Design and synthesis of 7-diethylaminocoumarin-based 1,3,4-oxadiazole derivatives with anti-acetylcholinesterase activities

Article abstract of DOI:10.1080/10286020.2020.1803293

Twelve novel 7-diethylaminocoumarin-based 1,3,4-oxadiazole derivatives were synthesized via iodine-mediated oxidative cyclisation and confirmed by ¹H NMR, ¹³C NMR and HRMS. The result of these derivatives' activities inhibiting acetylcholinesterase in?vitro showed that 4 g and 4i had moderate inhibitory activities with 69.19% and 65.06%, respectively. The preliminary structure-activity relationships revealed that introduction of halogen atom on the para-position of phenyl of 7-diethylaminocoumarin-based 1,3,4-oxadiazole derivatives could enhance their activities. Molecular docking study suggested that 4 g possessed an optimal docking pose with interactions inside AChE.

Full text of DOI:10.1080/10286020.2020.1803293

Journal of Asian Natural Products Research
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Design and synthesis of 7-diethylaminocoumarin-
based 1,3,4-oxadiazole derivatives with anti-
acetylcholinesterase activities
Xiang Yu , You-Fang Zhao , Guo-Juan Huang & Ya-Fang Chen
To cite this article: Xiang Yu , You-Fang Zhao , Guo-Juan Huang & Ya-Fang Chen (2020):
Design and synthesis of 7-diethylaminocoumarin-based 1,3,4-oxadiazole derivatives with
anti-acetylcholinesterase activities, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2020.1803293
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1803293
Design and synthesis of 7-diethylaminocoumarin-based
1,3,4-oxadiazole derivatives with anti-acetylcholinesterase
activities
Xiang Yua,b, You-Fang Zhaoa, Guo-Juan Huanga and Ya-Fang Chena,b
aCollege of Pharmacy, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China;
bResearch Center for Natural Medicine Chemistry, Guizhou University of Traditional Chinese
Medicine, Guiyang 550025, China
ABSTRACT
ARTICLE HISTORY
Received 29 April 2020
Accepted 26 July 2020
Twelve novel 7-diethylaminocoumarin-based 1,3,4-oxadiazole
derivatives were synthesized via iodine-mediated oxidative cyclisa-
tion and confirmed by H NMR, 13C NMR and HRMS. The result of
1
KEYWORDS
these derivatives’ activities inhibiting acetylcholinesterase in vitro
showed that 4 g and 4i had moderate inhibitory activities with
69.19% and 65.06%, respectively. The preliminary structure-activity
relationships revealed that introduction of halogen atom on the
para-position of phenyl of 7-diethylaminocoumarin-based 1,3,4-
oxadiazole derivatives could enhance their activities. Molecular
docking study suggested that 4 g possessed an optimal docking
pose with interactions inside AChE.
Coumarin; 1,3,4-oxadiazole;
acetylcholinesterase inhibi-
tor; molecular docking
1. Introduction
Alzheimer’s disease (AD) is a major age-related progressive neurodegenerative disease
characterised by the destruction of nerve cells, the rapid deterioration of memory and
CONTACT Ya-Fang Chen
chenyafang@gzy.edu.cn
College of Pharmacy, Guizhou University of Traditional
Chinese Medicine, Guiyang 550025, China
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2020.1803293.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
X. YU ET AL.
Figure 1. Structures of compounds I-III and the design of target derivatives.
other important cognitive functions, not only harms the health of patients, but also
brings a heavy burden to their families and society [1]. It is caused by an imbalance
between b-amyloid (Ab) production and clearance, resulting in increased amount of
Ab in various forms of brain [2]. Currently, the effective treatment for AD is inhibit-
ing brain acetylcholinesterase to increase lifetime of cerebral acetylcholine [3]. AChE,
enzymes that terminate cholinergic neurotransmission in the brain, act by catalysing
the hydrolysis of acetylcholine, so their inhibition can be used to alleviate memory
and cognitive deficits in AD [4,5]. Therefore, the development of highly effective
AChE inhibitors has been an object of research.
Coumarins, which are heterocyclic compounds with benzopyranone, commonly
occur in various biologically active natural products and synthetic compounds [6,7].
It was found that coumarins showed various interesting biological activities, including
anticancer, antimicrobial, antiviral, anti-inflammatory, antioxidant and other bio-
logical activities [8–11]. Recently, researchers had also reported that coumarin-based
derivatives had a significant role in the studies of AChE inhibitors [12]. In addition,
1,3,4-oxadiazoles, another important class of N-heterocyclic compounds with a wide
range of biological activities such as anticancer, antimicrobial, antiviral, analgesic
activities, were widely used in medicine and pesticides [13–15]. Several attempts
introducing hydrazone 1,3,4-oxadiazole moiety into natural products, such as sarisan
(I, Figure 1) [16], ursolic acid (II, Figure 1) [17] and fraxinellone (III, Figure 1) [18],
had been made to improve their biological activity. Encouraged by the above-men-
tioned interesting results, in this paper, we prepared a series of novel 7-diethylamino-
coumarin-based 1,3,4-oxadiazole derivatives by introduction of 1,3,4-oxadiazole
moiety into the coumarin skeleton. Besides, we have determined the anti-AChE activ-
ity of the target compounds by the method of Ellman and explored the possible
mechanisms via molecular modeling.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Scheme 1. Synthetic route of 7-diethylaminocoumarin-based 1,3,4-oxadiazole derivatives (4a-l).
2. Results and discussion
2.1. Chemistry
The synthesis of coumarin-based 1,3,4-oxadiazole derivatives was shown in Scheme 1
via the method reported in some literatures [16,18]. Oxidation of 7-diethylamino-4-
methylcoumarin (1) with SeO2 gave 4-formyl-7-diethylaminocoumarin (2).
Subsequently, compound 2 reacted with different arylhydrazide to afford the inter-
mediates (3a-l) in the presence of HOAc. After the second reaction was completed,
the solvent was evaporated under reduced pressure, and the products were not puri-
fied to save time and reagents, redissolved directly in dimethyl sulfoxide, followed by
the iodine-mediated oxidative cyclisation at refluxing to obtain twelve target com-
pounds containing 1,3,4-oxadiazole (4a-l). In order to prove whether the unpurified
intermediates could synthesize the target compounds, some individual intermediates
1
were purified and identified (see Supplementary Material). Comparison of H NMR
spectra of the intermediate 3j and the target compound 4j revealed that the protons
of H-10 (d, 8.53 ppm) and H-30 (d, 12.48 ppm) disappeared proving that this step
1
without purification was feasible. All target compounds were characterized by mp, H
NMR 13C NMRand HRMS (see Supplementary Materia).

4
X. YU ET AL.
Figure 2. The AChE inhibitory activities of titled compounds (4a-l) at 1 lmol/ml; Values represent
means SD; Tacrine was control and tested in 5 ꢀ 10ꢁ4 lmol/ml.
2.2. Biological activity
As shown in Figure 2, preliminary bioassay of these derivatives’ activities inhibiting
AChE was tested in vitro by method of Ellman at the concentration of 1 lmol/ml . It
was apparent from this figure that most of the target compounds have better inhibi-
tory activities against AChE compared with raw materials 1, especially 4 g and 4i
with an inhibitory rate of 69.19% and 65.06%, respectively, but did not exceed the
positive controls. Meanwhile, analysis of the structure-activity relationship of these
coumarin derivatives containing 1,3,4-thiadiazole moiety was also observed.
Introduction of halogen atom on the para-position of phenyl of 7-diethylaminocou-
marin-based 1,3,4-oxadiazole derivatives had higher activity (e.g., 4f, 4 g and 4i) in
comparison with the parent compound 1. On the contrary, no significant assistance
in introduction of alkyl group was found to increase activity (e.g., 14.31% for 4a). In
addition, when pyridine and furan heterocycle were introduced on 1,3,4-oxadiazole,
the compounds could increase the inhibitory activity appropriately. For examlpe, the
inhibition ratios of compounds 4j and 4k were 46.16% and 45.67%, respectively.
In order to find the lowest concentration of potent compounds, the inhibitory activities
of compounds 4d, 4g and 4i with inhibition rates higher than 50% were further evaluated
at concentrations of 1 lmol/ml, 0.1 lmol/ml and 0.01 lmol/ml, respectively. It could be
seen from the data in Figure 3 that the inhibitory activities of the compounds gradually
weaken as the concentration decreased. When the concentration reached 0.01 lmol/ml,
the three compounds showed little inhibitory activities. It demonstrated that the inhibitory
activities were positively correlated with the concentration of the compounds.
2.3. Molecular modeling
Molecular modeling studies of the possible binding mode of compound 4 g in the
active site of AChE were also performed to explore the possible inhibition mechanism

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 3. The AChE inhibitory activities of compounds 4d, 4 g, 4i in three concentrations; Values
represent means SD.
Figure 4. Docking pose of compound 4 g inside AChE. (a: The 3 D structure of the compound
binding model with AChE, the blue structure represents the derivative, the green structures repre-
sent amino acid residues. b: The 2 D structure of the compound binding model with AChE, the
blue dotted lines show the hydrogen bonds, the full lines show the C-H－p interactions).
of the potent compound. The 3 D structure of human AChE was selected from the
RCSB database (PDB code: 3LII) for docking studies. Interestingly, as illustrated in
Figure 4, the results obtained from the preliminary analysis of molecular ducking dis-
played that the coumarin portion of compound 4 g was located in the substrate of the
active channel of AChE, while the benzene ring with chlorine atoms was located at
the channel entrance (Figure 4, a). Simultaneously, the two nitrogen atoms of 1,3,4-
oxadiazole core of compound 4 g interacted with Tyr124 residue, located in the mid-
dle of the catalytic active channel of AChE, via two hydrogen bonds, oxygen atom on
the 1-position of coumarin core was bonded to Ser203 residue by hydrogen bonds,
and the benzene ring of coumarin maked C-H—p interaction with Gly121 residue,

6
X. YU ET AL.
located in the catalytic active site of AChE (Figure 4, b). Through these binding
actions, compound 4 g firmly occupied the catalytic site of AChE, while the chlorine
atom with a large atomic radius controlled the entrance of the catalytic channel,
which prevented acetylcholine from entering the catalytic center. This molecular
docking result, along with the biological assay data, suggested that compound 4 g was
a potential inhibitor of AChE.
In summary, a series of new 7-diethylaminocoumarin-based 1,3,4-oxadiazole deriv-
atives were designed, synthesised, and evaluated for their AChE inhibitory activities
in vitro. Among all the derivatives, 4 g and 4i showed moderate inhibitory activities
and the inhibitory activity was positively correlated with the concentration.
Furthermore, preliminary structure-activity relationships analysis revealed that the
introduction of halogen atom on the para-position of phenyl of 7-diethylaminocou-
marin-based 1,3,4-oxadiazole derivatives could lead a promising AChE inhibitor.
Molecular docking study of 4 g and AChE also suggested that compound 4 g was a
potential inhibitor of AChE.
3. Experimental
3.1. General experimental procedures
All reagents and solvents were of reagent grade or purified according to standard meth-
ods before use. Analytical thin-layer chromatography (TLC) and preparative thin-layer
chromatography (PTLC) were performed with silica gel plates using silica gel 60 GF254
(Qingdao Haiyang Chemical Co., Ltd.). Melting points were determined on a XT-4
digital melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) and
are uncorrected. Nuclear magnetic resonance spectra (NMR) were recorded on a Bruker
Avance NEO 400 or 600 MHz instrument (Bruker, Bremerhaven, Germany) in CDCl3 or
DMSO-d6 using TMS (tetramethylsilane) as the internal standard. Mass spectrometry
(MS) was carried out with a Waters XEVO TQ-D instrument (waters, Massachusetts,
USA). High-resolution mass spectrometry (HRMS) was carried out with a Xevo G2-S
QTOF instrument (waters, Massachusetts, USA).
3.2. Synthesis of compound 2
A solution of 7-diethylamino-4-methylcoumarin (1 mmol, 231.3 mg) in dioxane (10 ml)
was heated to 60 ꢂC. SeO2 (1.1 mmol, 122.1) was added to this solution. Then the tem-
perature was increased to 80 ꢂC. The reaction was checked by TLC. After 12-18 h, the
mixture was cooled to room temperature. The precipitates were filtered off and washed
with CH2Cl2 (20 ml). The organic phases were combined, concentrated and separated by
preparative thin-layer chromatography (PTLC) to give the pure compound 2.
3.3. General procedure for the synthesis of compounds 3a-l
A mixture of 2 (1 mmol, 245.3 mg), hydrazides or hydrazines (1 mmol), and two
drops of HOAc in EtOH (10 ml) was refluxed. When the reaction was completed by
checking with TLC after 3-7 h, the resulting reaction mixture was cooled to room

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
temperature until no more precipitate was observed. Then the solvent was removed
under reduced pressure to give the intermediates (3a-l). The product was not purified
and went straight to the next step. Compounds 3d and 3j were purified and charac-
1
terized by H NMR (600 MHz) and MS.
3.3.1. Compound 3d
1
Yield ¼ 74%, yellow solid, mp 236-238 ꢂC; H NMR (600 MHz, DMSO-d6, d, ppm)
12.25 (s, 1H, NH), 8.53 (s, 1H, H-10), 8.42 (d, J ¼ 9.0 Hz, 1H, H-5), 7.47-7.54 (m, 3H,
H-Ar), 7.20 (dd, J ¼ 8.4, 2.4 Hz, 1H, H-Ar), 6.77 (dd, J ¼ 9.0, 2.4 Hz, 1H, H-6), 6.57
(d, J ¼ 2.4 Hz, 1H, H-8), 6.29 (s, 1H, H-3), 3.85 (s, 3H, OCH3), 3.44 (q, J ¼ 7.2 Hz,
4H, N(CH2CH3)2), 1.13 (t, J ¼ 7.2 Hz, 6H, N(CH2CH3)2); MS (ESI): m/z
416.2 [M þ Na]þ.
3.3.2. Compound 3j
1
Yield ¼ 68%, yellow solid, mp 238-241 ꢂC; H NMR (600 MHz, DMSO-d6, d, ppm)
12.48 (s, 1H, NH), 8.82 (d, J ¼ 5.4 Hz, 2H, H-Ar), 8.53 (s, 1H, H-10), 8.41 (d,
J ¼ 9.0 Hz, 1H, H-5), 7.85 (d, J ¼ 6.0 Hz, 2H, H-Ar), 6.78 (dd, J ¼ 9.0, 2.4 Hz, 1H, H-
6), 6.57 (d, J ¼ 2.4 Hz, 1H, H-8), 6.33 (s, 1H, H-3), 3.44 (q, J ¼ 7.2 Hz, 4H,
N(CH2CH3)2), 1.13 (t, J ¼ 7.2 Hz, 6H, N(CH2CH3)2); MS (ESI): m/z 387.0 [M þ Na]þ.
3.4. General procedure for the preparation of 1,3,4-oxadiazole derivatives (4a-l)
The intermediates (3a-l) were redissolved in DMSO (10 ml), followed by addition of
K2CO3 (3.0 mmol, 414.6 mg), iodine (1.1 mmol, 279.4 mg) in sequence. The reaction
mixture was stirred at 100 ꢂC until the reaction was completed according to TLC ana-
lysis (2-6 h). After being cooled to room temperature, the mixture was treated with
saturated Na2S2O3 (20 ml) and extracted with ethyl acetate (3 ꢀ 20 ml). The combined
organic layer was washed with brine (3 ꢀ 20 ml), dried over anhydrous Na2SO4, fil-
tered and evaporated. The given residue was purified by PTLC to give the target
1
products 4a-l in 43-71% yield. All compounds were characterized by mp, H NMR
(400 or 600 MHz) and HRMS. The data for 4a-l were shown as follows.
3.4.1. 4-(5-Butyl-1,3,4-oxadiazol-2-yl)-7-(diethylamino)-2H-chromen-2-one (4a)
1
Yield ¼ 52%, yellow solid, mp 208-210 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.67
(d, J ¼ 9.2 Hz, 1H, H-5), 6.67 (dd, J ¼ 9.2, 2.6 Hz, 1H, H-6), 6.63 (s, 1H, H-3), 6.55 (d,
J ¼ 2.6 Hz, 1H, H-8), 3.45 (q, J ¼ 7.2 Hz, 4H, N(CH2CH3)2), 2.99 (t, J ¼ 7.6 Hz, 2H,
CH2CH2CH2CH3), 1.83-1.88 (m, 2H, CH2CH2CH2CH3), 1.45-1.51 (m, 2H,
CH2CH2CH2CH3), 1.24 (t, J ¼ 7.2 Hz, 6H, N(CH2CH3)2), 1.00 (t, J ¼ 7.2 Hz, 3H,
CH2CH2CH2CH3); 13C NMR (100 MHz, CDCl3, d, ppm): 167.8, 161.6, 161.2, 157.0,
151.1, 135.4, 128.7, 109.3, 108.8, 104.0, 97.6, 44.8 ꢀ 2, 28.4, 25.0, 22.1, 13.5, 12.4 ꢀ 2;
HRESIMS: m/z 342.1824 [M þ H]þ (calcd for C19H24N3O3, 342.1818).
3.4.2. 7-(Diethylamino)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)-2H-chromen-2-one (4 b)
1
Yield ¼ 68%, yellow solid, mp 205-207 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.73
(d, J ¼ 9.2 Hz, 1H, H-5), 8.17 (d, J ¼ 7.2 Hz, 2H, H-Ar), 7.62 (t, J ¼ 7.2 Hz, 1H, H-Ar),

8
X. YU ET AL.
7.58 (t, J ¼ 7.2 Hz, 2H, H-Ar), 6.77 (s, 1H, H-3), 6.71 (dd, J ¼ 9.2, 2.6 Hz, 1H, H-6),
6.57 (d, J ¼ 2.6 Hz, 1H, H-8), 3.46 (q, J ¼ 7.2 Hz, 4H, N(CH2CH3)2), 1.25 (t,
J ¼ 7.2 Hz, 6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3, d, ppm): 165.0, 161.4,
161.2, 157.0, 151.1, 135.3, 132.5, 129.3 ꢀ 2, 128.6, 127.3 ꢀ 2, 123.0, 109.4, 108.9, 104.1,
97.7, 44.8 ꢀ 2, 12.4 ꢀ 2; HRESIMS: m/z 362.1511 [M þ H]þ (calcd for
C21H20N3O3, 362.1505).
3.4.3. 7-(Diethylamino)-4-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)-2H-chromen-2-one (4c)
1
Yield ¼ 61%, yellow solid, mp 212-214 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.74
(d, J ¼ 9.2 Hz, 1H, H-5), 7.98 (s, 1H, H-Ar), 7.96 (d, J ¼ 7.6 Hz, 1H, H-Ar), 7.46 (t,
J ¼ 7.6 Hz, 1H, H-Ar), 7.42 (d, J ¼ 7.6 Hz, 1H, H-Ar), 6.78 (s, 1H, H-3), 6.71 (dd,
J ¼ 9.2, 2.6 Hz, 1H, H-6), 6.56 (d, J ¼ 2.6 Hz, 1H, H-8), 3.46 (q, J ¼ 7.2 Hz, 4H,
N(CH2CH3)2), 2.48 (s, 3H, CH3), 1.25 (t, J ¼ 7.2 Hz, 6H, N(CH2CH3)2); 13C NMR
(100 MHz, CDCl3, d, ppm): 165.2, 161.4, 161.3, 157.0, 151.1, 139.2, 135.3, 133.3,
129.2, 128.7, 127.8, 124.5, 122.8, 109.4, 108.8, 104.0, 97.7, 44.8 ꢀ 2, 21.3, 12.4 ꢀ 2;
HRESIMS: m/z 376.1664 [M þ H]þ (calcd for C22H22N3O3, 376.1661).
3.4.4. 7-(Diethylamino)-4-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-2H-chromen-
2-one (4d)
1
Yield ¼ 68%, yellow solid, mp 212-214 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.72
(d, J ¼ 9.2 Hz, 1H, H-5), 7.74 (d, J ¼ 7.6 Hz, 1H, H-Ar), 7.67 (s, 1H, H-Ar), 7.48 (t,
J ¼ 8.0 Hz, 1H, H-Ar), 7.13-7.15 (m, 1H, H-Ar), 6.77 (s, 1H, H-3), 6.71 (dd, J ¼ 9.2,
2.6 Hz, 1H, H-6), 6.56 (d, J ¼ 2.6 Hz, 1H, H-8), 3.92 (s, 3H, OCH3), 3.46 (q,
J ¼ 7.2 Hz, 4H, N(CH2CH3)2), 1.25 (t, J ¼ 7.2 Hz, 6H, N(CH2CH3)2); 13C NMR
(100 MHz, CDCl3, d, ppm): 165.0, 161.4, 161.2, 160.1, 157.0, 151.1, 135.3, 130.4,
128.6, 124.1, 119.7, 119.1, 111.8, 109.4, 108.9, 104.0, 97.7, 55.6, 44.8 ꢀ 2, 12.4 ꢀ 2;
HRESIMS: m/z 392.1617 [M þ H]þ (calcd for C22H22N3O4, 392.1610).
3.4.5. 4-(5-(4-Cyanophenyl)-1,3,4-oxadiazol-2-yl)-7-(diethylamino)-2H-chromen-2-
one (4e)
1
Yield ¼ 56%, yellow solid, mp 204-206 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.67
(d, J ¼ 9.2 Hz, 1H, H-5), 8.30 (d, J ¼ 8.4 Hz, 2H, H-Ar), 7.89 (d, J ¼ 8.4 Hz, 2H, H-
Ar), 6.76 (s, 1H, H-3), 6.71 (dd, J ¼ 9.2, 2.6 Hz, 1H, H-6), 6.57 (d, J ¼ 2.6 Hz, 1H, H-
8), 3.47 (q, J ¼ 7.2 Hz, 4H, N(CH2CH3)2), 1.25 (t, J ¼ 7.2 Hz, 6H, N(CH2CH3)2); 13C
NMR (100 MHz, CDCl3, d, ppm): 163.4, 162.1, 161.0, 157.1, 151.3, 134.8, 133.0 ꢀ 2,
128.4, 127.8 ꢀ 2, 126.9, 117.6, 116.0, 109.5, 109.2, 103.8, 97.7, 44.8 ꢀ 2, 12.4 ꢀ 2;
HRESIMS: m/z 387.1458 [M þ H]þ (calcd for C22H19N4O3, 387.1457).
3.4.6. 7-(Diethylamino)-4-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-2H-chromen-2-
one (4f)
1
Yield ¼ 54%, yellow solid, mp 232-234 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.71
(d, J ¼ 9.2 Hz, 1H, H-5), 8.18 (dd, J ¼ 8.8, 5.2 Hz, 2H, H-Ar), 7.25-7.28 (m, 2H, H-
Ar), 6.75 (s, 1H, H-3), 6.70 (dd, J ¼ 9.2, 2.6 Hz, 1H, H-6), 6.56 (d, J ¼ 2.6 Hz, 1H, H-
8), 3.46 (q, J ¼ 7.1 Hz, 4H, N(CH2CH3)2), 1.25 (t, J ¼ 7.1 Hz, 6H, N(CH2CH3)2); 13C
NMR (100 MHz, CDCl3, d, ppm): 166.5, 164.2, 161.4, 161.2, 157.1, 151.2, 135.2,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
129.8, 129.7, 128.6, 119.4, 116.8, 116.6, 109.4, 108.9, 104.0, 97.6, 44.8 ꢀ 2, 12.4 ꢀ 2;
HRESIMS: m/z 380.1416 [M þ H]þ (calcd for C21H19N3O3F, 380.1410).
3.4.7. 4-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-7-(diethylamino)-2H-chromen-2-
one (4 g)
1
Yield ¼ 60%, yellow solid, mp 254-256 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.70
(d, J ¼ 9.2 Hz, 1H, H-5), 8.10 (d, J ¼ 8.5 Hz, 2H, H-Ar), 7.56 (d, J ¼ 8.5 Hz, 2H, H-
Ar), 6.75 (s, 1H, H-3), 6.70 (dd, J ¼ 9.2, 2.6 Hz, 1H, H-6), 6.56 (d, J ¼ 2.6 Hz, 1H, H-
8), 3.46 (q, J ¼ 7.2 Hz, 4H, N(CH2CH3)2), 1.25 (t, J ¼ 7.2 Hz, 6H, N(CH2CH3)2); 13C
NMR (100 MHz, CDCl3, d, ppm): 164.2, 161.5, 161.1, 157.0, 151.2, 138.9, 135.1,
129.7 ꢀ 2, 128.6 ꢀ 3, 121.5, 109.4, 108.9, 103.9, 97.6, 44.8 ꢀ 2, 12.4 ꢀ 2; HRESIMS: m/z
396.1119 [M þ H]þ (calcd for C21H19N3O3Cl, 396.1115).
3.4.8. 4-(5-(3-Bromophenyl)-1,3,4-oxadiazol-2-yl)-7-(diethylamino)-2H-chromen-2-
one (4 h)
1
Yield ¼ 71%, yellow solid, mp 192-194 ꢂC; H NMR (400 MHz, CDCl3, d, ppm): 8.69
(d, J ¼ 9.2 Hz, 1H, H-5), 8.31 (t, J ¼ 1.6 Hz, 1H, H-Ar), 8.10 (dt, J ¼ 7.9, 1.6 Hz, 1H,
H-Ar), 7.72-7.75 (m, 1H, H-Ar), 7.46 (t, J ¼ 7.9 Hz, 1H, H-Ar), 6.76 (s, 1H, H-3),
6.71 (dd, J ¼ 9.2, 2.6 Hz, 1H, H-6), 6.56 (d, J ¼ 2.6 Hz, 1H, H-8), 3.46 (q, J ¼ 7.1 Hz,
4H, N(CH2CH3)2), 1.25 (t, J ¼ 7.1 Hz, 6H, N(CH2CH3)2); 13C NMR (100 MHz,
CDCl3, d, ppm): 163.7, 161.7, 161.0, 157.0, 151.0, 135.5, 135.0, 130.8, 130.2, 128.6,
125.8, 124.8, 123.3, 109.6, 109.3, 104.2, 98.0, 45.0 ꢀ 2, 12.4 ꢀ 2; HRESIMS: m/z
440.0612 [M þ H]þ (calcd for C21H19N3O3Br, 440.0610).
3.4.9. 4-(5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl)-7-(diethylamino)-2H-chromen-2-
one (4i)
1
Yield ¼ 66%, yellow solid, mp 220-222 ꢂC; H NMR (400 MHz, CDCl3, d, ppm): 8.70
(d, J ¼ 9.2 Hz, 1H, H-5), 8.03 (d, J ¼ 8.6 Hz, 2H, H-Ar), 7.72 (d, J ¼ 8.6 Hz, 2H, H-
Ar)), 6.75 (s, 1H, H-3)), 6.70 (dd, J ¼ 9.2, 2.4 Hz, 1H, H-6), 6.56 (d, J ¼ 2.4 Hz, 1H,
H-8), 3.46 (q, J ¼ 7.1 Hz, 4H, N(CH2CH3)2), 1.25 (t, J ¼ 7.1 Hz, 6H, N(CH2CH3)2);
13C NMR (100 MHz, CDCl3, d, ppm): 164.3, 161.6, 161.1, 157.1, 151.2, 135.1,
132.7 ꢀ 2, 128.7, 128.6 ꢀ 2, 127.4, 121.9, 108.4, 109.0, 103.9, 97.7, 44.8 ꢀ 2, 12.4 ꢀ 2;
HRESIMS: m/z 440.0613 [M þ H]þ (calcd for C21H19N3O3Br, 440.0610).
3.4.10.
7-(Diethylamino)-4-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)-2H-chromen-2-
one (4j)
1
Yield ¼ 52%, yellow solid, mp 259-261 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.90
(d, J ¼ 6.0 Hz, 2H, H-Ar), 8.67 (d, J ¼ 9.2 Hz, 1H, H-5), 8.02 (d, J ¼ 6.0 Hz, 2H, H-
Ar), 6.77 (s, 1H, H-3), 6.71 (dd, J ¼ 9.2, 2.6 Hz, 1H, H-6), 6.57 (d, J ¼ 2.6 Hz, 1H, H-
8), 3.47 (q, J ¼ 7.1 Hz, 4H, N(CH2CH3)2), 1.25 (t, J ¼ 7.1 Hz, 6H, N(CH2CH3)2); 13C
NMR (100 MHz, CDCl3, d, ppm): 163.2, 162.3, 160.9, 157.1, 151.3, 151.1 ꢀ 2, 134.8,
130.1, 128.4, 120.5 ꢀ 2, 109.5, 109.3, 103.8, 97.7, 44.8 ꢀ 2, 12.4 ꢀ 2; HRESIMS: m/z
363.1462 [M þ H]þ (calcd for C20H19N4O3, 363.1458).

10
X. YU ET AL.
3.4.11.
7-(Diethylamino)-4-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)-2H-chromen-2-
one (4k)
1
Yield ¼ 55%, yellow solid, mp 215-217 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.70
(d, J ¼ 9.2 Hz, 1H, H-5), 7.73 (d, J ¼ 1.7 Hz, 1H, H-Ar), 7.33 (d, J ¼ 3.6 Hz, 1H, H-
Ar), 6.75 (s, 1H, H-3), 6.70 (dd, J ¼ 9.2, 2.6 Hz, 1H, H-6), 6.68 (dd, J ¼ 3.6, 1.7 Hz,
1H, H-Ar), 6.56 (d, J ¼ 2.6 Hz, 1H, H-8), 3.46 (q, J ¼ 7.2 Hz, 4H, N(CH2CH3)2), 1.24
(t, J ¼ 7.2 Hz, 6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3, d, ppm): 161.1, 160.7,
157.7, 157.0, 151.2, 146.6, 138.7, 134.9, 128.6, 115.7, 112.5, 109.4, 109.0, 103.9, 97.6,
44.8 ꢀ 2,
12.4 ꢀ 2;
HRESIMS:
m/z
352.1306
[M þ H]þ
(calcd
for
C19H18N3O4, 352.1297).
3.4.12. 7-(Diethylamino)-4-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl)-2H-chromen-2-
one (4 l)
1
Yield ¼ 43%, yellow solid, mp 223-225 ꢂC; H NMR (600 MHz, CDCl3, d, ppm): 8.71 (d,
J¼ 9.2 Hz, 1H, H-5), 7.90 (dd, J¼ 3.8, 1.2 Hz, 1H, H-Ar), 7.66 (dd, J¼ 5.0, 1.2 Hz, 1H,
H-Ar), 7.23-7.25 (m, 1H, H-Ar), 6.74 (s, 1H, H-3), 6.70 (dd, J¼ 9.2, 2.6 Hz, 1H, H-6),
6.56 (d, J¼ 2.6 Hz, 1H, H-8), 3.46 (q, J¼ 7.2 Hz, 4H, N(CH2CH3)2), 1.25 (t, J¼ 7.2 Hz,
6H, N(CH2CH3)2); 13C NMR (100 MHz, CDCl3, d, ppm): 161.3, 161.2, 160.8, 157.0,
151.1, 135.0, 131.4, 131.0, 128.6, 128.5, 124.2, 109.4, 108.8, 104.0, 97.6, 44.8 ꢀ 2, 12.4 ꢀ 2;
HRESIMS: m/z 368.1077 [M þ H]þ (calcd for C19H18N3O3S, 368.1069).
3.5. In vitro biological assay
The anti-AChE activity of the 7-diethylaminocoumarin-based 1,3,4-oxadiazole deriva-
tives was determined by the method of Ellman. The in vitro inhibition assays of
AChE from electric eel run in phosphate buffer 0.1 M, at pH 7.4. Acetylthiocholine
iodide was used as substrates, and 5,50-Dithiobis(2-nitrobenzoic acid) (DTNB) was
used as the chromophoric reagent. To a 96-well plate of the sample solution (10 ll),
phosphate buffer solution (PBS) (40 ll, 0.1 M, pH ¼ 7.4), DTNB (20 ll, 2.5 mM in
0.1 M PBS, pH ¼ 8.0) and AChE solution (10 ll, 0.2 U/ml in 0.1 M PBS, pH ¼ 7.4)
were added sequentially, shaken well, and incubated at 37 ꢂC for 10 min. Then, ace-
tylthiocholine iodide (20 ll, 10 mM in 0.1 M PBS, pH ¼ 7.4) was added, shaken well
again, and incubated at 37 ꢂC for 10 min. The absorbance at 405 nm of the samples
was measured using a spectrophotometer, the sample solution was set to 1 lmol/ml
and the experiment was repeated three times. Tacrine was used as a positive control.
Inhibitory effect (%)¼ [OD0ꢁ(OD1ꢁOD2)]/OD0ꢀ100%, where OD0 is the absorbance
of blank group; OD1 is the absorbance of sample group; OD2 is the absorbance of
sample blank group.
3.6. Molecular docking
The 3 D-structure of human AChE was downloaded from the RCSB database (PDB
ID: 3LII), a chain A of the structure was used for docking study. Then, Autodock
Tool was used to determine the atom types and calculate the partial charges of the
protein, and the pdbqt file was generated for docking.The 2 D structure of compound

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
4 g was drawn by ChemDraw15.0, and saved as cdx file. Then, MM2 force field in
Chem3D 15.0 was used to optimize the 3 D structure of the ligand. Again, Autodock
Tool was used to determine the atom types and calculate the partial charges of the
ligand, and pdbqt file was generated for docking. Docking of the ligand was carried
out using the Autodock vina 1.1.2 program. A sphere of 20 Å around the carbonyl
group of Gly122 was defined as the binding site for the ligand docking and 250 con-
firmations was allowed. Pymol was used to analyze the docking solutions, and the
conformation of lowest affinity value was chosen for further analysis in ligplot.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Science and Technology Fund of Guizhou [QKH-
PTRC(2017)5735-22], Guizhou Provincial Natural Science Foundation [QKH-J(2020)1Y070]
and the Youth Talent Development Project of Guizhou Provincial Department of
Education [2017]169.
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