Propafenone analogue with additional H-bond acceptor group shows increased inhibitory activity on P-glycoprotein

Article abstract of DOI:10.1002/ardp.201900269

P-glycoprotein (P-gp) is an ATP-dependent efflux pump that has a marked impact on the absorption, distribution, and excretion of therapeutic drugs. As P-gp inhibition can result in drug–drug interactions and altered drug bioavailability, identifying molec

Full text of DOI:10.1002/ardp.201900269

Received: 12 September 2019
DOI: 10.1002/ardp.201900269
Revised: 21 November 2019
Accepted: 8 December 2019
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F U L L P A P E R
PropafenoneanaloguewithadditionalH‐bondacceptorgroup
shows increased inhibitory activity on P‐glycoprotein
Anna Cseke1*
Kerstin Vogl1
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Theresa Schwarz1*
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Sankalp Jain1,2
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Simon Decker1
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Ernst Urban1 Gerhard F. Ecker1
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1Department of Pharmaceutical Chemistry,
University of Vienna, Vienna, Austria
Abstract
2National Center for Advancing Translational
Sciences, National Institutes of Health,
Rockville, MD, USA
P‐glycoprotein (P‐gp) is an ATP‐dependent efflux pump that has a marked impact on
the absorption, distribution, and excretion of therapeutic drugs. As P‐gp inhibition can
result in drug–drug interactions and altered drug bioavailability, identifying molecular
properties that are linked to inhibition is of great interest in drug development. In
this study, we combined chemical synthesis, in vitro testing, quantitative
structure–activity relationship analysis, and docking studies to investigate the role
of hydrogen bond (H‐bond) donor/acceptor properties in transporter–ligand
interaction. In a previous work, it has been shown that propafenone analogs with a
4‐hydroxy‐4‐piperidine moiety exhibit a generally 10‐fold higher P‐gp inhibitory
activity than expected based on their lipophilicity. Here, we specifically expanded the
data set by introducing substituents at position 4 of the 4‐phenylpiperidine moiety to
assess the importance of H‐bond donor/acceptor features in this region. The results
suggest that indeed an H‐bond acceptor, such as hydroxy and methoxy, increases the
affinity by forming a H‐bond with Tyr310.
Correspondence
Gerhard F. Ecker, Department of
Pharmaceutical Chemistry, University of
Vienna, Althanstrasse 14, 1090 Vienna,
Austria.
Email: gerhard.f.ecker@univie.ac.at
Funding information
European Union’s Horizon 2020 Research and
Innovation Programme, Grant/Award Number:
681002 (EU‐ToxRisk); Austrian Science Fund,
Grant/Award Numbers: #F03502, SFB35
K E Y W O R D S
inhibitor, molecular docking, P‐glycoprotein, propafenone, quantitative structure–activity
relationship
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1
INTRODUCTION
barrier, where it mediates the uptake and elimination of endogenous
compounds and xenobiotics.[4] In addition, overexpression of P‐gp in
P‐glycoprotein (P‐gp) is an extensively studied efflux pump belonging
to the ABC (ATP binding cassette) transporter superfamily.[1] This
transporter protein is integrated into the plasma membrane and uses
the energy of adenosine triphosphate (ATP) hydrolysis to extrude a
wide variety of substances from the intracellular to the extracellular
compartment.[2,3] In the human body, P‐gp is physiologically
expressed in tissues with barrier and/or excretory functions,
including the liver, kidney, gastrointestinal tract and blood–brain
cancerous cells has been recognized as a major cellular mechanism of
multidrug resistance.[5]
On the basis of its expression profile and broad substrate
specificity, P‐gp is involved in the absorption, distribution, and
excretion of therapeutic drugs.[6] Co‐administration of a transported
drug and a P‐gp inhibitor can lead to elevated drug levels in the
plasma and in the organs defended by blood–tissue barriers, which
poses the risk of organ toxicity.[7,8] On this account, both the US Food
and Drug Administration (FDA) and the European Medicines Agency
(EMA) recommend drug candidates to be routinely screened for their
*Anna Cseke and Theresa Schwarz contributed equally to this work.
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This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
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© 2020 The Authors. Archiv der Pharmazie published by Wiley-VCH Verlag GmbH & Co. KGaA on behalf of Deutsche Pharmazeutische Gesellschaft
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Arch Pharm Chem Life Sci. 2020;e1900269.
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https://doi.org/10.1002/ardp.201900269

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SCHEME 1 Synthetic route to compounds 4a–g
capacity to inhibit P‐gp and the analogous breast cancer‐resistant
protein BCRP.[9–11]
the 4‐hydroxy‐4‐phenylpiperidine moiety. To this end, we synthe-
sized six novel propafenone derivatives sharing the same scaffold
with 4g, but bearing varying functional groups in position 4 of the
4‐phenylpiperidine substituent and tested their inhibitory activity
on P‐gp in vitro.
Over the past four decades, a considerable effort has been made
to characterize the molecular basis of the interaction between P‐gp
and its inhibitors.[12,13] Although within analogous sets of compounds
(e. g. propafenone, tetrahydroisoquinoline, tariquidar and chalcone
derivatives, alkaloids, and flavonoids), a clear structure–activity
relationship (SAR) pattern was observed, a general and conclusive
model is still missing.[14–19] Numerous SAR studies suggested that
there is a strong correlation between lipophilicity and P‐gp inhibitory
potency and that active compounds commonly possess at least one
aromatic ring, a basic nitrogen atom, and several hydrophobic
regions.[12] Preference of P‐gp toward lipophilic compounds can be
explained by the widely accepted model of substrate transport, which
proposes that P‐gp extracts its ligands directly from the inner leaflet
of the plasma membrane.[20,21] In addition, ligand‐ and structure‐
based approaches suggest that hydrogen‐bond (H‐bond) acceptor
properties are important for P‐gp inhibitors.[21,22]
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2
RESULTS AND DISCUSSION
Synthesis
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2.1
The propafenone derivatives 3a–g were synthesized by ring‐opening
reaction of oxirane 1 with appropriate substituted 4‐phenylpiperidines
2a–g and subsequently transformed to their hydrochlorides 4a–g,
as described previously by Chiba et al.[24] (see Scheme 1). While 4‐
phenylpiperidines 2d–g were commercially available, 4‐phenylpiperidines
2a–c were synthesized either from 4‐hydroxy‐4‐phenylpiperidine 6 or
1‐benzyl‐4‐piperidone 10 (see Schemes 2 and 3).
Propafenone analogs are potent inhibitors of P‐gp mediated
drug efflux, exhibit a well‐defined SAR pattern, and therefore,
represent an excellent tool for investigating the molecular
features triggering P‐gp inhibition.[23] Chiba et al.[24] performed
quantitative structure–activity relationship (QSAR) analyses on a
Following Barker et al.,[26] the commercially acquired amine 6 was
protected by Boc2O to yield carbamate 7. The free hydroxy group of 7
was activated using sodium hydride and methylated by methyl iodide to
OH
OH
series of highly related propafenone analogs and found
a
Ph
Ph
N
tBuO
N
significant correlation between lipophilicity and inhibitory activity
(log[1/EC50]). They also revealed that piperazine and piperidine
H
O
6
7
analogous propafenone derivatives, which bear
a 4‐hydroxy‐
4‐phenylpiperidine moiety, are generally 10‐fold more active than
equilipophilic compounds without a hydroxy group in position 4 of
the piperidine ring. On the basis of the docking study of a small set
of propafenone analogs into P‐gp homology models, Klepsch
et al.[23] proposed the formation of an H‐bond between the
4‐hydroxy group of the 4‐hydroxy‐4‐phenylpiperidine compound
GPV062 (4g) and the Y310 moiety of P‐gp. This additional H‐bond
could account for a stronger interaction with P‐gp, and thus for the
high relative activity of 4g when compared to its calculated logP
value (EC50 = 0.07 µM and logP = 3.98[24]).[25] In the present study,
we further investigate the structural requirements for P‐gp
inhibition, focusing on the role of potential H‐bond acceptors at
OMe
OMe
Ph
Ph
7
tBuO
tBuO
N
N
H
O
8
2a
F
F
Ph
Ph
7
N
N
H
O
9
2b
SCHEME 2 Synthesis of 4‐methoxy‐4‐phenylpiperidine and
4‐fluoro‐4‐phenylpiperidine

CSEKE ET AL.
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SCHEME 3 Synthesis of 4‐methyl‐4‐
phenylpiperidine
CH3
OH
CH3
Ph
CH3
Ph
O
Ph
N
Ph
N
Ph
N
N
H
10
11
12
2c
give 8. For deprotection of carbamates, the method of DeGoey et al.[27]
proved to give the best results. Thus, 8 was deprotected by HCl in
dioxane to yield 4‐methoxy‐4‐phenylpiperidine (2a).
2.3
QSAR analysis
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As previously reported,[24]
a hydroxy group located at the
Substitution of the hydroxy group in carbamate 7 by diethylami-
nosulfur trifluoride led to 4‐fluoro‐4‐phenylpiperidine 11. Deprotec-
tion of 11 by HCl in dioxane yielded 4‐fluoro‐4‐phenylpiperidine (2b).
1‐Benzyl‐4‐piperidone 10 was used as a starting material to
synthesize 4‐methyl‐4‐phenylpiperidine (2c). The addition of methyl-
lithium to 10 gave 1‐benzyl‐4‐methylpiperidine‐4‐ol (11), which was
activated by aluminum chloride and reacted with benzene to yield
the Friedel–Crafts alkylation product 12. Finally, the protective
group was removed by catalytic transfer hydrogenation in accor-
dance with Salon et al.[28] to give 4‐methyl‐4‐phenylpiperidine (2c).
4‐phenylpiperidine position of propafenone derivatives increases
P‐gp inhibitory activity, compared to other analogs with the same
lipophilicity. Docking studies of analog 4g into a protein homology
model of P‐gp indicated that this affinity increase is possibly due to
an H‐bond formed between the hydroxy group and Y310.[23]
However, as all four compounds synthesized by Chiba et al.[24] bear
a 4‐phenyl‐4‐hydroxy moiety, other reasons than hydrogen bonding
might be responsible for the affinity increase. Thus, to broaden the
chemical space of the substituent at this position, we synthesized a
set of compounds with different substituents on the 4‐
phenylpiperidine moiety. Substituents included the two H‐bond
acceptors methoxy and acetyl, as well as hydrogen, fluoro, methyl,
and cyano, which are devoid of H‐bonding capabilities.
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2.2
In vitro studies
To evaluate the SAR of our newly synthesized compound set
(4a–f) and 4g in a broader context, we involved the propafenone
analogs 5a–p synthesized and characterized by Chiba et al.[24] in
the analysis (Table 2 and Scheme 4). A previous work showed that
the activities (IC50 values) of the sodium channel blocker
propafenone and its analogs exhibit an excellent correlation with
their octanol/water partition coefficient (logP value).[31] As it is
To evaluate the P‐gp inhibitory activity of 4a–g, the intracellular
accumulation of daunorubicin in the P‐gp overexpressing CCRF
VCR1000 cells was measured by flow cytometry. As shown in
Table 1, all compounds proved to be strong P‐gp inhibitors with IC50
values in the low nanomolar range (see Supporting Information for
Figure S1 showing the dose–response curves). Among the new
compounds, 4a and 4d showed the highest activity with IC50 values
of 0.03 0.01 and 0.03 0.00 µM, respectively, while the cyano
derivative 4f exhibited somewhat lower values (0.15 0.04 µM). The
IC50 value of 4g (0.05 0.01 µM) was in good agreement with IC50
values reported earlier for this compound (0.07,[24] 0.06,[29] and
0.06 µM[30]), which demonstrates a high comparability with earlier
experiments on series of propafenone analogs and thus allows to
pool all compounds into one data set for subsequent QSAR analysis.
TABLE 2 Calculated logP and IC50 values of compounds 5a–p
(Scheme 4)[24]
Compound
clogPa
3.54
3.89
4.46
2.82
5.29
4.62
4.62
4.62
4.78
4.83
4.1
IC50 (µM)b
1.08
0.68
0.34
3.75
0.05
0.11
0.12
0.18
0.21
0.14
0.42
3.84
6.84
2.83
0.30
0.19
5a
5b
5c
5d
5e
5f
TABLE 1 Results of in vitro IC50 evaluation of P‐glycoprotein
inhibition for the compounds 4a–g (Scheme 1)
Compound
clogPa
4.65
4.90
5.61
5.31
5.13
4.86
4.01
Rb
IC50 (µM)c
0.03 0.01
0.06 0.01
0.05 0.01
0.03 0.00
0.04 0.01
0.15 0.04
0.05 0.01
5g
5h
5i
4a
4b
4c
4d
4e
4f
OMe
F
CH3
H
5j
5k
5l
COCH3
CN
2.73
2.31
1.73
2.43
3.63
5m
5n
5o
5p
4g
OH
aCalculated partition coefficient logP(octanol/water); determined for newly
synthesized compounds using the MarvinSketch software (ChemAxon).
bSubstituent.
cIC50 values represent the mean standard deviation of n = 3 indepen-
dent experiments performed in triplicate; IC50 values were calculated as
described in Section 4.
aCalculated partition coefficient logP(octanol/water) using the
MarvinSketch software (ChemAxon).
bIC50 values experimentally determined by Chiba et al.[24]

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CSEKE ET AL.
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OH
OH
OH
OH
O
H
N
H
N
O
O
N
O
O
N
CH3
O
O
O
O
5a
5b
5c
5d
OMe
MeO
N
OH
N
OH
N
OH
OH
N
OMe
O
N
CH3
O
N
O
N
O
N
O
O
O
O
5e
5f
5g
5h
F
F
F
OH
N
N
OH
N
OH
N
OH
O
N
O
N
O
N
O
N
O
O
O
O
CH3
5k
5i
5j
5l
OH
OH
OH
OH
OH
OH
OH
N
O
N
O
N
O
O
N
O
CH3
O
CH3
O
CH3
O
5m
5o
5p
5n
SCHEME 4 Chemical structures of compounds 5a–p[24]
difficult to quantify the H‐bonding capacity, we used an indicator
variable for groups at the 4‐piperidine position with H‐bond
donor or acceptor properties (I = 1; else I = 0) as a descriptor for
multiple linear regression analysis:
pIC50 = 0.66(±0.04)clogP + 0.73(±0.10)I − 2.29(±0.19),
(r2 = 0.93; SD = 0.20; n = 23).
(1)
The results indicate that indeed an H‐bond donor or acceptor
in position 4 of the 4‐phenylpiperidine is favorable for activity,
which is apparent in the case of the 4‐hydroxy‐4‐piperidines
5n–p and 4g, as well as for the 4‐methoxy‐4‐piperidine derivative
4a (Figure 1). However, it is less pronounced for the acetyl analog
4e. This might be due to a less favorable positioning of the
carboxy oxygen in the binding site. We therefore docked
compounds 4a and 4e into the binding pocket of a homology
model of P‐gp.
FIGURE 1 Correlation of P‐glycoprotein inhibitory activity
(expressed as pIC50 values) and calculated logP values. Propafenone
analogs (•) bearing and (▪) lacking a hydrogen‐bond donor or
acceptor group

CSEKE ET AL.
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basic nitrogen atom of a 4‐phenylpiperidine moiety. In this contribu-
tion, synthesis of a set of compounds with varied substituents at
position 4 of the piperidine, followed by biological testing and ligand‐
and structure‐based modeling studies further support this hypoth-
esis. QSAR studies performed also once more outline the importance
of lipophilicity as a main basic descriptor for activity, which might
compensate for missing interaction features. Thus, SAR of compound
series derived for P‐gp should always be discussed in light of
pIC50/logP ratios rather than on pIC50 values alone.
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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
All moisture‐sensitive reactions were conducted in anhydrous
solvents (Sigma‐Aldrich) under dry argon atmosphere. All solvents
and reagents were obtained from Sigma‐Aldrich, ABCR, TCI, or Acros
and used without further purification. 1‐(2‐((Oxiran‐2yl)methoxy)-
phenyl‐3‐phenylpropan‐1‐one was synthesized following a literature
procedure.[24] All other reagents are commercially acquired and were
used without further purification. Thin‐layer chromatography (TLC)
testings were done on Macherey–Nagel TLC sheets ALUGRAM Xtra
SIL G/UV254. TLC silica gel 60 with fluorescent indicator UV254 was
visualized with UV‐light (254 nm) or rather ninhydrin and/or Seebach
stain. Silica gel 60 M 0.040–0.63 mm from Macherey–Nagel was used
for flash chromatography to optimize yields. The InChI codes of the
investigated compounds and the biological activity data are provided
as Supporting Information Material.
FIGURE 2 Interactions of the propafenone derivatives 4a (blue),
4e (green) and 4g (yellow) with P‐gp. The methoxy group of 4a and
the hydroxy group of 4g formed an H‐bond with Tyr310
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2.4
Molecular docking analysis
Due to the unavailability of the crystal structure of human P‐gp, we
used our recently published homology model[32] for docking
experiments. As can be seen from Figure 2, both the methoxy analog
4a and the acetyl derivative 4e are positioned in the same way as
reported previously for the hydroxy compound 4g.[23] However,
though the methoxy‐oxygen is perfectly located for an H‐bond with
Tyr310, the position of the acetyl‐oxygen is less suited for exploiting
the full potential of a H‐bond. This might be the reason for 4e not
perfectly fitting the regression line for 4‐hydrox‐4‐phenylpiperidines
(IC50 measured, 0.04 µM; IC50 predicted, 0.015 µM). Further evi-
dence could be obtained from binding affinity score predictions
obtained from LigandScout 4.4,[33] which provided a score of −46.27
for 4e, and a slightly better score of −48.16 for 4a. The hydroxy
analog 4g showed a binding affinity score of −45.67, which is in line
with the ranking observed in the biological experiments. These
results thus strengthen the hypothesis that indeed hydrogen bonding
with Tyr310 is the main driving factor for the increase of the
pIC50/logP ratio observed for 4‐hydroxy/alkoxy‐4‐phenylpiperidine
analogous propafenone derivatives.
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4.1.2
Nuclear magnetic resonance spectroscopy
1H‐ and 13C‐NMR‐spectra were measured by Bruker Avance 500
Nuclear Magnetic Resonance spectrometer at 500 MHz using
deuterated chloroform (CDCl3) as the solvent. Chemical shifts are
specified in parts per million (ppm) relative to tetramethylsilane and
coupling constants (J) are given in Hertz (Hz). Multiplicities are
described as s (singlet), brs (broad singlet), d (doublet), t (triplet), q
(quadruplet), quin (quintuplet), septet or m (multiplet). Spectra were
adjusted to the solvent signal of CDCl3, δ(1H) = 7.26 ppm and δ
(
13C) = 77.00 ppm, respectively. The 1H‐ and 13C‐NMR‐spectra of
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3
CONCLUSIONS
newly synthesized compounds are given in the Supporting
Information. For data of compounds 4g and 5a–p.[24]
The ABC transporter P‐gp is one of the driving molecular mechan-
isms for the development of multiple drug resistance in tumor
therapy. Although thousands of compounds are known to inhibit the
transporter, knowledge of the molecular basis of interaction for
distinct inhibitor classes is still far from being complete. In the light of
our studies on propafenone‐type inhibitors of P‐gp, we previously
hypothesized the importance of an H‐bond acceptor close to the
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4.1.3
High‐resolution electrospray ionization mass
spectrometry (HR‐ESI‐MS)
High‐resolution mass spectra (HRMS) were obtained on a Bruker-
Daltonics maXis HD ESI‐Qq‐TOF mass spectrometer using the direct

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CSEKE ET AL.
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infusion. The ESI ion source was operated as follows: capillary
voltage: 1.0 to 4.0 kV (individually optimized), nebulizer: 0.4 bar (N2),
dry gas flow: 4 l/min (N2), and dry temperature: 200°C. Mass spectra
were recorded in the range of m/z 50–1,550 in the positive‐ion mode.
The sum formulas were determined using Bruker Compass Data
Analysis 4.2 based on the mass accuracy (Δm/z ≤ 2 ppm) and isotopic
pattern matching (SmartFormula algorithm).
128.22 (Cq), 128.39 (6C, CH), 128.89 (2C, CH), 130.49 (CH), 133.51
(CH), 141.65 (Cq), 144.19 (d, J(13C–19F) = 20.9 Hz, Cq), 157.80 (Cq),
and 201.21 (Cq) ppm. HRMS (ESI): calcd for C29H33FNO3 m/z
462.2439 [M+H]+, found m/z 462.2439 [M+H]+.
1‐(2‐(2‐Hydroxy‐3‐(4‐methyl‐4‐phenylpiperidine‐1‐yl)propoxy)-
phenyl)‐3‐phenylpropan‐1‐one (3c)
Phenylpiperidine 2c (95 mg; 0.54 mmol, 1 eq.) and oxirane 1 (124 mg;
0.54 mmol, 1 eq.) in methanol (8 ml) gave after chromatographic
purification (silica gel, DCM, MeOH, conc. NH3 200 + 0 + 1 to
200 + 5 + 1) 136 mg 3c, yellow oil, 94% yield. 1H‐NMR (500 MHz,
CDCl3): δ = 1.23 (s, 3H), 1.76 (m, 2H), 2.12 (m, 2H), 2.27 (m, 1H), 2.34
(m, 1H), 2.43 (m, 2H), 2.54 (m, 1H), 2.64 (m, 1H), 3.02 (t, J = 7.8 Hz,
2H), 3.35 (t, J = 8.1 Hz, 2H), 4.00 (m, 1H), 4.04 (m, 1H), 4.08 (m, 1H),
6.95 (d, J = 8.2 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 7.16 (m, 1H), 7.21 (m,
1H), 7.23 (m, 4H), 7.34 (m, 2H), 7.35 (m, 2H), 7.44 (td, J = 7.8, 2.0 Hz,
1H), and 7.71 (dd, J = 7.7, 1.8 Hz, 1H) ppm. 13C‐NMR (125 MHz,
CDCl3): δ = 29.68 (CH2), 30.26 (CH2), 36.00 (Cq), 37.08 (2C, CH2),
45.58 (CH2), 50.29 (2C, CH2), 60.85 (CH2), 65.06 (CH), 70.91 (CH2),
112.55 (CH), 120.96 (CH), 125.72 (3C, CH), 125.86 (CH), 128.21 (Cq),
128.34 (4C, CH), 128.40 (2C, CH), 130.42 (CH), 133.47 (CH), 141.62
(Cq), 148.70 (Cq), 157.78 (Cq), and 201.30 (Cq) ppm. HRMS (ESI):
calcd for C30H36NO3 m/z 458.2690 [M+H]+, found m/z 458.2690
[M+H]+.
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4.1.4
Synthesis of propafenone derivates
(general procedure)
Oxirane 1 and the 4‐phenylpiperidine derivates were dissolved in
MeOH under argon. The mixture was stirred at reflux for 2 hr. Then
the solvent was removed under reduced pressure.
1‐(2‐(2‐Hydroxy‐3‐(4‐methoxy‐4‐phenylpiperidine‐1‐yl)propoxy)-
phenyl)‐3‐phenylpropan‐1‐one (3a)
Phenylpiperidine 2a (200 mg; 1.05 mmol; 1 eq.) and oxirane 1
(290 mg; 1.03 mmol; 1.02 eq.) in methanol (5 ml) gave after
chromatographic purification (silica gel, DCM, MeOH, conc. NH3
200 + 0 + 1 to 200 + 5 + 1) 400 mg 3a, yellow oil, 82% yield. 1H‐NMR
(500 MHz, CDCl3): δ = 1.93 (m, 1H), 1.95 (m, 1H), 2.00 (m, 1H), 2.01
(m, 1H), 2.37 (t, J = 11.0 Hz, 1H), 2.49 (m, 1H), 2.53 (m, 2H), 2.68
(td, J = 11.3, 3.0 Hz, 1H), 2.75 (d, J = 11.0 Hz, 2H), 2.97 (s, 3H), 3.04
(t, J = 7.7 Hz, 2H), 3.38 (td, J = 7.8, 3.3 Hz, 2H), 4.08 (m, 1H), 4.09
(m, 2H), 6.97 (d, J = 8.2 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 7.18 (t,
J = 6.9 Hz, 1H), 7.27 (m, 4H), 7.30 (m, 1H), 7.38 (m, 2H), 7.41 (m, 2H),
7.45 (t, J = 7.9 Hz, 1H), and 7.72 (d, J = 7.9 Hz, 1H) ppm. 13C‐NMR
(125 MHz, CDCl3): δ = 30.27 (CH2), 34.70 (CH2), 34.86 (CH2), 45.55
(CH2), 47.85 (CH2), 49.38 (CH3), 50.76 (CH2), 60.66 (CH2), 65.21
(CH), 70.91 (CH2), 75.19 (Cq), 112.58 (CH), 120.97 (CH), 125.85
(CH), 125.94 (2C, CH), 127.26 (CH), 128.25 (Cq), 128.35 (2C, CH),
128.39 (4C, CH), 130.44 (CH), 133.46 (CH), 141.63 (Cq), 144.38 (Cq),
157.81 (Cq), and 201.37 (Cq) ppm. HRMS (ESI): calcd for C30H35NO4
m/z 474.2639 [M+H]+, found m/z 474.2641 [M+H]+.
1‐(2‐(2‐Hydroxy‐3‐(4‐phenylpiperidine‐1‐yl)propoxy)phenyl)‐
3‐phenylpropan‐1‐one (3d)
Phenylpiperidine 2d (200 mg; 1.24 mmol; 1 eq.) and oxirane 1 (360 mg;
1.26 mmol; 1 eq.) in methanol (5 ml) gave after chromatographic
purification (silica gel, DCM, MeOH, conc. NH3 200 + 0 + 1 to
200 + 5 + 1) 190 mg 3d, white to pale yellow oil, 35% yield. 1H‐NMR
(500 MHz, CDCl3): δ = 1.72 (m, 1H), 1.77 (m, 1H), 1.80 (m, 1H), 1.82 (m,
1H), 1.93 (t, J = 11.8 Hz, 1H), 2.31 (td, J = 11.5, 2.4 Hz, 2H), 2.48 (m, 1H),
2.50 (m, 1H), 2.52 (m, 1H), 2.76 (d, J = 11.3 Hz, 1H), 2.99 (d, J = 11.7 Hz,
1H), 3.04 (t, J = 7.7 Hz, 2H), 3.38 (td, J = 7.9, 3.6 Hz, 2H), 4.07 (m, 2H),
4.11 (m, 1H), 6.97 (d, J = 8.2 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 7.18 (t,
J = 6.8 Hz, 1H), 7.23 (m, 1H), 7.24 (m, 2H), 7.25 (m, 2H), 7.28 (m, 2H),
7.33 (t, J = 7.6 Hz, 2H), 7.45 (t, J = 7.7 Hz, 1H), and 7.73 (d, J = 7.9 Hz,
1H) ppm. 13C‐NMR (125 MHz, CDCl3): δ = 30.30 (CH2), 33.34 (CH2),
33.69 (CH2), 42.33 (CH), 45.73 (CH2), 52.65 (CH2), 55.99 (CH2), 61.13
(CH2), 65.23 (CH), 70.86 (CH2), 112.55 (CH), 120.99 (CH), 125.89 (CH),
126.25 (CH), 126.79 (2C, CH), 128.17 (Cq), 128.38 (2C, CH), 128.41
(2C, CH), 128.46 (2C, CH), 130.48 (CH), 133.52 (CH), 141.71 (Cq),
146.07 (Cq), 157.92 (Cq), and 201.33 (Cq) ppm. HRMS (ESI): calcd for
C29H34NO3 m/z 444.2533 [M+H]+, found m/z 444.2536 [M+H]+.
1‐(2‐(3‐(4‐Fluoro‐4‐phenylpiperidine‐1‐yl)‐2‐hydroxypropoxy)-
phenyl)‐3‐phenylpropan‐1‐one (3b)
Phenylpiperidine 2b (124 mg; 0.54 mmol; 1 eq.) and oxirane 1
(324 mg; 1.15 mmol; 1.5 eq.) in methanol (8 ml) gave after chromato-
graphic purification (silica gel, DCM, MeOH, conc. NH3 200 + 0 + 1 to
200 + 5 + 1) 70.9 mg 3b, yellow oil, 27% yield. 1H‐NMR (500 MHz,
CDCl3): δ = 1.98 (m, 1H), 1.99 (m, 1H), 2.08 (m, 1H), 2.13 (m, 1H), 2.35
(t, J = 11.5 Hz, 1H), 2.56 (m, 2H), 2.58 (m, 1H), 2.68 (t, J = 10.9 Hz, 1H),
2.82 (d, J = 11.0 Hz, 1H), 3.05 (t, J = 7.9 Hz, 2H), 3.38 (m, 2H), 4.09 (m,
1H), 4.10 (m, 2H), 6.98 (d, J = 8.2 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 7.18
(t, J = 6.8 Hz, 1H), 7.26 (m, 4H), 7.32 (m, 1H), 7.40 (m, 4H), 7.47 (t,
J = 7.3 Hz, 1H), and 7.73 (d, J = 7.9 Hz, 1H) ppm. 13C‐NMR (125 MHz,
CDCl3): δ = 30.30 (CH2), 36.80 (d, J(13C–19F) = 21.6 Hz, CH2), 37.11
(d, J(13C–19F) = 20.9 Hz, CH2), 45.70 (CH2), 47.82 (CH2), 50.98 (CH2),
60.78 (CH2), 65.40 (CH), 70.85 (CH2), 112.63 (CH), 121.05 (CH),
123.89 (d, J(13C–19F) = 9.2 Hz, 2C, CH), 125.93 (CH), 127.64 (CH),
1‐(2‐(3‐(4‐Acetyl‐4‐phenylpiperidine‐1‐yl)‐2‐hydroxypropoxy)-
phenyl)‐3‐phenylpropan‐1‐one (3e)
Phenylpiperidine 2e (254 mg; 1.06 mmol; 1 eq.) and oxirane 1 (300 mg;
1.06 mmol; 1 eq.) in methanol (8 ml) gave after chromatographic
purification (silica gel, DCM, MeOH, conc. NH3 200 + 0 + 1 to
200 + 5 + 1) 303 mg 3e, yellow oil, 57% yield. 1H‐NMR (500 MHz,
CDCl3): δ = 1.92 (s, 3H), 2.02 (m, 2H), 2.16 (t, J = 10.4 Hz, 1H), 2.39

CSEKE ET AL.
7 of 11
|
(m, 1H), 2.43 (m, 2H), 2.45 (m, 2H), 2.47 (m, 1H), 2.75 (m, 1H), 3.03 (t,
J = 7.9 Hz, 2H), 3.35 (td, J = 8.2, 3.9 Hz, 2H), 4.02 (m, 1H), 4.04 (m, 2H),
6.95 (d, J = 8.2 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 7.1 Hz, 1H),
7.23 (m, 2H), 7.26 (m, 2H), 7.28 (m, 1H), 7.30 (m, 2H), 7.38 (t, J = 7.7 Hz,
2H), 7.44 (t, J = 7.9 Hz, 1H), and 7.70 (d, J = 7.6 Hz, 1H) ppm. 13C‐NMR
(125 MHz, CDCl3): δ = 25.61 (CH3), 30.25 (CH2), 32.79 (CH2), 32.91
(CH2), 45.52 (CH2), 50.11 (CH2), 51.61 (CH2), 54.43 (Cq), 60.60 (CH2),
65.23 (CH), 70.84 (CH2), 112.62 (CH), 121.00 (CH), 125.78 (CH), 126.34
(2C, CH), 128.24 (5C, CH und Cq), 127.27 (CH), 128.95 (2C, CH),
130.42 (CH), 133.45 (CH), 141.68 (2C, Cq), 157.81 (Cq), 201.27 (Cq),
and 209.29 (Cq) ppm. HRMS (ESI): calcd for C31H36NO4 m/z 486.2639
[M+H]+, found m/z 486.2638 [M+H]+.
1‐(2‐(2‐Hydroxy‐3‐(4‐methyl‐4‐phenylpiperidine‐1‐yl)propoxy)-
phenyl)‐3‐phenylpropan‐1‐one hydrochloride (4c)
3c (90.0 mg; 0.20 mmol; 1 eq.), ether (8 ml), HCl in ether (0.20 ml;
2 M; 2 eq.) gave 4c (79 mg white powder, 88% yield). HRMS (ESI):
calcd for C30H36NO3 m/z 458.2690 [M+H]+, found m/z 458.2694
[M+H]+.
1‐(2‐(2‐Hydroxy‐3‐(4‐phenylpiperidine‐1‐yl)propoxy)phenyl)‐
3‐phenylpropan‐1‐one hydrochloride (4d)
3d (55.0 mg; 0.21 mmol; 1 eq.), ether (8 ml), HCl in ether (0.12 ml;
2 M; 2 eq.) gave 4d (49.6 mg white powder, 90% yield). HRMS (ESI):
calcd for C29H34NO3 m/z 444.2533 [M+H]+, found m/z 444.2533
[M+H]+.
1‐(2‐Hydroxy‐3‐(2‐(3‐phenylpropanoyl)phenoxy)propyl)‐4‐
phenylpiperidine‐4‐carbonitrile (3f)
1‐(2‐(3‐(4‐Acetyl‐4‐phenylpiperidine‐1‐yl)‐2‐hydroxypropoxy)-
phenyl)‐3‐phenylpropan‐1‐one hydrochloride (4e)
3e (240 mg; 0.48 mmol; 1 eq.), ether (8 ml), HCl in ether (0.50 ml; 2 M;
2 eq.) gave 4e (225 mg white to gray powder, 88% yield). HRMS (ESI):
calcd for C31H36NO4 m/z 486.2639 [M+H]+, found m/z 486.2640
[M+H]+.
Phenylpiperidine 2f (236 mg; 1.06 mmol;
1 eq.) and oxirane 1
(300 mg; 1.06 mmol; 1 eq.) in methanol (8 ml) gave after chromato-
graphic purification (silica gel, DCM, MeOH, conc. NH3 200 + 0 + 1 to
200 + 10 + 1) 69 mg 3f, yellow oil, 13% yield. 1H‐NMR (500 MHz,
CDCl3): δ = 2.09 (td, 4H), 2.44 (td, J = 12.1, 2.1 Hz, 1H), 2.59 (m, 2H),
2.74 (m, 2 H), 2.99 (d, J = 12.0 Hz, 1H), 3.05 (t, J = 8.5 Hz, 2H), 3.37 (m,
2H), 4.08 (m, 1H), 4.09 (m, 2H), 6.97 (d, J = 8.5 Hz, 1H), 7.03 (t,
J = 7.4 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.25 (m, 2H), 7.28 (m, 2H), 7.36
(t, J = 7.4 Hz, 1H), 7.43 (m, 2H), 7.46 (m, 1H), 7.50 (d, J = 7.9 Hz, 2H),
and 7.73 (dd, J = 7.6, 1.2 Hz, 1H) ppm. 13C‐NMR (125 MHz, CDCl3):
δ = 30.29 (CH2), 36.45 (CH2), 36.69 (CH2), 42.44 (Cq), 45.61 (CH2),
49.36 (CH2), 52.31 (CH2), 60.50 (CH2), 65.54 (CH), 70.78 (CH2),
112.77 (CH), 121.12 (CH), 121.79 (CN), 125.55 (2C, CH), 125.95 (2C,
CH), 128.21 (Cq), 128.40 (2C, CH), 128.43 (2C, CH), 129.08 (2C, CH),
130. 47 (CH), 133.52 (CH), 139.78 (Cq), 141.64 (Cq), 157.83 (Cq), and
201.16 (Cq) ppm. HRMS (ESI): calcd for C30H33N2O3 m/z 469.2486
[M+H]+, found m/z 469.2485 [M+H]+.
1‐(2‐Hydroxy‐3‐(2‐(3‐phenylpropanoyl)phenoxy)propyl)‐
4‐phenylpiperidine‐4‐carbonitrile hydrochloride (4f)
3f (69.0 mg; 0.13 mmol; 1 eq), ether (8 ml), HCl in ether (0.14 ml; 2 M;
2 eq.) gave 4f (35 mg white to gray powder, 51% yield). HRMS (ESI):
calcd for C30H33N2O3 m/z 469.2486 [M+H]+, found m/z 469.2486
[M+H]+.
|
4.1.6
Synthesis of substituted 4‐phenylpiperidines
tert‐Butyl 4‐hydroxy‐4‐phenylpiperidine‐1‐carboxylate (7)
4‐Hydroxy‐4‐phenylpiperidine 6 (15 g, 84.6 mmol; 1 eq.) and di‐tert‐
butyl dicarbonate (20.25 g, 93.15 mmol; 1.1 eq.) were dissolved in
DCM (450 ml) under argon and stirred at room temperature for 2 hr.
Then saturated aqueous NaHCO3 solution was added. The aqueous
phase was washed three times with DCM and the organic phases
were dried over Na2SO4, filtered and concentrated. Yield: 24.53 g 7,
yellow to light brown oil, 105% (was used without further
purification). 1H‐NMR (500 MHz, CDCl3): δ = 1.48 (s, 9H), 1.73 (d,
J = 12.6 Hz, 2H), 2.00 (td, J = 13.2, 4.7 Hz, 2H), 3.24 (td, J = 12.9,
2.1 Hz, 2H), 4.03 (dt, J = 13.2, 2.0 Hz, 2H), 7.29 (t, J = 7.6 Hz, 1H), 7.37
(t, J = 7.6, 2H), and 7.48 (d, J = 7.9 Hz, 2H) ppm. 13C‐NMR (125 MHz,
CDCl3): δ = 28.46 (3C, CH3), 30.08 (2C, CH2), 39.84 (2C, CH2), 71.53
(Cq), 79.49 (Cq), 124.40 (2C, CH), 127.26 (CH), 128.47 (2C, CH),
147.94 (Cq), and 154.87 (Cq) ppm.
|
4.1.5
Synthesis of hydrochlorides
(general procedure)
The propafenone derivates were dissolved in ether under argon then
HCl in ether (2 M) was added and stirred for 1 hr at room
temperature. The mixture was filtered, washed with ether, and dried.
1‐(2‐(2‐Hydroxy‐3‐(4‐methoxy‐4‐phenylpiperidine‐1‐yl)propoxy)-
phenyl)‐3‐phenylpropan‐1‐one hydrochloride (4a)
3a (100 mg; 0.21 mmol; 1 eq.), ether (6 ml), HCl in ether (0.21 ml; 2 M;
2 eq.) gave 4a (73 mg white to gray powder, 61% yield). HRMS (ESI):
calcd for C30H36NO4 m/z 474.2639 [M+H]+, found m/z 474.2642
[M+H]+.
1‐(2‐(3‐(4‐Fluoro‐4‐phenylpiperidine‐1‐yl)‐2‐hydroxypropoxy)-
phenyl)‐3‐phenylpropan‐1‐one hydrochloride (4b)
tert‐Butyl 4‐methoxy‐4‐phenylpiperidine‐1‐carboxylate (8)
tert‐Butyl 4‐hydroxy‐4‐phenylpiperidine‐1‐carboxylate
7
(2 g,
3b (71.0 mg; 0.15 mmol; 1 eq.), ether (8 ml), HCl in ether (0.15 ml; 2 M; 2
eq.) gave 4b (39.5 mg white powder, 56% yield). HRMS (ESI): calcd for
C29H33FNO3 m/z 462.2439 [M+H]+, found m/z 462.2442 [M+H]+.
7.2 mmol; 1 eq.) was dissolved in THF (60 ml) under argon. Sodium
hydride (0.18 g, 7.5 mmol; 1.04 eq.) was added portionwise. The
mixture was stirred at room temperature for 1 hr. Methyl iodide

8 of 11
CSEKE ET AL.
|
(0.64 ml, 10.8 mmol; 1.5 eq.) was added and the reaction was stirred
overnight at room temperature. On the next day, more sodium
hydride (0.15 g, 6.3 mmol, 0.88 eq.) and methyl iodide (0.33 ml;
5.2 mmol, 0.72 eq.) were added, and the mixture was stirred
overnight at room temperature again. Saturated aqueous NaCl
solution was added to the reaction, and the aqueous phase was
washed three times with EtOAc. The organic phases were dried over
Na2SO4, filtered, and concentrated. Yield: 2.06 g 8, yellow oil, 98%
yield. 1H‐NMR (500 MHz, CDCl3): δ = 1.47 (s, 9H), 1.84 (td, J = 13.2,
4.4 Hz, 2H), 2.01 (d, J = 13.2 Hz, 2H), 2.98 (s, 3H), 3.17 (t, J = 12.5 Hz,
2H), 3.97 (d, J = 12.9 Hz, 2H), 7.26–7.31 (m, 1H), and 7.34–7.40 (m,
4H) ppm. 13C‐NMR (125 MHz, CDCl3): δ = 28.45 (3C, CH3), 34.52 (2C,
CH2), 39.60 (2C, CH2), 49.87 (CH3), 75.62 (Cq), 79.38 (Cq), 125.91
(2C, CH), 127.34 (CH), 128.34 (2C, CH), 144.19 (Cq), and 154.95
(Cq) ppm.
(Cq), 94.23 (d, J(13C–19F) = 174.5 Hz, Cq), 123.81 (d, J(13C–19F)
= 1.9 Hz, 2C, CH), 127.68 (d, J(13C–19F) = 1.3 Hz, CH), 128.39 (d, J
(
13C–19F) = 1.0 Hz, 2C, CH), 144.01 (d, J(13C–19F) = 21.3 Hz, Cq), and
154.78 (Cq) ppm.
4‐Fluoro‐4‐phenylpiperidine (2b)
Hydrogen chloride solution (0.97 ml; 2 M) was added to tert‐butyl‐4‐
fluoro‐4‐phenylpiperidine‐1‐carboxylate 9 (0.20 g, 0.72 mmol) in dioxane
(5 ml) under argon and stirred for 4 hr at room temperature. Because
NMR showed there was no reaction, hydrogen chloride solution (5 ml;
2 M) was added and stirred overnight at room temperature. Afterward,
the mixture was concentrated. Yield: 0.17 g 2b, dark brown, olive oil, 86%
yield. 1H‐NMR (500 MHz, CDCl3): δ = 2.16 (dd, J = 14.2, 8.5 Hz, 2H), 2.66
(dt, J = 38.3, 13.3 Hz, 2H), 3.38 (d, J = 7.9 Hz, 2H), 3.58 (d, J = 10.7 Hz, 2H),
7.29–7.44 (m, 5H) and 9.65 (brs, 1H) ppm. 13C‐NMR (125 MHz, CDCl3):
δ = 33.30 (d, J(13C–19F) = 22.7 Hz, 2C, CH2), 40.36 (2C, CH2), 92.15 (d, J
4‐Methoxy‐4‐phenylpiperidine (2a)
(
13C–19F) = 177.8 Hz, Cq), 123.65 (d, J(13C–19F) = 9.2 Hz, 2C, CH), 128.30
(CH), 128.65 (2C, CH), and 141.99 (d, J(13C–19F) = 21.4 Hz, Cq) ppm.
tert‐Butyl 4‐methoxy‐4‐phenylpiperidine‐1‐carboxylate 8 (2.06 g,
7.07 mmol; 1 eq.) was dissolved in dioxane and HCl (4.7 ml, 4 M;
2.5 eq.) and stirred for 4 hr under argon. More HCl (1 ml, 6 M; 0.85
eq.) was added and the mixture was stirred overnight at room
temperature. Then saturated aqueous NaHCO3 solution was
added. The aqueous phase was washed three times with EtOAc
and the organic phases were dried over Na2SO4, filtered, and
concentrated.[27] Yield: 580 mg 2a, yellow oil, 45% yield. 1H‐NMR
(500 MHz, CDCl3): δ = 1.94 (td, J = 12.5, 4.2 Hz, 2H), 2.04
(d, J = 13.6 Hz, 2H), 2.97 (s, 3H), 2.97–3.06 (m, 2H), 3.11
(t, J = 11.8 Hz, 2H), 7.27 (t, J= 7.3 Hz, 1H), 7.36 (t, J = 7.6 Hz, 2H),
and 7.39 (d, J = 7.9 Hz, 2H) ppm. 13C‐NMR (50 MHz, CDCl3):
δ = 33.24 (2C, CH2), 40.77 (2C, CH2), 49.74 (CH3), 74.78 (Cq),
125.79 (2C, CH), 127.51 (CH), 128.48 (2C, CH), and 143.40
(Cq) ppm. HRMS (ESI): calcd for C12H18NO m/z 192.1383 [M+H]+,
found m/z 192.1382 [M+H]+.
1‐Benzyl‐4‐methylpiperidine‐4‐ol (11)
Methyllithium (30.4 ml, 48.6 mmol) was added dropwise to a solution
of 1‐benzyl‐4‐piperidone 10 (5 ml; 27 mmol) in THF (40 ml) at −78°C
and stirred for 1 hr 30 min. Ether and water were added and the
phases were separated. The aqueous phase was extracted with
diethyl ether, combined with the organic phase, dried with Na2SO4
and concentrated. The product was purified by flash chromatography
with petroleum ether and EtOAc 3 + 1 to 1 + 2. Yield: 2.4 g 11, dark
brown oil, 43% yield. 1H‐NMR (500 MHz, CDCl3): δ = 1.24 (s, 3H),
1.58 (d, J = 13.2 Hz, 2H), 1.70 (dd, J = 13.4, 10.9, 3.3 Hz, 2H), 2.39
(t, J = 9.9, 2H), 2.55–2.64 (m, 2H), 3.54 (s, 2H), and 7.24−7.37
(m, 5H) ppm. 13C‐NMR (125 MHz, CDCl3): δ = 29.90 (CH3), 38.55
(2 C, CH2), 49.60 (2C, CH2), 62.99 (CH2), 67.83 (Cq), 127.04 (CH),
128.19 (2C, CH), 129.26 (2C, CH), and 138.08 (Cq) ppm.
tert‐Butyl‐4‐fluoro‐4‐phenylpiperidine‐1‐carboxylate (9)
1‐Benzyl‐4‐methyl‐4‐phenylpiperidine (12)
tert‐Butyl‐4‐hydroxy‐4‐phenylpiperidine‐1‐carboxylate
7
(2.00 g;
1‐Benzyl‐4‐methylpiperidine‐4‐ol 11 (1.0 g; 4.9 mmol) and aluminum
chloride (3.24 g; 24.3 mmol) were dissolved in benzene (21 ml) under
argon and stirred at reflux for 18 hr. Benzene (10 ml) was added
again and the mixture was poured cautiously into ice water. The pH
of the aqueous phase was adjusted to 11–12 by the addition of
aqueous sodium hydroxide solution (6 M) at a temperature of 0°C.
The aqueous phase was extracted with EtOAc, the organic fractions
were combined, dried over Na2SO4 and concentrated. The product
was purified by flash chromatography with petroleum ether and
diethyl ether 19 + 1 to 1 + 1. Yield: 0.550 g 12, light brown oil, 43%
yield. 1H‐NMR (500 MHz, CDCl3): δ = 1.22 (s, 3H), 1.74–1.85 (m, 2H),
2.10–2.20 (m, 2H), 2.36–2.46 (m, 2H), 2.47–2.57 (m, 2H), 3.47 (s, 2H),
7.19 (t, J = 7.1 Hz, 1H), 7.22–7.27 (m, 1H), and 7.29–7.37 (m, 8H) ppm.
13C‐NMR (125 MHz, CDCl3): δ = 29.39 (CH3), 36.12 (Cq), 37.00 (2C,
CH2), 50.25 (2C, CH2), 63.37 (CH2), 125.48 (CH), 125.82 (2C, CH),
126.86 (CH), 128.13 (2C, CH), 128.25 (2C, CH), 129.16 (2C, CH),
7.2 mmol; 1.2 eq.) in DCM (25 ml) was cooled to −78°C under argon.
A solution of diethylaminosulfur trifluoride (0.81 ml; 6.1 mmol; 1 eq.) in
DCM (3 ml) was added dropwise. The mixture was stirred between
−70 and −90°C for 1 hr. Then it was warmed to room temperature and
stirred for another 30 min. Saturated aqueous NaHCO3 solution was
added, and the organic phases were washed with brine. Then,
3‐chloroperoxybenzoic acid (0.375 g; 2.2 mmol) was added and the
reaction was stirred for 30 min at room temperature. Saturated
aqueous NaHCO3 solution was added and the organic phase was
washed with saturated aqueous NaHCO3 solution, water, and brine.
The organic phases were dried with Na2SO4, tested for peroxide and
concentrated. Yield: 1.56 g 9, light brown oil, 77% yield. 1H‐NMR
(500 MHz, CDCl3): δ = 1.49 (d, J = 3.8 Hz, 9H), 1.74 (d, J = 12.6 Hz, 2H),
1.92–2.07 (m, 2H), 3.21 (dtd, J = 35.7, 12.9, 2.2 Hz, 2H), 4.07 (dd,
J = 42.2, 13.6 Hz, 2H), 7.27–7.34 (m, 1H), 7.34–7.39 (m, 2H), 7.43 (dd,
J = 50.9, 7.4 Hz, 2H) ppm. 13C‐NMR (50 MHz, CDCl3): δ = 28.43 (3C,
CH3), 36.50 (d, J(13C–19F) = 23.2 Hz, 2C, CH2), 39.68 (2C, CH2), 79.71
138.67 (Cq), and 149.23 (Cq) ppm. HRMS (ESI): calcd for C24H26
N
m/z 328.2060 [M+H]+, found m/z 328.2062 [M+H]+.

CSEKE ET AL.
9 of 11
|
4‐Methyl‐4‐phenylpiperidine (2c)
for 5 min at 37°C with 15 µl of 2% DMSO in RPMI alone (DMSO
control) or containing different concentrations of test compound
solutions. Incubation was done by placing the 96‐well plate (nerbe plus
Methanolic formic acid solution (12 ml; 4.4 wt%; 5.5 eq.) was freshly
prepared and added to 1‐benzyl‐4‐methyl‐4‐phenylpiperidine 12
(550 mg; 2.07 mmol; 1 eq.) and Pd on activated carbon (350 mg; 10
wt%) and stirred overnight. The mixture was filtered over celite and
washed with MeOH, water, DCM, and MeOH, and concentrated. The
aqueous residue was washed three times with DCM, dried over Na2SO4
and concentrated. Yield: 0.307 g 2c, light brown oil, 85% yield. 1H‐NMR
(500 MHz, CDCl3): δ = 1.25 (s, 3H), 1.66–1.79 (m, 2H), 2.02–2.14 (m,
2H), 2.78–2.89 (m, 2H), 2.90–2.99 (m, 2H), 7.14–7.24 (m, 1H), and
7.29–7.41 (m, 4H) ppm. 13C‐NMR (125 MHz, CDCl3): δ = 29.64 (CH3),
36.60 (Cq), 38.07 (2C, CH2), 42.91 (2C, CH2), 125.60 (CH), 125.67 (2C,
CH), 128.35 (2C, CH), and 149.30 (Cq) ppm. HRMS (ESI): calcd for
C12H18N m/z 176.1434 [M+H]+, found m/z 176.1433 [M+H]+.
GmbH, Winsen/Luhe, Germany) into an Eppendorf ThermoMixer®
C
device (Eppendorf, Hamburg, Germany). Thereafter, cells were loaded
with 30 µl of daunorubicin, for 30 min at 37°C, to reach a final
daunorubicin concentration of 3 µM. The final DMSO concentration
was 0.5%. Loading was stopped by chilling the cells on watery ice for
5 min followed by the addition of 120 µl ice‐cold PBS. Cells were
pelleted (300 g, 5 min, 4°C), the supernatant was aspirated and cell
pellet was resuspended in 120 µl ice‐cold PBS. Until measurement cells
were kept on ice and in the dark. Immediately before cell fluorescence
measurement by flow cytometry (MACSQuant, Miltenyi Biotec GmbH,
Bergisch Gladbach, Germany), 40 µl of DAPI solution (4 µg/ml in PBS)
was added to gate out dead cells. Cells were kept at 4°C during
measurement. Verapamil (100 µM) was included as a positive control
for P‐gp inhibition. The background fluorescence of the cell suspension
was measured in the presence of 0.5% DMSO.
|
4.2
In vitro inhibition studies
Materials
|
4.2.1
|
IC50 value measurements and calculations
Rosewell Park Memorial Institute‐1640 (RPMI‐1640) cell culture media
was purchased from Thermo Fisher Scientific (Waltham, MA). Supple-
ments for cell culture, including fetal bovine serum (FBS), the antibiotics
penicillin and streptomycin, as well as vincristine for selection of the
CCRF VCR1000 cells were obtained from Sigma‐Aldrich (St. Louis, MO).
The fluorescent substrate daunorubicin as well as verapamil, DMSO,
phosphate‐buffered saline (PBS) and all compounds used for RPMI
buffer preparation were also purchased from Sigma‐Aldrich.
4.2.4
Flow cytometry data were analyzed using FlowJo software (Tree
Star, Ashland, OR). IC50 apparent values were estimated measuring
eight different compound concentrations and performing nonlinear
regression analyses (GraphPad Prism 6; “log(Agonist) vs.
response–Variable slope” GraphPad Software, La Jolla, CA), for
which the following equation was used:
Top − Bottom
Y = Bottom +
,
(2)
1 + 10(Log(IC
)−X)∙Hillslope
50 apparent
|
4.2.2
Cell culture
where X is the log of compound concentration, Y is the response in
fluorescent intensity units, “bottom” and “top” are the lower and the
higher plateaus of the nonlinear fit curve, respectively, and HillSlope
is a factor that describes the steepness of the curve.
The human T‐lymphoblast cell line CCRF VCR1000, overexpressing P‐
gp, was generated by stepwise selection of CCRF‐CEM cells in the
vincristine‐containing medium[34] and was kindly provided by V. Gekeler
(Altana‐Pharma AG, formerly Byk–Gulden, Konstanz, Germany). CCRF
VCR1000 cells were cultured in RPMI‐1640 medium containing 20%
FBS and were treated regularly with vincristine (1 µg/ml) for 72 hr,
followed by centrifugation (300 g, 5 min, RT) and resuspension in a
normal cell culture medium. Cells were maintained at 37°C in an
atmosphere containing 5% CO2 with 95% relative humidity.
To correct for the dependency of IC50 apparent values on the
expression level of P‐gp and the pump‐leak kinetics as reviewed by
Stein,[35] the final IC50 values were calculated using the following
Equation:
Bottom
IC50 = IC50 apparent
∙
,
(3)
Top
where “bottom” and “top” are the lower and the higher plateaus of the
nonlinear fit curve, respectively, and therefore they refer to the
fluorescence intensity at zero and infinite inhibitor concentration,
respectively.[36] The mean IC50 values standard deviations given were
calculated from three independent experiments for each compound.
|
4.2.3
Steady‐state daunorubicin accumulation
experiments
For P‐gp inhibition studies, the steady‐state accumulation of daunor-
ubicin (3 µM) was performed as previously described,[14] optimized to
a 96‐well plate format. Cells were harvested, pelleted (300 g, 5 min,
4°C), and diluted to a concentration of 12 × 106 cells/ml in RPMI
(10 mM Hepes, 120 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 0.04 mM
CaCl2, 10 mM glucose, 10 mM NaHCO3, 5 mM Na2HPO4, pH 7.4 with
NaOH). For each data point, 15 µl of cell suspension was preincubated
|
4.3
Calculation of lipophilicity and QSAR studies
The logP values were calculated using the “consensus” method of
the MarvinSketch software (ChemAxon).[37] The performance of the

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CSEKE ET AL.
|
“consensus” method was verified on a set of 19 propafenone
derivatives whose distribution coefficient was experimentally deter-
mined by Chiba et al.,[31] using high‐performance liquid chromato-
graphy (HPLC). The experimentally obtained and the calculated
values were in good agreement (r = 0.98). QSAR studies carried out
comprised multiple linear regression analyses performed in MS Excel.
ORCID
Anna Cseke
http://orcid.org/0000-0002-1801-0317
Gerhard F. Ecker
http://orcid.org/0000-0003-4209-6883
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