First biological evaluation of developed 3-benzyloxyfluorenes as novel class of MDR modulators

Article abstract of DOI:10.1016/j.ejmech.2010.02.024

A series of 3-benzyloxy-1-aza-9-oxafluorenes has been synthesized and biologically evaluated as novel MDR modulators. The concentration dependent inhibition of the efflux pump ABCB1 (P-glycoprotein) has been characterized and is discussed in relation to calculated lipophilicity data. Instead of the molecular lipophilicity the exact positioning of functional groups was found decisive for the biological activities.

Full text of DOI:10.1016/j.ejmech.2010.02.024

European Journal of Medicinal Chemistry 45 (2010) 2683–2688
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
First biological evaluation of developed 3-benzyloxyfluorenes as novel class
of MDR modulators
a
a
a
a
b
a,
*
´
´
Martin Krug , Burkhardt Voigt , Christiane Baumert , Ralf Lu¨pken , Josef Molnar , Andreas Hilgeroth
a Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck Str. 4, D-06120 Halle, Germany
b
´
Department of Medical Microbiology, University of Szeged, Dom ter 10, 6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-benzyloxy-1-aza-9-oxafluorenes has been synthesized and biologically evaluated as novel
MDR modulators. The concentration dependent inhibition of the efflux pump ABCB1 (P-glycoprotein) has
been characterized and is discussed in relation to calculated lipophilicity data. Instead of the molecular
lipophilicity the exact positioning of functional groups was found decisive for the biological activities.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 26 November 2009
Received in revised form
4 February 2010
Accepted 8 February 2010
Available online 13 February 2010
Keywords:
ABCB1 inhibitor
Efflux pump
MDR modulator
Binding site
Lipophilicity calculation
1. Introduction
structures should show high activities in first screening studies thus
providing effective modulators at low application concentrations.
MDR (multidrug resistance) is one main obstacle in cancer
therapy [1,2]. MDR-causative multidrug efflux pumps transport
structurally differing anticancer drugs as efflux pump substrates
out of the cells so that the intracellular drug levels are insufficient
for an effective cancer treatment [1,3,4]. One reason for the struc-
tural diversity of the efflux pump substrates has been the existence
of several substrate binding sites within the architecture frame-
work of the transmembrane domains of the various efflux pump
types [1,5,6]. The use of inhibitors of the efflux pump activities is
still the most effective strategy to overcome MDR because alter-
native strategies like a transcriptional control of the efflux pump
expression disappointed so far [4,7].
The use of such inhibitors in clinical studies has been strongly
dose-limited because of toxic side effects which are observed in
clinically necessary MDR reversal concentrations. A structure-
dependent development of an inhibitor of an efflux pump is still
not possible because of a missing of highly solved structures of the
efflux pumps. So the development of novel inhibitor classes depends
on the discovery of novel lead structures and the investigation of
structure-activity relationships (SARs). Moreover, novel lead
We discovered 3-benzyloxy-1-aza-9-oxafluorenes as a novel
class of strong inhibitors of the efflux pump ABCB1 (P-glycoprotein)
which plays a central role in the MDR cancer treatment [1,7].
Systematic hydrophobic substitutions of the aromatic residues
have been made to discuss SARs of the biological activities related
to calculated lipophilicity data.
2. Results and discussion
2.1. Chemistry
The 3-benzyloxy substituted pyridine compounds 1a–c have
been given by a benzylation of 3-hydroxypyridine in dried THF
under strong alkaline conditions using n-tetrabutylammonium
bromide as phase transfer catalyst (Scheme 1) [8]. The chlorosub-
stituted grignard reagents for the N-acetyl 1,4-dihydropyridine
formations were yielded by the reaction of isopropyl magnesium
chloride and the corresponding 3- or 4-substituted iodobenzene in
THF at low temperature without isolation (Scheme 1).
The N-acetyl 1,4-dihydropyridines 2a–i resulted as a mixture of
E/Z isomers from a primary acetylation reaction of the 3-benzyloxy-
pyridines 1a–c in dried THF to non-isolated N-acetyl pyridinium salts
and a following regioselective 4-arylation using copper(I) iodide as
catalyst and the prepared grignard reagents [9].
* Corresponding author. Tel.: þ49 345 55 25168; fax: þ49 345 55 27207.
E-mail address: andreas.hilgeroth@pharmazie.uni-halle.de (A. Hilgeroth).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.024

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M. Krug et al. / European Journal of Medicinal Chemistry 45 (2010) 2683–2688
R2
The unsubstituted 3-benzyloxy-1-aza-9-oxafluorene 4a was
much more active at 10
M with a FAR value of 34.49 ꢀ 5.61 than
the verapamil control with a FAR value of 4.35 ꢀ 0.01 at the twofold
concentration of 20 M (Table 1). The corresponding increase in the
resulting mean fluorescence intensity (MIF) for compound 4a made
M in the P-gp
R1
m
OH
O
m
a
N
44.54 ꢀ 4.65 at 1
m
M and 801.70 ꢀ 19.95 at 10
m
N
expressing cells if compared to the inhibitor-untreated cells with
a MIF of 24.30 ꢀ 5.59.
1 a-c
R3
R4
R3
R4
The introduction of a chloro atom into the benzylic substituent
mainly increased the inhibitory activity in compounds 4b and 4c at
the higher inhibitor concentrations if compared to compound 4a.
Chloro substituents have a lipophilic character and thus their
introduction into the benzylic residue shall increase the lip-
ophilicity of the compound. We calculated the lipophilicity of the
compounds with various calculation programs and finally the log P
values were given as shown in Table 1. Indeed we found increased
log P values with the introduction of the chloro atom into the
benzylic residue of compounds 4b and 4c.
b
c
I
MgCl
R3
R4
O
R2
R1
O
O
Close structure-activity studies in many MDR modulators
classes suggested that increased lipophilicities of the compounds
increased the biological activities as MDR modulators [11]. This was
reasoned with a better membrane penetration of the modulators to
interact with the transmembrane efflux pump protein.
When the chloro atom was introduced into the phenyl substit-
uent in compounds 4d and 4e were found interesting results. An
introduction into the 4-position of the phenyl ring significantly
increased the inhibitory activities of derivative 4d at both the lower
and the higher inhibitor concentrations with a FAR value of
+
N
O
H3C
2 a-i
d
R3
R4
O
R2
R1
HO
95.58 ꢀ 6.18 at 10
mM.
If the chloro atom was introduced into the neighbouring
O
R3
N
3-position of the phenyl ring in compound 4e we surprisingly
found a mainly reduced activity at 10 mM with a comparably poor
R4
O
R2
O
H3C
R4
3
FAR value of 10.47 ꢀ 5.14.
R3
R1
HO
The lipophilicity data of the latter chlorophenyl substituted
derivatives were consistent with those of the chlorobenzyl deriv-
atives, so that it can be stated that an increased lipophilicity of
a MDR modulator within one modulator class does not conse-
quently correlate with an increased biological activity. This is a new
insight in the SAR discussion of MDR modulators.
R2
R1
O
N
+
4 a-i
O
+
HO
OH
Obviously the exact positioning within the phenyl ring is the
decisive criterion for influencing the activity of the monochloro
derivatives.
The introduction of a second chloro atom in compounds 4f–4i
again led to increases in the lipophilicities of the compounds.
However, significant differences in activities were found. First, all
activities were below the activity of the unsubstituted derivative
4a. Again this fact impressively proves that the lipophilicity within
N
5 a-i
Scheme 1. Reagents and conditions: (a) ArCH2Cl, KOH, n-Bu4NBr, THF, 70 ꢁC;
(b) (H3C)2CHMgBr, THF, ꢂ40 ꢁC; (c) Cu(I)I, LiCl, H3CCOCl, THF, ꢂ40 ꢁC; (d) HClO4 (2%),
dioxane, rt.
The 1-aza-9-oxafluorene target compounds 4a–i have been
given by a primary reaction of p-benzoquinone with the N-acetyl
1,4-dihydropyridines 2a–i in dioxane under perchloric acid catal-
ysis to non-isolated tetrahydro 1-aza-9-oxafluorene intermediates
3 [9] which underwent a following oxidation by an excess of
p-benzoquinone. The formation of the target compounds 4a–i was
accompanied by an oxidation procedure to pyridine compounds
5a–i.
Table 1
Lipophilicity data and ABCB1 inhibition of 3-benzyloxy-1-aza-9-oxafluorene target
compounds 4a–i.
R1 R2 R3 R4 log P
FAR valuesa
1
m
M
10 mM
2.2. Biological activities
4a
4b
4c
4d
4e
4f
4g
4h
H
Cl
H
H
H
Cl
H
Cl
H
H
H
Cl
H
H
H
Cl Cl
H
Cl
H
H
H
Cl
H
Cl
H
H
H
H
5.38 ꢀ 0.71 1.75 ꢀ 0.29 34.49 ꢀ 5.61
5.97 ꢀ 0.76 1.23 ꢀ 0.01 56.85 ꢀ 3.67
5.97 ꢀ 0.76 1.26 ꢀ 0.01 57.42 ꢀ 3.71
5.97 ꢀ 0.76 3.04 ꢀ 0.01 95.58 ꢀ 6.18
The evaluation of the ABCB1 inhibiting properties has been
carried out in a mouse T lymphoma cell line which exclusively
overexpresses ABCB1 after mdr1-retroviral gene transfection of the
parental cell line [10]. The uptake of the fluorescent ABCB1
substrate rhodamine 123 has been determined in both the ABCB1
overexpressing cell line and the parental cell line by flow cytom-
etry. The ABCB1 inhibiting activities were determined as fluores-
cence activity ratios (FAR values) in both cell lines with each
fluorescence values related to those of the untreated controls.
Cl 5.97 ꢀ 0.76 1.35 ꢀ 0.01 10.47 ꢀ 5.14
H
H
6.59 ꢀ 0.80 1.11 ꢀ 0.01
6.59 ꢀ 0.80 1.53 ꢀ 0.25 21.48 ꢀ 3.50
2.61 ꢀ 0.61
Cl 6.59 ꢀ 0.80 1.00 ꢀ 0.01 17.14 ꢀ 8.41
n.d.b
4.35 ꢀ 0.01
5.92 ꢀ 2.87
H
H
Cl 6.59 ꢀ 0.80 1.81 ꢀ 0.01
4i
Verapamil (20 mM)
a
Mean of two determinations.
Not determined.
b

M. Krug et al. / European Journal of Medicinal Chemistry 45 (2010) 2683–2688
2685
a class of MDR modulators does not consequently lead to increased
biological activities.
We found an interesting structure-activity correlation within
the series of the twofold chlorosubstituted derivatives. Both meta
chlorobenzyl derivatives 4g and 4i were much more active than
both para chlorobenzyl derivatives 4f and 4h.
Similar to the series of monochloro derivatives we found within
the series of dichloro derivatives the 4-chlorophenyl compounds 4f
and 4g to be more active than the 3-chlorophenyl compounds 4h
and 4i.
So the positioning of the chloro atom within the phenyl residue
is decisive for the biological activity and if such a chloro atom is
present in the phenyl ring the positioning of a second chloro atom
in the benzylic residue either in the para or the meta position leads
to significant differences in activity.
alkalined with a 10 M sodium hydroxide solution to pH ¼ 8 and
then extracted with 75 mL chloroform for three times. After drying
over sodium sulfate and filtration the organic layer was removed in
vaccuum and the resulting product was purified by column chro-
matography using chloroform/ethyl acetate/methanol (85/15/0.5).
4.1.1.1. 3-Benzyloxypyridine 1a. Yield 5.75 g (59%); yellow oil; 1H
NMR (acetone-d6)
d
8.37 (d, 1H, J ¼ 2.9 Hz), 8.18 (dd, 1H, J ¼ 4.6,
1.3 Hz), 7.57–7.50 (m, 2H), 7.41–7.31 (m, 4H), 7.26 (ddd, 1H, J ¼ 8.4,
4.6, 0.6 Hz), 5.18 (s, 2H); MS (ESI), m/z ¼ 186 [M þ Hþ]; IR (ATR):
3062, 2931, 2872, 1574, 1475, 1454, 1424, 1260, 1227, 1048 cmꢂ1
.
Anal. (C12H11NO) C, H, N.
4.1.1.2. 3-(4-Chlorobenzyloxy)pyridine 1b. Yield 5.53 g (48%); yellow
oil; 1H NMR (acetone-d6)
d
8.35 (d,1H, J ¼ 2.8 Hz), 8.20(dd,1H, J ¼ 4.8,
1.2 Hz), 7.43 (dd, 2H, J ¼ 8.6, 2.0 Hz), 7.51 (dd, 2H, J ¼ 8.6, 2.0 Hz), 7.40
(ddd,1H, J ¼ 8.4, 4.3,1.3 Hz), 7.28 (ddd,1H, J ¼ 8.4, 4.6, 0.6 Hz), 5.21 (s,
2H); MS (ESI), m/z ¼ 220 [M þ Hþ]; IR (ATR): 3056, 2926, 2873, 1573,
1491, 1475, 1262, 1227, 1086 cmꢂ1. Anal. (C12H10Cl NO) C, H, N.
3. Conclusions
In summary, the systematically varied substitution of both
benzylic and phenylic substituents of the highly active ABCB1
inhibitor with the 3-benzyloxy-1-aza-9-oxafluorene scaffold led to
interesting insights in the SARs of MDR modulators. It could be
demonstrated that an increased lipophilicity does not lead to
increased biological activities. The opposite effect was observed for
the most lipophilic dichloro derivatives which were all less active
than the lowest lipophilic unsubstituted derivative. The exact
positioning of the chloro atom in the 4-position of the phenyl ring
and if in combination with this additionally in the meta position of
the benzylic residue led to derivatives with each best activities of
the monochloro and dichloro derivatives, respectively. Beside their
contribution to lipophilicity chloro atoms may serve as hydrogen
bond acceptor functions. The high sensitivity of their positioning
within the phenyl ring suggests that they interact with amide
functions of the protein backbone at the respective protein binding
site. Highest activity was found for the 4-monochloro phenyl
substituted derivative 4d. This activity exceeded the activity of the
standard compound verapamil by a factor of about 50 if related to
the concentrations. Further varied hydrogen bond acceptor func-
tions at the 4-phenyl ring within this new class of highly active
ABCB1 inhibitors will help to understand the potential binding to
the respective ABCB1 binding site.
4.1.1.3. 3-(3-Chlorobenzyloxy)pyridine 1c. Yield 5.87 g (51%);
yellow oil; 1H NMR (acetone-d6)
d
8.37 (d, 1H, J ¼ 2.9 Hz), 8.18 (dd,
1H, J ¼ 5.8, 1.2 Hz), 7.55–7.52 (m, 1H), 7.47–7.35 (m, 4H), 7.28 (ddd,
1H, J ¼ 8.4, 4.6, 0.5 Hz), 5.22 (s, 2H); MS (ESI), m/z ¼ 220 [M þ Hþ];
IR (ATR): 3059, 1573, 1474, 1423, 1259, 1227, 1096 cmꢂ1. Anal.
(C12H10Cl NO) C, H, N.
4.1.2. General procedure for the N-acetyl 1,4-dihydropyridine
formation of 2a–i
3-Benzyloxypyridine (1 equiv, 5 mmol) 1 was dissolved in dried
THF under argon atmosphere. After addition of copper(I) iodide
(0.1 equiv, 0.5 mmol) and lithium chloride (0.2 equiv, 1 mmol) the
solution was stirred at rt for 30 min. Then acetyl chloride (1.1 equiv,
5.5 mmol) was added dropwise over 15 min at ꢂ40 ꢁC. Meanwhile
the corresponding substituted iodobenzene (1.2 equiv, 6 mmol)
was dissolved in another flask in 50 mL of dried THF, cooled down
to ꢂ40 ꢁC and then isopropyl magnesium bromide (1.1 eqiv.
5.5 mmol) was added as solution in THF. After stirring of this
solution for 1 h at ꢂ40 ꢁC it was added dropwise under stirring to
the prepared pyridinium salt solution and stirring was continued
for 30 min. The solution was left stirring until rt was reached. After
additional 15 min, 50 mL of ammonium chloride solution in
ammonia (20%) and 50 mL of diethyl ether were added. The phases
were separated and the water phase was extracted with 50 mL of
diethyl ether for two times. The collected organic layer was
extracted as follows with each 50 mL of ammonium chloride
solution in ammonia (20%), water, twice with hydrochloric acid
(10%), water and saturated sodium chloride solution. After drying
over sodium sulfate and filtration the organic layer was removed in
vaccuum and the oily residue of 2 was purified by column chro-
matography using silica gel and a cyclohexane/ethyl acetate
mixture as eluent. The purified compound 2 was recrystallized
from methanol.
4. Experimental protocols
4.1. Chemistry
Commercial reagents were used without further purification.
The 1H NMR spectra (400 MHz) were measured using tetrame-
thylsilane as internal standard. TLC was performed on E. Merck
5554 silica gel plates. The EI mass spectra were measured with an
AMD 402 mass spectrometer, the ESI spectra were recorded on
a Finnigan LCQ Classic mass spectrometer. IR spectra were recorded
on a FT-IR spectrometer. Elemental analysis indicated by the
symbols of the elements was within ꢀ0.4% of the theoretical values
and was performed using a Leco CHNS-932 apparatus.
4.1.2.1. (E/Z)-1-Acetyl-3-benzyloxy-4-phenyl-1,4-dihydropyridine
2a. Yield 0.89 g (58%); mp 78–82 ꢁC; 1H NMR (CDCl3)
d 7.33–
4.1.1. General procedure for the formation of 3-benzyloxypyridines
1a–c
7.20 (m, 17H), 7.13–7.10 (m, 4H), 6.98 (d, 1H, J ¼ 1.3 Hz), 6.67 (dt,
1H, J ¼ 8.2, 1.3 Hz), 6.10 (d, 1H, J ¼ 1.3 Hz), 5.15 (dd, 1H, J ¼ 8.2,
4.3 Hz), 5.07 (dd, 1H, J ¼ 8.2, 4.3 Hz), 4.77 (d, 2H, J ¼ 11.8 Hz),
4.74 (d, 2H, J ¼ 11.9 Hz), 4.31 (d, 2H, J ¼ 4.3 Hz), 2.26 (s, 3H), 2.16
(s, 3H); MS (EI), m/z ¼ 305 [Mþ]; IR (ATR): 3045, 2936, 1672,
1653, 1586, 1366, 1315 cmꢂ1. Anal. (C20H19NO2) C, H, N.
3-Hydroxypyridine (5 g, 52.6 mmol), powdered KOH (5.9 g,
110 mmol) and n-tetrabutylammonium bromide (0.85 g, 2.6 mmol)
were dissolved in 150 mL dried THF. After addition of the corre-
sponding benzylchloride (1.6 equiv, 84 mmol) the solution was
stirred for 12 h under reflux. Then 200 mL of distilled water were
added and extraction followed twice with 100 mL of hydrochloric
acid solution in water (10%). The united water phases were
4.1.2.2. (E/Z)-1-Acetyl-3-(4-chlorobenzyloxy)-4-phenyl-1,4-dihydro-
pyridine 2b. Yield 0.97 g (57%); mp 68–72 ꢁC; 1H NMR (acetone-

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M. Krug et al. / European Journal of Medicinal Chemistry 45 (2010) 2683–2688
d6)
d
7.37–7.13 (m, 19H), 6.94 (s, 1H), 6.90 (d, 1H, J ¼ 8.2 Hz), 6.50 (s,
(acetone-d6)
d
7.35 (d, 1H, J ¼ 8.3 Hz), 7.29–7.19 (m, 10H), 7.16–7.11
1H), 5.14 (dd, 1H, J ¼ 8.2, 4.2 Hz), 5.04 (dd, 1H, J ¼ 8.1, 4.2 Hz), 4.94
(d, 1H, J ¼ 12.4 Hz), 4.89 (d, 1H, J ¼ 12.6 Hz), 4.83 (d, 1H, J
¼ 12.4 Hz), 4.74 (d, 1H, J ¼ 12.6 Hz), 4.36 (d, 2H, J ¼ 4.2 Hz), 2.25 (s,
3H), 2.22 (s, 3H); MS (EI), m/z ¼ 339 [Mþ]; IR (ATR): 1672, 1635,
1411, 1376, 1315 cmꢂ1. Anal. (C20H18ClNO2) C, H, N.
(m, 2H), 7.10–7.06 (m, 2H), 7.04–6.69 (m, 2H), 6.94 (s, 1H), 6.70 (d,
1H, J ¼ 8.2 Hz), 6.09 (s, 1H), 5.11 (dd, 1H, J ¼ 8.2, 4.2 Hz), 5.02 (dd,
1H, J ¼ 8.2, 4.2 Hz), 4.78 (d, 2H, J ¼ 12.1 Hz), 4.70 (d, 2H, J ¼ 12.1 Hz),
4.30 (d, 2H, J ¼ 4.1 Hz), 2.28 (s, 3H), 2.19 (s, 3H); MS (EI), m/z ¼ 373
[Mþ]; IR (ATR): 1674, 1635, 1487, 1376, 1316 cmꢂ1
. Anal.
(C20H17Cl2NO2) C, H, N.
4.1.2.3. (E/Z)-1-Acetyl-3-(3-chlorobenzyloxy)-4-phenyl-1,4-dihy-
dropyridine 2c. Yield 0.93 g (55%); mp 88–96 ꢁC; 1H NMR
4.1.3. General procedure for the formation of the 1-aza-9-
(acetone-d6)
d
7.49–7.16 (m, 19H), 6.94 (s, 1H), 6.89 (d, 1H,
oxafluorenes 4a–i
J ¼ 8.1 Hz), 6.50 (s, 1H), 5.13 (dd, 1H, J ¼ 8.3, 4.3 Hz), 5.03 (dd, 1H,
J ¼ 8.1, 4.2 Hz), 4.91 (d,1H, J ¼ 12.2 Hz), 4.86 (d,1H, J ¼ 12.3 Hz), 4.79
(d, 1H, J ¼ 12.2 Hz), 4.74 (d, 1H, J ¼ 12.3 Hz), 4.33 (d, 2H, J ¼ 4.2 Hz),
2.25 (s, 3H), 2.22 (s, 3H); MS (EI), m/z ¼ 339 [Mþ]; IR (ATR): 1673,
1636, 1432, 1377, 1316 cmꢂ1. Anal. (C20H18ClNO2) C, H, N.
N-Acetyl 1,4-dihydropyridine
2
(1 equiv, 1.5 mmol) was
dissolved in a mixture of dried dioxane (19.6 mL) and perchloric
acid (70%, 0.4 mL). p-Benzoquinone (1.2 equiv, 1.8 mmol) was
added to the solution. After stirring for 24 h at rt, the reaction was
controlled by tlc in cyclohexane/ethyl acetate (60/40) until no more
detectable intermediate 3 [9] was observed. Then the solution was
neutralized with a 1 M sodium hydroxide solution to pH 7–8 and
extracted with chloroform for three times. The united organic layer
was dried over sodium sulfate, filtered and the solution was
evaporated to dryness leaving a yellow oil which was purified by
column chromatography using silica gel and an eluent mixture of
cyclohexane/ethyl acetate (60/40). The collected fractions were
dried over sodium sulfate, filtered and then the organic layer was
removed in vacuum. The oily residue was dissolved in diethyl ether
from which the target compound 4 crystallized. The pyridine side
product 5 was also isolated within the purification procedure by
collecting the compound containing fractions and drying over
sodium sulfate. After filtration and removing of the organic layer
the final crystallization took place in diethyl ether under cooling.
4.1.2.4. (E/Z)-1-Acetyl-3-benzyloxy-4-(4-chlorophenyl)-1,4-dihy-
dropyridine 2d. Yield 0.59 g (35%); mp 74–77 ꢁC; 1H NMR (CDCl3)
d
7.33 (d, 1H, J ¼ 8.3 Hz), 7.17 (d, 4H, J ¼ 8.2 Hz), 7.14–7.10 (m, 14H),
6.97 (s, 1H), 6.68 (d, 1H, J ¼ 8.2 Hz), 6.10 (s, 1H), 5.10 (dd, 1H, J ¼ 8.3,
4.3 Hz), 5.01 (dd,1H, J ¼ 8.2, 4.1 Hz), 4.79 (d, 2H, J ¼ 11.6 Hz), 4.71 (d,
2H, J ¼ 11.6 Hz), 4.29 (d, 2H, J ¼ 4.3 Hz), 2.27 (s, 3H), 2.17 (s, 3H); MS
(EI), m/z ¼ 339 [Mþ]; IR (ATR): 1672, 1635, 1411, 1376, 1315 cmꢂ1
.
Anal. (C20H18ClNO2) C, H, N.
4.1.2.5. (E/Z)-1-Acetyl-3-benzyloxy-4-(3-chlorophenyl)-1,4-dihy-
dropyridine 2e. Yield 0.34 g (20%); mp 61–67 ꢁC; 1H NMR (CDCl3)
d
7.37–7.34 (m, 5H), 7.31–7.25 (m, 6H), 7.23–7.20 (m, 4H), 7.15–7.11
(m, 4H), 6.98 (s, 1H), 6.69 (d, 1H, J ¼ 8.2 Hz), 6.11 (s, 1H), 5.10 (dd, 1H,
J ¼ 8.2, 4.3 Hz), 5.02 (dd, 1H, J ¼ 8.2, 4.2 Hz), 4.83 (d, 1H, J ¼ 12.0 Hz),
4.82 (d, 2H, J ¼ 12.0 Hz), 4.78 (d, 1H, J ¼ 12.0 Hz), 4.29 (d, 2H,
J ¼ 4.8 Hz), 2.27 (s, 3H), 2.17 (s, 3H); MS (EI), m/z ¼ 339 [Mþ]; IR
(ATR): 1673,1635,1496,1376,1319 cmꢂ1. Anal. (C20H18ClNO2) C, H, N.
4.1.3.1. 3-Benzyloxy-4-phenyl[1]benzofuro[2,3-b]pyridine-6-ol
4a. Yield 0.07 g (13%); mp 220–224 ꢁC; 1H NMR (dmso-d6)
d 9.34 (s,
1H), 8.32 (s,1H), 7.60–7.54 (m, 5H), 7.49 (d,1H, J ¼ 8.9 Hz), 7.31–7.26
(m, 5H), 6.92 (dd, 1H, J ¼ 8.9, 2.7 Hz), 6.51 (d, 1H, J ¼ 2.7 Hz), 5.17 (s,
2H); MS (EI), m/z ¼ 367 [Mþ]; IR (KBr): 3401, 1359, 1276 cmꢂ1. Anal.
(C24H17NO3) C, H, N.
4.1.2.6. (E/Z)-1-Acetyl-3-(4-chlorobenzyloxy)-4-(4-chlorophenyl)-1,4-
dihydropyridine 2f. Yield 0.58 g (31%); mp 93–97 ꢁC; 1H NMR
(CDCl3)
d
7.34 (d, 1H, J ¼ 8.0 Hz), 7.28–7.22 (m, 8H), 7.16 (d, 4H,
J ¼ 8.4 Hz), 7.03 (d, 4H, J ¼ 8.4 Hz), 6.94 (s, 1H), 6.68 (d, 1H,
J ¼ 8.4 Hz), 6.08 (s, 1H), 5.10 (dd, 1H, J ¼ 8.4, 4.0 Hz), 5.01 (dd, 1H,
J ¼ 8.2, 4.2 Hz), 4.76 (d, 2H, J ¼ 11.6 Hz), 4.70 (d, 2H, J ¼ 11.6 Hz), 4.27
(d, 2H, J ¼ 3.2 Hz), 2.27 (s, 3H), 2.18 (s, 3H); MS (EI), m/z ¼ 373 [Mþ];
IR (ATR): 1673, 1636, 1488, 1377, 1316 cmꢂ1. Anal. (C20H17Cl2NO2) C,
H, N.
4.1.3.2. 3-(4-Chlorobenzyloxy)-4-phenyl[1]benzofuro[2,3-b]pyridine-
6-ol 4b. Yield 0.12 g (20%); mp 208–211 ꢁC; 1H NMR (dmso-d6)
d
9.40 (s, 1H), 8.36 (s, 1H), 7.62–7.57 (m, 4H), 7.46 (d, 1H, J ¼ 8.9 Hz),
7.35–7.29 (m, 5H), 7.01 (dd, 1H, J ¼ 8.9, 2.6 Hz), 6.69 (d, 1H,
J ¼ 2.6 Hz), 5.19 (s, 2H); MS (ESI), m/z ¼ 402 [M þ Hþ]; IR (KBr):
3429, 1472, 1354, 1184 cmꢂ1. Anal. (C24H16ClNO3) C, H, N.
4.1.2.7. (E/Z)-1-Acetyl-3-(3-chlorobenzyloxy)-4-(4-chlorophenyl)-
4.1.3.3. 3-(3-Chlorobenzyloxy)-4-phenyl[1]benzofuro[2,3-b]pyridine-
1,4-dihydropyridine 2g. Yield 0.83 g (44%); mp 63–70 ꢁC; 1H NMR
6-ol 4c. Yield 0.08 g (14%); mp 222–226 ꢁC; 1H NMR (dmso-d6)
(CDCl3)
d
7.34 (d, 1H, J ¼ 8.3 Hz), 7.28 (d, 4H, J ¼ 8.4 Hz), 7.25–7.22
d
9.34 (s, 1H), 8.28 (s, 1H), 7.49–7.48 (m, 4H), 7.47 (d, 1H, J ¼ 8.9 Hz),
(m, 2H), 7.21–7.19 (m, 2H), 7.18 (d, 4H, J ¼ 8.4 Hz), 7.07–7.03 (m, 2H),
7.01–6.97 (m, 2H), 6.93 (s, 1H), 6.69 (d, 1H, J ¼ 8.2 Hz, 1 H), 6.07 (s,
1H), 5.11 (dd, 1H, J ¼ 8.2, 4.1 Hz), 5.01 (dd, 1H, J ¼ 8.2, 4.2 Hz), 4.77
(d, 2H, J ¼ 12.1 Hz), 4.68 (d, 2H, J ¼ 12.1 Hz), 4.30 (d, 2H, J ¼ 4.1 Hz),
2.27 (s, 3H), 2.18 (s, 3H); MS (EI), m/z ¼ 373 [Mþ]; IR (ATR): 1675,
1636, 1487, 1376, 1317 cmꢂ1. Anal. (C20H17Cl2NO2) C, H, N.
7.27–7.24 (m, 5H), 6.90 (dd, 1H, J ¼ 8.9, 2.6 Hz), 6.48 (d, 1H,
J ¼ 2.6 Hz), 5.13 (s, 2H); MS (ESI), m/z ¼ 402 [M þ Hþ]; IR (KBr):
3392, 1493, 1470, 1354 cmꢂ1. Anal. (C24H16ClNO3) C, H, N.
4.1.3.4. 3-Benzyloxy-4-(4-chlorophenyl)[1]benzofuro[2,3-b]pyridine-
6-ol 4d. Yield 0.12 g (20%); mp 211–214 ꢁC; 1H NMR (dmso-d6)
d
9.39 (s, 1H), 8.33 (s, 1H), 7.65 (d, 2H, J ¼ 8.4 Hz), 7.59 (d, 2H,
4.1.2.8. (E/Z)-1-Acetyl-3-(4-chlorobenzyloxy)-4-(3-chlorophenyl)-
J ¼ 8.4 Hz), 7.50 (d, 1H, J ¼ 8.9 Hz), 7.37–7.25 (m, 5H), 6.92 (dd, 1H,
J ¼ 8.9, 2.5 Hz), 6.55 (d, 1H, J ¼ 2.5 Hz), 5.18 (s, 2H); MS (ESI), m/
1,4-dihydropyridine 2h. Yield 0.50 g (27%); mp 88–93 ꢁC; 1H NMR
(acetone-d6)
d
7.30 (d, 1H, J ¼ 7.2 Hz), 7.32–7.21 (m, 16H), 6.96 (s,
z ¼ 402 [M þ Hþ]; IR (ATR): 3140, 1472, 1185 cmꢂ1
. Anal.
1H), 6.94 (d, 1H, J ¼ 8.3 Hz), 6.55 (s, 1H), 5.14 (dd, 1H, J ¼ 8.4, 4.4 Hz),
5.04 (dd, 1H, J ¼ 8.4, 4.4 Hz), 4.89 (d, 2H, J ¼ 12.4 Hz), 4.78 (d, 2H,
J ¼ 12.4 Hz), 4.39 (d, 2H, J ¼ 4.0 Hz), 2.26 (s, 3H), 2.24 (s, 3H). MS
(C24H16ClNO3) C, H, N.
4.1.3.5. 3-Benzyloxy-4-(3-chlorophenyl)[1]benzofuro[2,3-b]pyridine-
(EI), m/z ¼ 373 [Mþ]; IR (ATR): 1674, 1634, 1491, 1376, 1318 cmꢂ1
.
6-ol 4e. Yield 0.12 g (20%); mp 215–217 ꢁC; 1H NMR (dmso-d6)
Anal. (C20H17Cl2NO2) C, H, N.
d 9.40 (s, 1H), 8.35 (s, 1H), 7.65–7.61 (m, 3H), 7.55–7.49 (m, 2H),
7.35–7.25 (m, 5H), 6.94 (dd, 1H, J ¼ 8.9, 2.5 Hz), 6.52 (d, 1H,
J ¼ 2.5 Hz), 5.19 (s, 2H); MS (ESI), m/z ¼ 402 [M þ Hþ]; IR (ATR):
3132, 1499, 1472, 1186 cmꢂ1. Anal. (C24H16ClNO3) C, H, N.
4.1.2.9. (E/Z)-1-Acetyl-3-(3-chlorobenzyloxy)-4-(3-chlorophenyl)-
1,4-dihydropyridine 2i. Yield 0.48 g (26%); mp 88–92 ꢁC; 1H NMR

M. Krug et al. / European Journal of Medicinal Chemistry 45 (2010) 2683–2688
2687
4.1.3.6. 3-(4-Chlorobenzyloxy)-4-(4-chlorophenyl)[1]benzofuro[2,3-
1H, J ¼ 4.8 Hz), 7.55–7.53 (m, 1H), 7.50 (dd, 2H, J ¼ 8.6, 2.0 Hz), 7.39
(dd, 2H, J ¼ 8.6, 2.0 Hz), 7.30 (dd, 2H, J ¼ 8.6, 1.9 Hz), 7.21 (dd, 2H,
J ¼ 8.6, 1.9 Hz), 5.10 (s, 2H); MS (ESI), m/z ¼ 331 [M þ Hþ]; IR (ATR):
1493, 1093, 1070 cmꢂ1. Anal. (C18H13Cl2NO) C, H, N.
b]pyridine-6-ol 4f. Yield 0.13 g (20%); mp 219–222 ꢁC; 1H NMR
(dmso-d6)
d
9.39 (s, 1H), 8.33 (s, 1H), 7.66 (d, 2H, J ¼ 8.4 Hz), 7.59 (d,
2H, J ¼ 8.4 Hz), 7.51 (d, 1H, J ¼ 8.9 Hz), 7.39 (d, 2H, J ¼ 8.4 Hz), 7.31
(d, 2H, J ¼ 8.4 Hz), 6.93 (dd, 1H, J ¼ 8.8, 2.6 Hz), 6.55 (d, 1H,
J ¼ 2.6 Hz), 5.18 (s, 2H); MS (ESI), m/z ¼ 437 [M þ Hþ]; IR (ATR):
3079, 1495, 1182, 1025 cmꢂ1. Anal. (C24H15Cl2NO3) C, H, N.
4.1.3.16. 3-(3-Chlorobenzyloxy)-4-(4-chlorophenyl)pyridine 5g. Yield
0.28 g (57%); mp 77–82 ꢁC; 1H NMR (CDCl3)
d 8.38 (s,1H), 8.33 (d,1H,
J ¼ 4.7 Hz), 7.52 (dd, 2H, J ¼ 8.7, 2.0 Hz), 7.41 (dd, 2H, J ¼ 8.7, 2.0 Hz),
7.28–7.25 (m, 3H), 7.18–7.14 (m, 1H), 5.11 (s, 2H); MS (ESI), m/z ¼ 331
[M þ Hþ]; IR (KBr): 1601,1472,1094 cmꢂ1 Anal. (C18H13Cl2NO) C, H, N.
4.1.3.7. 3-(3-Chlorobenzyloxy)-4-(4-chlorophenyl)[1]benzofuro[2,3-
b]pyridine-6-ol 4g. Yield 0.05 g (7%); mp 228–232 ꢁC; 1H NMR
(dmso-d6)
d
9.40 (s, 1H), 8.69 (s, 1H), 7.66 (d, 2H, J ¼ 8.6 Hz), 7.59 (d,
2H, J ¼ 8.6 Hz), 7.51 (d, 1H, J ¼ 8.9 Hz), 7.39–7.31 (m, 2H), 7.27–7.21
(m, 2H), 6.93 (dd, 1H, J ¼ 8.9, 2.6 Hz), 6.56 (d, 1H, J ¼ 2.5 Hz), 5.19 (s,
2H); MS (ESI), m/z ¼ 437 [M þ Hþ]; IR (ATR): 3100, 1506, 1472, 1187,
1086 cmꢂ1. Anal. (C24H15Cl2NO3) C, H, N.
4.1.3.17. 3-(4-Chlorobenzyloxy)-4-(3-chlorophenyl)pyridine 5h. Yield
0.33 g (68%); mp 111–115 ꢁC; 1H NMR (CDCl3)
d 8.39 (s, 1H), 8.33 (d,
1H, J ¼ 4.8 Hz), 7.60–7.58 (m, 1H), 7.44–7.42 (m, 1H), 7.37–7.35 (m,
2H), 7.32–7.29 (m, 2H), 7.28–7.26 (m, 1H), 7.23–7.22 (m, 2H), 5.12 (s,
2H); MS (ESI), m/z ¼ 331 [M þ Hþ]; IR (KBr): 1492, 1466, 1085,
1032 cmꢂ1 Anal. (C18H13Cl2NO) C, H, N.
4.1.3.8. 3-(4-Chlorobenzyloxy)-4-(3-chlorophenyl)[1]benzofuro[2,3-
b]pyridine-6-ol 4h. Yield 0.07 g (10%); mp 209–212 ꢁC; 1H NMR
(dmso-d6)
d
9.38 (s, 1H), 8.31 (s, 1H), 7.64 (dd, 2H, J ¼ 8.4, 2.0 Hz),
4.1.3.18. 3-(3-Chlorobenzyloxy)-4-(3-chlorophenyl)pyridine 5i. Yield
7.57 (dd, 2H, J ¼ 8.4, 2.0 Hz), 7.48 (d, 1H, J ¼ 8.9 Hz), 7.36–7.30 (m,
2H), 7.24–7.17 (m, 2H), 6.91 (dd, 1H, J ¼ 8.9, 2.5 Hz), 6.54 (d, 1H,
J ¼ 2.5 Hz), 5.17 (s, 2H); MS (ESI), m/z ¼ 437 [M þ Hþ]; IR (ATR):
3088, 1499, 1471, 1185, 1019 cmꢂ1. Anal. (C24H15Cl2NO3) C, H, N.
0.33 g (70%); mp 93–95 ꢁC; 1H NMR (CDCl3)
d 8.39 (s,1H), 8.33 (d,1H,
J ¼ 4.8 Hz), 7.60–7.58 (m, 1H), 7.44–7.42 (m, 1H), 7.37–7.35 (m, 2H),
7.32–7.29 (m, 2H), 7.28–7.26 (m, 1H), 7.23–7.22 (m, 2H), 5.12 (s, 2H);
MS (ESI), m/z ¼ 331 [M þ Hþ]; IR (KBr): 1588, 1464, 1098 cmꢂ1 Anal.
(C18H13Cl2NO) C, H, N.
4.1.3.9. 3-(3-Chlorobenzyloxy)-4-(3-chlorophenyl)[1]benzofuro[2,3-
b]pyridine-6-ol 4i. Yield 0.13 g (20%); mp 199–202 ꢁC; 1H NMR
4.2. Lipophilicity calculations
(dmso-d6)
d 9.40 (s, 1H), 8.36 (s, 1H), 7.67–7.63 (m, 3H), 7.56–7.50
(m, 2H), 7.36–7.34 (m, 2H), 7.31–7.28 (m, 1H), 7.26–7.21 (m, 1H),
Molecules were constructed and log P values were determined
with nine programs (ALOGPS, AC logP, AB/LogP, miLogP, ALOGP,
MLOGP, KOWWIN, XLOGP2 and XLOGP3) [12]. The resulting mean
values were given by the ALOGPS 2.1 program [12].
6.94 (dd, 1H, J ¼ 8.9, 2.6 Hz), 6.54 (d, 1H, J ¼ 2.6 Hz), 5.22 (s, 2H); MS
(ESI), m/z ¼ 473 [M þ Hþ]; IR (ATR): 3175, 1580, 1470, 1190 cmꢂ1
.
Anal. (C24H15Cl2NO3) C, H, N.
4.1.3.10. 3-Benzyloxy-4-phenylpyridine 5a. Yield 0.21 g (55%); mp
68–72 ꢁC; 1H NMR (acetone-d6)
d
8.50 (s,1H), 8.28 (d,1H, J ¼ 4.8 Hz),
4.3. Biological activity
7.67–7.64 (m, 2H), 7.47–7.27 (m, 9H), 5.27 (s, 2H); MS (EI), m/z ¼ 261
[Mþ]; IR (ATR): 1248, 1235 cmꢂ1. Anal. (C18H15NO) C, H, N.
Verapamil as IsoptinÒ injection solution was purchased from
Abbott GmbH & Co. KG (Wiesbaden, Germany). Other chemicals for
cell culture were purchased from Sigma–Aldrich (Steinheim,
Germany) unless otherwise specified.
4.1.3.11. 3-(4-Chlorobenzyloxy)-4-phenylpyridine 5b. Yield 0.25 g
(57%); mp 72–74 ꢁC; 1H NMR (CDCl3)
d 8.37 (s, 1H), 8.32 (d, 1H,
J ¼ 4.8 Hz), 7.60–7.56 (m, 2H), 7.47–7.42 (m, 2H), 7.42–7.36 (m, 1H),
7.31–7.26 (m, 1H), 7.27 (d, 1H, J ¼ 4.8 Hz), 7.25–7.22 (m, 2H), 7.18–
7.13 (m, 1H), 5.09 (s, 2H); MS (ESI), m/z ¼ 296 [M þ Hþ]; IR (KBr):
1479, 1415, 1076, 1014 cmꢂ1. Anal. (C18H14ClNO) C, H, N.
4.3.1. Cell culture
The mouse T lymphoma cell line L5178Y and the L5178Y mdr
subline were grown in serum-free McCoys 5A medium with 10%
heat-inactivated horse serum albumine, 2 mM glutamine, peni-
4.1.3.12. 3-(3-Chlorobenzyloxy)-4-phenylpyridine 5c. Yield 0.26 g
cillin (100 U/ml) and streptomycin (100 mg/ml) [13]. The cell culture
(59%); mp 81–85 ꢁC; 1H NMR (CDCl3)
d
8.36 (s, 1H), 8.31 (d, 1H,
medium for the MDR resistant subline was supplemented with
colchicine to a final concentration of 60 ng/ml to ensure a stable
ABCB1 expression.
J ¼ 4.8 Hz), 7.60–7.55 (m, 2H), 7.46–7.38 (m, 1H), 7.30 (dd, 2H,
J ¼ 8.6,1.9 Hz), 7.30–7.26 (m, 3H), 7.25–7.18 (m, 2H), 5.07 (s, 2H); MS
(ESI), m/z ¼ 296 [M þ Hþ]; IR (KBr): 1492, 1416, 1085, 1012 cmꢂ1
.
Anal. (C18H14ClNO) C, H, N.
4.3.2. ABCB1 inhibition assay
Cultured cells from both mouse T lymphoma parental cell line
L5178 and the ABCB1 expressing subline L5178Y mdr were adjusted
to a concentration of 2 ꢃ106 cells per mL in serum-free McCoys 5A
medium [14]. To each 0.5 mL of the cell suspensions the test
compounds were added from stock solutions in DMSO. Incubation
time lasted for 10 min at rt. The fluorescent ABCB1 substrate
4.1.3.13. 3-Benzyloxy-4-(4-chlorophenyl)pyridine 5d. Yield 0.28 g
(62%); mp 51–56 ꢁC; 1H NMR (CDCl3)
d 8.41 (s, 1H), 8.31 (d, 1H,
J ¼ 5.0 Hz), 7.52 (dd, 2H, J ¼ 8.7, 2.0 Hz), 7.48–7.44 (m, 1H), 7.38 (dd,
2H, J ¼ 8.7, 2.0 Hz), 7.36–7.27 (m, 5H), 5.15 (s, 2H); MS (ESI), m/z ¼ 296
[M þ Hþ]; IR (KBr): 1598,1481,1092 cmꢂ1. Anal. (C18H14ClNO) C, H, N.
rhodamine 123 was added reaching a final concentration of 5.2 mM.
4.1.3.14. 3-Benzyloxy-4-(3-chlorophenyl)pyridine 5e. Yield 0.29 g
After an additional incubation period of 20 min at 37 ꢁC the cells
were washed twice with phosphate-buffered saline (PBS). Then
they were resuspended in PBS and each 1 ꢃ104 cells were
measured by flow cytometry. Fluorescence activity ratios have been
calculated as the relation of the determined fluorescence in the
inhibitor treated ABCB1 expressing subline to the parental cell line
with each fluorescence values corrected to those of the untreated
control.
(64%); mp 78–80 ꢁC; 1H NMR (CDCl3)
d 8.41 (s, 1H), 8.31 (d, 1H,
J ¼ 4.8 Hz), 7.63–7.60 (m, 1H), 7.48–7.44 (m, 2H), 7.38–7.28 (m, 5H),
7.26–7.23 (m, 2H), 5.16 (s, 2H); MS (ESI), m/z ¼ 296 [M þ Hþ]; IR
(KBr): 1470, 1085, 1015 cmꢂ1. Anal. (C18H14ClNO) C, H, N.
4.1.3.15. 3-(4-Chlorobenzyloxy)-4-(4-chlorophenyl)pyridine 5f. Yield
0.14 g (29%); mp 107–110 ꢁC; 1H NMR (CDCl3)
d 8.38 (s, 1H), 8.32 (d,

2688
M. Krug et al. / European Journal of Medicinal Chemistry 45 (2010) 2683–2688
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Acknowledgement
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218–227.
Financial support of the work was given from the Studien-
stiftung des Deutschen Volkes and the country Saxony-Anhalt to
Martin Krug and Burkhardt Voigt, the BMBF within a DAAD project
and the DFG to Christiane Baumert and Andreas Hilgeroth.
[6] G. St. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, R. Zhuo, P.M. Harrell,
Y.T. Trinh, I.L. Urbatsch, Science 323 (2009) 1718–1722.
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