Investigating 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole as scaffold of butyrylcholinesterase-selective inhibitors with additional neuroprotective activities for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.05.062

Due to the role of butyrylcholinesterase (BChE)in acetylcholine hydrolysis in the late stages of the Alzheimer's disease (AD), inhibitors of butyrylcholinesterase (BChE)have been recently envisaged, besides acetylcholinesterase (AChE)inhibitors, as candidates for treating mild-to-moderate AD. Herein, synthesis and AChE/BChE inhibition activity of some twenty derivatives of 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (HHAI)is reported. Most of the newly synthesized HHAI derivatives achieved the inhibition of both ChE isoforms with IC₅₀s in the micromolar range, with a structure-dependent selectivity toward BChE. Apparently, molecular volume and lipophilicity do increase selectivity toward BChE, and indeed the N²-(4-phenylbutyl)HHAI derivative 15d, which behaves as a mixed-type inhibitor, resulted the most potent (IC₅₀ 0.17 μM)and selective (>100-fold)inhibitor toward either horse serum and human BChE. Moreover, 15d inhibited in vitro self-induced aggregation of neurotoxic amyloid-β (Aβ)peptide and displayed neuroprotective effects in neuroblastoma SH-SY5Y cell line, significantly recovering (P < 0.001)cell viability when impaired by Aβ_1-42 and hydrogen peroxide insults. Overall, this study highlighted HHAI as useful and versatile scaffold for developing new small molecules targeting some enzymes and biochemical pathways involved in the pathogenesis of AD.

Full text of DOI:10.1016/j.ejmech.2019.05.062

Accepted Manuscript
Investigating 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole as scaffold of
butyrylcholinesterase-selective inhibitors with additional neuroprotective activities for
Alzheimer's disease
Rosa Purgatorio, Modesto de Candia, Marco Catto, Antonio Carrieri, Leonardo Pisani,
Annalisa De Palma, Maddalena Toma, Olga A. Ivanova, Leonid G. Voskressensky,
Cosimo D. Altomare
PII:
S0223-5234(19)30479-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.062
EJMECH 11372
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 March 2019
Revised Date: 13 May 2019
Accepted Date: 23 May 2019
Please cite this article as: R. Purgatorio, M. de Candia, M. Catto, A. Carrieri, L. Pisani, A. De
Palma, M. Toma, O.A. Ivanova, L.G. Voskressensky, C.D. Altomare, Investigating 1,2,3,4,5,6-
hexahydroazepino[4,3-b]indole as scaffold of butyrylcholinesterase-selective inhibitors with additional
neuroprotective activities for Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.05.062.
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ACCEPTED MANUSCRIPT
Investigating 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole as scaffold of butyrylcholinesterase-
selective inhibitors with additional neuroprotective activities for Alzheimer’s disease
Rosa Purgatorio,a Modesto de Candia,a* Marco Catto,a Antonio Carrieri,a Leonardo Pisani,a Annalisa De
Palma,b Maddalena Toma,a Olga A. Ivanova,c Leonid G. Voskressensky,d and Cosimo D. Altomarea
aDepartment of Pharmacy-Drug Sciences, University of Bari Aldo Moro, Via E. Orabona 4, 70125 Bari, Italy.
bDepartment of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Via E.
Orabona 4, 70125 Bari, Italy. cDepartment of Chemistry, M. V. Lomonosov Moscow State University, Leninskie
Gory 1-3, Moscow 119991, Russian Federation. dOrganic Chemistry Department, RUDN University, Miklukho-
Maklai St, 6, Moscow 117198, Russian Federation.
Graphical abstract
N
NH
15d
IC50 (hBChE) = 170 nM
IC50 (hAChE) = 20.0 µM
SH-SY5Y cells cytoprotection of 15d (5 µM) after treatment
with Aβ1-42(5 µM).

ACCEPTED MANUSCRIPT
Investigating 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole as scaffold of
butyrylcholinesterase-selective inhibitors with additional neuroprotective
activities for Alzheimer’s disease
Rosa Purgatorio,a Modesto de Candia,a* Marco Catto,a Antonio Carrieri,a Leonardo Pisani,a
Annalisa De Palma,b Maddalena Toma,a Olga A. Ivanova,c Leonid G. Voskressensky,d and Cosimo
D. Altomarea
aDepartment of Pharmacy-Drug Sciences, University of Bari Aldo Moro, Via E. Orabona 4, 70125
Bari, Italy. bDepartment of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari
Aldo Moro, Via E. Orabona 4, 70125 Bari, Italy. cDepartment of Chemistry, M. V. Lomonosov
Moscow State University, Leninskie Gory 1-3, Moscow 119991, Russian Federation. dOrganic
Chemistry Department, RUDN University, Miklukho-Maklai St, 6, Moscow 117198, Russian
Federation.
Author Contributions:
M.d.C., R.P., L.P., O.I.A. and L.G.V. contributed to chemistry and physicochemical data. M.d.C.
and R.P. performed enzymes’ inhibition measurements. M.C., A.D. and M.T. carried out cell assays
and analyzed the biological data analysis. A.C. performed the molecular modeling study. M.d.C.
designed the research project, interpreted the SARs and wrote the manuscript. C.D.A. supervised
the project and revised the manuscript. All the authors approved the final version of the manuscript.
*Corresponding
Author:
Phone
+39-080-5443573;
fax
+39-080-5442230;
e-mail
modesto.decandia@uniba.it
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ACCEPTED MANUSCRIPT
ABSTRACT
Due to the role of butyrylcholinesterase (BChE) in acetylcholine hydrolysis in the late stages of the
Alzheimer’s disease (AD), inhibitors of butyrylcholinesterase (BChE) have been recently
envisaged, besides acetylcholinesterase (AChE) inhibitors, as candidates for treating mild-to-
moderate AD. Herein, synthesis and AChE/BChE inhibition activity of some twenty derivatives of
1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (HHAI) is reported. Most of the newly synthesized
HHAI derivatives achieved the inhibition of both ChE isoforms with IC50s in the micromolar range,
with a structure-dependent selectivity toward BChE. Apparently, molecular volume and
lipophilicity do increase selectivity toward BChE, and indeed the N2-(4-phenylbutyl) HHAI
derivative 15d, which behaves as a mixed-type inhibitor, resulted the most potent (IC50 0.17 µM)
and selective (> 100-fold) inhibitor toward either horse serum and human BChE. Moreover, 15d
inhibited in vitro self-induced aggregation of neurotoxic amyloid-β (Aβ) peptide and displayed
neuroprotective effects in neuroblastoma SH-SY5Y cell line, significantly recovering (P < 0.001)
cell viability when impaired by Aβ1-42 and hydrogen peroxide insults. Overall, this study
highlighted HHAI as useful and versatile scaffold for developing new small molecules targeting
some enzymes and biochemical pathways involved in the pathogenesis of AD.
2

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Keywords:
Alzheimer’s disease
Azepino[4,3-b]indole
Acetylcholinesterase
Butyrylcholinesterase
Amyloid-β aggregation
Neuroprotection
Abbreviations
AD, Alzheimer Disease; ACh, Acetylcholine; AChE, Acetylcholinesterase; BChE,
Butyrylcholinesterase; CAS, Catalytic anionic site; CNS, Central Nervous System; DCM,
dichloromethane; PAS, Peripheral anionic site; PB, Phosphate Buffer; PBS, Phosphate Buffered
Saline; SAR, Structure-activity relationship; SH-SY5Y, human neuroblastoma cell line; TBAB,
Tetrabutyl ammonium bromide.
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1. Introduction
Alzheimer’s disease (AD) is the prevailing neurodegenerative and devastating disorder, which
accounts for most elderly-related dementias. As summarized in World Alzheimer Report 2018 [1],
people affected by AD has been estimated to increase to more than 131 million up to 2050. The
histopathological hallmarks of AD are deposition of extracellular neurotoxic amyloid-β (Aβ)
peptide aggregates that, with intracellular neurofibrillary tangles of hyperphosphorylated τ-protein,
generates senile plaques, by triggering oxidative stress, perturbation of cellular metabolism, and
finally synaptic and neuronal loss. In addition, a lower level of the neurotransmitter acetylcholine
(ACh) into hippocampus, and progressively into the whole brain cortex, typically contributes to the
AD-related cognitive and memory impairment and decline [2,3].
Despite the efforts (and financial resources employed as well) aimed at identifying disease-
modifying molecules, only few drugs have been approved for the symptomatic treatment of mild-to-
moderate AD (Chart 1), which include the acetylcholinesterase (AChE) inhibitors rivastigmine (1),
galantamine (2), and donepezil (3), and the N-methyl-D-aspartate receptor (NMDAR) antagonist
memantine (4). Galantamine (2) and donepezil (3) are reversible ChE inhibitors, whereas
rivastigmine (1) is a pseudo-irreversible inhibitor that transfers a carbamate moiety to the catalytic
serine residue (followed by slow hydrolysis) in the active sites of both AChE and
butyrylcholinesterase (BChE). Regarding memantine (4), it has been established that AD patients
may further benefit from reduction of NMDAR glutamate-induced Ca2+-mediated excitotoxicity
[4].
ACh is mainly hydrolyzed by AChE in the synaptic cleft of the cholinergic neurons, but at higher
concentrations it could be also hydrolyzed by BChE, an α-glycoprotein produced in liver and
primarily distributed in plasma but also detected in central and peripheral nervous systems. Plasma
BChE may be likely responsible for detoxification of xenobiotics [5], whereas BChE expressed and
secreted in CNS glial cells and neurons likely acts therein as a co-regulator of cholinergic
neurotransmission [6].
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O
NH2
O
H
O
N
O
N
O
N
O
N
O
Memantine (4)
Rivastigmine (1)
Galantamine (2)
Donepezil (3)
Chart 1. Currently available drugs for the management of AD.
In healthy brain AChE is the main enzyme responsible for ACh regulation, whereas in brain areas
of AD patients, a decline of AChE level and a significant increase (30-60%) of BChE expression
and activity have been observed [7-9]. Overall, this evidence suggests a role of BChE in AD
progression, and then the importance of developing inhibitors targeting BChE, endowed with other
neuroprotective activities, as useful drugs for treating AD [10,11].
AChE and BChE share about 70% of structural homology but differ in their three-dimensional
structures. AChE contains a 20 Å deep and narrow gorge, and five regions are involved in the
ligand binding: (i) the catalytic triad residues (Ser203, His447, Glu334, human species numbering);
(ii) the ‘oxyanion hole’ inside the active center, that stabilizes the transient tetrahedral enzyme-
substrate complex; (iii) the ‘anionic site’ (AS), where Trp86 (conserved in both ChEs) is involved
in the orientation and stabilization of trimethylammonium head of ACh, through cation-π
interactions; (iv) the ‘acyl pocket’ interacting with the substrate acyl group; (v) the ‘peripheral
anionic site’ (PAS), located on the rim of the active site gorge [12]. The main differences between
AChE and BChE occur in the ‘acyl pocket’ and PAS, where two Phe residues (Phe295, Phe297) in
the AChE ‘acyl pocket’, which prevent the access of bulkier molecules to the catalytic site, are
replaced by two aliphatic residues (Leu286, Val288) in BChE. Furthermore, six out of the fourteen
aromatic residues lining the AChE gorge rim and PAS are replaced by aliphatic residues in BChE.
Consequently, the BChE cavity is about 200 Å3 larger than the AChE gorge [13-15].
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Besides their catalytic function, both enzymes exhibit several noncholinergic (nonenzymatic)
functions, related in AD physiopathology. Indeed, both enzymes proved to be involved in processes
leading to formation and deposition of Aβ fibrils [16-18]. The peripheral site of AChE interacts
with Aβ protein, by enhancing its deposition and aggregation. In contrast, limited knowledge has
been reported on mechanism linking amyloidogenesis and BChE, even if a putative role has been
associated to plaques formation and maturation. Indeed, BChE was found to be colocalized with Aβ
into the senile plaques [7], and selective BChE inhibitors, such as N1-phenetylnorcymserine (5,
Chart 2) [6,19-21], proved to reduce fibrils deposition in some cerebral areas (amygdala,
hippocampal structures, thalamus and basal ganglia), and ameliorate cognitive dysfunction induced
by Aβ40 peptide in animal models of AD, likely through a BChE involvement in glia-mediated
neuroinflammation associated to AD [22,23].
H
O
N
H
N
O
N
O
N
N
H
6
PEC, 5
Chart 2. Structures of BChE-selective inhibitors.
Selective BChE inhibitors usually contain polycyclic structures [24-27], such as compound 5,
ethopropazine [28,29], quinazolinimine-based derivatives [30,31], carbazole and indolpiperidines
[32,33]. Previously, some of us reported efficient syntheses of partially hydrogenated pyrrole- and
indole-fused azaheterocycles with six-to-eight-membered ring size [34-36], as scaffolds of novel
bioactive compounds, such as antiplatelet [37,38], antimicrobial [39], antioxidant agents [40,41]
and AChE inhibitors [42-44]. More recently, some N6-substituted partially hydrogenated
azepino[4,3-b]indole derivatives showed selective inhibition of BChE [45]. Azepino-indoles could
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be considered homologues of carbazoles and carbolines, exploited as binder moieties in a number of
dual-site AChE inhibitors, and multitarget-directed ligands (MTDL) addressing the pathogenic
pathways underlying AD. In particular, the 2,3,4,5-tetrahydroazepino[4,3-b]indole(1H)-2-one
derivative 6 (Chart 2) exhibited nanomolar and selective BChE inhibition (IC50s vs AChE and
BChE 20 and 0.020 µM, respectively), and showed protective activity against NMDA-induced
excitotoxicity in neuronal cell line (SH-SY5Y), higher than the NMDAR antagonist memantine 4
[45]. Unfortunately, the lactam derivative 6 suffered from low aqueous solubility, whereas its
reduction to the more soluble amino derivative did decrease BChE inhibition potency and increase
toxicity toward neuronal cells. Herein, with the aim of better characterizing the azepino[4,3-
b]indole nucleus as a template for novel MTDLs for treating AD, about twenty N2-substituted
1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (HHAI) derivatives were synthesized and tested as
AChE/BChE inhibitors. The most active ChE inhibitors were then evaluated as Aβ-antiaggregating
agent and neuroprotectant against Aβ and oxidative stress insults in neuroblastoma cell line (SH-
SY5Y).
2. Results and discussion
2.1. Chemistry
Compounds 8a-c, 12a-b, and 13b-c were previously published or prepared using known procedures
[46-48], according to Scheme 1. The HHAI compounds 8a-c were prepared by reduction of
respective lactam derivatives 7a-c [45,47] with LiAlH4 in refluxing 1,4-dioxane. The azepine
intermediates 8a-c were treated with formaldehyde, and the N-formyl were catalytically reduced to
yield the N-methyl derivatives 12a-b [48]. Compounds 8a-c, reacted with suitable benzoyl- or
phenylalkanoyl chlorides, leading to compounds 9-11. A reduction reaction, followed by
crystallization of the HHAIs as hydrochloride salts, led to 13a-h, 15a and 15c-d in satisfactory
yields. For preparing the N6-methyl derivative 15b, the phenylacetyl amide 9e underwent reaction
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with MeI and TBAB in a biphasic mixture (1:1 v/v of DCM/25% NaOH) to yield 14a, which was in
turn reduced to 15b with LiAlH4.
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NH2
H
H
N
O
HN
O
N
a-d
Scheme 1.a
e
R1
R1
+
O
N
N
H
R1
H
7a (R1= H)
7b (R1= CH3)
7c (R1= F)
8a (R1= H)
8b (R1= CH3)
8c (R1= F)
f, or g
O
R1
R1
R1
R3
R2
( )n
N
h
e
N
N
.
HCl
N
N
N
H
H
H
12a (R1= H)
12b (R1= CH3)
9a-g (R1= H)
10a-b (R1= CH3)
11a-d (R1= F)
13a-h (n = 1; R1= H, CH3, F, CH2NRR)
15a,c-d (n = 2-4; R1= H)
9e
R1=H
R2=CH2(C6H5)
i
O
N
N
.
e
HCl
N
N
H3C
H3C
14a
15b
a Reagents and conditions: a) Water, r.t., overnight, 85%; b) TFA, reflux, 24 h, 65-70%; c) NH2OH×HCl, AcONa, EtOH/H2O 2/1 v/v, reflux, 24h, 85-90%; d) 110 °C-preheated PPA,
30 min, 70%; e) 1. LiAlH4, dry dioxane, reflux (50-75%); 2. HCl sat. MeOH solution; f) HCHO, EtOH/H2O; g) acyl halides, TEA, dry CH2Cl2, overnight; h) H2, PtO2, EtOH; i)
CH3I, TBAB, 25% NaOH/DCM 1/1 v/v, r.t., 48 h, 55%.
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2.2. Inhibition of cholinesterases
The in vitro inhibitory activity of the investigated compounds toward electric eel (ee) AChE and
horse serum (hs) BChE were determined by the Ellman colorimetric assay [49]. The half maximal
inhibitory concentration values (IC50) are summarized in Tables 1-3 (galantamine 2 and donepezil 3
used as the positive controls).
Table 1. Half maximal inhibitory concentration (IC50) values of eeAChE and hsBChE of HHAI
derivatives 8a-c and 12a,b.
IC50 (µM)a
Cmpd
R1
eeAChE
70.0 ± 5.0
45.0 ± 2.5
16.0 ± 1.4
7.1 ± 1.7
hsBChE
20 ± 3
H
CH3
F
8a
R1
NH
8b
8c
8.0 ± 2.1
4.3 ± 1.2
25.0 ± 2.0
N
H
R1
H
12a
N
N
H
CH3
25.0 ± 3.0
7.1 ± 0.3
12b
Galantamine, 2
Donepezil, 3
0.56 ± 0.10
12.0 ± 0.30
2.3 ± 0.12
0.021 ± 0.002
aSeven different concentrations were tested for determining IC50 values by regression of the sigmoid dose-response
curves through GraphPad Prism software (vers. 5.01); data are means ± SEM of at least three independent
measurements.
Compounds 8a-c and 12a-b (Table 1) proved to be inhibitors of both ChEs with a potency in the
micromolar range. No HHAI derivative resulted in vitro more potent than the standard references 2
and 3 toward AChE. Except for the N2-Me derivative 12a, all these compounds showed a
preference toward BChE, with inhibition potencies in the low micromolar range.
A net increase of ChEs’ inhibition activity was achieved by introducing benzyl groups on the
azepine nitrogen (Table 2). The effect of N2-benzyl groups, also bearing substituents exerting
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opposite electronic effects, were stronger on BChE than on AChE inhibition. Higher inhibitory
activity and ten-fold BChE selectivity ratio was attained by 13d (R3 = 4’-CH3) and 13e (R3 = 4’-F).
Table 2. IC50 values of eeAChE and hsBChE of N2-benzyl HHAI derivatives 13a-h.
IC50 (µM)b
Cmpd
R1
R3
eeAChE
hsBChE
H
CH3
F
H
13a
13b
13c
13d
13e
13f
8.70 ± 0.40 2.00 ± 0.30
6.95 ± 1.30 1.20 ± 1.30
4.96 ± 1.20 2.13 ± 0.20
H
H
R1
R3
H
4’-CH3
4’-F
10.0 ± 2.0
12.0 ± 2.1
0.95 ± 0.10
1.20 ± 0.20
N
N
H
H
F
4’-F
4.10 ± 0.70 2.90 ± 1.00
4.80 ± 1.60 1.40 ± 0.60
7.90 ± 1.10 4.30 ± 1.20
F
4’-CH2NEt2
4’-CH2N(CH2)4
13g
13h
F
aSee footnote in Table 1; galantamine (2) and donepezil (3) as positive controls.
In contrast with the 9-methyl derivative (13b), the insertion of fluorine on C-9 (13c, BChE IC50
2.13 µM) did not improve BChE inhibition potency, whereas diminished the selectivity ratio (13c
vs 13a). Among the 9-F congeners, the replacement of 4’-F (13f) with the basic 4’-N,N’-
diethylamino-methyl group (13g) did slightly increase the BChE (and not AChE) inhibition
potency. This might be due to similar withdrawing effect exerted by fluorine and N,N’-
diethylamino-methyl group protonated at physiological pH, even if the volume and lipophilicity of
alkylamino group seems to play a role, as accounted for the lower activity of the pyrrolidine-methyl
congener 13h.
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A SAR comparison (Table 3) of 13c with the N-benzoyl analog (11a) and the previously reported
[3,4-b] fusion isomer 16 [35] suggests that azepine basic nitrogen and [4,3-b] fusion should be
preferred features for ChEs’ inhibition.
Table 3. Cholinesterases’ inhibition IC50s for SAR comparison of 13c with 11a and 16.
O
N
F
F
F
N
IC50 (µM)
N
N
N
N
H
H
H
11a
13c
16
eeAChE
hsBChE
10.6 ± 1.20
6.00 ± 1.20
4.96 ± 1.20
2.13 ± 0.20
10.7 ± 1.10
3.40 ± 1.40
The effect on ChEs’ inhibition potency of the length of alkyl chain in N2-phenylalkyl HHAI
derivatives, as expressed as the number of CH2 units (n), was finally explored (Table 4).
Table 4. Half maximal inhibitory concentration (IC50) values of eeAChE and hsBChE by N2-
phenylalkyl HHAI derivatives 13a and 15a-d (R1 = R3 = H).
IC50 (µM)a
Cmpd
n
eeAChE
8.7 ± 0.4
hsBChE
2.00 ± 0.3
0.70 ± 0.2
0.86 ± 0.10
0.28 ± 0.05
0.17 ± 0.02
1
2
2
3
4
13a
15a
15bb
15c
9.00 ± 1.5
12.0 ± 1.9
20.0 ± 2.1
20.0 ± 1.8
( )n
N
N
H
15d
aSee footnote in Table 1; galantamine (2) and donepezil (3) as positive controls. bN6-methyl derivative.
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The AChE inhibition activity remained unchanged (n = 1, 2) or decreased (n = 3, 4), whereas BChE
inhibition enhanced by increasing the alkyl chain length till 3-4 CH2 units. The methylation of the
indole nitrogen in 15b did not significantly affect ChE inhibition (IC50s close to those of 15a). Fig.
1 shows the effect of the linker elongation on the BChE/AChE selectivity ratio.
2.5
2.0
1.5
1.0
0.5
0.0
0
1
2
3
4
n
Figure 1. BChE selectivity ratios of N2-phenylalkyl HHAI derivatives, expressed in log units, as a
function of the number of CH2 units (n) in the alkyl chain.
The mechanism of the BChE inhibition of the most potent hsBChE inhibitor 15d was studied. The
Lineweaver-Burk curves were outlined using a fixed amount of BChE and varying concentrations
of the substrates between 25 and 300 µM, in the absence or presence of inhibitor at different
concentrations (0.1-0.5 µM). Binding of 15d to BChE changed both Vmax and Km values, a trend
that is generally ascribed to mixed-type inhibition (Fig. 2). A replot of the slopes versus the
corresponding inhibitor concentrations provided a Ki value of 0.098 µM.
The inhibitory activity of 15d was also evaluated against human (hu) ChEs, which share > 80%
homology with eeAChE and hsBChE (Table 4), and any noteworthy species-dependent ChEs'
inhibition activity was observed.
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3 1002
×
2 1002
×
6
4
ò no inhibitor
¢ 1.0×10-4 M
Å 2.5×10-4 M
© 5.0×10-4 M
1 1002
×
-5.0 10-7
0
5.0 10-7
×
×
[I], M
2
0
0.000
0.005
0.010
0.015
0.020
0.025
1/[S] µM
Figure 2. Inhibition kinetics of 15d using Lineweaver–Burk plots (r2 ≥ 0.975) with hsBChE (0.18
U/mL) and different substrate (butyrylthiocholine iodide) concentrations (50-300 µM); replot (r2 ≥
0.978) of the slopes versus [I] to determine Ki (0.098 µM) as the x-axis intercept is shown in the
upper left insert.
Table 4. IC50 values of the most potent derivative 15d against human AChE and BChE.
IC50 (µM)
huAChE
huBChE
0.199 ± 0.01
4.80 ± 0.50
20.0
15d
Donepezil 0.016 ± 0.002
2.3 Molecular modeling
The binding mode of compound 15d to huBChE was investigated by molecular docking simulation.
As shown in Fig. 3, the whole molecular bundle is merged into the deeper gorge of the BChE active
site, with a largely extended ligand-enzyme contact surface (717.52 Å2). The HHAI moiety is
oriented towards the top of the choline-binding site via a parallel π-stacking engaging the indole
moiety of the ligand and the aromatic side chain of Trp82, whereas the pending 4-phenylbutyl
group is laying on the bottom of the acyl-binding pocket through an edge-to-face π-stacking with
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Trp231 side chain. The lack or greater weakness of the aromatic interaction of the N2-benzyl
derivative 13a may explain, at least in part, its lower inhibitory potency with respect to 15d toward
huBChE, as also supported by Free Energy Perturbation (FEP) calculation (see Supp. Info). In
addition, two hydrogen bonds further stabilize the ligand-enzyme complex; the first between the
positively charged N-2 of 15d (with assistance of a water molecule) and the Oγ of Thr120, and the
second one between the indole NH and C=O of His438 in the backbone. It is worth noting that the
highest-scored docking pose of 15d resembles, regarding the location HHAI moiety, that observed
in the X-ray complex of tacrine with BChE (pdb code: 4BDS; Supp. Info.).
Figure 3. Binding mode of 15d into the human BChE binding site (the free energy of binding
calculated with hydration force field of AutoDock is -8.08 kcal/mol, while the contact surface area
measures 717.52 Å2).
The same docking calculation protocol was applied to simulate the binding mode of 15d to
huAChE. The observed selectivity was substantially justified by docking results (see Supp. Info.),
which showed lower stabilizing interactions with Trp286 in huAChE.
15

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2.5 Additional anti-AD properties
2.5.1. Aβ1-40 aggregation assay
In current approaches, small molecules able to inhibit ChEs and Aβ aggregation/neurotoxicity
simultaneously may have therapeutic potential as anti-AD agents [50-52]. Herein, the most potent
BChE-selective inhibitors (Table 4) were assayed for their ability to inhibit self-induced
aggregation of Aβ1-40 through a test measuring thioflavin T (ThT) fluorescence [53]. Quercetin, as
strong in vitro inhibitor of Aβ aggregation, was used as positive control. All the tested compounds,
significantly less active than quercetin, behaved as moderate inhibitors of Aβ aggregation, with %
inhibition at 100 µM concentration between 38 and 54%, without apparent dependence upon the
length N2-phenylalkyl group (Fig. 4). Several aza-heterocyclic derivatives endowed with in vitro
antiaggregating activity were reported in literature, and the anti-Aβ pharmacophore consists in a
planar heterocyclic scaffold (that is the intercalating moiety for β-sheet disruption) suitably
decorated with small polar and H-bonding groups (OH, OMe, etc.) [53]. Most likely, compared to
quercetin and heterocyclic analogs, the lower activity of the HHAI derivatives may be mainly due
to the lack of OH groups in suitable positions.
100
80
60
***
***
***
***
***
40
20
0
Qc
13a
15a
15b
15c
15d
Figure 4. Percent inhibition of Aβ40 aggregation (mean ± SD) of N-phenylalkyl-HHAI derivatives
at 100 µM concentration; quercetin (Qc, 100 µM) used as positive control. Significant difference
from positive control (ANOVA): *** P < 0.001
16

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2.5.2. Neuroprotective activity against Aβ1-42 and ROS
The in vitro neuroprotection in SH-SY5Y cell line against Aβ42-induced cytotoxicity for 15d was
measured through an MTT assay on a cell model, without considering the molecular events
triggering neurotoxicity [53, 54]. As shown in Fig 5, the aggregates formed by 5 µM Aβ in cultured
SH-SY5Y cells produced around 50% of cell death in two days. According to the observed anti-Aβ
aggregation activity, cells co-incubated with equimolar (5 µM) Aβ42 and 15d exhibited a
significantly reduced decline of cell viability, after 24 h (35% viability recovery) and 48 h (30%
viability recovery) as hallmark of neuroprotection. Compound 15d, tested alone at the same
concentration, showed own cytotoxicity (ca. 30% decrease in cell viability) only after 48 h, while
maintaining a significant protecting effect against Aβ-induced toxicity, which resulted slightly
lower than that observed at 24 h. The observed time-dependent effect may be ascribed to physical
and/or chemical (metabolic) stability of 15d.
Figure 5. Cytoprotection in SH-SY5Y cells of compound 15d (5 µM) as assessed by MTT cell
viability assay, after treatment with Aβ1-42 (5 µM). Values are expressed as mean ± S.E.M. of six
replicates; significantly different from untreated cells and Aβ1-42 (5 µM) alone, as estimated by
ANOVA: #P < 0.05 vs untreated cells; ***P < 0.001 vs Aβ1-42 (5 µM) alone.
17

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Cytoprotective effect against oxidative stress cell damage was also investigated in H2O2-induced
oxidation SH-SY5Y cell model (Fig. 6). Reactive oxygen species (ROS) production was detected
by means of a spectrofluorometric measure of the fluorescent probe 2′,7′-dichlorofluorescein
(DCFH) [53]. Quercetin, a well-known natural antioxidant, was used as reference compound.
H2O2
(100 µM)
1400
1200
1000
800
600
400
200
0
*
**
**
**
**
**
**
H2O2
100
-
+
1
5
10
20
40
60
100
+quercetin
quercetin
(100 µM)
H2O2 (100 µM)+15d (µM)
Figure 6. Radical scavenging activity of increasing concentrations of 15d on SH-SY5Y cell line,
after H2O2 (100 µM) induced cell damage (DCFH-DA) assay; (-) untreated cells; (+) H2O2-treated
cells. Values are expressed as mean ± S.E.M. from six replicates; significantly different from H2O2-
treated cells: *P < 0.01, **P < 0.0001.
A dose-dependent ROS-scavenging effect of 15d was observed after coincubation of the test
compound at different concentrations (1-100 µM) with 100 µM H2O2. Interestingly, the protection
activity of 15d from H2O2-induced oxidative stress was statistically significant at concentrations
higher than 5 µM, and comparable to that of 100 µM quercetin at even lower concentrations (40
µM). The calculated IC50 was equal to 10.1 ± 1.2 µM. The data suggested an antioxidant activity of
15d, an effect which should not be surprising, considering that tertiary amines, like 15d, react in
aqueous solvents with peroxides leading to formation of quaternary N-oxides [55], including some
bioreductive drugs [56].
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The aqueous solubility of compound 15d (as well as 15a and 15c), experimentally measured
[57,58], was found higher than 1 mM. The same compounds were predicted by the ‘admetSAR’
software [59] as potentially able to cross blood brain barrier (BBB) [60,61], non-hepatotoxic and
well absorbed through human intestine [62]. Physicochemical descriptors and ADMET parameters
are reported in Supporting Information.
3. Conclusions
About twenty derivatives of 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (HHAI) were evaluated in
vitro as inhibitors of ChEs and amyloid Aβ aggregation. A structure-activity relationship study,
mostly exploring substitutions at the azepine nitrogen with groups having different size and
lipophilicity, led to find out N2-phenylalkyl HHAI derivatives (15) which achieved inhibition of
hsBChE at submicromolar concentrations and ten-to-hundred selectivity over eeAChE. The most
potent N2-phenylbutyl compound 15d behaved as a mixed-type inhibitor and showed in vitro
inhibitory activity (IC50 ca. 0.2 µM) and selectivity (> 100-fold) also towards human BChE.
Molecular docking calculation of 15d into the binding site of BChE suggested π-stacking
interactions (involving Trp82 and Trp231) and two hydrogen bonds (with Thr120 side chain and
His438 carbonyl in the backbone) as important for the good inhibitory activity. Finally, 15d
exhibited significant (P < 0.001) protection activity on neuroblastoma SH-SY5Y cell line against
cytotoxicity induced by Aβ42 peptide and oxidative stress (hydrogen peroxide). Taking these
properties into account, the HHAI moiety can be considered a useful template for developing new
molecules inhibiting BChE, which is a promising drug target in advanced AD. Compound 15d,
showing additional anti-Aβ and anti-oxidative stress in neuronal cell culture, can be a candidate for
advancing in molecular optimization and in vivo pharmacological studies aimed at assessing its
potential as MTDL in the management of multifactorial AD-related neurodegeneration.
4. Experimental methods
19

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Starting materials and all chemicals and solvents were purchased from Sigma-Aldrich and Alfa
Aesar. Melting points were determined by using the capillary method on a Stuart Scientific SMP3
electrothermal apparatus and are uncorrected. Final compound purities were assessed by elemental
analyses (C, H, N), performed on Euro EA3000 analyzer (Eurovector, Milan, Italy) by the
Analytical Laboratory Service of the Department of Pharmacy-Drug Sciences of the University of
Bari (Italy), and the results agreed to within ±0.40% of theoretical values. Mass spectra were
obtained by Agilent 1100 Series LC-MSD Trap System VL, equipped with ESI (electrospray
ionization) source (Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Milan, Italy). The
high-resolution molecular masses of test compounds were assessed by Agilent 6530 Accurate Mass
Q-TOF (Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Milan, Italy). IR spectra (KBr
disks) were recorded on a Perkin-Elmer Spectrum One Fourier transform infrared
spectrophotometer (Perkin-Elmer Ltd., Buckinghamshire, U.K.), and the most significant
1
absorption bands are listed. H NMR spectra, Unless otherwise stated, were recorded at 300 MHz
on a Varian Mercury 300 instrument. Chemical shifts are expressed in δ and the coupling constants
J are in hertz (Hz); the following abbreviations are used: s, singlet; d, doublet; dd, doublet-doublet;
t, triplet; m, multiplet. Signals due to NH and OH protons were located by deuterium exchange with
D2O. Chromatographic separations were performed on silica gel 60 for column chromatography
(Merck 70-230 mesh, or alternatively 15-40 mesh for flash chromatography).
Tetrahydro- (7a-c), and hexahydroazepinoindoles (8a-c), and compounds 10a-b, 11a-d, 13a-c, and
16 were synthesized according to procedures reported by us or described elsewhere
[35,45,46,47,48]. Analytical and spectral data of newly synthesized and tested compounds (13d-h,
and 15a-d), and their intermediates, are described below, and in Supporting Information.
4.1 General procedure A: preparation of 1,2,3,4,5,6-hexahydroazepino[4,3-b]indoles (8a-c)
4.1.1 Synthesis of 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (8a),
20

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The preparation of 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (8a), previously synthesized,[46,47]
has been reported as a representative example. To a stirred solution of 3,4,5,6-
tetrahydroazepino[4,3-b]indol-1(2H)-one 7a[45] (300 mg, 1.5 mmol), preheated and refluxed in 60
mL of dry 1,4-dioxane up to complete solubilization, was added portionwise lithium aluminium
hydride (625 mg, 16.5 mmol), and mixture was refluxed under nitrogen atmosphere, until
disappearance of starting material was observed (TLC, about 20 hours). After cooling, mixture was
quenched by adding 20 mL of Na2SO4 saturated aqueous solution, and stirred 30 minutes at room
temperature. Residue was filtered off and washed with 1,4-dioxane. The filtrates were diluted with
100 mL of water, and the organic/aqueous mixture was extracted with chloroform (3 × 50 mL).
Collected organic phases were washed twice with 20 mL of brine, dried (anhydrous Na2SO4),
filtered and concentrated under reduced pressure, to obtain compound 8a, as a brown solid, which
was further used without purification. Spectral data were in agreement with those of literature.
1
Yield: 90% (GC). IR (KBr) ν: 3416, 3276, 1621, 1448 cm-1; H NMR (300 MHz, DMSO-d6) δ:
10.74 (br, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 6.97 (t, J = 8.0 Hz, 1H), 6.92 (t, J
= 8.0 Hz, 1H), 3.88 (s, 2H), 3.06 (dd, J1 = 6 Hz, J2 = 7.5 Hz, 2H), 3.03 (br, 1H), 2.90 (dd, J1 =
6Hz, J2 = 7 Hz, 2H), 1.76 (m, 2H).
9-fluoro-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole
(8b)
and
9-methyl-1,2,3,4,5,6-
hexahydroazepino[4,3-b]indole (8c) were prepared by following the same synthetic conditions;
spectral data were in agreement with those of literature.[36,46]
4.2 General procedure B: preparation of 2-(phenacyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole
(9-10-11)
4.2.1. Synthesis of 2-(1-phenyl-2-(3,4,5,6-tetrahydroazepino[4,3-b]indol-2(1H)-yl)ethanone (9e)
The preparation of 2-(1-phenyl-2-(3,4,5,6-tetrahydroazepino[4,3-b]indol-2(1H)-yl)ethanone (9e),
has been reported as a representative example.
21

ACCEPTED MANUSCRIPT
To a solution of phenylacetic acid (570 mg, 4.17 mmol), in 10 mL of fresh distilled dry THF was
added thionyl chloride (SOCl2, 0.30 mL, 4.30 mmol). The mixture was refluxed 3 hours, and after
cooling dried under N2 stream. The crude residue was dissolved in 10 mL of fresh distilled dry
methylene chloride (DCM) and added dropwise to an ice bath cooled solution of compound 8a (518
mg, 2.78 mmol), and TEA (0.80 mL, 5.73 mmol). Mixture was stirred overnight at room
temperature, and then diluted with 20 mL of DCM. The organic phase was washed with saturated
aqueous Na2CO3 (3×15 mL), 1N diluted HCl (3×15 mL), and brine (3×15 mL), then dried
(anhydrous Na2SO4), filtered and concentrated under reduced pressure. The residue oil was purified
by chromatography on silica gel (ethyl acetate/n-hexane 70/30 v/v) to afford compound 9e. Yield:
37% (315 mg), pale brown oil; IR (KBr) ν: 3278, 2923, 1614, 1232, 851, 742 cm-1; 1H-NMR (300
MHz, CDCl3) δ: 7.93 (s, 1H), 7.46 (dd, J1 = 3.3 Hz, J2 = 6 Hz, 1H), 7.38 - 7.21 (m, 6H), 7.17 - 7.12
(m, 2H), 4.62 (s, 2H), 3.90 (t, J = 5.8 Hz, 2H), 3.70 (s, 2H), 2.94 (t, J = 6.0 Hz, 2H), 2.10 - 2.00 (m,
2H).
4.3 Synthesis of 6-methyl-2-(3-phenylpropanoyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (14a)
TBAB (276 mg, 0.85 mmol), methyl iodide (0.20 mL, 2.85 mmol) and 5.0 mL of 50% m/v NaOH
were added to a solution of 9e (175 mg, 0.57 mmol) in 5 mL of dry DCM. The mixture was stirred
overnight at room temperature, and then diluted with 20 mL of DCM and 20 mL of water. The
collected organic phase was washed twice with 20 mL of brine, dried (Na2SO4), filtered and
concentrated under reduced pressure. The residue was purified by chromatography on silica gel
(ethyl acetate/n-hexane 80/20 v/v, as eluent), to afford compound 14a. Yield: 79% (150 mg), pale
yellow oil; IR (KBr) ν: 3029, 2938, 1635, 1471, 910, 734 cm-1; 1H-NMR (300 MHz, CDCl3) δ: 7.48
(d, J = 6.8 Hz, 1H), 7.35 - 7.5 (m, 8H), 4.62 (s, 2H), 3.94 (t, J = 5.8 Hz, 2H), 3.67 (s, 3H), 2.98-2.85
(m, 2H), 2.16-2.10 (m, 2H).
22

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4.4 General procedure C: preparation of 2-(phenylalkyl)-1,2,3,4,5,6-hexahydroazepino[4,3-
b]indole as hydrochloride salts (13a-g, 15a-d)
4.4.1. Synthesis of 2-(2-phenylethyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole hydrochloride
(15a).
The preparation of compound 15a, has been reported as a representative example. To a 0 °C ice
bath cooled solution of compound 9e (330 mg, 1.09 mmol) in 50 mL of fresh distilled dry THF
anidro, LiAlH4 (227 mg, 5.97 mmol) was added portionwise, and the mixture was refluxed
overnight, until starting material disappearance. After cooling, mixture was quenched by adding 10
mL of Na2SO4 saturated aqueous solution and stirred 30 minutes at room temperature. Residue was
filtered off and washed with 1,4-dioxane. The filtrates were collected and dried under reduced
pressure. The obtained oil residue was suspended in 50 mL of distilled water, and aqueous mixture
was extracted with chloroform (3 × 50 mL). Collected organic phases were washed twice with 20
mL of brine, dried (anhydrous Na2SO4), filtered and concentrated under reduced pressure. The
residue oil was purified by chromatography on silica gel (ethyl acetate/MeOH 70/30 v/v) to afford a
brown oil, which was stirred 2 hours in HCl saturated methanol solution. After solvent removal, and
crystallization of solid residue (EtOH/Ethyl acetate) compound 15a, as hydrochloride salt. Yield:
56% (200 mg); brown solid, mp 125-127 °C; IR (KBr): ν = 3399, 3261, 2695, 2616, 1629, 1456,
747 cm-1; 1H-NMR (300 MHz, d6-DMSO) δ: 11.46 (s, 1H), 10.77 (s, 1H), 7.63 (dd, J1 = 6.0 Hz, J2
= 2.0 Hz, 1H), 7.40 - 6.90 (m, 8H), 4.69 (d, J = 14.5 Hz, 1H), 4.59 (dd, J1 = 14.5 Hz, J2 = 5.0 Hz,
1H), 3.68 (dd, J1 = 12.0 Hz, J2 = 9.0 Hz, 1H), 3.62-3.35 (m, 3H), 3.20 - 2.80 (m, 4H), 2.20 - 1.90
(m, 2H); 13C-NMR (300 MHz, d6-DMSO) δ: 140.77,137.54, 129.16, 128.42, 127.15, 121.15,
119.88, 117.54, 111.34, 101.17, 79.69, 57.14, 55.30, 53.85, 48.76, 30.24, 26.37, 22.19; HRMS
calcd for C20H23N2 [M+H]+ 291.1856, found 291.1844. Anal C20H22N2 × HCl (C, H, N).
4.4.2. Synthesis of 2-(benzyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole hydrochloride (13a)
23

ACCEPTED MANUSCRIPT
Compound 13a was obtained as described for compound 15a, by following the general procedure
C. The residue oil was purified by chromatography on silica gel (ethyl acetate/MeOH 70/30 v/v).
Obtained hydrochloride salt of 13a. Yield: 76% (80 mg); brown solid, mp 105-107 °C; IR (KBr) ν:
1
3432, 1624, 1440, 1380, 800, 740 cm-1; H-NMR (300 MHz, d6-DMSO) δ: 11.38 (s, 1H), 9.60 (s,
1H), 7.60-7.40 (m, 3H), 7.18-7.10 (m, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.06 - 6.99 (m, 2H), 4.29 (dt,
J1 = 4.0 Hz, J2 =15 Hz, 2H), 3.70 (d, J = 15.0 Hz, 2H), 3.05-2.90 (m, 2H), 2.20 - 2.10 (m, 2H),
2.10-2.00 (m, 2H); 13C-NMR (300 MHz, d6-DMSO) δ: 140.06, 138.72, 134.60, 129.10 (2C),
128.75, 128.49 (2C), 127.16, 120.01, 118.58, 117.06, 111.05, 109.88, 59.03, 58.36, 49.92, 28.00,
25.14; HRMS calcd for C19H21N2 [M+H]+ 277.1699, found 277.1702. Anal C19H20N2 × HCl (C, H,
N).
4.4.3. Synthesis of 2-(4-methylbenzyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole hydrochloride
(13d)
Compound 13d was obtained as described for compound 15a, by following the general procedure
C. The residue oil was purified by chromatography on silica gel (ethyl acetate/MeOH 70/30 v/v).
Obtained hydrochloride salt of 13d. Yield: 78% (84 mg); brown solid, mp 105-106 °C; IR (KBr) ν:
3433, 1626, 1442, 1384, 876, 742 cm-1; 1H-NMR (300 MHz, d6-DMSO) δ: 11.40 (s, 1H), 10.30 (s,
1H), 7.43 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.06 - 6.99 (m,
2H), 4.64 (d, J = 15.0 Hz, 1H), 4.29 (dt, J1 = 4.0 Hz, J2 =15 Hz, 2H), 4.20 (d, J = 15.0 Hz, 1H),
3.70 - 3.45 (m, 2H), 2.99 (t, J = 5.5 Hz, 2H), 2.33 (s, 3H), 2.20 - 2.10 (m, 1H), 2.10-2.00 (m, 1H);
13C-NMR (300 MHz, d6-DMSO) δ: 140.76, 139.30, 134.83, 131.65, 129.71, 128.24, 127.67,
121.10, 119.54, 117.32, 111.60, 101.03, 56.66, 56.40, 48.61, 26.19, 22.45, 21.29; HRMS calcd for
C20H23N2 [M+H]+ 291.1856, found 291.1854. Anal C20H22N2 × HCl (C, H, N).
4.4.4. Synthesis of 2-(4-fluorobenzyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole hydrochloride
(13e)
24

ACCEPTED MANUSCRIPT
Compound 13e was obtained as described for compound 15a, by following the general procedure C.
The residue oil was purified by chromatography on silica gel (ethyl acetate/MeOH 70/30 v/v).
Obtained hydrochloride salt of 13e. Yield: 40% (160 mg); brown solid, mp 100-102 °C; IR (KBr) ν:
1
3430, 2937, 2586, 1605, 1432, 1228, 876, 744 cm-1; H-NMR (300 MHz, d6-DMSO) δ: 11.42 (s,
1H), 10.33 (s, 1H), 7.65 - 7.60 (m,2H), 7.37 - 7.20 (m, 2H), 7.02 (quintet, J = 8.0 Hz, 2H), 4.62 (d,
J = 14.0 Hz, 1H), 4.35 - 4.20 (m, 3H), 3.59 (dd, J1 = 12.0 Hz, J2 = 9.0 Hz, 1H), 3.38 (dd, J1 = 12.0
13
Hz, J2 = 9.0 Hz, 1H), 3.05 - 2.95 (m, 2H), 2.25-2.10 (m, 1H), 2.10-1.90 (m, 1H) ); C-NMR (300
MHz, d6-DMSO) δ: 164.00, 162.04, 140.91, 134.79, 134.16, 128.21, 127.07, 121.11, 119.57,
117.30, 116.14, 115.97, 111.61, 100.85, 56.46, 55.73, 48.47, 26.43, 22.31; HRMS calcd for
C19H20N2F [M+H]+ 195.1605, found 295.1611. Anal C19H19N2F × HCl (C, H, N).
4.4.5.
Synthesis
of
9-fluoro-2-(4-fluorobenzyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole
hydrochloride (13f)
Compound 13f was obtained as described for compound 15a, by following the general procedure C.
The residue oil was purified by chromatography on silica gel (ethyl acetate/MeOH 70/30 v/v).
Obtained hydrochloride salt of 13f. Yield: 40% (155 mg); brown solid, mp 96-99 °C; IR (KBr)
ν:3271, 2956, 2750, 1237, 830, 701 cm-1H-NMR (300 MHz, d6-DMSO) δ: 11.35 (s, 1H), 10.29 (s,
1H), 7.32 (dd, J1 = 4.0 Hz, J2 = 9.0 Hz, 1H), 7.22 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 9.0 Hz, 1H),
7.05 (t, J = 8.0 Hz, 2H), 6.88 (t, J = 9.0 Hz, 1H), 4.60 (s, 2H), 4.05 (t, J = 5.0 Hz, 2H), 3.06 (t, J =
13
5.5 Hz, 2H), 2.20 - 2.10 (m, 2H); C-NMR (300 MHz, d6-DMSO) δ: 160.79, 158.28, 141.25,
135.99, 131.31, 130.81 (2C),128.96, 115.28 (2C), 111.87, 110.34, 107.90, 102.03, 57.91, 57.71,
49.75, 28.06, 24.73; HRMS calcd for C19H19N2F2 [M+H]+ 33.1511, found 313.1521. Anal
C19H18N2F2 × HCl (C, H, N).
4.4.6. Synthesis of N,N-diethyl-N-{4-[(9-fluoro-1,2,3,4,5,6-hexahydroazepino[4,3-b]indol-2(1H)-
yl)methyl]benzyl}amine dihydrochloride (13g)
25

ACCEPTED MANUSCRIPT
Compound 13g was obtained as described for compound 15a, by following the general procedure
C. The residue oil was purified by chromatography on silica gel (DCM/MeOH 90/10 v/v). Obtained
dihydrochloride salt of 13g. Yield: 50% (180 mg); black oil; IR (KBr) ν: 3331, 3275, 2750, 1200,
810, 700 cm-1. 1H-NMR (300 MHz, d6-DMSO) δ: 11.70 (s, 1H), 10.50 (s br, 1H), 10.38 (s br, 1H),
7.80-7.55 (m, 4H), 7.30 (dd, J1 = 4.5 Hz, J2 = 9.0 Hz, 1H), 7.05 (d, J = 9.0 Hz, 1H), 6.87 (t, J = 9.0
Hz, 1H), 4.52 (d, J = 15.0 Hz, 1H), 4.45-4.05 (m, 9H), 3.15-2.90 (m, 4H), 2.20-2.00 (m, 2H), 1.24
13
(t, J = 7.0 Hz, 6H) ); C-NMR (300 MHz, d6-DMSO) δ: 158.47, 156.63, 143.15, 133.14, 131.93
(2C), 131.62, 131.47, 131.19 (2C), 128.76, 112.55, 109.80, 102.55, 56.44, 56.32, 55.71, 52.99,
52.82, 48.55, 26.43, 23.02 (2C), 22.05; HRMS calcd for C24H31N3F [M+H]+ 380.2497, found
380.2499.
4.4.7. Synthesis of 2-[4-(pyrrolidin-1-ylmethyl)benzyl]-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole
dihydrochloride (13h)
Compound 13h was obtained as described for compound 15a, by following the general procedure
C. The residue oil was purified by chromatography on silica gel (DCM/MeOH 90/10 v/v). Obtained
dihydrochloride salt of 13h. Yield: 37% (135 mg); pale brown oil; IR (KBr) ν: 3290, 3277, 2755,
1205, 807, 705 cm-1; 1H-NMR (300 MHz, d6-DMSO) δ: 11.49 (s, 1H), 10.49 (s br, 1H), 10.22 (s br,
1H), 7.73-7.55 (m, 4H), 7.32 (dd, J1 = 4.5 Hz, J2 = 9.0 Hz, 1H), 7.09 (dd, J1 = 3.0 Hz, J2 = 9.0 Hz,
1H), 6.89 (t, J1 = 9.0 Hz, 1H), 4.65 (d, J1 = 14.0 Hz, 1H), 4.45-4.25 (m, 9H), 3.55-3.45 (m, 2H),
13
3.10-2.90 (m, 2H), 2.09-1.78 (m, 6H); C-NMR (300 MHz, d6-DMSO) δ: 158.49, 156.81, 143.12,
133.11, 132.00 (2C), 131.60, 131.47, 131.15 (2C), 128.81, 112.63, 109.77, 102.59, 57.42, 57.30,
52.11, 48.70, 45.71, 42.87, 28.77, 26.43, 18.32, 18.09; HRMS calcd for C24H29N3F [M+H]+
378.2340, found 378.2341.
4.4.8.
Synthesis
of
6-methyl-2-(2-phenylethyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole
hydrochloride (15b)
26

ACCEPTED MANUSCRIPT
Compound 15b was obtained as described for compound 15a, by following the general procedure
C. The residue oil was purified by chromatography on silica gel (ethyl acetate/MeOH 75/25 v/v).
Obtained hydrochloride salt of 15b. Yield: 66% (70 mg); brown solid, mp 98-100 °C; IR (KBr) ν:
3399, 2695, 2616, 1629, 1456, 747 cm-1; 1H-NMR (300 MHz, d6-DMSO) δ: 10.75 (s, 1H), 7.65 (dd,
J1 = 6.0 Hz, J2 = 2.0 Hz, 1H), 7.40 - 6.90 (m, 8H), 4.70 (d, J = 14.5 Hz, 1H), 4.61 (dd, J1 = 14.5
Hz, J2 = 5.0 Hz, 1H), 3.70 (s, 3H), 3.65 (dd, J1 = 12.0 Hz, J2 = 9.0 Hz, 1H), 3.62-3.35 (m, 3H),
13
3.20-2.80 (m, 4H), 2.20-1.85 (m, 2H); C-NMR (300 MHz, d6-DMSO) δ: 140.70,137.52, 129.15,
128.45, 127.25, 121.11, 119.90, 117.52, 111.34, 101.19, 79.69, 57.12, 55.32, 53.83, 48.82, 30.29,
29.03, 26.35, 22.18; HRMS calcd for C21H25N2 [M+H]+305.2012, found 305.2013. Anal C21H24N2
× HCl (C, H, N).
4.4.9. Synthesis of 2-(3-phenylpropyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole hydrochloride
(15c)
Compound 15c was synthesized as described for compound 15a, by following the general
procedure C. The residue oil was purified by chromatography on silica gel (ethyl acetate/MeOH
90/10 v/v), and converted into the corresponding hydrochloride salt of 15c. Yield 56% (202 mg),
1
brown solid; m.p. 143-144 °C; IR (KBr): ν; 3330, 1628, 738, 713, 700 cm-1; H-NMR (300 MHz,
d6-DMSO) δ: 11.38 (s, 1H), 10.28 (s, 1H), 7.51 (d, J= 7 Hz, 1H), 7.35-7.20 (m, 3H), 7.22-7.10 (m,
3H), 7.08-6.88 (m, 2H), 4.60-4.40 (m, 2H), 3.70-3.55 (m, 1H), 3.50-3.35 (m, 1H), 3-05-2.85 (m,
4H), 2.53 (t, J=7Hz, 2H), 2.10-1.90 (m, 2H), 1.62-1.43 (m, 2H); 13C-NMR (300 MHz, d6-DMSO) δ:
140.70, 136.43, 134.80, 128.82 (2C), 128.62 (2C), 126.51, 122.83, 121.14, 119.66, 117.45, 111.97,
111.58, 57.13, 52.78, 48.65, 38.26, 32.47, 26.37, 23.90; HRMS calcd for C21H25N2
[M+H]+305.2012, found 305.2011. Anal C21H24N2 × HCl (C, H, N).
4.4.10. Synthesis of 2-(4-phenylbutyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole hydrochloride
(15d)
27

ACCEPTED MANUSCRIPT
Compound 15d was synthesized as described for compound 15a, by following the general
procedure C. The residue oil was purified by chromatography on silica gel (ethyl acetate/MeOH
90/10 v/v), and converted into the corresponding hydrochloride salt of 15d. Yield 66% (315 mg),
dark brown solid; m.p. 165-167 °C; IR (KBr) ν: 3430, 3152, 2952, 2579, 1621, 1523, 1452, 1384,
1
1354 cm-1; H-NMR (300 MHz, d6-DMSO) δ: 11.40 (s, 1H), 10.27 (s, 1H), 7.50 (d, J= 7 Hz, 1H),
7.32-7.21 (m, 3H), 7.20-7.10 (m, 3H), 7.10-6.90 (m, 2H), 4.60-4.40 (m, 2H), 3.70-3.55 (m, 1H),
3.50-3.35 (m, 1H), 3-05-2.85 (m, 4H), 2.53 (t, J=7Hz, 2H), 2.10-1.90 (m, 2H), 1.71 (quint., J=7.5
13
Hz, 2H), 1.62-1.43 (m, 2H); C-NMR (300 MHz, d6-DMSO) δ: 141.99, 140.67, 134.80, 128.69,
128.39, 126.26, 121.11, 119.64, 117.33, 111.53, 101.14, 57.08, 52.60, 48.38, 35.03, 28.36, 26.41,
23.75, 22.08; HRMS calcd for C22H27N2 [M+H]+ 319.2169, found 319.2167x. Anal C22H26N2 × HCl
(C, H, N).
4.5. Molecular modeling
The molecular skeletons of compounds 13a and 15d, with standard values of bond lengths and
valence angles, were built using the Maestro software package (Schrödinger Release 2018-4,
Maestro, Schrödinger, LLC, New York, NY, 2018), and for the most active inhibitor a
conformational sampling, ensuring an energetically stable puckering of the hexahydroazepino ring,
was after performed by means of OMEGA (rel. 3.0.0.1, OpenEye Scientific Software, Santa Fe,
NM), generating a total of 2217 conformers, hereafter docked into the BChE binding site.
According to the high homology (ca. 90%) between the horse serum and human sequence, the
recently published X-ray data of BChE in complex with a N-propargyl piperidine nanomolar
inhibitor (pdb code 6F7Q) [63] was used as biomolecular target.
Chain A of the enzyme structure was passed to the Protein Preparation Wizard interface of
MAESTRO for removing water molecules, and hydrogen atoms added, optimizing their position,
and determining the protonation states of residues according to PROPKA prediction at pH 7.0.
AMBER UNITED force field electrostatic charges [64] were applied to protein structure, whereas
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