Synthesis and biological evaluation of some tacrine analogs: Study of the effect of the chloro substituent on the acetylcholinesterase inhibitory activity

Article abstract of DOI:10.1007/s00706-015-1641-2

New tetrahydroquinoline derivatives were synthesized, characterized, and evaluated for their in vitro and in vivo acetylcholinesterase inhibitory activity as well as hepatotoxicity using tacrine as a reference standard. The obtained results revealed that

Full text of DOI:10.1007/s00706-015-1641-2

Monatsh Chem (2016) 147:539–552
DOI 10.1007/s00706-015-1641-2
ORIGINAL PAPER
Synthesis and biological evaluation of some tacrine analogs: study
of the effect of the chloro substituent on the acetylcholinesterase
inhibitory activity
Hanan M. Ragab1 Hayam M. A. Ashour1 Amal Galal2,3
•
•
•
Asser I. Ghoneim3,4 Hassan R. Haidar3
•
Received: 23 June 2015 / Accepted: 22 December 2015 / Published online: 1 February 2016
Ó Springer-Verlag Wien 2016
Abstract New tetrahydroquinoline derivatives were
synthesized, characterized, and evaluated for their in vitro
and in vivo acetylcholinesterase inhibitory activity as well
as hepatotoxicity using tacrine as a reference standard. The
obtained results revealed that most of the compounds
comprising a chloro substituent displayed higher activity
when compared with the other analogs. Among the newly
synthesized compounds, four analogs displayed in vitro
and in vivo acetylcholinesterase inhibitory activity com-
parable to or slightly higher than tacrine. Among these
compounds, the 2-chlorotetrahydroquinoline derivative
emerged with hepatotoxicity results comparable to saline.
Graphical abstract
Keywords Alzheimer’s disease Á
Acetylcholinesterase inhibitory activity Á Tacrine Á
Chlorinated analogs Á Bioorganic synthesis
Introduction
Alzheimer’s disease (AD) is a progressive and neurode-
generative disorder which currently represents one of the
biggest threats for the human kind [1–4]. The disease is
characterized by progressive memory impairment related
to a disordered cognitive function [5, 6]. Although the
etiology of AD is still poorly understood, several factors
are thought to play significant roles in the pathology of the
disease such as accumulation of numerous amyloid plaques
of b-amyloid peptide [7], neurofibrillary tangles, dramatic
loss of synapses, shrinkage and death of neurons [8] in
addition to inflammatory changes marked by accumulation
of activated microglia [9]. Scientists have proposed several
theories explaining the mechanism of AD development
[10–14]. The oldest of the AD hypothesis is the cholinergic
hypothesis [10, 11, 15] which has become the leading
strategy for the development of cholinesterase inhibitors
(ChEIs) aimed to increasing levels of acetylcholine (ACh)
through inhibition of cholinesterases (ChEs) [16, 17].
Tacrine (THA, 9-amino-1,2,3,4-tetrahydroacridine) (I,
Fig. 1) [18] was the first drug approved by the FDA for the
treatment of cognitive impairment in AD [3, 19]. The
acetylcholinesterase inhibitory (AChEI) activity of tacrine
was thought to be mediated by its binding to a hydrophobic
area close to the active site [20, 21].
CN
Cl
N
& Hanan M. Ragab
tafida12@yahoo.com
1
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Alexandria University, Alexandria 21521, Egypt
2
3
4
Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Alexandria University, Alexandria, Egypt
Structure–activity relationship (SAR) studies of aminoa-
cridine-related derivatives indicated that 4-aminopyridine
(II, Fig. 1) and 4-aminoquinoline (III, Fig. 1) displayed a
very weak AChEI activity, although their basicities were
Department of Pharmacology and Therapeutics, Faculty of
Pharmacy, Beirut Arab University, Beirut, Lebanon
Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Damanhour University, Damanhour, Egypt
123

540
H. M. Ragab et al.
NH2
N
Based on the above-mentioned findings, we deemed it
interesting to synthesize a new set of 3-cyano-2-substituted
tetrahydroquinolines V in order to investigate whether the
introduction of a chloro group to the phenyl ring at position
4 of the tetrahydroquinoline skeleton would have the same
positive influence on activity as in case of chlorotacrines
VI. This was accomplished by triggering the synthesis of
two series of 3-cyano-2-substituted tetrahydroquinolines V;
one comprising a 4-chloro group on the phenyl ring and the
other lacking it. The synthesized compounds were evalu-
ated for their acetylcholinesterase inhibitory activity and
hepatotoxicity hoping to discover more potent and less
hepatotoxic tacrine analogs.
NH2
NH2
N
N
N
II
III
IV
Tacrine (I)
Fig. 1 Chemical structures of tacrine (I), 4-aminopyridine (II),
4-aminoquinoline (III), and tetrahydroacridine (IV)
NH2
CN
Cl
N
R
N
V
VI
Results and discussion
Chemistry
Fig. 2 Chemical structures of previously synthesized 3-cyano-2-
substituted tetrahydroquinoline derivatives (V) and chlorotacrines
(VI)
The proposed synthetic routes adopted to obtain the target
compounds are illustrated in Schemes 1 and 2. In
Scheme 1, the 4-aryl-3-cyano-2-hydroxy-5,6,7,8-tetrahy-
droquinolines 1 were synthesized by multi-component one-
pot reaction of cyclohexanone, the appropriate aldehyde
and ethyl cyanoacetate in boiling ethanol in presence of
excess ammonium acetate [30–32]. The IR spectra of these
compounds revealed absorption bands characteristic for the
cyano group in addition to absorption bands characteristic
for OH, NH, and C=O functions, indicating existence of the
products in a keto-enol tautomerism. This was further
almost equal to that of tacrine [22]. On the other hand, the
tetrahydroacridine derivative (IV, Fig. 1) was found to be
almost as active as tacrine despite being much weaker base
[23]. These findings indicated that the cyclohexyl ring is
important for activity [22]. Confirmation was reached by
examination of the X-ray crystal structure which revealed
that the 4-aminoquinoline portion of the tacrine molecule
was responsible for binding of the drug to AChE, while the
cyclohexyl ring acted mostly as a block to the substrate at the
active site [23]. However, tacrine was far from ideal due to its
low bioavailability, short half-life and significant hepato-
toxicity [24, 25]. Accordingly, several structural
modifications were conducted on tacrine with the aim of
producing potent compounds that are more selective for
AChE inhibition and with minimum toxicity (if any), to
provide clinically advantageous drugs [26]. In a trial to fulfill
this target, a series of 3-cyano-2-substituted tetrahydro-
quinolines (V, Fig. 2) was designed and synthesized in our
laboratory and their biological testing indicated their high
AChEI activity [27]. SAR studies of these derivatives indi-
cated that presence of a chloro group at the 2-position of the
pyridyl ring (compound V; R = Cl) resulted in the most
potent compound of the whole series [27]. Literature survey
on other chlorinated tacrine analogs indicated that intro-
duction of a chloro group to the phenyl ring of tacrine (VI,
Fig. 2), resulted in potent AChEIs [28].
1
confirmed by investigation of the H NMR spectra which
showed two D2O exchangeable singlets, one integrated for
1/4 H corresponding to the NH and the other integrated for
3/4 H attributed to the OH. Synthesis of the 2-amino analog
2 was achieved by heating malononitrile in dry benzene
utilizing a Dean-Stark head for water separation [33–35].
On the other hand, trials to synthesize the methyl car-
boxylate derivatives 3 according to a reported procedure
described for the synthesis of analogous compounds [36]
where the reaction was performed in presence of catalytic
amount of zinc oxide using water as a solvent failed to give
the required products. However, utilizing a procedure
similar to that used for preparation of compounds 1
resulted in the corresponding methyl 4-aryl-2-hydroxy-
5,6,7,8-tetrahydroquinoline-3-carboxylates 3. IR and 1H
NMR spectra for these compounds again indicated their
existence as keto-enol tautomers, in addition they revealed
signals characteristic for the ester group.
Correlating the results of both studies, it was clear that
presence of a chloro group in these compounds, whether on
the pyridyl or on the phenyl moieties had a positive
influence on the AChEI activity. This was explained by the
recent discovery that better binding of halogenated com-
pounds to receptors was attributed to their ability to form
halogen bonding with those receptors [29].
In Scheme 2, chlorination of the 2-hydroxy derivatives 1
was readily achieved through reflux in a mixture of POCl3/
PCl5 adopting reaction conditions reported for synthesis of
allied compounds [27]. The IR spectra of the chloro
derivatives 4 showed the CN band at 2223 and 2230 cm-1
123

Synthesis and biological evaluation of some tacrine analogs: Study of the effect of the…
541
Scheme 1
X
X
CN
O
CN
OH
N
H
N
1a 1b
,
X
X
i
H
O
O
NH
4OAc
ii
CN
CN
X
NH2
N
,
N
H
NH
2a 2b
iii
X
X
O
O
O
OMe
OMe
a
b
: X = H, : X = Cl
N
H
OH
3a 3b
N
,
Reagents and reaction conditions: (i) ethyl cyanoacetate, ethanol, reflux; (ii) malononitrile, benzene, reflux;
(iii) dimethyl malonate, ethanol, reflux
but lacked the OH, NH, and C = O absorption bands,
1
whereas, the H NMR spectra displayed signals for the
NMR spectra showed additional signals for the newly
introduced alkylamino or phenylhydrazino moieties.
eight protons of the cyclohexyl ring in addition to the
phenyl protons at their expected chemical shifts. Treatment
of the chloro derivatives 4 with excess hydrazine hydrate
98 % in boiling ethanol yielded the corresponding
3-amino-tetrahydropyrazolo[3,4-b]quinolines 5. The reac-
tion smoothly proceeded forming at first a clear solution
which gradually separated the product as a heavy yellow
solid. Complete deposition of the product was achieved by
leaving the reaction mixture for 24 h at room temperature.
The reaction involved nucleophilic replacement of the
chloro group by the hydrazine moiety giving the unsta-
ble intermediate 3-cyano-2-hydrazino-4-phenyl-5,6,7,8-
tetrahydroquinolines, which underwent internal cycload-
dition to form 5 (Fig. 3). The IR spectra proved this
proposed mechanism since it lacked the absorption bands
Acetylcholinesterase inhibitory activity
The synthesized compounds were evaluated for their
acetylcholinesterase inhibitory effects using in vitro phar-
macological experiments. Compounds showing promising
results were further investigated by in vivo pharmacolog-
ical experiment.
In vitro acetylcholinesterase inhibitory activity
The in vitro biological activity of the synthesized com-
pounds was evaluated through the measurement of the
contraction of frog’s Rectus abdominis [37], as an example
of a skeletal muscle that is known to be rich in AChE [38],
the enzyme responsible for the breakdown and deactivation
of ACh. 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 a cholinesterase inhibitor
before the addition of ACh to a muscle preparation leads to
accumulation of unhydrolyzed ACh with resultant increase
1
of the CN group, whereas the H NMR spectra provided
further confirmation of the assigned structures.
As for the synthesis of derivatives 6–10, a general
nucleophilic substitution reaction was employed in which
the chloro derivatives 4 were reacted with the appropriate
amine or phenyl hydrazine in boiling dioxane for 20 h. The
IR spectra of these compounds retained the absorption
bands corresponding to the CN group, whereas the 1H
123

542
H. M. Ragab et al.
Scheme 2
X
N
CN
X
N
X
N
OH
N
H
6a 6b
,
NH2
N
CN
iii
N
N
H
H
5a 5b
,
ii
7a 7b
,
iv
X
X
N
X
CN
Cl
CN
OH
CN
v
i
NH2
N
N
N
H
4a 4b
,
1a 1b
,
8a 8b
,
X
X
vii
vi
CN
CN
H
N
N
N
N
N
H
a
b
: X = H, : X = Cl
10a 10b
9a 9b
,
,
Reagents and reaction conditions: (i) POCl , PCl , reflux; (ii) NH NH (98%), ethanol, reflux;
3
5
2
2
(iii) ethanolamine, dioxane, reflux; (iv) butylamine, dioxane, reflux ; (v) ethylene diamine, dioxane, reflux;
(vi) phenylhydrazine, dioxane, reflux; (vii) diethylamine, dioxane, reflux
Fig. 3 Proposed mechanism for
the synthesis of compounds 5
X
X
N
X
NH2
N
N
H
CN
NH2NH2 H
.
N
C
2O
..
NHNH2
N
Cl
N
4a 4b
5a 5b
,
,
in the height of ACh-induced contractions. The difference
in response of the muscle to ACh before and after exposure
to a suspected cholinesterase inhibitor indicates the extent
of its inhibitory effect on the enzyme. The percentage
inhibition of ACh activity, as indicated by the percentage
potentiation of ACh-induced contractions, was determined.
123

Synthesis and biological evaluation of some tacrine analogs: Study of the effect of the…
543
Table 1 In vitro AChE inhibitory activity of the synthesized com-
pounds on frog’s rectus abdominus
Table 2 In vivo inhibition of AChE activity by the synthesized
compounds in rat brain using Ellman method
Compound
X
In vitro Increase
in contraction/%
Compound
X
In vivo AchE activity/%
inhibition
Control
Tacrine
1a
5.3 1.7
Control
Tacrine
1b
0.00 6.60
38.43 6.39*
34.43 4.31*
12.41 2.72
10.27 9.96
43.63 1.27*
11.17 9.82
6.39 3.06
160.0 34.9*
23.5 2.1
H
Cl
Cl
Cl
H
1b
Cl
H
111.7 11.8*
76.5 9.2*
2b
2a
3b
2b
Cl
H
178.8 58.9*
17.5 3.5
4a
3a
4b
Cl
H
3b
Cl
H
161.7 45.4*
240.0 42.7*
188.7 33.2*
170.0 7.1*
131.7 37.5*
62.5 10.6
5a
4a
5b
Cl
Cl
Cl
H
8.50 2.82
4b
Cl
H
6b
9.01 3.28
5a
7b
54.00 5.05*
7.43 9.86
5b
Cl
H
8a
6a
8b
Cl
H
2.25 7.57
6b
Cl
H
107.5 17.7*
66.0 5.7
9a
15.75 1.99*
20.23 1.58*
14.44 0.04*
42.55 5.35*
7a
9b
Cl
H
7b
Cl
H
227.5 24.7*
183.3 47.3*
233.3 66.6*
80.0 28.3*
183.3 68.1*
225.5 21.9*
81.5 2.1*
10a
10b
8a
Cl
8b
Cl
H
The tested compounds were administered at a dose of 10 mg/kg body
weight
9a
9b
Cl
H
Data are expressed as mean SD (n = 5–7) of at least three different
experiments
10a
10b
Data were analyzed using One factor ANOVA followed by post hoc
Tukey simultaneous comparison t-values (pairwise t tests)
Cl
The tested compounds were added at a concentration of 1 lg/cm3
* Denotes significant difference from control value at P \ 0.05
Data are expressed as mean SD (n = 5–7) of at least three different
experiments
potentiation of the contractions induced by Ach (2–9
times) compared to chloro free analogs except for com-
pounds 4, 5, and 10. Results of compounds 1 and 3 were
highly consistent with the assumed rationale where the
chlorinated analogs 1b and 3b showed almost 5- and
9-fold inhibition of the cholinesterase activity, respec-
tively. However, presence of two chloro groups
(compound 4b) unexpectedly showed lower inhibition
than the monochlorinated analog 4a. This might probably
Data were analyzed using One factor ANOVA followed by post hoc
Tukey simultaneous comparison t-values (pairwise t tests)
* Denotes significant difference from control value at P \ 0.05
The values standard deviation (at confidence levels of
95 %) are recorded in Table 1 and presented in Fig. 4.
Tacrine was used as a reference standard.
The results revealed that, in general, presence of a
4-chloro group in the test compounds caused significant
Fig. 4 Comparison between the
in vitro percentage
acetylcholinesterase inhibitory
300
250
activity of the two synthesized
series (chlorinated versus the
unchlorinated compounds) at a
dose of 1 lg/cm3. Values are
expressed as % increase in Ach-
induced contraction in the
200
150
100
50
0
isolated rectus abdominis
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
Compound No
Series a: Unchlorinated cpd. Series b: Chlorinated cpd
123

544
H. M. Ragab et al.
In vivo acetylcholinesterase inhibitory activity
be due to the introduction of a sort of competition
between the two halogens for the binding site of the
receptor. In addition, compounds 5 and 10 showed an
opposite behavior to the proposed rationale where the
non-chlorinated compounds showed higher in vitro
activity than the chlorinated ones.
Compounds showing in vitro cholinesterase inhibitory
activity that was significantly different from control values
(P \ 0.05) were further investigated by quantitative in vivo
anti-cholinesterase assay according to the method developed
by Ellman et al. [39]. The principle of the assay is based on
the fact that active enzyme hydrolyzes acetyl thiocholine
into free thiocholine which reacts with Ellman reagent
(DTNB; 5,50-dithiobis-(2-nitrobenzoic acid)) to form a yel-
low colored disulfide complex. The activity of AchE is
therefore, measured by the intensity of the yellow color
produced due to the amount of hydrolyzed thiocholine. In
other words, a decrease in the amount of produced thio-
choline than in the control would show a decreased AchE
activity and indicate a successful cholinesterase inhibitory
effect [40]. The results standard deviation (at confidence
levels of 95 %) are recorded in Table 2.
Table 3 Biochemical estimation of the liver enzyme SGPT level in
rats after administration of the tested compounds
Compound
X
SGPT/IU cm-3
Control (DMF)
204.3 26.5
304.7 30.9*
47.25 4.5*
145.5 6.3*
56.8 8.5*
Tacrine (in DMF)
Saline
1b
Cl
Cl
Cl
H
2b
3b
116 10.0*
63.2 25.7*
43.0 8.9*
The results revealed that most of the in vivo tested
compounds showed moderate to high cholinesterase inhi-
bitory activity and four of the synthesized compounds (1b,
4a, 7b, and 10b) showed the same or significantly higher
activity compared to tacrine.
4a
4b
Cl
H
5a
301.5 10.2
236.4 10.6
216.4 19.6
139.1 16.7*
158.5 9.8*
39.1 3.7*
5b
Cl
Cl
Cl
H
6b
7b
Hepatotoxicity testing
8a
8b
Cl
H
The hepatotoxicity of compounds showing in vitro and/or
in vivo cholinesterase inhibitory activity that was signifi-
cantly different from control values at P \ 0.05 was
evaluated through determination of serum glutamic pyruvic
transaminase (SGPT) levels using Ultraviolet (UV) kinetic
method in which SGPT was assayed based in enzyme
coupled system, where the enzyme is involved in the
transfer of an amino group from alanine to an a-ketoacid
(2-oxoglutarate). The formed pyruvate reacts in a system
using NADH which is oxidized to NAD and the rate of
oxidation is measured by determining the absorbance at
340 nm at 0, 1, 2, 3 min.
9a
136.9 17.2*
40.7 2.5*
9b
Cl
H
10a
10b
253.15 16.7
211.7 11.7
Cl
The tested compounds were administered at a dose of 7 mg/kg body
weight
Data are expressed as mean SD (n = 5–7) 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)
* Denotes significant difference from control (DMF) value at
P \ 0.05
Fig. 5 Comparison between the
SGPT levels in rats after
administration of the two
synthesized series (chlorinated
350
300
250
versus the unchlorinated
compounds) at a dose of 7 mg/
kg body weight
200
150
100
50
Series1
0
)
)
b
b
b
e
l
F
F
0a
1b
2b
3b
4a
4b
5a
5
6
7b
8a
8b
9a
9b
n
i
1
10
l
M
M
Sa
(D
n D
i
ro
(
t
n
ne
i
r
c
Co
Ta
Compound No.
123

Synthesis and biological evaluation of some tacrine analogs: Study of the effect of the…
545
The higher the values obtained for SGPT, the higher the
hepatotoxicity caused and vice versa [41, 42]. The
results standard deviation (at confidence levels of 95 %)
are recorded in Table 3 and presented in Fig. 5.
The results indicated that DMF showed considerably
high hepatotoxicity itself. However, most of the synthe-
sized compounds, unlike tacrine, were able to reduce this
hepatotoxic effect. In other words, the compounds showed
Fig. 6 a Binding mode of the
(a)
hAChE (PDB ID: 1B41) using
MOE software. b Binding mode
of compound 4a docked and
minimized in the hAChE
binding site using MOE
software. c Binding mode of
compound 4b docked and
minimized in the hAChE
binding site using MOE
software
inhibitor in the binding site of
Gly
149
Trp
Ser
Gly
151
Gly
150
83
238
Leu
479
Glu
237
His
480
Trp
472
Tyr
162
H
H
+
NH
N
H
H
Tyr
370
Phe
371
(b)
Gly
Thr
Tyr
149
154
162
Gly
150
Leu
Gly
155
159
N
Cl
N
Tyr
Trp
83
370
Tyr
71
Glu
237
His
480
Gly
481
123

546
H. M. Ragab et al.
Fig. 6 continued
His
480
(c)
Gly
481
Glu
237
Cl
N
Trp
83
Tyr
71
Cl
Tyr
N
162
Gly
150
Tyr
370
Leu
159
Thr
Gly
149
154
Gly
155
to some extent a hepatoprotective effect. Compounds 2b,
4a, 4b, 8b, and 9b almost brought SGPT level back to its
normal value despite the presence of DMF. However five
compounds (5a, 5b, 6b, 10a, and 10b) showed high SGPT
levels which were comparable to that of tacrine.
CN group and Thr 154 for both compounds. In addition,
both compounds displayed hydrophobic interactions with
the same amino acid residues as those found in the X-ray
crystal structure of AChE (Trp 83, Gly 149, Gly 150, Tyr
162, Glu 237, Tyr 370, and His 480). Therefore, it could
be concluded that the docking pattern of the test com-
pounds revealed different hydrogen bonding interaction
from that of the ligand whereas they showed nearly
identical hydrophobic binding pattern to that observed
with the inhibitor.
Docking studies
Docking study was carried out using the enzyme param-
eters obtained from the crystallographic structure of the
complex between AChE (PDB ID: 1DX4) with the co-
crystallized ligand tacrine derivative 9-(3-phenylmethy-
lamino)-1,2,3,4-tetrahydroacridine [43]. The docking
simulation for the ligand was carried out using molecular
operating environment (MOE) software supplied by the
Conclusion
The present study reports the synthesis and biological
investigation of acetylcholinesterase inhibitory activity of
chlorinated versus non chlorinated tetrahydroquinolines.
The obtained results clearly revealed that introduction of a
chloro group at the 4-position of the phenyl ring potenti-
ated the acetylcholinesterase inhibitory activity relative to
chloro free analogs. This was obvious on comparing
in vitro results for both chlorinated and non chlorinated
analogs of compounds 1, 2, 3, 6, 7, 8, and 9. The in vivo
results of the promising compounds indicated that most of
´
Chemical Computing Group, Inc., Montreal, QC [44]
(Fig. 6a). The X-ray crystal structure revealed that the NH
group in the ligand 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 6b and c
shows the binding mode of compounds 4a and 4b where a
hydrogen bond is observed between the nitrogen of the
123

Synthesis and biological evaluation of some tacrine analogs: Study of the effect of the…
547
by concentration under vaccum and the residue was left to
stand for 24 h to separate yellow crystals. These were
filtered, washed with ethanol, dried, and recrystallized from
ethanol.
the compounds retained their cholinesterase inhibitory
activity. Furthermore, most of these compounds showed
reduced hepatotoxicity compared to tacrine. Among the
synthesized compounds, compound 4a displayed high
cholinesterase inhibitory activity both in vitro and in vivo,
together with low hepatotoxicity. Collectively, the above
findings could establish a molecular basis for the devel-
opment of new tetrahydroquinolines with higher
cholinesterase inhibitory activity and lower hepatotoxicity
compared to tacrine. MOE-based molecular docking results
suggested that compounds 4a and 4b showed a binding
pattern close to the pattern observed for the ligand
regarding the hydrophobic binding, whereas, the hydrogen
bonding interaction was different from that of the ligand.
Although compounds 4a and 4b showed similar binding
pattern, they differ in their biological activity. This dis-
crepancy in activity of compounds 4a and 4b might be
attributed to difference in halogen bonding capabilities of
both compounds to the target enzyme.
5,6,7,8-Tetrahydro-2-hydroxy-4-phenylquinoline-3-car-
bonitrile (1a) [30, 31]
Yield 35.7 %; m.p.: 260–262 °C (Ref. [26, 27]
267–268 °C).
4-(4-Chlorophenyl)-2-hydroxy-5,6,7,8-tetrahydroquino-
line-3-carbonitrile (1b)
1
Yield 29.6 %; m.p.: 296 °C; H NMR (300 MHz, DMSO-
d6): d = 1.66–1.70 (m, 2H, C6-H2), 2.02–2.07 (m, 2H, C7-
H2), 2.60–2.64 (m, 2H, C5-H2), 2.80–3.11 (m, 2H, C8-H2),
7.31, 7.59 (2d, J = 7.8 Hz, each 2H, chlorophenyl-H), 8.14
(s, 1/4H, NH, D2O-exchangeable), 11.75 (s, 3/4H, OH,
D2O-exchangeable) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 24.4, 26.3, 29.9, 31.1 (cyclohexyl-C), 116.8 (CN),
119.5, 121.6, 127.1, 129.6, 133.5, 135.6, 151.9, 157.5,
164.2 (Ar–C) ppm; IR (KBr): vꢀ ¼ 3400; 3220 (NH and
OH), 2250 (CN), 1695 (C = O), 1600, 1576, 1544, 1480
(C=N and C=C) cm-1
.
Experimental
2-Amino-4-aryl-5,6,7,8-tetrahydroquinoline-3-carboni-
triles 2a, 2b
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 for KBr
discs on Nicolet-800 FT-IR infrared spectrophotometer.
NMR spectra were scanned on a Bruker 300 ultrashield
spectrometer using tetramethylsilane (TMS) as internal
standard and DMSO-d6 as solvent (chemical shifts are
given in d/ppm). Splitting patterns were designated as
follows: s: singlet, br s: broad singlet, d: doublet, t: triplet,
and m: multiplet. Microanalyses were performed at the
regional Center for Mycology and Biotechnology, El Azhar
University and the found values were within 0.3 % of the
theoretical values. Follow up of the reactions and checking
the purity of the compounds was made by thin layer
chromatography (TLC) on silica gel-precoated aluminum
sheets (Type 60 GF254; Merck; Germany) and the spots
were detected by exposure to UV lamp at k = 254 nm for
few seconds.
A mixture of equimolar amounts of the appropriate aldehyde
(45 mmol), 4.41 g cyclohexanone (45 mmol), 2.97 g
malononitrile (45 mmol), and 6.93 g ammonium acetate
(90 mmol) in 25 cm3 dry benzene was heated under reflux
while stirring, using Dean Stark head for 5 h. The solvent
was distilled off and the residue was boiled in 15 cm3
ethanol. The clear solution was left to stand for 24 h to
separate yellow crystals. These were filtered, washed with
ethanol, dried, and recrystallized from ethanol.
2-Amino-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carboni-
trile (2a) [33]
Yield 24.3 %; m.p.: 233–235 °C (Ref. [33] 234–236 °C).
2-Amino-4-(4-chlorophenyl)-5,6,7,8-tetrahydroquinoline-
3-carbonitrile (2b) [34, 35]
Yield 33.7 %; m.p.: 263–266 °C (Ref. [34] 278-280 °C
and Ref. [35] 258–259 °C).
Methyl
4-aryl-2-hydroxy-5,6,7,8-tetrahydroquinoline-3-
carboxylates 3a, 3b
4-Aryl-2-hydroxy-5,6,7,8-tetrahydroquinoline-3-carboni-
triles 1a, 1b
Equimolar amounts of the appropriate aldehyde (45 mmol),
4.41 g cyclohexanone (45 mmol, 4.66 cm3), 5.94 g
dimethyl malonate (45 mmol, 5.15 cm3), and 6.93 g ammo-
nium acetate (90 mmol) were heated under reflux in 25 cm3
ethanol for 3 h. Most of the solvent was removed by
evaporation under vacuum and the residue was left to stand
for 24 h to separate white crystals that were filtered, washed
with ethanol, dried, and recrystallized from ethanol.
Equimolar amounts of the appropriate aldehyde
(45 mmol), 4.41 g cyclohexanone (45 mmol, 4.66 cm3),
5.08 g ethyl cyanoacetate (45 mmol, 4.78 cm3), and 6.93 g
ammonium acetate (90 mmol) were heated under reflux in
25 cm3 ethanol for 15 min. The mixture was then stirred at
room temperature for 3 h. Most of the solvent was removed
123

548
H. M. Ragab et al.
Methyl 2-hydroxy-4-phenyl-5,6,7,8-tetrahydroquinoline-3-
carboxylate (3a, C17H17NO3)
d = 1.61–1.66 (m, 2H, C6-H2), 1.73–1.78 (m, 2H, C7-H2),
2.25–2.31 (m, 2H, C5-H2), 2.81–2.86 (m, 2H, C8-H2),
7.22, 7.56 (2d, J = 7.8 Hz, each 2H, chlorophenyl-H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 21.5, 26.4,
30.6, 36.3 (cyclohexyl-C), 112.02 (CN), 104.5, 128.1,
130.3, 132.7, 134.4, 135.9, 150.4, 152.7, 160.9 (Ar–C)
ppm; IR (KBr): 2223 (CN), 1578, 1545, 1498 (C=N and
Yield 45.9 %; m.p.: 167–168 °C; 1H NMR (300 MHz,
DMSO-d6): d = 1.63–1.68 (m, 2H, C6-H2), 1.76–1.81 (m,
2H, C7-H2), 2.61–2.66 (m, 2H, C5-H2), 2.77–2.82 (m, 2H,
C8-H2), 3.59 (s, 3H, OCH3), 7.32–7.45 (m, 5H, phenyl-H),
7.81 (s, 1/4H, NH, D2O-exchangeable), 11.21 (s, 3/4H,
OH, D2O-exchangeable) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 22.4, 27.3, 31.2, 38.4 (cyclohexyl-C),
56.1 (OCH3), 119.4, 121.0, 126.4, 128.7, 132.6, 133.8,
152.1, 157.0, 161.8 (Ar–C), 164.3 (ester C = O) ppm; IR
(KBr): vꢀ = 3405 (NH or OH), 1743 (ester C = O), 1653
(amide C = O), 1599, 1519, 1498 (C=N and Ar–C=C)
Ar C=C) cm-1
.
3-Amino-4-aryl-5,6,7,8-tetrahydro-1H-pyrazolo[3,4-b]-
quinolines 5a, 5b
A mixture of the 2-chloro-5,6,7,8-tetrahydroquinolines 4
(3.7 mmol) and 0.93 g hydrazine hydrate (98 %,
18.6 mmol, 0.91 cm3) in 5 cm3 absolute ethanol was
heated under reflux for 4 h. The mixture became clear at
first, and then a heavy yellow solid gradually separated out.
The reaction mixture was left to stand at room temperature
for 24 h for complete deposition of the product which was
then filtered, washed with ethanol, dried, and crystallized
from ethanol.
cm-1
.
Methyl 4-(4-chlorophenyl)-2-hydroxy-5,6,7,8-tetrahydro-
quinoline -3-carboxylate (3b, C17H16ClNO3)
1
Yield 42.3 %; m.p.: 171 °C; H NMR (300 MHz, DMSO-
d6): d = 1.64–1.68 (m, 2H, C6-H2), 1.80–1.84 (m, 2H, C7-
H2), 2.62–2.67 (m, 2H, C5-H2), 2.76–2.81 (m, 2H, C8-H2),
3.32 (s, 3H, OCH3), 7.51, 7.59 (2d, J = 8.0 Hz, each 2H,
chlorophenyl-H), 7.89 (s, 1/4H, NH, D2O-exchangeable),
11.43 (s, 3/4H, OH, D2O-exchangeable) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 25.37, 26.29, 31.12, 38.22
(cyclohexyl-C), 56.5 (OCH3), 119.2, 120.6, 127.1, 128.6,
132.5, 132.9, 152.8, 157.0, 165.3 (Ar–C), 165.8 (ester
C = O) ppm; IR (KBr) vꢀ = 3420 (NH or OH), 1745
(C = O ester), 1659 (C = O amide), 1604, 1510, 1500
3-Amino-4-phenyl-5,6,7,8-tetrahydro-1H-pyrazolo[3,4-b]-
quinoline (5a) [27]
Yield 96 %; m.p.: 264–266 °C (Ref. [27] 264–266 °C).
3-Amino-4-(4-chlorophenyl)-5,6,7,8-tetrahydro-1H-pyra-
zolo[3,4-b]quinoline (5b, C16H15ClN4)
1
Yield 94.5 %; m.p.: 254 °C; H NMR (300 MHz, DMSO-
d6): d = 1.63–1.68 (m, 2H, C6-H2), 1.85–1.90 (m, 2H, C7-
H2), 2.46–2.52 (m, 2H, C5-H2), 2.75–2.81 (m, 2H, C8-H2),
5.62 (s, 2H, NH2, D2O-exchangeable), 7.22, 7.47 (2d,
J = 8.2 Hz, each 2H, chlorophenyl-H), 11.60 (s, 1H, NH,
D2O-exchangeable) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 22.4, 27.7, 29.7, 35.9 (cyclohexyl-C), 91.7, 128.6,
129.4, 132.9, 133.3, 135.9, 146.5, 150.7, 152.3, 157.6 (Ar–
C) ppm; IR (KBr): vꢀ = 3450–3178 (NH), 1599, 1560,
(C=N and Ar–C=C) cm-1
.
2-Chloro-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carboni-
trile (4a) [27]
A solution of 5.0 g 5,6,7,8-tetrahydro-2-hydroxyquinoline
1a (20 mmol) in 15 cm3 phosphorus oxychloride was
heated under reflux for 20 h in presence of catalytic
amount of phosphorus pentachloride. The reaction mixture
was cooled and poured onto crushed ice to give a yellowish
orange precipitate, which was filtered, dried, and crystal-
lized from ethanol. Yield 73 %; m.p.: 200–202 °C (Ref.
[27] 201–202 °C); IR (KBr): vꢀ = 2230 (CN), 1559, 1532,
1528, 1490 (C=N and Ar–C=C) cm-1
.
General procedure for 4-aryl-2-(substituted amino)-
5,6,7,8-tetrahydroquinoline-3-carbonitriles 6-8, 10 and
4-aryl-2-(2-phenylhydrazinyl)-5,6,7,8-tetrahydroquinoline-
3-carbonitriles 9a, 9b
1493 (C=N and Ar–C=C) cm-1
.
A
mixture of the 2-chlorotetrahydroquinolines
4
2-Chloro-4-(4-chlorophenyl)- 5,6,7,8-tetrahydroquinoline-
3-carbonitrile (4b, C16H12Cl2N2)
(3.7 mmol) and the appropriate amine or phenyl hydrazine
(7.4 mmol) in 10 cm3 dioxane was heated under reflux for
20 h. The reaction mixture was left to attain room
temperature, then poured onto crushed ice to give a
brownish precipitate, which was filtered, dried, and purified
using column chromatography.
A solution of 5.69 g 5,6,7,8-tetrahydro-2-hydroxyquinoline
1b (20 mmol) in 15 cm3 phosphorus oxychloride was
heated under reflux for 20 h in presence of catalytic
amount of phosphorus pentachloride. The reaction mixture
was cooled and poured onto crushed ice to give a yellowish
orange oil which was extracted using chloroform. The
chloroform extract was dried over anhydrous Na2SO4 and
evaporated to dryness. The produced oil was purified using
column chromatography (chloroform: methanol 9:1). Yield
87 %; m.p.: 117 °C; 1H NMR (300 MHz, DMSO-d6):
4-Aryl-2-(2-hydroxyethylamino)-5,6,7,8-tetrahydroquino-
line-3-carbonitriles 6a, 6b
Purified using column chromatography (methylene chlo-
ride: methanol: ammonium hydroxide, 5:1:0.1) as a
yellowish solid.
123

Synthesis and biological evaluation of some tacrine analogs: Study of the effect of the…
549
2-(2-Hydroxyethylamino)-4-phenyl-5,6,7,8-tetrahydroquino-
2-(Butylamino)-4-(4-chlorophenyl)-5,6,7,8-tetrahydro-
quinoline-3-carbonitrile (7b, C20H22ClN3)
line-3-carbonitrile (6a, C18H19N3O)
1
Yield 46 %; m.p.: 164 °C; H NMR (300 MHz, DMSO-
Yield 39 %; m.p.: 163 °C; 1H NMR (300 MHz, DMSO-d6):
d = 0.89 (t, J = 6.9 Hz, 3H, CH3), 1.30–1.36 (m, 2H,
CH2CH3), 1.44–1.50 (m, 2H, CH2CH2CH3), 1.56–1.62 (m,
2H, C6-H2), 1.64–1.69 (m, 2H, C7-H2), 2.16–2.21 (m, 2H, C5-
H2), 2.81–2.87 (m, 2H, C8-H2), 3.36 (t, J = 6.9 Hz, 2H,
NCH2), 6.51 (s, 1H, NH, D2O-exchangeable), 7.41, 7.67 (2d,
J = 8.2 Hz, each 2H, chlorophenyl-H) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 13.7 (CH3), 19.5 (NH-CH2CH2-
CH2CH3), 21.5, 21.9, 29.8, 30.7, 35.1 (cyclohexyl-C and NH-
CH2CH2CH2CH3), 41.2 (NH-CH2CH2CH2CH3), 95.6 (pyr-
idine-C3), 115.67 (CN), 125.9, 128.4, 129.6, 133.3, 137.2,
150.6, 159.1, 162.9 (Ar–C) ppm; IR (KBr): vꢀ = 3319 (NH),
d6): d = 1.66–1.70 (m, 2H, C6-H2), 1.76–1.81 (m, 2H, C7-
H2), 2.10–2.16 (m, 2H, C5-H2), 2.87–2.92 (m, 2H, C8-H2),
3.37 (t, J = 6.9 Hz, 2H, NCH2), 3.46 (t, J = 6.9 Hz, 2H,
OCH2), 4.55 (s, 1H, OH, D2O-exchangeable), 6.43 (s, 1H,
NH, D2O-exchangeable), 7.18-7.53 (m, 5H, phenyl-H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 21.3, 25.2,
29.8, 36.1 (cyclohexyl-C), 45.3 (NCH2), 59.2 (OCH2), 95.4
(pyridine-C3), 115.3 (CN), 125.7, 126.8, 128.1, 128.6,
137.2, 150.6, 159.3, 161.2 (Ar–C) ppm; IR (KBr):
vꢀ = 3458 (OH), 3289 (NH), 2230 (CN), 1593, 1552,
1543, 1478 (C=N and Ar–C=C) cm-1
.
2235 (CN), 1590, 1486, 1466 (C=N and Ar–C=C) cm-1
.
4-(4-Chlorophenyl)-2-(2-hydroxyethylamino)-5,6,7,8-te-
trahydroquinoline-3-carbonitrile (6b, C18H18ClN3O)
1
Yield 46 %; m.p.: 143 °C; H NMR (300 MHz, DMSO-
2-(2-Aminoethylamino)-4-aryl-5,6,7,8-tetrahydroquino-
line-3-carbonitriles 8a, 8b
d6): d = 1.65–1.70 (m, 2H, C6-H2), 1.79-1.84 (m, 2H, C7-
H2), 2.19–2.24 (m, 2H, C5-H2), 2.51–2.56 (m, 2H, C8-H2),
3.41 (t, J = 6.9 Hz, 2H, NCH2), 3.47 (t, J = 6.9 Hz, 2H,
OCH2), 4.46 (s, 1H, OH, D2O-exchangeable), 6.43 (s, 1H,
NH, D2O-exchangeable), 7.30, 7.59 (2d, J = 7.8 Hz, each
2H, chlorophenyl-H) ppm; 13C NMR (75 MHz, DMSO-
d6): d = 21.9, 25.1, 29.7, 35.9 (cyclohexyl-C), 45.7
(NCH2), 59.3 (OCH2), 95.7 (pyridine-C3), 115.7 (CN),
126.1, 128.4, 129. 5, 132.4, 134.7, 151.3, 159.6, 161.5 (Ar–
C) ppm; IR (KBr): vꢀ = 3423 (OH), 3254 (NH), 2245 (CN),
Purified using column chromatography (chloroform:
methanol: ammonium hydroxide, 9:1:0.1) as a yellow
solid.
2-(2-Aminoethylamino)-4-phenyl-5,6,7,8-tetrahydroquino-
line-3-carbonitrile (8a, C18H20N4)
1
Yield 43 %; m.p.: 130 °C; H NMR (300 MHz, DMSO-
d6): d = 1.60–1.66 (m, 2H, C6-H2), 1.74–1.80 (m, 2H, C7-
H2), 2.20–2.26 (m, 2H, C5-H2), 2.74 (t, J = 6.2 Hz, 2H,
NCH2), 2.90–2.96 (m, 2H, C8-H2), 3.38 (t, J = 6.2 Hz, 2H,
NCH2), 6.30 (br s, 2H, NH2, D2O-exchangeable), 6.52 (br
s, 1H, NH, D2O-exchangeable), 7.23–7.56 (m, 5H, Ar–H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 21.4, 22.1,
30.7, 36.4 (cyclohexyl-C), 45.7, 49.5 (NCH2), 90.7
(pyridine-C3), 116.4 (CN), 125.8, 127.8, 128.3, 128.6,
136.1, 153.9, 159.1, 160.3 (Ar–C) ppm; IR (KBr):
vꢀ = 3394, 3294 (NH), 2208 (CN), 1595, 1586, 1489,
1584, 1551, 1524, 1485 (C=N and Ar–C=C) cm-1
.
4-Aryl-2-(butylamino)-5,6,7,8-tetrahydroquinoline-3-car-
bonitriles 7a, 7b
Purified using column chromatography (chloroform:
methanol: ammonium hydroxide, 9:1:0.1) as a pale yellow
solid.
1465 (C=N and Ar–C=C) cm-1
.
2-(Butylamino)-4-phenyl-5,6,7,8-tetrahydroquinoline-3-
carbonitrile (7a, C20H23N3)
2-(2-Aminoethylamino)- 4-(4-chlorophenyl)-5,6,7,8-te-
Yield 41 %; m.p.: 209–210 °C; 1H NMR (300 MHz,
DMSO-d6): d = 0.91 (t, J = 6.9 Hz, 3H, CH3), 1.25–
1.31 (m, 2H, CH2CH3), 1.47–1.52 (m, 2H, CH2CH2CH3),
1.54–1.60 (m, 2H, C6-H2), 1.63–1.68 (m, 2H, C7-H2),
2.07–2.13 (m, 2H, C5-H2), 2.69–2.74 (m, 2H, C8-H2), 3.43
(t, J = 6.9 Hz, 2H, NCH2), 6.45 (s, 1H, NH, D2O-
exchangeable), 7.38–7.61 (m, 5H, phenyl-H) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 13.50 (CH3), 19.20
(CH2CH3), 21.3, 21.9, 29.9, 30.5, 35.6 (cyclohexyl-C and
NH-CH2CH2CH2CH3), 40.31 (NH-CH2CH2CH2CH3), 95.4
(pyridine-C3), 115.3 (CN), 124.1, 127.7, 129.1, 129.5,
137.2, 150.0, 158.7, 162.8 (Ar–C) ppm; IR (KBr):
vꢀ = 3325 (NH), 2240 (CN), 1585, 1474, 1455 (C=N and
trahydroquinoline-3-carbonitrile (8b, C18H19ClN4)
Yield 41 %; m.p.: 152–155 °C; 1H NMR (300 MHz,
DMSO-d6): d = 1.60–1.64 (m, 2H, C6-H2), 1.70–1.76
(m, 2H, C7-H2), 2.29–2.35 (m, 2H, C5-H2), 2.79 (t,
J = 6.2 Hz, 2H, NCH2), 2.98–3.14 (m, 2H, C8-H2), 3.37
(t, J = 6.2 Hz, 2H, NCH2), 6.34 (br s, 2H, NH2, D2O-
exchangeable), 6.50 (br s, 1H, NH, D2O-exchangeable),
7.33, 7.59 (2d, J = 8.2 Hz, each 2H, chlorophenyl-H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 21.5, 22.4,
30.8, 36.5 (cyclohexyl-C), 45.3, 51.8 (NCH2), 88.8
(pyridine-C3), 117.6 (CN), 125.9, 128.7, 129.2, 134.6,
136.7, 155.4, 158.6, 160.45 (Ar–C) ppm; IR (KBr):
vꢀ = 3389, 3302 (NH), 2228 (CN), 1590, 1579, 1492,
Ar–C=C) cm-1
.
1460 (C=N and Ar–C=C) cm-1
.
123

550
H. M. Ragab et al.
4-Aryl-2-(2-phenylhydrazinyl)-5,6,7,8-tetrahydroquino-
line-3-carbonitriles 9a, 9b
(m, 2H, C5-H2), 2.94–3.00 (m, 2H, C8-H2), 3.67 (q,
J = 6.9 Hz, 4H, 2NCH2), 7.37, 7.58 (2d, J = 7.8 Hz, each
2H, chlorophenyl-H) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 13.0(CH3), 22.3, 26.9, 30.7, 36.1 (cyclohexyl-C), 44.3
(NCH2), 92.4 (pyridine-C3), 115.7 (CN), 124.5, 127.9,
128.8, 132.7, 135.9, 150.2, 161.8, 163.6 (Ar–C) ppm; IR
(KBr): vꢀ = 2230 (CN), 1595, 1578, 1489, 1476 (C=N and
Purified using column chromatography (methylene chlo-
ride:methanol:petroleum ether, 12:1:0.5) as a light beige
solid.
2-(2-Phenylhydrazinyl)-4-phenyl-5,6,7,8-tetrahydroquino-
line-3-carbonitrile (9a, C22H20N4)
Ar–C=C) cm-1
.
Yield 65 %; m.p.: 134–136 °C; 1H NMR (300 MHz,
DMSO-d6): d = 1.64–1.69 (m, 2H, C6-H2), 1.72–1.77
(m, 2H, C7-H2), 2.38–2.43 (m, 2H, C5-H2), 2.85–2.90 (m,
2H, C8-H2), 7.15–7.55 (m, 12H, Ar–H and NH) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 21.5, 23.2, 30.6, 36.3
(cyclohexyl-C), 88.8 (pyridine-C3), 112.02 (CN), 116.3,
119.6, 127.4, 128.7, 129.4, 129.8, 130.3, 136.2, 151.4,
152.6, 156.5, 158.8 (Ar–C) ppm; IR (KBr): vꢀ = 3210,
3163 (NH), 2211 (CN), 1580, 1500, 1488 (C=N and Ar–
Animals
Frogs for the in vitro acetylcholinesterase testing were
obtained from a local supplier, used within 2 days of their
purchase and kept hydrated throughout the 2 days. Male
albino rats (weighing 250–330 g) for the in vivo acetyl
cholinesterase and hepatotoxicity testing were obtained
from the animal house of Faculty of Pharmacy, Beirut Arab
University. The animals were housed under standard con-
ditions as per the rules and regulations of the Faculty
animal ethics committee (IRB) (approval code. 2014A-
002-P-R-0004).
C=C) cm-1
.
4-(4-Chlorophenyl)-2-(2-phenylhydrazinyl)-5,6,7,8-te-
trahydroquinoline-3-carbonitrile (9b, C22H19ClN4)
Yield 60 %; m.p.: 179–181 °C; 1H NMR (300 MHz,
DMSO-d6): d = 1.65–1.70 (m, 2H, C6-H2), 1.74–1.79 (m,
2H, C7-H2), 2.39–2.45 (m, 2H, C5-H2), 2.88–2.94 (m, 2H,
C8-H2), 7.18–7.60 (m, 11H, Ar–H and NH) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 22.1, 26.9, 30.9, 36.2 (cyclo-
hexyl-C), 89.6 (pyridine-C3), 114.7 (CN), 118.6, 120.2,
128.8, 129.3, 129.7, 130.5, 133.8, 137.4, 150.8, 151.6, 155.4,
157.8 (Ar–C) ppm; IR (KBr): vꢀ = 3219, 3180 (NH), 2226
In vitro acetylcholinesterase inhibitory activity [37]
All synthesized compounds were evaluated for their
in vitro cholinesterase inhibitory effects on acetylcholine-
induced contractions of the frog rectus abdominis. The
triangular muscle of the frog was isolated and suspended in
an organ bath of 10 cm3 capacity containing aerated
Ringer’s solution which has been kept at room temperature.
The contractions induced by acetylcholine (65 lg/cm3)
were recorded by a force–displacement transducer con-
nected to a Kymograph recorder. A submaximal dose of
acetylcholine was determined using a contact time of
1.5 min in 3 min cycles. The solution of the tested com-
pounds in dimethylformamide (1 lg/cm3) was added
10 min before adding the submaximal dose of acetyl-
choline. Five to seven observations were recorded for each
compound. The percentage inhibition of acetyl-
cholinesterase activity, as indicated by the percentage
potentiation of acetylcholine-induced contractions, and the
standard deviation were calculated as recorded in Table 1.
(CN), 1585, 1515, 1491 (C=N and Ar–C=C) cm-1
.
4-Aryl-2-(diethylamino)-5,6,7,8-tetrahydroquinoline-3-
carbonitriles 10a, 10b
Purified using column chromatography (chloroform:
methanol: ammonium hydroxide, 9:1:0.1) as a white solid.
2-(Diethylamino)-4-phenyl-5,6,7,8-tetrahydroquinoline-3-
carbonitrile (10a, C20H23N3)
Yield 46 %; m.p.: 167–169 °C; 1H NMR (300 MHz,
DMSO-d6): d = 1.24 (t, J = 6.9 Hz, 6H, 2CH3), 1.67–
1.72 (m, 2H, C6-H2), 1.75–1.81 (m, 2H, C7-H2), 2.31–2.36
(m, 2H, C5-H2), 2.95–3.01 (m, 2H, C8-H2), 3.62 (q,
J = 6.9 Hz, 4H, 2NCH2), 7.38–7.56 (m, 5H, Ar–H) ppm;
13C NMR (75 MHz, DMSO-d6): d = 12.5 (CH3), 21.8, 26.7,
30.7, 35.9 (cyclohexyl-C), 43.8 (NCH2), 88.9 (pyridine-C3),
115.3 (CN), 125.2, 127.7, 128.5, 129.6, 137.2, 151.7, 160.0,
162.8 (Ar–C) ppm; IR (KBr): vꢀ = 2223 (CN), 1597, 1568,
In vivo acetylcholinesterase inhibitory activity
The rats were fasted overnight, and then given the solutions
of the tested compounds at a dose of 10 mg/kg body
weight, via intraperitoneal route. One hour after drug
administration, the rats were sacrificed by decapitation.
The whole brain was removed, immediately homogenized
in 9 volumes of ice-cold 0.1 M sodium–potassium phos-
phate buffer (pH 8.0). The homogenate was centrifuged at
1492, 1460 (C=N and Ar–C=C) cm-1
.
4-(4-Chlorophenyl)-2-(diethylamino)-5,6,7,8-tetrahydro-
quinoline-3-carbonitrile (10b, C20H22ClN3)
Yield 42 %; m.p.: 159–160 °C; 1H NMR (300 MHz,
DMSO-d6): d = 1.23 (t, J = 6.9 Hz, 6H, 2 CH3), 1.69–
1.75 (m, 2H, C6-H2), 1.78–1.84 (m, 2H, C7-H2), 2.35–3.01
123

Synthesis and biological evaluation of some tacrine analogs: Study of the effect of the…
551
5000 rpm for 30 min and the supernatant was used for
estimation of cholinesterase activity spectrophotometri-
cally as reported by Ellman et al. [39].
1,2,3,4-tetrahydroacridine. The ligand molecules were
constructed using the builder module and were energy
minimized. The active site of AChE was generated using
the MOE-Alpha Site Finder, and then ligands were docked
within this active site using the MOE Dock. The lowest
energy conformation was selected, and the ligand interac-
tions (hydrogen bonding and hydrophobic interaction) with
AChE were recorded.
Measurement of acetylcholinesterase activity
Acetyl thiocholine reaction was initiated by mixing
0.1 cm3 of the brain homogenate supernatant with 0.5 mM
acetyl thiocholine and 0.33 mM 5,50-dithiobis(2-nitroben-
zoic acid) (DTNB) in phosphate buffer (pH 8.0). The
produced thiocholine reacted with the DTNB to form a
colored disulfide product whose intensity was measured
kinetically over 10 min at 412 nm, using a Perkin Elmer
type (Lambda 2S) spectrophotometer. Five to seven read-
ings were recorded for each compound. The percent
inhibition of acetyl cholinesterase activity and standard
deviation were recorded in Table 2.
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