New azepino[4,3-b]indole derivatives as nanomolar selective inhibitors of human butyrylcholinesterase showing protective effects?against NMDA-induced neurotoxicity

Article abstract of DOI:10.1016/j.ejmech.2016.09.037

Several 6-substituted 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (THAI) derivatives were synthesized and evaluated for their activity as cholinesterase (ChE) inhibitors. The most potent inhibitors were identified among 6-(2-phenylethyl)-THAI derivatives, and in particular compounds 12b and 12d proved to be very active against human BChE (IC₅₀?=?13 and 1.8?nM, respectively), with 1000-fold selectivity over AChE. Structure-activity relationships highlighted critical features (e.g., ring fusion [4,3-b], integrity of the lactam CONH function) and favorable physicochemical properties of the 6-(2-phenylethyl) group (i.e., optimal position, size and lipophilicity of phenyl substituents). The effects of a number of compounds against NMDA-induced SH-SY5Y neuronal cell injury were also evaluated. Treatment with 12b increased cell viability in SH-SY5Y cells pretreated with 250?μM NMDA, with significant effects (P?

Full text of DOI:10.1016/j.ejmech.2016.09.037

Accepted Manuscript
New Azepino[4,3-b]indole derivatives as nanomolar selective inhibitors of human
butyrylcholinesterase showing protective effects against NMDA-induced neurotoxicity
Modesto de Candia, Giorgia Zaetta, Nunzio Denora, Domenico Tricarico, Maria
Majellaro, Saverio Cellamare, Cosimo D. Altomare
PII:
S0223-5234(16)30767-X
10.1016/j.ejmech.2016.09.037
EJMECH 8903
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 July 2016
Revised Date: 9 September 2016
Accepted Date: 12 September 2016
Please cite this article as: M. de Candia, G. Zaetta, N. Denora, D. Tricarico, M. Majellaro, S. Cellamare,
C.D. Altomare, New Azepino[4,3-b]indole derivatives as nanomolar selective inhibitors of human
butyrylcholinesterase showing protective effects against NMDA-induced neurotoxicity, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.037.
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ACCEPTED MANUSCRIPT
September 9, 2016
New Azepino[4,3-b]indole Derivatives as Nanomolar Selective Inhibitors of
Human Butyrylcholinesterase Showing Protective Effects Against NMDA-
induced Neurotoxicity
Modesto de Candia, Giorgia Zaetta, Nunzio Denora, Domenico Tricarico, Maria Majellaro, Saverio
Cellamare and Cosimo D. Altomare*
Department of Pharmacy – Drug Sciences, University of Bari ‘‘A. Moro’’, Via E. Orabona 4, 70125
Bari, Italy.
*Corresponding Author: Phone +39-080-5442781; fax +39-080-5442230; e-mail
cosimodamiano.altomare@uniba.it
Author Contributions: M.d.C., G.Z., M.M. and S.C. contributed to chemistry and performed enzymes’
inhibition measurements. N.D. and D.T. carried out cytotoxicity assays and biological data analysis.
C.D.A. and M.d.C. designed the research project and interpreted SAR data. All the authors approved
the final version of the manuscript.
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Graphical abstract
H
O
N
100
***
N
80
60
40
**
*
cntrl NMDA 0,1
0,5
1
5
12b
NMDA + 12b [µM]
IC50 (hBChE) = 13 nM
IC50 (hAChE) = 12.6 µM
Significant concentration-related reduction of NMDA-mediated
toxicity on SH-SY5Y neuronal cells at low µM concentration.
Cytotoxicity on liver HepG2 cells at GI50 = 120 µM.
Highlights
• New azepino[4,3-b]indol-1(2H)-one derivatives were synthesized as anti-Alzheimer agents
• Compound 12b proved to be very potent as inhibitor of human BChE (IC50 = 13 nM)
• At micromolar concentrations, 12b showed protective effects against NMDA in SH-SY5Y cells
• At the active doses, 12b did not exhibit cytotoxicity on liver HepG2 cells
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ABSTRACT
Several 6-substituted 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (THAI) derivatives were
synthesized and evaluated for their activity as cholinesterase (ChE) inhibitors. The most potent
inhibitors were identified among 6-(2-phenylethyl)-THAI derivatives, and in particular compounds 12b
and 12d proved to be very active against human BChE (IC50 = 13 and 1.8 nM, respectively), with
1000-fold selectivity over AChE. Structure-activity relationships highlighted critical features (e.g., ring
fusion [4,3-b], integrity of the lactam CONH function) and favorable physicochemical properties of the
6-(2-phenylethyl) group (i.e., optimal position, size and lipophilicity of phenyl substituents). The
effects of a number of compounds against NMDA-induced SH-SY5Y neuronal cell injury were also
evaluated. Treatment with 12b increased cell viability in SH-SY5Y cells pretreated with 250 ꢀM
NMDA, with significant effects (P < 0.05) at concentrations between 0.5 and 5 ꢀM. These findings
suggest that THAI can be used as a scaffold for developing new drug leads for the treatment of
Alzheimer-type neurodegeneration syndrome.
Keywords:
Alzheimer’s disease
Azepino[4,3-b]indole
Butyrylcholinesterase
N-Methyl
D-aspartate receptor
Neuroprotection
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Abbreviations
AD, Alzheimer Disease; ACh, Acetylcholine; AChE, Acetylcholinesterase; BChE,
Butyrylcholinesterase; CAS, Catalytic anionic site; CNS, Central Nervous System; DCM,
dichloromethane; DMEM, Dulbecco’s modified eagle’s medium; DMSO, Dimethyl sulfoxide; FBS,
Fetal bovine serum; 5-FU, 5-fluorouracil; HB, Hydrogen bond; HepG2, human hepatocellular
carcinoma cell line; LAH, Lithium Aluminum Hydride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; MCF-7, human breast carcinoma cell line; NMDA, N-methyl-
D-
aspartate; NMDAR, N-methyl- -aspartate receptor; PAS, Peripheral anionic site; PB, Phosphate
D
Buffer, PBS, Phosphate Buffered Saline; SAR, Structure-activity relationship; SH-SY5Y, human
neuroblastoma cell line; TBAB, Tetrabutyl amoniumbromide; TFA, Trifluoro acetic acid; U-87, human
glioblastoma cell line.
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1. Introduction
Neurodegeneration in Alzheimer’s disease (AD), a devastating disorder accounting for the majority of
the dementias, is primarily characterized by histopathological hallmarks, namely the presence of
intracellular neurofibrillary tangles of hyperphosphorilated τ-protein and extracellular β-amyloid
protein deposits contributing to senile plaques, and lower levels of the neurotransmitter acetylcholine
(ACh) [1]. Despite the advances achieved over the last two decades in understanding the pathogenic
mechanisms underlying AD, only a few drugs have been approved. The currently available treatments
(Chart 1) include acetylcholinesterase (AChE, EC 3.1.1.7) inhibitors 1-3 (rivastigmine, galantamine,
and donepezil), approved for the symptomatic relief of mild to moderate AD, and the N-methyl-
D-
aspartate receptor (NMDAR) antagonist 4 (memantine) [2].
Galantamine and donepezil are reversible cholinesterase (ChE) inhibitors, whereas rivastigmine is a
pseudo-irreversible inhibitor which transfers a carbamate moiety to the Ser residue (followed by slow
hydrolysis) in the active sites of AChE and butyrylcholinesterase (BChE, EC 3.1.1.8). The impairment
of ACh neurotransmission is a downstream process in the cognitive deficit accompanying AD;
therefore a role of ChE inhibitors leading to a recovery of the cholinergic neurotransmission and related
beneficial effects on cognition capacity has long been postulated [3]. AD patients may further benefit
from reduction of glutamate-induced, Ca2+-mediated excitotoxicity by the NMDAR antagonist
memantine (4). Based on the available data, it may be supposed that new small molecules which
simultaneously target both cholinergic transmission and glutamate-induced excitotoxicity may improve
the AD pharmacological treatment [4,5].
[Insert Chart 1, about here]
ACh is mainly hydrolyzed by AChE in the synaptic cleft of the cholinergic neurons, but at higher
concentrations is also hydrolyzed by BChE, which is produced in liver and found primarily in plasma
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where it is also responsible for detoxification of xenobiotics [6,7]. BChE is also localized in CNS glial
cells and neurons, where it works as a co-regulator of cholinergic neurotransmission [8]. It has been
found that BChE can compensate AChE catalytic functions in synaptic cleft and its activity increases
by 30-60% during the time course in AD [9-12].
Selective BChE inhibitors usually contain tri- or polycyclic structures (Chart 2). For instance,
physostigmine has been taken as a scaffold to synthesize selective irreversible BChE inhibitors, such as
N1-phenetylnorcymserine (irreversible) [8,13], whereas phenothiazine derivatives (e.g., ethopropazine)
[14] and some quinazolinimine-based compounds proved to be selective reversible BChE inhibitors
[15].
[Insert Chart 2, about here]
AChE and BChE, which share about 70% of homology and retain the same key residues in the choline
binding pocket, differ for their three-dimensional (3D) structures. AChE contains a 20 Å deep and
narrow gorge, in which five regions are involved in the ligand binding: (i) the catalytic triad residues
(Ser203, His447, Glu334); (ii) the ‘oxyanion hole’ inside the active center, that stabilizes the transient
tetrahedral enzyme-substrate complex by accommodation of negatively charged carbonyl oxygen; (iii)
the ‘anionic site’ (AS), where Trp86 (a residue conserved in all ChEs), involved in the orientation and
stabilization of trimethylammonium group 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 [16]. The main differences between AChE and BChE occur in the ‘acyl pocket’
and PAS. Two Phe residues (Phe295, Phe297) in the ‘acyl pocket’, which prevent the access of bulkier
molecules to the catalytic site in AChE, are replaced by two aliphatic residues (Leu286, Val288) in
BChE, whereas globally six of the fourteen AChE aromatic residues encompassing the ‘acyl pocket’
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and PAS in AChE are replaced by aliphatic residues in BChE. This ultimately results in ca. 200 Å3
larger gorge in BChE than in AChE [17-20
In previous work, we reported the ChEs’ inhibitory activity of a number of diverse ester derivatives of
annulated tetrahydroazocines, showing that some compounds containing two isomeric 2,3,6,11-
tetrahydro-1H-azocino[4,5-b]indole and 2,3,6,7-tetrahydro-1H-azocino[5,4-b]indole scaffolds
preferentially inhibited AChE over BChE, with potency in the low micromolar range [21,22]. In this
study, in order to obtain more information on ChEs’ inhibition activity and selectivity of indole-
containing tricycle derivatives, we focus on a series of compounds bearing partially hydrogenated
azepino[4,3-b]indole as core structure. In particular, 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(6H)-one
(THAI) [22] was taken as scaffold, and a number of diverse substituents on indole nitrogen (N6) and
chemical modifications of the annulated heterocyclic core were investigated.
The THAI scaffold investigated herein is structurally similar to the heterocyclic core of the known drug
dimebon (latrepirdine), that is a 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole derivative showing
multimodal activity with rather controversial results in clinical studies of AD patients [23]. Developed
as antihistamine drug, dimebon exhibited in vitro activities against ion channels (e.g., L-type calcium
channels), receptors and neurotransmitter systems (e.g., AMPA, NMDA, glutamate, α-adrenergic and
serotoninergic 5-HT2C, 5-HT5A, 5-HT6 receptors), and ChEs inhibition (preference for BChE) with
potencies in the micromolar range [24-27
Taking in mind the neuroprotective effects of the NMDAR antagonists (e.g., memantine 4) and the
structural resemblance between the THAI scaffold and the heterocyclic core of dimebon, which also
showed NMDAR antagonist properties, a number of synthesized compounds, exhibiting elevated
BChE inhibition potency, were also assayed for their activity in an NMDA-induced neuronal cells
death model.
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2. Chemistry
Most of the 6-substituted derivatives of 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (10a) and
3,4,5,6-tetrahydroazepino[3,2-b]indol-2(1H)-one (16) were synthesized as shown in Scheme 1.
As previously reported by others [28], phenylhydrazine or (4-fluorophenyl)hydrazine were reacted with
1,3-cyclohexanedione to provide the respective hydrazones which, by treatment with refluxing
trifluoroacetic acid (TFA) under Fisher conditions, afforded the 1,2,3,9-tetrahydro-4H-carbazol-4-one
derivatives 8a and 8b (65-70% yields). Compounds 8a,b were converted into the corresponding
ketoximes 9a,b, which then underwent a selective Beckmann rearrangement by treatment with
preheated PPA at 110 °C to provide the [4,3-b] THAI products 10a (unsubstituted) and 10b (9-F
substituted) in high yield (> 70%), as shown by a triplet signal (δ 7.43 ppm) of the lactam NH in the
1HNMR spectrum; under these reaction conditions negligible amounts of the [3,2-b] ring fusion
isomers were found.
Regioselective alkylation by suitable alkyl halides was achieved at the indole nitrogen (N6), and not at
the lactam nitrogen (N1) of 10a and 10b, as shown by the 1HNMR disappearance of N6-H proton
singlet (δ 11.40 ppm), in the presence of TBAB as the phase transfer catalyst in stirred DCM/25%
NaOH biphasic mixtures (24-72 h at rt or 40 °C), affording 11a-d and 12a-h (X = H), and 12i (X = F).
Then, nitrile reduction of 11d by CoCl2 and NaBH4 gave the 6-(2-aminoethyl)-THAI derivative 11e,
which was then reacted with ethyl chloroformate to afford the ethylcarbamate 11f. Methylation reaction
of 6-(2-phenylethyl)-THAI 12b with methyl iodide in the presence of NaH yielded 13, whereas
reduction of the lactam group by lithium aluminum hydride (LAH) gave the basic compound 14,
isolated as hydrochloride salt.
[Insert Scheme 1, about here]
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The THAI derivative 17, that is the [3,2-b] ring fusion isomer of 12b, was synthesized starting from 9a,
which was first treated with tosyl choride, and the produced sulphonyl oxime 15 refluxed in a toluene
suspension with aluminum oxide to afford 16 [29], that was finally alkylated by 2-phenethyl bromide
under the above conditions.
The synthesis of the ketoxime derivative 19 was accomplished as shown in Scheme 2.
[Insert Scheme 2, about here]
Compound 22, designed as ring-opened analogue of 12b, was also synthesized (Scheme 3). The
commercially available methyl 1H-indole-3-carboxylate was first alkylated by reaction with phenethyl
bromide to afford 20, which underwent subsequent ester hydrolysis (21) and coupling with n-
propylamine to provide the amide derivative 22.
[Insert Scheme 3, about here]
3. Results and discussion
The 6-substituted THAI-based synthesized compounds were tested for their inhibitory activity toward
electric eel AChE and horse serum BChE, using the Ellmann colorimetric assay (galantamine as the
reference standard) [22,30]. The half maximal inhibitory concentration (IC50) values or the inhibition
percentages at 10 µM concentration are reported in Tables 1 and 2.
The THAI derivatives bearing diverse moieties on the indole nitrogen (Table 1) selectively inhibited
BChE. The 6-(2-phenylethyl) group (12b) proved to inhibit BChE in the nanomolar range (IC50 = 24
nM) with 830-fold selectivity over AChE. Compound 12b proved to be not only much more potent
than the lower (12a) and higher (12c) homologues, bearing 6-benzyl and 6-(3-phenylpropyl) groups,
respectively, but also more potent than other 6-substitued derivatives bearing isopropyl (11a), ester
(11b,c) and carbamate (11f) groups. A fluorine atom in the meta-position (and not in para) of phenyl
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(12d) led to a 16-fold additional improvement over 12b in BChE binding affinity on IC50 scale (1.5
nM), which is translated into about 7 kJ·mol-1 free energy change, and remarkable decrease in AChE
inhibitory activity. With Cl in place of F no activity improvement was observed, but a similar position-
related trend appeared (12f, m-Cl > 12g, p-Cl).
[Insert Table 1, about here]
The introduction of a p-Me group (12h) led to a sharp decrease in BChE inhibition.
Lineweaver-Burk plots outlined for the most active compound 12d (Figure 1), using fixed amounts of
BChE and varying concentrations of the substrates between 25 and 250 µM, in the absence or presence
of inhibitor at different concentrations (0.5-5 nM), clearly showed a competitive type inhibition. A
replot of the slopes versus the corresponding inhibitor concentrations provided a Ki value equal to 0.7
nM.
[Insert Figure 1, about here]
Some further modifications around the 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (THAI)
tricyclic scaffold were explored, choosing the 6-(2-phenylethyl) group as the constant feature (Table 2).
The 9-F THAI analog 12i resulted almost equipotent with the unsubstituted parent compound 12b. The
N-methylation of the lactam (13) led to a > 400-fold decrease of BChE binding affinity (> 15 kJ·mol-1
free energy increase) and a selectivity reversal, whereas the reduction of the lactam -CONH- to -
CH2NH- amine derivative 14 caused about 50-fold loss of BChE inhibition potency.
The comparison between the activities of the ring fusion isomers 12b and 17 is noteworthy. As a matter
of fact, the reversal of the directionality of -CONH- (12b) into -NHCO- (17), due to the different ring
fusion between azepan-2-one and indole rings, resulted in a marked drop of BChE (and AChE as well)
binding affinity.
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[Insert Table 2, about here]
The enzyme inhibition data of 17, compared with those of 12b, pointed out that the lactam -CONH-
group of THAI core should be involved in directional HB interactions within the BChE binding site. In
addition, spectrophotometric evidence led us to exclude that the fusion isomer lactam derivatives 12b
and 17 may undergo any (different) inactivating BChE-catalyzed amide hydrolysis [31], remaining
both stable when incubated with the enzyme for 1 h (see Supplementary material). We could therefore
reasonably hypothesize that HB interactions with the BChE binding pocket should be more efficiently
achieved with the [4,3-b] ring fusion in 12b and other analogs, which become critical to well allocate
the 6-(2-phenylethyl)-THAI inhibitors into the BChE binding site [32]. At the best of our knowledge,
the role of such a kind of HB interactions has not been pointed out for other tricyclic BChE-selective
reversible inhibitors.
Finally, the decrease of BChE inhibition potency shown by the 4H-carbazol-4-one derivatives 18 and
19 is somehow in line with expectations, taking in mind that BChE active site, compared with that of
AChE, should better accommodate larger scaffolds. The N-propylcarboxamide 22 as the ring opening
analog of 12b, likely due to entropic factors, proved to be about 500-fold less potent BChE inhibitor,
while increasing inhibition activity toward AChE. The main SARs of 6-substituted THAI-based
inhibitors are summarized in Figure 2.
[Insert Figure 2, about here]
The THAI derivatives 12b, 12d and 12e were also tested as inhibitors of human ChEs, which are
known to share high (> 80%) structural homology with electric eel AChE and horse serum BChE
(Table 3). Interestingly, compounds 12b and 12d did not exhibit any noteworthy species-dependent
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ChEs’ inhibition activity and selectivity, whereas the 4’F-phenethyl THAI derivative 12e proved less
potent and selective in human ChEs.
[Insert Table 3, about here]
Finally, four active BChE inhibitors (12b, 12d, 12e and 14) were tested for their effects on cell viability
by exposing human neuroblastoma (SH-SY5Y) cell line [33-35] to the compounds at increasing
concentrations from 0.1 ꢀM to 1 mM during 72 hours. Cell viability was assessed by (3,4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The NMDAR antagonist
memantine (4) [36,37] was also tested in this assay. The test compounds affected SH-SY5Y cell
viability in a dose dependent manner (plots in Supplementary material), and the respective
concentration for half maximal inhibition of cell proliferation (GI50) values are reported in Table 4.
[Insert Table 4, about here]
Compound 12d and the amino derivative 14 caused a 50%-reduction of SH-SY5Y cell viability at
concentrations in the low micromolar range (GI50 values equal 2.61 and 5.60 ꢀM, respectively). In
contrast, 12b (GI50 = 138 ꢀM) and 12e (GI50 = 251 ꢀM) showed cytotoxicity on human neuroblastoma
cells at doses close to or higher than 4 (GI50 = 145 ꢀM).
Compounds 12b, 12d, 12e and 14 were further tested in a panel of cultured tumor cells, namely human
glioblastoma (U-87), human breast carcinoma (MCF-7) and human hepatocellular carcinoma (HepG2)
cell lines, using 5-fluorouracil (5-FU) as the positive control (Table 4). Compound 14 proved to be
toxic enough with GI50’s in the low micromolar range (25-41 ꢀM), whereas the lactam derivatives 12b,
12d and 12e showed significant cytotoxicity in the three cell lines at doses >100 µM, their GI50’s being
of the same order of magnitude as those reported for tacrine (a known AChE inhibitor) in HepG2 cells
[33].
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Finally, compounds 12b, 12d, 12e, 14 and memantine (4) were assayed in vitro for their ability to
counteract the NMDA-induced toxicity in SH-SY5Y cells. The neuronal cells were exposed to NMDA
(250 µM, i.e. a concentration close to its GI50; see Supplementary material) in co-treatment with
various concentrations of test compounds (0.1, 0.5, 1 and 5 µM), and cell viability was determined by
MTT assay. As shown in Figure 3, NMDA decreased viable cells, which resulted ca. 40% lower than
the NMDA-untreated cells. Memantine (4) showed statistically significant protective effects against
NMDA-induced injury only at 5 µM (68.0% viability, P < 0.01; Fig. 3e).
[Insert Figure 3, about here]
Treatment with 12b increased cell viability (Fig. 3a), exhibiting significant effects at 0.5 ꢀM and higher
protection at 5 ꢀM concentration (76.9% viable cells, P < 0.001). A similar trend (Fig. 3c) was shown
by the 4’F-phenyl THAI derivative 12e (76.6% viable cells, P < 0.001, at 5 ꢀM). In contrast,
compound 12d (3’F-phenylethyl-bearing THAI derivative; Fig. 3b) and the amino derivative 14 (Fig.
3d) showed maximal protective activity at 0.5 ꢀM (70% and 76% viable cells, respectively) with a
decrease of activity at higher doses. Both compounds did not exhibit significant SH-SY5Y protection at
5 ꢀM concentration. This trend could most likely be explained taking into account the observed
intrinsic toxicity of 12d and 14 in SH-SY5Y neuronal cells (GI50 values equal 2.61 and 5.60 ꢀM,
respectively).
In summary, the above findings led us to identify 12b as potent and selective inhibitor of human BChE
(IC50 = 13 nM; 1000-fold selectivity over hAChE), endowed with moderate protective effects in SH-
SY5Y cell-based model of NMDA-induced toxicity. In this assay, 12b proved to be even more active
than memantine (4). Moreover, 12b did not show in vitro remarkable hepatotoxicity, assessed as cell
viability in HepG2 cell [38], whereas, compared to the stronger hBChE inhibitor 12d (IC50 = 1.8 nM;
>>5000-fold selectivity over hAChE) 12b proved to be itself much less neurotoxic, as shown by cell
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viability measures in SH-SY5Y cell line (GI50 equals to 138 and 2.61 ꢀM for 12b and 12d,
respectively).
4. Conclusions
A new class of selective BChE inhibitors was identified which contain the azepinoindole heterocyclic
core. A number of 6-substituted 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (THAI) derivatives
achieved nanomolar IC50 values against BChE and remarkable selectivity over AChE. The ring fusion
isomerism appeared as a critical feature, given that [4,3-b] ring fusion between azepan-2-one and
indole (12b) is required for BChE inhibitory activity, whereas [3,2-b] ring fusion (17) leads to activity
loss. Besides the critical importance of the directionality of the lactam -CONH- group, SARs
highlighted the following major features that allow THAI-based inhibitors to achieve efficient and
selective binding into the BChE active site: (i) integrity (e.g., no lactam reduction, N-methylation, ring
opening) of the azepan-2-one moiety, and (ii) optimal size and lipophilicity of the 6-(2-phenylethyl)
group. Two 6-(2-phenylethyl)-THAI derivatives, namely 12b and 12d, showed competitive type
inhibition, nanomolar potency against BChE and remarkable selectivity over AChE.
Toxicity investigations on liver HepG2 cells and neuronal SH-SY5Y cells, as well as a preliminary
screening of neuroprotection effect against NMDA-induced toxicity in SH-SY5Y cells, led to identify
12b as an attractive hit for further pharmacological studies on animal models of neurodegeneration and
AD. Indeed, compound 12b, which did not display any hepatoxicity and neurotoxicity in cell models
up to 10 ꢀM, proved to be a highly potent and selective inhibitor of human BChE (IC50 = 13 nM and
1000-fold selectivity over hAChE) showing also moderate (20-25%) protective effects against NMDA-
induced neurotoxicity in an SH-SY5Y cell model at concentrations in the low micromolar range (0.5-5
ꢀM).
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In conclusion, this study provides data to support the 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one
as a tricyclic scaffold for designing new potent BChE-selective inhibitors useful on one hand as
molecular tools for better investigating the role of BChE in the later stages of AD and on the other for
developing new multipotent drug leads for the treatment of AD-type neurodegeneration.
5. Experimental section
5.1 Chemistry
Compounds 8a, 9a, 10a and 16 were synthesized according to known procedures with slight
modifications [28,29,39,40]; their melting points and spectral data were in full agreement with those
reported in literature, and no effort was made at this stage to optimize the yields. Unless otherwise
stated, starting materials and all chemicals and solvents were purchased from Sigma-Aldrich. 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 were recorded via KBr disks on a Perkin-Elmer Spectrum One
Fourier transform infrared spectrophotometer (Perkin-Elmer Ltd., Buckinghamshire, U.K.), and the
most significant absorption bands are listed. 1H NMR spectra 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,
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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).
Synthesis procedures, analytical and spectral data of 8a, 9a, 10a, 11e,f, 12b, 13-16, 20-22 are described
below, whereas experimental details on synthesis, analysis and spectra of the other compounds (8b, 9b,
10b, 11a-d, 12a, 12c-i, 14, 17-19) are reported in Supplementary material.
5.1.1. 1,2,3,9-Tetrahydro-4H-carbazol-4-one (8a). A solution of phenylhydrazine (0.9 mL, 8.92
mmol) in 10 mL of water was added dropwise to 1,3-cyclohexanedione (1.0 g, 8.92 mmol) in 50 mL of
distilled water, and the solution was stirred overnight at room temperature. The precipitate was isolated
by filtration, washed with 100 mL of water and dried, yielding 1.65 g (Yield 90%) of cyclohexane-1,3-
dione phenylhydrazone (m.p. 176-178 °C; lit. 175-177 °C) [28,29,40], which was then refluxed in 15
mL of TFA for 24 h. After cooling, the solvent was removed under N2 gas stream and the residue was
treated with 100 mL of water. Compound 8a as a brown solid (1.30 g, yield 70%, crude) was isolated
by filtration, washed with 100 mL of water, dried and used in the preparation of the corresponding
oxime without further purification. Spectral data were in agreement with those of literature. IR (KBr,
cm-1): 3162, 1610, 1580, 1460. 1H NMR (300 MHz, CDCl3) δ ppm11.70 (s, br, 1H), 8.10-8.00 (m, 1H),
7.45-7.38 (m, 1H), 7.15-7.00 (m, 2H), 2.97 (t, J = 7.0 Hz, 2H), 2.44 (t, J = 7.0 Hz, 2H), 2.15 (quintet, J
= 7.0 Hz, 2H).
5.1.2. 1,2,3,9-Tetrahydro-4H-carbazol-4-one oxime (9a). Sodium acetate (1.33 g, 16.2 mmol) and
hydroxylamine hydrochloride (1.13 g, 16.2 mmol) were added to 8a (2.0 g, 10.8 mmol) in 30 mL of
EtOH/water 2/1 v/v, and the mixture was refluxed 24 h under nitrogen atmosphere. After cooling, the
solvent was removed and the residue was suspended in 150 mL of water and triturated, until
16

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precipitation of the desired oxime product as brown solid, which was collected by filtration and
recrystallized from EtOH and water (1.73 g, Yield 80%). Analytical and spectral data were in
agreement with those of literature (m.p. 205-9 °C, dec.) [29,40]. IR (KBr, cm-1): 3418, 3373, 1607,
1578, 1464, 1251, 1177, 753, 747. 1H NMR (300 MHz, DMSO-d6) δ ppm 11.22 (s, br, 1H) 10.26 (s,
1H), 7.92 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz,
1H), 2.82 (t, J = 6.5 Hz, 2H), 2.70 (t, J = 6.5 Hz, 2H), 1.94 (quintet, J = 6.5 Hz, 2H).
5.1.3. 3,4,5,6-Tetrahydroazepino[4,3-b]indol-1(2H)-one (10a). Compound 9a (2.5 g, 12.5 mmol) of
was added portionwise to 95 g of preheated (110 °C) PPA under vigorous stirring, and the mixture was
stirred at this temperature for 30 min. Then, 200 g of ice were carefully poured into the mixture and
triturated until complete dissolution of PPA and formation of a grey precipitate, which was collected by
filtration under reduced pressure, washed with 100 mL of water, 10 mL of 5% diluted ammonia and
further 100 mL of water. The solid was then suspended into 50 mL of MeOH and, after addition of 1.0
g of vegetal carbon, refluxed for 1 h. After cooling, the suspension was filtered on a Celite pad and the
solvent removed to furnish 10a as pale brown solid (1.75 g, 70% Yield). Analytical and spectral data
were in good agreement with those of literature [29,40]; m.p. 208-10 °C (lit. 209-211 °C). IR (KBr, cm-
1): 3302, 1626, 1591, 1480. 1H NMR (300 MHz, DMSO-d6) δ ppm 11.45 (br, 1H), 8.23 (d, J = 8.0 Hz,
1H), 7.43 (t, J = 7.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H),
3.24 (q, J = 7.0 Hz, 2H), 3.14 (t, J = 7.0 Hz, 2H), 2.10-2.00 (m, 2H).
5.1.4. 6-Substituted derivatives of the 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one. The
preparation method of the N-phenethyl substituted compound 12b is reported as a representative
example of the synthesis procedures of 11a-d, 12a-i, 17 and 18.
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5.1.4.1.
6-(2-Phenylethyl)-3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one
(12b).
TBAB
(0.443 g, 1.38 mmol), phenethyl bromide (0.65 mL, 2.50 mmol) and 5.0 mL of 25 % m/m NaOH
solution were added to a solution of 10a (0.25 g, 1.275 mmol) in 15 mL of dry DCM. The mixture was
stirred at rt for 48 h, 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, eluting with ethyl acetate, to afford
12b as pale brown solid (0.270 g, 70% yield). m.p. 202-203 °C. IR (KBr, cm-1): 3265, 3173, 3026,
2919, 1633, 1528, 1461, 813, 745, 702. 1H NMR (300 MHz, DMSO-d6) δ ppm 8.23 (dd, J1 = 2.5 Hz, J2
= 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.44 (t br, J = 5.5 Hz, 1H), 7.30-7.00 (m, 7H), 4.32 (t, J = 7.0
13
Hz, 2H), 3.10-3.00 (m, 2H), 2.96 (t, J = 7.0 Hz, 2H), 2.77 (t, J = 7.0 Hz, 2H), 1.95-1.80 (m, 2H). C
NMR (CDCl3): 26.5, 27.0, 35.6, 41.4, 45.0, 106.1, 108.8, 121.6, 122.5, 122.7, 126.9, 128.3, 128.7 (2
C), 128.9 (2 C), 135.8, 138.0, 142.4, 169.1 HRMS (ESI) m/z [M+H]+ calcd for C20H21N2O+, 305.1648;
found, 305.1644. Anal. Calcd for C20H20N2O: C, 78.92; H, 6.62; N, 9.20. Found: C, 79.05; H, 6.82; N,
9.33.
5.1.4.2.
6-(3-Aminopropyl)-3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (11e). NaBH4
(0.223 g, 5.90 mmol) was added portionwise to a cooled (0 °C) solution of 11d (0.150 g, 0.59 mmol)
and CoCl2×6H2O (0.280 g, 1.18 mmol) in 15 mL of methanol, and the mixture was stirred 1 h at rt.
After quenching with 10 mL of 3N HCl, the solution was stirred at rt until disappearance of precipitate,
and then concentrated under reduced pressure. The residue was suspended in 20 mL of water, and 2N
NaOH was added to alkaline pH. The obtained aqueous suspension was extracted with DCM (3×20
mL), and the collected organic phases were dried (Na2SO4), filtered and concentrated, to yield 11e as
brown oil (0.10 g, Yield 66%). IR (KBr, cm-1): 3343, 3261, 1627, 1523, 1465, 783, 754. 1H NMR (300
MHz, DMSO-d6) δ ppm 8.21 (d, J = 8.0 Hz, 1H), 7.60 (s br, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.15 (t, J =
18

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7.0 Hz, 1H), 7.07 (t, J = 7.0 Hz, 1H), 4.26 (t, J = 7.5 Hz, 2H), 3.21-3.16 (m, 2H), 3.07 (t, J = 6.5 Hz,
2H), 2.89-2.80 (m, 2H), 2.65 (s, br, 2H), 2.05-1.90 (m, 4H). MS (ESI) m/z [M+H]+ 258.
5.1.4.3.
Ethyl 3-(1-oxo-2,3,4,5-tetrahydroazepino[4,3-b]indol-6(1H)-yl)propylcarbamate (11f).
Triethylamine (0.2 mL, 1.6 mmol) and, dropwise, ethyl chloroformate (0.1 mL, 0.8 mmol) were added
to a solution of 7e (0.10 mg, 0.4 mmol) in 15 mL of dry THF. The mixture was stirred overnight at rt
and, after solvent removal, the residue was partitioned between 50 mL of ethyl acetate and 20 mL of
brine. The organic phase was dried (Na2SO4), filtered and concentrated under reduced pressure, and the
obtained residue was recrystallyzed from ethyl acetate to leave 11f as white solid (0.08 g, Yield 60%).
m.p. 134-135 °C. IR (KBr, cm-1): 3266, 3173, 3023, 2979, 1718, 1623, 1525, 1246, 1042, 781, 763. 1H
NMR (300 MHz, DMSO-d6) δ ppm 8.21 (d, J = 7.0 Hz, 1H), 7.48 (t, J = 5.0 Hz, 1H), 7.42 (d, J = 8.0
Hz, 1H), 7.24 (t, J = 5.0 Hz, 1H), 7.13 (t, J = 7.0 Hz, 1H), 7.07 (t, J = 7.0 Hz, 1H), 4.13 (t, J = 7.0 Hz,
2H), 3.99 (q, J = 7.0 Hz, 2H), 3.16 (dd, J1 = 3.0 Hz, J2 = 5.0 Hz, 2H), 3.10-2.95 (m, 4H), 2.10-1.95 (m,
+
2H), 1.78 (quintet, J = 7.0 Hz, 2H). HRMS (ESI) m/z [M+H]+ calcd for C18H24N3O3 , 330.1812; found,
330.1808. Anal. Calcd for C18H23N3O3: C, 65.63; H, 7.04; N, 12.76. Found: C, 65.70; H, 7.22; N,
12.78.
5.1.4.4.
2-Methyl-6-(2-phenylethyl)-3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (13). NaH
in 60% mineral oil dispersion (0.02 g) was added to a solution of 12b (0.08 g, 0.26 mmol) in 3 mL of
anhydrous DMF. After 10 min of stirring at rt, CH3I (0.1 mL, 1.5 mmol) was added, and the mixture
was stirred at rt overnight, whereupon further 20 mg of NaH and 0.1 mL of CH3I were added, and
stirring prolonged for 6 h until disappearance of the starting material (TLC). The mixture was diluted
with 50 mL of water and aqueous phase was extracted with 3×15 mL of ethyl acetate. The collected
organic phases were dried (Na2SO4), filtered and concentrated under reduced pressure. The oil residue
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was purified by chromatography on silica gel (ethyl acetate /n-hexane 95/5 v/v) to afford compound 13
as pale brown oil (0.03 g, Yield 36%). IR (KBr, cm-1): 2918, 1632, 1525, 1458, 810, 747, 700.1H NMR
(300 MHz, DMSO-d6) δ ppm 8.20 (dd, J1 = 2.5 Hz, J2 = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.42 (t
br, J = 5.5 Hz, 1H), 7.28-7.00 (m, 7H), 4.33 (t, J = 7.0 Hz, 2H), 3.12 (s, 3H), 3.10-3.00 (m, 2H), 2.94 (t,
J = 7.0 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H), 1.95-1.80 (m, 2H). HRMS (ESI) m/z [M+H]+ calcd for
C21H23N2O+, 319.1805; found, 319.1802. Anal. Calcd for C21H22N2O: C, 79.21; H, 6.96; N, 8.80.
Found: C, 79.45; H, 7.04; N, 8.79.
5.1.4.5.
6-(2-Phenylethyl)-1,2,3,4,5,6-hexahydroazepino[4,3-b]indole
hydrochloride
(14).
LiAlH4 (0.127 g, 3.33 mmol) was added to a solution of 12b (0.145 g, 0.48 mmol) in 20 mL of dry
dioxane, and the mixture was refluxed until disappearance of the starting material (TLC). After
cooling, the reaction was quenched by adding 10 mL of a saturated solution of sodium sulfate (Na2SO4)
and stirred at rt for 30 min. The mixture was then filtered and the precipitate was washed with 20 mL
of dioxane. The collected filtrates were concentrated and the residue, suspended in 50 mL of distilled
water, was extracted with 3×50 mL of CHCl3. The organic fractions were dried (Na2SO4), filtered and
concentrate under reduced pressure, and the oil residue was dissolved in 5 mL of 1.25 M HCl
methanolic solution and stirred at rt for 4h. After solvent removal, the solid residue was recrystallyzed
from ethyl acetate and few drops of ethanol, to afford compound 14·HCl salt as dark brown solid
1
(0.070 g, Yield 50%). m.p. 241-242 °C. IR (KBr, cm-1): 3230, 1520, 1460, 810, 742, 700. H NMR
(300 MHz, DMSO-d6) δ ppm 9.17 (s, 2H), 7.56 (d, J = 7.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.39-7.08
(m, 6H), 7.07 (t, J = 8.5 Hz, 2H), 4.45-4.25 (m, 4H), 3.40-3.25 (m, 2H), 2.95-2.75 (m, 4H), 1.95-1.75
(m, 2H). 13C NMR (CDCl3): 25.4, 26.5, 31.6, 42.2, 42.8, 46.0, 110.1, 120.9, 121.9, 122.5, 122.7, 126.7,
+
128.0, 128.3 (2 C), 128.5 (2 C), 135.0, 137.3, 139.2 HRMS (ESI) m/z [M + H]+ calcd for C20H23N2 ,
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291.1856; found, 291.1852. Anal. Calcd for C20H23ClN2×H2O: C, 69.63; H, 7.31; N, 8.12. Found: C,
69.85; H, 7.54; N, 8.09.
5.1.4.6.
N-{[(4-Methylphenyl)sulfonyl]oxy}-1,2,3,9-tetrahydro-4H-carbazol-4-imine (15).
DMAP (1.83 g, 15.0 mmol) and, dropwise, a solution of tosyl chloride (1.05 g, 15.0 mmol) in 10 mL of
dry DCM were added to a solution of 9a (1.00 g, 5 mmol) in 15 mL of dry DCM cooled at 0 °C. The
mixture was stirred at rt under N2 atmosphere for 48 h. The reaction mixture was then washed with 1N
HCl (3×20 mL) and brine (3×20 mL), dried (Na2SO4), filtered and concentrated under reduced
pressure, to afford the desired product 15 (0.625 g, Yield 70%), whose analytical data were in good
agreement with those of literature [29]; m.p. 138 °C (lit. 135-138 °C). 1H NMR (300 MHz, DMSO-d6)
δ ppm 10.16 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H), 7.28
(d, J = 8.5 Hz, 2H), 7.08 (t, J = 8.0 Hz, 1H), 7.02 (t, J = 8.0 Hz, 1H), 2.82 (t, J = 6.5 Hz, 1H), 2.70 (t, J
= 6.5 Hz, 1H), 2.33 (s, 3H), 1.94 (quintet, J = 6.5 Hz, 2H). MS (ESI) m/z [M+H]+ 355.
5.1.4.6.
3,4,5,6-Tetrahydroazepino[3,2-b]indol-2(1H)-one (16). Compound 15 (1.37 g, 3.86
mmol) dissolved in 25 mL of chloroform was added to 60 g of Al2O3, previously activated with
hexane, in 100 mL of toluene, and the suspension was stirred at rt for 48 and then refluxed for 8 h.
After cooling, the heterogeneous mixture was filtered and the filtrate concentrated under reduced
pressure, to yield the desired product 16 (0.230 g, Yield 30%), whose analytical data were in good
agreement with those of literature [29]; m.p. 245 °C (lit. 243-244 °C). IR (KBr, cm-1): 3261, 3190,
1648, 783, 754. 1H NMR (300 MHz, DMSO-d6) δ ppm 10.64 (s, 1H), 9.40 (s, 1H), 7.67 (d, J = 7.5 Hz,
1H), 7.17 (d, J = 8.0 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.88 (t, J = 8.0 Hz, 1H), 2.94 (t, J = 6.5 Hz, 2H),
2.43 (t, J = 6.5 Hz, 2H), 2.05-1.95 (m, 2H). MS (ESI): 223 (M+Na)+.
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5.1.4.7.
Methyl 1-(2-phenylethyl)-1H-indole-3-carboxylate (21). NaH (0.10 g, 4.17 mmol) was
added to a solution of methyl indole-3-carboxylate (0.50 g, 2.86 mmol) in 10 mL of dry DMF. After 10
min of stirring at rt, a solution of phenethyl bromide (0.50 mL, 3.43 mmol) in 5 mL of dry DMF was
added dropwise, and the mixture was stirred at rt for 24 h. The reaction mixture was then poured into
200 mL of water, and the aqueous suspension was extracted with 3×30 mL of ethyl acetate. The
collected organic phases were dried (Na2SO4), filtered and concentrated under reduced pressure, and
the oil residue was purified by chromatography on silica gel, using ethyl acetate as the eluent, to afford
20 as white solid (0.250 g, Yield 46%). m.p. 103-105 °C. IR (KBr, cm-1): 1696, 1535, 1267, 1225,
1154, 1096, 775, 752, 738, 703. 1H NMR (300 MHz, DMSO-d6) δ ppm 7.66 (s, 1H), 7.40-7.20 (m,
7H), 7.07 (dd, J1 = 1.5 Hz, J2 = 7.5 Hz, 1H), 4.37 (t, J = 7.0 Hz, 2H), 3.89 (s, 3H), 3.17 (t, J = 8.0 Hz,
2H). MS (ESI): 302 (M+Na)+.
5.1.4.8.
1-(2-Phenylethyl)-1H-indole-3-carboxylic acid (21). 10 mL of 4N NaOH was added to a
solution of 20 (1.0 g, 3.58 mmol) in 20 mL of methanol, and the reaction mixture was stirred overnight
at rt. After concentration under reduced pressure the aqueous suspension was acidified to pH 2, and
then extracted with 3×20 mL of ethyl acetate. The collected organic phases were dried (Na2SO4),
filtered and concentrated under reduced pressure, to yield 21 as colorless oil (0.550 g, Yield 60%). IR
(KBr, cm-1): 3025, 2933, 1661, 1525. 1275, 1177, 748, 698. 1H NMR (300 MHz, DMSO-d6) δ ppm
10.00 (s, 1H), 7.70 (s, 1H), 7.30-7.00 (m, 9H), 4.25 (t, J = 7.0 Hz, 2H), 3.13 (t, J = 7.0 Hz, 2H). MS
(ESI): 264 (M-H)-.
5.1.4.9.
1-(2-Phenylethyl)-N-propyl-1H-indole-3-carboxamide (22). DCC (230 mg, 1.12 mmol)
and HOBt (0.152 g, 1.12 mmol) were added to a solution of 21 (0.300 g, 1.12 mmol) in 10 mL of dry
DCM. After 30 min of stirring at rt, n-propylamine (0.1 mL, 1.12 mmol) was added and the reaction
22

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mixture was stirred at rt overnight and then filtered. The organic phase was washed twice with 10%
m/m Na2CO3, 1N HCl and brine, dried (Na2SO4), filtered and concentrated under reduced pressure. The
oil residue was purified by chromatography on silica gel (eluent: 80% ethyl acetate/20% methanol v/v)
to afford 22 as white solid (0.150 g, Yield 45%). m.p. 98-100 °C. IR (KBr, cm-1): 3383, 1627, 1541,
1223, 740, 695. 1H NMR (300 MHz, DMSO-d6) δ 7.95 (t, J = 7.0 Hz, 1H), 7.50 (s, 1H), 7.35-7.20 (m,
5H), 7.07 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 2H), 5.87 (s br, 1H), 4.36 (t, J = 7.0 Hz, 2H), 3.45 (q, J = 7.0
Hz, 2H), 3.12 (t, J = 7.5 Hz, 2H), 1.69 (quintet, J = 6.0 Hz, 2H), 1.01 (t, J = 7.5 Hz, 3H). HRMS (ESI)
m/z [M+H]+ calcd for C20H23N2O+ 307.1805; found, 307.1800 Anal. Calcd for C20H22N2O·1/4 H2O: C,
77.25; H, 7.29; N, 9.10. Found: C, 77.37; H, 7.43; N, 9.29.
5.2 Cholinesterase inhibition assay
The test compounds were assayed for their inhibitory activity toward AChE and BChE from electric eel
and horse serum, respectively, and human cholinesterases as well (Sigma-Aldrich), following the
Ellman’s method [30]. The BChE activity was determined in a reaction mixture containing 100 µL of a
solution of BChE (0.9 U/mL in 0.1 M pH 8.0 phosphate buffer, PB), 100 µL of a solution of 5,5-dithio-
bis-(2-nitrobenzoic) acid (DTNB 3.3 mM in 0.1 M pH 7.0 PB, containing 0.1 mM NaHCO3), 100 µL
of a solution of the test compound (five to seven concentrations, ranging from 1×10-4 to 1×10-9 M in
0.1 M pH 8.0 PB), and 600 µL of pH 8.0 PB. After incubation for 20 min at 25 °C, butyrylthiocholine
iodide (100 µL of 0.05 mM water solution) was added as the substrate, and the hydrolysis rates of the
substrate monitored at 412 nm for 5.0 min at 25 °C. The concentration of compound which produced
50% inhibition of the BChE activity (IC50) was calculated by nonlinear regression of the
response/concentration (log) curve, by using Prisma GraphPad software (vers. 5.01). AChE inhibitory
activity was determined similarly, by using a solution of AChE (0.415 U/mL in 0.1 M pH 8.0 PB), and
acetylthiocholine iodide (0.05 mM) as the substrate. The inhibition data are reported as means of IC50’s
23

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determined at least in three independent measurements. To determine the type of inhibition for the most
potent BuChE inhibitor 12d, the Lineweaver-Burk eq (1/v vs 1/[S]) was fitted for varying
concentrations of substrates (25−250 ꢀM) in the absence or presence of inhibitor at four different
concentrations, ranging from 0.5 to 5.0 nM, and by using fixed amounts of enzymes (0.09 U×mL-1).
Replotting the slopes of the above plots against the inhibitor 12d concentration (0 nM, r2 = 0.9988; 0.5
nM, r2 = 0.9837; 2.0 nM, r2 = 0.9840; 5.0 nM, r2 = 0.9833) yielded the Ki value as the X-axis intercept.
5.3 Cell cultures and treatment
The human neuroblastoma SH-SY5Y, the human glioblastoma U-87, the human breast carcinoma
MCF-7, and the human liver hepatocellular carcinoma HepG2 cell lines were purchased from ATCC.
Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), 2 mM L-glutamine, 50 U/mL penicillin and 50 ꢀg/mL streptomycin in a humid
atmosphere of 95% air and 5% CO2 at 37 °C, and grown to 80% of confluence. Prior to cell treatment,
the medium was replaced with fresh medium containing test compounds in the range 0.1-500 ꢀM on U-
87, MCF-7 and HepG2 cells. Toxicity studies on SH-SY5Y cells were carried out using a reduced
serum medium (2% FBS), and the test compounds were prepared as a stock solution of 20 mM in
DMSO and were used at concentrations ranging from 0.1 to 1000 ꢀM. In the cytoprotection assay, SH-
SY5Y cells were co-incubated with NMDA at a concentration of 250 ꢀM and test compounds at six
different concentrations (0.1-50 ꢀM).
5.4 Cell viability
The number of living cells was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. Briefly, 100 ꢀL of cell suspension were plated in 96-well plates at a density of
~5,000 cells/well. After 1 day incubation at 37 °C in a humid atmosphere with 5% CO2, the culture
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medium was replaced with 150 ꢀL of fresh medium or medium containing different concentrations of
the test compounds. Untreated cells were used as positive control and cells incubated with a 2% (w/v)
SDS solution were used as negative control. After the incubation period of 48 h, 10 ꢀL of a 0.5% (w/v)
MTT/PBS solution were added to each well and the incubation was prolonged further for 4 h. After this
period, medium was removed and replaced with 150 ꢀL of a DMSO/ethanol (1/1 v/v) solution per well.
The absorbance of the individual well was measured by a microplate reader (Wallac Victor3, 1420
Multilabel Counter, Perkin-Elmer). Each compound concentration was tested in triplicate, and results
presented as percentage of the control value.
For the neuroprotection assay, SH-SY5Y cells were seeded in 96-well plates at the same density used
for cytotoxicity assay. Cells were exposed for 48 h to NMDA at a final concentration of 250 ꢀM in
reduced-serum medium in the absence or presence of test compounds at concentrations ranging from
0.1 to 50 ꢀM at 37 °C. Cell viability was also measured by the MTT assay.
Data are presented as the mean ± SEM. Statistical comparisons were performed by one-way ANOVA
followed by multiple comparison tests (Dunnett’s test) using the statistical package in the GraphPad
Prism software vers. 5.01; values of P < 0.05 were considered statistically significant.
Conflict of interest
We declare that we have no conflict of interest.
Appendix A. Supplementary data
Supplementary data related to this article can be found at …
Acknowledgements
The financial support from MIUR, Italy (Grant PRIN 2009ESXPT2_005) is gratefully acknowledged.
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