9-Substituted acridine derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors possessing antioxidant activity for Alzheimer's disease treatment

Article abstract of DOI:10.1016/j.bmc.2017.09.028

We investigated the inhibitory activity of 4 groups of novel acridine derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) using the methods of enzyme kinetics and molecular docking. Antioxidant activity of the compounds was determined using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^[rad]+) radical decolorization assay as their ability to scavenge free radicals. Analysis of the esterase profiles and antiradical activities of the acridine derivatives showed that 9-aryl(heteroaryl)-N-methyl-9,10-dihydroacridines have a high radical-scavenging activity but low potency as AChE and BChE inhibitors, whereas 9-aryl(heteroaryl)-N-methyl-acridinium tetrafluoroborates effectively inhibit cholinesterases but do not exhibit antiradical activity. In contrast, a group of derivatives of 9-heterocyclic amino-N-methyl-9,10-dihydroacridine has been found that combine effective inhibition of AChE and BChE with rather high radical-scavenging activity. The results of molecular docking well explain the observed features in the efficacy, selectivity, and mechanism of cholinesterase inhibition by the acridine derivatives. Thus, in a series of acridine derivatives we have found compounds possessing dual properties of effective and selective cholinesterase inhibition together with free radical scavenging, which makes promising the use of the acridine scaffold to create multifunctional drugs for the therapy of neurodegenerative diseases.

Full text of DOI:10.1016/j.bmc.2017.09.028

Accepted Manuscript
9-Substituted acridine derivatives as acetylcholinesterase and butyrylcholines-
terase inhibitors possessing antioxidant activity for Alzheimer's disease treat-
ment
Galina F. Makhaeva, Sofya V. Lushchekina, Natalia P. Boltneva, Olga G.
Serebryakova, Elena V. Rudakova, Alexey A. Ustyugov, Sergey O. Bachurin,
Alexander V. Shchepochkin, Oleg N. Chupakhin, Valery N. Charushin, Rudy
J. Richardson
PII:
S0968-0896(17)31509-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.09.028
BMC 13988
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 July 2017
17 September 2017
18 September 2017
Please cite this article as: Makhaeva, G.F., Lushchekina, S.V., Boltneva, N.P., Serebryakova, O.G., Rudakova, E.V.,
Ustyugov, A.A., Bachurin, S.O., Shchepochkin, A.V., Chupakhin, O.N., Charushin, V.N., Richardson, R.J., 9-
Substituted acridine derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors possessing antioxidant
activity for Alzheimer's disease treatment, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.09.028
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9-Substituted acridine derivatives as acetylcholinesterase and
butyrylcholinesterase inhibitors possessing antioxidant activity for
Alzheimer's disease treatment
Galina F. Makhaevaa, Sofya V. Lushchekinaa,b, Natalia P. Boltnevaa, Olga G. Serebryakovaa,
Elena V. Rudakovaa, Alexey A. Ustyugova, Sergey O. Bachurina, Alexander V. Shchepochkinc,d,
Oleg N. Chupakhinc,d, Valery N. Charushinc,d, Rudy J. Richardsone,f
aInstitute of Physiologically Active Compounds Russian Academy of Sciences,
Chernogolovka, 142432, Russia
bEmanuel Institute of Biochemical Physics Russian Academy of Sciences, Moscow, 119334,
Russia
cInstitute of Organic Synthesis of the Russian Academy of Sciences, S. Kovalevskaya Str., 22,
Ekaterinburg, 620041, Russia.
dUral Federal University, Mira St. 19, Ekaterinburg, 620002, Russia
eDepartment of Environmental Health Sciences, University of Michigan, Ann Arbor, MI 48109
USA
fDepartment of Neurology, University of Michigan, Ann Arbor, MI 48109 USA
*Corresponding author:
Dr. Rudy J. Richardson
Computational Toxicology Laboratory
Toxicology Program
Department of Environmental Health Sciences
University of Michigan
Ann Arbor, Michigan 48109-2029 USA
E-mail:
Tel:
Fax:
rjrich@umich.edu
+1-734-936-0769
+1-734-763-8095
1

Abstract
We investigated the inhibitory activity of 4 groups of novel acridine derivatives against
acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) using
the methods of enzyme kinetics and molecular docking. Antioxidant activity of the compounds
was determined using the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS.+) radical
decolorization assay as their ability to scavenge free radicals. Analysis of the esterase profiles
and antiradical activities of the acridine derivatives showed that 9-aryl(heteroaryl)-N-methyl-
9,10-dihydroacridines have a high radical-scavenging activity but low potency as AChE and
BChE inhibitors, whereas 9-aryl(heteroaryl)-N-methyl-acridinium tetrafluoroborates effectively
inhibit cholinesterases but do not exhibit antiradical activity. In contrast, a group of derivatives
of 9-heterocyclic amino-N-methyl-9,10-dihydroacridine has been found that combine effective
inhibition of AChE and BChE with rather high radical-scavenging activity. The results of
molecular docking well explain the observed features in the efficacy, selectivity, and mechanism
of cholinesterase inhibition by the acridine derivatives. Thus, in a series of acridine derivatives
we have found compounds possessing dual properties of effective and selective cholinesterase
inhibition together with free radical scavenging, which makes promising the use of the acridine
scaffold to create multifunctional drugs for the therapy of neurodegenerative diseases.
Keywords
9-heterocyclic aminо-N-methyl-9,10-dihydroacridines; esterase profile; acetylcholinesterase
inhibitors; butyrylcholinesterase inhibitors; molecular docking; antioxidant activity
2

1. Introduction
Acridines are considered privileged scaffolds in drug discovery for protozoan and
neurodegenerative diseases 1. Acridine derivatives have a broad spectrum of therapeutic
applications as antibacterial 2, antimalarial 3, antileishmanial and antitrypanosomal 4, antiviral 5,
anticancer 6 and antiprion 7-9 agents. They have also been reported to have anti-inflammatory,
anti-diabetic 10, 11 and anti-Alzheimer activity 12-14. Recently they demonstrated an anti-TDP-43
aggregation effect in ALS disease models 15. Acridine derivatives are optimal starting points for
the design of novel hybrid and dimeric multitarget lead and drug candidates 1.
Alzheimer's disease (AD), the most widely encountered type of dementia in older people, is a
multifactorial and fatal neurodegenerative disorder, which is characterized by an inexorable
decline in cognitive function and memory that progresses to the complete degradation of
personality. AD involves degeneration of cholinergic neurons and diminishing cholinergic
transmission 16. Anticholinesterase drugs are used to compensate for deficiency of the
neurotransmitter acetylcholine 17. They replenish the acetylcholine deficit in the brain by
inhibiting cholinesterases, thereby increasing the duration of acetylcholine action on postsynaptic
receptors, thus enhancing cholinergic transmission. In a normal brain, acetylcholine is
predominantly (80%) hydrolyzed by AChE, whereas BChE plays a supplementary role.
However, with progression of AD, the AChE activity decreases, whereas the activity of BChE
gradually increases 18, 19. This phenomenon enhances the significance of BChE as an additional
therapeutic target for reducing the cholinergic deficiency inherent in AD 20-22
.
Currently, AD therapy is mainly founded on cholinesterase inhibitors, which are able to
increase acetylcholine levels in cholinergic synapses. To date, the number of approved drugs is
limited to only three cholinesterase inhibitors (rivastigmine, donepezil, and galantamine), and the
N-methyl-D-aspartate (NMDA) receptor antagonist, memantine 23, 24
.
The multifactorial nature of AD is commonly recognized, implying the involvement a
number of neurobiological targets in the development of this neurodegenerative disease. In this
context, the concept of multitarget drugs having an integrated action on a number of biological
targets involved in pathogenesis of the disease currently appears to be highly promising in the
design of drugs for treating AD 25, 26
.
Oxidative stress leading to oxidative damage of cell membranes, mitochondria, lipids and
proteins, may be one of the possible causes of neuronal death 27. Oxidative stress is characterized
3

as an imbalance between biochemical processes leading to the production of reactive oxygen
species (ROS) and their removal 28. The efficiency of the brain’s antioxidant system gradually
declines with age, and this decline is more pronounced in AD patients. This fact substantiates the
use of antioxidants in AD therapy 29, and the development of cholinesterase inhibitors with
attendant antioxidant properties is a modern trend in research directed toward efficient therapy of
AD 30-32
.
It is well known that compounds of the acridine family are able to inhibit AChE and BChE 33
34, 35. Tacrine (9-amino-1,2,3,4-tetrahydroacridine), a potent reversible inhibitor of AChE and
BChE, was the first drug approved by the FDA for treatment of Alzheimer’s disease 36.
However, it was withdrawn from clinical use because of its hepatotoxicity 37. Nevertheless, there
is a continuing interest in the tacrine template to design new hybrid molecules that might be safer
and more effective AD drugs than tacrine 38-41. Although the precise mechanism of
hepatotoxicity of tacrine has not been elucidated, oxidative stress appears to be involved to some
degree. Therefore, derivatization of acridine-based anticholinesterase compounds in a manner
that confers antioxidant activity would be expected to ameliorate this toxic concern.
The aim of the present study was to investigate the inhibitory properties of novel acridine
derivatives against the key enzymes of the cholinergic nervous system AChE and BChE, using
both kinetic and computational molecular modeling methods, as well to assess the ability of
compounds to scavenge free radicals using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS) assay. In addition, we have determined the inhibitory activity of the synthesized
acridines toward carboxylesterase (CaE, EC 3.1.1.1), a serine hydrolase structurally related to
cholinesterases that catalyzes the hydrolysis of many therapeutically important agents bearing
ester and other hydrolysable groups 42, 43. The ability of anticholinesterase compounds used for
AD therapy to inhibit CaE could lead to undesirable drug-drug interactions 44. Consequently, in
this investigation we were seeking to find compounds with anticholinesterase and antiradical
activity but lacking anti-CaE activity.
4

2. Results and discussion
2.1. Synthesis of acridine derivatives
Acridine derivatives can be obtained using three main approaches. The first one is based on
construction of the acridine ring system through the reaction of the corresponding carboxylic
acid with diphenylamine in the presence of ZnCl2 at elevated temperatures (200-260 оС) 45, 46
The second approach entails functionalization of acridine using metal-catalyzed cross-coupling
reactions 47. However, these two methods have some disadvantages, such as the necessity to
incorporate good leaving groups, the formation of byproducts due to side reactions of
organometallic reagents, and some difficulties in eliminating catalysts and auxiliary ligands.
Fortunately, there is a third approach involving metal-free methods for direct C-H
functionalization of acridines, based on nucleophilic aromatic substitution of hydrogen, the so-
called SNH reactions 48-54
.
A mechanism that is commonly accepted for the SNH reactions involves two steps. The first
one is addition of a nucleophile to an aromatic ring, thus leading to the formation of ϭH-adducts.
Oxidative aromatization of ϭH-adducts is realized at the second step by action of an outer-sphere
oxidant (Scheme 1). The ability of ϭH-adducts to undergo aromatization into SNH products varies
greatly: from very unstable and hardly spectroscopically detectable ϭH-adducts to rather stable
compounds, derived from the reactions of N-methylacridinium salts. In the latter case,
dihydroacridines can be easily isolated for studying their biological activity. Moreover, it is
possible to carry out aromatization of dihydroacridines in order to estimate how these structural
changes affect their biological properties.
Nu
Nu
H
H
Nu-
[O]
-H-
H
SN
N
N+
N+
-
-
BF4
CH3
бH-adduct
CH3
CH3
BF4
H-=2e-+H+
Scheme 1. Mechanism of SNH reactions.
Chemical structures of the studied compounds are given in Fig.1. All compounds can be
classified into 4 groups, depending on the structure of the core acridine nucleus and substituents
5

in the 9-position: 9-Aryl-N-methyl-9,10-dihydroacridines (compounds 1a-j), 9-Aryl-N-
methylacridinium tetrafluoroborates (compounds 2a-m), 9-heterocyclic aminо derivatives of 10-
methyl-9,10-dihydroacridine (compounds 3a-f), and 9-amino-N-methyl acridinium
tetrafluoroborates (compounds 4a,b).
R
R
R
HN
H
N
N
H
N+
N
N+
-
-
BF4
CH3
CH3
BF4
CH3
CH3
1a-j
2a-m
3a-f
4a,b
Fig. 1. Structures of the studied acridine derivatives.
Synthetic pathways to these compounds have previously been reported 55-57
.
Dihydroacridines 1a,b,f,g,j have been prepared through the reaction of 10-methyl-acridinium
iodide with the corresponding magnesium aryl bromides. Compounds 1h,i are derived from the
reaction of the 10-methylacridinium ion with sodium phenolates in diethyl ether at room
temperature. Aniline proved to react with the acridinium cation in DMSO to form the
corresponding amino compound 1c. Carbamoyl derivatives 1d and 1e were obtained by
treatment of the amino compound 1c with the corresponding anhydrides.
Acridinium salts 2a-m have been obtained by electrochemical oxidation of dihydroacridines
according to the previously reported procedure 55. The reaction of NH-heterocyclic compounds
with the 10-methylacridinium ion takes place smoothly in the presence of a base, thus affording
the corresponding acridines 3a-f 56. Compounds 4a and 4b have been obtained by
electrochemical amination of 10-methylacridinium tetrafluoroborate 58.
2.2. Inhibition of AChE, BChE and CaE. Structure-activity relationships
For all 31 acridine derivatives, we have determined their esterase profiles, i.e., the ability to
inhibit several esterases, including AChE, BChE, and CaE. This approach enables one to
estimate both the primary pharmacological effects of the tested compounds and their possible
6

adverse effects 59-63. AChE from human erythrocytes was used along with two enzymes of non-
human origin: equine serum BChE and porcine liver CaE. These sources of BChE and CaE were
used because of their relatively low cost, high sequence identity to human enzymes 60, 62, and the
exploratory character of this work.
The inhibitory activity against the esterases was characterized as the percentage of control
inhibition at 20 µM or as the IC50 value, i.e., the inhibitor concentration required to reduce the
enzyme activity by 50%. Bis-4-nitrophenyl phosphate (BNPP), which is a selective CaE
inhibitor, and tacrine, which is an effective AChE and BChE inhibitor, were used as positive
controls in the study of enzyme inhibition. The results of the inhibitory activity of 4 groups of
acridine derivatives against AChE, BChE and CaE, characterizing the esterase profile of
compounds, are presented in Table 1.
7

Table 1. Esterase profiles and ABTS-radical scavenging activity of acridine derivatives.
Inhibitory activity against AChE, BChE and
CaE
ABTS•+ scavenging
IC50 SEM (µM) or inhibition % at 20 µM
Comp.
R
IC50 SEM
(µM)
AChE
BChE
CaE
TEAC*
R
H
N
CH3
9-Aryl-N-methyl-9,10-dihydroacridines
n.a
62.1 6.7
n.a
1.02
0.93
18.9 1.5
22.4 1.6
1a
1b
CH3
NH2
O
> 20
(5.1 0.9%)
> 20
(10.6 2.1%)
n.a.
n.a.
n.a.
66.5 5.9
n.a.
n.a.
0.97
0.91
19.6 1.5
1c
CF3
HN
> 20
(7.1 1.3%)
21.05 1.6
1d
O
HN
CH3
> 20
(10.8 1.9%)
> 20
(7.2 1.3%)
n.a.
n.a.
0.99
20.3 1.4
1e
F
> 20
(11.1 1.9%)
n.a.
n.a.
0.89
0.95
24.7 1.5
20.4 1.3
1f
CH3
O
> 20
(9.3 1.8%)
> 20
(11.6 2.1%)
1g
8

> 20
(13.4 2.2%)
n.a.
n.a.
0.88
23.2 1.9
1h
OH
OH
> 20
(10.4 1.9%)
> 20
(23.6 3.3%)
n.a.
n.a.
1.0
19.6 1.1
22.9 1.5
1i
1j
> 20
(12.3 2.2%)
> 20
(11.1 2.1%)
S
0.92
R
N+
-
BF4
CH3
9-Aryl-N-methylacridinium tetrafluoroborates
> 20
(22.6 2.9%)
61.3 5.5
41.5 3.3
51.9 5.7
40.7 4.3
0.03
0.03
n.d.
n.d.
2a
2b
CH3
> 20
(16.3 2.9%)
NH2
> 20
(18.6 3.1%)
7.62 0.69
25.2 2.3
39.1 4.3
0.14
0.04
n.d.
n.d.
2c
N
3.35 0.34
n.a.
2d
O
HN
CF3
> 20
(7.7 1.3%)
> 20
(7.2 1.4%)
36.5 2.9
0.02
n.d.
2e
9

O
CH3
HN
54.3 4.8
32.3 2.9
13.7 1.2
35.1 2.9
n.a.
0.03
0.03
0.02
n.d.
n.d.
n.d.
2f
2g
2h
F
> 20
(8.2 1.5%)
> 20
(5.9 1.1%)
CH3
O
> 20
(7.8 1.4%)
58.5 5.7
> 20
(16.7 2.0%)
> 20
(31.2 2.8%)
2.74 0.24
61.8 6.6
0.37
0.43
n.d.
n.d.
2i
2j
OH
OH
CH3
H3C
49.8 3.9
60.1 5.4
n.a.
n.a.
CH3
> 20
(14.2 2.5%)
0.01
0.03
n.a.
n.d.
n.d.
n.d.
2k
2l
CH3
H3C
> 20
(7.3 1.3%)
> 20
(8.9 1.6%)
50.8 4.5
58.3 5.2
OH
> 20
(6.1 1.1%)
S
40.9 4.3
2m
N
H
N
CH3
9-Heterocyclic aminо derivatives of N-methyl-9,10-dihydroacridine
O
> 20
(16.4 1.8%)
12.3 1.1
0.46 0.05
0.83
25.6 1.9
3a
N
10

S
> 20
(5.0 0.9%)
11.8 0.6
18.8 1.1
16.7 1.3
0.84 0.05
0.81 0.09
1.17 0.09
0.74
0.65
0.50
29.7 2.1
36.5 2.8
47.2 3.2
3b
3c
3d
N
N
> 20
(5.4 1.0%)
N
N
O2N
N
> 20
(10.6 1.8%)
N
N
N
> 20
(15.3 1.8%)
N
12.4 1.3
1.08 0.09
13.0 1.2
0.59
0.80
40.6 2.4
27.0 1.2
3e
3f
N
> 20
(5.6 0.9%)
n.a.
N
R
HN
N+
-
BF4
CH3
9-Amino-N-methylacridinium tetrafluoroborates
H
39.5 3.5
4.83 0.39
15.7 1.5
n.a.
n.a.
0.09
0.05
n.d.
n.d.
4a
4b
> 20
(16.8 1.7)%
n-Bu
0.60 0.05
n.a.
0.0290 0.0002
n.а.
1.80 0.11
n.d.
Tacrine
BNPP
n.d.
n.d.
1.0
n.d.
n.d.
n.a.
n.d.
n.d.
21.6 1.4
Trolox
Esterase profiles: n.a. – not active at 20 µM.
Values expressed as % correspond to % inhibition at 20 µM.
Values without units of measurement correspond to IC50 values in µM.
ABTS.+- scavenging activity: TEAC values are expressed as Trolox equivalents calculated from
(A0–Atest)/(A0–ATrolox) at 20 µM concentrations, where A0 is the absorbance of a control lacking any
radical scavenger, Atest and ATrolox are the absorbances of the remaining ABTS.+ in the presence of the test
compound or Trolox, respectively. IC50 values (compound concentration required for 50 % reduction of
ABTS-radical) were determined for the most active compounds.
n.d. = not determined
n.a. = no activity
11

It follows from the data given in Table 1 that compounds of the first group, derivatives of 9-
aryl-N-methyl-9,10-dihydroacridines 1a-j (aryl substituent at C-9 and uncharged nitrogen atom
in the ring), proved to be only slight inhibitors of the cholinesterases.
It is worth noting that their structural aromatic analogues, 9-aryl(hetaryl)-N-
methylacridinium tetrafluoroborates (group 2), which carry a positive charge, demonstrated
higher anticholinesterase activity: compare compounds 1b and 2b, 1c and 2c, 1d and 2e, 1e and
2f, 1f and 2g, 1g and 2h, 1h and 2i. The activity and selectivity of these compounds toward
AChE and BChE depended on the structure of the 9-aryl substituents. The presence of a nitrogen
atom in the para-position of the aryl substituent (compounds 2c and 2d) facilitated inhibitory
activity against both enzymes. In addition, the high anti-BChE activity of compound 2i is
noteworthy.
Compounds of the third group, namely derivatives of 9-heterocyclic aminо-N-methyl-9,10-
dihydroacridine (compounds 3a-f), appeared to be the most promising inhibitors of
cholinesterases. These compounds were moderate inhibitors of AChE (IC50 values are about 10-5
М), while they more effectively inhibited BChE with IC50 values in the range of 10-6 – 10-7 М.
Compounds of this series were more selective toward BChE in comparison with AChE; namely,
9-morpholino (3a), 9-thiomorpholino (3b) and 9-(1,2,4-triazolyl) (3c) substituted
dihydroacridines were the best BChE inhibitors with IC50 values of 0.46 0.05, 0.84 0.05 and
0.81 0.09 µМ, respectively.
Derivatives of 9-amino-N-methyl-9,10-dihydroacridinium tetrafluoroborate, compounds 4a
and 4b, were also found to be more effective inhibitors of BChE relative to AChE.
2.3. Kinetic studies of AChE and BChE inhibition
Detailed kinetic studies of cholinesterase inhibition with the most active acridine derivatives
demonstrated that these compounds are predominantly reversible inhibitors of the mixed type
(Table 2). It is interesting to note that the mechanism of inhibiting AChE switched from mixed
to competitive when the morpholino substituent in compound 3a (Fig. 2A) was replaced with the
thiomorpholino analogue 3b (Fig. 3A). In contrast, only a mixed mechanism was exhibited by
both compounds for inhibiting BChE (Fig. 2B and 3B). The same situation was observed for the
pair of compounds with 1,2,3-benzotriazolyl (3e) and 1,2,4-triazolyl (3c) substituents (Table 2).
12

Table 2. Inhibition constants for active acridine derivatives toward AChE and BChE a.
AChE BChE
Comp.
R
Ki (µM)
Ki (µM)
αKi (µM)
αKi (µM)
NH2
2.66 0.24
8.95 0.91
19.0 1.7
55.4 4.9
2c
N
13.2 1.4
28.0 2.5
0.94 0.08
4.22 0.34
2d
n.d.
n.d.
1.46 0.15
4.75 0.42
2i
OH
O
4.01 0.36
19.6 1.6
0.41 0.03
1.17 0.11
3a
N
S
3.75 0.33
3.34 0.31
n.a.
n.a.
0.35 0.03
0.16 0.02
1.31 0.14
0.72 0.06
3b
3c
N
N
N
N
N
N
6.43 0.57
17.9 1.6
0.49 0.04
2.13 0.23
3e
N
a Values for Ki (competitive inhibition constant) and αKi (non-competitive inhibition constant) were
determined from analysis of slopes of 1/V versus 1/S at various inhibitor concentrations. The values
(mean SEM) were taken from at least three experiments.
n.d. = not determined.
n.a. = not applicable. These compounds were competitive inhibitors of AChE; therefore, only Ki applies.
13

0.030
0.025
0.020
0.015
0.010
0.005
20 µM
10 µM
5 µM
no inhibitor
0.000
-1
-4
-3
-2
0
-1
1
2
3
4
5
6
-1
[S] mM
A
0.1 µM
5 µM
2.5 µM
0.018
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
no inhibitor
0.000
-1
-4
-3
-2
0
1
2
3
4
5
6
-1
-1
[S] mM
B
Fig. 2. Steady state inhibition of AChE (A) and BChE (B) by compound 3a. Lineweaver-Burk double-
reciprocal plots of initial velocity and substrate concentrations in the presence of inhibitor 3a (three
concentrations) and without inhibitor are presented. Plots A and B show mixed-type inhibition.
14

20 µM
10 µM
0.0245
0.0210
0.0175
0.0140
0.0105
0.0070
0.0035
5 µM
no inhibitor
0.0000
-1
-4
-3
-2
0
1
2
-1
3
4
5
6
-1
[S] mM
A
0.020
0.018
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
1 µM
0.5 µM
0.25 µM
no inhibitor
0.000
-1
-4
-3
-2
0
1
2
3
4
5
6
-1
-1
[S] mM
B
Fig. 3. Steady state inhibition of AChE (A) and BChE (B) by compound 3b. Lineweaver-Burk double-
reciprocal plots of initial velocity and substrate concentrations in the presence of inhibitor 3b (three
concentrations) and without inhibitor are presented. Plot A shows competitive inhibition and plot B
exhibits mixed-type inhibition.
15

2.4. Molecular modeling studies
To explain the varied efficacy of the studied acridine derivatives toward cholinesterases and
to elucidate their inhibitory mechanism, molecular docking studies were carried out for all
compounds as ligands with AChE and BChE as receptors.
Quantum-chemical optimization of these compounds revealed differences in geometries
between dihydroacridine and acridinium derivatives, which have a conjugated π-system and
positive charge (Fig. 4). In spite of conjugation between the aryl substituent and the acridinium
moiety, these molecules are not fully planar due to steric conflicts between the acridine ring
system and benzene rings, though they are aligned along one axis. Dihydroacridine derivatives
have a sp3-hybridized carbon atom that determines the bend in their structure. The results of
quantum-chemical optimization were well correlated with experimental X-ray diffraction data 55.
Fig. 4. Geometries of acridinium (upper) and dihydroacridine compounds (lower) rows. Atom colors:
carbon = cyan (upper row) or magenta (lower row); nitrogen = blue; oxygen = red; hydrogen = white. The
С-9 atom is sp3-hybridized in compound 1h, but due to the symmetry of the dihydroacridine fragment,
compound 1h does not have enantiomers. The C-9 atom in compound 2i is sp2-hybridized. The different
hybridization of this atom in compounds 2i and 1h determines the dissimilar configurations of these
ligands.
16

Results of molecular docking revealed that some compounds can nearly reach the bottom of
the active site gorge of cholinesterases (catalytic active site, CAS), AChE or BChE, while others
bind at the peripheral anionic site (PAS), or at both CAS and PAS, thus explaining differences in
the mode of inhibition and selectivity of these compounds. As typically seen for cholinesterases,
positively charged ligands generally show better inhibitory activity compared to uncharged
analogues 64. For compounds that bind at the bottom of the gorge, a positive charge on the
acridinium fragment (or the N-protonated piperidine moiety in dihydroacridines) plays an
important role in binding due to cationic π-interactions with Trp86(82) and charge-pairing with
Glu202(197), where the first number refers to the AChE sequence and the second number in
parentheses refers to the BChE sequence. As an example, both compounds 1h and 2i bind at the
active site of BChE, but the absence of positively charged groups in 1h, like in other 9-aryl-9,10-
dihydro-N-methylacridines, lessens their computed binding affinity and experimentally
measured inhibitory activity (Fig. 5).
Fig. 5. Binding modes for the acridinium salt 2i (left; cyan carbons) and dihydroacridine 1h (right;
magenta carbons) at the bottom of the BChE gorge. The structural formulae of 2i and 1h are shown on the
far right. Active site amino acid residues: carbon = green; nitrogen = blue; oxygen = red; hydrogen =
white; for clarity, some hydrogens are not shown. Secondary structures of BChE are shown in white and
gray in the background. Dashed lines depict cationic π-interaction with Trp82 and charge-pairing with
Glu197 for the 2i ligand.
17

Due to the presence of aromatic fragments at the PAS of AChE, a positive charge in the
ligand molecule is also important, given that it contributes to binding, although this effect is not
greatly pronounced. Both compounds 2h and 1g bind at the PAS of AChE (Fig. 6) and the
positive charge of 2h has π-cation interactions with Trp286, thus resulting in a slightly better
inhibitory activity compared to that of 1g.
Fig. 6. Binding modes for the acridinium salt 2h (left; cyan carbons) and dihydroacridine 1g (right;
magenta carbons) at the PAS of AChE. White dashes show possible contacts due to conformational
change and protein accommodation, as has been shown previously in molecular dynamics studies for
positively charged compounds binding at the AChE PAS 65. Interacting amino acid residues are shown
with carbon = tan; nitrogen = blue; hydrogen = white; some hydrogens are not shown to enhance clarity.
Structural formulae of 2h and 1g are shown on the far right.
Some compounds can reach the bottom of the wider gorge found in BChE, but not the
narrower gorge found in AChE, as shown for compound 2i, which contains bulky tert-butyl
groups (Fig. 7).
18

When the size of substituents in the acridinium ring allows these compounds to reach the
bottom of the AChE gorge, tighter and more specific interactions provide a higher inhibitory
activity toward AChE rather than BChE, as observed for compound 2c (Fig. 8).
Fig. 7. Binding positions of compound 2i in the active site of BChE (left; cyan carbons; amino acid
residues shown with carbon = green; nitrogen = blue; oxygen = red; hydrogen = white; some hydrogens
not shown for clarity) and at the PAS of AChE (right; cyan carbons; amino acid residues shown with
carbon = tan; nitrogen = blue; oxygen = red; hydrogen = white; some hydrogens not shown for clarity).
Dashed lines depict cationic π-interaction with Trp82 and charge-pairing with Glu197 for the 2i ligand in
BChE and cationic π-interaction with Tryp286 in AChE. Background shows secondary structure of
protein in white and gray ribbon view. The structural formula of 2i is shown on the far right.
19

Fig. 8. Binding positions of compound 2c in the active site of BChE (left; cyan carbons; amino acid
residues shown with carbon = green; nitrogen = blue; oxygen = red; hydrogen = white; some hydrogens
not shown for clarity) and AChE (right; cyan carbons; amino acid residues shown with carbon = green;
nitrogen = blue; oxygen = red; hydrogen = white; some hydrogens not shown for clarity). The ligand has
a few more specific interactions with AChE amino acid residues than it does with BChE, resulting in a
slightly higher computed binding affinity and experimental inhibitory activity toward AChE (Table 1).
Dashed lines depict cationic π-interactions, charge-pairing, and hydrogen bonds. Background shows
secondary structure of protein in white and gray ribbon view. The structural formula of 2c is shown on the
far right.
Although dihydroacridines are generally less active than acridinium derivatives as
cholinesterase inhibitors due to the absence of a positive charge in the dihydroacridines, their
amino derivatives (9-heterocyclic amino-N-methyl-9,10-dihydroacridines) have enhanced
activity due to the presence of a tertiary nitrogen atom, which can be protonated (calculated pKa
~7-8), thus providing more specific interactions. Additionally, these specific interactions might
mediate the change in mechanism of inhibition (i.e., mixed-type vs. competitive), as exemplified
by compounds 3a and 3b). As shown in Fig. 9, compound 3b has a calculated pKa of 7.86, and
its docking poses reside in the AChE active site. In contrast, compound 3a has a computed pKa
of 6.65, and its docking poses occupy both the active site and the PAS of AChE.
20

2.5. Antioxidant activity of acridine derivatives
Antioxidant activity of compounds was determined using the 2,2'-azinobis-(3-
ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) radical decolorization assay 66 as their ability to
scavenge free radicals. Here, antioxidant activity is reported as Trolox equivalent antioxidant
capacity (TEAC) values by comparing (A0–Atest) of a test compound with (A0–ATrolox) of the
Trolox standard after 1 h of reaction time, where A0 is the absorbance of a control lacking any
radical scavenger, and Atest and ATrolox are the absorbances of the remaining ABTS.+ in the
presence of the test compound or Trolox, respectively. For the most active compounds, we also
determined IC50 values (compound concentration required for 50% reduction of the ABTS
radical). Lower IC50 values are indicative of higher ABTS free radical scavenging ability. The
results are presented in Table 1.
It follows from the data given in Table 1 that derivatives of 9-aryl-9,10-dihydro-N-methyl-
acridines (group 1) bind ABTS•+ most effectively relative to other acridine derivatives. All
representatives of this group of compounds (1a-j) exhibit a high antiradical activity, which is
compared with the standard antioxidant, Trolox.
21

Fig. 9. Docked poses from one docking run for compounds 3b (right; pink carbons) and 3a (left; magenta
carbons) with AChE (amino acid residues shown with carbon = green; nitrogen = blue; oxygen = red;
hydrogen = white; some hydrogens not shown for clarity; secondary structures shown in background as
white and gray ribbon). Structural formulae of 3b and 3a are shown on the far right.
In contrast, the corresponding charged analogues (group 2), 9-aryl(hetaryl)-N-methyl-
acridinium tetrafluoroborates 2a-m, exhibit very low antiradical activity. The moderate activity
of two of these compounds, 2i and 2j, may be due to the phenolic R' moieties, which are
structures known to have antioxidant properties 66.
Compounds of the third group, derivatives of 9-heterocyclic aminо-N-methyl-9,10-
dihydroacridine 3a-f, exhibit good ABTS•+ scavenging activity, although their potencies are
somewhat lower than that of Trolox (0.6 < TEAC < 1). Leaders in this group were compounds
3a and 3f.
9-Amino-N-methylacridinium tetrafluoroborates (group 4), compounds 4a and 4b, as well as
charged 9-aryl(hetaryl)-N-methyl-acridinium tetrafluoroborates (group 2), proved not to bind the
ABTS•+ radical.
Using the ABTS assay, we evaluated the intrinsic property of compounds to bind free
radicals as a “primary antioxidant”, i.e., those which actively inhibit oxidation reactions 67, 68
.
The ABTS•+ indicator radical may be neutralized either by direct reduction via single electron
transfer (SET reaction) or by radical quenching via hydrogen atom transfer (HAT), both of
which are applicable to the antioxidant action of dihydroacridines. During the process of their
oxidation, these compounds transiently form free radicals, which would be capable of binding
the ABTS•+ radical 69, 70. These antioxidant results are in good agreement with the relatively
facile oxidation of dihydroacridines of groups 1 and 3. Antioxidant compounds can act as
reducing agents and, in solutions, they tend to be easily oxidized on inert electrodes. Indeed,
cyclic voltammetry data indicate that compounds of groups 1 and 3 are relatively easily oxidized
and characterized by well-defined peaks of irreversible oxidations 55, 56. Low oxidation potentials
(about 0.65 V versus Ag/AgNO3 in acetonitrile) are associated with the ability of the
dihydroacridine system to donate electron(s) and, thus, to act as antioxidants.
22

3. Conclusions
Our studies of the esterase profiles and estimation of antioxidant activities performed for 4
groups of acridine derivatives demonstrated that 9-aryl(hetaryl)-N-methyl-9,10-dihydroacridines
(compounds 1a-j) exhibit a high radical-scavenging activity (at the level of Trolox) and have low
inhibitory potencies against AChE and BChE, while 9-aryl(hetaryl)-N-methyl-acridinium
tetrafluoroborates (compounds 2a-m) proved to be effective inhibitors of cholinesterases but
lacking in antiradical activity. However, 9-Heterocyclic amino- N-methyl-9,10-dihydroacridines
(compounds 3a-f), combine effective inhibition of AChE and BChE with rather high radical-
scavenging activity. Results of molecular docking well explain the observed features in the
efficacy, selectivity, and mechanism of cholinesterase inhibition by the acridine derivatives.
Thus, effective inhibitors of cholinesterases possessing radical scavenging capability were found
in a series of acridine derivatives, thereby demonstrating that the acridine scaffold is a promising
starting point for the development of multifunctional therapeutics against AD and perhaps other
neurodegenerative diseases.
4. Experimental section
4.1. Synthesis of compounds
All compounds were synthesized according to established procedures already well
documented in the literature 55, 56, 58, 71
.
4.2. Biological assay
4.2.1. In vitro AChE, BChE, and CaE inhibition
Acetylcholinesterase (AChE, EC 3.1.1.7, from human erythrocyte), butyrylcholinesterase
(BChE, EC 3.1.1.8, from equine serum), carboxylesterase (CaE, EC 3.1.1.1, from porcine liver),
acetylthiocholine iodide (ATCh), butylthiocholine iodide (BTCh), 5,5´-dithiobis-(2-nitrobenzoic
acid) (DTNB), and 4-nitrophenol acetate (4-NPA), were purchased from Sigma-Aldrich
(Germany).
The AChE and BChE activities were measured by the Ellman method as described earlier 72.
The assay solution consisted of 0.1 M K/Na phosphate buffer (pH 7.5, 25°C) with the addition of
0.33 mM DTNB, 0.02 unit/mL of AChE or BChE, and 1 mM of substrate (ATCh or BTCh,
respectively). The assays were carried out with a reagent blank containing all components except
23

AChE or BChE in order to account for non-enzymatic hydrolysis of substrate. In addition, an
enzyme blank was included that contained all components except substrate to account for non-
substrate sulfhydryl groups.
The activity of CaE was determined spectrophotometrically by the release of 4-nitrophenol at
405 nm 73. The assay solution consisted of 0.1 M K/Na phosphate buffer (pH 8.0, 25°C) with the
addition of 1 mM 4-nitrophenyl acetate and 0.02 unit/mL of CaE. The assays were carried out
with a blank containing all components except CaE.
The test compounds were dissolved in DMSO; the incubation mixture contained 2% (v/v) of
the solvent, a concentration determined not to affect the activity of each enzyme on its own (data
not shown). The primary evaluation of inhibitory activity of compounds was carried out by
determining the degree of inhibition of enzymes at a 20 µM concentration of test compound. For
this, the enzyme of interest was incubated with a test compound for 10 min, and then the residual
activity of the enzyme was measured. Each experiment was carried out in triplicate.
The IC50 values, a concentration of the inhibitor required to decrease enzyme activity by
50%, were determined for the most active compounds. To determine the IC50 of the inhibition of
AChE and BChE, a sample of the corresponding enzyme was incubated with eight different
concentrations of the test compounds in the range of 10-11-10-4 M. Inhibitor concentrations were
selected in order to obtain enzyme inhibitions of 20% to 80% of control. The test compounds
were added to the assay solution and pre-incubated at 25oC with the enzymes for 10 min
followed by the addition of the substrate. A parallel control was made for the assay solution with
no inhibitor. Absorbance measurements were carried out using a BioRad Benchmark Plus
microplate spectrophotometer (France). Each experiment was performed in triplicate. The results
were expressed as the mean SEM calculated with GraphPad Prism 6.05 software for Windows.
The reaction rates in the presence and absence of inhibitor were compared, and the percentage of
residual enzyme activity due to the presence of test compounds was calculated. The IC50 values
were determined graphically from inhibition curves (log inhibitor concentration vs. percentage of
residual enzyme activity) using Origin 6.1 software for Windows.
4.2.2. Kinetic analysis of AChE and BChE inhibition. Determination of steady-state inhibition
constants
24

To elucidate the inhibition mechanisms for the active compounds, the AChE and BChE
residual activities were determined in the presence of 3 increasing concentrations of the test
compounds and 6 decreasing concentrations of the substrates. The test compounds were pre-
incubated with the enzymes at 25°C for 10 min, followed by the addition of the substrates.
Parallel controls were made to find the rate of hydrolysis of the same concentrations of substrates
in the solutions with no inhibitor. The kinetic parameters of substrate hydrolysis were
determined. The measurements were carried out using a BioRad Benchmark Plus microplate
spectrophotometer (France). Each experiment was performed in triplicate. The results were fitted
into Lineweaver-Burk double-reciprocal kinetic plots of 1/V versus 1/[S] and the values of
inhibition constants Ki (competitive component) and αKi (noncompetitive component) were
calculated using Origin 6.1 software for Windows.
4.2.3. ABTS radical cation scavenging activity assay
Radical scavenging activity of the test compounds was assessed using an ABTS.+ radical
decolorization assay 66 with some modifications as described below.
ABTS (2,2ʹ-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) was
purchased from TCI (Tokyo, Japan). Potassium persulfate (di-potassium peroxodisulfate),
Trolox® (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid, and ascorbic acid were
obtained from Sigma-Aldrich (St. Louis, MO, USA). Ethanol was HPLC grade. Aqueous
solutions were prepared using deionized water.
Trolox was used as the antioxidant standard. A 5 mM solution of Trolox was prepared in
DMSO for use as the stock. Fresh working solutions of standard Trolox concentrations (1-100
µM) were prepared on the day of experiments and used for calibration as well as positive
controls for ABTS•+ scavenging activity.
ABTS was dissolved in deionized water to a 7 mM concentration. The solution of ABTS
radical cation (ABTS.+) was produced by mixing 7 mM ABTS stock solution with 2.45 mM
potassium persulfate aqueous solution in equal quantities and allowing them to react for 12–16 h
at room temperature in the dark. At the time of activity, ABTS.+ solution was diluted with
ethanol to adjust the absorbance value to 0.80 0.02 at 734 nm. Fresh working ABTS.+ solution
was prepared for each assay.
25

The radical scavenging capacity of the test compounds was analyzed by mixing 10 µl of
compound with 240 µl of ABTS.+ working solution. The decrease in absorbance was measured
spectrophotometrically at 734 nm after 1 h of mixing the solutions using a microplate UV/VIS
spectrophotometer BioRad xMark (Japan). Ethanol blanks were run in each assay. Values were
obtained from three replicates of each sample and three independent experiments.
The antioxidant activity was reported as Trolox equivalent antioxidant capacity (TEAC)
values by comparing (A0–Atest) of the test antioxidant with (A0–ATrolox) of the Trolox standard at
aconcentration of 20 µM after reaction time of 1 h, where A0 is the absorbance of a control
lacking any radical scavenger, Atest is the absorbance of the remaining ABTS.+ in the presence of
the test compound, and ATrolox is is the absorbance of the remaining ABTS.+ in the presence of
Trolox:
TEAC = (A0–Atest)/(A0–ATrolox).
For the most active compounds, we also determined the IC50 values (test compound
concentration required for 50% reduction of the ABTS radical). The IC50 values were calculated
using Origin 6.1 for Windows.
4.2.4. Molecular modeling
4.2.4.1. Ligand and protein structure preparation
To determine the protonation state of ionizable groups of the compounds, Marvin 14.9.1.0
(ChemAxon, http://www.chemaxon.com) was used to estimate pKa values. Geometries of the
ligands were quantum-mechanically (QM) optimized in the Gamess-US package 74 using DFT
method B3LYP and basis set 6-31G*. Partial atomic charges were taken from QM results
according to the Mulliken scheme 75. These optimized geometries and partial charges were used
for molecular docking.
For AChE, there are several structures available in the Protein Data Bank (PDB) of the
human enzyme (hAChE) in the apo state or in complex with several ligands (PDB ID 4EY4-
4EY8, 76). All X-ray structures as well as the water-saturated structure of apo-hAChE optimized
with molecular mechanics (MM) were used for molecular docking as described previously 64.
Similar to results obtained in 64, the best binding affinities were obtained with structure PDB ID
4EY7 (hAChE co-crystallized with Donepezil, resolution = 2.35Å) and were comparable to
results obtained with the water-saturated MM-optimized structure of apo-hAChE.
26

For BChE, the X-ray structure of the human enzyme (PDB ID 1P0I 77) was used. Previously,
the importance of saturation of the BChE gorge with water molecules was demonstrated 78.
Consequently, the structure was saturated with water molecules and optimized using a QM/MM
method as reported previously 78, 79
.
4.2.4.2. Molecular docking
Molecular docking with a Lamarckian Genetic Algorithm (LGA) 80 was performed using
Autodock 4.2.6 81 and AutoDock Vina 1.1.2 82 software. The rectangular prism grid box for
docking included the whole active site and the gorge of AChE (22.5 Å×22.5 Å×22.5 Å grid box
dimensions) and BChE (15Å × 20.25Å × 18Å grid box dimensions) with grid spacing of 0.375
Å. The main selected LGA parameters were 256 runs, 25×106 evaluations, 27×104 generations
and population size of 300. For AutoDock Vina the same grid boxes were used along with an
exhuastiveness setting of 40. Autodock Vina results were in very good agreement. Structural
images were prepared with PyMOL (Schrödinger, LLC).
27

Acknowledgements
This work was partly supported by grant No 17-03-00984 of the Russian Foundation for
Basic Research and program “Medicinal Chemistry” of the Russian Academy of Sciences. We
thank the Lomonosov Supercomputer center 83 for computational time. We are also grateful to
the Center of the Collective-Access Equipment of the Institute of Physiologically Active
Compounds, Russian Academy of Sciences for use of equipment (agreement no. 14.621.21.0008,
ID no. RFMEFI62114X0008). The funding sources had no role in the study design; collection,
analysis and interpretation of data; writing of the manuscript; or the decision to submit the article
for publication.
28

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