Structure-activity relationship study of tryptophan-based butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112766

A series of tryptophan-based selective nanomolar butyrylcholinesterase (BChE) inhibitors was designed and synthesized. Compounds were optimized in terms of potency, selectivity, and synthetic accessibility. The crystal structure of the inhibitor 18 in complex with BChE revealed the molecular basis for its low nanomolar inhibition (IC₅₀ = 2.8 nM). The favourable in vitro results enabled a first-in-animal in vivo efficacy and safety trial, which demonstrated a positive impact on fear-motivated and spatial long-term memory retrieval without any concomitant adverse motor effects. Altogether, this research culminated in a handful of new lead compounds with promising potential for symptomatic treatment of patients with Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2020.112766

European Journal of Medicinal Chemistry 208 (2020) 112766
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structure-activity relationship study of tryptophan-based
butyrylcholinesterase inhibitors
a
a
b
c
ꢀ
Anze Meden , Damijan Knez , Natalia Malikowska-Racia , Xavier Brazzolotto ,
Florian Nachon , Jurij Svete , Kinga Sałat , Uros Groselj **, Stanislav Gobec a
c
d
b
d,
, *
ꢀ
ꢀ
a
ꢀ
ꢀ
University of Ljubljana, Faculty of Pharmacy, Askerceva 7, SI-1000, Ljubljana, Slovenia
b Department of Pharmacodynamics, Chair of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna St., 30-688,
Krakow, Poland
c
ꢁ
ꢁ
ꢁ
ꢁ
Departement de Toxicologie et Risques Chimiques, Institut de Recherche Biomedicale des Armees, 91223, Bretigny sur Orge, France
d
ꢀ
University of Ljubljana, Faculty of Chemistry and Chemical Technology, Vecna Pot 113, SI-1000, Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tryptophan-based selective nanomolar butyrylcholinesterase (BChE) inhibitors was designed
and synthesized. Compounds were optimized in terms of potency, selectivity, and synthetic accessibility.
The crystal structure of the inhibitor 18 in complex with BChE revealed the molecular basis for its low
nanomolar inhibition (IC50 ¼ 2.8 nM). The favourable in vitro results enabled a first-in-animal in vivo
efficacy and safety trial, which demonstrated a positive impact on fear-motivated and spatial long-term
memory retrieval without any concomitant adverse motor effects. Altogether, this research culminated in
a handful of new lead compounds with promising potential for symptomatic treatment of patients with
Alzheimer’s disease.
Received 20 July 2020
Received in revised form
14 August 2020
Accepted 15 August 2020
Available online 22 August 2020
Keywords:
Cholinesterase inhibitors
Alzheimer’s disease
Butyrylcholinesterase
Neurodegenerative diseases
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
Neurotransmitter acetylcholine (ACh) plays an essential role in
the formation and retrieval of memories, judgement, and orienta-
Dementia is a grievous neurological condition that severely
debilitates the patient’s quality of life. By some estimates, over 50
million people in the world are beset by Alzheimer’s disease (AD)
and this number is expected to triple by the year 2050, mostly due
to the aging of human population [1]. The pathological processes
begin even 20 years before the first clinical symptoms appear, in the
prodromal phase, when only different biomarkers indicate the
presence of pathological processes. Abnormal proteins interfere
with neuronal communication, metabolism, intracellular transport,
and cause chronic inflammation. The disease progresses to mild
cognitive impairment, e.g. diminished formation of new memories,
memory loss, changes in personality, mood, etc. that do not yet
hinder patient’s day-to-day activities. The last phase of AD, com-
plete dementia, when the patient is utterly dependent on others, is
followed by death, usually 8e10 years after diagnosis [2,3].
tion. In AD, cholinergic regions, e.g. Meynert’s nucleus basalis and
its projections to the frontal cortex, undergo significant neuro-
degradation and loss of cholinergic signalling [4e6].
This, combined with cognitive improvement after applying
cholinergic agonists or cholinesterase (ChE) inhibitors, gives evi-
dence to the so-called cholinergic hypothesis of AD etiology [7].
However, cholinergic dysfunction is only one of many pathophys-
iological changes in AD, in addition to extracellular amyloid
b (Ab)
plaques, dystrophic neurites with intracellular neurofibrillary tan-
gles of aggregated protein tau, neuronal loss, amyloid angiopathy,
oxidative damage, calcium imbalance, and neuroinflammation
[3,8,9].
Despite tremendous efforts, no new drugs for AD have been
approved since 2003 and even those currently available in clinical
practice e three ChE inhibitors (donepezil, galantamine, riva-
stigmine) and a NMDA antagonist memantine e do not halt disease
progression. Recent review of 132 agents in anti-AD clinical trials
shows that they are predominantly focused on achieving disease
modification. While novel agents are still being introduced, many
trials have since failed due to the lack of efficacy or serious side
* Corresponding author.
** Corresponding author.
ꢀ
E-mail addresses: uros.groselj@fkkt.uni-lj.si (U. Groselj), stanislav.gobec@ffa.uni-
lj.si (S. Gobec).
https://doi.org/10.1016/j.ejmech.2020.112766
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

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A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
effects [8,10]. Amyloid deposition precedes taupathy, which is un-
derstandable in the light of recent discoveries [11]. Moreover,
accumulation of neurofibrillary tangles, not amyloid plaques,
positively correlates with AD severity [4]. Because many trials were
focused solely on A
casts doubt on the classic amyloid hypothesis of AD and necessi-
tates better understanding of A
0s physiological role and novel
b clearance in patients with progressed AD, this
b
approaches in AD drug development [8,12,13]. Improving cognitive
functions via ChE inhibition therefore remains a time-tested ther-
apeutic option for palliative treatment of AD.
Two ChEs, acetyl- (AChE) and butyrylcholinesterase (BChE),
terminate cholinergic signalling by hydrolysing ACh. AChE is the
main ChE that performs 80% of hydrolytic activity in the brain,
being attached to the postsynaptic membrane. BChE, located at the
glial cells, can function as an auxiliary ChE, taking over at high ACh
concentrations when substrate inhibition of AChE takes place.
While its physiological role and substrates remain to be elucidated,
BChE hydrolyses many exogenous esters in plasma, e.g. succinyl-
choline, acetylsalicylic acid, heroin, etc., and performs additional
non-cholinergic roles as well, for example, in embryonal develop-
ment [14e16]. As AD progresses, not only cholinergic transmission
but also the AChE activity subsides. To compensate, BChE takes over
and terminates even up to 90% of the cholinergic signalling in the
brain [14,17]. Additionally, both enzymes are deposited in senile
plaques and AChE is implicated in Ab aggregation through its pe-
ripheral anionic binding site [18]. Several selective BChE inhibitors,
e.g. bisnorcymserine [19] and naphtha(sulfon)amide-based leads
[20,21], in vivo ameliorated cognitive performance in aged rats or in
scopolamine-treated mice, respectively. Additionally, due to the
selective inhibition of BChE over AChE, these compounds do not
elicit peripheral parasympathomimetic side effects that are typical
for AChE inhibitors. Altogether, this provides the rationale for
further development of new BChE-selective ligands to function as
chemical probes [22] and inhibitors for symptomatic treatment of
AD [23e25].
Fig. 1. Superimposed active sites of hBChE (PDB 6XTA) and hAChE (PDB 6O4W[30])
devoid of ligands. The key amino acid residues are shown in light green (hBChE) and
dark green (hAChE, labels in parentheses). The internal surface of the hAChE active site
is shown in dark grey, whereas light grey areas denote enlarged active site of hBChE
due to six substituted residues (down left). The catalytic triad Ser-His-Glu is located at
the bottom of the active site gorge next to the choline-binding pocket with Trp82(86)
(down right). (For interpretation of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)
Human BChE (hBChE), a 574 amino acid-long protein with a
number of glycosylation sites and three disulfide bridges, has a
hydrophobic active site, comprised of 55 residues that form a 20 Å
deep gorge (Fig. 1) [15]. Near the entrance to the active site residues
Phe329, Asp70, and Tyr332 direct cationic substrates deeper into
the active site [26]. The catalytic triad of Ser198, His438, and
Glu325 is located at the bottom of the gorge. Nucleophilic hydroxyl
group of Ser198 attacks the substrate’s ester group, forming a
tetrahedral intermediate that is stabilized with hydrogen bonds to
amide NH of Gly116, Gly117, and Ala199 in the oxyanion hole. The
acylated enzyme is regenerated by hydrolysis with a water mole-
focused library of additional tryptophan derivatives that expand
the previously established structure-activity relationships (SARs).
Furthermore, the ability of compound 1 to attenuate learning and
memory disorders caused by cholinergic deficit was assessed in an
in vivo mouse model of scopolamine-induced AD-like amnesia. In
addition to the cognitive tasks, a preliminary in vivo safety phar-
macology study was performed. The in vivo evaluation, coupled
with the exhaustive SAR profile, therefore concludes the lead
generation process in this series of new hBChE inhibitors.
cule. Cationic substrates form a cation-
p
interaction with Trp82 in
1.1. Chemistry
the choline-binding pocket, while the other portion of the molecule
binds in the acyl-binding pocket [27]. The active site of hBChE is
approximately 200 Å3 larger than that of human AChE (hAChE) due
to 6 amino acid substitutions. Most importantly, two aromatic
residues Phe295 and Phe297 in acyl-binding site of hAChE are
replaced by Leu286 and Val288 in hBChE (Fig. 1) [28,29]. This
broadens the enzyme’s substrate specificity since its acyl-binding
pocket can accommodate larger side chains (e.g. as in butyrylcho-
line) and allows for design of selective inhibitors.
In a previous communication [31] we disclosed preliminary
results of a new series of tryptophan-based selective hBChE in-
hibitors with nanomolar affinity, which was designed from
(þ)-isocampholenic-acid-derived tryptophan-amide hit (Fig. 2).
Through medicinal chemistry-based approach, the hit was opti-
mized towards lead-like compound 1 that exhibited low cytotox-
icity, high predicted permeability of the blood-brain barrier, and
sp3-rich character.
The required 2-((aza)cycloalkyl)ethan-1-amine building blocks
were either commercially available or synthesized from the cor-
responding cyclic ketone 2a in a three-step sequence (Scheme 1,
only the representative examples are depicted). Firstly, Hor-
nereWadswortheEmmons olefination with diethyl cyanomethyl-
phosphonate led to
a,b-unsaturated nitrile 2b, which was
catalytically hydrogenated to the substituted acetonitrile 2c, and
finally reduced with lithium aluminum hydride (LiAlH4) to afford
the primary amine 2d. In order to prepare the N-alkylated ana-
logues, the N-Boc-protected 3- or 4-(cyanomethyl)piperidine or
-azepane 2c was deprotected to the secondary amine 2e by acid-
olysis in trifluoroacetic acid (TFA). 2e was acylated with an aroyl
chloride or N-acylimidazole 2f, and the resulting tertiary amide 2g
was reduced with excess LiAlH4 to afford the N-substituted 2-
(piperidin-3(4)- or azepan-4-yl)ethan-1-amine 2h. In the case of
perfluorobenzoyl amide, the reduction of amide was accompanied
by the loss of the para-fluorine. Arguably, due to the high electron-
Here, we report the synthesis and biochemical evaluation of a

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
3
Fig. 2. Previously reported structure-activity optimization from the initial hit compound (left) to the lead-like compound 1.
Scheme 1. General synthetic routes to the required 2-((aza)cycloalkyl)ethan-1-amines.
Scheme 2. The two complementary synthetic routes to N-butylated a-aminoamide inhibitors.
withdrawing character of the perfluorinated ring, the SNAr reaction
with hydride precedes the amide reduction [32,33].
used (Scheme 2). The parent amino acid 3a (e.g. L-Trp, L-Phe) was
first reductively alkylated with butyraldehyde and sodium boro-
hydride in methanol [34], and the resulting secondary amino group
For the preparation of N-butylated amide inhibitors derived
from natural
a-amino acids, two complementary strategies were
Boc-protected
with
di-tert-butyl
dicarbonate.
1,10-

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A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
Carbonyldiimidazole (CDI)-mediated amidation of 3b with the
2. Results and discussion
respective amine (see Scheme 1) and the following acidolytic
deprotection with TFA afforded the N-butylated
a
-aminoamide 3c
2.1. In vitro biological evaluation
(Scheme 2, Path A). Alternatively, the N-Boc protected parent amino
acid 3d (e.g. N-Boc-L-Leu, N-Boc-L-Trp) was first amidated with the
respective amine using CDI as the activating agent, and then tert-
butyl carbamate cleaved with TFA. The resulting primary a-ami-
noamide 3e was subsequently reductively butylated with butyral-
In the previous communication [31] we have shown that the
high selectivity of tryptophan-derived series of compounds for
hBChE over hAChE lies in their indole moiety that occupies the
hydrophobic acyl-binding pocket, T-stacked with Phe329 and
dehyde and sodium triacetoxyborohydride in dichloromethane
Trp231. To confirm the necessity of this p-p interaction, com-
(DCM) to furnish the desired N-butylated
(Scheme 2, Path B).
a
-amino amide 3c
pounds 8 and 9 were synthesized (Fig. 3). Replacing tryptophan’s
indole ring with leucine’s isopropyl group greatly reduced inhibi-
tory potency for three orders of magnitude, while replacing indole
with the smaller phenyl ring also diminished potency, although to a
lesser extent. Interestingly, the selectivity for hBChE over hAChE
was retained.
The two
g-aminoamides 4 and 5 were prepared in five steps
from N-Boc-L-tryptophanal [35]. Wittig olefination of the aldehyde
with benzyl(triphenylphosphoranylidene)acetate, followed by cat-
alytic hydrogenation of benzyl acrylate 4a gave N-Boc g-amino acid
4b, which was amidated with 2-cycloheptylethan-1-amine. The so
obtained tert-butyl carbamate 4c was acidolytically cleaved to
primary amine 4d, which was finally reductively n-butylated with
butyraldehyde and sodium triacetoxyborohydride in DCM, which
afforded both the mono- and the dialkylated products 4 and 5,
respectively (Scheme 3).
The 1,4-diaminobutane derivatives 6 and 7 were prepared in
five steps from 3-indolepropanoic acid: CDI-mediated amidation
with n-butylamine, reduction with LiAlH4, and microwave-assisted
acylation of the secondary amine with succinic anhydride, followed
by amidation with 2-cycloheptylethan-1-amine gave succindia-
mide 6, which was subsequently reduced with LiAlH4 to the cor-
responding 1,4-butanediamine 7 (Scheme 4).
Based on the previous observation [31] that the choline-binding
pocket can accommodate 2-cycloheptylethyl, 3-cycloheptylpropyl,
and even 2-cyclododecylethyl substituents on the amide nitrogen,
inhibitors designed to reach the spatial limits for the accommo-
dation of larger substituents were prepared and evaluated (Fig. 4).
Compared with the parent compound 1, elongated i.e.
-aminoamide derivatives 4 and 5 exhibited slightly diminished
inhibition of hBChE. Compound 7, as a less rigid 1,4-diaminobutane
analogue of the -aminoamides 4 and 5, was almost fourfold less
g-instead of
a
g
potent than 1. Centrally rigidized succindiamide analogue 6 per-
formed even worse with inhibition in high nanomolar range.
Introducing a rigid turn in the form of a dipeptide with L-Pro
(angular compound 10) led to comparable inhibitory potency as
seen with 1.
Scheme 3. Synthesis of
g
-aminoamides 4 and 5.

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
5
Scheme 4. Synthetic route to compounds 6 and 7.
Fig. 3. In vitro ChE inhibitory potencies of compounds 1, 8e9, given as IC50 SEM, i.e. standard error of the mean. Data are average of two independent experiments, each per-
formed in triplicate. RA e residual activity, compounds are considered inactive when RA is above 50%.
As evident from the previously resolved crystal complexes [31],
the amide moiety of 1 is located below Tyr332 (Fig. 7, 1 coloured
orange). In order to reduce the molecular mass, provide further
donor on the far end of the ring could reach down to the Trp82 and
form an interaction e finally our efforts were met with success (18,
IC50(hBChE) ¼ 2.8 nM). The existence of
p-p interaction was
rigidization, and to establish a
p
-p
interaction with Tyr332, phenyl
confirmed in the crystal complex of 18 with hBChE as described in
detail below. To thoroughly sample the available conformational
space, ring sizes, tether lengths, and substitution patterns were
varied. The following structure-activity relationships (SARs) could
be extrapolated (Table 1): simply attaching the phenyl substituent
onto the cyclohexyl ring (13) or introducing the N-benzyl residue at
the 1,3-position of piperidine ring (14) improved the inhibitory
potency. However, the observed improvement could have resulted
either from filling hydrophobic environment above Trp82 (as seen
in the previous work, no size discrimination was seen going from
ring was introduced in the vicinity of the amide moiety e either in
the form of N-(2-phenylpyrrolidine) (11) or N-(4-benzylpiperidine)
(12). However, these attempts failed to improve the inhibitory
potency and were therefore not pursued further (Fig. 5).
In the previous series [31], the introduction of the endo- and the
exocyclic tertiary amine on the far end of the six- or seven-
membered ring did not result in improved inhibition. As it was
later evident from resolved crystal complexes, the designed cation-
p
interaction with Trp82 could not be formed since the afore-
mentioned ring resided in a hydrophobic environment above
Trp82. We postulated that a flexible sidechain with cation or
six-to twelve-membered ring system) or from less than optimal
p-
p
p interaction with Trp82. N-Benzyl, N-phenethyl or N-phenyl

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A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
Fig. 4. In vitro ChE inhibitory potencies of elongated compounds 4e7, 10.
Fig. 5. In vitro ChE inhibitory potencies of compounds 11e12.
substituents in the 1,4-position of the piperidine or the azepane
ring (compounds 15e18) produced comparably potent hBChE in-
hibitors, with N-benzyl group being the optimal one (compound 15,
electron-withdrawing 2,3,5,6-tetrafluorophenyl bioisoster 19 was
synthesized, however, the interaction did not benefit from the
additional charge-transfer. The introduction of strongly electron-
donating para-methoxy group in compound 20 decreased the in-
hibition of hBChE. This could have also been due to steric hindrance
in the choline-binding pocket. Lastly, compound 21 was designed
to function as a cation donor analogue of the above compounds.
Surprisingly, the result was loss of affinity for three orders of
IC50(hBChE) ¼ 2.0 nM). The
p-p interaction with Trp82 was
tentatively assigned to these derivatives based on the crystal
complex 18/hBChE, comparable potency, and similar conformation
adopted by the piperidine and the azepane ring. To evaluate two
diametral cases of electronic effects on p-p interaction, the strongly

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
7
Table 1
In vitro ChE inhibitory potencies of compounds 13e21.
Compound
hBChE
hAChE
IC50 SEMa [nM]
RAb at 100 M [%] or IC50 [nM] SEMa
m
General structure:
13d
6.6 1.7
34 5%c
14d
15
9.0 0.8
2.0 0.4
3.8 0.9
43 1%c
25400 6600
77 1%
16
17
5.6 1.2
49 6%
18d
2.8 0.8
16000 4000
19d
5.9 1.3
20 3%c
20d
100
7
37 5%c
21
6600 2000
29700 8300
a
SEM e standard error of the mean, data are the average of two independent experiments, each performed in triplicate.
RA e residual activity.
nonspecific inhibition at the concentration tested (100 mM) due to solubility issues, inhibition disappears upon dilution (30 mM).
b
c
d
mixture of diastereomers.
magnitude, possibly due to the steric discomplementarity of the
non-planar N-methylpiperidine ring with the lower portion of the
choline-binding pocket around Trp82, coupled with unfavourable
desolvation. Interestingly, compounds 4e7, 15, 18, and 21 exhibit a
modest micromolar affinity for hAChE. To elucidate the structural
reasons for decreased selectivity, a docking study to hAChE was
performed (see Supporting Information, Figure S8). The examina-
tion of predicted binding poses reveals that these inhibitors occupy
an extended quasilinear conformation, with one part of the mole-
cule protruding towards the bulk solvent, and the other reaching
downwards into the narrower active site of hAChE, interacting with
the aromatic residues in the choline-binding pocket and peripheral
binding site of hAChE. This binding mode concurs with the avail-
able crystal structures of other rod-shaped hAChE inhibitors, e.g.
donepezil [36] and bistacrine dimer from PDB 5EI5 [37].

8
A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
Waals hydrophobic contacts are formed between 18 and Phe329,
Gly116, and Gly117. The acyl-binding pocket of hBChE is bigger than
acyl-binding pocket of hAChE and is therefore able to accommodate
larger ligands (Fig. 1), thus explaining the compound 18 selective
inhibition of hBChE over hAChE. The central N-butyl sidechain
protrudes out of the active site towards the bulk solvent, tethered
by hydrophobic contacts with Gln119, Ala277, Thr284, Ser287, and
Asn289 along the entrance to the active site gorge. The amide
hydrogen of 18 donates a hydrogen bond to the nearby backbone
amide carbonyl oxygen of Pro285 (distance of 2.6 Å), with a slight
out-of-plane deviation (18.8ꢀ). The voluminous azepane ring oc-
cupies the bulk of the active site, imposing a suitable orientation
whilst retaining the necessary flexibility to accommodate the
phenyl ring in the proper position. The N-benzyl sidechain on the
right is engaged in a p-p interaction with Trp82 located 4.1 Å away
on the bottom of the choline-binding pocket.
Comparing with the crystal structure [31] of the compound 1,
both ligands adopt a similar conformation (Fig. 7). However, the
p-
p
interaction of compound 18 benzyl group with Trp82 forces the
seven-membered ring, which had previously resided in the vicinity
of hydrophobic residues (i.e. Ala328, Trp430, Tyr332, Met437,
Tyr440), to the position above Trp82 and towards the active site
entrance, where the flexible N-butyl sidechain of 18 exits the active
site under a different angle. As predicted in the previous commu-
nication [31], adding the N-benzyl substituent on the far side of the
azepane ring provided the necessary tether to reach Trp82 at the
bottom of the choline-binding pocket. This also expels the DMSO
molecule (present in the four previously reported crystal structures
[31]), that had occupied the voluminous choline-binding pocket e
thus optimizing the available spatial and interaction potential.
Fig. 6. Crystal structure of compound 18 (displayed in red-coloured stick model)
bound to hBChE (shown as grey-coloured internal surface, at resolution of 2.5 Å, PDB
6XTA). The (S,R)-diastereomer of (S)-N-(2-((R)-1-benzylazepan-4-yl)ethyl)-2-(butyla-
mino)-3-(1H-indol-3-yl)propanamide was observed in the crystal structure. Key amino
acid residues in the proximity of 18 are coloured green, shown as sticks, and hydrogen
bond is shown in yellow dashes. Indole ring of 18 occupies the acyl-binding pocket, the
azepane ring resides above the choline-binding pocket, wherein the N-benzyl group
forms a p-p interaction with Trp82. The central N-butyl sidechain is oriented out of the
active site gorge. (For interpretation of the references to colour in this figure legend,
the reader is referred to the Web version of this article.)
2.3. In vivo evaluation
A
nonselective muscarinic receptor (mAChR) antagonist,
scopolamine, is used as a research tool that induces cholinergic
deficits and AD-like amnesia in animal models, resembling the
cognitive decline observed in AD patients [38]. ChE inhibitors in-
crease synaptic levels of ACh, and consequently displace scopol-
amine from mAChR, thus reversing the scopolamine-induced
cognitive deficits [39]. As in our previous studies [20,21], comple-
mentary cognitive assays were selected to provide differential
stimuli and evaluate various aspects of memory functions. Passive
avoidance task (PA), Morris water maze (MWM), and Barnes maze
(BM) were used to assess the impact of the compound 1 on animals’
learning skills, i.e. memory acquisition and retrieval.
2.3.1. Passive avoidance task
This behavioural test assesses the long-term memory based on
negative reinforcement by an aversive stimulus, i.e. an electric
shock to the feet. There were no significant differences in the step-
through latency between vehicle-treated mice and scopolamine-
treated control group, as well as between scopolamine-treated
control mice and compound 1-treated memory-impaired mice in
the acquisition trial (Fig. 8, day 1). As expected, there were statis-
tically significant differences between the vehicle-treated mice and
scopolamine-treated control mice in the retention trial on day 2.
Gratifyingly, the step-through latency of compound 1-treated
memory-impaired mice compared to the scopolamine-treated
control mice was significantly extended at 10 mg/kg (p < 0.05)
and 30 mg/kg doses (p < 0.01) in the retention trial (Fig. 8, day 2).
This significant prolongation of the step-through latency from
the acquisition trial to the retention trial in vehicle-treated subjects
confirms the unimpaired learning and memory functions, while the
significantly reduced latency in scopolamine-treated control vs.
Fig. 7. Overlay of crystal structures of compounds 1 [31] (orange) and 18 (red) in
hBChE active site (shown as grey-coloured internal surface, key amino acid residues
are shown as green sticks). Both ligands adopt a similar conformation, however the
p-
p
interaction of 18 benzyl with Trp82 pushes the azepane ring from hydrophobic
environment above Trp82 (shown as teal-coloured sticks on the right-hand side) back
towards the active site opening. (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)
2.2. Crystal structure determination
To confirm the hypothesized
p-p interaction of the aromatic
side chain of inhibitors 13e20 with Trp231, the crystal structure of
18ehBChE complex was resolved (Fig. 6, Table S1). The indole ring
of 18 indeed resides in the acyl-binding pocket of the hBChE active
site, forming a T-stacking
p-p interaction with Trp231 at distance of
4.3 Å, which is, however, longer than typically observed. Van der

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
9
to assess the effect of the pharmacological treatment on working
memory. In this phase BChE inhibitor 1 did not manifest any effect
on working memory (Figure S5). Same conclusion could be drawn
also from the number of primary errors, where compound 1 did not
exert beneficial effects on the working memory in the acquisition
phase of BM task (Figure S6 e between days; Figure S7 e between
individual trials of the day).
The memory retrieval (the drug-off) trials were conducted on
days 5 and 12. On day 5, the one-way ANOVA did not reveal sig-
nificant effects of the treatment on the latency to finding the former
escape box location (F[2,21] ¼ 2.689, p ¼ 0.0913), or on the number
of errors made (F[2,22] ¼ 2.254, p ¼ 0.1287) (Fig. 9A and B). In
contrast to this, day 12 trial confirmed the cognitive deficit of
scopolamine-treated group, showing both in the increased time
needed to find the former target location, and in the number of
primary errors made (F[2,22] ¼ 8.099, p < 0.01 and F[2,22] ¼ 10.86,
p < 0.001, respectively). Both the vehicle-treated and the scopol-
amine and compound 1-treated mice had significantly shorter la-
tencies to finding the former target location than the mice that
were treated only with scopolamine (p < 0.01, Fig. 9C). Additionally,
both test groups made fewer primary errors when compared to the
scopolamine-treated control group (statistically significant with
p < 0.001 and p < 0.05, respectively, Fig. 9D).
Fig. 8. The effect of the compound 1 on fear-motivated memory formation in PA task
in scopolamine-induced memory-impaired CD-1 mice (n ¼ 8e9). Results are shown as
mean step-through latency SEM to enter the dark compartment in the acquisition
trial (day 1) and the retention trial (day 2). Statistical analysis: repeated-measures
ANOVA, followed by Dunnett’s post hoc comparison. The overall effects of treatment
(F[4,74] ¼ 3.856; p < 0.01) and day of trial (F[1,74] ¼ 23.34; p < 0.0001) were found to
be significant, whereas drug ꢁ time interaction was not (F[4,74] ¼ 2.030; p > 0.05).
Significance vs. scopolamine-treated control in the respective trial: *p
**p < 0.01.
< 0.05,
Taken together, the results obtained in the BM task show that
although compound 1 did not enhance learning abilities and
working memory in the acquisition phase or the short-term
memory retrieval on day 5, it did significantly improve long-term
memory retrieval, as observed on day 12 of the BM task.
vehicle-treated mice demonstrates the scopolamine-induced
amnesia in this mouse model. The application of compound 1
significantly prolonged the step-through latencies in memory-
impaired mice, therefore establishing a positive effect of com-
pound 1 on mitigating the scopolamine-induced impaired memory
formation. Considering these results, the dose of 30 mg/kg was
chosen for further experiments assessing spatial learning skills and
memory.
2.3.4. Preliminary safety pharmacology
This part of the in vivo study aimed to assess the potential
neurological complications, such as impaired motor coordination
and reduced or increased locomotor activity, due to the adminis-
tration of compound 1. ChE inhibitors can cause peripheral adverse
effects due to the unrestrained ACh build-up in the peripheral
nervous system, the parasympathetic autonomic system, and the
basal ganglia [41]. Furthermore, the drug-induced altered loco-
motor activity or impaired motor coordination may directly
confound the results of behavioural tests that assess the drug’s
effect on cognition.
We therefore evaluated the impact of compound 1 at its highest
dose in the locomotor activity and rotarod tests. hBChE inhibitor
1 at the dose of 30 mg/kg in mice neither altered the number of
light-beam interruptions (Fig. 10) in the locomotor activity test nor
induced any motor deficits at 6, 18 and 24 rpm in the rotarod test.
These results exclude the potential source of false results and
confirm that the compound 1 does not induce adverse motor ef-
fects in vivo. Additionally, no acute adverse cholinergic effects
following the administration of compound 1 were observed in
mice. This was expected, as selective BChE inhibitors in vivo do not
induce peripheral (parasympathomimetic) adverse effects [21].
2.3.2. Morris water maze
MWM is
a hippocampus-dependent memory and spatial
learning task that explores the test compound’s effects on working,
short-term memory (decreased escape latency, shorter overall
distance swum before reaching the escape platform), and memory
retrieval. During the ensuing 6 training days of the acquisition
phase the escape latencies of all test groups progressively
decreased, indicating learning (Figure S1). Significant differences
were found between the vehicle- and the scopolamine-treated
groups, while the compound 1-treated memory-impaired mice
did not outperform the scopolamine-treated control mice. Similar
results were observed on day 7 (the drug-off retrieval phase), with
no statistical significance in spatial memory retention parameters
in compound 1-treated memory-impaired mice vs. scopolamine-
treated control group (Figures S2eS3). For detailed results of
MWM assay, the interested reader is referred to the Supplementary
Information.
2.3.3. Barnes maze
3. Conclusion
BM task is a land maze-based spatial learning and memory
assay, complementary to the water-based MWM, and is used to
assess the test compound’s effects on working, short-term and
long-term memory [40]. In the memory acquisition phase (days
1e4) post hoc analysis of the latency to target showed a statistically
significant difference between vehicle-treated and scopolamine-
treated groups on days 3 and 4. The compound 1 also reduced
the latency to target in scopolamine-induced memory-impaired
mice, albeit not enough to reach the statistical significance
(Figure S4). The comparison of latencies to reach the target in
separate trials on individual days of the acquisition phase was made
We presented a follow-up study of a new series of tryptophan-
based selective hBChE inhibitors, recently disclosed in a short
communication [31]. Through chemical space exploration, accom-
panied with structure-guided rational design using X-ray crystal
structures, the disclosed series was now optimized for inhibitory
potency. Structural modifications presented in this study revealed
the elements responsible for recognition and selectivity, as well as
limitations of the chosen scaffold. The favourable physicochemical
properties and in vitro experiments had predicted high perme-
ability of the blood-brain barrier [31], allowing the inhibitors to

10
A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
Fig. 9. The effect of the compound 1 on memory retrieval as measured in the BM task on days 5 (above) and 12 (below) in C57BL/6J mice (n ¼ 7e9). Results are shown as the mean
latency SEM to finding the former target location (left) and the mean number of primary errors made SEM (right). Statistical analysis: one-way ANOVA, followed by Dunnett’s
post hoc comparison. Significance vs. scopolamine-treated control mice: *p < 0.05, **p < 0.01, ***p < 0.001.
selective hBChE inhibitors and well-established therapeutic benefit
of registered ChE inhibitors, this new series of low nanomolar se-
lective hBChE inhibitors with prominent sp3 character, provides a
source of new lead compounds with promising therapeutic po-
tential for developing a symptomatic treatment of patients with
AD.
4. Experimental
4.1. Inhibition of cholinesterases
The inhibitory potencies of the compounds against the ChEs
were determined using the method of Ellman following the pro-
cedure described previously [21]. Briefly, compound stock solutions
in DMSO were incubated with Ellman’s reagent and the ChEs (final
concentrations: 370 mM Ellman’s reagent, approximately 1 nM or
Fig. 10. The effect of the compound 1 on locomotor activity. Results are shown as mean
number of light-beam interruptions SEM counted during the 60-min testing period
(n ¼ 8). Statistical analysis: Student’s t-test: p > 0.05.
50 pM hBChE or hAChE, respectively) in 0.1 M phosphate buffer pH
8.0 for 5 min at 20 ꢀC. The reactions were started by the addition of
the substrate (final concentration, 500 mM butyrylthiocholine io-
access to the site of action in the brain. Therefore, the first-in-
animal trial was made to assess the ability of the lead compound
1 to attenuate learning and memory deficits caused by cholinergic
deficit in AD mouse model. The behavioural tasks were carefully
selected to provide orthogonal and complementary evidence to
cognitive amelioration mediated by compound 1 as a representa-
tive of this class of hBChE inhibitors. A significant impact on fear-
motivated long-term memory and spatial long-term memory
retrieval was observed. Additionally, a preliminary in vivo safety
pharmacology study excluded potential neurological side effects.
Coupled with the low peripheral adverse effects, potential of
dide or acetylthiocholine iodide for hBChE and hAChE, respec-
tively). The final content of DMSO was always 1%. The increase in
absorbance at 412 nm was monitored for 1 min using a 96-well
microplate reader (Synergy H4, BioTek Instruments, VT, USA). The
initial velocities in the presence (vi) and absence (vo) of the test
compounds were calculated. The inhibitory potencies were
expressed as the residual activities, according to RA ¼ (vi e b)/(vo e
b), where b is the blank value using phosphate buffer without ChEs.
For IC50 determinations, at least seven different concentrations for
each compound were used. The IC50 values were obtained by
plotting the residual ChE activities against the applied inhibitor

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
11
concentrations, with the experimental data fitted to a four-
parameter logistic function (GraphPad Prism 8.0). Tacrine and
donepezil were used as positive controls.
used for data analysis. The numerical results from behavioural tests
are reported as means SEM. For the statistical analysis, Student’s
t-test, one-way analysis of variance (ANOVA), or two-way repeated
measures ANOVA followed by Dunnett’s post hoc comparison, were
used. P < 0.05 was considered significant.
4.2. Crystallization
Recombinant hBChE was produced in Chinese hamster ovary
cells as described previously [42] and purified with BChE-specific
affinity (Hupresin, CHEMFORASE, Rouen, France) and size exclu-
sion (Superdex 200, GE Healthcare) chromatographies [43]. hBChE
crystals were obtained using the hanging drop method at 293 K
using a 12 mg/mL protein solution and 0.1 M MES pH 6.5, 2.15 M
(NH4)2SO4 as crystallization buffer. 18£HCl was solubilized in wa-
ter (at 0.1 M) and protein complex was obtained by crystal soaking
at 1 mM final ligand concentration (0.1 M MES pH 6.5, 2.15 M
(NH4)2SO4). Crystals were cryo-protected in a solution of 0.1 M MES
pH 6.5, 2.15 M (NH4)2SO4, 20% glycerol, 1 mM ligand before flash
cooling into liquid nitrogen.
4.5. In vivo evaluation e behavioural tests
4.5.1. Passive avoidance task
The passive avoidance apparatus (Panlab Harvard Apparatus,
Spain) consists of a large white-painted illuminated compartment
(26 cm ꢁ 26 cm ꢁ 34 cm) and a small black-painted dark
compartment (13 cm ꢁ 7.5 cm ꢁ 7.5 cm) separated by a guillotine
gate (5 cm ꢁ 5 cm). The mice were subjected to two separate trials:
an acquisition trial (the ‘conditioning phase’), followed by a
retention trial (the ‘testing phase’) 24 h later.
During the acquisition trial, each mouse was initially placed into
the lit compartment for 30 s, then the guillotine gate was opened,
and the time elapsed before entering the dark chamber was
recorded (the ‘step-through latency’). As soon as the mouse entered
the dark compartment, the gate closed and an electrical shock
(intensity: 0.2 mA, duration: 2 s) was administered through the
grid floor. If the mouse did not cross over to the dark compartment,
the latency of 180 s was assumed.
On the next day, the retention (drug off) trial was conducted.
The mice were placed in the white compartment for 30 s as before,
and the latency from the gate opening until the mouse entered the
black compartment was measured. If the mouse did not enter the
dark compartment during the 180-s testing period, it was assumed
that it remembered the electrical shock from acquisition trial
[51,52].
4.3. X-ray structure determination
X-ray diffraction data were collected on the PROXIMA-1 beam-
line of the SOLEIL Synchrotron (Saint Aubin, France) at 100 K. Im-
ages recorded on an EIGER 16 M detector were processed with XDS
[44] using CC1/2 and I/sigma>1 as selection criteria. Data analysis
was realized using the Phenix software suite [45]. Initial model was
obtained by molecular replacement using Phaser-MR and the
hBChE structure (PDB entry 1P0I) devoid of any ligand, glycans or
water molecules. Electron density was observed in the active site
gorge and allowed unambiguous fitting of 18. Ligand geometry
restraints were processed with Phenix eLBOW [46] using the semi-
empirical quantum mechanical method (AM1) and assuming the
calculated charged positions at pH 6.5 [47]. The model was refined
by iterative cycles of Phenix.refine and model building using Coot.
[48] Human BChE structure in complex with 18 was deposited into
the Protein Data Bank under accession number 6XTA. The struc-
tures were visualized using PyMol [49].
4.5.2. Morris water maze
The Morris water maze is a circular, grey-painted plastic pool
(diameter, 120 cm; height, 60 cm), filled with water (up to about
12 cm below the edge to prevent escape) thermostated at 23 1 ꢀC,
and lit with the intensity of 45 lx. The pool was divided into four
equal quadrants (points of the compass: NE, NW, SE, SW) by a
computerized video tracking system (SMART, ver. 3.0; Panlab,
Spain). An escape platform (diameter, 11 cm; height, 47 cm) of
transparent Plexiglas (invisible to the swimming animal) was
immersed 1 cm below the surface in the center of the NW quadrant
(the target quadrant).
During 6 consecutive days (the spatial acquisition trial) the mice
were assigned to 4 training sessions a day held 90 min apart, where
they were trained to escape from water by reaching the hidden
platform. The location of hidden platform could be identified by
distal extra-maze cues (A4-sized sheets of black laminated paper
with colour geometric symbols) that were attached to the room
walls to provide navigation points [53]. These visual cues were of
different colours and dimensions, and were kept constant during
the experiment. Additionally, the experimenter remained station-
ary at a fixed location, therefore providing another distal cue for the
swimming animals. In each trial the subject was placed in the water
in a different randomly chosen quadrant without the platform,
whereas the platform was always positioned in the NW quadrant.
The time needed for the swimming subject to reach the hidden
platform (‘escape latency’) was measured. After 60 s, the animal
which failed to reach the hidden platform was gently placed on the
platform for 15 s.
4.4. In vivo evaluation e general information
Adult male Albino Swiss CD-1 (Animal Breeding Farm, Faculty of
Pharmacy, Jagiellonian University Medical College) and C57BL/6J
(Animalab, Poland) mice (18e22 g) were used. The CD-1 Albino
mice perform poorly in spatial memory tests due to their visual
impairments [40,50], therefore the C57BL/6J strain was used for
MWM and BM. The mice were housed in groups of 10 per cage with
tree bedding (Transwior, Poland) and cage enrichment and were
maintained at 22
2
ꢀC under a light/dark (12h:12h) cycle. The
mice were given ad libitum access to food and water. Groups of
7e10 randomly selected mice were used for the behavioural tests.
The mice were immediately euthanized by cervical dislocation after
the experiments. The procedures for maintenance and treatment of
laboratory animals were approved by the Local Ethics Committee of
the Jagiellonian University in Krakow (No 250/2019) and were in
full accordance with the EU Directive 2010/63/EU for animal
experiments.
Scopolamine hydrobromide (Sigma Aldrich, Poland), dissolved
in 0.9% saline (Polfa Kutno, Poland) was administered subcutane-
ously at the dose of 1 mg/kg 30 min before the first trial on each day
of the acquisition phase of respective cognitive assays to induce
memory impairment. The test compound 1 was suspended in 1%
Tween 80 (Baxter, Poland) and administered intraperitoneally (i.p.)
60 min before the trial, while the control group mice received 1%
Tween 80 (i.p.) 60 min before testing.
On the 7th day (24 h after last training session) the platform was
removed from the pool and a probe trial was performed (without
drug treatment). Subjects were released from a different starting
point and allowed to swim for 60 s. If a mouse did not find the
former platform location (‘target zone’) within 60 s, it was given a
GraphPad Prism (version 8, GraphPad Software, CA, USA) was

12
A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
latency score of 60 s. In addition to the number of target crossings,
the time spent in the target quadrant, the total distance, the dis-
tance in NW quadrant and the number of entries in NW quadrant
were measured [21].
dried over anhydrous sodium sulfate. Reactions were monitored
using analytical thin-layer chromatography (TLC) on silica gel 60
F254 Al plates. Developed plates were inspected under UV light and,
if necessary, visualized with ninhydrin, vanillin/sulfuric acid, Dra-
gendorff’s or potassium permanganate stains. Melting points were
determined with Stanford Research Systems OptiMelt MPA100 e
Automated Melting Point System (uncorrected). Nuclear magnetic
resonance spectra were recorded on a Bruker Avance III 500 MHz,
Bruker Avance III 400 MHz, or Bruker Avance DPX 300 MHz spec-
trometer at 500 MHz (400 MHz, 300 MHz) for 1H, 126 MHz
(100 MHz, 75 MHz) for 13C, and 376 MHz for 19F nucleus, respec-
tively, using DMSO‑d6 or CDCl3 with TMS as the internal standard,
as solvents. Chemical shifts are reported in parts per million (ppm),
TMS peak was calibrated to 0 ppm or, alternatively, the central peak
of the residual solvent resonance was used as the internal standard
i.e. for CDCl3 at 7.27 ppm for 1H and 77.16 ppm for 13C and DMSO‑d6
at 2.50 ppm for 1H and 39.52 ppm for 13C, respectively. 19F spectra
were not calibrated. The multiplicities are reported as follows: s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd
(doublet of doublets), ddd (doublet doublet of doublets), td (triplet
of doublets), qd (quartet of doublets), and br (broad), number of
equivalent nuclei (by integration), coupling constants (J) quoted in
Hertz (Hz). Mass spectra were recorded on Agilent 6224 Accurate
Mass TOF LC/MS and Thermo Scientific Q Executive Plus LC-MS/MS
spectrometers, IR spectra on Bruker ALPHA FT-IR and Thermo
Nicolet FT-IR spectrophotometers and optical rotation was
measured on PerkinElmer 241 MC Polarimeter with specific rota-
tion values given in 10ꢂ1 deg cm2 gꢂ1. Column chromatography was
performed on silica gel (Silica gel 60, particle size:
0.035e0.070 mm, Merck). UPLC analyses were performed on
Thermo Scientific Dionex UltiMate 3000 modular system (Thermo
Fisher Scientific Inc.). The general method (method I) used a Waters
4.5.3. Barnes maze
Barnes maze apparatus (Panlab Harvard Apparatus, Spain)
consists of a circular, dry, open platform surface with a cylindrical
dark start chamber in the middle and 18 holes located around the
perimeter, only one of them leading to a small dark chamber
(‘escape box’) located underneath. Visual cues (coloured geometric
figures with contrast background) placed 1 m away from the
platform were provided to facilitate learning. Weak aversive stimuli
(600 lx light and fan) were applied to further motivate the animals
to escape from the circular platform [54].
The mice first underwent the adaptation period, in which they
were individually placed in the start chamber, released after 10 s
with light and fan turned on, gently guided towards the escape box,
and left there for the next 120 s. Afterwards, the spatial acquisition
trials were performed and continued for the next 4 days for four
times a day in 15-min intervals. The test compound or the vehicle,
followed by scopolamine, were administered daily before the first
trial of the day as described under In vivo evaluation e general in-
formation. In the trial, the test subject was released from the start
chamber after 10 s and allowed to explore the maze for 180 s. The
number of primary errors, i.e. the number of errors before reaching
the escape box, and latencies were measured during 180 s, after-
wards the mice which did not find the escape box were gently
guided to it, and were allowed to stay inside for 60 s. On the 5th day,
the reference short-term memory was assessed with a drug-off
trial. The escape box was now removed, and the opening closed
with a plastic plate. The mice were released from the start chamber
and left to explore the maze for 90 s, while primary errors and
latencies to reach the former escape box location were recorded
and analysed. The same procedure was repeated on day 12 to assess
the effect of test compound 1 and scopolamine on the long-term
memory [40,54,55].
Acquity UPLC® HSS C18 SB column (2.1 ꢁ 50 mm, 1.8
mostated at 40 ꢀC, with: injection volume,
L; sample,
0.1e0.2 mg/mL in MeOH; flow rate, 0.4 mL/min; detector , 220 and
mm) ther-
4
m
l
254 nm; mobile phase A: 0.1% TFA (v/v) in water; mobile phase B:
MeCN. Gradient: 0e2 min, 20% B; 2e5 min, 20%e90% B; 5e8 min,
90% B. Method II had the same conditions except the gradient:
0e2 min, 5% B; 2e9 min, 5%e90% B; 9e10 min, 90% B. The purities
of the tested compounds were established to be ꢃ 95%, as deter-
mined by UPLC, unless indicated otherwise.
General procedure 1 (GP1) e amide formation using CDI: Car-
boxylic acid (1.0 eq.) was azeotropically dried with toluene, dis-
solved in anhydrous THF under argon and 1,10-carbonyldiimidazole
(CDI) (1.1 eq.) was added. After stirring the reaction mixture at r.t.
for 1.5 h, amine (1.1 eq., neat) was added and stirring continued at
r.t. for 12 h. Volatile components were evaporated in vacuo and the
residue was purified by column chromatography to afford pure
product.
4.5.4. Locomotor activity assay
The
test
was
performed
using
activity
cages
(40 cm ꢁ 40 cm ꢁ 31 cm; with horizontal and vertical IR beam
emitters; Activity Cage 7441, Ugo Basile, Italy). The test compound 1
or the vehicle were administered i.p. and the mice placed in the
activity cages within a sound attenuated room for 60 min (the
habituation period). Afterwards, the number of light-beam in-
terruptions was counted during the next 60 min and analysed by
software [56].
4.5.5. Rotarod test
Before the rotarod test, the animals were trained for 3 consec-
utive days on the rotarod apparatus (May Commat RR0711, Turkey;
rod diameter, 2 cm) that was rotating at 18 rotations per minute
(rpm). 24 h after the last training session, the test compound 1 or
the vehicle were administered i.p., and the mice’s performance was
evaluated after 60 min on the rod revolving at 6, 18, and 24 rpm.
Motor impairment was defined as the inability to remain on the
rotarod apparatus for 60 s and was expressed as the mean time
spent on the rotarod.
General procedure 2 (GP2) e Boc-deprotection: Starting com-
pound was dissolved in DCM (approx. 4 mL/mmol reactant), cooled
on an icebath and then trifluoroacetic acid (TFA) (0.5e1 eq. by
volume) was slowly added. The icebath was then removed and the
resulting mixture was stirred at r.t. for 2 h or until total conversion
was achieved (monitored by TLC). The reaction mixture was made
alkaline with 1 M NaOH(aq) and the product was extracted into DCM
(2 x 20 mL), the combined organic phase was dried over anhydrous
sodium sulfate, filtered, and volatile components evaporated in
vacuo. The product was purified by column chromatography.
General procedure 3 (GP3) e reductive alkylation: Primary amine
(1.0 mmol) and an appropriate aldehyde (1.1 eq.) were dissolved in
DCM (10 mL) and stirred at r.t. for 10 min before sodium tri-
acetoxyborohydride (1.5 eq.) was added. The reaction mixture was
stirred for 6 h at r.t., then NaHCO3(aq., sat.) (5 mL) was added and the
resulting mixture extracted with DCM (2 x 20 mL). The combined
4.6. Chemistry
General information: The reagents and solvents were used as
received from commercial suppliers. Tetrahydrofuran (THF) was
distilled from sodium/benzophenone and stored under Ar over 4 Å
molecular sieves prior to use. After extraction organic phases were

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
13
organic phase was dried over anhydrous sodium sulfate, filtered
and volatile components evaporated in vacuo. The product was
isolated by column chromatography.
General procedure 4 (GP4) e catalytic hydrogenation: Starting
compound (5.0 mmol) was dissolved in MeOH (25 mL), flushed
with Ar, then 10% Pd/C (50 mg) was added and the resulting
mixture vigorously stirred at r.t. under H2 (balloon) for 12 h. The
catalyst was removed by filtration, washed with MeOH and volatile
components evaporated in vacuo to afford the product.
258 mg) in THF, isolated by column chromatography on silica (PE/
EtOAc ¼ 1:1), and immediately deprotected following GP2 to afford
4d. Yield: 361 mg (1.017 mmol, 52.1% over three steps) of yellowish
oil. ESI-HRMS: m/z ¼ 356.2697 (MHþ); C22H34N3O requires: m/
z ¼ 356.2696 (MHþ); nmax 3274, 2914, 2850, 1637, 1544, 1456, 1340,
r.t.
1222, 1012, 1010, 919, 738 cme1. [
(500 MHz, CDCl3):
a]
¼ ꢂ7.1 (c 0.35, DCM). 1H NMR
D
d 1.15e1.22 (m, 2H); 1.34e1.72 (m, 16H);
1.93e2.03 (m, 1H); 2.30e2.38 (m, 2H); 2.66 (dd, J ¼ 8.5; 14.3 Hz,
1H); 2.97 (dd, J ¼ 4.5; 14.2 Hz,1H); 3.07e3.16 (m,1H); 3.22e3.28 (m,
2H); 5.98 (t, J ¼ 5.6 Hz,1H, NH); 7.03 (d, J ¼ 1.9 Hz,1H); 7.10e7.15 (m,
1H); 7.18e7.23 (m, 1H); 7.36e7.40 (m, 1H); 7.60 (d, J ¼ 7.9 Hz, 1H);
General procedure 5 (GP5) e reduction with lithium aluminum
hydride: To a solution of the starting compound (amide, nitrile,
ester) (1.0 mmol) in anhydrous THF (1 mL), LiAlH4 solution (2.4 M in
THF, 3.0 eq. (1.25 mL) for amides and nitriles or 1.0 eq. (0.42 mL) for
esters) was added slowly with stirring under argon. The reaction
mixture was stirred at 50 ꢀC for 12 h and then, while being cooled
on an icebath, cautiously quenched with brine. The resulting grey
solids were suspended in Et2O (20 mL), sonicated in an ultrasonic
cleaning bath for 5 min, filtered, and washed with Et2O (2 x 10 mL).
The combined ethereal extracts were dried over sodium sulfate,
filtered, and volatile components evaporated in vacuo to afford the
product (amine or alcohol).
General procedure 6 (GP6) e RP-CC purification: Compounds
were purified by reversed-phase column chromatography (RP-CC)
(Isolera Biotage One Flash Chromatography system, SNAP Biotage
KP-C18-HS column, 30 g) using a gradient of 0.1% TFA in deionized
water and MeCN as eluent (gradient 0% MeCN for 2 column vol-
umes; 10e100% MeCN in 15 column volumes; 100% MeCN for 5
column volumes). After the RP-CC, fractions containing the product
were combined and organic volatiles were evaporated in vacuo. The
remaining aqueous solution was made alkaline (pH 10) with 1 M
NaOH(aq) and extracted with DCM (2 x 30 mL). The combined
organic phase was dried over anhydrous sodium sulfate, filtered,
and volatile components evaporated in vacuo to afford the pure
product.
8.51 (s, 1H, NH). 13C NMR (126 MHz, CDCl3):
d 26.43, 28.58, 33.41,
34.01, 34.50, 34.77, 37.01, 37.78, 37.93, 51.41, 53.56, 111.37, 112.99,
118.97, 119.39, 122.08, 122.81, 127.73, 136.53, 173.11.
Following GP3, 4 and 5 were prepared from 4d (345 mg,
0.972 mmol), n-butanal (1.1 eq., 110
mL), and sodium triacetox-
yborohydride (1.5 eq., 309 mg). The products were isolated by
column chromatography on silica (1. DCM/MeOH ¼ 50:1; 2. DCM/
MeOH ¼ 20:1) and additionally purified following GP6.
Compound 4 elutes first from the column. Yield: 79.1 mg
(0.169 mmol, 17.4%) of yellowish oil. ESI-HRMS: m/z ¼ 468.3949
(MHþ); C30H50N3O requires: m/z ¼ 468.3948 (MHþ); nmax 3276,
2921, 2852, 1642, 1551, 1457, 1353, 1106, 1010, 738 cme1
.
r.t.
[a
]
¼ ꢂ1.5 (c 0.52, DCM). Purity: UPLC (254 nm): tr ¼ 5.337 min,
D
94.8% total area. 1H NMR (500 MHz, CDCl3):
d
0.96 (t, J ¼ 7.2 Hz, 6H);
1.14e1.23 (m, 2H); 1.25e1.75 (m, 23H); 1.75e1.87 (m, 1H);
2.00e2.14 (m, 1H); 2.26e2.35 (m, 1H); 2.42e2.58 (m, 3H);
2.57e2.73 (m, 2H); 2.90 (br s, 1H); 3.07e3.14 (m, 1H); 3.14e3.22 (m,
2H); 5.83 (br s, 1H, NH); 7.00 (d, J ¼ 2.2 Hz, 1H, 1H of Arl); 7.12 (t,
J ¼ 7.4 Hz, 1H, 1H of Arl); 7.20 (t, J ¼ 7.5 Hz, 1H, 1H of Arl); 7.38 (d,
J ¼ 8.1 Hz, 1H, 1H of Arl); 7.58 (d, J ¼ 7.9 Hz, 1H, 1H of Arl); 8.26 (br s,
1H, NH). 13C NMR (126 MHz, CDCl3):
d 14.33, 20.85, 24.34, 26.44,
27.27, 28.62, 31.84, 34.52, 34.76, 37.06, 37.82, 37.85, 49.94, 60.59,
111.43, 114.36, 118.55, 119.21, 121.91, 122.47, 127.67, 136.49, 173.73.
Compound 5 elutes second from the column. Yield: 95.8 mg
(0.233 mmol, 24%) of yellowish oil. ESI-HRMS: m/z ¼ 412.3321
(MHþ); C26H42N3O requires: m/z ¼ 412.3322 (MHþ); nmax 3272,
4.6.1. (R)-N-(2-Cycloheptylethyl)-4-(dibutylamino)-5-(1H-indol-3-
yl)pentanamide (4) and (R)-4-(Butylamino)-N-(2-
cycloheptylethyl)-5-(1H-indol-3-yl)pentanamide (5)
2921, 2853, 1644, 1528, 1456, 1353, 1232, 1175, 1093, 1009,
r.t.
tert-Butyl (S)-(1-(1H-indol-3-yl)-3-oxopropan-2-yl)carbamate
[35] (2594 mg, 9.0 mmol) and benzyl(triphenylphosphoranylidene)
acetate (4618 mg, 1.25 eq.) in anhydrous DCM (50 mL) were stirred
at r.t. for 24 h. Volatile components were evaporated in vacuo and
4a was isolated by column chromatography on silica (1. PE/
EtOAc ¼ 3:1, 2. PE/EtOAc ¼ 2:1). Yield: 3708 mg (8.83 mmol, 98%) of
beige solid. mp ¼ 132e134 ꢀC; ESI-HRMS: m/z ¼ 421.2116 (MHþ);
737 cme1. [
a]
¼ ꢂ1.44 (c 1.2, DCM). Purity: UPLC (254 nm):
D
tr ¼ 4.860 min, 97.5% total area. 1H NMR (500 MHz, CDCl3):
d 0.87 (t,
J ¼ 7.3 Hz, 3H); 1.14e1.24 (m, 2H); 1.24e1.33 (m, 2H); 1.36e1.72 (m,
16H); 1.72e1.81 (m, 1H); 1.82e1.91 (m, 1H); 2.24e2.40 (m, 2H);
2.59e2.72 (m, 2H), 2.82e2.99 (m, 3H); 3.17e3.26 (m, 2H); 6.61 (t,
J ¼ 5.5 Hz, 1H, NH); 6.98 (d, J ¼ 2.0 Hz, 1H, 1H of Arl); 7.11 (t,
J ¼ 7.5 Hz, 1H, 1H of Arl); 7.19 (t, J ¼ 7.5 Hz, 1H, 1H of Arl); 7.38 (d,
J ¼ 8.0 Hz, 1H, 1H of Arl); 7.59 (d, J ¼ 7.9 Hz, 1H, 1H of Arl); 9.00 (s,
C
25H29N2O4 requires: m/z ¼ 421.2112 (MHþ); nmax 3348, 3324, 2978,
1707, 1693, 1656, 1544, 1457, 1312, 1288, 1274, 1171, 1063, 904, 867,
1H, NH). 13C NMR (126 MHz, CDCl3):
d 14.01, 20.49, 26.36, 28.52,
735 cme1. [
a
]
¼ þ1.6 (c 1.1, DCM). Two isomers in a ratio of 88:12.
29.70, 29.83, 32.27, 33.49, 34.43, 34.44, 36.97, 37.69, 37.88, 46.72,
57.63, 111.48, 112.16, 118.66, 119.16, 121.86, 122.93, 127.63, 136.53,
173.57.
r.t.
D
1H NMR (400 MHz, CDCl3) for the major isomer:
d
1.39 (s, 9H);
2.88e3.10 (m, 2H); 4.41e4.90 (m, 2H); 5.10e5.18 (m, 2H); 5.93 (d,
J ¼ 15.7 Hz,1H); 6.92e7.05 (m, 2H); 7.06e7.12 (m,1H); 7.13e7.21 (m,
1H); 7.28e7.38 (m, 6H); 7.56 (d, J ¼ 7.9 Hz, 1H); 8.25 (s, 1H, NH). 1H
4.6.2. N1–(3-(1H-Indol-3-yl)propyl)-N1-butyl-N4-(2-
cycloheptylethyl)butane-1,4-diamine (7)
NMR (400 MHz, CDCl3) for the minor isomer:
d
1.36 (s, 9H);
3.10e3.20 (m, 1H); 5.88 (dd, J ¼ 1.0; 11.7 Hz, 1H); 7.71 (d, J ¼ 7.8 Hz,
N-Butyl-3-(1H-indol-3-yl)propanamide [57] was prepared
following GP1 from 3-(1H-indol-3-yl)propanoic acid (1892 mg,
10 mmol), CDI (1.1 eq., 1839 mg), and butan-1-amine (1.1 eq.,
1.087 mL) in THF, and isolated by column chromatography on silica
(PE/EtOAc ¼ 1:2). Yield: 2200 mg (9.00 mmol, 90%) of white solid.
1H). 13C NMR (101 MHz, CDCl3) for the major isomer:
d
28.42, 30.59,
51.89, 66.36, 79.91, 110.46, 111.33, 118.92, 119.69, 120.63, 122.25,
123.04, 127.65, 128.27, 128.30, 128.64, 135.99, 136.32, 149.31, 155.27,
166.18. 13C NMR (101 MHz, CDCl3) for the minor isomer:
d 30.08,
49.73, 111.23, 119.44, 122.21, 123.24, 127.79, 128.41, 136.46, 151.95,
165.67.
1H NMR (500 MHz, CDCl3):
d
0.88 (t, J ¼ 7.3 Hz, 3H, Me); 1.18e1.28
(m, 2H); 1.32e1.41 (m, 2H); 2.58 (t, J ¼ 7.4 Hz, 2H); 3.15 (t, J ¼ 7.4 Hz,
2H); 3.18e3.24 (m, 2H); 5.31 (br s, 1H, NH); 7.05 (s, 1H, 1H of Arl);
7.13e7.17 (m, 1H, 1H of Arl); 7.19e7.25 (m, 1H, 1H of Arl); 7.39 (d,
J ¼ 8.1 Hz, 1H, 1H of Arl); 7.63 (d, J ¼ 7.8 Hz, 1H, 1H of Arl); 8.02 (s,
Benzyl (S,E)-4-((tert-butoxycarbonyl)amino)-5-(1H-indol-3-yl)
pent-2-enoate (819 mg, 1.95 mmol) was reduced following GP4 to
4b, which was immediately used further without purification.
4c was prepared following GP1 from 4b (553 mg, 1.65 mmol),
CDI (1.1 eq., 294 mg), and 2-cycloheptylethan-1-amine (1.1 eq.,
1H, NH). 13C NMR (126 MHz, CDCl3):
d 13.87, 20.09, 21.56, 31.73,
37.66, 39.34, 111.32, 115.22, 118.85, 119.50, 121.85, 122.22, 127.24,

14
A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
136.46, 172.69.
111.25, 111.43, 114.72, 115.56, 118.63, 118.77, 119.05, 119.21, 121.45,
121.65, 121.81, 121.98, 127.28, 127.47, 136.49, 136.53, 171.69, 171.70,
172.65, 172.76.
6 (125 mg, 0.276 mmol) was reduced following GP5 to afford 7
that was purified by column chromatography on silica (1.
N-Butyl-3-(1H-indol-3-yl)propanamide (2200 mg, 9.0 mmol)
was reduced following GP5 to afford N-(3-(1H-indol-3-yl)propyl)
butan-1-amine. Yield: 1660 mg (7.20 mmol, 80%) of yelþlowish solid.
mp ¼ 96.4e96.7 ꢀC. ESI-HRMS: m/z ¼ 231.1860 (MH ); C15H23N2
requires: m/z ¼ 231.1856 (MHþ); nmax 3056, 2956, 2925, 2869, 2829,
1620, 1577, 1506, 1451, 1373, 1354, 1337, 1241, 1219, 1106, 1083, 1041,
DCM:MeOH
¼
20:1;
2.
DCM:MeOH
¼
10:1;
3.
DCM:MeOH:Et3N ¼ 5:2:1). Yield: 70 mg (0.1644 mmol, 59.6%) of
orange oil. ESI-HRMS: m/z ¼ 213.6954 ((Mþ2H)2þ); C28H49N3 re-
quires: m/z ¼ 213.6958 ((Mþ2H)2þ); nmax 3229, 2920, 2852, 2804,
1456, 1354, 1301, 1230, 1095, 1074, 1009, 803, 736 cmꢂ1. Purity:
UPLC (220 nm): tr ¼ 4.247 min, 99.5% total area. 1H NMR (500 MHz,
1007, 949, 889, 796, 735 cmꢂ1. 1H NMR (500 MHz, CDCl3):
d 0.92 (t,
J ¼ 7.3 Hz, 3H, Me); 1.29e1.38 (m, 2H); 1.43e1.51 (m, 2H); 1.81 (br s,
1H, NH); 1.94 (p, J ¼ 7.5 Hz, 2H); 2.59e2.64 (m, 2H); 2.70e2.74 (m,
2H); 2.81 (t, J ¼ 7.5 Hz, 2H); 6.96 (s, 1H, 1H of Arl); 7.09e7.13 (m, 1H,
1H of Arl); 7.16e7.21 (m, 1H, 1H of Arl); 7.31e7.36 (m, 1H, 1H of Arl);
7.59e7.63 (m, 1H, 1H of Arl); 8.33 (s, 1H, NH). 13C NMR (126 MHz,
CDCl3):
d
0.92 (t, J ¼ 7.3 Hz, 3H, Me); 1.12e1.74 (m, 24H); 1.83e1.94
(m, 2H); 2.40e2.50 (m, 4H); 2.54e2.59 (m, 2H); 2.60e2.68 (m, 4H);
2.77 (t, J ¼ 7.6 Hz, 2H); 6.98 (s, 1H, 1H of Arl); 7.11 (t, J ¼ 7.4 Hz, 1H,
1H of Arl); 7.19 (t, J ¼ 7.6 Hz, 1H, 1H of Arl); 7.36 (d, J ¼ 8.0 Hz, 1H, 1H
of Arl); 7.62 (d, J ¼ 7.9 Hz, 1H, 1H of Arl); 8.75 (s, 1H, NH). 13C NMR
CDCl3):
d 14.15, 20.64, 23.08, 30.50, 32.33, 49.85, 50.01, 111.19,
116.30, 118.98, 119.13, 121.32, 121.90, 127.60, 136.47.
N-(3-(1H-Indol-3-yl)propyl)butan-1-amine (231 mg, 1.0 mmol)
and succinic anhydride (300 mg, 3.0 mmol) in THF (3 mL) and
pyridine (1 mL) were irradiated in a microwave reactor at 60 ꢀC for
3 h. The reaction mixture was dissolved in EtOAc (60 mL) and
washed with 1 M NaHSO4(aq) (3 x 10 mL). Volatile components of
the organic phase were evaporated in vacuo, the residue was dis-
solved in THF (10 mL) and 2 M LiOH(aq) (3 mL). The reaction mixture
was stirred at r.t. for 24 h. Volatile components were evaporated in
vacuo, the residue was acidified by the addition of 1 M NaHSO4(aq)
(10 mL), and the product was extracted with EtOAc (60 mL). The
combined organic phase was dried over sodium sulfate, filtered and
volatile components evaporated in vacuo to afford 4-((3-(1H-indol-
3-yl)propyl)(butyl)amino)-4-oxobutanoic acid. Yield: 300 mg
(0.901 mmol, 91%) of beige semisolid. ESI-HRMS: m/z ¼ 331.2016
(MHþ); C19H27N2O3 requires: m/z ¼ 331.2016 (MHþ); nmax 3293,
2956, 2929, 2871,1712,1603,1457,1419,1377,1339,1306, 1197,1173,
1009, 908, 891, 801, 739, 635 cmꢂ1. Two conformers in a 1:1 ratio.
(126 MHz, CDCl3):
d 14.19, 20.86, 23.18, 24.99, 26.44, 27.22, 27.91,
28.55, 28.99, 34.69, 37.45, 37.96, 47.96, 49.87, 53.76, 53.82, 54.03,
111.22, 116.20, 118.87, 118.92, 121.40, 121.69, 127.58, 136.48.
4.6.3. (S)-2-(Butylamino)-N-(2-cycloheptylethyl)-4-
methylpentanamide (8)
tert-Butyl
oxopentan-2-yl)carbamate was prepared following GP1 from
(tert-butoxycarbonyl)- -leucine (347 mg, 1.61 mmol), CDI (1.1 eq.,
(S)-(1-((2-cycloheptylethyl)amino)-4-methyl-1-
L
288 mg), and 2-cycloheptylethan-1-amine (1.1 eq., 252 mg) in THF,
isolated by column chromatography on silica (PE/EtOAc ¼ 10:1),
and immediately deprotected following GP2 to afford (S)-2-amino-
N-(2-cycloheptylethyl)-4-methylpentanamide, which was imme-
diately used further without purification.
Following GP3, (S)-2-(butylamino)-N-(2-cycloheptylethyl)-4-
methylpentanamide was prepared from (S)-2-amino-N-(2-
cycloheptylethyl)-4-methylpentanamide (199 mg, 0.782 mmol),
1H NMR (500 MHz, CDCl3) for both conformers:
d 0.85e0.94 (m, 3H,
Me); 1.21e1.32 (m, 2H); 1.42e1.56 (m, 2H); 1.93e2.05 (m, 2H);
2.41e2.47 (m, 1H); 2.53e2.58 (m, 1H); 2.62e2.72 (m, 2H);
2.72e2.82 (m, 2H); 3.18e3.24 (m, 1H); 3.25e3.34 (m, 2H);
3.40e3.46 (m, 1H); 6.99 and 7.01 (2 x s, 1H, 1H of Arl); 7.07e7.14 (m,
1H, 1H of Arl); 7.14e7.22 (m, 1H, 1H of Arl); 7.33 and 7.37 (2 x d,
J ¼ 8.1 Hz, 1H, 1H of Arl); 7.53e7.60 (m, 1H, 1H of Arl); 8.18 and 8.29
(2 x s, 1H, NH). 13C NMR (126 MHz, CDCl3) for both conformers:
n-butanal (1.1 eq., 79 mL) and sodium triacetoxyborohydride (1.5
eq., 249 mg). 8 was isolated by column chromatography on silica (1.
PE/EtOAc ¼ 3:1; PE/EtOAc ¼ 1:1). Yield: 151 mg (0.486 mmol,
30.2%) of beige oil. ESI-HRMS: m/z ¼ 311.3056 (MHþ); C19H39N2O
requires: m/z ¼ 311.3057 (MHþ); nmax 3302, 2920, 2853, 1641, 1526,
r.t.
1460, 1366, 1223, 1170, 1137, 734 cme1. [
a
]
D
¼ ꢂ22.8 (c 0.19, DCM).
Purity: UPLC (220 nm): tr ¼ 4.497 min, 95.1% total area. 1H NMR
d
13.86, 13.93, 20.18, 20.30, 22.40, 22.63, 27.79, 28.05, 28.15, 29.02,
(300 MHz, CDCl3): d 0.83e1.01 (m, 9H); 1.11e1.25 (m, 2H); 1.26e1.76
29.87, 30.27, 30.98, 46.34, 46.52, 47.67, 48.14, 111.29, 111.53, 114.66,
115.44, 118.65, 118.80, 119.18, 119.46, 121.69, 121.92, 122.24, 127.27,
127.52, 136.50, 136.58, 172.16, 172.25, 176.12.
(m, 21H); 2.41e2.62 (m, 2H); 3.03 (dd, J ¼ 9.2, 4.4 Hz,1H); 3.12e3.35
(m, 2H); 7.22 (t, J ¼ 6.1 Hz, 1H, NH). 13C NMR (75 MHz, CDCl3):
d
14.06, 20.52, 22.03, 23.40, 25.34, 26.52, 28.58, 32.60, 34.54, 36.97,
N1-(3-(1H-Indol-3-yl)propyl)-N1-butyl-N4-(2-cycloheptylethyl)
succinimide (6) was prepared following GP1 from 4-((3-(1H-indol-
37.13, 37.87, 43.25, 48.89, 61.87, 175.00.
3-yl)propyl)(butyl)amino)-4-oxobutanoic
acid
(165
mg,
4.6.4. (S)-2-(Butylamino)-N-(2-cycloheptylethyl)-3-
0.55 mmol), CDI (1.1 eq., 98 mg), and 2-cycloheptylethan-1-amine
(1.1 eq., 85 mg) in THF, and isolated by column chromatography
on silica (EtOAc). Yield: 140 mg (0.309 mmol, 61%) of colourless oil.
Two conformers in a 1:1 ratio. ESI-HRMS: m/z ¼ 454.3425 (MHþ);
phenylpropanamide (9)
Butyl- -phenylalanine [34] (2210 mg, 10 mmol) was dissolved in
L
a mixture of THF (10 mL) and 1 M NaOH(aq) (10 mL), then potassium
carbonate (1.0 eq., 1380 mg) and di-tert-butyl dicarbonate (1.5 eq.,
3270 mg) were added and the reaction mixture was stirred at r.t. for
12 h. THF was then removed in vacuo, the residue was acidified with
dilute hydrochloric acid (pH 3) and extracted with EtOAc (3 x
20 mL). The combined organic phase was dried over anhydrous
sodium sulfate, filtered, and volatile components evaporated in
vacuo. N-(tert-butoxycarbonyl)-N-butyl-L-phenylalanine 9a was
isolated by column chromatography on silica (1. PE/EtOAc ¼ 3:1; 2.
PE/EtOAc ¼ 1:1). Yield: 1790 mg (5.576 mmol, 55.8%) of beige oil.
ESI-HRMS: m/z ¼ 222.1487 ((M-Boc)Hþ); C13H20NO2 requires: m/
z ¼ 222.1489 ((M-Boc)Hþ). nmax 2961, 1693, 1455, 1418, 1367, 1295,
1251, 1165, 1112, 1079, 1031, 966, 861, 751, 699, 664 cmꢂ1. Two
conformers in a 1:1 ratio (CDCl3). 1H NMR (500 MHz, CDCl3) for
C
28H43N3O2 requires: m/z ¼ 454.3428 (MHþ); nmax 3293, 2920,
2853, 1620, 1551, 1457, 1430, 1354, 1253, 1223, 1169, 1101, 1009,
738 cmꢂ1. Purity: UPLC (220 nm): tr ¼ 5.403 min, 98.7% total area.
1H NMR (500 MHz, CDCl3) for both conformers:
d 0.85e0.93 (m,
3H); 1.09e1.19 (m, 2H); 1.21e1.29 (m, 1H); 1.31e1.70 (m, 16H);
1.90e2.02 (m, 2H); 2.42e2.47 (m, 1H); 2.52 (t, J ¼ 6.3 Hz, 2H); 2.65
(t, J ¼ 6.4 Hz, 1H); 2.74 (q, J ¼ 7.1 Hz, 2H); 3.16e3.23 (m, 3H);
3.25e3.33 (m, 2H); 3.38e3.44 (m, 1H); 6.30 and 6.36 (2 x br s, 1H,
NH); 6.97 (dd, J ¼ 2.1; 13.2 Hz, 1H, 1H of Arl); 7.05e7.13 (m, 1H, 1H of
Arl); 7.14e7.19 (m,1H,1H of Arl); 7.31e7.37 (m,1H,1H of Arl); 7.56 (t,
J ¼ 7.2 Hz, 1H, 1H of Arl); 8.44 and 8.55 (2 x s, 1H, NH). 13C NMR
(126 MHz, CDCl3) for both conformers:
d 13.85, 13.94, 20.15, 20.30,
22.41, 22.74, 26.38, 28.06, 28.56, 29.07, 29.10, 29.95, 31.05, 31.89,
34.42, 34.44, 36.90, 37.63, 37.65, 37.93, 45.96, 46.30, 47.48, 47.88,
both conformers:
(s, 9H, tBu); 2.41e2.63 (m, 1H); 3.03e3.41 (m, 3H); 3.92e4.14 (m,
d
0.79 (t, J ¼ 7.3 Hz, 3H); 1.04e1.35 (m, 4H); 1.47

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
15
1H); 7.13e7.34 (m, 5H, Ph); 9.02 (br s, 1H, NH). 13C NMR (126 MHz,
CDCl3) for two conformers: 13.86, 13.92, 19.89, 20.06, 27.53, 28.50,
30.20, 30.66, 35.15, 36.33, 49.36, 49.58, 62.83, 63.85, 81.02, 126.78,
128.59, 128.67, 129.38, 129.45, 137.91, 138.16, 154.67, 156.34, 175.75,
177.33.
32.57, 32.61, 34.34, 34.48, 34.51, 37.00, 37.08, 37.67, 37.74, 37.94,
38.13, 46.70, 47.14, 48.02, 48.11, 59.84, 60.31, 60.57, 62.33, 111.17,
111.21, 111.36, 111.53, 118.43, 118.99, 119.45, 119.55, 122.08, 122.26,
123.09, 123.21, 127.22, 127.45, 136.23, 170.74, 171.65, 175.43, 175.46.
d
tert-Butyl
(S)-butyl(1-((2-cycloheptylethyl)amino)-1-oxo-3-
4.6.6. (2S)-2-(Butylamino)-3-(1H-indol-3-yl)-1-(2-
phenylpropan-2-yl)carbamate was prepared following GP1 from
9a (470 mg, 1.47 mmol), CDI (1.1 eq., 262 mg), and 2-
cycloheptylethan-1-amine (1.1 eq., 228 mg) in THF, isolated by
column chromatography on silica (PE/EtOAc ¼ 3:1), and immedi-
ately deprotected following GP2. 9 was purified by column chro-
matography on silica (1. PE/EtOAc ¼ 2:1; 2. PE/EtOAc ¼ 1:2). Yield:
305 mg (0.886 mmol, 60.3% over two steps) of colourless oil. ESI-
phenylpyrrolidin-1-yl)propan-1-one (11)
tert-Butyl ((2S)-3-(1H-indol-3-yl)-1-oxo-1-(2-phenylpyrrolidin-
1-yl)propan-2-yl)(butyl)carbamate was prepared following GP1
from
Na-(tert-butoxycarbonyl)-Na-butyl-L-tryptophan
[31]
(213 mg, 0.591 mmol), CDI (1.1 eq., 105 mg), and 2-
phenylpyrrolidine (1.1 eq., 96 mg) in THF, isolated by column
chromatography on silica (PE/EtOAc ¼ 5:1), and immediately
deprotected following GP2. 11 was purified by column chroma-
tography on silica (1. PE/EtOAc ¼ 1:1; 2. EtOAc). Yield: 45.8 mg
(0.118 mmol, 20.0% over two steps) of beige semisolid. ESI-HRMS:
m/z ¼ 390.2541 (MHþ); C25H32N3O requires: m/z ¼ 390.254
HRMS: m/z
¼
345.2898 (MHþ); C22H37N2O requires: m/
z ¼ 345.2900 (MHþ). nmax 3308, 2919, 2852, 1646, 1522, 1496, 1454,
r.t.
1376, 1242, 1125, 1080, 1031, 744, 698 cme1. [
a
]
¼ e 69.3 (c 0.153,
D
DCM). Purity: UPLC (220 nm): tr ¼ 4.587 min, 97.5% total area. 1H
NMR (500 MHz, CDCl3):
d
0.80 (t, J ¼ 7.3 Hz, 3H); 1.12e1.22 (m, 4H);
(MHþ). nmax 3271, 2956, 2926, 2871, 1623, 1423, 1340, 1104, 908,
r.t.
1.24e1.31 (m, 2H); 1.34e1.72 (m, 14H); 2.33e2.47 (m, 2H); 2.64 (dd,
J ¼ 9.5; 13.7 Hz, 1H); 3.16e3.34 (m, 4H); 7.18e7.33 (m, 6H, NH, Ph).
730, 699, 627 cme1. [
a]
¼ þ66.8 (c 0.25, acetone). Purity: UPLC
D
(254 nm): tr ¼ 4.280 min, 99.2% total area. A complex mixture of
13C NMR (126 MHz, CDCl3):
d
13.93, 20.25, 26.46, 28.57, 32.14, 34.47,
conformers (three major conformers). 1H NMR (400 MHz, CDCl3):
34.49, 36.93, 37.21, 37.76, 39.57, 48.64, 64.21, 126.89, 128.78, 129.14,
137.90, 173.75.
d 0.71e0.93 (m, 3H); 0.99e1.64 (m, 6H), 1.77e2.09 (m, 2H),
2.16e2.63 (m, 2H); 2.73e3.23 (m, 2H); 3.31e3.85 (m, 3H);
4.93e5.26 (m, 1H); 6.50e7.72 (m, 11H); 8.17 and 8.34 and 8.50 and
4.6.5. (S)-1-(N-Butyl-L-tryptophanyl)-N-[2-(cycloheptyl)ethyl]
pyrrolidine-2-carboxamide (10)
8.53 (s, 1H, NH). 13C NMR (126 MHz, CDCl3):
d 13.92, 14.00, 14.05,
20.41, 20.47, 20.56, 21.36, 21.39, 23.27, 28.45, 30.14, 31.00, 32.25,
32.38, 32.55, 33.68, 35.32, 36.31, 46.99, 47.12, 47.22, 47.41, 47.92,
59.88, 60.23, 61.12, 61.23, 61.38, 61.72, 110.97, 111.28, 111.36, 111.81,
111.89, 118.73, 118.93, 118.98, 119.37, 119.50, 121.51, 121.97, 122.18,
122.90, 123.15, 123.45, 125.45, 125.53, 125.89, 126.66, 127.24, 127.35,
127.44, 127.66, 127.78, 128.33, 128.52, 128.82, 136.19, 136.25, 136.31,
143.30, 143.47, 143.77, 174.14, 174.55, 175.23.
tert-Butyl (S)-2-((2-cycloheptylethyl)carbamoyl)pyrrolidine-1-
carboxylate was prepared following GP1 from (tert-butox-
ycarbonyl)-L-proline (215 mg, 1.0 mmol), CDI (1.1 eq., 178 mg), and
2-cycloheptylethan-1-amine (1.1 eq., 156 mg) in THF, isolated by
column chromatography on silica (PE/EtOAc ¼ 3:1), and immedi-
ately deprotected following GP2 to afford (S)-N-(2-
cycloheptylethyl)pyrrolidine-2-carboxamide. Yield: 209 mg
(0.877 mmol, 87.7% over two steps) of beige semisolid. ESI-HRMS:
m/z ¼ 239.212 (MHþ); C14H27N2O requires: m/z ¼ 239.2118
4.6.7. (S)-1-(4-Benzylpiperidin-1-yl)-2-(dibutylamino)-3-(1H-
indol-3-yl)propan-1-one (12)
(MHþ). nmax 3307, 2917, 2852, 1650, 1521, 1445, 1247, 1173, 1103,
tert-Butyl (S)-(1-(4-benzylpiperidin-1-yl)-3-(1H-indol-3-yl)-1-
oxopropan-2-yl)carbamate was prepared following GP1 from
(tert-butoxycarbonyl)-L-tryptophan (304 mg, 1.0 mmol), CDI (1.1
eq., 178 mg), and 4-benzylpiperidine (1.1 eq., 193 mg) in THF, iso-
lated by column chromatography on silica (PE/EtOAc ¼ 2:1), and
immediately deprotected following GP2 to afford (S)-2-amino-1-(4-
benzylpiperidin-1-yl)-3-(1H-indol-3-yl)propan-1-one, which was
immediately used further without purification.
r.t.
909, 826, 657 cme1. [
(300 MHz, CDCl3):
a
]
¼ ꢂ34.5 (c 0.57, MeOH). 1H NMR
D
d 1.12e1.26 (m, 2H); 1.32e1.76 (m, 14H);
1.84e1.98 (m, 1H); 2.04e2.22 (m, 3H); 2.84e2.94 (m, 1H);
2.97e3.07 (m, 1H); 3.23 (q, J ¼ 6.4 Hz, 2H); 3.74 (dd, J ¼ 5.3; 9.1 Hz,
1H); 7.55 (br s, 1H, NH).
tert-Butyl
N-butyl-((S)-1-((S)-2-((2-cycloheptylethyl)carba-
moyl)pyrrolidin-1-yl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carba-
mate was prepared following GP1 from Na-(tert-butoxycarbonyl)-
Na-butyl-L-tryptophan [31] (300 mg, 0.832 mmol), CDI (1.1 eq.,
Following GP3, 12 was prepared from (S)-2-amino-1-(4-
benzylpiperidin-1-yl)-3-(1H-indol-3-yl)propan-1-one (324 mg,
148
mg),
and
(S)eN-(2-cycloheptylethyl)pyrrolidine-2-
0.898 mmol), n-butanal (1.1 eq., 90 mL) and sodium triacetoxybor-
carboxamide (1.1 eq., 198 mg) in THF, isolated by column chroma-
tography on silica (1. PE/EtOAc ¼ 3:1; 2. PE/EtOAc ¼ 1:1), and
immediately deprotected following GP2. 10 was purified by column
ohydride (1.5 eq., 286 mg), and isolated by column chromatography
on silica (1. PE/EtOAc ¼ 2:1; PE/EtOAc ¼ 1:1). Yield: 71 mg
(0.150 mmol, 16.7%) of beige oil. ESI-HRMS: m/z ¼ 474.3477 (MHþ);
chromatography on silica (1. DCM/MeOH
¼
50:1; 2. DCM/
C
31H44N3O requires: m/z ¼ 474.3479 (MHþ); nmax 3273, 2922, 2852,
MeOH ¼ 20:1). Yield: 124 mg (0.285 mmol, 31.0%) of beige solid.
1614, 1454, 1356, 1262, 1230, 1164, 1095, 1055, 1010, 965, 808, 737,
r.t.
mp ¼ 186e187 ꢀC. ESI-HRMS: m/z ¼ 481.3535 (MHþ); C29H45N4O2
698 cme1. [
a]
¼ þ66.8 (c 0.25, acetone). Purity: UPLC (254 nm):
D
requires: m/z ¼ 481.3537 (MHþ). nmax 3253, 2921, 2852, 1666, 1615,
tr ¼ 4.783 min, 98.7% total area. Two conformers in approximately
1550,1441,1343,1251,1098,1009, 740, 682, 620 cme1. [
a]
¼ ꢂ24.6
1:1 ratio. 1H NMR (300 MHz, CDCl3) for both major conformers:
r.t.
D
(c 0.38, acetone). Purity: UPLC (254 nm): tr ¼ 4.587 min, 96.9% total
d
ꢂ0.11e0.11 (m, 0.5H); 0.63e0.80 (m, 0.5H); 0.87e1.01 (m, 6H);
area. Two conformers in a ratio of 62:38 in CDCl3. 1H NMR
1.04e1.74 (m, 13H); 2.12e2.55 (m, 4H); 2.58e2.82 (m, 4H);
2.96e3.09 (m,1H); 3.37e3.51 (m,1H); 3.71 (d, J ¼ 13.9 Hz, 0.5H); 3.90
(dd, J ¼ 2.7; 10.6 Hz, 1H); 4.01 (dd, J ¼ 3.0; 10.7 Hz, 0.5H); 4.51e4.67
(m, 1H); 6.93e7.04 (m, 2H); 7.06e7.41 (m, 7H); 7.57e7.69 (m, 1H);
8.27 (s, 1H, NH). 13C NMR (75 MHz, CDCl3) for both major con-
(500 MHz, CDCl3) for both conformers:
d 0.83e0.95 (m, 3H);
1.09e1.21 (m, 2H); 1.26e1.80 (m, 19H); 2.02e2.37 (m, 2H);
2.45e2.58 (m, 2H); 2.78e2.85 (m, 1H); 2.97e3.32 (m, 5H);
3.38e3.45 (m, 1H); 3.78 (dd, J ¼ 5.5; 8.9 Hz, 1H); 4.50e4.62 (m, 1H);
5.88 and 6.80 (2 x t, J ¼ 5.7 Hz, 1H, NH); 6.95 (d, J ¼ 2.1 Hz, 1H);
7.03e7.20 (m, 2H); 7.32 (dd, J ¼ 2.7; 8.2 Hz, 1H); 7.52 and 7.64 (2 x d,
J ¼ 7.9 Hz, 1H); 8.63 and 8.88 (2 x s, 1H, NH). 13C NMR (126 MHz,
formers: d 14.23, 14.31, 20.19, 20.65, 20.81, 22.87, 24.04, 31.31, 31.48,
31.88, 31.98, 32.03, 32.56, 38.02, 38.25, 42.37, 42.87, 43.29, 45.40,
46.10, 51.09, 51.29, 61.08, 62.45, 111.29, 113.17, 113.38, 118.33, 118.66,
119.08, 119.33, 121.57, 121.74, 123.46, 123.74, 125.95, 126.05, 127.77,
127.84, 128.25, 128.32, 129.16, 129.19, 136.20, 136.32, 140.21, 170.66.
CDCl3) for both conformers:
d 14.08, 14.12, 20.53, 20.60, 22.07,
25.02, 26.34, 26.40, 27.25, 28.51, 28.53, 28.57, 29.75, 30.11, 30.64,

16
A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
4.6.8. (2S)-2-(Butylamino)-3-(1H-indol-3-yl)-N-(2-(3-
phenylcyclohexyl)ethyl)propanamide (13)
4.6.9. (2S)eN-(2-(1-Benzylpiperidin-3-yl)ethyl)-2-(butylamino)-3-
(1H-indol-3-yl)propanamide (14)
Sodium hydride (60 wt% suspension in mineral oil) (1.2 eq.,
480 mg) was added portionwise to diethyl cyanomethylphospho-
nate (1.3 eq., 2100 mL) in anhydrous THF (40 mL) under argon at r.t.
and stirred for 15 min, before 3-phenylcyclohexan-1-one [58]
(1740 mg, 1.0 eq., 10 mmol) was added and the reaction mixture
was stirred at r.t. for 12 h. Volatile components were evaporated in
vacuo, residue dissolved in water (30 mL), and extracted with Et2O
(2 x 20 mL). The organic phase was dried over anhydrous sodium
sulfate, filtered, and volatile components evaporated in vacuo. 2-(3-
Phenylcyclohexylidene)acetonitrile was isolated by column chro-
Sodium hydride (60 wt% suspension in mineral oil) (1.2 eq.,
610 mg) was added portionwise to diethyl cyanomethylphospho-
nate (1.3 eq., 2.67 mL) in anhydrous THF (80 mL) under argon at r.t.
and stirred for 15 min, before tert-butyl 3-oxopiperidine-1-
carboxylate (2541 mg, 1.0 eq., 12.7 mmol) was added and the re-
action mixture was stirred at r.t. for 12 h. Water (50 mL) was added,
volatile components were evaporated in vacuo, residue extracted
with Et2O (2 x 30 mL). The organic phase was dried over anhydrous
sodium sulfate, filtered, and volatile components evaporated in
vacuo. tert-Butyl 3-(cyanomethylene)piperidine-1-carboxylate was
isolated by column chromatography on silica (1. PE/EtOAc ¼ 3:1; 2.
matography on silica (PE/EtOAc
¼
20:1). Yield: 1688 mg
(8.56 mmol, 85.6%) of beige oil. ESI-HRMS: m/z ¼ 198.1274 (MHþ);
PE/EtOAc ¼ 1:1) as
a mixture of isomers. Yield: 2054 mg
C
14H16N requires: m/z ¼ 198.1277 (MHþ). nmax 3028, 2936, 2859,
(9.25 mmol, 72.8%) of beige solid, which was immediately used
further. ESI-HRMS: m/z ¼ 223.1439 (MHþ); C12H19N2O2 requires: m/
z ¼ 223.1441 (MHþ).
2215, 1630, 1494, 1448, 1324, 1030, 808, 754, 698 cmꢂ1. Mixture of
diastereomers in a ratio of 1:1. 1H NMR (500 MHz, CDCl3) for both
diastereomers:
d
1.48e1.62 (m, 1H); 1.67 (qd, J ¼ 2.9; 12.5 Hz, 1H);
2-(Piperidin-3-yl)acetonitrile [59] was prepared from tert-butyl
1.96e2.40 (m, 4H); 2.42e2.59 (m, 1H); 2.63e2.76 (m, 1H);
2.97e3.16 (m, 1H), 5.13 and 5.15 (2 x s, 1H); 7.17e7.28 (m, 3H, 3H of
Ph); 7.30e7.37 (m, 2H, 2H of Ph). 13C NMR (126 MHz, CDCl3) for both
3-(cyanomethylene)piperidine-1-carboxylate
9.20 mmol) following GP4 and GP2. Yield: 1085 mg (8.75 mmol,
95.1%) of beige semisolid. 1H NMR (400 MHz, CDCl3):
1.05e1.31
(2044
mg,
d
diastereomers:
d
26.96, 27.30, 32.67, 33.44, 33.47, 35.46, 40.38,
(m, 1H); 1.32e1.34 (m, 3H); 1.40e2.01 (m, 4H); 2.26e2.61 (m, 2H);
2.93e3.14 (m, 1H).
43.29, 45.28, 45.77, 93.01, 93.04, 116.90, 117.01, 126.71, 126.78,
126.81, 128.73, 128.76, 144.94, 145.03, 167.33, 167.43.
Benzoyl chloride (1.0 eq., 498 mL) was slowly added to a solution
Following GP4 and GP5, 2-(3-phenylcyclohexylidene)acetoni-
trile (1600 mg, 8.12 mmol) was reduced to 2-(3-phenylcyclohexyl)
ethan-1-amine. Yield: 1527 mg (7.52 mmol, 92.6%) of colourless oil.
of 2-(piperidin-3-yl)acetonitrile (532 mg, 4.29 mmol) and trie-
thylamine (2 mL) in DCM (20 mL). After stirring at r.t. for 12 h, the
volatile components were removed in vacuo and 2-(1-
benzoylpiperidin-3-yl)acetonitrile [60] was isolated by column
chromatography on silica (1. PE/EtOAc ¼ 1:1, 2. PE/EtOAc ¼ 1:2).
Yield: 430 mg (1.883 mmol, 43.9%) of beige oil. ESI-HRMS: m/
z ¼ 227.1282 (MeHe); C14H15N2O requires: m/z ¼ 227.1190 (M-H-).
nmax 2963, 2913, 2813, 2363, 2218, 1633, 1600, 1492, 1465, 1436,
1384, 1365, 1338, 1276, 1255, 1234, 1214, 1183, 1154, 1134, 1100,
1079, 1028, 1005, 983, 952, 906, 829, 811, 794, 767, 733, 698, 552,
ESI-HRMS: m/z
¼
204.1754 (MHþ); C14H22
N requires: m/
z ¼ 204.1747 (MHþ). nmax 3291, 2918, 2849, 1601, 1493, 1447, 1326,
1068, 1029, 753, 697 cmꢂ1. Mixture of two diastereomers in a ratio
of 76:24. 1H NMR (500 MHz, CDCl3) for both diastereomers:
d
0.91e1.01 (m, 1H); 1.12 (q, J ¼ 12.2 Hz, 1H); 1.21e1.97 (m, 11H);
2.49e2.59 (m, 1H); 2.73 (t, J ¼ 7.3 Hz, 2H); 7.10e7.39 (m, 5H). 13
C
NMR (126 MHz, CDCl3):
d 21.50, 26.62, 30.15, 30.94, 32.95, 33.97,
34.27, 35.73, 36.42, 38.13, 38.26, 39.68, 40.66, 41.31, 41.67, 44.45,
125.80, 125.92, 126.87, 127.01, 128.34, 128.37, 147.49, 147.77.
528 cmꢂ1
2.13e2.54 (m, 2H); 2.78 (s, 1H); 2.99 (s, 1H); 3.69 (s, 1H); 4.52 (s,
1H); 7.34e7.42 (m, 5H). 13C NMR (101 MHz, CDCl3):
21.47, 24.12,
. d 1.37e2.11 (m, 5H);
1H NMR (400 MHz, CDCl3):
tert-Butyl
((2S)-3-(1H-indol-3-yl)-1-oxo-1-((2-(3-
d
phenylcyclohexyl)ethyl)amino)propan-2-yl)(butyl)carbamate was
prepared following GP1 from Na-(tert-butoxycarbonyl)-Na-butyl-L-
tryptophan [31] (183 mg, 0.508 mmol), CDI (1.1 eq., 91 mg), and 2-
(3-phenylcyclohexyl)ethan-1-amine (1.1 eq., 113 mg) in THF, iso-
lated by column chromatography on silica (PE/EtOAc ¼ 3:1), and
immediately deprotected following GP2. 13 was isolated by column
chromatography on silica (1. PE/EtOAc ¼ 1:1; 2. PE/EtOAc ¼ 1:2)
and additionally purified following GP6. Yield: 144.5 mg
(0.324 mmol, 63.9% over two steps) of beige semisolid. ESI-HRMS:
m/z ¼ 446.3155 (MHþ); C29H40N3O requires: m/z ¼ 446.3166
25.19, 29.96, 32.66, 33.63, 42.49, 46.77, 48.01, 52.25, 117.66, 126.91,
128.64, 129.90, 135.76, 170.66.
Following GP5, 2-(1-benzoylpiperidin-3-yl)acetonitrile (400 mg,
1.75 mmol) was reduced to 2-(1-benzylpiperidin-3-yl)ethan-1-
amine, which was immediately used further. Yield: 328 mg
(1.51 mmol, 86%) of beige oil. ESI-HRMS: m/z ¼ 219.1854 (MHþ);
C
14H23N2 requires: m/z ¼ 219.1856 (MHþ).
tert-Butyl (S)-(1-((2-(1-benzylpiperidin-3-yl)ethyl)amino)-3-
(1H-indol-3-yl)-1-oxopropan-2-yl)(butyl)carbamate was prepared
following GP1 from Na-(tert-butoxycarbonyl)-Na-butyl-
L
-trypto-
(MHþ). nmax 3271, 2921, 2851, 1651, 1524, 1455, 1341, 1232, 1125,
phan [31] (324 mg, 0.9 mmol), CDI (1.1 eq., 161 mg), and 2-(1-
benzylpiperidin-3-yl)ethan-1-amine (1.1 eq., 216 mg) in THF, iso-
lated by column chromatography on silica (1. PE/EtOAc ¼ 1:1, 2.
EtOAc), and immediately deprotected following GP2 e however
deprotection was made in neat TFA. 14 was purified following GP6.
Yield: 116 mg (0.252 mmol, 28.0% over two steps) of yellowish oil.
r.t.
1010, 908, 735, 698 cme1. [
a]
¼ ꢂ31.3 (c 0.21, acetone). Purity:
D
UPLC (254 nm): tr ¼ 4.803 min, 99.2% total area. Mixture of ste-
reoisomers in a ratio of 70:30. 1H NMR (500 MHz, CDCl3):
d
0.77e0.84 (m, 3H); 0.91e1.03 (m, 1H); 1.09e1.97 (m, 15H);
2.39e2.58 (m, 3H); 2.90e2.98 (m, 1H); 3.26e3.48 (m, 4H); 7.05 (d,
J ¼ 2.2 Hz, 1H); 7.10e7.16 (m, 1H); 7.18e7.28 (m, 4H); 7.29e7.36 (m,
2H); 7.36e7.46 (m, 2H); 7.64e7.72 (m,1H); 8.93 (s,1H, NH). 13C NMR
ESI-HRMS: m/z
¼
461.3269 (MHþ); C29H41N4O requires: m/
z ¼ 461.3275 (MHþ). nmax 3262, 2924, 1650, 1523, 1453, 354, 1231,
1112, 1074, 737, 698, 572 cme1. [
a
]
¼ ꢂ34.1 (c 0.11, acetone). Pu-
r.t.
(126 MHz, CDCl3):
d
13.86, 13.89, 20.19, 20.24, 21.35, 26.24, 29.41,
D
29.44, 29.78, 29.83, 30.37, 30.99, 31.84, 32.04, 32.06, 32.13, 32.18,
32.57, 32.59, 33.74, 33.76, 34.11, 35.86, 35.88, 36.70, 36.72, 37.08,
37.11, 37.51, 37.86, 37.91, 38.08, 38.10, 41.02, 44.30, 44.32, 48.70,
48.72, 63.48, 63.51, 111.36, 111.41, 118.80, 118.83, 119.41, 122.07,
123.05, 125.82, 125.94, 126.82, 126.99, 127.47, 128.33,128.36, 136.57,
147.15, 147.18, 147.50, 174.66, 174.73.
rity: UPLC (220 nm): tr ¼ 2.907 min, 98.9% total area (mixture of
diastereomers). A mixture of diastereomers in a ratio of 57:43. 1H
NMR (400 MHz, CDCl3) for both diastereomers:
d 0.77 and 0.78 (t,
J ¼ 7.3 Hz, 3H); 0.86 (qd, J ¼ 12.3; 4.1 Hz, 1H); 1.10e1.40 (m, 7H);
1.44e1.56 (m, 2H); 1.58e1.74 (m, 3H); 1.85e1.94 (m, 1H); 2.38e2.47
(m, 2H); 2.75e2.85 (m, 2H); 2.87e3.00 (m, 1H); 3.15e3.31 (m, 3H);

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
17
3.32e3.38 (m, 1H); 3.41e3.55 (m, 2H); 6.97 (d, J ¼ 2.2 Hz, 1H);
7.06e7.35 (m, 9H); 7.62 and 7.64 (d, J ¼ 4.1 Hz, 1H); 8.82 and 8.95 (s,
tryptophan [31] (721 mg, 2.0 mmol), CDI (1.1 eq., 357 mg), and 2-(1-
benzylpiperidin-4-yl)ethan-1-amine (1.1 eq., 480 mg) in THF, iso-
lated by column chromatography on silica (1. PE/EtOAc ¼ 1:1, 2.
EtOAc), and immediately deprotected following GP2 e however
deprotection was made in neat TFA. 15 was purified following GP6.
Yield: 261 mg (0.567 mmol, 28.3% over two steps) of yellowish oil.
1H, NH). 13C NMR (101 MHz, CDCl3) for both diastereomers:
d 13.91,
13.92, 20.26, 20.28, 25.26, 28.98, 29.33, 30.62, 30.72, 32.23, 32.25,
33.40, 33.64, 34.17, 36.44, 36.62, 48.68, 48.73, 54.11, 54.14, 60.17,
60.20, 63.55, 63.59, 63.70, 63.76, 111.42, 111.44, 111.62, 118.75,
118.88, 119.51, 122.07, 122.15, 123.13, 123.28, 127.06, 127.10, 127.60,
127.81, 128.27, 128.29, 129.29, 129.35,136.49, 136.58, 138.34, 138.39,
174.56, 174.60.
ESI-HRMS: m/z
¼
461.3267 (MHþ); C29H41N4O requires: m/
z ¼ 461.3275 (MHþ). nmax 3279, 2922, 1651, 1524, 1453, 1342, 1257,
r.t.
1232, 1118, 1076, 1011, 975, 909, 736, 698, 615 cme1. [
a]
¼ ꢂ36.6 (c
D
0.25, acetone). Purity: UPLC (220 nm): tr ¼ 3.200 min, 97.6% total
4.6.10. (S)eN-(2-(1-Benzylpiperidin-4-yl)ethyl)-2-(butylamino)-3-
(1H-indol-3-yl)propanamide (15)
area. 1H NMR (400 MHz, CDCl3):
d
0.76 (t, J ¼ 7.2 Hz, 3H); 1.13 (dq,
J ¼ 7.0; 14.2 Hz, 2H); 1.19e1.28 (m, 6H); 1.38 (q, J ¼ 7.0 Hz, 2H);
1.58e1.66 (m, 2H); 1.90 (t, J ¼ 10.9 Hz, 2H); 2.37e2.45 (m, 2H);
2.82e2.92 (m, 3H); 3.17e3.28 (m, 1H); 3.28e3.39 (m, 3H); 3.48 (s,
2H); 7.01 (d, J ¼ 2.2 Hz, 1H); 7.06e7.11 (m, 1H); 7.14e7.19 (m, 1H);
7.21e7.36 (m, 7H); 7.64 (d, J ¼ 7.9 Hz, 1H); 8.77 (s, 1H, NH). 13C NMR
Sodium hydride (60 wt% suspension in mineral oil) (1.2 eq.,
1925 mg) was added portionwise to diethyl cyanomethylphosph-
onate (1.3 eq., 8.42 mL) in anhydrous THF (80 mL) under argon at r.t.
and the mixture was stirred for 15 min. Then, tert-butyl 4-
oxopiperidine-1-carboxylate (8000 mg, 1.0 eq., 40.1 mmol) was
added and the reaction mixture was stirred at r.t. for 12h. Water
(50 mL) was added, volatile components were evaporated in vacuo
and residue extracted with Et2O (2 x 50 mL). The combined organic
phase was dried over anhydrous sodium sulfate, filtered, and vol-
atile components evaporated in vacuo. tert-Butyl 4-(cyano-
methylene)piperidine-1-carboxylate [61] was purified by
crystallization (EtOAc/Et2O). Yield: 6430 mg (28.9 mmol, 72.1%) of
beige crystals.
(101 MHz, CDCl3):
d 13.90, 20.26, 29.38, 32.20, 32.22, 33.57, 36.30,
36.65, 48.75, 53.79, 63.53, 63.57, 111.38, 111.59, 118.89, 119.52,
122.17, 122.99, 127.01, 127.57, 128.22, 129.36, 136.56, 138.40, 174.60.
4.6.11. (S)-2-(Butylamino)-3-(1H-indol-3-yl)-N-(2-(1-
phenethylpiperidin-4-yl)ethyl)propanamide (16)
Sodium hydride (60 wt% suspension in mineral oil) (1.2 eq.,
756 mg) was added portionwise to diethyl cyanomethylphospho-
nate (1.3 eq., 3.31 mL) in anhydrous THF (40 mL) under argon at r.t.
and the mixture was stirred for 15 min. Then, 1-
phenethylpiperidin-4-one [64] (3200 mg, 1.0 eq., 15.7 mmol) was
added and the reaction mixture was stirred at r.t. for 12h. Water
(50 mL) was added, volatile components were evaporated in vacuo,
and the residue was extracted with Et2O (2 x 30 mL). The combined
organic phase was dried over anhydrous sodium sulfate, filtered,
concentrated in vacuo to approx. 10 mL and cooled to ꢂ20 ꢀC. The
precipitate was collected by filtration to give 2-(1-
tert-Butyl 4-(cyanomethyl)piperidine-1-carboxylate was pre-
pared from tert-butyl 4-(cyanomethylene)piperidine-1-carboxylate
(5000 mg, 22.5 mmol) following GP4. Yield: 4931 mg (22.0 mmol,
97.7%) of beige solid, which was immediately used further. 1H NMR
(400 MHz, CDCl3):
d 1.16e1.30 (m, 2H); 1.42 (s, 9H); 1.71e1.87 (m,
3H); 2.28 (d, J ¼ 6.5 Hz, 2H); 2.68 (t, J ¼ 12.1 Hz, 2H); 4.12 (s, 2H). 13
C
NMR (101 MHz, CDCl3):
118.18, 154.67.
d 24.13, 28.47, 31.34, 33.39, 43.37, 79.72,
2-(Piperidin-4-yl)acetonitrile [62] was prepared from tert-butyl
4-(cyanomethyl)piperidine-1-carboxylate following GP2 the
amine could be obtained after extraction with heavy losses in yield
due to its high aqueous solubility, therefore neutralized solution
obtained after TFA deprotection was immediately used further. A
mixture of conformers in a ratio of 76:24. 1H NMR (400 MHz, CDCl3)
phenethylpiperidin-4-ylidene)acetonitrile.
Yield:
2575
mg
e
(11.4 mmol, 72.3%) of beige crystals. mp ¼ 78.8e79.5 ꢀC. ESI-HRMS:
m/z ¼ 227.1540 (MHþ); C15H19N2 requires: m/z ¼ 227.1543 (MHþ).
nmax 3020, 2940, 2900, 2814, 2792, 2768, 2218, 1628, 1468, 1354,
1318, 1293, 1246, 1122, 1084, 1026, 1001, 915, 842, 819, 801, 769, 751,
702, 601, 505 cmꢂ1. 1H NMR (400 MHz, CDCl3):
d
2.37e2.43 (m, 2H);
2.57e2.67 (m, 8H); 2.77e2.84 (m, 2H); 5.10 (s, 1H); 7.17e7.23 (m,
3H); 7.25e7.32 (m, 2H). 13C NMR (101 MHz, CDCl3):
32.73, 33.95,
for the major conformer:
d 1.34e1.49 (m, 2H); 1.72e2.00 (m, 3H);
2.31 (d, J ¼ 6.6 Hz, 2H); 2.70 (t, J ¼ 12.2 Hz, 2H); 3.24 (d, J ¼ 12.6 Hz,
d
2H); 3.60 (br s, 1H). 1H NMR (400 MHz, CDCl3) for the minor
35.23, 53.76, 54.11, 59.75, 93.21, 116.71, 126.26, 128.55, 128.77,
140.19, 165.06.
2-(1-Phenethylpiperidin-4-ylidene)acetonitrile
11.1 mmol) was reduced following GP4 to 2-(1-phenethylpiperidin-
conformer:
d
2.28 (d, J ¼ 6.8 Hz, 2H); 2.79e2.88 (m, 2H); 3.41 (d,
J ¼ 10.9 Hz, 1H).
(2520
mg,
Benzoyl chloride (2.0 eq., 1404
mL) was slowly added to aqueous
solution of 2-(piperidin-4-yl)acetonitrile (after deprotection of
6.04 mmol of tert-butyl 4-(cyanomethyl)piperidine-1-carboxylate)
and potassium carbonate (2000 mg) in THF (30 mL). After vigorous
stirring at r.t. for 12h, the volatile components were removed in
vacuo and 2-(1-benzoylpiperidin-4-yl)acetonitrile was isolated by
column chromatography on silica (1. PE/EtOAc ¼ 1:1, 2. EtOAc).
Yield: 958 mg (4.196 mmol, 69.5%) of colourless solid, which was
4-yl)acetonitrile. Yield: 2491 mg (10.9 mmol, 98.3%) of off-white
solid. 1H NMR (400 MHz, CDCl3):
d
1.42 (qd, J ¼ 3.8; 12.1 Hz, 2H);
1.63e1.76 (m, 1H); 1.80e1.88 (m, 2H); 2.03 (td, J ¼ 2.3; 11.8 Hz, 2H);
2.29 (d, J ¼ 6.9 Hz, 2H); 2.56e2.62 (m, 2H); 2.77e2.83 (m, 2H);
2.99e3.06 (m, 2H); 7.17e7.22 (m, 3H); 7.25e7.31 (m, 2H). 13C NMR
(101 MHz, CDCl3):
d 24.14, 31.79, 33.36, 33.86, 53.24, 60.71, 118.62,
126.16, 128.50, 128.78, 140.42.
immediately used further. 1H NMR (400 MHz, CDCl3):
d
1.20e2.04
2-(1-Phenethylpiperidin-4-yl)acetonitrile (2491 mg, 10.9 mmol)
was reduced following GP5 to 2-(1-phenethylpiperidin-4-yl)ethan-
1-amine. Yield: 2221 mg (9.56 mmol, 88.7%) of beige oil. ESI-HRMS:
m/z ¼ 233.2009 (MHþ); C15H25N2 requires: m/z ¼ 233.2012 (MHþ).
(m, 5H); 2.35 (d, J ¼ 6.6 Hz, 2H); 2.80 (br s, 1H); 3.00 (br s, 1H); 3.83
(br s, 1H); 4.78 (br s, 1H); 7.36e7.43 (m, 5H). 13C NMR (101 MHz,
CDCl3):
d 24.14, 31.43, 31.91, 33.61, 41.95, 47.46, 118.00, 126.99,
128.67, 129.88, 135.99, 170.60.
1H NMR (400 MHz, CDCl3):
d 1.12e1.44 (m, 7H); 1.66e1.73 (m, 2H);
Following GP5, 2-(1-benzoylpiperidin-4-yl)acetonitrile (900 mg,
3.94 mmol) was reduced to 2-(1-benzylpiperidin-4-yl)ethan-1-
amine [63], which was immediately used further. Yield: 713 mg
(3.27 mmol, 83%) of colourless oil. ESI-HRMS: m/z ¼ 219.1854
(MHþ); C14H23N2 requires: m/z ¼ 219.1856 (MHþ).
1.94e2.03 (m, 2H); 2.53e2.59 (m, 2H); 2.73 (t, J ¼ 7.1 Hz, 2H);
2.77e2.84 (m, 2H), 2.96e3.03 (m, 2H), 7.16e7.22 (m, 3H), 7.25e7.31
(m, 2H). 13C NMR (101 MHz, CDCl3):
d
32.57, 33.50, 33.94, 39.76,
40.85, 54.09, 61.21, 126.07, 128.47, 128.81, 140.73.
tert-Butyl
(S)-(3-(1H-indol-3-yl)-1-oxo-1-((2-(1-
tert-Butyl (S)-(1-((2-(1-benzylpiperidin-4-yl)ethyl)amino)-3-
(1H-indol-3-yl)-1-oxopropan-2-yl)(butyl)carbamate was prepared
phenethylpiperidin-4-yl)ethyl)amino)propan-2-yl)(butyl)carba-
mate was prepared following GP1 from Na-(tert-butoxycarbonyl)-
following
GP1
from
Na-(tert-butoxycarbonyl)-Na-butyl-L-
Na-butyl-
-tryptophan [31] (721 mg, 2.0 mmol), CDI (1.1 eq.,
L

18
A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
357 mg), and 2-(1-phenethylpiperidin-4-yl)ethan-1-amine (1.1 eq.,
511 mg) in THF, isolated by column chromatography on silica (1. PE/
EtOAc ¼ 1:1; 2. EtOAc), and immediately deprotected following GP2
e however deprotection was made in neat TFA. 16 was purified
following GP6. Yield: 163 mg (0.343 mmol, 17.2% over two steps) of
(1-Benzoylazepan-4-yl)acetonitrile 18a was purified by column
chromatography on silica (PE/EtOAc ¼ 1:2) to afford colorless oil.
Yield: 807 mg (3.33 mmol, 83.3% over two steps). ESI-HRMS: m/
z ¼ 243.1492 (MHþ); C15H19N2O requires: m/z ¼ 243.1492 (MHþ).
nmax 2923, 2874, 2244, 1618, 1576, 1421, 1377, 1357, 1300, 1111, 1027,
1005, 920, 784, 731, 703,633 cmꢂ1. Mixture of two conformers in a
ratio of 58:42. 1H NMR (500 MHz, CDCl3) for both conformers:
beige-yellowish oil. ESI-HRMS: m/z
C
¼
238.1750 ((Mþ2H)2þ);
30H44N4O requires: m/z ¼ 238.1752 ((Mþ2H)2þ). nmax 3264, 2923,
2853, 1652, 1524, 1453, 1354, 1235, 1119, 1011, 976, 908, 733, 698,
d
1.27e2.14 (m, 7H); 2.25e2.44 (m, 2H); 3.20e3.65 (m, 3H);
3.81e4.05 (m,1H); 7.31e7.43 (m, 5H). 13C NMR (126 MHz, CDCl3) for
both conformers: 25.14, 25.34, 25.65, 27.55, 31.83, 33.46, 33.78,
r.t.
566 cme1. [
a]
¼ ꢂ33.9 (c 0.59, acetone). Purity: UPLC (220 nm):
D
tr ¼ 3.517 min, 95.6% total area. 1H NMR (400 MHz, CDCl3):
d
0.76 (t,
d
J ¼ 7.2 Hz, 3H); 1.14 (dq, J ¼ 7.0; 14.2 Hz, 2H); 1.20e1.32 (m, 6H); 1.40
(q, J ¼ 6.9 Hz, 2H); 1.69 (d, J ¼ 8.7 Hz, 2H); 1.96 (t, J ¼ 10.4 Hz, 2H);
2.37e2.45 (m, 2H); 2.54e2.60 (m, 2H); 2.77e2.83 (m, 2H); 2.89 (dd,
J ¼ 9.1; 14.2 Hz, 1H); 2.99 (d, J ¼ 10.8 Hz, 2H); 3.19e3.30 (m, J ¼ 7.1,
13.1 Hz, 1H); 3.30e3.41 (m, 3H); 7.02 (d, J ¼ 2.2 Hz, 1H); 7.06e7.11
(m, 1H); 7.14e7.20 (m, 4H); 7.24e7.29 (m, 2H); 7.31e7.37 (m, 2H);
7.64 (d, J ¼ 7.9 Hz, 1H); 9.04 (s, 1H, NH). 13C NMR (101 MHz, CDCl3):
35.41, 35.71, 36.98, 44.04, 46.02, 47.54, 49.77, 118.53, 118.71, 126.54,
126.70, 128.61, 128.69, 129.53, 136.80, 136.90, 171.72, 171.84.
2-(1-Benzylazepan-4-yl)ethan-1-amine 18b was prepared
following GP5 from 18a (800 mg, 3.33 mmol) e 5.0 eq. of LiAlH4
were used, and used further without purification. Yield: 494 mg
(2.13 mmol, 63.8%) of colourless oil. ESI-HRMS: m/z ¼ 233.2011
(MHþ); C15H25N2 requires: m/z ¼ 233.2012 (MHþ).
d
13.86, 20.21, 29.40, 32.17, 32.19, 32.22, 33.57, 33.74, 36.28, 36.59,
(2S)eN-(2-(1-Benzylazepan-4-yl)ethyl)-2-(butylamino)-3-(1H-
indol-3-yl)propanamide was prepared following GP1 from Na-(tert-
48.72, 53.84, 61.01, 63.50, 111.36, 111.42, 118.83, 119.42, 122.08,
122.99, 126.01, 127.52, 128.40, 128.70, 136.57, 140.51, 174.63.
butoxycarbonyl)-Na-butyl-L-tryptophan
[31]
(295
mg,
0.818 mmol), CDI (1.1 eq., 146 mg), and 18b (1.1 eq., 209 mg) in THF,
isolated by column chromatography on silica (1. DCM/
MeOH ¼ 50:1; 2. DCM/MeOH ¼ 10:1), and immediately depro-
tected following GP2 e however deprotection was made in neat
TFA. 18 was purified by column chromatography on silica (1. DCM/
MeOH ¼ 50:1; 2. DCM/MeOH ¼ 5:1). Yield: 124.5 mg (0.262 mmol,
32.1%) of colourless oil. ESI-HRMS: m/z ¼ 475.3426 (MHþ);
4.6.12. (S)-2-(Butylamino)-3-(1H-indol-3-yl)-N-(2-(1-
phenylpiperidin-4-yl)ethyl)propanamide (17)
2-(1-Phenylpiperidin-4-ylidene)acetonitrile [65] (850 mg,
4.29 mmol) was reduced following GP4 and GP5 to afford 2-(1-
phenylpiperidin-4-yl)ethan-1-amine. Yield: 694 mg (3.40 mmol,
79.2%) of brownish oil, which was used further without purifica-
tion. ESI-HRMS: m/z ¼ 205.1699 (MHþ); C13H21N2O requires: m/
z ¼ 205.1699 (MHþ).
C
30H43N4O requires: m/z ¼ 475.3405 (MHþ), m/z ¼ 238.1751
((Mþ2H)2þ); C30H44N4O requires: m/z ¼ 238.1752 ((Mþ2H)2þ). nmax
tert-Butyl
(S)-(3-(1H-indol-3-yl)-1-oxo-1-((2-(1-
3272, 2922, 1650, 1524, 1494, 1454, 1353, 1233, 1103, 1010, 907, 738,
r.t.
phenylpiperidin-4-yl)ethyl)amino)propan-2-yl)(butyl)carbamate
was prepared following GP1 from Na-(tert-butoxycarbonyl)-Na-
butyl-L-tryptophan [31] (541 mg, 1.5 mmol), CDI (1.1 eq., 268 mg),
and 2-(1-phenylpiperidin-4-yl)ethan-1-amine (1.1 eq., 337 mg) in
THF, isolated by column chromatography on silica (1. PE/
EtOAc ¼ 1:1; 2. EtOAc), and immediately deprotected following GP2
e however deprotection was made in neat TFA. 17 was purified
following GP6. Yield: 36.5 mg (0.082 mmol, 5.4% over two steps) of
dark yellow oil. ESI-HRMS: m/z ¼ 447.3113 (MHþ); C28H39N4O re-
quires: m/z ¼ 447.31184 (MHþ). nmax 3279, 2923, 1651, 1597, 1525,
697 cme1. [
a
]
¼ ꢂ29.3 (c 1.15, acetone). Purity: UPLC (220 nm):
D
tr ¼ 3.420 min, 95.1% total area. Two diastereomers in a 1:1 ratio. 1H
NMR (500 MHz, CDCl3) for both diastereomers:
d 0.64e0.70 (m,
3H); 1.00e1.10 (m, 2H), 1.12e1.22 (m, 3H); 1.24e1.33 (m, 4H);
1.39e1.52 (m, 3H); 1.54e1.67 (m, 3H); 2.28e2.37 (m, 2H);
2.40e2.65 (m, 4H); 2.78e2.87 (m, 1H); 3.07e3.25 (m, 3H);
3.26e3.31 (m, 1H); 3.54 (s, 2H); 6.92 (d, J ¼ 2.2 Hz, 1H); 6.98 (t,
J ¼ 7.5 Hz, 1H); 7.03e7.08 (m, 1H); 7.12e7.16 (m, 1H); 7.17e7.27 (m,
5H); 7.53 (d, J ¼ 7.8 Hz, 1H); 7.26 and 7.33 (2 x s, 1H, NH). 13C NMR
(126 MHz, CDCl3):
d 13.85, 20.19, 26.24, 26.36, 29.18, 29.31, 32.13,
1496, 1457, 1383, 1340, 1233, 1195, 1124, 990, 909, 732, 691,
33.11, 33.13, 33.61, 33.65, 35.60, 35.71, 36.87, 36.94, 37.32, 37.36,
48.65, 53.43, 53.48, 55.70, 55.85, 62.81, 62.90, 63.47, 111.06, 111.13,
111.46, 118.63, 118.68, 119.27, 121.89, 121.92, 123.17, 123.21, 127.04,
127.06, 127.47, 127.53, 128.23, 129.04, 129.08, 136.53, 136.56, 138.73,
174.60, 174.62.
r.t.
526 cme1. [
a
]
¼ ꢂ28.7 (c 0.20, acetone). Purity: UPLC (220 nm):
D
tr ¼ 3.74 min, 96.3% total area. 1H NMR (400 MHz, CDCl3):
d 0.77 (t,
J ¼ 7.2 Hz, 3H); 1.08e1.48 (m, 10H); 1.72e1.80 (m, 2H); 2.43 (td,
J ¼ 2.7; 6.9 Hz, 2H); 2.64 (t, J ¼ 11.0 Hz, 2H); 2.90 (dd, J ¼ 9.1;
14.4 Hz, 1H); 3.22e3.42 (m, 4H); 3.60e3.68 (m, 2H); 6.80e6.85 (m,
1H); 6.90e6.96 (m, 2H); 7.02 (d, J ¼ 2.2 Hz, 1H); 7.07e7.13 (m, 1H);
7.16e7.20 (m, 1H); 7.21e7.28 (m, 2H); 7.30e7.36 (m, 2H); 7.65 (d,
4.6.14. (2S)-2-(Butylamino)-3-(1H-indol-3-yl)-N-(2-(1-(2,3,5,6-
tetrafluorobenzyl)azepan-4-yl)ethyl)propanamide (19)
J ¼ 7.9 Hz, 1H); 8.55 (s, 1H, NH). 13C NMR (101 MHz, CDCl3):
d
13.92,
Pentafluorobenzoyl chloride (1.0 eq., 344 mL) was slowly added
20.28, 29.39, 32.06, 32.09, 32.23, 33.55, 36.31, 36.55, 48.77, 50.02,
63.52, 111.37, 111.66, 116.63, 118.94, 119.43, 119.60, 122.25, 122.96,
127.58, 129.13, 136.56, 151.87, 174.61.
to a solution of 2-(azepan-4-yl)acetonitrile (330 mg, 2.39 mmol)
and triethylamine (1 mL) in DCM (20 mL). After stirring at r.t. for
12h, the volatile components were removed in vacuo and 2-(1-
(perfluorobenzoyl)azepan-4-yl)acetonitrile 19a was isolated by
column chromatography on silica (1. PE/EtOAc ¼ 3:1, 2. PE/
EtOAc ¼ 1:1). Yield: 549 mg (1.65 mmol, 69.1%) of yellowish oil. ESI-
4.6.13. (2S)eN-(2-(1-Benzylazepan-4-yl)ethyl)-2-(butylamino)-3-
(1H-indol-3-yl)propanamide (18)
tert-Butyl 4-(cyanomethyl)azepane-1-carboxylate [31] (953 mg,
4.0 mmol) was deprotected following GP2 to afford 2-(azepan-4-yl)
acetonitrile as colourless oil, which was immediately used further
without purification.
HRMS: m/z
¼
333.1015 (MHþ); C15H14F5N2O requires: m/
z ¼ 333.1026 (MHþ). nmax 2931, 2245, 1648, 1521, 1499, 1434, 1377,
1314, 1239, 1186, 1102, 987, 803, 734, 655, 582, 536 cmꢂ1. Two
conformers in a ratio of 60:40. 1H NMR (400 MHz, CDCl3) for a
Benzoyl chloride (1.0 eq., 445
m
L) was slowly added to a solution
mixture of two conformers:
3.09e3.53 (m, 3H); 3.74e3.99 (m, 1H). 13C NMR (101 MHz, CDCl3)
for a mixture of two conformers: 24.47, 24.70, 25.13, 26.40, 31.13,
32.78, 33.20, 34.21, 35.07, 36.41, 43.45, 45.53, 46.65, 48.53,
109.18e112.87 (m), 118.15, 118.26, 135.82e136.67 (m),
138.35e139.16 (m), 139.79e140.61 (m), 140.75e141.48 (m),
d 1.20e2.07 (m, 7H); 2.17e2.35 (m, 2H);
of 2-(azepan-4-yl)acetonitrile (530 mg, 3.83 mmol) and triethyl-
amine (1 mL) in DCM (20 mL). After stirring at r.t. for 1 h, the re-
action mixture was successively extracted with 0.1 M NaOH (20 mL)
and 10% aqueous citric acid (20 mL). The organic phase was dried
with sodium sulfate and volatile components removed in vacuo. 2-
d

A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
19
142.57e143.09 (m), 143.23e143.93 (m), 158.05, 158.12.
chromatography on silica (1. DCM/MeOH
¼
50:1; 2. DCM/
19a (500 mg, 1.506 mmol) was reduced following GP5 (5 eq.
LiAlH4) to 2-(1-(2,3,5,6-tetrafluorobenzyl)azepan-4-yl)ethan-1-
amine 19b (377 mg, 1.24 mmol), which was immediately used
further without purification. ESI-HRMS: m/z ¼ 305.1633 (MHþ);
MeOH ¼ 20:1), and immediately deprotected following GP2 e
however deprotection was made in neat TFA. 20 was purified
following GP6. Yield: 148 mg (0.293 mmol, 45.7% over two steps) of
beige oil. ESI-HRMS: m/z ¼ 505.3528 (MHþ); C31H45N4O2 requires:
m/z ¼ 505.3537 (MHþ). nmax 3261, 2922,1651,1611,1510,1454,1353,
C
15H21N2F4 requires: m/z ¼ 305.1635 (MHþ).
tert-Butyl
((2S)-3-(1H-indol-3-yl)-1-oxo-1-((2-(1-(2,3,5,6-
1299, 1241, 1177, 1104, 1035, 910, 832, 736, 647, 615, 585, 550,
r.t.
tetrafluorobenzyl)azepan-4-yl)ethyl)amino)propan-2-yl)(butyl)
carbamate was prepared following GP1 from Na-(tert-butox-
ycarbonyl)-Na-butyl-L-tryptophan [31] (215 mg, 0.597 mmol), CDI
(1.1 eq., 106 mg), and 19b (1.1 eq., 200 mg) in THF, isolated by col-
520 cme1. [
a
]
D
¼ ꢂ31.4 (c 0.77, acetone). Purity: UPLC (220 nm):
tr ¼ 4.030 min, 98.4% total area. A 1:1 mixture of two di-
astereomers. 1H NMR (400 MHz, CDCl3) for both diastereomers:
d
0.79 (td, J ¼ 1.6; 7.2 Hz, 3H); 1.09e1.80 (m, 14H); 2.36e2.75 (m,
umn chromatography on silica (1. PE/EtOAc
¼
2:1, 2. PE/
6H); 2.89e3.02 (m, 1H); 3.18e3.46 (m, 4H); 3.57 (s, 2H); 3.78 (s,
3H); 6.80e6.89 (m, 2H); 7.04 (d, J ¼ 1.6 Hz, 1H); 7.09 (t, J ¼ 7.4 Hz,
1H); 7.14e7.21 (m, 1H); 7.22e7.28 (m, 2H); 7.29e7.39 (m, 2H); 7.65
(d, J ¼ 7.8 Hz, 1H); 9.49 and 9.54 (2 x s, 1H, 2xNH). 13C NMR
EtOAc ¼ 1:1), and immediately deprotected following GP2 e
however deprotection was made in neat TFA. 19 was purified
following GP6. Yield: 108 mg (0.198 mmol, 33.1% over two steps) of
yellowish oil. ESI-HRMS: m/z ¼ 547.3047 (MHþ); C30H39F4N4O re-
quires: m/z ¼ 547.3055 (MHþ). nmax 3279, 2924, 2855, 1651, 1500,
(101 MHz, CDCl3) for both diastereomers and conformers:
d 13.80,
20.15, 26.57, 26.69, 28.78, 28.80, 29.13, 29.18, 29.30, 29.35, 32.06,
32.10, 33.12, 33.14, 33.99, 34.04, 35.56, 35.68, 36.75, 36.83, 36.87,
36.95, 37.37, 37.39, 37.42, 37.45, 48.51, 48.62, 53.37, 53.43, 55.14,
55.64, 55.79, 62.25, 62.34, 63.40, 63.49, 111.02, 111.11, 111.39, 113.48,
118.59, 118.65, 119.21, 121.83, 121.87, 123.15, 127.43, 127.51, 129.98,
130.04, 131.46, 131.53, 136.53, 158.44, 158.46, 174.51, 174.53, 174.58,
174.60.
1455, 1342, 1251, 1171, 1105, 998, 843, 739, 711, 645 cme1
.
r.t.
[
a]
¼ ꢂ25.0 (c 0.34, acetone). Purity: UPLC (220 nm):
D
tr ¼ 3.273 min, 97.4% total area. Two diastereomers in a 1:1 ratio. 1H
NMR (400 MHz, CDCl3) for both diastereomers:
d
0.76 (t, J ¼ 7.2 Hz,
3H); 1.03e1.45 (m, 8H); 1.47e1.61 (m, 2H); 1.62e1.81 (m, 3H);
2.35e2.48 (m, 2H); 2.51e2.70 (m, 3H); 2.70e2.80 (m, 1H); 2.89
(ddd, J ¼ 3.4; 9.0; 14.2 Hz, 1H); 3.13e3.41 (m, 4H); 3.81 (s, 2H);
6.92e7.01 (m, 1H); 7.03 (d, J ¼ 2.2 Hz, 1H); 7.07e7.14 (m, 1H); 7.18 (t,
J ¼ 7.5 Hz, 1H); 7.23e7.29 (m, 1H); 7.32e7.40 (m, 1H); 7.65 (d,
J ¼ 7.9 Hz, 1H); 8.58 and 8.61 (2 x s, 1H, 2xNH). 13C NMR (101 MHz,
4.6.16. (S)-2-(Butylamino)-3-(1H-indol-3-yl)-N-(2-(1-((1-
methylpiperidin-4-yl)methyl)piperidin-4-yl)ethyl)propanamide
(21)
CDCl3) for both diastereomers:
d
13.87, 20.26, 26.37, 26.41, 29.31,
tert-Butyl
dine-1-carboxylate was prepared following GP1 from 1-(tert-
butoxycarbonyl)piperidine-4-carboxylic acid (1000 mg,
4-(4-(cyanomethyl)piperidine-1-carbonyl)piperi-
29.37, 32.22, 33.17, 33.93, 33.94, 35.77, 35.83, 37.01, 37.05, 37.39,
48.74, 49.34, 52.99, 53.03, 55.18, 55.26, 63.54, 105.03 (t, J ¼ 22.8 Hz),
111.37, 111.51e111.70 (m), 118.07 (td, J ¼ 2.5, 18.3 Hz), 118.89, 119.53,
122.18, 123.05, 127.59, 127.61, 136.57, 136.58, 143.93e144.21 (m),
144.38e144.73 (m), 146.36e146.67 (m), 146.85e147.21 (m), 174.60,
4.36 mmol), CDI (1.1 eq., 778 mg), and 2-(piperidin-4-yl)acetoni-
trile (1.1 eq., 596 mg) in THF, and isolated by column chromatog-
raphy on silica (1. PE/EtOAc ¼ 1:1; 2. EtOAc). Yield: 1247 mg
(3.72 mmol, 85.3%) of beige semisolid. ESI-HRMS: m/z ¼ 336.2276
(MHþ); C18H30N3O3 requires: m/z ¼ 336.2282 (MHþ). nmax 2917,
2240, 1685, 1616, 1475, 1442, 1413, 1365, 1255, 1231, 1161, 1063,
1025, 981, 951, 869, 775, 728, 560 cmꢂ1. 1H NMR (400 MHz, CDCl3):
174.62. 19F NMR (376 MHz, CDCl3):
22.6 Hz); ꢂ139.49 (ddd, J ¼ 8.6; 13.6; 22.1 Hz).
d
ꢂ142.21 (ddd, J ¼ 9.3; 13.3;
4.6.15. (2S)-2-(Butylamino)-3-(1H-indol-3-yl)-(2-(1-(4-
methoxybenzyl)azepan-4-yl)ethyl)propanamide (20)
d
1.26 (q, J ¼ 7.1, 7.7 Hz, 2H); 1.44 (s, 9H); 1.57e1.79 (m, 4H);
p-Anisoyl chloride (1.0 eq., 370 mg) was slowly added to a so-
lution of 2-(azepan-4-yl)acetonitrile (300 mg, 2.17 mmol) and
triethylamine (1 mL) in DCM (20 mL). After stirring at r.t. for 12h,
the volatile components were removed in vacuo and 2-(1-(4-
methoxybenzoyl)azepan-4-yl)acetonitrile 20a isolated by column
chromatography on silica (1. PE/EtOAc ¼ 1:1, 2. EtOAc). Yield:
566 mg (2.08 mmol, 95.9%) of yellowish oil. ESI-HRMS: m/
z ¼ 273.1595 (MHþ); C16H21N2O2 requires: m/z ¼ 273.1598 (MHþ).
nmax 3330, 2927, 2243, 1607, 1574, 1513, 1423, 177, 1358, 1299, 1246,
1.79e2.00 (m, 3H); 2.33 (dd, J ¼ 6.4,11.5 Hz, 2H); 2.48e2.66 (m, 2H);
2.68e2.82 (m, 2H); 3.05 (t, J ¼ 12.5 Hz, 1H); 3.96 (d, J ¼ 13.6 Hz, 1H);
4.13 (br s, 2H); 4.69 (d, J ¼ 12.6 Hz, 1H). 13C NMR (101 MHz, CDCl3):
d
24.08, 28.55, 31.30, 32.29, 33.60, 38.64, 41.60, 45.13, 79.71, 117.96,
154.81, 172.95.
tert-Butyl
4-(4-(cyanomethyl)piperidine-1-carbonyl)piperi-
dine-1-carboxylate (1200 mg, 3.58 mmol) was reduced following
GP5 e 10.0 eq. of LiAlH4 were used to afford 2-(1-((1-
methylpiperidin-4-yl)methyl)piperidin-4-yl)ethan-1-amine 21b
(654 mg, 2.73 mmol, 76.3%), which was used further without pu-
rification. ESI-HRMS: m/z ¼ 240.2429 (MHþ); C14H30N3 requires: m/
z ¼ 240.2434 (MHþ).
e
1174, 1110, 1028, 841, 794, 764, 717, 675, 593, 519 cmꢂ1
.
1H NMR
(400 MHz, CDCl3) for both conformers: 1.10e2.12 (m, 7H);
d
2.24e2.46 (m, 2H); 3.14e3.66 (m, 3H); 3.72e4.06 (m, 4H);
6.85e6.95 (m, 2H); 7.29e7.40 (m, 2H). 13C NMR (101 MHz, CDCl3)
tert-Butyl (S)-(3-(1H-indol-3-yl)-1-((2-(1-((1-methylpiperidin-
4-yl)methyl)piperidin-4-yl)ethyl)amino)-1-oxopropan-2-yl)(butyl)
carbamate was prepared following GP1 from Na-(tert-butox-
ycarbonyl)-Na-butyl-L-tryptophan [31] (411 mg, 1.14 mmol), CDI
(1.1 eq., 203 mg), and 21b (1.1 eq., 300 mg) in THF, the reaction
mixture was poured into ice water (20 mL), decanted, and the
precipitate was washed with ice water (5 mL). The remaining solid
was dissolved in EtOAc (20 mL), dried with sodium sulfate, filtered
and volatile components removed in vacuo to afford crude product,
which was immediately deprotected following GP2 e however
deprotection was made in neat TFA under Ar overnight on an ice-
bath. 21 was purified following GP6. Yield: 161 mg (0.334 mmol,
29.3% over two steps) of beige oil. ESI-HRMS: m/z ¼ 241.6960
((Mþ2H)2þ); C29H49N5O requires: m/z ¼ 241.6963 ((Mþ2H)2þ). nmax
3282, 2921, 2794, 1653, 1524, 1450, 1377, 1356, 1267, 1143, 1011, 909,
for both conformers: d 24.44, 25.11, 25.41, 26.95, 27.13, 28.36, 31.29,
31.68, 32.88, 33.16, 33.63, 34.80, 34.99, 36.34, 36.77, 43.58, 44.23,
45.61, 45.90, 47.22, 47.85, 49.38, 50.57, 54.97, 113.24, 113.33, 118.38,
128.07, 128.16, 128.50, 159.90, 160.05, 171.01.
20a (250 mg, 0.919 mmol) was reduced following GP5 e how-
ever 5.0 eq. of LiAlH4 were used e to 2-(1-(4-methoxybenzyl)aze-
pan-4-yl)ethan-1-amine 20b (185 mg, 0.706 mmol), which was
immediately used further without purification. ESI-HRMS: m/
z ¼ 263.2115 (MHþ); C16H27N2O requires: m/z ¼ 263.2118 (MHþ).
tert-Butyl ((2S)-3-(1H-indol-3-yl)-1-((2-(1-(4-methoxybenzyl)
azepan-4-yl)ethyl)amino)-1-oxopropan-2-yl)(butyl)carbamate
was prepared following GP1 from Na-(tert-butoxycarbonyl)-Na-
butyl-L-tryptophan [31] (231 mg, 0.641 mmol), CDI (1.1 eq.,
114 mg), and 20b (1.1 eq., 185 mg) in THF, isolated by column

20
A. Meden et al. / European Journal of Medicinal Chemistry 208 (2020) 112766
r.t.
731 cme1. [
a
]
D
¼ ꢂ22.7 (c 0.29, acetone). Purity (method II): UPLC
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CDCl3):
d
0.76 (t, J ¼ 7.2 Hz, 3H); 1.08e1.49 (m, 13H); 1.59e1.66 (m,
2H); 1.72 (d, J ¼ 11.9 Hz, 2H); 1.81 (t, J ¼ 10.4 Hz, 2H); 1.89 (td,
J ¼ 11.8; 2.2 Hz, 2H); 2.12 (d, J ¼ 7.0 Hz, 2H); 2.25 (s, 3H); 2.37e2.46
(m, 2H); 2.78e2.91 (m, 5H); 3.22 (dq, J ¼ 7.2; 13.1 Hz,1H); 3.27e3.39
(m, 3H); 7.04 (d, J ¼ 2.2 Hz, 1H); 7.08e7.13 (m, 1H); 7.16e7.21 (m,
1H); 7.27 (t, J ¼ 5.6 Hz,1H); 7.36 (d, J ¼ 8.1 Hz,1H); 7.66 (d, J ¼ 7.9 Hz,
1H); 8.62 (s, 1H, NH). 13C NMR (101 MHz, CDCl3):
d 13.94, 20.31,
T. Saito, T. Saido, C.D. Keene, K. Jiao, E.D. Roberson, H. Xu, Q. Wang,
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b
-Amyloid
/tau
29.42, 31.32, 32.28, 32.33, 32.36, 33.03, 33.80, 36.40, 36.67, 46.61,
48.80, 54.55, 54.57, 55.99, 63.57, 65.39, 111.34, 111.86, 119.01, 119.63,
122.29, 122.93, 127.64, 136.57, 174.51.
b
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Declaration of competing interest
loid-b peptide hint at new ways to treat Alzheimer’s disease, Front. Aging
Neurosci. 10 (2018), https://doi.org/10.3389/fnagi.2018.00118.
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The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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genetic variants, history of use in the clinic, and potential therapeutic uses,
Acknowledgements
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Abbreviations
A
ACh
b
amyloid
acetylcholine
(h)AChE (human) acetylcholinesterase
b
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
[20] U. Kosak, B. Brus, D. Knez, S. Zakelj, J. Trontelj, A. Pislar, R. Sink, M. Jukic,
ꢀ
M. Zivin, A. Podkowa, F. Nachon, X. Brazzolotto, J. Stojan, J. Kos, N. Coquelle,
AD
Alzheimer’s disease
K. Sałat, J.-P. Colletier, S. Gobec, The magic of crystal structure-based inhibitor
optimization: development of a butyrylcholinesterase inhibitor with pico-
molar affinity and in vivo activity, J. Med. Chem. 61 (1) (2018) 119e139,
https://doi.org/10.1021/acs.jmedchem.7b01086.
BM
Barnes maze
(h)BChE (human) butyrylcholinesterase
CDI
1,10-carbonyldiimidazole
cholinesterase(s)
muscarinic acetylcholine receptor
2-(N-morpholino)ethanesulfonic acid
Morris water maze
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
[21] U. Kosak, B. Brus, D. Knez, R. Sink, S. Zakelj, J. Trontelj, A. Pislar, J. Slenc,
ChE(s)
mAChR
MES
MWM
NMDA
PA
ꢀ
ꢀ
M. Gobec, M. Zivin, L. Tratnjek, M. Perse, K. Sałat, A. Podkowa, B. Filipek,
F. Nachon, X. Brazzolotto, A. Wie˛ckowska, B. Malawska, J. Stojan, I.M. Rascan,
ꢀꢀ
J. Kos, N. Coquelle, J.-P. Colletier, S. Gobec, Development of an in-vivo active
reversible butyrylcholinesterase inhibitor, Sci. Rep. 6 (2016) 39495.
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human serum based on a fluorophore with specific binding affinity for human
serum albumin, Chem. Commun. 55 (97) (2019) 14574e14577, https://
doi.org/10.1039/C9CC07737E.
N-methyl-D-aspartate
passive avoidance
RA
residual activity
ꢁ
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SEM
TFA
standard error of the mean
trifluoroacetic acid
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