Design, synthesis and biological activity of 1H-indene-2-carboxamides as multi-targeted anti-Alzheimer agents

Article abstract of DOI:10.1080/14756366.2016.1186019

The aim of this study was to design new molecules and evaluate their anticholinesterase and amyloid beta (Aβ_1–42) inhibition activities as multifunctional drug candidates for the treatment of Alzheimer’s disease (AD). A series of 5,6-dimethoxy-1H-indene-2-carboxamides (1–22) was synthesized; cholinesterase inhibitory activities of the compounds were measured according to Ellman’s colorimetric assay, while the thioflavin T assay was used for measuring the inhibition of Aβ_1–42 aggregation. The results revealed that most compounds showed higher inhibitory activity against BuChE than AChE. Compounds 20 and 21 were found to be the most potent BuChE inhibitors with respective IC₅₀ values of 1.08 and 1.09 μM. Compounds 16, 20, 21 and 22 exhibited remarkable inhibition of Aβ_1–42 aggregation. Kinetic analysis showed that the most potent BuChE inhibitor (20) acted as a noncompetitive inhibitor. Docking studies suggested that inhibitor 20 displayed many potential hydrogen-bondings with the PAS of BuChE. These results suggest that compound 20 may be an especially promising multifunctional drug for the prevention and treatment of AD.

Full text of DOI:10.1080/14756366.2016.1186019

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and biological activity of 1H-
indene-2-carboxamides as multi-targeted anti-
Alzheimer agents
Mehmet Koca, Kadir Ozden Yerdelen, Baris Anil, Zeynep Kasap, Handan
Sevindik, Ibrahim Ozyurek, Gulsen Gunesacar & Kubra Turkaydin
To cite this article: Mehmet Koca, Kadir Ozden Yerdelen, Baris Anil, Zeynep Kasap, Handan
Sevindik, Ibrahim Ozyurek, Gulsen Gunesacar & Kubra Turkaydin (2016): Design, synthesis
and biological activity of 1H-indene-2-carboxamides as multi-targeted anti-Alzheimer agents,
Journal of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1186019
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1186019
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–11
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1186019
!
RESEARCH ARTICLE
Design, synthesis and biological activity of 1H-indene-2-carboxamides
as multi-targeted anti-Alzheimer agents
Mehmet Koca1, Kadir Ozden Yerdelen1, Baris Anil2, Zeynep Kasap1, Handan Sevindik3, Ibrahim Ozyurek2,
Gulsen Gunesacar1, and Kubra Turkaydin1
1Department of Pharmaceutical Chemistry, 2Department of Organic Chemistry, and 3Department of Pharmacognosy, Ataturk University, Erzurum,
Turkey
Abstract
Keywords
The aim of this study was to design new molecules and evaluate their anticholinesterase and
amyloid beta (Ab1–42) inhibition activities as multifunctional drug candidates for the treatment
of Alzheimer’s disease (AD). A series of 5,6-dimethoxy-1H-indene-2-carboxamides (1–22) was
synthesized; cholinesterase inhibitory activities of the compounds were measured according to
Ellman’s colorimetric assay, while the thioflavin T assay was used for measuring the inhibition of
Ab1–42 aggregation. The results revealed that most compounds showed higher inhibitory
activity against BuChE than AChE. Compounds 20 and 21 were found to be the most potent
BuChE inhibitors with respective IC50 values of 1.08 and 1.09 mM. Compounds 16, 20, 21 and 22
exhibited remarkable inhibition of Ab1–42 aggregation. Kinetic analysis showed that the most
potent BuChE inhibitor (20) acted as a noncompetitive inhibitor. Docking studies suggested
that inhibitor 20 displayed many potential hydrogen-bondings with the PAS of BuChE. These
results suggest that compound 20 may be an especially promising multifunctional drug for the
prevention and treatment of AD.
AChE, Alzheimer, Ab1–42, BuChE, molecular
modeling
History
Received 3 March 2016
Revised 28 March 2016
Accepted 30 March 2016
Published online 23 May 2016
Introduction
aggregation of amyloid peptide is a critical stage in AD
development; accumulation of the Ab peptide in neurons may
induce biochemical events leading to neuronal dysfunctions12. In
addition, recent studies have shown that oxidative damage may
increase the incidence of amyloid plaques in the brains of AD
patients13. Another hypothesis, known as the metal hypothesis,
Alzheimer’s disease (AD), which is a type of mental health
decline or dementia, is a complex neurodegenerative disorder
with highest prevalence in the elderly population. Causes of and
effective treatments for this disease remain uncertain, requiring
complex attempts to develop new, effective anti-AD drugs. In
recent years, the most popular pathophysiological approaches
have suggested that AD is characterized by low levels of
acetylcholine (ACh), increased oxidative stress, high levels of
indicates that high levels of metal ions (Fe2+, Cu2+ and Zn2+
)
cause severe and fatal neurologic disorders. The accumulation of
metal ions is closely associated with abnormal clumps of Ab
plaques that are a hallmark of AD13–16. Several AChE inhibitors
such as donepezil, galantamine, and rivastigmine have been used
for the treatment of AD17. Among these drugs, donepezil was
approved for the treatment of mild to moderate dementia in AD
by the United States Food and Drug Administration in 200618.
Many studies have demonstrated that donepezil analogues (I and
II) are able to interact with the catalytic anionic site (CAS) and
the peripheral anionic site (PAS) of AChE, with their highest
inhibitory activities at nanomolar concentrations (Figure 1)3. In
our recent docking studies work, we have synthesized a series of
tertiary diamide (2-butendiamide and ethanediamide) derivatives
and described their possible hydrogen-bonding interactions with
the –C¼O and –NH groups between the amide functional group
and both active sites of AChE19–23. The positive contributions on
cholinesterase inhibition activity of halogenated (F, Cl and Br)
phenyl rings have also been investigated in these studies.
some metal ions, and overproduction and aggregation of Ab1–3
.
The oldest and most popular current strategy for AD treatment is
based on the cholinergic hypothesis and specifically on acetyl-
cholinesterase (AChE) inhibition to improve cholinergic neuro-
transmission in the brain by increasing the levels of ACh4–6
.
AChE’s sister enzyme, butyrylcholinesterase (BuChE), has also
been found to be capable of compensating for the missing AChE
catalytic functions in the synaptic cleft7,8. This function has
driven increased attention on BuChE, due to its ability to
hydrolyze ACh and other esters. Inhibition of both cholinesterase
enzymes (ChEs) should provide additional contributions in the
treatment of AD9–11
.
The second main therapeutic strategy for AD is based on
preventing Ab aggregation. According to this amyloid hypothesis,
Address for correspondence: Kadir Ozden Yerdelen, Assist. Prof.,
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk
University, 25240 Erzurum, Turkey. Tel: +90 4422315220. Fax: +90
4422360962. E-mail: dadasozden@gmail.com
In this study, the 5,6-dimethoxyindanone structure of donepe-
zil, which is the structural part responsible for binding AChE to
the PAS, was taken as the main structural moiety to design new

2
M. Koca et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Figure 1. Structures of some AChE inhibitors: donepezil and indanol- and indene-based derivatives (I and II) reported as AChE inhibitors.
donepezil analogues. The indanone ring structure of donepezil and cooled to 0 ꢀC. Ice-water (85 mL) was added slowly in
was modified to 1H-indene to measure the possible contributions reaction medium to quench the excess AlCl3. The layers were
on ChE inhibition. For this purpose, we have designed a new separated, and the aqueous phase was extracted with four portions
series of secondary 5,6-dimethoxy-1H-indene-2-carboxamide of CH2Cl2 (4 ꢁ 85 mL). The combined organic phases were dried
derivatives containing ortho-, meta-, and para-substituted primary over Na2SO4, filtered and concentrated by rotary evaporation. The
anilines and evaluated their inhibitory activities on ChEs and crude solid was dissolved in ethylacetate and treated with
Ab1–42 aggregation and their metal chelation abilities. In silico activated carbon, and stirred overnight. The mixture was filtered,
studies were used to provide preliminary assessment of the and the filtrate was concentrated. And the target compound
potential interactions against AChE, while molecular docking (5,6-dimethoxy-indane-1-one) was recrystallized from ethyl acet-
studies were performed using Sybyl X 2.0 (Tripos Associates ate and obtained as yellow solid in 75% yield.
(St Louis, MO) for the most potent AChE/BuChE inhibitors.
Synthesis procedure of 5,6-dimethoxy-1-oxo-indan-2-car-
boxylic acid methyl ester
Materials and methods
NaH (2.4 g, 60 mmol, 60% in mineral oil) was added slowly to the
solution of 5,6-dimethoxy-indane-1-one (4 g, 20 mmol) in
Materials
dimethylcarbonate (22 mL), and the suspension was refluxed at
90 ꢀC for 2–3 h. The resulting solid was cooled to 0 ꢀC with an ice
bath. After cooling, cold water (80 mL) was added carefully. The
aqueus layer was extracted with CH2Cl2 (3 ꢁ 80 mL). The
combined organic extracts were dried over Na2SO4 and
concentrated. The desired product (white solid, 60% yield) were
obtained by recrystallization from ethanol24.
All chemical reagents, starting materials and solvents used in this
study were purchased from Merck & Co. (Kenilworth, NJ), Fluka
Chemical Corp. (Milwaukee, WI) and Sigma Aldrich (St. Louis,
MO). The melting points were measured on an Electrothermal
9100 melting-point apparatus (Bibby Scientific Limited, Stone,
UK) and are uncorrected. The NMR spectra were recorded on a
Bruker FT-400(100) MHz spectrometer (Bruker corporation,
Billerica, MA). Coupling constants were given in Hertz (Hz).
High resolution mass spectra (HRMS) were obtained on a Waters
LCT Premier XE Mass Spectrometer equipped with an ESI (+)
method, also coupled to an AQUITY Ultra Performance Liquid
Chromatography system (Waters Corporation, Milford, MA).
General microwave-assisted synthesis of 5,6-dimethox-
yindane-1-one-2-carboxamides
The suspension of 5,6-dimethoxy-1-oxo-indane-2-carboxylic acid
methyl ester (1.2 mmol) and appropriate aniline (1.2 mmol) in
dioxane (3 mL) was sonicated for 1 min. The reaction tube was
subjected to microwave irradiation under the following condi-
tions: power, 300 W; pressure, 15 psi; temperature, 170 ꢀC;
reaction time, 10 min. The reaction solvent was evaporated under
vacuum and the crude product was purified by recrystallization
with ethanol.
Chemistry
Synthesis procedure of 5,6-dimethoxy-indane-1-one
For the synthesis of 5,6-dimethoxy-indane-1-one, a solution of
3-(3,4-dimethoxy-phenyl)-propionic acid (4.98 g, 23.7 mmol) in
dry dichloromethane (72.5 mL), N,N-dimethylformamide
(1.39 mmol) and oxalyl chloride (8.03 mL, 95 mmol) were
stirred at room temperature for 8 h. The reaction mixture was
concentrated by rotary evaporation. A solution of crude 3-
(3,4-dimethoxy-phenyl)-propionyl chloride in anhydrous dichlor-
omethane (82 mL) was cooled with an ice bath to 0 ꢀC and a
powder of AlCl3 (5.73 g, 43 mmol) was added portionwise over
30 min. The reaction was then stirred at room temperature for 3 h
General synthesis of 5,6-dimethoxy-1H-indene-2-
carboxamides
Appropriate 5,6-dimethoxy-1-oxo-indane-2-carboxamide (0.69 mmol)
was dissolved in dry tetrahydrofuran (4 mL) and 0.4 mL
methanol was added in reaction solution. NaBH4 (1.035 mmol)
was added portionwise at 0 ꢀC and the mixture was stirred

DOI: 10.1080/14756366.2016.1186019
1H-indene-2-carboxamides as anti-Alzheimer agents
3
Scheme 1. Reagents and conditions: (a) oxalyl chloride, CH2Cl2, room temperature, 12 h; (b) AlCl3, CH2Cl2, 0 ꢀC, ice bath; (c) Dimethyl carbonate,
NaH, 90 ꢀC, reflux; (d) appropriate aniline, dioxane, microwave; (e) NaBH4, THF, MeOH, 2 h; (f) MeOH, PTSA, reflux, 25 min. *R1, R2, R3
substituent groups at the o-, m- and p- positions: H, CH3, C2H5, OCH3, OC2H5, F, Cl and Br.
at room temperature for 2 h. The reaction solvent was evaporated Ar-Hg), 6.98 (s, 1H, Ar-Hd), 6.90 (s, 1H, Ar-He), 3.88 (s, 3H,
and a solution of aqueous HCl (10%, 5 mL) was added in to the Ar-OCH3) 3.85 (s, 3H, Ar-OCH3), 3.64 (s, 2H, Ha and Hb), 2.29
reaction mixture and it was stirred a few minutes. The reaction (s, 3H, Ph-CH3). 13C-NMR (100 MHz, CDCl3) ꢁ: 163.23, 149.55,
mixture was extracted with chloroform (3 ꢁ 5 mL). The combined 148.89, 139.96, 137.53, 136.90, 135.84, 135.70, 133.96, 129.70,
organic layers were dried over Na2SO4 and concentrated. The 120.37, 107.79, 105.90, 56.33, 56.28, 38.55 and 21.11.
obtained white solid product was dissolved in MeOH (4 mL) and HRMS (ESI) m/z [M + H]+ 310.1330 (calcd for C19H19NO3
was added catalytic amount of PTSA (p-toluenesulfonic acid) in [M + H]+ 310.1365).
reaction mixture. The reaction was refluxed for 25 min and
monitored by TLC. The solvent was evaporated under reduced 5,6-Dimethoxy-1H-indene-2-carboxylic acid (4-ethyl-phenyl)-
pressure and the resulting mixture was washed with water, and amide (3)
extracted with chloroform (3 ꢁ 5 mL) and dried over Na2SO4 and
DMSO
maks
Yield 72%; m.p. 184–186 ꢀC; ꢀ
(log e) 336 nm (4.36).
concentrated. The crude white solid was recrystallized from
ethanol to obtain 5,6-dimethoxy-1H-inden-2-carboxamide deriva-
tives. The synthesis pathway is shown in Scheme 1.
1H-NMR (400 MHz, CDCl3) ꢁ: 7.64 (s, 1H, –NH), 7.56 (d, 1H,
J ¼ 8.42 Hz, Ar-Hf), 7.50 (s, 1H, Hc), 7.17 (d, 2H, J ¼ 8.42 Hz,
Ar-Hg), 7.06 (s, 1H, Ar-Hd), 6.99 (s, 1H, Ar-He), 3.93 (s, 3H,
Ar-OCH3) 3.91 (s, 3H, Ar-OCH3), 3.74 (s, 2H, Ha and Hb), 2.62
(q, 2H, CH2–CH3), 1.22 (t, 3H, CH2–CH3). 13C-NMR (100 MHz,
5,6-Dimethoxy-1H-indene-2-carboxylic acid phenylamide (1)
DMSO
maks
Yield 73%; m.p. 194–196 ꢀC; ꢀ
(log e) 336 nm (4.37). CDCl3) ꢁ: 162.95, 149.65, 148.98, 139.99, 137.49, 136.70,
1H-NMR (400 MHz, CDCl3) ꢁ: 7.81 (s, 1H, –NH), 7.64 (d, 2H, 135.86, 135.66, 133.96, 128.60, 120.26, 107.85, 105.94, 56.38,
J ¼ 8.05 Hz, Ar-Hf), 7.52 (s, 1H, Hc), 7.33 (dd, 2H, J ¼ 7.69 Hz, 56.36, 38.56, 28.55 and 15.89. HRMS (ESI) m/z
J ¼ 7.69 Hz, Ar-Hg), 7.13–7.09 (m, 1H, Ar-Hh), 7.04 (s, 1H, [M + H]+ 324.1518 (calcd for C20H21NO3 [M + H]+ 324.1521).
Ar-Hd) 6.96 (s, 1H, Ar-He), 3.91 (s, 3H, Ar-OCH3) 3.89 (s, 3H,
Ar-OCH3), 3.69 (d, 2H, Ha and Hb). 13C-NMR (100 MHz, CDCl3) 5,6-Dimethoxy-1H-indene-2-carboxylic acid (4-methoxy-phenyl)-
ꢁ: 163.14, 149.69, 148.97, 139.82, 138.31, 137.55, 137.01, amide (4)
135.59, 129.27, 124.41, 120.18, 107.82, 105.94, 56.37, 56.34
DMSO
maks
Yield 60%; m.p. 205–207 ꢀC; ꢀ
(log e) 337 nm (4.35).
and 38.56. HRMS (ESI) m/z [M + H]+ 296.1209 (calcd for
1H-NMR (400 MHz, CDCl3) ꢁ: 7.77 (s, 1H, –NH), 7.53 (d, 2H,
J ¼ 9.14 Hz, Ar-Hf), 7.49 (s, 1H, Ar-Hc), 7.02 (s, 1H, Ar-Hd), 6.94
(s, 1H, Ar-He), 6.85 (d, 2H, J ¼ 8.48 Hz, Ar-Hg), 3.91 (s, 3H,
Ar-OCH3) 3.88 (s, 3H, Ar-OCH3), 3.77 (s, 3H, Ph-OCH3), 3.66
C18H17NO3 [M + H]+ 296.1208).
5,6-Dimethoxy-1H-indene-2-carboxylic acid p-tolylamide (2)
Yield 67%; m.p. 204–207 ꢀC; ꢀ
(log e) 336 nm (4.45). (s, 2H, Ha and Hb). 13C-NMR (100 MHz, CDCl3) ꢁ: 163.08,
DMSO
maks
1H-NMR (400 MHz, CDCl3) ꢁ: 7.97 (s, 1H, –NH), 7.53 (d, 2H, 156.56, 149.57, 148.93, 139.87, 137.46, 136.78, 135.69, 131.39,
J ¼ 8.42 Hz, Ar-Hf), 7.49 (s, 1H, Hc), 7.09 (d, 2H, J ¼8.05 Hz, 122.12, 114.36, 107.82, 105.90, 56.35, 56.32, 55.68 and 38.54.

4
M. Koca et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
HRMS (ESI) m/z [M + H]+ 326.1314 (calcd for C19H19NO4 HRMS (ESI) m/z [M + H]+ 310.1365 (calcd for C19H19NO3
[M + H]+ 326.1314).
[M + H]+ 310.1365).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (4-ethoxy-phenyl)-
amide (5)
5,6-Dimethoxy-1H-indene-2-carboxylic acid (3-ethyl-phenyl)-
amide (10)
DMSO
maks
DMSO
maks
Yield 47%; m.p. 227–229 ꢀC; ꢀ
(log e) 337 nm (4.35). Yield 55%; m.p. 161–164 ꢀC; ꢀ
(log e) 337 nm (3.94).
1H-NMR (400 MHz, CDCl3) ꢁ: 7.52 (s, 1H, –NH), 7.50 (d, 2H, 1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.76 (s, 1H, –NH), 7.70 (s, 1H,
J ¼ 8.78 Hz, Ar-Hf), 7.49 (s, 1H, Ar-Hc), 7.07 (s, 1H, Ar-Hd), 7.01 Ar-Hc), 7.59 (s, 1H, Ar-Hf), 7.57 (d, 1H, J ¼ 9.13 Hz, Ar-Hi), 7.21
(s, 1H, Ar-He), 6.88 (d, 2H, J ¼ 9.17 Hz, Ar-Hg), 4.02 (q, 2H, (d, 1H, J ¼ 7.76 Hz, Ar-Hh), 7.19 (s, 1H, Ar-Hd), 7.17 (s, 1H,
CH2–CH3) 3.93 (s, 3H, Ar-OCH3), 3.92 (s, 3H, Ar-OCH3), 3.71 Ar-He), 6.89 (d, 1H, J ¼ 7.76 Hz, Ar-Hg), 3.79 (s, 3H, Ar-OCH3),
(s, 2H, Ha and Hb), 1.41 (t, 3H, CH2–CH3). 13C-NMR (100 MHz, 3.78 (s, 3H, Ar-OCH3), 3.67 (s, 2H, Ha and Hb), 2.57 (q, 2H,
CDCl3) ꢁ: 162.85, 155.98, 149.62, 148.99, 139.93, 137.43, CH2CH3), 1.17 (t, 3H, CH2CH3). 13C-NMR (100 MHz, DMSO-
136.59, 135.67, 131.18, 121.95, 115.05, 107.86, 105.93, 63.92, d6) ꢁ: 163.38, 149.63, 149.06, 144.73, 140.85, 139.95, 137.83,
56.39, 56.36, 38.54 and 15.08. HRMS (ESI) m/z 137.09, 135.94, 129.13, 123.40, 119.99, 118.07, 108.92, 107.01,
[M + H]+ 340.1467 (calcd for C20H21NO4 [M + H]+ 340.1471).
56.39 (2ꢁ Ar-OCH3), 39.06, 29.00 and 16.24. HRMS (ESI) m/z
[M + H]+ 324.1507 (calcd for C20H21NO3 [M + H]+ 324.1521).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (4-fluoro-phenyl)-
amide (6)
5,6-Dimethoxy-1H-indene-2-carboxylic acid (3-methoxy-phenyl)-
amide (11)
1
DMSO
maks
Yield 80%; m.p. 217–219 ꢀC; ꢀ
(log e) 336 nm (4.29). H-
DMSO
maks
Yield 35%; m.p. 159–162 ꢀC; ꢀ
(log e) 337 nm (4.33).
NMR (400 MHz, CDCl3) ꢁ: 7.74 (s, 1H, –NH), 7.59 (d, 1H,
J ¼ 8.42 Hz, Ar-Hf), 7.51 (s, 1H, Hc), 7.04 (s, 1H, Ar-Hd), 7.00
(d, 2H, J ¼ 8.42 Hz, Ar-Hg), 6.97 (s, 1H, Ar-He), 3.92 (s, 3H,
Ar-OCH3) 3.89 (s, 3H, Ar-OCH3), 3.69 (s, 2H, Ha and Hb).
13C-NMR (100 MHz, CDCl3) ꢁ: 163.06, 158.32, 149.78, 149.03,
139.52, 137.53, 137.12, 135.53, 134.27, 121.97, 116.00, 115.77,
107.83, 105.97, 56.37, 56.34 and 38.52. HRMS (ESI) m/z
[M + H]+ 314.1120 (calcd for C18H16FNO3 [M + H]+ 314.1114).
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.79 (s, 1H, –NH), 7.71 (s, 1H,
Ar-Hc), 7.45 (s, 1H, Ar-Hf), 7.31 (d, 1H, J ¼ 8.22 Hz, Ar-Hi), 7.21
(d, 1H, J ¼ 8.22 Hz, Ar-Hg), 7.19 (s, 1H, Ar-Hd), 7.18 (s, 1H,
Ar-He), 6.62 (dd, 1H, J ¼ 8.22 Hz, J¼8.22 Hz, Ar-Hh), 3.79 (s,
3H, Ar-OCH3), 3.78 (s, 3H, Ar-OCH3), 3.73 (s, 3H, Ph-OCH3),
3.67 (s, 2H, Ha and Hb). 13C-NMR (100 MHz, DMSO-d6) ꢁ:
163.48, 160.07, 149.68, 149.08, 141.19, 140.75, 137.87, 137.22,
135.90, 130.00, 112.83, 109.36, 108.93, 107.04, 106.24, 56.42,
56.40 and 55.63. HRMS (ESI) m/z [M + H]+ 326.1315 (calcd for
C19H19NO4 [M + H]+ 326.1314).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (4-chloro-phenyl)-
amide (7)
DMSO
maks
Yield 71%; m.p. 222–224 ꢀC; ꢀ
(log e) 338 nm (4.38).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (3-ethoxy-phenyl)-
amide (12)
1H-NMR (400 MHz, CDCl3) ꢁ: 7.78 (s, 1H, –NH), 7.58 (d,
1H, J ¼ 8.78 Hz, Ar-Hf), 7.51 (s, 1H, Hc), 7.27 (d, 2H,
J ¼ 8.78 Hz, Ar-Hg), 7.03 (s, 1H, Ar-Hd), 6.96 (s, 1H, Ar-He),
3.91 (s, 3H, Ar-OCH3) 3.89 (s, 3H, Ar-OCH3), 3.67 (s, 2H,
Ha and Hb). 13C-NMR (100 MHz, CDCl3) ꢁ: 160.08, 149.85,
149.04, 139.39, 137.59, 137.34, 136.88, 135.46, 129.32,
129.24, 121.39, 107.82, 105.99, 56.37, 56.34 and 38.51.
HRMS (ESI) m/z [M + H]+ 330.0817 (calcd for C18H16ClNO3
[M + H]+ 330.0819).
DMSO
maks
Yield 45%; m.p. 167–169 ꢀC; ꢀ
(log e) 337 nm (4.36).
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.76 (s, 1H, –NH), 7.70 (s, 1H,
Ar-Hc), 7.44 (s, 1H, Ar-Hf), 7.29 (d, 1H, J ¼ 8.68 Hz, Ar-Hi), 7.19
(d, 1H, J ¼ 7.76 Hz, Ar-Hg), 7.18 (s, 1H, Ar-Hd), 7.17 (s, 1H,
Ar-He), 6.60 (dd, 1H, J ¼ 7.76 Hz, J ¼ 7.76 Hz, Ar-Hh), 3.98 (q,
2H, OCH2CH3), 3.79 (s, 3H, Ar-OCH3), 3.78 (s, 3H, Ar-OCH3),
3.67 (s, 2H, Ha and Hb), 1.32 (t, 3H, OCH2CH3). 13C-NMR
(100 MHz, DMSO-d6) ꢁ: 163.47, 159.34, 149.69, 149.09, 141.16,
140.78, 137.87, 137.19, 135.91, 129.97, 112.72, 109.95, 108.94,
107.05, 106.69, 63.55, 56.42, 56.41, 39.05 and 15.37. HRMS (ESI)
m/z [M + H]+ 340.1468 (calcd for C20H21NO4 [M + H]+ 340.1471).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (4-bromo-phenyl)-
amide (8)
DMSO
maks
Yield 70%; m.p. 228–230 ꢀC; ꢀ
(log e) 339 nm (4.41).
1H-NMR (400 MHz, CDCl3) ꢁ: 7.80 (s, 1H, –NH), 7.54 (s, 1H,
Hc), 7.51 (d, 2H, J ¼7.68 Hz, Ar-Hf), 7.42 (d, 2H, J ¼ 8.78 Hz,
Ar-Hg), 7.03 (s, 1H, Ar-Hd), 6.95 (s, 1H, Ar-He), 3.91 (s, 3H, Ar-
OCH3) 3.88 (s, 3H, Ar-OCH3), 3.67 (d, 2H, Ha and Hb). 13C-NMR
(100 MHz, CDCl3) ꢁ: 163.09, 149.85, 149.03, 139.37, 137.60,
137.40, 136.84, 135.45, 132.18, 121.72, 116.92, 107.81, 105.99,
56.37, 56.35 and 38.50. HRMS (ESI) m/z [M + H]+ 374.0314
(calcd for C18H16BrNO3 [M + H]+ 374.0314).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (3-fluoro-phenyl)-
amide (13)
1
DMSO
maks
Yield 32%; m.p. 161–164 ꢀC; ꢀ
(log e) 338 nm (4.39). H-
NMR (400 MHz, DMSO-d6) ꢁ: 10.06 (s, 1H, –NH), 8.01 (s, 1H,
Ar-Hf), 7.76 (s, 1H, Ar-Hc), 7.72 (d, 1H, J¼8.22 Hz, Ar-Hi), 7.31
(d, 1H, J¼7.76 Hz, Ar-Hg), 7.23 (dd, 1H, J¼8.21 Hz, J¼8.22 Hz,
Ar-Hh), 7.19 (s, 1H, Ar-Hd), 7.18 (s, 1H, Ar-He), 3.79 (s, 3H,
Ar-OCH3), 3.78 (s, 3H, Ar-OCH3), 3.67 (s, 2H, Ha and Hb).
13C-NMR (100 MHz, DMSO-d6) ꢁ: 163.66, 161.55, 149.81,
149.11, 141.75, 140.32, 137.99, 137.72, 135.79, 130.86, 130.77,
116.20, 110.11, 108.89, 107.28, 107.08, 56.40 (2ꢁ Ar-OCH3) and
39.01. HRMS (ESI) m/z [M + H]+ 314.1121 (calcd for
C18H16FNO3 [M + H]+ 314.1114).
5,6-Dimethoxy-1H-indene-2-carboxylic acid m-tolylamide (9)
DMSO
maks
Yield 50%; m.p. 159–161 ꢀC; ꢀ
(log e) 336 nm (4.42).
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.63 (s, 1H, –NH), 7.68 (s, 1H,
Ar-Hc), 7.59 (s, 1H, Ar-Hf), 7.54 (d, 1H, J ¼ 8.60 Hz, Ar-Hi), 7.20
(s, 1H, Ar-Hd), 7.18–7.16 (m, 2H, Ar-Hh and Ar-He), 6.86 (d, 1H,
J ¼ 7.36 Hz, Ar-Hg), 3.80 (s, 3H, Ar-OCH3), 3.79 (s, 3H,
Ar-OCH3), 3.68 (s, 2H, Ha and Hb), 2.29 (s, 3H, Ph-CH3).
13C-NMR (100 MHz, DMSO-d6) ꢁ: 163.19, 149.69, 149.14,
5,6-Dimethoxy-1H-indene-2-carboxylic acid (3-chloro-phenyl)-
amide (14)
140.79, 139.70, 138.09, 137.77, 136.76, 135.96, 128.78, 124.34, Yield 30%; m.p. 160–163 ꢀC; ꢀ
(log e) 339 nm (4.28).
DMSO
maks
121.08, 117.72, 109.19, 107.29, 56.44, 56.41, 38.85 and 21.61. 1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.97 (s, 1H, –NH), 7.93 (s, 1H,

DOI: 10.1080/14756366.2016.1186019
1H-indene-2-carboxamides as anti-Alzheimer agents
5
Ar-Hf), 7.73 (s, 1H, Ar-Hc), 7.66 (d, 1H, J¼8.22 Hz, Ar-Hi), 7.33 5,6-Dimethoxy-1H-indene-2-carboxylic acid (2-ethoxy-phenyl)-
(dd, 1H, J¼8.22 Hz, J¼8.22 Hz, Ar-Hh), 7.20 (s, 1H, Ar-Hd), 7.19 amide (19)
(s, 1H, Ar-He), 6.88 (d, 1H, J¼7.76 Hz, Ar-Hg), 3.79 (s, 3H,
DMSO
maks
Yield 45%; m.p. 179–182 ꢀC; ꢀ
(log e) 338 nm (4.25).
Ar-OCH3), 3.78 (s, 3H, Ar-OCH3), 3.67 (s, 2H, Ha and Hb).
13C-NMR (100 MHz, DMSO-d6) ꢁ: 163.64, 149.86, 149.14,
141.52, 140.27, 138.02, 137.83, 135.81, 131.60, 130.96, 123.49,
119.92, 118.85, 108.93, 107.13, 56.43, 56.41 and 39.01. HRMS
(ESI) m/z [M + H]+ 330.0815 (calcd for C18H16ClNO4
[M + H]+ 330. 0819).
1H-NMR (400 MHz, DMSO-d6) ꢁ: 8.78 (s, 1H, –NH), 8.01 (d, 1H,
J ¼ 7.31 Hz, Ar-Hi), 7.64 (s, 1H, Ar-Hc), 7.20 (s, 1H, Ar-Hd), 7.19
(s, 1H, Ar-He), 7.06–7.05 (m, 2H, Ar-Hg and Ar-Hh), 6.91 (d, 1H,
J¼8.05 Hz, Ar-Hf), 4.10 (q, 2H, OCH2CH3), 3.79 (s, 3H,
Ar-OCH3), 3.77 (s, 3H, Ar-OCH3), 3.68 (s, 2H, Ha and Hb),
1.39 (t, 3H, OCH2CH3). 13C-NMR (100 MHz, DMSO-d6) ꢁ:
162.70, 149.72, 149.60, 149.05, 140.42, 137.74, 137.44, 135.89,
128.08, 125.11, 122.39, 121.01, 112.78, 108.93, 107.05, 64.72,
56.39, 56.34, 38.55 and 15.34. HRMS (ESI) m/z
5,6-Dimethoxy-1H-indene-2-carboxylic acid (3-bromo-phenyl)-
amide (15)
Yield 34%; m.p. 155–157 ꢀC; ꢀ
(log e) 339 nm (4.29). [M + H]+ 340.1467 (calcd for C20H21NO4 [M + H]+ 340.1471).
DMSO
maks
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.96 (s, 1H, –NH), 8.07 (s, 1H,
Ar-Hf), 7.73 (s, 1H, Ar-Hc), 7.70 (d, 1H, J¼8.22 Hz, Ar-Hi), 7.29 5,6-Dimethoxy-1H-indene-2-carboxylic acid (2-fluoro-phenyl)-
(d, 1H, J¼8.22 Hz, Ar-Hg), 7.23 (dd, 1H, J¼9.59 Hz, J¼8.22 Hz, amide (20)
Ar-Hh), 7.19 (s, 1H, Ar-Hd), 7.18 (s, 1H, Ar-He), 3.79 (s, 3H,
DMSO
maks
Yield 38%; m.p. 167–170 ꢀC; ꢀ
(log e) 333 nm (4.22).
Ar-OCH3), 3.77 (s, 3H, Ar-OCH3), 3.67 (s, 2H, Ha and Hb).
13C-NMR (100 MHz, DMSO-d6) ꢁ: 163.59, 149.82, 149.10,
141.64, 140.23, 138.00, 137.83, 135.77, 131.26, 126.36, 122.74,
122.08, 119.20, 108.89, 107.10, 56.41, 56.39 and 39.00. HRMS
(ESI) m/z [M + H]+ 374.0322 (calcd for C18H16BrNO4
[M + H]+ 374.0314).
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.63 (s, 1H, –NH), 7.73 (s, 1H,
Ar-Hc), 7.65 (d, 1H, J ¼ 8.02 Hz, Ar-Hi), 7.25 (dd, 1H,
J ¼ 8.22 Hz, J ¼ 8.22 Hz, Ar-Hh), 7.24–7.21 (m, 2H, Ar-Hf and
Ar-Hg), 7.19 (s, 1H, Ar-Hd), 7.18 (s, 1H, Ar-He), 3.79 (s, 3H,
Ar-OCH3), 3.78 (s, 3H, Ar-OCH3), 3.67 (s, 2H, Ha and Hb).
13C-NMR (100MHz, DMSO-d6) ꢁ: 163.36, 149.77, 149.09,
139.90, 137.92, 135.87, 127.18, 126.99, 126.91, 126.61, 124.93,
124.89, 116.47, 116.27, 108.93, 107.10, 56.40 (2ꢁ Ar-OCH3) and
5,6-Dimethoxy-1H-indene-2-carboxylic acid o-tolylamide (16)
Yield 48%; m.p. 197–200 ꢀC; ꢀ
(log e) 329 nm (4.31). 38.89. HRMS (ESI) m/z [M + H]+ 314.1121 (calcd for
DMSO
maks
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.41 (s, 1H, –NH), 7.66 (s, 1H, C18H16FNO3 [M + H]+ 314.1114).
Ar-Hc), 7.32 (d, 1H, J ¼ 7.31 Hz, Ar-Hi), 7.26–7.21 (m, 2H, Ar-Hh
and Ar-Hg), 7.19 (s, 1H, Ar-Hd), 7.18 (s, 1H, Ar-He), 7.12 (d, 1H,
J ¼ 6.85 Hz, Ar-Hf), 3.79 (s, 3H, Ar-OCH3), 3.78 (s, 3H,
Ar-OCH3), 3.67 (s, 2H, Ha and Hb), 2.23 (s, 3H, Ph-CH3).
13C-NMR (100 MHz, DMSO-d6) ꢁ: 163.30, 149.57, 149.54,
140.63, 139.94, 137.68, 137.08, 135.99, 133.80, 130.94, 126.78,
126.61, 126.22, 108.94, 106.97, 56.39 (2ꢁ Ar-OCH3) and 39.00,
18.59. HRMS (ESI) m/z [M + H]+ 310.1360 (calcd for C19H19NO3
[M + H]+ 310.1365).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (2-chloro-phenyl)-
amide (21)
Yield 41%; m.p. 156–159 ꢀC; ꢀ
(log e) 333 nm (4.24).
DMSO
maks
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.49 (s, 1H, –NH), 7.72 (s, 1H,
Ar-Hc), 7.68 (d, 1H, J ¼ 8.22 Hz, Ar-Hi), 7.56 (dd, 1H,
J ¼ 7.76 Hz, J ¼ 7.76 Hz, Ar-Hh), 7.34 (dd, 1H, J ¼ 7.76 Hz,
J ¼ 7.76 Hz, Ar-Hg), 7.23 (d, 1H, J ¼ 7.76 Hz, Ar-Hf), 7.20 (s,
1H, Ar-Hd), 7.19 (s, 1H, Ar-He), 3.79 (s, 3H, Ar-OCH3), 3.78 (s,
3H, Ar-OCH3), 3.68 (s, 2H, Ha and Hb). 13C-NMR (100 MHz,
DMSO-d6) ꢁ: 163.28, 149.79, 149.09, 139.88, 137.94, 137.90,
135.84, 135.71, 130.15, 128.87, 128.10, 127.96, 127.39, 108.92,
107.09, 56.38, 56.34 and 38.89. HRMS (ESI) m/z
[M + H]+ 330.0827 (calcd for C18H16ClNO3 [M + H]+ 330.0819).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (2-ethyl-phenyl)-
amide (17)
DMSO
maks
Yield 42%; m.p. 196–200 ꢀC; ꢀ
(log e) 329 nm (4.21).
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.40 (s, 1H, –NH), 7.66 (s, 1H,
Ar-Hc), 7.31 (d, 1H, J ¼ 6.39 Hz, Ar-Hi), 7.25 (dd, 1H, J ¼ 5.94 Hz,
J ¼ 6.39 Hz, Ar-Hg), 7.19 (s, 1H, Ar-Hd), 7.18 (s, 1H, Ar-He), 7.16
(d, 1H, J ¼ 8.05 Hz, Ar-Hf), 3.79 (s, 3H, Ar-OCH3), 3.77 (s, 3H, Ar-
OCH3), 3.75 (s, 2H, Ha and Hb), 2.57 (q, 2H, CH2CH3), 1.11 (t, 3H,
CH2CH3). 13C-NMR (100 MHz, DMSO-d6) ꢁ: 163.67, 149.54,
149.02, 140.63, 139.94, 137.68, 136.96, 136.41, 135.98, 129.09,
127.77, 126.74, 126.60, 108.92, 106.95, 56.38 (2ꢁ Ar-OCH3),
39.02, 24.64 and 14.75. HRMS (ESI) m/z [M + H]+ 324.1526
(calcd for C20H21NO3 [M + H]+ 324.1521).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (2-bromo-phenyl)-
amide (22)
Yield 45%; m.p. 173–175 ꢀC; ꢀ
(log e) 333 nm (4.24).
DMSO
maks
1H-NMR (400 MHz, DMSO-d6) ꢁ: 9.44 (s, 1H, –NH), 7.72 (s, 1H,
Ar-Hc), 7.68 (d, 1H, J ¼ 8.68 Hz, Ar-Hi), 7.66 (d, 1H, J ¼ 8.68 Hz,
Ar-Hf), 7.39 (dd, 1H, J ¼ 7.76 Hz, J ¼ 7.76 Hz, Ar-Hh), 7.20
(s, 1H, Ar-Hd), 7.19 (s, 1H, Ar-He), 7.15 (d, 1H, J ¼ 7.31 Hz, Ar-
Hg), 3.79 (s, 3H, Ar-OCH3), 3.78 (s, 3H, Ar-OCH3), 3.68 (s, 2H,
Ha and Hb). 13C-NMR (100 MHz, DMSO-d6) ꢁ: 163.22, 149.80,
149.11, 139.93, 137.88, 137.08, 135.84 (2ꢁ Aromatic C), 133.29,
128.73, 128.34, 127.90, 126.88, 108.94, 107.11, 56.38 (2ꢁ
Ar-OCH3) and 38.87. HRMS (ESI) m/z [M + H]+ 374.0325
(calcd for C18H16BrNO4 [M + H]+ 374.0314).
5,6-Dimethoxy-1H-indene-2-carboxylic acid (2-methoxy-phenyl)-
amide (18)
DMSO
maks
Yield 47%; m.p. 206–209 ꢀC; ꢀ
(log e) 337 nm (4.26).
1H-NMR (400 MHz, DMSO-d6) ꢁ: 8.88 (s, 1H, –NH), 7.93 (d, 1H,
J ¼ 8.22 Hz, Ar-Hi), 7.67 (s, 1H, Ar-Hc), 7.19 (s, 1H, Ar-Hd), 7.17
(s, 1H, Ar-He), 7.10–7.05 (m, 2H, Ar-Hg, Ar-Hh), 6.94 (d, 1H,
J ¼ 8.68 Hz, Ar-Hf), 3.85 (s, 3H, Ar-OCH3), 3.79 (s, 3H,
Ar-OCH3), 3.77 (s, 3H, Ph-OCH3), 3.68 (s, 2H, Ha and Hb).
13C-NMR (100 MHz, DMSO-d6) ꢁ: 162.90, 150.83, 149.68,
149.05, 140.44, 137.79, 137.39, 135.93, 127.80, 125.39, 123.14,
120.97, 111.80, 108.93, 107.02, 56.47, 56.39, 56.36 and 38.73.
HRMS (ESI) m/z [M + H]+ 326.1320 (calcd for C19H19NO4
[M + H]+ 326.1314).
Inhibition studies on AChE and BuChE
The inhibitory activities of 5,6-dimethoxy-1H-indene-based sec-
ondary amide derivatives were evaluated against AChE (E.C.
3.1.1.7, Type VI-S, Electrophorus electricus) and BuChE (E.C.
3.1.1.8, equine serum) spectrophotometrically by the method of
Ellman with slight modifications using commercially available
donepezil hydrochloride as the reference compound25. Stock

6
M. Koca et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
solutions were dissolved in dimethylsulfoxide and then diluted in of the reaction mixture was 3 mL, and the final concentrations of
a 50 mM Tris buffer (pH 8.0) to provide a final concentration the test compounds and metals were 100 mM.
range. In a 96-well polystyrene photometric microplates, the assay
medium in each well consisted of 50 mL of a Tris buffer, 125 mL of Molecular modeling procedure
3 mM 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), 25 mL of
The simulation system was built on the crystallographic structures
0.2 U/mL enzyme (AChE or BuChE) and a 15 mM substrate
of 1EVE and 1P0I, which were obtained from the Protein Data
acetylthiocholine iodide (ATCI) or butyrylthiocholine iodide
Bank. The molecular modelling studies were performed via
(BTCI). The assay mixture containing the enzyme, buffer,
Surflex-Dock in Sybyl-X 2.0 by Tripos Associates (St Louis,
DTNB and 25 mL of the inhibitor compound was preincubated
MO). 3D structures of compounds 20 and 21 were constructed
for 15 min at 37 ꢀC before the substrate was added to begin the
using the Sybyl sketcher module. The structures were minimized
reaction. All test compounds were prepared at seven different
using the Powell method until the gradient was 0.05 kcal/mol,
concentrations: 0.09, 0.195, 0.39, 0.78, 3.13, 12.5 and 50 mg/mL.
max iterations: 1000 with the Tripos force field with the
The absorbance of the reaction mixture was then measured three
Gasteiger–Marsili charge. At the commencement of docking, all
times at 412 nm every 45 s using a microplate reader (Bio-Tek
the water and ligands were removed and the random hydrogen
ELx800, Winooski, VT). The IC50 values of the compounds
atoms were added. Docking calculations using Surflex-Dock for
showing half of the maximal inhibitory concentration, the
1EVE and 1P0I were performed through protomol generation by
measurements and the calculations were determined with
ligand. The parameters used were threshold 0.5 and bloat 0.
GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA).
Results were expressed as mean SD and all experiments were
Result and discussion
performed in triplicate.
Chemistry
The mechanism of AChE and BuChE inhibition
5,6-Dimethoxy-1H-indene-2-carboxamide
derivatives
were
synthesized via the pathway outlined in Scheme 1. In the first
step, 3-(3,4-dimethoxy-phenyl)-propionic acid (I), that was used
as the starting material, was converted to 3-(3,4-dimethoxy-
phenyl)-propionyl chloride (II) in the presence of oxalyl chloride,
in mixing CH2Cl2 at the room temperature for 12 h. In the second
step, 5,6-dimethoxy-1-indanone (III) was occurred by a ring-
closure reaction between 3-(3,4-dimethoxy-phenyl)-propionyl
chloride and AlCl3 as a Lewis acid. The indanone ring was then
reacted with dimethylcarbonate in the presence of sodium hydride
and resulted in 5,6-dimethoxy-1-oxo-indane-2-carboxylic acid
methyl ester (IV). Microwave heating has been used to synthesis
of secondary amides by direct irradiation of para, meta and ortho-
substituted primary anilines–carboxylic acid methyl esters. The
5,6-dimethoxy-1-oxo-indane-2-carboxamide derivatives (V) were
obtained with good yields only in a few minutes. In the next step,
5,6-dimethoxyindanone ring system was reduced to obtaine the
intermediate reduction product, 1-hydroxy-5,6-dimethoxy-indane-
2-carboxamide (VI), by using a mild reducing agent (NaBH4) for
2 h in an ice bath (0 ꢀC) with 100% yield. And in the final step, 1-
hydroxy-5,6-dimethoxy-indane-2-carboxamide was refluxed with
catalytic amount of p-toluenesulfonic acid (PTSA) in methanol for
2.5h to obtaine the 5,6-dimethoxy-1H-inden-2-carboxamides (1–
22) as the final products, in moderate to good yields (30–80%).
The structures of the compounds were confirmed by using the
proton nuclear magnetic resonance (1H-NMR), the carbon nuclear
magnetic resonance (13C-NMR) and HRMS.
The compounds 20 and 21 were selected for kinetic measurements
because they were found to be the highest inhibitory activity
against BuChE and AChE, respectively. For this purpose, the rate
of the enzyme activity was carried out without the inhibitor and in
5, 10 and 20 mM concentrations of the inhibitor for AChE and 5,
10 and 20 mM concentrations for BuChE using different concen-
tration of the substrates (from 0.1 to 1.5 mM). The obtained data
were used to create substrate-velocity curves, which were
transformed in GraphPad Prism program to Lineweaver–Burk
plots. Each experiment was performed in triplicate.
Inhibition of Ab1–42 self-induced aggregation
Thioflavin T (ThT)-based fluorometric assay is a well-known
method for measuring the inhibition of Ab1–42 aggregation26. For
this method, we selected a natural reference product, curcumin,
that inhibits Ab1–42 aggregation27. ThT, Curcumin, and
1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) were purchased from
Sigma Aldrich. Ab1–42 samples (Anaspec Inc., Fremont, CA)
were dissolved in HFIP (1 mg/mL) and incubated for 24 h at room
temperature. After solvent evaporation, HFIP pretreated Ab1–42
was resolubilized in dry DMSO to a final stock concentration of
200 mM. Stock solutions of each test compound were prepared in
DMSO and diluted with sodium phosphate buffer (pH 7.4) at
different concentrations ranging from 10 to 100 mM. Then, 20 mL
of 25 mM Ab1–42 sample was added to each well for the incubation
with 20 mL of test compounds in dark at 37 ꢀC for 48 h with no
agitation. After the incubation period, 160 mL of 5 mM ThT in
50 mM glycine-NaOH buffer (pH 8.0) was added to each well.
Each assay was run in triplicate. Fluorescence was measured on a
(Bio-Tek FLx800) with excitation and emission wavelengths of
446 and 490 nm, respectively.
In 1H-NMR spectra, the resonance signals of the amide (–NH)
proton appeared at ꢁ 10.06–7.52 ppm for the compounds. The
peaks for aromatic protons appeared in the range of ꢁ 8.07–
6.60 ppm. The 13C-NMR spectra showed the characteristic reson-
ance frequencies of the carbonyl signals of the secondary amide
functional group in the range of 163.67–160.08 ppm. Also the
aromatic carbons appeared at 161.55–105.90 ppm. Spectral data of
the compounds are documented in Supplementary material.
Spectrophotometric measurement of metal-chelation
capacity
Inhibitory activity against AChE and BuChE
The metal complexation capacities of the most potent cholin-
esterase and Ab1–42 inhibitors was studied for some metals such
as Cu2+, Fe2+ and Zn2+ in dimethylsulfoxide (DMSO) at room
temperature by using a Ultraviolet-visible (UV–Vis) spectropho-
tometer with a wavelength ranging from 190 to 380 nm28,29. The
UV absorption of test compounds 20 and 21, in the absence or
presence of CuSO4, FeSO4 and ZnSO4, was recorded in a 1 cm
quartz cuvette after 20 min at room temperature. The final volume
The AChE and BuChE activities of all the compounds (1–22)
were evaluated and compared with the anti-Alzheimer drug
donepezil as the reference compound. The activities were
measured in vitro by the spectrophotometric method developed
by Ellman, with slight modifications25. The IC50 values (mM) for
ChE inhibition and the selectivity indexes (SIs) of the compounds
are summarized in Table 1, which displays how the compounds

DOI: 10.1080/14756366.2016.1186019
1H-indene-2-carboxamides as anti-Alzheimer agents
7
Table 1. Anticholinesterase activity, inhibition of self-induced Ab1–42 aggregation of the compounds (1–22).
Selectivity
for BuChEz
Ab1–42 aggregation
inhibitionô,x (%)
Compound
R1
R2
R3
IC50 (mM) SDx
Log PI
MRI
AChE*,x
BuChEy,x
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
H
CH3
C2H5
OCH3
OC2H5
F
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
C2H5
OCH3
OC2H5
F
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
6.42 1.685
4.17 0.207
5.16 3.158
6.47 0.33
5.27 0.127
3.69 0.08
3.85 0.141
3.17 0.105
4.45 0.197
4.63 0.007
4.5 0.197
5.87 1.332
2.86 0.172
2.29 0.574
4.34 0.10
3.51 0.022
3.50 3.568
3.95 0.06
4.87 0.038
2.33 0.021
1.33 0.062
3.02 0.072
0.042 0.041
1.23 0.125
1.45 0.07
1.48 0.12
5.07 0.103
3.78 0.042
1.38 0.012
2.8 0.41
2.64 0.01
2.05 0.059
4.9 0.47
3.05 0.112
2.41 0.19
2.23 0.121
3.49 0.068
5.57 0.27
1.28 0.059
1.72 0.13
3.71 0.125
4.32 0.072
1.08 0.011
1.27 0.032
1.09 0.31
0.54 0.017
5.2
2.9
3.5
1.3
1.4
2.7
1.4
1.2
2.2
0.9
1.5
2.4
1.3
0.7
0.8
2.7
2.0
1.1
1.1
2.2
1.0
2.8
0.1
34.6 0.1
40.3 1.4
31.5 2.5
5.4 0.7
2.85
3.34
3.75
2.72
3.06
3.01
3.41
3.68
3.34
3.75
2.72
3.06
3.01
3.41
3.68
3.34
3.75
2.72
3.06
3.01
3.41
3.68
86.89
92.78
97.38
94.14
98.94
87.29
91.49
94.58
92.78
97.38
94.14
98.94
87.29
91.49
94.58
92.78
97.38
94.14
98.94
87.29
91.49
94.58
9.8 1.9
42.5 2.2
30.3 3.7
33.4 1.1
12.7 1.3
8.4 2.7
13.8 1.5
22.6 2.7
26.9 1.0
10.1 2.8
3.8 3.1
46.4 4.0
37.6 0.3
14.8 2.7
11.3 3.1
50.3 2.4
45.7 3.5
48.2 0.8
CH3
C2H5
OCH3
OC2H5
F
H
H
Cl
Br
H
Donepezil
Curcumin
42.3 2.6
*50% Inhibitory concentration of AChE.
y50% Inhibitory concentration of BuChE.
zSelectivity for BuChE ¼ IC50 (AChE)/IC50 (BuChE).
ôInhibition of self-induced Ab1–42 aggregation (25 mM) by tested inhibitors (25 mM).
xValues are expressed as mean standart error of the mean of three independent experiments and each performed in triplicate (SD ¼ standart
deviation).
ICalculated by ChemDraw 2015.
MR: Molar Refractivity.
(1–22) clearly showed moderate to good potency and similar beneficial effects on BuChE than on AChE inhibitory activities.
inhibitory activity against both ChEs at micromolar concentra- In these compounds, para- and meta-CH3-substituted compounds
tions. The synthesized compounds exhibited inhibitory activities 16 and 9 showed the most significant BuChE inhibition. The
against AChE, with IC50 values ranging from 1.33 to 6.47 mM. positive effects of the electron-withdrawing atoms (F, Cl and Br)
Generally, most compounds were found to be similarly effective were seen in BuChE inhibition, as they were with AChE
inhibitors of both ChEs, with low SIs.
inhibition. It can be stated that the AChE inhibition mechanism
Among them, meta- and ortho-chloro-substituted analogues of the synthesized compounds depends highly on the electro-
(14 and 21) displayed the highest AChE inhibition, with IC50 negative properties of the substituent groups. However, based on
values of 2.29 and 1.33 mM, respectively. The other potent AChE the BuChE inhibition results, not only electronegativity but also
inhibitors, 13 and 20 (meta- and ortho-fluoro analogues), showed hydrophobic or electrostatic interactions may be responsible for
similar activity, with IC50 values of 2.86 and 2.33 mM, respect- BuChE inhibition of these compounds. Conversely, in the overall
ively. The comparison of non-substituted compound 1 and the evaluation of the results, no statistically significant correlation
other substituted compounds demonstrated that substitution of any was found between the calculated LogP and molar refractivity
groups, except the p-methoxy substitution, at the ortho, meta and values of the synthesized compounds and either of their ChE
para positions of the phenyl ring increased anti-AChE activity inhibition activities (Table 1). In the series, the most potent
between 1.09 and 4.82 times. The results indicated that substi- BuChE (20) and AChE (21) inhibitors were selected for kinetic
tution at any position on the phenyl ring makes a significant analysis to investigate the type of inhibition.
contribution to anti-AChE activity. Specifically, substituting the
fluorine and chlorine groups at the meta and ortho positions of the
Kinetic study of AChE and BuChE
phenyl ring appeared to have a crucial effect on AChE inhibition.
This positive contribution might be sourced from the high
electronegativity of the halogen atoms.
For deep understanding of the mechanism of inhibition, we
choosed the most potent ChE inhibitors, 20 and 21, for kinetic
studies. The steady-state inhibition data of the compounds for
both ChEs are shown in Figure 2. Kinetic analysis of the
Lineweaver–Burk plots of AChE inhibitory activity showed that
there was an increasing slope and an increasing intercept at higher
concentrations. The result indicates mixed-type inhibition mech-
anism as the result of binding to both active sites (CAS and PAS)
of AChE for the compound 21. The kinetic study of compound 20
on BuChE inhibitory activity showed that lines crossing the x-axis
in the same point (unchanged Km) and decreased Vmax with
The IC50 values of compounds 1–22 showed that all
compounds were good BuChE inhibitors (1.08–5.57 mM).
Compounds 1 (non-substituted), 20 (p-fluoro-substituted), and
22 (p-chloro-substituted) exhibited the most powerful effects on
BuChE inhibition in series, with IC50 values of 1.23, 1.08 and
1.09 mM and SIs of 5.2, 2.2 and 2.8, respectively (Table 1).
According to the anti-BuChE results, the electron-donating
groups CH3, C2H5, OCH3 and OC2H5, except compound 10, at
all positions (o-, m- and p-) on the phenyl ring provided more

8
M. Koca et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Figure 2. Lineweaver–Burk plots of inhibition kinetics of the compounds 20 and 21.
Figure 3. Inhibition of self-induced Ab1–42 aggregation by the test compounds and reference curcumin at concentration 25 mM.
increasing inhibitor concentrations. This result indicates that promote beneficial results in AD patients, so the chelation
5,6-dimethoxy-1H-indene-2-carboxamide derivatives exhibit non- abilities for biometals of the most potent BuChE inhibitor (20)
competitive inhibition mechanism on BuChE.
were determined by use of a spectrophotometer. The UV
absorption of the compounds in dimethyl sulfoxide changed
with the titration of CuSO4, FeSO4, and ZnSO4. Compound 20
(Figure 4) showed decreased absorption peaks after adding with
all metal ions used. Alterations in the absorption intensity, after
adding CuSO4, FeSO4 and ZnSO4, indicated the formation of
biometal complexes for compound 20.
Inhibition of Ab1–42 self-induced aggregation
In order to investigate the inhibition of Ab1–42 aggregation, a
thioflavin T (ThT)-based fluorometric assay was performed26.
Curcumin, known to be a natural product that inhibits self-
induced Ab aggregation, was used as the reference com-
pound30,31. The effects on Ab1–42 peptide aggregation of the
compounds at a concentration of 25 mM are summarized in Table
1 and Figure 3. In the ThT assay, the inhibition results showed that
some of the compounds prevented Ab1–42 peptide aggregation
with different inhibition values (3.8–55.3%). The most potent
AChE inhibitors (16, 20 and 22) exhibited good Ab1–42 inhibition
potencies with respective inhibition ratios of 46.4, 50.3 and
48.2%, which are higher than curcumin’s inhibition ratio (42.3%).
In silico docking studies
The most potent ChE inhibitors are docked at the binding site of
acetyl- and butyrylcholinesterase enzymes, we performed
molecular modeling studies by utilizing the surflex-dock simu-
lation program. According to the simulation results, the most
potent AChE inhibitor 21 displayed multiple binding patterns with
Torpedo californica (TcAChE) (Figure 5). In the 1EVE-21
complex, all structural main moieties (5,6-dimethoxy-indanone,
carboxamide and phenyl ring) occupied the CAS and PAS of
enzyme and dominated several hydrogen bondings with residues
Tyr130, Tyr121, Gly117 and ꢂ–ꢂ stacking interactions with
residues Phe330 and Tyr334. The aromatic OCH3 groups of the
Studies of metal chelating effect
High levels of metal accumulation are frequently encountered in
the brains of AD patients. Additionally, studies have postulated
that chelation of some biometal ions (Cu2+, Fe2+ and Zn2+) might

DOI: 10.1080/14756366.2016.1186019
1H-indene-2-carboxamides as anti-Alzheimer agents
9
Figure 4. (a) UV absorption spectra of
compound 20 (100 mM in DMSO) alone, (b)
UV absorption spectra of the mixture com-
pound 20 (100 mM) and CuSO4 (100 mM), (c)
UV absorption spectra of the mixture com-
pound 20 (100 mM) and FeSO4 (100 mM) and
(d) UV absorption spectra of the mixture
compound 20 (100 mM) and ZnSO4
(100 mM).
Figure 5. 3D representation of the binding
mode of the most potent inhibitor 21 at the
active sites of AChE.
compound 21 exhibited two hydrogen bond interactions with OH (Figure 6). Two hydrogen bond interactions occurred between the
˚
˚
group of Tyr130 residue in CAS (distances 1.94 and 2.75 A). The C¼O group of ligand 20 and the NH groups of Asn289 (2.43 A)
˚
other hydrogen bonding interactions occurred between two and Gln119 (1.90 A). The OCH3 group in position six of the
methoxy groups of ligand 21 and NH group of Gly117 residue indanone ring of the ligand 20 occurred a hydrogen bond with
˚
in oxyanion hole within the distances 1.95 and 2.68 A. Also phenolic OH group of Thr120 (2.63 A) in PAS of HuBuChE. In
˚
ligand 21 created ꢂ–ꢂ stacking interaction with indole moiety of peripheric anionic site of 1P0I, fluorine atom at the ortho position
˚
Trp84 in CAS (distance 3.8–4.7 A). In peripheric anionic site of on phenyl ring created a hydrogen bond with NH group of Ala277
˚
˚
1-EVE, the C¼O group of amide moiety is bridged to phenolic residue (2.30 A) and NH group of Asn68 residue (2.33 A). These
˚
OH group of Tyr121 residue via hydrogen bonding (2.71 A). In several hydrogen bonding interactions supports high anti-BuChE
the complex, 2-chlorophenyl ring is stacked against Phe330 and activity of this compound.
Tyr334, located at the PAS. A ꢂ–ꢂ stacking interaction occurred
between the phenyl group of Tyr334 residue and 2-fluorophenyl
Conclusion
˚
ring (2.97–2.99 A). The simulation study of compound 21–1EVE
In this study, a new series of 5,6-dimethoxy-1H-indene-2-
carboxamide derivatives (1–22) was developed. This study
originated with an effective anti-Alzheimer drug, donepezil,
which is a selective AChE inhibitor with very poor Ab anti-
aggregation activity. The aim of this research was to obtain new
complex has illuminated for its potent AChE inhibitory activity
and mixed-type inhibition.
On the other hand, the most potent BuChE inhibitor 20, showed
many hydrogen bonds with Thr120, Ala277, Asn68, Asn289 and
Gln119 residues of Human butyrylcholinesterase enzyme (1P0I)

10 M. Koca et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Figure 6. 3D representation of the binding
mode of the most potent inhibitor 20 at the
active sites of BuChE.
disease: synthesis, biological assessment, and molecular modeling.
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amyloid inhibition activities. Therefore, one of the pharmaco-
phore moieties (5,6-dimethoxyindanone) of donepezil was
modified to 5,6-dimethoxy-1H-indene;
a secondary amide
bridge was constructed with different substituted primary anilines
to obtain 1H-inden-based secondary carboxamide analogues to
measure the possible effects on ChE inhibition. Among the
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to be the most potent inhibitor for Ab1–42 aggregation (50.3% 2.4
at 25mM). Docking simulations also supported the cholinesterase
inhibition study of the most potent BuChE inhibitor (20), which
exhibited many hydrogen-bonding interactions with amino acid
residues in the PAS of BuChE. Further studies to synthesize 5,6-
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Acknowledgements
This research work was supported by Ataturk University Research Fund
(Project No: 2015/326), Turkey. The measurement of cholinesterase
inhibitions was performed at the Faculty of Pharmacy and Department of
Pharmaceutical Chemistry, University of Ataturk, Turkey, under the
supervision of Prof. Dr. H. Inci Gul.
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Declaration of interest
The authors have declared no conflicts of interest with the
presented data from this article.
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Supplementary material available online