Development of 2-substituted-N-(naphth-1-ylmethyl) and N- benzhydrylpyrimidin-4-amines as dual cholinesterase and Aβ-aggregation inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2011.07.091

A group of 2-substituted N-(naphth-1-ylmethyl)pyrimidin-4-amines (6a-k) and N-benzhydrylpyrimidin-4-amines (7a-k) in conjunction with varying steric and electronic properties at the C-2 position were designed, synthesized and evaluated as dual cholinester

Full text of DOI:10.1016/j.bmcl.2011.07.091

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5881–5887
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of 2-substituted-N-(naphth-1-ylmethyl) and N-benzhydrylpyrim-
idin-4-amines as dual cholinesterase and Ab-aggregation inhibitors: Synthesis
and biological evaluation
Tarek Mohamed a,b, Jacky C.K. Yeung a,b, Praveen P.N. Rao b,
⇑
a Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
b School of Pharmacy, Health Sciences Campus, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of 2-substituted N-(naphth-1-ylmethyl)pyrimidin-4-amines (6a–k) and N-benzhydrylpyrimidin-
4-amines (7a–k) in conjunction with varying steric and electronic properties at the C-2 position were
designed, synthesized and evaluated as dual cholinesterase and amyloid-b (Ab)-aggregation inhibitors.
The naphth-1-ylmethyl compound 6f (2-(4-cyclohexylpiperazin-1-yl)-N-(naphth-1-ylmethyl)pyrimi-
Received 4 July 2011
Revised 22 July 2011
Accepted 25 July 2011
Available online 30 July 2011
din-4-amine) exhibited optimum dual ChE (AChE IC50 = 8.0
moted Ab-aggregation inhibition (30.8% at 100 M), whereas in the N-benzhydryl series, compound 7f
(N-benzhydryl-2-(4-cyclohexylpiperazin-1-yl)pyrimidin-4-amine) exhibited optimum combination of
dual ChE (AChE IC50 = 10.0 M, BuChE IC50 = 7.6 M) and hAChE-promoted Ab-aggregation inhibition
(32% at 100 M). These results demonstrate that a 2,4-disubstituted pyrimidine ring serves as a suitable
lM, BuChE IC50 = 3.9 lM) and hAChE-pro-
l
Keywords:
Cholinesterase inhibitors (ChEIs)
Acetylcholinesterase (AChE)
Butyrylcholinesterase (BuChE)
Human acetylcholinesterase (hAChE)
Structure-activity relationship (SAR)
Amyloid-b (Ab)
l
l
l
template to target multiple pathological routes in AD, with a C-2 cyclohexylpiperazine substituent pro-
viding dual ChE inhibition and potency whereas a C-4 diphenylmethane substituent provides Ab-aggre-
gation inhibition.
5,50-Dithiobis(2-nitrobenzoic acid) (DTNB)
Ó 2011 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a devastating neurodegenerative
disease that targets the cholinergic regions of the central nervous
system (CNS) associated with cognitive ability and spatial aware-
ness.1 The pathological initiation and progression of AD is highly
complex and its prevalence is on the rise with significant socioeco-
nomic impact that places a heavy burden on patients and their care
providers.2,3 Some molecular and physical characteristics of AD in-
clude the progressive loss of cholinergic neurons leading to cogni-
tive dysfunction, the accelerated generation and aggregation of
amyloid-b (Ab) fibrils and the formation of neurofibrillary tangles
(NFTs).2–6 These findings support the basis for the cholinergic,
amyloid and tau hypotheses of AD etiology.
The cholinergic dysfunction hypothesis attributes the pathology
of AD to the systemic collapse of acetylcholine (ACh) mediated
neurotransmission in the cortical regions of the CNS.7 Its action
in the synapse is terminated by cholinesterase enzymes; acetyl-
and butyrylcholinesterase (AChE and BuChE), respectively.8,9
Impairment of the ACh synthesizing enzyme (choline acetlytrans-
ferase – ChAT) in AD patients also contributes to the overall decline
of ACh concentration in the CNS.9–12 Furthermore, recent evidence
suggests that AChE plays a vital role in the early stages of AD; how-
ever, as the disease progresses BuChE, with a wider distribution
within the body, acts as the major ACh degrading enzyme indicat-
ing the need to develop dual AChE and BuChE inhibitors.13,14
According to the amyloid hypothesis, the accelerated genera-
tion of Ab1–40/42-peptides and their rapid oligomerization and
aggregation to toxic Ab-plaques is a major factor for AD etiol-
ogy.15–17 Furthermore, recent studies have implicated metal-
ions18–24 and the peripheral anionic site (PAS) of AChE with facili-
tating the aggregation of those Ab-fibrils.25–29 These multiple fac-
tors signify the need to develop small molecule therapies that
could potentially target multiple pathways in AD pathology.
Research efforts aimed at developing cholinesterase inhibitors
(ChEIs) has led to the development of several fused and nonfused
ring systems with a wide range of inhibitory profiles. Some exam-
ples are tacrine (1),30 donepezil (AriceptÒ, 2), a piperidine-based
AChE inhibitor31 and propidium (3), a PAS specific AChE inhibitor
(Fig. 1).32 In addition, recent work by DeLisa et al. examined s-tri-
azine based ring templates (4) for their ability to inhibit the aggre-
gation of Ab1–42 plaques.33 In this regard, we previously reported
the design, synthesis and evaluation of a group of heterocyclic,
nonfused small molecules based on a 2,4-disubstituted pyrimidine
ring template with ChE and Ab-aggregation inhibitory profiles.34,35
Herein we expand on our efforts with the development of 2-
⇑
Corresponding author. Tel.: +1 519 888 4567x21317; fax: +1 519 888 7910.
E-mail address: praopera@uwaterloo.ca (P.P.N. Rao).
substituted-N-(naphth-1-ylmethyl)-pyrimidin-4-amines
(6a–k)
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.091

5882
T. Mohamed et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5881–5887
tuted-N-benzyhydrylpyrimidin-4-amines (7a–k) in moderate to
NH2
O
good yield (50–80%) (Scheme 1).34,35,40 The deprotection of the
tert-butoxycarbonyl (t-Boc) group of 6h [tert-butyl 4-(4-[(naphth-
1-ylmethyl)amino]pyrimidin-2-yl)piperazine-1-carboxylate] and
7h [tert-butyl 4-(4-[benzhydrylamino]pyrimidin-2-yl)piperazine-
1-carboxylate] was accomplished using 50% v/v trifluoroacetic acid
(TFA) to yield N-(naphth-1-ylmethyl)-2-(piperazin-1-yl)pyrimi-
din-4-amine (6i) and N-benzhydryl-2-(piperazin-1-yl)pyrimidin-
4-amine (7i), respectively in good yield (60%) (Scheme 1).
MeO
MeO
N
Donepezil 2
( )
Tacrine (1)
N
Me
Et
Et
Bn
N
Ph
O
HN
N
NH2
N
2
The ChE inhibitory (ChEI) profiles of the 2,4-disubstituted
pyrimidine derivatives (6a–k and 7a–k) were determined using
an in vitro assay based on a modified version of the Ellman proto-
col.41 The IC50 values, selectivity index (S.I.), partition coefficient
(C log P) and molecular volume (Å3) are reported in Tables 1 and
2 along with tacrine (1), donepezil (2) and galantamine as controls.
The SAR studies indicated that ChEI and selectivity were sensitive
to the steric and electronic parameters at both C-2 and C-4 posi-
tions of the pyrimidine ring. These derivatives exhibited a broad
range of inhibition (C-4 naphth-1-ylmethyl series, AChE
N
Me
N
H2N
NH2
N
(4)
N
Me
Propidium (3)
Me
Figure 1. Structures of ChEIs (1, 2) and Ab-aggregation inhibitors (3, 4).
and 2-substituted-N-benzhydrylpyrimidin-4-amines (7a–k) to ex-
plore their ChE and Ab inhibitory potential.36 In vitro ChE inhibi-
tion (ChEI)37 and structure–activity relationship (SAR) data are
discussed, along with their ability to inhibit hAChE-induced and
self-induced aggregation of Ab1–40 fibrils38 and some molecular
modeling investigations on their binding modes are described.
The synthesis of target derivatives (6a–k and 7a–k) was accom-
plished in two to three steps (Scheme 1). Initially, N-(naphth-1-yl-
methyl)-2-chloropyrimidin-4-amine (6) and N-benzhydryl-2-
chloropyrimidin-4-amine (7) intermediates were synthesized from
the 2,4-dichloropyrimidine starting material (5) by a nucleophilic
aromatic substitution reaction at the C-4 position using either
napth-1-ylmethanamine or benzhydrylamine in presence of N,N-
diisopropylethylamine (DIPEA). The reaction was run in EtOH at
80–85 °C and refluxed for 4 h. Intermediates 6 and 7 were obtained
in moderate to good yields ranging from 55% to 75%
(Scheme 1).34,35,39 In the second step, the C-2 chlorine was dis-
placed by various substituted cyclic amines (1-methylpiperazine,
4-methylpiperidine, N-isopropylpiperazine, 4-isopropylpiperidine,
N-propylpiperazine, 1-cyclohexylpiperazine, 1-acetylpiperazine,
t-butyl piperazine-1-carboxylate, N-(2-hydroxyethyl)piperazine
or 1-(2-methoxyethyl)piperazine). This reaction was run in n-buta-
nol under rigorous conditions (145–150 °C) for 50–60 min in a
sealed pressure vessel (PV) to afford the target 2-substituted-N-
(naphth-1-ylmethyl)pyrimidin-4-amines (6a–k) and 2-substi-
IC50 = 8.0–50.8
C-4 N-benzhydryl series, AChE IC50 = 10.0 to >100
BuChE IC50 = 7.6 to >100
M range).35
l
M range; BuChE IC50 = 2.2 to >100
l
M range and
lM range;
l
Among the naphth-1-ylmethyl series of derivatives (6a–k), the
substituent electronic and steric properties at C-2 position modu-
lated ChE inhibition (Table 1). The SAR of C-2 piperazine-substi-
tuted derivatives were explored by incorporating a wide range of
terminal 4-alkyl, cycloalkyl, acyl and alkoxy substituents (6a, c
and e–k). The presence of a methyl, isopropyl or N-propyl group
in 6a, c or e (X–R2 = 1-methylpiperazine, N-isopropylpiperazine
and N-propylpiperazine, respectively) provided equipotent AChE
inhibitory activity (IC50 = 17.5, 15.8 and 19.0 lM, respectively). In
contrast, BuChE inhibition and potency was dependent on the nat-
ure of the alkyl group attached. The presence of a smaller methyl
group in 6a provided potent and selective BuChE inhibition
(IC50 = 2.6
lM), whereas the presence of a branched isopropyl in
6c (IC50 = 7.6
l
M) or N-propyl side chain in 6e (IC50 = 18.1 M) re-
l
sulted in a ꢀthreefold decrease in potency. Replacing the isopropyl
group in 6c with a cycloalkyl group as in 6f (X–R2 = 1-cyclohexyl-
piperazine) resulted in a ꢀtwofold increase in both AChE and
BuChE inhibitory potency (IC50 = 8.0 and 3.9 lM, respectively) rel-
ative to 6c. In addition, 6f exhibited near equipotent BuChE inhibi-
tion compared to the reference drug donepezil (IC50 = 3.6 M). The
l
NH
NH
Cl
NH
NH
4
b
a
N
N
N
N
N
or
or
2
N
N
N
N
N
Cl
N
Cl
N
Cl
X
X
R2
R2
iii
5
( )
(6)
(7)
(6a-k)
(7a-k)
g, X = N; R2 = Acetyl
h X = N; R2 = -Boc
a, X = N; R2 = Methyl
b, X = CH; R2 = Methyl
c, X = N; R2 = i-Propyl
,
t
i X = N; R2 = H
,
d X = CH; R2
=
Propyl
j X = N; R2 = EtOH
,
i-
,
e X = N; R2 = n-Propyl
,
k, X = N; R2 = EtOMe
f X = N; R2 = Cyclohexyl
,
Scheme 1. Reagents and conditions: (a) DIPEA, naphth-1-ylmethanamine or diphenylamine, EtOH, 0 °C to 80–85 °C and reflux for 4 h; (b) various cyclic amines; 1-
methylpiperazine, 4-methylpiperidine, N-isopropylpiperazine, 4-isopropylpiperidine, N-propylpiperazine, 1-cyclohexylpiperazine, 1-acetylpiperazine, t-butyl piperazine-1-
carboxylate, N-(2-hydroxyethyl)piperazine or 1-(2-methoxyethyl)piperazine, respectively, n-BuOH, 145–150 °C, 50–60 min; (iii) TFA, CH2Cl2, 0 °C to rt, 2 h.

T. Mohamed et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5881–5887
5883
Table 1
ChEI activity profile, C log P and molecular volume data for compounds 6a–k
d
Compound
IC50
(l
M) SEMa
EqBuChE
S.I.b
C log Pc
Vol. (Å3)
X–R2 group
hAChE
6a35
6b35
17.5 1.8
25.8 2.6
2.6 0.3
2.2 0.2
6.7
11.7
3.9
5.2
230
232
N–Me
HC–Me
6c
15.8 2.1
16.7 1.7
7.6 0.8
9.1 0.9
2.1
1.8
4.7
6.2
255
258
6d
6e
6f
19.0 1.9
8.0 1.9
18.1 1.8
3.9 0.4
1.1
2.1
4.9
5.8
256
284
6g
6h
13.8 1.4
50.8 5.1
32.9 0.4
>100
0.4
2.9
5.3
244
293
<0.5
6i
6j
17.5 1.8
9.8 0.8
25.4 8.9
17.9 1.8
0.7
0.5
3.3
3.3
216
247
N–H
6k
11.7 2.5
26.5 2.7
0.4
3.4
0.009
0.3
4.1
260
Tacrine
Donepezil
Galantamine
0.077 0.008
0.032 0.003
3.2 0.7
0.021 0.001
3.6 0.4
12.6 1.3
3.3
4.6
1.0
138
271
179
(1)
(2)
–
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).
b
Selectivity Index = hAChE IC50/BuChE IC50
.
c
C log P was determined using ChemDraw Ultra 12.0. CambridgeSoft Company.
d
Molecular volume (Å3) was determined using a minimization protocol using the molecular properties calculator in the Discovery Studio program from Accelrys Inc. (San
Diego, CA).
presence of polar groups such as a hydroxyethyl in 6j (X–R2 = N-(2-
obtain dual ChE inhibition. In addition, the ChE inhibitory potency
was sensitive to substituents at C-2 position. The cyclohexylpiper-
azine compound 6f was identified as the most potent AChE inhib-
hydroxyethyl)piperazine; AChE IC50 = 9.8
17.9 M) or
methoxyethyl in 6k (X–R2 = 1-(2-methoxy-
ethyl)piperazine; AChE IC50 = 11.7 M; BuChE IC50 = 26.5 M)
lM; BuChE IC50 =
l
a
l
l
itor among this series (AChE IC50 = 8.0
lM) with dual ChE
resulted in better AChE inhibition compared to the alkyl-pipera-
inhibition (BuChE IC50 = 3.9 M). In addition, the presence of polar
l
zines 6a, c and d (Table 1). The introduction of an acetyl group in
C-2 groups in 6j and k provided better AChE inhibition. Further-
more, the presence of alkyl groups in 6a–d at C-2 position within
this series modulated ChE activity in favor of BuChE and the po-
tency was of the order: 4-methylpiperidine 6b >1-methylpipera-
zine 6a >N-isopropylpiperazine 6c >4-isopropylpiperidine 6d. In
addition, derivatives 6c, d and f were ꢀ1.5 to threefold more po-
tent BuChE inhibitors compared to the reference compound galan-
6g (X–R2 = 1-acetylpiperazine; AChE IC50 = 13.8
l
M; BuChE
IC50 = 32.9
lM) resulted in AChE activity comparable to 6k with a
decrease in BuChE potency. In contrast the presence of a bulky t-
Boc group in 6h (X–R2 = tert-butyl piperazine-1-carboxylate,
molecular volume = 293 Å3) led to a significant decrease in AChE
potency (IC50 = 50.8) compared to all the other compounds from
the naphth-1-ylmethyl series (Table 1). In addition, 6h exhibited
tamine (BuChE IC50 = 12.6 lM).
a loss of BuChE activity (IC50 >100
l
M). Interestingly, once the t-
Among the N-benzhydryl series of derivatives (7a–k), the pres-
ence of a methyl or N-propyl group in 7a or e (X–R2 = 1-methylpi-
perazine or N-propylpiperazine, Table 2) provided similar AChE
Boc group was hydrolyzed to generate the free piperazine ring as
in 6i (X–R2 = piperazine), dual ChE inhibition was restored (AChE
IC50 = 17.5
parable to the methylated piperazine compound 6a (AChE
IC50 = 17.5 M); however, 6i exhibited a significant decrease in
BuChE inhibitory potency compared to 6a (BuChE IC50 = 2.6 M).
In addition, replacing the methylpiperazine group (6a) with a
methylpiperidine bioisostere in 6b provided comparable BuChE
l
M; BuChE IC50 = 25.4
l
M) and AChE potency was com-
inhibitory activity (IC50 = 13.7 and 14.6
ever, 7a was less potent as a BuChE inhibitor compared to 7e
lM, respectively); how-
l
(IC50 = 23.8 and 17.5 lM, respectively). Replacing the N-propyl
l
chain with the branched isopropyl group in 7c (X–R2 = N-isopro-
pylpiperazine) resulted in a ꢀ1.4-fold decrease in AChE potency
and
9.7
a
ꢀ1.8-fold increase in BuChE potency (IC50 = 20.3 and
inhibition with greater selectivity (AChE IC50 = 25.8
l
M; BuChE
lM, respectively) relative to 7e. When compared to 7a, the iso-
IC50 = 2.2
l
M, S.I. = 11.7) relative to 6a.35 Furthermore, the bioisos-
propyl derivative (7c) exhibited a ꢀ1.5-decrease in AChE potency
and a ꢀ2.5-fold increase in BuChE potency. The presence of a bulk-
ier cycloalkyl group as in 7f (X–R2 = 1-cyclohexylpiperazine,
molecular volume = 303 Å3, Table 2) resulted in a ꢀtwofold in-
crease in AChE potency and a ꢀ1.3-fold increase in BuChE potency
teric replacement of C-2 isopropylpiperazine in 6c with an isopro-
pylpiperidine bioisostere in 6d provided dual ChE inhibition
similar to 6c (AChE IC50 = 16.7 lM; BuChE IC50 = 9.1 lM).
In naphth-1-ylmethyl series, all the compounds evaluated (6a–
k), except 6h exhibited dual AChE and BuChE inhibition indicating
that the presence of a C-4 naphth-1-yl group is a requirement to
(AChE IC50 = 10.0 and BuChE IC50 7.6 lM, respectively) relative to
7c and was the most potent dual ChE inhibitor in the N-benzhydryl

5884
T. Mohamed et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5881–5887
Table 2
ChEI activity profile, C log P and molecular volume data for compounds 7a–k
d
Compound
IC50
(l
M) SEMa
BuChE
S.I.b
C log Pc
Vol. (Å3)
X–R2 group
AChE
7a
7b
13.7 1.4
32.2 5.4
23.8 2.4
33.8 1.6
0.6
0.9
4.1
5.4
248
251
N–Me
HC–Me
7c
20.3 2.1
42.5 4.3
9.7 1.0
2.1
0.5
4.9
6.3
279
282
7d
87.0 8.7
7e
7f
14.6 1.5
10.0 0.9
17.5 1.8
7.6 0.1
0.8
1.3
5.1
6.0
272
303
7g
7h
29.0 2.9
>100
>100
>100
<0.3
–
3.1
5.5
260
311
7i
7j
31.3 3.1
21.6 2.2
>100
<0.3
0.4
3.5
3.5
235
265
N–H
59.5 6.0
7k
39.2 3.9
28.4 2.8
1.4
3.4
0.009
0.3
4.2
285
Tacrine
Donepezil
Galantamine
0.077 0.008
0.032 0.003
3.2 0.7
0.021 0.001
3.6 0.4
12.6 1.3
3.3
4.6
1.0
138
271
179
(1)
(2)
–
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).
b
Selectivity Index = hAChE IC50/BuChE IC50
.
c
C log P was determined using ChemDraw Ultra 12.0. CambridgeSoft Company.
d
Molecular volume (Å3) was determined using a minimization protocol using the molecular properties calculator in the Discovery Studio program from Accelrys Inc. (San
Diego, CA).
series. When compared to the N-propyl group in 7e (AChE
ꢀ1.3-fold greater potency against BuChE compared to galantamine
(BuChE IC50 = 12.6 M). Derivative 7f with a C-2 cyclohexylpiper-
azine was identified as the most potent dual ChE inhibitor (AChE
IC50 = 10.0 and BuChE IC50 7.6 M; S.I. = 1.3) with slight selectivity
toward BuChE. It was interesting to note that the presence of a C-2
cyclohexylpiperazine substituent in both naphth-1-ylmethyl (6f)
and N-benzhydryl series (7f) provided ChE inhibition and superior
potency for both AChE and BuChE, indicating that a C-2 cyclohexyl-
piperazine substituent could be a potential ChE pharmacophore for
2,4-disubstituted pyrimidines.
The ability of naphth-1-ylmethyl and N-benzhydrylpyrimidin-
4-amines (6a–k and 7a–k) to prevent both hAChE-induced and
self-induced Ab1–40 aggregation was evaluated by a thioflavin T
(ThT) fluorescence assay (Table 3).35 In the hAChE-induced aggre-
gation assay, the anti-Ab1–40 aggregation activity ranged from no
activity to 31.8% inhibition. Among the naphth-1-ylmethyl series
of compounds (6a–k), the presence of a C-2 N-isopropylpiperazine
(6c), N-propylpiperazine (6e), cyclohexylpiperazine (6f) and 2-
hydroxyethylpiperazine (6j) provided inhibition of hAChE-induced
Ab1–40 aggregation. Compound 6f exhibited superior inhibition
IC50 = 14.6
groups such as a hydroxyethyl in 7j (X–R2 = 2-hydroxyethylpiper-
azine; AChE IC50 = 21.6 M; BuChE IC50 = 59.5 M) or a methoxy-
ethyl in 7k
(X–R2 = 2-methoxyethylpiperazine;
IC50 = 39.2 M; BuChE IC50 = 28.4
l
M; BuChE IC50 = 17.5
l
M), the presence of polar
l
l
l
l
AChE
l
l
M) resulted in reduced ChE
inhibitory potency. On the other hand, the introduction of an acetyl
group in 7g (X–R2 = 1-acetylpiperazine; AChE IC50 = 29.9
M;
BuChE IC50 >100 M) resulted in moderate AChE activity and led
l
l
to a loss of BuChE activity. The presence of a bulky t-Boc group
in 7h (X–R2 = tert-butyl piperazine-1-carboxylate) led to a loss of
ChE activity (IC50 >100 lM). Interestingly, once the t-Boc group
was hydrolyzed to generate the free piperazine ring as in 7i
(X–R2 = piperazine), AChE inhibition was restored (AChE
IC50 = 31.3
(IC50 >100
l
M) although 7i did not exhibit BuChE inhibition
lM). In addition, replacing the methylpiperazine group
(7a) with a methylpiperidine bioisostere in 7b resulted in a dual,
nonselective ChE inhibitory profile (AChE IC50 = 32.2 M; BuChE
IC50 = 33.8
M, S.I. ꢀ1.0) relative to 7a. Furthermore, the bioisos-
teric replacement of C-2 isopropylpiperazine in 7c with corre-
sponding isopropylpiperidine bioisostere in 7d resulted in
ꢀtwofold decrease in AChE potency and weak BuChE inhibition
(AChE IC50 = 42.5 M; BuChE IC50 = 87.0 M) compared to 7c.
l
l
a
(30.8% inhibition at 100
potent compared to propidium (82% at 100
note that 6f is the most potent AChE inhibitor (IC50 = 8.0 l
l
M) compared to 6c, e and j and was less
M). It is interesting to
M) in the
l
l
l
It is noteworthy that the molecular volumes (Å3) of N-benzhy-
dryl derivatives (7a–k) are ꢀ6–9% greater than those of their naph-
th-1-ylmethyl derivatives (6a–k) (Tables 1 and 2). The majority of
N-benzhydryl derivatives were not as potent as their naphth-1-yl-
methyl derivatives with the exception of 7a and e (ꢀ1.3-fold in-
crease in AChE inhibition compared to 6a and e, respectively).
Compound 7c was identified as a selective BuChE inhibitor with
naphth-1-ylmethyl series, which supports its ability to inhibit PAS
mediated Ab1–40 aggregation. This is further supported by the fact
that 6f was not active in the self-induced Ab1–40 aggregation assay
(Table 3). These studies indicate that for the naphth-1-ylmethyl
series of compounds, the hAChE-induced aggregation inhibition
is sensitive to the steric and electronic factors of substituents at
the C-2 position of the pyrimidine ring. In the N-benzhydryl series

T. Mohamed et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5881–5887
5885
Table 3
Ab1–40 aggregation (ꢀ32% inhibition toward hAChE-induced Ab-
aggregation). These observations indicate the effect of C-4 aromatic
ring structures on Ab1–40 aggregation. For example, a C-4 diph-
enylmethane substituent could potentially interact with Ab1–40
peptides and prevent them from stacking/aggregating and could
serve as a suitable pharmacophore to prevent both self-induced
as well as hAChE-induced Ab1–40 aggregation as compared to a pla-
nar C-4 naphth-1-yl substituent. Furthermore, the ability of syn-
thesized compounds to reach the central nervous system was
correlated by calculating the theoretical C log P values (Tables 1
and 2). They exhibited a wide range from 2.9 to 6.3. In this regard,
compound 6f that exhibited a good combination of dual ChE and
Ab-aggregation inhibition exhibited a C log P value of 5.8 which
is comparable with the marketed anti-AD compound donepezil
(C log P = 4.6) indicating it’s potential to reach the central nervous
system.
Percent inhibition of hAChE-induced and self-induced Ab1–40 aggregation by 6a–k and
7a–k at 100 M
Compound
Inhibition of Ab1–40 aggregation (%) SDa
hAChE-induced
Self-induced
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
7a
7b
7c
7d
7e
NA
NA
NA
NA
17.1 1.7
NA
12.1 1.2
NA
22.0 2.2
30.8 3.1
NA
NA
NA
NA
NA
NA
NA
NA
13.4 1.3
NA
NA
NA
24.1 2.4
14.8 1.4
20.8 2.1
31.8 3.2
23.1 2.3
32.0 3.2
10.3 1.0
13.6 1.4
NA
18.8 1.9
10.4 1.0
82.1 8.2
17.0 1.7b
ND
24.8 1.2
18.9 1.9
21.8 2.2
11.8 1.2
18.2 1.8
27.6 2.8
15.8 1.6
11.4 1.1
20.8 2.1
15.0 1.5
16.3 1.6
ND
The ligand-enzyme binding interactions of the potent dual ChE
inhibitor, 6f (hAChE IC50 = 8.0
lM; equine serum BuChE
IC50 = 3.9 M, S.I. = 2.1), were investigated by molecular modeling
l
7f
7g
7h
7i
7j
7k
studies. The docking study of 6f within the active site of hAChE
(Fig. 2) indicates that the pyrimidine ring was positioned midway
through the active site gorge (ꢀ8.0 Å away from the catalytic triad
His447 residue at the bottom of the active site and ꢀ7.0 Å away
from the gorge entry, with N-3 oriented toward the entry). The ring
was stacked close to Phe295, Phe297 and Phe338 (distance ꢀ4.5–
7.5 Å). The C-4 naphth-1-yl ring was stacked between Trp86 and
a tyrosine pocket comprised of Tyr124, Tyr337 and Tyr341 (dis-
tance ꢀ3.8–5.8 Å). The C-4 NH group was undergoing hydrogen
bonding with the hydroxyl group of Tyr124 (distance ꢀ2.9 Å) and
the OH of Tyr124 was also undergoing hydrogen bonding interac-
tion with the C-4 pyrimidin-4-amine nitrogen (distance ꢀ3.1 Å).
The cyclohexylpiperazine C-2 group of 6f was oriented toward
the PAS, where the cyclohexyl ring was perpendicularly stacked
over Trp286 (distance ꢀ4.1 Å) and was ꢀ4.8 Å away from
Leu289. The piperazine ring was stacked over Ser293 and Val294
(distance ꢀ4.7 Å). It is significant to note that the linear conforma-
tion allows 6f, to span both the catalytic active site (CAS) and PAS
that contributes to its superior binding to hAChE. In general the
molecular volumes of naphth-1-ylmethyl derivatives were ꢀ6–9%
Propidium iodide
Donepezil. HCl
Galantamine. HBr
ND
ꢀ48c
a
b
c
The result (% inhibition) is the mean of two separate experiments (n = 4).
Previously reported [Ref. 35].
Previously reported [Ref. 45], NA, not active; SD, standard deviation; ND, not
determined.
(7a–k), it was interesting to note that all the compounds except 7i
exhibited activity in both hAChE-promoted as well as self-induced
Ab1–40 aggregation assay (inhibition range ꢀ10–32%) (Table 3).
This clearly indicates that the presence of a C-4 diphenylmethane
group is a major contributing factor involved in the inhibition
Ab1–40 aggregation. Compounds 7d (4-isopropylpiperidine) and f
(cyclohexylpiperazine) exhibited almost equipotent inhibition of
Figure 2. Docking of 2-(4-cyclohexylpiperazin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine (6f) in the active site of hAChE (PBD code: 1B41). Red lines represent
hydrogen-bond interactions. Hydrogen atoms are not shown for clarity.

5886
T. Mohamed et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5881–5887
Figure 3. Docking of 2-(4-cyclohexylpiperazin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine (6f) in the active site of hBuChE (PBD code: 1P0I). Red lines represent
hydrogen-bond interactions. Hydrogen atoms are not shown for clarity.
smaller (Å3) compared to their N-benzhydryl counterparts 7a–k
and this resulted in better anti-ChE profile for the naphth-1-yl-
methyl derivatives.
ies, compound 7f (N-benzhydryl-2-(4-cyclohexylpiperazin-1-
yl)pyrimidin-4-amine) exhibited optimum combination of dual
ChE (AChE IC50 = 10.0
l
M, BuChE IC50 = 7.6
lM) and hAChE-pro-
On the other hand, the docking study of 6f within the active site
of hBuChE (Fig. 3) indicates that the pyrimidine ring was posi-
tioned midway through the active site gorge (ꢀ9.1 Å away from
the catalytic triad His438 residue at the bottom of the active site
and ꢀ9.5 Å away from the gorge entry) with N-1 directly facing
Ala277 at the active site entrance. The pyrimidine ring was stacked
between Pro285 and Ile69/Asp70 (distance ꢀ8.0 and ꢀ4.4 Å,
respectively). The C-4 naphth-1-yl ring was stacked between
Trp82 and Phe329 (distance ꢀ4.2 and 6.0 Å, respectively) and posi-
tioned in an aromatic pocket comprised of Trp430 (distance
ꢀ4.4 Å), Tyr440 (distance ꢀ5.3 Å) and Tyr332 (distance ꢀ3.9 Å).
The C-4 NH group was undergoing hydrogen bonding with the
OH group of Tyr332 (distance ꢀ3.1 Å) and the COOH side chain
of Asp70 (distance ꢀ3.1 Å). The cyclohexylpiperazine C-2 group
of 6f was oriented toward a hydrophobic pocket, where the piper-
azine ring was in close proximity to Gly116/117, Gln119 and
Thr120 (distance <5.0 Å) and the cyclohexyl ring was in close prox-
imity to Ser198, Trp231, Leu286, Ser287, Val288 and Phe398 (dis-
tance <5.0 Å). Although the overall U-shape conformation of 6f in
BuChE does not facilitate multiple hydrogen bonds, the strong
hydrophobic interactions with the C-2 group and the positioning
of the C-4 naphth-1-yl ring in close proximity to Trp82 contributes
its superior binding toward BuChE.
moted Ab-aggregation inhibition (32% at 100
l
M). Results of the
biological screening and SAR studies demonstrate that the 2,4-
disubstituted pyrimidine ring could potentially serve as a suitable
template to design small molecules that could target multiple
pathological routes in AD, such as dual inhibition of AChE and
BuChE, coupled with anti-Ab-aggregation activity.
Acknowledgments
The authors would like to thank the Department of Biology and
the School of Pharmacy at the University of Waterloo for support-
ing this research project.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.091.
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In conclusion, our studies indicate that the presence of a C-4
naphth-1-ylmethyl substituent generally provides dual AChE and
BuChE inhibitors with 6f (2-(4-cyclohexylpiperazin-1-yl)-N-(naph-
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T. Mohamed et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5881–5887
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(3 ꢁ 5 mL) and the organic layer was dried over anhydrous MgSO4 then
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7
with
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J = 6.0 Hz, 1H), d 7.24–7.33 (m, 10H), d 5.92 (br s, 1H), d 5.61 (d, J = 6.0 Hz, 1H), d
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36. General procedure for the synthesis of 4-substituted-2-chloropyrimidin-4-amines
(6–7): To a mixture of 2,4-dichloropyrimidine (5) (5.00 g, 33.60 mmol) and
primary amines (R1 = naphth-1-ylmethanamine and diphenylmethanamine;
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 min and was refluxed at 80–85 °C for 4 h. After cooling to 25 °C, 20 mL of
EtOAc was added and solution was neutralized with drop-wise addition of
ꢀ6 M HCl (pH = 7–7.5), washed with a saturated NaHCO3 and NaCl solution
(1 ꢁ 50 mL). Aqueous layer was re-washed with EtOAc (3 ꢁ 25 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 or both of the following methods: (1) Method A: Silica
gel column chromatography using EtOAc: hexanes twice (3:1 and 1:3 v/v,
respectively) or 9:1 DCM: EtOAc to afford solid products (60–65%); (2) Method
B: The collected organic layers were evaporated in vacuo and the oily residue
was vigorously mixed with a solution of hexanes to afford a precipitate that
was dried on filter paper at 80–85 °C for ꢀ2–3 h to afford solid products.
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k and 7a–k): To a solution of 6 or 7 (0.68–0.74 mmol) in 3 mL of n-BuOH kept
in a PV with stirring, cyclic amines (1.02–1.11 mmol) was added. The sealed PV
was placed in an oil bath at 145–150 °C and stirred for 50–60 min. The solvent
was evaporated in vacuo with the aid of DCM and the residue was re-dissolved
in 2:1 EtOAc/DCM and washed successively with saturated NaHCO3 and NaCl
solution (1 ꢁ 15 mL), respectively. The aqueous layer was washed with EtOAc
test compound causing 50% inhibition (IC50
concentration-log% inhibition response curve.
38. hAChE-induced and self-induced Ab1–40 aggregation inhibition assay: The
lM) was calculated from the log
thioflavin
T (ThT) method was used to detect amyloid oligomers and
fibrils.42–44 The Ab1–40 HFIP was purchased from Anaspec (Cat. 64128-1),
human recombinant AChE lyophilized powder and ThT were purchased from
Sigma (Cat. C1682 and T3516; respectively) and the hAChE-induced assay was
run using propidium iodide as a control. Ab1–40 was dissolved in DMSO and
sonicated for 30 min to obtain a 232
215 mM sodium phosphate buffer (pH 8.0) to obtain a 4.69
the hAChE-induced assay, 4 L of Ab1–40 were incubated with 20
to give a final concentration of 23.2 M of Ab1–40 and 2.35 M of hAChE. For co-
incubation experiments, 16 L of test samples (100 M) in 215 mM sodium
phosphate buffer pH 8.0 solution (6% DMSO) were used. For the self-induced
assay, 4 L of Ab1–40 were incubated with 16 L of test samples (100 M) in
215 mM sodium phosphate buffer pH 8.0 solution (6% DMSO). 96-well plates
were incubated at room temperature for 24 h and 150 L of 15 M of thioflavin
in 50 mM glycine-NaOH buffer (pH 8.5) was added. Fluorescence was
monitored at 446 nm and emission 490 nm using Molecular Devices
l
M solution. hAChE was dissolved in
M solution. For
L of hAChE
l
l
l
l
l
l
l
l
l
l
l
l
T
a
SpectraMax spectrofluorometer. The fluorescence intensities in the presence
and absence of inhibitors before and after the incubation period were
compared and the percentage inhibition was calculated with equation: 100%
control value (i.e. no inhibitor)
– [(IFi–IFo)] where IFi and IFo are the
fluorescence intensities in the presence of ThT and absence of ThT before
24 h incubation, respectively.35
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