Design, Synthesis and in vitro Evaluation of Indolotacrine Analogues as Multitarget-Directed Ligands for the Treatment of Alzheimer's Disease

Article abstract of DOI:10.1002/cmdc.201500383

Novel indolotacrine analogues were designed, synthesized, and evaluated as potential drugs for the treatment of Alzheimer's disease. By using a multitarget-directed ligand approach, compounds were designed to act simultaneously as cholinesterase (ChE) and

Full text of DOI:10.1002/cmdc.201500383

DOI: 10.1002/cmdc.201500383
Communications
Design, Synthesis and in vitro Evaluation of Indolotacrine
Analogues as Multitarget-Directed Ligands for the
Treatment of Alzheimer’s Disease
Ondrej Benek,[a, b, c] Ondrej Soukup,[b, c] Marketa Pasdiorova,[a, c] Lukas Hroch,[c, d]
Vendula Sepsova,[a, c] Petr Jost,[a, c] Martina Hrabinova,[a, c] Daniel Jun,[a] Kamil Kuca,[c, g]
Dominykas Zala,[e] Rona R. Ramsay,[e] JosØ Marco-Contelles,*[f] and Kamil Musilek*[c, g]
Novel indolotacrine analogues were designed, synthesized,
and evaluated as potential drugs for the treatment of Alzheim-
er’s disease. By using a multitarget-directed ligand approach,
compounds were designed to act simultaneously as cholines-
terase (ChE) and monoamine oxidase (MAO) inhibitors. The
compounds were also evaluated for antioxidant, cytotoxic,
hepatotoxic, and blood–brain barrier (BBB) permeability prop-
erties. Indolotacrine 9b (9-methoxy-2,3,4,6-tetrahydro-1H-
indolo[2,3-b]quinolin-11-amine) showed the most promising re-
sults in the in vitro assessment; it is a potent inhibitor of ace-
tylcholinesterase (AChE IC50: 1.5 mm), butyrylcholinesterase
(BChE IC50: 2.4 mm) and MAO A (IC50: 0.49 mm), and it is also
a weak inhibitor of MAO B (IC50: 53.9 mm). Although its cytotox-
ic (IC50: 5.5Æ0.4 mm) and hepatotoxic (IC50: 1.22Æ0.11 mm) pro-
files are not as good as those of the standard 7-methoxyta-
crine (IC50: 63Æ4 and 11.50Æ0.77 mm, respectively), the overall
improvement in the inhibitory activities and potential to cross
the BBB make indolotacrine 9b a promising lead compound
for further development and investigation.
The cholinergic hypothesis asserts that the decreased level
of ACh in the brain leads to cognitive and memory deficits,
and that sustaining or recovering cholinergic function should
therefore result in amelioration of the symptoms.[7–9] Accord-
ingly, current AD therapy is based mainly on acetylcholinester-
ase (AChE) inhibitors (AChEIs), which are able to increase ACh
levels in cholinergic synapses. To date, the number of ap-
proved drugs is limited to three AChEIs (rivastigmine, donepe-
zil, and galantamine) and an N-methyl-d-aspartic acid (NMDA)
antagonist (memantine). However, these drugs cannot prevent
or cure the disease, but afford only symptomatic treat-
ment.[9,10]
The “one-target, one-compound” paradigm has been highly
successful for many common diseases because their underly-
ing molecular mechanisms were understood, allowing biolo-
gists to define the key target for a particular disease. Once the
target was identified, medicinal chemists strategically designed
a molecule to interact selectively with such a target, with a po-
tential drug as the outcome. However, it is apparent that this
target-based approach does not always guarantee success.
Drugs directed to a single target might not always modify
complex multifactorial diseases such as AD, even if they act in
the way they are expected to proceed.[11] It is now widely ac-
cepted that a more effective therapy would result from the
use of multipotent compounds able to intervene simultane-
ously in the different pathological events underlying the etiolo-
gy of AD.[12,13]
Alzheimer’s disease (AD) is an age-related neurodegenerative
disorder characterized by progressive and irreversible cognitive
impairment and memory loss.[1] Despite enormous efforts, the
etiology of AD has not yet been elucidated, and the disease re-
mains incurable.[2] According to current knowledge, b-amyloid
(Ab) aggregates,[3] t-protein phosphorylation,[4] oxidative
stress,[5] and deficits in acetylcholine (ACh)[6] are considered to
play significant roles in AD pathophysiology.
Monoamine oxidase (MAO; EC 1.4.3.4) is another important
target that was considered for the treatment of AD because
some symptoms of AD are caused by alterations in the dopa-
[a] O. Benek, M. Pasdiorova, Dr. V. Sepsova, P. Jost, M. Hrabinova, Dr. D. Jun
Department of Toxicology and Military Pharmacy
Department of Epidemiology, Faculty of Military Health Sciences
University of Defense
[e] D. Zala, Dr. R. R. Ramsay
School of Biology, Biomolecular Sciences Building
University of St. Andrews, North Haugh, St. Andrews, KY16 9ST (UK)
[f] Prof. J. Marco-Contelles
Trebesska 1575, 500 01 Hradec Kralove (Czech Republic)
Laboratory of Medicinal Chemistry (IQOG, CSIC)
Juan de la Cierva 3, 28006 Madrid (Spain)
E-mail: iqoc21@iqog.csic.es
[b] O. Benek, Dr. O. Soukup
National Institute of Mental Health
Topolova 748, 250 67 Klecany (Czech Republic)
[g] Prof. K. Kuca, Dr. K. Musilek
[c] O. Benek, Dr. O. Soukup, M. Pasdiorova, L. Hroch, Dr. V. Sepsova, P. Jost,
M. Hrabinova, Prof. K. Kuca, Dr. K. Musilek
Department of Chemistry, Faculty of Science, University of Hradec Kralove
Rokitanskeho 62, 500 03 Hradec Kralove (Czech Republic)
E-mail: kamil.musilek@gmail.com
University Hospital, Sokolska 581, 500 05 Hradec Kralove (Czech Republic)
[d] L. Hroch
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500383.
Department of Pharmaceutical Chemistry and Drug Control
Faculty of Pharmacy in Hradec Kralove, Charles University in Prague
Akademika Heyrovskeho 1203, 500 05 Hradec Kralove (Czech Republic)
This article is part of a Special Issue on Polypharmacology and
Multitarget Drugs. To view the complete issue, visit:
http://onlinelibrary.wiley.com/doi/10.1002/cmdc.v11.12/issuetoc.
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minergic, serotoninergic, and other monoaminergic
neurotransmitter systems.[14,15] Moreover, MAO-cata-
lyzed oxidative deamination gives rise to the produc-
tion of hydrogen peroxide and, consequently, reac-
tive oxygen species (ROS) that have also been impli-
cated in the progress of AD.[16] MAO inhibitors
(MAOIs) should increase monoaminergic neurotrans-
mission and decrease the formation of ROS; both ef-
fects are potentially valuable for the treatment of
AD.[13,15] Therefore, in this context, multipotent mole-
cules that are able to bind both ChEs and MAOs
have been investigated.[17–20]
Figure 2. Design of indolotacrine series compounds 9a,b and 13.
The aim of the work reported herein was to devel-
op novel multitarget-directed ligands (MTDLs) that
act primarily as MAO and cholinesterase (ChE) inhibitors (ChEIs
). For this purpose we chose structural motifs contained in pre-
viously described MAO and/or ChEIs and incorporated them
into the scaffold of the novel compounds. Two distinct series
of molecules were designed. The first series, containing a 2-
aminoindole-3-carbonitrile scaffold (referred to as the “indole”
series; compounds 4a–c and 8c), uses an indole ring, which is
a structural core feature in several MAOIs and dual-acting com-
pounds that target both MAO and ChEs, such MBA236
(Figure 1),[21,22] as well as the b-aminonitrile motif found in
some previously identified MAOIs.[23] Compounds 4b,c also
contain the propargylamine moiety, which is an essential part
of many neuroprotective, irreversible MAOIs (Figure 1).[24] Origi-
nally, only compounds 4a,b had been designed; however,
during the synthesis of 4b a side-product 4c was isolated. Be-
cause of the low yield obtained, 4c was tested only for its in-
hibitory activity against MAO. Compound 8c was synthesized
later to explore whether the N-allyl or N-propargyl substitution
on the amino group at position 2 is important for MAO inhibi-
tion and also to validate the im-
pounds 9a,b and 13) to improve the unsatisfactory anti-ChE
activity of the indole series. For this purpose, the 2-aminoin-
dole-3-carbonitrile scaffold of the indole series was fused with
the structure of the potent ChEI tacrine or 7-methoxytacrine
(7-MEOTA). Moreover, the resulting indolotacrines also resem-
ble b-carboline alkaloids (e.g., harmine), which are known
MAOIs (Figure 2).[25,26] Because compound 4c, with an N-prop-
argyl substituent at position 1, was found to be the most
potent MAOI of the indole series, we decided to preserve this
potentially favorable motif in designing compound 13 with
benzyl substitution, analogous to the former N-propargyl
moiety.
5-Hydroxy-1H-indole-3-carbonitrile derivatives 4a–c were
prepared in three steps (Scheme 1). At first malononitrile (1)
was treated with ethanol in diethyl ether saturated with gas-
eous hydrochloric acid to obtain 3-amino-3-ethoxyacrylonitrile
(2). In the next step acrylonitrile 2 was treated with the corre-
sponding alkylamine to give N-alkylated 3,3-diaminoacryloni-
triles 3. Lastly, diaminoacrylonitriles 3 were treated with p-ben-
portance of the phenolic group
for the antioxidant activity of
other compounds in the series
(discussed below).
The second series was then
designed using the 2,3,4,6-tetra-
hydro-1H-indolo[2,3-b]quinolin-
11-amine scaffold (referred to as
Scheme 1. Synthesis of indole series 4a–4c. Reagents and conditions: a) HCl, EtOH, Et2O, 08C!RT, 4 h, 18%; b) al-
kylamine, EtOH, RT, overnight, 61–77%; c) p-benzoquinone, EtOH, RT, 1 h, 10–22%.
the “indolotacrine” series; com-
zoquinone to give 2-(alkylamino)-5-hydroxy-1H-indole-3-car-
bonitriles 4a,b.[27] Moreover, a byproduct whose structure was
assigned as alkylated at position 1 (compound 4c), was also
isolated from the reaction of 3-amino-3-(prop-2-yn-1-ylami-
no)acrylonitrile (3b).
Indole 8c and indolotacrines 9a,b were prepared in two to
four steps using a similar synthetic approach (Scheme 2). The
synthesis of compound 9b started from commercial 2-iodo-4-
methoxy-1-nitrobenzene (5), which was reduced using iron
powder and ammonium chloride to the corresponding aniline
derivative 6b. Intermediates 6a and 6c were obtained com-
Figure 1. Design of indole series compounds 4a–c and 8c.
mercially. From this point, the synthesis proceeded identically
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Scheme 2. Synthesis of indolotacrines 9a, 9b, 13 and indole 8c. Reagents and conditions: a) Fe, NH4Cl, MeOH/H2O (3:1), 508C, 2 h, 79%; b) trifluoroacetic an-
hydride, Et3N, THF, À78C!RT, overnight, 97–99%; c) malononitrile, l-proline, K2CO3, CuI, DMSO/H2O (1:1), 608C, overnight, 48–90%; d) cyclohexanone, AlCl3,
1,2-dichloroethane, microwave, 958C, 2 h, 16–54%; e) benzaldehyde, MeOH, RT, overnight, 97%; f) NaBH3CN, AcOH/MeOH, 08C!RT, overnight, 75%; g) malo-
nonitrile, picolinic acid, K2CO3, CuI, DMSO, microwave, 908C,
12 h, 26%.
Table 1. Inhibition of MAO A and MAO B.
for all three compounds. The 2-iodoaniline deriva-
tives 6a–6c were treated with trifluoroacetic anhy-
dride to give the trifluoracetamides 7a–7c, which
were then used for the copper iodide catalyzed cycli-
zation with malononitrile to obtain the correspond-
ing indole derivatives 8a–8c.[28] Finally, indolotacrines
9a and 9b were prepared by microwave-assisted
Friedländer reaction[29] of the corresponding indoles
8a and 8b with cyclohexanone.
Indolotacrine 13 was prepared by a slightly differ-
ent synthetic procedure involving four steps
(Scheme 2). Firstly, 2-iodoaniline 6 was treated with
benzaldehyde to give imine 10, which was then re-
duced to the corresponding amine 11 using sodium
cyanoborohydride. In next step cyclization of amine
11 with malononitrile gave indole 12.[30] In the final
step, Friedländer reaction[29] of 12 with cyclohexa-
none gave indolotacrine 13.
Compd
IC50 [mm][a]
SI[b]
30’ IC50 [mm][c]
MAO A MAO B
MAO A
MAO B
4a
4b
4c
8c
9a
9b
13
2.32Æ0.26
1.32Æ0.12
0.68Æ0.08
2.80Æ0.40
11.40Æ1.10
0.49Æ0.05
2.02Æ0.56
1.70Æ0.40
1.62Æ0.35
3.89Æ0.02
0.9
1.78Æ0.33 10.86Æ0.78
1.3
2.4
1.4
1.80Æ0.56
0.45Æ0.03
–
2.48Æ0.32
0.87Æ0.10
–
–
–
–
–
–
>100
8.8 30.0Æ1.9
53.90Æ10.70 110.0
–
–
–
–
>100
>100
–
22.5
13.9
tacrine
7-MEOTA
14.07Æ1.47
317.2Æ201.0
7.10Æ0.03
98.61Æ14.63
[a] Values are the meanÆSD of three independent measurements. [b] Selectivity
index: (IC50 MAO B)/(IC50 MAO A). [c] Determined after 30 min pre-incubation of
enzyme with inhibitor; values are the meanÆSEM of three independent measure-
ments.
pounds 4b,c bearing the N-propargylamine moiety, which is
For biological evaluations, all final products (Figure 3) were
transformed into better water-soluble hydrochlorides by stir-
ring them in diethyl ether saturated with gaseous hydrochloric
acid. Both series were assayed in vitro for their inhibitory activi-
ty against membrane-bound MAO A and MAO B (Table 1). All
indoles were found to be potent and unselective MAOIs, with
4c being the best inhibitor of both isozymes in the series. In-
doles 4a–c were evaluated for irreversible inhibition and, unex-
pectedly, none of compounds showed significantly lower IC50
values after 30 min pre-incubation with enzyme, despite com-
present in many known irreversible MAOIs (e.g., deprenyl, clor-
gyline, and rasagiline). This could be due to the change in elec-
tron density on the triple bond of the N-propargyl motif, as its
connecting nitrogen atom is part of the aromatic system in
contrast to the known irreversible inhibitors in which the N-
propargylamine moiety is separated from the aromatic system,
usually by an alkyl linker. Alternatively, steric hindrance from
the carbonitrile substituent could prevent the generation of
the reactive intermediate or its modification of the enzyme.
Based on this finding, we decided to investigate whether the
N-allyl or N-propargyl substitution is necessary for MAO inhibi-
tion, and so we synthesized compound 8c. Evaluation revealed
that indole 8c, devoid of any N-alkyl substituent on the amino
group at position 2, retains the inhibitory activity at level simi-
lar to that of other indoles, showing that the propargyl moiety
does not contribute to binding.
Indolotacrine 9b retained the inhibitory activity for both
MAO isozymes; however, 9a inhibited only MAO A, and 13,
with an extra N-benzyl substituent, showed no inhibition of
either MAO isozyme. It could be assumed that the extended
steric bulk of 13 would prevent entry into the active site of
Figure 3. a) Indole and b) indolotacrine analogues prepared in this study.
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MAO enzymes.[31] In addition, compound 9a was tested for in-
activation of MAO A, but it showed the expected reversible
mode of inhibition. Unlike the unselective indole analogues, in-
dolotacrines 9a,b both exerted some selectivity toward MAO A
inhibition, with 9b being the most potent MAO A inhibitor
among all the compounds tested (IC50: 0.49 mm). Standards ta-
crine and 7-MEOTA showed only moderate activity, being
poorer inhibitors of both MAO isozymes than the indolotacrine
9b.
All final compounds, with the exception of 4c (which was
a byproduct of synthesis and, due to low yield, was tested
only for MAO inhibition) and 8c (prepared subsequently to en-
hance SAR information on MAO inhibition and antioxidant ac-
tivity), were assayed in vitro for their inhibitory activity against
human recombinant acetylcholinesterase (AChE) and human
plasma butyrylcholinesterase (BChE) (Table 2).
Table 3. Antioxidant activity (EC50) and cytotoxicity (IC50) of prepared
compounds.
Compd
EC50 [mm][a]
IC50 [mm][a]
>1000
>1000
113 Æ29
4a
4b
8c
9a
9b
13
37.86Æ5.01
25.82Æ1.35
731.70Æ27.17
>5000
13.0Æ1.4
5.5Æ0.4
7.0Æ0.7
248Æ11
63Æ4
–
>5000
3827.0Æ227.1
tacrine
7-MEOTA
N-acetylcysteine
trolox
>5000
>5000
27.91Æ1.82
16.20Æ0.42
–
[a] Values are the meanÆSEM of three independent measurements.
sumption we synthesized compound 8c, in which the phenolic
group is replaced with chlorine. Evaluation supported our hy-
pothesis, in that indole 8c exerts more than 20-fold weaker an-
tioxidant activity than phenolic compounds 4a and 4b. Nei-
ther the indolotacrines, tacrine, nor 7-MEOTA showed any sig-
nificant antioxidant activity, which is not surprising, as they all
lack the phenolic group responsible for this activity, as demon-
strated for the indoles. Introduction of the phenolic moiety
therefore presents a possible improvement of the indolota-
crine compounds for future studies.
Table 2. Inhibition of AChE and BChE.
Compd
IC50 [mm][a]
SI[b]
AChE
BChE
4a
4b
9a
9b
319.2Æ15.9
101.9Æ5.4
11.6Æ0.6
1.5Æ0.1
>1000
>1000
3.1
9.8
0.4
1.6
0.001
0.3
1.8
4.7Æ0.1
2.4Æ0.1
13
>1000
1.09Æ0.07
0.088Æ0.001
17.6Æ0.8
tacrine
7-MEOTA
0.32Æ0.01
10.0Æ1.0
The cytotoxicity of the compounds was next evaluated by
using an MTT assay on the CHO-K1 cell line (Table 3). The in-
doles were found to possess very low toxicity, with IC50 values
above the measurable range (>1000 mm) in the case of 4a,b
and in the high micromolar range for 8c. All indolotacrines ex-
erted similar levels of cytotoxicity, with IC50 values ~10 mm.
Standards 7-MEOTA and tacrine were both found to be less
toxic, with tacrine being the least toxic compound among the
series in vitro. This could be considered quite a surprising
result, as it is known that in vivo tacrine is more toxic than 7-
MEOTA.[35]
[a] Values are the meanÆSEM of three independent measurements.
[b] Selectivity index: (IC50 BChE)/(IC50 AChE).
No significant inhibitory activity against AChE or BChE was
detected for indoles 4a,b. Both compounds exerted only poor
inhibition of AChE in the high micromolar range and were
found to be inactive against BChE at the highest concentration
tested (50 mm). A possible explanation for this observation is
that compounds 4a,b lack the structural complexity of other
indoles or indanes, which are capable of ChE inhibition (e.g.,
the extra N-benzylpiperidine moiety present in donepezil,
ASS234, and MBA236 or the carbamate moiety of ladostigil).[22]
Conversely, indolotacrines 9a,b were found to be potent unse-
lective inhibitors of both ChE enzymes, with IC50 values in low
micromolar range, and compound 13 was found to be a selec-
tive BChEI. None of the compounds were better than tacrine,
but compound 9b was a better inhibitor of both ChEs than 7-
MEOTA. IC50 values obtained for standard inhibitors tacrine and
7-MEOTA were in good agreement with previously published
results.[32]
Assuming that the principal target of tacrine toxicity in vivo
is the liver, we decided to evaluate tacrine and 7-MEOTA to-
gether with the most promising indolotacrine 9b for their hep-
atotoxicity on the HepG2 cell line by using the MTT assay
(Table 4).[36] Compound 9b was found to be more hepatotoxic
than 7-MEOTA and tacrine. As with the cytotoxicity evaluation,
tacrine showed lower in vitro hepatotoxicity than 7-MEOTA,
which is at odds with the in vivo results.[35] A possible explana-
tion for this discrepancy is that the hepatotoxicity is not
caused by tacrine itself, but by its metabolites, products of cy-
tochrome P450 oxidation.[37] Therefore, it is difficult to draw
a conclusion regarding the compounds’ toxicity in vivo (e.g.,
9b) based on the results of in vitro testing; these cytotoxicity
and hepatotoxicity assessments have, in this case, only gener-
ally informative character.
Additionally, as ROS are likely to play a part in the develop-
ment and progression of AD,[33] the compounds were evaluat-
ed for their antioxidant activity using a DPPH assay (Table 3).
Indoles 4a,b showed promising antioxidant properties, similar
to that of the standard N-acetylcysteine and only slightly
weaker than that of trolox. We hypothesized that this could be
due to the presence of phenolic group, which is a key structur-
al motif common of many antioxidants.[34] To prove this as-
Penetration across the blood–brain barrier (BBB) is an essen-
tial property for compounds targeting the central nervous
system (CNS) and should always be considered during drug
development. To predict passive BBB penetration, modification
of the parallel artificial membrane permeation assay (PAMPA)
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Acknowledgements
Table 4. Hepatotoxicity evaluation.
Compd
IC50 [mm][a]
This study was supported by the Czech National Institute of
Mental Health (NIMH–CZ) project no. ED2.1.00/03.0078, the Min-
istry of Education, Youth and Sports of the Czech Republic
(LD14009), MH CZ–DRO (UHHK, 00179906), the University of De-
fense of the Czech Republic (long-term development plan),
MINECO (Government of Spain, grant SAF2012-33304), the
School of Biology at the University of St. Andrews, and by COST
Action CM1103.
9b
tacrine
7-MEOTA
1.22Æ0.11
17.28Æ0.76
11.50Æ0.77
[a] Values are the meanÆSEM of three independent measurements.
was used based on a published protocol.[38] As summarized in
Table 5, it is clear that compound 9b has high potential for
availability in the CNS. Data obtained for the new compound
Keywords: Alzheimer’s disease · cholinesterases · cytotoxicity ·
inhibitors · monoamine oxidase · multitarget-directed ligands
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7.3Æ0.9
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rivastigmine
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hydrocortisone
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(+)
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(À)
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Published online on October 2, 2015
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