Synthesis and application of β-carbolines as novel multi-functional anti-Alzheimer's disease agents

Article abstract of DOI:10.1016/j.bmcl.2016.11.067

The design, synthesis and assessment of β-carboline core-based compounds as potential multifunctional agents against several processes that are believed to play a significant role in Alzheimer's disease (AD) pathology, are described. The activity of the compounds was determined in Aβ self-assembly (fibril and oligomer formation) and cholinesterase (AChE, BuChE) activity inhibition, and their antioxidant properties were also assessed. To obtain insight into the mode of action of the compounds, HR-MS studies were carried out on the inhibitor-Aβ complex formation and molecular docking was performed on inhibitor-BuChE interactions. While several compounds exhibited strong activities in individual assays, compound 14 emerged as a promising multi-target lead for the further structure-activity relationship studies.

Full text of DOI:10.1016/j.bmcl.2016.11.067

Accepted Manuscript
Synthesis and Application of β-Carbolines as Novel Multi-functional Anti-Alz-
heimer’s Disease Agents
William Horton, Abha Sood, Swarada Peerannawar, Nandor Kugyela, Aditya
Kulkarni, Rekha Tulsan, Chris D. Tran, Jessica Soule, Harry LeVine III, Béla
Török, Marianna Török
PII:
S0960-894X(16)31220-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.067
BMCL 24462
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 October 2016
21 November 2016
22 November 2016
Please cite this article as: Horton, W., Sood, A., Peerannawar, S., Kugyela, N., Kulkarni, A., Tulsan, R., Tran, C.D.,
Soule, J., LeVine, H. III, Török, B., Török, M., Synthesis and Application of β-Carbolines as Novel Multi-functional
Anti-Alzheimer’s Disease Agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.11.067
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Synthesis and Application of β-Carbolines as Novel Multi-functional Anti-
Alzheimer’s Disease Agents
William Horton,a Abha Sood,a Swarada Peerannawar,a Nandor Kugyela,a Aditya Kulkarni,a Rekha Tulsan,a
Chris D. Tran,a Jessica Soule,a Harry LeVine III,b Béla Török,a Marianna Töröka,*
a University of Massachusetts Boston, 100 Morrissey Blvd. Boston, MA, USA. Fax: (+1)-617-287-6030; Tel: (+1)-617-287-6159
b Department of Molecular and Cellular Biochemistry, Chandler School of Medicine, and Center on Aging, University of Kentucky, Lexington, KY 40536, USA
* To whom all correspondence should be addressed: bela.torok@umb.edu, marianna.torok@umb.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
The design, synthesis and assessment of β-carboline core-based compounds as potential
multifunctional agents against several processes that are believed to play a significant role
in Alzheimer’s Disease (AD) pathology, are described. The activity of the compounds was
determined in Aβ self-assembly (fibril and oligomer formation) and cholinesterase (AChE,
BuChE) activity inhibition, and their antioxidant properties were also assessed. To obtain
insight into the mode of action of the compounds, HR-MS studies were carried out on the
inhibitor-Aβ complex formation and molecular docking was performed on inhibitor-
BuChE interactions. While several compounds exhibited strong activities in individual
assays, compound 14 emerged as a promising multi-target lead for the further structure-
activity relationship studies.
Revised
Accepted
Available online
Keywords:
Keyword_1 Alzheimer’s disease
Keyword_2 amyloid beta
Keyword_3 cholinesterase inhibition
Keyword_4 antioxidant
Keyword_5 β-carbolines
2009 Elsevier Ltd. All rights reserved
.
Due to the rapidly aging population and the ever greater
areas of the pathogenesis should be more effective than drug
candidates that would only alter single pathogenic contributors.18
occurrence of Alzheimer’s disease (AD)
a multitude of
therapeutic approaches have been investigated. 1,2,3,4,5,6,7 The most
common ones that resulted in clinical effects are based on the
cholinergic8 and amyloid cascade hypotheses.9 The first strategy
Building upon our recent efforts19,20,21,22,23,24,25,26,27 potential
multitarget inhibitors were designed based on the β-carboline
core structure. Herein, we describe the synthesis, and
biochemical evaluation of these compounds. The β-carboline
core or sub-unit appears in a large number of biologically active
compounds. Such compounds possess a variety of biological
effects including anticancer28, antiprotozoal29, or anti-
leishmanial30 activities. Compounds with the β-carboline core
structure have also been described targeting a variety of
neurological disorders and neurodegenerative diseases and being
provides
a symptomatic treatment via the inhibition of
cholinesterase enzymes that successfully addressed the low level
of acetylcholine neurotransmitter.10 In fact, the currently
available AD drugs are mostly based on this approach, namely
the inhibition of the acetylcholinesterase enzyme (AChE).11
Similarly to AChE the activity of butyrylcholinesterase (BuChE)
also has a negative effect on the abundance of neurotransmitters.
12
However, it is widely accepted that the accumulation of
tau
phosphorylation
inhibitors31,
channel
blockers32
neurotoxic protein assemblies of the amyloid β peptide (Aβ) in
the form of soluble oligomers and insoluble fibrillar deposits are
among the significant instigators of AD.13 This initiated extensive
efforts on the development of Aβ self-assembly inhibitors.14 The
most recent potential therapeutic tried for AD therapy, the
antibody Aducanumab, targets the Aβ deposits resulting in their
clearance and possible cognitive benefits.15 As recent studies
suggest, there may be a connection between the cholinergic and
Aβ targets. It was proposed that AChE peripheral binding site
may initiate the Aβ self-assembly.16 To further highlight the
complex nature of AD, metal ions and oxidative stress were also
suggested to contribute to the progression of AD.17 The network
of symptoms and potential causative sources of the disease
suggest that the development of compounds that target multiple
cholinesterase inhibitors33 GABAA receptor modulators,34, or
antioxidants.35 Based on these preliminaries an initial set of β-
carbolines have been designed and synthesized with the aim of
incorporating structural features that could present the
opportunity to apply these compounds as multitarget modulators
of several processes thought to play significant roles in the
development and progression of AD.
The design of the structures was based on the following major
factors. The structural analysis of a large set of Aβ self-assembly
inhibitors14 indicated the importance of the aromatic structure as
well as the presence of H-donor and H-acceptor units. The basic
β-carboline skeleton fulfills this criterion. Analyzing the
structures of cholinesterase inhibitors it appears paramount to
have a relatively extended structure that is able to span the active

center of the cholinesterases involving a variety of hydrophobic
units. For this reason the original three ring system has been
extended with an additional aromatic ring either directly or via a
carbonyl linker to test the role of molecular flexibility on the
efficacy of anti-cholinesterase activity. The extended aromatic
structures are also expected to contribute to the potential
antioxidant activity. The compounds that were designed using the
above principals have been synthesized using our previously
developed environmentally benign method.36 The synthetic
procedure and preliminary set of β-carbolines are shown in Fig. 1.
Table 1. Effect of β-carbolines on the self-assembly of Aβ and the activity of
the AChE and BuChE enzymes.
Compound
AChE BuChE
inhibition inhibition
BuChE
IC50
(µM)
Aβ fibril Aβ oligomer
inhibition inhibition
(%)a
(%)b
(%)
(%)
1
2
- 4
- 7
19
10
12
37
34
17
20
11
- 5
6
79
6
- 12
- 9
-10
-15
-7
46
54
64
32
34
79
-
> 10
3.06 1.27
4.48 0.27
> 10
3
58
63
-
4
5
> 10
R
R
R
6
22
82
45
-
-3
4.27 1.30
-
> 10
Pd/C/K-10
MW, 130
7
-
NH
NH2
+
N
C
8
-9
7
N
H
N
H
N
H
O
Ar
Ar
9
-7
-6
11
69
46
1
> 10
10
11
12
13
14
GAL
-
1.29 0.25
1.42 0.73
> 10
tetrahydro-
β-carboline
H
Ar
84
82
30
58
-
-9
-9
β
-carboline
R
H
Ar
- 12
39
-
-10
-11
50
-
-
R
1
C6H5
95
29
0.22 0.03
10
CH3
CH3O
CH3O
CH3O
H
CH3
H
N
2
3
4
C6H5
C6H5
a Aβ:inhibitor=1:1 ratio at 100 µM Aβ concentration.
b Aβ:inhibitor = 0.0002 ratio at 0.01 µM Aβ concentration
N
H
C6H4-4-CH3
C6H4-4-F
C6H4-4-F
C6H4-4-CF3
C6H4-2-CH3
Ar
5
β
-carboline
c
6
7
β-carbolines were tested at 2 µM concentration for AChE and 10 µM
concentration for BuChE; GAL - galantamine
- data not measured due to solubility problems
8
O=C-C6H4
H
9
10
11
12
13
14
H
O=C-C6H3-4-CF3
CH3O
CH3O
H
O=C-C6H3-4-CF3
O=C-C6H3-4-F
cyclohexyl
As soluble Aβ oligomers are more neurotoxic than their
insoluble fibril counterparts, the activity of the compounds in the
inhibition of oligomer formation was also assessed by the
biotinyl-Aβ single-site streptavidin-based assay.38 Similarly to
anti-fibrillogenesis assays, the intensity of the inhibited samples
was normalized to the inhibitor-free control sample and the
inhibition was expressed in % compared to the uninhibited
sample. The data are summarized in Table 1.
α-naphthyl
H
Figure 1. Synthesis and structure of the designed β-carboline derivatives
The compounds were synthesized by a 3-step-one pot domino
reaction by using a special mixture of commercially available 5%
Pd/C and K-10 montmorillonite as a bifunctional catalyst. The
first condensation step between the aldehyde and tryptamine was
catalyzed by the solid acid K-10. The imine formed immediately
underwent a cyclization also by K-10 catalysis. The resulting
tetrahydro-β-carbolines were dehydrogenated by the Pd metal to
provide the aromatic β-carbolines. Each product was
characterized by 1H-and 13C-NMR spectroscopy and mass
spectrometry (GC-MS). The spectroscopic characterization of the
compounds was in agreement with their structures and the
literature data.
As the data show the compounds were highly active in
preventing the formation of soluble Aβ oligomers. A majority of
the compounds produced more than 50% inhibition (1, 3, 4, 7,
11, 12 and 14). The activity comparison of the compounds in the
Aβ fibril and oligomer formation inhibition assays are in
agreement with previous findings namely that a compound is
either a fibril inhibitor or an oligomer inhibitor as shown by the
respective behaviors of 1, 11 or 12.39 However, compounds 7 and
14 appear to provide a reasonable protection against both forms
of self-assembled Aβ.
After the completion of the synthesis the compounds were
evaluated in several assays to test the design hypothesis. Assays
included the inhibition of Aβ fibrillogenesis and oligomer
formation, modulation of cholinesterase activity of AChE and
BuChE enzymes, the determination of the antioxidant properties,
high resolution mass spectrometry and molecular docking.
+5
+6
+4
0:1
2:1
To determine the activity profile of the compounds they were
first subjected to Aβ fibrillogenesis assays. The quantitative
Thioflavin-T (THT) fluorescence assay was applied to determine
the antifibrillogenic potency of the compounds.37 The data were
0:1 1:1
0:1 1:1
compared to the fluorescence of the inhibitor-free control (Icontrol
)
and the observed decrease in fluorescence in the presence of the
inhibitors was normalized to a scale of 0-100% and was tabulated
as % inhibition in Table 1.
The data indicate that the compounds had a moderate effect on
the fibril formation of Aβ. While based on the limited number of
compounds a clear structure-activity relationship cannot be
drawn, it appears that the larger structures (6-10, 14) act as better
inhibitors. The best performance was shown by 6 (39%) and 14
(40%), which has an approximately 110 µM EC50 value.
Figure 2. High resolution mass spectrum of the Aβ-peptide-14 mixture
(30µM to 150 µM). The relevant signals indicate 1:1 and 1:2 complex
formation between the peptide and inhibitor compound. The intervals
highlight the relevant peaks of the charged Aβ carrying 4 to 6 positive
charges.

High resolution mass spectrometry data reveal a convincing
evidence that 14, which is able to block both fibril and oligomer
formation of Aβ, forms a complex with the peptide in the
solution (Fig. 2). The most typical complex appears to be the 1:1
ratio of Aβ:14, although another complex with somewhat higher
ratio (1:2) can be observed. The spectrum, however, shows that
Aβ is still overwhelmingly in an uncomplexed form, indicating
that a limited amount of inhibitor could modify the self-assembly
process by partially complexing/blocking the peptide.
In order to observe the potential multifunctional behavior of
the compounds, the β-carbolines were tested as cholinesterase
inhibitors as well. The compounds were subjected to the Ellman
assay using both AChE and BuChE, respectively (Table 1). The
compounds were assayed at the respective IC50 of galantamine
that was used as a reference compound (2 µM in AChE and 10
µM in BuChE inhibition).40, 41
Figure 4. Superimposition of molecule 14 (blue) with donepezil (red) and
galantamine (dark green) in the active site of huBChE (PDB ID: 1P0I).
(hydrogens are concealed for clarity)
The analysis of the docked structures revealed that while 11 is
extended through the active site of BuChE the reference
compounds galantamine and donepezil appear to bind in the right
side of the active site in 1P0I (Figs. 3-4). The indole -NH of 11
shows a hydrogen bonding interaction with the Pro285 and
Thr120 residues, respectively. In addition to that, the methoxy
oxygen of 11 interacts with the Glu197 residue of the active site.
In contrast, compound 14 appears to bind o the enzyme very
similarly to donepezil, which is another known inhibitor of AChE
as well as BuChE. The binding of 14 stretched through the active
site explains the IC50 that is an order of magnitude lower than that
of 11. The indole -NH of 14 shows hydrogen bonded interaction
with the hydroxyl group of residue Ser198. In addition to that
two π―π stacking interactions were observed, first between the
phenyl ring of indole and residue Trp231 and the second one
between the naphthalene ring of compound 14 and residue
His438. Likewise, a cation-π interaction was noticed between
quaternary nitrogen of 14 and residue Trp231. Since the
compounds appear to act as selective BuChE inhibitors a docking
study was carried out with 12 and AChE to observe whether the
compound-enzyme interaction would reveal the reasons (Fig. 5).
As shown the β-carbolines have negligible effect on the
activity of AChE. In contrast, the compounds were highly active
in the inhibition of BuChE; over 60% of the studied compounds
appear to be a better inhibitor of BuChE than galanthamine.
Several of them (e.g. 6, 14) show above 80% inhibition of the
enzyme at 10 µM concentration.
In order to compare the potency of the compounds to others in
the literature the IC50 values of compounds that showed >50%
inhibition at 10 µM concentration were determined. The
following IC50 values were obtained: 2 – 3.06 1.27µM, 3 –
4.48 0.27µM, 6 – 4.27 1.30µM, 10 – 1.29 0.25µM, 11 –
1.42 0.73µM, 14 – 0.225 0.03µM. The data show that, as
expected, these compounds in fact possess a better IC50 than the
reference compound. Compound 14 was found to be the best
inhibitor of BuChE; its 225 nM value is of practical importance
for further lead development. While at this level of the research it
is difficult to make structure-activity relationship predictions it
appears that the presence of an electron-donating substituent (Me,
OMe) on the β-carboline ring positively affects the BuChe
inhibition. In addition, considering the lower, additional ring, the
bulkier the group, the better the effect. Compound 14 with the
quite large naphthyl group was found to be by far the most
effective inhibitor.
With the aim of understanding the butyrylcholinesterase
inhibition property of these molecules, two among the best
compounds (11 and 14) were docked in the active site of BuChE
(PDB code: 1P0I42) using the Glide module of the Schrodinger
package.43 The superimposition of compound 11 and 14 with
donepezil and galantamine (known BuChE inhibitors) in the
active site of the enzyme is shown in Figs. 3, 4.
Figure 5. Superimposition of molecule 12 (purple) with donepezil (red)
and galanthamine (dark green) in the active site of huAChE (PDB ID-4EY7)
(hydrogen's are concealed for clarity).
Fig. 5 shows that the orientation of 12 is significantly different
from those of donepezil and galantamine. The molecule streches
completely through the active site while galantamine only
occupies the right side of the pocket and donepezil also appears
on the right side and turns back to the center. Although 12 shows
hydrogen bonding interaction with residues Phe295, Tyr124 as
well as π―π interaction with Trp286, The338 and Tyr337
residues, it is likely, that several of these interactions do not
block residues that possess a role in the catalytic action.
Figure 3. Superimposition of molecule 11 (blue) with donepezil (red) and
galantamine (dark green) in the active site of huBChE (PDB ID: 1P0I).
(hydrogens are concealed for clarity)
Oxidative stress caused by free radicals also plays an
important role in the development of AD. Thus, the potential
antioxidant character of the compounds was assessed in three

assays including the scavenging of the 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH assay), 2,2'-azino-bis(3-
ethylbenzo-thiazoline-6-sulphonic acid) (ABTS assay) and the
peroxyl (ORAC assay) free radicals.44 The data are compared to
those obtained with reference compounds ascorbic acid45,
resveratrol,46 and trolox,47 all of which are well-known
antioxidants (Table 2). The data show that while the compounds
have negligible effect in scavenging the large stable DPPH
radical, they exhibited low to moderate scavenging activity
against the also large ABTS radical.
the molecules (1, 3, 4, 7, 8, 11, 12, 14) had a much stronger
effect in the inhibition of Aβ oligomer formation. This is in line
with our earlier observations: a compound group is either a
strong fibril or oligomer formation inhibitor.37 Although the
compounds were inactive in AChE inhibition, they exhibited a
highly selective and efficient inhibition of the BuChE enzyme.
As shown, compounds 10, 11 and 14 exhibited the highest
efficiency, 14 having one order of magnitude lower IC50 (225
nM) than the other compounds. The structural comparisons
indicate that the added Ar part (Fig. 1.) appears highly important;
the large Ar groups, such as naphthyl in 14, result in significant
activity in the assays except the antioxidant tests.
Table 2. Radical scavenging activity of β-carbolines (10 µM) in the
DPPH, ABTS and ORAC antioxidant assays. Ascorbic acid, resveratrol and
trolox that are well-known antioxidants were used as reference.
In conclusion, a variety of β-carbolines with an extended
aromatic ring system were synthesized and tested with the aim of
identifying potential multitarget agents, that can interfere with
Aβ self-assembly and cholinesterase activity while exhibiting
promising antioxidant properties, for AD treatment. Based on the
analysis of the data compound 14 emerged as a potential lead
compound for further structure activity relationship studies. This
molecule exhibited moderate to high activity in a range of assays
suggesting that further modification of its basic ring system could
yield a truly efficient candidate to develop effective drugs for
disease management. To improve the drug-like properties of the
compound the introduction of hydrophilic units such as NH (in
the form of primary or secondary amines) or OH are proposed as
the presence of these groups would improve water solubility
(increased polarity), and antioxidant activity (presence of X-H
bond) and likely would initiate further interactions with the
cholinesterases that could improve the inhibition.
% radical scavenging
Compound
DPPH
ABTS
ORAC
1
-7
2
12
10
16
13
15
22
-
10
10
35
28
41
54
-
2
3
-6
-12
-13
-3
-
4
5
6
7
8
0
15
6
58
6
9
10
-3
-1
2
7
-10
5
11
12
3
-4
-
5
5
13
14
-
-
Acknowledgments
-6
15
28
23
14
28
88
26
0
ascorbic acid
resveratrol
trolox
15
91
90
Financial support provided by the University of Massachusetts
Boston through the 2013 Joseph P. Healey Research grant and
National Institute of Health (R21AG028816-01 to H. L.) is
gratefully acknowledged. Thanks are due to Alnylam
Pharmaceutical Inc. for their help with the HR-MS
measurements.
- data not measured due to solubility problems
Several compounds showed comparable activity to the
reference compounds ascorbic acid and trolox. The β-carbolines
were most active against the much smaller peroxyl radical used
in the ORAC assay. Since this radical is one of the naturally
occurring reactive oxygen species, these data are encouraging.
Supplementary Material
The synthesis of β-carbolines, as well as, the detailed
description of the biochemical assays are provided.
The analysis of the above data reveals that several of the
synthesized substituted β-carbolines show promising properties
in the AD related assays. Compounds 6, 7, 14 were able to inhibit
Aβ fibril formation to a meaningful extent, while the majority of
11 Larner, A.J. Expert Rev. Neurotherapeut. 2010, 10, 1699; Wollen, K. A.
Alt. Med. Rev. 2010, 15, 223; Francis, P. T.; Ramirez, M. J.; Lai, M. K.
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Synthesis and Application of -Carbolines as
Novel Multi-functional Anti-Alzheimer’s
Disease Agents
William Horton, Abha Sood, Swarada Peerannawar, Nándor Kugyela, Aditya Kulkarni, Rekha Tulsan, Chris D.
Tran, Jessica Soule, Harry LeVine III, Béla Török, Marianna Török*