Design, Synthesis, and Biological Evaluation of 1-Benzylamino-2-hydroxyalkyl Derivatives as New Potential Disease-Modifying Multifunctional Anti-Alzheimer's Agents

Article abstract of DOI:10.1021/acschemneuro.7b00461

The multitarget approach is a promising paradigm in drug discovery, potentially leading to new treatment options for complex disorders, such as Alzheimer's disease. Herein, we present the discovery of a unique series of 1-benzylamino-2-hydroxyalkyl deriva

Full text of DOI:10.1021/acschemneuro.7b00461

Subscriber access provided by READING UNIV
Article
Design, synthesis and biological evaluation of 1-
benzylamino-2-hydroxyalkyl derivatives as new potential
disease-modifying multifunctional anti-Alzheimer’s agents
Dawid Panek, Anna Wi#ckowska, Jakub Jo#czyk, Justyna Gody#, Marek W. Bajda,
Tomasz Wichur, Anna Pasieka, Damijan Knez, Anja Pislar, Jan Korabecny, Ondrej Soukup,
Vendula Sepsova, Raimon Sabaté, Janko Kos, Stanislav Gobec, and Barbara Malawska
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00461 • Publication Date (Web): 18 Jan 2018
Downloaded from http://pubs.acs.org on January 19, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Neuroscience is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Design, synthesis and biological evaluation of 1ꢀbenzylaminoꢀ2ꢀhydroxyalkyl derivatives
as new potential diseaseꢀmodifying multifunctional antiꢀAlzheimer’s agents
a1
a1
a
a
a
Dawid Panek , Anna Więckowska , Jakub Jończyk , Justyna Godyń , Marek Bajda ,
a
a
b
b
c,d
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Tomasz Wichur , Anna Pasieka , Damijan Knez , Anja Pišlar , Jan Korabecny ,
c,d
c,d
e,f
b
b
Ondrej Soukup , Vendula Sepsova , Raimon Sabaté , Janko Kos , Stanislav Gobec ,
a
Barbara Malawska *
a
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9, 30ꢀ688 Kraków, Poland
b
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
c
Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05
Hradec Kralove, Czech Republic
d
Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences,
University of Defense, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
e
Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty
of
Pharmacy
and
Food
Science,
University
of
Barcelona,
Av Joan XXIII 27ꢀ31, 08028 Barcelona, Spain
f
Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona, Av Joan
XXIII, S/N, 08028 Barcelona, Spain
*Correspondence: mfmalaws@cyfꢀkr.edu.pl
1
These authors contributed equally to this work
1
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 2 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
The multitarget approach is a promising paradigm in drug discovery, potentially leading to
new treatment options for complex disorders, such as Alzheimer’s disease. Herein, we present
the discovery of a unique series of 1ꢀbenzylaminoꢀ2ꢀhydroxyalkyl derivatives combining
inhibitory activity against butyrylcholinesterase, βꢀsecretase, βꢀamyloid and tau protein
aggregation, all related to mechanisms which underpin Alzheimer’s disease. Notably,
diphenylpropylamine derivative 10 showed balanced activity against both diseaseꢀmodifying
targets – inhibition of βꢀsecretase (IC50 hBACEꢀ1 = 41.60 ꢁM), inhibition of amyloid β
aggregation (IC50 Aβ = 3.09 ꢁM), inhibition of tau aggregation (55% at 10 ꢁM), as well as
against symptomatic targets: butyrylcholinesterase inhibition (IC50 hBuChE = 7.22 ꢁM). It might
represent an encouraging starting point for development of multifunctional diseaseꢀmodifying
antiꢀAlzheimer’s agents.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Keywords: Alzheimer’s disease; multifunctional agents; butyrylcholinesterase inhibitors;
BACEꢀ1 inhibitors; Aβ aggregation; tau aggregation; molecular docking
1. Introduction
Nearly 36 million are affected people today, and three times more projected to be affected in
2050; these alarming numbers show that Alzheimer's disease (AD) remains a serious
1
,2
economic and social problem. Search for novel drugs and effective therapies against AD is
3
one of the largest challenges for modern medicine and pharmacotherapy. Although the cause
of the disease is not fully understood, a variety of mechanisms that might be involved in its
pathogenesis have been described. Among them, deposition of amyloid β (Aβ) peptide,
hyperphosphorylation and aggregation of the tau protein, oxidative stress, and cholinergic
2
ACS Paragon Plus Environment

Page 3 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
4
–7
neurotransmission deficiency are the main players in the etiopathogenetic spectacle. The
cholinergic system is linked with cognitive processes in the brain; therefore, impaired
8
–10
cholinergic neurotransmission leads to memoryꢀassociated symptoms of AD.
Current
research has revealed that the deficiency of acetylcholine (ACh) is not the primary cause of
AD but a consequence contributing to symptoms related to neurodegeneration,.
Physiologically, the activity of ACh is terminated in the synapses by acetylcholinesterase
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
AChE, E.C. 3.1.1.7) and butyrylcholinesterase (BuChE, E.C. 3.1.1.8). While AChE
hydrolyses the majority of acetylcholine in a healthy brain, with the progression of the
disease, BuChE takes over the role of AChE, reaching almost 80% of the overall
11,12
cholinesterase activity.
Three of the four currently available drugs registered between
1
997 and 2003 (donepezil, rivastigmine, galantamine) act as AChE and/or BuChE
13–15
inhibitors.
The effectiveness of these drugs is limited since they are able to ameliorate the
symptoms only in the early stages of the disease. However, cholinesterases still represent
valuable targets in the search for new antiꢀAD agents.
A widely accepted amyloid hypothesis have postulated the formation and aggregation of
1
6,17
the βꢀamyloid (Aβ) peptide as the main cause of AD.
Aβ is a neurotoxic peptide
composed of 37ꢀ42 amino acids that is formed as a consequence of an enzymatic cleavage of
the amyloid precursor protein (APP) by βꢀsecretase (βꢀsite APPꢀcleaving enzyme 1, BACEꢀ1)
and γꢀsecretase. Aβ peptide, particularly the 42ꢀaminoꢀacid form, has high propensity for
aggregation leading to neuroinflammation, oxidative stress, loss of neurons and finally to
cognitive and psychological impairment. Accordingly, reduction of Aβ levels in the central
nervous system (CNS) is considered a rational therapeutic approach in the search for new
18–21
antiꢀAlzheimer’s agents.
The formation of neurofibrillary tangles (NFTs) is another pathological process occurring
in the course of AD. NFTs are composed of hyperphosphorylated microtubuleꢀassociated tau
3
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 4 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
proteins. In the healthy brain, tau stabilizes microtubules, but when hyperphosphorylated, it
forms paired helical filaments that aggregate into NFTs. NFTs promote the instability of
microtubules and impair axonal transport, leading to neurotoxicity and cell death. Ever since
the correlation between tau aggregation, neuron death and clinical dementia was proven, it has
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6,22,23
become one of the key targets in AD.
Despite the very important role of tau protein in
the development of AD, there is a limited number of compounds with tauꢀinhibitory
2
4–26
activity.
Several studies have provided evidence that the formation of tau tangles can be
2
7–29
induced by Aβ.
Therefore, compounds with inhibitory activities against Aβ and tauꢀ
aggregation might have diseaseꢀmodifying effects, which are of special interest for modern
drug discovery.
Taken together, AD is a complex disease of a multiꢀfactorial nature, and there are
multiple therapeutic targets that could be the key to the effective treatment of the disease.
Therefore, AD is considered a disease that requires combination therapy or, as an alternative,
development of multiꢀtarget directed ligands (MTDLs) for its effective therapy. MTDLs are
molecules that can interact simultaneously with more than one biological target. Design and
discovery of new multipotent ligands as potential antiꢀAD drugs is an attractive and widely
30–32
explored approach in drug discovery, as reflected in a number of current research papers.
Researchers are still exploring new and emerging routes to develop effective therapies for
AD. The appropriate selection of biological targets, including those designated as being
diseaseꢀmodifying, is crucial for the design of potent MTDLs. It is, however, challenging to
obtain compounds with balanced activities against the chosen targets that concomitantly retain
the drugꢀlike properties. Herein, we present the results of our research that meet all the above
criteria. Indeed, 1ꢀbenzylaminoꢀ2ꢀhydroxyalkyl derivatives represent a novel class of MTDLs
purposely designed as antiꢀAD agents, inhibitors of AChE, BuChE, BACEꢀ1, amyloid β and
tau aggregation. Due to their balanced activity profile against the selected targets they could
4
ACS Paragon Plus Environment

Page 5 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
display potential symptomatic as well as diseaseꢀmodifying effects. Their druglike properties
were calculated prior to their synthesis as a part of preliminary data (see Supplementary data).
Molecular modelling studies were performed to explain enzymeꢀligand interactions and to
support the structureꢀactivityꢀrelationship discussion.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
.
Results and discussion
.1
Design
The presented research is a continuation of our previous investigation dedicated to the search
33
for MTDLs with cholinesteraseꢀ, BACEꢀ1ꢀ and amyloidꢀβꢀ inhibitory activities. Recently,
we have reported a series of donepezilꢀrelated compounds with alicyclic amines containing
two basic nitrogen atoms in their structures (compound I, Fig. 1.). We assumed that such a
basic core would interact with the catalytic dyad of BACEꢀ1 consisting of two aspartic acid
34
residues: Asp32 and Asp228. In the compounds presented herein, we replaced the alicyclic
amines with 2ꢀhydroxyethyleneamine, a transition state analogue, in order to improve their
inhibitory properties against BACEꢀ1. The designed structures were based on a benzylamine
scaffold taken from donepezil that could interact with the catalytic active site (CAS) of AChE,
3
5
as reported previously. Moreover, substituted benzylamines are commonly used in potent
3
4,36
37,38
BACEꢀ1 inhibitors NVPꢀBXDꢀ552
and GRLꢀ8234
(Fig. 1.), among others.
5
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 6 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Design strategy of new MTDLs targeting cholinesterases and BACEꢀ1.
The benzyl fragment (Fig. 2, fragment A) was connected through a 2ꢀhydroxyethyleneamine
linker (Fig. 2, fragment B) with the fragments selected from the virtualꢀscreening results of
the building blocks available from Sigma Aldrich against BACEꢀ1, AChE and BuChE (Fig. 2,
fragment C). The fragment data based included primary and secondary amines with aliphatic
or aromatic substituents. Building blocks were directed to occupy the hydrophobic S1, S3 and
polar S2, S4 subꢀpockets of BACEꢀ1 as well as the peripheral active sites of AChE and
39
BuChE. From the topꢀscored docking poses, we selected 4ꢀ(diphenylmethyl)piperazine and
6
ACS Paragon Plus Environment

Page 7 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
4
ꢀ(bis(4ꢀfluorophenyl)methyl)piperazine, as well as their elongated analogues 2,2ꢀ
diphenylethanamine or 3,3ꢀdiphenylpropanꢀ1ꢀamine, and truncated analogues 4ꢀ
phenylpiperazine, 4ꢀbenzylpiperazine or 4ꢀ(pentanꢀ2ꢀyl)piperazine.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
fragment A
fragment B
fragment C
N R1
F
F
N
1
R =
R
N
n1
H
OH
N
n2
H
R = H, 4ꢀOCH , 3ꢀtertꢀbutyl
n1 = 1, 2
n2 = 1, 2
3
Figure 2. Combinations of different fragments yielding novel multipotent hybrids to confront
AD
We expected that the designed molecules could also have antiꢀaggregation properties
against neurotoxic βꢀamyloid because our recent studies showed that several series of
40–43
substituted benzylamine derivatives possess Aβ antiꢀaggregation potency.
Analysis of a
set of over 100 compounds revealed that more than 40% of them have Aβ antiꢀaggregation
activity at a 10 ꢁM screening concentration. Molecular docking revealed that the compounds
containing a benzylamine moiety are able to anchor to the amyloid fibre along its axis
4
4
between two betaꢀsheet fragments. The benzyl fragment formed hydrophobic interactions
with alanine (A21), leucine (L34) and valine (V36), while the protonated amine group formed
ionic interactions with the carboxyl group of aspartic acid (D23). Therefore, we decided to
evaluate Aβ antiꢀaggregation properties of the described novel benzylamine derivatives. It
was also shown for several moieties and compounds that inhibition of Aβ aggregation
4
5,46
correlates with inhibition of tau protein aggregation.
propensity of our novel hybrids to inhibit tau aggregation.
Accordingly, we explored the
7
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 8 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Considering the importance of physicochemical properties of compounds in relation to
their drugꢀlikeness and success in the drug development process we calculated the most
4
7
important parameters of the newly designed MTDLs . A majority of the compounds meet the
48
criteria set by Lipinski’s rule of five and the requirements of bioavailability.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.2
Chemistry
We obtained 24 final compounds that can be classified into two groups: 1,3ꢀdiaminopropanꢀ2ꢀ
ol derivatives (7‒17) and 1,4ꢀdiaminobutanꢀ2ꢀol derivatives (28‒38, 45, 46).
The first class, bearing 1,3ꢀdiaminopropanꢀ2ꢀol, was synthesized according to the
Scheme 1. In the first step,
of nonꢀsubstituted or appropriately substituted (4ꢀOCH
chloride. The secondary amine groups in the resulting products were then
Next, the reaction with ꢀbromosuccinimide led to the appropriate bromohydrins that, in the
N
ꢀallylbenzylamine derivatives (1‒3) were obtained by alkylation
, 3ꢀtertꢀbutyl) benzylamines with allyl
ꢀBOC protected.
3
N
N
conditions used, spontaneously formed oxazolidinꢀ2ꢀone rings that served as protecting
groups for the amino and hydroxyl groups. This yielded the compounds (4‒6) used in the next
step as alkylating agents for different Nꢀsubstituted amines. The final compounds (7‒17) were
prepared by alkaline hydrolysis of oxazolidinꢀ2ꢀones, that led to the 1,3ꢀdiaminopropanꢀ2ꢀol
moiety in the core.
8
ACS Paragon Plus Environment

Page 9 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2 3
Scheme 1. Reagents and conditions: i) allyl chloride, MeCN, K CO , 80 °C, 16 h; ii) diꢀtertꢀ
butyl dicarbonate, TEA, DCM, R.T., 4 h; iii)
iv) 4ꢀ(pentanꢀ2ꢀyl)piperazine/4ꢀphenylpiperazine/4ꢀbenzylpiperazine/
diphenylmethyl)piperazine/4ꢀ(bis(4ꢀfluorophenyl)methyl) piperazine/2,2ꢀ
diphenylethanamine/3,3ꢀdiphenylpropanꢀ1ꢀamine, MeCN/ toluene/DMF (microwaveꢀ
assisted), K CO , 60–80 °C, 1‒16 h; v) ethanol, NaOH, 78 °C, 6 h.
Nꢀbromosuccinimide, water, MeCN, R.T., 16 h;
N
ꢀ
(
2
3
To obtain 1,4ꢀdiaminobutanꢀ2ꢀol derivatives, we used the synthetic route depicted in
Scheme 2. First, the hydroxyl group of butꢀ3ꢀenꢀ1ꢀol was protected with a tertꢀbutyl dimethyl
silyl group. Then, the double bond was oxidized with metaꢀchloroperoxybenzoic acid. The
resulting epoxide 18 was used as an alkylating agent in the reaction with the appropriate
benzylamines to provide 1,2ꢀaminoalcohols 19‒21. In the next step the amino and hydroxyl
groups were protected by transformation to oxazolidinꢀ2ꢀone derivatives using
carbonyldiimidazole. Then, the tertꢀbutyl dimethyl silyl group was removed from 22‒24 by
tetraꢀnꢀbutylammonium fluoride and the resulting alcohol was reacted with methanesulfonyl
chloride. Mesylates 25‒27 were used to alkylate the appropriate primary or secondary amines.
Some of these alkylations were carried out using microwave radiation, which reduced the
9
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 10 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
time of reaction from 16 hours to 1 hour. The final compounds 28‒38 were obtained after
alkaline hydrolysis of oxazolidinꢀ2ꢀone.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Reagents and conditions: i) tertꢀbutylchlorodimethylsilane, DCM, imidazole, R.T.,
5 h, ii) metaꢀchloroperoxybenzoic acid, DCM, R.T. 16 h, iii) phenylmethanamine/(4ꢀ
methoxyphenyl)methanamine/(3ꢀ(tertꢀbutyl)phenyl)methanamine,
C, iv) carbonyldiimidazole, DCM, TEA, R.T., 5 h, v) tetraꢀ ꢀbutylammonium fluoride, THF,
R.T., 16 h; vi) methanesulfonyl chloride, DCM, TEA, R.T., 2 h; vii) 4ꢀ(pentanꢀ2ꢀ
yl)piperazine/4ꢀphenylpiperazine/ ꢀbenzylpiperazine/ ꢀ(diphenylmethyl)piperazine/4ꢀ(bis(4ꢀ
nꢀpropanol, pyridine, 97
°
n
N
N
fluorophenyl)methyl)piperazine/2,2ꢀdiphenylethanamine/3,3ꢀdiphenylpropanꢀ1ꢀamine,
MeCN/toluene/DMF (microwave assisted), K CO , 80 °C, 1‒16 h; viii) ethanol, NaOH, 78
2
3
°
C, 6 h.
Compounds 45 and 46, with the fourꢀcarbon fragment B, (Fig. 2) were prepared by a
slightly modified synthetic pathway presented in Scheme 3. The principal difference was the
1
0
ACS Paragon Plus Environment

Page 11 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
order of amine substitution; herein, ω, ω’ꢀdiphenylalkylamines were alkylated prior to
attachment of the benzylamine moiety in the last step.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Reagents and conditions: i) 2,2ꢀdiphenylethanamine/3,3ꢀdiphenylpropanꢀ1ꢀamine,
n
ꢀpropanol, pyridine, 97 °C, ii) carbonyldiimidazole, DCM, TEA, R.T., 5 h, iii) tetraꢀ
butylammonium fluoride, THF, R.T., 16 h; iv) methanesulfonyl chloride, DCM, TEA, R.T., 2
h; v) phenylmethanamine, MeCN, K CO , 80 °C, 16 h; vi) ethanol, NaOH, 78 °C, 6 h.
BuChE, BACEꢀ1 and Aβ aggregation by 7‒17,
nꢀ
2
3
Table 1. Inhibition of eeAChE, eqBuChE,
h
h
a
28‒38, 45, 46.
eeAChE
eqBuChE
hBuChE
hBACE1
Inh. of Aβ aggr.
Cmp.
R
1
R
2
n
1
n
2
IC50 [µM]/
IC50 [µM]/
IC50 [µM]/
IC50 [µM]/
IC50 [µM]/
b
c
d
e
f
%
inh.
% inh.
% inh.
% inh.
% inh.
h
11
12
13
14
15
16
17
32
33
34
35
4ꢀOMe
4ꢀOMe
4ꢀOMe
H
pentanꢀ2ꢀyl
phenyl
1
1
<10%
<10%
20.03% ± 0.57
39.57% ± 2.27
24.91% ± 1.39
30.72% ± 2.55
24.50% ± 0.70
16.61 ± 0.35
7.12 ± 0.15
n.d.
31.08% ± 4.14
23.67% ± 4.43
24.65% ± 1.66
43.55% ± 2.53
47.45% ± 2.98
41.59% ± 4.83
79.68 ± 2.72
15.9% ± 1.1
<10%
h
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
n.d.
h
benzyl
<10%
n.d.
<10%
h
diphenylmethyl
diphenylmethyl
diphenylmethyl
bis(4ꢀfluorophenyl)methyl
pentanꢀ2ꢀyl
<10%
n.d.
21.08% ± 0.5
16.3% ± 5.2
77.2% ± 2.1
21.8% ± 10.5
23.7% ± 8.9
<10%
h
4ꢀOMe
3ꢀtertꢀbutyl
4ꢀOMe
4ꢀOMe
4ꢀOMe
4ꢀOMe
H
<10%
n.d.
14.13% ± 4.03
14.04% ± 2.32
3.62 ± 0.07
<10%
<50%
<50%
<50%
9.36 ± 0.08
35.03% ± 5.52
22.43% ± 2.37
34.88% ± 7.73
42.95% ± 3.60
h
phenyl
<10%
n.d.
h
benzyl
36.62% ± 1.85
17.08% ± 7.92
35.55% ± 0.64
21.01% ± 5.13
n.d.
32.3% ± 14.0
24.2% ± 9.9
h
diphenylmethyl
n.d.
1
1
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 12 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
h
3
3
3
6
7
8
4ꢀOMe
diphenylmethyl
1
1
1
2
2
2
13.63% ± 0.98
<10%
31.52% ± 3.05
9.16 ± 0.24
n.d.
33.89% ± 3.28
41.43% ± 3.68
101.90 ± 2.76
15.2% ± 8.1
81.4% ± 7.9
<10%
h
3ꢀtertꢀbutyl
4ꢀOMe
phenylmethyl
n.d. .
h
bis(4ꢀfluorophenyl)methyl
25.08% ± 1.10
45.57% ± 1.76
n.d.
7
8
9
H
2,2ꢀdiphenylethylamine
3,3ꢀdiphenylpropylamine
2,2ꢀdiphenylethylamine
1
1
1
1
1
1
17.94% ± 3.13
<10%
1.49 ± 0.03
1.59 ± 0.05
1.55 ± 0.05
<50%
76.47 ± 3.60
33.6% ± 1.2
53.3% ± 2.7
88.7% ± 5.5
H
6.76 ± 0.18 55.42% ± 7.36
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3ꢀtertꢀbutyl
<10%
7.22 ± 0.32
64.48 ± 2.55
8
8.2% ± 5.9
10
3ꢀtertꢀbutyl
3,3ꢀdiphenylpropylamine
1
1
21.78% ± 5.46
2.92 ± 0.1
5.74 ± 0.26
41.60 ± 1.47
3.09 ± 1.64
29.7% ± 6.3
<10%
28
H
2,2ꢀdiphenylethylamine
3,3ꢀdiphenylpropylamine
2,2ꢀdiphenylethylamine
1
1
1
2
2
2
13.22% ± 0.42
<10%
2.90 ± 0.04
6.74 ± 0.12
2.09 ± 0.08
5.18±0.16
63.76 ± 3.27
39.68 ± 2.11
2
9
0
H
<50%
3
3ꢀtertꢀbutyl
<10%
2.69±0.12 49.24% ± 16.11
87.6% ± 3.1
8
4.9% ± 0.8
.22 ± 0.89
31
3ꢀtertꢀbutyl
3,3ꢀdiphenylpropylamine
1
2
<10%
<10%
5.61 ± 016
<50%
<50%
32.17 ± 1.46
1
45
ꢀ
ꢀ
2,2ꢀdiphenylethylamine
3,3ꢀdiphenylpropylamine
2
2
1
1
6.64 ± 0.086
2.34 ± 0.044
36.24% ± 2.96
44.8% ± 1.3
45.4% ± 5.0
46
<10%
4.99±0.18 22.26% ± 13.86
References
0.023±0.0004
0.011±0.0002
h
Tacrine
0.015±0.0001
1.83±0.04
n.d.
h
Donepezil
n.d.
13.80% ± 6.8
78.50 ± 3.1
g
h
h
Inhibitor IV
n.d.
n.d.
0.046 ± 0.001
Resveratrol
█
ꢀ the most interesting values
a
Values are expressed as the means ± the standard error of the mean (SEM) of at least three
experiments (n = 3), each performed in triplicate (eeAChE, eqBuChE and hBACEꢀ1
inhibition).
b
IC50 inhibitory concentration of AChE from electric eel or percent inhibition with inhibitor
at 10 ꢁM.
c
IC50 inhibitory concentration of BuChE from horse serum or percent inhibition with
inhibitor at 10 ꢁM.
d
IC50 inhibitory concentration of human recombinant BuChE or percent inhibition with
inhibitor at 10 ꢁM.
e
IC inhibitory concentration of human recombinant BACEꢀ1 and substrate (Rhꢀ
50
EVNLDAEFKꢀquencher) or percent inhibition with inhibitor at 50 mM.
1
2
ACS Paragon Plus Environment

Page 13 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
f
IC inhibitory concentration of selfꢀinduced Aβ aggregation or percent inhibition with
50
1–42
inhibitor at 10 ꢁM.
g
Calbiochem, Merck; Nottingham, UK.
h
Not determined.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.3
Inhibition of eeAChE, eqBuChE and
hBuChE
We evaluated the abilities of the synthesized compounds to inhibit AChE from electric eel
49
(
eeAChE) and BuChE from equine serum (eqBuChE) using Ellman’s method. Initially, we
performed a screening assay using a single concentration of inhibitors (10 ꢁM) and
determined their activity as a percentage of enzyme inhibition. Based on the preliminary data,
the compounds with inhibitory potencies higher than 50% were established, and their
inhibitory potencies were expressed as IC50 values. Tacrine and donepezil were used as
positive reference compounds. The results of the assay are presented in Table 1.
Among the tested compounds, only compound 32 displayed inhibitory activity against
both eeAChE and eqBuChE, with IC50 values of 3.62 ꢁM and 9.36 ꢁM, respectively. At the
same time, we found some potent inhibitors of eqBuChE with IC50 values ranging from 1.49
to 16.61 ꢁM: 7‒10, 16, 17, 28‒31, 37, 45, and 46, with 7 being the most promising. Since
these compounds were almost inactive against eeAChE they are considered as being selective
eqBuChE inhibitors. A structureꢀactivity relationship analysis revealed that the most active
compounds contain a 2,2ꢀdiphenylethylamine or 3,3ꢀdiphenylpropylamine moiety in their
structures (Fig. 2, fragment C). Replacement of these fragments with 4ꢀpiperazine resulted in
a noticeable reduction of their inhibitory activity (14 vs. 7 and 8, 35 vs. 28 and 29), although
this trend was not observed in the compounds with a 3ꢀtertꢀbutylꢀbenzylamine fragment (Fig.
2, fragment A) (16 vs. 9 and 10, 37 vs. 30 and 31). The tertꢀbutyl substituent improved the
activity of
Nꢀ(diphenylmethyl)piperazine derivatives as compared to unsubstituted ones (14
1
3
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 14 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
vs. 16, 35 vs. 37) but it did not have the same effect on ω, ω’ꢀdiphenylalkylamine derivatives
(7 vs. 9, 8 vs. 10, 28 vs. 30, 29 vs. 31). Regarding the length of an alkylene core (Fig. 2,
fragment B), we observed slightly higher activities for 1,3ꢀdiaminopropanꢀ2ꢀol derivatives
than for 1,4ꢀdiaminobutanꢀ2ꢀol derivatives (28 vs. 7, 30 vs. 9, 29 vs. 8, 31 vs. 10). Based on
the results we can say that a secondary ω, ω’ꢀdiphenylalkylamine group (Fig. 2, fragment C)
is more beneficial here than the two tertiary amine groups of the piperazine fragment (Fig. 2,
fragment C).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The selectivity of the presented compounds against BuChE over AChE may be a
consequence of the size of fragment C (Fig. 2). Larger fragments, such as those containing
benzhydryl moieties, are too bulky to fit into the narrow gorge of the AChE catalytic site. On
the other hand, the BuChE active site is more spacious and accepts larger molecules;
therefore, the presented compounds are selective BuChE inhibitors. The only exception is
compound 32, with a smaller and more flexible 4ꢀ(pentꢀ2ꢀyl)piperazine fragment, which is an
inhibitor of both AChE and BuChE. Similar observations have already been described in
41,50,51
previously published papers.
For the most potent inhibitors of eqBuChE we also evaluated their inhibitory potencies
against human BuChE ( BuChE). We found that six of the tested compounds (8‒10, 28, 30,
6) inhibit this enzyme more than 50%, and for those six compounds, we established IC50
values ranging from 2.69 to 7.22 ꢁM. Only the compounds with 2,2ꢀdiphenylethylamine or
,3ꢀdiphenylpropylamine moieties displayed activities against BuChE. Additionally, the
h
4
3
h
activities were lower than they were for eqBuChE. The effect of the tertꢀbutyl substituent in
the benzyl ring keeps us from drawing an unambiguous SAR. The tertꢀbutyl substituent
significantly improved the activity of 9 compared with unsubstituted 7 but improved the
activities of 10 and 30 only to a small extent compared to 8 and 28, respectively. The role of
the linker length was crucial for the inhibition of hBuChE. Derivatives of 1,3ꢀdiaminopropanꢀ
1
4
ACS Paragon Plus Environment

Page 15 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
2
ꢀol with 3,3ꢀdiphenylpropylamine (8, 10) exhibited higher inhibition potency compared to
their 2,2ꢀdiphenylethylamine counterparts (7 and 9), while derivatives of 1,4ꢀdiaminobutanꢀ2ꢀ
ol (28, 30) were more active in combination with 2,2ꢀdiphenylethylamine than with the 3,3ꢀ
diphenylpropylamine fragment (29, 31).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In the search for new antiꢀAlzheimer’s agents, both AChE and BuChE are important
biological targets. With the progression of the disease the role of BuChE in the regulation of
cholinergic neurotransmission systematically grows at the expense of AChE. Additionally,
there is evidence for nonꢀenzymatic functions of BuChE that are relevant for AD treatment,
5
2–56
e.g., reduction of Aβ levels.
Due to the above facts, MTDLs with inhibitory potency
toward BuChE presented in this study may be relevant for the treatment of both the symptoms
57
and the causes of the disease .
2.4 Kinetic studies of eqBuChE inhibition
A
B
Compound 10
Compound 10
3
30
0 ꢀM
2
ꢀM
ꢀM
0.3 mM BTC
3
0.18 mM BTC
0.12 mM BTC
0.06 mM BTC
20
5 ꢀM
7.5 ꢀM
2
1
10 ꢀM
10
-30
-20
-10
10
20
30
-15
-10
-5
5
10
15
/ S [mM]ꢀ1
I [ꢁM]
1
-
10
-1
-2
-20
Figure 3. LineweaverꢀBurk (A) and CornishꢀBowden (B) plots illustrating mixed types of
eqBuChE inhibition by compound 10. S = butyrylthiocholine;
V = initial velocity rate; I =
inhibitor concentration.
The kinetic studies of eqBuChE inhibition were performed with compound 10 as one of the
most potent eqBuChE inhibitors presented herein. Although the LineweaverꢀBurk reciprocal
1
5
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 16 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
plots are ambiguous (Fig. 3A), the calculated K values and Cornish–Bowden plots are
m
characteristic for the mixed types of inhibition (Fig. 3B). Mixed types of enzyme inhibition
m
with increasing K at increasing concentrations of 10 indicates a higher affinity of the
inhibitor to free enzyme than to the enzymeꢀsubstrate complex and suggests preferential
interaction of the inhibitor with the catalytic active site than with the peripheral anionic site of
eqBuChE; however, both interactions are possible at the same time.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To date, a number of selective pseudoꢀirreversible BuChE inhibitors have been described,
58
59
including carbamate analogues of cymserine or isosorbide. However, the pseudoꢀ
irreversible mechanism of inhibition may cause nonꢀspecific or offꢀtarget reactivityꢀbased
60,61
toxicities.
Therefore, selective reversible BuChE inhibitors are now of great interest, due
52,62–65
to their potential for use in AD treatment. Such inhibitors have already been reported
,
however, compound 10 as the selective reversible BuChE inhibitor with mixed types of
enzyme inhibition and a broad multifunctional profile of biological activity, seems to be
unique.
2.5 Inhibition of BACEꢀ1
We determined the inhibitory activities against human recombinant BACEꢀ1 (hBACEꢀ1) of
all the compounds in the study using a spectrofluorometric assay (fluorescence resonance
energy transfer, FRET based). This assay is based on the cleavage of a modified peptide
substrate (analogue of the APP sequence with the Swedish mutation), leading to an increase in
66
fluorescence. For the reference compound, inhibitor IV, we established an IC50 value of
67
0.046 ꢁM, which corresponds to the literature data. First, we tested the ability of the new
compounds to inhibit BACEꢀ1 at a single concentration of inhibitors (50 ꢁM). For the
h
selected compounds we evaluated IC50 values ranging from 32.17 to 101.90 ꢁM (Table 1).
The most potent compounds contain 2,2ꢀdiphenylethylamine (7, 9, 28), 3,3ꢀ
1
6
ACS Paragon Plus Environment

Page 17 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
diphenylpropylamine (8, 10, 29, 31) or 4ꢀ(bis(4ꢀfluorophenyl) methyl)piperazine (17 and 38)
fragments.
The starting point for the design of the MTDLs presented herein was the selective BACEꢀ1
inhibitor NVPꢀBXDꢀ552. To expand the activity profile of the compound towards
cholinesterases, we discarded the sulfone moiety embedded in the cyclic hydroxyethylamine
scaffold. Although it creates significant interactions with the flap of BACEꢀ1 (Hꢀbond with
Thr72 and Gly73), it does not provide optimal interactions with cholinesterases. The obtained
compounds displayed significantly reduced activity against BACEꢀ1 when compared to
selective BACEꢀ1 inhibitors, but they displayed wellꢀbalanced activity towards all biological
targets what is an important feature of MTDLs.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.6 Inhibition of Aβ aggregation
We determined the Aβ antiꢀaggregation profiles of the obtained compounds by a ThioflavinꢀT
68
assay. First, we carried out a screening assay at a 10 ꢁM concentration of each compound.
Seven of the tested compounds inhibited Aβ aggregation by more than 50% (8‒10, 16, 30, 31,
37) with comparable or higher activities to the reference compound resveratrol. The IC50
values were established for the two selected compounds: 10 (IC = 3.09 ꢁM) and 31 (IC =
5
0
50
1.22 ꢁM). To exclude the interference of the compounds with the readout of Thioflavin T
fluorescence and to confirm the inhibitory activity, we also performed a dot blot assay for
these two derivatives. Both reduced the formation of Aβ fibrils, which was detected using a
fibrilꢀspecific LOC antibody (46.0% ± 3.8% and 40.6% ± 4.6% inhibition of Aβ fibril
formation for 10 and 31, respectively). Analysis of the results (Table 1.) showed that all the
active compounds contained a diphenylmethyl moiety in their structures. The tertꢀbutyl
substituent in the benzylamine fragment has a major impact on the activity of the compounds,
as exemplified by the comparisons of 7 vs. 9, 8 vs. 10, 14 vs. 16, 28 vs. 30 and 35 vs. 37. The
1
7
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 18 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
results obtained confirm our expectation that the target compounds may inhibit betaꢀamyloid
aggregation. This is a valuable property of these molecules, which can further inhibit the
formation of amyloid plaques.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.7 Inhibition of tau aggregation
The role of tau proteins in the aetiopathogenesis of AD is unambiguous, and the inhibition of
its aggregation may modify the course of the disease. Based on their inhibitory activities
against Aβ aggregation we selected four compounds (9, 10, 31 and 37) and tested their
abilities to inhibit tau aggregation. We tested the compounds using a ThioflavinꢀTꢀbased
69
assay using truncated and fullꢀlength forms of tau protein. All the tested compounds
displayed inhibitory potencies ranging from 45% to 70% at 10 ꢁM concentration (Table 2).
The most potent compound 37 inhibited both truncated and fullꢀlength tau aggregation by
6
8%.
Table 2. Inhibition of fullꢀlength and truncated tau aggregation by compounds 9, 10, 30 and
7.
3
Compound Inhibition of
Inhibition of
fullꢀlenght tau truncated tau
aggregationa
aggregationa
[% ± SEM]
54.5 ± 2.5
53.8 ± 5.0
59.7 ± 2.3
68.7 ± 3.7
[
% ± SEM]
55.1 ± 3.6
44.4 ± 4.5
62.7 ± 1.2
68.0 ± 3.4
9
1
3
3
0
0
7
a
10 ꢁM compound concentration, 40 ꢁM tau concentration, experiments performed in
triplicate.
1
8
ACS Paragon Plus Environment

Page 19 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Anti-aggregation activity
100
90
80
70
60
50
40
30
20
10
0
IC = 3.09 µM
IC = 1.22 µM
50
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
Aβ aggregation
tau aggregation
Figure 4. The effect of the synthesized compounds on Aβ and tau aggregation.
Among multitarget agents with inhibitory activities towards symptomatic and diseaseꢀ
modifying targets in AD, only a few groups of compounds possess activities towards the
inhibition of aggregation of both Aβ and tau. Compared with the data presented herein (Fig.
4
), the potencies of these inhibitors are similar or lower against tau aggregation and lower
against Aβ aggregation. Furthermore, some of these compounds with similar multifunctional
profiles are relatively large, with a high molecular weight, large numbers of condensed
70,71,25
aromatic rings and very high lipophilicities
. On the other hand, there is a number of
aggregation inhibitors with high hydrophilicity and therefore low bioavailability e.g.
24,72
polyphenols, anthraquinones, phenylthiazolylhydrazides and benzothiazoles
. Not rare
73
aggregation inhibitors can be classified as PAINS (Pan Assay Interference Compounds) so
they can nonꢀspecifically react with a number of undesirable proteins. Herein, we have
proposed nonꢀPAINS with antiꢀaggregation properties, relatively small molecular weights and
optimized druglike properties which constitutes an interesting approach for further
development.
1
9
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 20 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
.8 Blood brain barrier (BBB) permeation
Since one of the basic features of antiꢀAD drugs is their ability to reach therapeutic targets in
CNS, it is important to evaluate this parameter at the earliest stage of the development of
novel antiꢀAD agents. To evaluate whether our compounds are centrally active, we used a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
74
parallel artificial membrane permeability assay (PAMPA). The data obtained for the new
compounds were compared to those of standard drugs with known CNS permeability, which
were also confirmed using PAMPA. Prediction of BBB penetration is summarized in Table 3.
e
All the tested compounds (7, 8, 9, 17, 31, 32, 37, 45) displayed P values higher than 4.0, thus
showing high probability for penetration of the BBB via passive diffusion. One of the major
challenges in the design of MTDLs is optimization of their druglike properties. These
preliminary results are optimistic forecasts for advanced pharmacokinetic studies and the
possibility of further development of these compounds as antiꢀAD agents.
Table 3. In vitro bloodꢀbrain barrier permeation assay for compounds 7, 8, 9, 17, 31, 32, 37
and 45.
BBB penetration estimation
Compound
–
6
–1
a
P
e
± SEM (*10 cm s )
CNS (+/–)
7
8
9
8.9 ± 3.3
5.2 ± 0.2
7.4 ± 1.4
7.1 ± 1.2
5.7 ± 1.4
6.5 ± 0.9
9.1 ± 0.2
4.9 ± 0.7
7.3 ± 0.9
6.6 ± 0.5
5.3 ± 0.19
11.3 ± 1.6
5.6 ± 0.6
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
1
7
1
2
7
3
3
3
45
Donepezil
Rivastigmine
Tacrine
Testosterone
Chlorpromazine
2
0
ACS Paragon Plus Environment

Page 21 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
Hydrocortisone
Piroxicam
2.85 ± 0.1
2.2 ± 0.15
1.07 ± 0.18
1.02 ± 0.37
CNS (+/–)
CNS (+/–)
CNS (–)
Theophylline
Atenolol
CNS (–)
a
ꢀ6
ꢀ1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CNS (+) (high BBB permeation predicted), P
e
(10 cm s ) > 4.0; CNS () (low BBB
ꢀ6
ꢀ1
e e
permeation predicted), P (10 cm s ) ≤ 2.0; CNS (+/) (BBB permeation uncertain), 4.0 ≥ P
ꢀ6
ꢀ1
75
(
10 cm s ) > 2.0.
2.9 Docking studies on BACEꢀ1 and BuChE
Molecular modelling studies helped us better understand the binding modes of the active
compounds within the active sites of BACEꢀ1 and BuChE. All BACEꢀ1 inhibitors
h
h
presented one dominant conformation when docked to the enzyme. We chose the most active
ligand, 31, to present the observed interactions (Figure 5). The hydroxyethylamine group,
used as a transition state analogue, was the main grip point of molecule 31. It formed
characteristic ionic and hydrogen bonds with the catalytic dyad (Asp32 and Asp228) and
additional hydrogen bonds with Gly34, Thr220 and the ligand. The benzylamine fragment
provided interactions within the S1’ and S2’ subꢀpockets of the enzyme. A substituted
benzylamine fragment together with the hydroxyethylamine moiety allows for effective
pharmacophore binding to the BACEꢀ1 active site. The positions that these fragments take in
the enzyme are the same as those observed in the Xꢀray structures of potent BACEꢀ1
36,76,37,38
inhibitors, including the previously mentioned NVPꢀBXDꢀ552 and GRLꢀ8234.
The
diphenylmethyl moiety was located within the confines of the aromatic S1 and S3 subꢀpockets
and interacted with the s10 loop. These interactions are crucial for the effective inhibition of
the enzyme both by strong, single target inhibitors of BACEꢀ1 and by the presented MTDLs.
Ligands with smaller substituents like benzyl or phenyl fragments, occupying only one subꢀ
pocket, were less active. Additionally, the position of the diphenylmethyl fragment enables
2
1
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 22 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
further extension of the compounds in the S2 and S3 subꢀpockets and makes the enhancement
of their inhibitory activity possible.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Subsite color legend:
ꢀ S4'
ꢀ S3'
ꢀ S2'
ꢀ S1'
ꢀ S1
ꢀ S2
ꢀ S3
ꢀ S4
Figure 5. Predicted binding mode of compound 31 (green) with BACEꢀ1. The general view
presents the position of the ligand in the active site of the enzyme with coloured subsites S4'–
77
S4. In the detailed representation, the most important amino acids are marked in yellow; red
dots represent hydrogen bonds.
Considering the results of the docking studies performed with hBuChE, we divided the
ligands into two groups: more potent derivatives with bent conformations (2,2ꢀ
diphenylethylamine and 3,3ꢀdiphenylpropylamine derivatives) and less potent derivatives
with extended conformations (piperazine derivatives). A bent conformation allows the
formation of additional CH−π interactions between the benzyl ring and Trp231, as presented
in Figure 6. In the extended conformation, the benzyl ring is situated at the entrance of the
active site, where it creates only weak interactions with the amino acids of PAS. Additionally,
the nitrogen atoms in 2,2ꢀdiphenylethylamine and 3,3ꢀdiphenylpropylamine derivatives are
2
2
ACS Paragon Plus Environment

Page 23 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
preferentially protonated compared to the piperazine nitrogen atom and provide cation−π
interactions with Trp82 and Tyr332. The diphenylmethyl moiety in all the compounds creates
hydrophobic interactions with Trp82 and Trp430. In addition, the hydroxyethylamine
fragment provides strong interactions with Pro285 by forming two hydrogen bonds.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Subsite color legend:
ꢀ anionic subsite
ꢀ
ꢀ oxyanion hole
acyl pocket
ꢀ PAS
ꢀ catalitic triad
Figure 6. Predicted binding mode of compound 30 (green) with BuChE. The general view
presents the position of the ligand in the active site of the enzyme with coloured subsites. In
the detailed representation, the most important amino acids are marked in yellow; red dots
7
8
represent hydrogen bonds.
3
Conclusions
2
3
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 24 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
With a rapidly growing number of patients with AD, a lack of effective treatments has
rendered AD one of the largest issues in current pharmaceutical research. Due to the
multifactorial nature of the disease, designing multifunctional ligands is considered a rational
strategy in the search for new antiꢀAlzheimer’s treatments. In the selection of biological
targets for the novel MTDLs presented herein, we turned our attention mainly to diseaseꢀ
modifying mechanisms. As a starting point, we used compounds on the basis of their
cholinesterase inhibition and modified them to treat the symptoms of the disease. Based on
the structure of BACEꢀ1 inhibitor (NVPꢀBXDꢀ552) and on our previous studies, we designed,
synthesized and evaluated 24 novel multitargetꢀdirected ligands targeting AChE, BuChE,
BACEꢀ1 and the aggregation of Aβ and tau proteins. Generally, we found that the compounds
exhibited potency towards all chosen biological targets in the micromolar range, and some of
them even showed broad and wellꢀbalanced profiles of activities directed at all of the targets.
For the representative compounds, we confirmed the ability to permeate the BBB by the
BBBꢀPAMPA assay.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We highlight compounds 9 and 10 as being the most interesting due to their potencies as well
as their broad and wellꢀbalanced spectra of biological activities. Both compounds selectively
inhibit eqBuChE and hBuChE over eeAChE. However, more importantly, 9 and 10 are also
able to inhibit diseaseꢀmodifying targets BACEꢀ1 and the aggregation of both Aβ and tau
79
proteins. Compared to previously reported compounds we have broadened the profile in
these new multifunctional agents, conferring upon them a broader spectrum of activities and
improved potencies against BACEꢀ1 and Aβ aggregation. We have tried to compile the results
with the current knowledge about AD. The pathogenesis based on amyloidopathy and
tauopathy starts can even start 25 years before the first symptoms. The appearance of amyloid
in the CSF and brain slightly precede the occurrence of tau pathology, nevertheless these
processes are progressing simultaneously therefore they are just as important. There is
2
4
ACS Paragon Plus Environment

Page 25 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
continued progress in the diagnostics of AD, and perhaps, soon, it will be possible to diagnose
AD long before the first symptoms. Therefore, the dual Aβ and tau antiꢀaggregation profile in
combination with the inhibition of BACEꢀ1 is of much interest for diseaseꢀmodifying antiꢀ
Alzheimer’s agents. However, if the first memory disorders are already present, the patients
should be treated with cholinesterase inhibitors, for instance; even at that stage, it is worth
inhibiting pathological processes leading to the formation of both senile plaques and
neurofibrillary tangles to try to stop the progression of AD. To the best of our knowledge,
compounds combining inhibitory activities against BuChE, BACEꢀ1, tau and Aβ aggregation
have not been published to date. Therefore, this is the first report of multiꢀtargeted compounds
combining these functionalities and taking into account potential diseaseꢀmodifying and
symptomatic activities. The presented results will be a good starting point for further
development of analogues derived from compound 10 and for further studies of these valuable
candidates.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
Methods
4.1
Chemistry
4.1.1 General methods
1
13
H NMR and C NMR spectra were recorded on Varian Mercury 300 at 300 MHz. The
1
1
chemical shifts for H NMR are referenced to TMS via residual solvent signals ( H, CDCl
3
at
7
.26 ppm, DMSOꢀd at 2.50 ppm). Mass spectra (MS) were recorded on UPLCꢀMS/MS
6
®
®
system consisting of a Waters ACQUITY UPLC (Waters Corporation, Milford, MA, USA)
coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESIꢀtandem
quadrupole). Analytical thin layer chromatography (TLC) was done using aluminum sheets
precoated with silica gel 60 F254. Column chromatography was performed on Merck silica gel
60 (63–200 ꢁm). For the TLC and column chromatography following solvents were used:
2
5
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 26 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dichloromethane (DCM), methanol (MeOH), petroleum ether, chloroform, ethyl acetate, 25%
ammonia water solution. The purity of the final compounds was determined using an
analytical RPLCꢀMS on Waters Acquity TQD using an Aquity UPLC BEH C18 column (1.7
3 2
ꢁm, 2.1 x 100 mm) at 214 nm and 254 nm. CH CN/H O gradient with 0.1% HCOOH was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
used as the mobile phase at a flow rate of 0.3 mL/min. All the compounds showed purity
above 95%. All of the reagents were purchased from commercial suppliers and were used
without further purification. Tetrahydrofuran (THF) and DCM were distilled under nitrogen
immediately before use. The drying agent used for THF was sodium/benzophenone ketyl, and
for DCM, calcium hydride.
80
The following compounds: Nꢀbenzylpropꢀ2ꢀenꢀ1ꢀamine (1) , Nꢀ(4ꢀmethoxybenzyl)propꢀ2ꢀ
8
0
81
enꢀ1ꢀamine (2) , 1ꢀ(4ꢀbenzhydrylpiperazinꢀ1ꢀyl)ꢀ3ꢀ(benzylamino)propanꢀ2ꢀol (14) , tertꢀ
82
butylꢀdimethyl(2ꢀ(oxiranꢀ2ꢀyl)ethoxy)silane (18) have been reported previously.
4.1.2 Procedure for the synthesis of compounds: 1‒3 (procedure A)
2 3
To a mixture of benzylamine derivative (1.5 equiv.) and K CO (3.0 equiv.) refluxed in
acetonitrile (MeCN) a solution of allyl chloride (1.0 equiv.) in MeCN was added dropwise
and the reaction mixture was refluxed for 16 h. After the mixture was cooled to the room
temperature, the solvent was removed and the residue was treated with saturated aqueous
solution of NaHCO
anhydrous Na SO , filtered and concentrated under reduced pressure. The resulting residue
was then purified by flash column chromatography.
3
and extracted with DCM. The combined organic extracts were dried over
2
4
4.1.2.1 NꢀBenzylpropꢀ2ꢀenꢀ1ꢀamine (1)
Following the procedure A, reaction of benzylamine (0.428 mL, 3.92 mmol) dissolved in 10
mL MeCN with allyl chloride (0.214 mL, 2.61 mmol) dissolved in 5 mL MeCN in the
2 3
presence of K CO (1.080 g, 7.84 mmol) was performed. Extraction: 10 mL sat. NaHCO3(aq),
2
6
ACS Paragon Plus Environment

Page 27 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
3
× 10 mL DCM. Purification: column chromatography in DCM/MeOH/NH ·H O
3 2
(9.8/0.2/0.05, v/v/v). Yield: 180 mg (47%), TLC (DCM/MeOH/NH ·H O, 9.8/0.2/0.05, v/v/v)
3
2
+
1
R
f
= 0.13. MW 147.22. Formula C10
CDCl ) δ 7.22 ꢀ 7.37 (m, 5H), 5.94 (ddt, J = 17.18, 10.19, 5.93, 5.93 Hz, 1H), 5.20 (dq, J =
7.18, 1.71 Hz, 1H), 5.12 (dq, J = 10.26, 1.45 Hz, 1H), 3.80 (s, 2H), 3.28 (dt, J = 5.96, 1.51
Hz, 2H), 1.51 (br. s, 1H).
H13N. MS m/z 148.13 (M+H ). H NMR (300 MHz,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4.1.2.2
Nꢀ(4ꢀMethoxybenzyl)propꢀ2ꢀenꢀ1ꢀamine (2)
Following the procedure A, reaction of 4ꢀmethoxybenzylamine (0.512 mL, 3.92 mmol)
dissolved in 10 mL MeCN with allyl chloride (0.214 mL, 2.61 mmol) dissolved in 5 mL
MeCN in the presence of K
2 3
CO (1.080 g, 7.84 mmol) was performed. Extraction: 10 mL sat.
NaHCO3(aq)
,
3
×
10 mL DCM. Purification: column chromatography in
DCM/MeOH/NH ·H O (9.8/0.2/0.05, v/v/v). Yield: 221 mg (48%), TLC
3
2
(
DCM/MeOH/NH
3
·H
2
O, 9.8/0.2/0.05, v/v/v) R
f
= 0.16. MW 177.24. Formula C11
) δ 7.21 ꢀ 7.27 (m, 2H), 6.83 ꢀ 6.89 (m, 2H),
.85 ꢀ 6.01 (m, 1H), 5.19 (dq, J = 17.28, 1.68 Hz, 1H), 5.10 (dq, J = 10.26, 1.45 Hz, 1H), 3.79
s, 3H), 3.73 (s, 2H), 3.26 (dt, J = 5.96, 1.51 Hz, 2H), 1.42 (br. s, 1H).
H15NO. MS
+
1
m/z 178.04 (M+H ). H NMR (300 MHz, CDCl
3
5
(
4.1.2.3
Nꢀ(3ꢀ(Tertꢀbutyl)benzyl)propꢀ2ꢀenꢀ1ꢀamine (3)
Following the procedure A, reaction of (3ꢀ(tertꢀbutyl)phenyl)methanamine (1.000 g, 6.13
mmol) dissolved in 20 mL MeCN with allyl chloride (0.312 g, 4.08 mmol) dissolved in 10
mL MeCN in the presence of K
2 3
CO (1.693 g, 12.25 mmol) was performed. Extraction: 20
mL sat. NaHCO3(aq), 3 × 20 mL DCM. Purification: flash chromatography in DCM/MeOH
(9.5/0.5, v/v). Yield: 507 mg (61%), TLC (DCM/MeOH, 9.8/0.2 v/v) R
f
= 0.17. MW 203.32.
) δ 7.12 ꢀ 7.38 (m,
+
1
Formula C14
H
21N. MS m/z 204.09 (M+H ). H NMR (300 MHz, CDCl
3
2
7
ACS Paragon Plus Environment

ACS Chemical Neuroscience
Page 28 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
H), 5.87 ꢀ 6.04 (m, 1H), 5.22 (dd, J = 17.29, 1.47 Hz, 1H), 5.13 (dd, J = 10.26, 1.47 Hz, 1H),
3.80 (s, 2H), 3.31 (dt, J = 6.15, 1.32 Hz, 2H), 1.58 (br. s, 1H), 1.33 (s, 9H).
4.1.3 General procedure for the synthesis of compounds: 4‒6 (procedure B)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To an iceꢀcooled solution of the amine 4‒6 (1.1 equiv.) and anhydrous TEA (3.0 equiv.) in
anhydrous THF a solution of diꢀtertꢀbutyl dicarbonate (1.2 equiv.) in THF was added
dropwise and the reaction mixture was stirred at room temperature for 4 h. Then the solvent
was removed and the resulting residue was treated with water and extracted with DCM. The
2 4
combined organic extracts were dried over anhydrous Na SO , filtered and concentrated. To
the resulting residue dissolved in MeCN and cooled on an iceꢀbath, a solution of Nꢀ
bromosuccinimide (1.2 equiv.) in water was added dropwise, and the reaction mixture was
stirred at room temperature for 16 h. Then the MeCN was evaporated and water phase was
extracted with ethyl acetate. The combined organic extracts were dried over anhydrous
Na
2 4
SO , filtered and concentrated under the reduced pressure. The resulting residue was
purified by flash column chromatography in 20‒60% ethyl acetate in petroleum ether.
4.1.3.1
3ꢀBenzylꢀ5ꢀ(bromomethyl)oxazolidinꢀ2ꢀone (4)
Following the procedure B, solution of diꢀtertꢀbutyl dicarbonate (1.636 g, 7.50 mmol) in 5 mL
anhydrous THF was added dropwise to a solution of Nꢀbenzylpropꢀ2ꢀenꢀ1ꢀamine (1) (0.920 g,
6.25 mmol) in 15 mL anhydrous THF in the presence of anhydrous TEA (2.620 mL, 18.75
mmol). Extraction: 3 × 20 mL DCM. To the resulting tertꢀbutyl allyl(benzyl)carbamate (1.761
g, 7.12 mmol) dissolved in 13 mL MeCN, a solution of Nꢀbromosuccinimide (1.521 g, 8.54
mmol) in 20 mL water was added. Extraction 3 × 20 mL ethyl acetate. Yield: 760 mg (38%),
TLC (petroleum ether/ethyl acetate, 5/5, v/v) R
f
= 0.49. MW 270.12. Formula C11
2
H12BrNO .
1
MS m/z 270.16 (M+), 272.15 (M+2). H NMR (300 MHz, CDCl
3
) δ 7.25 ꢀ 7.41 (m, 5H), 4.63
ꢀ
4.74 (m, 1H), 4.35 ꢀ 4.51 (m, 2H), 3.38 ꢀ 3.59 (m, 3H), 3.27 (dd, J = 9.38, 5.86 Hz, 1H).
2
8
ACS Paragon Plus Environment

Page 29 of 78
ACS Chemical Neuroscience
1
2
3
4
5
6
7
8
9
4
.1.3.2
5ꢀ(Bromomethyl)ꢀ3ꢀ(4ꢀmethoxybenzyl)oxazolidinꢀ2ꢀone (5)
Following the procedure B, solution of diꢀtertꢀbutyl dicarbonate (0.993 g, 4.55 mmol) in 3 mL
anhydrous THF was added dropwise to a solution of Nꢀ(4ꢀmethoxybenzyl)propꢀ2ꢀenꢀ1ꢀamine
(2) (0.672 g, 3.79 mmol) in 10 mL anhydrous THF in the presence of anhydrous TEA (1.588
mL, 11.37 mmol). Extraction: 3 × 15 mL DCM. To the resulting tertꢀbutyl
allyl(benzyl)carbamate (1.215 g, 4.38 mmol) dissolved in 10 mL MeCN, a solution of Nꢀ
bromosuccinimide (0.936 g, 5.26 mmol) in 15 mL water was added. Extraction 3 × 15 mL
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f
ethyl acetate. Yield: 669 mg (59%), TLC (petroleum ether/ethyl acetate, 5/5, v/v) R = 0.46.
1
MW 300.15. Formula C12
MHz, CDCl
H
14BrNO
3
. MS m/z 299.98 (M), 301.98 (M+2). H NMR (300
3
) δ 7.16 ꢀ 7.25 (m, 2H), 6.83 ꢀ 6.92 (m, 2H), 4.59 ꢀ 4.72 (m, 1H), 4.28 ꢀ 4.45 (m,
H), 3.77 ꢀ 3.84 (m, 3H), 3.48 ꢀ 3.56 (m, 2H), 3.40 (dd, J = 10.52, 7.69 Hz, 1H), 3.24 (dd, J =
.98, 5.90 Hz, 1H).
2
8
4
.1.3.3
5ꢀ(Bromomethyl)ꢀ3ꢀ(3ꢀ(tertꢀbutyl)benzyl)oxazolidinꢀ2ꢀone (6)
Following the procedure B, solution of diꢀtertꢀbutyl dicarbonate (0.330 g, 1.51 mmol) in 1.5
mL anhydrous THF was added dropwise to a solution of Nꢀ(3ꢀ(tertꢀbutyl)benzyl)propꢀ2ꢀenꢀ1ꢀ
amine (3) (0.256 g, 1.26 mmol) in 5 mL anhydrous THF in the presence of anhydrous TEA
(0.526 mL, 3.78 mmol). Extraction: 3 × 8 mL DCM. To the resulting tertꢀbutyl allyl(3ꢀ(tertꢀ
butyl)benzyl)carbamate (0.378 g, 1.25 mmol) dissolved in 5 mL MeCN, a solution of Nꢀ
bromosuccinimide (0.266 mg ,1.50 mmol) in 10 mL water was added. Extraction 3 × 10 mL
f
ethyl acetate. Yield: 211 mg (51%), TLC (petroleum ether/ethyl acetate, 5/5, v/v) R = 0.62.
1
MW 326.23. Formula C H BrNO . MS m/z 325.96 (M), 327.97 (M+2). H NMR (300
15
20
2
MHz, CDCl ) δ 7.25 ꢀ 7.38 (m, 3H), 7.06 ꢀ 7.11 (m, 1H), 4.63 ꢀ 4.75 (m, 1H), 4.43 (s, 2H),
3
3
.49 ꢀ 3.58 (m, 2H), 3.45 (dd, J= 10.55, 7.03 Hz, 1H), 3.28 (dd, J = 8.79, 5.86 Hz, 1H), 1.32
(s, 9H).
2
9
ACS Paragon Plus Environment