Synthesis, characterization, crystal structure and evaluation of four carbazole-coumarin hybrids as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.molstruc.2020.127897

Coupling of two distinct pharmacophores, carbazole and coumarin, endowed with different biological properties, afforded four hybrid compounds. The structures of the carbazole-coumarin hybrids were characterized by FT-IR, NMR, HRMS and single-crystal X-ray diffraction studies. All of these compounds exhibited significant acetylcholinesterase inhibitory activities. Among them, compound 4-((5-(9H-carbazol-9-yl)pentyl)oxy)-2H-chromen-2-one (3d) exhibited the best inhibition activity with IC₅₀ of 3.75 μM for acetylcholinesterase from electric eel and 70.51 μM for human recombinant acetylcholinesterase. Moreover, the compound 7-(3-(9H-carbazol-9-yl propoxy)-4-methyl-2H-chromen-2-one (3a) had the best antioxidant activity. The docking studies demonstrated that compound 3d could interact with both the catalytic active site and the peripheral anionic site of acetylcholinesterase. These attributes imply carbazole-coumarin hybrids as multifunctional agents for the Alzheimer's disease treatment.

Full text of DOI:10.1016/j.molstruc.2020.127897

Journal Pre-proof
Synthesis, characterization, crystal structure and evaluation of four carbazole-
coumarin hybrids as multifunctional agents for the treatment of Alzheimer's disease
Da-Hua Shi, Wei Min, Meng-qiu Song, Xin-Xin Si, Ming-Cheng Li, Zhao-yuan Zhang,
Yu-Wei Liu, Wei-Wei Liu
PII:
S0022-2860(20)30221-0
DOI:
https://doi.org/10.1016/j.molstruc.2020.127897
MOLSTR 127897
Reference:
To appear in: Journal of Molecular Structure
Received Date: 15 October 2019
Revised Date: 1 February 2020
Accepted Date: 12 February 2020
Please cite this article as: D.-H. Shi, W. Min, M.-q. Song, X.-X. Si, M.-C. Li, Z.-y. Zhang, Y.-W. Liu, W.-
W. Liu, Synthesis, characterization, crystal structure and evaluation of four carbazole-coumarin hybrids
as multifunctional agents for the treatment of Alzheimer's disease, Journal of Molecular Structure (2020),
doi: https://doi.org/10.1016/j.molstruc.2020.127897.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. 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.
©
2020 Published by Elsevier B.V.

Credit author statement
Da-Hua Shi: Conceptualization, Methodology, Software. Wei Min:
synthesis, Writing- Original draft preparation. Meng-qiu Song:
ChE-inhibition activity. Xin-Xin Si: ChE-inhibition activity.
Ming-Cheng Li: antioxidant activity. Zhao-yuan Zhang: synthesis.
Yu-Wei Liu: Molecular modeling study. Wei-Wei Liu: X-ray diffraction
analysis.

Synthesis, characterization, crystal structure and evaluation of four
carbazole-coumarin hybrids as multifunctional agents for the
treatment of Alzheimer's disease
a,b,
ꢀ
a
a
a
a
Da-Hua Shi
, Wei Min , Meng-qiu Song , Xin-Xin Si , Ming-Cheng Li ,
a
a
a
Zhao-yuan Zhang , Yu-Wei Liu and Wei-Wei Liu
a School of Pharmacy, Jiangsu Ocean University, Lianyungang 222005, People’s
Republic of China
b Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Lianyungang
2
22005, People’s Republic of China
Abstract
Coupling of two distinct pharmacophores, carbazole and coumarin, endowed with
different biological properties, afforded four hybrid compounds. The structures of the
carbazole-coumarin hybrids were characterized by FT-IR, NMR, HRMS and
single-crystal X-ray diffraction studies. All of these compounds exhibited significant
acetylcholinesterase
-((5-(9H-carbazol-9-yl)pentyl)oxy)-2H-chromen-2-one (3d) exhibited the best
inhibition activity with IC of 3.75 ꢀM for acetylcholinesterase from electric eel and
inhibitory
activities.
Among
them,
compound
4
50
7
0.51 ꢀM for human recombinant acetylcholinesterase. Moreover, the compound
-(3-(9H-carbazol-9-yl propoxy)-4-methyl-2H-chromen-2-one ( 3a ) had the best
7
antioxidant activity. The docking studies demonstrated that compound 3d could
interact with both the catalytic active site and the peripheral anionic site of
acetylcholinesterase. These attributes imply carbazole-coumarin hybrids as
multifunctional agents for the Alzheimer's disease treatment.
Keywords: carbazole-coumarin hybrids, acetylcholinesterase, antioxidant,
Alzheimer’s disease
ꢀ
Correspondent. E-Mailꢁshidahua@jou.edu.cn
1

1
. Introduction
Alzheimer's disease (AD, MIM No. 104300) is an irreversible brain-producing
disorder and the most common type of dementia (60-80%)[1]. Although the exact
cause of AD remains unknown, several factors including low levels of
neurotransmitter acetylcholine (ACh), the aggregation of β-amyloid (Aβ) peptide,
dyshomeostasis of biometals, hyperphosphorylation of tau protein, and oxidative
stress, are suggested to play important roles in the pathogenesis of AD[2, 3].The
numerous results achieved for AD treatment, but few goes to clinic trials or offers
satisfied outcomes.
Cholinesterase inhibitors (ChEIs) are the well-studied compounds for the
treatment of AD based on the cholinergic hypothesis. These drug candidates are prior
options for patients with mild to moderate AD, and can alleviate symptom of
cognitive dysfunction[4, 5]. The main function of Cholinesterases (ChE) including
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), is to hydrolyze
cholinergic neurotransmitters[6]. AChE can also induce Aβ aggregation (another
marker of AD) through direct interaction of its PAS with Aβ peptide. According to
X-ray structural analysis, there is a narrow and deep canyon in the active site of AChE.
Two different binding sites are included: the catalytic active site (CAS) at the bottom
of the canyon and the peripheral anion binding site (PAS) at the entrance to the
canyon[7]. Accordingly, AChE inhibitors can be categorized as single- and two-site
inhibitors[7, 8]. It is believed that AChE inhibitors interacting with both CAS and
PAS appear to be more beneficial for AD treatment[8, 9].
Antioxidants have drawn much attention recently in AD treatment. Increases of
damaged macromolecules such as nucleic acids, proteins, lipids have been observed
in AD brains. These findings support reactive oxygen species (ROS) plays a pivotal
role in the pathogenesis of AD[10]. Therefore, antioxidants should have great benefits
in the treatment of AD[11].
Due to the complex nature of AD, most of the single-target directed drug
candidates failed to reach clinical trials. Thus, multi-target-directed ligands (MTDLs)
are considered as an effective alternative for the treatment of AD[12, 13]. Various
2

coumarin-based hybrids possessing diverse medicinal attributes were synthesized[14].
Studies have also shown that both naturally occurring and synthetic coumarin
analogues exhibit potent AChE, BuChE, dual AChE/BuChE and monoamine oxidase
(MAO) inhibitory activity[15-17]. More and more coumarin core contained MTDLs
with AChE inhibitory activity have been designed and synthesized [2, 18-22].
Carbazoles are naturally occurring phytochemicals in many plants with a wide range
of biological activities. Some carbazole derivatives possessed AD-related biological
activities[23]. It is reported that naturally occurring carbazole derivatives have strong
antioxidant capacity and can directly scavenge ROS[24]. In addition, studies have
shown that carbazole derivatives can inhibit Aβ aggregation[25]. It is reported that
carbazole derivatives which could be regarded as the D-ring opened analogs of
galantamine could inhibit ChE and protect neurons from the toxicity induced by
β-amyloid[26]. Many carbazole derivatives were designed as MTDLs for the
treatment of AD[27-29]. In the present study, we synthesized and evaluated four
carbazole-coumarin hybrids as MTDLs for the treatment of AD.
2
. Experimental
2
.1. General instrumentations and materials
All synthetic reagents with AR grade were purchased from Aladdin Industrial
Corporation and were used as received. AChE from electric eel (eeAChE) and human
recombinant AChE (hAChE), BuChE from equine serum (eqBuChE) and human
serum (hBuChE) were purchased from Sigma-Aldrich. TLC was performed on the
glassbacked silica gel sheets (Silica Gel 60 GF254) and visualized in UV light (254
nm). All the melting points were determined by Kohler melting point apparatus. The
NMR spectra were recorded in CDCl solvent on a Bruker Avance 600 MHz
3
instrument. Infrared (IR) spectra were recorded on a Thermo Scientific Nicolet iS10
spectrometer in KBr. High resolution mass spectra (HRMS) were recorded using
HPLC 1260-6230 TOF MASS. The X-ray single crystal diffraction data were
recorded on a Bruker SMART APEX-II CCD diffractometer.
2
.2. General procedure for the synthesis of compounds 3a – 3d
The synthetic routes to final compounds 3a – 3d were depicted in Schemes 1. A
3

mixture of carbazole (1.79 mmol), α,ω- dibromoalkane (7.2 mmol),
tetrabutylammonium bromide (TBAB) (1.79 mmol) were combined in benzene (4 mL)
and NaOH solution (0.5 M, 4 mL). The reaction mixture was stirred at 55 � for 6 h.
After cooling, ethyl acetate (20 mL) was added to the solution, then the solution
washed with saturated sodium chloride (20 mL) solution three times. The ethyl
acetate phase was dried over anhydrous sodium sulfate and evaporated under reduced
pressure to yield the crude product that was purified by column chromatography using
petroleum ether and ethyl acetate (4:1) as an eluent to obtain 2a and 2b. A mixture of
2
a or 2b (1.2 mmol), K CO (0.6 mmol), coumarin (3.6 mmol) were combined in
2 3
DMF (4 mL). The mixture was stirred at 55 � for 3 h and ethyl acetate (20 mL) was
added to the solution, then the solution washed with saturated sodium chloride (20 mL)
solution three times. The ethyl acetate phase was dried over anhydrous sodium sulfate
and evaporated under reduced pressure to yield the crude product that was purified by
column chromatography using petroleum ether and ethyl acetate (2:1) as an eluent to
obtain compound 3a – 3d.
Schemes 1. Synthesis of final compounds 3a – 3d. Reagents and conditions: (a)
α
,ω- dibromoalkane, TBAB, NaOH, benzene, 55℃, 6h; (b) coumarin, anhydrous
K CO , DMF, 55℃, 3h.
2
3
4

2
.2.1. 9-(3-bromopropyl)-9H-carbazole (2a)
White solid; yield 56%; mp 40 – 42 � ; IR (KBr): 3425, 3046, 2963, 2359, 2358,
-1 1
2
339, 1928, 1891, 1769, 1626 cm . H NMR (500 MHz, Chloroform-d) δ 8.10 (d, J =
.8 Hz, 2H), 7.51 – 7.44 (m, 4H), 7.25 – 7.21 (m, 2H), 4.49 (t, J = 6.5 Hz, 2H), 3.37 (t,
7
13
J = 6.2 Hz, 2H), 2.42 (p, J = 6.4 Hz, 2H). C NMR (126 MHz, Chloroform-d) δ 140.3,
1
25.8, 122.9, 120.4, 119.1, 108.6, 40.9, 31.9, 30.9.
2
.2.2. 9-(5-bromopentyl)-9H-carbazole (2b)
White solid; yield 60%; mp 53 – 55 � ; IR (KBr): 3440, 3047, 2924, 2859, 2359,
-
1 1
2
7
4
–
340, 1936, 1898, 1782, 1625 cm . H NMR (500 MHz, Chloroform-d) δ 8.09 (d, J =
.7 Hz, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.22 (t, J = 7.4 Hz, 2H),
.28 (t, J = 7.2 Hz, 2H), 3.32 (t, J = 6.7 Hz, 2H), 1.85 (dp, J = 14.3, 7.1 Hz, 4H), 1.53
1
3
1.46 (m, 2H). C NMR (126 MHz, Chloroform-d) δ 140.3, 125.7, 122.8, 120.4,
118.8, 108.6, 42.8, 33.3, 32.5, 28.2, 25.9.
2
.2.3. 7-(3-(9H-carbazol-9-yl propoxy)-4-methyl-2H-chromen-2-one (3a)
White powder; yield 64%; mp 197 – 199 � ; IR (KBr): 3059, 2931, 2877, 2360,
-
1 1
2
7
6
1
341, 1706, 1610 cm . H NMR (600 MHz, Chloroform-d) δ 8.10 (d, J = 7.8 Hz, 2H),
.48 (d, J = 8.9 Hz, 1H), 7.42 – 7.37 (m, 4H), 7.22 (ddd, J = 7.9, 6.0, 2.1 Hz, 2H),
.83 (dd, J = 8.8, 2.4 Hz, 1H), 6.74 (d, J = 2.4 Hz, 1H), 6.16 – 6.12 (d, J = 1.1 Hz,
1
3
H), 4.57 (t, J = 6.5 Hz, 2H), 3.93 (t, J = 5.6 Hz, 2H), 2.40 (m, 5H). C NMR (151
MHz, Chloroform-d) δ 161.6, 161.3, 155.2, 152.5, 140.4, 125.8, 125.6, 122.9, 120.4,
19.1, 113.8, 112.3, 112.1, 108.4, 101.6, 65.1, 39.2, 28.5, 18.7. HRMS: (ESI, m/z):
1
+
[
M+Na] calcd for C H NO 406.1414, found 406.1429.
25 21 3
2
.2.4. 7-(5-(9H-carbazol-9-yl)butoxy)-4-methyl-2H-chromen-2-one (3b)
White solid; yield 61%; mp 150 – 152 � ; IR (KBr): 3094, 2942, 2867, 2358,
-
1 1
2
7
6
1
1
337, 1704, 1608 cm . H NMR (600 MHz, Chloroform-d) δ 8.10 (d, J = 7.7 Hz, 2H),
.48 – 7.43 (m, 3H), 7.41 (d, J = 8.2 Hz, 2H), 7.23 (ddd, J = 7.9, 7.0, 0.9 Hz, 2H),
.77 (dd, J = 8.8, 2.5 Hz, 1H), 6.74 (d, J = 2.5 Hz, 1H), 6.12 – 6.11 (d, J = 1.2 Hz,
H), 4.34 (t, J = 7.1 Hz, 2H), 3.94 (t, J = 6.3 Hz, 2H), 2.37 (d, J = 1.2 Hz, 3H), 1.98 –
1
3
.93 (m, 2H), 1.85 – 1.81 (m, 2H), 1.56 (m, 2H). C NMR (151 MHz, Chloroform-d)
δ 161.9, 161.3, 155.1, 152.5, 140.3, 125.6, 125.4, 122.8, 120.3, 118.7, 113.4, 112.4,
5

+
111.8, 108.5, 101.3, 68.1, 42.8, 28.8, 28.7, 23.7, 18.6. HRMS: (ESI, m/z): [M+Na]
calcd for C H NO 434.1727, found 434.1741.
2
7
25
3
2
.2.5. 4-(3-(9H-carbazol-9-yl)propoxy)-2H-chromen-2-one (3c)
White solid; yield 52%; mp 200 – 202 � ; IR (KBr): 3068, 2933, 2882, 2360,
-1 1
2
337, 1705, 1626 cm . H NMR (600 MHz, CDCl ) δ 8.08 (d, J = 7.7 Hz, 2H), 7.77
3
(dd, J = 7.9, 1.6 Hz, 1H), 7.58 (ddd, J = 8.6, 7.2, 1.6 Hz, 1H), 7.42 – 7.28 (m, 6H),
7
5
1
1
3
2
.22 (ddd, J = 7.9, 6.6, 1.5 Hz, 2H), 5.46 (s, 1H), 4.59 (t, J = 6.5 Hz, 2H), 4.02 (t, J =
13
.7 Hz, 2H), 2.49 (p, J = 6.1 Hz, 2H). C NMR (151 MHz, Chloroform-d) δ 165.1,
62.7, 153.3, 140.2, 132.5, 125.9, 124.0, 123.0, 122.7, 120.6, 119.3, 116.9, 115.5,
+
08.2, 90.8, 66.3, 39.3, 28.0. HRMS: (ESI, m/z): [M+Na] calcd for C H NO
24
19
3
92.1257, found 392.1262.
.2.6. 4-((5-(9H-carbazol-9-yl)pentyl)oxy)-2H-chromen-2-one (3d )
White solid; yield 61%; mp 140 – 142 � ; IR (KBr): 3061, 2946, 2923, 2859,
-
1 1
2
2
2
1
–
1
6
4
2
358, 2339, 1716, 1620 cm . H NMR (600 MHz, CDCl ) δ 8.01 (dd, J = 7.7, 1.0 Hz,
3
H), 7.54 (dd, J = 7.9, 1.6 Hz, 1H), 7.47 – 7.43 (m, 1H), 7.37 (td, J = 7.6, 7.1, 1.2 Hz,
H), 7.32 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.6 Hz, 1H), 7.16 – 7.12 (m, 3H), 5.49 (s,
H), 4.28 (t, J = 6.9 Hz, 2H), 3.94 (td, J = 6.2, 2.4 Hz, 2H), 1.95 – 1.89 (m, 2H), 1.82
1
3
1.78 (m, 2H), 1.51 – 1.46 (m, 2H). C NMR (151 MHz, Chloroform-d) δ 164.4,
61.9, 152.2, 139.2, 131.2, 124.6, 122.8, 121.7, 119.3, 117.8, 115.6, 114.5, 107.4, 89.3,
+
7.8, 41.6, 27.6, 27.2, 22.6. HRMS: (ESI, m/z): [M+Na] calcd for C H NO
26
23
3
20.1570, found 420.1584.
.3. Single crystal XRD studies
Crystals of compound 3d was grown by slow evaporation of methanol at room
temperature. Diffraction intensities for the compounds were collected at 296(2) K
using a Bruker SMART APEX-II CCD area-detector with MoK radiation (λ =
.71073 Å). The collected data were reduced with the SAINT program , and
α
[30]
0
multi-scan absorption corrections were performed using the SADABS program[31].
2
Structures were solved by direct methods. The compounds were refined against F by
full-matrix least-squares methods using the SHELXTL package[32]. All of the
non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed in
6

calculated positions and constrained to ride on their parent atoms. The Mercury
programs[33] were used to describe the molecular structures.
2
.4. Hirshfeld surfaces analysis
Analysis of Hirshfeld surfaces and their associated two dimensional fingerprint
plots of compound 3d were calculated by using CrystalExplorer17[34]. The Hirshfeld
surfaces are mapped with different properties dnorm, shape index, curvedness. The
dnorm is normalized contact distance, defined in terms of d , d and the vdW radii of the
e
i
atoms. The combination of d and d in the form of a 2D fingerprint plot displays
e
i
summary of intermolecular contacts in the crystal.
2
.5. In vitro ChE activity assay
The ChE inhibitory activities of test compounds 3a – 3d were measured using
Ellman’s colorimetric method with a slight modification[35]. The Ellman's assays
were performed in 96 well microplate using eeAChE, hAChE, eqBuChE, hBuChE.
An AChE and BuChE was dissolved in 0.1 M phosphate-buffered saline (PBS, pH 8.0)
to obtain a solution of 0.35 U/mL. The compounds were dissolved in methanol and
diluted with the 0.1 M phosphate-buffered saline (PBS, pH 8.0) to yield
corresponding test concentrations (methanol less than 5%). In assays, 20 ꢀL of AChE
or BuChE were incubated with 10 ꢀL of tested compounds and 130 ꢀL of 0.1 M PBS
(pH 8.0) for 10 min in 96-well microplates before addition of 20 ꢀL of 3.33 mM
DTNB solution and 20 ꢀL of 5.30 mM ATCI (or BTCI) solution. After the addition of
DTNB and ATCI (or BTCI), the 96-well microplates were read at 412 nm with a
microplate reader (SPECTRAFLUOR, TECAN, Sunrise, Austria) for 15 min One
triplicate sample without inhibitors was always present to yield 100% of AChE or
BuChE activity. The reaction rates were compared and the percent inhibition due to
the presence of tested compounds was calculated. Tacrine was used as a positive
control. All samples were assayed in triplicate.
2
.6. Molecular Modeling Evaluations
The pdb structure of AChE (PDB ID: 2CMF) was obtained from the RCSB
Protein Data Bank (www.rcsb.org/pdb). Docking simulations were performed on the
compound 3a - 3d with Molecular Operating Environment (MOE)[36] using the
7

CHARMm force field. Enzyme structure of 2CMF was checked for missing atoms,
bonds and contacts. Hydrogens and partial charges were added using the protonate 3D
application in MOE. The compound 3a - 3d was drawn in MOE then protonated using
the protonate 3D protocol and energy was minimized using the MMFF94×force field
in MOE. After the enzyme and the ligand were ready for the docking study,
compound 3a - 3d was docked into the active site of the protein by the “Triangle
Matcher” method. The Dock scoring in MOE software was done using ASE scoring
function and forcefield was selected as the refinement method. The best 10 poses of
molecules were retained and scored. After docking, the geometry of resulting complex
was studied using the MOE's pose viewer utility.
2
.7. Antioxidant activity assay
The antioxidant potency was evaluated by the oxygen radical absorbance
capacity-fluorescein
-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and 2,2′-Azobis
amidinopropane) dihydrochloride (AAPH) was purchased from Aladdin Industrial
(ORAC-FL)
assay[37].
Fluorescein
(FL),
6
(
Corporation. All the assays were performed with 75 mM phosphate buffer (pH = 7.40),
and the final reaction mixture was 200 ꢀL. FL (120 ꢀL, 150 nM final concentration)
and antioxidant (20 ꢀL) were added in the wells of a black 96-well plate using Trolox
as a standard (1–8 ꢀM, final concentration). The plate was incubated for 20 min at
3
7 °C and AAPH solution (60 ꢀL, 12 mM final concentration) was added rapidly.
Then the plate was placed in a microplate reader (SPECTRAFLUOR, TECAN,
Sunrise, Austria) and the fluorescence recorded every minute for 90 min with
excitation at 485 nm and emission at 535 nm. The plate was automatically shaken
prior to each reading. A blank (FL + AAPH) using phosphate buffer instead of
antioxidant and Trolox calibration were carried out in each assay. The samples were
measured at different concentrations (1–10 ꢀM). All the reaction mixture was
prepared in duplicate, and at least three independent assays were performed for each
sample. Antioxidant curves (fluorescence versus time) were normalized to the curve
of the blank in the same assay, and then the area under the fluorescence decay curve
(AUC) was calculated. The net AUC of a sample was obtained by subtracting the
8

AUC of the blank. ORAC-FL values were expressed as Trolox equivalents by using
the standard curve calculated for each sample, where the ORAC-FL value of Trolox
was taken as 1, indicating the antioxidant potency of the tested compounds.
3
. Result and discussion
3
.1. Spectral characterization
The route for the synthesis of the desired compounds 3a-d is illustrated in
Scheme 1. Carbazole dimers in the first steps of the synthesis were observed. To
increase the yields of the compound 2a and 2b and decrease the yields of dimers
excessive α,ω- dibromoalkanes were used. The yields of the compound 2a and 2b
were 56% and 60% respectively. All crude products 3a-3d were purified by silica gel
column chromatography. Purity of the obtained compounds was checked by the TLC.
The compounds were characterized by FT-IR, NMR, HRMS techniques and X-ray
crystallography. All the result substantiates the structures proposed.
The FT-IR spectrum of the prepared compounds are showed by a strong broad
-1
absorption at the range of 1608-1626 cm which can be assigned to the υ (C=O)
-1
stretches. The absorption bands falling in the range of 2859-2946 cm are
corresponding to the υ (C-H) of the alkane chain for compounds 3a, 3b, 3c and 3d.
-1
The mode of υ (C-H) appears at the range of 3059-3094 cm suggests the presence of
1
the aromatic benzene rings of the three compounds. In the H NMR spectrum of the
prepared compounds, displayed the proton of the lactone ring of the coumarin ring at
6
.14 ppm, 6.12 ppm, 5.46 ppm and 5.49 ppm respectively for compound 3a, 3b, 3c
and 3d, and that proton is coupled with a methyl group on the lactone ring and split
into a doublet respectively for compound 3a and 3b. And for 3c and 3d, that proton is
a single peak. The appeared signals of all the protons of the compound 3a-3d were
1
3
found as to be in their expected region. In the C NMR spectra the characteristic
phenyl signals are observed at 107.4 ppm to 165.1 ppm for compounds 3a, 3b, 3c and
3
d. The signals of the carbonyl are found at 161.6 ppm, 161.9 ppm, 165.1ppm and
64.4 ppm for compounds 3a, 3b, 3c and 3d respectively. The carbons of alkyl chains
1
are observed at 18.6 ppm to 68.1 ppm for the four compounds. The HRMS spectrum
+
of compound 3a-3d show [M+H] peak at m/z : 384.1593, 412.1902, 370.1439 and
9

+
+
3
98.1755 corresponding to C H NO [M+H] , C H NO [M+H] , C H NO
25 21 3 27 25 3 24 19 3
+
+
[
M+H] and C H NO [M+H] for compound 3a, 3b, 3c and 3d respectively.
26 23 3
3
.2. Crystal structure of the compound 3d
The structure of compounds 3d was demonstrated by X-ray diffraction analysis.
The crystal data was presented in Table 1, Figure 1 and Figure 2 has been deposited
at the Cambridge Crystallographic Data Centre as supplementary publication no.
CCDC 1961036. The compound 3d had the crystal system of monoclinic and the
space group of P2 /c. The selected bond lengths and angles of the compound were
1
listed in Table 2. The bond lengths of O(3)-C(3) and O(3)-C(10) were 1.3413(19) Å
and 1.4373(19) Å respectively. The bond length of N(1)-C(14) for compound 3d was
1
.457(2) Å. The packing diagram of the compound 3d viewed along the axis is
showed in Figure 2. The crystal packing diagrams of the compound showed that there
is no hydrogen bond for the compound.
Table 1. Crystallographic data for the compound 3d.
Compound
Formula
3d
C H NO
3
26
23
FW
397.45
Crystal shape/colour
Crystal size (mm)
T (K)
plate/colourless
0.24×0.22× 0.12
296(2)
λ (MoKα) (Å)
Crystal system
Space group
a (Å)
0.71073
monoclinic
P2 /c
1
9.174(3)
23.827(7)
9.558(3)
90
b (Å)
c (Å)
α (º)
β
γ
(º)
(º)
101.444(5)
90
1
0

3
V (Å )
2047.8(11)
4
Z
–
1
µ
(MoKα) (mm )
0.084
Tmin
Tmax
0.9521
0.9622
1.289
–
3
D (g cm )
c
Measured reflections
12325
Unique reflections and Rint
Observed reflections
4661 and 0.0417
1740
data/restraints/parameters
4661/0/271
0.966
2
Goodness of fit on F
R1 [I ≥ 2
σ
(I)]
(I)]
0.0462
0.0983
0.1037
0.1195
wR [I ≥ 2
σ
2
R1 (all data)
wR (all data)
2
–
3
Large diff. peak and hole (e Å )
0.13/-0.18
Figure 1. ORTEP of the compound 3d with thermal ellipsoids drawn at 50%
probability.
11

Figure 2. Packing diagrams of the compound 3d viewed along the axis.
o
Table 2. Selected bond lengths (Å) and angles ( ) for the compound 3d.
Bond lengths
O(1)-C(1)
1.380(2)
1.374(2)
1.207(2)
1.3413(19)
O(3)-C(10)
N(1)-C(14)
N(1)-C(15)
N(1)-C(26)
1.4373(19)
1.457(2)
1.389(2)
1.382(2)
O(1)-C(5)
O(2)-C(1)
O(3)-C(3)
Bond angles
C(1)-O(1)-C(5)
O(1)-C(1)-O(2)
C(2)-C(1)-O(2)
C(3)-O(3)-C(10)
121.47(14)
116.26(18)
126.1(2)
C(2)-C(3)-O(3)
125.74(16)
125.64(15)
125.81(15)
108.42(14)
C(14)-N(1)-C(15)
C(14)-N(1)-C(26)
C(15)-N(1)-C(26)
117.08(13)
3
.3. Hirshfeld surface analysis
Hirshfeld surfaces and finger print plots were generated for the compound 3d to
understand the different intermolecular interactions and packing modes. The dnorm
mapped on Hirshfeld surface and selected 2D finger print plots of different interaction
of the compound are shown in Figure 3 and Figure 4. For the compound 3d, the red
regions mainly indicate C···H and O···H interactions. The dnorm map of compound
shows four pairs of adjacent deep-red regions which demonstrate the C-H···O and
1
2

C-H···C intermolecular interactions forming corresponding crystal packing pattern.
One pairs of the deep-red regions demonstrate the strong C-H···O intermolecular
interactions between the carbonyl of the coumarin part and the carbazole part of
another molecule. One pairs of the deep-red regions demonstrate the strong C-H···O
intermolecular interactions between the carbonyl of the coumarin part and the
coumarin part of another molecule. The other two pairs of the deep-red regions
demonstrate the C-H···C intermolecular interactions of the carbazole part with the
carbazole part and the linker respectively.
The 2D fingerprint plots of the main intermolecular contacts for compound 3d
are showed in Figure 4. H···H intermolecular contact with contribution of 47.7% of
the overall contacts for the compound is the major contributor to the Hirshfeld surface.
The C···H intermolecular contact being one of the strong contacts with contribution
of 32.0% due to the presence of C-H···C intermolecular interactions. The O···H
intermolecular contact is another strong contact for compound 3d with the
contribution of 15.0% due to the presence of C-H···O intermolecular interactions.
Besides the contacts mentioned above, the presence of C···C, N···H, C···O and other
interactions are summarized in Table 3.
Figure 3 Hirshfeld surface mapped with dnorm for the compound 3d.
1
3

Figure 4. The 2D fingerprint plots of the compounds 3d.
Table 3.
The percentages of the various interactions contributions to the total Hirshfeld surface
area of the compound 3d.
Interactions
Percentage (%)
…
H H
47.7
32.0
15.0
0.9
…
C H
…
O H
…
N H
…
C C
3.2
…
C O
0.8
…
C N
0.0
1
4

…
O N
0.0
0.5
0.0
…
O O
…
N N
3
.4 In vitro inhibition of ChEs
The inhibitory activities against ChEs were determined according to Ellman's
method using tacrine as reference compounds[35]. The obtained IC values (in ꢀM)
5
0
were presented in Table 4. Four of the compounds showed the eeAChE-inhibition
activities and could inhibit the eeAChE dose dependently (Figure 5). Compound 3d
with IC50 of 3.75 ꢀM showed the best activity against eeAChE. While the hAChE
inhibitory activities of the four compounds were weak. Only compound 3b and 3d
could inhibited hAChE more than 30% at the concentration of 50 ꢀM with the IC of
5
0
7
6.63 ꢀM and 70.51 ꢀM respectively (Figure 6). Compound 3d showed potent
eeAChE inhibitory activity which was 18.8-fold more efficient for inhibition of
hAChE. It is reported that most ChEIs exhibit the inhibitory activity for hChEs in the
same range as that for animal ChEs. While the inhibitory activities on hChEs and
animal ChEs of some compounds are very different[2, 18, 38]. None of the four could
inhibit eqBuChE or hBuChE more than 30% at the concentration of 50 ꢀM. So,
compound 3b and 3d showed high selectivity for AChE over BuChE. These
compounds would result in lesser degree of associated side effects and might be more
beneficial for AD treatment because that BuChE was mainly localized in the
peripheral tissues and very small amount was present in the brain region[39]. It can be
seen that the inhibitory activities of the carbazole-coumarin hybrids on AChE is
related to both the type of coumarin substituents and the length of the alkyl chain. It is
reported that the linker length of hybrids connected the CAS and PAS binding
moieties is critical for ChE inhibition[18, 40]. In this study, the inhibitory activities of
the hybrids on AChE generally increases as the alkyl chain becomes longer. When the
alkyl chain length is 5, the compounds (3b and 3d) possessed better AChE inhibitory
activities than that of compounds with alkyl chain length of 3 (3a and 3c). The AChE
inhibitory activities of hybrids with 4-hydroxycoumarin substituted product are better
than that of 4-methylumbelliferone substitution. It is also reported that the compounds
1
5

with phenylpiperazine substitutions on the positions 4 of coumarin ring are better than
that of the 6-substituted coumarins[41].
Figure 5. Dose-dependent inhibition of 3a (A), 3b (B), 3c (C) and 3d (D) against
eeAChE. Values are means ± SD, n=5.
Figure 6. Dose-dependent inhibition of 3b (A) and 3d (B) against hAChE. Values are
means ± SD, n=5.
Table 4. ChEs inhibitory activities and ORAC of target compounds.
a
IC (ꢀM)
5
0
c
Compd. n
ORAC
eeAChE
eqBuChE
hAChE
hBuChE
b
3
3
3
3
a
b
c
d
3
5
3
5
32.31±3.18 N/A
4.37±0.02 N/A
5.36±0.61 N/A
3.75±0.12 N/A
N/A
N/A
0.96±0.018
0.86±0.008
0.86±0.001
0.81±0.003
76.63±7.51 N/A
N/A N/A
70.51±4.66 N/A
1
6

Tacrine
-
-
0.18±0.01 N/A
0.23±0.07 0.04±0.002 -
Trolox
-
-
-
-
1
a
The 50% inhibitory concentration (means ± SD of three experiments) of ChE.
The compound could not inhibit the ChE more than 30% at the concentration of 50
M.
b
ꢀ
c
Results are expressed as ꢀM of Trolox equivalent/ꢀM of tested compounds.
.5 Antioxidant activity
3
The antioxidant activities of all synthesized target compounds were determined
by following the well-established ORAC-FL method (oxygen radical absorbance
capacity by fluorescein) [37] and the results were shown in Table 4. Trolox, a vitamin
E analogue, was used as the standard, and the results were expressed as Trolox
equivalents. As expected, there were no oxygen radical-trapping groups in all of the
compound structures, and the tested compounds did not exhibit favorable antioxidant
capacity.
3
.6 Molecular modeling study
To understand the binding mode of the hybrids 3a - 3d with the AChE, the
compounds were docked with AChE (PDB code:2CMF) with MOE program. The
molecule docking of the compound 3a - 3d with AChE showed that the compound 3b
and 3d could interact with the CAS and the PAS of AChE as that of bis-tacrine
(Figure 7B and 7D) with docking score of −34.80, and −34.65 kcal/mol respectively.
While, compound 3a and 3c only interacted with the CAS of the enzyme with
docking scores of −27.20 and −32.76 kcal/mol respectively (Figure 7A and 7C). The
docking results are along with the in vitro AChEs-inhibition activities of the hybrids.
It can be seen from the docking results that the length of the alkyl chain effected the
activities of the hybrids. When the alkyl chain length is 5, the hybrids (3b and 3d)
could interact with both the CAS and the PAS of AChE and possessed potent
AChE-inhibition activities. When the alkyl chain length is 3, the hybrids (3a and 3c)
only interacted with the CAS of AChE and showed weak AChE inhibitory activities.
When compound 3d which possessed the best AChE inhibitory activity was docked
with AChE, the compound could interact with both the CAS and the PAS of the
1
7

enzyme like the bis-tacrine AChE (Figure 8, bis-tacrine was showed in green color).
The carbazole part and coumarin part of the hybrids bind with CAS and PAS,
respectively. The carbazole moiety was located between TRP84 and PHE330 in the
PAS of the enzyme via the arene-arene interaction. The coumarin moiety could bind
to the PAS between TRP279 and Tyr70 as reported previously[7, 42]. The carbonyl of
the coumarin part could interact with TYR70 via the hydrogen bonds. The benzene
ring of the coumarin part could interact with TRP279 via arene-arene interactions. It
is reported that the AChEI that interacted with PAS can interfere Aβ aggregation.
Therefore, the hybrid 3a-3b should be able to enhance cholinergic tone by reducing
the hydrolytic activity of AChE and decreasing the deposition of Aβ fibrils[28].
1
8

1
9

Figure 7. The AChE interaction map displaying the binding and interactions of
compound of 3a (A), 3b (B), 3c (C) and 3d (D) with AChE (PDB ID 2CMF).
Figure 8. Close-up depiction of the docking pose of compound 3d showing
different types of ligand-enzyme interactions in the binding site of 2CMF (Bis-tacrine
was showed in green color).
2
0

4
. Conclusion
Four carbazole-coumarin hybrids were synthesized with simple ways and high
yields. The structures of the four compounds were determined by IR, NMR, HRMS
and X-ray Crystallography. The ChE inhibitory activities and antioxidant activities of
the carbazole-coumarin hybrids were evaluated in vitro. The compound 3d, which
possessed the best AChE-inhibition activity, could interact with both the CAS and the
PAS of AChE, and shown antioxidant capacity. This compound could be a promising
lead candidate for the treatment of AD.
Supporting Information Summary
The Supporting Information contains the spectral data for compounds.
Acknowledgements
We gratefully acknowledge financial supports of graduate research and
innovation projects of Jiangsu province (KYCX18_2579), natural science foundation
of Jiangsu province (BK20191470), national natural science foundation of China
(81703557) and project funded by the priority academic program development of
Jiangsu higher education institutions.
Reference
[
[
1] 2015 Alzheimer's disease facts and figures, Alzheimer's & Dementia 11(3’ (2015’ 332-384.
2] S.S. Xie, X. Wang, N. Jiang, W. Yu, K.D. Wang, J.S. Lan, Z.R. Li, L.Y. Kong, Multi-target
tacrine-coumarin hybrids: cholinesterase and monoamine oxidase B inhibition properties
against Alzheimer's disease, Eur J Med Chem 95 (2015’ 153-65.
[
3] B. Li, R. Pai, M. Di, D. Aiello, M.H. Barnes, M.M. Butler, T.F. Tashjian, N.P. Peet, T.L.
Bowlin, D.T. Moir, Coumarin-based inhibitors of Bacillus anthracis and Staphylococcus aureus
replicative DNA helicase: chemical optimization, biological evaluation, and antibacterial
activities, J. Med. Chem. 55(24’ (2012’ 10896-908.
[
4] J.S. Birks, R.J. Harvey, Donepezil for dementia due to Alzheimer's disease, Cochrane
2
1

Database of systematic reviews (6’ (2018’.
[
5] J.T. Olin, L. Schneider, Galantamine for Alzheimer's disease, Cochrane database of
systematic reviews (3’ (2002’.
[
6] J.S. Lan, Y. Ding, Y. Liu, P. Kang, J.W. Hou, X.Y. Zhang, S.S. Xie, T. Zhang, Design,
synthesis and biological evaluation of novel coumarin-N-benzyl pyridinium hybrids as
multi-target agents for the treatment of Alzheimer's disease, Eur J Med Chem 139 (2017’
48-59.
[
7] M. Alipour, M. Khoobi, A. Moradi, H. Nadri, F. Homayouni Moghadam, S. Emami, Z.
Hasanpour, A. Foroumadi, A. Shafiee, Synthesis and anti-cholinesterase activity of new
7-hydroxycoumarin derivatives, Eur J Med Chem 82 (2014’ 536-44.
[
8] H. Akrami, B.F. Mirjalili, M. Khoobi, H. Nadri, A. Moradi, A. Sakhteman, S. Emami, A.
Foroumadi, A. Shafiee, Indolinone-based acetylcholinesterase inhibitors: synthesis, biological
activity and molecular modeling, Eur. J. Med. Chem. 84 (2014’ 375-81.
[
9] C. Wang, Z. Wu, H. Cai, S. Xu, J. Liu, J. Jiang, H. Yao, X. Wu, J. Xu, Design, synthesis,
biological evaluation and docking study of 4-isochromanone hybrids bearing N-benzyl
pyridinium moiety as dual binding site acetylcholinesterase inhibitors, Bioorg. Med. Chem. Lett.
25(22’ (2015’ 5212-6.
[
10] M. Rosini, E. Simoni, A. Milelli, A. Minarini, C. Melchiorre, Oxidative stress in Alzheimer's
disease: are we connecting the dots?, J. Med. Chem. 57(7’ (2014’ 2821-31.
[
11] L. Wang, Y. Wang, Y. Tian, J. Shang, X. Sun, H. Chen, H. Wang, W. Tan, Design,
synthesis, biological evaluation, and molecular modeling studies of chalcone-rivastigmine
hybrids as cholinesterase inhibitors, Bioorganic & Medicinal Chemistry 25(1’ (2017’ 360-371.
2
2

[
12] Z. Sang, K. Wang, H. Wang, L. Yu, H. Wang, Q. Ma, M. Ye, X. Han, W. Liu, Design,
synthesis and biological evaluation of phthalimide-alkylamine derivatives as balanced
multifunctional cholinesterase and monoamine oxidase-B inhibitors for the treatment of
Alzheimer's disease, Bioorganic & medicinal chemistry letters 27(22’ (2017’ 5053-5059.
[
13] H.L. Yang, P. Cai, Q.H. Liu, X.L. Yang, S.Q. Fang, Y.W. Tang, C. Wang, X.B. Wang, L.Y.
Kong, Design, synthesis, and evaluation of salicyladimine derivatives as multitarget-directed
ligands against Alzheimer's disease, Bioorg Med Chem 25(21’ (2017’ 5917-5928.
[
14] H. Singh, J.V. Singh, K. Bhagat, H.K. Gulati, M. Sanduja, N. Kumar, N. Kinarivala, S.
Sharma, Rational approaches, design strategies, structure activity relationship and
mechanistic insights for therapeutic coumarin hybrids, Bioorg Med Chem 27(16’ (2019’
3477-3510.
[
15] S.K. Yusufzai, M.S. Khan, O. Sulaiman, H. Osman, D.N. Lamjin, Molecular docking
studies of coumarin hybrids as potential acetylcholinesterase, butyrylcholinesterase,
monoamine oxidase A/B and beta-amyloid inhibitors for Alzheimer's disease, Chem Cent J
12(1’ (2018’ 128.
[
16] L.G. de Souza, M.N. Renna, J.D. Figueroa-Villar, Coumarins as cholinesterase inhibitors:
A review, Chem Biol Interact 254 (2016’ 11-23.
[
17] S. Hamulakova, L. Janovec, M. Hrabinova, K. Spilovska, J. Korabecny, P. Kristian, K.
Kuca, J. Imrich, Synthesis and biological evaluation of novel tacrine derivatives and
tacrine-coumarin hybrids as cholinesterase inhibitors, J Med Chem 57(16’ (2014’ 7073-84.
[
18] S.S. Xie, J.S. Lan, X. Wang, Z.M. Wang, N. Jiang, F. Li, J.J. Wu, J. Wang, L.Y. Kong,
Design, synthesis and biological evaluation of novel donepezil-coumarin hybrids as
2
3

multi-target agents for the treatment of Alzheimer's disease, Bioorg Med Chem 24(7’ (2016’
1528-39.
[
19] A. Hiremathad, K. Chand, R.S. Keri, Development of coumarin-benzofuran hybrids as
versatile multitargeted compounds for the treatment of Alzheimer's Disease, Chem Biol Drug
Des 92(2’ (2018’ 1497-1503.
[
20] Q. Sun, D.Y. Peng, S.G. Yang, X.L. Zhu, W.C. Yang, G.F. Yang, Syntheses of
coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against
butylcholinesterase, Abeta aggregation, and beta-secretase, Bioorg Med Chem 22(17’ (2014’
4784-91.
[
21] C. Zhang, K. Yang, S. Yu, J. Su, S. Yuan, J. Han, Y. Chen, J. Gu, T. Zhou, R. Bai, Design,
synthesis and biological evaluation of hydroxypyridinone-coumarin hybrids as multimodal
monoamine oxidase B inhibitors and iron chelates against Alzheimer's disease, Eur. J. Med.
Chem. 180 (2019’ 367-382.
[
22] Z. Najafi, M. Mahdavi, M. Saeedi, E. Karimpour-Razkenari, N. Edraki, M. Sharifzadeh, M.
Khanavi, T. Akbarzadeh, Novel tacrine-coumarin hybrids linked to 1, 2, 3-triazole as
anti-Alzheimerꢀs compounds: In vitro and in vivo biological evaluation and docking study,
Bioorg. Chem. 83 (2019’ 303-316.
[
23] S. Thiratmatrakul, C. Yenjai, P. Waiwut, O. Vajragupta, P. Reubroycharoen, M. Tohda, C.
Boonyarat, Synthesis, biological evaluation and molecular modeling study of novel tacrineꢁ
carbazole hybrids as potential multifunctional agents for the treatment of Alzheimer's disease,
Eur. J. Med. Chem. 75 (2014’ 21-30.
[
24] Y.-Z. Tang, Z.-Q. Liu, Free-radical-scavenging effect of carbazole derivatives on
2
4

AAPH-induced hemolysis of human erythrocytes, Biorg. Med. Chem. 15(5’ (2007’ 1903-1913.
[
25] W. Yang, Y. Wong, O.T. Ng, L.P. Bai, D.W. Kwong, Y. Ke, Z.H. Jiang, H.W. Li, K.K. Yung,
M.S. Wong, Inhibition of BetaꢂAmyloid Peptide Aggregation by Multifunctional Carbazoleꢂ
Based Fluorophores, Angew. Chem. Int. Ed. 51(8’ (2012’ 1804-1810.
[
26] L. Fang, X. Fang, S. Gou, A. Lupp, I. Lenhardt, Y. Sun, Z. Huang, Y. Chen, Y. Zhang, C.
Fleck, Design, synthesis and biological evaluation of D-ring opened galantamine analogs as
multifunctional anti-Alzheimer agents, Eur J Med Chem 76 (2014’ 376-86.
[
27] L. Fang, M. Chen, Z. Liu, X. Fang, S. Gou, L. Chen, Ferulic acid-carbazole hybrid
compounds: Combination of cholinesterase inhibition, antioxidant and neuroprotection as
multifunctional anti-Alzheimer agents, Bioorg Med Chem 24(4’ (2016’ 886-93.
[
28] S. Thiratmatrakul, C. Yenjai, P. Waiwut, O. Vajragupta, P. Reubroycharoen, M. Tohda, C.
Boonyarat, Synthesis, biological evaluation and molecular modeling study of novel
tacrine-carbazole hybrids as potential multifunctional agents for the treatment of Alzheimer's
disease, Eur J Med Chem 75 (2014’ 21-30.
[
29] G.F. Makhaeva, E.F. Shevtsova, N.P. Boltneva, S.V. Lushchekina, N.V. Kovaleva, E.V.
Rudakova, S.O. Bachurin, R.J. Richardson, Overview of novel multifunctional agents based on
conjugates of gamma-carbolines, carbazoles, tetrahydrocarbazoles, phenothiazines, and
aminoadamantanes for treatment of Alzheimer's disease, Chem Biol Interact 308 (2019’
224-234.
[
30] Bruker, SMART (Version 5.628’ and SAINT (Version 6.02’; Bruker AXS Inc: Madison, WI
(
1998’.
[
31] G.M. Sheldrick, SADABS. Program for Empirical Absorption Correction of Area Detector;
2
5

University of G¨ottingen: G¨ottingen, Germany (1996’.
[
32] G.M. Sheldrick, SHELXTL V5.1. Software Reference Manual; Bruker AXS: Madison, WI
(
1997’.
[
33] C.F. Macrae, I.J. Bruno, J.A. Chisholm, P.R. Edgington, P. McCabe, E. Pidcock, L.
Rodriguez-Monge, R. Taylor, J. van de Streek, P.A. Wood, Mercury CSD 2.0 - new features
for the visualization and investigation of crystal structures, Journal of Applied Crystallography
41(2’ (2008’ 466-470.
[
34] J.J.M. M.J. Turner, S.K. Wolff, D.J. Grimwood, P.R. Spackman, D. Jayatilaka, M.A.
Spackman, CrystalExplorer 17, University of Western Australia 2017 (2017’.
[
35] G.L. Ellman, K.D. Courtney, V. Andres Jr, R.M. Featherstone, A new and rapid
colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7(2’ (1961’
88-95.
[
[
36] Molecular Operating Environment (MOE’, Version 2015.10.
37] Y. Li, X. Qiang, L. Luo, X. Yang, G. Xiao, Q. Liu, J. Ai, Z. Tan, Y. Deng, Aurone Mannich
base derivatives as promising multifunctional agents with acetylcholinesterase inhibition,
anti-beta-amyloid aggragation and neuroprotective properties for the treatment of Alzheimer's
disease, Eur. J. Med. Chem. 126 (2017’ 762-775.
[
38] Q. He, J. Liu, J.S. Lan, J. Ding, Y. Sun, Y. Fang, N. Jiang, Z. Yang, L. Sun, Y. Jin, S.S. Xie,
Coumarin-dithiocarbamate hybrids as novel multitarget AChE and MAO-B inhibitors against
Alzheimer's disease: Design, synthesis and biological evaluation, Bioorg Chem 81 (2018’
512-528.
[
39] N. Jiang, Q. Huang, J. Liu, N. Liang, Q. Li, Q. Li, S.S. Xie, Design, synthesis and biological
2
6

evaluation of new coumarin-dithiocarbamate hybrids as multifunctional agents for the
treatment of Alzheimer's disease, Eur J Med Chem 146 (2018’ 287-298.
[
40] S.S. Xie, J.S. Lan, X.B. Wang, N. Jiang, G. Dong, Z.R. Li, K.D. Wang, P.P. Guo, L.Y.
Kong, Multifunctional tacrine-trolox hybrids for the treatment of Alzheimer's disease with
cholinergic, antioxidant, neuroprotective and hepatoprotective properties, Eur J Med Chem 93
(
2015’ 42-50.
[
41] X. Zhou, X.B. Wang, T. Wang, L.Y. Kong, Design, synthesis, and acetylcholinesterase
inhibitory activity of novel coumarin analogues, Bioorg Med Chem 16(17’ (2008’ 8011-21.
[
42] A. Asadipour, M. Alipour, M. Jafari, M. Khoobi, S. Emami, H. Nadri, A. Sakhteman, A.
Moradi, V. Sheibani, F. Homayouni Moghadam, A. Shafiee, A. Foroumadi, Novel
coumarin-3-carboxamides bearing N-benzylpiperidine moiety as potent acetylcholinesterase
inhibitors, Eur J Med Chem 70 (2013’ 623-30.
2
7