Donepezil–melatonin hybrids as butyrylcholinesterase inhibitors: Improving binding affinity through varying mode of linking fragments

Article abstract of DOI:10.1002/ardp.201800194

Hybrid inhibitors of acetyl- and butyrylcholinesterase are compounds that combine structural motifs of two different classical inhibitors, leading to a dual binding ligand. A rapidly growing collection of those compounds involves a wide diversity of structural motifs, but the way of linking two active fragments and its impact on the affinity toward cholinesterases usually remains beyond the extent of investigation. We present hereby a detailed analysis of this aspect using melatonin–donepezil hybrids. A new series of compounds, in which two fragments are connected using a carbamate linker, exhibits excellent activity and selectivity toward butyrylcholinesterase.

Full text of DOI:10.1002/ardp.201800194

Received: 3 July 2018
Revised: 7 September 2018
Accepted: 12 September 2018
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DOI: 10.1002/ardp.201800194
FULL PAPER
Donepezil–melatonin hybrids as butyrylcholinesterase
inhibitors: Improving binding affinity through varying
mode of linking fragments
Iwona Łozińska1
Alwin M. Hartman2
Aleksandra Świerczyńska1
Zuzanna Molęda1
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Anna K. H. Hirsch2,3
Zbigniew Czarnocki1
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1 Faculty of Chemistry, University of Warsaw,
Warsaw, Poland
Abstract
2 Department of Drug Design and
Optimization, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS) –
Helmholtz Centre for Infection Research
(HZI), Saarbrücken, Germany
Hybrid inhibitors of acetyl- and butyrylcholinesterase are compounds that combine
structural motifs of two different classical inhibitors, leading to a dual binding ligand. A
rapidly growing collection of those compounds involves a wide diversity of structural
motifs, but the way of linking two active fragments and its impact on the affinity toward
cholinesterases usually remains beyond the extent of investigation. We present hereby
a detailed analysis of this aspect using melatonin–donepezil hybrids. A new series of
compounds, in which two fragments are connected using a carbamate linker, exhibits
excellent activity and selectivity toward butyrylcholinesterase.
3 Department of Pharmacy, Medicinal
Chemistry, Saarland University, Saarbrücken,
Germany
Correspondence
Prof. Zbigniew Czarnocki, Faculty of
Chemistry, University of Warsaw, ul. Pasteura
1, 02-093 Warsaw, Poland.
K E Y W O R D S
Email: czarnoz@chem.uw.edu.pl
biological activity, drug design, hydrolases, medicinal chemistry, structure–activity relationship
Funding information
Helmholtz-Association's Initiative and
Networking Fund
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1
INTRODUCTION
BChE inhibitors, as well as detailed studies of the structure and
function of this enzyme.[3–5]
Cholinesterases are an important drug target. The corresponding
inhibitors are promising agents in the therapy of Alzheimer's disease
(AD), acting as modulators of acetylcholine levels and halting β-amyloid
deposition.[1] From the two sister enzymes, acetylcholinesterase
(AChE) was for years the better studied one as it functions in synapses
hydrolyzing acetylcholine. Some of its inhibitors have been developed
and introduced successfully to the market (rivastigmine, galantamine,
and donepezil). Meanwhile, it has emerged that better effects can be
reached by the inhibition of butyrylcholinesterase (BChE), which is
much less known than its sister enzyme. It was shown that BChE also
hydrolyzes acetylcholine, induces β-amyloid aggregation,[2] and
enhances its toxicity. At the advanced stages of AD, the levels of
AChE drop dramatically, which may explain the fact, that its inhibitors
do not show therapeutic effects at this point. In contrast, levels of
BChE remain very high. Those facts account for an intensive search for
Both AChE and BChE contain a binding pocket, constituted of two
binding sites – the catalytic active site (CAS) and the peripheral anionic
site (PAS). The classical inhibitors can bind to either CAS or PAS.
In recent years, so-called “hybrid compounds” as cholinesterase
inhibitors have been developed.[6,7] These molecules are built
from two motifs able to bind to CAS and PAS, linked with a bridge
of diverse length and character. The effect is the dual binding to the
enzyme which guarantees high efficacy of halting acetylcholine
hydrolysis. Moreover, blocking the PAS is believed to hamper
β-amyloid aggregation.[8] Therefore, it is expected that the new
cholinesterase inhibitors will be designed to interact simultaneously
with both binding sites of the enzyme.
Taking into account other factors connected with the develop-
ment of AD, it is strongly desirable that the potential drug exhibits
additional therapeutic effects, i.e., neuroprotective antioxidant
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Arch Pharm Chem Life Sci. 2018;e1800194.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800194

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properties. In this respect, melatonin (1) seems to be a perfect
candidate for inspiring the design of new hybrid molecules. This
neurohormone plays a crucial role in the regulation of circadian
rhythm, it is also an extremely potent free-radical scavenger and
activator of antioxidant enzymes.[9] The indolyl moiety of the
melatonin scaffold was shown to bind effectively to the PAS of
cholinesterases,[10] and melatonin-derived fragments have been
successfully incorporated into new hybrid compounds. Those
involve hybrids of melatonin with tacrine,[11] berberine,[12] or
(−)-meptazinol,[13] which, apart from dual binding to cholinesterases,
maintain the antioxidant properties. Advances in the development
of melatonin-derived multitarget hybrids have been recently
reviewed.[14]
donepezil are linked may improve the affinity toward BChE, by analogy
to melatonin–tacrine hybrids. To verify this hypothesis and understand
the origin of the selectivity for BChE, we designed and synthesized a
group of compounds possessing a melatonin moiety bridged to
donepezil through a carbamate bond linked directly to the aromatic
system (Figure 2).
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2
RESULTS
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2.1 Chemistry
The compounds of interest were synthesized in a coupling reaction of
alkylamine-substituted N-benzylpiperidine derivatives 10–14 with a
carbonate derivative of N-acetylserotonin 15 (Scheme 1).
Despite the introduction of new methods in medicinal chemistry
for more effective screening of series of compounds, hybrid
cholinesterase inhibitors are usually developed by classical synthesis
of a lead compound followed by laborious structural modifications
aimed at optimizing the biological activity and/or properties. Those
modifications usually concern substituents on the aromatic moieties
and the length of the linker chain, while the type of the linker seems to
be chosen arbitrarily.
Melatonin-derived building block 15 was obtained according to a
previously developed procedure from N-acetylserotonin and
4-nitrophenyl chloroformate, in the presence of N-methylmorpho-
line.[16] N-Benzyl piperidine derivatives 10 and 11 are commercially
available. Compounds 12–14 were prepared as presented in Scheme 2.
Starting with a diterminal bromo- or chloroalcohol (16a–c)
possessing one carbon fewer than the side chain of the desired
derivative, we protected the hydroxyl group with the THP-ether. The
pyridine moiety was then introduced simultaneously with the side
chain elongation in the reaction with the lithium salt of γ-picoline,
leading to compounds 18a–c. After coupling with benzyl bromide,
the pyridinium salts were selectively reduced to N-benzylpiperidine
derivatives 20a–c. Deprotection under acidic conditions, azide
formation, and Staudinger reaction allowed us to easily obtain the
desired products 12–14.
In the case of tacrine–melatonin hybrids, a comparison between
two different modes of linking active moieties is possible. The first
compounds of this class connect a melatonin-derived moiety through
an amide bond, using the aliphatic tail of the melatonin fragment
(Figure 1a).[15] Previously, we demonstrated that the modification of
the linker to a carbamate bond connected to the aromatic system of
the melatonin moiety generates an excellent selectivity toward BChE
(Figure 1b).[16]
Melatonin was recently connected to donepezil 2, a molecule
which is often successfully incorporated into multitarget hybrids,[17]
creating a group of potent dual-binding cholinesterase inhibitors with
strong antioxidant properties and impact on halting β-amyloid
deposition.[18] We reasoned that changing the way melatonin and
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2.2 Biological evaluation
The inhibitory activity against human acetylcholinesterase (hAChE)
and butyrylcholinesterase (hBChE) derived from erythrocytes and
FIGURE 1 Melatonin (1) and two different types of tacrine–melatonin heterodimers[15,16]

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FIGURE 2 Donepezil (2) and donepezil–melatonin heterodimers. (a) Compounds synthesized by Wu et al.[18] (b) Linkage modification
presented in this work
blood serum, respectively, was determined using spectrophotometric
Ellman's assay.[19] The results are presented in Table 1 and compared
to the most active known melatonin–donepezil hybrid, 4.
connected directly to the carbamate bridge. In case of compounds
6–9, elongation of the linker chain introduces additional aliphatic
interactions, which improves the affinity toward both cholinester-
ases. All compounds with longer linkers exhibit significant
activity.
As can be seen, the only compound lacking activity is
compound 5, in which the N-benzylpiperidinyl skeleton is
SCHEME 1 Synthesis of compounds 5–9. (a) DMAP, DCM, RT, 71–92%

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SCHEME 2 Synthesis of compounds 12–14. (a) PPTS, DCM, RT, 97–100%; (b) nBuLi, THF, RT, 61–91%; (c) toluene, 60°C; (d) H2, PtO2,
EtOH, RT; (e) HClaq., MeOH, EtOH, RT, 53–68%; (f) DPPA, DBU, NaN3, DMF, RT, 76–80%; (g) PPh3, H2O, THF, RT, 80–95%
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2.3 Modeling studies
evaluate the docking results, the program SeeSAR, with the in-built
scoring function HYDE, was used.[21]
To be able to investigate the binding modes, for the different ways in
which donepezil (2) and melatonin (1) can be linked, we used two
computer programs. The X-ray crystal structures of hAChE (PDB code:
4EY7) and hBChE (PDB code: 4TPK) in complex with donepezil (2) and
a naphthamide derivative, are used, respectively, for our docking
studies. The docking module FlexX in the LeadIT suite was used to
dock the compounds in the active site of hAChE and hBChE.[20] To
As compared to compound 4 reported by Wang et al.,[18] we
observe a slightly different binding mode, showing a dependence of
the pose on the way donepezil (2) and melatonin (1) are linked.
Predominately for hAChE, the docked inhibitors show π–π stacking
interactions of W286 and the indolyl part and W86 with the benzene
ring, where W86 as well as Y337 are engaged in a cation–π interaction
with the tertiary amine. Our compounds resemble more the melatonin
structure to which donepezil is linked via a carbamate.
Figure 3 shows the overlap of the best poses for each of our
dimers, in the pocket of hAChE with donepezil as a reference ligand
(see also Figure 4). It can be seen that the melatonin part of our
compounds is predicted to remain in the CAS site, having interactions
with W86, E202, and S203. The donepezil part, the piperidinyl and
benzyl moieties, are directed toward Y341 and W286. Due to the
increased linker length, the donepezil part is gradually more solvent-
exposed.
TABLE 1 Inhibitory activity of the melatonin−donepezil hybrids 5–9
against human acetyl- and butyrylcholinesterase
IC50 [μM]a
hAChE
hBChE
Selectivity indexb
>0.9
5
>10
9.034 1.753
0.176 0.017
0.015 0.002
0.018 0.001
0.003 0.0003
0.056 0.01
6
2.222 0.248
0.753 0.036
0.725 0.024
0.621 0.016
0.273 0.05
0.079
7
0.020
From the docking studies the structures can be ranked according
to their increasing affinity: 5 ≈ 7 < 6 ≈ 8 ≈ 9.
8
0.025
9
0.005
For hBChE it is expected that the benzene part predominately
interacts with G117, V288, L286, W231, and F398, the indolyl moiety
will most probably show lipophilic interactions with W82. It is also
expected that the NH of the piperidinyl will form a hydrogen bond with
4[17]
0.21
aValues are the mean SD obtained from three experiments.
bIC50 (hBuChE)/IC50 (hAChE).

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FIGURE 3 Cocrystal structure of hAChE with donepezil (PDB
code: 4EY7) and the best pose of each dimer. Color code: protein
surface: gray; interacting residues: sticks; inhibitor skeletons:
donepezil: light blue; 5: red; 6: green; 7: blue; 8: yellow; 9: magenta.
Figures of this type were generated with PyMOL (Schrödinger)
FIGURE 5 Cocrystal structure of hBChE with a naphthamide
derivative (PDB code: 4TPK) and the best pose of each dimer. Color
code: protein surface: gray; interacting residues: sticks; inhibitor
skeletons: donepezil: light blue; 5: red; 6: green; 7: blue; 8: yellow;
9: magenta
the carbonyl oxygen of P285 (Figures 5 and 6). Compared to the amide
in structure 4 of Wang et al.,[18] in hBChE, it is expected that the
activity of the carbamate does not change much. The extra binding
activity might arise from additional interactions stemming from the
amide linker, from the melatonin moiety. This part is not present in
the parent compound reported by Wu et al.[18] The rest of the molecule
shows a similar pose, the indolyl is directed toward W82 and the
benzene toward G117.
pocket, near the PAS site, while in the case of compound 4, the PAS is
occupied by the indolyl moiety and the donepezil part interacts with
Y337 and W86 located in the pocket. A conclusion can be drawn that
linking the melatonin fragment through its phenyl oxygen atom
notably reduces possible interactions with AChE.
This effect is not observed in case of BChE. Compounds 6–9
exhibit very strong inhibitory activity, which can be explained by the
fact that the interactions similar to those of compound 4 are enhanced
by additional binding of the acetyl moiety, not present in the
compounds of Wang et al.[18] The strongest inhibition of BChE was
achieved in case of compound 9, which additionally exhibits excellent
selectivity for this type of enzyme.
From the docking studies, the structures can be ranked according
to their increasing affinity: 5 ≤ 6 < 9 ≤ 7 ≤ 8.
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3
DISCUSSION
Derivatives 5–9 present a significantly lower affinity toward AChE
than the previously described donepezil–melatonin hybrids.[18] This
arises probably from the different orientation of the ligand – in this
case the donepezil part of the hybrids is located at the end of the
FIGURE 4 Cocrystal structure of hAChE with donepezil (PDB
code: 4EY7) and the best pose of 9. Color code: protein surface:
gray; interacting residues: sticks; inhibitor skeletons: C: yellow; N:
blue; O: red; donepezil: light blue; H-bonds, below 3.2 Å: dashed
lines
FIGURE 6 Cocrystal structure of hBChE with a naphthamide
derivative (PDB code: 4TPK) and the best pose of 9. Color code:
protein surface: gray; interacting residues: sticks; inhibitor skeletons:
C: yellow; N: blue; O: red; naphthamide: light blue; H-bonds, below
3.2 Å: dashed lines

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4
CONCLUSIONS
method. Column chromatography was performed on silica gel MN-
Kieselgel 100–200 mesh ASTM (Macherey-Nagel GmbH & Co. KG,
Düren, Germany) and aluminum oxide (Fluka, St. Louis, MO, USA)
using the indicated eluents. The progress of the reactions was
followed by thin-layer chromatography using plates with silica gel
and aluminum oxide with methanol, methylene chloride, ethyl
acetate, and 20% aqueous ammonia as eluents. Anhydrous magne-
sium sulfate or sodium sulfate was used as drying agent for the
organic phases. Organic solvents were removed under reduced
pressure at 40°C. The melting points were measured with Kofler's
apparatus (Boetius HMK type).
New potent and selective inhibitors of BChE have been developed. A
detailed study of their binding mode allowed us to understand the
origin of this phenomenon. It was demonstrated that in case of
melatonin–donepezil hybrids, the modification of the linker to a
carbamate bond connected to the aromatic system of the melatonin
moiety improves the affinity toward BChE in the same way as in the
case of melatonin–tacrine hybrids. These results open new promising
perspectives for the search for new neurotherapeutics based on
melatonin-derived fragments.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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5
EXPERIMENTAL
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5.1 Chemistry
5.1.2 Synthesis of compounds 15, 11, and 23
3-(Acetamidomethyl)-1H-indol-5-yl 4-nitrophenylcarbonate (15) was
obtained according to the procedure described in our previous
work.[16] N-Benzylpiperidine derivative 11 was prepared according
to Scheme 3. Ethyl (2E)-3-(pyridin-4-yl)prop-2-enoate (23) was
prepared according to the methods reported in the literature, from
the commercially available isonicotinaldehyde.[22]
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5.1.1 General
Serotonin hydrochloride was obtained from Alfa Aesar GmbH & Co.
KG (Karlsruhe, Germany). Other reagents were obtained from Sigma–
Aldrich Co. (St. Louis, MO, USA). The 1H NMR and 13C NMR spectra
were recorded on a Bruker AVANCE apparatus, operating at 300 MHz
(1H NMR) and 75 MHz (13C NMR) and VARIAN Unity Plus (respectively
200, 50 MHz) using tetramethylsilane (TMS) as an internal standard.
Chemical shifts are given in ppm. The following abbreviations were
used to indicate the peak multiplicity and shape: s, singlet; d, doublet; t,
triplet; m, multiplet; br, broad. High-resolution mass spectra were
recorded on a Quatro LC AMD 604 apparatus using TOF MS ES+
1-Benzyl-4-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyridin-1-ium
bromide (24)
To ethyl (2E)-3-(pyridin-4-yl)prop-2-enoate (23) (2 g; 11.3 mmol)
dissolved in toluene (30 mL), (bromomethyl)benzene (1.47 mL;
2.12 g; 12.4 mmol) was added. The reaction was left to stir for 14 h
SCHEME 3 Synthesis of compound 11. (a) K2CO3, EtOH, RT, 90%; (b) toluene, RT, 77%; (c) NaBH4, EtOH, RT, 56%; (d) H2, PtO2, EtOH,
RT, 99%; (e) LiAlH4, THF, RT, 99%; (f) DPPA, DBU, NaN3, DMF, RT, 82%; (g) PPh3, H2O, THF, RT, 97%

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at 60°C, under an inert atmosphere. The product precipitated and was
recrystallized from a mixture of dichloromethane and ethyl acetate.
Compound 24 was obtained as a yellow solid in 77% yield (2.3 g).
1H NMR (300 MHz, CDCl3) δ (ppm): 9.74–9.60 (m, 2H, 2Harom);
8.17–8.07 (m, 2H, 2Harom); 7.82–7.68 (m, 2H, 2Harom); 7.60 (d,
slowly. The reaction mixture was stirred for 30 min and the
corresponding compound 17a–c (10 mmol) dissolved in THF (5 mL)
was added dropwise. The reaction was left to stir for 1 h at −78°C, it
was allowed to reach room temperature. It was stirred for an additional
20 h. After that time, aqueous solution of NH4Cl was added and the
solution was extracted three times with ethyl acetate. The combined
organic layers were dried over anhydrous MgSO4 and filtered. The
product was purified by column chromatography (SiO2, 20–60%
EtOAc in hexane).

J = 16.0 Hz; 1H, β-CH ); 7.40–7.32 (m, 3H, 3Harom), 6.83 (d,

J = 16.0 Hz, 1H, α-CH ), 6.31 (s, 2H, CH2Ph), 4.28 (q, J = 7.1 Hz,
2H, OCH2CH3), 1.34 (t, J = 7.1 Hz, 3H, OCH2CH3). 13C NMR (75 MHz,
CDCl3) δ (ppm): 164.68; 150.37; 145.64; 137.20; 133.17; 130.10;
130.05; 129.83; 129.75; 126.18; 63.78; 61.86; 14.29. MS EI(+) (m/z):
268 [M]+.
18a: The product was obtained as yellow oil in 70% yield (1.75 g).
1H NMR (300 MHz, CDCl3) δ (ppm): 8.51–8.42 (m, 2H, 2Harom);
7.13–7.05 (m, 2H, 2Harom); 4.55 (t, J = 3.5 Hz, 1H); 3.91–3.66 (m, 2H);
3.55–3.30 (m, 2H); 2.62 (t, J = 7.5 Hz, 2H); 1.98–1.29 (m, 12H). MS EI
(+) (m/z): 250 [M+H]+.
Ethyl (2E)-3-(1-benzyl-1,2,3,6-tetrahydropyridin-4-yl)prop-2-
enoate (25)
To
1-benzyl-4-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyridin-1-ium
18b: The product was obtained as yellow oil in 61% yield (1.61 g).
Analytical data are in agreement with the literature reports.[27]
18c: The product was obtained as yellow oil in 91% yield (2.53 g).
1H NMR (300 MHz, CDCl3) δ (ppm): 8.44 (d, J = 6.0 Hz, 1H, Harom); 8.43
(d, J = 3.0 Hz, 1H, Harom); 7.06 (d, J = 6.0 Hz, 1H, Harom); 7.05 (d,
J = 3.0 Hz, 1H, Harom); 4.53 (t, J = 3.0 Hz, 1H); 3.87–3.78 (m, 1H);
3.74–3.64 (m, 1H); 3.51–3.41 (m, 1H); 3.39–3.29 (m, 1H); 2.55 (t,
J = 6.0 Hz, 2H); 1.70–1.43 (m, 10H); 1.27–1.37 (m, 6H). 13C NMR
(75 MHz, CDCl3) δ (ppm): 151.7; 149.5; 123.8; 98.8; 67.5; 62.3; 35.2;
30.7; 30.2; 29.6; 29.2; 29.0; 26.1; 25.4; 19.7.
bromide 24 (0.83 g; 2.5 mmol) dissolved in ethanol (15 mL), NaBH4
(0.19 g; 5.0 mmol) was added slowly (over 0.5 h) at 0°C. The reaction
was left to stir for 90 min. The solvent was evaporated by half and
DCM (30 mL) was added. The solution was washed with water and
brine, then dried over anhydrous Na2SO4, and filtered. The product
was purified by column chromatography (SiO2, 15% EtOAc in hexane)
to give 25 as a colorless oil in 56% yield (0.38 g).
1H NMR (300 MHz, CDCl3)
δ
(ppm): 7.45–7.09 (m, 6H,


5Harom + CH ); 6.04 (t, J = 3.5 Hz, 1H, CH ); 5.73 (d, J = 15.8 Hz,

1H, CH ); 4.16 (q, J = 7.2 Hz, 2H, OCH2CH3); 3.56 (s, 2H, CH2Ph);
3.10 (br.d, J = 3.2 Hz, 2H); 2.60 (t, J = 5.7 Hz, 2H); 2.27–2.17 (m, 2H);
1.25 (t, J = 7.1 Hz, 3H, OCH2CH3). 13C NMR (75 MHz, CDCl3) δ (ppm):
172.8; 146.41; 138.16; 135.03; 133.41; 129.34; 128.53; 127.41;
116.13; 62.81; 60.49; 53.57; 49.39; 25.38; 14.53. MS EI(+) (m/z): 272
[M+H]+.
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5.1.5 General procedure for the synthesis of
compounds 19a–c
To the corresponding compound 18 (3.0 mmol), dissolved in toluene
(10 mL), benzyl bromide (1.1 eq) was added. The reaction was left to
stir overnight at 60°C. The solvent was evaporated and the product
was used for the next step without purification.
Ethyl 3-(1-benzylpiperidin-4-yl)propanoate (26)
Ethyl (2E)-3-(1-benzyl-1,2,3,6-tetrahydropyridin-4-yl)prop-2-enoate
25 (310 mg; 1.29 mmol) was dissolved in ethanol (30 mL) and PtO2
(50 mg) was added. Air was replaced by hydrogen and the reaction was
left to stir overnight. The reaction mixture was filtered through Celite.
The solvent was evaporated and the product was obtained as a
colorless oil in 99% yield (351 mg). Analytical data are in agreement
with the literature reports.[23]
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5.1.6 General procedure for the synthesis of
compounds 20a–c
The corresponding crude compound 19a–c (3.0 mmol) was dissolved in
absolute ethanol (100 mL) and PtO2 (300 mg) was added. Air was
replaced with hydrogen and the reaction was left to stir for 3 h. The
reaction mixture was filtered through Celite and the solvent was
evaporated by half. Ten percent HClaq was added dropwise until pH = 3
was reached. After 1 h, NaHCO3aq was added, and the mixture was
extractedfourtimeswithchloroform. Thecombinedorganicphaseswere
washed with water, brine, dried over anhydrous MgSO4, and filtered.
20a: The product was obtained as a yellowish oil in 60% yield
(470 mg) over three steps. Analytical data are in agreement with the
literature reports.[28]
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5.1.3 Synthesis of compounds 27, 28, and 11 and
precursors 17a–c
Compounds 27, 28, and 11 were prepared according to the methods
reported in the literature.[24] The precursors 17a–c were prepared
according to the methods reported in the literature.[25,26]
20b: The product was obtained as a yellowish oil in 68% yield
(561 mg) over three steps. Analytical data are in agreement with the
literature reports.[28]
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5.1.4 General procedure for the synthesis of
compounds 18a–c
Freshly distilled and dried over sodium carbonate γ-picoline (1.1 eq)
was dissolved in anhydrous THF (25 mL), cooled down to −78°C under
inert atmosphere (Ar) and n-BuLi (2.5 M in hexane, 1.5 eq) was added
20c: The product was obtained as in 53% yield (460 mg) over three
steps; 1H NMR (300 MHz, CDCl3) δ (ppm): 7.31–7.27 (m, 5H, 5Harom);
3.60 (t, J = 6.0 Hz, 1H, CH2OH); 3.47 (s, 2H, CH2Ph); 2.85 (d,

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J = 12.0 Hz, 2H); 1.90 (t, J = 12.0 Hz, 2H); 1.67–1.47 (m, 4H); 1.36–1.13
(m, 14H). 13C NMR (75 MHz, CDCl3) δ (ppm): 138.26; 129.33; 128.11;
126.91; 63.51; 68.98; 53.95; 36.57; 35.68; 32.80; 32.32; 29.82; 29.43;
26.72; 25.74. MS EI(+) (m/z): 290 [M+H]+.
13: The product was obtained as a yellowish oil in 80% yield
(88 mg); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.33–7.23 (m, 5H,
5Harom); 3.49 (s, 2H, CH2Ph); 2.87 (d, J = 12.0 Hz, 2H); 2.71 (br.s, 2H,
CH2N3); 2.60 (br.s, 2H, NH2); 1.92 (t, J = 12.0 Hz, 2H); 1.63 (m, 2H);
1.48 (m, 2H); 1.35–1.15 (m, 11H). 13C NMR (75 MHz, CDCl3) δ (ppm):
138.17; 129.38; 128.12; 126.95; 63.53; 53.94; 50.59; 41.50; 36.50;
35.66; 32.28; 29.58; 26.78; 26.70. MS EI(+) (m/z): 275 [M+H]+.
14: The product was obtained as a yellowish oil in 82% yield
(94 mg); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.31–7.22 (m, 5H,
5Harom); 3.48 (s, 2H, CH2Ph); 2.86 (d, J = 12.0 Hz, 2H); 2.67 (t,
J = 7.0 Hz, 2H, CH2N3); 2.02 (br.s, 2H, NH2); 1.91 (t, J = 12.0 Hz, 2H);
1.61 (m, 2H); 1.43 (m, 2H); 1.27–1.20 (m, 13H). 13C NMR (75 MHz,
CDCl3) δ (ppm): 138.43; 129.31; 128.09; 126.87; 63.58; 53.99; 42.07;
36.60; 35.73; 32.39; 29.84; 29.48; 26.87; 26.75; 18.45. MS EI(+) (m/z):
289 [M+H]+.
|
5.1.7 General procedure for the synthesis of
compounds 21a–c
To the corresponding compound 20 (0.5 mmol) dissolved in DMF
(10 mL) and stirred at 0°C, the other reagents were added in sequence:
DPPA (3 eq), DBU (3 eq) and after 30 min, NaN3 (3 eq) were added. The
reaction was let to stir for 4 h at 90°C. After that time, the solvent was
evaporated, DCM was added, and the solution was washed with water
and brine. The product was purified by column chromatography (SiO2,
10–50% EtOAc in hexane).
21a: The product was obtained as a colorless oil in 80% yield
(114 mg); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.31–7.26 (m, 5H,
5Harom); 3.48 (s, 2H, CH2Ph); 3.25 (t, J = 7.0 Hz, 2H, CH2N3); 2.86 (br.d,
J1 = 9.0 Hz, 2H); 1.91 (br.t, J = 9.0 Hz, 2H); 1.64–1.54 (m, 4H); 1.33–
1.21 (m, 9H). 13C NMR (75 MHz, CDCl3) δ (ppm): 138.35; 129.34;
128.10; 126.89; 63.58; 62.80; 53.96; 36.58; 35.66; 32.80; 32.38;
26.62; 26.02. MS EI(+) (m/z): 287 [M+H]+.
|
5.1.9 General procedure for the synthesis of
compounds 5–9
To the corresponding amine 10–14 (0.25 mmol) dissolved in anhy-
drous THF (10 mL), DMAP (1.2 eq) was added. To this mixture ester 15
(1 eq) was added. The reaction was let to stir in anhydrous conditions,
under an inert atmosphere (Ar), at room temperature, for 12 h. The
product was purified by column chromatography (SiO2, 20% MeOH in
CHCl3, saturated by NH3 for 5–7, 5% MeOH in DCM for 8 and 9).
5: The product was obtained as white glass-like solid in 92% yield
(100 mg); 1H NMR (300 MHz, CD3OD) δ (ppm): 7.45–7.20 (m, 7H,
5Hbenzyl + 2Hindolyl); 7.13 (d, J = 1.0 Hz, 1H, Hindolyl); 6.84 (dd,
J1 = 8.6 Hz, J2 = 2.2 Hz, 1H, Hindolyl); 3.57 (s, 2H, CH2Ph); 3.56–3.48
(m, 1H, CH); 3.45 (t, J = 7.2 Hz, 2H, CH2NHCOCH3); 3.04–2.82 (m, 4H),
2.19 (t, J = 11.6 Hz, 2H); 2.03–1.93 (m, 2H); 1.92 (s, 3H, CH3); 1.75–
1.51 (m, 2H). 13C NMR (75 MHz, CD3OD) δ (ppm): 171.88; 156.39;
144.03; 136.88; 134.37; 129.40; 127.94; 127.58; 127.12; 123.54;
115.50; 112.24; 111.01; 110.30; 62.51; 51.89; 51.89; 40.16; 31.18;
24.72; 21.20. HR MS ES(+) (m/z) calculated for C25H30N4O3Na
([M+Na]+) 457.2216. Found: 457.2233.
21b: The product was obtained as a yellowish oil in 78% yield
(117 mg); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.37–7.16 (m, 5H,
5Harom); 3.47 (br.s, 2H, CH2Ph); 3.23 (t, J = 6.0 Hz, 2H, CH2N3); 2.85 (d,
J = 9.0 Hz, 2H); 1.90 (t, J = 9.0 Hz, 2H); 1.65–1.52 (m, 4H); 1.36–1.15
(m, 11H). 13C NMR (75 MHz, CDCl3) δ (ppm): 138.06; 129.87; 128.11;
125.62; 63.58; 54.00; 51.49; 36.51; 35.72; 32.43; 29.40; 28.84; 26.73;
26.65. MS EI(+) (m/z): 301 [M+H]+.
21c: The product was obtained as a yellowish oil in 76% yield
(119 mg); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.32–7.27 (m, 5H,
5Harom); 3.46 (s, 2H, CH2Ph); 3.23 (t, J = 6.0 Hz, 2H, CH2N3); 2.84 (d,
J = 9.0 Hz, 2H); 1.86 (t, J = 9.0 Hz, 2H); 1.66–1.52 (m, 4H); 1.35–1.15
(m, 13H). 13C NMR (75 MHz, CDCl3) δ (ppm): 138.64; 129.23; 128.08;
126.8; 63.59; 54.02; 51.47; 36.59; 35.74; 32.46; 29.70; 29.15; 28.83;
26.70; 26.68. MS EI(+) (m/z): 315 [M+H]+.
6: The product was obtained as white glass-like solid in 83% yield
(99 mg); 1H NMR (300 MHz, CD3OD) δ (ppm): 7.44–7.22 (m, 7H,
5Hbenzyl + 2Hindolyl); 7.12 (s, 1H, Hindolyl); 6.84 (dd, J1 = 8.7 Hz,
J2 = 2.3 Hz, 1H, Hindolyl); 3.63 (s, 2H, CH2Ph); 3.44 (t, J = 7.2 Hz, 2H,
CH2NHCOCH3); 3.17 (t, J = 7.0 Hz, 2H); 2.98 (br.d, J = 11.4 Hz, 2H);
2.90 (t, J = 7.30 Hz, 2H); 2.16 (t, J = 11.3 Hz, 2H); 1.91 (s, 3H, CH3);
1.85–1.50 (m, 4H); 1.50–1.10 (m, 5H). 13C NMR (75 MHz, CD3OD) δ
(ppm): 171.86; 157.12; 144.12; 135.54; 134.35; 129.80; 128.05;
128.05; 127.49; 123.58; 115.53; 112.24; 111.09; 110.32; 62.51;
53.15; 40.75; 40.17; 34.75; 33.11; 31.06; 26.65; 24.76; 21.29. HR MS
ES(+) (m/z) calculated for C28H36N4O3H ([M+H]+) 477.2860. Found:
477.2859.
|
5.1.8 General procedure for the synthesis of
compounds 12–14
To the corresponding azide 21 (0.4 mmol) dissolved in THF (10 mL),
PPh3 (1.5 eq) and water (4 eq) were added, the reaction was left to stir
for 2.5 h under reflux. The solvent was evaporated and the product
was purified by column chromatography (SiO2, 0–20% MeOH in
CHCl3 to 20% MeOH in CHCl3 saturated with NH3).
12: The product was obtained as a yellowish oil in 95% yield
(99 mg); 1H NMR (300 MHz, CDCl3) δ (ppm): 7.37–7.18 (m, 5H,
5Harom); 3.48 (s, 2H, CH2Ph); 2.87 (d, J = 11.1 Hz, 2H); 2.69 (t,
J = 7.0 Hz, 2H, CH2N3); 2.00 (br.s, 2H, NH2); 1.92 (t, J = 10.8 Hz, 2H);
1.84–1.57 (m, 2H); 1.35–1.37 (m, 2H); 1.36–1.16 (m, 9H). 13C NMR
(75 MHz, CDCl3) δ (ppm): 138.61; 129.22; 128.08; 126.82; 63.58;
54.00; 42.03; 36.60; 35.70; 33.42; 32.45; 27.11; 26.64. MS EI(+) (m/z):
261 [M+H]+, 283 [M+Na]+.
7: The product was obtained as a colorless oil in 79% yield
(100 mg); 1H NMR (300 MHz, CDCl3) δ (ppm): 8.48 (s, 1H, NHindolyl);
7.44–7.17 (m, 7H, 5Hbenzyl + 2Hindolyl); 7.01–6.83 (m, 2H, 2Hindolyl);
5.69 (br.s, 1H, NH); 5.13 (t, J = 5.9 Hz; 1H, NHcarbam); 3.62 (s, 2H,
CH2Ph); 3.52–3.40 (m, 2H); 3.29–3.22 (m, 2H); 3.04–2.71 (m, 4H);

ŁOZIŃSKA ET AL.
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|
1.96 (t, J = 10.7 Hz, 2H); 1.90 (s, 3H, CH3); 1.75–1.54 (m, 4H); 1.46–
1.06 (m, 9H). 13C NMR (75 MHz, CDCl3) δ (ppm): 170.29; 162.31;
155.81; 144.44; 134.08; 129.44; 128.20; 127.65; 127.10; 123.40;
116.58; 113.09; 111.59; 111.03; 63.37; 53.85; 39.79; 36.41; 35.55;
32.14; 32.14; 29.89; 26.99; 26.43; 25.16; 23.32. HR MS ES(+) (m/z)
calculated for C30H40N4O3H ([M+H]+) 505.3179. Found: 505.3172.
8: The product was obtained as a colorless oil in 79% yield
(102 mg); 1H NMR (300 MHz, CDCl3) δ (ppm): 8.66 (s, 1H, NHindolyl);
7.31–7.18 (m, 7H, 5Hbenzyl + 2Hindolyl); 6.91–6.85 (m, 2H, 2Hindolyl);
5.76 (br.s, 1H, NH); 5.19 (t, J = 5.19 Hz; 1H, NHcarbam); 3.48 (s, 2H,
CH2Ph); 3.47–3.43 (m, 2H); 3.28–3.22 (m, 2H); 2.91–2.77 (m, 4H);
1.92 (t, J = 10.7 Hz, 2H); 1.88 (s, 3H, CH3); 1.67–1.51 (m, 4H); 1.40–
1.15 (m, 11H). 13C NMR (75 MHz, CDCl3) δ (ppm): 170.55; 156.08;
144.54; 138.63; 134.28; 129.49; 128.31; 127.80; 127.08; 123.64;
116.65; 113.11; 111.80; 111.15; 63.74; 54.17; 41.51; 39.96; 36.72;
35.88; 32.56; 30.06; 29.70; 26.97; 26.88; 25.28; 23.45. HR MS ES(+)
(m/z) calcd. for C31H42N4O3H ([M+H]+) 519.3330. Found: 519.3327.
9: The product was obtained as a colorless oil in 71% yield (95 mg);
1H NMR (300 MHz, CDCl3) δ (ppm): 8.94 (s, 1H, NHindolyl); 7.29–7.17
(m, 7H, 5Hbenzyl + 2Hindolyl); 6.92–6.85 (m, 2H, 2Hindolyl); 5.82 (br.s, 1H,
NH); 5.24 (t, J = 5.9 Hz; 1H, NHcarbam); 3.50 (s, 2H, CH2Ph); 3.50–3.41
(m, 2H); 3.23 (q, J = 6.0 Hz, 2H); 2.89–2.77 (m, 4H); 1.93 (m, 2H); 1.91
(s, 3H, CH3); 1.65–1.49 (m, 4H); 1.37–1.13 (m, 13H). 13C NMR
(75 MHz, CDCl3) δ (ppm): 170.30; 155.84; 144.37; 138.55; 134.17;
129.28; 128.10; 127.63; 126.85; 123.52; 116.45; 112.90; 111.63;
110.96; 63.59; 54.02; 41.33; 39.03; 36.61; 35.74; 32.44; 29.90; 29.79;
29.2; 26.79; 26.74; 25.15; 23.30. HR MS ES(+) (m/z) calcd. for
log concentration. The inhibitory activity was calculated as IC50
values.
|
5.3 Docking experiments
For the docking, the binding site in the protein was restricted to 6.5 Å
around the co-crystallized ligand, and the 30 top-scored FlexX
solutions were retained, subsequently post-scored with the program
SeeSAR.[20]
ACKNOWLEDGMENT
A.K.H.H. gratefully acknowledges funding from the Helmholtz-
Association's Initiative and Networking Fund.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
ORCID
Zuzanna Molęda
Alwin M. Hartman
Anna K. H. Hirsch
Zbigniew Czarnocki
http://orcid.org/0000-0002-0868-2216
http://orcid.org/0000-0002-0987-9088
http://orcid.org/0000-0001-8734-4663
http://orcid.org/0000-0002-6581-9282
C
32H44N4O3H ([M+H]+) 533.3486. Found: 533.3490.
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