Novel indanone derivatives as MAO B/H3R dual-targeting ligands for treatment of Parkinson's disease

Article abstract of DOI:10.1016/j.ejmech.2018.02.015

The design of multi-targeting ligands was developed in the last decades as an innovative therapeutic concept for Parkinson's disease (PD) and other neurodegenerative disorders. As the monoamine oxidase B (MAO B) and the histamine H₃ receptor (H₃R) are promising targets for dopaminergic regulation, we synthetized dual-targeting ligands (DTLs) as non-dopaminergic receptor approach for the treatment of PD. Three series of compounds were developed by attaching the H₃R pharmacophore to indanone-related MAO B motifs, leading to development of MAO B/H₃R DTLs. Among synthesized indanone DTLs, compounds bearing the 2-benzylidene-1-indanone core structure showed MAO B preferring inhibition capabilities along with nanomolar hH₃R affinity. Substitution of C5 and C6 position of the 2-benzylidene-1-indanones with lipophilic substituents revealed three promising candidates exhibiting inhibitory potencies for MAO B with IC₅₀ values ranging from 1931 nM to 276 nM and high affinities at hH₃R (K_i < 50 nM). Compound 3f ((E)-5-((4-bromobenzyl)oxy)-2-(4-(3-(piperidin-1-yl)propoxy)benzylidene)-2,3-dihydro-1H-inden-1-one, MAO B IC₅₀ = 276 nM, hH₃R K_i = 6.5 nM) showed highest preference for MAO B over MAO A (SI > 36). Interestingly, IC₅₀ determinations after preincubation of enzyme and DTLs revealed also nanomolar MAO B potency for 3e (MAO B IC₅₀ = 232 nM), a structural isomer of 3f, and 3d (MAO B IC₅₀ = 541 nM), suggesting time-dependent inhibition modes. Reversibility of inhibition for all three compounds were confirmed by dilution studies in excess of substrate. Thus, indanone-substituted derivatives are promising lead structures for the design of MAO B/hH₃R DTLs as novel therapeutic approach of PD therapy.

Full text of DOI:10.1016/j.ejmech.2018.02.015

Accepted Manuscript
Novel indanone derivatives as MAO B/H R dual-targeting ligands for treatment of
3
PARKINSON’S disease
Anna Affini, Stefanie Hagenow, Aleksandra Zivkovic, Jose Marco-Contelles, Holger
Stark
PII:
S0223-5234(18)30137-5
10.1016/j.ejmech.2018.02.015
EJMECH 10192
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 September 2017
Revised Date: 5 February 2018
Accepted Date: 6 February 2018
Please cite this article as: A. Affini, S. Hagenow, A. Zivkovic, J. Marco-Contelles, H. Stark, Novel
indanone derivatives as MAO B/H R dual-targeting ligands for treatment of PARKINSON’S disease,
3
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.015.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
NOVEL INDANONE DERIVATIVES AS MAO B/H R DUAL-TARGETING LIGANDS FOR
3
TREATMENT OF PARKINSON’S DISEASE
a1
a1
a
b
a*
Anna Affini , Stefanie Hagenow , Aleksandra Zivkovic , Jose Marco-Contelles , Holger Stark
a
Heinrich Heine University Duesseldorf, Institute of Pharmaceutical and Medicinal Chemistry,
Universitaetstr. 1, 40225 Duesseldorf, Germany
b
Laboratory of Medicinal Chemistry (IQOG, CSIC), C/ Juan de la Cierva 3, 28006-Madrid, Spain
*
Corresponding author: e-mail: stark@hhu.de, Fax +49 211 8113359, phone: +49 211 8110478.
These authors have contributed equally to this work.
1
1

ACCEPTED MANUSCRIPT
Abstract
The design of multi-targeting ligands was developed in the last decades as an innovative therapeutic
concept for Parkinson’s disease (PD) and other neurodegenerative disorders. As the monoamine
oxidase B (MAO B) and the histamine H receptor (H R) are promising targets for dopaminergic
3
3
regulation, we synthetized dual-targeting ligands (DTLs) as non-dopaminergic receptor approach for
the treatment of PD. Three series of compounds were developed by attaching the H R
3
pharmacophore to indanone-related MAO B motifs, leading to development of MAO B/H R DTLs.
3
Among synthesized indanone DTLs, compounds bearing the 2-benzylidene-1-indanone core structure
showed MAO B preferring inhibition capabilities along with nanomolar hH R affinity. Substitution of C5
3
and C6 position of the 2-benzylidene-1-indanones with lipophilic substituents revealed three promising
candidates exhibiting inhibitory potencies for MAO B with IC50 values ranging from 1931 nM to 276 nM
and high affinities at hH R (K < 50 nM). Compound 3f ((E)-5-((4-bromobenzyl)oxy)-2-(4-(3-(piperidin-
3
i
1
-yl)propoxy)benzylidene)-2,3-dihydro-1H-inden-1-one, MAO B IC50 = 276 nM, hH R K = 10 nM)
3 i
showed highest preference for MAO B over MAO A (SI > 36). Interestingly, IC50 determinations after
preincubation of enzyme and DTLs revealed also nanomolar MAO B potency for 3e (MAO B
IC50 = 232 nM), a structural isomer of 3f, and 3d (MAO B IC50 = 541 nM), suggesting time-dependent
inhibition modes. Reversibility of inhibition for all three compounds were confirmed by dilution studies
in excess of substrate. Thus, indanone-substituted derivatives are promising lead structures for the
design of MAO B/hH R DTLs as novel therapeutic approach of PD therapy.
3
Abbreviations: AChE acetylcholinesterase, anal. calc. elementary analysis calculated, BuChE Butyrylcholinesterase, Calcd
calculated, CDCl
enhancement spectroscopy, DMSO-d
3
deuterated chloroform, CI confidence interval, CNS central nervous system, NOESY nuclear overhauser
deuterated dimethyl sulfoxide, DTL dual-targeting ligand, eq equivalent, ESI-MS
6
electronspray ionization mass spectrometry, FAD flavine adenine dinucleotide, GPCR G-protein coupled receptor, HMT
histamine methyltransferase, HPLC high performance liquid chromatography, HRMS high resolution mass spectrometry, MAO
monoamine oxidase, mp melting point, NMR nuclear magnetic resonance, 6-OHDA 6-Hydroxydopamine, PD Parkinson´s
disease, ppm parts per million, RFU relative fluorescence units, SD standard deviation, SI selectivity index, TLC thin layer
chromatography
Keywords
Multi-targeting, neurodegenerative diseases, histamine H receptor antagonist, monoamine oxidase
3
inhibitor, neurotransmitter dysregulation, Parkinson’s disease
2

ACCEPTED MANUSCRIPT
Introduction
Parkinson's disease (PD) is one of the most common central nervous system (CNS) disorder affecting
1.5 % of the global population older than 65 years. Recognizing PD as high complex and multifactorial
dysregulation forced researchers to come up with more and more imaginative approaches for
medicinal therapy. Challenging the “one-drug one-target” paradigm, showing mostly inadequate
therapeutical efficacy, the development of multi-targeting ligands attracted notice over the last
decades, were only a few entered the drug market. Due to the fact that multi-targeting drugs,
addressing more than one target simultaneously, promise several improvements compared to
selective drugs, i.e. potential synergistic efficacy, less drug-drug interactions, easier dosing schedule,
and unified pharmacokinetic profile [1–4], great effort has been brought to the design of such ligands
for treatment of PD. As a common strategy in PD therapy monoamine oxidase (MAO) B inhibitors are
approved as mono- and add-on therapy [5,6]. MAOs are mitochondrial membrane-bound FAD-
containing enzymes playing a key role in neurotransmitter degradation. In addition to their localizations
in brain regions, the two isoforms A and B differ in substrate and inhibitor specificities. While MAO A
inhibitors are used in treatment of depression, the MAO B isoform predominantly degrades dopamine
in human brain, thus, representing a well-established target for PD therapy. Among irreversible
inhibitors like L-deprenyl or rasagiline, safinamide is the only approved reversible MAO B inhibitor in
PD therapy. As dual-targeting approach for PD treatment, ligands addressing MAO B and adenosine
A2A receptors, a G-protein coupled receptor (GPCR), were recently suggested as indirect non-
dopaminergic therapy [7]. The human histamine H receptor (hH R), a class A GPCR predominantly
3
3
expressed in the CNS, serves as pan-neurotransmitter modulator in the human brain due to its
heteroreceptor activity. Activation of presynaptic H Rs may decrease the release of dopamine and
3
other neurotransmitters [8,9], meaning that H R inverse agonism/antagonism might provide a hitherto
3
hardly investigated opportunity for the treatment of PD [10]. Although their exact utility in PD treatment
can be hardly estimated so far, H R antagonists show promising features to improve motor but
3
especially non-motor symptoms, i.e. cognitive or sleep impairment [11,12]. As H Rs are co-expressed
3
with dopamine D1 and D2 receptors in the basal ganglia [12], Ferrada et al. demonstrated the
potentiation of dopamine agonist-induced locomotor activation by the H R antagonist thioperamide
3
[
13], indicating a potential benefit of H R antagonists on motor control in PD patients when applied
3
with dopamine agonists. Recently, thioperamide was also proven to counteract memory and sleep
impairment in a 6-OHDA PD mouse model [14], while wake-promoting effects for thioperamide and
numerous other H R antagonists could be also shown previously in vivo and clinical trials [15,16]. To
3
®
date, pitolisant (Wakix ) is the only H R inverse agonist/antagonist on the drug market approved for
3
the treatment of narcolepsy with and without catalepsy and already in Phase III clinical trials for
excessive daytime sleepiness in PD patients (clinicaltrials.gov). Numerous H R ligands are described,
3
showing additional affinity to other GPCRs, ion channels, transporters or even enzymes [2].
Additionally, the dopamine modulation capacities of MAO B and H R as well as their co-localization
3
especially in the substantia nigra [12,13], which is predominantly affected by loss of dopaminergic
neurons in PD patients, strongly encouraged us to design MAO B/H R dual-targeting ligands (DTL) as
3
possible and innovative approach to modulate the dopamine imbalance in PD patients. To overcome
drawbacks associated with irreversible MAO inhibition, such as slow recovery of MAO expression
levels after inactivation, we have focussed on reversible MAO B inhibition strategies. Numerous
structural classes of MAO B inhibitors are described, while especially structural related
(
benzylidene-)chromones and coumarine derivatives [17] as well as recently described (2-
benzylidene-)indanones [18,19] showed highly selective and reversible inhibition properties in
nanomolar concentration ranges. Compounds I and II (Figure 1) are potent and selective reversible
MAO B inhibitors, showing IC50 values in low nanomolar concentrations ranges [18,19]. We recently
found ciproxifan (Figure 1), a common reference H R inverse agonist/antagonist, to have MAO B
3
preferring inhibition potency (MAO B IC = 2.1 µM) [20], while the probability of MAO inhibition by
5
0
already described H R ligands could be also shown elsewhere [21]. Preserving the alkyloxyphenyl
3
linker of ciproxifan, the imidazole moiety, associated with adverse side effects, was replaced by
piperidine as shown for UCL2190 [22] (Figure 1) fitting a pharmacologically more favourable H R
3
pharmacophore [22,23]. Accordingly, three series of DTLs were synthetized by attaching piperidine- or
3

ACCEPTED MANUSCRIPT
pyrrolidine(alkyloxy)phenyl H R structural elements to already described (2-benzylidene-)indanone
3
MAO B inhibitor scaffolds in different positions. The first series of MAO B/H R DTLs (1a-e) were
3
synthesized by attachment of the H R motif to the C5 or C6 position of an indanone core structure
3
related to compound I. Variations in alkyl chain length between the aromatic core A and the amine as
well as in size of the aliphatic heterocycle (piperidino or pyrrolidino group) were performed. A second
set of DTLs were obtained by introduction of the H R pharmacophore in either C5 or in C6 position of
3
the more lipophilic 2-benzylidene-indanone derivative II (2a-c). A third series of compounds (3a-f) was
received by connecting the H R pharmacophore to the benzylidene ring B at the C4´ position [19].
3
1
25
3 3 i
Figure 1. H R antagonists/inverse agonists ciproxifan [20] and UCL2190 (hH R K = 1.6 nM, [ I]iodoproxyfan
replacement studies [24]) as well as potent reversible (2-benzylidene-)indanone MAO B inhibitors (I and II) [18,19]
taken as lead structures for three series of potential dual-targeting ligands (DTLs).
Chemistry
In the present study, the commercially available 5- and 6-methoxy-1-indanone were used for the
synthesis of the corresponding hydroxy derivatives. The demethylation of phenol ethers was achieved
with yields higher than 90% [25]. Precursors were then synthesized by alkylation of piperidine with 2-
chloroethan-1-ol or 3-chloropropan-1-ol [26]. UCL2190 was obtained with 60% yield via nucleophilic
exchange between 3-(piperidin-1-yl)propan-1-ol and cyclopropyl 4-fluorophenyl methanone
(
Scheme 1) [22,27]. Precursor were obtained after chlorination of the alcohol derivatives in
quantitative yield [28,29]. Williamson ether reaction was then performed to alkylate 6- or 7-hydroxy-1-
indanone with 1-(2-chloroethyl)piperidine/pyrrolidine or 1-(3-chloropropyl)piperidine/pyrrolidine [30].
The first series of compounds, 1a-e, was obtained with a yield ranging from 72% to 85% (Scheme 1).
Afterwards, some of the latter compounds were combined with benzaldehyde via aldol condensation
to obtain the corresponding 2-benzylidene-1-indanone derivatives 2a-c in good yields (80-90%)
4

ACCEPTED MANUSCRIPT
(
Scheme 2) [31]. The synthesis of compounds 3a-f started with the alkylation of the commercially
available 4-hydroxybenzaldehyde with 1-(3-chloropropyl)piperidine hydrochloride. The obtained
aldehyde was converted into several 2-benzylidene-1-indanone derivatives via aldol condensation with
the corresponding commercially available indanones (Scheme 2). For the synthesis of these
compounds the same procedure described for the second series of products were used. For 2-
benzylidene-1-indanone derivatives the isomers were determined to be in E-conformation using 2D
1
13
NMR (NOESY) [32]. The structures of final compounds were confirmed by H-NMR, C-NMR and
ESI-HRMS, while their purities were verified by elementary analysis.
O
NH
I
II
OH
n
N
N
O
m
UCL2190
NH
III
O
O
IV
N
OH
n
H3CO
HO
m
V
O
6
5
O
n
N
m
1a-e
Scheme 1. General procedure for the synthesis of first series of indanone DTLs 1a-e (m=1,2; n=2,3). (I):
Piperidine or pyrrolidine, 2-chloroethan-1-ol or 3-chloropropan-1-ol, K CO , KI, acetone, reflux, 72h; (II)
Cyclopropyl 4-fluorophenyl methanone, acetonitrile NaH, N , r.t.→reflux (III): SOCl , toluene, N , 0 °C→60 °C, 3h;
IV) AlCl , toluene, N , reflux, 3h; (V) K CO , KI, acetone, reflux, 24h.
2
3
,
2
2
2
(
3
2
2
3
Scheme 2. Synthetic route for the design of second and third series of 2-benzylidene-1-indanone DTLs 2a-c
(n=2,3) and 3a-f. Compounds 2a-c: I) NaOH, ethanol, r.t., 30 min. Compound 3a-f: II) K
2
CO
3
, KI, acetone, reflux,
2
4h; III) corresponding indanone, NaOH, ethanol, r.t., 30 min.
Pharmacology
5

ACCEPTED MANUSCRIPT
All compounds were screened for MAO A/B inhibition at one concentration (usually 10 µM) and
revealed mostly MAO B preferring inhibitory potencies, while series three DTLs (3a-f) were the most
potent ones (1a-e < 2a-c < 3a-f) (Table 1). Concerning their affinity at hH R, first series of compounds
3
(
1a-e) showed only poor hH R affinity (K > 100 nM). This could be improved in second (2a-c) and
3 i
third series (3a-f) of DTLs yielding desired nanomolar H R affinities. DTLs providing more than 76%
3
(
+++) inhibition for MAO B and high affinity for hH R (K < 100 nM) were further characterized to obtain
3 i
IC50 values for both isoforms as well as modes of inhibition (Table 2).
Table 1. H
3
R and MAO screening data of novel MAO B/H
3
R dual-targeting ligands.
1a-e
2a-c
3a-f
*
K [nM]
i
Inhibition at 10 µM
Pos.
n
2
3
2
2
3
2
3
3
m
1
2
1
2
2
R
[95% CI]
MAO A
MAO B
hH R
3
1a
6
6
5
5
5
6
6
5
-
+
6118
3129, 11963]
[
1b
-
+
-
++
-
304
122, 758]
[
1c
345
103, 1148]
[
1d
+
205
81, 520]
[
[
1
2
e
-
+
159
37, 678]
a
-
+++
++
++
++
++
++
696
302, 1605]
[
2b
-
39
8.9, 169]
[
2
3
c
-
3.9
0.6, 26]
[
[
[
a
-
2.1
0.5, 10]
3b
6
5
5
6
5
-OCH3
-OCH3
-F
++
-
11
3.5, 38]
3c
33
12, 89]
[
a
a
3d
++
+++
2.2
0.6, 8.1]
[
a
a
3e
-OCH -Ph-4-Br
+
++
32
13, 81]
2
[
a
a
3f
-OCH -Ph-4-Br
+
+++
6.5
1.5, 29]
2
[
K
i
values for the hH
3
R are given as mean within the 95% confidence interval (CI). * MAO inhibition was calculated
6

ACCEPTED MANUSCRIPT
as percentages related to control at a test concentration of 10 µM and given as ≤25% (-), 26-50% (+), 51-75%
a
(
++), 76-100% (+++); Fluorimetric assay with a test concentration of 10, 2 and 5 µM for 3d, 3e and 3f,
respectively; Ph = phenyl.
Discussion
In our study, we could obtain the final products in high yields, by performing only few synthetic steps.
In general, synthesis of precursor molecules was the most laborious part, while combining the H R
3
pharmacophore with the indanone or the benzylic moiety could be easily performed by Williamson
ether reaction. For lead structure optimization, based on our prior description of ciproxifan possessing
only moderate MAO and hH R activities [20], its analogue UCL2190 bearing a piperidine moiety
3
instead of an imidazole was synthesized and investigated for its MAO inhibition properties.
Surprisingly, UCL2190
provides a more favourable selectivity profile for MAO B in addition to an about 30-fold higher hH R
3
affinity (K = 11 nM; 95% CI = [3.5, 33], Figure 1) compared to ciproxifan, representing an optimized
i
lead structure for synthesis of MAO B/H R DTLs. It has been shown previously, that indanone
3
derivatives substituted in C6 and C5 position are potent inhibitors of the human MAO B [18]. Within the
first series of indanone DTLs (1a-e), most of the compounds exhibit inhibition potency for both MAO
isoforms < 50% at 10 µM, with 1b showing highest MAO B inhibition properties (72 ± 4.3%) (Table 1).
Furthermore, none of these compounds show the desired hH R affinity, most probably due to the lack
3
of an additional lipophilic group in the molecule, as frequently observed in the arbitrary region of H R
3
antagonists [23].
Table 2. H
indanone MAO B/H
3
R Affinity and MAO IC50 values (with and without preincubation) for most potent 2-benzylidene-1-
R dual-targeting ligands.
3
b
Pre-
incubation
37°C
MAO
SI
LE
IC5 [nM]
0
a
[95% CI]
(n)
Compound
[
min]
MAO A
MAO B
MAO B
0
9178
1931
[926, 4025]
(4)
4.8
3.7
0.28
[
4169, 20207]
(4)
3
d
e
30
541
362, 807]
0.31
0.22
0.25
0.25
0.25
0.35
[
(5)
0
5514
3567, 8522]
1455
[840, 2522]
(4)
[
(7)
3
30
232
70, 769]
[
(5)
0
>10 000
276
>36
>12
[
[
197, 385]
(6)
(3)
3f
30
262
185, 372]
(5)
0
>50 000
3884
[1816, 8311]
(3)
UCL2190
(3)
7

ACCEPTED MANUSCRIPT
0
>50 000
53
20, 141]
(4)
>940
>710
0.45
0.48
0.72
0.82
[
(4)
Safinamide
30
21
[
[
13, 33]
(4)
0
>30 000
42
23, 74]
(4)
(4)
L-Deprenyl
30
3.9
2.2, 6.8]
[
(5)
K
i
and IC50 values for human H R and MAO A/B, respectively, are given as mean within the 95% confidence
3
a
interval (CI) of n independent experiments each performed at least in duplicates; Selectivity index (SI) = IC50
MAO A/ IC50 MAO B; LE = pIC50/HA (heavy atoms), LE ≥ 0.3, LE was calculated as previously described [33].
b
The more lipophilic 2-benzylidene-1-indanone DTLs 2a-c (Table 1), showed more preferred and
improved MAO B inhibition with highest inhibition found for 2a (81 ± 4.5%), which still lacks H R
3
affinity. Thus, as it could be previously shown, that substitution of the benzylidene moiety in the C4´
position with lipophilic groups (e.g. halogens or alkyl chains) or basic/polar moieties (e.g. tertiary
amines) is tolerated by MAO B [19], in the series three DTLs (3a-f) the H R pharmacophore was
3
attached to the C4´ position of the benzylidene (Table 1). All of these compounds showed inhibition
capability for MAO B > 60% and hH R affinity in low nanomolar concentration ranges (K < 50 nM).
3
i
The DTLs with most potent MAO B inhibition properties 3d (94 ± 8.7%), 3e (64 ± 6.5%) and 3f
87 ± 3.8%) at 10, 2 and 5 µM, respectively, were further evaluated. All three showed IC50 values in
(
low micromolar to nanomolar concentration ranges (Table 2), while highest inhibition properties for
MAO B with optimized preference (SI > 36) over MAO A was found for DTL 3f (Figure 2).
1
40
20
00
1
1
8
0
0
0
0
0
6
4
2
-10
-9
-8
-7
-6
-5
-4
-3
-
1
3
f, log (c [mol l ])
Figure 2. Monoamine oxidase A (□) and B (▼) IC50 curves of compound 3f. Data are given as mean (normalized
to control) ± standard deviation (SD) of one representative experiment performed in duplicates.
To investigate time dependency and reversibility of inhibition two setups were performed for DTLs
3
d-f: (a) IC50 shift experiments after pre-incubation of enzyme and inhibitors (30 min, 37°C) (Table 4)
and (b) 50x dilution experiments after pre-incubation of enzyme and inhibitors (30 min and 60 min,
7°C) with excess of substrate (Figure 3). Interestingly, the IC50 values do not show significant
3
difference to non-preincubated ones for 3f, while a 3.5-fold and 6.3-fold shift of IC50 to nanomolar
concentrations was shown for 3d and 3e, respectively, suggesting a slow reversible or tight binding
inhibition mode within our test conditions. The dilution experiments under saturated substrate
conditions, however, revealed a reversible mode of inhibition for all three DTLs, indicated by recovery
of enzyme activity after dilution. This would be consistent with findings for structural related (E)-3-
heteroarylidenechroman-4-ones and (E)-3-benzylidenechroman-4-ones showing comparable
behaviour and differences within a series of compounds [34,35]. Tight binding inhibitors which do not
form covalent bonds with the active site can behave similar to typical covalent binding “suicide
inhibitors” like L-deprenyl in reversibility studies [17,34]. In vivo they might have a safer
8

ACCEPTED MANUSCRIPT
pharmacological profile with longer duration times than reversible inhibitors, but lacking the suicide
inactivation of MAOs. Reversible inhibitors are known to have less severe side effects, such as
hypertensive crisis or slow recovery of MAO expression levels after suicide inactivation [17,36]
associated with irreversible and non-selective MAO inhibitors. However, since they do not inactivate
the enzyme, reversible MAO inhibitors need higher target affinities to compete with the endogenous
ligands, which remains a demanding step in MAO B drug design [37].
Figure 3. Reversibility of inhibition after preincubation of MAO B and inhibitors for 0, 30 and 60 min prior to 50x
dilution, measured under saturated substrate assay conditions. Data are given as mean (normalized to each
control) ± standard deviation (SD) of at least two independent experiments, each performed in duplicates (global
fit).
For early evaluation of drug-likeness of most promising DTLs, ligand efficacy (LE), ligand efficiency
dependent lipophilicity (LELP) and ligand-lipophilicity efficiency (LipE) were calculated for hH R and
3
MAO B [33] (Supporting Information). The high lipophilic DTLs 3e and 3f showed improved MAO B
affinity but at the cost of LE due to the high molecular weight (Table 2). Instead, DTL 3d displays best
drug-like physicochemical parameters for both targets representing the most lead-like DTL within this
small series. Regarding proposed multi-targeting properties, the overlapping of pharmacophoric
elements of H R antagonists and AChE/BuChE inhibitors (e.g. piperidinopropyloxy element) most
3
probably result in potential AChE/BuChE inhibition potency for our MAO B/H R DTLs. Recently, the
3
multi-target ligand contilisant, combining affinity at H Rs, MAOs and cholinesterase in vitro,
3
demonstrated a pro-cognitive effect in mice [38]. Additionally, potent antioxidative 2-benzylidene-1-
indanones were described previously, combining nanomolar AChE and Aβ aggregation inhibition as
possible drugs for Alzheimer´s disease (AD) therapy [32]. Finding some additional AChE/BuChE
inhibition properties, however, might result in a beneficial multi-targeting ligand (MTL) profile, where
several MTLs are already described especially for treatment of cognitive impairments and AD [38–40].
From structural point of view our 2-benzylidene-1-indanone DTLs may act as Michael acceptors, which
are able to form adducts with proteins, especially with cysteine residues [41,42]. To address this
problem, compound 3d was incubated with an excess of acetylcysteine at physiological pH and
monitored by ESI-MS for up to three days. No thiol adduct was observed, suggesting that formation of
adducts with proteins are unlikely for our 2-benzylidene-1-indanone DTLs.
In conclusion, we were able to design hH R/MAO B DTLs by attaching a generally accepted H R
3
3
antagonist pharmacophore in different position to previously described indanone and 2-benzylidene-
-indanone MAO B inhibitor motifs. Within this study three 2-benzylidene-1-indanone DTLs were
1
obtained, showing promising capabilities with MAO B inhibition properties (IC50 = 232 – 541 nM after
preincubation) and affinity at the human H R (K = 2.2 – 32 nM). These DTLs (3d, 3e and 3f) were
3
i
obtained by attaching the H R pharmacophore to the C4´ position of the benzylidene ring B,
3
substituted either with fluoride (3d) or a bulky, lipophilic element (3e, 3f) on the indanone ring A.
Compound 3f were the most MAO B preferring inhibitor (MAO SI > 36) showing a reversible inhibition
mode, while 3d and 3e provide interesting, more balanced MAO profiles with presumed tight binding,
but still reversible inhibition mode. Thus, we successfully described promising MAO B/H R DTLs,
3
suitable as a starting point, for further optimization and development of potent DTLs or MTLs for the
treatment of Parkinson’s disease.
9

ACCEPTED MANUSCRIPT
Experimental part
Reagent and instrumentation
Reagents and solvents for synthesis were purchased from Sigma-Aldrich, VWR Chemicals, Fisher
Scientific, Panreac AppliChem, Alfa Aesar and Chemsolute and were used without further purifications
1
13
(
(
unless stated otherwise). H-NMR and C-NMR were recorded on a Bruker AMX spectrometer
Bruker, Germany) at 300 and 75 MHz respectively, where CDCl or DMSO-d were used as a solvent.
3
6
Tetramethylsilane was used as standard and chemical shifts are reported in part per million (ppm).
Spin multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quintet) or
m (multiplet). Approximate coupling constants (J) in Hertz (Hz). Number and assignment of protons
(
ax, axial; eq, equatorial; ph, phenyl; ind, indanone; prop, propyl; pip, piperidine; eth, ethyl; cycloprop,
cyclopropyl; pyr, pyrrolidine). Elementary analyses (C, H, N) were measured on a CHN-Rapid
Heraeus, Germany) and were within 0.4 % of the theoretical values for all final compounds.
(
Electrospray ionization mass spectrometry (ESI-MS) was performed on an amaZon speed (Bruker,
+
Germany) in positive polarity. Data are listed as mass number ([M+H ]) and relative intensity (%).
High-resolution mass spectra (HRMS) were run in electrospray ionization (ESI) mode. Melting points
(m.p., uncorrected) were determined on a M-564 Büchi melting point apparatus (Büchi, Germany).
Preparative column chromatography was performed on silica gel 60 M, 0.04-0.063 mm (Macherey-
Nagel, Germany) and thin-layer chromatography (TLC) was carried out using pre-coated silica gel 60
with fluorescence indicator at UV 254 nm (Macherey-Nagel, Germany).
Synthesis of precursors
1-(2-Chloroethyl or 3-chloropropyl)piperidine and 1-(2-chloroethyl)pyrrolidine as well as 4-(3-(piperidin-
1-yl)propoxy)benzaldehyde were synthetized as described previously [26,43,44]. Demethylation of the
5- or 6-hydroxy-1-indanone was achieved by using well described general methods [25,45]. The
precursors were used as starting point for the preparation of the final DTLs (series 1-2-3).
Synthesis of cyclopropyl(4-(3-(piperidin-1-yl)propoxy)phenyl)methanone hydrochloride
(
UCL2190) [22,27]
3-(Piperidine-1-yl)propan-1-ol (1 eq) was treated with NaH (5 eq, 60% suspension mineral oil) at room
temperature under inert atmosphere for 3 h. The freshly prepared 3-(piperidine-1-yl)propanolate was
heated with cyclopropyl-4-fluorophenyl methanone (2 eq) in acetonitrile for 24 h under reflux. The
mixture was concentrated under reduced pressure and the residue extracted with dichloromethane
and 2N NaOH solution. The organic extract was dried over MgSO , evaporated and transformed into
4
hydrogen chloride. The white solid was obtained in a yield of 62%: m.p. 174.6 °C.
1
H-NMR (300 MHz, DMSO-d ) δ 7.99-7.90 (m, 2H, ph-3,7H), 7.03-6.94 (m, 2H, ph-4,6H), 4.21-4.09 (t,
6
2
H, J=5.8, prop-1H ), 3.62-3.37 (m, 2H, prop-3H ), 3.29-3.16 (m, 2H, pip-2,6H ), 3.02-2.81 (m, 2H,
2 2 eq
pip-2,6H ), 2.79-2.69 (m, 1H, COCHCH ), 2.23-2.10 (m, 2H, prop-2H ), 2.01-1.30 (m, 6H, pip-3,5H
2,
ax
2
2
13
pip-4Heq/ax), 1.14-1.01 (m, 4H, Cycloprop-2,3H2); C-NMR (75 MHz, DMSO-d ) δ 204.6 (CO), 162.4
6
(
2
ph-5C), 129.7 (Ph-3,7C), 129.4 (ph-6,4C), 128.2 (Ph-2C), 73.1 (prop-1C), 58.5 (prop-3C), 57.1 (pip-
,6C) 27.7 (prop-2C), 25.9 (pip-3,5C), 24.5 (pip-4C), 18.1 (COCHCH ), 12.6 (Cycloprop-2,3(CH ) );
2
2 2
+
ESI-MS m/z: calcd for C H NO (MH ), 288.1964, found 288.1958.
18
25
2
Synthesis of the 1-indanone derivatives 1a-e [26,30]
The appropriately hydroxy phenols (1.2 eq) and chloride precursors (1 eq) were reflux in absolute
acetone together with potassium carbonate (3.2 eq) and potassium iodide (1 eq). After 24 h, the
reaction was cooled down. The inorganic materials were removed by filtration and the filtrate was
concentrated under vacuum. The crude product was taken up in methylene chloride and 2N NaOH
solution. The organic phase was then washed with saturated NaCl-solution and water, dryed over
sodium sulfate, and concentrated under vacuum. The resulting oil was purified by column
chromatography (CH Cl /MeOH, 9/1).
2
2
6-(2-(Pyrrolidin-1-yl)ethoxy)-2,3-dihydro-1H-inden-1-one hydrochloride (1a)
1
Yield: 76%; m.p. 195.2 °C. H-NMR (300 MHz, D O) δ 7.48-7.41 (d, 1H, J= 8.5, ph -7H), 7.31-7.25
2
ind
(
dd, 1H, J = 8.5, ph -5H), 7.13-7.08 (d, 1H, J= 2.5, ph -4H), 4.38-4.25 (t, 2H, J= 5.0, eth-1H ), 3.72-
ind ind 2
1
0

ACCEPTED MANUSCRIPT
3
2
1
.51 (m, 4H, COCH , COCH CH ), 3.20-3.05 (m, 2H, eth-2H ), 3.04-2.92 (m, 2H, pyr-2,5H ), 2.70-
2
2
2
2
eq
13
.57 (m, 2H, pyr-2,5H ), 2.21-1.80 (m, 4H, pyr-3,4H ); C-NMR (75MHz, DMSO-d ) δ 206.4 (CO),
ax
2
6
57.6 (ph -6C), 148.7 (COCph -7C), 138.4 (COCH CH C), 128.4 (ph -7C), 123.9 (ph -6C), 106.4
ind
ind
2
2
ind
ind
(
(
ph -4C), 64.3 (prop-1C), 54.0 (pyr-2,5C), 52.1 (prop-2C), 37.2 (COCH ), 25.3 (COCH CH ), 23.0
ind 2 2 2
pyr-3,4C); ESI-HRMS m/z: calcd for C H NO (MH ), 246.1494, found 246.1495. Anal. calc.: C,
15 19 2
+
61.96; H, 7.28; N, 4.82. Found: C, 62.43; H, 7.06; N, 4.98
6-(3-(Piperidin-1-yl)propoxy)-2,3-dihydro-1H-inden-1-one (1b)
1
Yield: 81%; m.p. 59.1 °C. H-NMR (300 MHz, DMSO-d ) δ 7.64-7.57 (d, 1H, J= 8.7, ph -7H), 7.28-
6
ind
7
1
3
3
1
.21 (dd, 1H, J= 8.4, ph -5H), 7.09-7.04 (m, 1H, J= 2.28, ph -4H), 4.07-3.97 (t, 2H, J= 6.4, prop-
ind ind
H ), 3.06-2.94 (t, 2H, J= 5.3, COCH ), 2.68-2.59 (m, 2H, COCH CH ), 2.42-2.33 (t, 2H, J = 6.9, prop-
2
2
2
2
H ), 2.32-2.20 (m, 4H, pip-2,6Heq/ax), 1.93-1.77 (q, 2H, J = 6.7, prop-2H ), 1.56-1.43 (m, 4H, pip-
2
2
1
3
,5H ), 1.42-1.30 (m, 2H, pip-4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 208.7 (CO), 160.9 (ph -6C),
2
6
ind
50.3 (COCph -7C), 140.5 (COCH CH C), 130.5 (ph -7C), 128.1 (ph -5C), 108.1 (ph -4C), 69.0
ind
2
2
ind
ind
ind
(
(
prop-1C), 57.7 (prop-3C), 56.8 (pip-2,6C), 39.3 (COCH ), 28.8 (COCH CH ), 28.2 (pip-3,5C), 27.3
2 2 2
+
prop-2C), 26.8 (pip-4C); ESI-HRMS m/z: calcd for C H NO (MH ), 274.1807, found 274.1800. Anal.
17
23
2
calc.: C, 74.68; H, 8.48; N, 5.12. Found: C, 74.76; H, 8.68; N, 5.06
5-(2-(Pyrrolidin-1-yl)ethoxy)-2,3-dihydro-1H-inden-1-one hydrochloride (1c)
1
Yield: 75%; m.p. 193.8 °C. H-NMR (300 MHz, D O) δ 7.59-7.53 (d, 1H, J= 8.6, ph -7H), 7.07-7.01
2
ind
(
m, 1H, ph -4H), 6.99-6.91 (dd, 1H, J= 8.6, ph -6H), 4.43-4.33 (t, 2H, J= 5.0, eth-1H ), 3.73-3.58 (m,
ind ind 2
4
H, COCH , COCH CH ), 3.24-3.07 (m, 2H, eth-2H ), 3.06-2.98 (m, 2H, pyr-2,5H ), 2.67-2.55 (m, 2H,
2 2 2 2 eq
13
pyr-2,5H ), 2.20-1.86 (m, 4H, pyr-3,4H ); C-NMR (75 MHz, DMSO-d ) δ 204.7 (CO), 163.3 (ph -
ax
2
6
ind
5C), 158.5 (COCph -7C), 130.8 (COCH CH C), 125.01(ph -7C), 116.1 (ph -4C), 111.4 (ph -6C),
ind 2 2 ind ind ind
6
4.1 (prop-1C), 54.1 (pyr-2,5C), 52.8 (prop-2C), 36.4 (COCH ), 25.8 (COCH CH ), 22.9 (pyr-3,4C);
2
2
2
+
ESI-HRMS m/z: calcd for C H NO (MH ), 246.1494, found 246.1484. Anal. calc.: C, 62.93; H, 7.22;
15
19
2
N, 4.89. Found: C, 62.69; H, 7.09; N, 5.03
5-(2-(Piperidin-1-yl)ethoxy)-2,3-dihydro-1H-inden-1-one hydrochloride (1d)
1
Yield: 72%; m.p. 194.4 °C. H-NMR (300 MHz, D O) δ 7.51-7.43 (d, 1H, J= 8.6, ph -7H), 7.28-7.21
2
ind
(
m, 1H, ph -4H), 6.96-6.88 (dd, 1H, J=8.5, ph -6H), 4.43-4.31 (t, 2H, J= 5.0, eth-1H ), 3.65-3.42 (m,
ind ind 2
4
1
1
H, COCH , COCH CH ), 3.10-2.88 (m, 4H, pip-2,6H , eth-2H ), 2.64-2.50 (m, 2H, pip-H2,6ax), 2.03-
2 2 2 eq 2
13
.30 (m, 6H, pip-3,5H , pip-4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 204.7 (CO), 163.3 (ph -5C),
2
6
ind
58.5 (COCph -7C), 130.7 (COCH CH C), 125.1 (ph -7C), 116.1 (ph -4C), 111.4 (ph -6C), 66.1
ind
2
2
ind
ind
ind
(
(
prop-1C), 54.9 (prop-2C), 52.9 (pip-2,6C), 36.4 (COCH ), 25.8 (COCH CH ), 22.6 (pip-3,5C), 21.5
2 2 2
+
pip-4C); ESI-HRMS m/z: calcd for C H NO (MH ), 260.1651, found 260.1642. Anal. calc.: C, 64.97;
16
21
2
H, 7.50; N, 4.74. Found: C, 64.52; H, 7.55; N, 4.88
5-(3-(Piperidin-1-yl)propoxy)-2,3-dihydro-1H-inden-1-one (1e)
1
Yield: 85%; m.p. 60.1 °C. H-NMR (300 MHz, DMSO-d ) δ 7.64-7.57 (d, 1H, J=8.5, ph-7H), 7.18-7.11
6
(
m, 1H, ph-4H), 7.05-6.96 (dd, 1H, J=8.5, ph-6H), 4.24-4.11 (t, 2H, J= 6.4, prop-1H ), 3.19-3.04 (t, 2H,
2
J= 6.0, COCH ), 2.76-2.61 (m, 2H, COCH CH ), 2.51-2.40 (t, 2H, J= 7.3, prop-3H ), 2.40-2.28 (m, 4H,
2
2
2
2
pip-2,6H
), 2.03-1.88 (q, 2H, J= 6.7, prop-2H ), 1.65-1.51 (m, 4H, pip-3,5H ), 1.50-1.38 (m, 2H, pip-
2 2
eq/ax
1
3
4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 204.6 (CO), 164.1 (ph-5C), 158.6 (COCph-7C), 130.4
6
(
(
COCH CH C), 124.9 (ph-7C), 115.9 (ph-4C), 111.1 (ph-6C), 66.3 (prop-1C), 53.4 (prop-3C), 52.2
2 2
pip-2,6C), 36.1 (COCH ), 25.8 (COCH CH ), 23.5 (prop-2C), 22.7 (pip-3,5C), 21.7 (pip-4C); ESI-
2
2
2
+
HRMS m/z: calcd for C H NO (MH ), 274.1807, found 274.1800. Anal. calc.: C, 74.69; H, 8.48; N,
1
7
23
2
5.12. Found: C, 74,82; H, 8.58; N, 5.01.
Synthesis of 2-benzylidene-1-indanone derivatives 2a-c [31]
A water solution of sodium hydroxide (1.5 eq) was added at room temperature to an ethanol solution
of the appropriately indanone (1 eq) and the commercially available benzaldehyde (1 eq). After 30
min, there was the formation of a precipitate. The solid was filtered off and extracted with ethyl acetate
and 2 N NaOH solution. The crude material was dried over sodium sulfate and concentrated under
vacuum. The resulting solid was elementary pure without further purification steps.
1
1

ACCEPTED MANUSCRIPT
(
E)-2-Benzylidene-6-(2-(piperidin-1-yl)ethoxy)-2,3-dihydro-1H-inden-1-one (2a)
1
1
Yield: 90%; m.p. 130.1 °C. H-NMR (300 MHz, DMSO-d ) δ 7.86-7.72 (m, 2H, ph -2,6H), 7.63-7.41
(
6
1
1
1
m, 5H, ph -CH, ph -5,7H, ph -3,5H ), 7.37-7.20 (m, 2H, ph -4H, ph -4H), 4.20-4.10 (t, 2H, J= 5.5,
i
n
d
i
n
d
eth-1H ), 4.04 (s, 2H, C=CCH ), 2.76-2.62 (t, 2H, J= 5.2, eth-2H ), 2.48-2.32 (m, 4H, pip-2,6Heq/ax),
2
2
2
13
1
(
1
1
2
7
.60-1.44 (m, 4H, pip-3,5H ), 1.43-1.29 (m, 2H, pip-4H
CO), 158.4 (ph -6C), 142.6 (COCph -7C), 138.3 (ph -CH), 135.8 (C=CCH C), 134.8 (COC=C),
32.7 (ph -1C), 130.7 (ph -2,6C), 129.8 (ph -4C), 128.9 (ph -3,5C), 127.5 (ph -7C), 123.9 (ph -5C),
06.3 (ph -4C), 66.0 (prop-1C), 57.2 (prop-2C), 54.3 (pip-2,6 C), 31.2 (C=CCH ), 25.5 (pip-3,5C),
5.9 (pip-4C); ESI-HRMS m/z: calcd for C H NO (MH ), 348.1964, found 348.1964. Anal. calc.: C,
9.51; H, 7.25; N, 4.03. Found: C, 79.23; H, 7.17; N, 3.88.
); C-NMR (75 MHz, DMSO-d ) δ 193.1
2
eq/ax
6
1
ind
ind
2
1
1
1
1
ind ind
ind
2
+
2
3
25
2
(
E)-2-Benzylidene-6-(3-(piperidin-1-yl)propoxy)-2,3-dihydro-1H-inden-1-one (2b)
1
1
Yield: 80%; m.p. 119.9 °C. H-NMR (300 MHz, DMSO-d ) δ 7.88-7.80 (m, 2H, ph -2,6H), 7.65-7.60 (d,
6
1
1
1
1
7
2
(
(
H, J = 8.5, ph -CH), 7.59-7.47 (m, 4H, ph-5,7H, ph -3,5H), 7.38-7.31 (dd, 1H, J = 8.3, ph -4H), 7.30-
.27 (d, 1H, J = 2.4, ph -4H), 4.18-4.10 (t, 2H, J = 6.4, prop-1H ), 4.09 (s, 2H, C=CCH ), 2.48-2.40 (t,
H, J = 7.3, prop-3H ), 2.40-2.30 (m, 4H, pip-2,6H
m, 4H, pip-3,5H ), 1.47-1.35 (m, 2H, pip-4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 193.1 (CO), 158.5
2 6
ph -6C), 142. (COCph -7C), 138.3 (ph -CH), 135.8 (C=CCH C), 134.8 (COC=C), 132.6 (ph -1C),
ind ind 2
30.7 (ph -2,6C), 129.8 (ph -4C), 128.9 (ph -3,5C), 127.5 (ph -7C), 123.8 (ph -5C), 106.1 (ph -
C), 66.4 (prop-1C), 55.0 (prop-3C), 54.1 (pip-2,6C), 31.1 (C=CCH ), 26.1 (prop-2C), 25.5 (pip-3,5C),
4.1 (pip-4C); ESI-HRMS m/z: calcd for C H NO (MH ), 362.2120, found 362.2117. Anal. calc.: C,
ind
2
2
), 1.99-1.87 (q, 2H, J = 6.8, prop-2H ), 1.61-1.48
2
2
eq/ax
1
3
1
1
1
1
1
1
4
2
7
ind ind ind
2
+
24
27
2
9.74; H, 7.53; N, 3.87. Found: C, 79.41; H, 7.31; N, 3.78.
(
E)-2-Benzylidene-5-(3-(piperidin-1-yl)propoxy)-2,3-dihydro-1H-inden-1-one (2c)
1
1
Yield: 86%; m.p. 110.7 °C. H-NMR (300 MHz, DMSO-d ) δ 7.80-7.74 (m, 2H, ph -2,6H), 7.74-7.63 (d,
6
1
1
1
1
H, J = 8.5, ph -CH), 7.56-7.41 (m, 4H, ph -4,7H, ph -3,5H), 7.19-7.14 (d, 1H, J = 1.9, ph -4H), 7.06-
ind
6
.98 (dd, 1H, J = 8.5, ph -6H), 4.18-4.11 (t, 2H, J = 6.4, prop-1H ), 4.07 (s, 2H, C=CCH ), 2.45-2.35
ind
2
2
(
t, 2H, J = 6.9, prop-3H ), 2.35-2.22 (m, 4H, pip-2,6H
), 1.98-1.82 (q, 2H, J = 6.7, prop-2H ), 1.55-
eq/ax 2
2
13
1
1
2
.45 (m, 4H, pip-3,5H ), 1.44-1.31 (m, 2H, pip-4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 191.5 (CO),
2
6
1
1
64. (ph -5C), 152.9 (COCph -7C), 135.7 (ph -CH), 135.0 (C=CCH C), 131.4 (COC=C), 130.5 (ph -
ind
ind
2
1
1
1
,6C), 130.3 (ph -1C), 129.5 (ph -4C), 128.9 (ph -3,5C), 125.4 (ph -7C), 115.7 (ph -4C), 110.6
ind
ind
(
(
ph -6C), 66.6 (prop-1C), 54.9 (prop-3C), 54.7 (pip-2,6C), 31.9 (C=CCH ), 26.1 (pip-3,5C), 25.6
ind 2
+
prop-2C), 24.1 (pip-4C); ESI-HRMS m/z: calcd for C H NO (MH ), 362.2120, found 362.2120. Anal.
24
27
2
calc.: C, 79.74; H, 7.53; N, 3.87. Found: C, 79.81; H, 7.33; N, 3.57.
Synthesis of 2-benzylidene-1-indanone derivatives 3a-f [31]
A water solution of sodium hydroxide (1.5 eq) was added at room temperature to an ethanol solution
of th appropriately commercially available indanones (1 eq) and 4-(3-(piperidin-1-yl)-propoxy)-
benzaldehyde (1 eq). The work up was performed as described for compounds 5a-f.
(
E)-2-(4-(3-(Piperidin-1-yl)propoxy)benzylidene)-2,3-dihydro-1H-inden-1-one (3a)
1
1
Yield 81%; m.p. 113.85 °C. H-NMR (300 MHz, DMSO-d ) δ 7.84-7.80 (d, 1H, J = 7.6, ph -CH), 7.61-
7
6
1
.45 (m, 5H, ph -2,3,5,6H, ph -7H), 7.39-7.31 (m, 1H, ph -6H), 6.94-6.86 (m, 2H, ph -4,5H), 4.07-
ind ind ind
3
.98 (t, 2H, J = 6.2, prop-1H ), 3.94 (s, 2H, C=CCH ), 2.64-2.51 (t, 2H, J = 7.7, prop-3H ), 2.51-2.31
2
2
2
(
2
(
1
m, 4H, pip-2,6H
), 2.10-1.96 (q, 2H, J = 6.7, prop-2H ), 1.69-1.54 (m, 4H, pip-3,5H ), 1.49-1.33 (m,
eq/ax 2 2
13
1
H, pip-4Heq/ax); C-NMR (75 MHz, CDCl ) δ 193.1 (CO), 158.5 (ph -4C), 142.5 (COCph -7C), 138.4
3
i
n
d
1
1
1
ph -CH), 135.8 (C=CCH C), 134.8 (ph -2,6C), 132.5 (COC=C), 130.7 (ph -1C), 129.7 (ph -7C),
2 ind
1
28.9 (ph -4C), 127.5 (ph -6C), 123.8 (ph -5C), 106.1 (ph -3,5C), 66.3 (prop-1C), 55.0 (prop-3C),
ind ind ind
5
4.1 (pip-2,6C), 31.2 (C=CCH ), 26.2 (prop-2C), 25.6 (pip-3,5C), 24.1 (pip-4C) ; ESI-HRMS m/z: calcd
2
+
for C H NO (MH ), 362.2120, found 362.2116. Anal. calc.: C, 79.74; H, 7.53; N, 3.87. Found: C,
24
27
2
79.72; H, 7.82; N, 3.58.
(
E)-6-Methoxy-2-(4-(3-(piperidin-1-yl)propoxy)benzylidene)-2,3-dihydro-1H-inden-1-one (3b)
1
1
Yield: 86%; m.p. 118.7 °C. H-NMR (300 MHz, DMSO-d ) δ 7.79.7.69 (d, 2H, J = 8.6, ph -2,6H), 7.62-
6
1
1
7
.55 (d, 1H, J = 8.3, ph -CH), 7.52-7.46 (m, 1H, ph -7H), 7.33-7.19 (d, 2H, J = 8.3, ph -3,5H), 7.11-
ind
1
2

ACCEPTED MANUSCRIPT
7
.0 (d, 2H, J = 8.5, ph -4,5H), 4.17-4.03 (t, 2H, J = 6.3, prop-1H ), 3.99 (s, 2H, C=CCH ), 3.83 (s, 3H,
ind
2
2
OCH ), 2.46-2.34 (t, 2H, J = 6.9, prop-3H ), 2.34-2.18 (m, 4H, pip-2,6H ), 1.97-1.79 (q, 2H, J = 6.6,
eq/ax
3
2
13
prop-2H ), 1.60-1.43 (m, 4H, pip-3,5H ), 1.43-1.25 (m, 2H, pip-4Heq/ax); C-NMR (75 MHz, DMSO-d )
δ 193.0 (CO), 160.0 (ph -4C), 159.1 (ph -6C), 142.4 (COCph -7C), 138.7 (ph -CH), 133.2
C=CCH C), 132.7 (ph -2,6C), 132.7 (COC=C), 127.4 (ph -1C), 127.3 (ph -7C), 123.1 (ph -4C),
2 ind ind
15.0 (ph -3,5C), 105.5 (ph -5C), 66.2 (prop-1C), 55.5 (prop-3C), 55.0 (OCH ), 54.1 (pip-2,6C), 31.2
2
2
6
1
1
ind
ind
1
1
(
1
(
1
i
n
d
3
+
C=CCH ), 26.2 (prop-2C), 25.6 (pip-3,5C), 24.1 (pip-4C); ESI-HRMS m/z: calcd for C H NO (MH ),
2 25 29 3
392.2226, found 392.2225. Anal. calc.: C, 76.70; H, 7.47; N, 3.58. Found: C, 76.57; H, 7.47; N, 3.39.
(
E)-5-Methoxy-2-(4-(3-(piperidin-1-yl)propoxy)benzylidene)-2,3-dihydro-1H-inden-1-one (3c)
1
1
Yield: 81%; m.p. 116.6 °C. H-NMR (300 MHz, CDCl ) δ 7.81-7.42 (d, 1H, J = 8.4, ph -CH), 7.58-
3
1
1
7
6
2
1
5
1
5
.47(m, 3H, ph -2,6H, ph -7H), 6.95-6.63 (m, 4H, ph -4H, ph -6H, ph -3,5H ), 4.07-3.97 (t, 2H, J =
ind ind ind
.3, prop-1H ), 3.88 (s, 2H, C=CCH ), 3.83 (s, 3H, OCH ), 2.56-2.47 (t, 2H, J = 7.2, prop-3H ), 2.46-
.33 (m, 4H, pip-2,6Heq/ax), 2.07-1.94 (q, 2H, J = 7.9, prop-2H ), 1.68-1.52 (m, 4H, pip-3,5H ), 1.47-
.34 (m, 2H, pip-4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 191.5 (CO), 164.7 (ph -4C), 159.8 (ph -
C), 152.7 (COCph -7C), 133.0 (ph -CH), 132.4 (ph -2,6C), 131.5 (C=CCH C), 130.7 (COC=C),
27.5 (ph -1C), 125.2 (ph -7C), 115.2 (ph -4C), 114.7 (ph -3,5C), 110.1 (ph -6C), 66.1(prop-1C),
5.7 (prop-3C), 55.0 (OCH ), 54.1 (pip-2,6C), 31.9 (C=CCH ), 26.2 (prop-2C), 25.6 (pip-3,5C), 24.1
pip-4C); ESI-HRMS m/z: calcd for C H NO (MH ), 392.2226, found 392.2222. Anal. calc.: C, 76.70;
25 29 3
2
2
3
2
2
2
1
3
1
6
1
ind
1
ind
2
1
1
ind
ind
ind
3
2
+
(
H, 7.47; N, 3.58. Found: C, 76.52; H, 7.61; N, 3.49.
(
E)-5-Fluoro-2-(4-(3-(piperidin-1-yl)propoxy)benzylidene)-2,3-dihydro-1H-inden-1-one (3d)
1
1
Yield: 80%; m.p. 123.2 °C. H-NMR (300 MHz, DMSO-d ) δ 7.88-7.79 (m, 1H, ph -CH), 7.58-7.50 (m,
6
1
3
6
2
(
(
H, ph -2,6H, ph -7H), 7.17-7.10 (dd, 1H, J = 8.4, ph -4H), 7.09-6.99 (td, 1H, J = 8.9, ph -6H),
ind ind ind
1
.96-6.86 (m, 2H, ph -3,5H), 4.10-3.96 (t, 2H, J = 6.3, prop-1H ), 3.93 (s, 2H, C=CCH ), 2.59-2.46 (t,
H, J = 7.7, prop-3H ), 2.46-2.28 (m, 4H, pip-2,6H
m, 4H, pip-3,5H ), 1.49-1.32 (m, 2H, pip-4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 192.6 (CO), 165.9
2 6
ph -4C), 160.4 (ph -5C), 152.3 (COCph -7C), 134.7(ph -CH), 133.9 (C=CCH C), 132.6 (ph -2,6C),
ind ind 2
2
2
), 2.08-1.91 (q, 2H, J = 7.1, prop-2H ), 1.69-1.51
2
2
eq/ax
1
3
1
1
1
1
1
1
31.8 (COC=C), 127.8 (ph -1C), 126.5 (ph -7C), 115.6 (ph -4C), 115.1 (ph -3,5C), 112.8 (ph -6C),
ind ind ind
6
6.6 (prop-1C), 55.8 (prop-3C), 54.6 (pip-2,6C), 32.4 (C=CCH ), 26.6 (prop-2C), 25.8 (pip-3,5C), 24.3
2
+
(
pip-4C) ; ESI-HRMS m/z: calcd for C H FNO (MH ), 380.2026, found 380.2017. Anal. calc.: C,
24 26 2
75.96; H, 6.91; N, 3.69. Found: C, 75.90; H, 6.71; N, 3.67.
(
E)-6-((4-Bromobenzyl)oxy)-2-(4-(3-(piperidin-1-yl)propoxy)benzylidene)-2,3-dihydro-1H-inden-1-
one (3e)
1
1
1
Yield: 87%; m.p. 159.5 °C. H-NMR (300 MHz, DMSO-d ) δ 7.58-7.51 (m, 3H, ph -2,6H, ph -CH),
6
2
7
7
.47-7.41 (m, 2H, ph - 3,5H), 7.40-7.35 (d, 1H, J = 8.4, ph -7H), 7.33-7.30 (d, 1H, J = 2.4, ph -4H),
ind ind
.28-7.21 (m, 2H, ph -2,6H), 7.19-7.14 (dd, 1H, J = 8.3, ph -6H), 6.93-6.85 (m, 2H, ph -3,5H), 4.99
s, 2H, ph -CH O), 4.09-3.96 (t, 2H, J = 6.2, prop-1H ), 3.86 (s, 2H, C=CCH ), 2.65-2.50 (t, 2H, J =
2
1
ind
2
(
2
2
2
7
.7, prop-3H ), 2.50-2.30 (m, 4H, pip-2,6H
), 2.13-1.94 (q, 2H, J = 6.9, prop-2H ), 1.73-1.55 (m, 4H,
eq/ax 2
2
13
1
pip-3,5H ), 1.49-1.33 (m, 2H, pip-4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 194.1 (CO), 160.2 (ph -4C),
2
6
1
2
158.4 (ph -6C), 142.7 (C=CCH C), 139.5 (ph -CH), 135.5 (ph -1C), 133.8 (COC=C), 133.1 (ph -7C),
ind
1
2
ind
2
2
1
132.6 (ph -2,6C), 131.7 (ph -3,5C), 129.1 (ph -2,6C), 128.1 (COCph -7C), 127.0 (ph -1C), 124.0
ind
2
1
2
(
(
ph -5C), 122.0 (ph -4C), 114.9 (ph -3,5C), 104.94 (ph -4C), 69.5 (prop-1C), 66.3 (ph -CH O), 55.7
ind ind 2
prop-3C), 54.1 (pip-2,6C), 31.8 (C=CCH ), 26.1 (prop-2C), 25.3 (pip-3,5C), 23.9 (pip-4C); ESI-HRMS
2
+
m/z: calcd for C H BrNO (MH ), 546.1644, found 546.1643. Anal. calc.: C, 68.13; H, 5.90; N, 2.56.
31
32
3
Found: C, 67.84; H, 5.80; N, 2.36.
(
E)-5-((4-Bromobenzyl)oxy)-2-(4-(3-(piperidin-1-yl)propoxy)benzylidene)-2,3-dihydro-1H-inden-1-
one (3f)
1
1
Yield: 85%; m.p. 133.8 °C. H-NMR (300 MHz, CDCl ) δ 7.81-7.74 (d, 1H, J= 8.6, ph -CH), 7.57-7.47
3
2
1
(
m, 4H, ph - 3,5H, ph -2,6H), 7.44-7.38 (td, 1H, J = 1.3, ph -7H), 7.33-7.26 (d, 1H, J = 1.1, ph -4H),
.23-7.16 (t, 1H, J = 7.6, ph -6H), 6.98-6.85 (m, 4H, ph -2,6H, ph -3,5H), 5.05 (s, 2H, ph -CH O),
.05-3.95 (t, 2H, J = 6.2, prop-1H ), 3.87 (s, 2H, C=CCH ), 2.52-2.42 (t, 2H, J = 7.7, prop-3H ), 2.41-
ind ind
2
1
2
7
4
2
1
i
n
d
2
2
2
2
.30 (m, 4H, pip-2,6Heq/ax), 2.03-1.89 (q, 2H, J = 6.9, prop-2H ), 1.62-1.51 (m, 4H, pip-3,5H ), 1.43-
2
2
1
3
1
.34 (m, 2H, pip-4Heq/ax); C-NMR (75 MHz, DMSO-d ) δ 191.5 (CO), 163.5 (ph -4C), 159.8 (ph -
6
ind
1
3

ACCEPTED MANUSCRIPT
1
2
1
2
2
5
5
(
(
C), 152.6 (C=CCH C), 139.2 (ph -CH), 132.9 (ph -1C), 132.4 (ph -2,6C), 131.6 (ph -3C), 131.0 (ph -
2
2
2
C), 130.9 (ph -2C), 130.7 (ph -6C), 130.3 (COC=C), 127.4 (ph -7C), 126.7 (COCph -7C), 125.3
ind
ind
1
2
1
ph -1C), 121.7 (ph -4C), 115.7 (ph -4C), 114.9 (ph -3,5C), 111.2 (ph -6C), 68.7 (prop-1C), 66.1
ind ind
2
ph -CH O), 55.0 (prop-3C), 54.0 (pip-2,6C), 31.9 (C=CCH ), 26.2 (prop-2C), 25.5 (pip-3,5C), 24.1
2 2
+
(
pip-4C); ESI-HRMS m/z: calcd for C H BrNO (MH ), 546.1644, found 546.1632. Anal. calc.: C,
31 32 3
68.13; H, 5.90; N, 2.56. Found: C, 68.06; H, 6.11; N, 2.48.
Human histamine H radioligand depletion assay
3
Radioligand depletion assay for human histamine H receptor were performed as described previously
3
[
46] with the following slight modifications. Briefly, HEK-293 cells stably expressing the human
histamine H receptor were washed and harvested in phosphate buffered saline (PBS) solution. They
3
were centrifuged (3 000 xg, 10 min, 4°C) and homogenized with an ULTRA-TURRAX® T 25 digital
(
IKA, Germany) in ice-cold binding buffer (12.5 mM MgCl , 100 mM NaCl and 75 mM Tris/HCl, pH
2
7.4). The cell membrane homogenate was centrifuged two times at 20 000 xg for 20 min (4°C). Crude
membranes, using 20 µg per well in a final volume of 0.2 ml binding buffer, were incubated with
3
-1
[
H]-N-α-methylhistamine (2 nM, 78.3 Ci mmol ) purchased from PerkinElmer (MA, USA) and various
concentrations of test compounds. Assays were performed at least in duplicates with at least seven
appropriate concentrations of test compounds. The incubation was performed for 90 min at room
temperature by continuous shaking using 10 µM Pitolisant for determination of non-specific binding.
Radioactivity was determined by liquid scintillation counting. Data were analyzed using GraphPad
PRISM 6 using implemented non-linear regression fit “one-site competition”, where K values were
i
calculated according to Cheng-Prusoff equation. Statistical analysis was performed on –log K values.
i
Mean values and confidence intervals (95%) were converted to micro- or nanomolar concentrations.
Monoamine oxidases inhibition assays
For assaying potential monoamine oxidase (MAO) A and B inhibition, compounds were included in
one-point screening for both isoforms predominantly using a continuous spectrophotometric method
as described previously [20] with the exception of 3d-f, where a discontinuous fluorimetric assay was
used (e.g. described in [47] with some modifications).
The spectrophotometric one-point measurements were performed in clear, flat-bottom 96 well plates
(
UV-Star®, No. 655801, greiner bio-one GmbH, Austria), measuring enzyme activity by
spectrophotometrical observation of 4-hydroxyquinoline (λmax=316 nm) formation over time as
described previously [20]. Initial velocities of substrate conversion (expressed as milli absorption units
per minute) were plotted against log inhibitor concentrations and fitted using the implemented non-
linear regression “log inhibitor vs. response (three parameters)”. For one-point measurements data
were calculated as percentage of control (product formation in absence of inhibitor) and expressed as
mean ± standard deviation (%) performing at least two independent experiments, each in duplicates.
The IC50 curves were determined in the discontinuous fluorimetric assay, allowing higher assay
sensitivity as well as time and cost savings. MAO inhibition assays were carried out using human
recombinant membrane-bound MAO A and MAO B purchased from Sigma-Aldrich (MO, USA).
Fluorimetric MAO assays were conducted in a total assay volume of 100 µL (max. 1% DMSO) using
black, flat-bottom 96 well plates (No. 655076, greiner bio-one GmbH, Austria), while pipetting was
partly automated using a EVO freedom pipetting robot (Tecan Trading AG, Switzerland). IC50 values
were obtained by measuring enzyme activity (determined as MAO-dependent product formation) with
inhibitor concentrations ranging from 0.001 µM to 100 µM in the presence of 2-fold K concentrations
M
of kynuramine (K = 30 µM for MAO A and K = 20 µM for MAO B). Reactions were started by addition
M
M
-1
-1
of MAO A (1.25 ng µL 900 units/mL) or MAO B (1.67 ng µL , 375 units/mL).
,
Shift of IC50 values were also measured after preincubating inhibitors with enzyme (30 min, 37°C),
while reactions were started by addition of substrate. For optimal enzyme activity conditions, reactions
were performed in pre-warmed potassium phosphate buffer (50 mM, pH = 7.4). After incubation (15 or
2
0 min, 37°C with and without preincubation IC50 setup, respectively) reactions were stopped by
manual addition of 35 µL sodium hydroxide (2 N) and enzyme activity was determined by detection of
-hydroxyquinoline (λ =320±20 nm, λEm=405±20 nm) using an infinite M1000 Pro microplate reader
4
Ex
(
Tecan Trading AG, Switzerland). Data were analysed using GraphPad PRISM 6. Enzyme activity,
expressed as relative fluorescence units (RFU), were plotted against log inhibitor concentrations and
fitted using the implemented non-linear regression “log inhibitor vs. response (three parameters)”.
1
4

ACCEPTED MANUSCRIPT
Since few compounds do not reach the lower plateau in IC50 curves (e.g. poor soluble compounds 3e
and 3f), the bottom for non-linear regression was set to zero. Data were obtained from at least three
independent experiments, each performed at least in duplicates.
-1
Reversibility of inhibition was assayed by preincubation of MAO B (10 ng µL ) and inhibitors (10x IC )
50
for 0, 30 and 60 min at 37°C prior to 50x dilution in potassium phosphate buffer to give a final
concentration of 0.05xIC50 of inhibitor. Remained enzyme activity were measured fluorimetrically as
described above under substrate saturating (10xK ) conditions. Data were calculated as percentage
M
of control (without inhibitor for each time point) and expressed as mean ± standard deviation (%) of at
least two independent experiments, each performed in duplicates (global fit).
Michael acceptor capacity determinations
The Michael acceptor capacity of DTL 3d were evaluated by appearance of probably formed adducts
between 3d and acetylcysteine under physiological conditions (Supporting Information, Figure 37).
-1
Accordingly, an aqueous solution containing 3d (4 µg mL ) and an excess of acetylcysteine (20 mg
-1
mL ) was prepared. The pH was adjusted to 7.2 using diluted (0.01 M) sodium hydroxide solution.
Electron Spray Ionisation (ESI) mass spectrometry (MS) was used to observed possible adducts
formed at several time points up to three days after mixture. To allow protonation in ESI-Spectra, the
samples were diluted with the same amount of water containing 0.1% of formic acid. All the
measurements were performed in ESI-MS (+) mode.
Author contributions
AA performed organic synthesis and analysis. SH performed human histamine H R radioligand
3
depletion studies and MAO enzyme inhibition studies. AZ, AA and SH performed Michael acceptor
capacity studies. AA and SH wrote the main manuscript. HS contributed to compound and
experimental design and supervised the project. JMC contributed to experimental design. All authors
approved and reviewed the manuscript.
Conflict of interest
The authors do not declare any conflict of interest.
Acknowledgements
This project was kindly supported by EU COST actions CM1103 and CA15135 as well as DFG INST
208/664–1 FUGG (Germany), and by SAF2012-33304 (MINECO, Spain).
References
[
[
[
1]
2]
3]
A. Anighoro, J. Bajorath, G. Rastelli, Polypharmacology: Challenges and opportunities in drug
discovery., J. Med. Chem. 57 (2014) 7874–7887.
M.A. Khanfar, A. Affini, K. Lutsenko, K. Nikolic, S. Butini, H. Stark, Multiple Targeting
Approaches on Histamine H3 Receptor Antagonists, Front. Neurosci. 10 (2016) 1–17.
S. Butini, K. Nikolic, S. Kassel, H. Brückmann, S. Filipic, D. Agbaba, S. Gemma, S. Brogi, M.
Brindisi, G. Campiani, H. Stark, Polypharmacology of dopamine receptor ligands, Prog.
Neurobiol. 142 (2016) 68–103.
[
[
[
[
4]
5]
6]
7]
R. Leon, A.G. Garcia, J. Marco-Contelles, Recent Advances in the Multitarget-Directed Ligands
Approach for the Treatment of Alzheimer’s Disease, Med. Res. Rev. 33 (2011) 139–189.
R.R. Ramsay, Monoamine oxidases: The biochemistry of the proteins as targets in medicinal
chemistry and drug discovery, Curr. Top. Med. Chem. 12 (2012) 2189–2209.
R.R. Ramsay, Molecular aspects of monoamine oxidase B, Prog. Neuropsychopharmacol. Biol.
Psychiatry. 69 (2016) 81–89.
X. Wang, C. Han, Y. Xu, K. Wu, S. Chen, M. Hu, L. Wang, Y. Ye, Synthesis and Evaluation of
Phenylxanthine Derivatives as Potential Dual A2AR Antagonists/MAO-B Inhibitors for
Parkinson’s Disease, Molecules. 22 (2017) 1010.
1
5

ACCEPTED MANUSCRIPT
[
[
[
[
8]
M. Berlin, C.W. Boyce, M. de Lera Ruiz, Histamine H3 Receptor as a Drug Discovery Target, J.
Med. Chem. 54 (2011) 26–53.
9]
K. Sander, T. Kottke, H. Stark, Histamine H3 receptor antagonists go to clinics., Biol. Pharm.
Bull. 31 (2008) 2163–2181.
10]
11]
J.C. Schwartz, The histamine H3 receptor: From discovery to clinical trials with pitolisant, Br. J.
Pharmacol. 163 (2011) 713–721.
G. Nieto-Alamilla, R. Márquez-Gómez, A.-M. García-Gálvez, G.-E. Morales-Figueroa, J.-A.
Arias-Montaño, The histamine H3 receptor: structure, pharmacology and function, Mol.
Pharmacol. 3964 (2016) 649–673.
[
[
12]
13]
W. Hu, Z. Chen, The roles of histamine and its receptor ligands in central nervous system
disorders: An update, Pharmacol. Ther. 175 (2017) 116–132.
C. Ferrada, S. Ferré, V. Casadó, A. Cortés, Z. Justinova, C. Barnes, E.I. Canela, S.R.
Goldberg, R. Leurs, C. Lluis, R. Franco, Interactions between histamine H3 and dopamine D2
receptors and the implications for striatal function, Neuropharmacology. 55 (2008) 190–197.
[
[
14]
15]
D. Masini, C. Lopes-Aguiar, A. Bonito-Oliva, D. Papadia, R. Andersson, A. Fisahn, G. Fisone,
The histamine H3 receptor antagonist thioperamide rescues circadian rhythm and memory
function in experimental parkinsonism, Transl. Psychiatry. 7 (2017) e1088.
R. Parmentier, C. Anaclet, C. Guhennec, E. Brousseau, D. Bricout, T. Giboulot, D. Bozyczko-
Coyne, K. Spiegel, H. Ohtsu, M. Williams, J.S. Lin, The brain H3-receptor as a novel
therapeutic target for vigilance and sleep-wake disorders., Biochem. Pharmacol. 73 (2007)
1157–1171.
[
[
[
[
[
[
16]
17]
18]
19]
20]
21]
J. Lin, O.A. Sergeeva, H.L. Haas, Histamine H3 Receptors and Sleep-Wake Regulation, J.
Pharmacol. Exp. Ther. 336 (2011) 17–23.
S. Carradori, R. Silvestri, New frontiers in selective human MAO-B inhibitors, J. Med. Chem. 58
(
2015) 6717–6732.
S. Mostert, A. Petzer, J.P. Petzer, Indanones As high-potency reversible inhibitors of
monoamine oxidase, ChemMedChem. 10 (2015) 862–873.
M.S. Nel, A. Petzer, J. Petzer, L.J. Legobabe, 2-Benzylidene-1-indanone derivatives as
inhibitors of monoamine oxidase, Bioorg. Med. Chem. Lett. 26 (2016) 4599–4605.
S. Hagenow, A. Stasiak, R.R. Ramsay, H. Stark, Ciproxifan, a histamine H3 receptor
antagonist, reversibly inhibits monoamine oxidase A and B, Sci. Rep. 7 (2017) 40541.
A. Olejarzk, U. Cichon, D. Lazewska, T. Karcz, H. Stark, K. Kiec-Kononowicz, Monoamine
oxidase B inhibition activity of novel analogs and derivatives of 1-[3-(4-tert-butyl-
phenoxy)propyl] piperidine, in: VII Meet. Paul Ehrlich Euro-PhD Network, B. Abstr., 2017: p. 26.
[
22]
G. Meier, J. Apelt, U. Reichert, S. Grassmann, X. Ligneau, S. Elz, F. Leurquin, C.R. Ganellin,
J.C. Schwartz, W. Schunack, H. Stark, Influence of imidazole replacement in different structural
classes of histamine H(3)-receptor antagonists, Eur. J. Pharm. Sci. 13 (2001) 249–259.
[
[
23]
24]
K. Wingen, H. Stark, Scaffold variations in amine warhead of histamine H3 receptor
antagonists, Drug Discov. Today Technol. 10 (2013) e483–e489.
T. Mikó, X. Ligneau, H.H. Pertz, J.-M. Arrang, C.R. Ganellin, J.-C. Schwartz, W. Schunack, H.
Stark, Structural variations of 1-(4-(phenoxymethyl)benzyl)piperidines as nonimidazole
histamine H3 receptor antagonists., Bioorg. Med. Chem. 12 (2004) 2727–36.
[
[
25]
26]
S.A. Weissman, D. Zewge, Recent advances in ether dealkylation, Tetrahedron. 61 (2005)
7833–7863.
M. Tomasch, J.S. Schwed, L. Weizel, H. Stark, Novel chalcone-based fluorescent human
histamine h(3) receptor ligands as pharmacological tools., Front. Syst. Neurosci. 6 (2012) 1–
16.
[
27]
H. Stark, Convenient procedures for synthesis of ciproxifan, a histamine H3-receptor
1
6

ACCEPTED MANUSCRIPT
antagonist, Arch. Pharm. Pharm. Med. Chem. 333 (2000) 315–316.
[
[
28]
29]
K. Sander, Y. Von Coburg, J.-C. Camelin, X. Ligneau, O. Rau, M. Schubert-Zsilavecz, J.
Schwartz, H. Stark, Acidic elements in histamine H3 receptor antagonists, Bioorg. Med. Chem.
Lett. 20 (2010) 1581–1584.
B. Sadek, J.S. Schwed, D. Subramanian, L. Weizel, M. Walter, A. Adem, H. Stark, Non-
imidazole histamine H3 receptor ligands incorporating antiepileptic moieties., Eur. J. Med.
Chem. 77 (2014) 269–279.
[
[
30]
31]
A. Williamson, About the theory of the formation of ethers, Ann. Chem. 77 (1851) 37–49.
N. Morales-Camilo, C.O. Salas, C. Sanhueza, C. Espinosa-Bustos, S. Sepúlveda-Boza, M.
Reyes-Parada, F. Gonzalez-Nilo, M. Caroli-Rezende, A. Fierro, Synthesis, biological
evaluation, and molecular simulation of chalcones and aurones as selective MAO-B inhibitors,
Chem. Biol. Drug Des. 85 (2015) 685–695.
[
[
32]
33]
L. Huang, H. Miao, Y. Sun, F. Meng, X. Li, Discovery of indanone derivatives as multi-target-
directed ligands against Alzheimer’s disease, Eur. J. Med. Chem. 87 (2014) 429–439.
A.L. Hopkins, G.M. Keserü, P.D. Leeson, D.C. Rees, C.H. Reynolds, The role of ligand
efficiency metrics in drug discovery., Nat. Rev. Drug Discov. 13 (2014) 105–21.
doi:10.1038/nrd4163.
[
[
[
34]
35]
36]
N. Desideri, A. Bolasco, R. Fioravanti, L. Proietti Monaco, F. Orallo, M. Yáñez, F. Ortuso, S.
Alcaro, Homoisoflavonoids: Natural scaffolds with potent and selective monoamine oxidase-B
inhibition properties, J. Med. Chem. 54 (2011) 2155–2164.
N. Desideri, L. Proietti Monaco, R. Fioravanti, M. Biava, M. Yanez, S. Alcaro, F. Ortuso, (E)-3-
Heteroarylidenechroman-4-ones as potent and selective monoamine oxidase-B inhibitors, Eur.
J. Med. Chem. 117 (2016) 292–300.
J.P.M. Finberg, Update on the pharmacology of selective inhibitors of MAO-A and MAO-B:
focus on modulation of CNS monoamine neurotransmitter release., Pharmacol. Ther. 143
(
2014) 133–152.
[
[
37]
38]
D.S. Johnson, E. Weerapana, B.F. Cravatt, Strategies for discovering and derisking covalent,
irreversible enzyme inhibitors, Future Med. Chem. 2 (2010) 949–964.
Ó.M. Bautista-Aguilera, S. Hagenow, A. Palomino-Antolin, V. Farré-Alins, L. Ismaili, P.-L.
Joffrin, M.L. Jimeno, O. Soukup, J. Janočková, L. Kalinowsky, E. Proschak, I. Iriepa, I.
Moraleda, J.S. Schwed, A. Romero Martínez, F. López-Muñoz, M. Chioua, J. Egea, R.R.
Ramsay, J. Marco-Contelles, H. Stark, Multitarget-Directed Ligands Combining Cholinesterase
and Monoamine Oxidase Inhibition with Histamine H3R Antagonism for Neurodegenerative
Diseases, Angew. Chem. Int. Ed. 56 (2017) in press. doi:10.1002/anie.201706072.
[
[
39]
40]
G. Petroianu, K. Arafat, B.C. Sasse, H. Stark, Multiple enzyme inhibitions by histamine H3
receptor antagonists as potential procognitive agents, Pharmazie. 61 (2006) 179–182.
L. Huang, C. Lu, Y. Sun, F. Mao, Z. Luo, T. Su, H. Jiang, W. Shan, X. Li, Multitarget-directed
benzylideneindanone derivatives: Anti-ß-amyloid (Aß) aggregation, antioxidant, metal
chelation, and monoamine oxidase B (MAO-B) inhibition properties against Alzheimer’s
disease, J. Med. Chem. 55 (2012) 8483–8492.
[
[
41]
42]
A. Autelitano, A. Minassi, A. Pagani, O. Taglialatela-Scafati, G. Appendino, The reaction of
cinnamaldehyde and cinnam(o)yl derivatives with thiols, Acta Pharm. Sin. B. 7 (2017) 523–526.
doi:10.1016/j.apsb.2017.06.005.
C. Avonto, O. Taglialatela-Scafati, F. Pollastro, A. Minassi, V. Di-Marzo, L. De-Petrocellis, G.
Appendino, An NMR spectroscopic method to identify and classify thiol-trapping agents:
Revival of michael acceptors for drug discovery?, Angew. Chem. - Int. Ed. 50 (2011) 467–471.
[
[
43]
44]
S. Searles, V.P. Gregory, The reaction of trimethylene oxide with amines, J. Am. Chem. Soc.
76 (1954) 2789–2790.
G. Doherty, T. Burnett, Synthesis of aminoalkylisothiuronium salts and their conversion to
1
7

ACCEPTED MANUSCRIPT
mercaptoalkylguanidines, J. Am. Chem. Soc. 3878 (1957) 5–9.
[
[
[
45]
46]
47]
L.W.L. Woo, N.M. Howarth, A. Purohit, H.A.M. Hejaz, M.J. Reed, B.V.L. Potter, Steroidal and
nonsteroidal sulfamates as potent inhibitors of steroid sulfatase, J. Med. Chem. 41 (1998)
1068–1083.
T. Kottke, K. Sander, L. Weizel, E.H. Schneider, R. Seifert, H. Stark, Receptor-specific
functional efficacies of alkyl imidazoles as dual histamine H3/H4 receptor ligands, Eur. J.
Pharmacol. 654 (2011) 200–208.
L. Meiring, J.P. Petzer, A. Petzer, Inhibition of monoamine oxidase by 3,4-dihydro-2(1H)-
quinolinone derivatives, Bioorg. Med. Chem. Lett. 23 (2013) 5498–5502.
1
8

ACCEPTED MANUSCRIPT
Highlights
1
2
3
4
. Promising inhibitory MAO B potency in nanomolar concentration range
. Promising histamine H receptor affinity in low nanomolar concentration range
3
. Dual-targeting ligands were designed for the treatment approach on Parkinson’s disease.
. Best compounds 3d, 3e and 3f with MAO B and H R activity were obtained by attaching a general
3
accepted H R pharmacophore to 2-benzylidene-1-indanone MAO motifs.
3
5. Different C5 and C6 substitution patterns of the indanone moiety strongly influence MAO B inhibition,
selectivity and time dependency of inhibition.