Design, synthesis and biological activity of multifunctional α,β-unsaturated carbonyl scaffolds for Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2013.02.103

A series of compounds containing an α,β-unsaturated carbonyl moiety, such as chalcones and coumarins were designed, synthesized and tested in a variety of assays to assess their potential as anti-Alzheimer's disease (AD) agents. The investigations include

Full text of DOI:10.1016/j.bmcl.2013.02.103

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2614–2618
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological activity of multifunctional
a,b-unsaturated carbonyl scaffolds for Alzheimer’s disease
Seema Bag a, Sanjukta Ghosh a, Rekha Tulsan a, Abha Sood a, Weihong Zhou a, Christine Schifone a,
Michelle Foster a, Harry LeVine III b, Béla Török a, Marianna Török a,
⇑
a Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Blvd, Boston MA 02125-3393, USA
b Department of Molecular and Cellular Biochemistry, Chandler School of Medicine, Center on Aging, University of Kentucky, Lexington, KY 40536, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of compounds containing an a,b-unsaturated carbonyl moiety, such as chalcones and coumarins
Received 3 December 2012
Revised 15 February 2013
Accepted 25 February 2013
Available online 14 March 2013
were designed, synthesized and tested in a variety of assays to assess their potential as anti-Alzheimer’s
disease (AD) agents. The investigations included the inhibition of cholinesterases (AChE, BuChE), the
inhibition of amyloid beta (Ab) self-assembly and the disassembly of preformed Ab oligomers. Several
compounds showed excellent potential as multifunctional compounds for AD. Docking studies for 16 that
performed well in all the assays gave a clear interpretation of various interactions in the gorge of AChE.
Based on the results, the long-chain coumarin scaffold appears to be a promising structural template for
further AD drug development.
Keywords:
Alzheimer’s disease
Cholinesterase inhibition
Amyloid beta
Ó 2013 Elsevier Ltd. All rights reserved.
Coumarins
Chalcones
Alzheimer’s disease (AD) is associated with a loss of presynaptic
markers of the cholinergic system in the areas of the brain related
to memory and learning, and is also characterized by the presence
of amyloid deposits and neurofibrillary tangles in the brain.1 The
deterioration of cholinergic neurotransmission is responsible for
the decline in memory and cognition in patients suffering from
AD.2 One of the ways to enhance cholinergic function by preserving
acetylcholine (ACh) levels is to inhibit acetylcholinesterase (AChE)
responsible for the metabolic breakdown of ACh. Preclinical exper-
iments and clinical trials have shown butyrylcholinesterase
(BuChE) to be an important contributor for the occurrence, symp-
toms, progression, and responses to treatment in dementia.3,4 Sev-
eral anti-cholinergic drugs have been launched including tacrine,
rivastigmine, donepezil, and galanthamine.5 It has also been shown
that dual AChE/BuChE agents might be superior to the selective
AChE inhibitors.2
AChE has a long and narrow gorge (20 Å) with two binding
sites; a catalytic site (active site) and a peripheral anionic site
(PAS).6 It was reported that the peripheral site of AChE could pro-
mote the formation of amyloid beta (Ab) deposits.7,8 The formation
of such deposits is a hallmark of AD pathology.9,10 Detailed stud-
ies11 indicated that both fibrillar and oligomeric species of Ab are
neurotoxic.12,13 Thus the design of multifunctional inhibitors,
which can control the hydrolytic activity of cholinesterases and
interfere with the self-assembly of Ab and disassemble the pre-
formed aggregates is highly desirable.14–16
Continuing our earlier work on anti-AD agents,17–21 we describe
novel compounds with an a,b-unsaturated carbonyl moiety (chal-
cones and coumarins) that can act as multifunctional anti-cholin-
esterase/Ab agents. Chalcone22 and coumarin23 derivatives have
been described as AChE and BuChE inhibitors. While coumarins
have also been applied as Ab aggregation inhibitors,24 chalcones
are more commonly used as Ab imaging agents.25,26 There are no
reports that such compounds synthesized or applied as multifunc-
tional agents inhibiting both cholinesterases and Ab self-assembly.
We designed
chain (chalcones) and cyclic (coumarins) structures. In order to
have dual binding property in the gorge of AChE the ,b-unsatu-
a,b-unsaturated carbonyl compounds with open
a
rated carbonyl unit was linked to different amines. The designed
structures were then screened through the 4-point pharmacophore
developed in our group27 and molecules that matched 3 of the 4
points of our pharmacophore (Fig. 1) were synthesized.
The syntheses of the compounds are summarized in Schemes 1
and 2.28 The synthesis of the target compounds applied readily
available starting materials and was completed using well estab-
lished methods.25
The compounds were tested by the Ellman colorimetric assay
for cholinesterase inhibition.29–31 For comparison, galanthamine
(GAL), a known cholinesterase inhibitor, was used. The experi-
⇑
Corresponding author. Tel.: +1 617 287 6199.
E-mail address: marianna.torok@umb.edu (M. Török).
ments were carried out with 2 and 10 lM inhibitor concentration,
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.103

S. Bag et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2614–2618
2615
60
50
40
30
20
10
0
(A)
Figure 1. Pharmacophore features of two of the designed compounds. Light blue
sphere-H-acceptor; red sphere-H-donor; ring—aromatic ring and dark blue
sphere—positively charged center.
Inhibitors
O
O
HN
NBS
70
60
50
40
30
20
10
0
(B)
Br
CCl4
reflux, 3h
CH2Cl2
reflux, 2h
O
O
Ar-CHO
Na
X
N
y HCl
n
X
N
n
OH, EtO
H
Ar
0 °C,1
h; rt 3h
(1-15)
8: Ar = 4-OCH3-Ph, X = N, n = 1
X = N, C
n = 0, 1
1: Ar = Ph, X = -, n = -
9
2
: Ar = 3-OCH3-Ph, X = -, n = -
10: Ar = 3-OCH3-Ph, X = C, n = 0
11
: Ar = Ph, X = N, n = 1
3: Ar = naphthalene, X = -, n = -
4
: Ar = 3-OCH3-Ph, X = N, n = 1
12: Ar = 4-F-Ph, X = -, n = -
: Ar = 3,4-di-OCH3-Ph, X = -, n = -
5: Ar = 3,4-di-OCH3-Ph, X = N, n = 1
13
6
: Ar = 4-Cl-Ph, X = -, n = -
14: Ar = 4-Br-Ph, X = -, n = -
15
: Ar = 4-OCH3-Ph, X = -, n = -
7: Ar = 4-OCH3-Ph, X = C, n = 0
: Ar = 4-Ph-Ph, X = -, n = -
Scheme 1. Synthesis and structures of chalcone derivatives used.
Inhibitors
Figure 2. Inhibition of (A) AChE and (B) BuChE hydrolytic activity by chalcones and
O
O
O
O
coumarins. The experiments were carried out at 2 lM (A) and 10 lM (B) inhibitor
OH
concentration and 0.02 U/mL enzyme concentration.29
Br2
O
CHCl3
rt, 2h
piperidine
0°C, 1h; rt, 3h
O
O
O
CHO
than that of GAL except 15 with an IC50 of 8.27 lM. While the ini-
O
O
Br
HN
tial data suggested 11 being a slightly better inhibitor than GAL,
the IC50 determination did not confirm this likely due to the devi-
ation of the data.
In order to understand the interactions of 16 with the enzyme, it
was docked in the active site of AChE (PDB code: 1E66) (Fig. 3) using
SP mode of Glide module of Schrödinger.32 For comparison com-
pound 16 was superimposed with galanthamine and another
well-known AChE inhibitor, donepezil (Fig. 3). Unlike galantha-
R
O
O
O
DMF, Na2CO3
rt, 2h
O
O
O
(16-22)
N
19: R =
HN
N
16: R =
HN
N
N
20: R =
HN
N
O
17: R = HN
N
21
: R = HN
HN
N
18
: R =
22
: R =
HN
Scheme 2. Synthesis and structures of coumarin derivatives used.
respectively (IC50 of GAL for AChE and BuChE) (Fig. 2).6,7 GAL was
selected due to its low toxicity and ready commercial availability.
The data (Fig. 2) indicate that the compounds possess apprecia-
ble dual cholinesterase inhibition. Coumarins are potent AChE
inhibitors while chalcones show stronger activity in BuChE inhibi-
tion. In AChE inhibition most inhibitors showed comparable but
somewhat weaker inhibition than that of the reference compound
(GAL). Compound 16 exhibited higher activity than GAL and
showed excellent (57%) inhibition at 2
IC50 of 1.76 M.
lM (IC50 of GAL) with an
l
The compounds behaved similarly in BuChE inhibition assays.
Chalcones possess activity similar to GAL and a few compounds
showed more than 50% inhibition at 10
lM (IC50 of GAL), however,
Figure 3. Superimposition of 16 (pink, ball and stick) with donezepil (green, ball
most of their IC50 values were determined to be slightly higher
and stick) and galanthamine (brown, ball and stick) in the active site of 1E66.

2616
S. Bag et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2614–2618
80
70
60
50
40
30
20
10
0
mine, 16 spanned the active site and peripheral anionic site of the
enzyme similarly to donepezil. As shown, 16 is oriented properly
in the active site (Fig. 3). The coumarin ring was stabilized by
hydrophobic interactions with Trp 333, Phe 290 and Phe 288 resi-
dues. The phenyl ring at the amino end was stabilized by hydropho-
bic and p–p interactions through Trp 84 and Trp 130. The methoxy
group of coumarin ring formed a hydrogen bond (Phe 288) and the
–NH of the piperazine ring formed a hydrogen bond with Ser 122.
These observations explain the potency of 16 in AChE inhibition.
As fibrillar and oligomeric aggregates of Ab are neurotoxic, the
activity of the compounds was determined against the formation
of these species and in the disassembly of preformed oligomers.
The compounds were tested by standard methods, the biotinyl-
Ab(1–42) single-site streptavidin assay for oligomer formation
and disassembly33,34 (Fig. 4) and the thioflavine T-fluorescence as-
say for fibril formation (Fig. 5).35–37
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Inhibitors
Figure 5. Effect of the designed compounds on the fibrillogenesis of Ab (100
inhibitor, 100 M Ab).
lM
l
In the oligomer assembly assay coumarins (16–22) are excellent
inhibitors (>65%, Fig. 4A) while chalcones showed little or no inhi-
bition (Fig. 4A). The concentration dependence studies revealed the
is worth noting that the compounds did not show significant
intrinsic fluorescence thus their presence in the assay did not affect
the fluorescence readings.
Atomic force microscopy (AFM) was used to confirm the fibril-
logenesis inhibition (Fig. 6). The images revealed the expected
dense network of long fibrils in the control sample. While fibrils
are still visible in the inhibited samples the density is significantly
lower in agreement with the assay results. It also can be observed
that shorter assemblies formed in the presence of 16.
following IC50 values: 16 (36
(1 M), 21 (29 M), 22 (36 M).
The same group of compounds (16–22) effectively (up to 95%)
disassembled the preformed oligomers as well (Fig. 4B). The IC50
data for oligomer disassembly are: 17 (29 M), 18 (6.5 M), 20
(4 M) and 21 (5 M).
The compounds exhibited similar behavior in the inhibition of
lM), 17 (14 lM), 18 (21 lM), 20
l
l
l
l
l
l
l
Ab fibril formation (Fig. 5). Both chalcones (1–15) coumarins
(16–22) were reasonable fibrillogenesis inhibitors (ꢀ30–70%). It
Chalcones and coumarins have already been investigated to
modulate cholinesterase activity or inhibit Ab self-assembly,
respectively.22–26 In an attempt to combine these activities in a
single chemical structure and develop multifunctional anti-AD
compounds we synthesized 22 compounds with chalcone and
coumarin head and varied tail groups. The aim was to build
compounds of sufficient length to span the active center of the cho-
linesterases and at the same time interfere with Ab self-assembly.
The structure–activity relationship reveals important information
for future design. It appears that the above results obtained with
coumarins support the hypothesis. Chalcones showed strong
activity in BuChE and fibrillogenesis inhibition only. They were
relatively weak inhibitors of AChE and Ab oligomer formation. In
contrast, coumarins performed more consistently showing signifi-
cant inhibition in all assays, except against BuChE where they were
moderate to weak inhibitors. At this point the coumarin deriva-
tives were limited to one head group and different tail groups pos-
sessing aryl-substituted piperidine, piperazine and morpholine
units. Compounds with the two nitrogen containing piperazine
ring (16, 18–20) performed the best overall. While the observed
differences in activity within this subgroup are not decisive, the
data suggest that the most beneficial tail groups are phenyl and
benzyl piperazines (16, 18). It appears that the presence of basic
nitrogens in the middle ring is advantageous; however, too many
nitrogens (e.g., pyrimidinyl-piperazine group, 20), result in a slight
decrease in activity in all assays.
100
80
60
40
20
0
(A)
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Inhibitors
-20
100
80
60
40
20
0
(B)
Comparing the anti-oligomer and anti-fibril assays one may no-
tice that while coumarins almost completely inhibit the formation
of oligomers the same compounds are moderate inhibitors (ꢀ65%)
of the fibril formation. It is most likely due to the fact that there are
multiple pathways involved in the amyloid formation and the olig-
omers are not obligatory precursors to the fibrils.38
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Inhibitors
In conclusion, a variety of compounds with
a,b-unsaturated
-20
-40
moiety were synthesized with the aim of preparing multifunc-
tional compounds with anticholinesterase and anti-Ab assembly
activities for AD. It was observed that while the open chain deriv-
atives (chalcones) were inactive in two important assays the cyclic
coumarins showed excellent to reasonable effects in several assays,
for example cholinesterase activity and Ab self-assembly. Based on
Figure 4. Effect of the designed compounds on the (A) oligomer assembly of Ab and
(B) disassembly of preformed Ab oligomers (50 M inhibitor; 0.01 M Ab-inhibi-
tion; 0.0028 M Ab-dissociation).
l
l
l

S. Bag et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2614–2618
2617
Figure 6. Illustrative images of the control and selected samples in the presence of compounds 15, 16 and 19, respectively.
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promising leads for further inhibitor design. The structures of our
lead compounds represent a relatively nonpolar character with
an extended network of hetero- and carbocyclic rings. This feature
suggests favorable membrane permeability, which is an important
factor for drug candidates.
Acknowledgments
Financial support provided by the University of Massachusetts
Boston, and National Institute of Health (R-15 AG025777-03A1
and R21AG028816-01 to H.L.) is gratefully acknowledged.
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.103.
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