Rational design and synthesis of dihydropyrimidine based dual binding site acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2016.10.002

Based on the pharmacological importance of dihydropyrimidine (DHPM) scaffold, substituted DHPMs linked with acetamide linker to substituted aromatic anilines were synthesized and evaluated for their potency as acetylcholinesterase (AChE) and butyrylcholin

Full text of DOI:10.1016/j.bioorg.2016.10.002

Accepted Manuscript
Rational design and synthesis of dihydropyrimidine based dual binding site
acetylcholinesterase inhibitors
Suffyan Ahmad, Fatima Iftikhar, Farhat Ullah, Abdul Sadiq, Umer Rashid
PII:
S0045-2068(16)30189-4
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.10.002
YBIOO 1957
Reference:
Bioorganic Chemistry
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8 October 2016
Please cite this article as: S. Ahmad, F. Iftikhar, F. Ullah, A. Sadiq, U. Rashid, Rational design and synthesis of
dihydropyrimidine based dual binding site acetylcholinesterase inhibitors, Bioorganic Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bioorg.2016.10.002
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Rational design and synthesis of dihydropyrimidine based dual binding site
acetylcholinesterase inhibitors
Suffyan Ahmad 1, Fatima Iftikhar 1, Farhat Ullah 2, Abdul Sadiq 2, Umer Rashid 1,3, *
1 Department of Chemistry, Hazara University, Mansehra 21120, Pakistan
2 Department of Pharmacy, University of Malakand, Chakdara 18000 Dir (L), Pakistan
3 Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
*Corresponding author. E-mail: umerrashid@ciit.net.pk; umer_rashid39@hotmail.com
1

Abstract
Based on the pharmacological importance of dihydropyrimidine (DHPM) scaffold, substituted DHPMs
linked with acetamide linker to substituted aromatic anilines were synthesized and evaluated for their
potency as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The good AChE
inhibitory activity of 4-dihydropyrimidine-2-thione (4a-h) and 2-amino-1,4-dihyropyrimidines (5a-h)
series was observed with compound 4a and 4d identified as the most potent compounds with IC50 values of
0.17±0.01 and 0.39±0.04 µM respectively. The inhibition of BChE was found in a broader range of
concentrations (2.37-56.32 µM). To explore the binding insights into the enzyme, molecular docking study
was carried out using GOLD software. The binding mode analysis indicated that all of these inhibitors are
well accommodated in the active site and interact with the key amino acid residues of Catalytic anionic site
(CAS) and peripheral anionic site (PAS). Furthermore, in silico ADMET predictions suggest that these
compounds are non-AMES toxic with good blood brain barrier (BBB) penetration, human intestinal
absorption.
Key words: Acetylcholinesterase, butyrylcholinesterase, dihydropyrimidines, Blood brain barrier, Dual
binding site
2

INTRODUCTION:
Peter Davies in 1976, proposed the cholinergic hypothesis by showing a connection between memory loss
and choline deficiency in brain of Alzheimer's disease (AD) patients. According to this hypothesis
disturbance of cholinergic system of the brain results in the gradual loss of cholinergic neurotransmission
which ultimately leads to neuronal apoptosis i.e. neuronal death. This disturbance may be caused by any
one of these reasons, either a decreased production of ACh or abnormal acetylcholinesterase (AChE)
activity. The cholinergic hypothesis led to the emergence of several strategies for the cholinergic
enhancement. Cholinergic pharmacotherapies are considered as the most consistent strategies among them.
These pharmacotherapies enhance the synaptic level of ACh by inhibiting cholinesterase (ChE) [1-3].
Cholinesterases (ChE) are a group of enzymes that catalyze the hydrolysis of ACh into choline and acetic
acid. Two isoforms of ChE, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are the
important targets for the treatment of AD. Acetylcholinesterase (AChE, EC 3.1.1.7), found in the blood and
neural synapses, specifically hydrolyses ACh. It has proven to be the most achievable target for the
medicinal chemists to design the therapeutics for the treatment of AD. The option for the treatment of the
AD are limited and mostly rely on the symptomatic treatment by using FDA approved cholinergic
pharmacotherapies such as tacrine, donepezil, rivastigmine and galathamine. In the last few years, there are
a steady stream of investigational drugs but unfortunately accompanied by high number of halted outcomes
[1, 4-5].
Dual AChE inhibitors have drawn the attention of many researchers now a days, as they are apparently,
able to address the cholinergic and non-cholinergic targets by fitting into Catalytic anionic site (CAS) and
peripheral anionic site (PAS) simultaneously. The successful strategy to design a dual binding site inhibitor
is to join the two molecules with a spacer (rigid or flexible) that can bind simultaneously with CAS and
PAS. The structures of dual binding site AChE inhibitors i.e. Donepezil and bis-tacrine are shown in
Figure 1. The orientation of donepezil in the binding site of AChE is the example of dual binding site
inhibitor. 3D structure of donepezil-AChE complex showed that it interacts with Trp84 in CAS and Trp279
in PAS [6]. Another successful example of the dual binding site inhibitor is heptylene-linked tacrine dimer
known as bis(7)-tacrine which is 149-fold more potent as AChE inhibitor than tacrine [7]. Recently, many
3

dual binding site AChEIs have been investigated that showed promising anti-cholinesterase inhibitory
activities with IC50 values in nano-molar range. Apart from tacrine-donepezil hybrids, a large variety of
classes of compounds like coumarin derivatives linked to benzyl pyridinium group, 8-imino-2-oxo-2H, 8H-
pyrano[2,3-f]chromene analogs and triazole-quinolines have been recently reported in literature as
promising dual binding site AChEIs [8-10]. Similarly, there are a number of drugs targeting both AChE
and BuChE, but some are more selectivity than others [11-13]. Dihydropyrimidines are the most attractive
derivatives of pyrimidines for a medicinal chemist. There are a number of factors that make this scaffold
pharmacologically important. One of the most important factor is the presence of multiple diversity points
where structural modifications can be incorporated. Valasani et al identified potent 2H-thiazolo[3,2-
a]pyrimidines and 5H-thiazolo[3,2-a]pyrimidines as dual binding site AChE inhibitors in a structure based
virtual screening experiment [14]. Canto et al reported potent AChE inhibitors by merging organoselenium
moiety at C-6 position of DHPMs [15]. Elumalai et al. reported a novel series of pyrazinamide condensed
1,2,3,4-tetrahydropyrimidines AChE and BChE inhibitors by structural modification at C-5 position of the
scaffold [16].
heptamethylene linker
O
HN
N
NH
N
N
O
O
Donepezil
bis(7)-tacrine
Figure 1: Chemical structures of dual binding site AChE inhibitors: Donepezil and bis-tacrine.
We have been working to uncover new structural paradigms of DHPM scaffold as potential drug candidates
to combat various diseases such as leishmaniasis, bacterial infections, urease related problems and cancer
[17-21]. Inspired by the multiple diversity points present on DHPMs scaffold, we directed our attention to
design and synthesize potent compounds having DHPM framework as dual binding site AChE inhibitors.
We have been interested to join the two molecules with a spacer that can bind simultaneously with CAS
4

and PAS. Herein, we report synthesis and biological evaluation of potent DHPM scaffold linked to aryl
ring through acetamide spacer (Figure 2).
R
O
H3C
N
R1
H
N
H3C N X
H
O
B
C
A
Ring
Ring
Spacer
Figure 2: The general structure of the designed DHPM derivatives
2.0. Results and Discussion
2.1. Starting point of our research: Search for AChE inhibitors from our data base of our
synthesized DHPMs.
To begin with a small data base of our synthesized DHPMs, docking was carried out using GOLD docking
program 5.4.1 [22]. The X-ray crystallographic structure AChE in complex with donepezil (PDB Code
1EVE) was used as enzyme structure. Based on the insights gained from the molecular docking, GOLD
fitness score, interaction with the key amino acid residues in CAS/PAS and derivatization feasibility, four
3,4-dihydropyrimidine-2-thiones (1a-e) and four 2-amino-1,4-dihyropyrimidines (2a-e) were considered
for synthesis and biological evaluation. These compounds were obtained through one pot Biginelli reaction
as reported earlier by our research group [17-19, 21]. The inhibitory potencies of these compounds were
determined and enlisted in Table 1. Analogues of 3,4-dihydropyrimidine-2-thiones (1a-e) showed AChE
5

inhibition with IC50 vales ranging between 28.1 and 87.6 µM. While, derivatives of 2-amino-1,4-
dihyropyrimidines (2a-e) exhibited potency between 39.5 and 68.3 µM. Top scored docking poses of
compounds 1c and 2a illustrated in Figure 3 showed that substituted phenyl rings at 4-position of the
pyrimidine ring are engaged in the π-π stacking interactions with Trp84 in CAS. Another π-π stacking
interactions were formed between benzyloxy ring and Phe330. Compound 1c-1EVE complex is further
stabilized by the formation of conventional hydrogen bond (HB) and sulfur atom at a distance of 2.2 Å and
π-S interactions. Compound 2a establishes interactions with Asp72, Ser81 and Tyr334.
Encouraged by these results, we have designed a molecular framework of our new AChE inhibitors with a
round of molecular modeling studies and analysis of GOLD fitness scores. We applied medicinal chemistry
chain extension approach to identify the new compounds for dual inhibition of AChE binding sites (Figure
2).
Table 1: Starting point of our research: In vitro AChE inhibition activity of dihydropyrimidines (1a-2e)
R
R
O
O
H3C
H3C
N
H3C
H3C
NH
N
H
NH2
N
S
H
2a-e
1a-e
Compound
R
Interacting Residues
Fitness score
GOLD
IC50 (µM) ±SEM
H
Trp84
Ser81, Trp84, Ser122
49.2215
55.5657
60.0495
55.4676
55.2981
58.2436
54.7724
59.6644
58.5540
53.9803
87.6±3.67
53.2±2.08
28.1±1.5
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
4-Br
4-(OCH2Ph)
4-CH3
4-Cl
Trp84, Phe288, Phe330, Phe331, Tyr334
Ser81, Trp84,Tyr334
55.9±1.96
59.9±2.19
41.3±1.36
58.0±2.06
39.5±1.65
41.9±2.10
68.3±3.36
Trp84,Tyr121, Ser122
H
Asp72, Ser81, Trp84, Tyr334
Ser81, Trp84, Glu 199, His440
Asp72, Ser81, Trp84, His440
Trp84, Ser81, Asp72
4-Br
4-(OCH2Ph)
4-CH3
4-Cl
Trp84, Ser81, Asp72
6

Donepzil (Standard drug)
Galantamine
87.1728
70.6802
0.03±0.01
4.0±0.10
Figure 3: Detailed molecular interactions of a) Compound 1c b) 2a showing interactions with Trp84 and Trp279.
2.2. Synthesis of the best predicted dihydropyrimidine derivatives
3,4-dihydropyrimidine-2-thiones (1a-d) and 2-amino-1,4-dihyropyrimidines (2a-d) were obtained through
one pot Biginelli reaction as reported earlier by our research group [18, 21]. Synthesis of N-substituted aryl
chloro acetamides (3a-d) were carried out by the reaction of substituted aniline with freshly distilled
chloroacetyl chloride at 0-5 oC as shown in Scheme 1.
O
NH2
O
HN
a
Cl
+
Cl
R1
Cl
R1
3a-c
3a) R1=H, 3b) R1=4-Cl, 3c) R1=4-CH3
Scheme 1: Synthesis of N-substituted aryl chloro acetamides. Reagents: (a) C6H6, 0-5 oC.
Finally Synthesis of N-substituted aryl chloro acetamide derivatives were carried out by the reaction of N-
substituted aryl chloro acetamides with 3,4-dihydropyrimidine-2-thiones (1a-d, Scheme 2) and 2-amino-
1,4-dihyropyrimidines (2a-d, Scheme 3). All the compounds were characterized by 1H NMR and LCMS.
7

R
R
O
O
O
HN
H3C
H3C
N
a
H
Cl
+
H3C
H3C
NH
N
N
S
H
N
S
O
R1
3a-c
R1
H
4a-h
1a-e
Scheme 2: Synthesis of N-substituted arylchloro acetamide derivatives from thiopyrimidines. Reagents: (a) DMF, USI,
40 oC.
R
R
O
O
O
HN
H3C
H3C
N
a
H
Cl
+
H3C
H3C
N
N
N
N
H
H
N
H
NH2
R1
3a-c
O
R1
5a-h
2a-e
Scheme 3: Synthesis of N-substituted arylchloro acetamide derivatives from 2-aminipyrimidines. Reagents: (a) DMF,
USI, 40 oC.
1
All the synthesized derivatives were characterized by melting points, H NMR and LC-MS. Purity of the
1
compounds were checked by thin layer chromatography. In H NMR spectrum (for 4a), two broad singlet
signals at 2.25 and 2.43 ppm designates the methyl proton at C-6 position and acetyl protons at C-5
position respectively. Similarly, a singlet with two proton integration, appears at 3.64 ppm, designates the
methylene group connected to sulfur atom (S-CH2). A broad singlet signal at 8.59 ppm designates the
amidic N-H protons. The aromatic protons appeared in the range of 7.13 and 7.52 ppm as multiplets. In the
1H NMR spectrum of 2-aminopyrimidines (5a-h), a doublet (J=5.1 Hz) observed with two proton
integration at 4.37 ppm designates to the methylene connecting amino group of DHPM ring. A triplet at
5.66 designates to NH proton. In LC-MS spectrum, a peak present at 380.2 confirms the protonated
molecular ion [M+H]+.
2.3. Acetylcholine inhibition assay
8

The synthesized compounds were assayed for AChE (Electrophorus electricus) and BChE inhibition
(equine serum) by using Ellman’s method [23]. The in vitro results (IC50 values) and selectivity index for
AChE and BChE inhibition are summarized in Table 2. Except 4c and 4h, all the 3,4-dihydropyrimidine-2-
thione derivatives showed good to moderate activity better than the standard drug galantamine (IC50
=
4.0±0.10 µM). Compound 4a was identified as the most potent ChE inhibitor exhibiting 14-fold selectivity
toward AChE (IC50 values for AChE 0.17±0.01 µM and for BChE 2.37±0.51 µM). Compound 4d
containing benzyloxy phenyl ring (Ring A, Figure 2) is also found to be more selective against AChE with
IC50 value of 0.39±0.04 µM (BChE IC50=5.69±2.19 µM, S.I=15). In contrast, presence of halogens
(Compounds 4e and 4f) on pyrimidine ring and methyl group on the second aromatic ring (C) resulted in
moderate AChE inhibition; however, these compounds showed very low BChE inhibitory effect (BChE
IC50=52.82±5.03 µM, and 49.95±7.19 µM, S.I=38 and 45 respectively). Compounds 4g also exhibited
moderate AChE activity with IC50 values of 2.01 µM. However, 4g exhibited very weak BChE inhibition
with IC50 value of 30 µM (S.I.=15). Substitution of methyl group at 4-positon of both the rings was found
to contribute least potent BChE inhibition with IC50 value 56.32 µM.
Amongst the 2-amino-1,4-dihyropyrimidine series (5a-h), 4-benzoxypehny derivative 5d is the most
potent and selective AChE inhibitor with IC50 value of 1.07±0.04 µM (S.I.= 17). With respect to
substitution pattern on aniline ring (C), 4-chloro substituted derivative 5b showed moderate AChE activity
with IC50 of 2.01±0.18 µM. Halogen substituted ring (A) also offers moderate inhibition. In contrast to
AChE inhibition, the IC50 value for BChE inhibition of the compounds of this series is found in a broader
range of concentrations (15.01-38.53 µM). Compound 5a is emerged as the most potent BChE inhibitor of
this series with the IC50 of 15.01 µM.
9

Table 2: Acetylcholinesterase (AChE) inhibitory activities, hydrophobicity and Gold fitness score of
synthesized compounds 4a-h and 5a-g.
R
O
H3C
H3C
N
H
N
N
X
H
O
R1
IC50 (µM ±SEM)
R1
X
Selectivity
Index b
R
GOLD
Compound
Fitness score a
eeAChE
eqBChE
H
H
H
4-Cl
H
S
S
83.0680
75.4698
70.7781
82.8694
81.0397
79.1115
79.0895
69.4923
71.0013
78.6381
67.1571
80.1001
72.4221
74.3088
70.0321
68.9843
2.37±0.51
16.41±1.06
24.54±3.19
5.69±2.19
52.82±5.03
49.95±7.19
30.71±3.7
56.32±0.35
15.01±0.98
27.21±2.44
38.53±3.35
18.37±1.10
20.89±2.51
26.41±1.96
16.75±1.17
29.41±2.20
5.4±0.27
14
8.3
3.0
15
4a
0.17±0.01
1.97±0.16
8.23±0.2
4b
4-Br
4-OBn
4-Br
4-Cl
4-Br
4-CH3
H
S
4c
H
S
4d
0.39±0.04
1.39±0.12
1.11±0.10
2.01±0.17
7.63±0.67
5.33±0.46
2.01±0.18
12.91±1.10
1.07±0.04
3.13±0.26
3.08±0.29
4.96±0.43
7.87±0.70
0.03±0.01
4.0±0.10
4-CH3
4-CH3
4- Cl
4-CH3
H
S
38
4e
S
45
4f
S
15
4g
S
7.4
2.8
13.5
3.0
17
4h
NH
NH
NH
NH
NH
NH
NH
NH
5a
H
4-Cl
H
5b
4-Br
4-OBn
4-Br
4-Cl
4-Br
4-CH3
5c
H
5d
4-CH3
4-CH3
4- Cl
4-CH3
7
5e
8.6
3.4
3.7
180
3.7
5f
5g
5h
Donepezil
Galantamine
15.0±0.67
a Gold fitness score of compounds towards the AChE (PDB Code 1EVE)
10

b Selectivity Index = IC50 ratio (BChE/AChE).
2.3. Computational Studies
Computational docking studies were carried out using GOLD (Genetic Optimization for Ligand Docking)
suit v5.4.1. The X-ray crystallographic structure AChE in complex with donepezil (PDB Code 1EVE) was
used as enzyme structure. Visual inspection of 3D docked binding orientations of all inhibitors within the
active site of AChE (1EVE) resulted in the following observations: All of these inhibitors are well
stabilized in the active site and interact with the key amino acid residues of CAS and PAS. With few
exceptions, the binding mode of best-scored inhibitors further suggests that the 4-arylpyrimidine moiety
(Ring A) is caged into the CAS, while, substituted aniline ring (Ring C) is embedded into PAS. The
superimposed binding modes of 4a (dark brown), 4c (purple), 4d (light green) and donepezil (magenta) are
shown in Figure 4a. In the PAS region, the most active compound 4a displays π-π stacking with the indole
ring of Trp279. Carbonyl oxygen of acetamide moiety forms two conventional hydrogen bonds (HB) with
Gly118 and Gly119. At the bottom of the gorge, phenyl ring (C) is engaged in the π-π stacking interaction
with the six membered ring of Trp84. Similar stacking interactions are also observed with His440. It is
important to note that sulfur atom also interacts with the aromatic rings of Tyr121 and Phe331 via π-sulfur
bonds (Figure 4b). The highest Gold fitness score of 4a (83.0680) towards the AChE may therefore
ascribed to these HB and π-π stacking interactions. Benzyloxy phenyl derivative 4d with Gold fitness score
of 82.8694 forms several π-π stacking and HB interactions (Figure 4c). Trp279 and Trp84 forms π-π
stacking interactions with second aromatic ring (C) and benzyloxy group on Ring A respectively. Phe300
forms π-π stacking interactions with Ring A. Pocket defined by Phe288 and Phe331 also establishes two
types of interactions with sulfur atom: one HB interactions with Phe288 and another π-sulfur interactions.
11

Figure 4: a) superimposed binding modes of 4a (dark brown), 4c (purple), 4d (light green) and donepezil (magenta); b)
3D Detailed molecular interactions of 4a in the binding pocket of 1EVE; c) 3D binding mode and interactions of
compound 4d.
Compound 4c with IC50 value of 8.23±0.2 µM showed many fold decrease in AChE inhibition than
unsubstituted 4a. The binding pose and interactions of the compound 4c are shown in Figure 5. Apart from
two classical π-π stacking interactions with Trp279 and Trp84, a halogen (Br in this case) ….O (of Tyr70)
was also formed. However, the low activity and interactions are also revealed by Gold fitness score value
of 70.7781 (Table 2).
12

Figure 5: Detailed molecular interactions 3D (a) and 2D (b) of 4c in the binding pocket of 1EVE
Binding mode analysis of 2-amino-1,4-dihyropyrimidine derivatives 5a-h were also carried out. 4-
Benzyloxy phenyl derivative 5d is emerged as the key compound of this series with IC50 value of
1.07±0.04 µM. As shown in Figure 6a, Trp84 and Gly117 forms π-π stacking interactions with aromatic
ring C. His440 forms conventional HB with the carbonyl oxygen of acetamide. Another conventional HB
was established by Phe288 present in the acyl pocket and carbonyl oxygen of 5-acetyl group on pyrimidine
ring. Benzyloxy and phenyl rings on pyrimidine ring make favorable π-π stacking interactions with Trp279.
Examination of 5b, another best ranked compound of this series, revealed that Asp72 and Tyr70 are
involved in the convention HB interactions with 2-aminopyrimidine and acetamide N-atoms (Figure 6b).
Figure 6: Detailed molecular 3D interactions of a) 5d; b) 5b in the binding pocket of 1EVE
13

A common binding feature of all the active compounds is their interaction with the residues of CAS and
PAS via π-π stacking interactions. Molecular modelling studies on the literature compounds having ChE
activity in nanomolar range established that anchoring aromatic rings with optimum length of linker is
essential to block the dual binding sites. Our present study reveals the ability of DHPM scaffold linked to
aryl ring through acetamide spacer to act as ChE inhibitors. We believe that these compounds deserve
further structural modifications for the development of novel ChE inhibitors.
2.4. Preliminary in silico pharmacokinetics
In silico ADMET predictions are practicable alternatives in drug discovery process since the in vivo and in
vitro experimental evaluations are laborious and costly. We employed here computational ADMET
(Absorption, Distribution, Metabolism, Excretion and Toxicity) predictions. LogP was calculated by using
ChemAxon software package (Marvin v15.4.6, 2015). It can be seen from Table 3 that lipophilicity of
compounds 4d and 5d increases by introduction of benzyl group. Addition of bromo group also increased
the lipophilicity. For targeting central nervous system, BBB penetration is a challenge. Therefore, the vital
condition for the efficient drug for AChE inhibition is the high BBB penetration. LogP values of all the
synthesize compounds were determined using ChemAxone software and are listed in Table 3. For BBB
penetration, online software was used to predict whether these compounds can cross blood brain barrier
(BBB+) or not (BBB-). Compounds having SVM_MACCSFP BBB Score greater than 0.02 were predicted
to cross the BBB. High BBB score of dihydropyrimidines-2-thiones (4a-h) indicated that these are BBB
penetrating agents. However with few exceptions, 2-amino-1,4-dihydropyrimidines (5a-h) are also
predicted to show good BBB penetration. Human Intestinal Absorption (HIA), Mutagenic potential of the
synthesized compounds (AMES test) and rodent carcinogenicity were computed using admetSAR, a web
base application. A positive HIA test indicates that all these compounds have good human intestinal
absorption. The negative predicted AMES test indicates that all the synthesized compounds are non-AMES
toxic and thus non-mutagenic.
14

Table 3: Predicted ADMET properties of compounds
Compound
Log P
3.56
4.08
4.36
4.94
4.82
4.55
4.87
4.50
3.56
3.42
3.70
4.43
4.16
3.89
4.21
3.84
BBB score
0.069
0.052
0.053
0.029
0.040
0.039
0.051
0.056
0.030
0.022
0.021
-0.004
0.011
0.012
0.022
0.020
HIA
+
AMES Toxicity
Carcinogens
-
a
4a
4b
4c
4d
4e
4f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
4g
4h
5a
5b
5c
5d
5e
5f
+
+
+
+
+
+
+
+
+
5g
+
5h
a Non-AMES toxic
3.0. Conclusion
In conclusion, rational design of dihydropyrimidines led to the discovery of potent and selective AChE
inhibitors. Some of them showed the inhibition in sub-micromolar range. Inhibitors containing thio group
at 2-position of pyrimidine ring exhibited good in vitro AChE inhibition than 2-amino group. High BBB
score of dihydropyrimidines-2-thiones (4a-h) indicated that these are BBB penetrating agents. However
with few exceptions, 2-amino-1,4-dihydropyrimidines are also predicted to show good BBB penetration.
Moreover, inhibitory effect towards BChE was also determined. The inhibition was found in a broader
15

range of concentrations (2.37-56.32 µM). Molecular modeling studies revealed that we are successful in
designing dual binding site AChE inhibitors from starting compounds (i.e. simple DHPMs) through
structural modification. The dual binding site inhibition of all the compounds suggested that these
compounds may be the promising candidates for the treatment of Alzheimer’s disease.
4.0. Experimental
Chemicals/solvents were purchased from vendors and were used without any further purification. 1H and
13C NMR spectra were recorded on a 300 MHz and 75 MHz Bruker spectrometer at using TMS as internal
standard and DMSO-d6 as solvent. Mass spectra were recorded on Agilent technologies 6890N. Gas
chromatography (GC) and an inert mass selective detector 5973 GC-mass spectrometer. Sonication was
performed in Elma E 30 H and E 60 H (Germany) ultrasonic cleaner with a nominal power of 250 W and
500W respectively [18].
4.1. Synthesis of Dihydropyrimidines-2-thiones (1a-e)
Dihydropyrimidines-2-thiones were synthesized by using three component Biginelli reaction. Aldehydes,
acetylacetone and thiourea were reacted in a test tube under ultrasonic irradiation by using SnCl2 as catalyst
and acetonitrile (ACN) as solvent [18].
4.1.1. 5-Aceto-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-thione (1a)
1a was prepared according to the general procedure by using benzaldehyde, thiourea, acetylacetone. Yield
69%. m.p. 183-185 ºC [18]. 1H NMR (300 MHz, DMSO-d6): δ 2.20 (s, 3H, CH3), 2.29 (s, 3H, CH3CO),
5.10 (d, 1H, J=3.3 Hz, CH), 7.0 (m, 5H, Ar-H), 8.87 (br s, 1H, NH), 10.00 (br s, 1H, NH). EIMS calculated
for C13H14N2OS (M+•) 246.
4.1.2. 1-(4-(4-bromophenyl)-6-methyl-2-thioxo-3,4-tetrahydropyrimidin-5-yl)ethanone (1b)
1b was prepared according to the general procedures by using 4-bromobenzaldehyde, thiourea,
1
acetylacetone. Yield 81%. 174-176ºC. H NMR (300 MHz, DMSO-d6): δ 2.20 (s, 3H, CH3), 2.34 (s, 3H,
CH3CO), 5.11 (d, 1H, J=3.0 Hz, CH), 7.06 (d, 2H, J=8.4 Hz, Ar-H), 7.43 (d, 2H, J=8.4 Hz, Ar-H), 9.00 (br
s, 1H, NH), 10.10 (br s, 1H, NH); EIMS calculated for C13H13BrN2OS (M+•) 324.2.
4.1.3. 1-(4-(4-(benzyloxy)phenyl)-6-methyl-2-thioxo-3,4-tetrahydropyrimidin-5-yl)ethanone (1c)
1c was prepared according to the general procedure by using 4-benzyloxybenzaldehyde, thiourea,
1
acetylacetone. m.p. 208-210 ºC. H NMR (300 MHz, DMSO-d6): δ 2.23 (s, 3H, CH3), 2.37 (s, 3H,
16

CH3CO), 5.09 (d, 1H, J=3.0 Hz, CH), 5.26 (s, 2H, OCH2), 7.16 (m, 9H, Ar-H), 9.11 (br s, 1H, NH), 10.08
(br s, 1H, NH); EIMS calculated for C20H20N2O2S(M+•) 352.
4.1.4. 1-(6-methyl-2-thioxo-4-p-tolyl-3,4-tetrahydropyrimidin-5-yl)ethanone (1d)
1d was prepared according to the general procedure by using 4-methylbenzaldehyde, thiourea,
acetylacetone. Yield 78%; m.p. 179-181 ºC. 1H NMR (300 MHz, DMSO-d6): δ 2.17 (s, 3H, CH3), 2.39 (s,
6H, CH3), 5.10 (d, 1H, J=3.3 Hz, CH), 7.06 (m, 4H, Ar-H), 9.05 (bs, 1H, NH), 10.08 (br s, 1H, NH); EIMS
calculated for C14H16N2OS (M+•) 260.
4.1.5. 1-(4-(4-chlorophenyl)-6-methyl-2-thioxo-3,4-tetrahydropyrimidin-5-yl)ethanone (1e)
2e was prepared according to the general procedure by using 4-chlorobenzaldehyde, thiourea,
1
acetylacetone. m.p. 162-164 °C. H NMR (300 MHz, DMSO-d6): δ 2.16 (s, 3H, CH3), 2.31 (s, 3H, CH3),
5.08 (d, 1H, J=3.3 Hz, CH), 7.04 (d, 2H, J=8.4 Hz, Ar-H), 7.28 (d, 2H, J=8.4 Hz, Ar-H), 9.11 (bs, 1H,
NH), 9.98 (br s, 1H, NH); EIMS calculated for C13H13ClN2OS (M+•) 280.
4.2. Synthesis of 2-amino-1,4-dihydropyrimidines (2a-e)
A mixture of an aldehyde (10 mmol), guanidine hydrochloride (12 mmol), acetylacetone (10 mmol) and
sodium bicarbonate (3.35 gm, 40 mmol), in DMF (20 mL) was taken in a Pyrex tube and irradiated in an
o
ultrasonic bath at 60-70 C. The reaction was monitored by TLC. After cooling, the mixture was poured
onto crushed ice. The precipitate was filtered and thoroughly washed water. The crude product was
recrystallized from an appropriate solvent or purified with silica gel column chromatography. The purity of
compounds was checked by TLC in different solvent systems. All the products were confirmed by
comparing their melting points, elemental analysis, MS and 1HNMR data [15].
4.2.1. 1-(2-amino-6-methyl-4-phenyl-1,4-dihydropyrimidin-5-yl)ethanone (2a)
2a was prepared according to the general procedure by using benzaldehyde, guanidine HCl, acetylacetone.
Rf 0.69 (CHCl3/MeOH 5:1). Yield 83%; m.p. 214-216 ºC. 1H NMR (300 MHz, DMSO-d6): δ 2.14 (s, 3H,
CH3), 2.35 (s, 3H, CH3CO), 5.15 (s, 1H, CH), 5.66 (s, 2H, NH2), 7.00 (m, 5H, Ar-H), 9.69 (br s, 1H, NH),;
EIMS calculated for C13H15N3O (M+•) 229.
17

4.2.2. 1-(2-amino-4-(4-bromophenyl)-6-methyl-1,4-dihydropyrimidin-5-yl)ethanone (2b)
2b was prepared according to the general procedures by using 4-bromobenzaldehyde, guanidine HCl,
acetylacetone. Rf 0.49 (CHCl3/MeOH 5:1). Yield 89%. 154-156ºC. 1H NMR (300 MHz, DMSO-d6): δ 2.13
(s, 3H, CH3), 2.31 (s, 3H, CH3CO), 5.19 (s, 1H, CH), 5.73 (s, 2H, NH2), 7.18 (d, 2H, J=8.4 Hz, Ar-H), 7.51
(d, 2H, J=8.4 Hz, Ar-H), 9.10 (br s, 1H, NH). EIMS calculated for C13H14BrN3O (M+•) 308.2.
4.2.3. 1-(2-amino-4-(4-(benzyloxy)phenyl)-6-methyl-1,4-dihydropyrimidin-5-yl)ethanone (2c)
2c was prepared according to the general procedure by using 4-benzyloxybenzaldehyde, guanidine HCl,
1
acetylacetone. Rf 0.48 (CHCl3/MeOH 5:1). m.p. 218-220 ºC. H NMR (300 MHz, DMSO-d6): δ 2.17 (s,
3H, CH3), 2.34 (s, 3H, CH3CO), 5.08 (s, 1H, CH), 5.29 (s, 2H, OCH2), 5.81 (s, 2H, NH2), 7.11 (m, 9H, Ar-
H), 9.29 (br s, 1H, NH). EIMS calculated for C20H21N3O2 (M+•) 335.
4.2.4. 1-(6-methyl-2-thioxo-4-p-tolyl-3,4-tetrahydropyrimidin-5-yl)ethanone (2d)
2d was prepared according to the general procedure by using 4-methylbenzaldehyde, guanidine HCl,
acetylacetone. Rf 0.46 (CHCl3/MeOH 5:1). Yield 70%; m.p. 208-210 °C. 1H NMR (300 MHz, DMSO-d6):
δ 1.17 (s, 3H, CH3), 2.29 (s, 6H, CH3), 5.06 (s, 1H, CH), 6.01 (s, 2H, NH2), 7.01 (m, 4H, Ar-H), 9.17 (bs,
1H, NH). EIMS calculated for C14H17N3O (M+•) 243.
4.2.5. 1-(2-amino-4-(4-chlorophenyl)-6-methyl-1,4-dihydropyrimidin-5-yl)ethanone (2e)
2e was prepared according to the general procedure by using 4-chlorobenzaldehyde, guanidine HCl,
acetylacetone. Rf 0.54 (CHCl3/MeOH 5:1). Yield 77%; m.p. 176-178 °C. 1H NMR (300 MHz, DMSO-d6):
δ 2.16 (s, 3H, CH3), 2.31 (s, 3H, CH3CO), 5.08 (s, 1H, CH), 5.91 (s, 2H, NH2), 7.14 (d, 1H, J=8.4 Hz, Ar-
H), 7.26 (d, 1H, J=8.4 Hz, Ar-H), 9.17 (bs, 1H, NH). EIMS calculated for C13H14N3O (M+•) 263.
4.3. Synthesis of N-substituted aryl chloro acetamide (3a-c)
In a pyrex tube containing 5 ml dry benzene, aniline derivatives (1 mM) was added drop-wise and mixed.
o
Then freshly distilled chloroacetyl chloride was added. The reaction mixture was stirred at 0-5 C. The
reaction was monitored by TLC. After the completion of reaction (TLC), the reaction mixture was poured
into the 20 gm of ice with stirring, neutralized with NaHCO3. The product obtained was filtered thoroughly,
washed and recrystallized with ethanol.
18

4.3.1. Synthesis of 2-chloro-N-phenylacetamide (3a)
o
1
Brown solid; Yield 78%; m.p= 134 C; Rf =0.50 (n-Hexane/ethyl acetate 3 : 1). H NMR (300 MHz,
CDCl3): δ 8.73 (br s, 1H, NHCO), 7.19 (m, 5H, Ar-H), 4.65 (s, 2H, CH2).
4.3.2. Synthesis of 2-chloro-N-(4-chlorophenyl)acetamide (3b)
1
Light brown solid; Yield 83%; m.p= 126 oC; Rf =0.49 (n-Hexane/ethyl acetate 4 : 1). Yield 75%. H NMR
(300 MHz, CDCl3): δ 8.65 (brs, 1H, NHCO), 7.64 (d, 2H, J=7.2 Hz, Ar-H), 7.28 (d, 2H, J=7.2 Hz, Ar-H),
4.64 (s, 2H, CH2).
4.3.4. Synthesis of 2-chloro-N-p-tolylacetamide (3c)
Light yellow solid; Yield 91%; Rf =0.50 (n-Hexane/ethyl acetate 4 : 1). Rf =0.46. Yield 75%. 1H NMR (300
MHz, CDCl3): δ 8.66 (brs, 1H, NHCO), 7.41 (d, 2H, J=7.2 Hz, Ar-H), 6.98 (d, 2H, J=7.2 Hz, Ar-H), 4.64
(s, 2H, CH2), 2.34 (s, 3H, CH3).
4.4. Synthesis of N-substituted aryl chloro acetamide derivatives from thiopyrimidines (4a-h)
5-acetyl-2-thioxodihydropyrimidines (1a-d) was dissolved in DMF in a sealed Pyrex tube. N-substituted
aryl chloro acetamide (3a-d) was then added. The reaction mixture was then irradiated under ultrasonic
bath at 40 oC for 3-4 hr. The reaction was monitored by TLC. After the completion of reaction, the reaction
mixture was poured into the 20 gm of ice with stirring. The product obtained was filtered thoroughly,
washed and recrystallized with ethanol.
4.4.1. 2-(5-acetyl-6-methyl-4-phenyl-1,4-dihydropyrimidin-2-ylthio)-N-phenylacetamide (4a)
4a was prepared according to the general procedure by using phenyl-3,4-dihydropyrimidine-2-thione (1a)
and 2-chloro-N-phenylacetamide (3a). Light yellow crystals; Yield 87%; Rf =0.547 (n-Hexane/ethyl acetate
5 : 1); m.p. 179-181 ºC. 1H NMR (300 MHz, DMSO-d6): δ 2.25 (s, 3H, CH3), 2.43 (s, 3H, CH3CO), 3.64 (s,
2H, S-CH2), 5.08 (s, 1H, CH), 7.13 (m, 8H Ar-H), 7.52 (m, 2H, Ar-H), 8.59 (brs, 1H, NHCO), 9.77 (brs,
1H, pyrimidine-NH); LCMS calculated for C21H21N3O2S= 380.14 [M+H].
4.4.3. 2-((5-acetyl-6-methyl-4-phenyl-1,4-dihydropyrimidin-2-yl)thio)-N-(4-chlorophenyl)acetamide
(4b)
4b was prepared according to the general procedure by using 1-(6-methyl-4-phenyl-2-thioxo-3,4-
tetrahydropyrimidin-5-yl)ethanone (1a) and 2-chloro-N-chlorophenyl)acetamide (3b). Yellow crystals;
1
Yield 79%; Rf =0.51 (n-Hexane/ethyl acetate 5 : 1); m.p. 226-228ºC; H NMR (300 MHz, DMSO-d6): δ
19

2.23 (s, 3H, CH3), 2.39 (s, 3H, CH3CO), 3.65 (s, 2H, S-CH2), 5.14 (s, 1H, CH), 7.10 (m, 5H Ar-H), 7.29 (d,
2H, J=8.1 Hz, Ar-H), 7.61 (d, 2H, J=8.1 Hz, Ar-H), 8.66 (s, 1H, NHCO), 9.74 (brs, 1H, pyrimidine-NH).
LCMS calculated for C21H20ClN3O2S = 414.2 [M+H].
4.4.4.
(4c)
2-(5-acetyl-4-(4-bromophenyl)-6-methyl-1,4-dihydropyrimidin-2-ylthio)-N-phenylacetamide
4c was prepared according to the general procedure by using 4-bromophenyl-3,4-dihydropyrimidine-2-
thione (1b) and 2-chloro-N-acetamide (3a). Yellow crystals; Yield 71%; Rf =0.51 (n-Hexane/ethyl acetate
3 : 1); m.p. 194-196 ºC; 1H NMR (300MHz, DMSO-d6): δ 2.26 (s, 3H, CH3), 2.40 (s, 3H, CH3CO), 3.69 (s,
2H, S-CH2), 5.10 (s, 1H, CH), 7.08 (m, 1H, Ar-H), 7.16 (d, 2H, J=8.1 Hz, Ar-H), 7.28 (m, 2H, Ar-H), 7.43
(d, 2H, J=8.1 Hz, Ar-H), 7.59 (m, 2H, Ar-H), 8.79 (s, 1H, NHCO), 9.75 (brs, 1H, pyrimidine-NH); LCMS
calculated for C21H20BrN3O2S= 458.2 [M+H].
4.4.5.
2-(5-acetyl-4-(4-(benzyloxy)phenyl)-6-methyl-1,4-dihydropyrimidin-2-ylthio)-N-phenyl
acetamide (4d)
4d was prepared according to the general procedure by using 4-benzyloxyphenyl-3,4-dihydropyrimidine-2-
thione (1c) and 2-chloro-N-acetamide (3a). Yellow crystals; Yield 76%; Rf =0.49 (n-Hexane/ethyl acetate 5
: 1); m.p. 197 ºC; 1H NMR (300 MHz, DMSO-d6): δ 2.25 (s, 3H, CH3), 2.41 (s, 3H, CH3CO), 3.68 (s, 2H,
S-CH2), 5.10 (s, 1H, CH), 5.19 (s, 2H, OCH2), 6.85 (m, 4H, Ar-H), 7.09 (m, 1H, Ar-H), 7.25 (m, 7H, Ar-
H), 7.58 (m, 2H, Ar-H), 8.67 (s, 1H, NHCO), 9.74 (brs, 1H, pyrimidine-NH). LCMS calculated for
C28H27N3O3S 486.2 [M+H].
4.4.6.
(4e)
2-(5-acetyl-4-(4-bromophenyl)-6-methyl-1,4-dihydropyrimidin-2-ylthio)-N-p-tolylacetamide
4e was prepared according to the general procedure by using 4-bromophenyl-3,4-dihydropyrimidine-2-
thione (1b) and 2-chloro-N-p-tolylacetamide (3c). Yellow crystals; Yield 69%; Rf =0.45 (n-Hexane/ethyl
acetate 3 : 1); m.p. 211 ºC; 1H NMR (300MHz, DMSO-d6): δ 2.27 (s, 3H, CH3), 2.38 (s, 3H, CH3CO), 2.42
(s, 3H, CH3), 3.71 (s, 2H, S-CH2), 5.09 (s, 1H, CH), 6.95 (d, 2H, J=8.1 Hz, Ar-H), 7.19 (d, 2H, J=8.1 Hz,
Ar-H), 7.44 (d, 2H, J=8.1 Hz, Ar-H), 7.51 (d, 2H, J=8.1 Hz, Ar-H), 8.81 (s, 1H, NHCO), 9.79 (brs, 1H,
pyrimidine-NH); LCMS calculated for C22H22BrN3O2S= 473.2 [M+H].
20

4.4.7. 2-(5-acetyl-4-(4-chlorophenyl)-6-methyl-1,4-dihydropyrimidin-2-ylthio)-N-p-tolylacetamide (4f)
4f was prepared according to the general procedure by using 4-chlorophenyl-3,4-dihydropyrimidine-2-
thione (1e) and 2-chloro-N-p-tolylacetamide (3c). Yellow crystals; Yield 74%; Rf =0.48 (n-Hexane/ethyl
acetate 5 : 1); m.p. 183 ºC; 1H NMR (300MHz, DMSO-d6): δ 2.28 (s, 3H, CH3), 2.32 (s, 3H, CH3CO), 2.40
(s, 3H, CH3), 3.73 (s, 2H, S-CH2), 5.11 (s, 1H, CH), 7.01 (d, 2H, J=8.1 Hz, Ar-H), 7.15 (d, 2H, J=8.1 Hz,
Ar-H), 7.28 (d, 2H, J=8.1 Hz, Ar-H), 7.42 (d, 2H, J=8.1 Hz, Ar-H), 8.66 (s, 1H, NHCO), 9.77 (brs, 1H,
pyrimidine-NH). LCMS calculated for C22H22ClN3O2S= 411.1 [M+H].
4.4.8. 2-(5-acetyl-4-(4-bromophenyl)-6-methyl-1,4-dihydropyrimidin-2-ylamino)-N-(4-chlorophenyl)
acetamide (4g)
4g was prepared according to the general procedure by using 4-bromophenyl-3,4-dihydropyrimidine-2-
thione (1b) and 2-chloro-N-(4-chlorophenyl)acetamide (3b). Light yellow crystals; Yield 91%; Rf =0.49 (n-
Hexane/ethyl acetate 6 : 1); m.p. 155 ºC; 1H NMR (300MHz, DMSO-d6): δ 2.27 (s, 3H, CH3), 2.34 (s, 3H,
CH3CO), 3.76 (s, 2H, S-CH2), 5.07 (s, 1H, CH), 7.12 (d, 2H, J=8.1 Hz, Ar-H), 7.19 (d, 2H, J=8.1 Hz, Ar-
H), 7.53 (d, 2H, J=8.1 Hz, Ar-H), 7.63 (d, 2H, J=8.1 Hz, Ar-H), 8.61 (s, 1H, NHCO), 9.74 (brs, 1H,
pyrimidine-NH),. LCMS calculated for C21H19BrClN3O2S= 492.1 [M+H].
4.4.8. 2-(5-acetyl-6-methyl-4-p-tolyl-1,4-dihydropyrimidin-2-ylthio)-N-p-tolylacetamide (4h)
4h was prepared according to the general procedure by using 1-(6-methyl-2-thioxo-4-p-tolyl-3,4-
tetrahydropyrimidin-5-yl)ethanone (1d) and 2-chloro-N-p-tolylacetamide (3d). Light yellow crystals; Yield
91%; Rf =0.49 (n-Hexane/ethyl acetate 6 : 1); m.p. 155 ºC; 1H NMR (300MHz, DMSO-d6): δ 2.27 (s, 3H,
CH3), 2.33 (s, 3H, CH3CO), 2.41 (s, 6H, CH3), 3.76 (s, 2H, S-CH2), 5.07 (s, 1H, CH), 6.94 (d, 2H, J=7.2
Hz, Ar-H), 7.11 (brs, 4H, Ar-H), 7.40 (d, 2H, J=7.2 Hz, Ar-H), 8.61 (s, 1H, NHCO), 9.74 (brs, 1H,
pyrimidine-NH),. LCMS calculated for C23H25N3O2S= 408.2 [M+H].
4.5. Synthesis of N-substituted aryl chloro acetamide derivatives from 2-aminopyrimidines (5a-h)
2-amino-1,4-dihydropyrimidines (2a-e) was dissolved in DMF in a sealed Pyrex tube. N-substituted aryl
chloro acetamide (3a-c) was then added. The reaction mixture was then irradiated under ultrasonic bath at
o
40 C for 3-4 hr. The reaction was monitored by TLC. After the completion of reaction, the reaction
mixture was poured into the 20 gm of ice with stirring. The product obtained was filtered thoroughly,
washed and recrystallized with ethanol.
21

4.5.1. 2-(5-acetyl-6-methyl-4-phenyl-1,4-dihydropyrimidin-2-ylamino)-N-phenylacetamide (5a)
5a was prepared according to the general procedure by using 1-(2-amino-6-methyl-4-phenyl-1,4-
dihydropyrimidin-5-yl)ethanone (2a) and 2-chloro-N-phenylacetamide (3a). Pale yellow crystals; Yield
1
83%; Rf =0.50 (n-Hexane/ethyl acetate 5 : 1); m.p. 141ºC. H NMR (300 MHz, DMSO-d6): δ 2.29 (s, 3H,
CH3), 2.49 (s, 3H, CH3CO), 4.37 (d, 2H, J=5.1 Hz, NH-CH2), 5.11 (s, 1H, CH), 5.66 (t, 1H, J=5.1 Hz, Ar-
NH), 7.16 (m, 8H Ar-H), 7.54 (m, 2H, Ar-H), 8.80 (brs, 1H, NHCO), 9.85 (brs, 1H, pyrimidine-NH);
LCMS calculated for C21H22N4O2= 363.2 [M+H].
4.5.2. 2-(5-acetyl-6-methyl-4-phenyl-1,4-dihydropyrimidin-2-ylamino)-N-(4-chlorophenyl)acetamide
(5b)
5b was prepared according to the general procedure by using 1-(2-amino-6-methyl-4-phenyl-1,4-
dihydropyrimidin-5-yl)ethanone (2a) and 2-chloro-N-(4-chlorophenyl)acetamide (3b). Pale yellow crystals;
1
Yield 71%; Rf =0.43 (n-Hexane/ethyl acetate 5 : 1); m.p. 177 ºC; H NMR (300 MHz, DMSO-d6): δ 2.20
(s, 3H, CH3), 2.32 (s, 3H, CH3CO), 4.35 (d, 2H, J=5.1 Hz, NH-CH2), 4.98 (s, 1H, CH), 5.60 (t, 1H, J=5.1
Hz, Ar-NH), 7.04 (m, 5H Ar-H), 7.13 (d, 2H, J=8.1 Hz, Ar-H), 7.62 (d, 2H, J=8.1 Hz, Ar-H), 8.63 (s, 1H,
NHCO), 9.84 (brs, 1H, pyrimidine-NH). LCMS calculated for C21H21ClN4O2 = 397.2 [M+H].
4.5.3. 2-(5-acetyl-4-(4-bromophenyl)-6-methyl-1,4-dihydropyrimidin-2-ylamino)-N-phenylacetamide
(5c)
5c was prepared according to the general procedure by using 1-(2-amino-4-(4-bromophenyl)-6-methyl-1,4-
dihydropyrimidin-5-yl)ethanone (2b) and 2-chloro-N-phenylacetamide (3a). Pale yellow crystals; Yield
67%; Rf =0.5 (CHCl3/MeOH 5 : 1); m.p. 207 ºC; 1H NMR (300 MHz, DMSO-d6): δ 2.21 (s, 3H, CH3), 2.33
(s, 3H, CH3CO), 4.37 (d, 2H, J=5.1 Hz, NH-CH2), 4.96 (s, 1H, CH), 5.60 (t, 1H, J=5.1 Hz, Ar-NH), 7.11
(m, 1H, Ar-H), 7.19 (d, 2H, J=8.1 Hz, Ar-H), 7.26 (m, 2H Ar-H), 7.45 (d, 2H, J=8.1 Hz, Ar-H), 7.56 (m,
2H, Ar-H), 8.83 (s, 1H, NHCO), 9.83 (brs, 1H, pyrimidine-NH). LCMS calculated for C21H21BrN4O2 =
441.2 [M+H].
4.5.4.
2-(5-acetyl-4-(4-(benzyloxy)phenyl)-6-methyl-1,4-dihydropyrimidin-2-ylamino)-N-phenyl
acetamide (5d)
5d was prepared according to the general procedure by using 1-(4-(4-(benzyloxy)phenyl)-6-methyl-2-
thioxo-3,4-tetrahydropyrimidin-5-yl)ethanone (2c) and 2-chloro-N-(4-chlorophenyl)acetamide (3c). Pale
22

1
yellow crystals; Yield 70%; Rf =0.48 (n-Hexane/ethyl acetate 5 : 1); m.p. 188 ºC; H NMR (300 MHz,
DMSO-d6): δ 2.19 (s, 3H, CH3), 2.31 (s, 3H, CH3CO), 4.33 (d, 2H, J=5.1 Hz, NH-CH2), 4.93 (s, 1H, CH),
5.17 (s, 2H, OCH2), 5.61 (t, 1H, J=5.1 Hz, Ar-NH), 6.87 (m, 4H, Ar-H), 7.08 (m, 1H, Ar-H), 7.28 (m, 7H,
Ar-H), 7.59 (m, 2H, Ar-H), 8.64 (s, 1H, NHCO), 9.85 (brs, 1H, pyrimidine-NH). LCMS calculated for
C28H28N4O3 = 469.2 [M+H].
4.5.5. 2-(5-acetyl-4-(4-bromophenyl)-6-methyl-1,4-dihydropyrimidin-2-ylamino)-N-p-tolylacetamide
(5e)
5e was prepared according to the general procedure by using 1-(2-amino-4-(4-bromophenyl)-6-methyl-1,4-
dihydropyrimidin-5-yl)ethanone (2c) and 2-chloro-N-p-tolylacetamide (3c). Pale yellow crystals; Yield
1
63%; Rf =0.48 (CHCl3/MeOH 5 : 1); m.p. 211 ºC; H NMR (300 MHz, DMSO-d6): δ 2.18 (s, 3H, CH3),
2.34 (s, 3H, CH3CO), 2.41 (s, 3H, CH3), 4.36 (d, 2H, J=5.1 Hz, NH-CH2), 4.96 (s, 1H, CH), 5.61 (t, 1H,
J=5.1 Hz, Ar-NH), 6.91 (d, 2H, J=8.1 Hz, Ar-H), 7.26 (d, 2H, J=8.1 Hz, Ar-H), 7.44 (d, 2H, J=8.1 Hz, Ar-
H), 7.57 (d, 2H, J=8.1 Hz, Ar-H), 8.83 (s, 1H, NHCO), 9.81 (brs, 1H, pyrimidine-NH); LCMS calculated
for C22H23BrN4O2 = 455.2 [M+H].
4.5.6. 2-(5-acetyl-4-(4-chlorophenyl)-6-methyl-1,4-dihydropyrimidin-2-ylamino)-N-p-tolylacetamide
(5f)
5f was prepared according to the general procedure by using 1-(2-amino-4-(4-chloroyphenyl)-6-methyl-
1,4-dihydropyrimidin-5-yl)ethanone (2c) and 2-chloro-N-p-tolylacetamide (3c). Pale yellow crystals; Yield
79%; Rf =0.5 (n-Hexane/ethyl acetate 5 : 1); m.p. 171 ºC; 1H NMR (300 MHz, DMSO-d6): δ 2.19 (s, 3H,
CH3), 2.31 (s, 3H, CH3CO), 2.39 (s, 3H, CH3), 4.40 (d, 2H, J=5.1 Hz, NH-CH2), 4.98 (s, 1H, CH), 5.63 (t,
1H, J=5.1 Hz, Ar-NH), 7.01 (d, 2H, J=7.2 Hz, Ar-H), 7.15 (d, 2H, J=7.5 Hz, Ar-H), 7.25 (d, 2H, J=7.2 Hz,
Ar-H), 7.40 (d, 2H, J=7.5 Hz, Ar-H), 8.63 (s, 1H, NHCO), 9.84 (brs, 1H, pyrimidine-NH); LCMS
calculated for C22H23ClN4O2 = 411.2 [M+H].
4.5.6. 2-(5-acetyl-4-(4-bromophenyl)-6-methyl-1,4-dihydropyrimidin-2-ylamino)-N-(4-chlorophenyl)
acetamide (5g)
5g was prepared according to the general procedure by using 1-(2-amino-4-(4-bromophenyl)-6-methyl-1,4-
dihydropyrimidin-5-yl)ethanone (2c) and 2-chloro-N-(4-chlorophenyl)acetamide (3c). Pale yellow crystals;
1
Yield 79%; Rf =0.5 (n-Hexane/ethyl acetate 5 : 1); m.p. 190-192 ºC; H NMR (300 MHz, DMSO-d6): δ
23

2.21 (s, 3H, CH3), 2.33 (s, 3H, CH3CO), 4.40 (d, 2H, J=5.1 Hz, NH-CH2), 4.99 (s, 1H, CH), 5.65 (t, 1H,
J=5.1 Hz, Ar-NH), 7.01 (d, 2H, J=8.1 Hz, Ar-H), 7.21 (d, 2H, J=8.4 Hz, Ar-H), 7.50 (d, 2H, J=8.4 Hz, Ar-
H), 7.67 (d, 2H, J=8.1 Hz, Ar-H), 8.63 (s, 1H, NHCO), 9.88 (brs, 1H, pyrimidine-NH); LCMS calculated
for C21H20BrClN4O2 = 475.2 [M+H].
4.5.7. 2-(5-acetyl-6-methyl-4-p-tolyl-1,4-dihydropyrimidin-2-ylamino)-N-p-tolylacetamide (5h)
5h was prepared according to the general procedure by using 1-(2-amino-6-methyl-4-p-tolyl-1,4-
dihydropyrimidin-5-yl)ethanone (2d) and 2-chloro-N-p-tolylacetamide (3d). Pale yellow crystals; Yield
1
79%; Rf =0.5 (n-Hexane/ethyl acetate 6 : 1); m.p. 149-151 ºC; H NMR (300 MHz, DMSO-d6): δ 2.20 (s,
3H, CH3), 2.31 (s, 3H, CH3CO), 2.39 (s, 6H, CH3), 4.41 (d, 2H, J=5.1 Hz, NH-CH2), 5.01 (s, 1H, CH), 5.63
(t, 1H, J=5.1 Hz, Ar-NH), 6.95 (d, 2H, J=7.2 Hz, Ar-H), 7.16 (brs, 4H, Ar-H), 7.38 (d, 2H, J=7.2 Hz, Ar-
H), 8.61 (s, 1H, NHCO), 9.84 (brs, 1H, pyrimidine-NH); LCMS calculated for C23H26N4O2 = 391.2 [M+H].
4.6. Determination of AChE and BChE inhibitory activity
4.6.1. Chemicals and drugs
AChE (Electric eel type-VI-S, Sigma-Aldrich GmbH USA, code 1001596210), BChE (Equine serum
Lyophilized Sigma-Aldrich GmbH USA, code 101292670), Acetylthiocholine iodide (Sigma-Aldrich UK,
code 101303874), Butyrylthiocholine Iodide (Sigma-Aldrich Switzerland, code 101334643), DTNB (Sigma-
Aldrich Germany, code 101261619), Galantamine hydrobromide Lycoris Sp. (Sigma-Aldrich France, code
G1660). All the other chemicals used were of analytical grade.
4.6.2. In vitro AChE and BChE assay
The acetylcholinesterase (AChE) from Electric eel and butyrylcholinesterase (BChE) from equine serum
were used in the determination of anticholinesterase assays of compounds. Galantamine and donepezil were
used as reference drugs. The assay is based on the acetylcholinesterase promoted hydrolysis of
acetylthiocholine iodide ATCI) and butyrylthiocholine iodide (BTCI) by butyrylcholinesterase. The
hydrolysis processes was monitored by Ellman’s microplate method at 412 nm in the presence and absence of
various concentrations of compounds at 30 oC.
The synthesized compounds (each separately) were dissolved in a mixture of DMSO (1ml) and methanol (9
ml) and then diluted in KH2PO4/K2HPO4 buffer (0.1 M, pH 8.0) to obtain desired concentration. The
reaction mixture consisted of appropriate amount of phosphate buffer (pH 8.0), DTNB, test compounds, of
24

0.03 U/ml of enzymes (AChE and BChE). The mixture was pre-incubated at 30 °C for 10 min and followed
by adding 1mM ATCI or BTCI and incubated again for 15 min. The enzymatic hydrolysis was monitored
at 412 nm using µQuant microplate spectrophotometer (MQX200, BioTek USA). Instead of inhibitor,
DMSO was used as negative control to subtract its effect on activity. All reactions were carried out in
triplicate. The IC50 values were determined by plotting the inhibition against the sample solution
concentrations.
4.7. Computational studies
Computational docking studies were carried out using GOLD (Genetic Optimization for Ligand Docking)
suit v5.4.1. The X-ray crystallographic structure AChE in complex with donepezil (PDB Code 1EVE) was
used as enzyme structure. As a first step, the ability of the docking algorithm was validated to reproduce
the co-crystallized pose of thymine in the 1EVE pocket. This yielded a good agreement between the docked
and crystal structures as it further confirmed the interaction region of the ligand and a favorable interaction
with 1EVE. Gold fitness score performs a force field based scoring function and is made up of four
components: 1. Protein-ligand hydrogen bond energy (external H-bond); 2. Protein-ligand van der Waals
energy (external vdw); 3. Ligand internal van der Waals energy (internal vdw); 4. Ligand intramolecular
hydrogen bond energy (internal-H-bond). Binding site was defined as the residues within 6 Å from the
ligand. No water was present in any binding site. The default docking protocol was applied (1.09 auto
settings, 10 GA) and the best pose saved. Each experiment was then repeated 10 times. Other docking
parameters were set to the software’s default values. The view of the docking results and analysis of their
surface with graphical representations were done using Discovery Studio Visualizer [24].
LogP was calculated by using ChemAxon software package (Marvin v15.4.6, 2015) [25]. BBB penetration
was predicted by using online BBB predictor available at http://www.cbligand.org/BBB/. This predictor
was built by applying the support vector machine (SVM) and LiCABEDS17 algorithms on four types of
fingerprints of 1593 reported compounds [26]. Human Intestinal Absorption (HIA), Mutagenic potential of
the synthesized compounds (AMES test) and rodent carcinogenicity were computed using admetSAR [27].
Acknowledgments
The Higher Education Commission (HEC), Pakistan is thankfully acknowledged for providing financial
support to Umer Rashid for startup grant under IPFP program (HEC No: PM-IPFP/HRD/HEC/2011/346).
25

Dr Umer Rashid is also thankful to The Cambridge Crystallographic Data Centre (CCDC) for providing
license GOLD suit to COMSATS Institute of Information Technology.
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Rational design and synthesis of dihydropyrimidine based dual binding site
acetylcholinesterase inhibitors
Suffyan Ahmad 1, Fatima Iftikhar , Farhat Ullah, Abdul Sadiq, Umer Rashid *
*Corresponding author. E-mail: umerrashid@ciit.net.pk; umer_rashid39@hotmail.com
GRAPHICAL ABSTRACT
29