Synthesis, molecular docking studies, and biological evaluation of novel alkyl bis(4-amino-5-cyanopyrimidine) derivatives

Article abstract of DOI:10.1002/ardp.201900027

A series of bis(4-amino-5-cyano-pyrimidines) was synthesized and evaluated as dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). To further explore the multifunctional properties of the new derivatives, their antioxidant and antibacterial activities were also tested. The results showed that most of these compounds could effectively inhibit AChE and BChE. Particularly, compound 7c exhibited the best AChE inhibitory activity (IC₅₀ = 5.72 ± 1.53 μM), whereas compound 7h was identified as the most potent BChE inhibitor (IC₅₀ = 12.19 ± 0.57 μM). Molecular modeling study revealed that compounds 7c, 7f, and 7b showed a higher inhibitory activity than that of galantamine against both AChE and BChE. Anticholinesterase activity of compounds 7h, 7b, and 7c was significant in vitro and in silico for both enzymes, since these compounds have hydrophobic rings (Br-phenyl, dimethyl, and methoxyphenyl), which bind very well in both sites. In addition to cholinesterase inhibitory activities, these compounds showed different levels of antioxidant activities. Indeed, in the superoxide–dimethyl sulfoxide alkaline assay, compound 7j showed very high inhibition (IC₅₀ = 0.37 ± 0.28 μM). Also, compound 7l exhibited strong and good antibacterial activity against Staphylococcus epidermidis and Staphylococcus aureus, respectively. Taking into account the results of biological evaluation, further modifications will be designed to increase potency on different targets. In this study, the obtained results can be a new starting point for further development of multifunctional agents for the treatment of Alzheimer's disease.

Full text of DOI:10.1002/ardp.201900027

Received: 24 January 2019
Revised: 25 July 2019
Accepted: 26 July 2019
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|
DOI: 10.1002/ardp.201900027
F U L L P A P E R
Synthesis, molecular docking studies, and biological evaluation of
novel alkyl bis(4‐amino‐5‐cyanopyrimidine) derivatives
1
1
1,2
3
Imen Boualia
|
Chamseddine Derabli
|
Raouf Boulcina
|
Chawki Bensouici
|
4
5
6
1
Muhammet Yildirim
|
Arzu Birinci Yildirim
|
El Hassen Mokrani
|
Abdelmadjid Debache
1
Laboratory of Synthesis of Molecules with
Biological Interest, Mentouri–Constantine
University, Constantine, Algeria
Abstract
A series of bis(4‐amino‐5‐cyano‐pyrimidines) was synthesized and evaluated as dual
inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). To
further explore the multifunctional properties of the new derivatives, their
antioxidant and antibacterial activities were also tested. The results showed that
most of these compounds could effectively inhibit AChE and BChE. Particularly,
compound 7c exhibited the best AChE inhibitory activity (IC50 = 5.72 ± 1.53 μM),
2
Faculty of Technology, Batna 2 University,
Batna, Algeria
3
Biotechnology Research Center, Constantine,
Algeria
4
Department of Chemistry, Faculty of
Sciences and Arts, Abant Izzet Baysal
University, Bolu, Turkey
5
Department of Field Crops, Faculty of
whereas compound 7h was identified as the most potent BChE inhibitor (IC50
=
Agriculture and Natural Sciences, Abant İzzet
1
2.19 ± 0.57 μM). Molecular modeling study revealed that compounds 7c, 7f, and 7b
Baysal University, Bolu, Turkey
6
showed a higher inhibitory activity than that of galantamine against both AChE and
BChE. Anticholinesterase activity of compounds 7h, 7b, and 7c was significant in vitro
and in silico for both enzymes, since these compounds have hydrophobic rings (Br‐
phenyl, dimethyl, and methoxyphenyl), which bind very well in both sites. In addition
to cholinesterase inhibitory activities, these compounds showed different levels of
antioxidant activities. Indeed, in the superoxide–dimethyl sulfoxide alkaline assay,
compound 7j showed very high inhibition (IC50 = 0.37 ± 0.28 μM). Also, compound 7l
exhibited strong and good antibacterial activity against Staphylococcus epidermidis and
Staphylococcus aureus, respectively. Taking into account the results of biological
evaluation, further modifications will be designed to increase potency on different
targets. In this study, the obtained results can be a new starting point for further
development of multifunctional agents for the treatment of Alzheimer’s disease.
Department of Biochemistry and Cellular and
Molecular Biology, Laboratory of Applied
Biochemistry, Faculty of Natural and Life
Sciences, Mentouri–Constantine University,
Constantine, Algeria
Correspondence
Dr. Raouf Boulcina, Laboratory of Synthesis
of Molecules with Biological Interest,
Mentouri–Constantine University,
2
5000 Constantine, Algeria.
Email: r.boulcina@univ-batna2.dz
Funding information
Ministère de l’Enseignement Supérieur et de la
Recherche Scientifique (Algeria)
K E Y W O R D S
alkyl bis(4‐amino‐5‐cyano‐pyrimidines), antibacterial activity, anticholinesterase activity,
antioxidant effect, molecular docking studies
1
|
INTRODUCTION
hydrolysis of acetylcholine by blocking its metabolic enzyme acet-
ylcholinesterase (AChE) increases the ACh concentration and provides
Alzheimer’s disease (AD) as a neurodegenerative disorder is mani-
a possible symptomatic treatment option for AD. On the contrary,
butyrylcholinesterase (BuChE) has recently been considered as a
potential target because it also plays an important role in regulating
fested by progressive deterioration of intellectual and cognitive
[1,2]
functions, memory loss, and personality changes.
One of the major
[4]
therapeutic strategies adopted for primarily symptomatic AD is based
on the cholinergic hypothesis targeting cholinesterase enzymes
ACh level. However, due to the complex pathologies of AD, the
current AChE inhibitors such as donepezil and galantamine (Figure 1)
that modulate a single target can only improve clinical symptoms but
[3]
(
acetylcholinesterase and butyrylcholinesterase). Inhibition of the
Arch Pharm Chem Life Sci. 2019;e1900027.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900027

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BOUALIA ET AL.
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FIGURE 1 Acetylcholinesterase inhibitors in use
cannot mitigate the progression of the disease process underlying the
[
5]
SCHEME 1 Preparation of 2‐alkylisothiouronium bromides 3 and
arylidenemalononitriles 6
AD. As reported in some earlier studies, inhibition of butyrylcholin-
esterase (BuChE) represents an important therapeutic target for AD. It
has also been recently reported that dual inhibition of AChE and
BuChE might alleviate AD symptoms owing to the key role of BuChE
in hydrolysis of ACh. It is essential to ensure that the drug inhibits
[6]
both AChE and BuChE. Moreover, oxidative injury caused by free
radical formation and iron accumulation has been revealed to be
[7]
another factor in AD pathogenesis.
On the contrary, the pyrimidine core is found in many natural
products and pharmaceuticals, which display a range of significant
biological properties, such as phosphatidyl inositol 3‐kinase
[
8]
[9]
[10]
inhibitors, A2A receptor antagonists, anti‐HIV,
and anti-
[
11,12]
bacterial.
Therefore, many researchers have contributed to the develop-
ment of efficient strategies for their construction. Pyrimidines have
been synthesized via one‐pot three‐component coupling of terminal
SCHEME 2 Synthesis of bis(4‐amino‐5‐cyano‐pyrimidines) 7
[13]
alkynes, elemental sulfur, and carbodiimides.
dine derivatives have been obtained from a single‐step reaction
by a titanium‐catalyzed one‐pot multicomponent coupling reac-
In addition, pyrimi-
[14]
or
2‐arylidenemalononitriles 6 in the presence of a catalytic amount of
potassium carbonate in ethanol at room temperature (Scheme 1).
For the synthesis of compound 7, the condensation of the
precursors 3 and 6 was carried out using various catalysts under
different conditions. At the outset of our synthetic work, we
attempted to find a feasible and efficient pathway to pyrimidine
formation under mild conditions. In the first trial experiment, a
reaction between 2‐benzylidenemalononitriles 6a and 2‐propyliso-
thiouronium bromide 3a in ethanol was left at room temperature for
[
15]
tion.
Also, a new series of pyrimidine derivatives has been
[16]
synthesized via tandem aza‐Wittig and annulation reactions.
As per our continuous interest in the synthesis of the biologically
important heterocycles, herein, we further report a simple, and cost‐
effective, and high‐yielding method for synthesis of a novel series of
highly functionalized 2‐amino‐3‐cyano‐4‐aryl‐6‐sulfapyrimidines as
cholinesterase inhibitors. In addition, molecular docking analysis was
also performed to disclose the binding interaction template of the
most active inhibitors to the amino acid residues composing the
active sites of the AChE and butyrylcholinesterase (BChE) enzymes
and the findings are presented below. Antioxidant activity and
antibacterial effects of these compounds are also discussed.
2
4 hr. After simple work‐up, the corresponding product was isolated in
very poor amounts. The use of K CO as a catalyst in ethanol at reflux
facilitated the reaction to some extent, but none exceeded the yield of
0%. However, the reaction was most efficient when two equivalents
of K CO was used in isopropanol under reflux.
2
3
5
2
3
Using the most efficient condition established above, we next
attempted to extend the process to a series of 2‐alkylisothiouronium
bromides 3 and 2‐arylidenemalononitriles 6 having electron‐with-
drawing and electron‐donating groups. The results showed that most
of the reactions afforded the desired products (7a–p) in good yields,
2
2
|
RESULTS AND DISCUSSION
|
Synthesis
.1
The synthesis of the intermediates and target compounds was
accomplished according to the steps depicted in Schemes 1 and 2. The
present synthetic strategy begins with the synthesis of 2‐alkylisothiour-
onium bromides 3 that were not commercially available, and were thus
obtained from the condensation of thiourea 1 with the corresponding
alkyl bromides 2 by prolonged heating of the mixture in ethanol at reflux.
On the contrary, the condensation between various substituted
benzaldehydes 4 and malononitrile 5 gave rise to the formation of
2 3
indicating that the designed synthetic method using K CO as
catalyst was very appropriate (Table 1).
1
H‐NMR spectra of compounds 7 revealed the NH
groups protons were at δ
1.78–3.79 ppm, and aromatic protons were in the range of
7.01–8.63 ppm. The structures of all the newly synthesized compounds
2
protons to
be a singlet at δ 5.71–6.09 ppm, CH
2
δ
1
13
were confirmed by the infrared, H NMR, C NMR, and HRMS spectral
data, which were in full agreement with their structures.

BOUALIA ET AL.
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TABLE 1 Synthesis of bis(4‐amino‐5‐cyano‐pyrimidines) 7a–p
possessed much higher activity than did those compounds containing
bromo group. Among these compounds, 7c, bearing
m‐methoxyphenyl group at position 4 of the pyrimidine ring, showed
the highest AChE inhibitory activity, followed by 7f, with
a
a
a
Entry
Ar
n
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
Product
7a
Time (hr)
Yield (%)
65
1
2
3
4
5
6
7
8
9
C
6
H
5
1
3
4
3
3
4
3
4
4
4
3
4
2.5
3
4
3
a
4‐(Me)‐C
6
H
4
7b
7c
58
m‐nitrophenyl group at position 4 of the pyrimidine ring, with IC50
values of 5.72 ± 1.53 and 9.09 ± 1.66 μM, respectively.
3‐(MeO)‐C
6
H
4
4
67
4‐(MeO)‐C
6
H
7d
7e
64
Similarly, these compounds showed BChE inhibitory activity, with
IC50 values ranging from 12.19 ± 0.57 to 123.09 ± 1.14 μM. Com-
pound 7h bearing a p‐bromophenyl group at position 4 of the
pyrimidine ring, with an IC50 value of 12.19 ± 0.57, had a higher
inhibitory activity than that of galantamine (IC50 = 31.77 ± 2.22 μM),
whereas other compounds such as 7f and 7c had similar or
lower inhibitory effects with IC50 values of 32.32 ± 1.42 and
4‐NO
3‐NO
2
2
‐C
‐C
6
H
H
4
4
64
6
7f
79
4‐Cl‐C
6
H
4
7g
70
4‐Br‐C
6
H
4
7h
7i
69
C
6
H
5
63
1
1
1
1
1
1
1
0
1
2
3
4
5
6
4‐Me‐C
6
H
4
7j
62
44.41 ± 2.32 μM, respectively.
3‐(MeO)‐C
6
H
4
4
7k
7l
73
The inhibition selectivity of the newly synthesised compounds
4‐(MeO)‐C
6
H
68
was determined for AChE and BChE (Table 2). Overall, all of the
compounds had a good preference for AChE, among which three
compounds displayed a 5–10 times higher inhibition selectivity
toward AChE over BChE. The most selective AChE inhibitor was
compound 7k, followed by 7c and 7b, whose affinities were 10.11,
7.76, and 5.72 times higher than that for BChE, respectively. The
lowest inhibition selectivity, only by 0.77 and 1.6 times, was obtained
for compounds 7h and 7n, respectively.
4‐NO
3‐NO
2
2
‐C
‐C
6
H
H
4
4
7m
7n
7o
7p
68
6
69
4‐Cl‐C
6
H
4
60
4‐Br ‐C
6
H
4
72
a
Yields of pure isolated products.
2
.2 In vitro cholinesterases inhibition studies
|
[17]
In a very recent study, Xu et al.
studied the BACE1 activity of 4‐
2
.3 Molecular docking studies
|
amino‐5‐cyano‐pyrimidine compounds, and they found at least three
derivatives, which were very similar to our scaffolds, had very strong
BACE1 inhibition effects with very low IC50 levels. For this purpose,
we study herein the anti‐ChE activity of bis(4‐amino‐5‐cyanopyr-
imidine) derivatives.
Molecular docking calculations of the designed compounds into the
active sites of AChE and BChE were performed using FlexX, which is
based on an incremental construction of ligands.
First, the binding energy was predicted between the synthesized
compounds and both AChE and BChE active sites using molecular
docking. From Table 2 it appears that the experiment data of in vitro
ChE inhibitory activity showed a satisfactory agreement with the
molecular docking results. According to their binding energies (ΔG)
and their experimental IC50 values, compounds 7c, 7f, and 7b were
found to exhibit a higher inhibitory activity than that of galantamine
against both AChE and BChE (see Table 2).
We selected arbitrarily ten bis(4‐amino‐5‐cyano‐pyrimidines)
7
, which were tested for their in vitro inhibitory activities against
AChE from Electrophorus electricus (eel‐AChE) and BChE from
horse serum (eqBChE) according to the Ellman method with
[
18]
some modifications.
The efficacy is expressed as IC50 values,
representing the concentration of an inhibitor required to
decrease the enzymatic activity by 50%. The results were
compared with those obtained for galantamine, a clinically used
ChE inhibitor which acts as a competitive inhibitor.
The most promising inhibitors 7c and 7h from in vitro assays
were selected for further investigations of their binding mode with
AChE and BChE active sites respectively. As shown in Figure 2, 7c fit
well the entrance until the bottom of AChE‐active gorge making five
hydrogen bonds.
As shown in Table 2, derivatives of 7 exhibited AChE inhibitory
activities with IC50 values ranging from 5.72 ± 1.53 to 35.08 ± 0.55 μM.
The strongest inhibitory activity towards AChE and BChE was displayed
by bis‐pyrimidine hybrids with a three‐carbon atom linker. The
elongation of the tether led to a decreased activity, except for
compound 7k, which gave good activity.
The first pyrimidine ring was involved in three hydrogen bonds,
two of them by its carbonitrile group with Ser293 and one by its
amine group with Trp289. The amine group of the second ring forms
the fourth hydrogen bond with Tyr341 while the last hydrogen bond
was observed between the carbonitrile group of the same ring and
Phe295. It should be noted that the two methoxyphenyl rings
performed an important role in stabilization of 7c inside the pocket
by making several hydrophobic interactions with Tyr124, Tyr337,
Tyr341, Phe338, Phe297, Ser293, Val294, Gly342, and Glu292.
On the contrary, 7h covers the entire BChE active site pocket
making four hydrogen bonds (Figure 3). Two of them were observed
On the contrary, the chemical nature of the substituents as well
as their positions on the benzene ring greatly influenced the activity.
In general, the inhibitory potency of the bis‐pyrimidine derivatives
against AChE was as follows: meta‐substituted > para‐substituted.
However, the inhibitory potency against BChE was reversed: para‐
substituted > meta‐substituted. In addition, the inhibitory activity
against AChE was highly dependent on the nature of the ring
substituent. Compounds containing a methoxyl or nitro group

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TABLE 2 The inhibitory concentration (IC50), calculated binding energy and selectivity of selected bis(4‐amino‐5‐cyano‐pyrimidines) 7
investigated against AChE and BChE
AChE
BChE
a
b
a
c
Compound
IC50 (μM)
Selectivity
5.72
Binding energy (kJ/mol)
−21.88
IC50 (μM)
Selectivity
0.17
Binding energy (kJ/mol)
−25.68
7
7
7
7
7
7
7
7
7
7
b
11.37 ± 2.12
5.72 ± 1.53
32.43 ± 0.61
9.09 ± 1.66
15.92 ± 0.32
17,58 ± 0.07
9.40 ± 0.16
17.26 ± 0.60
35.08 ± 0.55
30.42 ± 0.11
21.82 ± 4.00
65.05 ± 1.14
44.41 ± 2.32
123.09 ± 1.14
32.32 ± 1.42
12.19 ± 0.57
80.33 ± 0.17
95.08 ± 0.56
73.95 ± 0.75
55.96 ± 0.07
61.78 ± 0.44
31.77 ± 2.22
c
7.76
−32.11
0.13
−33.39
e
f
3.80
−19.14
0.26
−18.94
3.56
−30.66
0.28
−34.52
h
j
0.77
−18.10
1.31
−33.78
4.57
−19.34
0.22
−20.58
k
m
n
p
10.11
4.28
−29.56
0.099
0.24
−20.88
−20.15
−19.85
1.60
−18.30
0.63
−32.48
2.03
−20.96
0.49
−18.84
Galantamine
5.54
−18.27
0.18
−20.97
Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholinesterase; SD, standard deviation.
a
IC50 values expressed are means ± SD of three parallel measurements (p < 0.05).
b
Selectivity for AChE defined as IC50(BChE)/IC50(AChE).
c
Selectivity for BChE defined as IC50(AChE)/IC50(BChE).
between the amine group of the first ring and the two residues
Ser287 and Thr284. The two other hydrogen bonds were formed
between the carbonitrile group of the second ring and the two
residues Gly116 and His448 of BChE. This last residue is considered
compounds with antioxidant potential has received great attention
[21]
from researchers.
Thus, the antioxidant capabilities of the
synthesized compounds were also evaluated using cupric reducing
antioxidant capacity (CUPRAC) assay, reducing power as well as
β‐carotene, and superoxide–DMSO alkaline methods. For CUPRAC,
compound 7p showed the most promising antioxidant effect with an
IC50 value of 25.57 ± 0.60 μM (Table 3), that is approximately equal in
efficacy to butylated hydroxytoluene (BHT; IC50 = 24.28 ± 3.22 μM)
and two‐fold more effective than butylated hydroxyanisole (BHA;
IC50 = 36.62 ± 0.27 μM), followed by 7j with IC50 = 26.50 ± 1.51 μM.
α‐Tocopherol and ascorbic acid were used as standards for inhibition
in reducing power assay and the results are given as the A0.50 value.
According to this method, all tested compounds did not show any
significant activity. On the contrary, 7f (IC50 = 4.91 ± 0.32 μM) and 7b
(IC50 = 9.5 ± 1.30 μM) for inhibition in β‐carotene assay, had the
[19]
as a critical member of the BChE catalytic triad.
h was well stabilized in the BChE active pocket because
7
of its two Br‐phenyl rings making hydrophobic interactions with
Tyr332, Asp70, Gly117, Gly116, Phe329, Phe398, Ser198, Pro285,
and His448.
2
.4 Antioxidant activity studies
|
Oxidative stress is critical to the etiology of many chronic and
degenerative diseases such as cancer, cardiovascular diseases,
[20]
diabetes, obesity, epilepsy, and AD.
Therefore, the synthesis of
FIGURE 2 Binding mode prediction of 7c into the acetylcholinesterase active site

BOUALIA ET AL.
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FIGURE 3 Binding mode prediction of 7h into the butyrylcholinesterase active site
highest IC50 values among the selected products. Also, the compound
j (IC50 = 0.37 ± 0.28 μM), followed by 7p (IC50 = 18.57 ± 1.22 μM)
been the prime purpose of researchers working in the area of
medicinal chemistry and drug industry for the last two decades. With
7
[24]
and 7m (IC50 = 23.23 ± 1.33 μM) showed the best inhibition for
superoxide–DMSO alkaline assay (Table 3).
this design, the Kirby–Bauer (disk diffusion) method
the evaluation of the susceptibility of the synthesized bis‐pyrimidines
7) to different bacteria. The antibacterial properties of compounds
a, 7d, 7g, 7i, 7l, and 7o were tested against 10 bacterial strains
Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium,
was used for
(
7
2
.5 Antibacterial activity studies
|
(
Recently, the emergence of bacterial resistance to famous antibiotics
β‐lactams, cephalosphorins, and quinolones) due to their prolonged
use has generated some risks for patients like high mortality, longer
Serratia marcescens, Proteus vulgaris, Enterobacter cloacae, Klebsiella
pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis , and
Streptococcus pyogenes; Table 4).
(
[22,23]
hospital stays and costs, and risk of secondary infections.
Therefore, development of new antimicrobial agent candidates has
According to the test results, none of the compounds (7)
exhibited antibacterial effects against any Gram‐negative bacteria.
TABLE 3 Antioxidant activity of selected bis(4‐amino‐5‐cyano‐pyrimidines) 7 using CUPRAC, reducing power, β‐carotene, and super-
oxide–DMSO alkaline assays
a
a
A
0.50 (µM)
IC50 (µM)
Compound
CUPRAC assay
238.42 ± 0.46
231.90 ± 0.22
201.64 ± 0.63
203.24 ± 1.66
338.36 ± 0.72
26.50 ± 1.51
179.53 ± 0.95
177.05 ± 0.15
400.71 ± 1.63
25.57 ± 0.60
24.28 ± 3.22
36.62 ± 0.27
46.24 ± 3.38
70.57 ± 0.51
Reducing power assay
270.38 ± 0.10′
>200
β‐Carotene assay
9.5 ± 1.30
Superoxide anion‐scavenging assay
238.96 ± 0.46
236.72 ± 0.74
57.19 ± 0.61
81.11 ± 1.89
210.35 ± 0.67
0.37 ± 0.28
7
7
7
7
7
7
7
7
7
7
b
c
15.48 ± 0.32
10.44 ± 0.82
4.91 ± 0.32
e
f
298.16 ± 0.22
222.81 ± 0.82
>200
h
j
32.01 ± 0.55
20.07 ± 1.06
160.95 ± 1.61
25.58 ± 0.25
80.75 ± 1.83
17.19 ± 0.38
4.77 ± 0.05
>200
k
m
n
p
259.49 ± 1.12
>200
410.25 ± 1.44
23.23 ± 1.33
331.02 ± 0.42
18.57 ± 1.22
>200
>200
213.30 ± 0.20
>200
b
b
BHT
BHA
44.32 ± 4.82
81.10 ± 5.53
38.44 ± 6.53
4.99 ± 0.11
>200
α‐Tocopherol
Ascorbic acid
4.15 ± 0.06
52.17 ± 5.87
27.76 ± 1.48
52.59 ± 1.98
Abbreviations: BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; CUPRAC, cupric reducing antioxidant capacity; DMSO, dimethyl
sulfoxide, SD, standard deviation.
a
IC50 and A0.50 values expressed are means ± SD of three parallel measurements (p < 0.05).
b
Reference compounds.

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However, only compound 7l exhibited a strong antibacterial effect
against S. epidermidis (17–18 mm) and a good antibacterial effect
against S. aureus (13–12 mm) among Gram‐positive bacteria. Com-
pound 7l exhibited a higher inhibition zone than standard antibiotic,
tetracycline (12 mm) against S. epidermidis but it exhibited a lower
inhibition zone than standard antibiotic, erytromycin (45 mm) against
S. epidermidis. Besides this, the inhibition zone of compound 7l
and S. aureus among all tested compounds. These comprehensive
studies demonstrated the potential application of 4‐disubstituted
pyrimidines as a suitable template to develop multitargeting agents
for the treatment of AD.
4
|
EXPERIMENTAL
(13–12 mm) against S. aureus was much lower than that of the
4
4
.1 Chemistry
|
antibiotics, tetracyline (33 mm) and erytromycin (32 mm) against
S. aureus. Regarding the antibacterial activity, results against
S. epidermidis and S. aureus show compound 7l can be a candidate
antimicrobial agent for the treatment of implanted prostheses
infections in human body and for the treatment of various localized
and diffused skin infections, respectively.
.1.1 | General
All the chemicals were purchased from the Sigma‐Aldrich and used
without further purification. All solvents used for spectroscopic and
synthesis studies were of reagent grade and further purified by literature
methods. Thin‐layer chromatography (TLC) was performed using a
mixture of EtOAc and hexane (1:2) as eluents and silica gel 60 F254
(
Merck) plates. Melting points (mp) were determined on an Electro-
3
|
CONCLUSION
1
13
thermal capillary fine‐control apparatus. H and C NMR spectra were
recorded on a Bruker Avance 400 instrument at 400 and 100 MHz
In the present study, we report the development of a novel library
of substituted bis(4‐amino‐5‐cyano‐pyrimidines) as multitargeting
agents that exhibit cholinesterase inhibition as well as antioxidant
properties. Ten of the target compounds 7 exhibited varying levels of
dual ChE inhibitions, whereas the other derivatives exhibited good
antioxidant properties.
respectively, in CDCl
3
6
or DMSO‐d . Chemical shifts (δ) are given in parts
1
13
per million downfield from TMS as an internal standard for H and
C
NMR. Coupling constants (J) values were indicated in Hz. The chemicals
were used as obtained commercially. High‐resolution mass spectra
(
HRMS) were recorded with a high‐resolution time‐of‐flight mass
spectrometer coupled with a liquid chromatography system (Agilent
200/6210) or MicroTof–Q 98. Elemental analyses were carried out on a
Compound 7c exhibited high AChE inhibition (IC50 = 5.72 ± 1.53
μM); compound 7h (IC50 = 12.19 ± 0.57 μM) was considered as the
most potent BChE inhibitor, whereas compound 7f exhibited dual
BChE inhibition (IC50 = 9.09 ± 1.66 μM) and very high antioxidant
activity (AA) in the β‐carotene assay (IC50 = 4.91 ± 0.32 μM). Mole-
cular modeling study revealed that compounds 7c, 7f, and 7b showed
a higher inhibitory activity than the galantamine against both AChE
and BChE. Among all tested compounds for AA in the super-
oxide–DMSO alkaline assay, compound 7j showed very high
inhibition (IC50 = 0.37 ± 0.28 μM). Besides this, only compound 7l
showed strong and good antibacterial effects against S. epidermidis
1
Microanalyzer Flash EA1112 CHNS/O Thermo Electron.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
4
2
.1.2 | General procedure for the preparation of
,2′‐(alkane‐1,3‐diyl)bis(thiouronium) (
3
)
A mixture of thiourea (2 mmol) and alkyl bromide (1.2 mmol) in EtOH
(5 ml) was stirred overnight under reflux, the mixture was cooled to
TABLE 4 Antibacterial activities of selected bis(4‐amino‐5‐cyano‐pyrimidines) 7
a
Mean diameter of inhibitory zones
Compounds
Positive controls
Negative control
7
–
–
–
–
–
–
–
–
–
–
a
7d
–
–
–
–
–
–
–
–
–
–
7g
–
–
–
–
–
–
–
–
–
–
7i
–
–
–
–
–
–
–
–
–
–
7l
7o
–
–
–
–
–
–
–
–
–
–
Erythromycin
Tetracycline
(dimethyl sulfoxide)
Bacteria
Escherichia coli
–
15
–
30
16
21
17
27
30
27
33
12
–
–
–
–
–
–
–
–
–
–
–
Pseudomonas aeruginosa
Salmonella typhimurium
Serratia marcescens
–
–
12
12
12
–
–
Proteus vulgaris
–
Enterobacter cloacae
Klebsiella pneumoniae
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus pyogenes
–
–
14
32
45
–
12–13
17–18
–
a
Data presented as zone of inhibition of bacterial growth in mm.

BOUALIA ET AL.
7 of 10
|
−
room temperature and evaporated. The final product was used in the
next step without further purification.
116.5, 113.9, 82.8, 55.5, 29.5, and 29. HRMS (APCI ) m/z 555.14636
+
[M−H] . Anal. calcd for C27
H
23 8
N O
S
2 2
: 555.13685.
Propane‐1,3‐diyldicarbamimidothioate dihydrobromide (3a)
2,2′‐(Propane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐(4‐methoxyphe-
1
Compound 3a was obtained as a white solid (95%). Mp: 218°C.
NMR (300 MHz, DMSO‐d ) δ (ppm): 9.11 (s, 8H, 4NH
H
nyl)pyrimidine‐5‐carbonitrile) (7d)
1
6
2
), 3.28
Compound 7d was obtained as a white solid (64%). Mp: >250°C.
NMR (400 MHz, DMSO‐d
(dd, J = 6.8, 2 Hz, 4H), 3.79 (s, 6H, 2CH
H
1
3
(t, J = 7.3 Hz, 2H, CH
2
), and 1.94 (q, J = 7.3 Hz, 4H, 2CH
2
). C NMR
6
) δ (ppm): 7.85 (dd, J = 6.8, 2 Hz, 4H), 7.03
(
75 MHz, DMSO‐d ) δ (ppm): 170.0, 29.2, and 29.0.
6
3
‐O), 3.23 (t, J = 6.8 Hz, 4H,
1
3
2CH
2
), and 2.09 (qt, J = 6.8 Hz, 2H, CH
2
). C NMR (100 MHz, DMSO‐
Butane‐1,4‐diyldicarbamimidothioate dihydrobromide (3b
Compound 3b was obtained as a white solid (97%). Mp: 232°C.
NMR (300 MHz, DMSO‐d ) δ (ppm): 9.08 (s, 8H, 4NH ), 3.24–3.21 (m,
H, 2CH ), and 1.70–1.68 (m, 4H, 2CH ). C NMR (75 MHz, DMSO‐
) δ (ppm): 170.3, 29.9, and 27.7.
)
6
d ) δ (ppm): 173.4, 166.7, 163.9, 162.1, 130.7, 128.4, 117.1, 114.3,
1
+
+
H
81.9, 55.9, and 29.6. HRMS (ESI ) m/z 595.1121 [M+K] . Anal. calcd
for C27 K: 595.1101.
6
2
24 8 2 2
H N O S
1
3
4
2
2
d
6
2,2′‐(Propane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐(4‐nitrophenyl)‐
pyrimidine‐5‐carbonitrile) (7e
Compound 7e was obtained as a brown solid (64%). Mp: >250°C.
NMR (250 MHz, DMSO‐d ) δ (ppm): 8.28 (d, J = 8.7 Hz, 4H), 8.05 (d,
J = 8.7 Hz, 4H), 5.79 (s, 4H, 2NH ), 3.20 (t, J = 6.1 Hz, 4H, 2CH ), and
). C NMR (62.9 MHz, DMSO‐d ) δ
(ppm): 173.9, 165.2, 163.2, 148.7, 141.9, 129.9, 123.5, 119.5, 115.8,
)
1
H
4
.1.3 | General procedure for the preparation of bis
6
(
4‐amino‐5‐cyano‐pyrimidines) (7)
2
2
1
3
In a 100 ml flask were introduced 2‐alkylthiouronium (1 mmol) and 2‐
arylidene)malononitrile derivative (2 mmol) in the presence of
CO (2 mmol). The mixture was refluxed in 10 ml of isopropanol.
2.11 (qt, J = 6.1 Hz, 2H, CH
2
6
(
−
+
K
2
3
83.3, 29.4, and 29.1. HRMS (APCI ) m/z 585.09539 [M−H] . Anal.
calcd for C25 : 585.09021.
The progress of the reaction was monitored by TLC. After completion
of the reaction, the mixture was cooled to room temperature and was
poured into 100 ml of water. The precipitate was obtained by vacuum
filtration. The resulting solid residue was recrystallized from ethanol.
17 10 4 2
H N O S
2,2′‐(Propane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐(3‐nitrophenyl)‐
pyrimidine‐5‐carbonitrile) (7f
Compound 7f was obtained as a brown solid (79%). Mp: 238°C.
NMR (250 MHz, DMSO‐d ) δ (ppm): 8.63 (t, J = 8.3 Hz, 2H), 8.37 (d,
J = 8.0 Hz, 2H), 8.34 (d, J = 7.7 Hz, 2H), 7.79 (t, J = 8.0 Hz, 2H), 6.02 (s,
)
1
H
2
,2′‐(Propane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐phenylpyrimi-
6
dine‐5‐carbonitrile) (7a
)
Compound 7a was obtained as a white solid (65%). Mp: 228–230°C;
2
4H, 2NH ), 3.09 (t, J = 6.2 Hz, 4H, 2CH
2
), and 2.17 (qt, J = 6.2 Hz, 2H,
1
13
H NMR (400 MHz, DMSO‐d
6
) δ (ppm): 7.81 (dd, J = 6.8, 1.6 Hz, 4H),
.57–7.48 (m, 6H), 3.23 (t, J = 6.8 Hz, 4H, 2CH ), and 2.10 (qt,
) δ (ppm): 173.9,
CH
2
). C NMR (62.9 MHz, DMSO‐d
6
) δ (ppm): 173.9, 164.5, 163.2,
7
2
147.6, 137.3, 134.5, 129.1, 125.3, 116.1, 115.8, 82.8, 29.3, and
13
−
+
J = 6.8 Hz, 2H, CH
2
). C NMR (100 MHz, DMSO‐d
6
29.1. HRMS (APCI ) m/z 585.09539 [M−H] . Anal. calcd for
: 585.09214.
1
67.7, 164.0, 136.4, 131.1, 128.9, 128.8, 116.7, 83.9, and 29.7. Anal.
calcd for C25 : C, 60.46; H, 4.06; N, 22.56; S, 12.91; Found: C,
9.86; H, 4.05; N, 22.07, S, 12.30.
25 17 10 4 2
C H N O S
20 8 2
H N S
5
2,2′‐(Propane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐(4‐chlorophenyl)‐
pyrimidine‐5‐carbonitrile) (7g)
1
2
,2′‐(Propane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐p‐tolylpyrimi-
Compound 7g was obtained as a white solid (70%). Mp: >250°C.
NMR (400 MHz, DMSO‐d
(dd, J = 6.8, 2.0 Hz, 4H), 3.22 (t, J = 6.8 Hz, 4H, 2CH
H
dine‐5‐carbonitrile) (7b
Compound 7b was obtained as a beige solid (58%). Mp: >250°C.
NMR (250 MHz, DMSO‐d ) δ (ppm): 7.89 (d, J = 8.2 Hz, 4H), 7.23 (d,
J = 8.2 Hz, 4H), 3.23 (t, J = 5.9 Hz, 4H, 2CH ), 2.23 (s, 6H, 2CH ), and
). C NMR (62.9 MHz, DMSO‐d ) δ
)
6
) δ (ppm): 7.83 (dd, J = 6.8, 2 Hz, 4H), 7.56
), and 2.08 (qt,
) δ (ppm): 173.8,
166.4, 163.6, 136.4, 135.1, 130.8, 129.0, 116.5, 82.9, 29.7, and 29.4.
1
H
2
1
3
6
J = 6.8 Hz, 2H, CH
2
). C NMR (100 MHz, DMSO‐d
6
2
3
1
3
+
+
2
.14 (qt, J = 5.9 Hz, 2H, CH
2
6
2 8 2
HRMS (ESI ) m/z 603.0128 [M+K] . Anal. calcd for C25H18Cl N S K:
(ppm): 173.7, 167.2, 164.1, 141.3, 135.9, 135.5, 135.1, 133.4, 129.1,
603.0110.
−
1
5
28.7, 119.8, 116.9, 82.6, 29.6, 29.4, and 20.9. HRMS (APCI ) m/z
+
23.15653 [M−H] . Anal. calcd for C27
23
H N
S
8 2
: 523.15379.
2,2′‐(Propane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐(4‐bromophenyl)
pyrimidine‐5‐carbonitrile) (7h)
1
2
,2′‐(Propane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐(3‐methoxyphe-
nyl)pyrimidine‐5‐carbonitrile) (7c)
Compound 7c was obtained as a white solid (67%). Mp: 222°C.
NMR (250 MHz, DMSO‐d ) δ (ppm): 7.75–7.72 (m, 6H), 7.14–7.12 (m,
), 3.79 (s, 6H, 2CH ‐O), 3.23 (t, J = 6.2 Hz, 4H,
), and 2.13 (qt, J = 6.2 Hz, 2H, CH
) δ (ppm): 173.5, 167.2, 163.5, 159.2, 137.4, 129.9, 116.9,
Compound 7h was obtained as a white solid (69%). Mp: >250°C.
NMR (250 MHz, DMSO‐d
J = 8.6 Hz, 4H), 5.71 (br s, 4H, 2NH
H
6
) δ (ppm): 7.77 (d, J = 8.6 Hz, 4H), 7.61 (d,
), 3.22 (t, J = 6.8 Hz, 4H, 2CH ),
) δ
(ppm): 173.5, 165.8, 163.3, 134.9, 131.3, 130.2, 124.9, 116.0, 82.3,
1
H
2
2
1
3
6
and 2.12 (qt, J = 6.8 Hz, 2H, CH
2
). C NMR (62.9 MHz, DMSO‐d
6
2
H), 6.0 (s, 4H, 2NH
CH
2
3
1
3
−
+
2
2
2
). C NMR (62.9 MHz,
29.2, and 29. HRMS (APCI ) m/z 650.94626 [M–H] . Anal. calcd for
17Br : 650.93832.
DMSO‐d
6
25
C H
2 8 2
N S

8
of 10
BOUALIA ET AL.
|
2
,2′‐(Butane‐1,4‐diylbis(sulfanediyl))bis(4‐amino‐6‐phenylpyrimi-
2H), 8.29 (d, J = 7.7 Hz, 2H), 7.80 (t, J = 8.0 Hz, 2H), 6.05 (br s, 4H,
13
dine‐5‐carbonitrile) (7i)
2NH
2 2 2
), 3.23 (m, 4H, 2CH ), and 1.84 (m, 4H, 2CH ). C NMR
1
Compound 7i was obtained as a white solid (63%). Mp: >250°C.
H
(62.9 MHz, DMSO‐d
6
) δ (ppm): 174.3, 165.1, 163.5, 148.0, 137.6,
−
NMR (400 MHz, DMSO‐d
6
)
δ
(ppm): 7.79 (d, J = 6.8 Hz, 4H),
135.1, 130.7, 126.0, 123.5, 83.2, 30.1, and 28.9. HRMS (APCI ) m/z
1
3
+
7.54–7.47 (m, 6H), 3.12 (s, 4H), and 1.78 (s, 4H).
C NMR
599.11104 [M–H] . Anal. calcd for C26
H
19
N O
10 4
2
S : 599.10126.
(
100 MHz, DMSO‐d
6
) δ (ppm): 173.9, 166.4, 163.8, 136.4, 135.4,
+
1
5
30.5, 128.9, 116.3, 82.7, 30.2, 28.9, and 26.0. HRMS (ESI ) m/z
2,2′‐(Butane‐1,4‐diylbis(sulfanediyl))bis(4‐amino‐6‐(4‐chlorophenyl)‐
pyrimidine‐5‐carbonitrile) (7o
Compound 7o was obtained as a white solid (60%). Mp: >250°C.
NMR (400 MHz, DMSO‐d ) δ (ppm): 7.84 (d, J = 6.8 Hz, 4H), 7.56 (d,
2
J = 6.8 Hz, 4H), 3.13 (s, 4H, 2CH ), and 1.78 (s, 4H, 2CH ). C NMR
+
49.1074 [M+K] . Anal. calcd for C26
22
H N
8 2
S K: 546.1046.
)
1
H
2
5
,2′‐(Butane‐1,4‐diylbis(sulfanediyl))bis(4‐amino‐6‐p‐tolylpyrimidine‐
‐carbonitrile) (7j)
6
13
2
1
Compound 7j was obtained as a beige solid (62%). Mp: 140°C.
NMR (250 MHz, DMSO‐d ) δ (ppm): 7.87 (d, J = 8.0 Hz, 4H), 7.44 (d,
J = 8.0 Hz, 4H), 3.23–3.18 (m, 4H, 2CH ), 2.31 (s, 6H, 2CH ), and
). C NMR (62.9 MHz, DMSO‐d ) δ (ppm):
73.5, 166.5, 161.3, 145.8, 130.7, 130.2, 129.3, 128.8, 116.3, 82.8,
H
(100 MHz, DMSO‐d
6
) δ (ppm): 174.1, 166.5, 163.6, 136.3, 135.2,
+
130.8, 129.0, 116.6, 82.9, 62.4, 30.1, 28.7, and 25.9. HRMS (ESI ) m/z
6
+
617.0297 [M+K] . Anal. calcd for C26
H
2
20Cl N
8 2
S K: 617.0266.
2
3
1
3
1
1
2
.82–1.79 (m, 4H, 2CH
2
6
2,2′‐(Butane‐1,3‐diylbis(sulfanediyl))bis(4‐amino‐6‐(4‐bromophenyl)‐
pyrimidine‐5‐carbonitrile) (7p)
−
+
9.9, 29.1, and 21.5. HRMS (APCI ) m/z 537.17218 [M−H] . Anal.
: 537.16936.
1
calcd for C28
25
H N
S
8 2
Compound 7p was obtained as a white solid (72%). Mp: 160°C. H NMR
(
250 MHz, DMSO‐d
4H), 5.86 (br s, 4H, 2NH
NMR (62.9 MHz, DMSO‐d
6
) δ (ppm): 7.87 (d, J = 8.4 Hz, 4H), 7.42 (d, J = 8.4 Hz,
13
2
,2′‐(Butane‐1,4‐diylbis(sulfanediyl))bis(4‐amino‐6‐(3‐methoxyphe-
2
), 3.87–3.84 (m, 4H), and 1.83–1.78 (m, 4H).
C
nyl)pyrimidine‐5‐carbonitrile) (7k)
6
) δ (ppm): 173.1, 165.6, 164.1, 133.6, 132.1,
1
+
Compound 7k was obtained as a white solid (73%). Mp: 226°C.
H
130.5, 126.2, 124.9, 116.8, 81.9, 29.5, and 29.2. HRMS (ESI ) m/z
+
NMR (250 MHz, DMSO‐d
6
) δ (ppm): 7.49–7.43 (m, 6H), 7.15–7.12 (m,
), 3.92 (s, 6H, 2CH ‐O), 3.14–3.11 (m, 4H,
). C NMR (62.9 MHz, DMSO‐d
666.94830 [M+H] . Anal. calcd for C26
H
21Br
2
N
8
S
2
: 666.96191.
2H), 5.97 (s, 4H, 2NH
2
3
1
3
2
CH ), and 1.81–1.78 (m, 4H, 2CH
2
2
6
)
4
.2 Method for cholinesterase activity assay
|
δ (ppm): 173.6, 167.2, 163.5, 159.2, 137.4, 129.9, 120.9, 119.8, 116.9,
−
1
16.5, 114, 82.7, 55.4, 29.9, and 28.5. HRMS (APCI ) m/z 569.16201
AChE and BChE inhibitory activity was measured by the spectro-
[18]
+
[M−H] . Anal. calcd for C28
25
H N
8 2
O S
2
: 569.15243.
photometric method developed by Ellman et al.
Briefly, 150 µl of
100 mM sodium phosphate buffer (pH 8.0), 10 µl of a sample solution
2
,2′‐(Butane‐1,4‐diylbis(sulfanediyl))bis(4‐amino‐6‐(4‐methoxyphe-
dissolved in methanol at different concentrations and 20 µl AChE
−
3
−3
nyl)pyrimidine‐5‐carbonitrile) (7l)
Compound 7l was obtained as a white solid (68%). Mp: >250°C. H NMR
400 MHz, DMSO‐d ) δ (ppm): 7.82 (d, J = 6.8 Hz, 4H), 7.01 (d, J = 6.8 Hz,
H), 3.87 (s, 6H, 2CH
NMR (100 MHz, DMSO‐d
28.2, 117.5, 114.4, 82.0, 55.8, 30.2, 28.9, and 25.8. HRMS (ESI ) m/z
(5.32 × 10 units) or BChE (6.85 × 10 units) solutions were mixed
and incubated for 15 min at 25°C, and 10 µl of 0.5 mM 5,5′‐dithio‐bis‐
(2‐nitrobenzoic) acid (DTNB) were added. The reaction was then
initiated by the addition of 10 µl of acetylthiocholine iodide
(0.71 mM) or butyrylthiocholine chloride (0.2 mM). The hydrolysis
of these substrates were monitored spectrophotometrically by the
formation of the yellow 5‐thio‐2‐nitrobenzoate anion, as the result of
the reaction of DTNB with thiocholine, released by the enzymatic
hydrolysis of acetylthiocholine iodide or butyrylthiocholine chloride,
respectively, at a wavelength of 412 nm, every 5 min for 15 min,
utilizing a 96‐well microplate reader (Multimode Plate Reader
EnSpire; Perkin Elmer) in triplicate experiments. Galantamine was
used as a reference compound. The results were given as 50%
inhibition concentration (IC50) and the percentage of inhibition of
AChE or BChE was determined by the comparison of reaction rates
of samples relative to the blank sample (methanol in phosphate
buffers, pH 8) using the formula
1
(
6
13
4
3
‐O), 3.12 (s, 4H, 2CH
2
), and 1.78 (s, 4H, 2CH
2
).
C
6
) δ (ppm): 173.9, 166.5, 163.7, 162.5, 130.3,
+
1
+
6
26 8 2 2
09.1284 [M+K] . Anal. calcd for C28H N O S K: 609.1257.
2
,2′‐(Butane‐1,4‐diylbis(sulfanediyl))bis(4‐amino‐6‐(4‐nitrophenyl)‐
pyrimidine‐5‐carbonitrile) (7m)
Compound 7m was obtained as a brown solid (68%). Mp: >250°C. H
NMR (250 MHz, DMSO‐d ) δ (ppm): 8.33 (d, J = 8.7 Hz, 4H), 8.06 (d,
J = 8.7 Hz, 4H), 6.09 (s, 4H, 2NH ), 3.06 (m, 4H, 2CH ), and 1.80 (m,
). C NMR (62.9 MHz, DMSO‐d ) δ (ppm): 173.9, 165.1,
63.2, 148.8, 137.6, 135.1, 130.2, 123.8, 119.6, 83.2, 30.2, and 28.7.
1
6
2
2
13
4
H, 2CH
2
6
1
−
+
HRMS (APCI ) m/z 599.11104 [M−H] . Anal. calcd for
: 599.10129.
26 19 10 4 2
C H N O S
E − S
E
2
,2′‐(Butane‐1,4‐diylbis(sulfanediyl))bis(4‐amino‐6‐(3‐nitrophenyl)‐
pyrimidine‐5‐carbonitrile) (7n)
Compound 7n was obtained as a brown solid (69%). Mp: >250°C. H
NMR (250 MHz, DMSO‐d ) δ (ppm): 8.63 (t, 2H), 8.37 (d, J = 7.7 Hz,
Inhibition of AChE or BChE (%) =
× 100,
1
where E is the activity of an enzyme without test samples and S is the
activity of an enzyme with test samples.
6

BOUALIA ET AL.
9 of 10
|
4
.3
|
Molecular docking procedures
4.4.3 | Cupric reducing antioxidant capacity
[
25]
The three‐dimensional (3D) coordinates of hAChE (ID: 4M0E)
and
The cupric reducing antioxidant capacity was determined according
[29]
[
19]
hBChE (ID: 5NN0)
were retrieved from the Protein Data Bank
to the CUPRAC method.
In each well, to the reaction mixture
(
https://www.rcsb.org), and prepared using the LeadIT 2.1.8 package
containing 40 μl of sample solution and 50 μl of a copper(II) chloride
solution, 50 μl of neocuproine alcoholic solution, and 60 μl of
ammonium acetate aqueous buffer at pH 7 was added to give a
final volume of 200 µl. After 30 min, the absorbance was measured at
450 nm. Results were recorded as absorbance compared with the
absorbance of BHA and BHT, which were used as antioxidant
standards.
(
www.biosolveit.com). For each enzyme, only the catalytic chain A
was kept. Then, their active sites were defined by selecting all
residues within a radius of 6.5 Å around the inhibitors in the crystal
structures. Thereafter, the protonation state and the side chain
orientations of their active site residues were inspected. On the
contrary, the 3D structures of the designed compounds were drawn,
minimized, and exported as mol2 files using Chem3D 16.0 (http://
www.cambridgesoft.com).
4
.4.4 | Antioxidant capacity by the β‐carotene
Molecular docking calculations of the designed compounds into
the active sites of AChE and BChE were performed using FlexX
bleaching assay
[
26]
2.1.8,
which is based on an incremental construction of ligands.
The β‐carotene bleaching activity of our synthetic compounds was
The selection of the base fragments was set to automatic mode and
the fragment placement used the standard algorithm. FlexX scoring
function, which gives scores as ΔG in kJ/mol, was used for ranking of
molecules.
evaluated using the β‐carotene‐linoleic acid system described by
[30]
Marco.
Thus, a solution of β‐carotene (0.5 mg) in 1 ml of chloro-
form was added to 25 μl of linoleic acid and 200 µl of Tween 40. After
evaporation in vacuum of the chloroform, 50 ml of hydrogen
2 2
peroxide (H O ) was added under vigorous agitation. The absorbance
of the solution was adjusted to 0.8–09 nm. Amounts of 160 µl of this
solution were added to 40 µl of solution of the studied synthetic
compounds at different concentrations. The absorbance was mea-
sured at 470 nm using a 96‐well microplate reader. The emulsion
system was incubated for 2 hr at 50°C. A blank, devoid of β‐carotene,
was prepared for background subtraction. BHA and BHT were used
as standards.
4
.4
|
Method for antioxidant activity assays
4
.4.1 | Superoxide radical scavenging assay
Superoxide was generated according to the alkaline DMSO
[
27]
method,
the reduction of NBT by superoxide was determined
in the presence and absence of extracts. The reaction mixture
containing NBT (1 mg/ml solution in distilled water) and 40 μl of
sample at various concentrations was added to 130 μl alkaline
DMSO (1.0 ml DMSO containing 5 mM NaOH in 0.1 ml water) to
give a final volume of 200 µl and the absorbance was measured at
The bleaching rate (R) of β‐carotene was calculated according to
the following equation:
a
lnb
R =
,
5
60 nm using a microplate reader. The decrease in the absor-
t
bance at 560 nm with antioxidants indicated the consumption of
the generated superoxide. The experiment was repeated in
triplicate and the percentage scavenging was calculated by using
the formula:
where ln is the natural log, a is the absorbance at time zero, and b is
the absorbance at time t (120 min).
The AA was calculated in terms of percent of inhibition relative to
the control, using the following equation:
Asample − Acontrol
Inhibition (%) =
× 100.
R
sample − Rcontrol
Asample
AA (%) =
× 100.
R
sample
4
.4.2 | Reducing power assay
4
.5 Statistical analysis
|
The reducing power of the tested compounds was determined
[28]
All data on AA test were the average of triplicate analyses. Data were
recorded as the mean ± standard deviation. Significant differences
between means were determined using Student’s t test; p < 0.05
were regarded as significant.
according to the previously described method.
To determine the
reducing power activity, 10 µl of serial diluted sample were added
into a 96‐well round‐bottom plate. Following this, 40 μl of 0.2 M
phosphate buffer (pH 6.6) and 50 μl of potassium ferricyanide (1%)
were added to each well and the plate was incubated at 50°C for
2
1
0 min. Finally, 50 μl of TCA (10%) and distilled water (40 μl) and
0 μl of ferric chloride (0.1%) were added into each well to measure
4
.6 Method for antibacterial activity assay
|
the reducing power activity. Afterward, the absorbance was
measured in a microplate reader at 700 nm. Higher absorbance of
the reaction mixture indicates greater reducing power.
The antibacterial activity of the selected compounds (7a, 7e, 7i, 7k,
7n, 7r) was determined by using the disc‐diffusion assay. Agar culture
[31]
plates were prepared as previously described.
Each bacterial

10 of 10
BOUALIA ET AL.
|
strain was grown on Tryptic Soy Agar plates and incubated for 2 days
at 37°C. The turbidity of each bacterial broth culture was adjusted to
equal that of the 0.5 McFarland standard and then the broth cultures
adjusted were separately inoculated on Mueller Hinton Agar plates
using cotton swabs. Thirty to thirty‐five milligrams of each compound
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(
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6
1
00,000 ppm. Thirteen microliters of each solution was applied to
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sterile filter paper discs (6 mm in diameter; Glass Microfiber filters,
®
Whatman ). Standard antibiotic disks (tetracycline [30 µg], erythro-
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the compounds were dissolved in DMSO. Inoculated plates with discs
were incubated at 37°C for 24 hr. After incubation, the inhibition
zone diameter (mm) was measured. Three independent experiments
were performed at different times.
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ACKNOWLEDGEMENT
The authors gratefully acknowledge le Ministère de l’Enseignement
Supérieur et de la Recherche Scientifique (Algeria) for financial
support of this research program.
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How to cite this article: Boualia I, Derabli C, Boulcina R, et al.
Synthesis, molecular docking studies, and biological
evaluation of novel alkyl bis(4‐amino‐5‐cyanopyrimidine)
derivatives. Arch Pharm Chem Life Sci. 2019;e1900027.
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