCRIPT
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
Scheme 2. Synthetic route of coumarin derivatives 1-28. Reagents and conditions: (c) alkyl dibromide,
K2CO3, dry acetone, reflux, 20-24 h; (d) imidazole / 2-methylimidazole / 4-methylimidazole /
2-phenylimidazole / benzimidazole, K2CO3, CH3CN, r.t., 20-24 h.
Scheme 3. Synthetic route of coumarin derivatives 29. Reagents and conditions: (e) Phthalimide
potassium salt, DMF, 100 °C, 20-24 h; (f) NH2NH2·H2O, CH3CH2OH, 80 °C, 6-8 h.
Scheme 4. Synthetic route of coumarin derivatives 30-33. Reagents and conditions: (c)
1,6-dibromohexane, K2CO3, dry acetone, reflux, 20-24 h; (d) triazole / piperazine / parazole /
piperidine, K2CO3, CH3CN, r.t., 20-24 h.
Scheme 5. Synthetic route of coumarin derivatives 34-39. Reagents and conditions: (c)
1,6-dibromohexane, K2CO3, dry acetone, reflux, 20-24 h; (d) imidazole / 2-methylimidazole /
4-methylimidazole / benzimidazole / triazole, K2CO3, CH3CN, r.t., 20-24 h.
Scheme 6. Synthetic route of (E)-2-octenoyl-acyl-N-acetylcysteamine. Reagents and conditions: (g)
N-Acetylcysteamine, DMAP, DIC, CH2Cl2, r.t., 24 h.

ACCEPTED MANUSCRIPT
Table 1. MIC values of coumarin derivatives 1-28.
Substituent group
MIC (µM)
Compound
Gram-negative
F.cloumnare E. coli
Gram-positive
R1
R2
S.agalactiae
S. aureus
>256.00
>256.00
>256.00
>256.00
>256.00
1
2
3
4
5
-
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
-C2H4-
-C4H8-
-C6H12-
-C8H16-
-Br
6
7
-
>256.00
>256.00
64.00
128.00
128.00
32.00
>256.00
>256.00
>256.00
8.00
>256.00
64.00
64.00
16.00
64.00
-C2H4-
-C4H8-
-C6H12-
-C8H16-
8
9
4.00
>256.00
>256.00
10
16.00
8.00
11
12
13
14
15
-
>256.00
>256.00
16.00
2.00
64.00
64.00
>256.00
>256.00
128.00
4.00
>256.00
256.00
64.00
-C2H4-
-C4H8-
-C6H12-
-C8H16-
8.00
>256.00
>256.00
16.00
8.00
8.00
64.00
16
17
18
19
20
-
>256.00
>256.00
32.00
16.00
16.00
>256.00
>256.00
128.00
4.00
>256.00
>256.00
32.00
-C2H4-
-C4H8-
-C6H12-
-C8H16-
16.00
4.00
>256.00
>256.00
8.00
16.00
8.00
32.00
21
22
23
24
-
>256.00
>256.00
>256.00
>256.00
128.00
32.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
-C2H4-
-C4H8-
-C6H12-
N
N
>256.00
>256.00
25
26
27
28
-
>256.00
>256.00
32.00
128.00
8.00
>256.00
>256.00
32.00
64.00
32.00
32.00
32.00
N
N
-C2H4-
-C4H8-
-C6H12-
>256.00
>256.00
8.00
256.00
Enrofloxacin
Norfloxacin
2.00
1.00
1.00
8.00
2.00
32.00
64.00
32.00
“-” Means none carbon atom.

ACCEPTED MANUSCRIPT
Table 2. MIC values of coumarin derivatives 29-39 .
Substituent group
R2
MIC (µM)
Compound
Gram-negative
Gram-positive
R1
R3
F.cloumnare
E. coli
S.agalactiae S. aureus
29
30
>256.00
>256.00
64.00
32.00
31
32
33
>256.00
>256.00
>256.00
>256.00
>256.00
>256.00
-C6H12-
H
34
35
36
-C6H12-
-Br
CH3
>256.00
4.00
>256.00
>256.00
>256.00
>256.00
8.00
>256.00
64.00
4.00
16.00
64.00
37
38
39
8.00
16.00
32.00
>256.00
>256.00
64.00
8.00
16.00
32.00
32.00
32.00
32.00
-C6H12-
CH3
N
N
Enrofloxacin
Norfloxacin
2.00
1.00
1.00
8.00
2.00
32.00
64.00
32.00