Design, synthesis and biological evaluation of new phthalimide and saccharin derivatives with alicyclic amines targeting cholinesterases, beta-secretase and amyloid beta aggregation

Article abstract of DOI:10.1016/j.ejmech.2016.09.078

The complexity of Alzheimer's disease (AD) calls for search of multifunctional compounds as potential candidates for effective therapy. A series of phthalimide and saccharin derivatives linked by different alicyclic fragments (piperazine, hexahydropyrimidine, 3-aminopyrrolidine or 3-aminopiperidine) with phenylalkyl moieties attached have been designed, synthesized, and evaluated as multifunctional anti-AD agents with cholinesterase, β-secretase and β-amyloid inhibitory activities. In?vitro studies showed that the majority of saccharin derivatives with piperazine moiety and one phthalimide derivative with 3-aminopiperidine fragment exhibited inhibitory potency toward acetylcholinesterase (AChE) with EeAChE IC₅₀values ranging from 0.83?μM to 19.18?μM. The target compounds displayed inhibition of human β-secretase-1 (hBACE1) ranging from 26.71% to 61.42% at 50?μM concentration. Among these compounds, two multifunctional agents (26, [2-(2-(4-benzylpiperazin-1-yl)ethyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide] and 52, 2-(2-(3-(3,5-difluorobenzylamino)piperidin-1-yl)ethyl)isoindoline-1,3-dione) have been identified. Compound 26 exhibited the highest inhibitory potency against EeAChE (IC₅₀?=?0.83?μM) and inhibitory activity against hBACE1 (33.61% at 50?μM). Compound 52 is a selective AChE inhibitor (IC_{50 AChE}?=?6.47?μM) with BACE1 inhibitory activity (26.3% at 50?μM) and it displays the most significant Aβ anti-aggregating properties among all the obtained compounds (39% at 10?μM). Kinetic and molecular modeling studies indicate that 26 may act as non-competitive AChE inhibitor able to interact with both catalytic and peripheral active site of the enzyme.

Full text of DOI:10.1016/j.ejmech.2016.09.078

Accepted Manuscript
Design, synthesis and biological evaluation of new phthalimide and saccharin
derivatives with alicyclic amines targeting cholinesterases, beta-secretase and
amyloid beta aggregation
Dawid Panek, Anna Więckowska, Tomasz Wichur, Marek Bajda, Justyna Godyń,
Jakub Jończyk, Kamil Mika, Jana Janockova, Ondrej Soukup, Damijan Knez, Jan
Korabecny, Stanislav Gobec, Barbara Malawska
PII:
S0223-5234(16)30816-9
10.1016/j.ejmech.2016.09.078
EJMECH 8944
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 August 2016
Revised Date: 23 September 2016
Accepted Date: 24 September 2016
Please cite this article as: D. Panek, A. Więckowska, T. Wichur, M. Bajda, J. Godyń, J. Jończyk, K.
Mika, J. Janockova, O. Soukup, D. Knez, J. Korabecny, S. Gobec, B. Malawska, Design, synthesis
and biological evaluation of new phthalimide and saccharin derivatives with alicyclic amines targeting
cholinesterases, beta-secretase and amyloid beta aggregation, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.078.
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ACCEPTED MANUSCRIPT
BACE-1
interactions
PAS
interactions
AChE IC50 = 0.83 μM
BACE1 inh. = 33.61% (50μM)
Aβ1-42 aggr. inh. = 18.6% (10μM)
CAS
interactions

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of new phthalimide
and saccharin derivatives with alicyclic amines targeting
cholinesterases, beta-secretase and amyloid beta aggregation
a,1
a,1
a
a
a
Dawid Panek , Anna Więckowska , Tomasz Wichur , Marek Bajda , Justyna Godyń ,
a
a
b
b
c
Jakub Jończyk , Kamil Mika , Jana Janockova , Ondrej Soukup , Damijan Knez , Jan
b
c
a
Korabecny , Stanislav Gobec , Barbara Malawska *
a
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian
University Medical College, Kraków, Poland; mfmalaws@cyf-kr.edu.pl
b
Biomedical Research Centre, University Hospital Hradec Kralove, Czech Republic;
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana,
Ljubljana, Slovenia
*
Correspondence: mfmalaws@cyf-kr.edu.pl
These authors contributed equally to this work.
1
Abstract
The complexity of Alzheimer’s disease (AD) calls for search of multifunctional compounds
as potential candidates for effective therapy. A series of phthalimide and saccharin derivatives
linked by different alicyclic fragments (piperazine, hexahydropyrimidine, 3-aminopyrrolidine
or 3-aminopiperidine) with phenylalkyl moieties attached have been designed, synthesized,
and evaluated as multifunctional anti-AD agents with cholinesterase, β-secretase and β-
amyloid inhibitory activities. In vitro studies showed that the majority of saccharin derivatives
with piperazine moiety and one phthalimide derivative with 3-aminopiperidine fragment
exhibited inhibitory potency toward acetylcholinesterase (AChE) with EeAChE IC values
5
0
ranging from 0.83 µM to 19.18 µM. The target compounds displayed inhibition of human β-
secretase-1 (hBACE1) ranging from 26.71% to 61.42% at 50 µM concentration. Among these
compounds,
two
multifunctional
agents
(26,
[2-(2-(4-benzylpiperazin-1-
and 52, 2-(2-(3-(3,5-
yl)ethyl)benzo[d]isothiazol-3(2H)-one
1,1-dioxide]
difluorobenzylamino)piperidin-1-yl)ethyl)isoindoline-1,3-dione) have been identified.
Compound 26 exhibited the highest inhibitory potency against EeAChE (IC = 0.83 µM) and
50
inhibitory activity against hBACE1 (33.61% at 50 µM). Compound 52 is a selective AChE
1

ACCEPTED MANUSCRIPT
inhibitor (IC50 AChE = 6.47 µM) with BACE1 inhibitory activity (26.3% at 50 µM) and it
displays the most significant Aβ anti-aggregating properties among all the obtained
compounds (39% at 10 µM). Kinetic and molecular modeling studies indicate that 26 may act
as non-competitive AChE inhibitor able to interact with both catalytic and peripheral active
site of the enzyme.
Key words: Alzheimer’s disease; multifunctional agents; acetylcholinesterase inhibitors;
BACE1 inhibitors; Aβ aggregation; molecular docking
1
.
Introduction
Alzheimer's disease (AD) is a chronic, progressive form of dementia that constitutes 50–75%
of all the cases. Patients with AD are primarily the elderly, 84% of them are over 74 years old.
According to the latest data, the number of people living with dementia today is estimated at
about 40 million worldwide, and this number is expected to triple by 2050 [1]. These numbers
are correlated with the observed aging of population, especially in the developed countries of
Europe and North America [2]. The analysis of epidemiological data does not allow drawing
optimistic conclusions for the future, especially in the light of the lack of the effective
treatment of the disease.
AD is a complex neurodegenerative disease with multifaceted pathomechanism, which
despite significant progress in the field, remains unclear [3]. Researchers came up with many
hypotheses that try to explain the disease process. Currently, there are few well recognized
and acceptable theories, one of which is β-amyloid hypothesis [4]. As the major cause of the
disease it indicates the accumulation of aberrant, misfolded β-amyloid (Aβ) peptide in the
central nervous system (CNS) [5,6]. Aβ is a toxic polypeptide built from 37–43 amino acids
that is formed by a proteolysis of the amyloid precursor protein (APP). APP can be
metabolized in two pathways: non-amyloidogenic and amyloidogenic. In the latter, APP is
firstly cleaved by β-secretase (β-site APP-cleaving enzyme 1, BACE1) into a soluble amyloid
precursor protein beta (sAPPβ) and polypeptide C-99. Subsequently γ-secretase cleaves
polypeptide C-99, yielding fibrillogenic β-amyloid peptides, that consist of 37–43 amino
acids [7–9]. The most toxic of them, 42-unit fragments, are able to aggregate and accumulate
extracellular, forming Aβ plaques and leading to multidirectional neurotoxicity and neuron
death. Exposition to Aβ causes oxidative stress and inflammatory damage [10,11]. Moreover,
some evidence indicate that intracellular tangles of tau proteins accompanying AD could be
created as a response to the formation of Aβ [12]. However, researchers are not sure which
2

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comes first, “the chicken or the egg” since there are as many reports suggesting primary role
of tau protein in the pathogenesis of AD [13–15]. Nonetheless, there is no doubt about the
critical role of Aβ in the ethiopathogenesis and development of AD. Therefore, preventing the
formation of Aβ seems to be the reasonable objective for treating of AD. If we analyze a
process of Aβ formation we can assume that BACE1, which is involved in the first and rate-
limiting step of this process, is particularly attractive biological target [16,17].
Cholinergic hypothesis assumes that memory impairments in AD are associated with
the damage of cholinergic neurons in the CNS [18,19]. This results in a reduction of
cholinergic neurotransmission leading to decline in memory, problems with communication,
time and space disorientation and others. In a healthy brain, the action of acetylcholine (ACh)
is terminated mainly by acetylcholinesterase and additionally by butyrylcholinesterase
(AChE, E.C. 3.1.1.7, and BuChE, E.C. 3.1.1.8, respectively). However, with the progression
of AD, the level of AChE significantly decreases. The activity of BuChE, on the other hand,
is increased, especially in hippocampus and temporal cortex [7,20]. Although AChE is
recognized as a symptomatic drug target, numerous studies have shown that this enzyme
plays also non-enzymatic roles in neurite growth, differentiation, adhesion and also synaptic
maintenance. Furthermore, according to some in vitro studies, AChE can initiate the
formation of Aβ fibrils and Aβ plaques [21,22]. This process is mediated by the interaction
between Aβ and the peripheral anionic site (PAS) of AChE. These findings led to the
development of dual-binding site AChE inhibitors, able to block both catalytic active site
(CAS) and peripheral binding site (PAS), as potential anti-AD agents [23–25].
Currently there are only four drugs used in the pharmacotherapy of AD: donepezil,
rivastigmine, galantamine and memantine [26]. Three of them are AChE inhibitors that
improve cholinergic neurotransmission in the brain by inhibition of acetylcholine degradation
[
(
27,28]. Memantine is used complementarily and acts by blocking N-methyl-D-aspartate
NMDA) receptors. These drugs do not cure the disease but only delay the progression of its
symptoms [29]. This makes AD treatment one of the biggest unmet medical needs and one of
the biggest challenges for pharmaceutical research. Regarding current status of anti-AD drugs
in clinical trials (phase II and III) most of them focus on β-amyloid as biological target. These
include vaccines, antibodies and inhibitors or modulators of γ- and β-secretases. It is
interesting to note that among small molecules in ongoing phase III clinical trials, two
compounds belong to the class of non-peptidomimetic inhibitors of BACE1 [30].
3

ACCEPTED MANUSCRIPT
Diseases with multifactorial pathophysiology, such as AD, may require treatment that
modulates more than one biological target simultaneously (polypharmacology). An alternative
approach for traditional cocktail of drugs and multicomponent drugs is the strategy that
utilizes multi-target-directed-ligands (MTDLs). MTDLs are compounds that can act on two or
more independent biological targets. Over the last years, a number of multifunctional ligands
for the potential treatment of AD have been developed [31–33]. Most of them are AChE
and/or BuChE inhibitors endowed with some additional biological properties such as: Aβ-
aggregation inhibition, antioxidant or metal-chelating activity. Although many of these
molecules showed promising activity in vitro, only some were active in vivo in preclinical
(
ladostigil [34], bis-(7)-tacrine [35] and memoquin [36]) or even clinical studies (ladostigil)
34,37–39].
[
Among MTDL, compounds with inhibitory activity towards symptomatic and disease
modifying targets - cholinesterases and BACE1 - seem to be of a special interest. In Figure 1
we present few examples of such compounds (I–IV) with balanced potencies against both
biological targets [40–43].
F
O
O
N
N
H
N
Cl
F
O
N
N
N
H
H
O
N
I
II
hAChE IC50= 7.16 nM
BACE1 IC50= 0.099 µM
hAChE IC50= 7.7 µM
EqBuChE IC50 = 2.5 µM
hBACE1 IC50= 0.6 µM
Cl
Br-
N+
N
O
O
O
O
O
H
N
N
8
N
H
H
O
OH
OH
III
IV
hAChE K = 81.0 nM
hAChE IC50= 2.39 nM
i
hBuChE K = 93.0 nM
BACE1 IC50= 0.31 µM
hBuChE IC50 = 513 nM
hBACE1 IC50= 80 nM
i
Figure 1. Previously described, selected MTDLs, inhibitors of AChE and BACE1.
In this paper we describe the design, synthesis and biological evaluation of
phthalimide or saccharin derivatives linked by different alicyclic fragments with
phenylalkylmoieties, as novel multi-target anti-AD agents. In vitro evaluation of the
4

ACCEPTED MANUSCRIPT
compounds included the assessment of AChE, BuChE, BACE1 and Aβ aggregation inhibitory
activity. Furthermore, molecular modeling studies were carried out to investigate binding
mode of the compounds with AChE and BACE1 and structure-activity relationship of these
new compounds.
2
.
Results and discussion
2
.1. Design
Our research interests focus on multi-functional compounds against Alzheimer’s disease [44–
7]. Based on previous experience we aimed at developing new MTDLs with cholinesterases
4
and BACE1 inhibitory activity as well as Aβ anti-aggregation activity. Recently, we have
reported two lead structures derived from donepezil - compounds V and VI (Figure 2).
Compound V [45] was found to be a potent and selective human AChE inhibitor (IC =
5
0
0
.268 µM) with anti-Aβ aggregation activity (65.96% at 10 µM) and neuroprotective effect
against Aβ toxicity at 1 µM and 3 µM. Compound VI [47] was the most potent and selective
human AChE inhibitor in the series (IC = 33 nM) with the ability to inhibit Aβ aggregation
5
0
(22.19% at 10 µM). Molecular modeling showed that compounds V and VI bind
concomitantly to both catalytic and peripheral active site of AChE. The phthalimide or
saccharin fragments were engaged in the interactions with PAS while the benzylamine
fragment created interactions with amino acids of CAS of AChE. Starting with these findings,
we modified structures of compounds V and VI with an idea of expanding their biological
activity on BACE1 (Figure 2). Instead of a simple alkyl chain we introduced
hexahydropyrimidine, piperazine or 3-aminopiperidine as diaminoalkyl motives. We assumed
that these alicyclic amines containing two basic nitrogen atoms would provide interactions
with the catalytic dyad of BACE1 that is composed of two aspartic acid residues: Asp32 and
Asp228. Piperazine and pyrimidine-diamine fragments were reported previously as
responsible for the interactions with BACE1 [43,48]. Benzylamine fragment was modified by
the introduction of one or two fluorine atoms that are abundant in BACE1 inhibitors [49].
5

ACCEPTED MANUSCRIPT
hAChE IC50 = 268 nM
Aβ = 65.96% (10 μM)
4
0
V
hAChE IC50 = 33 nM
Aβ = 22.19% (10 μM)
4
0
VI
β-secretase inhibition
R = 2-F, 3,5-diF
n1 = 1,2
n2 = 2,3,4,5
x = =CO / =SO2
Figure 2. Design strategy of new phthalimide and saccharin derivatives with alicyclic
amines targeting cholinesterases, BACE1 and Aβ aggregation.
2
.2. Chemistry
The general procedure for the synthesis of the target compounds 23–48 is presented in
Scheme 1. Compounds 1–6 were prepared by alkylation of saccharin and phthalimide salts
with the appropriate α,ω-dibromoalkanes. N-phenyl-alkyl-piperazine derivatives (7–12) were
obtained in a reaction of N-alkylation of piperazine with the appropriate fluoro-substituted
phenyl-alkyl halides. Hexahydropyrimidine derivatives (15 and 16) were synthesized in a
two-step pathway. In the first step, propane-1,3-diamine reacted with 1-(chloromethyl)-2-
6

ACCEPTED MANUSCRIPT
fluorobenzene or 1-(bromomethyl)-3,5-difluorobenzene to give compounds 13 and 14. In the
next step, the reaction of 13 and 14 with formaldehyde led to the formation of the
hexahydropyrimidine ring and gave compounds 15 and 16. To obtain piperidine-3-amine
derivatives 20–22 at first tert-butyl piperidin-3-ylcarbamate was alkylated with fluorine-
substituted or non-substituted benzyl halides to give intermediates 17–19. Boc-deprotection of
1
7–19 resulted in primary amines 20–22. Finally, compounds 1–6 were used as alkylating
agents in the reaction of nucleophilic substitution with N-phenyl-alkyl-heterocyclic
derivatives (7–12, 15, 16, 20–22) and provided products 23–48.
Cmp.
R
2-F
3,5-diF
2-F
H
2-F
3-F
4-F
3,5-diF
H
n
1
1
n
1
2
X
23
24
25
C=O
C=O
C=O
S=(O)2
S=(O)
S=(O)
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
-
1
1
1
1
1
1
1
2
3
4
5
2
3
4
5
1
1
-
O
O
1, X = C=O, n
2
= 1
26
27
28
29
30
31
2, X = S(=O)
3, X = S(=O)
4, X = S(=O) , n = 3
, n
, n
= 1
= 2
-
+
i
Br
2
2
2
N Me
N
X
n
2
2
2
2
X
S=(O)2
2
2
5
, X = S(=O) , n = 4
S=(O)
S=(O)
S=(O)
S=(O)
S=(O)
S=(O)
S=(O)
S=(O)
S=(O)
S=(O)
S=(O)
C=O
2
2
2
2
2
2
2
2
2
2
2
2
2
6
, X = S(=O) , n = 5
2
2
32
33
34
35
36
37
38
39
H
H
H
2-F
2-F
2-F
2-F
H
2-F
2-F
3,5-diF
2-F
3,5-diF
H
X
N
ii
iii
NH
NH
N
n2
R
R
HN
N
N
O
40
41
42
43
n
1
n
1
-
-
-
1
2
2
2
-
-
-
-
-
-
-
C=O
2
3 - 40
S=(O)
S=(O)
C=O
C=O
C=O
2
2
7
8
9
1
1
1
, R = 2-F, n1 = 1
, R = 3-F, n1 = 1
, R = 4-F, n1 = 1
0, R = 3,5-diF, n1 = 1
1, R = H, n1 = 2
44
45
46
47
48
H
2-F
3,5-diF
C=O
2, R = 2-F, n1 = 2
X
N
iv
v
vi
H
2
N
NH
2
N
NH
2
N
NH
N
N
R
H
R
R
O
1
1
3, R = 2-F
4, R = 3,5-diF
15, R = 2-F
16, R = 3,5-diF
4
1 - 44
vii
viii
O
O
R
R
HN
N
N
N
H
O
N
O
NH2
H
O
n1 = 1
17, R = H
8, R = 2-F
9, R = 3,5-diF
20, R = H
21, R = 2-F
22, R = 3,5-diF
n
1
1
R
ix
N
N
1
N
H
O
4
4
5, n1 = 0
6-48, n1 = 1
R
N
NH
2
Scheme 1. Synthesis of compounds 23–48. Reagents and conditions: i) α,ω-dibromoalkane,
acetonitrile, K CO , 80 °C, 24 h; ii) phenyl-alkyl-halide derivative, acetonitrile, R.T., 24 h;
2
3
iii) compounds 1–6, acetonitrile, K CO , 80 °C, 24 h; iv) 1-(chloromethyl)-2-
2
3
fluorobenzene/1-(bromomethyl)-3,5-difluorobenzene, acetonitrile, 0 °C/R.T., 20 h; v)
formaldehyde, acetonitrile/water, R.T., 1 h; vi) compounds 1 or 2, DMF, K CO , 110 °C, 24
2
3
7

ACCEPTED MANUSCRIPT
h; vii) benzyl halide derivative, acetonitrile, K CO , R.T., 44 h; viii) HCl, methanol, R.T., 24
2
3
h; ix) 1, acetonitrile, K CO , 80 °C, 24 h.
2
3
To obtain N-benzylpiperidine-3-amine derivatives 51 and 52 we utilized method depicted in
Scheme 2. At first, tert-butyl piperidin-3-ylcarbamate was alkylated with 2-(2-
bromoethyl)isoindoline-1,3-dione. The resulting compound 49 was then Boc-deprotected and
the obtained primary amine 50 was alkylated by 1-(chloromethyl)-2-fluorobenzene or 1-
(bromomethyl)-3,5-difluorobenzene to give final products 51 and 52.
The method described above failed in case of saccharin derivatives 64 and 65. One of
the semi-products contained a primary amine group and a saccharine moiety, what caused
self-aminolysis and, as a consequence, an opening of the saccharin ring. Therefore, we
developed a synthetic pathway where we introduced a saccharin moiety in the last possible
reaction step (Scheme 2). First, tert-butyl piperidin-3-ylcarbamate was alkylated with 2-
bromoethyl acetate to give compound 53. Deprotection and subsequent alkylation of the
resulting primary amine with either 1-(chloromethyl)-2-fluorobenzene or 1-(bromomethyl)-
3
,5-difluorobenzene gave compounds 54 and 55. In the next step secondary amine groups of
compounds 54 and 55 were Boc-protected in the reaction with di-tert-butyl dicarbonate. The
resulting esters 56 and 57 were then hydrolyzed to alcohols 58 and 59 which were then
reacted with methanesulfonyl chloride in anhydrous dichloromethane in the presence of
triethylamine. The obtained mesylates 60 and 61 were used to alkylate sodium saccharin to
get compounds 62 and 63. The final products 64 and 65 in form of hydrochloride salts were
obtained by Boc-deprotection with HCl in ethyl acetate. Purity and structures of the obtained
compounds were confirmed by chromatographic (TLC, LC-MS) and spectroscopic (NMR)
methods.
8

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of compounds 51, 52, 64 and 65. Reagents and conditions: i) 2-(2-
bromoethyl)isoindoline-1,3-dione, acetonitrile, K CO , NaI, 80 °C, 24 h, ii) TFA,
2
3
dichloromethane, R.T., 4 h, iii) 1-(chloromethyl)-2-fluorobenzene/1-(bromomethyl)-3,5-
difluorobenzene, acetonitrile, K CO , 80 °C, 24 h, iv) 2-bromoethyl acetate, acetonitrile,
2
3
K CO , 80 °C, 24 h; v) TFA, dichloromethane, R.T., 2 h; vi) di-tert-butyl dicarbonate, TEA,
2
3
dichloromethane, R.T., 2 h; vii) methanol-water mixture, K CO , 65 °C, 2 h, viii)
2
3
methanesulfonyl chloride, dichloromethane, TEA, R.T., 2 h; ix) sodium saccharin,
acetonitrile, K CO , 80 °C, 24 h; x) 1M HCl in ethyl acetate, R.T., 24 h.
2
3
2
2
.3. Biological activity
.3.1. Inhibition of cholinesterases and BACE1
We determined the cholinesterase inhibitory profiles of the synthesized derivatives using the
method established by Ellman et al [50]. Compounds were evaluated against AChE from
electric eel (EeAChE) and BuChE from equine serum (EqBuChE). In the first step, we
9

ACCEPTED MANUSCRIPT
performed assays using a 10 µM screening concentration of the tested compounds. We
determined IC value for compounds which exhibited inhibitory potency higher than 50% at
5
0
1
0 µM. Tacrine and donepezil were used as the reference compounds. Results of the
biological evaluation are presented in Table 1.
All the compounds were also evaluated against human recombinant BACE1
(hBACE1). A biochemical spectrofluorometric assay (fluorescence resonance energy transfer,
FRET-based), which utilizes the cleavage of a peptide substrate mimicking the human APP
sequence with the Swedish mutation, was used [17]. As a reference we used well-known
inhibitor of BACE1 – Inhibitor IV – for which we established the IC value of 0.046 µM
5
0
that is in accordance with the published data [51]. The compounds were tested at 50 µM
concentration and their activities were reported as percentage of hBACE1 inhibition (Table
1
).
Table 1. Inhibition of EeAChE, EqBuChE, hBACE1 by compounds 23–48, 51, 52, 64
a
and 65.
EeAChE
EqBuChE
hBACE1
Cmp.
R
X
n1
n2
IC [µM]/
IC [µM]/
50
50
d
%
inh.
b
c
%
inh.
% inh.
piperazine derivatives
2
2
2
2
2
2
2
3
3
3
3
4
5
6
7
8
9
0
1
2
2-F
=CO
1
1
1
1
1
1
1
1
2
3
1
1
2
1
1
1
1
1
1
1
41.82% ± 2.83
<10%
<10%
38.06% ± 7.98
45.00% ± 6.73
48.36% ± 7.44
33.61% ± 3.27
36.01% ± 2.01
28.61% ± 4.01
3,5-diF =CO
14.22% ± 4.21
20.28% ± 4.09
22.15% ± 2.11
16.12% ± 6.60
<10%
2-F
H
=CO
<10%
=S(O)
=S(O)
=S(O)
=S(O)
0.830 ± 0.02
17.80 ± 0.12
2.14 ± 0.08
6.28 ± 0.30
19.18 ± 0.11
2
2
2
2
2
2
2
2-F
3-F
4-F
47.49% ± 0.77 36.00% ± 10.09
3,5-diF =S(O)
11.00% ± 1.63
42.03% ± 4.78
50.70% ± 3.45
37.31% ± 5.21
H
H
=S(O)
=S(O)
34.16% ± 2.49 11.74% ± 2.20
3.01 ± 0.09 41.45% ± 0.99
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0

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
4
3
4
5
6
7
8
9
0
H
=S(O)
=S(O)
=S(O)
=S(O)
=S(O)
=S(O)
=S(O)
=S(O)
2
2
2
2
2
2
2
2
4
5
2
3
4
5
1
1
1
1
1
1
1
1
2
2
43.06% ± 2.43 30.39% ± 2.06
41.74% ± 1.03 41.8% ± 2.68
21.74% ± 0.23
45.44% ± 5.79
32.74% ± 4.96
28.84% ± 9.11
39.66% ± 3.33
36.17% ± 9.82
36.55% ± 4.08
36.71% ± 3.28
H
2-F
2-F
2-F
2-F
H
<10%
4.25 ± 0.17
7.36 ± 0.18
8.43 ± 0.28
<10%
12.01% ± 1.79
29.56% ± 4.46
21.94% ± 3.73
31.57% ± 3.79
18.43% ± 1.45
11.32% ± 3.61
2-F
<10%
hexahydropyrimidine derivatives
4
4
4
4
1
2
3
4
2-F
=CO
-
-
-
-
-
-
-
-
<10%
<10%
27.76% ± 3.91
<10%
31.72% ± 1.20
28.04% ± 4.37
32.96% ± 6.37
26.71% ± 4.76
3,5-diF =CO
2-F =S(O)
3,5-diF =S(O)
18.40% ± 2.84 21.60% ± 4.12
25.42% ± 3.68 16.41% ± 3.61
2
2
3
-aminopyrrolidine derivative
25.7% ± 1.36 21.38% ± 2.66
-aminopiperidine derivatives (Series A)
4
5
H
=CO
3
0
-
36.01% ± 2.61
4
4
4
6
7
8
H
=CO
=CO
1
1
1
-
-
-
<10%
18.95% ± 4.61 28.34% ± 5.37
<10% 14.19% ± 1.22
16.04% ± 2.03
37.04% ± 1.38
36.32% ± 3.82
36.34% ± 1.57
2-F
3,5-diF =CO
3
-aminopiperidine derivatives (Series B)
5
5
6
6
1
2
4
5
2-F
3,5-diF =CO
2-F =S(O)
3,5-diF =S(O)
=CO
-
-
-
-
-
-
-
-
15.28% ± 4.61
6.47 ± 0.25
<10%
<10%
20.01% ± 1.43
<10%
45.01% ± 1.38
26.30% ± 1.61
30.00% ± 6.40
61.42% ± 2.64
2
2
<10%
<10%
reference compounds
f
Tacrine
-
-
-
-
-
-
-
-
-
-
-
-
0.023±0.0004
0.011±0.0002
0.002±0.0001
1.83±0.04
n.d.
f
Donepezil
Inhibitor IV
n.d.
e
f
f
g
n.d.
n.d.
0.046 ± 0.001
a) Values are expressed as means ± the standard error of the mean (SEM) of at least
three experiments (n = 3), each performed in triplicate (EeAChE, EqBuChE and
hBACE1 inhibition).
b) IC inhibitory concentration of AChE from electric eel or percent inhibition with
5
0
inhibitor at 10 µM.
c) IC inhibitory concentration of BuChE from horse serum or percent inhibition with
5
0
inhibitor at 10 µM.
d) IC50 inhibitory concentration of human recombinant hBACE1 and substrate (Rh-
EVNLDAEFK-quencher) or percent inhibition with inhibitor at 50 µM.
1
1

ACCEPTED MANUSCRIPT
e) Calbiochem, Merck; Nottingham, UK
f) Not determined
g) IC value
5
0
We found that nine of piperazine and one of 3-aminopiperidine derivatives showed
activity against EeAChE with IC values ranging from 0.830 µM to 19.18 µM. Their potency
5
0
was comparable with the literature value of IC for galantamine (IC = 0.623–0.665 µM)
5
0
50
[
52,53] but lower in comparison with compounds V and VI as well as references tacrine and
donepezil. Other compounds were rather weak inhibitors (18.40% to 43.06% inhibition at 10
µM) or were deprived of activity. From the presented data several conclusions can be drawn
regarding: (1) the presence of saccharin or phthalimide, (2) length of the carbon linker
between saccharin/phthalimide moiety and alicyclic amine ring, (3) various effects of
substitution at the phenyl ring. As the majority of the active compounds belong to piperazine
derivatives, it can be concluded that among the tested alicyclic fragments, piperazine is the
optimal one. Furthermore, all the potent piperazine derivatives contain saccharin which is
superior to phthalimide. The effect of the length of carbon linker between saccharin and
piperazine fragment depends on the substitution pattern at the phenyl ring. Among the
unsubstituted compounds 26, 31–34, the most active were 26 with ethylene linker and 32 with
buthylene linker. Within 2-fluorine substituted compounds 27, 35–38 the most potent were
3
6, 37 and 38 with buthylene, pentylene and hexylene linkers, respectively. The most
significant decrease in activity was observed for compounds 31 and 35 with propylene
linkers. The most potent compound was 26 with unsubstituted phenyl ring. None of the
modifications within the phenyl ring (2-F, 3-F, 4-F, 3,5-di-F) brought beneficial effect to the
inhibitory potency. The slightest decrease of potency was observed for 3-F and 4-F
substituted compounds 28 and 29. A major decrease of activity was noticed when the linker
connecting piperazine and phenyl ring was elongated from methylene to ethylene (26 vs. 39
and 27 vs. 40). It is worth noting that the rearrangement of 3-aminopiperidine fragment by
setting the secondary amine in the direction of benzylamine brought activity to 3,5-difluoro
substituted compound 52 (Figure 3). All of the EeAChE inhibitors were selective for this
enzyme since none of the tested compounds reached 50% of inhibition at the screening
concentration (10 µM) against EqBuChE.
1
2

ACCEPTED MANUSCRIPT
Figure 3. Comparison of EeAChE pIC values of the most active compounds.
5
0
Inhibitory potencies of the tested compounds towards hBACE1 were ranging from
1.74% to 61.42% at 50 µM concentration with the most potent derivative of saccharine 65
2
bearing 3-aminopiperidine core and 3,5-difluoro substituted N-benzylamine moiety.
Considering rather similar results for all the compounds it is hard to discuss their structure-
activity relationship.
2
.3.2. Kinetic study of AChE inhibition
As compound 26 showed the highest potency toward EeAChE, it was selected as a
representative for kinetic studies to gain insight into the mode of inhibition. Analysis of the
Lineweaver-Burk reciprocal plot (1/V versus 1/S) (Figure 4A) showed that compound 26
displays linear non-competitive inhibition, as increased slopes and preserved intercepts at
increasing concentrations of the inhibitor were observed. Non-competitive type of inhibition
was further confirmed by Cornish – Bowden plot (S/V versus [I]) (Figure 4B). Non-
competitive inhibition indicates preferential interactions with the PAS rather than the CAS of
the enzyme and is also characteristic for donepezil [54].
1
3

ACCEPTED MANUSCRIPT
Compound 26
Compound 26
Figure 4. Lineweaver-Burk (A) and Cornish-Bowden (B) plots illustrating non-competitive
EeAChE inhibition by compound 26. S = acetylthiocholine; V = initial velocity rate;
I = inhibitor concentration.
2
.3.3. Inhibition of Aβ1-42 aggregation
The ability of the target compounds to inhibit Aβ aggregation was tested in Thioflavin-T
based assay [55]. As a screening concentration we used 10 µM, the same as in Ellman’s
assay. Although donepezil has been described as a weak inhibitor in this assay [45] we tested
it for the comparison with our compounds which were structurally derived from this drug.
Most of the tested compounds showed inhibitory activity less than 10%, but for five of them
(23, 27, 40, 44 and 52) we found activities ranging from 13.3% to 39.0%. These activities are
comparable or higher with that of donepezil but not so high as our previous inhibitor (V) [45].
Table 2. Inhibition of Aβ-aggregation by compounds 23, 27, 40, 44 and 52.
Aβ aggregation
Compound
a,b
%
inh
2
2
4
4
5
3
7
0
4
2
13.3 ± 6.5
18.6 ± 7.4
15.0 ± 2.7
13.5 ± 3.6
39.0 ± 3.4
13.80 ± 6.8
Donepezil
a) % Inhibition of Aβ1–42 aggregation (10 µM compound concentration, 1.5 µM Aβ1–42).
Experiment was performed in quadruplicates.
b) (p<0.05) statistically different compared to control experiments (Aβ1-42 alone); one-
way analysis of variance (ANOVA), followed by post hoc Bonferroni t-test
(SigmaPlot v 12.0).
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4

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2
.3.4. Blood-Brain Barrier (BBB) permeation
The CNS is the expected site of action of the compounds presented here therefore it is
essential to know if they permeate the BBB. The ability of the selected representative
compounds to cross BBB was studied using a parallel artificial membrane permeability assay
(PAMPA) [56,57]. Nine standard drugs with known BBB accessibility were used as the
–6
–1
references. The following ranges of permeability (P 10 cm s ) were established: P > 4.0
e
e
for compounds with predicted high BBB permeability, P ≤ 2.0 for compounds with predicted
e
low permeability and 2.0 > P ≥ 4.0 for compounds with uncertain BBB permeability (Table
e
3
). Compounds 32, 36 and 37 with the values of P over 4.0 showed high probability to cross
e
the BBB via passive diffusion. Compounds 26 and 29 fall into the interval of P values
e
between 2.0 and 4.0 therefore their ability to permeate through BBB is uncertain.
Table 3. In vitro blood-brain barrier permeability prediction for compounds: 26, 29, 32, 36
and 37.
BBB penetration estimation
Compound
–
6
–1
a
P ± SEM (*10 cm s ) CNS (+/–)
e
2
2
3
3
3
6
9
2
6
7
3.75 ± 0.7
3.65 ± 0.1
10.6 ± 1.1
11.0 ± 1.5
9.6 ± 1.4
CNS (+/–)
CNS (+/–)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+/–)
CNS (+/–)
CNS (–)
CNS (–)
Donepezil
Rivastigmine
Tacrine
Testosterone
Chlorpromazine
Hydrocortisone
Piroxicam
7.3 ± 0.9
6.6 ± 0.5
5.3 ± 0.19
11.3 ± 1.6
5.6 ± 0.6
2.85 ± 0.1
2.2 ± 0.15
1.07 ± 0.18
1.02 ± 0.37
Theophylline
Atenolol
a
-6
–1
CNS (+) (high BBB permeation predicted); P (10 cm s ) > 4.0
e
-6
–1
CNS (–) (low BBB permeation predicted); P (10 cm s ) ≤ 2.0
e
-6
–1
CNS (+/–) (BBB permeation uncertain); P (10 cm s ) ≥ 4.0 > 2.0
e
2
.4. Docking studies on AChE and BACE1
1
5

ACCEPTED MANUSCRIPT
Novel compounds were docked into the Torpedo californica AChE crystal structure as well as
to human BACE1 to find the possible binding mode and to explain the reasons for varied
potencies. We used the previously developed methods for docking to both targets and
assessment of binding modes [58,59]. In the case of AChE all derivatives were arranged along
the active gorge and bound to the catalytic active site as well as peripheral anionic site. The
binding mode of the most active inhibitor 26 is shown in the Figure 5. The benzyl moiety was
responsible for π-π stacking with Trp84 in the CAS. The protonated piperazine nitrogen atom
forms cation-π interactions with Phe330 and a hydrogen bond network with Tyr121 via a
water molecule. The other part of piperazine ring and short two carbon linker interacted with
aromatic side chains of Phe290, Phe331, and Tyr334 in the middle of the active gorge. The
saccharin fragment was engaged in π-π stacking with Trp279 and CH-π interactions with
Tyr70 in the PAS. The hydrogen bonds were formed by the carbonyl group with Tyr121 and
one oxygen atom from sulfone with main chain of Phe288 via water molecules. Such binding
mode provided the highest activity against AChE for this compound. It was observed that
other compounds from piperazine series gave worse fit to the active gorge, that might result in
lower potency. Introduction of one or two fluorine atoms on the benzyl or phenethyl moiety
did not provide any extra interactions. Moreover, 3,5-substitution led to unbeneficial
excessive occupation of limited space close to the catalytic triad (Ser200, His440, Glu327).
Phthalimide established weaker hydrogen bonds compared to saccharin and thus led to
diminished activity. With regard to the length of linker it seems that optimal tether between
phenyl ring and piperazine moiety is equal one methylene group. The chain between
piperazine and saccharin/phthalimide might be of different length due to its fitting to enzyme
by conformational changes. However, it seems that two carbon linker (compound 26 and 28)
and four carbon (32 and 36) gave a little bit better fit than the others. It is also worth to note
that linearity of molecules provided by di-substituted piperazine is very important for binding
to AChE. The compounds from series of hexahydropyrimidine and 3-aminopiperidine
derivatives were not linear. Such geometry of their molecules resulted in non-optimal fit into
the active site and brought significantly reduced activity. Moreover, in case of series A of 3-
aminopiperidine derivatives (47, 48) the protonation of secondary amine group led to the lack
of cation-π interaction with Phe330. The strength of binding with AChE was assessed by
ChemScore function. The most active derivative 26 obtained value equal to 44.84 which was
still lower than the score for donepezil (49.48). This stays in accordance with the results of the
biological assay.
1
6

ACCEPTED MANUSCRIPT
Figure 5. The binding mode of compound 26 within the active site of AChE. The red spheres
represent conserved water molecules engaged in the hydrogen bond network. Hydrogen bonds
marked by grey dotted line.
The investigated compounds revealed diverse binding modes within the active site of
BACE1. The basic nitrogen atoms of alicyclic amines such as hexahydropyrimidine,
piperazine or 3-aminopiperidine interacted with the active-site aspartate residues Asp32 and
Asp228 (catalytic dyad). The distance between these basic centers and catalytic aspartates was
different for more and less active compounds. In case of the more potent derivatives (65, 24,
2
5, 31, 34 and 51) it was shorter while the less active inhibitors (most of the remaining
compounds) were more distant from dyad. The remaining parts of molecules were located in
the neighboring pockets (S2’, S2 and S3). The binding mode of the most active inhibitor 65 is
shown in the Figure 6. The protonated secondary amine group created salt bridge with
Asp228 as well as hydrogen bond with Gly34. The substituted benzyl moiety occupied S2’
pocket and interacted with Tyr71, Val69 and Tyr198. The saccharin fragment was surrounded
by the following residues: Asn233 and Arg235 from S2 pocket as well as Thr231 at the edge
of S3 pocket. The low occupancy of S3 site may be the reason of relatively low activity due to
1
7

ACCEPTED MANUSCRIPT
the importance of binding within this pocket for high inhibition of BACE1 [60]. The other
compounds with at least 45% inhibition of enzyme , such as inhibitor 24, 25 or 51, presented
similar binding mode as compound 65 but one main change appeared. In these cases,
saccharin was replaced by phthalimide, and this moiety was slightly shifted towards the center
of S3 pocket. However, this did not improve the activity because the phthalimide was not
engaged in any highly specific interactions. In case of compounds with lower activity (e.g. 28,
4
0, 42, 44) the orientation of the molecules in the active site was reversed. The benzyl moiety
occupied the S3 pocket instead of S2’. This can be non-specific nature of binding interactions
and may be responsible for reduced potency. The strength of binding with BACE1 was
assessed by GoldScore function. The most active compound 65 obtained value 69.16 in
comparison with 102.69 for the reference ligand - NVP-BXD552 [60].
Figure 6. The binding mode of compound 65 within the active site of BACE1. Asp32 and
Asp228 constitute catalytic dyad. The 10s loop and flap are the most flexible parts of BACE1.
The S2’ pocket contains Tyr71, Val69 and Tyr198. Asn233 and Arg235 belong to the S2 site,
and Thr231 to the S3 pocket. Hydrogen bonds marked by grey dotted line.
1
8

ACCEPTED MANUSCRIPT
3
.
Conclusions
Multi-target-directed ligand approach is widely used in the search for new treatment of
Alzheimer’s disease that remains unresolved therapeutic problem. Our previous studies
resulted in obtaining saccharin and phthalimide derivatives as MTDLs with cholinesterase
inhibitory activity and anti-Aβ aggregation properties. In the project presented herein we have
tried to endow the selected lead compounds - V and VI - with BACE1 inhibitory activity.
Therefore we introduced in their structure fragments containing diaminoalkyl motives that
could provide interactions with the catalytic site of BACE1. The activity of all the synthesized
compounds was evaluated against eeAChe, eqBuChE and hBACE1, we have also determined
their Aβ anti-aggregating properties and ability to permeate BBB for the selected compounds.
The novel compounds display decreased anti-cholinesterase activity compering to the lead
structures but they all gained BACE1 inhibitory activity. Among the synthesized and tested
thirty compounds we identified piperazine derivative 26 and 3-aminopiperidine derivative 52
as interesting hits for further development. Both compounds are selective AChE inhibitors
(26: IC50 AChE = 0.83 µM; 52: IC50 AChE = 6.47 µM) with BACE1 inhibitory activity (26:
3
3.6%; 52: 26.3% at 50 µM). Additionally compound 52 displays the most significant Aβ
anti-aggregating properties (39% at 10 µM). The SAR analysis of these series of compounds
gives us directions for further development of novel multifunctional anti-AD agents.
Experimental section
3
.1. Chemistry
4
.1.1 General methods
1
H NMR spectra were recorded on Varian Mercury 300 at 300 MHz. The chemical shifts for
1
13
H NMR and C NMR are referenced to TMS via residual solvent signals (CDCl at 7.26
3
ppm and 77.16 ppm DMSO-d at 2.50 ppm and 39.52). Mass spectra (MS) were obtained on
6
®
®
an UPLC-MS/MS system consisting of a Waters ACQUITY UPLC (Waters Corporation,
Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization
mode ESI-tandem quadrupole). Analytical thin layer chromatography (TLC) was done using
aluminum sheets precoated with silica gel 60 F . Column chromatography was performed on
2
54
TM
Merck silica gel 60 (63–200 µm). Flash chromatography was performed on Isolera Spectra
Biotage). The purity of the final compounds was determined using an analytical RPLC-MS
on Waters Acquity TQD using an Aquity UPLC BEH C18 column (1.7 µm, 2.1x100 mm) at
(
1
9

ACCEPTED MANUSCRIPT
2
14 nm and 254 nm. CH CN/H O gradient with 0.1% HCOOH was used as the mobile phase
3
2
at a flow rate of 0.3 mL/min. All the compounds showed purity of >95%, as determined by
RPLC. All of the reagents were purchased from commercial suppliers and were used without
further purification. Tetrahydrofuran (THF) and dichloromethane (DCM) were distilled under
nitrogen immediately before use. The drying agent used for THF was sodium/benzophenone
ketyl, and for DCM, calcium hydride.
The following compounds: 2-(2-bromoethyl)isoindoline-1,3-dione
1
[62], 2-(2-
bromoethyl)benzo[d]isothiazol-3(2H)-one
bromopropyl)benzo[d]isothiazol-3(2H)-one
bromobutyl)benzo[d]isothiazol-3(2H)-one
bromopentyl)benzo[d]isothiazol-3(2H)-one
1,1-dioxide
1,1-dioxide
1,1-dioxide
1,1-dioxide
2
[63],
[63],
[63],
[63],
2-(3-
2-(4-
2-(5-
2-(6-
3
4
5
bromohexyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide 6 [63], have been previously reported.
4
.1.2. General procedure for the preparation of hydrochloride salts
All the final compounds were evaluated in vitro as hydrochloride salts. The hydrochloride
salts were prepared by dissolving the compounds in a minimum quantity of ethyl acetate.
Then the solution was treated with 5 M solution of HCl in propan-2-ol, evaporated under
reduced pressure and dried.
4
.1.3. Procedure for the synthesis of compounds 7–12 (procedure A)
To a solution of piperazine (4 equiv.) in acetonitrile a solution of the appropriate phenyl-
alkyl-halide derivative was added dropwise at 0 °C. After the addition the reaction mixture
was stirred at room temperature for 24 h. Concentration under reduced pressure resulted in
solid residue. The crude product was purified by silica gel column chromatography in
DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v).
4
.1.3.1. 1-(2-Fluorobenzyl)piperazine (7)
Following the procedure A, reaction of piperazine (237 mg, 2.76 mmol) with 1-
chloromethyl)-2-fluorobenzene (100 mg, 0.69 mmol) in acetonitrile (10+5 mL) was
(
performed. Purification by column chromatography gave the product 7 (120 mg, yield 89%)
as a colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v) R = 0.34. MW 194.25.
f
+
1
Formula: C H FN . MS m/z 195.22 (M+H ). H NMR (300 MHz, CDCl )
δ
ppm 7.38 (td,
1
1
15
2
3
J = 7.50, 1.67 Hz, 1H), 7.19 - 7.26 (m, 1H), 7.07 - 7.14 (m, 1H), 7.03 (ddd, J = 9.87, 8.34,
2
0

ACCEPTED MANUSCRIPT
1
.28 Hz, 1H), 3.58 (d, J = 1.54 Hz, 2H), 2.86 - 2.95 (m, 4H), 2.41 - 2.53 (m, 4H), 1.89 (br. s.,
1
H).
4
.1.3.2. 1-(3-Fluorobenzyl)piperazine (8)
Following the procedure A, reaction of piperazine (911.4 mg, 10.58 mmol) with 1-
bromomethyl)-3-fluorobenzene (500 mg, 2.65 mmol) in acetonitrile (30+15 mL) was
(
performed. Purification by column chromatography gave the product 8 (417 mg, yield 81%)
as a colorless oil. TLC in DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v) R = 0.40. MW 194.25.
f
+
1
Formula: C H FN . MS m/z 195.22 (M+H ). H NMR (300 MHz, CDCl ) δ ppm 7.21 - 7.30
1
1
15
2
3
(
m, 1H), 7.02 - 7.12 (m, 2H), 6.87 - 6.98 (m, 1H), 3.47 (s, 2H), 2.85 (t, J = 4.90 Hz, 4H), 2.41
t, J = 4.40 Hz, 4H), 1.71 (s, 1H).
(
4
.1.3.3. 1-(4-Fluorobenzyl)piperazine (9)
Following the procedure A, reaction of piperazine (1170 mg, 13.60 mmol) with 1-
chloromethyl)-4-fluorobenzene (414 µL, 3.4 mmol) in acetonitrile (40+15 mL) was
(
performed. Purification by column chromatography gave the product 9 (579 mg, yield 88%)
as a colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v) R = 0.28. MW 194.25.
f
+
1
Formula: C H FN . MS m/z 195.22 (M +H ). H NMR (300 MHz, CDCl ) δ ppm 7.26 (tt,
1
1
15
2
3
J = 8.80, 2.90 Hz, 2H), 6.98 (tt, J = 8.80, 1.80 Hz, 2H), 3.44 (s, 2H), 2.89 (t, J = 4.70 Hz, 4H),
2
.41 (t, J = 4.70 Hz, 4H), 2.13 (s, 1H).
4
.1.3.4. 1-(3,5-Difluorobenzyl)piperazine (10)
Following the procedure A, reaction of piperazine (166.4 mg, 1.932 mmol) with 1-
bromomethyl)-3,5-difluorobenzene (100 mg, 0.483 mmol) in acetonitrile (10+5 mL) was
(
performed. Purification by column chromatography gave the product 10 (79 mg, yield 77%)
as a colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v) R = 0.31. MW 212.24.
f
+
1
Formula: C H F N . MS m/z 213.16 (M+H ). H NMR (300 MHz, CDCl )
δ
ppm 6.85 -
.91 (m, 2H), 6.68 (tt, J = 8.98, 2.31 Hz, 1H), 3.46 (s, 2H), 2.89 - 2.96 (m, 4H), 2.24 - 2.55
m, 5H).
1
1
14
2
2
3
6
(
4
.1.3.5. 1-Phenethylpiperazine (11)
Following the procedure A, reaction of piperazine (1858 mg, 21.570 mmol) with (2-
bromoethyl)benzene (731 µL, 5.393 mmol) in acetonitrile (40+15 mL) was performed.
Purification by column chromatography gave the product 11 (791 mg, yield 77.1%) as a
2
1

ACCEPTED MANUSCRIPT
colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v) R = 0.41. MW 190.28. Formula:
f
+
1
C H N . MS m/z 191.14 (M+H ). H NMR (300 MHz, CDCl )
δ ppm 7.08 - 7.25 (m, 5H),
12
18
2
3
2
.84 (t, J = 5.30 Hz, 4H), 2.47 - 2.78 (br. s, 4H), 2.43 (br. s, 4H), 2.17 (s, 1H).
4
.1.3.6. 1-(2-Fluorophenethyl)piperazine (12)
Following the procedure A, reaction of piperazine (170 mg, 1.970 mmol) with 1-
bromoethyl)-2-fluorobenzene (100 mg, 0.492 mmol) in acetonitrile (10+5 mL) was
(
performed. Purification by column chromatography gave the product 12 (88 mg, yield 86%)
as a colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v) R = 0.40. MW 208.28.
f
+
1
Formula: C H FN . MS m/z 209.24 (M+H ). H NMR (300 MHz, CDCl ) δ ppm 7.13 - 7.25
1
2
17
2
3
(m, 2H), 6.96 - 7.10 (m, 2H), 2.97 (t, J = 4.90 Hz, 2H), 2.85 (t, J = 11.30 Hz, 2H), 2.46 - 2.64
(m, 9H).
4
.1.4. Procedure for the synthesis of compounds 13 and 14 (procedure B)
To a solution of propane-1,3-diamine (4 equiv.) in acetonitrile a solution of the appropriate
phenyl-alkyl-halide derivative was added dropwise at 0 °C. After the addition the reaction
mixture was stirred at room temperature for 24 h., Subsequently the mixture was concentrated
under reduced pressure. Resulting residue was dissolved in 20 mL of 1 M NaOH solution and
extracted with DCM (3 × 20 mL). The combined organic extracts were dried over anhydrous
Na SO , filtered, and concentrated under reduced pressure. The crude product was purified by
2
4
silica gel column chromatography using DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v), yielding a
colorless oil.
4
.1.4.1. N-(2-fluorobenzyl)propane-1,3-diamine (13)
Following the procedure B, reaction of propane-1,3-diamine (1.13 mL, 13.49 mmol) with 1-
chloromethyl)-2-fluorobenzene (0.40 mL, 3.37 mmol) in acetonitrile was performed.
(
Purification by column chromatography gave the product 13 (505 mg, yield 82%) as a
colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v) R = 0.19. MW 182.24.
f
+
1
Formula: C H FN . MS m/z 183.18 (M+H ). H NMR (300 MHz, CDCl )
δ
ppm 7.32 (td,
J = 7.57, 1.80 Hz, 1H), 7.17 - 7.25 (m, 1H), 7.06 - 7.13 (m, 1H), 7.02 (ddd, J = 10.00, 8.34,
.15 Hz, 1H), 3.83 (s, 2H), 2.76 (t, J = 6.80 Hz, 2H), 2.68 (t, J = 6.82 Hz, 2H), 1.65 (quin,
J = 6.86 Hz, 2H), 1.57 (s, 1H), 1.24 (s, 1H).
1
0
15
2
3
1
4
.1.4.2. N-(3,5-difluorobenzyl)propane-1,3-diamine (14)
2
2

ACCEPTED MANUSCRIPT
Following the procedure B, reaction of propane-1,3-diamine (1.13 mL, 13.49 mmol) with 1-
(bromomethyl)-3,5-difluorobenzene (0.44 mL, 3.37 mmol) in acetonitrile was performed.
Purification by column chromatography gave the product 14 (467 mg, yield 69%) as a
colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.1, v/v/v) R = 0.21. MW 200.23. Formula:
f
+
1
C H F N . MS m/z 201.14 (M+H ). H NMR (300 MHz, CDCl )
δ
ppm 6.80 - 6.91 (m, 1H),
.62 - 6.72 (m, 2H), 3.77 (s, 2H), 2.79 (t, J = 6.80 Hz, 2H), 2.67 (t, J = 6.92 Hz, 2H), 1.66
quin, J = 6.67 Hz, 2H), 1.61 (s, 1H), 1.24 (s, 1H).
10
14
2
2
3
6
(
4
.1.5. Procedure for the synthesis of compounds 15 and 16 (procedure C)
To a solution of the appropriate propane-1,3-diamine derivative (1 equiv.) in ethanol a 37%
solution of formaldehyde (1.7 equiv.) and 20% NaOH aq. solution (0.2 equiv.) were added at
0
°C. After the addition, the reaction mixture was stirred at room temperature for 1 h, and
concentrated under reduced pressure. To the resulting residue 5 mL of saturated solution of
NaHCO was added, and extracted with DCM (3 × 10 mL). The combined organic extracts
3
were dried over anhydrous Na SO , filtered and concentrated under reduced pressure. The
2
4
crude product was purified by silica gel column chromatography DCM/MeOH (9/1, v/v).
.1.5.1. 1-(2-Fluorobenzyl)-hexahydropyrimidine (15)
4
Following the procedure C, reaction of N-(2-fluorobenzyl)propane-1,3-diamine (13) (200 mg,
.1 mmol) with 37% formaldehyde solution (0.14 mL, 1.87 mmol) and 20% NaOH(aq)
1
solution (0.04 mL, 0.22 mmol) in ethanol (5 mL) was performed. Purification by extraction
and column chromatography gave product 15 (198 mg, yield 93%) as a colorless oil. TLC
DCM/MeOH (9/1, v/v) R = 0.51. MW 194.25. Formula: C H FN . MS: m/z 181.16 (M-
f
11 15
2
+
1
(
(
(
CH )+H ). H NMR (300 MHz, CDCl ) δ ppm 7.36 (td, J = 7.37, 1.92 Hz, 1H), 7.17 - 7.26
2
3
m, 1H), 7.10 (td, J = 7.44, 1.28 Hz, 1H), 6.98 - 7.06 (m, 1H), 3.49 (d, J = 1.54 Hz, 2H), 3.45
s, 2H), 2.81 (t, J = 5.40 Hz, 2H), 2.62 (t, J = 5.40 Hz, 2H), 1.73 (s, 1H), 1.61 (quin, J = 5.51
Hz, 2H).
4
.1.5.2. 1-(3,5-Difluorobenzyl)-hexahydropyrimidine (16)
Following the procedure C, reaction of N-(3,5-difluorobenzyl)propane-1,3-diamine (14) (200
mg, 1 mmol) with 37% formaldehyde solution (0.13 mL, 1.70 mmol) and 20% NaOH(aq)
solution (0.04 mL, 0.2 mmol) in ethanol (5 mL) was performed. Purification by extraction and
column chromatography gave the product 16 (185 mg, yield 87%) as a colorless oil. TLC
DCM/MeOH (9/1, v/v)R = 0.54. MW 212.24. Formula: C H F N . MS: m/z 199.21 (M-
f
11 14
2
2
2
3

ACCEPTED MANUSCRIPT
+
1
(
CH )+H ). H NMR (300 MHz, CDCl ) δ ppm 6.81 - 6.90 (m, 2H), 6.66 (tt, J = 8.94, 2.34
2
3
Hz, 1H), 3.47 (s, 2H), 3.27 (s, 2H), 2.66 (t, J = 5.10 Hz, 2H), 2.55 (t, J = 4.90 Hz, 2H), 1.78
br. s., 1 H), 1.63 (q, J = 5.60 Hz, 2H).
(
4
.1.6. Procedure for the synthesis of compounds 17—19 (procedure D)
To a solution of tert-butyl piperidin-3-ylcarbamate (1 equiv.) in acetonitrile the appropriate
benzyl halide derivative (1.1 equiv.) was added at room temperature. After the addition the
reaction mixture was stirred at room temperature for 44 h. When the reaction was finished, the
solvent was evaporated under reduced pressure, and the resulting residue was dissolved in 20
mL of saturated solution of NaHCO and extracted with DCM (2
×
20 mL). The organic
3
extracts were dried over anhydrous Na SO , filtered and concentrated under vacuum. The
2
4
crude product was purified by flash column chromatography in petroleum ether/ethyl acetate
8/2, v/v).
(
4
.1.6.1. Tert-butyl 1-benzylpiperidin-3-ylcarbamate (17)
Following the general procedure D reaction of tert-butyl piperidin-3-ylcarbamate (298 mg,
.49 mmol) with (chloromethyl)benzene (208 mg, 1.64 mmol) in acetonitrile (30 mL) was
1
performed. Purification gave product 17 (248 mg, yield 57.4%) as a colorless oil. TLC
petroleum ether/ethyl acetate (8/2, v/v) R = 0.22. MW 290.40. Formula: C H N O . MS m/z
f
17 26
2
2
+
1
2
3
91.22 (M+H ). H NMR (300 MHz, CDCl ) δ ppm 7.18 - 7.39 (m, 5H), 4.89 - 5.11 (m, 1H),
3
.67 - 3.83 (m, 1H), 3.47 (s, 2H), 2.49 (m., 4H), 1.50 - 1.76 (m, 4H), 1.43 (s, 9H).
4
.1.6.2. Tert-butyl 1-(2-fluorobenzyl)piperidin-3-ylcarbamate (18)
Following the general procedure D reaction of tert-butyl piperidin-3-ylcarbamate (401 mg,
.00 mmol) with 1-(chloromethyl)-2-fluorobenzene (318 mg, 2.20 mmol) in acetonitrile (50
2
mL) was performed. Purification gave product 18 (345 mg, yield 56%) as a colorless oil. TLC
petroleum ether/ethyl acetate (8/2, v/v) R = 0.16. MW 308.39. Formula: C H FN O . MS
f
17 25
2
2
+
1
m/z 309.28 (M+H ). H NMR (300 MHz, CDCl ) δ ppm 7.35 (dt, J = 1.80, 7.44 Hz, 1H), 7.18
3
-
7.25 (m, 1H), 7.07 - 7.13 (m, 1H), 7.01 (ddd, J = 1.28, 8.34, 9.87 Hz, 1H), 4.98 (br. s, 1H),
.75 (br. s, 1H), 3.55 (s, 2H), 2.51 - 2.64 (m, 1H), 2.18 - 2.50 (m, 3H), 1.47 - 1.78 (m, 4H),
.43 (s, 9H).
3
1
4
.1.6.3. Tert-butyl 1-(3,5-difluorobenzyl)piperidin-3-ylcarbamate (19)
2
4

ACCEPTED MANUSCRIPT
Following the general procedure D reaction of tert-butyl piperidin-3-ylcarbamate (401 mg,
2
.00 mmol) with 1-(bromomethyl)-3,5-difluorobenzene (455 mg, 2.20 mmol) in acetonitrile
50 mL) was performed. Purification gave product 19 (515 mg, yield 79%) as a colorless oil.
TLC petroleum ether/ethyl acetate (8:2, v/v) R = 0.32. MW 326.38. Formula: C H N F O .
(
f
17 24
2
2
2
+
1
MS m/z 327.30 (M+H ). H NMR (300 MHz, CDCl ) δ ppm 6.80 - 6.89 (m, 2H), 6.67 (tt,
3
J = 2.34, 8.94 Hz, 1H), 4.95 (br. s, 1H), 3.70 - 3.83 (m, 1H), 3.43 (s, 2H), 2.47 - 2.61 (m, 1H),
2
.18 - 2.46 (m, 3H), 1.50 - 1.76 (m, 4H), 1.44 (s, 9H).
4
.1.7. Procedure for the synthesis of compounds 20–22 (procedure E)
To the appropriate tert-butyl piperidin-3-ylcarbamate derivative 10% solution of HCl in
MeOH was added. The reaction mixture was stirred at room temperature for 24 h. When the
reaction was finished, the solvent and hydrogen chloride were evaporated under reduced
pressure, producing a residue that was then suspended in ethyl acetate with a small amount of
diethyl ether. The obtained hydrochloride salt was then dissolved in saturated solution of
NaHCO and extracted with DCM (6
×
20 mL). The organic extracts were combined, dried
3
over anhydrous Na SO , filtered and concentrated under vacuum.
2
4
4
.1.7.1. 1-Benzylpiperidin-3-amine (20)
Following the procedure E, reaction of tert-butyl 1-benzylpiperidin-3-ylcarbamate (17) (248
mg, 0.855 mmol) with hydrochloric acid (15 mL) was performed to give product 20 (148 mg,
yield 91.0%) as a colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.05, v/v/v) R = 0.15.
f
+
1
MW 190.28. Formula: C H N . MS m/z 191.14 (M+H ). H NMR (300 MHz, CDCl ) δ ppm
1
2
18
2
3
7
-
.18 - 7.35 (m, 5H), 3.47 - 3.52 (m, 2H), 2.81 - 2.92 (m, 1H), 2.74 (d, J = 10.55 Hz, 1H), 2.53
2.66 (m, 1H), 2.00 - 2.14 (m, 1H), 1.75 - 1.92 (m, 2H), 1.64 - 1.75 (m, 1H), 1.39 - 1.63 (m,
H).
4
4
.1.7.2. 1-(2-Fluorobenzyl)piperidin-3-amine (21)
Following the procedure E reaction of tert-butyl 1-(2-fluorobenzyl)piperidin-3-ylcarbamate
18) (935 mg, 3.03 mmol) with hydrochloric acid (30.3 mL) was performed to give product
1 (600 mg, yield 95%) as a colorless oil. TLC DCM/MeOH/25% NH3(aq) (9/1/0.05, v/v/v)
(
2
+
1
R = 0.27. MW 208.28. Formula: C H FN . MS m/z 209.18 (M+H ). H NMR (300 MHz,
f
12 17
2
CDCl ) δ ppm 7.36 (dt, J = 2.05, 7.44 Hz, 1H), 7.17 - 7.24 (m, 1H), 7.05 - 7.12 (m, 1H), 7.01
3
(ddd, J = 1.28, 8.34, 9.87 Hz, 1H), 3.57 (s, 2H), 2.81 - 2.91 (m, 1H), 2.73 - 2.81 (m, 1H), 2.58
2
5

ACCEPTED MANUSCRIPT
2.69 (m, 1H), 2.04 - 2.14 (m, 1H), 1.89 (t, J = 9.49 Hz, 1H), 1.47 - 1.83 (m, 3H), 1.25 - 1.31
m, 2H), 0.99 - 1.16 (m, 1H).
-
(
4
.1.7.3. 1-(3,5-Difluorobenzyl)piperidin-3-amine (22)
Following the procedure E, reaction of tert-butyl 1-(3,5-difluorobenzyl)piperidin-3-
ylcarbamate (19) (500 mg, 1.532 mmol) with hydrochloric acid (15 mL) was performed to
give product 22 (316 mg, yield 89%) as a colorless oil. TLC DCM/MeOH/25% NH3(aq)
+
1
(
9/1/0.05, v/v/v) R = 0.25. MW 226.27. Formula: C H F N . MS m/z 227.19 (M+H ). H
f 12 16 2 2
NMR (300 MHz, CDCl ) δ ppm: 6.77 - 6.87 (m, 2H), 6.63 (tt, J = 2.34, 8.94 Hz, 1H), 3.33 -
3
3
.47 (m, 2H), 2.78 - 2.92 (m, 1H), 2.62 - 2.73 (m, 1H), 2.46 - 2.58 (m, 1H), 1.97 - 2.10 (m,
H), 1.72 - 1.91 (m, 2H), 1.43 - 1.72 (m, 4H), 0.99 - 1.16 (m, 1H).
1
4
.1.8. Procedure for the synthesis of compounds 23–25 (procedure F)
2
-(2-Bromoethyl)isoindoline-1,3-dione (1) (1 equiv.) with the appropriate piperazine
derivative (7, 10 or 12) (1 – 1.1 equiv.) in the presence of K CO (1.2-2 equiv.) in acetonitrile
2
3
was refluxed for 24 h. When the reaction was finished the solvent was evaporated under
reduced pressure and the resulting residue was dissolved in 5 mL of ethyl acetate and washed
with saturated solution of NaHCO (3
×
5 mL) and saturated solution of NaCl (5 mL). The
3
organic extract was dried over anhydrous Na SO , filtered and concentrated under vacuum.
2
4
The crude product (23, 24 or 25) was purified by column chromatography (petroleum
ether/ethyl acetate 4.5/5.5, v/v for 23, 5/5, v/v for 24, 3/7, v/v for 25).
4
.1.8.1. 2-(2-(4-(2-Fluorobenzyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (23)
Following the procedure F, reaction of 1-(2-fluorobenzyl)piperazine (7) (60 mg, 0.310 mmol)
with 2-(2-bromoethyl)isoindoline-1,3-dione (1) (90 mg, 0.310 mmol) and K CO (86 mg,
2
3
0
.62 mmol) in acetonitrile (5 mL) was performed. Purification by extraction and column
chromatography gave product 23 (35 mg, yield 28%). TLC petroleum ether/ethyl acetate
+
1
(
4.5/5.5, v/v) R = 0.22. MW 367.42. Formula: C H N O . MS m/z 368.31 (M+H ). H
f 21 22 3 2
NMR (300 MHz, CDCl ) δ ppm 7.79 - 7.87 (m, 2H), 7.67 - 7.74 (m, 2H), 7.35 (dt,
3
J = 1.80,7.44 Hz, 1H), 7.17 - 7.24 (m, 1H), 7.05 - 7.11 (m, 1H), 7.00 (ddd, J = 1.28, 8.34, 9.87
Hz, 1H), 3.80 (t, J = 6.67 Hz, 2H), 3.55 (d, J = 1.28 Hz, 2H), 2.63 (t, J = 6.67 Hz, 2H), 2.38 -
1
3
2
1
.59 (m, 8H). C NMR (75 MHz, CDCl ) δ ppm 168.31, 161.35 (d, J = 246.2 Hz), 133.80,
3 C-F
32.17, 131.57 (d, JC-F = 4.6 Hz), 128.65 (d, JC-F = 8.2 Hz), 124.61 (d, JC-F = 14.7 Hz), 123.75
2
6

ACCEPTED MANUSCRIPT
(d, JC-F = 3.5 Hz), 123.15, 115.17 (d, JC-F = 22.3 Hz), 55.66, 55.13 (d, JC-F = 1.9 Hz), 52.99,
5
2.77, 35.28.
4
.1.8.2. 2-(2-(4-(3,5-Difluorobenzyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (24)
Following the procedure F, reaction of 1-(3,5-difluorobenzyl)piperazine (10) (78 mg, 0.370
mmol) with 2-(2-bromoethyl)isoindoline-1,3-dione (1) (93 mg, 0.370 mmol) and K CO (61
2
3
mg, 0.440 mmol) in acetonitrile (2 mL) was performed. Purification by extraction and column
chromatography gave the product 24 (75 mg, yield 53%). TLC petroleum ether/ethyl acetate
+
1
(
5/5, v/v) R = 0.41. MW 385.41. Formula: C H F N O . MS m/z 386.33 (M+H ). H NMR
f 21 21 2 3 2
(
300 MHz, CDCl ) δ ppm 7.80 - 7.88 (m, 2H), 7.67 - 7.75 (m, 2H), 6.79 - 6.89 (m, 2H), 6.66
3
(tt, J = 2.31, 8.98 Hz, 1H), 3.81 (t, J = 6.67 Hz, 2H), 3.42 (s, 2H), 2.64 (t, J = 6.67 Hz, 2H),
1
3
2
1
.31 - 2.60 (m, 8H); C NMR (75 MHz, CDCl ) δ ppm 168.31, 162.93 (dd, J = 247.69,
3 C-F
2.72 Hz), 142.71 (t, JC-F = 8.7 Hz), 133.81, 132.17, 123.15, 111.14 - 111.57 (m), 102.29 (t,
JC-F = 25.4 Hz), 62.01 (t, JC-F = 2.21 Hz), 55.64, 53.01, 52.95, 35.25.
4
.1.8.3. 2-(2-(4-(2-Fluorophenethyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (25)
Following the procedure F, reaction of 2-(2-fluorophenethyl)piperazine (12) (77 mg, 0.370
mmol) with 2-(2-bromoethyl)isoindoline-1,3-dione (1) (94 mg, 0.370 mmol) and K CO (61
2
3
mg, 0.440 mmol) in acetonitrile (2 mL) was performed. Purification by extraction and column
chromatography gave the product 25 (64 mg, yield 45.6%). TLC petroleum ether/ethyl acetate
+
1
(
3/7, v/v) R = 0.24. MW 381.44. Formula: C H N O . MS m/z 382.34 (M+H ). H NMR
f 22 24 3 2
(
300 MHz, CDCl ) δ ppm 7.80 - 7.87 (m, 2H), 7.67 - 7.74 (m, 2H), 7.12 - 7.22 (m, 2H), 6.94 -
3
13
7
.07 (m, 2H), 3.82 (t, J = 6.54 Hz, 2H), 2.78 - 2.86 (m, 2H), 2.46 - 2.68 (m, 12H); C NMR
(
75 MHz, CDCl ) δ ppm 168.31, 161.07 (d, JC-F = 244.9 Hz), 133.81, 132.15, 130.92 (d, JC-
3
=
4.9 Hz), 127.82 (d, JC-F = 8.0 Hz), 126.77 (d, JC-F = 16.0 Hz), 123.97 (d, JC-F = 3.6 Hz),
F
1
23.15, 115.18 (d, JC-F = 22.1 Hz), 58.45, 55.60, 52.82, 52.65, 35.18, 26.44 (d, JC-F = 2.0 Hz).
4
.1.9. Procedure for the synthesis of compounds 26–40, 45–49, 51, 52 (procedure G)
The appropriate saccharine derivative (2–6) or isoindoline-1,3-dione derivative (1, 50) (1
equiv.) with the appropriate amine derivative (1 equiv.) in the presence of K CO (1-3 equiv.)
2
3
in acetonitrile was heated at reflux for 24 h. When the reaction was finished the solvent was
evaporated under reduced pressure, producing a residue that was then dissolved in 5 mL of
ethyl acetate and extracted with saturated solution of NaHCO (2
×
5 mL) and saturated
3
solution of NaCl (5 mL). The organic extracts were dried over anhydrous Na SO , filtered and
2
4
2
7

ACCEPTED MANUSCRIPT
concentrated under vacuum. The crude product was purified by flash column chromatography
in petroleum ether/ethyl acetate (5/5 to 1/9, v/v) or DCM/MeOH (10/0.5, v/v).
4
.1.9.1. 2-(2-(4-benzylpiperazin-1-yl)ethyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (26)
Following the procedure G, reaction of N-benzylpiperazine (91 mg, 0.517 mmol) with 2-(2-
bromoethyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (2) (150 mg, 0.517 mmol) and K CO3
2
(107 mg, 0.776 mmol) in acetonitrile (5 mL) was performed. Purification by extraction and
column chromatography gave the product 26 (24 mg, yield 14%). TLC petroleum ether/ethyl
+
1
acetate (1/9, v/v) R = 0.36. MW 385.48. Formula: C H N O S. MS m/z 386.46 (M+H ). H
f
20 23
3
3
NMR (300 MHz, CDCl3) δ ppm 8.02 - 8.07 (m, 1H), 7.77 - 7.94 (m, 3H), 7.19 - 7.35 (m, 5H),
13
3
.89 (t, J = 6.90 Hz, 2H), 3.51 (s, 2H), 2.78 (t, J = 6.70 Hz, 2H), 2.40 - 2.69 (m, 8H). C
NMR (75 MHz, CDCl3)
δ ppm 158.90, 137.66, 134.69, 134.29, 129.46, 128.33, 128.28,
1
27.38, 127.30, 125.14, 120.91, 62.70, 55.46, 52.79, 52.62, 36.64.
4
.1.9.2. 2-(2-(4-(2-Fluorobenzyl)piperazin-1-yl)ethyl)benzo[d]isothiazol-3(2H)-one 1,1-
dioxide (27)
Following the procedure G, reaction of 1-(2-fluorobenzyl)piperazine (7) (60 mg, 0.310 mmol)
with 2-(2-bromoethyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (2) (90 mg, 0.310 mmol) and
K CO (86 mg, 0.620 mmol) in acetonitrile (5 mL) was performed. Purification by extraction
2
3
and column chromatography gave the product 27 (35 mg, yield 28%). TLC ethyl
acetate/petroleum ether (5/5, v/v) R = 0.22. MW 403.47. Formula: C H FN O S. MS m/z
f
20 22
3
3
+
1
4
7
2
04.34 (M+H ). H NMR (300 MHz, CDCl ) δ ppm 8.02 - 8.06 (m, 1H), 7.78 - 7.93 (m, 3H),
3
.36 (dt, J = 1.92, 7.50 Hz, 1H), 7.18 - 7.25 (m, 1H), 6.97 - 7.13 (m, 2H), 3.88 (t, J = 7.18 Hz,
1
3
H), 3.59 (s, 2H), 2.77 (t, J = 7.18 Hz, 2H), 2.46 - 2.67 (m, 8H); C NMR (75 MHz, CDCl )
3
δ ppm 161.35 (d, JC-F = 246.2 Hz), 158.82, 137.63, 134.63, 134.23, 131.59 (d, JC-F = 4.5 Hz),
28.73 (d, JC-F = 8.2 Hz), 127.37, 125.09, 124.39 (d, JC-F = 14.5 Hz), 123.78 (d, JC-F = 3.6
Hz), 120.85, 115.17 (d, JC-F = 22.3 Hz), 55.49, 55.04 (d, JC-F = 1.8 Hz), 52.90, 52.65, 36.60.
1
4
.1.9.3. 2-(2-(4-(3-Fluorobenzyl)piperazin-1-yl)ethyl)benzo[d]isothiazol-3(2H)-one 1,1-
dioxide (28)
Following the procedure G, reaction of 1-(3-fluorobenzyl)piperazine (8) (80 mg, 0.414 mmol)
with 2-(2-bromoethyl)benzo[d]isothiazol-3(2H)-one 1,1-dioxide (2) (120 mg, 0.414 mmol)
and K CO (86 mg, 0.621 mmol) in acetonitrile (5 mL) was performed. Purification by
2
3
extraction and column chromatography gave the product 28 (24 mg, yield 14%). TLC ethyl
2
8