Novel Multitarget-Directed Ligands Aiming at Symptoms and Causes of Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.8b00024

Alzheimer's disease (AD) is a major public health problem, which is due to its increasing prevalence and lack of effective therapy or diagnostics. The complexity of the AD pathomechanism requires complex treatment, e.g. multifunctional ligands targeting both the causes and symptoms of the disease. Here, we present new multitarget-directed ligands combining pharmacophore fragments that provide a blockade of serotonin 5-HT₆ receptors, acetyl/butyrylcholinesterase inhibition, and amyloid β antiaggregation activity. Compound 12 has displayed balanced activity as an antagonist of 5-HT₆ receptors (K_i = 18 nM) and noncompetitive inhibitor of cholinesterases (IC_50hAChE = 14 nM, IC_50eqBuChE = 22 nM). In further in vitro studies, compound 12 has shown amyloid β antiaggregation activity (IC₅₀ = 1.27 μM) and ability to permeate through the blood-brain barrier. The presented findings may provide an excellent starting point for further studies and facilitate efforts to develop new effective anti-AD therapy.

Full text of DOI:10.1021/acschemneuro.8b00024

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Article
Novel multi-target-directed ligands aiming at
symptoms and causes of Alzheimer’s disease
Anna Wi#ckowska, Tomasz Wichur, Justyna Gody#, Adam Bucki, Monika Marcinkowska, Agata
Siwek, Krzysztof Wi#ckowski, Paula Zar#ba, Damijan Knez, Monika G#uch-Lutwin, Grzegorz
Kazek, Gniewomir Latacz, Kamil Mika, Marcin Ko#aczkowski, Jan Korabecny, Ondrej Soukup,
Marketa Benkova, Katarzyna J. Kiec-Kononowicz, Stanislav Gobec, and Barbara Malawska
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00024 • Publication Date (Web): 31 Jan 2018
Downloaded from http://pubs.acs.org on February 2, 2018
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Novel multi-target-directed ligands aiming at symptoms and causes of Alzheimer’s disease
a*
a
a
b
b
Anna Więckowska , Tomasz Wichur , Justyna Godyń , Adam Bucki , Monika Marcinkowska , Agata
c
d
a
e
c
Siwek , Krzysztof Więckowski , Paula Zaręba , Damijan Knez , Monika GłuchꢀLutwin , Grzegorz
f
g
a
b
h,i
h,i
Kazek , Gniewomir Latacz , Kamil Mika , Marcin Kołaczkowski , Jan Korabecny , Ondrej Soukup ,
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Marketa Benkova , Katarzyna KiećꢀKononowicz , Stanislav Gobec , Barbara Malawska
a
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University
Medical College, 9 Medyczna Str. 30ꢀ688 Kraków, Poland
b
Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College,
9
Medyczna Str. 30ꢀ688 Kraków, Poland
c
Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, 9
Medyczna Str. 30ꢀ688 Kraków, Poland
d
Department of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, 9
Medyczna Str. 30ꢀ688 Kraków, Poland
e
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
f
Department of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University Medical
College, 9 Medyczna Str. 30ꢀ688 Kraków, Poland
g
Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian
University Medical College , Medyczna 9, 30ꢀ688 Kraków, Poland
h
Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, Trebesska
1575, 500 01 Hradec Kralove, Czech Republic
i
Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec
Kralove, Czech Republic
*
Corresponding Author: tel: (+48 12) 620 54 50, fax: (+48 12) 657 02 62; eꢀmail:
anna.wieckowska@uj.edu.pl
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ABSTRACT
Alzheimer’s disease (AD) is a major public health problem, which is due to its increasing prevalence
and lack of effective therapy or diagnostics. Complexity of AD pathomechanism requires complex
treatment e.g. multifunctional ligands targeting both causes and symptoms of the disease. Here, we
present new multiꢀtargetꢀdirected ligands combining pharmacophore fragments that provide blockade
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of serotonin 5ꢀHT
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receptors, acetyl/butyrylcholinesterase inhibition, and amyloid β antiꢀaggregation
receptors (K = 18
activity. Compound 12 has displayed balanced activity as an antagonist of 5ꢀHT
6
i
nM) and nonꢀcompetitive inhibitor of cholinesterases (IC50hAChE = 14 nM, IC50eqBuChE = 22 nM). In
further in vitro studies, compound 12 has shown amyloid β antiꢀaggregation activity (IC50 = 1.27 ꢁM)
and ability to permeate through the bloodꢀbrain barrier. The presented findings may provide and
excellent starting point for further studies and facilitate efforts to develop new effective antiꢀAD
therapy.
Keywords: Alzheimer’s disease, multiꢀtargetꢀdirected ligands, acetylcholinesterase inhibitors,
6
butyrylcholinesterase inhibitors, 5ꢀHT receptor antagonists, inhibition of βꢀamyloid aggregation
1. INTRODUCTION
Alzheimer’s disease (AD) is the leading cause of dementia and it affects 34 million people worldwide.
With the aging of the population, the prevalence of AD continues to increase, and the World Health
Organization (WHO) estimates that it can reach at least 95 million by 2050. Comprehensive care and
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treatment of dementia worldwide in 2015 generated costs that amounted to $818 billion. According to
WHO, such numbers, along with a great burden on families and caregivers, make AD one of the
leading social, medical and economic challenges faced by society today, and a global public health
2
priority.
AD is a neurodegenerative disease that is manifested by cognitive symptoms, e.g. decline in memory,
language and speech impairment or time and space disorientation. In 80% cases these are accompanied
by behavioural and psychological symptoms of dementia (BPSD), e.g. agitation, psychosis, depression
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and anxiety. Neurodegeneration in AD causes neuronal and synaptic loss, and synaptic impairment,
thus leading to neurochemical changes within a number of neurotransmitter systems, mainly
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cholinergic system. Adequately, currently available treatment includes acetylcholinesterase (AChE)
or/and butyrylcholinesterase (BuChE) inhibitors: donepezil, rivastigmine, galantamine, which raise
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acetylcholine levels in the brain,
and NMDA receptor antagonist memantine, which regulates the
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activity of glutamate in the brain. However, these drugs can only slow down the progress of the
disease and they are effective only for a limited time.
Despite making great efforts and growing investment in the pharmaceutical industry and in academia,
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no new drugs against AD have been developed in more than a decade. Among drug candidates in
advanced stages of clinical trials, 5ꢀHT receptor antagonists have been successfully evaluated in
Phase II clinical trials. Two antagonists, idalopirdine and intepirdine, improved cognitive functions in
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4,15,16
donepezilꢀtreated patients with mild to moderate AD.
that serotonergic 5ꢀHT receptors are not only linked to cognitive dysfunction, but also to affective
disorders, anxiety and depression, the very symptoms of BPSD observed in AD patients.
also reports indicating diseaseꢀmodifying potential of 5ꢀHT receptor antagonists related to synaptic
remodelling and neuronal hyperexcitability, which are regarded to be possible causes of AD.
Hence, 5ꢀHT antagonists may provide both causal and symptomatic treatment of AD.
Moreover, preclinical studies have shown
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There are
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Diseaseꢀmodifying treatment that could affect the underlying disease process, and therefore
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delay onset of the disease or slow its progress is the holy grail of medicine. However, it is a big
challenge in the case of AD since its pathogenesis is not completely explained and it is not clear which
of the mechanisms underlying the disease is the trigger. Compelling body of evidence supports
22,23
amyloid cascade hypothesis
according to which amyloidꢀβ (Aβ) aggregation is the major and
necessary factor that initiates other processes that lead up to neurodegeneration in AD. Amyloidꢀ
lowering therapies are therefore in the limelight of research aimed at AD pathomechanism. There is a
number of strategies undertaken to prevent Aβ aggregation, including inhibition or modulation of
enzymes responsible for Aβ production (βꢀsecretase and γꢀsecretase) and inhibition of Aβ aggregation
24,25
itself. However, with respect to the mechanismꢀbased side effects of the secretases inhibition,
the
inhibition of Aβ selfꢀaggregation seems to be a more promising strategy. The direct association of Aβ
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with pathomechanism of AD makes amyloid β antiꢀaggregation activity a frequently used additional
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mechanism in designing new agents against AD.
It is appetent that search for a drug that could act through only one of the potential biological targets
may not be enough to address the problem of effective AD treatment. The currently available drugs
that were developed in accordance with the ‘oneꢀdiseaseꢀoneꢀtarget’ paradigm are not effective
enough. This is most likely due to the complex nature of AD, and the multitude of factors involved in
its pathogenesis. As a consequence, in the last few decades, a multiꢀtargetꢀdirected ligands (MTDLs)
strategy has gained much attention in making attempts to find the right treatment of complex
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neurodegenerative diseases like AD. MTDLs are specifically and rationally designed to modulate
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multiple targets simultaneously, which results in higher clinical efficiency of the therapy. Taking all
the facts into consideration, we aimed at the synthesis of MTDLs that combine mechanism that would
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improve cognitive functions (ChE inhibition, 5ꢀHT antagonism), alleviate psychological symptoms
accompanying AD (5ꢀHT antagonism), and affect causative processes (Aβ inhibition) (Figure 1).
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Figure 1. Combination of the mechanisms providing disease modifying and symptomatic effects as
the way to improved treatment of Alzheimer’s disease.
2. RESULTS AND DISCUSSION
2.1 Design
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Recently, we reported on the first MTDLs that comprise cholinesterase inhibition and 5ꢀHT receptor
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antagonism. A hit structure selected from the study (Cmp. III, Figure 2) has displayed wellꢀbalanced
in vitro potencies (IC50 hAChE = 12 nM, IC50 hBuChE = 29 nM, Kb 5ꢀHT6 = 27 nM) and the ability to cross
BBB in in vitro and in vivo assays. Interesting biological activity of the compound is accompanied by
poor physicochemical properties. That means it exceeds limits of Lipinski’s ‘rule of five’ that
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evaluates druglikeness of a molecule. This fact may result in a number of complications in further
development of the molecule, mainly related to poor pharmacokinetics. To address this issue, we have
expanded the library of compounds with multifunctional profile of activity and improved selected
drugꢀlike properties.
As presented in Figure 2, in the design of the novel compounds we combined the pharmacophore
fragments of cholinesterase inhibitors (donepezil and tacrine) and 5ꢀHT
II). As fragments providing 5ꢀHT antagonistic activity we have chosen two pharmacophores of
proven strong interactions with 5ꢀHT
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antagonists (Cmp. I and Cmp.
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receptor: 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine (Cmp. I,
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series I, Figure 2) and 1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole (Cmp. II, series II, Figure 2).
Introduction of these moieties instead of the previously used 1ꢀ(phenylsulfonyl)ꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ
1Hꢀindole resulted in the reduction of the molecular weight by about 50–60 Da. Since molecular
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weight is considered to be critical for optimal pharmacokinetic properties, its reduction was the first
step in the gradual optimization of the compounds. Additionally, for some compounds of series I, we
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reduced aromatic ring count to the recommended three. As pharmacophore fragments targeting
AChE and/or BuChE, we used tacrine (a former AD drug) and derivatives of Nꢀbenzylamine.
Regardless of hepatotoxicity of tacrine itself, its derivatives are often devoid of toxic effects and
tacrine is still the most commonly used pharmacophore fragment in the design of MTLDs against
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AD. NꢀBenzylamines were chosen as lower molecular weight pharmacophores and equally important
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fragments used as cholinesterase inhibitors in the design of new MTDLs against AD. The selected
serotoninergic and cholinergic pharmacophore fragments were combined by different linkers to obtain
potential MTDLs. In order to find the right distance between the pharmacophore fragments that would
allow optimal interactions with their biological targets, we have chosen several flexible aliphatic
chains of different lengths.
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9–
Both donepezil and tacrine derivatives were reported before as inhibitors of amyloid β aggregation.
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Taking this fact into consideration, we assume that the novel compounds may also inhibit amyloid β
aggregation and therefore gain another mechanism that is important for antiꢀAD agents.
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Figure 2. The design of novel MTDLs ꢀ merging pharmacophores responsible for cholinesterase
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inhibition and 5ꢀHT antagonism.
2.2.Chemistry
The designed compounds were prepared in accordance with the previously developed and described
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synthetic procedures. Tacrine derivatives 9–18 were obtained by means of two methods shown in
Scheme 1. The first one comprised alkylation of tacrine with suitable dibromoalkanes and subsequent
use of the resulting bromoalkyl tacrines (5–8) in alkylation of (1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀ
methylphenyl)piperazine or 1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole). These reactions were carried out
2 3
in DMSO in the presence of KOH, or in acetonitrile in the presence of K CO /KI, and resulted in
obtaining compounds 11–14, 17 and 18. Another method was used to synthesize tacrine derivatives 9,
10, 15, 16 with shorter, ethylene and propylene linkers (Scheme 1). Here, 9ꢀchloroꢀ1,2,3,4ꢀ
tetrahydroacridine reacted with the suitable amino alcohols in pentanꢀ1ꢀol at 150 °C to give alcohols 1
and 2. Next, in a oneꢀpot reaction the alcohols were converted into mesylates, which were used to
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alkylate 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine or 1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole
providing the desired compounds.
Scheme 1. Synthesis of compounds 9–18.
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Reagents and conditions: (a) suitable dibromoalkane, KOH, dry DMSO, rt, 24 h (for 5–8); (b) suitable
amino alcohol, pentanꢀ1ꢀol, 150 °C, 24 h (for 1, 2); (c) methanesulfonyl chloride, TEA, dry DCM, 0
°
C, 30 min; (d) 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine, DCM, 0 °C–rt, 24 h; (e) 1ꢀ(3ꢀ
benzyloxy)ꢀ2ꢀmethylphenyl)piperazine, K CO , KI, acetonitrile, reflux, 24 h; (f) 1ꢀbenzylꢀ4ꢀ
piperazinꢀ1ꢀyl)ꢀ1Hꢀindole hydrochloride, DCM, 0 °C–rt, 24 h; (g) 1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀ
CO , KI, acetonitrile, reflux, 24 h.
(
(
2
3
indole, K
2
3
As outlined in the Scheme 2, compounds 25–31, 34 and 35 were prepared from commercially
available amines in the following sequence of reactions: Nꢀacylation – N’ꢀalkylation – reduction. The
Nꢀacylation of benzylamine, Nꢀmethylbenzylamine, Nꢀbenzylpiperidine and Nꢀbenzylpyrrolidine with
the appropriate haloacyl halides in dichloromethane (DCM) at 0 °C–rt yielded bromoꢀ or
chloroalkylamides 19–24, 32 and 33. These products were used in N’ꢀalkylation of 1ꢀ(3ꢀ(benzyloxy)ꢀ
2 3
2ꢀmethylphenyl)piperazine and 1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole in the presence of K CO in
acetonitrile. The resulting amides were reduced with lithium aluminium hydride (1 M solution in
THF) to give final compounds 25–31, 34 and 35.
Scheme 2. Synthesis of compounds 25–31, 34 and 35.
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0
Reagents and conditions: (a) 2ꢀbromoacetyl bromide (for 19, 20, 32 and 33), 3ꢀbromopropanoyl
chloride (for 21), 4ꢀchlorobutanoyl chloride (for 22), 5ꢀbromopentanoyl chloride (for 23 and 24),
DCM, 0 °C–rt, 24 h; (b) 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine, K
reflux, 24 h (see Experimental section for further details); (c) 1 M LiAlH
reflux, 24 h; (d) 1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole hydrochloride, K CO
2 3
CO , acetonitrile, 50 °C or
4
in THF, dry THF, 0 °C–
3
, DCM, 0 °C–rt, 24 h.
2
Compounds 37, 42–45 were prepared utilizing Gabriel synthesis and subsequent reductive amination
as shown in Scheme 3. Firstly, 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine and 1ꢀbenzylꢀ4ꢀ
(
piperazinꢀ1ꢀyl)ꢀ1Hꢀindole reacted with the appropriate bromoalkylphthalimides. The phthalimides
were cleaved in the reaction with 40% aqueous solution of methylamine at 50 °C followed by
hydrolysis with 1 M NaOH at room temperature yielding primary amines 36 and 38–41. The amines
then reacted with benzaldehyde in the presence of sodium cyanoborohydride and glacial acetic acid in
methanol to give the final compounds 37 and 42–45.
Scheme 3. Synthesis of compounds 37 and 42–45.
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Reagents and conditions: (a) 2ꢀ(4ꢀbromobutyl)isoindolineꢀ1,3ꢀdione (for 36 and 38) or 2ꢀ(5ꢀ
bromopentyl)isoindolineꢀ1,3ꢀdione (for 39), 2ꢀ(6ꢀbromohexyl)isoindolineꢀ1,3ꢀdione (for 40), 2ꢀ(7ꢀ
2 3
bromoheptyl)isoindolineꢀ1,3ꢀdione (for 41), K CO , acetonitrile, reflux, 24 h; (b) methylamine (40%
aq. sol.), 50 °C, 1 h, then 1 M NaOH, rt, 1 h; (c) benzaldehyde, glacial acetic acid, NaCNBH3,
methanol, 0 °C–rt, 24 h.
Compounds 48, 49, 52 and 53 were prepared according to the synthetic route shown in Scheme 4. In
the first step Nꢀmethylꢀ1ꢀphenylmethanamine and 1ꢀbenzylpiperidinꢀ4ꢀamine were alkylated with 3ꢀ
chloropropyl acetate or 4ꢀbromobutyl acetate respectively, in refluxing acetonitrile with K
2 3
CO . The
subsequent hydrolysis of the acetyl group in a methanol/water solution of K CO at 50 °C yielded the
2
3
desired alcohols. The alcohols were converted into mesylates and reacted with 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀ
methylphenyl)piperazine and 1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole. This led to obtaining the final
products 48, 49 and 53. Compound 53 required additional acidolysis of BOC protecting group that
was introduced to prevent side reactions of the secondary amine group. Compound 52 was synthesized
by starting with reaction of 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine with 1ꢀbromoꢀ3ꢀ
2 3
chloropropane in the presence of K CO in acetonitrile at 50 °C. The resulting 50 was then used as
alkylating agent in the reaction with 1ꢀbenzylpiperidinꢀ4ꢀamine, carried out by refluxing the reactants
in acetonitrile in the presence of K CO (Scheme 4).
2
3
Scheme 4. Synthesis of compounds 48, 49, 52 and 53.
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2 3
Reagents and conditions: (a) 3ꢀchloropropyl acetate, tetraꢀNꢀbutylammonium bromide, K CO ,
acetonitrile, reflux, 24 h; (b) K CO , methanol, H O, 50 °C, 2.5 h; (c) methanesulfonyl chloride, TEA,
2
3
2
dry DCM, 0 °C, 3 h; (d) 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine hydrochloride (for 48) or 1ꢀ
benzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole hydrochloride (for 49), K CO , acetonitrile, reflux, 24 h; (e) 1ꢀ
bromoꢀ3ꢀchloropropane, K CO , acetonitrile, 50 °C, 24 h; (f) 1ꢀbenzylpiperidinꢀ4ꢀamine, K CO
acetonitrile, reflux, 24 h; (g) 4ꢀbromobutyl acetate, K CO , acetonitrile, reflux, 24 h; (h) diꢀtertꢀbutyl
dicarbonate, TEA, dry THF, 0 °C–rt, 24 h; (i) 0.1 M HCl in ethyl acetate, ethyl acetate, rt, 24 h.
2
3
2
3
2
3
,
2
3
2.3.In vitro studies
Affinity and functional activity on 5ꢀHT receptor
6
6
The affinity of all the novel compounds for recombinant human 5ꢀHT receptor was evaluated in a
4
2
i i
radioligand binding assay and the results were presented as K values in Table 1 and 2. In series I, K
values ranged from 10 nM to 845 nM, and from 72 nM to 916 nM in series II. The results have shown
that the affinity of tacrine derivatives increased with the length of a linker up to five carbon atoms and
reached K
i
value of 10 nM for 11 and 72 nM for 17 and decreased for longer, seven (13) and eight (14)
carbon atom linkers (K of 234 nM and 240 nM, respectively). Among nonꢀtacrine derivatives, there
i
were only a few compounds with K
i
values lower than 100 nM: NꢀmethylꢀNꢀbenzylamine derivatives
26 and 48, 1ꢀbenzylpiperidinꢀ4ꢀamine derivative 53, and Nꢀbenzylamine derivative 44. From both
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series I and II we selected two representative derivatives for cellꢀbased functional studies in order to
verify their antagonistic mode of action. The K
b
values of 132 nM and 510 nM for 12 and 44,
receptor antagonists. Based on these results and
respectively, proved that the compounds act as 5ꢀHT
6
the fact that both applied pharmacophore fragments aiming at 5ꢀHT receptor are indeed strong
6
0
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antagonists of this receptor, we infer that their derivatives possess the same profile of functional
activity.
The above results indicate that the AChEꢀaiming fragment (tacrine or Nꢀbenzylamine) negatively
6 6
affects, but does not exclude binding of the 5ꢀHT ꢀaiming fragment in the orthosteric site of the 5ꢀHT
receptor. The binding mode of compounds with 5 and 6 carbon atoms linkers is disturbed the least.
This might be due to the observed Hꢀbonding of tacrine and Nꢀbenzylamine fragments with Asp7.36 in
6
the accessory site that allows maintaining key interactions characteristic for the 5ꢀHT receptor ligands
34,43
in the orthosteric site (Figure 3).
The latter include chargeꢀreinforced hydrogen bond between
piperazine moiety and Asp3.32, CHꢀπ aromatic stackings of methylphenyl (12) or indole (44) with
Phe6.52, and analogous interaction of benzyloxy (12) or benzyl (44) groups with Phe5.38 (Figure 3).
Due to unfavorable arrangement of molecules in the binding site and lack of the stabilizing Hꢀbonds
described above, the compounds with shorter (i.e. 2–4 carbon atoms) and longer (i.e 7–8 carbon
6
atoms) linkers display suboptimal binding mode in the 5ꢀHT receptor and therefore show lower
affinity.
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Figure 3. The predicted binding mode of compound 12 (orange) and 44 (green) in the binding site of
6
5ꢀHT receptor. The interaction network is consistent within the highꢀaffinity representatives of both
series, which applies for the 5ꢀHT receptorꢀaiming arylpiperazine fragment in the orthosteric site as
6
well as the AChEꢀaiming tacrine and Nꢀbenzylamine moieties bound in the accessory site. Amino acid
residues engaged in ligand binding (within 4 Å from the ligand atoms) are shown as sticks, whereas
residues identified as crucial for ligand binding, e.g. forming Hꢀbonds (dotted yellow lines) and CHꢀπ
stacking interactions (dotted cyan lines) are represented as thick sticks. ECL2 residues were hidden for
clarity.
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Cholinesterase (AChE/BuChE) inhibitory activity
4
4
The inhibitory activities of novel MTDLs were assessed in spectrophotometric Ellman’s assay using
AChE form electric eel (eeAChE) and BuChE form horse serum (eqBuChE). First, the compounds
were screened at the concentration of 10 ꢁM and for those with inhibition exceeding 50%, IC values
5
0
were determined. The most potent compounds were further tested on human AChE (hAChE)
following the same procedure. The results determined for the test compounds as well as for tacrine and
donepezil, used as references, are presented in Tables 1 and 2.
Among all the compounds the most potent eeAChE inhibitors were tacrine derivatives 9–18. Their
IC50 values have ranged from 5 to 44 nM, except for compounds 11 and 17 with 5 carbon atom linkers
that displayed reduced activity with IC50 values of 176 nM and 250 nM, respectively. It is worth
noting that the potencies of 13–16, and 18 were higher than tacrine itself (IC50 values of 5–17 nM, vs.
24 nM). This may be the result of additional interactions with the enzyme provided by the
arylpiperazine 5ꢀHT ꢀaiming fragments. Molecular docking studies predicted two distinctive binding
6
modes for shortꢀchain (2, 3 carbon atoms linker) compounds and longꢀchain (6–8 carbon atoms linker)
compounds (Figure 4). The tacrine fragment of the shortꢀchain compounds binds to the catalytic site of
6
AChE and interacts with Trp84 and Phe330 (aromatic πꢀπ interaction), whereas the 5ꢀHT fragment is
located in the peripheral site, forming cationꢀπ interactions between piperazine and Tyr334 and πꢀπ
stacking between methylphenyl (series I) or indole (series II) fragment and Trp279 (Figure 4A). The
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latter interactions are preserved in the longꢀchain compounds but piperazine forms cationꢀπ interaction
with Phe331 instead of Tyr334. Unlike in the shortꢀchain compounds, tacrine takes a perpendicular
position in the catalytic site and apart from aromatic πꢀπ interaction with Trp84 and Phe330 it forms πꢀ
π stacking interactions with Tyr334. It is worth noting that the compounds with 5 carbon atoms linker
0
1
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(
11 and 17) are less prone to form interactions with Trp279, Tyr334 or Phe331 that might explain their
lower inhibitory potency (Table 1 and 2 and Figure 4B). The benzyloxy (series I) and benzyl (series II)
fragments interact in nonspecific manner in the outer part of the enzyme's gorge, regardless of the
length of the alkyl linker (Figure 4).
Figure 4. The predicted binding mode of a shortꢀchain compound 10 (orange) and an alternative
positioning of longꢀchain compound 14 (green) in the active site of AChE. The binding modes show
equivalent interaction patterns, irrespective of the length of the alkyl linker (A). Compound 11 is
unable to form aromatic interactions with Trp279 or Phe331/Tyr334 residues due to unfavorable 5ꢀ
unit spacer (B). Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are
shown as sticks, whereas residues identified as crucial for ligand binding, e.g., forming πꢀπ stacking
interactions (dotted cyan lines) or cationꢀπ (dotted green lines) are represented as thick sticks.
In series I, among the nonꢀtacrine derivatives, compound 29, with Nꢀbenzylamine substituent and the
longest, 5 carbon atoms linker displayed IC50 value of 2.3 ꢁM. Similar activities (IC50 values ranging
from 3.1 ꢁM to 8.2 ꢁM) were recorded for Nꢀbenzylamine derivatives 42–45 from series II.
Comparison of binding pattern predicted for donepezil (which mimics the one evidenced in the crystal
45
structure 1EVE) and 29 unravels the possible reason for lower performance of Nꢀbenzylamine
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derivatives (Figure 5). The binding mode of donepezil includes πꢀπ aromatic interactions of Nꢀ
benzylpiperidine with Trp84 and cationꢀπ interactions with Phe330 and Tyr334, which allow the 2,3ꢀ
dihydroindenꢀ1ꢀon fragment for πꢀπ stacking with Trp279. On the other hand, compound 29 interacts
with Trp84 via cationꢀπ interaction, reinforced by its highly basic Nꢀbenzylamine fragment (calculated
1
1
1
1
1
1
1
1
1
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2
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A
pK = 9.67 vs. 8.54 of donepezil), which draws the compound deeper in the catalytic site, preventing
formation of the favorable aromatic interaction with Trp279 in the peripheral site. Although cationꢀπ
interactions with Phe330 and Tyr334 are preserved due to basic piperazine of 29, the overall binding
mode of Nꢀbenzylamine derivatives appears to be disadvantageous (Figure 5).
Figure 5. The predicted binding mode of compound 29 (purple) vs. donepezil (grey) in the active site
of AChE. Compound 29 is unable to reach Trp279 due to cationꢀπ interaction with Trp84 provided by
a high conformational flexibility of alkyl linker. Amino acid residues taking part in the ligand binding
(within 4 Å from the ligand atoms) are shown as sticks, whereas residues identified as crucial for
ligand binding, e.g., forming πꢀπ stacking interactions (dotted cyan lines) or cationꢀπ (dotted green
lines) are represented as thick sticks.
Additionally, we observed higher average activity of the compounds against eqBuChE than for
eeAChE. Again, tacrine derivatives 9–18 were the most potent inhibitors with IC50 values ranging
from 22 to 85 nM in series I and from 5 to 25 nM in series II. All except one (36) of the nonꢀtacrine
derivatives form series I and II, displayed inhibitory activities on eqBuChE in ranges from 0.196 ꢁM
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for compound 49 to 7.78 ꢁM for 34. It was demonstrated that BuChE, which plays rather supportive
4
6
role in the healthy brain, in the course of AD takes over the function of AChE. Therefore search for
both selective and nonꢀselective BuChE inhibitors is important and may benefit AD patients.
As the most active compounds from both series, the tacrine derivatives were tested against
recombinant human AChE. Their IC50 values have ranged from 3 to 62 nM and were lower than the
activity of unsubstituted tacrine (IC50 = 131 nM). We have tested two selected compounds against
human BuChE, tacrine derivative – compound 12 (IC50 = 22 nM) and nonꢀtacrine, selective BuChE
inhibitor – compound 49 (IC50 = 620 nM).
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
Table 1. Cholinesterase inhibitory potency and 5-HT receptor affinity of the hybrid compounds
6
of series I.
a
K
i
[µM]
IC50 [µM]
Cmpd.
9
n
R
b
c
d
h5-HT
6
R
eeAChE
hAChE
eqBuChE
2
3
5
6
7
8
2
3
4
5
2
3
0.232 ± 0.012
0.078 ± 0.002
0.010 ± 0.005
0.044 ± 0.0007
0.025 ± 0.0007
0.176 ± 0.003
0.043 ± 0.001
0.014 ± 0.0005
0.005 ± 0.0001
n.a
0.062 ± 0.0015 0.026 ± 0.0008
0.010 ± 0.0003 0.045 ± 0.0007
10
11
12
13
14
0.024 ± 0.0003 0.085 ± 0.002
0.022 ± 0.0006
f
N
N
0.018 ± 0.001e
0.132 ± 0.030
H
0.014 ± 0.0004
0.022 ± 0.0007
0.234 ± 0.022
0.240 ± 0.006
0.096 ± 0.002
0.157 ± 0.008
0.242 ± 0.016
0.140 ± 0.005
0.069 ± 0.002
0.060 ± 0.001
0.011 ± 0.0004 0.026 ± 0.0009
0.003 ± 0.0001 0.024 ± 0.0007
25
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4.554 ± 0.269
4.888 ± 0.242
5.605 ± 0.202
3.733 ± 0.114
4.497 ± 0.154
4.814 ± 0.096
27
37
29
26
n.a
n.a
2.340 ± 0.042
n.a
48
n.a
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5
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7
8
9
2
8
4
5
0.845 ± 0.043
n.a
n.a
ꢀ
ꢀ
6.091 ± 0.154
5.866 ± 0.181
30
34
36
0.300 ± 0.031
0.225 ± 0.003
2
n.a
ꢀ
7.782 ± 0.074
2
3
4
0.320 ± 0.026
n.a.
n.a
n.a
n.a
ꢀ
ꢀ
ꢀ
n.a
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
5
2
3
4.990 ± 0.200
4.461 ± 0.064
5
0.070 ± 0.002
0.003 ± 0.0002e
0.007± 0.0002
Cmp. I
n.a.
n.a.
1.830 ± 0.040
ꢀ
Donepe
zil
ꢀ
ꢀ
0.010 ± 0.0002
0.024 ± 0.0004
0.006 ± 0.0001
Tacrine
a
0.002 ± 0.0001 0.131 ± 0.002
IC50 values are expressed as the mean ± standard error of the mean (SEM) of at least three
b
c
d
experiments; AChE from the electric eel; recombinant human AChE; BuChE from equine serum;
e
f
K value; human recombinant BuChE.
b
Table 2. Cholinesterase inhibitory potency and 5-HT
6
receptor affinity of the hybrid compounds
of series II.
a
K
i
[µM]
IC50 [µM]
Cmpd.
n R
b
c
d
h5-HT R
eeAChE
hAChE
eqBuChE
6
1
1
1
5
6
7
2
3
5
6
4
5
6
7
0.094 ± 0.002
0.148 ± 0.014
0.072 ± 0.002
0.130 ± 0.001
0.275 ± 0.028
0.407 ± 0.012
0.011 ± 0.0003 0.026 ± 0.0007 0.005 ± 0.0002
0.013 ± 0.0003 0.038 ± 0.0007 0.011 ± 0.0002
N
N
0.25 ± 0.005
0.020 ±0.0001 0.025 ± 0.0005
H
18
0.017 ± 0.0002 0.013 ± 0.0003 0.015 ± 0.0003
4
4
4
4
2
3
4
5
7.285 ± 0.151
4.389 ± 0.070
8.217 ± 0.180
3.107 ± 0.075
ꢀ
ꢀ
ꢀ
ꢀ
0.534 ± 0.006
1.333 ± 0.030
1.562 ± 0.039
1.115 ± 0.013
0.079 ± 0.007e
0.510 ± 0.001
0.165 ± 0.005
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9
2
3
0.916 ± 0.071
n.a
n.a
ꢀ
ꢀ
1.027 ± 0.018
0
0
.196 ± 0.002
.620 ± 0.012
0.240 ± 0.013
f
Cmp. II
a
0.014 ± 0.003
n.a.
ꢀ
n.a.
IC50 values are expressed as the mean ± standard error of the mean (SEM) of at least three
0
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0
b
c
d
experiments; AChE from the electric eel; recombinant human AChE; BuChE from equine serum;
e
f
K value; human recombinant BuChE.
b
AChE/BuChE inhibition kinetic studies
The mechanism of cholinesterase inhibition was determined for two potent inhibitors of both AChE
and BuChE, compounds 12 and 18, the representatives of series I and II, respectively. As illustrated in
Figure 6 by LineweaverꢀBurk reciprocal plots, the compounds display nonꢀcompetitive mode of
action. This is indicated by increasing slopes (decreased Vmax) and preserved intercepts (unchanged
K ) at increasing concentrations of the enzyme inhibitor. The nest of line converges at the xꢀaxis that
m
is characteristic for inhibitors displaying equal affinity for the free enzyme and for the enzymeꢀ
substrate complex.
A
Compound 12
B
Compound 12
2
0
4
0
0
0
7
nM
.5 nM
0 nM
15 nM
25 nM
40 nM
75 nM
15 nM
0 nM
10
3
2
-
20
-10
10
20
-20
-10
10
20
30
1/BTC [mM]ꢀ1
1
/ATC [mM]ꢀ1
-20
-10
C
Compound 18
20
D
Compound 18
0
nM
7.5 nM
5 nM
0 nM
50 nM
10
0
nM
1
7.5 nM
3
15 nM
10
5
30 nM
-
20
-10
10
20
-
20
-10
10
20
30
/ATC [mM]ꢀ1
1/BTC [mM]ꢀ1
1
-10
-5
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Figure 6. Lineweaver – Burk plots illustrating hAChE and eqBuChE hydrolytic activity over a range
of substrate concentrations and in the presence of increasing concentration of inhibitor 12 (A, B) and
18 (C, D).
Inhibition of amyloidꢀβ aggregation
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The inhibitory effect of the novel compounds presented herein (9–18, 25–31, 34, 36, 42–45, 49, 52,
47
5
3) on Aβ1ꢀ42 aggregation was determined in thioflavine T (ThT) fluorescence assay. ThT is a
thioflavine dye that displays enhanced fluorescence emission at 482 nm when bound to amyloid
fibrils. Compounds with antiꢀaggregation properties cause a reduction of the fluorescence emission in
the assay. The results of the screening performed at a concentration of 10 ꢁM of each compound and
1.5 µM of Aβ1–42 are presented in Figure 7 as a percentage of inhibition. Inhibitory activity of the
compounds ranged from 48% to 95%. Ten compounds (9, 10, 12, 13, 15–18, 34, 45) exhibited equal
or better inhibitory potency than resveratrol used as a reference (79%–95% vs. 79%). The most active
compounds were tacrine derivatives 12, 13, 15–18 with inhibitory potency over 92%. Within tacrine
derivatives from series I, we observed similar SAR when it comes to a length of a linker as for 5ꢀHT
6
receptor affinity. The activity increases with the linker elongation to reach maximum of 94.4% for
compound 12 with 6 carbon atom linker and then decreases to 64.3% for 14 with 8 carbon atom linker.
For compounds 12 and 15 we tested Aβ1–42 aggregation kinetics at the decreasing concentrations of
each compound and determined IC50 values of 1.27 ꢁM and 1.29 ꢁM respectively.
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Figure 7. The effect of the synthesized compounds on Aβ aggregation. The antiꢀaggregation activity
was determined as reported in the experimental section in the presence of 10 ꢁM of the synthesized
compounds and 1.5 µM Aβ1–42. The activity of the new compounds was compared to that displayed by
resveratrol used as the reference. The data is expressed as a mean percentage of inhibition ± standard
deviation of at least three independent experiments; p < 0.05, statistically different compared to
control experiments (Aβ1–42 + DMSO); oneꢀway analysis of variance (ANOVA), followed by post hoc
Bonferroni tꢀtest (SigmaPlot v 12.0, GraphPad Prism 5).
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Bloodꢀbrain barrier penetration
In order to penetrate into the CNS and to reach their intended biological targets, the novel MTDLs
must pass the bloodꢀbrain barrier (BBB). To estimate their ability to cross BBB by passive diffusion
we have used parallel artificial membrane permeability assay for the BBB (PAMPAꢀBBB). The
48
method was described by Di in 2003 , and today it is commonly used as reliable, easy and fast in vitro
permeability prediction method. By using PAMPAꢀBBB, we have determined effective permeability
(
P
e
) for the selected compounds and for eight commercial drugs with known CNS penetration as
48
references (Table 3). According to Di, the permeability ranges were set at P > 4.0 for compounds
e
with high permeability, P
e
< 2.0 for compounds with low permeability, and 4.0 > P
e
> 2.0 for
values
compounds with uncertain permeability. As the tested compounds 15, 42, 49 and 12 display P
e
higher than 4.0, we predict that they should cross the BBB by passive diffusion.
e
Table 3. Permeability (P ) of the selected compounds and commercial drugs in the PAMPA-BBB
assay with prediction of their penetration in the CNS.
BBB penetration estimation
a
b
Compound
P
e
CNS (+/ꢀ)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
1
2
5.7 ± 1.4
7.6 ± 1.1
4.8 ± 0.3
5.0 ± 0.5
15
4
2
49
Donepezil
7.3 ± 0.9
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Rivastigmine
Tacrine
6.6 ± 0.5
5.3 ± 0.2
11.3 ± 1.6
5.6 ± 0.6
2.8 ± 0.1
1.1 ± 0.2
1.0 ± 0.4
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+/ꢀ)
CNS (ꢀ)
CNS (ꢀ)
Testosterone
Chlorpromazine
Hydrocortisone
Theophylline
Atenolol
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0
a
–6
–1
Data is expressed as a mean of four replicates (n = 4) ± SEM (10 cm s ).
b
CNS+, P
e
> 4.0, high permeability (high BBB permeation predicted); CNSꢀ, P
e
< 2.0, low
permeability (low BBB permeation predicted); CNS±, 4.0 > P
e
> 2.0, uncertain permeability.
In silico and in vitro metabolic stability
Biotransformation plays essential role in bioavailability and toxicity of drugs. The major site of
biotransformation is liver and liver microsomal enzymes. Therefore, preliminary assessment of
compounds’ metabolic stability in vitro is evaluated on liver microsomes.
To predict metabolic stability of the most promising compound 12, we have used in silico MetaSite
4
9
5.1.1 tool, which shows metabolic sites and structures of the possible metabolites. As depicted in
Figure 8, position 4 of tacrine moiety (marked in blue) was selected as the most sensitive site for
metabolic biotransformations. The program predicted three possible reactions at this site:
dehydrogenation, hydroxylation or oxidation. Other possible sites of metabolism were marked in red
(
for details see figure S1 in supplementary information).
Figure 8. In silico prediction of the most probable metabolic sites.
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In vitro metabolic stability of compound 12 was evaluated using human liver microsomes (HLMs).
After 120 minutes of incubation with HLMs compound 12 was metabolized in 50%. UPLCꢀMS
analysis led to the identification of the main metabolite – M1, and small amounts of additional
metabolites M2–M7 (Figure 9).
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Figure 9. The UPLC spectrum after 120 minutes of incubation of 12 with HLMs.
The MS analysis (see figure S2 in supporting information) allowed the determination of the molecular
masses of metabolites’ quasimolecular ions [M+H]+: m/z = 473.46 (M1, main metabolite), m/z =
3
13.28 (M2), m/z = 579.47 (M3), m/z = 597.55 (M4), m/z = 199.18 (M5), m/z = 283.31 (M6) and m/z
397.35 (M7). By using MetaSite, we have identified M1 as a product of ether bond hydrolysis
Figure 10).
=
(
Figure 10. The MS fragmentation analysis and the structure of the main metabolite M1.
Other minor metabolites were analysed and identified as products of compound 12 degradation and
hydroxylation (for further details see figures S3–S8 in supplementary information). We did not
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identify any of known metabolites responsible for hepatotoxic effect of tacrine. Although, we have
found traces of tacrine (M5) and metabolites M3 and M4 that contain hydroxylated tacrine fragments
(
figures S4–S6 in supporting information).
Influence on CYP3A4 activity
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Alzheimer's disease affects mainly elderly patients who usually receive more than one drug at the
time, and as a consequence they are exposed to drugꢀdrug interactions (DDI). A major mechanism
leading to DDI is an inhibition of cytochrome P450 (CYP). Among various CYP isoenzymes,
CYP3A4 is responsible for metabolism of 40–50% of all marketed drugs, and therefore preliminary
5
1
data is usually assessed by using this enzyme. We have tested the influence of compound 12 on
cytochrome P450 CYP3A4 isoform activity in a luminescence method provided by Promega®.The
method is based on the CYP3A4ꢀcatalysed conversion of the beetle Dꢀluciferin derivative into Dꢀ
52
luciferin. As shown in Figure 11, the inhibitory effect of compound 12 was 170ꢀfold weaker than for
ketoconazole used as a reference (IC50 = 17.35 ꢁM vs IC50 = 0.1 ꢁM).
KE
IC50 = 0.1 ꢁM
100
12
M
IC50 = 17.35 ꢀ
50
0
-9
-8
-7
-6
log M]
-5
-4
[
Figure 11. Effect of ketoconazole (KE) and compound 12 on CYP3A4 activity.
3. CONCLUSIONS
Last decade has shown how difficult it is to develop a new, safe and efficient drug to treat AD. One of
the major obstacles is still incomplete understanding of pathomechanisms leading to the disease, and
its complexity. Due to the latter, it is anticipated that we should be looking for a complex therapy
consisting of drugs with different mechanisms of action. As an alternative to the combination therapy,
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multiꢀtargetꢀdirected ligands approach is considered to make a significant contribution to the
development of new antiꢀAD treatment.
In the pursuit of an effective strategy of AD treatment we have designed new multiꢀtargetꢀdirected
ligands aiming at both symptoms and causes of the disease. Pharmacophore fragments that were
combined in these compounds provided them with significant in vitro cholinesterase inhibitory
0
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activity, 5ꢀHT receptor antagonistic potential and amyloid β antiꢀaggregation activity. As one of the
6
most potent compounds with wellꢀbalanced activity, we selected compound 12 with K
i
= 18 nM and
K
b
= 132 nM against 5ꢀHT , and IC50 values of 14 nM and 22 nM against AChE and BuChE,
6
respectively. The combination of pharmacophore fragments aiming at AChE and 5ꢀHT receptor in
6
this compound improved the hAChE inhibitory potency when compared to tacrine (IC50hAChE = 14 nM,
vs. 131 nM for tacrine). Based on molecular modelling studies, we assume this might be a result of the
additional interactions provided by 5ꢀHT
AChEꢀaiming fragment decreases the interactions of 5ꢀHT
resulting in decrease of affinity for the receptor. Therefore, it is important for further studies to choose
carefully the 5ꢀHT receptor pharmacophore fragment with the highest possible affinity for the
receptor, most preferably of K value below 1 nM. Although compound 12 is tacrine derivative, in in
6
ꢀaiming fragment within AChE. We have also observed that
6
ꢀaiming fragment with 5ꢀHT receptor
6
6
i
vitro metabolic stability study on human liver microsomes, we did not detect any metabolites that are
responsible for hepatotoxicity of tacrine. Another compound that attracted our attention is compound
49. This compound displays wellꢀbalanced activity against 5ꢀHT
6 i
receptor (K = 240 nM) and
butyrylcholinesterase (IC = 196 nM), and, what is particularly interesting, it is benzylamine, not a
5
0
tacrine derivative. This provides favourable physicochemical properties (lower molecular weight and
aromatic ring count) and therefore better potential for further development of this compound as a
pharmacological tool or antiꢀAD agent. A unique feature of the described compounds is that two
6
synergistic mechanisms ꢀ cholinesterase inhibition and 5ꢀHT receptor antagonism ꢀ were
complemented with a significant inhibitory activity against selfꢀinduced amyloid β aggregation
displayed by both compound 12 (94% at 10 ꢁM, IC50 = 1.27 ꢁM) and 49 (65% at 10 ꢁM). Neuritic
plaques formed from the aggregated amyloid β are considered to be a primary lesion in AD, and hence
inhibition of aggregation is of a special interest in the search for new antiꢀAD agents.
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To sum up, the study has revealed new multiꢀtargetꢀdirected ligands that interfere with the most
crucial mechanisms underlying AD, that may serve as an excellent starting point for further
development of a new antiꢀAD therapy.
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.
METHODS
Chemistry
.1.
.1.1. General procedures. Column chromatography was performed on silica gel 60 (63–200 ꢁm;
TM
Merck). Flash chromatography was performed on Isolera Spectra (Biotage). Analytical thin layer
chromatography was carried out using aluminium sheets precoated with silica gel 60 F254 (Merck).
Compounds were visualized with UV light and by solution of ninhydrin. Analytical RPLCꢀMS was
performed by tandem quadrupole mass spectrometry (Waters Acquity TQD), with detection by UV
using a diode array detector with a UPLC BEH C18 column (1.7 ꢁm, 2.1 × 100 mm, Aquity). An
2
acetonitrile/H O gradient with 0.1% HCOOH was used as the mobile phase, at a flow rate of 0.3
1
13
1
mL/min. H NMR and C NMR spectra were recorded on a Mercury 300 instrument (Varian), with H
13
at 300 MHz, and C at 75 MHz. The chemical shifts are reported in ppm and were referenced to the
1
13
residual solvent signals (CHLOROFORMꢀd H: 7.26 ppm, C: 77.16 ppm; DEUTERIUM OXIDE
1
1
6
H: 4.79 ppm, DMSOꢀd H: 2.50 ppm), coupling constants are reported in hertz (Hz). The purities of
the final compounds were determined using analytical RPLCꢀMS (Waters Acquity TQD) with a
UPLC BEH C18 column (1.7 ꢁm, 2.1 × 100 mm, Aquity), with detection at 214 nm and 254 nm. A
acetonitrile/H O gradient with 0.1% HCOOH was used as the mobile phase, at a flow rate of 0.3
2
mL/min. All of the compounds showed purities of >95%. 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.
3
1
The following compounds: Nꢀbenzylꢀ2ꢀbromoacetamide (19), Nꢀbenzylꢀ2ꢀbromoꢀNꢀmethylacetamide
31
31
31
(20), Nꢀbenzylꢀ4ꢀchloroꢀNꢀmethylbutanamide (22), Nꢀbenzylꢀ5ꢀbromopentanamide (23), Nꢀ
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benzylꢀ5ꢀbromoꢀNꢀmethylpentanamide (24), Nꢀ(1ꢀbenzylpyrrolidinꢀ3ꢀyl)ꢀ2ꢀbromoacetamide (32),
31
Nꢀ(1ꢀbenzylpiperidinꢀ4ꢀyl)ꢀ2ꢀbromoacetamide (33), 2ꢀ(1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)ethanol
31
31
(
1),
3ꢀ(1,2,3,4ꢀtetrahydroacridinꢀ9ꢀylamino)propanꢀ1ꢀol
(2),
Nꢀ(5ꢀbromopentyl)ꢀ1,2,3,4ꢀ
31
31
tetrahydroacridinꢀ9ꢀamine (5), Nꢀ(6ꢀbromohexyl)ꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀamine (6), Nꢀ(7ꢀ
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0
31
bromoheptyl)ꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀamine (7), Nꢀ(8ꢀbromooctyl)ꢀ1,2,3,4ꢀtetrahydroacridinꢀ9ꢀ
3
1
31
amine (8), 4ꢀ((1ꢀbenzylpiperidinꢀ4ꢀyl)(tertꢀbutoxycarbonyl)amino)butyl methanesulfonate (51), 1ꢀ
5
3
34,35
(
3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine,
previously reported.
.1.2. Synthesis of N-benzyl-3-bromopropanamide (21).
1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole
have been
4
To a solution of phenylmethanamine (535.8 mg, 5.0 mmol) in 40 mL of DCM cooled at 0–5 °C a
solution of 3ꢀbromopropanoyl chloride (985.7 mg, 5.75 mmol) in 20 mL of DCM was added
dropwise. The reaction mixture was stirred at room temperature for 24 h, then cooled to 0–5 °C and 24
mL of saturated solution of NaHCO
3
was added. The organic layer was washed with saturated solution
SO and concentrated under reduced pressure to give
of NaHCO (2 × 24 mL NaHCO ), dried over Na
3
3
2
4
a crude product that was purified using flash chromatography in 50% ethyl acetate in petroleum ether.
1
Yield: 1,1181 g (97,6%). H NMR (300 MHz, CHLOROFORMꢀd) δ 7.28 ꢀ 7.39 (m, 5H), 5.81 (br. s,
1
H), 4.50 (d, J=5.64 Hz, 2H), 3.69 (t, J=6.60 Hz, 2H), 2.80 (t, J=6.60 Hz, 2H). Formula: C10H12BrNO;
+
MS: m/z 243 (M+H ).
4
.1.3. General procedure for the synthesis of compounds 25–31, 34 and 35.
A mixture of 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine or 1ꢀbenzylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ1Hꢀindole
in form of a hydrochloride salt or as a free base) (1 equiv), corresponding bromide/chloride (1 equiv)
and K CO (1–2 equiv) in acetonitrile was stirred at 50 °C for 24 h. After the mixture was cooled to
room temperature, the solvent was removed, and the product was partitioned between saturated
solution of NaHCO and DCM. The combined organic fractions were dried over Na SO and
(
2
3
3
2
4
concentrated under reduced pressure to give a crude product which was purified according to the
methods described below. To an iceꢀbath cooled solution of the amide in anhydrous THF, 1M solution
of lithium aluminium hydride in THF was added (2.5 equiv) dropwise. The mixture was stirred at
room temperature for 12 h. When the reaction was completed, the mixture was iceꢀcooled, and diethyl
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ether (1 mL), water (10 mL) and 1 M solution of NaOH (0.1 mL) were added. The mixture then was
2 4
extracted with ethyl acetate (3 × 10 mL). The combined organic fractions were dried over Na SO and
evaporated under reduced pressure to give a crude product that was purified according to the methods
described below.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
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3
4
4
4
4
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4
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4
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5
5
5
5
5
6
0
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9
0
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9
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.1.3.1. NꢀBenzylꢀ2ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀyl)ethanꢀ1ꢀamine (25)
Compound 25 was prepared using 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine hydrochloride (159.4
mg, 0.50 mmol), Nꢀbenzylꢀ2ꢀbromoacetamide (19) (114.0 mg, 0.50 mmol), K CO (138.2 mg, 1.00
2
3
mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO
3
(sat.), 3 × 10 mL DCM. Purification: flash
1
chromatography in 40–70% ethyl acetate in petroleum ether. Yield: 140 mg (65%). H NMR (300
MHz, CHLOROFORMꢀd) δ 7.56 (br. s., 1H), 7.25 ꢀ 7.49 (m, 10H), 7.11 (t, J = 8.21 Hz, 1H), 6.68 (dd,
J = 3.98, 8.08 Hz, 2H), 5.07 (s, 2H), 4.52 (d, J = 5.90 Hz, 2H), 3.16 (s, 2H), 2.87 ꢀ 2.95 (m, 4H), 2.68 ꢀ
+
2.76 (m, 4H), 2.23 (s, 3H). Formula: C27
H
31
N
3
O
2
; MS: m/z 430 (M+H ). The Nꢀbenzylꢀ2ꢀ(4ꢀ(3ꢀ
(
benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀyl)acetamide obtained (100.0 mg, 0.225 mmol) was
dissolved in 1 mL THF and reduced using 1 M LiAlH
4
in THF (0.56 mL, 0.56 mmol). Purification:
1
column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 34.6 mg (37%). H
NMR (300 MHz, CHLOROFORMꢀd) δ 7.23 ꢀ 7.48 (m, 10H), 7.08 ꢀ 7.15 (m, 1H), 6.70 (t, J = 8.46
Hz, 2H), 5.07 (s, 2H), 3.83 (s, 2H), 2.83 ꢀ 2.99 (m, 5H), 2.65 ꢀ 2.81 (m, 3H), 2.58 (t, J = 6.03 Hz, 5H),
+
2
.23 (s, 3H). Formula: C27
H
33
N
3
O; MS: m/z 416 (M+H ).
4.1.3.2. NꢀBenzylꢀ2ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀyl)ꢀNꢀmethylethanꢀ1ꢀamine (26)
Compound 26 was prepared using 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine hydrochloride (159.4
mg, 0.50 mmol), Nꢀbenzylꢀ2ꢀbromoꢀNꢀmethylacetamide (20) (121.1 mg, 0.50 mmol), K CO (276.4
mg, 1.00 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO (sat.), 3 × 10 mL DCM.
2
3
3
Purification: flash chromatography in 40–70% ethyl acetate in petroleum ether. Yield: 146 mg (66%).
1
Rotamers in ratio 1:0.9, rotamer 1: H NMR (300 MHz, CHLOROFORMꢀd) δ 7.19 ꢀ 7.49 (m, 10H),
7.11 (dt, J = 2.82, 8.08 Hz, 1H), 6.65 ꢀ 6.75 (m, 2H), 5.08 (d, J = 1.28 Hz, 2H), 4.62 (s, 2H), 3.32 (d, J
=
1.28 Hz, 2H), 3.02 (s, 3H), 2.99 (t, J = 4.74 Hz, 4H), 2.73 (d, J = 15.64 Hz, 4H), 2.25 (s, 3H),
1
rotamer 2: H NMR (300 MHz, CHLOROFORMꢀd) δ 7.19 ꢀ 7.49 (m, 10H), 7.11 (dt, J = 2.82, 8.08
Hz, 1H), 6.65 ꢀ 6.75 (m, 2H), 5.08 (d, J = 1.28 Hz, 2H), 4.73 (s, 2H), 3.32 (d, J = 1.28 Hz, 2H), 2.89 ꢀ
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2
2
2
2
2
3
3
3
3
3
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5
5
5
5
5
5
5
6
+
2
.96 (m, 7H), 2.73 (d, J = 15.64 Hz, 4H), 2.23 (s, 3H). Formula: C28
H
33
N
3
O
2
; MS: m/z 444 (M+H ).
The
Nꢀbenzylꢀ2ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀyl)ꢀNꢀmethylacetamide obtained
(
105.0 mg, 0.24 mmol) was dissolved in 1 mL THF and reduced using 1 M LiAlH in THF (0.6 mL,
4
0
.60 mmol). Purification: column chromatography in DCM/methanol (9.5/0.5, v/v). Yield: 32.0 mg
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
(
31%). H NMR (300 MHz, CHLOROFORMꢀd) δ 7.22 ꢀ 7.48 (m, 10H), 7.11 (t, J = 8.21 Hz, 1H),
6
8
1
4
4
.70 (dd, J = 8.21, 10.52 Hz, 2H), 5.08 (s, 2H), 3.57 (s, 2H), 2.95 (t, J = 4.74 Hz, 4H), 2.53 ꢀ 2.76 (m,
13
H), 2.28 (s, 3H), 2.23 (s, 3H), C NMR (75 MHz, CHLOROFORMꢀd) δ 157.7, 152.6, 138.7, 137.6,
29.1, 128.5, 128.2, 127.7, 127.1, 127.1, 126.3, 121.2, 111.9, 107.0, 70.1, 62.8, 56.5, 54.5, 54.1, 51.8,
+
2.7, 11.0. Formula: C H N O; MS: m/z 430 (M+H ).
28
35
3
.1.3.3. NꢀBenzylꢀ3ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀyl)propanꢀ1ꢀamine (27)
Compound 27 was prepared using 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine (141.2 mg, 0.50
mmol), Nꢀbenzylꢀ3ꢀbromopropanamide (21) (121.1 mg, 0.50 mmol), K CO (69.1 mg, 0.50 mmol) in
2
3
5
mL acetonitrile. Extraction: 10 mL NaHCO
3
(sat.), 3 × 10 mL DCM. Purification: column
1
chromatography in 1–5% methanol in DCM with 0.25% NH3(aq). Yield: 220 mg (50%). H NMR (300
MHz, CHLOROFORMꢀd) δ 8.82 (br. s., 1H), 7.24 ꢀ 7.48 (m, 10H), 7.07 ꢀ 7.15 (m, 1H), 6.68 ꢀ 6.71
(
m, 1H), 6.52 ꢀ 6.59 (m, 1H), 5.07 (s, 2H), 4.45 (d, J = 5.39 Hz, 2H), 2.76 (br. s., 4H), 2.70 (t, J = 6.03
33 3 2
Hz, 2H), 2.62 (br. s, 4H), 2.47 (t, J = 6.03 Hz, 2H), 2.20 (s, 3H). Formula: C28H N O ; MS: m/z 444
+
(
M+H ). The Nꢀbenzylꢀ3ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀyl)propanamide obtained
(
4
180.0 mg, 0.40 mmol) was dissolved in 1 mL THF and reduced using 1 M LiAlH in THF (1.0 mL,
1
.0 mmol). Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v).
1
Yield: 78.0 mg (45%). H NMR (300 MHz, CHLOROFORMꢀd) δ 7.23 ꢀ 7.48 (m, 10H) 7.12 (t, J =
8
2
1
1
2
4
.08 Hz, 1H), 6.69 (dd, J = 7.82, 5.77 Hz, 2H), 5.08 (s, 2H), 3.82 (s, 2H), 2.93 (t, J = 4.74 Hz, 4H),
.74 (t, J = 6.80 Hz, 2H), 2.62 (br. s., 4H), 2.49 (t, J = 7.44 Hz, 2H), 2.24 (s, 3H), 2.06 (br. s., 1H),
13
.77 (quin, J = 6.99 Hz, 2H), C NMR (75 MHz, CHLOROFORMꢀd) δ 157.6, 152.6, 140.4, 137.6,
28.4, 128.3, 128.0, 127.6, 127.0, 126.8, 126.2, 121.1, 111.7, 106.9, 70.0, 57.1, 53.9, 53.7, 51.9, 48.0,
+
4.1, 11.0. Formula: C H N O; MS: m/z 430 (M+H ).
28
35
3
.1.3.4. NꢀBenzylꢀ4ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀyl)ꢀNꢀmethylbutanꢀ1ꢀamine (28)
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Compound 28 was prepared using 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine (141.2 mg, 0.50
2 3
mmol), Nꢀbenzylꢀ4ꢀchloroꢀNꢀmethylbutanamide (22) (112.9 mg, 0.50 mmol), K CO (69.1 mg, 0.50
mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO (sat.), 3 × 10 mL DCM. Purification: column
3
chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.025, v/v/v). Yield: 118 mg (50%). Rotamers in
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
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
ratio 1:0.8, rotamer 1: H NMR (300 MHz, CHLOROFORMꢀd) δ 7.15 ꢀ 7.48 (m, 10H), 7.07 ꢀ 7.14 (m,
1
2
7
H), 6.65 ꢀ 6.74 (m, 2H), 5.07 (s, 2H), 4.61 (s, 2H), 2.87 ꢀ 2.99 (m, 7H), 2.55 ꢀ 2.74 (m, 4H), 2.40 ꢀ
1
.53 (m, 4H), 2.24 (s, 3H),1.86 ꢀ 2.00 (m, 2H), rotamer 2: H NMR (300 MHz, CHLOROFORMꢀd) δ
.15 ꢀ 7.48 (m, 10H), 7.07 ꢀ 7.14 (m, 1H), 6.65 ꢀ 6.74 (m, 2H), 5.07 (s, 2H), 4.58 (s, 2H), 2.87 ꢀ 2.99
(
m, 7H), 2.55 ꢀ 2.74 (m, 4H), 2.40 ꢀ 2.53 (m, 4H), 2.23 (s, 3H), 1.86 ꢀ 2.00 (m, 2H). Formula:
+
C
30
H
37
N
3
O
2
; MS: m/z 472 (M+H ). The Nꢀbenzylꢀ4ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀ
yl)ꢀNꢀmethylbutanamide obtained (118.0 mg, 0.25 mmol) was dissolved in 1 mL THF and reduced
using 1 M LiAlH in THF (0.625 mL, 0.625 mmol). Purification: column chromatography in 10%
1
methanol in chloroform with 0.25% NH3(aq). Yield: 66.0 mg (58%). H NMR (300 MHz,
CHLOROFORMꢀd) δ 7.22 ꢀ 7.48 (m, 10H), 7.12 (t, J = 8.08 Hz, 1H), 6.72 (d, J = 12.82 Hz, 1H), 6.70
(
d, J = 13.08 Hz, 1H), 5.08 (s, 2H), 3.51 (s, 2H), 2.96 (t, J = 4.74 Hz, 4H), 2.63 (br. s., 4H), 2.37 ꢀ
13
2
.48 (m, 4H), 2.25 (s, 3H), 2.21 (s, 3H), 1.53 ꢀ 1.63 (m, 4H), C NMR (75 MHz, CHLOROFORMꢀd)
δ 157.7, 152.7, 139.0, 137.6, 129.1, 128.5, 128.2, 127.7, 127.1, 126.9, 126.3, 121.2, 111.8, 107.0, 70.1,
+
62.3, 58.6, 57.2, 53.7, 51.9, 31.5, 25.4, 24.7, 11.1. Formula: C30
H
39
N
3
O; MS: m/z 458 (M+H ).
4.1.3.5. NꢀBenzylꢀ5ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazinꢀ1ꢀyl)pentanꢀ1ꢀamine (29)
Compound 29 was prepared using 1ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀmethylphenyl)piperazine hydrochloride (159.4
mg, 0.50 mmol), Nꢀbenzylꢀ5ꢀbromopentanamide (23) (135.1 mg, 0.50 mmol), K CO (138.2 mg, 1.0
2
3
mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO
3
(sat.), 3 × 10 mL DCM. Purification: column
1
chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 112 mg (48%). H NMR (300
MHz, CHLOROFORMꢀd) δ 7.24 ꢀ 7.48 (m, 10H), 7.11 (t, J = 8.21 Hz, 1H), 6.68 (d, J = 8.21 Hz, 2H),
6.00 (br. s., 1H), 5.07 (s, 2H), 4.45 (d, J = 5.64 Hz, 2H), 2.90 ꢀ 2.93 (m, 4H), 2.59 (br. s., 4H), 2.39 ꢀ
2.47 (m, 2H), 2.27 (t, J = 7.18 Hz, 2H), 2.23 (s, 3H), 1.66 ꢀ 1.79 (m, 2H) ,1.52 ꢀ 1.65 (m, 2H).
+
Formula:
C
30
H
37
N
3
O
2
;
MS: m/z 472 (M+H ). The Nꢀbenzylꢀ5ꢀ(4ꢀ(3ꢀ(benzyloxy)ꢀ2ꢀ
methylphenyl)piperazinꢀ1ꢀyl)pentanamide obtained (112.0 mg, 0.24 mmol) was dissolved in 1 mL
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