Coumarin and 3,4-dihydroquinolinone derivatives: Synthesis, antidepressant activity, and molecular docking studies

Article abstract of DOI:10.1016/j.pharep.2019.07.011

Background: Coumarin and 3,4-dihydroquinolinone nuclei are two heterocyclic rings that are important and widely exploited for the development of bioactive molecules. Here, we designed and synthesized a series of 3,4-dihydroquinolinone and coumarin derivatives (Compounds 8, 9, 11, 14, 15, 18-20, 23, 24 and 28 are new compounds) and studied their antidepressant activities. Methods: Forced swimming test (FST) and tail suspension test (TST) were used to evaluate the antidepressant activity of the target compounds. The most active compound was used to evaluate the exploratory activity of the animals by the open-field test. 5-HT concentration was estimated to evaluate if the compound has an effect on the mouse brain, by using ELISA. A 5-HT_1A binding assay was also performed. The biological activities of the compounds were verified by molecular docking studies. The physicochemical and pharmacokinetic properties of the target compounds were predicted by Discovery Studio and ChemBioDraw Ultra. Results: Of all the compounds tested, compound 7 showed the best antidepressant activity, which decreased the immobility time by 65.52 s in FST. However, in the open-field test, compound 7 did not affect spontaneous activity. The results of 5-HT concentration estimation in vivo showed that compound 7 may have an effect on the mouse brain. Molecular docking results indicated that compound 7 showed significant interactions with residues at the 5-HT_1A receptor using homology modeling. The results show that compound 7 exhibits good affinity for the 5-HT_1A receptor. Conclusion: Coumarin and 3,4-dihydroquinolinone derivatives synthesized in this study have a significant antidepressant activity. These findings can be useful in the design and synthesis of novel antidepressants.

Full text of DOI:10.1016/j.pharep.2019.07.011

Accepted Manuscript
Title: Coumarin and 3,4-dihydroquinolinone derivatives:
synthesis, antidepressant activity, and molecular docking
studies
Authors: Shi-Ben Wang, Hui Liu, Guang-Yong Li, Jun Li,
Xiao-Jing Li, Kang Lei, Li-Chao Wei, Zhe-Shan Quan,
Xue-Kun Wang, Ren-Min Liu
PII:
S1734-1140(19)30303-2
DOI:
Reference:
https://doi.org/10.1016/j.pharep.2019.07.011
PHAREP 1089
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Received date:
Revised date:
Accepted date:
25 April 2019
8 July 2019
30 July 2019
Please cite this article as: Wang S-Ben, Liu H, Li G-Yong, Li J, Li X-Jing, Lei K, Wei L-
Chao, Quan Z-Shan, Wang X-Kun, Liu R-Min, Coumarin and 3,4-dihydroquinolinone
derivatives: synthesis, antidepressant activity, and molecular docking studies,
Pharmacological Reports (2019), https://doi.org/10.1016/j.pharep.2019.07.011
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Coumarin
and 3,4-dihydroquinolinone derivatives: synthesis,
antidepressant activity, and molecular docking studies
Running head title
Antidepressant activity of coumarin and 3,4-dihydroquinolinone derivatives
Shi-Ben Wang*1, Hui Liu2, Guang-Yong Li1, Jun Li1, Xiao-Jing Li1, Kang Lei1, Li-Chao Wei1,
Zhe-Shan Quan3, Xue-Kun Wang*1, Ren-Min Liu*1
1 College of pharmacy, Liaocheng University, Liaocheng, Shandong 252059, China
2 College of life sciences, Liaocheng University, Liaocheng, Shandong 252059, China
3 College of pharmacy, Yanbian University, Yanji, Jilin 133002, China
*Corresponding author: Shi-ben Wang, College of Pharmacy of Liaocheng University, 1 Hunan Road,
Liaocheng 252059, Shandong, China. Email: wangshiben110@163.com.
Graphical abstract
Coumarin and 3,4-dihydroquinolinone derivatives: synthesis, antidepressant activity, and
molecular docking studies.

Highlights


Derivatives of coumarin and 3,4-dihydroquinolinone were designed and synthesized.
Compound 7 showed the best antidepressant activity.

Compound 7 exhibits good affinity for the 5-HT1A receptor.
Abstract
Background: Coumarin and 3,4-dihydroquinolinone nuclei are two heterocyclic rings that are
important and widely exploited for the development of bioactive molecules. Here, we designed and
synthesized a series of 3,4-dihydroquinolinone and coumarin derivatives (Compounds 8, 9, 11, 14, 15,
18-20, 23, 24 and 28 are new compounds) and studied their antidepressant activities.
Methods: Forced swimming test (FST) and tail suspension test (TST) were used to evaluate the
antidepressant activity of the target compounds. The most active compound was used to evaluate the
exploratory activity of the animals by the open-field test. 5-HT concentration was estimated to evaluate
if the compound has an effect on the mouse brain, by using ELISA. A 5-HT1A binding assay was also
performed. The biological activities of the compounds were verified by molecular docking studies. The
physicochemical and pharmacokinetic properties of the target compounds were predicted by Discovery
Studio and ChemBioDraw Ultra.
Results: Of all the compounds tested, compound 7 showed the best antidepressant activity, which
decreased the immobility time by 65.52s in FST. However, in the open-field test, compound 7 did not
affect spontaneous activity. The results of 5-HT concentration estimation in vivo showed that
compound 7 may have an effect on the mouse brain. Molecular docking results indicated that
compound 7 showed significant interactions with residues at the 5-HT1A receptor using homology
modeling. The results show that compound 7 exhibits good affinity for the 5-HT1A receptor.
Conclusion: Coumarin and 3,4-dihydroquinolinone derivatives synthesized in this study have a
significant antidepressant activity. These findings can be useful in the design and synthesis of novel
antidepressants.
Keywords: coumarin; 3,4-dihydroquinolinone; molecular docking studies; antidepressant; 5-HT

Introduction
Depression is one of the most commonly encountered neurological disorders [1], and is caused by
many psychological, social, environmental, and genetic factors [2]. Current antidepressant drugs can
only alleviate some symptoms of depression, and the occurrence of side effects is common. Therefore,
the development of more effective and less toxic antidepressants has attracted significant attention in
recent years. Coumarin (2H-chromen-2-one) (Fig. 1), one of the most useful heterocyclic nuclei, has
gained prominence in medicinal chemistry owing to its diverse biological activities [3,4]. Several
studies have demonstrated the antidepressant potential of coumarin derivatives [5,6].
<Fig. 1 Insert here>
The 3,4-dihydroquinolinone nucleus is one of the most important and widely exploited
heterocyclic ring for the development of bioactive molecules (Fig. 1). The literature contains many
examples of 3,4-dihydroquinolinone derivatives that exhibit a range of biological properties, including
analgesic [7], antibacterial [8], antimalarial [9], anticancer [10,11], anti-inflammatory [12],
anticonvulsant [13,14], and antidepressant [15,16] activities.
In the field of depression research, molecules with carboxylic acid amide scaffolds display a broad
scope of pharmacological and biological activities and have been widely used as frameworks in drug
design. For example, many commercial amide antidepressants, such as Benzamide, Toloxatone,
Isocarboxazid, and Agomelatin (Fig. 2) showed high activity and were successfully used to control a
variety of depression symptoms.
<Fig. 2. Insert here>
Given these findings, we designed a formulation containing the combination of coumarin and
3,4-dihydroquinolinone framework with carboxamide units (Fig. 3) and examined their antidepressant
activities in a mouse model. Forced swimming test (FST) was used to evaluate the antidepressant
activity of the target compounds. Moreover, the most active compound was also tested using the tail
suspension test (TST). From the three series compounds, three compounds 7, 16, and 24 with the best
antidepressant activity in FST test were selected for binding assays. Binding affinity values (Ki) of the
compounds at 5-HT1A receptor were determined. The concentration of 5-HT in mouse brain was
determined to explore the possible mechanism of action for compound 7, which showed the highest
activity. A homology model of 5-HT1A receptor was constructed using Discovery Studio software, and

molecular docking study was performed by this model. We predicted the physicochemical and
pharmacokinetic properties of these compounds, and the newly synthesized target compounds were
1
characterized by infrared spectroscopy, high resolution mass spectrometry, and H-nuclear/13C-nuclear
magnetic resonance techniques.
<Fig. 3. Insert here>
Material and methods
Chemistry
An X-4 binocular microscope melting point apparatus was used to determine melting points (Mp).
High resolution mass spectra (HRMS) were measured by mass spectrometer (Bruker Daltonik,
Germany). All NMR (1H and 13C NMR) results were measured by AV-300 spectrometer (solvent:
CDCl3 or DMSO-d6; internal standard: tetramethylsilane). Most chemicals used for the synthesis were
commercial products without further purification.
Synthesis of compound 2
Appropriate amine (10.0 mmol), triethylamine (12.0 mmol), and 30 ml CH2Cl2 were placed in a
100 ml round-bottomed flask, and the resulting mixture was stirred for 0.5 h at room temperature. A
solution of ClCH2COCl (12.0 mmol) in 5 ml of CH2Cl2 was added drop wise to the mixture in an
ice-bath. After the reaction was completed, the solvent was removing under reduced pressure to obtain
a white solid.
Synthesis of target compounds 4-11, 13-20, and 22-28
7-Hydroxy-2H-chromen-2-one/4-hydroxy-2H-chromen-2-one/7-hydroxy-3,4-dihydroquinolin-2(1
H)-one (10.0 mmol), K2CO3 (10.0 mmol), and 50 ml acetone were combined in a 100 ml
o
round-bottomed flask, and the resulting mixture was stirred for 6-12 h at 80 C. After the reaction was
completed (TLC), the mixture was poured into water (50 ml), and then extracted with CH2Cl2. The
combined organic extracts were washed with water, dried (MgSO4), and concentrated to dryness in
vacuum. The residue was purified by chromatography on silica gel, eluting with CH2Cl2, to extract
target compounds 4-11, 13-20, and 22-28. Half of the compounds thus obtained were novel compounds
(Compounds 4, 5 and 6 are reported by reference [17]; Compounds 7, 13 and 16 are reported by
reference [18]; Compounds 10 and 17 are reported by reference [19]; Compounds 22, 25 and 27 are
reported by reference [20]). The spectral data of the synthesized known compounds were consistent
with the previously described papers [17-20]. The yield, melting point, and nuclear magnetic data of
the new target compounds are shown below.

2-[(2-Oxo-2H-chromen-7-yl)oxy]-N-phenylacetamide (4)
The spectral data of this compound can be found in reference [17].
N-benzyl-2-[(2-oxo-2H-chromen-7-yl)oxy]acetamide (5)
The spectral data of this compound can be found in reference [17].
2-[(2-Oxo-2H-chromen-7-yl)oxy]-N-phenethylacetamide (6)
The spectral data of this compound can be found in reference [17].
N-(4-chlorophenyl)-2-[(2-oxo-2H-chromen-7-yl)oxy]acetamide (7)
The spectral data of this compound can be found in reference [18].
2-[(2-Oxo-2H-chromen-7-yl)oxy]-N-[4-(trifluoromethyl)phenyl]acetamide (8)
1
An white solid. Yield: 47 %. Mp: 237-238 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 4.91 (s,
2H, CH2), 6.32 (d, 1H, J = 8.00 Hz, COCH=), 7.06 (s, 1H, =CH), 7.06-8.02 (m, 7H, Ar-H), 10.54 (s,
1H, NH). 13C-NMR (DMSO-d6, 100 MHz) δ: 67.69, 102.13, 113.18, 113.39, 120.00, 120.00, 123.43,
124.06, 126.13, 126.56, 126.60, 130.02, 142.39, 144.69, 155.64, 160.65, 161.33, 167.01. ESI-HRMS
+
calcd for C18H13F3NO4 ([M + H]+): 364.0791; found: 364.0798.
2-[(2-Oxo-2H-chromen-7-yl)oxy]-N-(p-tolyl)acetamide (9)
1
An white solid. Yield: 51 %. Mp: 231-232 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 2.25 (s,
3H, CH3), 4.83 (s, 2H, CH2), 6.32 (d, 1H, J = 8.00 Hz, COCH=), 7.04 (s, 1H, =CH), 7.04-8.02 (m, 7H,
Ar-H), 10.08 (s, 1H, NH). 13C-NMR (DMSO-d6, 100 MHz) δ: 20.93, 67.78, 102.09, 113.21, 113.30,
113.34, 120.16, 120.16, 129.62, 129.62, 130.00, 133.21, 135.24, 144.72, 155.64, 160.67, 161.41,
+
166.03. ESI-HRMS calcd for C18H16NO4 ([M + H]+): 310.1074; found: 310.1082.
N-(4-methoxyphenyl)-2-[(2-oxo-2H-chromen-7-yl)oxy]acetamide (10)
The spectral data of this compound can be found in reference [19].
N-cyclohexyl-2-[(2-oxo-2H-chromen-7-yl)oxy]acetamide (11)
An white solid. Yield: 53 %. Mp: 239-240 oC. 1H-NMR (DMSO-d6, 400 MHz, ppm) δ: 1.10-3.61
(m, 11H, cyclohexane-H), 4.59 (s, 2H, CH2), 6.31 (d, 1H, J = 8.00 Hz, COCH=), 6.95 (s, 1H, =CH),
6.95-8.02 (m, 3H, Ar-H), 8.01 (s, 1H, NH). 13C-NMR (DMSO-d6, 100 MHz) δ: 25.08, 25.08, 25.61,
32.71, 32.71, 47.98, 67.66, 102.06, 113.23, 113.27, 129.93, 144.73, 155.62, 160.68, 161.42, 166.25.
+
ESI-HRMS calcd for C17H20NO4 ([M + H]+): 302.1387; found: 302.1396.
3-[(2-Oxo-2H-chromen-4-yl)oxy]-N-phenylacetamide (13)
The spectral data of this compound can be found in reference [18].
N-benzyl-2-[(2-oxo-2H-chromen-4-yl)oxy]acetamide (14)
1
An white solid. Yield: 60 %. Mp: 250-251 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 4.39 (d,
2H, J = 8.00 Hz, CH2), 4.88 (s, 2H, CH2), 5.87 (s, 1H, COCH=), 7.25-8.07 (m, 9H, Ar-H), 8.79 (t, 1H,
J = 6.00 Hz, NH). 13C-NMR (DMSO-d6, 100 MHz) δ: 42.44, 68.16, 91.91, 115.47, 116.84, 124.07,

124.60, 127.34, 127.67, 128.77, 127.67, 128.77, 133.38, 139.56, 153.21, 161.89, 164.85, 166.35.
+
ESI-HRMS calcd for C18H16NO4 ([M + H]+): 310.1074; found: 310.1081.
2-[(2-Oxo-2H-chromen-4-yl)oxy]-N-phenethylacetamide (15)
1
An white solid. Yield: 48 %. Mp: 267-268 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 2.79 (t,
2H, J = 8.00 Hz, CH2), 3.41 (q, 2H, J = 6.80 Hz, CH2), 4.77 (s, 2H, CH2), 5.83 (s, 1H, COCH=),
7.21-8.00 (m, 9H, Ar-H), 8.31 (t, 1H, J = 6.00 Hz, NH). 13C-NMR (DMSO-d6, 100 MHz) δ: 35.49,
68.14, 91.82, 115.43, 116.84, 123.99, 124.58, 126.63, 128.82, 128.82, 129.12, 129.12, 133.37, 139.68,
+
153.20, 161.89, 164.76, 166.13. ESI-HRMS calcd for C19H18NO4 ([M + H]+): 324.1230; found:
324.1238.
N-(4-chlorophenyl)-2-[(2-oxo-2H-chromen-4-yl)oxy]acetamide (16)
The spectral data of this compound can be found in reference [18].
N-(4-fluorophenyl)-2-[(2-oxo-2H-chromen-4-yl)oxy]acetamide (17)
The spectral data of this compound can be found in reference [19].
2-[(2-Oxo-2H-chromen-4-yl)oxy]-N-[4-(trifluoromethyl)phenyl]acetamide (18)
1
An white solid. Yield: 63 %. Mp: 249-251 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 5.09 (s,
2H, CH2), 5.93 (s, 1H, COCH=), 7.40-7.97 (m, 8H, Ar-H), 10.65 (s, 1H, NH). 13C-NMR (DMSO-d6,
100 MHz) δ: 68.03, 91.80, 115.48, 116.69, 116.94, 119.30, 119.99, 119.99, 123.71, 124.46, 124.76,
+
126.68, 132.51, 133.44, 142.31, 153.25, 161.93, 165.03, 165.68. ESI-HRMS calcd for C18H13F3NO4
([M + H]+): 364.0791; found: 364.0798.
2-[(2-Oxo-2H-chromen-4-yl)oxy]-N-(p-tolyl)acetamide (19)
1
An white solid. Yield: 62 %. Mp: 234-236 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 2.25 (s,
3H, CH3), 5.01 (s, 2H, CH2), 5.89 (s, 1H, COCH=), 7.14-7.97 (m, 8H, Ar-H), 10.19 (s, 1H, NH).
13C-NMR (DMSO-d6, 100 MHz) δ: 20.93, 68.18, 91.70, 115.52, 116.92, 120.17, 120.17, 123.77,
124.73, 129.69, 129.69, 133.35, 133.42, 136.15, 153.24, 161.95, 164.72, 165.11. ESI-HRMS calcd for
+
C18H16NO4 ([M + H]+): 310.1074; found: 310.1077.
N-cyclohexyl-2-[(2-oxo-2H-chromen-4-yl)oxy]acetamide (20)
1
An grey solid. Yield: 67 %. Mp: 242-243 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 1.24-1.77
(m, 11H, cyclohexane-H), 4.77 (s, 2H, CH2), 5.78 (s, 1H, COCH=), 7.39-7.94 (m, 4H, Ar-H), 8.10 (d,
1H, J = 8.00 Hz, NH). 13C-NMR (DMSO-d6, 100 MHz) δ: 25.71, 25.71, 26.30, 33.35, 33.35, 48.82,
68.85, 92.29, 116.22, 117.58, 124.51, 125.37, 134.07, 153.91, 162.61, 165.77, 165.77. ESI-HRMS
+
calcd for C17H20NO4 ([M + H]+): 302.1387; found: 302.1393.
2-[(2-Oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy]-N-phenylacetamide (22)
The spectral data of this compound can be found in reference [20].
N-benzyl-2-[(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy]acetamide (23)

1
An white solid. Yield: 58 %. Mp: 234-235 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 2.47 (t,
2H, J = 8.00 Hz, COCH2), 2.85 (t, 2H, J = 8.00 Hz, CH2), 4.39 (d, 2H, J = 4.00 Hz, CH2), 4.53 (s, 2H,
CH2), 6.57-7.37 (m, 8H, Ar-H), 8.68 (t, 1H, J = 6.40 Hz, NH), 10.13 (s, 1H, NH). 13C-NMR (DMSO-d6,
100 MHz) δ: 24.50, 31.18, 42.24, 67.52, 102.72, 108.01, 116.87, 127.21, 127.67, 127.67, 128.67,
+
128.67, 128.83, 139.71, 139.78, 157.42, 168.14, 170.78. ESI-HRMS calcd for C18H19N2O3 ([M + H]+):
311.1390; found: 311.1398.
N-(4-chlorophenyl)-2-[(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy]acetamide (24)
o
1
An white solid. Yield: 71%. Mp: 246-248 C. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 2.41 (t,
2H, J = 6.00 Hz, COCH2), 2.79 (t, 2H, J = 6.00 Hz, CH2), 4.63 (s, 2H, CH2), 6.52-7.69 (m, 7H, Ar-H),
10.07 (s, 1H, NH), 10.23 (s, 1H, NH). 13C-NMR (DMSO-d6, 100 MHz) δ: 24.49, 31.14, 67.68, 102.57,
108.05, 116.88, 121.74, 121.74, 127.73, 128.89, 129.10, 129.10, 137.85, 139.74, 157.52, 167.18,
+
170.75. ESI-HRMS calcd for C17H16ClN2O3 ([M + H]+): 331.0844; found: 331.0850.
N-(4-fluorophenyl)-2-[(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy]acetamide (25)
The spectral data of this compound can be found in reference [20].
2-[(2-Oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy]-N-(p-tolyl)acetamide (26)
1
An white solid. Yield: 55 %. Mp: 253-254 oC. H-NMR (DMSO-d6, 400 MHz, ppm) δ: 2.25 (s,
3H, CH3), 2.41 (t, 2H, J = 6.00 Hz, COCH2), 2.79 (t, 2H, J = 6.00 Hz, CH2), 4.60 (s, 2H, CH2),
6.52-7.53 (m, 7H, Ar-H), 9.99 (s, 1H, NH), 10.08 (s, 1H, NH). 13C-NMR (DMSO-d6, 100 MHz) δ:
20.92, 24.49, 31.15, 67.70, 102.59, 108.05, 116.82, 120.20, 120.20, 128.87, 129.54, 129.54, 133.09,
136.34, 139.72, 157.58, 166.71, 170.78. ESI-HRMS calcd for C18H19N2O3+ ([M + H]+): 311.1390;
found: 311.1396
N-(4-methoxyphenyl)-2-[(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy]acetamide (27)
The spectral data of this compound can be found in reference [20].
N-cyclohexyl-2-[(2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy]acetamide (28)
An white solid. Yield: 73 %. Mp: 242-243 oC. 1H-NMR (DMSO-d6, 400 MHz, ppm) δ: 1.09-3.60
(m, 11H, cyclohexane-H), 2.78 (t, 2H, J = 8.00 Hz, CH2), 4.35 (s, 2H, CH2), 6.46-7.06 (m, 3H, Ar-H),
7.84 (d, 1H, J = 4.00 Hz, NH), 10.06 (s, 1H, NH). 13C-NMR (DMSO-d6, 100 MHz) δ: 24.48, 25.15,
25.15, 25.62, 31.16, 32.70, 32.70, 47.90, 67.56, 102.68, 108.00, 116.74, 128.80, 139.66, 157.58, 166.84,
+
170.78. ESI-HRMS calcd for C17H23N2O3 ([M + H]+): 303.1703; found: 303.1710.
Pharmacology
All the target compounds were submitted for evaluation in vivo. All the target compounds were
dissolved in dimethyl sulfoxide or 0.5% methylcellulose before the experiment, and Fluoxetine
(racemic mixture) was used as the positive control. Kunming mice (body weight, 18–22 g) were used
as experimental animals. Before the experiment, all the mice were allowed free access to food and

water. Two experimental tests, FST and TST, were used to evaluate the preliminary antidepressant
activity of the target compounds. The 5-HT enzyme-linked immunosorbent assay (ELISA) kits were
used to determine the concentrations of 5-HT in mice. The binding affinity for 5-HT1A receptor was
determined by competition binding studies with [3H] 8-OH-DPAT using racemic 8-OH-DPAT as the
reference compound [21].
Antidepressant activity
According to the method reported in the literature, the preliminary antidepressant activities of the
target compounds were determined by the FST [22,23]. According to the preliminary screening results,
compound 7 was selected for further evaluation of its antidepressant activity using the TST model
[24,25]. Preliminary tests were performed with compound 7 to evaluate the exploratory activity of the
animals by the open-field test [26]. 5-HT concentration was tested using ELISA to determine whether
compound 7 has an effect on the mouse brain. 5-HT1A binding assay was then carried out to determine
the binding capacity of compound 7 to receptors, serotonin was used to define nonspecific binding
[21].
Molecular docking studies and prediction of ADME properties
In this study, the FASTA sequence of 5-HT1A receptor was selected from RCSB-PDB (web site:
https://www.rcsb.org/), and structure crystals with high homology (PDB: 4IAR, 4IAQ, 5V54, and 6G79)
were used to construct a homology model of 5-HT1A receptor by Discovery Studio software. The
structures of the ligands were sketched on ChemBioDraw Ultra 14.0. LibDock protocol was executed
and the top LibDock Score was used for further analysis. The docking results were analyzed by
Discovery Studio.
The structures of the ligands were sketched on ChemBioDraw Ultra 14.0 and their
physicochemical properties were calculated by Discovery Studio or ChemBioDraw Ultra 14.0. The
ADME properties of these compounds were predicted by Discovery Studio and ChemBioDraw Ultra
14.0, including molecular number of hydrogen donors (nHBD), acceptors (nHBA), weight (MW),
number of rotatable bonds (RotB), polar surface area (tPSA), and cLogP. The absorption (ABS) level
and blood-brain barrier (BBB) permeability level were also calculated using the Discovery Studio.
Statistical analysis
Data are expressed as mean ± standard deviation. One-Way analysis of variance (ANOVA) was
performed using GraphPad Prism 5.0 statistical software. 0.01 < p < 0.05 indicated statistically

significant differences. p < 0.01 indicated highly significant differences.
Results
Chemistry
For
the
preparation
of
all
compounds
described
in
this
paper,
7-hydroxy-2H-chromen-2-one/4-hydroxy-2H-chromen-2-one/7-hydroxy-3,4-dihydroquinolin-2(1H)-on
e were used as the starting material (Scheme 1). A chemical reaction with ClCOCH2Cl and an
appropriate amine, triethylamine was set at room temperature to obtain intermediate compound 2.
Treatment
of
compound
2
with
7-hydroxy-2H-chromen-2-one/4-hydroxy-2H-chromen-2-one/7-hydroxy-3,4-dihydroquinolin-2(1H)-on
e in acetone yielded target compounds 4-11, 13-20, and 22-28 in good amounts. The structures of the
target compounds were characterized by spectral methods.
< Scheme 1. Insert here>
Pharmacology
FST
The synthesized compounds were then evaluated for the antidepressant activity. Most of the
compounds showed antidepressant activity at 40 mg/kg body weight, and seven compounds 4, 5, 13, 18,
20, 21, and 27 showed significant antidepressant activity compared with the control group (0.01 < p <
0.05); four compounds 7, 8, 16, and 24 showed highly significant antidepressant activity compared
with the control group (p < 0.01).
<Table 1. Insert here>
The antidepressant activity tests were then performed with 10, 20, and 40 mg/kg body weight of
the most active compound 7 and Fluoxetine (Table 2). At the dose of 40 mg/kg, both, compound 7 and
Fluoxetine showed the best antidepressant activity, and reduced the immobility time of the mice to
61.28 s and 67.21s, respectively.
<Table 2. Insert here>
TST
After FST, TST was performed, and we noted that compound 7 showed a highly significant
antidepressant activity compared to the control group (p < 0.01), and the immobility time of the mice
was reduced to 64.31 s, which was superior to Fluoxetine which reduced the immobility time of the
mice to 71.35 s) (Table 3).

<Table 3. Insert here>
Open-field test
In the open-field test, there were no significant differences in effects seen upon treatment with
compound 7 (p > 0.05, motor activity: crossing, rearing, and grooming) as compared with the control
group. The results suggest that compound 7 did not affect the spontaneous locomotor activity in mice
(Fig. 4).
<Fig. 4. Insert here>
Determination of 5-HT concentration
The estimation of 5-HT concentration by ELISA showed that the 5-HT content in the brain tissue
of compound 7 and Fluoxetine group (40 mg/kg body weight) was significantly (0.01 < p < 0.05) higher
than that of the control group (Table 4).
<Table 4. Insert here>
5-HT1A binding assay
Compounds 7, 16, and 24, which showed the most potent antidepressant activity, were used for
analyzing the binding with 5-HT1A receptor. Binding affinity analysis showed that compound 7 exhibits
better affinity with the 5-HT1A receptor (Ki = 1.4 nM), serotonin has a 1.5 nM Ki for 5-HT1A receptor.
<Table 5. Insert here>
Docking study
Docking investigation showed that compound 7 interacts with amino acid residues at the 5-HT1A
receptor active site, by chemical interactions, such as the hydrogen bond (Thr121, Thr196, and Ser 199)
and π-π stacking (Phe362 and Tyr195) interactions (Fig. 5).
<Fig. 5. Insert here>
Prediction of ADME properties
We further observed that none of the compounds violated Lipinski’s “rule of 5” (drug-like
compounds have MW ≤ 500, nHBA ≤ 10, nHBD ≤ 5, clogP ≤ 5, and RotB ≤ 10), as shown in Table 6.
Moreover, we observed that tPSA of all the synthesized compounds was < 140. In addition, most
compounds showed favorable BBB permeability and good absorption rates.
<Table 6. Insert here>
Discussion
FST is an effective method for screening antidepressants. A variety of antidepressants have been

developed using this method. In this study, we used the FST model to perform a preliminary
antidepressant activity test on all the synthesized target compounds, with Fluoxetine used as a positive
control. The antidepressant activity data are shown in Table 1. Among all the compounds, compound 7
showed the best antidepressant activity, which was observed to be better than the positive control.
The structure-activity relationship was obtained based on the pharmacological results shown in
Table 1. Overall, the antidepressant activity of coumarin derivatives was slightly better than that of
quinolinone derivatives. Coumarin (-COO-) shows higher fat-solubility than 3,4-dihydroquinolinone
(-CONH-), and hence is more likely to be absorbed and transported in the body, thereby exerting
stronger biological activity. The activity of the compound derived from the 7th position of the coumarin
is not significantly different from the activity of the compound derived from the 4th position. At the
same time, we also noted a significant antidepressant activity of the compounds with Cl atom that
showed superior activity as compared to the compounds substituted by other groups, such as F, CF3,
CH3, and OCH3. This indicates that Cl atoms have a certain influence on improving antidepressant
activity. The antidepressant activity of compound 7 was observed to be better than that of other
compounds, which could be due to the charge distribution of the compound after the introduction of Cl
atom, and is more suitable for enhancing the electrical binding effect with the receptor. At the same
time, the introduction of Cl atoms on the benzene ring can increase the fat-solubility, thereby allowing
the compound to exert better biological activity.
To investigate whether the antidepressant activity of the compounds was dose-dependent,
antidepressant activity of compound 7 and Fluoxetine were performed. Pharmacological results showed
that the antidepressant activity of both compound 7 and fluoxetine increased with increasing dose.
The TST model is another effective way to screen antidepressants. Table 3 shows that compound
7 showed a highly significant antidepressant activity compared with the control group (p < 0.01), which
was superior to Fluoxetine.
The open-field test is a classical animal experimental model for assessing the autonomic effects of
drugs and the general activities of animals. Since the shortening of animal immobility time in
behavioral despair and depression animal models may be caused by the excited sympathetic nerves of
the drug, the open-field experiment was performed to examine the central excitability of compound 7
[27,28]. In this study, we used an open-field test to determine whether compound 7 affected the
spontaneous locomotor activity in mice. Compared with the control group, there was no significant

difference in animals treated with compound 7 (p > 0.05), which excluded the false positive results of
the mice due to central activity excitability.
Studies show that monoamine neurotransmitter pathways primarily control the body's
physiological activities, and changes in transmitters affect monoamine-based transmitter pathways
result in a variety of clinical depressive symptoms. At present, most people recognize that monoamine
neurotransmitters such as 5-HT may be involved in the neurobiochemical mechanisms of depression.
The results of pathological autopsy of depression showed a decrease in 5-HT levels in the brainstem
and frontal lobe, and a decrease in the total amount of 5-HT receptors in the hippocampus [29]. The
effect of compound 7 on the content of 5-HT in brain tissue of mice was determined here by ELISA.
The results showed that compound 7 and Fluoxetine significantly increased the 5-HT content in the
brain.
The 5-HT1A receptor plays a role in the pathogenesis of various mental and neurological diseases.
Activation of postsynaptic 5-HT1A receptors is important for the favorable response to antidepressants
[30,31]. In 5-HT1A binding assay, compound 7, 16 and 24 exhibited higher affinities for the 5-HT1A
receptor, at or below the level of the native neurotransmitter, serotonin (Ki= 1.5 nM).
Molecular docking is an important means to study the possible mechanisms of action for
biologically active compounds. In the present study, we docked the compound 7 to the constructed
5-HT1A receptor by homology modeling. Molecular docking and results analysis were performed using
Discovery Studio, and the docking results are shown in Fig. 5. According to the literature [32], amino
acid residues at the active site of 5-HT1A receptor homology model are Ala93, Ala263, Ala365, Ala383,
Asn386, Asp116, Cys120, Thr379, Gln97, Gly382, Ile113, Ile124, Ile167, Phe112, Phe361, Phe362,
Ser199, Thr196, Thr121, Thr200, and Val117. Docking results showed that compound 7 interacts with
amino acid residues at the 5-HT1A receptor active site by hydrogen bond and π-π stacking interactions.
The drug-like properties of designed compounds based on Lipinski’s “rule of five” are an effective
way of discovering new antidepressant drugs. Statistically, extremely few drugs violate the “rule of
five,” because compliance with this rule enables the drugs to be bioavailable. In addition, tPSA is
closely related to the bioavailability of drugs; compounds with a tPSA > 140 tend to have a lower oral
bioavailability [33]. In addition, BBB permeability is an important factor affecting drug activity in the
CNS. We used Discovery Studio software to predict BBB permeability of the target compounds.
Treatment of depression, Parkinson's disease, epilepsy, and brain tumors require the drugs to cross the

BBB. Due to the complexity of the BBB, it is particularly important to design compounds with suitable
physicochemical properties to penetrate the BBB. According to previous reports [34,35], penetration of
the BBB by drugs is closely related to the clogP, tPSA, and MW of the compounds. Pajouhesh and
Lenz studied the physicochemical properties of the marketed CNS drugs, and highlighted that a
successful CNS drug should have the following physical and chemical properties: clogP < 5, tPSA < 70,
and MW < 450 [36]. In this study, the clogP, tPSA, and MW of the compounds we designed are
consistent with the physicochemical properties of CNS drugs. The absorption of the synthesized
compounds was predicted using Discovery Studio. As shown in Table 6, most compounds showed
favorable BBB permeability and good absorption.
Conclusions
In the present study, a series of coumarin and 3,4-dihydroquinolinone derivatives were
synthesized. Two experimental methods, FST and TST, were used to evaluate the antidepressant
activity of the target compounds. The pharmacological results showed that most of the target
compounds exhibited significant antidepressant activity in the FST model. Among these compounds,
compound 7, N-(4-chlorophenyl)-2-[(2-oxo-2H-chromen-7-yl)oxy] acetamide, was noted to be the
most potent compound. In the open-field test, compound 7 did not affect spontaneous activity. The
results of in vivo 5-HT concentration estimation showed that compound 7 may have an effect on the
mouse brain. In the 5-HT1A binding assay, the results showed that compound 7 exhibits strong affinity
for the 5-HT1A receptor. Molecular docking results revealed that compound 7 showed significant
interactions with residues at the 5-HT1A receptor by homology modeling. The results of molecular
properties prediction tests showed that all the compounds exhibited BBB permeability and favorable
bioavailability via the oral route.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
The work was supported by Natural Science Foundation of Shandong (No. ZR2017BH037),
Doctoral Foundation of Liaocheng University (No. 318051517), National Natural Science Foundation
of China (No. 21675071, 81402512, and 81803360), A Projet of Shandong Province Higher Education
Science and Technology Program (J13LK17).
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Captions:
Fig. 1. The chemical structure of coumarin and 3,4-dihydroquinolinone.
Fig. 2. Representative structures of carboxylic acid amide antidepressants.
Fig. 3. Design strategy for target molecules.
Fig. 4. Exploratory activity (counts) in the open-field test. The behavioral parameters were recorded for
3 min. Locomotion: number of line crossings; rearing: number of times seen standing on hind legs;
grooming: number of modifications; 7 (40 mg/kg) was administered 60 min before the test. The values
represent the mean ± SEM (n =8).
Fig. 5. (a) 3D model of 5-HT1A receptor homology model; (b) Ramachandran plot; (c) Compound 7

show interactions with residues at the 5-HT1A receptor.
Scheme 1. Synthesis of the target compounds 4-11, 13-20 and 22-28. Reagents and conditions: (a)
triethylamine, CH2Cl2, rt, 3-8h; (b) K2CO3, acetone, reflux, 6-12h.

Table 1. Antidepressant activities of compounds 4-11, 13-20 and 22-28 in FST.
Antidepressant activitiesa
Compds
R
Duration of immobility (s)
(mean ± SEM)b
Change from control
(%)
84.41 ± 18.81*
94.82 ± 15.38*
128.83 ± 9.87
65.52 ± 9.70**
75.27 ± 17.29**
154.17 ± 14.01
125.95 ± 20.38
100.82 ± 19.38
86.38 ± 22.21*
128.56 ± 22.88
109.72 ± 19.21
71.91 ± 15.38**
130.12 ± 12.01
89.21 ± 14.78*
151.69 ± 15.48
85.16 ± 15.51*
96.83 ± 18.96*
137.32 ± 21.09
76.45 ± 17.87**
134.92 ± 21.40
122.01 ± 21.79
91.25 ± 17.88*
131.31 ± 11.07
68.15 ± 9.41**
167.92 ± 22.09
49.73
43.53
23.27
60.98
55.17
8.18
4
Ph
5
CH2Ph
6
CH2CH2Ph
(4-Cl) Ph
(4-CF3) Ph
(4-CH3) Ph
(4-OCH3) Ph
Cyclohexane
Ph
7
8
9
24.99
39.96
48.55
23.44
34.66
57.17
22.51
46.87
9.66
10
11
13
14
15
16
17
18
19
20
22
23
24
25
26
27
CH2Ph
CH2CH2Ph
(4-Cl) Ph
(4-F) Ph
(4-CF3) Ph
(4-CH3) Ph
Cyclohexane
Ph
49.28
42.33
18.22
54.47
19.65
27.34
45.65
21.80
59.41
-
CH2Ph
(4-Cl) Ph
(4-F) Ph
(4-CH3) Ph
(4-OCH3) Ph
28
Cyclohexane
-
-
Fluoxetine
Control
a Target compounds and Fluoxetine were administered at 40 mg/kg. b Values represent the mean ± SEM (n = 8).
* significantly compared to control (0.01＜p＜0.05). ** very significantly compared to control (p＜0.01).

Table 2. Antidepressant activities of compounds 7 and Fluoxetine in FST at different doses.
Antidepressant activitiesa
Dose (mg/kg)
Duration of immobility(s)
(mean ± SEM)b
Change from control
(%)
Compds
7
83.22 ± 17.21*
68.32 ± 13.88**
61.28 ± 11.79**
47.46
56.87
60.97
10
20
40
85.76 ± 16.81*
72.54 ± 15.67**
67.21 ± 13.34**
45.86
54.29
57.57
10
20
40
Fluoxetine
Control
-
158.41 ± 18.61
-
a
b
Compound 7 and Fluoxetine were administered at 40, 20, 10 mg/kg, respectively. Values represent the
mean ± SEM (n = 8). * significantly compared to control (0.01＜p＜0.05). ** very significantly compared to
control (p＜0.01).
Table 3. Antidepressant activities of compounds 7 and Fluoxetine in the TST test.
Antidepressant activitiesa
Dose (mg/kg)
Duration of immobility (s)
(mean ± SEM)b
Change from control
(%)
Compds
64.31 ± 15.72**
71.35 ± 14.48**
144.31 ± 21.07
54.88
50.55
-
7
40
40
-
Fluoxetine
Control
a Compound 7 and Fluoxetine were administered at 40 mg/kg. b Values represent the mean ± SEM (n = 8).
* significantly compared to control (0.01＜p＜0.05). ** very significantly compared to control (p＜0.01).
Table 4. Effect of compounds 7 and Fluoxetine on brain 5-HT level in mice.
Compds
Dose (mg/kg)
5-HT (ng/mg)a
1.314 ± 0.113*b
1.298 ± 0.095*
1.023 ± 0.107
7
40
40
-
Fluoxetine
Control
a Compounds 7 and Fluoxetine were analyzed (The concentration of 5-HT in the mice brain) 1 hour after oral
administration. b Represent the mean ± SEM (n = 8). * significantly compared to control (0.01＜p＜0.05).

Table 5. 5-HT1A receptor binding of selected compounds and reference serotonin.
Compds
5-HT1A (Ki [nM] ± SEM)a
7
16
1.4 ± 0.1
2.5 ± 0.2
24
6.3 ± 0.4
Serotonin
1.5 ± 0.1
a Ki values were obtained from 8 concentrations of the compound, each in duplicate.
Table 6. Drug-likeness parameters of target compounds.
BBB
ABS
Comds
R
MWa
ClogP nHBD nHBA tPSA nRotB
(level) (level)
4
Ph
295.08
309.10
323.11
329.04
363.07
309.10
325.09
2.36
2.38
2.60
3.33
3.69
2.86
2.43
2.53
2.50
2.52
2.74
3.47
2.90
3.83
2.99
2.67
1.98
2.01
2.95
2.38
2.48
2.06
2.16
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
4
4
4
4
4
4
5
4
4
4
4
4
4
4
4
4
3
3
3
3
3
4
3
64.63
64.63
64.63
64.63
64.63
64.63
73.86
64.63
64.63
64.63
64.63
64.63
64.63
64.63
64.63
64.63
67.43
67.43
67.43
67.43
67.43
76.66
67.43
4
5
6
4
5
4
5
4
4
5
6
4
4
5
4
4
4
5
4
4
4
5
4
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
3
3
2
3
2
3
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
CH2Ph
6
CH2CH2Ph
(4-Cl) Ph
(4-CF3) Ph
(4-CH3) Ph
(4-OCH3)Ph
7
8
9
10
11
13
14
15
16
17
18
19
20
22
23
24
25
26
27
Cyclohexane 301.13
Ph
295.08
309.10
323.11
329.04
313.07
363.07
309.10
CH2Ph
CH2CH2Ph
(4-Cl) Ph
(4-F) Ph
(4-CF3) Ph
(4-CH3) Ph
Cyclohexane 301.13
Ph
296.11
310.13
330.07
314.10
310.13
326.12
CH2Ph
(4-Cl) Ph
(4-F) Ph
(4-CH3) Ph
(4-OCH3)Ph
28
Cyclohexane 302.16
a
MW, molecular weight; CLogP, Calculated lipophilicity; nHBD, number of hydrogen bond donors; nHBA,
number of hydrogen bond acceptors; tPSA, topological polar surface area; nRotB, number of rotatable bonds;
BBB-Level, 0 (very high penetration), 1 (high penetration), 2 (medium penetration), 3 (weak penetration);
Absorption-Level, 0 (very well absorption), 1 (well absorption), 2 (poorly absorption), 3 (very poorly absorption).