Chlorinated tacrine analogs: Design, synthesis and biological evaluation of their anti-cholinesterase activity as potential treatment for Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2019.02.033

In search of potent acetyl cholinesterase inhibitors with low hepatotoxicity for the treatment of Alzheimer's disease, introduction of a chloro substitution to tacrine and some of its analogs has proven to be beneficial in maintaining or potentiating the

Full text of DOI:10.1016/j.bioorg.2019.02.033

Accepted Manuscript
Chlorinated Tacrine Analogs: Design, Synthesis and Biological Evaluation of
Their Anti-cholinesterase Activity as Potential Treatment for Alzheimer's Dis-
ease
Hanan.M. Ragab, Mohamed Teleb, Hassan R. Haidar, Noha Gouda
PII:
S0045-2068(18)31168-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.033
YBIOO 2808
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 October 2018
13 February 2019
13 February 2019
Please cite this article as: Hanan.M. Ragab, M. Teleb, H.R. Haidar, N. Gouda, Chlorinated Tacrine Analogs: Design,
Synthesis and Biological Evaluation of Their Anti-cholinesterase Activity as Potential Treatment for Alzheimer's
Disease, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chlorinated Tacrine Analogs: Design, Synthesis and Biological
Evaluation of Their Anti-cholinesterase Activity as Potential Treatment
for Alzheimer's Disease
Hanan. M. Ragaba*, Mohamed Teleba, Hassan R. Haidarb, Noha Goudac
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria
21521, Egypt, b Hassan R. Haidar; Department of Pharmacology and Therapeutics, Faculty of
Pharmacy, Beirut Arab University, Beirut, Lebanon, c Noha Gouda; Department of Pharmaceutics,
Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
*Corresponding author details: Hanan. M. Ragab(); Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt. E-mail: tafida12@yahoo.com.
Tel.: +20 1002646716, Fax: ‏+20 34873273.
Abstract: In search of potent acetyl chlolinesterase inhibitors with low hepatotoxicity for the
treatment of Alzheimer's disease, introduction of a chloro substitution to tacrine and some of its
analogs has proven to be beneficial in maintaining or potentiating the cholinesterase inhibitory
activity. Furthermore, it was found to be able to reduce the hepatotoxicity of the synthesized
compounds, which is the main target of the study. Accordingly, a series of new 4-
(chlorophenyl)tetrahydroquinoline derivatives, was synthesized and characterized. The
synthesized compounds were evaluated for their in vitro and in vivo anti-cholinesterase activity
using tacrine as a reference standard. Furthermore, they were investigated for their
hepatotoxicity compared to tacrine. The obtained biological results revealed that all synthesized
compounds displayed equivalent or significantly higher anti-cholinesterase activity and lower
hepatotoxicity in comparison to tacrine. In addition, in silico drug-likeness of the synthesized
compounds were predicted and their practical logP were assessed indicating that all synthesized
compounds can be considered as promising hits/leads. Furthermore, docking study of the
compound showing the highest in vitro anticholinesterase activity was performed and its binding
mode was compared to that of tacrine.
Key words: Alzheimer's disease, anti-cholinesterase activity, tacrine, chlorinated phenyl
tetrahydroquinolines, hepatotoxicity.
Funding: The research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.

1. Introduction:
Alzheimer's disease (AD) is an age-related chronic neurodegenerative disorder occurring in
middle or late life [1-4]. The disease is associated with progressive dementia leading to severe
disability in performing the daily life activities [5]. Memory deterioration, loss of cognitive
function and privation of personality are common symptoms of the disease [6]. AD is
progressive and irreversible leading to abnormal changes in the brain that interferes with many
aspects of brain functions and worsens over time [7]. AD progresses in stages ranging from mild
forgetfulness and cognitive impairment to great loss of mental abilities. In highly advanced
stages of AD, the patient becomes helpless and dependant on others for aspects of everyday life
activities [8]. A family history may play a role in increasing one's risk of developing AD [9].
Postmortem brain tissue samples from AD patients revealed progressive accumulation of -
amyloid proteins (A) together with tau ()-protein aggregation leading to shrinkage and death
of neurons [10, 11]. Several theories have been proposed to explain the mechanism of AD
development [12, 13], among which the cholinergic hypothesis has become the most leading
theory to explain the etiology of the disease. This was based on the observation of cholinergic
neurons loss in the brain area involved in cognitive and behavioral functions of AD patients.
Furthermore, several centrally active anticholinergic drugs were found to induce dose-related
cognitive deficits in humans [14]. Accordingly, increasing levels of acetylcholine (ACh) in
cholinergic synapses in the brain of AD patients would be expected to relieve symptoms
associated with the disease [15-18]. Consequently, inhibition of acetyl cholinesterase (AChE),
the enzyme responsible for the hydrolysis of acetylcholine, has become the most targeted
approach for the development of agents active against AD [19-24]. However, it is well
established that the use of acetyl cholinesterase inhibitors (AChEIs) for the treatment of AD can
only alleviate the symptoms. Unfortunately no clinically approved drug has been discovered that
can reverse the progress of the disease.
Tacrine (9-amino-1,2,3,4-tetrahydroacridine) (I, Fig. 1) was the first AChEI to be approved by the
Food and Drug Administration (FDA) for the treatment of AD [25-28]. However, despite its good
AChE inhibitory activity, tacrine was far from ideal due to its low bioavailability and short half-
life. Furthermore, frequent administration of tacrine was found to be associated with significant
hepatotoxicity [29, 30]. Accordingly, several structural modifications were performed on tacrine
aiming at producing potent AChEIs (equivalent to or more potent than tacrine) with minimum
hepatotoxic side effects to provide clinically advantageous drugs [5]. Among the accomplished
structural modifications, introduction of a chloro substitution to the phenyl ring of tacrine
resulted into a group of chlorinated tetrahydroacridines (THA) (II, Fig. 1) showing high AChE
inhibitory activity [31, 32].
One other successful structural modification which was affected in our laboratories, was the
design and synthesis of a group of 3-cyano-2-substituted tetrahydroquinolines having a phenyl
ring at the 4-position (III, Fig. 1). Structure-activity relationship (SAR) studies of these
compounds indicated that the presence of a chloro substituent at the 2-position of the pyridine
ring resulted in a lead compound that was more potent AChEI and less hepatotoxic than tacrine
[1, 5]. Furthermore, studying the significance of the chloro substitution in tacrine analogs, Ragab
et al. have previously synthesized two series of 4-phenyltetrahydroquinolines (IV; X = H, Cl, Fig.
1) and evaluated their anti-cholinesterase activity and hepatotoxicity. The chlorinated
compounds (IV; X = Cl) showed higher AChE inhibitory activity and lower hepatotoxicity relative

to the unchlorinated analogs (IV; X = H) [5]. Correlating all the previously mentioned results, it
was obvious that the presence of a chloro group on any of the aryl rings, whether pyridyl or
phenyl, present in the synthesized tetrahydroquinolines (THQ) resulted in potent AChEIs with
lower hepatotoxicity [5].
X
NH2
NH2
CN
R
CN
Cl
Cl
N
N
N
N
Tacrine (I)
chlorinated THA (II)
THQ (III)
chlorinated THQ (IV)
X = H, Cl
Figure 1: Chemical structure of tacrine (I), chlorinated tacrine (II), 2-chlorotetrahydroquinoline (III), 4-aryltetrahydroquinoline (THQ;
IV).
Further investigation of the synthesized chlorinated THQ compounds (IV where X=Cl) revealed
that nature of the substituent at the 2-position was found to possess great influence on the
AChE inhibitory activity and the hepatotoxicity of the products. Presence of an amino group,
whether free or mono substituted, at the 2-position (V; R1 = alkyl and R2 = H, Fig. 2) resulted into
compounds having the highest AChE inhibitory activity and least hepatotoxicity. However, the
introduction of a second alkyl substituent on the 2-amino group (V; R1 = R2 = alkyl, Fig. 2) totally
abolished the activity. Furthermore, fusing the pyridine ring to a pyrazolo nucleus (VI, Fig. 2)
resulted in a promising activity [5].
In addition, in a trial to improve AChE activity and decrease hepatotoxicity, Reddy et al.
introduced an amide moiety in the form of acidhydrazide at the 2-position of tacrine (VII, Fig. 2).
The produced compound was found to be totally safe implying that the introduction of an amide
group might be beneficial[33].
Cl
Cl
NH2
O
NH2
N
CN
NH2
N
H
R1
N
N
N
N
N
H
R2
V
VI
VII
Figure
2:
Chemical
structure
of
2-alkylamino-3-cyano-4-(p-chlorophenyl)tetrahydroquinolines
(V)
and
4-(p-
chlorophenyl)pyrazolo[3,4-b]tetrahydroquinoline (VI) and 2-carbohydrazide derivative of tacrine (VII).
As an investment of the aforementioned findings, a novel group of chlorinated-2-amino-THQ
derivatives with various functionalities and/or fused to different heterocyclic ring systems (VIII
and IX, Fig. 3) was synthesized and evaluated for their AChE inhibitory activity and
hepatotoxicity aiming at discovering potential potent AChEIs with lower (if any) hepatotoxicity
than tacrine. The substitution pattern of the synthesized compounds was carefully chosen so as
to examine the effect of different N-substituents at the 2-position having various electronic and
lipophilic characters (VIII, Fig. 3). Furthermore, the effect of introduction of an amide group at
the 2- and 3-positions of the synthesized analogs has been investigated. In addition, in case of

fused heterocycles (IX, Fig. 3), it was targeted to study how increasing the size of the ring fused
to the pyridine ring; a pyrimidine rather than a pyrazole, would affect the biological results of
the synthesized compounds.
Cl
Cl
Y
Z
Z
X
N
Y
N
N
H
N
N
H
VIII
IX
Figure 3: Newly designed 2-alkylamino-4-(p-chlorophenyl)tetrahydroquinolines (VIII) and 5-(p-chlorophenyl)pyrimido[4,5-
b]tetrahydroquinoline (IX).
Furthermore, in silico physicochemical characters and pharmacokinetic parameters for the
synthesized compounds were predicted using dedicated computer software [34-36] to ensure
successful in vivo therapeutic activity. Practical logP testing was performed and compared to the
theoretical values obtained. Finally, a docking study of compound 7 (the most in vitro active
compound) was performed and its binding mode was compared to that of tacrine.
2. Results and discussion
2.1.Chemistry
The proposed synthetic routes adopted to obtain the target compounds are illustrated in Fig.4-6.
In Fig. 4, the 2-amino-4-(4-chlorophenyl)-5,6,7,8-tetrahydroquinoline-3-carboxamide (2) was
synthesized through hydrolysis of its 3-cyano precursor (1) upon heating on a water bath with
70% sulfuric acid according to a reported procedure [37, 38]. In addition, reaction of the 2-amino-
3-cyano analog (1) with the appropriate aryl isothiocyanates resulted in the corresponding
substituted tricyclic thiones (3a-c) instead of the expected open chain thiourea derivative. The
cyclization of the obtained products was proven by their IR spectra which were missing the CN
absorption band. The spectra also possessed new absorption bands for the C=S group.
Furthermore, 13C-NMR of the synthesized derivatives indicated the appearance of a signal at 
above 180 ppm corresponding to the C=S. Condensation of 1 with the appropriate aromatic
aldehydes yielded the corresponding Schiff's bases (4a, b) which lacked the NH2 absorption bands
present in its starting material. Finally, reaction of 1 with benzoic acid hydrazide in pyridine
resulted in the benzoylhydrazide derivative (5) with its IR spectrum showing the absorption
bands corresponding to both the carbonyl and NH-NH of the hydrazide group.

Cl
Cl
O
CN
N
Cl
R2
NH2
i
iii
iv
N
N
2
NH2
4a,b
CN
N
1
NH2
Cl
Cl
ii
R1
NH
N
CN
NH
N
N
H
S
N
HN
3a-c
5
O
Reagents and reaction conditions: i: 70% H2SO4, heat over waterbath, 5h; ii: R1-C6H4-
NCS, pyridine, reflux, 10h; iii: R2-C6H4-CHO, pyridine, reflux, 6h; iv: C6H5-CONHNH2, fusion
at 180-2000C, 1h.
Figure 4: Synthesis of the target compounds 2-5
Fig. 5 illustrates the synthesis of target compounds 6-9, where acylation of the amino group at
the 2 position of compound 1 with benzoyl chloride yielded the amide 6 showing the expected
absorption bands for newly formed amide functional group in its IR spectrum. The spectrum also
retained the absorption band for the CN group. Its 13C-NMR retained the signal for the CN group
at  = 115 ppm in addition to the appearance of a new signal at  = 167 ppm corresponding to
the C=O.
While alkylation of compound 1 with different alkyl halides such as benzyl chloride, cyclohexyl
bromide and phenacyl bromide yielded the designed alkylated amines 7-9. 13C-NMR of
compound 9 retained the signal for the CN group at  = 115 ppm in addition to the appearance of
a new signal at  = 168 ppm corresponding to the C=O.

Cl
Cl
CN
NH
CN
NH
Cl
i
iii
iv
N
8
N
6
O
CN
N
1
NH2
Cl
Cl
ii
CN
NH
CN
NH
Cl
N
7
N
9
O
Reagents and reaction conditions: i: C6H5COCl, dioxan, reflux, 10h; ii: C6H5CH2Cl,
dioxan, reflux, 10h; iii: C6H10Br, dioxan, reflux, 5h; iv: ClC6H4COCH2Br, dioxan, reflux, 6h.
Figure 5: Synthesis of the target compounds 6-9
Finally, in Fig. 6 fusion of compound 1 with urea and thiourea at different reaction temperatures
resulted in different products. Using moderate temperatures in this reaction resulted in the open
chain urea and thiourea derivatives (10 and 12 respectively). This was proved by the presence of
an absorption band characteristic for the cyano group at the 3-position in their IR spectra.
Furthermore, 13C-NMR of both compounds retained the signal for the CN group at  = 116 ppm in
addition to the appearance of a new signal at  = 156 and 185 ppm corresponding to the C=O and
C=S respectively.
While utilizing higher temperatures for the same reaction resulted in cyclization of the side chain
into the pyrimidinone and pyrimidinethione derivatives (11 and 13 respectively) which has been
verified by the disappearance of the nitrile absorption bands in their IR spectra. In addition, their
13C-NMR of compound 13 lacked the signal for the CN group and revealed the appearance of a
new signal at  = 156 and 182 ppm corresponding to the C=O and C=S respectively.

Cl
Cl
CN
NH
CN
NH
Cl
i
iii
iv
N
N
10
12
O
NH2
S
NH2
CN
N
1
NH2
Cl
Cl
ii
NH2
N
NH2
N
N
N
H
S
N
N
H
O
13
11
Reagents and reaction conditions: i: urea, fusion at 180-2000C, 1h; ii: urea, fusion at 280-
3000C, 1h; iii: thiourea, fusion at 180-2000C, 1h; iv: thiourea, fusion at 280-3000C.
Figure 6: Synthesis of the target compounds 10-13
2.2.Biological evaluation
2.2.1. Acetylcholinesterase inhibitory activity
AChE is the enzyme responsible for the hydrolysis of ACh into acetyl CoA and choline thus
terminating its effect at the cholinergic receptors. Accordingly, its inhibition would be expected
to result in increasing the amount of ACh available for neural transmission at the receptor site
and thus will most likely be successful in relieving some cognitive as well as behavioral symptoms
of AD. Accordingly, all newly synthesized compounds were evaluated for their AChE inhibitory
effect using both in vitro and in vivo pharmacological experiments.
2.2.1.1. In vitro Acetylcholinesterase inhibitory activity
The in-vitro biological activities of the synthesized target compounds were evaluated through
measuring the percentage increase in the contraction of frog’s Rectus abdominis [39], a skeletal
muscle that is known to be rich in AChE [40]. Such muscle contractions could be measured using
force displacement transducer (FTO 3C) connected to a grass polyview 16 data acquisition and
analysis system version 1 Grass technology WARWICK, RI, USA. Therefore, administration of an
AChEI before the addition of ACh to a muscle preparation should result in accumulation of
unhydrolyzed ACh with resultant increase in the height of ACh induced contractions. The
difference in height of ACh induced contractions of the muscle before and after exposure to a
suspected AChEI is considered as an indication of the extent of its inhibitory effect on the
enzyme.

The percentage inhibition of AChE activity was determined. The values ± standard deviation (at
confidence levels of 95 %) are recorded in Table 1 and presented in Fig. 7. Tacrine was used as a
reference standard.
Table 1: In vitro and in vivo AChE inhibitory activity, biochemical estimation of the liver enzymes; serum glutamic pyruvated
transaminase (SGPT) and reduced glutathione (GSH) levels in liver homogenate level.
In vivo AChE
In vitro Increase in
SGPT(IU/mL)c
GSH (g/mL)c
Compound
activity %
contraction (%)a
inhibitionb
Saline
47.25±4.5
0.24±0.027
Control
5.3±1.7
0.00±6.60
9.0±0.5
204.3±26.5
304.7±30.9
47.25±4.5
0.15±0.042
0.12±0.026
0.16±0.011
Tacrine
2
37.5±3.5
41.2±4.5
28.9±1.6
11.9±1.3
34.2±1.2
44.9±7.7
36.01±2.7
13.96±1.4
21.6±7.1
71.3±1.2
46.8±1.0
48.7±1.7
48.9±3.9
48.3±4.8
50.6±1.0
29.1±1.6
15.8±0.04
17.6±1.2
22.7±1.1
18.9±1.5
16.3±1.6
17.3±1.3
22.7±1.6
17.8±1.9
17.3±0.6
16.9±1.2
21.6±1.4
14.8±1.3
18.4±1.3
14.8±1.3
16.5±1.2
3a
3b
3c
4a
4b
5
158.08.5±6.3
70.0±8.5
0.19±0.012
176.75±10.0
46.08±25.7
74.66±8.9
0.21±0.019
0.23±0.012
196.0±10.2
191.0±10.6
146.41±9.8
127.75±16.7
141.16±9.8
106.16±3.7
187.25±17.2
167.41±2.5
201.25±16.7
6
7
8
9
10
11
12
13
aThe tested compounds were added at a concentration of 1 mol.
bThe tested compounds were added at a concentration of 1 mol /Kg body weight.
cThe tested compounds were added at a concentration of 7 mg/Kg body weight.
Data are expressed as means± SD (n=5) of at least three different experiments.
Data were analyzed using One factor ANOVA followed by post hoc Tukey simultaneous comparison t-values (pairwise t-tests).

Figure 7: (A): In vitro percentage AChE inhibitory activity of the synthesized compounds at a dose 1 mol. Values are expressed as %
increase in acetyl choline induced contraction in the isolated rectus abdominis. (B): In vivo percentage AChE inhibitory activity of the
synthesized compounds at a dose 1 mol /Kg body weight. Values are expressed as % increase in AChE activity in rat brain using
Ellman method. (C): SGPT levels of the synthesized compounds at a dose of 7mg/Kg body weight. (D): GSH levels of the selected
compounds at a dose of 7mg/Kg body weight
Results, in general, revealed that all tested compounds except 3b, 5 and 6 showed promising
AChE inhibitory activities. Correlating these results with structures of the tested compounds
indicated that nature of substituents at the 3-position of the pyridine ring doesn't affect
biological activity. However, the type of substituents at the 2-position has a noticeable impact on
activity where the presence of an amino group whether free (2) or substituted by an alkyl group
(4a,b and 7-9) resulted in equivalent or higher activity than tacrine (36% -71% in vitro increase in
contraction relative to 37.5% in case of tacrine) with the 2-benzylamino substituent (7) showing
the highest activity (71.3%). On the other hand, introduction of an amide group at the 2-position
of the pyridine ring either through acylation of the amino group (5) or incorporation of an
acylated hydrazine moiety (6) resulted in a noticeable reduction in the inhibitory activity.
Nevertheless, if the amide group is a part of a urea (10) or thiourea (12) side chains, the products
maintained high activity (almost 1.5 fold tacrine's activity). Furthermore, cyclization of the urea
side chains into a pyrimidine nucleus (11) maintained the high activity while cyclization of the
thiourea side chains (3a-c and 13) either decreased or abolished the activity.
2.2.1.2. In vivo acetyl cholinesterase inhibitory activity
All synthesized target compounds were further investigated by the quantitative in-vivo anti-
cholinesterase assay according to the method developed by Ellman et al [41]. The principle of the
assay relies on the fact that the active enzyme hydrolyzes acetyl thiocholine releasing free
thiocholine which upon reaction with Ellman reagent [DTNB; 5,5’-dithiobis-(2-nitrobenzoic acid)]

will form a yellow colored disulfide complex. The intensity of the yellow color produced due to
the amount of hydrolyzed thiocholine is therefore considered as a reliable measure of the activity
of AChE. In other words, a decrease in the intensity of the yellow color is proportional to the
amount of produced thiocholine relative to the control indicating a decrease in AChE activity and
thus a successful anti-cholinesterase effect [42]. The results ± standard deviation (at confidence
levels of 95 %) are recorded in Table 1 and presented in Fig. 7. Tacrine was used as a reference
standard.
The results revealed that all compounds showed high anti-cholinesterase activity with percentage
inhibition ranging from 14.8% to 22.7% relative to 9% in case of tacrine. The fact that the in vitro
inactive compounds (3b, 5 and 6) showed promising in vivo AChE inhibitory activity indicates that
they can be regarded as pro-drugs where they are metabolized in the body into active
metabolites.
2.2.2. Hepatotoxicity testing
All newly synthesized compounds were evaluated for their hepatotoxicity through determination
of their serum glutamic pyruvic transaminase (SGPT) levels. Compounds showing promising
results were further investigated through determination of their reduced glutathione (GSH) levels
in rat's liver.
2.2.2.1. Alanine transaminase (ALT ) "Serum glutamic pyruvated transaminase (SGPT)" assay
Since all target compounds showed either in vitro and/or in vivo anti-AChE inhibitory activity that
was significantly different from control values at P<0.05, thus hepatotoxicity of all compounds
was tested through determination of SGPT levels using Ultraviolet (UV) kinetic method in which
SGPT was assayed based on enzyme coupled system. SGPT is an enzyme that is involved in the
transfer of an amino group from alanine to anketoacid (2-oxoglutarate) resulting into the
formation of a pyruvate moiety. The formed pyruvate is reduced into L-lactate by NADH which is
itself oxidized to NAD and the rate of oxidation is measured through determination of the
absorbance at 340 nm at 0,1,2,3 minutes.
The higher the values obtained for SGPT, the higher the hepatotoxicity caused and vice versa [43,
44]. The results ± standard deviation (at confidence levels of 95 %) are recorded in Table 1 and
presented in Fig. 7.
The results indicated that dimethyl formamide (DMF, the solvent used) showed considerably high
hepatotoxicity itself thus SGPT levels for saline were calculated to determine its normal levels. All
the synthesized compounds, unlike tacrine, lacked any hepatotoxic effect (SGPT levels ranging
from 46 to 201 IU/mL relative to 304 IU/mL in case of tacrine and 204 IU/mL for the control).
Compounds containing the amide linkage in the open chain substituent at the 2-position (5, 6)
didn't show any hepatotoxicity and maintained SGPT levels similar to that of the control.
However, if the amide group is a part of a urea (10) or thiourea (12) side chains, the products
were capable of reducing the hepatotoxic effect of the solvent (106 and 167 IU/mL). On the other
hand, cyclization of the urea or thiourea side chains into a pyrimidine nucleus (11, 13) resulted
in compounds that didn't show any hepatotoxicity but were incapable of reducing the
hepatotoxicity of the solvent showing SGPT levels almost the same as DMF. Nevertheless,

arylation of the N3 of the fused pyrimidine ring slightly improved the hepatoprotective effect of
the synthesized compounds against the solvent used. To conclude, all compounds except 5, 6, 11
and 13 were capable of reducing the hepatotoxic effect of the solvent used DMF. In addition,
compounds 2, 3a, 4a and 4b almost brought SGPT levels back to its normal values (saline values)
despite the presence of DMF. In other words, the compounds not only lacked a hepatotoxic
effect, they were to some extent hepatoprotective against the effect of the solvent used.
2.2.2.2. Reduced glutathione (GSH) assay
Furthermore, reduced GSH in rats' livers was evaluated for the four compounds that showed a
somewhat hepatoprotective effect against the solvent's toxicity and brought SGPT levels to its
normal saline values (2, 3b, 4a and 4b). The major objective was to investigate the effect of GSH
depletion and oxidative stress on the toxicity induced by tacrine and selected compounds in liver
homogenate. Reduced GSH as a major antioxidant in hepatocytes acts as a scavenger that can
protect hepatocytes against oxidative stress through its ability to conjugate to a variety of
electrophiles leading to their detoxication [45]. The balance of glutathione redox cycle is an
important mechanism for cell protection. Literature survey indicated that depletion of cellular
GSH has been demonstrated in the toxicity associated with tacrine. The production of the
undesired protein reactive metabolites was significantly reduced by GSH. Reduced levels of
cellular GSH resulted in pathophysiological formation of reactive oxygen species and cellular
damage occurred [46].
The lower the values obtained for reduced GSH, the higher the hepatotoxicity caused and vice
versa. Tacrine was found to cause reduction in GSH levels in rats' liver. This observation was
consistent with other studies that reported the reduction of hepatic GSH level in rats given
tacrine [47].
The results ± standard deviation (at confidence levels of 95 %) are recorded in Table 1 and
presented in Fig. 7.
The results again indicated that DMF showed considerably high hepatotoxicity itself thus GSH
levels for saline were calculated to determine its normal levels. Tacrine caused further reduction
of GSH levels indicating higher hepatotoxicity than DMF. All the tested compounds, unlike
tacrine, lacked any hepatotoxic effect with GSH levels ranging from 0.16 to 0.23 µg/mL relative to
0.15 µg/mL in case of DMF and 0.12 µg/mL for the standard drug used (tacrine). Compounds 4b
almost brought GSH level back to its normal values (0.23 µg/mL reduced GSH relative to a value
of 0.24 µg/mL in case of saline) despite the presence of DMF.
2.3.Physicochemical properties and pharmacokinetic parameters determination.
2.3.1. Theoretical in silico prediction of physicochemical properties and pharmacokinetic
parameters.
Research over the last few decades indicated that compounds having good pharmacological
activities may fail to become good drug candidates due to low bioavailability [48]. Thus
physicochemical properties and pharmacokinetic parameters of newly synthesized compounds
are criteria that have to be carefully considered in the drug discovery and development
processes. Absorption, distribution, metabolism and excretion (ADME) of a drug candidate are

crucial for successful in vivo therapeutic activity [49]. These properties can be predicted using
computer software. In silico ADME computational studies are used nowadays to select the most
promising compound and thus reduce costly late stage failures in drug development processes
[50]. Through virtual ADMET screening software [36], the different physicochemical properties of
the newly synthesized compounds were calculated such as partition coefficient (logP), solubility
(S) and topological polar surface area (PSA). Furthermore, pharmacokinetic parameters such as
percentage of human intestinal absorption (HIA), plasma protein binding (PPB), as well as blood
brain barrier penetration (BBB) were determined using the preADMET software [35]. Finally, a
collective numerical representation of physicochemical properties, pharmacokinetics and
pharmacodynamics for each compound was thus computed and expressed as drug likeness score
[34].
All computed descriptors were listed in Table 2.
Table 2: In silicophysicochemical and pharmacokinetic parameters.
Lipinski's
violation
(practical)
Drug-
likeness
model
score
1.06
0.86
0.53
0.45
0.56
0.87
1.14
1.31
1.09
0.76
0.92
1.73
1.37
1.45
1.08
Lipinski's
violation
(theoretical)
Comp.
No.
Practical
logPb
logPa
Sc
TPSAd
HIAe
BBBf
PPBg
2
3a
3b
3c
4a
4b
5
6
7
8
9
3.09
4.11
4.56
4.79
6.07
6.01
5.19
5.43
5.79
6.30
6.22
3.67
3.68
4.21
4.03
3.099
4.900
3.570
4.837
4.866
4.807
4.400
2.972
3.750
2.435
4.814
4.438
4.686
4.026
4.685
1.83
82.01
57.47
57.47
57.47
49.05
69.28
77.81
65.78
94.77 0.80
100
0
0
0
0
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2.4*10-4
7.1*10-5
3.4*10-5
8.9*10-3
1.7*10-2
1.9*10-2
1.2*10-2
93.75 8.34 90.81
93.84 8.95 88.89
94.14 9.14 94.95
97.66 0.50
96.58 0.67
95.43 2.69
100
100
100
96.61 0.51 98.19
5.99*10-3 48.71
96.78 3.68
96.42 6.83
97.13 1.24
94.52 0.17 89.84
94.58 0.38 96.84
100
100
100
7.9*10-3
2.3*10-4
3.1*10-1
1.1*10-1
2.2*10-2
1.6*10-2
48.71
65.78
91.81
84.67
74.73
67.60
10
11
12
13
95.63 0.46
95.66 0.65
100
100
aLOgP: logarithm of compound partition coefficient between n-octanol and water.
bData are expressed as means of at least three different experiments.
cS: solubility.
dTPSA: topological polar surface area.
eHIA: percentage human intestinal absorption.
fBBB: blood brain barrier penetration is the ratio of steady state concentration in brain to those in blood.
gPPB: plasma protein binding.
From the previous calculations, eight compounds obeyed Lipinski's rule of five [51] which is
considered as an important parameter to select drug-like candidates. Where logP values
represent the ratio of concentration of a compound in two immiscible phases at equilibrium thus
it is a measure of drug affinity towards lipophilic bilayer plasma membranes. Large logP values
reflect higher level of lipophilicity. Other parameters such as topological polar surface area
(TPSA) and solubility are regarded as vital descriptors for oral bioavailability and blood brain
barrier (BBB) penetration, where high solubility is considered as a prerequisite for a rapid
absorption and higher bioavailability of a drug candidate. As for TPSA, it is regarded as a critical
parameter for human intestinal absorption, cell permeability and blood brain barrier (BBB)
penetration. Results indicated that all synthesized compounds showed TPSA values less than 140

Å2 indicating good permeablity through intestine, which was further proven by their HIA values
which were all above 90%. Furthermore, the TPSA values for all synthesized compounds, except
compound 10, were less than 90 Å2 implying reasonable BBB penetration which was further
proven by their BBB values where all compounds showed values above 0.1. Compounds 3a-c, 5, 7
and 8 showed BBB values higher than 2 indicating high CNS absorption. Compounds 3a-c and 8
exhibited outstanding BBB penetration values (6.83-9.14) indicating their stupendous CNS
absorption.
It is worth mentioning that some researchers [52, 53] have limited CNS drugs activity to
compounds having logP in the range 2-4, however, they eventually concluded that this is just a
preferred range and that it remains acceptable for CNS drugs to have logP values in the range 2-
5. This was highly consistent with the results obtained in the present study, where all the
synthesized compounds showed in vivo ACHE inhibitory activities (even the compounds that
were inactive in vitro) despite the fact that their logP values of most of them were higher than 4.
Screening plasma protein binding revealed that all investigated compounds were strongly bound
to plasma proteins except compounds 3b and 10 which were moderately bound to plasma
proteins (88.89 and 89.94% respectively). Nine compounds (2, 4a,b, 5, 7-9, 12, 13) out of the
fifteen synthesized compounds showed 100% plasma protein binding capabilities. However, all
Lipinski's violations were due to logP values more than 5. In conclusion, the majority of the
synthesized compounds showed reasonable to high pharmacokinetic properties. Finally, the
drug-likeness score of the synthesized compound was predicted, the higher the value of the
score is, the higher the probability that the particular molecule will be regarded as drug-like
candidate. Results revealed that all synthesized compounds possessed a positive drug-like score
value and thus considered as drug-like. Furthermore, Eight out of the synthesized compounds (2,
5-7, 10-13) showed drug-likeness values above 1. Other important issues, such as compliance
with Lipinski's rule of five, acceptable HIA, PPB and BBB penetration advocated these compounds
to be drug-like candidates.
2.3.2. Experimental determination of LogP.
Theoretical calculations indicated that seven out of the fifteen synthesized compounds showed logP
values higher than 5 and thus violating Lipinski's rule of five. However, these results were not
consistent with the fact that all synthesized compounds showed high in vivo anti-cholinesterase
activities. Accordingly, practical logP testing was performed. Determination of the partition coefficient
(LogP) was accomplished using capacity factor calculation method, as previously described [54].
Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was used for the determination.
In brief, the octanol/water partition coefficient of the synthesized compounds was determined from
the qualitative analysis of its capacity factor 'k' which was calculated through determination of
retention time and dead time (the average time needed by a solvent molecule to pass the column)
[55]. Four reference standards, benzoic acid, toluene, anthraquinone and benzyl benzoate, which are
structurally related to the compounds being tested, were selected to cover LogP range from 1.9 to 4.
The results are recorded in Table 2. Capacity factor (k) for each compound was determined using the
following equation:
tR – t0
t0
k =
Where k is the capacity factor, tR is the retention time of the test compound, and t0 is the dead-
time. Unretained thiourea was used to measure the dead time t0. The results indicated that all

compounds had logP values less than 5 and thus are promising hits and /or leads as anti-
cholinesterase inhibitors.
2.4.Docking studies
Docking study was carried out using the enzyme parameters obtained from the crystallographic
structure of the complex between AChE (PDB ID: 6g1u) with the co-crystallized ligand tacrine
derivative 6-chloroanyl-10-methyl-1,2,3,4-tetrahydroacridin-10-ium-9-amine [56]. The docking
simulation for the ligand (tacrine) was carried out using molecular operating environment (MOE)
software supplied by the Chemical Computing Group, Inc., Montre´al, QC [44] (Fig. 8A). The X-ray
crystal structure revealed that the NH group in the ligand tacrine is hydrogen-bonded to His 480,
while the phenyl ring displayed arene-arene interactions with Trp 83 and Tyr 370 residues.
Besides, it showed hydrophobic interactions with Trp 83, Gly 149, Gly 150, Tyr 162, Glu 237, Tyr
370 and His 480. Figure 8B shows the binding mode of compounds 7 indicating a superior binding
score (-3.3252) relative to the ligand used (tacrine -2.7680). Furthermore, a hydrogen bond is
observed between the side chain NH and ASP93 and another hydrogen bond is present between
the nitrogen of the CN group and Tyr96. In addition, compound 7 displayed hydrophobic
interactions with Leu31, Gly32, Trp58, Asn59, Thr63, Pro64, Asn65 and Ser91.
(A)
(B)
Figure 8: (A) Binding mode of the inhibitor (tacrine) in the binding site of hAChE (PDB ID: 6g1u) using MOE software and (B) Binding
mode of compound 7 docked and minimized in the binding site of hAChE (PDB ID: 6g1u) using MOE software
3. Conclusion
Due to the hepatotoxicity associated with the use of tacrine as AChEI in the treatment of AD, this
study portrays the synthesis of fifteen chlorinated tetrahydroquinolines to serve as tacrine
analogs with lower hepatotoxicity. The in vitro testing of the synthesized compounds as AChEIs
revealed that the 2-benzylamino derivative (7) showed twice the inhibitory activity of tacrine.
Other 2-alkylamino compounds showed cholinesterase inhibitory activities comparable to or
slightly higher than tacrine. However, results revealed that incorporation of an amide bond in the

side chain at the 2-position is not preferable except if it is a part of a urea or thiourea side chains.
Fusion of the pyridine ring to a pyrimidine nucleus was regarded in most cases unsuccessful in
producing promising AChEIs. The in vivo screening of the synthesized compounds as AChEIs
revealed that all compounds showed inhibitory effect equivalent to or significantly higher (2.5
fold) than tacrine with compounds 3b, 5 and 9 showing the highest in vivo percentage inhibition
(>20% AChE inhibition compared to 9% in case of tacrine). It could be concluded that hydrolysis
of the amide bondage and opening of the fused pyrimidine ring takes place during metabolism in
vivo resulting in retaining the abolished activity of some products (3b and 5). In addition,
hepatotoxicity screening of the compounds was estimated by determination of levels of SGPT
and reduced GSH. SGPT testing indicated that all compounds were safe to the liver with ten
compounds possessing hepatoprotective effect against the toxic effect of the solvent used
(DMF). Furthermore, four compounds; 2, 3b, 4a and 4b were able to completely reverse the
effect of the solvent and result in SGPT values almost similar to that of saline. Finally, the four
most promising compounds that brought SGPT values back to normal were further investigated
through determination of reduced GSH levels in rat liver after administration of the tested
compounds. The results indicated that these compounds showed no hepatic injury and less GSH
depletion than tacrine. Therefore, the hypothesis that the presence of a chloro group in tacrine
related analogs results in encouraging anti-cholinesterase activity and most importantly reduced
hepatotoxicity deserves further investigation. Furthermore, in silico physicochemical properties
and pharmacokinetic parameters for the synthesized compounds were calculated using
computer software and their practical logP determined. In addition, their drug-likeness score was
calculated. Results indicated that all drugs are promising drug-like candidates. Finally, docking
simulation of the in vitro most active compound 7 indicated superior binding to the receptor over
tacrine.
4. Experimental
4.1.Chemistry
All reagents and solvents were purchased from commercial suppliers and were dried and purified
when necessary by standard techniques. Melting points were determined in open glass
capillaries using Thomas-Hoover melting point apparatus. Infrared spectra (IR) were recorded,
using KBr discs, by a Perkin-Elmer 1430 Infrared spectrophotometer. Nuclear magnetic
resonance (1H-NMR and 13C-NMR) were determined using a Bruker 300 ultrashield
spectrophotometer. The data were reported as chemical shifts or δ values (ppm) relative to
tetramethylsilane (TMS) as internal standard. Elemental microanalyses were performed at the
regional center for Mycology and Biotechnology, Al-Azhar University and the values were within
± 0.4% of the theoretical values. Reaction progress was monitored by thin-layer chromatography
(TLC) on silica gel sheets (60 GF254, Merck). The spots were visualized by exposure to iodine
vapor or UV-lamp at λ 254 nm for few seconds.
Compounds 1, 2 and 7 were prepared according to reported procedures [5, 37, 38, 57, 58].
4.1.1. General procedure for the preparation of 5-(4-chlorophenyl)-3,4,6,7,8,9-hexahydro-4-
imino-3-arylpyrimido[4,5-b]quinoline-2(1H)-thione (3a-c)
A mixture of 1 (1 mmol; 0.28 g) and the appropriate isothiocyanate (1 mmol) in pyridine (10 ml)
was heated under reflux for 10 h. The reaction mixture was cooled then poured onto ice-water.

The obtained solid was filtered, washed several times with water, dried and recrystallized from
acetic acid. IR (KBr, cm-1): 3327-3180 (NH), 1210 (C=S).
4.1.1.1. 5-(4-chlorophenyl)-3,4,6,7,8,9-hexahydro-4-imino-3-phenylpyrimido[4,5-b]quinoline-
2(1H)-thione (3a)
1
Brownish yellow crystals (0.36 g, 86%); m.p. 134-1360C. H-NMR (300 MHz, CDCl3): δ 1.57-1.61
(m, 2H, C6-H2), 1.71-1.76 (m, 2H, C7-H2), 2.38-2.45 (t, J = 8.7 Hz, 2H, C5-H2), 2.87-2.93 (t, J = 8.7 Hz,
2H, C8H2), 7.001-7.47 (m, 10H,Ar-H and NH) and 11.77 (s, 1H, NH, D2O-exchangeable); 13C-NMR
(75 MHz, [D6]DMSO): δ 22.8, 26.3, 32.4, 33.3 (cyclohexyl-C), 117, 125, 127, 127.7, 128.6, 129,
129.5, 135, 138, 154, 157, 162 (Ar-C), 167.5 (C=NH) and 180 (C=S); Anal. calcd. For C23H19ClN4S
(418.94): C, 65.94; H, 4.57; N, 13.37. Found: C, 65.67; H, 4.81; N, 13.45.
4.1.1.2. 5-(4-chlorophenyl)-3,4,6,7,8,9-hexahydro-4-imino-3-p-tolylpyrimido[4,5-b]quinoline-
2(1H)-thione (3b)
1
Brownish yellow crystals (0.37 g, 85%); m.p. 146-1480C. H-NMR (300 MHz, [D6]DMSO): δ 1.58-
1.80 (m, 4H, C6-H2 and C7-H2), 2.39-2.40 (dist. t, 5H, C5-H2 and CH3), 2.75-2.78 (dist. t, 2H, C8-H2),
7.44-7.61 (m, 8H, Ar-H), 10.56 (s, 1H, NH, D2O-exchangeable) and 11.22 (s, 1H, NH, D2O-
exchangeable); 13C-NMR (75 MHz, [D6]DMSO): δ 22, 24.8, 26.5, 32.4, 33.5 (cyclohexyl-C and 4-Ph-
CH3), 117, 127, 128, 128.5, 129, 129.8, 132, 135, 138, 153.9, 157.2, 161.8 (Ar-C), 167.3 (C=NH)
and 181.4 (C=S); Anal. calcd. for C24H21ClN4S (432.97): C, 66.58; H, 4.89; N, 12.94. Found: C, 66.65;
H, 4.99; N, 12.81.
4.1.1.3. 3,5-bis(4-chlorophenyl)-3,4,6,7,8,9-hexahydro-4-iminopyrimido[4,5-b]quinoline-2(1H)-
thione (3c)
Yellow crystals (0.39 g, 86%); m.p. 120-1220C. 1H-NMR (300 MHz, [D6]DMSO): δ1.70-1.73 (m, 2H,
C6-H2), 1.84-1.87 (m, 2H, C7-H2), 2.51-2.53 (t, J = 4.5 Hz,2H, C5-H2), 2.55.2.58 (t, J = 4.5 Hz,2H, C8-
H2), ), 7.00-7.47 (m, 8H, Ar-H), 8.49 (s, 1H, NH, D2O-exchangeable) and 10.13 (s, 1H, NH, D2O-
exchangeable); 13C-NMR (75 MHz, [D6]DMSO): δ 22.1, 26.4, 32.5, 33.1 (cyclohexyl-C), 117.6, 127,
127.9, 128.3, 129, 129.4, 132, 135, 138, 153.9, 157.2, 161.9 (Ar-C), 167.4 (C=NH) and 181 (C=S);
Anal. calcd. for C23H18ClN4S (453.39): C, 60.93; H, 4.00; N, 12.36. Found: C, 60.75; H, 4.21; N,
12.56.
4.1.2. General procedure for the preparation of 2-(arylideneamino)-4-(4-chlorophenyl)-
5,6,7,8-tetrahydro-quinoline-3-carbonitrile (4a,b)
A mixture of 1 (1 mmol; 0.28 g) and the appropriate aromatic aldehyde (1 mmol) in pyridine (10
ml) was heated under reflux for 6 h. The reaction mixture was cooled then poured onto ice-
water. The obtained solid was filtered, washed several times with water, dried and recrystallized
from dimethylformamide/water.
4.1.2.1. 2-(benzylideneamino)-4-(4-chlorophenyl)-5,6,7,8-tetrahydroquinoline-3-carbonitrile
(4a)
Yellow crystals (0.33 g, 90%); m.p. 116-1180C. 1H-NMR (300 MHz, CDCl3): δ 1.79-1.82 (m, 2H, C6-
H2), 1.96-1.98 (m, 2H, C7-H2), 2.66-2.69 (t, J = 4.5 Hz, 2H, C5-H2), 3.22-3.28 (t, J = 4.5 Hz, 2H, C8-H2),

7.26-7.45 (m, 9H, Ar-H) and 8.7 (s, 1H, N = CH); 13C-NMR (75 MHz, CDCl3):  25.37, 26.29, 27.32,
28.22 (cyclohexyl-C), 124.1 (CN), 128.66, 129.45, 130.6, 131.53, 132.9, 136.8, 137.3, 140.17,
145.7, 148.2, 154.4 (Ar-C); IR (KBr, cm-1): 2237 (CN); Anal. calcd. for C23H18ClN3 (371.86): C, 74.29;
H, 4.88; N, 11.30; Found: C, 74.15; H, 4.69; N, 11.23.
4.1.2.2. 2-(2-hydroxybenzylideneamino)-4-(4-chlorophenyl)-5,6,7,8-tetrahydroquinoline-3-
carbonitrile (4b)
Yellow crystals (0.34 g, 87%); m.p. 136-1380C. 1H-NMR (300 MHz, [D6]DMSO): δ 1.73-1.74 (m, 2H,
C6-H2), 1.9-1.93 (m, 2H, C7-H2), 2.58-2.61 (t, J = 4.5 Hz, 2H, C5-H2), 3.67-3.69 (dist t,2H, C8-H2),
7.30-7.48 (m, 8 H, Ar-H), 8.66 (s, 1H, N = CH) and 10.02 (s, 1H, OH, D2O-exchangeable); 13C-NMR
(75 MHz, CDCl3):  23.57, 26.59, 27.92, 28.22 (cyclohexyl-C), 124.5 (CN), 114, 119, 129.45,
131.53, 136.8, 137.3, 140.17, 145.7, 148.2, 154.4, 161 (Ar-C); IR (KBr, cm-1): 3565 (OH),2220 (CN);
Anal. calcd. for C23H18ClN3O (387.86): C, 71.22; H, 4.68; N, 10.83. Found: C, 71.11; H, 4.53; N,
10.65.
4.1.3. N'-(-4-(4-chlorophenyl)-3-cyano-5,6,7,8-tetrahydroquinolin-2-yl)benzohydrazide (5)
A mixture of 1 (1 mmol; 0.28 g) and benzoic acid hydrazide (1 mmol; 0.136 g) was fused at 280-
2810C using a sand bath for 1 h. The reaction mixture was cooled then triturated with ethanol.
The obtained solid was filtered, washed several times with water, dried and recrystallized from
1
dimethylformamide/water. Brownish crystals (0.34 g, 84%); m.p. 181-1830C. H-NMR (300 MHz,
[D6]DMSO): δ1.66-1.69 (m, 2H, C6-H2), 1.84.83 (m, 2H, C7-H2), 2.51-2.54 (dist. t, 2H, C5-H2), 3.64-
3.65 (dist. t, 2H, C8-H2), ), 7.26-7.52 (m, 9H, Ar-H), 10.13 (s, 1H, NH, D2O-exchangeable) and 13.24
(s, 1H, NH, D2O-exchangeable); 13C-NMR (75 MHz, [D6]DMSO): , 21.9, 26.4, 31.9, 32.6
(cyclohexyl-C), 115 (CN), 124.6, 128.6, 128.4, 129.2, 129.5, 133.4, 135.1, 139.4, 140.1, 153.5,
156.2, 159.7 (Ar-C) and 165.4 (C=O); IR (KBr, cm-1): 3561-3250 (NH), 2229 (CN), 1644 (C = O);
Anal. calcd. for C23H19ClN4O (402.88): C, 68.57; H, 4.75; N, 13.91. Found: C, 68.29; H, 4.53; N,
13.65.
4.1.4. N-(-4-(4-chlorophenyl)-3-cyano-5,6,7,8-tetrahydroquinolin-2-yl)benzamide (6)
A mixture of 1 (1 mmol; 0.28 g) and benzoyl chloride (1 mmol; 0.14 g) in dioxan(10 ml) was
heated under reflux for 10 h. The reaction mixture was cooled then poured onto ice-water. The
obtained solid was filtered, washed several times with water, dried and recrystallized from
dimethylformamide/water. Yellowish white crystals (0.34 g, 88%); m.p. 271-2720C. 1H-NMR (300
MHz, [D6]DMSO): δ 1.69-1.70 (m, 2H, C6-H2), 1.85-1.87 (m, 2H, C7-H2), 2.42-2.43 (dist. t, 2H, C5-
H2), 3.01-3.03 (dist. t, 2H, C8-H2), 7.05-7.42 (m, 9H, Ar-H), and 12.778 (s, 1H, NH, D2O-
exchangeable); 13C-NMR (75 MHz, [D6]DMSO): , 21.8, 26.4, 31.7, 32.8 (cyclohexyl-C), 115.4
(CN), 124.7, 128.0, 128.3, 129.0, 129.2, 133.0, 135.1, 139.4, 140.2, 153.7, 156.2, 161.7 (Ar-C) and
167.4 (C=O); IR (KBr, cm-1): 3565-3230 (NH), 2239 (CN), 1654 (C = O); Anal. calcd. for C23H18ClN3O
(387.86): C, 71.22; H, 4.68; N, 10.83. Found: C, 71.15; H, 4.43; N, 10.76.
4.1.5. General procedure for the preparation of 2-(3alkylamino)-4-(4-chlorophenyl)-5,6,7,8-
tetrahydroquinoline-3-carbonitrile (8,9)

A mixture of 1 (1 mmol; 0.28 g) and the appropriate alkyl halide (1 mmol) in dioxin (10 ml) was
heated under reflux for 5-10 h. The reaction mixture was cooled then poured onto ice-water. The
obtained solid was filtered, washed several times with water, dried and recrystallized from
dimethylformamide/water.
4.1.5.1. 4-(4-chlorophenyl)-2-(cyclohexylamino)-5,6,7,8-tetrahydroquinoline-3-carbonitrile (8)
1
Brown crystals (0.25 g, 69%); m.p. 160-1620C. H-NMR (300 MHz, [D6]DMSO): δ 1.711-1.895 (m,
4H, C6-H2 and C7-H2), 2.4044 (dist. t, 2H, C5-H2), 2.50.2.54 (dist. t, 2H; N-cyclohexyl-C4-H2), 2.56-
2.68 (dist. t, 2H, C8-H2), 2.9874 (dist. t, 4H, N-cyclohexyl-C3-H2 and C5-H2), 3.62-3.63 (dist. t, 4H, N-
cyclohexyl-C2-H2 and C6-H2), and 7.41-7.60 (m, 5H, Ar-H); 13C-NMR (75 MHz, [D6]DMSO):  18.5,
24.1, 24.3, 30.6, 31.9, 45.5, 46.1, 51.8 (cyclohexyl-C), 113.8 (CN), 124.7, 127.9, 130.5, 131.3,
136.2, 157.2, 161.8, 167.2 (Ar-C); IR (KBr, cm-1): 2237 (CN); Anal. calcd. for C22H24ClN3 (365.90): C,
72.22; H, 6.61; N, 11.48. Found: C, 72.34; H, 6.45; N, 11.41.
4.1.5.2. 2-(4-(4-chlorophenyl)-3-cyano-5,6,7,8-tetrahydroquinolin-2-yl)amino-1-(4-
chlorophenyl)ethanone (9)
Yellow crystals (0.39 g, 89%); m.p. 172-1740C. 1H-NMR (300 MHz, [D6]DMSO): δ 1.67-1.72 (m, 2H,
C6-H2), 1.80-1.83 (m, 2H, C7-H2), 2.44-2.50 (t, J =9 Hz, 2H, C5-H2), 2.91-2.96 (t, J =9 Hz, 2H, C8-H2),
4.0192 (s, 2H, N-CH2), 7.33-7.56 (m, 8H, Ar-H), and 11.8 (s, 1H, NH, D2O-exchangeable); 13C-NMR
(75 MHz, [D6]DMSO): 24.1, 26.4, 32.4, 33.1 (cyclohexyl-C), 61.3 (N-CH2-C=O), 103.5 (pyridinyl-
C3), 115.4 (CN), 119.1, 123.1,127.1, 127.8, 128.3, 129, 132,135, 135.1, 138.4, 153.9, 156.8, 161.6
(Ar-C) and 168.8 (C=O); IR (KBr, cm-1): 3330 (NH), 2236 (CN), 1736 (C=O); Anal. calcd. for
C24H19Cl2N3O (436.33): C, 66.06; H, 4.39; N, 9.63. Found: C, 66.23; H, 4.34; N, 9.43.
4.1.6. General procedure for the preparation of 1-4-(4-chlorophenyl)-3-cyano-5,6,7,8-
tetrahydroquinolin-2-yl)urea or thiourea (10, 12)
A mixture of 1 (1 mmol; 0.28 g) and either urea (5mmol; 0.3 g) or thiourea (5 mmol; 0.4 g) was
fused at 180-2000Cusing a sand bath for 1 h. The reaction mixture was cooled then triturated
with ethanol. The obtained solid was filtered, washed several times with water, dried and
recrystallized from dimethylformamide/water.
4.1.6.1. 1-4-(4-chlorophenyl)-3-cyano-5,6,7,8-tetrahydroquinolin-2-yl)urea (10)
Yellow crystals (0.25 g, 76%); m.p. 180-1820C. 1H-NMR (300 MHz, [D6]DMSO): δ 1.24-1.87 (m, 4H,
C6-H2 andC7-H2), 2.29-2.35 (t, J =9 Hz, 2H, C5-H2), 3.00-3.06 (t, J =9 Hz, 2H, C8-H2), 7.08-7.52 (m,
4H, Ar-H), 8.20 (s, 2H, NH2, D2O-exchangeable) and 9.42 (s, 1H, NH, D2O-exchangeable); 13C-NMR
(75 MHz, [D6]DMSO): 22.2, 22.5, 25.8, 33.3 (cyclohexyl-C), 88.8 (pyridinyl-C3), 116.4 (CN),
127.8, 128.3, 128.6, 129.2, 136.2, 153.9 (Ar-C), 156.1 (C=O) and 160.3, 163.5 (pyridinyl-C2 and
C6); IR (KBr, cm-1): 3334 (NH2), 3266 (NH), 2221 (CN), 1691 (C = O); Anal. calcd. for C17H15ClN4O
(326.78): C, 62.48; H, 4.63; N, 17.15. Found: C, 62.23; H, 4.46; N, 17.08.
4.1.6.2. 1-4-(4-chlorophenyl)-3-cyano-5,6,7,8-tetrahydroquinolin-2-yl)thiourea (12)
Yellow crystals (0.28 g, 83%); m.p. 206-2080C. 1H-NMR (300 MHz, [D6]DMSO): δ 1.66-1.84 (m, 4H,
C6-H2 and C7-H2), 2.43-2.53 (t, J = 9.6 Hz, 2H, C5-H2), 2.89-2.96 (t, J = 9.6 Hz, 2H, C8-H2),4.03 (s, 2H,

NH2, D2O-exchangeable), 7.32-7.57 (m, 4H, Ar-H), and 11.84 (s, 1H, NH, D2O-exchangeable); 13C-
NMR (75 MHz, [D6]DMSO): 22.1, 22.5, 25.9, 33.2 (cyclohexyl-C), 88.9 (pyridinyl-C3), 114 (CN),
127.9, 128.2, 128.7, 129.1, 136.3, 154 (Ar-C), and 160.2, 164 (pyridinyl-C2 and C6) and 185 (C=S);
IR (KBr, cm-1): 3432 (NH2), 3266 (NH), 2221 (CN), 1214 (C=S);Anal. calcd. for C17H15ClN4S (342.85):
C, 59.56; H, 4.41; N, 16.34. Found: C, 59.29; H, 4.43; N, 16.15.
4.1.7. General procedure for the preparation of 4-amino-5-(4-chlorophenyl)-6,7,8,9-
tetrahydropyrimido[4,5-b]quinolin-2(1H)-oneorthione (11, 13)
A mixture of 1 (1 mmol; 0.28 g) and either urea (5mmol; 0.3 g) or thiourea (5 mmol; 0.4 g) was
fused at 2800Cusing a sand bath for 1 h. The reaction mixture was cooled then triturated with
ethanol. The obtained solid was filtered, washed several times with water, dried and
recrystallized from dimethylformamide/water.
4.1.7.1. 4-amino-5-(4-chlorophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-2(1H)-one (11)
Brown crystals (0.21 g, 64%); m.p. 195-1970C. 1H-NMR (300 MHz, CDCl3): δ 1.69-1.89 (m, 4H, C6-
H2 and C7-H2), 2.35-2.42 (t, J = 10.5 Hz, 2H, C5-H2), 3.11-3.17 (t, J = 10.5 Hz, 2H, C8-H2), 7.09-7.47
(m, 4H, Ar-H), 7.77 (s, 2H, NH2, D2O-exchangeable) and 11.01 (s, 1H, NH, D2O-exchangeable); 13C-
NMR (75 MHz, [D6]DMSO): 21.6, 21.8, 24, 26.9 (cyclohexyl-C), 112.0, 127.7, 128.5, 129.3,
130.6, 132.1, 133.7, 150.4, 156, 159.6, 160.2, 160.9 (Ar-C and C=O); IR (KBr, cm-1): 3346 (NH2),
3234 (NH), 1691 (C = O); Anal. calcd. for C17H15ClN4O (326.78): C, 62.48; H, 4.63; N, 17.15. Found:
C, 62.34; H, 4.55; N, 17.26.
4.1.7.2.
(13)
4-amino-5-(4-chlorophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-2(1H)-thione
Brown crystals (0.25 g, 74%); m.p. 226-2280C. 1H-NMR (300 MHz, [D6]DMSO): δ 1.59-1.84 (m, 4H,
C6-H2 and C7-H2), 2.22-2.25 (dist. t, 2H, C5-H2), 2.95-2.98 (dist. t, 2H, C8-H2), 7.21-7.50 (m, 6H, Ar-H
and NH2, D2O-exchangeable) and 13.68 (s, 1H, NH, D2O-exchangeable); 13C-NMR (75 MHz,
[D6]DMSO): 20.6, 21.3, 24.8, 26.8 (cyclohexyl-C), 112.0, 127.7, 128.5, 129.3, 130.6, 132.1,
133.7, 150.4, 159.6, 160.2, 160.9 (Ar-C) and 181.9 (C=S); IR (KBr, cm-1): 3443 (NH2), 3265 (NH),
1234 (C=S); Anal. calcd. for C17H15ClN4S (342.85): C, 59.56; H, 4.41; N, 16.34. Found: C, 59.38; H,
4.31; N, 16.44.
4.2.Biological evaluation
4.2.1. Animals
Frogs for the in vitro AChE testing were obtained from a local supplier, used within 2 days of their
purchase. The frogs were kept hydrated throughout the 2 days. Male albino rats (weighing 250-
330 g) for the in vivo AChE and hepatotoxicity testing were obtained from the animal house of
Beirut Arab University. The animals were housed under standard conditions as per the rules and
regulations of the Faculty animal ethics committee (IRB) (approval code 2014A-002-P-R-0004)
4.2.2. In vitro AChE inhibitory activity

All synthesized compounds were evaluated for their in-vitro AChE inhibitory effects on
acetylcholine-induced contractions of the frog rectus abdominis. After isolation of the triangular
muscle of the frog was performed, it was suspended in an organ bath of 10 mL capacity
containing aerated Ringer’s solution which has been kept at room temperature. The contractions
induced by acetylcholine (at a dose of 65 g/mL) were recorded by a force-displacement
transducer connected to a Kymograph recorder. The submaximal dose of acetylcholine was
determined after a contact time of 1.5 minutes in 3 minute cycles. The solution of the tested
compounds in DMF (1 µmol/mL) was added 10 minutes before adding the submaximal dose of
acetylcholine. Five observations were recorded for each compound.
Calculations of the percentage inhibition of acetyl cholinesterase activity, as indicated by the
percentage potentiation of acetylcholine-induced contractions, together with the standard
deviation were done and recorded in Table 1.
4.2.3. In-vivo acetyl cholinesterase inhibitory activity
The rats were fasted overnight then given the solutions of the tested compounds at a dose
1mol/Kg body weight, via intraperitoneal route. One hour after drug administration, the rats
were sacrificed by decapitation. The whole brain of each rat was removed, immediately and
homogenized in 9 volumes of ice-cold 0.1M sodium-potassium phosphate buffer (pH 8.0). The
homogenate was centrifuged at 5000 rpm for 30 min. The resultant supernatant was used for
estimation of cholinesterase activity spectrophotometrically as reported by Ellman et al [41].
4.2.3.1. Measurement of AChE activity
Acetyl thiocholine reaction was accomplished by mixing 0.1 mL of the brain homogenate
supernatant with 0.5 mM acetyl thiocholine and 0.33 mM 5,5’-dithiobis(2-nitrobenzoic acid)
“DTNB” in phosphate buffer (pH 8.0). The intensity of the colored disulfide product resulting
from the reaction of the free thiocholine with the Ellman reagent “DTNB” was measured
kinetically over 10 min at 412 nm, using a Perkin Elmer type (Lambda 2S) spectrophotometer.
Five readings were recorded for each compound. The percent inhibition of acetyl cholinesterase
activity and standard deviation were calculated and recorded in Table 1.
4.2.4. Hepatotoxicity testing
4.2.4.1. Alanine transaminase (ALT )"Serum glutamic pyruvated transaminase (SGPT)" assay
The rats were given the solutions of the tested compounds in dimethylformamide, in a
concentration of 7 mg/Kg body weight, via intraperitoneal route. Control animals were given
dimethylformamide or saline. 24 Hours after administering the drug, a blood sample was
collected in sterilized dry centrifuge tube and allowed to coagulate for 10 minutes at 370C. The
clear serum was separated at 6000 rpm for 10 minutes and subjected to biochemical
investigation of SGPT. The kit used was obtained from Medilab (Beirut, Lebanon) and contains L-
Alanine, buffer pH7.8, NADH, LDH and oxoglutarate.
The reaction was performed by mixing 0.1 mL of the serum sample with 1 mL of the
reconstituted kit. The produced solution was well mixed resulting in a colored product whose
intensity was measured at 340 nm, using a Perkin Elmer type (Lambda 2S) spectrophotometer.
Five readings were recorded for each compound. The values are recorded in Table 1.

4.2.4.2. Reduced glutathione (GSH) assay
The rats were given the solutions of the tested compounds in dimethylformamide, in a
concentration of 7 mg/Kg body weight, via intraperitoneal route. Control animals were given
dimethylformamide or saline. 24 Hours after drug administration, the abdomen of each rat was
opened and one lobe of the liver was quickly removed, and immediately homogenized in 9
volumes of ice-cold phosphate buffer (0.1 M, pH 8) for GSH measurement.
The reaction was affected by mixing 0.5 ml of the homogenate with 0.5 mL of the precipitating
solution followed by centrifugation at 2000 rpm for 5 min. To 0.2 mL of the resulting
supernatant, 1.7 mL phosphate buffer (0.1 M, pH 8) and 0.1 mL Ellman reagent were added
giving a colored product whose intensity was measured at 412 nm, using a Perkin Elmer type
(Lambda 2S) spectrophotometer. The concentration of GSH in the test samples was expressed
g/mL using standard curve constructed using a serial dilution of GSH. Five readings were
recorded for each compound. The values are recorded in Table 1.
4.2.5. Experimental determination of LogP.
The reference substances, benzoic acid, toluene, anthraquinone and benzyl benzoate were dissolved
in HPLC grade methanol. Their retention time on RP-C18 column was determined. The HPLC analysis
employed reversed-phase C18 column (Agilent HC-C18(2), 5 μm, 150 mm x 4.6 mm, Agilent
Technologies, Inc., USA) using photodiode detector (Agilent 1260 Infinity DAD, Agilent Technologies,
Inc., USA). For analysis, an isocratic mobile phase of 3:1 (v/v) methanol and water was employed. A
volume of 20.0 μL (Agilent 1260 Infinity High performance autosampler) was injected with a flow rate
of 0.6 mL/min (Agilent 1260 Infinity Quaternary Pump). Temperature of the column oven was kept at
25 ºC (Agilent 1260 Infinity Thermostatted Column Compartment). A calibration curve of log k versus
Log P of the reference compounds was constructed to obtain its regression equation. The test
compound dissolved in the mobile phase as a solvent was injected into the column. The partition
coefficient of the test compound was calculated from interpolation its capacity factor value in the
calibration curve regression equation. Log P of the tested compounds was calculated by extrapolating
its tR using regression equation (r2= 0.955). Three readings were recorded for each compound. The
values are recorded in Table 2.
4.3.
Docking study
Computer-assisted simulation docking experiments were carried out under an MMFF94X force field
using Molecular Operating Environment (MOEDock 2009) software, Chemical Computing Group,
Montreal, QC.
Docking protocol
The coordinates from the X-ray crystal structure of AChE used in this simulation were obtained from
the Protein Data Bank (PDB ID: 6g1u), where the active site is bound to the inhibitor tacrine derivative
6-chloroanyl-10-methyl-1,2,3,4-tetrahydroacridin-10-ium-9-amine. The ligand molecules were
constructed using the builder molecule and were energy minimized. The active site of AChE was
generated using the MOE-Alpha Site Finder, and then ligand was docked within this active site using
the MOE Dock. The lowest energy conformation was selected, and the ligand interactions (hydrogen
bonding together with other hydrophobic interactions) with AChE were recorded.

5. References
1.
El-Tombary A, Omar A, Eshba N, Rostom S, Ragab H, Saad E. Synthesis and
Acetylcholinesterase Inhibitory Activity of Novel Tetrahydroquinoline and Pyrazolo
[3, 4-b]-tetrahydroquinoline Derivatives As Potential Agents for Treatment of
Alzheimer's Disease. ALEXANDRIA JOURNAL OF PHARMACEUTICAL SCIENCES 22(1),
61 (2008).
2.
3.
Mohamed LW, Abuel-Maaty SM, Mohammed WA, Galal MA. Synthesis and
biological evaluation of new oxopyrrolidine derivatives as inhibitors of acetyl
cholinesterase and β amyloid protein as anti–Alzheimer’s agents. Bioorganic
chemistry 76 210-217 (2018).
Liu W, Lang M, Youdim MB et al. Design, synthesis and evaluation of novel dual
monoamine-cholinesterase inhibitors as potential treatment for Alzheimer’s disease.
Neuropharmacology 109 376-385 (2016).
4.
5.
Yeong KY, Liew W-L, Murugaiyah V, Ang CW, Osman H, Tan SC. Ethyl nitrobenzoate:
A novel scaffold for cholinesterase inhibition. Bioorganic chemistry 70 27-33 (2017).
Ragab HM, Ashour HM, Galal A, Ghoneim AI, Haidar HR. Synthesis and biological
evaluation of some tacrine analogs: study of the effect of the chloro substituent on
the acetylcholinesterase inhibitory activity. Monatshefte für Chemie-Chemical
Monthly 147(3), 539-552 (2016).
6.
Gurjar AS, Darekar MN, Yeong KY, Ooi L. In silico studies, synthesis and
pharmacological evaluation to explore multi-targeted approach for imidazole
analogues as potential cholinesterase inhibitors with neuroprotective role for
Alzheimer’s disease. Bioorganic & medicinal chemistry (2018).
7.
8.
Mayo
foundation
for
medical
education
and
research.
http://www.Mayoclinic.com.htm. (June 2018).
Brody TM, Larner J, Minneman KP, Wecker L. Brody's human pharmacology:
molecular to clinical. Elsevier Mosby, (2005).
9.
10.
Rang HP. Rang and Dale’s pharmacology. Churchill Livingstone Elsevier 727 (2007).
Joubert J, Foka GB, Repsold BP, Oliver DW, Kapp E, Malan SF. Synthesis and
evaluation of 7-substituted coumarin derivatives as multimodal monoamine
oxidase-B and cholinesterase inhibitors for the treatment of Alzheimer's disease.
European journal of medicinal chemistry 125 853-864 (2017).
11.
Vafadarnejad F, Mahdavi M, Karimpour-Razkenari E et al. Design and synthesis of
novel coumarin-pyridinium hybrids: In vitro cholinesterase inhibitory activity.
Bioorganic chemistry 77 311-319 (2018).
12.
13.
Hardy J. The amyloid hypothesis for Alzheimer’s disease: a critical reappraisal.
Journal of neurochemistry 110(4), 1129-1134 (2009).
Webber KM, Raina AK, Marlatt MW et al. The cell cycle in Alzheimer disease: a
unique target for neuropharmacology. Mechanisms of ageing and development
126(10), 1019-1025 (2005).
14.
15.
Więckowska A, Kołaczkowski M, Bucki A et al. Novel multi-target-directed ligands for
Alzheimer's disease: Combining cholinesterase inhibitors and 5-HT6 receptor
antagonists. Design, synthesis and biological evaluation. European journal of
medicinal chemistry 124 63-81 (2016).
Zemek F, Drtinova L, Nepovimova E et al. Outcomes of Alzheimer's disease therapy
with acetylcholinesterase inhibitors and memantine. Expert opinion on drug safety
13(6), 759-774 (2014).

16.
17.
18.
Rizzo S, Rivi Re CL, Piazzi L et al. Benzofuran-based hybrid compounds for the
inhibition of cholinesterase activity, β amyloid aggregation, and Aβ neurotoxicity.
Journal of medicinal chemistry 51(10), 2883-2886 (2008).
Saeed A, Mahesar PA, Zaib S et al. Synthesis, cytotoxicity and molecular modelling
studies of new phenylcinnamide derivatives as potent inhibitors of cholinesterases.
European journal of medicinal chemistry 78 43-53 (2014).
Ragab HM, Kim JS, Dukat M, Navarro H, Glennon RA. Aryloxyethylamines: Binding at
α7 nicotinic acetylcholine receptors. Bioorganic & medicinal chemistry letters 16(16),
4283-4286 (2006).
19.
20.
De Souza LG, Rennó MN, Figueroa-Villar JD. Coumarins as cholinesterase inhibitors:
a review. Chemico-biological interactions 254 11-23 (2016).
Sang Z, Wang K, Wang H et al. Design, synthesis and biological evaluation of
phthalimide-alkylamine derivatives as balanced multifunctional cholinesterase and
monoamine oxidase-B inhibitors for the treatment of Alzheimer’s disease.
Bioorganic & medicinal chemistry letters 27(22), 5053-5059 (2017).
21.
22.
23.
24.
25.
26.
Rayatzadeh A, Saeedi M, Mahdavi M et al. Synthesis and evaluation of novel
oxoisoindoline derivatives as acetylcholinesterase inhibitors. Monatshefte für
Chemie-Chemical Monthly 146(4), 637-643 (2015).
Saeed A, Zaib S, Ashraf S et al. Synthesis, cholinesterase inhibition and molecular
modelling studies of coumarin linked thiourea derivatives. Bioorganic chemistry 63
58-63 (2015).
Elumalai K, Ali MA, Munusamy S, Elumalai M, Eluri K, Srinivasan S. Novel
pyrazinamide condensed azetidinones inhibit the activities of cholinesterase
enzymes. Journal of Taibah University for Science 10(5), 643-650 (2016).
Sarfraz M, Sultana N, Rashid U, Akram MS, Sadiq A, Tariq MI. Synthesis, biological
evaluation and docking studies of 2, 3-dihydroquinazolin-4 (1H)-one derivatives as
inhibitors of cholinesterases. Bioorganic chemistry 70 237-244 (2017).
Romero A, Cacabelos R, Oset-Gasque MJ, Samadi A, Marco-Contelles J. Novel
tacrine-related drugs as potential candidates for the treatment of Alzheimer’s
disease. Bioorganic & medicinal chemistry letters 23(7), 1916-1922 (2013).
Liu Z, Fang L, Zhang H, Gou S, Chen L. Design, synthesis and biological evaluation of
multifunctional tacrine-curcumin hybrids as new cholinesterase inhibitors with
metal ions-chelating and neuroprotective property. Bioorganic & medicinal
chemistry 25(8), 2387-2398 (2017).
27.
28.
Khoobi M, Ghanoni F, Nadri H et al. New tetracyclic tacrine analogs containing
pyrano [2, 3-c] pyrazole: efficient synthesis, biological assessment and docking
simulation study. European journal of medicinal chemistry 89 296-303 (2015).
Martins C, Carreiras MC, León R et al. Synthesis and biological assessment of
diversely substituted furo [2, 3-b] quinolin-4-amine and pyrrolo [2, 3-b] quinolin-4-
amine derivatives, as novel tacrine analogues. European journal of medicinal
chemistry 46(12), 6119-6130 (2011).
29.
30.
31.
32.
Soukup O, Jun D, Zdarova-Karasova J et al. A resurrection of 7-MEOTA: a comparison
with tacrine. Current Alzheimer Research 10(8), 893-906 (2013).
Alldredge BK, Corelli RL, Ernst ME, Guglielmo B, Jacobson P, Kradjan W. Koda-Kimble
& Young's applied therapeutics. (2013).
Erdelyi M. Halogen bonding in solution. Chemical Society Reviews 41(9), 3547-3557
(2012).
Gregor VE, Emmerling MR, Lee C, Moore CJ. The synthesis and in vitro
acetylcholinesterase and butyrylcholinesterase inhibitory activity of tacrine
(Cognex®) derivaties. Bioorganic & medicinal chemistry letters 2(8), 861-864 (1992).

33.
Reddy EK, Remya C, Mantosh K et al. Novel tacrine derivatives exhibiting improved
acetylcholinesterase inhibition: Design, synthesis and biological evaluation.
European journal of medicinal chemistry 139 367-377 (2017).
34.
35.
36.
37.
Molsoft. http://molsoft.com/mprop. (June 2018).
Preadmet. http://preadmet.bmdrc.org. (June 2018).
Molinspiration. http://www.molinspiration.com. (June 2018).
Kambe S, Saito K, Sakurai A, Midorikawa H. A simple method for the preparation of
2-amino-4-aryl-3-cyanopyridines by the condensation of malononitrile with aromatic
aldehydes and alkyl ketones in the presence of ammonium acetate. Synthesis
1980(05), 366-368 (1980).
38.
Wan Y, Yuan R, Zhang F-R et al. One-pot synthesis of N2-substituted 2-amino-4-aryl-
5, 6, 7, 8-tetrahydroquinoline-3-carbonitrile in basic ionic liquid [bmim] OH.
Synthetic Communications 41(20), 2997-3015 (2011).
39.
40.
41.
Barlow R, Crawford T, Perry W. Pharmacological experiments on isolated
preparations. Churchill Livingstone, New York 58 (1974).
Brunton LL, Chabner B, Knollmann BC. Goodman & Gilman's the pharmacological
basis of therapeutics. (2011).
Ellman GL, Courtney KD, Andres Jr V, Featherstone RM. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochemical pharmacology 7(2), 88-
95 (1961).
42.
43.
44.
Burtis CA, Bruns DE. Tietz Fundamentals of Clinical Chemistry and Molecular
Diagnostics-E-Book. Elsevier Health Sciences, (2014).
Kumar R, Kumar A, Sharma R, Baruwa A. Pharmacological review on natural ACE
inhibitors. Der Pharmacia Lettre 2(2), 273-293 (2010).
Raju NJ, Sreekanth N. Investigation of hepatoprotective activity of Ficus retusa
(Moraceae). International Journal of Research in Ayurveda and Pharmacy (IJRAP)
1(1), 166-169 (2010).
45.
46.
47.
48.
49.
Ganesan K, Sukalingam K, Xu B. Solanum trilobatum L. Ameliorate Thioacetamide-
Induced Oxidative Stress and Hepatic Damage in Albino Rats. Antioxidants 6(3), 68
(2017).
Madden S, Woolf TF, Pool WF, Park BK. An investigation into the formation of stable,
protein-reactive and cytotoxic metabolites from tacrine in vitro: studies with human
and rat liver microsomes. Biochemical pharmacology 46(1), 13-20 (1993).
Park SM, Ki SH, Han NR et al. Tacrine, an oral acetylcholinesterase inhibitor, induced
hepatic oxidative damage, which was blocked by liquiritigenin through GSK3-beta
inhibition. Biological and Pharmaceutical Bulletin 38(2), 184-192 (2015).
Bose PP, Chatterjee U, Hubatsch I et al. In vitro ADMET and physicochemical
investigations of poly-N-methylated peptides designed to inhibit Aβ aggregation.
Bioorganic & medicinal chemistry 18(16), 5896-5902 (2010).
Mälkiä A, Murtomäki L, Urtti A, Kontturi K. Drug permeation in biomembranes: in
vitro and in silico prediction and influence of physicochemical properties. European
Journal of Pharmaceutical Sciences 23(1), 13-47 (2004).
50.
51.
Cheng F, Li W, Zhou Y et al. admetSAR: a comprehensive source and free tool for
assessment of chemical ADMET properties. (2012).
Ntie-Kang F, Onguéné PA, Lifongo LL, Ndom JC, Sippl W, Mbaze LMA. The potential
of anti-malarial compounds derived from African medicinal plants, part II: a
pharmacological evaluation of non-alkaloids and non-terpenoids. Malaria journal
13(1), 81 (2014).
52.
Pajouhesh H, Lenz GR. Medicinal chemical properties of successful central nervous
system drugs. NeuroRx 2(4), 541-553 (2005).

53.
54.
55.
56.
57.
Brain
penetration
(work
in
progress).
https://www.cambridgemedchemconsulting.com/resources/ADME/brian_penetrati
on.html. (february 2019).
Abdelsalam MA, Aboulwafa OM, M Badawey E-SA et al. Design, synthesis, anticancer
screening, docking studies and in silico ADME prediction of some β-carboline
derivatives. Future medicinal chemistry (0), (2018).
Valkó K. Application of high-performance liquid chromatography based
measurements of lipophilicity to model biological distribution. Journal of
chromatography A 1037(1-2), 299-310 (2004).
Galdeano C, Coquelle N, Cieslikiewicz-Bouet M et al. Increasing Polarity in Tacrine
and Huprine Derivatives: Potent Anticholinesterase Agents for the Treatment of
Myasthenia Gravis. Molecules 23(3), 634 (2018).
Maharani S, Almansour AI, Kumar RS, Arumugam N, Kumar RR. Synthesis of
cycloalkano [b] pyridines by multicomponent strategy: ring-size mediated product
selectivity, substitution-induced axial chirality and influence of the 14 N quadrupole-
relaxation. Tetrahedron 72(30), 4582-4592 (2016).
58.
El-Salam O, Ella D, Ismail N, Abdullah M. Synthesis, docking studies and anti-
inflammatory activity of some 2-amino-5, 6, 7, 8-tetrahydroquinoline-3-carbonitriles
and related compounds. Die Pharmazie-An International Journal of Pharmaceutical
Sciences 64(3), 147-155 (2009).

Highlights

Chlorinated tacrine analogs were designed, synthesized and evaluated for their in vitro and
in vivo anti-cholinesterase activity.


All compounds showed activities either comparable to or significantly higher than tacrine.
Hepatotoxicity of compounds was evaluated by determining SGPT and reduced GSH levels
and the results indicated that all compounds showed a safe hepatic profile.
In silico physicochemical characters and pharmacokinetic parameters together with practical
logP testing were assessed indicating that all synthesized compounds are promising
hits/leads for treatment of Alzheimer's disease.


Graphical abstract