Design, synthesis and structure-activity relationship (SAR) studies of 2,4-disubstituted pyrimidine derivatives: Dual activity as cholinesterase and Aβ-aggregation inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.02.030

A novel class of 2,4-disubstituted pyrimidines (7a-u, 8a-f, 9a-e) that possess substituents with varying steric and electronic properties at the C-2 and C-4 positions, were designed, synthesized and evaluated as dual cholinesterase and amyloid-β (Aβ)-aggregation inhibitors. In vitro screening identified N-(naphth-1-ylmethyl)-2-(pyrrolidin-1-yl)pyrimidin-4-amine (9a) as the most potent AChE inhibitor (IC₅₀ = 5.5 μM). Among this class of compounds, 2-(4-methylpiperidin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin- 4-amine (9e) was identified as the most potent and selective BuChE inhibitor (IC₅₀ = 2.2 μM, selectivity index = 11.7) and was about 5.7-fold more potent compared to the commercial, approved reference drug galanthamine (BuChE IC₅₀ = 12.6 μM). In addition, the selective AChE inhibitor N-benzyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine (7d), exhibited good inhibition of hAChE-induced aggregation of Aβ_1-40 fibrils (59% inhibition). Furthermore, molecular modeling studies indicate that a central pyrimidine ring serves as a suitable template to develop dual inhibitors of cholinesterase and AChE-induced Aβ aggregation thereby targeting multiple pathological routes in AD.

Full text of DOI:10.1016/j.bmc.2011.02.030

Bioorganic & Medicinal Chemistry 19 (2011) 2269–2281
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and structure–activity relationship (SAR) studies
of 2,4-disubstituted pyrimidine derivatives: Dual activity as cholinesterase
and Ab-aggregation inhibitors
Tarek Mohamed a,b, Xiaobei Zhao c, Lila K. Habib d, Jerry Yang c, Praveen P. N. Rao b,
⇑
a Department of Biology, University of Waterloo, Waterloo, Ontario, Canada
b School of Pharmacy, Health Sciences Campus, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
c Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA
d Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of 2,4-disubstituted pyrimidines (7a–u, 8a–f, 9a–e) that possess substituents with varying
steric and electronic properties at the C-2 and C-4 positions, were designed, synthesized and evaluated as
dual cholinesterase and amyloid-b (Ab)-aggregation inhibitors. In vitro screening identified N-(naphth-1-
Received 6 December 2010
Revised 11 February 2011
Accepted 16 February 2011
Available online 1 March 2011
ylmethyl)-2-(pyrrolidin-1-yl)pyrimidin-4-amine (9a) as the most potent AChE inhibitor (IC50 = 5.5
Among this class of compounds, 2-(4-methylpiperidin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine
(9e) was identified as the most potent and selective BuChE inhibitor (IC50 = 2.2 M, selectivity
index = 11.7) and was about 5.7-fold more potent compared to the commercial, approved reference drug
galanthamine (BuChE IC50 = 12.6 M). In addition, the selective AChE inhibitor N-benzyl-2-(4-methylpi-
lM).
l
Keywords:
Cholinesterase inhibitors (ChEIs)
Acetylcholinesterase (AChE)
Butyrylcholinesterase (BuChE)
Human acetylcholinesterase (hAChE)
Structure–activity relationship (SAR)
Amyloid-b (Ab)
l
perazin-1-yl)pyrimidin-4-amine (7d), exhibited good inhibition of hAChE-induced aggregation of Ab1–40
fibrils (59% inhibition). Furthermore, molecular modeling studies indicate that a central pyrimidine ring
serves as a suitable template to develop dual inhibitors of cholinesterase and AChE-induced Ab aggrega-
tion thereby targeting multiple pathological routes in AD.
5,50-Dithiobis(2-nitrobenzoic acid) (DTNB)
Thioflavin T (ThT)
Ó 2011 Elsevier Ltd. All rights reserved.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT)
1. Introduction
ylcholinesterase (AChE and BuChE, respectively) enzymes act on
ACh to terminate its actions in the synaptic cleft by hydrolyzing
Neurological disorders inflict a heavy burden on healthcare costs,
affected individuals and their caregivers. Alzheimer’s disease (AD) is
classified as a progressive, neurodegenerative disease that affects
the cholinergic regions of the central nervous system (CNS) that
associate with cognitive function and spatial awareness.1 This dev-
astating neurological disease targets the elderly populations and
its prevalence is on the rise.2,3 The hallmark characteristics of AD in-
clude the rapid loss of cholinergic neurotransmission, accelerated
aggregation of amyloid-b (Ab) peptides and formation of neurofibril-
lary tangles (NFTs) of hyperphosphorylated tau protein.2–6 These
characteristics establish the basis for the cholinergic, amyloid and
tau hypotheses for AD pathology, respectively.
the neurotransmitter to choline and acetate.8,9 Deficiencies in the
ACh synthesizing enzyme (choline acetyltransferase—ChAT) also
contributes to the overall decline of ACh concentration in the cor-
tical regions of the brain.9–12 Furthermore, recent studies have
shown that the ratio of AChE to BuChE is dependent on the stage
of pathogenesis. In the CNS, AChE plays a vital role in the early
stages of AD. However, as the disease progresses and cholinergic
neurons are depleted, BuChE, which has a wider distribution with-
in the body, acts as the major degrading enzyme.13,14
In contrast, the amyloid hypothesis suggests that the progres-
sion of AD is attributed to the accelerated accumulation of toxic
forms of self-induced and/or AChE-promoted toxic aggregates of
Ab peptides.15–17 These toxic peptides arise from the cleavage of
the amyloid-precursor protein (APP) by the b-secretase (BACE-1)
According to the cholinergic hypothesis, the pathology of AD is
attributed to the rapid decline in neurotransmission in the cholin-
ergic regions of the CNS that house acetylcholine (ACh) utilizing
sympathetic and parasympathetic neurons.7 The acetyl and butyr-
and c-secretase enzymes. In this regard, recent studies indicate a
link between the cholinergic and amyloid hypotheses.18,19 AChE
is known to induce amyloid-b (Ab) formation leading to the highly
toxic AChE-Ab peptide complexes.20–22 These multiple factors in
AD pathology mandate the need to develop small molecule thera-
⇑
Corresponding author. Tel.: +1 519 888 4567x21317; fax: +1 519 888 7910.
E-mail address: praopera@uwaterloo.ca (P.P.N. Rao).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.030

2270
T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
R1
R1
pies that exhibit dual ChE inhibition as well as reduce the forma-
tion of neurotoxic Ab-aggregates.
NH
Cl
N
NH
b
a
Research into the cholinergic hypothesis has led to the develop-
ment of several fused and non-fused ring systems as ChE inhibitors
(ChEIs) (Fig. 1). For example, tacrine (1), an acridine derivative, was
one of the earliest ChEI developed to treat AD.23 Bis-7-tacrine (2) is
a potent dual AChE and BuChE inhibitor.24 Propidium (3) binds
specifically to the peripheral anionic site (PAS) of AChE.25 The
anti-Alzheimer drug donepezil (4), is highly selective toward AChE
and its binding conformation spans both the catalytic and periph-
eral sites of AChE.26 Recent work by DeLisa and co-workers exam-
ined s-triazine based ring templates (5) for their ability to inhibit
the aggregation of Ab1–42 plaques.27
N
N
N
N
R2
7a-r, 8a-f, 9a-e
)
Cl
=
N
Cl
6
( )
7-9
(
(
)
R1
or
or
9
8
7
As part of our research program, we previously reported the
development of a group of heterocyclic, non-fused ChEIs based
on a 2,4-disubstituted pyrimidine ring template.28 Here we report
the design, synthesis and evaluation of heterocycles 7a–u, 8a–f,
9a–e containing a 2,4-disubstituted pyrimidine ring scaffold as no-
vel small molecules possessing dual activity as cholinesterase
inhibitors and as inhibitors of hAChE-induced Ab1–40 aggregation.
R2
a,
=
HN
h,
i,
HN
N
Cl
n,
HN
HN
HN
b,
HN
HN
HN
HN
HN
HN
N
O
HN
HN
HN
HN
HN
Br
F
o,
p,
N
c,
i-Pr
i-Pr
N
N
N
S
N
2. Results and discussion
2.1. Chemistry
j,
k,
l,
d,
e,
f,
Me
The synthesis of target 2,4-disubstituted pyrimidine derivatives
(7a–r, 8a–f and 9a–e) was accomplished in two steps. In the first
step, the N-benzyl, N-phenethyl and naphth-1-ylmethyl-2-chloro-
pyrimidin-4-amine intermediates (7, 8 and 9, respectively) were
synthesized from the 2,4-dichloropyrimidine starting material (6)
by a nucleophilic aromatic substitution reaction at C-4 using a base
such as N,N-diisopropylethylamine (DIPEA). The reaction was run
in EtOH at 75–85 °C and refluxed for 3 h. The intermediates 7, 8
and 9 were obtained in moderate to good yield ranging from 60%
to 75% (Scheme 1).28,29
In the second step, the C-2 chlorine was displaced by various
cyclic amines (R2 = pyrrolidine, morpholine, thiomorpholine,
1-methylpiperazine, 4-methylpiperidine, acetylpiperazine, t-butyl
piperazine-1-carboxylate, cyclohexylpiperazine, isopropylpiper-
azine, isopropylpiperidine, N-propylpiperazine, N-hydroxyethyl-
piperazine, N-methoxyethylpiperazine, 4-chloro, bromo, fluoro
and trifluoromethylbenzylpiperazine, 4-amino-1-benzylpiperi-
dine, Scheme 1). This reaction was run under rigorous conditions
N
CF3
q,
r,
HN
n-Pr
Me
Ac
N
N
N
H2N
OH
g,
m,
N
N
Boc
OMe
Scheme 1. Reagents and conditions: (a) DIPEA, phenylmethanamine (7), 2-phen-
ylethanamine (8) or (naphthalene-1-yl)methanamine (9), EtOH, 0 °C to 75–85 °C
and reflux for 3 h.; (b) R2 = pyrrolidine, morpholine, thiomorpholine, 1-methylpi-
perazine, 4-methylpiperidine, acetylpiperazine, t-butyl piperazine-1-carboxylate,
cyclohexylpiperazine, isopropyl piperazine, isopropylpiperidine, N-propylpiper-
azine, N-hydroxyethyl piperazine, N-methoxyethylpiperazine, 4-chloro, bromo,
fluoro and trifluoromethylbenzylpiperazine, 4-amino-1-benzylpiperidine, n-BuOH,
145–150 °C, 30–40 min.
(145–150 °C) for 30–40 min in a sealed pressure vessel (PV) using
n-butanol as a solvent to afford the target 2,4-disubstituted pyrim-
idine derivatives (7a–r, 8a–f, 9a–e) in moderate to good yield
(50–90%) (Scheme 1).28,30
NH2
N
N
N-Benzyl-2-thiomorpholinopyrimidin-4-amine (7c) was oxi-
dized to 4-[4-(benzylamino)pyrimidin-2-yl]thiomorpholine 1-
oxide (7s) in good yield (75%) using meta-chloroperoxybenzoic acid
(MCPBA) (Scheme 2) and to 4-[4-(benzylamino) pyrimidin-2-yl]thi-
omorpholine 1,1-dioxide (7t) in good yield (75%) using potassium
peroxymonosulfate (OxoneÒ) as the oxidizing agent (Scheme 2).
N
H
N
N
1
( )
7H
2
( )
I
N
Me
Et
Et
MeO
OMe
I
O
N
H2N
NH2
(3)
(4)
N
HN
N
HN
N
HN
N
a
b
O
N
NH2
HN
Me N
O
N
N
N
N
N
N
N
N
(7c)
(7t)
(7s)
S O
O
S
S
O
Me
Me
5
( )
Scheme 2. Reagents and conditions: (a) mCPBA, 7c, 1,4-dioxane, 0 °C to rt, 3 h; (b)
OxoneÒ, 7c, MeOH, H2O and 1,4-dioxane, 0 °C to 70–75 °C, 1 h then rt, 4 h.
Figure 1. Structures of ChEIs (1, 2 and 4) and inhibitors of Ab-aggregation (3 and 5).

T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
2271
The deprotection of the tert-butoxycarbonyl (t-Boc) group of 7g
[tert-butyl 4-(4-(benzylamino)pyrimidin-2-yl)piperazine-1-car-
possessed a terminal alkyl or alkoxy group (7d, 7f–g and 7k–m) gen-
erally exhibited weak or a lack of BuChE inhibition (BuChE
boxylate] was accomplished by using 50% v/v trifluoroacetic acid
(TFA) to yield N-benzyl-2(piperazin-1-yl)pyrimidin-4-amine (7u)
in good yield (75%) (Scheme 3).
IC50 = 59.9
more, the unsubstituted C-2 piperazine containing compound 7u
exhibited selective AChE inhibition (IC50 = 15.5 M) with no BuChE
inhibition up to 100 M. It was interesting to note that the presence
of a cycloalkyl ring (7h) provided superior BuChE inhibition and
selectivity (AChE IC50 = 22.9 M; BuChE IC50 = 7.6 M, SI = 3.01)
lM to >100 lM range) with the exception of 7i. Further-
l
l
2.2. Cholinesterase inhibition and SAR studies
l
l
The ability of the synthesized derivatives (7a–u, 8a–f and 9a–e)
to inhibit both hAChE and equine BuChE was evaluated in vitro
(IC50 values, Table 1).28,31 Structure–activity relationship (SAR)
studies indicated the cholinesterase inhibition and selectivity were
sensitive to steric and electronic parameters at C-2 and C-4 posi-
tions of the central pyrimidine ring. They exhibited a broad range
among the piperazine derivatives. In contrast to terminal alkyl or
alkoxy substituted piperazines, the presence of a C-2 benzylpipera-
zine possessing halogen atoms (Cl, Br or F) (7n–p) or electron with-
drawing groups (CF3) (7q) at the para-position exhibited dual ChE
inhibition (AChE IC50 = 20.2 to 28.8
lM range; BuChE IC50 = 7.3 to
14.3 M range) with the exception of 7q (BuChE IC50 >100
l
l
M).
of inhibition (AChE IC50 = 5.5 to 28.8
to >100 M range).
lM range; BuChE IC50 = 2.2
The AChE activity was of the order: Cl > F > Br > CF3, whereas the
presence of a fluorine atom at para-position provides superior
l
Among the N-benzyl series of derivatives (7a–u), the substitu-
ent electronic and steric factors at C-2 position modulated ChE
inhibition. The presence of five-membered heterocycloalkyl C-2
substituent such as a pyrrolidine (7a) provided ChE inhibition with
BuChE inhibition (BuChE IC50 = 7.3
the order: F > Cl > Br > CF3 (inactive at 100
of increasing electronegativity with superior BuChE inhibition. In
addition, replacing the C-2 4-methylpiperazine (7d) with a methyl-
piperidine bioisostere in 7e provided dual ChE inhibition along with
l
M). The BuChE activity was of
lM) establishing the role
superior potency and selectivity toward AChE (AChE IC50 = 8.7
BuChE IC50 = 26.4
morpholine provides 7b that exhibited weak AChE (IC50 = 14
and BuChE inhibition (IC50 = 68.3
the presence of a C-2 thiomorpholine ring in 7c provided superior
BuChE inhibition and selectivity (AChE IC50 = 23.2 M; BuChE
IC50 = 6.1 M, SI = 3.8). This observation indicates that the differ-
lM;
l
M). Replacing the C-2 with a six-membered
superior BuChE inhibition and selectivity (AChE IC50 = 18.4
BuChE IC50 = 3.4 M, SI = 5.41) relative to 7d. In addition to its supe-
rior BuChE inhibitory potency, 7e was about 3.7-fold more potent
relative to the reference drug galanthamine (BuChEIC50 = 12.6 M),
lM;
l
M)
l
l
M) compared to 7a. In contrast,
l
l
although it was not as potent as bis-tacrine (BuChEIC50 = 10 nM).
Furthermore, the bioisosteric replacement of C-2 4-isopropylpiper-
azine in 7i with an isopropylpiperidine bioisostere in 7j provided
dual ChE inhibition similar to 7i, although with a 2.2-fold loss in
BuChE inhibitory potency (BuChE IC50 = 6.5
in AChE inhibitory potency (AChE IC50 = 14.2
derivative 7r, the benzylpiperidine pharmacophore present in
donepezil was incorporated into a pyrimidine diamine template.
This modification provided dual ChE inhibition and exhibited supe-
rior BuChE inhibition (BuChE IC50 = 8.2
drug galanthamine (BuChE IC50 = 12.6
l
ences in the electronic properties of oxygen and sulfur could mod-
ulate the binding modes within the active sites of both ChEs. This
was further explored by oxidizing the sulfur in 7c to either a sulf-
oxide (7s) or a sulfone (7t). Both 7s and 7t exhibited AChE inhibi-
tion, with 7s exhibiting about 1.8-fold more potent AChE inhibition
lM) and a 1.7-fold gain
lM) compared to 7i. In
(IC50 = 12.6
the unsubstituted terminal sulfur atom present in 7c) did not exhi-
bit BuChE inhibition up to 100 M.
lM) relative to 7c. Interestingly, 7s and 7t (both lack
l
lM) relative to the reference
The SAR of C-2 piperazine-substituted N-benzylpyrimidine
derivatives were explored by incorporating a wide range of terminal
4-alkyl, alkoxy, acyl, cycloalkyl and substituted-benzyl rings (7d, 7f–
i, 7k–q). The incorporation of terminal 4-alkylpiperazine and 4-alk-
oxy substituents (7d, 7f–g, 7i and 7k–m) provided AChE inhibition.
The presence of a methyl group in 7d (R2 = 4-methylpiperazine) pro-
lM).
From the N-benzyl series (7a–u), 7a was identified as the most
potent AChE inhibitor (IC50 = 8.7 M); 7s as the most selective
AChE inhibitor (SI <0.12); 7e and 7i as equally potent BuChE inhib-
itors (IC50 = 3.40 M), where 7i was also the most selective BuChE
l
l
inhibitor (SI = 7.35).
vided moderate AChE inhibition (IC50 = 24.9
ing the chain length to propyl (7k, R2 = 4-propylpiperazine) led to
superior AChE inhibition (IC50 = 15.3 M). In contrast, the presence
l
M), whereas increas-
In the N-phenethyl C-4 substituted pyrimidines (8a–f) evalu-
ated, the presence of a five-membered pyrrolidine ring in 8a led
l
to dual ChE inhibition (AChE IC50 = 9.8
and similar dual ChE inhibition was seen with 8e that possessed
a six-membered methylpiperidine ring at C-2 (AChE IC50 = 8.8 M;
BuChE IC50 = 17.7 M; Table 1). In contrast, the presence of C-2 six-
lM; BuChE IC50 = 13.8 lM)
of a branched, sterically demanding alkyl chain such as an isopropyl
in 7i (R2 = 4-isopropylpiperazine) exhibited similar AChE inhibition
l
(AChE IC50 = 25.0
BuChE inhibitor (BuChE IC50 = 3.4
ence of alkoxy groups such as hydroxyethyl in 7l (AChE
IC50 = 26.4 M) or a methoxyethyl in 7m (AChE IC50 = 26.7 M)
exhibited comparable AChE inhibition relative to 7d
(IC50 = 24.9
M). Interestingly, C-2 piperazine R2 substituents that
l
M) relative to 7d. However, 7i was a selective
l
l
M; SI = 7.35). Similarly, the pres-
membered rings such as morpholine (8b), thiomorpholine (8c),
methylpiperazine (8d) and acetylpiperazine (8f) was detrimental
to BuChE inhibition (IC50 values >100 lM; Table 1). It was interest-
ing to note that the naphth-1-ylmethyl series of C-4 substituted
l
l
l
pyrimidines (9a–e) evaluated exhibited dual ChE inhibition (AChE
IC50 = 5.5 to 25.8
Table 1). For example, the presence of a C-2 five-membered pyrrol-
idine in 9a provided superior BuChE inhibition (IC50 = 8.9 M) rel-
ative to donepezil (BuChE IC50 = 12.6 M) although it was a less
potent AChE inhibitor relative to tacrine (AChE IC50 = 0.093 M)
lM range; BuChE IC50 = 2.2 to 34.7 lM range;
l
l
l
HN
N
HN
N
a
or bis-tacrine (AChE IC50 = 4 nM). Replacing the C-2 five-membered
pyrrolidine with either a six-membered morpholine (9b, AChE
N
N
IC50 = 14.7
IC50 = 12.8
l
M; BuChE IC50 = 28
l
M) or thiomorpholine (9c, AChE
N
N
lM; BuChE IC50 = 34.7
lM; Table 1) led to a decline in
N
O
NH
(7g)
(7u)
ChE inhibitory potency relative to 9a. In contrast, the presence of
either a C-2 4-methylpiperazine (9d) or the corresponding bioiso-
stere methylpiperidine (9e) provided superior BuChE inhibitory
potency and selectivity. Compound 9e exhibited about 5.7-fold
O
Me
Me
Me
Scheme 3. Reagents and conditions: (a) TFA, 7g, DCM, 0 °C to rt, 2 h.
superior BuChE inhibition (IC50 = 2.2 lM) and selectivity

2272
T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
Table 1
AChE and BuChE inhibitory activities and C log P data for 2,4-disubstituted pyrimidines (7a–u, 8a–f and 9a–e)
NH
NH
NH
N
N
N
R2
N
8a-f
R2
N
9a-e
R2
N
7a-u
R2
IC50
(
lM)
Selectivity indexb (SI)
C log Pc
a,b
Compd
AChE
8.70
BuChE
26.40
N
N
N
N
N
N
N
N
7a
7b
7c
7d
7e
7f
0.33
2.97
2.14
2.98
2.71
4.05
1.73
4.12
4.65
O
14.0
68.30
6.10
0.21
S
23.20
24.90
18.40
16.60
18.80
22.90
3.80
>100
3.40
<0.25
5.41
<0.17
<0.19
3.01
N Me
Me
>100
>100
7.60
N
N
N
Ac
7g
7h
Boc
Me
N
N
7i
7j
25.0
3.40
6.50
7.35
2.19
3.54
4.97
Me
Me
N
N
N
N
14.20
Me
N
N
N
7k
7l
15.30
26.40
26.70
59.90
>100
>100
0.26
3.76
2.13
2.89
Me
<0.26
<0.27
OH
7m
OMe
Cl
7n
7o
7p
20.20
25.50
21.60
10.70
14.30
7.30
1.89
1.78
2.96
5.14
5.29
4.57
N
N
N
N
N
N
Br
F

T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
2273
Table 1 (continued)
a,b
Compd
R2
IC50
(
lM)
Selectivity indexb (SI)
C log Pc
AChE
28.80
BuChE
>100
CF3
7q
7r
<0.29
1.51
5.31
4.60
N
N
S
12.40
8.20
H
N
N
O
7s
7t
N
N
12.60
24.20
>100
>100
<0.13
<0.24
1.26
1.18
O
S
O
7u
8a
8b
8c
8d
8e
8f
N
N
N
N
N
N
NH
15.50
9.80
>100
13.80
>100
>100
>100
17.70
>100
8.90
0.16
2.13
3.62
2.79
3.63
3.35
4.69
2.37
4.14
3.32
4.15
3.88
5.22
0.71
O
S
19.70
26.40
20.40
8.80
<0.20
<0.26
<0.20
0.50
N Me
Me
N
N
N
Ac
19.90
5.50
<0.20
0.62
9a
9b
9c
9d
9e
N
N
N
N
O
S
14.70
12.80
17.50
25.80
28.0
0.53
34.70
2.60
0.37
N Me
Me
6.73
2.20
11.73
Tacrine. HCl
Figure 1 (1)
Figure 1 (2)
ꢁ
0.093
0.004
3.80
0.019
0.010
12.60
3.60
9.11
0.171
0.274
3.27
10.10
1.03
4.59
Bis(7)-tacrine
GalanthamineꢀHBr
Donepezil. HCl
0.032
a
The in vitro test compound concentration required to produce 50% inhibition of hAChE and equine BuChE.
The result (IC50) is the mean of two separate experiments (n = 4) and the deviation from the mean is <10% of the mean value. Selectivity index = hAChE IC50/BuChE IC50
C log P was determined using ChemDraw Ultra 12.0. CambridgeSoft Company.
b
c
.
(SI = 11.7) relative to the reference drug galanthamine (BuChE
IC50 = 12.6 M; SI = 0.27) and was much more potent compared
to donepezil (BuChE IC50 = 3.6 M) whereas the corresponding C-
2 methylpiperidine 9d exhibited similar BuChE inhibition (BuChE
IC50 = 2.6 M) and superior AChE inhibition (AChE IC50 = 17.5 M)
relative to 9e (AChE IC50 = 25.8 M). In addition, the theoretical
partition coefficient values (C log P) of various synthesized pyrim-
idines (Table 1) compares well with the reference compound ta-
crine (C log P = 3.27) favoring their use as agents to treat
disorders of the central nervous system (CNS).
a thioflavin T (ThT) fluorescence method.32 The anti-aggregating
activity of eight 2,4-disubstituted pyrimidines along with refer-
ence compounds propidium iodide and donepezil are presented
l
l
in Table 2. At a concentration of 100 lM, 2,4-disubstituted pyrim-
l
l
idines exhibited varying degrees of inhibition (inactive to 59% inhi-
bition of aggregation). In compound 7a, the presence of a smaller
five-membered pyrrolidine at C-2 did not provide anti-aggregation
activity (Table 2). Similarly, the presence of a C-2 thiomorpholine
ring in 7c, 8c and 9c provided either weak or no anti-aggregation
activity. Among the 2,4-disubstituted pyrimidines evaluated, 7d
with a combination of C-4 N-benzyl and a C-2 methylpiperazine
substituent exhibited good activity against hAChE-induced aggre-
gation of Ab1–40 peptides (59% inhibition). This compound was
3.4-fold more potent compared to donepezil (17% inhibition) and
was not as potent as propidium iodide (85% inhibition), which is
known to bind to the PAS in AChE.20,25 Interestingly, when the
l
2.3. Inhibition of hAChE-induced Ab aggregation
Some 2,4-disubstituted pyrimidines that exhibit a range of cho-
linesterase inhibition activities ( 7a, 7c, 7d, 7g, 7s, 7t, 8c and 9c)
were evaluated to prevent hAChE-induced Ab1–40 aggregation by

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T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
Table 2
Table 3
Inhibition of hAChE-induced aggregation of Ab1–40 by 2,4-disubstituted pyrimidines
(7a, 7c, 7d, 7g, 7s, 7t, 8c and 9c)
MTT reduction cytotoxicity assay in SH-SY5Y neuroblastoma cells of 2,4-disubstituted
pyrimidines (7a, 7c, 7d, 7g, 7s, 7t, 8c and 9c)
Compd
Inhibition at 100
lM
SEMa (%)
Compd
Cell Viability at 40
l
M
SEM (%)a
7a
7c
7d
7g
7s
7t
8c
9c
NA
NA
7a
7c
7d
7g
7s
7t
8c
9c
100.3 16.4b
97.8 11.5
98.3 10.5
102.3 0.5c
89.7 11.5
91.8 2.2
59.0 2.9
26.7 17.0
55.9 6.3
44.1 11.0
NA
38 25
17.4 8.1
84.9 4.1
104.5 4.6d
89.3 2.9
Donepezil
Propidium iodide
Tacrine
Galanthamine
89.0 6.3
97.1 12.9
a
a
The values are the mean of two independent measurements each performed in
The values are the mean of measurements performed in quadruplicates,
triplicates, SEM = standard error of mean. NA = not active.
SEM = standard error of mean. 100% cell viability represents the viability of SH-
SY5Y cells measured in the absence of small molecules, as determined by MTT
reduction. Statistical analysis using an independent two-tailed student’s t-tests
were performed using Origin 7.0 (Microcal Software, Inc., Northhampton, MA) to
evaluate the statistical significance of the difference between the viability of control
cells (measured in the absence of test molecules) and experimental mean values. A
p-value of <0.05 was defined as statistically significant.
C-2 thiomorpholine substituent of 7c was oxidized to a polar sulf-
oxide (7s) or a sulfone (7t), it provided anti-aggregation activity of
about 56% and 44% inhibition, respectively (Table 1). This suggests
that small molecules with polar functional groups could poten-
tially exhibit superior binding within the AChE active which has
polar pockets in its catalytic site (Ser203, His447, Glu202) as well
as the active site gorge entry (Trp286, Tyr124). This justifies our re-
sults that the presence of polar functional groups at C-2 in 7d, 7s
and 7t led to superior inhibition of AChE-promoted Ab aggregation
compared to 7g that has a lipophilic boc group at C-2. These stud-
ies indicate that the substituent electronic and steric effects both at
the C-2 and C-4 positions can be manipulated to develop 2,4-
disubstituted pyrimidines that bind favorably within the PAS pres-
ent in AChE. Further studies are in progress to acquire SAR data on
the role of various C-2 substituents and their effect on AChE-pro-
moted Ab aggregation.
b
p-Value = 0.97.
p-Value = 0.30.
p-Value = 0.55.
c
d
(Figure 2). These investigations indicate that the central pyrimidine
ring was approximately located midway through the active site
gorge (ꢂ6 Å away from the catalytic triad His447 residue at the bot-
tom of the active site and ꢂ7 Å away from the PAS Trp286). The ring
was also, (a) perpendicularly oriented between Trp86 and Phe297,
(b) ꢂ5 Å away from the hydrophobic pocket (Gly120–122) and (c)
equidistantly stacked between Tyr124 and Tyr337. The latter fea-
ture allowed for two hydrogen bonding interactions between the
tyrosine hydroxyl groups and the C-4 NH and two between the tyro-
sine hydroxyl groups and the pyrimidine N-3 (distances <3.5 Å). The
C-4 naphth-1-yl ring was tightly stacked between Tyr337 and Trp86
(distance ꢂ3.5 Å) and was in close proximity to His447 (distances
3.5–4 Å). The small C-2 pyrrolidine substituent was oriented toward
an aromatic region close to the PAS (distance to Trp286 is ꢂ4.5 Å)
and was stacked beneath Tyr341 (distance ꢂ3.5 Å). It is noteworthy
that, although the catalytic site is relatively exposed in this binding
pattern, the presence and location of the bulky C-4 naphth-1-yl ring
most likely attribute to this derivative’s potency considering the role
that Trp86 plays in stabilizing the substrate ACh. This binding pat-
tern is not seen in the related N-benzyl derivative 7a (AChE
2.4. SH-SY5Y neuroblastoma cell toxicity
The cytotoxicity of 2,4-disubstituted pyrimidine derivatives 7a,
7c, 7d, 7g, 7s, 7t, 8c and 9c was evaluated using a 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) - based
colorimetric assay. The cell viability of SH-SY5Y neuroblastoma
cells after exposure to 2,4-disubstituted pyrimidines derivatives
was compared with reference compounds tacrine and galantha-
mine (Table 3). At 40 lM, all the tested pyrimidine derivatives
maintained efficient cell viability as measured by MTT reduction
and were relatively nontoxic (Table 3). The N-benzyl derivative
7d that exhibited selective AChE inhibition (IC50 = 24.9 lM) and
IC50 = 8.7 lM), which re-enforces the unique dynamics offered by
the naphth-1-yl methyl series of derivatives.
A similar docking experiment was conducted to investigate the
binding interactions of the most potent and selective BuChE inhib-
itor 9e [2-(4-methylpiperidin-1-yl)-N-(naphth-1-ylmethyl)pyrimi-
excellent anti-aggregation activity (59% inhibition) was nontoxic
(98% cell viability). For comparison, the viability of SH-SY5Y cells
was 89% in the presence of the reference compound tacrine and
97% in the presence of the reference compound galanthamine un-
der the same experimental conditions. In addition, the C-2 oxidized
compounds 7s (56% inhibition of Ab-aggregation) and 7t (44% inhi-
bition of Ab-aggregation) were only slightly toxic under these con-
ditions, with cell viabilities of 90% and 92%, respectively. These
studies indicate that the 2,4-disubstituted pyrimidines were non-
toxic or only slightly toxic to SH-SY5Y cells and serve as a suitable
template to develop ChEIs that exhibit activity against AChE-in-
duced aggregation of Ab.
din-4-amine hAChE IC50 = 25.8 lM; BuChE IC50 = 2.2 lM,
SI = 11.73) within the active site of hBuChE (Fig. 3). These studies
indicate that the central pyrimidine ring was oriented (a) much
closer to the catalytic active site (distance to His438 <3.5 Å), (b)
was close to the hydrophobic pocket (Gly115–117; distance
ꢂ5 Å) and (c) was also perpendicularly stacked between Trp82
and Trp231. The C-4 naphth-1-yl methylamine group was oriented
toward the acyl pocket (Leu286, Ser287 and Val288; distance ꢂ4Å)
and the naphth-1-yl ring was oriented toward an aromatic cage
comprised of Trp231, Phe398 and Phe329. The C-4 NH was in close
proximity to the catalytic residues (Ser198 and His438) and was
undergoing hydrogen bonding interactions (distance <3 Å). The
C-2 4-methylpiperidine substituent was oriented in an aromatic
pocket comprised of Tyr440, Tyr332, Trp430 and Trp82. The short-
est distance calculated between 9e and the gorge entry residue
2.5. Molecular modeling (docking) studies
Among the naphth-1-yl methyl series, the enzyme-ligand bind-
ing interactions of the most potent hAChE inhibitor 9a [N-(naphth-
1-ylmethyl)-2-(pyrrolidin-1-yl)pyrimidin-4-amine; hAChE IC50
5.5 M; equine serum BuChE IC50 = 8.9 M, SI = 0.62] within the
active site of hAChE was investigated by molecular modeling studies
=
l
l

T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
2275
Figure 2. Docking of N-(naphthalen-1-ylmethyl)-2-(pyrrolidin-1-yl)pyrimidin-4-amine (9a, ball and stick) in the active site of hAChE. Red dotted lines represent hydrogen
bonding. Hydrogen atoms are not shown for clarity.
Figure 3. Docking of 2-(4-methylpiperidin-1-yl)-N-(naphthalen-1-yl methyl)pyrimidin-4-amine (9e, ball and stick) in the active site of hBuChE. Red dotted lines represent
hydrogen bonding. Hydrogen atoms are not shown for clarity.
(Ala277) was ꢂ10 Å, which reiterates the proximity of 9e to the
buried active site.
consistent with the fact that 9e (volume = 231.9 Å3, Supplementary
data) has a bulkier C-4 (naphth-1-yl) substituent whereas 7e con-
sists of a less bulky phenyl ring at C-4 explaining the superior
BuChE inhibitory potency exhibited by 9e compared to 7e.
Furthermore, the similar BuChE inhibitory profiles of 9e and its
N-benzyl relative (7e, BuChE IC50 = 3.4; SI = 5.4) were investigated
by superimposing the binding modes of both derivatives in hBuChE
(Fig. 4). The superimposition revealed similar binding orientations
within the active site where the larger naphth-1-ylmethyl C-4
group in 9e occupies more room in the glycine hydrophobic pock-
et. Also, the C-2 methylpiperidine ring is planar to the pyrimidine
ring in 7e but perpendicular to the pyrimidine ring in 9e. This is
3. Conclusions
The SAR data obtained on these novel class of 2,4-disubstituted
pyrimidines indicates that (i) simple and efficient synthetic meth-
ods can be used to synthesize 2,4-disubstituted pyrimidines; (ii)

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T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
Figure 4. Superimposition of the binding modes of N-benzyl-2-(4-methylpiperidin-1-yl)pyrimidin-4-amine (7e, red) and 2-(4-methylpiperidin-1-yl)-N-(naphthalene-1-
ylmethyl)pyrimidin-4-amine (9e, blue) within the active site of hBuChE.
the cholinesterase inhibition activity was sensitive to substituent
steric and electronic properties at both C-2 and C-4 positions of
the central pyrimidine ring; (iii) compound 9a [N-(naphth-1-yl-
methyl)-2-(pyrrolidin-1-yl)pyrimidin-4-amine] was identified as
4.2. General procedure for the synthesis of 4-substituted-2-
chloropyrimidin-4-amines (7–9)28
To a mixture of 2,4-dichloropyrimidine (6) (5.00 g, 33.60 mmol)
and primary amines (33.60 mmol) in 65 mL of EtOH, kept at 0 °C
(ice-bath), DIPEA (6.08 mL, 36.80 mmol) was added. The reaction
was allowed to stir on the ice-bath for 5 minutes and was refluxed
at 75–80 °C for 3 h. After cooling to 25 °C, the EtOH was evaporated
in vacuo and the residue was re-dissolved in a solvent mixture of
EtOAc and dichloromethane (DCM) in ꢂ3:1 ratio and successfully
the most potent AChEI (IC50 = 5.5
peridin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine] was the
most potent and selective BuChEI (IC50 = 2.2 M, S.I = 11.73); (iv)
lM), whereas 9e [2-(4-methylpi-
l
7d containing a C-4 phenyl and a C-2 methylpiperidine acts as
an AChEI and inhibits AChE-induced Ab-aggregation; (v) 2,4-disub-
stituted pyrimidines could potentially serve as a suitable ring tem-
plate to develop ChEIs that possess anti-Ab aggregation activity
and thus could target multiple pathological routes in AD.
washed with
a
concentrated NaHCO3 and NaCl solution
(1 ꢃ 15 mL). Aqueous layer was washed with EtOAc (3 ꢃ 15 mL)
and the combined organic layer was dried over anhydrous MgSO4
and filtered. The organic layer is evaporated in vacuo and the
resulting residue was further purified using either one of the fol-
lowing two methods: (1) Method A: Silica gel column chromatog-
raphy using EtOAc/hexanes twice (3:1 and 1:3, respectively) to
afford solid products (60–65%); (2) Method B: Differential melting
point separation—the collected organic layers are evaporated in va-
cuo and the oily residue is washed with a solution of hexanes and
ether (ꢂ3:1) to afford a precipitate that was dried on filter paper at
80–85 °C for ꢂ2 h to afford solid products. Some physical and spec-
troscopy data are provided below for 7, 8 and 9.
4. Experimental section
4.1. General
Melting points were determined on a Fisher–Johns apparatus
and are uncorrected. Infrared (IR) spectra were recorded as films
on NaCl plates using a Perkin–Elmer FT-IR spectrometer. 1H NMR
spectra were recorded on a Bruker Avance 300 MHz series spec-
trometer (CDCl3 as solvent). Coupling constants (J values) are in
hertz (Hz). The following abbreviations are used for multiplicity
of NMR signals: s = singlet, d = doublet, t = triplet, q = quartet,
dd = double doublet, m = multiplet, br = broad. High-resolution
electron ionization mass spectral (HREIMS) analysis was recorded
using a JEOL HX110 double focusing mass spectrometer. Com-
pound 7c was synthesized from a previous literature report.28
Combustion analysis was carried out by Midwest Microlab, LLC
(Indianapolis, IN) and C, H, N of tested compounds 7a–t, 8a–f
and 9a–e were within 0.4% of theoretical values for all elements
listed indicating a purity of >95%. Silica gel column purification
was performed using Merck 230–400 mesh Silica Gel 60. Com-
pounds 7a–u, 8a–f and 9a–e showed single spot on thin-layer
chromatography (TLC) performed on Merck 60F254 silica gel plates
(0.2 mm) using three different solvent systems (9:1 EtOAc/MeOH;
3:1 EtOAc/hexanes and 1:3 DCM/EtOAc) and spots were visualized
with UV 254 nm or iodine. All other solvents and reagents were ob-
tained from various vendors (Acros Organics, Sigma–Aldrich and
Alfa Aesar) with a minimum purity of 95% and were used without
further purification.
4.2.1. N-Benzyl-2-chloropyrimidin-4-amine (7)
The product was obtained as a white/light yellow solid after
coupling with phenylmethaneamine (3.67 mL, 33.60 mmol). Meth-
od A—4.78 g, 65%; Method B—5.31 g, 72%. Mp: 130–132 °C; IR
(film, CH2Cl2): 3434 cmꢁ1 (NH); 1H NMR (300 MHz, CDCl3): d
8.00 (d, J = 6.0 Hz, 1H), d 7.28–7.34 (m, 5H), d 6.20 (d, J = 6.0 Hz,
1H), d 4.53 (br s, 2H). HREIMS calcd for C11H10ClN3 (M+) m/z
219.6702, observed 219.0498. Anal. Calcd for: C11H10ClN3ꢀ0.32
H2O; C, 58.55; H, 4.43, N, 18.63. Found: C, 58.61; H, 4.76; N, 18.64.
4.2.2. 2-Chloro-N-phenethylpyrimidin-4-amine (8)
The product was synthesized after coupling with 2-phenyle-
thanamine (4.35 mL, 33.60 mmol). The residue is re-dissolved in
a solvent mixture of EtOAc, DCM and MeOH in ꢂ4:2:1 ratio. The
resulting oily residue was further purified by silica gel column
chromatography (Method A) to afford a white/off-white solid
(4.70 g, 60%): mp: 75–77 °C. IR (film, CH2Cl2): 3433 cmꢁ1 (NH);

T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
2277
1H NMR (300 MHz, CDCl3): d 7.98 (d, J = 6.0 Hz, 1H), d7.17–7.33 (m,
5H), d 6.16 (d, J = 6.0 Hz, 1H), d 3.60 (br s, 2H), d 2.88–2.92 (m, 2H).
HREIMS calcd for
233.0606.
85%). Mp: 150–153 °C. IR (film, CH2Cl2): 3454 cmꢁ1 (NH); 1H
NMR (300 MHz, CDCl3): d 7.86 (d, J = 6.0 Hz, 1H), d 7.27–7.32 (m,
5H), d 5.66 (d, J = 6.0 Hz, 1H), d 4.87 (br s, 1H), d 4.50 (d,
J = 6.0 Hz, 2H), d 3.77 (t, J = 6.0 Hz, 4H), d 2.42 (t, J = 6.0 Hz, 4H), d
2.31 (s, 3H). HREIMS calcd for C16H21N5 (M+) m/z 283.3714, found
283.1804. Anal. Calcd for C16H21N5ꢀ0.6EtOAc: C, 65.74; H, 7.74, N,
20.83. Found: C, 65.64; H, 7.75; N, 20.67.
C
12H12ClN3 (M+) m/z 233.6968, observed
4.2.3. 2-Chloro-N-(naphth-1-ylmethyl)pyrimidin-4-amine (9)
The product was obtained after coupling with naphthalen-1-
ylmethanamine (4.95 mL, 33.60 mmol) and was a light orange/
brown solid (Method A—5.43 g, 60%; Method B—6.83 g, 75%):
mp: 158–160 °C. IR (film, CH2Cl2): 3432 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 8.01 (d, J = 6.0 Hz, 1H), d 7.82–7.96 (m, 3H),
d 7.40–7.56 (m, 4H), d 6.23 (d, J = 6.0 Hz, 1H) d 4.70 (br s, 2H). HRE-
IMS calcd for C15H12ClN3 (M+) m/z 269.7289, observed 269.0737.
Anal. Calcd for: C17H18ClN3ꢀ0.17H20: C, 66.79; H, 4.48, N, 15.58.
Found: C, 66.04; H, 4.56; N, 15.40.
4.3.5. N-Benzyl-2-(4-methylpiperidin-1-yl)pyrimidin-4-amine
(7e)
The product was obtained after coupling 7 with methylpiperi-
dine (0.14 mL, 1.12 mmol) to afford a whitish pink solid (0.22 g,
85%). Mp: 83–85 °C. IR (film, CH2Cl2): 3433 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 7.85 (d, J = 6.0 Hz, 1H), d 7.28–7.32 (m, 5H),
d 5.61 (d, J = 6.0 Hz, 1H), d 4.84 (br s, 1H), d 4.63–4.67 (m, 2H), d
4.50 (d, J = 6.0 Hz, 2H), d 2.73–2.81 (m, 2H), d 1.62–1.66 (m, 2H),
d 1.22–1.26 (m, 1H), d 1.10–1.18 (m, 2H), d 0.91 (d, J = 6.0 Hz,
4.3. General procedure for the synthesis of 2,4-disubstituted-
pyrimidin-4-amines (7a–r, 8a–f, 9a–e)28
3H). HREIMS calcd for
C
17H22N4 (M+) m/z 282.3834, found
282.2376. Anal. Calcd for C17H22N4: C, 72.31; H, 7.85, N, 19.84.
Found: C, 72.18; H, 7.85; N, 19.60.
To a solution of 7, 8 or 9 (0.74–0.91 mmol) in 3 mL of n-BuOH
kept in a PV with stirring, cyclic amines (1.12 mmol) was added.
The sealed PV was placed in an oil bath at 145–150 °C and stirred
for 30–40 min. n-BuOH was evaporated in vacuo and the residue
was re-dissolved in 3:1 EtOAc/DCM and washed successively with
saturated NaHCO3 and NaCl solution (1 ꢃ 15 mL), respectively. The
aqueous layer was washed with EtOAc (3 ꢃ 5 mL) and the organic
layer was dried over anhydrous MgSO4 then filtered. The solution
was evaporated in vacuo to afford either solid or semisolid product.
Some physical and spectroscopy data are provided below for 7a–r,
8a–f, 9a–e.
4.3.6. 1-[4-(4-(Benzylamino)pyrimidin-2-yl)piperazin-1-
yl]ethanone (7f)
The product was obtained after coupling 7 with acetylpiper-
azine (0.15 g, 1.12 mmol) to afford
a yellowish white solid
(0.25 g, 86%). Mp: 150–153 °C. IR (film, CH2Cl2): 3437 cmꢁ1 (NH);
1H NMR (300 MHz, CDCl3): d 7.87 (d, J = 6.0 Hz, 1H), d 7.28–7.31
(m, 5H), d 5.70 (d, J = 6.0 Hz, 1H), d 4.93 (br s, 1H), d 4.49 (d,
J = 6.0 Hz, 2H), d 3.72–3.79 (m, 4H), d 3.61–3.64 (m, 4H), d 3.44–
3.48 (m, 2H), d 2.11 (s, 1H). HREIMS calcd for C17H21N5O (M+) m/
z 311.3815, found 311.1746. Anal. Calcd for C17H21N5Oꢀ0.4EtOAc:
C, 64.46; H, 7.04, N, 20.21. Found: C, 64.43; H, 7.04; N, 20.15.
4.3.1. N-Benzyl-2-(pyrrolidin-1-yl)pyrimidin-4-amine (7a)
The product was obtained as a light brown solid after coupling 7
with pyrrolidine (0.15 g, 65%). Mp: 103–105 °C. IR (film, CH2Cl2):
3435 cmꢁ1 (NH); 1H NMR (300 MHz, CDCl3): d 7.87 (d, J = 6.0 Hz,
1H), d 7.26–7.32 (m, 5H), d 5.62 (d, J = 6.0 Hz, 1H), d 4.86 (br s,
1H), d 4.51 (d, J = 6.0 Hz, 2H), d 3.49–3.53 (m, 4H), d 1.90–1.94
(m, 4H). HREIMS calcd for C15H18N4 (M+) m/z 254.3302, found
254.1964. Anal. Calcd for: C15H18N4ꢀ1H2O; C, 66.15; H, 7.40, N,
20.57. Found: C, 66.15; H, 7.40, N, 20.57.
4.3.7. tert-Butyl 4-[4-(Benzylamino)pyrimidin-2-yl]piperazine-
1-carboxylate (7g)
The product was obtained after coupling 7 with tert-butyl
piperazine-1-carboxylate (0.22 g, 1.12 mmol). Product was puri-
fied using a 3:1 EtOAc/hexanes silica gel chromatography to afford
a light yellowish solid (0.31 g, 90%). Mp: 115–117 °C. IR (film,
CH2Cl2): 3438 cmꢁ1 (NH); 1H NMR (300 MHz, CDCl3): d 7.80 (d,
J = 6.0 Hz, 1H), d 7.21–7.30 (m, 5H), d 5.65 (d, J = 6.0 Hz, 1H), d
4.91 (br s, 1H), d 4.49 (d, J = 6.0 Hz, 2H), d 3.66–3.69 (m, 4H), d
3.37–3.40 (m, 4H), d 1.43 (s, 9H). HREIMS calcd for C20H27N5O
(M+) m/z 369.4607, found 369.2163. Anal. Calcd for C20H27N5O2:
C, 65.02; H, 7.37, N, 18.96. Found: C, 65.54; H, 7.33; N, 18.76.
4.3.2. N-Benzyl-2-morpholinopyrimidin-4-amine (7b)
The product was obtained after 7 coupling with morpholine
(0.10 mL, 1.12 mmol) to afford an orange/light brown solid
(0.20 g, 80%): mp: 93–95 °C. IR (film, CH2Cl2): 3434 cmꢁ1 (NH);
1H NMR (300 MHz, CDCl3): d 7.87 (d, J = 6.0 Hz, 1H), d 7.29–7.31
(m, 5H), d 5.69 (d, J = 6.0 Hz, 1H), d 4.91 (br s, 1H), d 4.50 (d,
J = 6.0 Hz, 2H), d 3.70–3.75 (m, 8H). HREIMS calcd for C15H18N4O
(M+) m/z 270.3296, found 270.1605. Anal. Calcd for C15H18N4O: C,
66.64; H, 6.71, N, 20.73. Found: C, 66.45; H, 6.72; N, 20.46.
4.3.8. N-Benzyl-2-(4-cyclohexylpiperazin-1-yl)pyrimidin-4-
amine (7h)
The product was obtained after coupling 7 with cyclohexylpip-
erazine (0.20 g, 1.12 mmol) and was purified using a 3:1 EtOAc/
hexanes silica gel chromatography to afford an orange solid
(0.21 g, 66%). Mp: 60–62 °C. IR (film, CH2Cl2): 3435 cmꢁ1 (NH);
1H NMR (300 MHz, CDCl3): d 7.82 (d, J = 6.0 Hz, 1H), d 7.21–7.32
(m, 5H), d 5.61 (d, J = 6.0 Hz, 1H), d 5.07 (br s, 1H), d 4.46 (d,
J = 6.0 Hz, 2H), d 3.72–3.75 (m, 4H), d 2.54–2.57 (m, 4H), d 2.25
(s, 1H), d 1.86 (br s, 2H), d 1.75 (br s, 2H), d 1.58–1.61 (m, 1H), d
4.3.3. N-Benzyl-2-thiomorpholinopyrimidin-4-amine (7c)28
The product was obtained after 7 coupling with thiomorpholine
(0.11 mL, 1.12 mmol) to afford an orange/light brown solid (0.20 g,
77%): mp: 85–87 °C. IR (film, CH2Cl2): 3258 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 7.84 (d, J = 5.7 Hz, 1H), d 7.24–7.33 (m, 5H),
d 5.66 (d, J = 5.7 Hz, 1H), d 5.03 (br s, 1H), d 4.47 (d, J = 5.6 Hz,
2H), d 4.03–4.07 (m, 4H), d 2.54–2.58 (m, 4H). HREIMS calcd for
1.13–1.19 (m, 5H). HREIMS calcd for
C
21H29N5 (M+) m/z
351.4885, found = 351.2259. Anal. Calcd for C21H29N5ꢀ0.5DCM: C,
C
15H18N4S (M+) m/z 286.3952, found 286.1872. Anal. Calcd for
65.55; H, 7.68, N, 17.78. Found: C, 65.77; H, 7.70; N, 17.85.
C15H18N4S: C, 62.91; H, 6.33, N, 19.56. Found: C, 62.62; H, 6.33;
N, 19.31.
4.3.9. N-Benzyl-2-(4-isopropylpiperazin-1-yl)pyrimidin-4-
amine (7i)
4.3.4. N-Benzyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine
(7d)
The product was obtained after coupling 7 with methylpipera-
zine (0.13 mL, 1.12 mmol) to afford a light yellow solid (0.22 g,
The product was obtained after coupling 7 with isopropylpiper-
azine (0.17 mL, 1.12 mmol) and was purified using a 3:1 EtOAc/
hexanes silica gel chromatography to afford a white solid (0.14 g,
50%). Mp: 103–105 °C. IR (film, CH2Cl2): 3438 cmꢁ1 (NH); 1H

2278
T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
NMR (300 MHz, CDCl3): d 7.85 (d, J = 6.0 Hz, 1H), d 7.24–7.34 (m,
5H), d 5.63 (d, J = 6.0 Hz, 1H), d 4.93 (br s, 1H), d 4.48 (d,
J = 6.0 Hz, 2H), d 3.73–3.76 (m, 4H), d 2.62–2.71 (m, 1H), d 2.50–
2.53 (m, 4H), d 1.03 (d, J = 6.0 Hz, 6H). HREIMS calcd for C18H25N5
(M+) m/z 311.4246, found 311.2552. Anal. Calcd for C18H25N5: C,
69.42; H, 8.09, N, 22.49. Found: C, 69.69; H, 8.31; N, 22.15.
55%). Mp: 88–90 °C. IR (film, CH2Cl2): 3437 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3) d 7.85 (d, J = 6.0 Hz, 1H), d 7.24–7.30 (m, 9H), d
5.65 (d, J = 6.0 Hz, 1H), d 4.87 (br s, 1H), d 4.48 (d, J = 6.0 Hz, 2H),
d 3.73–3.76 (m, 4H), d 3.47 (s, 2H), d 2.41–2.44 (m, 4H). HREIMS
calcd for C22H24ClN5 (M+) m/z 393.9125, found 393.2443. Anal.
Calcd for C22H24ClN5: C, 67.08; H, 6.14, N, 17.78. Found: C, 67.44;
H, 6.13; N, 17.68.
4.3.10. N-Benzyl-2-(4-isopropylpiperidin-1-yl)pyrimidin-4-
amine (7j)
The product was obtained after coupling 7 with isopropylpi-
peridine (0.17 mL, 1.12 mmol) and was purified using 3:1 ether/
4.3.15. N-Benzyl-2-[4-(4-bromobenzyl)piperazin-1-yl]pyrimidin-
4-amine (7o)
The product was obtained after coupling 7 with 4-bromoben-
zylpiperazine (0.29 g, 1.12 mmol) to afford a yellowish solid
(0.20 g, 50%). Mp: 90–93 °C. IR (film, CH2Cl2): 3434 cmꢁ1 (NH);
1H NMR (300 MHz, CDCl3) d 7.85 (d, J = 6.0 Hz, 1H), d 7.41–7.44
(m, 2H), d 7.24–7.34 (m, 5H), d 7.19–7.22 (m, 2H), d 5.65 (d,
J = 6.0 Hz, 1H), d 4.87 (br s, 1H), d 4.48 (d, J = 6.0 Hz, 2H), d 3.73–
3.76 (m, 4H), d 3.45 (s, 2H), d 2.41–2.44 (m, 4H). HREIMS calcd
hexanes silica gel chromatography to afford
a yellow solid
(0.16 g, 55%). Mp: 58–60 °C. IR (film, CH2Cl2): 3438 cmꢁ1 (NH);
1H NMR (300 MHz, CDCl3): d 7.86 (d, J = 6.0 Hz, 1H), d 7.22–7.32
(m, 5H), d 5.61 (d, J = 6.0 Hz, 1H), d 4.84 (br s, 1H), d 4.71–4.75
(m, 2H), d 4.49 (d, J = 6.0 Hz, 2H), d 2.66–2.74 (m, 2H), d 1.65–
1.69 (m, 2H), d 1.38–1.48 (m, 1H), d 1.06–1.21 (m, 5H), d 0.85–
0.88 (m, 6H). HREIMS calcd for C19H26N4 (M+) m/z 310.4365, found
310.3086. Anal. Calcd for C19H26N4: C, 73.51; H, 8.44, N, 18.05.
Found: C, 73.51; H, 8.57; N, 17.91.
C
22H24BrN5 (M+) m/z 438.3635, found 438.2430. Anal. Calcd for
C22H24BrN5ꢀ0.2EtOAc: C, 60.06; H, 5.66, N, 15.36. Found: C, 60.07;
H, 5.65; N, 15.39.
4.3.11. N-Benzyl-2-(4-propylpiperazin-1-yl)pyrimidin-4-amine
(7k)
4.3.16. N-Benzyl-2-[4-(4-fluorobenzyl)piperazin-1-
yl]pyrimidin-4-amine (7p)
The product was obtained after coupling 7 with n-propylpiper-
azine (0.15 g, 1.12 mmol) and was purified using a 3:1 EtOAc/hex-
anes silica gel chromatography to afford a light orange solid
(0.16 g, 55%). Mp: 93–95 °C. IR (film, CH2Cl2): 3436 cmꢁ1 (NH);
1H NMR (300 MHz, CDCl3): d 7.85 (d, J = 6.0 Hz, 1H), d 7.21–7.30
(m, 5H), d 5.64 (d, J = 6.0 Hz, 1H), d 4.93 (br s, 1H), d 4.48 (d,
J = 6.0 Hz, 2H), d 3.71–3.75 (m, 4H), d 2.39–2.42 (m, 4H), d 2.27–
2.30 (m, 2H), d 1.49–1.56 (m, 2H), d 0.87–0.91 (m, 3H). HREIMS
calcd for C18H25N5 (M+) m/z 311.4246, observed 311.2008. Anal.
Calcd for C18H25N5ꢀ0.6H2O: C, 67.09; H, 8.20, N, 21.73. Found: C,
67.11; H, 7.85; N, 21.55.
The product was obtained after coupling 7 with 4-fluorobenzyl-
piperazine (0.22 g, 1.12 mmol) to afford an orange semi-solid
(0.22 g, 65%). IR (film, CH2Cl2): 3439 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 7.84 (d, J = 6.0 Hz, 1H), d 7.26–7.29 (m, 7H),
d 6.96–7.02 (m, 3H), d 5.63 (d, J = 6.0 Hz, 1H), d 5.17 (br s, 1H), d
4.47 (d, J = 6.0 Hz, 2H), d 3.74–3.76 (m, 4H), d 3.46 (s, 2H), d 2.40
(t, J = 6.0 Hz, 4H). HREIMS calcd for C22H24FN5 (M+) m/z 377.4579,
found 377.1980. Anal. Calcd for C22H24FN5ꢀ0.5DCM: C, 64.36; H,
6.00, N, 16.68. Found: C, 63.95; H, 5.97; N, 16.54.
4.3.17. N-Benzyl-2-[4-(4-trifluoromethylbenzyl)piperazin-1-
yl]pyrimidin-4-amine (7q)
4.3.12. 2-[4-(4-(Benzylamino)pyrimidin-2-yl)piperazin-1-
yl]ethanol (7l)
The product was obtained after coupling 7 with 4-trifluorome-
thybenzylpiperazine (0.24 mL, 1.12 mmol) to afford a yellowish or-
ange solid (0.25 g, 64%). Mp: 88–90 °C. IR (film, CH2Cl2): 3437 cmꢁ1
(NH). 1H NMR (300 MHz, CDCl3): d 7.85 (d, J = 6.0 Hz, 1H), d 7.55–
7.58 (m, 2H), d 7.44–7.47 (m, 2H), d 7.24–7.34 (m, 5H), d 5.65 (d,
J = 6.0 Hz, 1H), d 5.00 (br s, 1H), d 4.48 (d, J = 6.0 Hz, 2H), d 3.75–
3.78 (m, 4H), d 3.55 (s, 2H), d 2.43–2.46 (m, 4H). HREIMS calcd
The product was obtained after coupling 7 with hydroxyethyl-
piperazine (0.14 mL, 1.12 mmol). Residue was re-dissolved in 1:1
EtOAc/DCM and was purified using a 3:1 EtOAc/hexanes silica gel
chromatography to afford a brownish yellow solid (0.14 g, 50%).
Mp: 103–105 °C. IR (film, CH2Cl2): 3439 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 7.84 (d, J = 6.0 Hz, 1H), d 7.24–7.34 (m, 5H),
d 5.65 (d, J = 6.0 Hz, 1H), d 5.01 (br s, 1H), d 4.48 (d, J = 6.0 Hz,
2H), d 3.73–3.76 (m, 4H), d 3.60–3.64 (m, 2H), d 2.94 (s, 1H), d
2.51–2.55 (m, 2H), d 2.47–2.50 (m, 4H). HREIMS calcd for
C
C
23H24F3N5 (M+) m/z 427.4654, found 427.2203. Anal. Calcd for
23H24F3N5: C, 64.62; H, 5.66, N, 16.38. Found: C, 64.39; H, 5.64;
N, 16.12.
C
C
17H23N5O (M+) m/z 313.3974, found 313.1637. Anal. Calcd for
17H23N5Oꢀ0.3DCM: C, 60.20; H, 6.78, N, 20.65. Found: C, 60.03;
4.3.18. N4-Benzyl-N2-(1-benzylpiperidin-4-yl)pyrimidine-2,4-
diamine (7r)
H, 6.89; N, 20.10.
The product was obtained after coupling 7 with 4-amino-1-ben-
zylpiperidine (0.23 mL, 1.12 mmol) to afford a dark orange solid
(0.20 g, 59%). Mp: 88–90 °C. IR (film, CH2Cl2): 3438 cmꢁ1 (NH).
1H NMR (300 MHz, CDCl3): d 7.78 (d, J = 6.0 Hz, 1H), d 7.21–7.34
(m, 10H), d 5.66 (d, J = 6.0 Hz, 1H), d 4.92 (br s, 1H), d 4.46 (d,
J = 6.0 Hz, 2H), d 3.76–3.82 (m, 1H), d 3.48 (s, 2H), d 2.76–2.80
(m, 2H), 2.09–2.16 (m, 2H), 1.95–1.99 (m, 3H), 1.42–1.55 (m,
2H). HREIMS calcd C23H27N5 (M+) m/z 373.4940, found 373.2008.
Anal. Calcd for C23H27N5ꢀDCM: C, 62.88; H, 6.38, N, 15.28. Found:
C, 62.88; H, 6.38; N, 15.28.
4.3.13. N-Benzyl-2-[4-(2-methoxyethyl)piperazin-1-
yl]pyrimidin-4-amine (7m)
The product was obtained after coupling 7 with methoxyethyl-
piperazine (0.17 mL, 1.12 mmol) to afford an orange semi-solid
(0.17 g, 57%). Mp: 63–65 °C. IR (film, CH2Cl2): 3433 cmꢁ1 (NH);
1H NMR (300 MHz, CDCl3): d 7.82 (d, J = 6.0 Hz, 1H), d 7.21–7.31
(m, 5H), d 5.62 (d, J = 6.0 Hz, 1H), d 5.06 (br s, 1H), d 4.46 (d,
J = 6.0 Hz, 2H), d 3.74–3.77 (m, 4H), d 3.48–3.52 (m, 2H), d 3.32
(s, 3H), d 2.54–2.58 (m, 2H), d 2.45–2.48 (m, 4H). HREIMS calcd
for C18H25N5O (M+) m/z 327.4240, found 327.2040.
4.3.19. N-Phenethyl-2-(pyrrolidin-1-yl)pyrimidin-4-amines (8a)
The product was obtained by coupling 8 with pyrrolidine to af-
ford a light yellowish brown solid (0.15 g, 65%). Mp: 85–87 °C. IR
(film, CH2Cl2): 3436 cmꢁ1 (NH); 1H NMR (300 MHz, CDCl3): d
7.86 (d, J = 6.0 Hz, 1H), d 7.18–7.32 (m, 5H), d 5.59 (d, J = 6.0 Hz,
1H), d 4.56 (br s, 1H), d 3.55 (t, J = 6.0 Hz, 2H), d 3.50 (t, J = 6.0 Hz,
4H), d 2.86–2.91 (m, 2H), d 1.90–1.93 (m, 4H). HREIMS C16H20N4
4.3.14. N-Benzyl-2-[4-(4-chlorobenzyl)piperazin-1-yl]pyrimidin-
4-amine (7n)
The product was obtained after coupling 7 with 4-chlorobenzyl-
piperazine (0.22 mL, 1.12 mmol). The sample was purified using a
9:1 EtOAc/DCM silica gel column to afford a yellowish solid (0.20 g,

T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
2279
(M+) m/z 268.3568, found 268.2125. Anal. Calcd for
CH2Cl2): 3433 cmꢁ1 (NH); 1H NMR (300 MHz, CDCl3) d 8.05 (d,
J = 6.0 Hz, 1H), d 7.78–7.91 (m, 3H), d 7.38–7.51 (m, 4H), d 5.64
(d, J = 6.0 Hz, 1H), d 4.96 (d, J = 6.0 Hz, 2H), d 4.79 (br s, 1H), d
3.53–3.57 (m, 4H), d 1.91–1.95 (m, 4H). HREIMS calcd C19H20N4
(M+) m/z 304.3889, found 304.2086. Anal. Calcd for
C
16H20N4ꢀ0.5H2O: C, 69.29; H, 7.63, N, 20.2. Found: C, 69.51; H,
7.32; N, 20.11.
4.3.20. 2-Morpholino-N-phenethylpyrimidin-4-amine (8b)
The product was obtained after coupling 8 with morpholine
(0.10 mL, 1.11 mmol) to afford a light brown solid (0.21 g, 86%).
Mp: 93–95 °C. IR (film, CH2Cl2): 3433 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 7.85 (d, J = 6.0 Hz, 1H), d 7.18–7.32 (m, 5H),
d 5.65 (d, J = 6.0 Hz, 1H), d 4.59 (br s, 1H), d 3.68–3.75 (m, 8H), d
3.55–3.59 (m, 2H), d 2.86–2.90 (m, 2H). HREIMS calcd C16H20N4O
(M+) m/z 284.3562, found 284.1725. Anal. Calcd for C16H20N4O: C,
67.58; H, 7.09, N, 19.70. Found: C, 67.71; H, 7.14; N, 19.42.
C
19H20N4ꢀ0.2H2O: C, 74.02; H, 6.49, N, 18.18. Found: C, 74.09; C,
6.68; N, 18.19.
4.3.26. 2-Morpholino-N-(naphth-1-ylmethyl)pyrimidin-4-
amine (9b)
The product was obtained after coupling 9 with morpholine
(0.08 mL, 0.96 mmol) to afford a light yellow solid (0.18 g, 75%).
Mp: 170–172 °C. IR (film, CH2Cl2): 3437 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 8.01 (d, J = 6.0 Hz, 1H), d 7.79–7.90 (m, 3H),
d 7.39–7.52 (m, 4H), d 5.71 (d, J = 6.0 Hz, 1H), d 4.92 (d, J = 6.0 Hz,
2H), d 4.77 (br s, 1H), d 3.69–3.77 (m, 8H). HREIMS calcd
4.3.21. N-Phenethyl-2-thiomorpholinopyrimidin-4-amine (8c)
The product was obtained after coupling 8 with thiomorpholine
(0.11 mL, 1.11 mmol) to afford a brown solid (0.21 g, 81%). Mp: 60–
62 °C. IR (film, CH2Cl2): 3437 cmꢁ1 (NH); 1H NMR (300 MHz, CDCl3)
d 7.84 (d, J = 6.0 Hz, 1H), d 7.18–7.30 (m, 5H), d 5.61 (d, J = 6.0 Hz,
1H), d 4.58 (br s, 1H), d 3.98 (t, J = 6.0 Hz, 4H), d 3.54 (t, J = 6.0 Hz,
2H), d 2.86–2.90 (m, 2H), d 2.61 (t, J = 6.0 Hz, 4H). HREIMS calcd
C
C
19H20N4O (M+) m/z 320.3883, found 320.1825. Anal. Calcd for
19H20N4O: C, 71.23; H, 6.29, N, 17.49. Found: C, 71.28; H, 6.37;
N, 17.08.
4.3.27. N-(Naphth-1-ylmethyl)-2-thiomorpholinopyrimidin-4-
amine (9c)
C
16H20N4S (M+) m/z 284.3562, found 284.1725. Anal. Calcd for
C16H20N4S: C, 63.97; H, 6.71, N, 18.65. Found: C, 64.12; H, 6.85;
N, 18.46.
The product was obtained after coupling 9 thiomorpholine
(0.10 mL, 0.96 mmol) to afford a yellowish brown solid (0.19 g,
76%). Mp: 105–107 °C. IR (film, CH2Cl2): 3439 cmꢁ1 (NH); 1H
NMR (300 MHz, CDCl3): d 8.01 (d, J = 6.0 Hz, 1H), d 7.73–7.87 (m,
3H), d 7.39–7.51 (m, 4H), d 5.68 (d, J = 6.0 Hz, 1H), d 4.93 (d,
J = 6.0 Hz, 2H), d 4.82 (br s, 1H), d 4.05–4.12 (m, 4H), d 2.59–2.65
(m, 4H). HREIMS calcd C19H20N4S (M+) m/z 336.4539, found
336.2171. Anal. Calcd for C19H20N4S: C, 67.83; H, 5.99, N, 16.65.
Found: C, 67.78; H, 5.86; N, 16.50.
4.3.22. 2-(4-Methylpiperazin-1-yl)-N-phenethylpyrimidin-4-
amine (8d)
The product was obtained after coupling 8 with methylpipera-
zine (0.12 mL, 1.11 mmol) to afford a light yellow semi-solid
(0.18 g, 69%). Mp: 58–60 °C IR (film, CH2Cl2): 3436 cmꢁ1 (NH); 1H
NMR (300 MHz, CDCl3): d 7.85 (d, J = 6.0 Hz, 1H), d 7.18–7.30 (m,
5H), d 5.62 (d, J = 6.0 Hz, 1H), d 4.57 (br s, 1H), d 3.76–3.79 (m,
4H), d 3.52–3.56 (m, 2H), d 2.86–2.90 (m, 2H), d 2.41–2.44 (m,
4H), d 2.31 (s, 3H). HREIMS calcd C17H23N5 (M+) m/z 297.3980,
found 297.1958. Anal. Calcd for: C17H23N5ꢀ13 H2O; C, 68.05; H,
7.67, N, 23.35. Found: C, 68.12; H, 7.82; N, 23.36.
4.3.28. 2-(4-Methylpiperazin-1-yl)-N-(naphth-1-
ylmethyl)pyrimidin-4-amine (9d)
The product was obtained after coupling 9 with methylpipera-
zine (0.11 mL, 0.96 mmol) to afford a yellow solid (0.23 g, 80%).
Mp: 118–120 °C. IR (film, CH2Cl2): 3436 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 8.03 (d, J = 6.0 Hz, 1H), d 7.78–7.90 (m, 3H),
d 7.39–7.52 (m, 4H), d 5.67 (d, J = 6.0 Hz, 1H), d 4.94 (d, J = 6.0 Hz,
2H), d 4.80 (br s, 1H), d 3.81–3.85 (m, 4H), d 2.41–2.45 (m, 4H), d
2.32 (s, 3H). HREIMS calcd C20H23N5 (M+) m/z 333.4301, found
333.1961. Anal. Calcd for C20H23N5ꢀ0.5EtOAc: C, 70.01; H, 7.21, N,
18.55. Found: C, 69.72; H, 7.25; N, 18.21.
4.3.23. 2-(4-Methylpiperidin-1-yl)-N-phenethylpyrimidin-4-
amine (8e)
The product was obtained after coupling 8 with 4-methylpiper-
idine (0.13 mL, 1.11 mmol) to afford a light brown solid (0.20 g,
79%). Mp: 65–67 °C. IR (film, CH2Cl2): 3439 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 7.84 (d, J = 6.0 Hz, 1H), d 7.18–7.33 (m, 5H),
d 5.57 (d, J = 6.0 Hz, 1H), d 4.64–4.69 (m, 2H), d 4.53 (br s, 1H), d
3.52–3.56 (m, 2H), d 2.86–2.90 (m, 2H), d 2.73–2.82 (m, 2H), d
1.63–1.68 (m, 3H), d 1.33–1.40 (m, 1H), d 1.08–1.16 (m, 3H), d
4.3.29. 2-(4-Methylpiperidin-1-yl)-N-(naphth-1-
ylmethyl)pyrimidin-4-amine (9e)
0.92 (d, J = 6.0 Hz, 3H). HREIMS calcd
C
18H24N4 (M+) m/z
The product was obtained after coupling 9 with 4-methylpiper-
idine (0.11 mL, 0.96 mmol). The residue was purified using 3:1
ether/hexanes column to afford an off-white/light yellow semi-so-
lid (0.14 g, 55%). IR (film, CH2Cl2): 3439 cmꢁ1 (NH); 1H NMR
(300 MHz, CDCl3): d 8.03 (d, J = 6.0 Hz, 1H), d 7.78–7.91 (m, 3H),
d 7.38–7.53 (m, 4H), d 5.62 (d, J = 6.0 Hz, 1H), d 4.94 (d, J = 6.0 Hz,
2H), d 4.75 (br s, 1H), d 4.68–4.72 (m, 3H), d 2.76–2.84 (m, 2H), d
1.64–1.68 (m, 2H), 1.57–1.60 (m, 1H), 1.10–1.14 (m, 4H), 0.92 (d,
J = 6.0 Hz, 3H). HREIMS calcd C21H24N4 (M+) m/z 332.4421, found
332.2246. Anal. Calcd for C21H24N4ꢀ0.5EtOAc: C, 73.38; H, 7.50, N,
14.88. Found: C, 73.07; H, 7.52; N, 14.64.
296.4100, found 296.2380. Anal. Calcd for C18H24N4: C, 72.94; H,
8.16, N, 18.90. Found: C, 72.38; H, 7.99; N, 18.34.
4.3.24. 1-[4-(4-(Phenethylamino)pyrimidin-2-yl)piperazin-1-
yl]ethanone (8f)
The product was obtained after coupling 8 with acetylpiperazine
(0.14 g, 1.11 mmol) to afford a yellow solid (0.24 g, 86%). Mp:
150–152 °C. IR (film, CH2Cl2): 3437 cmꢁ1 (NH); 1H NMR (300 MHz,
CDCl3): d 7.85 (d, J = 6.0 Hz, 1H), d 7.19–7.33 (m, 5H), d 5.66 (d,
J = 6.0 Hz, 1H), d 4.61 (br s, 1H), d 3.73–3.80 (m, 4H), d 3.65 (t,
J = 6.0 Hz, 2H), d 3.55 (t, J = 6.0 Hz, 2H), d 3.46–3.50 (m, 2H), d
2.86–2.90 (m, 2H), d 2.12 (s, 3H). HREIMS calcd C18H23N5O (M+) m/z
325.4081, found 325.1913. Anal. Calcd for C18H23N5ꢀ0.6EtOAc: C,
64.78; H, 7.41, N, 18.52. Found: C, 64.66; H, 7.43; N, 18.30.
4.4. Preparation of 4-[4-(benzylamino)pyrimidin-2-
yl]thiomorpholine-1-oxide (7s)
To a mixture of N-benzyl-2-thiomorpholinopyrimidin-4-amine
(7c) (0.20 g, 0.70 mmol) in 4 mL of 1,4-dioxane, kept at 0 °C (ice-
bath), mCPBA (0.19 g, 1.12 mmol) was added dropwise. The reac-
tion is allowed to stir on the ice-bath for 5 min and then was kept
at rt for 3 h. DCM was added to the mixture to aid in the 1,4-
4.3.25. N-(Naphth-1-ylmethyl)-2-(pyrrolidine)pyrimidin-4-
amines (9a)
The product was obtained after coupling 9 with pyrrolidine to
afford a light brown solid (0.16 g, 70%). Mp: 105–107 °C. IR (film,

2280
T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
dioxane in vacuo evaporation. The residue was re-dissolved in a
St. Louis, MO) and equine serum BuChE (product number C1057,
Sigma-Aldrich, St. Louis, MO) was determined using Ellman’s
method (IC50 values, lM) using appropriate reference agents
solvent mixture of EtOAc and DCM in ꢂ3:1 ratio and successfully
washed with
a
concentrated NaHCO3 and NaCl solution
(1 ꢃ 15 mL). Aqueous layer was washed with EtOAc (3 ꢃ 15 mL)
and the combined organic layer was dried over anhydrous MgSO4
and filtered. The organic layer was evaporated in vacuo and the
resulting solid residue was further purified by silica gel column
chromatography using 3:1 EtOAc/hexanes to afford a light yellow
solid (0.16 g, 75%). Mp: 78–80 °C. IR (film, CH2Cl2): 3439 cmꢁ1
(NH); 1H NMR (300 MHz, CDCl3): d 7.87 (d, J = 6.0 Hz, 1H), d
7.24–7.35 (m, 5H), d 5.74 (d, J = 6.0 Hz, 1H), d 4.99 (br s, 1H), d
4.49 (d, J = 6.0 Hz, 2H), d 4.37 (t, J = 6.0 Hz, 1H), d 4.32 (t,
J = 6.0 Hz, 1H), 4.10–4.19 (m, 2H), 2.69–2.73 (m, 4H). HREIMS calcd
tacrine hydrochloride (item number 70240, Cayman Chemical,
Ann Arbor, MI), bis(7)-tacrine (item number 10005836, Cayman
Chemical, Ann Arbor, MI), and galanthamine hydrobromide (prod-
uct number G1660, Sigma, St. Louis, MO). Stock solutions of test
compounds were dissolved in a minimum volume of DMSO (1%)
and were diluted using the buffer solution (50 mM Tris–HCl, pH
8.0, 0.1 M NaCl, 0.02 M MgCl2ꢀ6H2O). In 96-well plates, 160
5,50-dithiobis(2-nitrobenzoic acid) (1.5 mM DTNB), 50
L of AChE
(0.22 U/mL prepared in 50 mM Tris–HCl, pH 8.0, 0.1% w/v bovine
serum albumin, BSA) or 50 L of BuChE (0.06 U/mL prepared in
50 mM Tris–HCl, pH 8.0, 0.1% w/v BSA) were incubated with
lL
l
l
C
C
15H18N4OS (M+) m/z 302.3946, found 302.1259. Anal. Calcd for
15H18N4OSꢀ0.3DCM: C, 56.05; H, 5.72, N, 17.09. Found: C, 56.38;
10
100
l
l
L
of various concentrations of test compounds (0.001–
M) at room temperature for 5 min followed by the addition
H, 5.74; N, 17.22.
of the substrates (30 lL) acetylthiocholine iodide (15 mM ATCl)
4.5. Preparation of 4-[4-(benzylamino)pyrimidin-2-
yl]thiomorpholine-1,1-dioxide (7t)
or S-butyrylthiocholine iodide (15 mM BTCI) and the absorbance
was measured at different time intervals (0, 60, 120 and 180 s) at
a wavelength of 405 nm BioTek ELx800 microplate reader. Percent
inhibition was calculated by the comparison of compound treated
to various control incubations that included 1% DMSO. The concen-
tration of the test compound causing 50% inhibition (IC50, lM) was
calculated from the concentration–inhibition response curve on
logarithmic scale (duplicate to quadruplicate determinations).
To a mixture of N-benzyl-2-thiomorpholinopyrimidin-4-amine
(7c) (0.20 g, 0.70 mmol) in 5 mL of MeOH, kept at 0 °C (ice-bath),
potassium peroxymonosulphate (0.23 g, 1.54 mmol) in H2O was
added dropwise. The reaction was allowed to stir on the ice-bath
for 5 min and then was kept at 70–75 °C for 1 h. Four milliliters
of 1,4-dioxane were added to the mixture and the reaction vessel
was moved to rt for 4 h. MeOH and 1,4-dioxane were evaporated
in vacuo and the residue was re-dissolved in 3:1 EtOAc/DCM and
successfully washed with a concentrated NaHCO3 and NaCl solu-
tion (1 ꢃ 15 mL). Aqueous layer was washed with EtOAc
(3 ꢃ 15 mL) and the combined organic layer was dried over anhy-
drous MgSO4 and filtered. The organic layer was evaporated in va-
cuo and the resulting solid residue was further purified by silica gel
column chromatography using 3:1 EtOAc/hexanes to afford a light
orange solid (0.17 g, 75%). Mp: 65–67 °C. IR (film, CH2Cl2): 3464 cm
4.8. hAChE-induced Ab aggregation inhibition studies
Thioflavin T (ThT) is a benzothiazole salt frequently used in the
detection of amyloid plaque formation. As Ab peptides start to
aggregate into oligomers and fibrils, ThT binds to the beta sheets
formed as a result of the aggregation and the detectable change
in its emission spectrum is used to quantify the degree of aggrega-
tion.32–34 Ab1–40 was purchased from GL Biochem Ltd. Human re-
combinant AChE lyophilized powder was purchased from Sigma
Aldrich (product number C1682) and ThT was purchased from
Fisher Scientific. Ab1–40 was prepared as previously described.32
Lyophilized Ab1–40 was dissolved in DMSO to obtain a 2 mM solu-
ꢁ1
(NH); 1H NMR (300 MHz, CDCl3): d 7.87 (d, J = 6.0 Hz, 1H), d
7.25–7.35 (m, 5H), d 5.79 (d, J = 6.0 Hz, 1H), d 5.14 (br s, 1H), d
4.49 (d, J = 6.0 Hz, 2H), d 4.21–4.25 (m, 4H), d 2.88–2.92 (m, 4H),
4.10–4.19 (m, 2H), 2.69–2.73 (m, 4H). HREIMS calcd C15H10N4O2S
(M+) m/z 318.3946, found 318.1310. Anal. Calcd for
C15H18N4OS2ꢀ0.2DCM: C, 51.95; H, 5.28, N, 15.94. Found: C,
52.04; H, 5.34; N, 15.71.
tion and then diluted into 500
phosphate buffer (pH 8.0). Aliquots (2
with 16 L of hAChE, which was dissolved in 0.215 M sodium
phosphate buffer (pH 8.0), to give a final concentration of 50
of Ab1–40 and 230 M of hAChE. For co-incubation experiments,
L of the test compounds in 0.215 M sodium phosphate buffer
pH 8.0 solution (final concentration 100 M) were added into ali-
quots (2 L) of Ab1–40 (final concentration 50 M) along with
16 L of hAChE (final concentration 230 M). The reaction mix-
tures were incubated at room temperature for 24 h and 100 L of
ThT (20 M) in 50 mM glycine-NaOH buffer (pH 8.5) was added.
lM solution with 0.215 M sodium
lL) of Ab1–40 were incubated
l
lM
l
4.6. Preparation of N-benzyl-2-(piperazin-1-yl)pyrimidin-4-
amine (7u)
2 l
l
l
l
To a mixture of tert-butyl 4-(4-(benzyl amino)pyrimidin-2-
yl)piperazine-1-carboxylate (7g) (0.20 g, 0.54 mmol) in 4 mL of
DCM, kept at 0 °C (ice-bath), TFA (4 mL, 53.83 mmol) was added
dropwise. The reaction was allowed to stir on the ice-bath for
5 min and then was kept at rt for 2 h. DCM is evaporated in vacuo
and the residue was re-dissolved in 3:1 EtOAc/DCM and successfully
washed with a concentrated NaHCO3 and NaCl solution (1 ꢃ 15 mL).
Aqueous layer was washed with EtOAc (3 ꢃ 15 mL) and the com-
bined organic layer was dried over anhydrous MgSO4 and filtered.
The organic layer was evaporatedin vacuo to afford a light yellowso-
lid (0.11 g, 75%). Mp: 70–72 °C. IR (film, CH2Cl2): 3438 cmꢁ1 (NH); 1H
NMR (300 MHz, CDCl3): d 7.86 (d, J = 6.0 Hz, 1H), d7.24–7.34 (m, 5H),
d 5.66 (d, J = 6.0 Hz, 1H), d 4.91 (br s, 1H), d 4.49 (d, J = 6.0 Hz, 2H), d
3.73–3.76 (m, 4H), 3.09 (s, 1H), d 2.88–2.91 (m, 4H). HREIMS calcd
l
l
l
l
Fluorescence was monitored at 442 nm and emission at 490 nm
using a Proton Technology International (PTI, Birmingham, NJ)
spectrofluorometer. Each assay was run in triplicates along with
donepezil (item number 13245, Caymen Chemical, Ann Arbor,
MI) and propidium iodide (product number C4170, Sigma–Aldrich,
St. Louis, MO) as reference agents. The fluorescence intensities in
the presence and absence of inhibitors were compared using
appropriate controls containing 1% DMSO and the percentage of
inhibition was calculated using the equation: 100 ꢁ (IFi/IFo ꢃ 100)
where IFi and IFo are the fluorescence intensities obtained for Ab1–
40 + hAChE in the presence and absence of inhibitor, respectively.
C
15H19N5 (M+) m/z 269.3449, found 269.1953.
4.9. SH-SY5Y neuroblastoma cell toxicity studies
4.7. Cholinesterase inhibition assay
The cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide, MTT (Product No. 30-1010K, from
American Type Culture Collection, Manassas, VA) was measured
The ability of the test compounds (7a–u, 8a–f and 9a–e) to
inhibit human AChE (product number C3389, Sigma-Aldrich,

T. Mohamed et al. / Bioorg. Med. Chem. 19 (2011) 2269–2281
2281
by detecting a purple formazan intermediate at 570 nm. By incu-
bating the cells with varying concentrations of test compounds,
their toxicity attributes can be determined directly by determining
the %reduction of MTT or indirectly by reporting the %viability of
the cell line used.35,36 The SH-SY5Y neuroblastoma cells were pla-
support of this research project. This work was also partially
supported by the Alzheimer’s Association (NIRG-08-91651) and
the Alzheimer’s Disease Research Center (NIH 3P50 AG005131).
The NSF is also acknowledged for a CAREER Award to JY (CHE-
0847530).
ted at a density of 50,000 cells per well in 100 lL of complete med-
ia consisting of a 1:1 mixture of Eagle’s Minimum Essential
Medium (EMEM) and Ham’s F12, supplemented with 10% Fetal Bo-
vine Serum (FBS). The cells were incubated overnight before treat-
Supplementary data
Supplementary data [a table of molecular volume (Å3) for some
compounds are given] associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.02.030.
ment with 100 lL of test sample solutions and select controls
(tacrine hydrochloride, item number 70240, Caymen Chemical,
Ann Arbor, MI and galanthamine hydrobromide, product number
G1660 Sigma–Aldrich, St. Louis, MO) at various concentrations
References and notes
(0–160 lM) in 2% DMSO for 24 h at 37 °C (n = 4). A 20 lL of the
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4.10. Molecular modeling (docking) studies
Docking experiments were performed using Discovery Studio
Client v2.5.0.9164 (2005-09), Accelrys Software Inc. running on a
HP xw4600 workstation (Processor x86 family 6 model 23 stepping
10 GenuineIntel 2999 MHz). The coordinates for the X-ray crystal
structure of the enzyme hAChE and hBuChE were obtained from
the RCSB Protein Data Bank and hydrogens were added. The ligand
molecules were constructed using the Build Fragment tool and en-
ergy minimized for 1000 iterations reaching a convergence of
0.01 kcal/mol Å. The docking experiment on hAChE was carried
out by superimposing the energy minimized ligand in the PDB file
1B41 after which fasciculin was deleted. The coordinates for
hBuChE was obtained from PDB file 1P0I and the energy minimized
ligand was superimposed and the resulting ligand–enzyme com-
plex was subjected to docking using the Libdock command in the
receptor–ligand interactions protocol of Discovery Studio after
defining subsets of the enzyme within 10 Å sphere radius of the li-
gand. The force field, Chemistry at HARvard Macromolecular
Mechanics (CHARMM) was employed for all docking purposes.
The ligand–enzyme assembly was then subjected to a molecular
dynamics (MD) simulation using Simulation protocol at a constant
temperature of 300 K with a 100 step equilibration for over 1000
iterations and a time step of 1 fs using a distance dependent
dielectric constant 4r. The optimal binding orientation of the
ligand–enzyme assembly obtained after docking was further min-
imized for 1000 iterations using the conjugate gradient method
until a convergence of 0.001 kcal/mol Å was reached after which
Eintermolecular (kcal/mol) of the ligand–enzyme assembly was
evaluated.
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Dickerson, T. J.; Janda, K. D. Mol. Pharm. 2006, 3, 773.
Acknowledgments
The authors would like to thank the Department of Biology and
the School of Pharmacy at the University of Waterloo for financial
35. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
36. Vellonen, K.; Honkakoski, P.; Urtti, A. Eur. J. Pharm. Sci. 2004, 23, 181.