Structure-activity relationships, ligand efficiency, and lipophilic efficiency profiles of benzophenone-type inhibitors of the multidrug transporter P-glycoprotein

Article abstract of DOI:10.1021/jm201705f

The drug efflux pump P-glycoprotein (P-gp) has been shown to promote multidrug resistance (MDR) in tumors as well as to influence ADME properties of drug candidates. Here we synthesized and tested a series of benzophenone derivatives structurally analogous to propafenone-type inhibitors of P-gp. Some of the compounds showed ligand efficiency and lipophilic efficiency (LipE) values in the range of compounds which entered clinical trials as MDR modulators. Interestingly, although lipophilicity plays a dominant role for P-gp inhibitors, all compounds investigated showed LipE values below the threshold for promising drug candidates. Docking studies of selected analogues into a homology model of P-glycoprotein suggest that benzophenones show an interaction pattern similar to that previously identified for propafenone-type inhibitors.

Full text of DOI:10.1021/jm201705f

Article
pubs.acs.org/jmc
Structure−Activity Relationships, Ligand Efficiency, and Lipophilic
Efficiency Profiles of Benzophenone-Type Inhibitors of the Multidrug
Transporter P-Glycoprotein
Ishrat Jabeen,† Karin Pleban,† Uwe Rinner,‡ Peter Chiba,§ and Gerhard F. Ecker*,†
†University of Vienna, Department of Medicinal Chemistry, Althanstrasse 14, 1090, Vienna, Austria
‡University of Vienna, Department of Organic Chemistry, Wahringerstrasse 38, 1090, Vienna, Austria
̈
§Medical University of Vienna, Institute of Medical Chemistry, Wahringerstrasse 10, 1090, Vienna, Austria
̈
S
* Supporting Information
ABSTRACT: The drug efflux pump P-glycoprotein (P-gp) has been shown to
promote multidrug resistance (MDR) in tumors as well as to influence ADME
properties of drug candidates. Here we synthesized and tested a series of
benzophenone derivatives structurally analogous to propafenone-type
inhibitors of P-gp. Some of the compounds showed ligand efficiency and
lipophilic efficiency (LipE) values in the range of compounds which entered
clinical trials as MDR modulators. Interestingly, although lipophilicity plays a
dominant role for P-gp inhibitors, all compounds investigated showed LipE
values below the threshold for promising drug candidates. Docking studies of
selected analogues into a homology model of P-glycoprotein suggest that
benzophenones show an interaction pattern similar to that previously identified for propafenone-type inhibitors.
and stresses the need for a more detailed knowledge on the
structure and function of these proteins and the molecular basis
of their interaction with small molecules.17 The latter has been
approached by numerous SAR and QSAR studies, which
revealed that high lipophilicity seems to be a general
prerequisite for high P-gp inhibitory potency, valid across
different chemical scaffolds. This is also in line with recent
structure-based studies, which indicate an entry pathway via the
membrane bilayer.18,19
In recent years the concepts of “Binding energy of the ligand
per atom” or ligand efficiency (LE)20−22 and lipophilic
efficiency (LipE),23,24 which combines both “potency and
lipophilicity,” have been shown to be useful tools in the lead
optimization process.25,26 In the light of our extensive SAR and
QSAR studies on propafenone analogues27,28 (Figure 1) and
related compounds, we also utilized benzophenone-based
probes, which contain a photoactive arylcarbonyl group as
part of the pharmacophore. This led to the identification of key
amino acid residues interacting with these ligands.29,30 Within
this study, we extended the set of benzophenones in order to
identify compounds with higher potency, utilizing also the
concepts of LE and LipE. In addition, docking studies of
selected compounds into a homology model of P-gp were
performed to shed light on the potential binding mode of these
compounds and to compare it with the binding hypothesis
derived for analogous propafenones.17
INTRODUCTION
■
Membrane transporters are increasingly recognized for playing
a key role in safety profiles of drug candidates, predominantly
by their involvement in drug−drug interactions.1,2 One of the
most intensively studied families in this context is the ATP-
binding cassette (ABC) transporter superfamily.3−5 Several
members of these ATP-driven transporters are expressed at
tissue barriers and thus influence uptake and elimination of
drugs and drug candidates.6 Originally they have been linked to
development of multidrug resistance (MDR) in tumor therapy,
as they transport a wide variety of natural product toxins such
as anthracyclines, vincristine, and taxanes out of tumor cells.7,8
Thus, P-glycoprotein (P-gp/ABCB1), discovered in 1976 and
considered the paradigm ABC transporter,9,10 shows a
remarkably broad substrate pattern, transporting numerous
structurally and functionally diverse compounds across cell
membranes.3 P-gp is expressed at the blood−brain barrier
(BBB), the blood−cerebrospinal fluid (B-CSF) barrier, and the
intestinal barrier, thus modulating the absorption and excretion
of xenobiotics across these barriers.6 P-gp and its ligands
(substrates and inhibitors) are therefore extensively studied
both with respect to reversing multidrug resistance in tumors
and for modifying ADME-Tox properties of drug candidates,11
such as central nervous system (CNS) active agents.12,13 Within
the past two decades, numerous modulators of P-gp mediated
drug efflux have been identified14,15 and several entered clinical
studies up to phase III. However, up to now no compound
achieved approval, which is mainly due to severe side effects
and lack of efficacy. This further emphasizes the physiological
role of efflux transporters in general and P-gp in particular16
Received: December 19, 2011
Published: March 27, 2012
© 2012 American Chemical Society
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of at least three independently performed experiments.
Generally, interexperimental variation was below 20%.
Structure−Activity Relationships. Table 1 shows the P-
gp inhibitory potency of compounds 6−24. The IC50 values
cover a broad range, spanning from 0.05 μM for the dimer 23
up to 13.37 μM for the morpholine analogue 15. Besides the
ortho-benzophenone dimer 23, also the ortho analogues
showing an arylpiperazine moiety (6, 9) are highly active.
Interestingly, the heterodimer 24 is one of the least active
compounds in the data set, together with the morpholine
derivatives 15 and 16. With respect to substitution pattern at
the central aromatic benzene moiety, the rank order for
arylpiperazine substituted compounds generally is ortho > meta
> para. An analogous trend has also been observed for
propafenone analogues.33 However, for compounds bearing
piperidine or morpholine moieties, this trend is partly reversed.
In the case of piperidine derivatives, the para-derivative is
slightly more active than the meta analogue (1.20 vs 3.55 vs
2.18). Interestingly, also for the morpholine analogues, the
para-derivative is by a factor of 2 more active than ortho-
derivative (P = 0.01). Thus, the influence of the substitution
pattern at the central aromatic ring seems to be more
pronounced if the vicinity of the nitrogen comprises large,
lipophilic moieties. This is in line with our previous findings
using hydrophobic moments as descriptors in QSAR studies.34
To assess the role of lipophilicity as a general predictor for
high potency, we also calculated logP values using the software
Bio-Loom version 1.535 and correlated them with pIC50 values
(Figure 2). Boi-Loom, which calculates logP values by a
fragment-based approach, was validated against experimental
logP values by Sakuratani et al.36 The r2 value of 0.56 indicates
that also in the series of benzophenones biological activity
increases with the lipophilicity of the compounds. This is in
agreement with the notion that compounds most probably
enter the binding cavity of P-gp directly from the membrane
bilayer.18 This is additionally supported by the recent X-ray
structure of mouse P-gp, which shows a large inner cavity
accessible from the membrane via putative entry ports
composed of transmembrane helices 4/6 on one side and
10/12 on the other side.19
Figure 1. Selected propafenone analogues used in this study.
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of benzophenone analogues 6−24
was carried out in analogy to the synthesis of propafenone
derivatives.27 Briefly, the respective ortho-, meta-, or para-
hydroxy benzophenone (1a−c) was alkylated with epichlor-
ohydrine yielding ortho-, meta-, and para-oxiranes 2a−c.
Subsequent nucleophilic oxirane ring-opening with primary or
secondary amines (R1) gave target compounds 6−19.
Excessive amount of oxirane 2a upon nucleophilic ring-opening
with piperazine yielded the homodimer 23, whereas equimolar
amounts of both partners predominantly gave the piperazine
analogue 5 (Scheme 1).
Further treatment of piperazine analogue 5 with phenyl-
isocyanate and its thio-analogue as described by Pitha et al.31
yielded 21 and 22, respectively. O-Alkylation of 2-hydroxy-5-
methyl acetophenone (3) with epichlorohydrine yielded 4,
which, upon subsequent nucleophilic ring-opening by piper-
azine 5 yielded the heterodimer 24 (Scheme 2).
Biological Activity. Biological activity of target compounds
6−24 was assessed using the daunorubicin efflux protocol as
described previously.32 Briefly, multidrug resistant CCRF-CEM
vcr 1000 cells were preloaded with daunorubicin and efflux was
monitored by time-dependent decrease in mean cellular
fluorescence in the absence and presence of various
concentrations of compounds. IC50 values were calculated
from concentration−response curves derived from first-order
rate constant of transport (i.e.Vmax/Km) as a function of
compound concentration.32 Thus, the effect of different
modulators on the transport rate is measured in a direct
functional assay. Values are given in Table 1 and are the mean
The 4-hydroxy-4-phenyl-piperidine analogue 19 is located
above the clogP/pIC50 correlation line (pIC50, 5.76 calcd vs
6.51 obs), which further confirms our previous results on the
a
Scheme 1
a
Reagents and conditions: (i) NaOH, epicholorohydrine, reflux for 24 h; (ii) methanol, respective amine (R1), reflux for 24 h (5−19, 23).
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a
Scheme 2
a
Reagents and conditions: (i) NaOH, epicholorohydrine, reflux for 24 h; (iv) p-tolyl isocyanate, CH2Cl2, stirring 2 h (X = O), p-tolylisothiocyanate,
CH2Cl2, stirring 2 h (X = S); (v) methanol, reflux 5 h.
Table 1. Chemical Structure, Ligand Efficiency (LE), Lipophilic Efficiency (LipE), and Pharmacological Activity of Compounds
6−24
a
Position of the side chain at central aromatic ring.
importance of the 4-hydroxy-4-phenyl-piperidine moiety for
high biological activity of propafenone derivatives.37 These
results were recently supported by extensive docking studies of
propafenone analogues.17 It is also interesting to note that the
homodimer 23 is about one log unit more potent than
predicted by the clogP/pIC50 plot (pIC50, 6.10 calcd vs 7.27
obs). A pairwise comparison of equilipophilic compounds 23 vs
21 (clogP, 4.27 vs 4.28; IC50, 0.05 vs 0.48 μM) and 19 vs 20
(clogP, 3.65 vs 3.64; IC50, 0.31 vs 1.21 μM) indicates that
mutual activity differences might also be due to difference in
molecular size. The dimer 23 (44 heavy atoms) is about 1 order
of magnitude more active then 21 (35 heavy atoms). Similarly,
19 (32 heavy atoms) is about a factor of 4 more active than 20
(24 heavy atoms). This also points toward a commonly
observed phenomenon in lead optimization programs, i.e.,
activity increases with the size of the molecules. Therefore,
ligand efficiency (LE)20−22 and lipophilic efficiency (LipE)23,24
profiles of inhibitors/substrates of P-gp have been used to
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described by Reynolds et al.21,22 to retrieve a size-independent
ligand efficiency value (LE_Scale). This was achieved by fitting
the top ligand efficiency versus heavy atom count to a simple
exponential function, as outlined by Reynolds et al.,21 (eq 1;
Figure 3). Subsequently, the ratio of ligand efficiency over
normalized ligand efficiency scale gives a scoring function called
“Fit Quality” (FQ) (eq 2). According to Reynolds et al., fit
quality scores close to 1.0 or above indicate near optimal ligand
binding, while low fit quality scores are indicative of suboptimal
binding.
−0.037 HA
*
(1)
LEScale = 0.104 + 0.65e
FQ = LE/LEScale
(2)
Use of this criterion shows that most of the compounds
under clinical investigation show FQ scores above 1, including
zosuquidar, ONT093, elacridar, and tariquidar, along with
benzophenones 6 and 23, as well as propafenone and its
analogues GPV062 and GPV576 (Figure 4; Table 2).
Figure 2. Correlation of P-gp inhibitory potency of compounds 6−24
(expressed as pIC50 values) vs calculated logP values of the ligands.
identify the derivatives with the best activity/size (or logP)
ratio, which should provide further insights for the design of
new ligands.25,26
It is interesting to note that especially those compounds
which were specifically designed as P-gp inhibitors (ONT093,
zosuquidar, elacridar, tariquidar) show higher FQ values than
those originating from drug repurposing attempts (verapamil,
cyclosporine, and its analogue valspodar). With respect to
propafenone analogues, GPV576 is the hitherto most active
analogue we synthesized showing a highly lipophilic but quite
compact substituent at the nitrogen atom (4-tolylpiperazine).
Interestingly, the top ranked benzophenone analogue 6 also has
a 4-tolylpiperazine moiety. This might point toward the
tolylpiperazine substituent for being a privileged substructure
for P-gp inhibitors. GPV062 bears a 4-hydroxy-4-phenyl-
piperidine moiety, which has been shown to influence
biological activity independent of lipophilicity, resulting in an
almost 10-fold increase of inhibitory potency when compared
to compounds having other substituents at the nitrogen atom.
This points toward a distinct additional interaction mediated by
the 4-hydroxy group, most probably in the form of a hydrogen
bond. Finally, propafenone itself shows a very good value, thus
retrospectively demonstrating its validity as starting point for
structural modifications. However, it should be noted that
during its catalytic cycle the transporter undergoes a major
conformational change and that it might well be that some
inhibitors exert their effect by slow off kinetics rather than by
strong binding to the apo state. In this case, LE values derived
from IC50 values for transport inhibition should be taken
cautiously.
Ligand Efficiency (LE). LE, most commonly defined as the
ratio of free energy of binding to the number of heavy atoms, is
a simple metric for assessing whether a ligand derives its
potency from optimal fit with the target protein or simply by
virtue of making many contacts.38 To get more information on
the most promising P-gp inhibitors and to compare them to
well established P-gp inhibitors/substrates, we calculated ligand
efficiency values of benzophenones 6−24, selected propafenone
analogues (Figure 1), as well as P-gp inhibitors which entered
clinical studies. Ligand efficiencies were calculated as described
in the Materials and Methods section. For benzophenones,
small ligands such as the N-propyl derivative 20 and the
piperidine analogue 12 show higher efficiency values (0.35;
0.33) than the large dimers 23 and 24 (0.23; 0.18). For the
whole data set, it can be observed that ligand efficiencies drop
dramatically when the size of the ligands increases above 50
heavy atoms (Figure 3).
A similar trend has been observed in the literature, with LE
showing generally a dependency on ligand size.21 As LE in
principle is supposed to normalize for the size of the ligand,
various proposals have been made to solve this problem.22,39 As
the heavy atom count of the ligands in our data set varies from
24 to 86 (20; valspodar), LE values were subsequently scaled as
As already outlined, lipophilicity has been shown in
numerous studies to be a general predictor for high P-gp
inhibitory potency. This most probably is due to the proposed
access path of the compounds, which seems to be directly from
the membrane bilayer. On the other hand, high lipophilicity is
very often associated with poor oral drug-like properties. This
led to the assumption that clogP values between 2 and 3 are
considered optimal in an oral drug program and prompted
Leeson et al., to introduce the concept of lipophilic efficiency.23
Liphophilic Efficiency (LipE). LipE is a parameter that
combines both potency and lipophilicity and is defined as a
measure of how efficiently a ligand exploits its lipophilicity to
bind to a given target. Briefly, in a lead optimization series,
there is a greater likelihood of achieving good in vivo
performance when potency can be increased without increasing
logP or logD values. To explore this concept also for P-gp
inhibitors, we calculated LipE values for the whole set of
Figure 3. Plot of ligand efficiency vs heavy atom count for
benzophenone analogues, compounds which entered clinical studies
and selected propafenones.
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Figure 4. Fit quality scores vs heavy atom counts of benzophenones 6−24, compounds which entered clinical studies, and selected propafenones.
FQ score around 1 indicate a near optimal ligand binding affinity for a given number of heavy atoms.
Table 2. Pharmacological Activities, Ligand Efficiency (LE),
and Lipophilic Efficiency (LipE) Profiles of Selected
Propafenones and P-gp Inhibitors Which Entered in Clinical
Studies
To study in more detail whether the unique access path of P-
gp inhibitors directly from the membrane bilayer is linked to
this unexpectedly low LipE values, we studied the distribution
of LipE profiles for a set of targets showing different access
pathways of their ligands: P-glycoprotein (via the membrane
bilayer), the serotonin transporter SERT (from the extracellular
environment), and the hERG potassium channel (from the
cytoplasm) (Figure 6). LipE values of inhibitors of SERT
(extracted from the ChemBL database),40 hERG blockers,41
and propafenone-type inhibitors of P-gp (in-house data) were
calculated as described in the Materials and Methods section.
The LipE distribution profile of SERT inhibitors extracted
from the ChemBL database identified about 13% of the
compounds that cross the LipE threshold of 5 (Figure 7).
These compounds cover a wide range of IC50 (0.01 nM to 10
mM) and clogP (−3.42 to 4.66) (SM Figure 1). Moreover, 15
SERT inhibitors have been identified with clogP ∼ 2.5, LipE >
5, and IC50 < 10 nM, none of them was listed as a marketed
drug. In the case of hERG, only 2.5% of the compounds cross
the LipE threshold of 5 that showed a potency distribution
from 5 nM to 18 μM and clogP values between −0.77 and 2.21
(SM Figure 1). Only two compounds, almokalant and
dofetilide, complied with the desired profile (clogP ∼ 2.5,
LipE > 5, potency values <10 nM). Dofetilide is a registered
class III antiarrhythmic agent, while almokalant is in phase II
clinical investigations.42,43
compd
pIC50
HA
LE
clogP
LipE
Verapamil
Elacridar
6.24
7.14
7.48
7.23
7.50
6.30
6.99
6.15
6.48
8.25
7.24
6.22
33
42
48
39
37
86
85
45
25
35
34
27
0.27
0.24
0.22
0.26
0.29
0.10
0.12
0.20
0.37
0.33
0.30
0.33
4.47
4.21
5.55
4.96
7.30
15.09
14.36
7.80
3.64
6.02
4.15
4.38
1.77
2.93
Tariquidar
Zosuquidar
ONT093
1.93
2.27
0.19
Valspodar
Cyclosporine A
Niguldipine
Propafenone
GPV576
−8.79
−7.37
−1.65
2.84
2.23
GPV062
3.09
GPV005
1.84
benzophenones as well as for the compounds used for the LE
study (Table 2). The clogP values vary from 2.66 to 15.09,
leading to a lipophilic efficiency range between −8.79 and
+3.08. This is somewhat surprising, as it has been reported that
a lipophilic efficiency greater than 5 combined with clogP
values between 2 and 3 is considered optimal for a promising
drug candidate.23,24 None of the clinically tested P-gp inhibitors
fulfils these requirements. Only the 4-hydroxy-4-phenyl-
piperidine analogous propafenone GPV062 as well as the
dimer 23 exhibit values slightly higher than 3. All other
compounds show values lower than 3 (Figure 5). It is tempting
to speculate whether this is due to the unique entrance pathway
directly from the membrane bilayer, which requires a different
logP profile than for compounds which access their binding site
directly from the extracellular or intracellular aqueous compart-
ment.
LipE profiles of P-gp inhibitors could not identify any
compound that reaches the standard threshold value of 5. Most
of the ligands fall in the LipE range of 1−2 (39%) or 2−3
(28%), with wide a range in distribution of their clogP (0.40 to
6.02) as well as IC50 (5.60 nM to 1.20 mM) values (SM Figure
1). Thus, the LipE threshold for ligands of P-gp needs to be
reconsidered. Nevertheless, from the benzophenone data set
presented here, compounds 16, 19, 20, and 23 might be the
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Figure 5. Plot of clogP vs biological activity of inhibitors of P-gp; LipE values higher than 5 are considered to be the threshold for compounds of
■
●
▲
compounds which entered clinical trials.
clinical interest; benzophenones, propafenones,
Figure 6. Schematic representation of access of inhibitors/substrates to the binding sites of P-gp, SERT, and hERG along three different pathways.
Ligands of P-gp approach the binding cavity via the membrane bilayer; in SERT the ligands get access from the extracellular environment, while in
hERG this access occurs via the cytoplasm.
most promising ones as their LipE values are between 2 and 3,
a range where most of the compounds which in the past
entered clinical trials are located.
Docking into a Homology Model of P-Glycoprotein.
To get insights into the potential binding mode of
propafenone-type benzophenones, we selected compounds 6,
19, and 20 and the dimer 23 for further in silico studies.
Compounds 19, 20, and 23 were selected as they are ranked
high both in LipE and FQ scores, and 6 was additionally
included as it is top ranked with respect to FQ. Interestingly,
this selection resembles the key features observed for
propafenone analogues: compound 6 shows a 4-tolylpiperazine
substituent (analogous to GPV576), compound 19 is analogous
to GPV062 (4-hydroxy-4-phenyl-piperidine), and derivative 20
is the direct propafenone analogue (N-propyl). The docking
protocol follows those previously published44 and is provided in
detail in the Materials and Methods section.
The analysis of the interaction pattern of selected docking
poses indicates that the benzophenone scaffold interacts with
F343 and F303 near the entry gate, whereas the lipophilic
Figure 7. LipE distribution profiles of ligands of P-gp, SERT, and the
hERG potassium channel.
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Figure 8. Ligand−protein interaction profile of the best scored pose of benzophenone dimer 23. Blue circle represent the putative position of
benzopyrano[3,4-b][1,4]oxazines having 4aS,10bR configuration, while the green circle indicates the position of diastereoisomers with 4aR,10bS
configuration.
Figure 9. (A) Ligand protein interaction pattern of clustered pose of 19. (B) Overlaid clustered poses (green, 23; blue, 19; yellow, 20; brown, 6).
Two important hydrophobic binding cavities have been formed by amino acid residues of TM 7, 8, 9, and 12, indicated by two circles.
substituents in the vicinity of the basic nitrogen atom are
surrounded by hydrophobic amino acid residues L724, I720,
V981, I840, I836, and I765 located at TM 7, 8, 9, and 12
(Figure 8). This further supports the importance of high
lipophilicity and also is in line with previous studies performed
by Pajeva and Wiese, who showed that for a series of inhibitors
of P-gp hydrophobicity represents a space directed molecular
property rather than a simple overall descriptor.45 The top
ranked cluster of poses are in close vicinity of our previously
purposed binding positions for benzopyrano[3,4-b][1,4]-
oxazines, where compounds having 4aS,10bR configuration
interact mainly with amino acid residues of TM 4, 5, and 6 near
the entry gate, while compounds having 4aR,10bS configuration
are positioned deeper inside the binding cavity, being mainly
surrounded by hydrophobic amino acid residues of TM 7, 8, 9,
and 12.44 Interestingly, the top scored dimer 23 is positioned in
a way to bridge these two positions (Figure 8). Moreover, this
pose might also aid in the explanation for the activity
differences of homodimer 23 (0.05 μM) and heterodimer 22
(9.48 μM): The additional benzene ring in the best scored pose
of homodimer 23 is surrounded by several hydrophobic amino
acids (I836, L720, I840, and L724).
A representative docking pose of the 4-hydroxy-4-phenyl-
piperidine derivative 19 showed an H-bond interaction
between the 4-hydroxy group and A985 (Figure 9A). This
further supports our SAR data and strengthens the importance
of 4-hydroxy-4-phenyl-piperidine moieties for high inhibitory
potency of propafenones and benzophenones. Furthermore,
A985 was also identified as interacting with verapamil and the
cyclic peptide (AQZ59-SSS) cocrystallized in mouse P-gp.19
A
binding pocket of 4.5 Å around interacting amino acid residues
of TM 7, 8, 9, and 12 showed two small hydrophobic cavities
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mL), treated with 2.04 g (51 mmol) of sodium hydroxide, and refluxed
for 24 h. Then, after cooling, the residue was filtered off and washed
with diethyl ether. Subsequently, the solvent was removed by rotary
evaporation. The remaining oil was taken up in diethyl ether and
washed with water (175 mL). The organic phase was then dried over
anhydrous sodium sulfate. After removal of the solvent by rotary
evaporation, yellow oil was obtained, yield 12.81 g (98.85%). 1H NMR
(CDCl3) δ 2.37−2.66 (m, 2H, CH2-O-CH), 2.96−3.00 (m, 1H, CH),
3.92−4.15 (m, 2H, Ar-O-CH2), 6.97−7.81 (m, 9H, arom H). 13C
NMR (CDCl3) δ 44.03 (CH-CH2-O), 49.59 (CH), 68.62 (Ar-O-
CH2), 112.72, 121.13, 128.05 (arom C), 129.00 (Ar-CO), 129.38,
129.69, 131.97, 132.66 (arom C), 137.93 (Ph-CO), 156.08 (Ar-O),
196.15 (CO).
General Procedure for the Preparation of (3-Oxiranylmethoxy-
phenyl)-phenyl-methanone C16H14O3 (2b). First, 5 g (25.25 mmol)
of 3-hydroxy-benzophenone was dissolved in epichlorohydrine (60
mL), treated with 1.01 g (25.25 mmol) of sodium hydroxide, refluxed
for 6 h, and stirred overnight. Then the residue was filtered off and
washed with diethyl ether. After removal of the solvents under reduced
pressure, the resulting oil was taken up in diethyl ether and washed
with water several times. The organic layers were combined, dried over
anhydrous sodium sulfate, and evaporated to dryness yielding yellow
oil. For further purification a column chromatography (silica gel,
ether/petrol ether, 70 + 30) was performed. Subsequent removal of
the solvents under reduced pressure gave white opalescent oil, yield 6
g (93.6%). 1H NMR (CDCl3) δ 2.71−2.74 (m, 1H, HA), 2.86 (t, 1H, J
= 4.42, HB), 3.29−3.37 (m, 1H, CH), 3.93 (dd, 1H, J = 5.94/11.12,
HX), 4.28 (dd, H, J = 2.90/10.98, HY), 7.10−7.78 (arom H). 13C NMR
(CDCl3) δ 44.34 (CH2−O), 49.81 (CH), 68.76 (Ar-O-CH2), 114.93,
119.17, 123.10, 128.11, 129.18, 129.81, 132.31, (arom C), 137.28,
138.70 (C), 158.24 (Ar-O), 196.08 (CO).
General Procedure for the Preparation of (4-Oxiranylmethoxy-
phenyl)-phenyl-methanone C16H14O3 (2c). first, 6 g (30.30 mmol) of
4-hydroxy-benzophenone was dissolved in epichlorohydrine (50 mL),
treated with 2 g (50 mmol) of sodium hydroxide, refluxed for 5 h, and
stirred overnight. The residue was filtered off and washed with diethyl
ether. Subsequently, the solvent was removed by rotary evaporation.
The remaining opalescent oil was taken up in diethyl ether and washed
with water several times. The organic phase was then dried over
anhydrous sodium sulfate. After removal of the solvents by rotary
evaporation, a white solid was obtained, yield 6.7 g (87.54%). 1H
NMR (CDCl3) δ 2.76 (dd, 1H, J = 2.66/4.8, HA), 2.92 (t, 1H, J = 4.64,
HB), 3.35−3.40 (m, 1H, CH), 3.96−4.0 (dd, 1H, J = 5.81−11.12, HX),
4.29−4.36 (dd, 1H, J = 2.91/11.11, HY), 6.95−7.00 (m, 2H, H-3, H-
5), 7.42−7.56 (m, 5H, H-2, H-6, H-3′, H-4′, H-5′), 7.72−7.83 (m, 2H,
H-2′, H-6′). 13C NMR (CDCl3) δ 44.51 (CH2-O), 49.84 (CH), 68.82
(Ar-O-CH2), 114.07, 128.14, 129.65, 131.90, 132.46, (arom C), 130.54
(Ar-CO), 138.06 (Ph-CO), 161.93 (Ar-O), 195.41 (CO).
General Procedure for the Preparation of [2-(2-Hydroxy-3-
piperazine-1-yl-propoxy)-phenyl]-phenyl-methanone (5). First,
1.82 g (7.20 mmol) of (2-oxiranylmethoxy-phenyl)-phenyl-methanone
(2a) was dissolved in 20−30 mL of methanol, 1.6 g (18.6 mmol) of
piperazine was added, and then the reaction mixture was refluxed for 5
h. After removal of the solvent by rotary evaporation, a column
chromatography was performed (silica gel, CH2Cl2/methanol/
concentrated NH3, 100/10/1) subsequent evaporation to dryness
yielded 1.88 g (77.33%) of yellow oil which was solidified on cooling.
1H NMR (CDCl3) δ 2.01−2.42 (m, 6H, CH2-N-(CH2)2), 2.77−2.81
(encircled in Figure 9B), occupying the hydrophobic
substituents at the basic nitrogen atom of the ligands. A closer
look of the overlaid poses shows that the benzophenone
substituent in dimer 23 fits well in the hydrophobic pockets,
which might explain its high FQ score.
Overall, benzophenones shared a similar interaction profile as
propafenones. Amino acids S952, F434, F336, L724, and Y307
have been identified as common interacting amino acid residues
of all three classes of propafenone type inhibitors of P-gp (SM
Figure 3). Selected benzophenone analogues have been
previously used as photoaffinity ligands to characterize the
drug-binding domain of propafenone-type analogues. In these
studies, TM 3, 5, 6, 8, 10, 11, and 12 were identified as potential
interacting helices.30,46,47 This is well in line with our docking
studies, which show main interactions with TM 5 and 6 near
the entry gate and TM 7, 8, 9, and 12 deeper inside the cavity
(SM Figure 4). No significant cluster of poses has been
identified on the second wing (2/11 interface), which might be
due to the asymmetry in the template used for building the
homology model of P-gp, thus narrowing the available space at
this side.
CONCLUSIONS
■
Calculation of ligand efficiency and lipophilic efficiency values
for a set of P-gp inhibitors shows that ligands of P-gp exhibit
LipE values below the threshold of 5 considered to be optimal
for clinical candidates. This might be due to the unique
entrance pathway of these classes of compounds, taking a route
directly from the membrane bilayer. However, LipE and LE
values of benzophenones 6, 19, and 20, as well as of the dimer
23, are close to compounds which entered clinical studies, thus
qualifying them for further lead optimization cycles. Docking
studies further strengthen the evidence provided by QSAR
studies that the benzophenones bind to the same region as
propafenone-type inhibitors. Moreover, the dimer 23 seems to
bridge the two distinct binding sites recently proposed for
benzopyrano[3,4-b][1,4]oxazines. This further supports the
general assumption of a binding zone with distinct, but
overlapping binding sites for individual scaffolds as a basis for
the promiscuity of P-gp.
EXPERIMENTAL SECTION
■
Chemistry. Material and Methods. The data set used consists
of a set of previously published benzophenones 937 and 12, 19,
and 2029 as well as a series of newly synthesized analogues.
Melting points were determined on Leica Galen III (ser. no.
1413 WT) and are uncorrected. Purity of the compounds was
checked by elemental analysis, and all values were within
0.3%. Elemental analysis was performed at Microanalytical
Laboratory of the Institute of Physical Chemistry (Mag.
Johannes Theiner), University of Vienna. The equipment
used was a 2400 CHN-Elemental Analyzer from Perkin-Elmer.
Mass spectra were recorded on a Maldi-TOF, Kratos
Instruments, matrix assisted laser-desorption-ionization time-
of-flight, reflection mass spectrometer. NMR spectra were
(m, 4H, (CH2)2-N), 3.68−3.75 (m, 1H, CH), 3.90−3.94 (m, 2H, O−
CH2), 6.96−7.79 (m, 9H, arom H). 13C NMR (CDCl3) δ 45.86 (N-
(CH2)2), 54.30 ((CH2)2-NH), 60.79 (CH2-N), 65.27 (CH), 70.76
(O-CH2), 112.56, 121.05, 128.28, 129.51, 130.07, 132.33, 132.83
(arom C), 128.79 (Ar-CO), 138.36 (Ph-CO), 156.55 (arom C-O),
196.56 (CO).
(2-{3-[4-(2,3-Xylyl)-piperazine-1-yl]-2-hydroxy-propoxy}-phenyl)-
phenyl-methanone (6). First, 700 mg (2.75 mmol) of (2-
oxiranylmethoxy-phenyl)-phenyl-methanone (2a) was dissolved in
15 mL of methanol and treated with 526 mg (2.75 mmol) of 1-(2,3-
xylyl)-piperazine. The mixture was refluxed for 24 h. Subsequent
removal of the solvent yielded yellow oil, crystallization from ethyl
1
recorded on a Bruker spectrospin for 200 MHz H NMR and
50 MHz for 13C NMR. CDCl3 and DMSO at room temperature
were used as internal standards. Column chromatographic
separations were performed by using silica gel 60 (Particle size
40−63 μm, 230−300 mesh) from J. T. Baker or Merck. Thin
layer chromatography (TLC) was performed on silica gel
60F254 TLC plates from Merck.
General Procedure for the Preparation of (2-Oxiranylmethoxy-
phenyl)-phenyl-methanone C16H14O3 (2a). First, 10 g (51 mmol) of
2-hydroxy-benzophenone was dissolved in epichlorohydrine (120
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acetate/diethylether gave 904 mg (73.8%) white crystals; mp 116−118
°C. 1H NMR (CDCl3) δ 2.19−2.27 (m, 2H, CH2), 2.21 (s, 3H, CH3),
2.27 (s, 3H, CH3), 2.42−2.47 (m, 2H, CH2), 2.58−2.66 (m, 2H,
CH2), 2.83−2.88 (m, 4H, (CH2)2)-N-Xyl), 3.77−3.83 (m, 1H, CH),
3.91−4.02 (m, 2H, O-CH2), 6.90 (dd, 2H, J = 2.90/7.83 Hz, arom H),
6.99−7.12 (m, 3H, arom H), 7.44−7.58 (m, 5H, arom H), 7.79−7.83
(m, 2H, arom H). 13C NMR (CDCl3) δ 13.93 (CH3-ortho), 20.61
(CH3-meta), 51.97 (N-(CH2)2), 53.79 (CH2-N), 60.36 ((CH2)2-N-
Ph), 65.42 (CH), 70.75 (O-CH2), 112.64, 116.56, 121.12, 125.01,
125.81, 128.33, 128.86, 129.58, 130.12, 132.37, 132.87 (arom C),
137.99 (Ar-CO), 138.43 (Ph-CO), 151.29 (Ar-N), 156.58 (Ar-O). MS
m/e 556.25 (M+, 100%). Anal. Calcd for C28H32N2O3: C, 74.89; H,
7.29; N, 6.24. Found: C, 74.73; H, 7.59; N, 6.32.
oxiranylmethoxy-phenyl)-phenyl-methanone (2c) was dissolved in 15
mL of methanol, treated with 496 mg (2.75 mmol) of p-F-phenyl-
piperazine, and refluxed for 5 h. Then removal of solvent under
reduced pressure left a yellow oil which was crystallized from isopropyl
alcohol, yielding 1 g (83.6%) white crystals; mp 96−100 °C. 1H NMR
(CDCl3) δ 2.61−2.69 (m, 4H, N-(CH2)2), 2.80−2.91 (m, 2H, CH2-
N), 3.12−3.17 (m, 4H, (CH2)2-N-Ph), 3.56 (br, 1H, OH), 4.11−4.19
(m, 3H, CH2-CH), 6.89−7.85 (m, 13H, arom H). 13C NMR (CDCl3)
δ 50.24, 53.28, 60.28 (CH2-N-(CH2)4), 65.40 (CH), 70.33 (O-CH2),
114.07, 115.31, 115.75, 117.81, 117.96, 128.17, 129.71, (arom C),
130.42 (Ar-CO), 131.92, 132.52 (arom C), 138.15 (Ph-CO), 147.77
(Ar-N), 162.28 (Ar-O), 195.50 (CO). MS m/e 435.3 (M+, 100%).
Anal. Calcd for C26H27FN2O3: C, 71.87; H, 6.26; N, 6.45. Found: C,
71.61; H, 6.43; N, 6.41.
(3-{3-[4-(2,3-Xylyl)-piperazine-1-yl]-2-hydroxy-propoxy}-phenyl)-
phenyl-methanone (7). First, 560 mg (2.20 mmol) of 3-
oxiranylmethoxy-phenyl)-phenyl-methanone (2b) was dissolved in
20 mL of methanol and treated with 418 mg (2.20 mmol) of 1-(2,3-
xylyl)-piperazine. Then the mixture was refluxed for 6 h. After removal
of the solvent under reduced pressure, a yellow oil was obtained which
was crystallized from ethyl acetate/diethyl ether, yielding 960 mg
[3-(2-Hydroxy-3-piperidine-1-yl-propoxy)-phenyl]-phenyl-metha-
none (13). First, 700 mg (2.75 mmol) of (3-oxiranylmethoxy-phenyl)-
phenyl-methanone (2b) was dissolved in 15 mL of methanol and
treated with 234 mg (2.75 mmol) of piperidine. Then the mixture was
refluxed for 4 h and stirred at room temperature overnight. After
removal of the solvent under reduced pressure, 336 mg (36%) yellow
1
1
oil was obtained; mp 64 °C. H NMR (CDCl3) δ 1.45−1.59 (m, 6H,
(98%) white crystals; mp 92−99 °C. H NMR (CDCl3) δ 2.23, 2.27
CH2), 2.37−2.50 (m, 4H, N-(CH2)2), 2.56−2.62 (m, 2H, CH2-N),
4.02−4.13 (m, 3H, O-CH2-CH), 7.13−7.81 (m, 9H, arom H). 13C
NMR (CDCl3) δ 24.17, 26.05 (CH2), 54.67 (N-(CH2)2), 60.89 (CH2-
N), 65.18 (CH), 70.64 (O-CH2), 115.11, 119.28, 122.99, 128.23,
129.22 130.00, 132.39 (arom C), 137.54, 138.82 (C), 158.75 (Ar-O),
196.44 (CO). MS m/e 340.43 (M+, 100%). Anal. Calcd for
C21H25NO3: C, 73.53; H, 7.46; N, 4.08. Found: C, 73.51; H, 7.71;
N, 4.11.
[4-(2-Hydroxy-3-piperidine-1-yl-propoxy)-phenyl]-phenyl-metha-
none (14). First, 700 mg (2.75 mmol) of (4-oxiranylmethoxy-phenyl)-
phenyl-methanone (2c) was dissolved in 10 mL of piperidine and
refluxed for 2.5 h. Then after removal of piperidine under reduced
pressure, a yellow oil was obtained. For further purification, a column
chromatography was performed (silica gel, CH2Cl2/methanol/
concentrated NH3, 95/5/1 after removal of piperidine the percentage
of methanol was increased to 80/20/1). Solvents were removed by
rotary evaporation to yield light-yellow oil, which crystallized from
ethyl acetate. Further recrystallization from methanol/diethyl ether
yielded 400 mg (42.8%) white crystals; mp 106 °C. 1H NMR (CDCl3)
δ 1.49−1.72 (m, 6H, CH2), 2.58−2.73 (m, 6H, CH2-N-(CH2)2),
4.05−4.08 (m, 2H, O-CH2), 4.20−4.29 (m, 1H, CH), 4.32 (s, 1H,
OH), 6.97 (d, 2H, J = 8.85, H-3, H-5), 7.45−7.82 (m, 7H, arom H).
13C NMR (CDCl3) δ 23.65, 25.33 (CH2), 54.78 (N-(CH2)2), 61.21
(2s, 6H, 2CH3), 2.62−2.66 (m, 4H, N-(CH2)2), 2.83−2.93 (m, 6H,
CH2-N, (CH2)2-N-Ph), 4.08−4.18 (m, 3H, O-CH2-CH), 6.92 (d, 2H,
J = 7.07, H-4, H-6), 7.05−7.20 (m, 2H, arom H), 7.37−7.79 (m, 6H,
arom H), 7.80−7.83 (m, 2H, arom H). 13C NMR (CDCl3) δ 13.92
(CH3-ortho), 20.60 (CH3-meta), 52.15 (N-(CH2)2), 53.77 ((CH2)2-
N), 60.38 (CH2-N), 65.38(CH), 70.49(O-CH2), 115.07, 116.58,
119.30, 123.09, 125.03, 125.81, 128.24, 129.26, 130.00 (arom C),
131.19 (C), 132.42 (arom C), 137.52, 137.97, 138.86, 151.31 (C),
158.70 (Ar-O), 196.41(CO). MS m/e 444.68 (M, 90%). Anal. Calcd
for C28H32N2O3·0.3H2O: C, 74.64; H, 7.31; N, 6.22. Found: C, 74.76;
H, 7.56; N, 6.08.
(4-{3-[4-(2,3-Xylyl)-piperazine-1-yl]-2-hydroxy-propoxy}-phenyl)-
phenyl-methanone (8). First, 700 mg (2.76 mmol) of (4-
oxiranylmethoxy-phenyl)-phenyl-methanone (2c) was dissolved in
15 mL of methanol, treated with 523.6 mg (2.756 mmol) of 1-(2,3
xylyl)-piperazine, and the mixture was refluxed for 5 h. Then, removal
of the solvent under reduced pressure yielded white crystals which
were recrystallized from methanol giving 1.06 g (94.62%) white
1
crystals; mp 122−126 °C. H NMR (CDCl3) δ 2.23−2.27 (2s, 6H,
2CH3), 2.55−2.70 (m, 4H, (CH2)2-N-Ph), 2.70−3.00 (m, 6H, CH2-N-
(CH2)2), 3.40−3.70 (bs, 1H, OH), 4.10−4.17 (m, 3H, O-CH2-CH),
6.90−7.03 (m, 5H, arom H), 7.44−7.51 (m, 3H, arom H), 7.74−7.81
(m, 4H, arom H). 13C NMR (CDCl3) δ 13.90 (CH3-ortho), 20.58
(CH3-meta), 52.15, 53.76 (CH2-N-(CH2)2), 60.38 ((CH2)2-N), 65.31
(CH), 70.44 (O-CH2), 114.08, 116.57, 125.05, 125.81, 128.15, 129.69
(arom C), 130.37, 131.88 (C), 131.88, 132.50 (arom C), 137.97,
138.18 (C), 151.29 (Ar-N), 162.34 (Ar-O), 195.50 (CO). MS m/e
445.4 (M+, 100%). Anal. Calcd for C28H32N2O3: C, 75.65; H, 7.26; N,
6.30. Found: C, 75.61; H, 7.48; N, 6.33.
(CH2-N), 64.89 (CH), 70.64 (O-CH2), 114.06, 128.15, 129.67 (arom
Cr), 130.38 (Ar-O), 131.89, 132.48 (arom C), 138.13 (Ph-CO),
162.21 (Ar-O), 195.49 (CO). MS m/e 340.3 (M+, 100%). Anal. Calcd
for C21H25NO3: C, 71.77; H, 7.55; N, 3.99. Found: C, 71.68; H, 7.51;
N, 4.02.
[2-(2-Hydroxy-3-morpholine-4-yl-propoxy)-phenyl]-phenyl-
methanone (15). First, 1 g (4.33 mmol) of (2-oxiranylmethoxy-
phenyl)-phenyl-methanone (2a) was dissolved in 20 mL of methanol
and treated with 500 mg (5.75 mmol) of morpholine. Then the
mixture was refluxed for 4 h. Removal of the solvents gives yellow oil,
which was purified via column chromatography (silica gel, CH2Cl2/
methanol/concentrated NH3, 200/10/1). Subsequent removal of the
solvents under reduced pressure yielded 400 mg (27%) of light-yellow
oil; mp 185−193 °C. 1H NMR (CDCl3) δ 1.97−2.46 (m, 6H, CH2-N-
(CH2)2), 3.73 (t, 2H, J = 4.67, O-CH2), 3.71−3.80 (m, 4H, O-
(CH2)2), 6.95−7.10 (m, 2H, arom H), 7.41−7.58 (m, 5H, arom H),
7.75−7.79 (m, 5H, arom H). 13C NMR (CDCl3) δ 53.57, 60.68 (CH2-
N-(CH2)2), 62.24 (CH), 66.68 (O-(CH2)2), 70.60 (O-CH3), 112.54,
121.07, 128.25 (arom C), 128.72 (Ar-CO), 129.48, 130.04, 132.31,
132.80 (arom C), 138.31 (Ph-CO), 156.45 (Ar-O), 196.41 (CO). MS
m/e 341.50 (M, 20%). Anal. Calcd for C20H23NO4: C, 68.55; H, 6.90;
N, 4.00. Found: C, 68.30; H, 6.62; N, 4.29.
(3-{3-[4-(4-Fluoro-phenyl)-piperazine-1-yl]-2hydroxy-propoxy}-
phenyl)-phenyl-methanone (10). First, 500 mg (1.97 mmol) of (3-
oxiranylmethoxy-phenyl)-phenyl-methanone (2b) was dissolved in 15
mL of methanol, treated with 360 mg (2 mmol) of p-F-phenyl-
piperazine, and the mixture was refluxed for 6 h. Then the solution was
allowed to cool down and stirred at room temperature overnight. The
obtained solid was filtered off and washed with diethyl ether, giving
690 mg (80.76%) white crystals; mp 131−133 °C. 1H NMR (CDCl3)
δ 2.60−2.85 (m, 6H, CH2-N-(CH2)2), 3.12−3.17 (m, 4H, (CH2)2-N-
Ph), 4.05−4.18 (m, 3H, O-CH2-CH-OH), 6.87−6.97 (m, 4H, arom
H), 7.19−7.25 (m, 1H, arom H), 7.37−7.60 (m, 6H, arom H), 7.78−
7.82 (m, 2H, arom H). 13C NMR (CDCl3) δ 50.18, 53.28, 60.30
(CH2-N-(CH2)4), 65.46 (CH), 70.36 (O-CH2), 115.04, 115.29,
115.73, 117.81, 117.97, 119.28, 123.15, 128.24, 129.99, 132.43
(arom C), 137.48 (Ar-CO), 138.87 (Ph-CO), 147.71 (Ar-N),
157.22 (J = 239 Hz, C-F), 158.64 (Ar-O), 196.39 (CO). MS m/e
434.77 (M, 100%). Anal. Calcd for C26H27FN2O3: C, 71.87; H, 6.26;
N,6.45. Found: C, 71.65; H, 6.45; N, 6.46.
[4-(2-Hydroxy-3-morpholine-4-yl-propoxy)-phenyl]-phenyl-
methanone (16). First, 700 mg (2.75 mmol) of (4-oxiranylmethoxy-
phenyl)-phenyl-methanone (2c) was dissolved in 15 mL of methanol,
treated with 240 mg of morpholine, and refluxed for 7 h. Then
(4-{3-[4-(4-Fluoro-phenyl)-piperazine-1-yl]-2-hydroxy-propoxy}-
phenyl)-phenyl-methanone (11). First, 700 mg (2.75 mmol) of (4-
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subsequent removal of the solvent under reduced pressure left 910 mg
(96.8%) of yellow oil; mp 135−141 °C. H NMR (CDCl3) δ 2.44−
methane was added dropwise. The mixture was stirred at room
temperature and monitored by TLC. After evaporation to dryness,
yellow oil was obtained, which was further purified by column
chromatography (silica gel, CH2Cl2/methanol/concentrated NH3,
100/10/1). Subsequent removal of the solvents under reduced
pressure yielded 181 mg (62.92%) of a light-yellow oil. 1H NMR
(CDCl3) δ 2.12−2.20 (m, 2H, CH2-N), 2.33 (s, 3H, CH3), 3.39−2.47
(m, 4H, N-(CH2)2), 3.74−3.79 (m, 5H, (CH2)2-N, CH), 3.96 (d, 2H,
J = 4.9, O-CH2), 6.96−7.80 (m, 14H, arom H, NH). 13C NMR
(CDCl3) δ 20.89 (CH3), 49.02 (N-(CH2)2), 52.55 ((CH2)2-N), 59.78
(CH2-N), 65.88 (CH), 70.74 (O-CH2), 112.73, 121.15, 123.47,
125.45, 128.31, (arom C), 128.71 (Ar-CO), 129.53, 129.63, 130.12,
132.38, 132.93 (arom H), 135.11 (Ar-CH3), 137.34 (NH-Ar), 138.25
(Ph-CO), 156.51 (arom CO), 183.26 (CS), 196.47 (CO). MS m/e
489.5 (M, 15%). Anal. Calcd for C28H31N3O3S: C, 68.69; H, 6.38; N,
8.58. Found: C, 68.60; H, 6.53; N, 8.47.
[2-(3-{Benzoyl-phenoxy)-2-hydroxyl-propyl]-piperazine-1-yl}-2-
hydroxy-propoxy)-phenyl]-phenyl-methanone (23). First, 356 mg
(1.40 mmol) of (2-oxiranylmethoxy-phenyl)-phenyl-methanone (2a)
was dissolved in 20−30 mL of methanol and then 50.8 mg (0.59
mmol) of piperazine was added and the reaction mixture was refluxed
for 5 h. Then after removal of the solvent by rotary evaporation, a
column chromatography was performed (silica gel, CH2Cl2/methanol/
concentrated NH3, 100/10/1). Subsequent evaporation to dryness
gave yellow oil, which crystallized from isopropyl alcohol to leave 424
mg (51%) of white solid; mp 125−135 °C. 1H NMR (CDCl3) δ 1.98−
2.42 (m, 12H, CH2-N-(CH2)2-N-CH2), 3.10 (s, 1H, OH), 3.70−3.76
(m, 2H, 2CH), 3.94−4.07 (m, 5H, O-CH2, OH), 6.99−7.83 (m, 18H,
arom H). 13C NMR (CDCl3) δ 53.13 (N-(CH2)4-N), 60.06 (CH2-
NH), 65.47 (CH), 70.78 (O-CH2), 112.62, 121.10, 128.28, 129.53,
130.09, 132.33, 132.82 (arom C), 128.81 (Ar-CO), 138.34 (Ph-CO),
156.55 (Ar-O), 196.46 (CO). MS m/e 594.7 (M, 100%). Anal. Calcd
for C36H38N2O6: C, 70.57; H, 7.14; N, 4.22. Found: C, 70.91; H, 7.02;
N, 4.26.
1
2.71 (m, 6H, CH2-N-(CH2)2), 3.73 (t, 4H, J = 4.56, (CH2)2-O), 4.06−
4.17 (m, 3H, O-CH2), 6.98 (d, 2H, J = 8.82, H-3, H-5), 7.42−7.56 (m,
3H, arom H), 7.72−7.84 (m, 4H, arom H). 13C NMR (CDCl3) δ
53.69, 60.83 (CH2-N-(CH2)2), 65.18 (CH), 66.94 ((CH2)2-O), 70.28
(O-CH2), 114.04, 128.15, 129.68 (arom C), 130.40 (Ar-CO), 131.90,
132.49 (arom C), 138.13 (Ph-CO), 162.24 (Ar-O), 195.48 (CO). MS
m/e 342.30 (M+, 95%). Anal. Calcd for C20H23NO4: C, 69.15; H, 6.87;
N, 4.03. Found: C, 69.16; H, 7.07; N, 4.18.
{3-[2-Hydroxy-3-(4-o-tolyl-piperazine-1-yl)-propoxy]-phenyl}-
phenyl-methanone (17). First, 700 mg (2.75 mmol) of (3-
oxiranylmethoxy-phenyl)-phenyl-methanone (2b) was dissolved in
15 mL of methanol and treated with 500 mg (2.84 mmol) of o-tolyl-
piperazine, and the mixture was refluxed for 6 h. Then subsequent
removal of the solvent yielded 1.07 g (90.29%) of yellow oil; mp 173−
1
179 °C. H NMR (CDCl3) δ 2.31 (s, 3H, CH3), 2.61−2.66 (m, 4H,
N(CH2)2), 2.28−2.96 (m, 6H, CH2-N, (CH2)2)-N-Phe), 4.05−4.18
(m, 3H, O-CH2-CH), 6.99−7.05 (m, 2H, arom H), 7.37−7.60 (m,
6H, arom H), 7.79−7.83 (m, 2H, arom H). 13C NMR (CDCl3) δ
17.82 (CH3), 51.71 (N-(CH2)2), 53.74 (CH2-N), 60.34 ((CH2)2-N-
Ph), 65.37(CH), 70.46 (O-CH2), 115.05, 118.92, 119.28, 123.07,
123.18, 126.53, 128.23, 129.23, 131.02, 132.40 (arom C), 132.40 (Ar-
CO), 138.83 (Ph-CO), 151.23 (Ar−N), 158.68 (Ar-O), 196.40 (CO).
MS m/e 430.52 (M, 20%). Anal. Calcd for C27H30N2O3: C, 73.78; H,
7.11; N, 6.37. Found. C, 73.71; H, 7.31; N, 6.24.
{4-[2-Hydroxy-3-(4-o-tolyl-piperazine-1-yl)-propoxy]-phenyl}-
phenyl-methanone (18). First, 700 mg (2.75 mmol) of (4-
oxiranylmethoxy-phenyl)-phenyl-methanone (2c) was dissolved in
15 mL of methanol and treated with 500 mg (2.84 mmol) of o-tolyl-
piperazine, and the mixture was refluxed for 5 h. Then subsequent
removal of the solvent under reduced pressure yield 1.1 g (92.83%) of
white crystals, which were recrystallized from methanol; mp 107−114
°C. 1H NMR (CDCl3) δ 2.31 (s, 3H, CH3), 2.63−2.67 (m, 4H,
N(CH2)2), 2.84−2.97 (m, 6H, CH2-N, (CH2)2)-N-Ph), 4.10−4.22
(m, 3H, O-CH2-CH), 6.99−7.03 (m, 4H, arom H), 7.15−7.21 (m,
2H, arom H), 7.48−7.58 (m, 3H, arom H), 7.74−7.86 (m, 4H, arom
H). 13C NMR (CDCl3) δ 17.82 (CH3), 51.74 (N-(CH2)2), 53.74
(CH2-N), 60.36 ((CH2)2-N-Ph), 65.29 (CH), 70.42 (O-CH2), 114.09,
118.94, 123.24, 126.57, 128.17, 129.71, (arom C), 130.38 (Ar-CO),
131.06, 131.90, 132.52 (arom C), 138.18 (Ph-CO), 151.22 (Ar-N),
162.34 (Ar-O), 195.51 (CO); MS m/e 431.4 (M+, 100%). Anal. Calcd
for C27H30N2O3: C, 75.32; H, 7.02; N, 6.51. Found: C, 75.11; H, 7.12;
N, 6.39.
1-[2-(3-{4-[3-(2-Benzoyl-phenoxy)-2-hydroxyl-propyl]-piperazine-
1-yl}-2-hydroxy-propoxy)-5-methyl-phenyl]-ethanone (24). First,
500 mg (2.43 mmol) of 1-(5-methyl-2-oxiranylmethoxy-phenyl)-
ethanone (4) was dissolved in 10 mL of methanol, treated with
857.1 mg (2.52 mmol) of [2-(2-hydroxy-3-piperazine-1-yl-propoxy)-
phenyl]-phenyl-methanone (5), and refluxed for 5 h. Then after
removal of solvent under reduced pressure, a column chromatography
was performed (silica gel, CH2Cl2/methanol/concentrated NH3, 120/
10/1). Subsequent removal of the solvents under reduced pressure
1
yielded 1.20 g (90.67%) of an orange oil: mp 89−93 °C. H NMR
(CDCl3) δ 2.05−2.13 (m, 2H, CH2-N), 2.29 (s, 3H, CH3), 2.33−2.55
(m, 10H, N-(CH2)4-N-CH2), 2.62 (s, 3H, CO-CH3), 2.98−3.01 (m,
1H, OH), 3.69−4.10 (m, 6H, 2O-CH2-CH), 6.83−7.80 (m, 12H,
arom H). 13C NMR (CDCl3) δ 20.22 (CH3-Ar), 31.81 (CH3-CO),
53.20 (N-(CH2)2), 60.03, 60.49 (N-CH2), 65.43, 65.51 (CH), 70.75,
71.12 (O-CH2), 128.03, 128.82 (Ar-CO), 130.25 (CH3-Ar), 112.61,
112.79, 121.10, 128.27, 129.51, 130.08, 130.59, 132.32, 132.81, 134.12
(arom C), 138 (Ph-CO), 156.00, 156.55 (Ar-O), 196.46 (CO), 199.88
(Ar-CO-CH3). MS m/e 594.7 (M+, 100%). Anal. Calcd for
C32H38N2O6: C, 61.82; H, 6.65; N, 4.51. Found: C, 61.60; H, 6.91;
N, 4.37.
Computational Studies. Ligand Efficiency (LE). Ligand
efficiency (LE = Δg) values of the data were calculated by
normalizing binding free energy of a ligand for number of heavy
atoms. Free energy calculation was carried out as described by
Hopkins et al. (eq 3). According to Hopkins et al., IC50 from
percentage inhibition can be substituted for Kd (dissociation
constant potency)48 which was further confirmed by
experimental results of Kuntz and co-workers.38
4-[3-(2-Benzoyl-phenoxy)-2-hydroxy-propyl]-piperazine-1-car-
boxylic Acid p-Tolylamide (21). First, 186 mg (0.54 mmol) of [2-(2-
hydroxy-3-piperazine-1-yl-propoxy)-phenyl]-phenyl-methanone (5)
was dissolved in 15 mL of dichloromethane, and then a solution of
75 mg (0.57 mmol) of 4-methyl phenyl isocyanate in dichloromethane
was added dropwise. The mixture was stirred at room temperature and
monitored by TLC. After evaporation to dryness, a yellow oil was
obtained which was purified by column chromatography (silica gel,
CH2Cl2/methanol/concentrated NH3, 100/10/1). Subsequent remov-
al of the solvents under reduced pressure yielded 136 mg (52.56%) of
a colorless oil. 1H NMR (CDCl3) δ 2.41−2.53 (m, 6H, CH2-N-
(CH2)2), 2.43 (s, 3H, CH3), 3.13 (s, 1H, OH), 3.51−3.53 (m, 4H,
(CH2)2-N), 3.86−3.95 (m, 1H, CH), 4.09 (d, 2H, J = 4.80, O-CH2),
7.08−7.96 (m, 14H, arom H, NH). 13C NMR (CDCl3) δ 20.57
(CH3), 43.70 ((CH2)2-N-CO), 52.76 (N-(CH2)2), 59.97 (CH2-N),
65.60 (CH), 70.60 (O-CH2), 112.56, 120.32, 120.96, 128.20, 29.07,
129.42, 129.88, 132.27, 132.85 (arom C), 128.54 (Ar-CO), 132.30
(Ar-CH3), 136.38 (NH-Ar), 138.08 (Ph-CO), 155.24 (N-CO-NH),
156.39 (arom C−O), 196.54 (CO). MS m/e 473.8 (M, 18%). Anal.
Calcd for C28H31N3O4: C, 71.02; H, 6.60; N, 8.87. Found: C, 70.77;
H, 6.89; N, 8.64.
ΔG = − RT ln K
(3)
d
4-[3-(2-Benzoyl-phenoxy)-2-hydroxy-propyl]-piperazine-1-carbo-
thioic Acid p-Tolylamide (22). First, 200 mg (0.58 mmol) of [2-(2-
hydroxy-3-piperazine-1-yl-propoxy)-phenyl]-phenyl-methanone (5)
was dissolved in 10 mL of dichloromethane, and then a solution of
91 mg (0.61 mmol) of 4-methyl phenyl isothiocyanate in dichloro-
Ligand efficiency calculations was done for a temperature of 310 K
and given in kcal per heavy atom (eq 4).
LE = (Δg) = −ΔG/HA
(non‐hydrogen atom)
(4)
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Article
A size independent fit quality score was obtained as described by
Reynolds et al.21 by fitting the maximum LE over a large range of
molecular size. All calculations regarding ligand efficiency were done
by using Excel spreadsheet. IC50 values of the propafenone type
inhibitors (GPV576, GPV005, GPV062, and propafenone) were
determined experimentally by a daunorubicin efflux essay.37,49
Inhibition of rhodamine 123 efflux in the transfectant mouse
lymphoma line L5178 VMDR1 C.06 were used to characterize the
MDR-modulating activity values of verapamil, niguldipine, and
cyclosporine A. IC50 values of tariquidar,50 elacridar,51,52 valdapodar,53
zosuquidar,54,55 and ONT-09356 were taken from literature (Table 2).
IC50 values for most of the compounds in clinical studies were
reported by using rhodamine 123 efflux essays. We use these values, as
there is a direct correlation between the IC50 values from daunorubicin
and rhodamine 123 efflux essays.57
least an order of magnitude below the Km values published for P-
gp.58,59
Briefly, cells were sedimented, the supernatant was removed by
aspiration, and the cells were resuspended at a density of 1 × 106/mL
in RPMI 1640 medium containing daunorubicin (Sigma Chemical Co.,
St. Louis, MO) at a final concentration of 3 μmol/L. Cell suspensions
were incubated at 37 °C for 30 min. Tubes were chilled on ice and
centrifuged at 500g in an Eppendorf 5403 centrifuge (Eppendorf,
Hamburg, Germany). Supernatants were removed, and the cell pellet
was resuspended in medium prewarmed to 37 °C containing either no
inhibitor or compounds at various concentrations ranging from 20 to
200 μM, depending on the solubility and expected potency of the
inhibitor. Eight concentrations (serial 1:3 dilution) were tested for
each inhibitor. After 60, 120, 180, and 240 s, aliquots of the incubation
mixture were transferred to tubes containing an equal volume of ice-
cold stop solution (RPMI medium containing GPV31 at a final
concentration of 5 μmol/L). Zero time points were determined by
immediately pipetting daunorubicin-preloaded cells into ice-cold stop
solution. Samples drawn at the respective time points were kept in an
ice water bath and measured within 1 h on a Becton Dickinson FACS
Calibur flow cytometer (Becton Dickinson, Vienna, Austria). Viable
cells were selected by setting appropriate gates for forward and side
scatter. The excitation and emission wavelengths were 482 and 558
nm, respectively. Five thousand gated events were accumulated for the
determination of mean fluorescence values.
Lipophilic Efficiency (LipE). LipE of benzophenones were calculated
(eq 5) and compared with the compounds which reached clinical
studies (verapamil, tariquidar, valspodar, elacridar, zosuquidar, ONT-
093, niguldipine, and cyclosporine A) as well as with selected
propafenone analogues.
LipE = LLE = pIC − clogP
(5)
50
clogP values of the data set were computed by using the Bio-Loom
software package,35 and the LipE calculations were performed by using
Excel spreadsheet. To compare the standard threshold of LipE along
three different entry pathways of ligands into respective binding
pockets of P-gp, hERG and SERT, a data set from literature was used.
It includes 744 SERT inhibitors extracted from the ChEMBL
database,40 313 hERG blockers41 from literature, and 372 inhibitors
of P-gp mediated daunorubicin efflux (in-house data). The data sets
are available at our homepage (pharminfo.univie.ac.at) and from
Chemspider (www.chemspider.com).
Docking. Compounds 6, 19, 20, and 23 were docked in their
neutral form into an open state homology model of human P-gp17
based on the X-ray structure of mouse P-gp (PDB: 3G5U)19 by using
the software package GOLD. To avoid any bias, we considered the
whole transmembrane domain region as binding pocket. Then 100
poses per ligand were obtained, and finally ligand protein complexes
were minimized by LigX, a minimization tool implemented in MOE,
by using the MMFF94 force field.
A complete work flow of poses selection has been provided in
Supporting Information (SM Workflow 1). Briefly, agglomerative
Hierarchical Cluster analysis of the consensus rmsd matrix based on
the common scaffold of the ligands identified two interesting clusters
of poses containing all four ligands. However, additional five clusters
have been identified containing three out of four ligands. All seven
clusters were occupying the center of the binding cavity mainly
interacting with amino acid residues of TM 1, 5, 6, 7, 8, 10, and 11
(SM Figure 2A). For a more detailed analysis of the ligand−protein
interaction profiles of selected ligands, we used the two clusters
containing all four ligands (SM Figure 2B).
To prioritize among the two clusters, a rescoring of all docking
poses by using four different scoring functions in MOE (ASE, affinity
dG, Alpha HB, London dG) was performed. Subsequently, for each
ligand, the top 10 ranked poses according to consensus scoring were
taken and analyzed. Out of these 40 poses, seven poses were present in
cluster 1 while only one showed up in cluster 2. In addition, taking
only the top ranked pose per ligand, two (6, 23) out of four ligands
were located in cluster 1 (SM Figure 2B). Additionally, cluster 1 was
supported by photo affinity labeling and SAR data of the series.
Therefore interaction position of cluster 1 was supposed to be the
most likely one for benzophenones.
ASSOCIATED CONTENT
* Supporting Information
■
S
LipE profiles of inhibitors of P-gp, SERT, and hERG; docking
workflow; clustered docking poses; overlap of interacting
amino acid residues of propafenone-type inhibitors of P-gp;
photolabeled drug binding domains of propafenone-type
inhibitors of P-gp. This material is available free of charge via
the Internet at http://pubs.acs.org.
Accession Codes
PDB ID: 3G5U.
AUTHOR INFORMATION
Corresponding Author
*Phone: +43-1-4277-55110. Fax: +43-1-4277-9551. E-mail:
gerhard.f.ecker@univie.ac.at.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the Austrian Science Fund for financial
support (grant SFB F35). Ishrat Jabeen thanks the Higher
Education Commission of Pakistan for financial support.
ABBREVIATIONS USED
■
P-gp, P-glycoprotein; LipE, lipophilic efficiency; LE, ligand
efficiency; MDR, multidrug resistance; ABC, ATP binding
cassette; QSAR, quantitative structure−activity relationship
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