Application of quinazoline and pyrido[3,2-: D] pyrimidine templates to design multi-targeting agents in Alzheimer's disease

Article abstract of DOI:10.1039/c7ra02889j

A quinazoline and pyrido[3,2-d]pyrimidine based compound library was designed, synthesized and evaluated as multi-targeting agents aimed at Alzheimer's disease (AD). The SAR studies identified compound 8h (8-chloro-N²-isopropyl-N⁴-ph

Full text of DOI:10.1039/c7ra02889j

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Application of quinazoline and pyrido[3,2-d]
pyrimidine templates to design multi-targeting
Cite this: RSC Adv., 2017, 7, 22360
agents in Alzheimer's disease†
Tarek Mohamed, Mandeep K. Mann and Praveen P. N. Rao *a
ab
a
A quinazoline and pyrido[3,2-d]pyrimidine based compound library was designed, synthesized and
evaluated as multi-targeting agents aimed at Alzheimer's disease (AD). The SAR studies identified
2
4
compound 8h (8-chloro-N -isopropyl-N -phenethylquinazoline-2,4-diamine) as a potent inhibitor of
Ab40 aggregation (IC50 ¼ 900 nM) which was 3.6-fold more potent compared to the reference agent
curcumin (Ab40 IC50 ¼ 3.3 mM). It also exhibited dual ChE inhibition (AChE IC50 ¼ 8.6 mM; BuChE IC50
¼
4
2
2
.6 mM). Compound 9h (8-chloro-N -(3,4-dimethoxyphenethyl)-N -isopropylquinazoline-2,4-diamine)
was identified as the most potent Ab42 aggregation inhibitor (IC50 ꢀ 1.5 mM). Transmission electron
microscopy (TEM) imaging demonstrates their anti-Ab40/Ab42 aggregation properties. Compound 8e
was identified as a potent BuChE inhibitor (BuChE IC50 ¼ 100 nM) which was 36-fold more potent
2
compared to donepezil (BuChE IC50 ¼ 3.6 mM). The pyrido[3,2-d]pyrimidine bioisostere 10b (N -
Received 9th March 2017
Accepted 17th April 2017
4
isopropyl-N -phenethylpyrido[3,2-d]pyrimidine-2,4-diamine) exhibited good anti-Ab activity (Ab40 IC50
¼
1.1 mM), dual ChE inhibition and iron-chelating properties (23.6% chelation at 50 mM). These
DOI: 10.1039/c7ra02889j
investigations demonstrate the usefulness of either a quinazoline or a pyrido[3,2-d]pyrimidine based ring
scaffold in the design of multi-targeting agents to treat AD.
rsc.li/rsc-advances
including metal-induced neurotoxicity in AD disease patho-
physiology, highlights the need to design and develop novel
Alzheimer's disease (AD) is a neurodegenerative disorder with therapies to target multiple pathways.
no cure in sight. It is anticipated that with an aging population
As part of our research program aimed at designing multi-
1
Introduction
6–10
and increasing lifespan, the social and economic burden is targeting small molecules, we have investigated the develop-
going to multiply in the years to come. Current AD research ment of novel agents including quinazolines that exhibit Ab
indicates that many factors are involved in its pathophysiology. aggregation inhibition, dual acetylcholinesterase (AChE) and
Due to the ever-growing complexity of AD, it is no surprise that butyrylcholinesterase (BuChE) inhibition and possess radical
11,12
the shi toward multi-targeting small molecule candidates scavenging activity.
In this regard, dual inhibition of both
which can counter the various facets of the disease, as the next AChE and BuChE is known to be benecial as BuChE can
1–3
13
generation AD treatments is increasing. While current phar- process ACh in advance stages of AD. Building on our recent
macotherapy options rely on cholinesterase inhibitors (done- efforts on multi-targeting small molecules, herein we present
pezil, rivastigmine and galantamine) that reduce the the structure-activity relationship (SAR) studies of substituted
degradation of the neurotransmitter acetylcholine (ACh) and an quinazolines and its bioisostere, pyrido[3,2-d]pyrimidine ring
N-methyl-D-aspartate antagonist (memantine), these were not template (Fig. 1). Several compounds from this series exhibited
designed as multi-targeting agents and provide only symptom-
4,5
atic relief. The role of beta-amyloid (Ab) which tends to form
neurotoxic aggregates, cholinesterases (ChE) that degrade ACh
which facilitates neurotransmission and oxidative stress
a
School of Pharmacy, Health Sciences Campus, University of Waterloo, Waterloo,
Ontario, Canada N2L 3G1. E-mail: praopera@uwaterloo.ca; Tel: +1-519-888-4567
ext. 21317
b
Department of Chemistry and School of Pharmacy, University of Waterloo, Waterloo,
Ontario, Canada N2L 3G1
1
†
Electronic supplementary information (ESI) available: Includes H NMR spectra.
Fig. 1 SAR studies of quinazoline and pyrido[3,2-d]pyrimidines as
See DOI: 10.1039/c7ra02889j
multi-targeting anti-AD agents.
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ꢁ
ꢁ
3
, 0–105 C, reflux, 14–16 h.
Scheme 1 Reagents and conditions: (a) urea, pressure vial, 150–155 C, 2 h; (b) toluene, N,N-diethylaniline, POCl
desirable multi-targeting ability including dual Ab/ChE inhibi-
tion and iron chelation properties. The road toward this library
of 32 derivatives (8–15) started by rst reacting the substituted-
2
Results and discussion
2.1 Inhibition of Ab aggregation
chloro-2-aminobenzoic or picolinic acids (1a–d, Scheme 1) with The results of the SAR investigation on the anti-Ab aggregation
urea to afford the quinazoline or pyrido[3,2-d]pyrimidine ring properties of chloroquinazolines and pyrido[3,2-d]pyrimidines
template (2a–d), followed by 2,4-dichlorination with POCl to is shown in Table 1. Examining the SAR of C4 phenethylamine-
3
obtain 2,4,6-, 2,4,7- or 2,4,8-trichloroquinazolines (3a–c) or 2,4- based chloroquinazolines (8a–i), shows that chlorine placement
dichloropyrido[3,2-d]pyrimidine (3d) respectively (65–80% on the quinazoline ring had an effect on anti-Ab activity with the
1
2
yield, Scheme 1).
8-Cl placement exhibiting superior Ab aggregation inhibition.
The N -substituted dichloroquinazolines (4a–c and 5a–c) or The activity order was 8-Cl > 7-Cl > 6-Cl with respect to both
4
4
N -substituted-2-chloropyrido[3,2-d]pyrimidine (6a and 7a) Ab40/Ab42 inhibition. While 8a–c was ineffective toward Ab42,
intermediates were obtained in moderate to good yields (ꢀ75– others in the group showed inhibition ranging from ꢀ3.9–12
9
0%) by coupling 3a–d with phenethylamine or 3,4-dimethox- mM, with 8h being the most potent Ab42 inhibitor. Signicant
yphenethylamine in the presence of N,N-diisopropylethylamine Ab40 aggregation inhibition was observed with compounds 8g–i
DIPEA) under reux for 4 hours (Scheme 2). Displacement of (ꢀ900 nM to 3.7 mM), with 8h (8-Cl, C2 i-Pr) being the most
the C2 chlorine in 4a–c, 5a–c, 6a and 7a was readily achieved by potent inhibitor (Ab40 IC ¼ 900 nM) and was 3.6-fold more
(
5
0
heating with respective amines (n-propylamine, isopropyl- potent compared to the reference agent curcumin (Ab40 IC50
¼
amine, cyclopropylamine or N,N-dimethylamine) with 1,4- 3.3 mM, Table 1). The addition of a C4 3,4-dimethox-
dioxane in the presence of DIPEA, in a pressure vial for 2 h to ylphenethylamine substituent in compounds (9a–i) generally
afford the quinazoline (Qnz) and pyrido[3,2-d]pyrimidine (Ppd) provided dual inhibition of both Ab40 and Ab42 aggregation
based compound library 8a–i, 9a–i, 10a–c, 11a–c, 12a–c, 13a–c, except for the 7-chloro derivative 9e (Table 1). Compound 9h (8-
14a and 15a with yields ranging from 68–78% (Scheme 1). The chloro, C2 i-Pr) was identied as a good inhibitor of both Ab40
synthesized quinazoline derivatives were evaluated for their and Ab42 (Ab40 IC50 ꢀ 4.0 mM; Ab42 IC ꢀ 1.5 mM). Signi-
50
ability to modulate the aggregation kinetics of Ab40 and Ab42 cantly, it was ꢀ6.6-fold and 10-fold more potent toward Ab42
1
2,14
using the thioavin T (ThT) uorescence assay.
Their compared to reference agents resveratrol (Ab42 IC ¼ 15.3) and
5
0
cholinesterase (hAChE/hBuChE) inhibition activity was deter- curcumin (Ab42 IC50 ¼ 9.9 mM). Replacing the quinazoline ring
12,15
mined using the Ellman's protocol. We identied few template with a pyrido[3,2-d]pyrimidine bioisostere was gener-
chloroquinazoline and pyrido[3,2-d]pyrimidines that exhibited ally ineffective. In this group of compounds, the C4 phene-
excellent Ab aggregation inhibition, dual ChE inhibition and thylamine derivatives 10a–c were better than the corresponding
iron-chelation properties thereby demonstrating multi- C4 3,4-dimethoxyphenethylamine derivatives 11a–c with
targeting ability.
compound 10b identied as the most potent Ab40 aggregation
ꢁ
Scheme 2 Reagents and conditions: (a) phenethylamine or 3,4-dimethoxyphenethylamine, DIPEA, ethanol, reflux, 80–85 C, 4 h; (b) alkylamine,
ꢁ
DIPEA, 1,4-dioxane, 150–155 C, pressure vial, 2 h.
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Table 1 Amyloid-b (Ab40/Ab42) inhibition activity of quinazolines (8, Derivative 8h (8-chloro-N -isopropyl-N -phenethylquinazoline-
9
, 12 and 13) and pyrido[3,2-d]pyrimidines (10, 11, 14 and 15)
2
,4-diamine) was the most potent Ab40 aggregation inhibitor
4
a,b
(IC50
ꢀ
900 nM), while 9h (8-chloro-N -(3,4-dimethox-
IC50 (mM)
2
Chloride
yphenethyl)-N -isopropylquinazoline-2,4-diamine) was the most
potent Ab42 aggregation inhibitor (IC50 ꢀ 1.5 mM).
1
Compd
R
substitution
Ab40
Ab42
The ThT-based Ab40 aggregation kinetics data in the pres-
ence of the most active inhibitors (8h and 10b for Ab40) is
shown in Fig. 2. This reveals that, their modes of aggregation
inhibition do differ from one another. As seen in Fig. 2 (panel
A), 8h was exhibiting a concentration-dependent reduction in
the aggregation with complete inhibition of Ab40 aggregation
seen at 25 mM. On the other hand, 10b (Fig. 2, panel C) was also
exhibiting a concentration-dependent reduction in aggregation.
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
a
b
c
d
e
f
g
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
—
C6
C6
C6
C7
C7
C7
C8
C8
C8
C6
C6
C6
C7
C7
C7
C8
C8
C8
—
>25
>25
>25
>25
4.6 ꢂ 0.9
7.2 ꢂ 1.6
11.7 ꢂ 2.0
6.5 ꢂ 0.4
3.9 ꢂ 0.5
6.8 ꢂ 1.3
8.6 ꢂ 0.7
8.7 ꢂ 0.9
6.9 ꢂ 1.4
6.1 ꢂ 1.3
4.8 ꢂ 1.0
3.7 ꢂ 0.5
0.9 ꢂ 0.1
1.3 ꢂ 0.2
4.9 ꢂ 0.5
5.8 ꢂ 0.7
8.9 ꢂ 0.9
3.1 ꢂ 0.3
10.6 ꢂ 0.9
5.2 ꢂ 0.6
4.9 ꢂ 0.5
4.0 ꢂ 0.4
4.6 ꢂ 0.5
NA
1.1 ꢂ 0.1
6.8 ꢂ 1.0
NA
NA
>25
6.5 ꢂ 0.9
5.1 ꢂ 1.7
1.9 ꢂ 0.3
2.5 ꢂ 0.3
4.3 ꢂ 0.4
2.3 ꢂ 0.2
NA
10.7 ꢂ 1.1 In addition, unlike compound 8h, it was able to delay the onset
7.7 ꢂ 0.8
9.8 ꢂ 0.8
12.4 ꢂ 1.1
>25
22.5 ꢂ 1.9
2.3 ꢂ 0.3
1.5 ꢂ 0.2
2.2 ꢂ 0.2
13.7 ꢂ 2.0
>25
of aggregation by delaying the lag phase further. Investigating
the Ab morphology by TEM imaging further conrms the ability
of both compounds 8h and 10b to prevent Ab aggregation
(Fig. 2). Analysis of the Ab42 aggregation kinetics in the pres-
ence of the most active inhibitors (9h and 13c for Ab42, Fig. 2)
revealed similar observations. Both derivatives, which were
nearly equipotent against Ab42 aggregation, shared similar
modes of action. As seen in Fig. 2 (panel B), 9h was able to
reduce the overall load of Ab42 bril formation in a concentra-
tion dependent manner with almost complete inhibition seen
at 25 mM. A similar trend was seen with 13c (Fig. 2, panel D).
Furthermore, TEM morphology studies corroborate aggregation
kinetics data observed (Fig. 2).
h
i
0a
0b
0c
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
5a
—
—
—
—
11.5 ꢂ 1.5
>25
>25
>25
>25
>25
>5.8 ꢂ 0.7
5.6 ꢂ 0.7
9.4 ꢂ 0.9
1.8 ꢂ 0.3
12.7 ꢂ 2.0
>25
—
C6
C7
C8
C6
C7
C8
—
—
—
—
—
—
—
—
—
—
—
—
The molecular docking study of best two Ab40 aggregation
inhibitors, 8h and 10b, in the Ab dimer model (Fig. 3, panel A)
16
derived from the solution structure of Ab brils (pdb id:2LMN)
indicated that both the 8-chloroquinazoline and the pyrido[3,2]
pyrimidine templates were oriented between C- and N-terminal
region whereas the C4-phenethylamine substituent was closer
>25
Resveratrol
Curcumin
1.1 ꢂ 0.1
15.3 ꢂ 1.9
˚
—
3.3 ꢂ 0.4
9.9 ꢂ 0.4
to the turn region Asp23-Gly29 (distance ꢀ 6–8 A). Hydrophobic
a
interactions were the dominating force involved. In the pyrido
IC50 values were calculated using the ThT-based uorescence
[3,2-d]pyrimidine compound 10b, the presence of an additional
spectroscopy assay (excitation ¼ 440 nm, emission ¼ 490 nm).
b
Values are mean of triplicate readings for three independent ring nitrogen (N5) led to a hydrogen bonding contact with the
experiments. NA ¼ not active.
˚
amide backbone of Val24 (distance ꢀ 3 A). In the bril model,
Fig. 3, panel B, both 8h and 10b had similar binding modes
where the C4-phenethylamine group was oriented toward the
inhibitor (IC50 ꢀ 1.1 mM). In the next step, the impact of
a tertiary dimethylamine moiety at the C2-position was inves-
tigated. The presence of a C4 phenethylamine, increased
potency toward Ab40 12a–c; 6-Cl < 7-Cl < 8-Cl while only 12c was
active toward Ab42. On the other hand, with a 3,4-dimethox-
yphenethylamine at the C4 position, potency toward Ab40
ranged from ꢀ2–4 mM with chlorine placement at C6 being
˚
center region of the bril core (Ile32-Met35, distance ꢀ 5–8 A).
Both core templates were undergoing hydrophobic interactions
˚
with Ile31-Val38 (distance ꢀ 5–8 A) at the C-terminal end. These
studies support the ability of 8h and 10b to reduce bril
aggregation.
equipotent to C8 placement. The Ab42 aggregation inhibitory 2.2 Inhibition of cholinesterases
potency increased from 7-Cl (13b, IC50 ꢀ 9.4 mM) to 6-Cl (13a,
With respect to cholinesterase inhibition, the SAR data is shown
IC50 ꢀ 5.6 mM) to 8-Cl (13c, IC50 ꢀ 1.8 mM). Combining the C2
dimethylamine group with a C4 phenethylamine or 3,4-dime-
thoxyphenethylamine on a pyrido[3,2-d]pyrimidine scaffold
resulted in either weak or no inhibition of Ab aggregation
in Table 2. While examining the rst cluster of C4
phenethylamine-based chloroquinazolines (8a–i), it became
evident that chlorine placement at either C6, C7 or C8 was able
to modulate the ChE inhibitory activity with 7-Cl exhibiting
better ChE inhibition. The ChE inhibition can be ranked as 7-Cl
(compounds 14a and 15a, Table 1). With respect to the Ab
aggregation inhibition SAR, ꢀ22% of the derivatives screened
>
6-Cl > 8-Cl with respect to both ChEs. Most noteworthy was the
surpassed the activity level of curcumin toward Ab40, while
outcome from 8e (AChE IC50 ꢀ 6.6 mM, BuChE IC50 ꢀ 100 nM),
ꢀ
44% surpassed the activity level of curcumin toward Ab42.
which was the most potent AChE inhibitor in this group, and
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Fig. 2 Ab40/Ab42 (5 mM) aggregation kinetics study with or without 1, 5 or 25 mM of 8h (panel A), 9h (panel B), 10b (panel C) or 13c (panel D) along
with corresponding TEM morphology in the presence of 25 mM of 8h, 9h, 10b or 13c. Aggregation kinetics were monitored by ThT-fluorescence
ꢁ
spectroscopy (excitation ¼ 440 nm, emission ¼ 490 nm) for 24 h at 37 C in phosphate buffer at pH 7.4. TEM image scale: white bars represent
5
00 nm.
Fig. 3 Ab molecular docking studies. Binding modes of the quinazoline compound 8h (red) and pyrido[3,2-d]pyrimidine compound 10b (green)
in Ab-dimer model (panel A) and Ab-fibril model (panel B). Hydrogen atoms were removed for clarity.
was the most potent BuChE inhibitor overall (about 36-fold template with a pyrido[3,2-d]pyrimidine template (10a–c and
more potent compared to donepezil, IC50 ¼ 3.60 mM). The 11a–c), led to weak or no inhibitory activity toward BuChE. With
addition of the 3,4-dimethoxyphenethyl substituent in the respect to AChE inhibition, they were on par with the chlor-
second cluster of derivatives (9a–i) exhibited similar AChE oquinazolines (8a–i and 9a–i) with activity range AChE IC50 ꢀ 7–
inhibition range as per compounds 8a–i. However, it was 8 mM, Table 2. The incorporation of a N,N-dimethylamine at the
detrimental for BuChE inhibition, although one key derivative C2 position was explored to investigate the role of a tertiary
stood out from the pack (9e, BuChE IC50 ꢀ 4 mM) and was amine instead of a secondary amine toward ChE inhibition
equivalent to donepezil. It is interesting to point out that both (12a–c and 13a–c). The chlorine placement played a role as 12b
compounds 8e and 9e that exhibited signicant BuChE inhi- (7-Cl, BuChE IC50 ꢀ 1.5 mM) was ꢀ3.6-fold more potent
bition possessed a C2 isopropylamine substituent which compared to its 6-chloro isomer (12a). In addition, 12b was
promoted BuChE inhibition. Replacing the quinazoline more potent compared to compound 13b (BuChE IC50 > 50 mM).
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Table 2 Cholinesterase inhibition (AChE and BuChE) of quinazolines
(8, 9, 12 and 13) and pyrido[3,2-d]pyrimidines (10, 11, 14 and 15)
a
IC50 (mM)
Chloride
1
Compd
R
substitution
hAChE
hBuChE
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
a
b
c
d
e
f
g
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
—
C6
C6
C6
C7
C7
C7
C8
C8
C8
C6
C6
C6
C7
C7
C7
C8
C8
C8
—
8.9 ꢂ 0.6
8.7 ꢂ 0.5
8.7 ꢂ 0.9
7.2 ꢂ 0.7
6.6 ꢂ 0.6
7.3 ꢂ 0.6
7.7 ꢂ 0.7
8.6 ꢂ 0.9
7.6 ꢂ 0.7
6.2 ꢂ 0.4
6.8 ꢂ 0.7
8.0 ꢂ 0.7
7.2 ꢂ 0.7
7.9 ꢂ 0.8
8.7 ꢂ 0.8
6.5 ꢂ 0.5
6.8 ꢂ 0.7
6.6 ꢂ 0.7
8.1 ꢂ 0.8
7.8 ꢂ 0.8
7.6 ꢂ 0.6
7.2 ꢂ 0.8
7.4 ꢂ 0.7
7.2 ꢂ 0.5
7.7 ꢂ 0.8
5.8 ꢂ 0.7
7.5 ꢂ 0.8
7.2 ꢂ 0.7
13.4 ꢂ 1.1
8.1 ꢂ 0.8
6.8 ꢂ 0.7
6.7 ꢂ 0.5
0.03 ꢂ 0.002
2.6 ꢂ 0.6
4.8 ꢂ 0.3
1.5 ꢂ 0.1
9.1 ꢂ 0.7
4.4 ꢂ 0.4
0.1 ꢂ 0.02
3.5 ꢂ 0.4
6.0 ꢂ 0.6
2.6 ꢂ 0.2
10.9 ꢂ 0.9
>50
Fig. 4 Cholinesterase molecular docking studies. Panel (A): Binding
modes of 9a (red) and 12b (green) in hAChE. Panel (B): Binding modes
of 8e (red) and 12b (green) in hBuChE. Hydrogen atoms removed for
clarity.
20.3 ꢂ 1.5
>50
>50
3.9 ꢂ 0.3
>50
>50
binding interactions, explaining their near-equipotent inhibi-
h
i
19.3 ꢂ 1.1
tion of the AChE (IC50 ꢀ 6 mM). In both derivatives, the 7-
>50
chloroquinazoline ring scaffold was stacked parallel against
0a
0b
0c
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
5a
30.3 ꢂ 2.8
˚
—
—
—
—
29.3 ꢂ 2.0 Trp86 (distance ꢀ 4–6 A) and the C4-benzylamino groups were
>50
>50
>50
>50
5.4 ꢂ 0.6
1.5 ꢂ 0.1
>50
extending from the catalytic site toward the peripheral anionic
site (PAS), where the 3,4-dimethoxy moiety of compound 9a was
undergoing favorable interactions with Trp286 (distance ꢀ 3–4
—
˚
A). The C6 and C7 chlorine atoms were oriented toward the
C6
C7
C8
C6
C7
C8
—
—
—
—
˚
—
—
—
—
—
—
—
—
catalytic triad (distance ꢀ 6–8 A), while the C2-alkylamine
groups underwent hydrophobic interactions with Trp86
31.2 ꢂ 3.4
˚
(
distance ꢀ 3–5 A). The binding orientation of compounds 8e
>50
and 12b that exhibited superior inhibitory potency toward
BuChE was quite different (Fig. 4, panel B). The most potent
BuChE inhibitor 8e underwent three hydrogen-bonding inter-
actions centered around the C2 isopropylamine-NH, the 7-
>50
>50
>50
Donepezil
Galantamine
3.6 ꢂ 0.4
—
>50
chloroquinazoline-N1 with His447 and Ser203 duo (distance ꢀ
a
˚
IC50 values were an average ꢂ SD of triplicate readings for two to three 2.5–3.5 A). The core ring scaffold was also oriented in the
independent experiments.
catalytic triad, with the C7 chlorine oriented toward the acyl
˚
pocket (Leu286-Val288, distance
ꢀ
4–5 A). The C4-
phenethylamine substituent was oriented perpendicular to
This trend also extended to the AChE proles, where 12b (AChE Trp82 (Fig. 4, panel B). On the other hand, derivative 12b had its
IC50 ꢀ 5.8 mM) was ꢀ2.3-fold more potent compared to 13b, it core scaffold and C4-phenethylamine substituent directly
was also ꢀ1.3-fold more potent compared to its 6- and 8-chloro interacting with Trp82 (distance ꢀ 3–4 A˚ ), while the N,N-dime-
isomers (12a and 12c). These studies show that combining the thylamine was oriented toward the catalytic triad (distance ꢀ 4–
N,N-dimethylamine group at C2 with phenethylamine or 3,4- 5 A˚ ) which explains their superior inhibition prole.
dimethoxyphenethylamine on a pyrido[3,2-d]pyrimidine scaf-
fold resulted in equipotent and selective AChE inhibition (14a
2
.3 Iron chelation properties
and 15a, AChE IC50 ꢀ 6.7 mM, BuChE IC50 > 50 mM, Table 2). The
2
4
The presence of an additional ring nitrogen in the pyrido[3,2-d]
pyrimidine class of compounds (10a–c, 11a–c, 14a and 15a)
provides a chelation center.
promoting oxidative stress in AD
formation of neurotoxic Ab plaques and neurobrillary tangles
quinazoline
derivative
8e
(7-chloro-N -isopropyl-N -
phenethylquinazoline-2,4-diamine) was the most potent BuChE
19,20
2
2
Due to the role of iron in
and its contribution in the
inhibitor with an IC5 ꢀ 100 nM, while 12b (7-chloro-N ,N -
0
21–23
4
dimethyl-N -phenethylquinazoline-2,4-diamine) was the most
potent AChE inhibitor with an IC50 ꢀ 5.8 mM.
2
+
(NFTs), we assessed the Fe -chelation potential of the pyrido
The binding interaction of quinazoline derivatives was
investigated by using the X-ray crystal structures of human
[3,2-d]pyrimidines (Table 3) in ferrozine based assay. The
17,18
known iron chelators clioquinol and deferroxamine were used
as reference agents. These studies show that, the C4 phene-
thylamine substituted compounds 10a–c exhibited better
AChE (pdb id:1B41) and BuChE (pdb id:1P0I) enzymes.
binding modes of two best quinazoline based AChE inhibitors
a and 12b in hAChE (Fig. 4, panel A) revealed quite similar
The
9
chelation
capacity
compared
to
their
C4
3,4-
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Table 3 Iron (Fe ) chelation studies of pyrido[3,2-d]pyrimidines
RSC Advances
2+
further purication. Melting points were determined using
1
a Fisher-Johns melting point apparatus and are uncorrected. H
2+
%
Fe
13
1
a
NMR (300 MHz) and C NMR spectra (100 MHz) were recorded
Compd
R
R
Chelation
on a Bruker Avance NMR spectrometer in DMSO-d . Coupling
6
1
1
1
1
1
1
1
1
0a
0b
0c
1a
1b
1c
4a
5a
Phenethyl
Phenethyl
Phenethyl
3,4-DiOMe-phenethyl
3,4-DiOMe-phenethyl
3,4-DiOMe-phenethyl
Phenethyl
3,4-DiOMe-phenethyl
—
n-Pr
i-Pr
c-Pr
n-Pr
i-Pr
c-Pr
—
—
—
—
29.3 ꢂ 4.3
23.6 ꢂ 4.0
37.0 ꢂ 5.6
24.1 ꢂ 3.6
21.8 ꢂ 3.3
26.7 ꢂ 4.0
22.9 ꢂ 3.4
23.0 ꢂ 3.0
39.0 ꢂ 5.8
86.7 ꢂ 11.0
constants (J values) were recorded in hertz (Hz) and the
following abbreviations were used to represent multiplets of
NMR signals: s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet,
br ¼ broad. Carbon multiplicities (C, CH, CH
2 3
and CH ) were
assigned by DEPT 90/135 experiments. Low-resolution mass
spectra (LRMS) was obtained using an Agilent 6100 series single
quad LCMS whereas high-resolution mass spectra (HRMS) were
recorded on a Thermo Scientic Q Exactive™ mass spectrom-
eter with an ESI source, Department of Chemistry, University of
Clioquinol
Deferoxamine
—
a
2+
%
Fe chelation values were an average ꢂ SD of duplicate readings for Waterloo. Compound purity was assessed (ꢀ95% purity) using
two independent experiments. The ferrozine absorbance was measured
at 562 nm.
an Agilent 6100 series single quad LCMS equipped with an
Agilent 1.8 mm Zorbax Eclipse Plus C18 (2.1 ꢃ 50 mm) running
ꢄ1
50 : 50 water/ACN with 0.1% FA at a ow rate of 0.5 mL min
with detection at 254 nm by UV. Compounds 3a–d and 4a were
dimethoxyphenethyl dimethoxyphenethylamine counterparts
24–26
previously reported.
.1.1 General procedure for the synthesis of compounds
a–d. These compounds were synthesized as per previously re-
11a–c, 14a and 15a (inactive at 50 mM).
4
Overall, these pyrido[3,2-d]pyrimidine compounds exhibited
3
weak to moderate (22–37% chelation) iron-chelation capacity.
Interestingly, the presence of a C2 cyclopropyl substituent
provided the best chelation capacity compared to other C2
substituents explored with compound 10c exhibiting equi-
potent iron-chelation capability (37% chelation at 50 mM)
compared to the reference agent clioquinol (39% inhibition at
24,26
ported method.
In a 350 mL round pressure vial 16–18 g urea
ꢁ
was heated at 150–155 C till it melted. To the liquid urea
solution 0.1 eq. of 1a, or 1b, or 1c or 1d was added. The pressure
ꢁ
vial was sealed and heated at 150–155 C for 2 h, cooled to room
temperature, followed by the addition of water (100 mL) aer
ꢁ
which the reaction mixture was heated at 100–105 C for 1 h.
50 mM, Table 3). More signicantly, the corresponding quina-
Aer cooling to room temperature, the reaction mixture was
diluted with ꢀ80 mL EtOAc and washed with brine solution (50
mL ꢃ 4). The combined aqueous layers were washed with ꢀ35
zoline class of compounds did not exhibit any iron-chelation
capacity highlighting the requirement of a pyrido[3,2-d]pyrim-
idine ring scaffold to display iron-chelation properties.
4
mL EtOAc, dried over MgSO and the organic solvent was
removed in vacuo. The solid obtained (compounds 2a–d) was
straightaway carried to the next step (yield 70–85%) without
further purication. In a 250 mL RBF, 29.24 mmol of either 2a
or 2b or 2c or 2d was suspended in 25 mL of anhydrous toluene
3
Conclusions
In summary, we synthesized and optimized the structure of
quinazoline based ring scaffold as multi-targeting agents aimed
at the amyloid, cholinergic, and oxidative stress pathways of AD
pathophysiology. A number of agents synthesized exhibited
dual Ab, ChE inhibition and iron-chelation capacity. The SAR
acquired shows that compound 8h was the most potent Ab40
aggregation inhibitor (IC50 ¼ 900 nM) and compound 8e
provided dual Ab (IC50 Ab40 ¼ 6.1 mM; Ab42 IC50 ¼ 7.2 mM) and
ChE inhibition (IC50 AChE ¼ 6.6 mM; BuChE IC50 ¼ 100 nM).
The presence of a pyrido[3,2-d]pyrimidine bioisostere instead of
a quinazoline was essential to exhibit iron-chelation properties
and allowed to stir on an ice bath. To this, 5 eq. of POCl was
3
added in small aliquots followed by the slow addition of 5 eq. of
DEA. The solution was kept on the ice bath for 10 min before
moving to room temperature and allowed to stir for 1 h prior to
ꢁ
reuxing at 105–110 C for 14–16 h. Upon cooling to room
temperature, the reaction mixture was added in small aliquots
to a double-ice-water bath while stirring. The quenching solu-
tion was le stirring at room temperature for 5 h before vacuum

ltering the yellowish-grey precipitate. The precipitate was
stirred for 1 h in saturated NaHCO solution and was ltered.
3
(22–37% chelation) with compounds 10b and 10c exhibiting
This neutralization process was carried out 2–3 times until the
multi-targeting activity toward Ab, ChE and iron-chelation
properties. The data presented herein highlights the dual
anti-Ab40/b42 aggregation, ChE inhibition, and metal-chelation
capabilities of quinazoline and pyrido[3,2-d]pyrimidine class of
compounds as multi-targeting agents to treat AD.
bicarbonate solution maintains a neutral to slight basic pH. The

nal precipitate was dissolved in DCM and puried by a silica
gel column chromatography using 100% DCM as the eluent to
afford white to light grey solids 3a–d (65–80%).
4.1.2 General procedure for the synthesis of compounds
4
a–c, 5a–c, 6a and 7a. These compounds were synthesized as
27,28
per previously reported method.
To a 30 mL solution of
4
Materials and methods
ethanol in a 100 mL round-bottom ask on ice, ꢀ21.46–
4.1 General information
2
5.13 mmol of either 3a or 3b or 3c or 3d was added followed by
All chemicals and reagents used were purchased from either slow addition of 1.3 eq. (27.90–32.66 mmol) of the corre-
Sigma-Aldrich, USA or Alfa Aesar, USA and was used without sponding primary amine. Contents were stirred on an ice bath
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while 2.0 eq. of diisopropyl-ethylamine (DIPEA, 42.92–50.25 and 5 min). Appropriate controls, without the test compounds
mmol) was added in drop wise fashion. The solution was then and ChE were included. The inhibitory concentration (IC50
ꢁ
heated at 80–85 C under reux for 3–4 h. The reaction contents values) was calculated from the concentration-inhibition dose
were cooled to room temperature and precipitated residues response curve on a logarithmic scale based on two indepen-
were vacuum-ltered with EtOAc. The organic supernatant was dent experiments run in triplicates.
concentrated in vacuo followed by two rounds of extraction
using EtOAc and saturated brine solution (40–50 mL each
4.3 Experimental procedure for thioavin T based Ab
respectively). The combined organic layers were dried over
aggregation kinetics
4
MgSO , evaporated in vacuo and puried (1–2 times) using silica
The anti-Ab aggregation activity of test compounds (8a–i, 9a–i,
gel column chromatography with 5 : 1 EtOAc : MeOH as the
elution solvent to afford 4a–c, 5a–c, 6a and 7a as white to beige
solids with yields ranging from 75–90%.
1
0a–c, 11a–c, 12a–c, 13a–c, 14a and 15a), was evaluated using
11,14
the ThT-based uorescence assay.
The Ab40 and Ab42
hexauoro-2-propanol (HFIP) (rPeptide, Geogia, USA) stock
4
.1.3 General procedure for the synthesis of compounds
a–i, 9a–i, 10a–c, 11a–c, 12a–c, 13a–c, 14a and 15a. The target
compounds were synthesized as per previously reported
solution was prepared by dissolving in 1% NH
4
OH solution, to
8
ꢄ1
a 1 mg mL stock solution, followed by dilution to 50 mM in
phosphate buffer (215 mM, pH 7.4). Stock solutions of test
compounds were prepared in DMSO, diluted in phosphate
buffer (pH 7.4), and were sonicated for 30 min. The nal DMSO
concentration per each well was 1% v/v or lower. The ThT
1
2,28
method.
dichloro, 2,7-dichloro or 2,8-dichloro-N-substituted quinazolin-
-amine or 2-chloro-N-substituted pyrido[3,2-d]pyrimidine-2,4-
diamine (ꢀ0.66–0.83 mmol) was combined with 2 eq. (ꢀ1.32–
.66 mmol) of primary amine (methyl-, ethyl-, n-propyl-, iso-
Briey in a 50 mL pressure vial (PV), 0.25 g of 2,6-
4

uorescent dye stock solution (15 mM) was prepared in 50 mM
1
glycine buffer (pH 7.4). The aggregation kinetics assay was
carried out using a Corning® 384-well at, clear bottom black
plates. Each well contains 44 mL of ThT, 20–35 mL of phosphate
buffer (pH 7.4), 8 mL of test compounds in different concen-
trations (1, 5, 10 and 25 mM, nal concentration) and 8 mL of
either Ab40 or Ab42 (5 mM nal concentration). The plate was
propyl- or cyclopropylamine) or dimethylamine which was dis-
solved in 5 mL of 1,4-dioxane followed by the addition of 3 eq. of
DIPEA (ꢀ1.98–2.40 mmol). Pressure vial was sealed and stirred
ꢁ
in an oil bath at 150–155 C for 2 h. Upon completion and
cooling to room temperature, the reaction mixture was diluted
with ꢀ40 mL of EtOAc and washed with brine solution (25 mL ꢃ
ꢁ
incubated at 37 C with a plate cover under shaking and uo-
2
). The combined aqueous layer was washed with ꢀ25 mL of
rescence was measured every 5 min using a BioTek Synergy H1
microplate reader multimode plate reader (excitation ¼ 440 nm
and emission ¼ 490 nm) over a period of 24 h. Appropriate
control experiments that contain either Ab40/42 and test
compound alone were evaluated. The known Ab aggregation
inhibitors curcumin and resveratrol were used as reference
agents. The IC50 value (mM) was calculated using the equation
EtOAc. The combined EtOAc layers were dried over MgSO
4
before removing EtOAc in vacuo to yield a solid product that was
puried by silica gel column chromatography using 5 : 1
EtOAc : MeOH as the eluent to afford pale yellow to brown
solids (yield ꢀ 68–78%).
100% control – [(IFi–IFo)] where 100% control indicates no
4.2 Experimental procedure for cholinesterase (hAChE/
inhibitor, IFi and IFo are the uorescence intensities in the
presence and absence of ThT. The results were expressed as IC50
values ꢂ SD based on two separate experiments in triplicate
measurements.
hBuChE) inhibition
Quinazoline and pyrido[3,2-d]pyrimidine derivatives (8a–i, 9a–i,
10a–c, 11a–c, 12a–c, 13a–c, 14a and 15a) were evaluated for ChE
12,15,29
inhibitory activity using Ellman's method.
The test
compounds compete with substrates acetylthiocholine iodide
ATChI) and S-butyrylthiocholine iodide (BuTChI) for either
human AChE (Sigma-Aldrich, St. Louis, MO) or human BuChE
Sigma-Aldrich, St. Louis, MO), respectively. Known ChE In Costar 96-well, round-bottom plates were added 80 mL of
4
.4 Experimental procedure for transmission electron
(
microscopy (TEM)
(
inhibitors donepezil and rivastigmine were used as reference 215 mM phosphate buffer, 20 mL of 10ꢃ test compound dilu-
agents for comparison. Quinazoline and pyrido[3,2-d]pyrimi- tions (250 mM – prepared in the same way as for the ThT assay)
dine derivatives were prepared in DMSO (maximum concen- and 100 mL of Ab40 or Ab42 respectively (50 mM each). For the
tration used 1% v/v) and 10 mL each (0.001–25 mM nal control wells, 2 mL of DMSO and 18 mL of phosphate buffer was
concentration range), was incubated for 5 minutes at room added. Final Ab: test compound ratio was 1 : 1 (25 mM : 25 mM).
ꢄ1
ꢁ
temperature with 160 mL of 1.5 mM DTNB, 50 mL 0.22 U mL
Plates were incubated on a Fisher plate incubator set to 37 C
AChE (in 50 mm Tris$HCl, pH 8.0, 0.1% w/v bovine serum and the contents were shaken at 730 cpm for 24 h. To prepare
ꢄ1
albumin, BSA) or 50 mL 0.12 U mL BuChE (in 50 mm Tris$HCl, the TEM grids, ꢀ20 mL droplet was added using a disposable
pH 8.0, 0.1% w/v bovine serum albumin, BSA). Aer the incu- Pasteur pipette over the formvar-coated copper grids (400
bation period, 30 mL of ATChI (15 mM, prepared in ultra pure mesh). Grids were air-dried for about 3 h before adding two
water) or BuTChI (15 mM, prepared in ultra pure water) were droplets (ꢀ40 mL, using a disposable Pasteur pipette) of ultra-
added to 96-well plates. The ChE inhibition was measured at pure water and using small pieces of lter paper to wash out
4
12 nm wavelength using a microplate reader (BioTek Synergy precipitated buffer salts. Aer air-drying for ꢀ15–20 min, the
H1 microplate reader) at various time intervals (t ¼ 0, 1, 2, 3, 4 grids were negatively stained by adding a droplet (ꢀ20 mL, using
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a disposable Pasteur pipette) of 2% phosphotungstic acid (PTA) as average % iron-chelation ꢂ SD in triplicate measurements
and immediately aer the grids were dried using small pieces of based on two independent experiments.

lter paper. Grids were further air-dried overnight. The scan-
ning was carried out using a Philips CM 10 transmission elec-
tron microscope at 60 kV (Department of Biology, University of
Waterloo) and micrographs were obtained using a 14-megapixel
Acknowledgements
The authors would like to thank the Faculty of Science, Office of
Research, the School of Pharmacy at the University of Waterloo,
Ontario Mental Health Foundation (graduate scholarship for
TM), NSERC-USRA (for MM), NSERC-Discovery (RGPIN: 03830-
11,30
AMT camera.
4.5 Experimental procedure for molecular docking studies
The modeling experiments were carried out using Discovery 2014), Canada Foundation for Innovation (CFI-JELF), Ontario
Studio (DS), Structure-Based-Design version 4.0, soware Research Fund (ORF) and Early Researcher Award, Ministry of
program from BIOVIA Inc, San Diego, USA. Quinazoline and Research and Innovation, Government of Ontario, Canada (PR)
pyrido[3,2-d]pyrimidine derivatives 8e, 8h, 9a, 10b and 12b were for nancial support of this research project.
built using the small molecules module in DS. For ChE docking
studies, the X-ray coordinates of human AChE (pdb id:1B41)
and BuChE (pdb id:1P0I) were obtained from protein data bank.
The enzymes were prepared for docking using the macromole-
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