Development of small-molecule P-gp inhibitors of the N-benzyl 1,4-dihydropyridine type: Novel aspects in SAR and bioanalytical evaluation of multidrug resistance (MDR) reversal properties

Article abstract of DOI:10.1016/j.bmc.2012.10.041

Novel series of N-benzyl 1,4-dihydropyridines have been prepared by facile syntheses. All relevant substituents of the molecular scaffold have been varied. The resulting compounds were biologically evaluated as P-glycoprotein (P-gp) inhibitors. Substitutions of the N-benzyl residue favour biological activity beside respective 3-ester functions. Most active compounds were further evaluated as multidrug resistance (MDR) modulators to restore the cytotoxic properties of varying daunorubicin applications.

Full text of DOI:10.1016/j.bmc.2012.10.041

Bioorganic & Medicinal Chemistry 21 (2013) 166–177
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of small-molecule P-gp inhibitors of the N-benzyl
1,4-dihydropyridine type: Novel aspects in SAR and bioanalytical evaluation
of multidrug resistance (MDR) reversal properties
Christiane Baumert a, Marianne Günthel a, Sören Krawczyk a, Marc Hemmer a, Tom Wersig a,
Andreas Langner a, Joséf Molnár b, Hermann Lage c, Andreas Hilgeroth a,
⇑
a Institute of Pharmacy, Martin Luther University, 06120 Halle, Germany
b Department of Medical Microbiology, University of Szeged, 6720 Szeged, Hungary
c Institute of Pathology, University Hospital Charité, 10117 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel series of N-benzyl 1,4-dihydropyridines have been prepared by facile syntheses. All relevant sub-
stituents of the molecular scaffold have been varied. The resulting compounds were biologically evalu-
ated as P-glycoprotein (P-gp) inhibitors. Substitutions of the N-benzyl residue favour biological activity
beside respective 3-ester functions. Most active compounds were further evaluated as multidrug resis-
tance (MDR) modulators to restore the cytotoxic properties of varying daunorubicin applications.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 27 September 2012
Accepted 25 October 2012
Available online 3 November 2012
Keywords:
Structure–activity relationships (SAR)
Multidrug resistance (MDR)
P-glycoprotein (P-gp)
Inhibitor
Efflux pump inhibition
1. Introduction
of their originally pharmacological properties as antihypertensive
or immunosuppressive drugs, second generation inhibitors with
Multidrug resistance (MDR) against various anticancer drugs of
different compound classes has maintained the central problem in
anticancer drug therapies over the last decades.1–3 Although recent
anticancer drug developments resulted in novel compound classes
like tyrosine kinase inhibitors as monoclonal antibodies or small-
molecule inhibitors, the improvement in drug-resistant therapies
remained limited because these novel compounds were also af-
fected by the MDR phenomenon.4–6 Multidrug efflux pumps of var-
ious types have been identified to be mainly causative agents in
the multidrug affected process of MDR by transporting anticancer
drugs out of the cells so that effective intracellular drug levels are
no longer reached.3,7,8 As the hopes of novel drugs to overcome the
MDR phenomenon have been disappointed in general, other strat-
egies have been followed over the last years.2,4,9 The inhibition of
the efflux pump activity by drugs turned out as the most perspec-
tive approach to combate MDR because successful alternatives of
influencing the efflux pump protein formation by a transcriptional
control are still limited to in vitro studies.2,10 Drugs of various fam-
ilies have early been observed to modulate the MDR phenome-
non.9,11 With a limited application range of these drugs because
reduced original effects were developed. However, they showed a
partial loss of their MDR modulating properties.9,11,12 Following
inhibitors which presently undergo clinical trials suffer from
observed toxic problems.4,13
Intensive efforts have been taken to develop small-molecule
inhibitors with a promising favourable resorption in contrast to re-
cent complex natural compounds with high and critical molecular
weights which are unfavourable also because the synthetic access
to such natural compounds is difficult and expensive.4,14,15
We recently described pyridine-2-ones as novel class of
MDR modulators.16 They have been developed from our earlier
N-acyloxy-1,4-dihydropyridines which suffered from a critical
hydrolysis sensitivity of the N-acyloxy substituent (Fig. 1).4
The pyridine-2-ones partly showed merely in vitro activities
in the range of verapamil which was used as one of the best
described in vitro inhibitors. The N-acyloxy function of our former
1,4-dihydropyridines was placed within the dihydropyridine
scaffold leading to a 1,2-dihydro structure of the pyridine-2-ones
instead of the former 1,4-dihydro structure.
We now present 1,4-dihydropyridines without a ring-carbonyl
function. We maintained the N-benzyl function of the pyridine-2-
ones. In our novel series we varied the substitution patterns of
the 4-phenyl and the N-benzyl residue to find out which structural
⇑
Corresponding author. Tel.: +49 345 55 25168; fax: +49 345 55 27207.
E-mail address: andreas.hilgeroth@pharmazie.uni-halle.de (A. Hilgeroth).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.041

C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
167
R2- R4
O
ing or of the otherwise commercially available Grignard reagents
were added dropwise to a stirring suspension of the N-benzyl
pyridinium salts 3a and b at low temperatures of À20 °C. The cat-
alyst directed the nucleophilic aryl residue into the 4-position of
the pyridinium nucleus so that the 4-aryl 1,4-dihydropyridines
4a–l were yielded. This method has been successfully used for
the preparation of corresponding 4-phenyl substituted N-acetyl
1,4-dihydropyridines.17 However, the isolated reaction products
after the workup procedure were mainly not pure and made an-
other column chromatography necessary.
R2
Aryl
N
R1
O
COR
OR1
N
Bn
COOPh
N
Pyridine-2-ones
N-Acyloxy-1,4-di-
hydropyridines
R5- R7
As the described favourable two-step procedure led to impure
reaction products which afforded additional purifications we prac-
ticed a second access to the 1,4-dihydropyridines target structures
by a simple two component reaction of previously prepared com-
pounds 7a–j of both the benzylamine and the varying ester moie-
ties and of 9a and b bearing the later 4-aryl substituent of the
resulting 1,4-dihydropyridines 10a–j (Scheme 2).
The b-enaminocarbonyl compounds 7a–j were given by a quan-
titative reaction of the propiolic acid esters 5a and 5b with the
respective benzylamines 6a–e in dried THF under stirring for 5 h
at rt. After evaporation of the solvent the residual oil of compounds
7a–j was used without further purification. The compounds were
given as a 1:3 mixture of E/Z isomers according to the 1H NMR data
which showed coupling constants of the methine protons of the E
isomer in the range of 3J = 13 Hz while the neighboured methine
protons in the Z isomer had a coupling constant of about 3J = 8 Hz.
The aldehyde compounds 9a and 9b were given by the reaction
of the corresponding aryl iodide 8a and 8b with acrolein diethyl
acetale in DMF at 90 °C in a palladium catalyzed Heck coupling
reaction using palladium(II) acetate as primary catalyst, tetrabutyl
ammonium acetate as phase transfer catalyst and, finally, potas-
sium carbonate as basic component.
The resulting aldehyde compounds 9a and 9b exclusively con-
sisted of the E isomer with high coupling constants of the methine
protons of about 3J = 16 Hz according to a selective trans formation
of the double bond within the complex reaction pathway in a ther-
modynamically controlled reaction as described in literature.18
The final 1,4-dihydropyridine formation took place under
smooth reaction conditions of 40 °C using iron(III) chloride hexa-
hydrate as lewis acid for the activation of the non-saturated alde-
hyde components 9a and 9b to react in a following michael
addition with the b-enaminocarbonyl components 7a–j. A con-
cluding condensation reaction led to the 1,4-dihydropyridine ring
closure giving the 1,4-dihydropyridine target structures 10a–j.
N-Benzyl-1,4-dihydropyridine
Target Structure
Figure 1. Pyridine-2-ones starting structure, N-acyloxy-1,4-dihydropyridine pre-
cursor structure and N-benzyl 1,4-dihydropyridine target structure.
element is more sensitive to substitution with respect to the bio-
logical activity. Moreover, we varied the 3-ester substituent.
2. Results and discussion
2.1. Chemistry
The synthesis of the 4-aryl substituted 1,4-dihydropyridines
4a–l followed a simple two-step procedure starting with the N-
alkylation of the commercially available pyridines 2a and b
(Scheme 1).
Both pyridines 2a and b were dissolved in a minimum volume
of isopropanol. Then 1.5 equiv of benzyl bromide were added drop-
wise under stirring which continued for 18 h. The resulting partly
oily products of 3a and b were washed with diethyl ether and then
evaporated in vaccuum. After additional washing of the formed
semi-solid compounds with diethyl ether they were stored in vac-
uum to be used for the following reactions without further
purification.
The 4-arylation of the N-benzyl pyridinium ions of the crude
compounds 3a and b was carried out in dried THF with 1.5 equiv
of Grignard reagents under copper(I) iodide catalysis and under
addition of lithium chloride to improve the catalyst solubility in
the reaction mixture.
In the case of the 3-halogen substituted derivatives 4a–f, the
Grignard reagents were freshly prepared from isopropyl magne-
sium chloride and the corresponding 3-chloro, 3-bromo and 3-tri-
fluoromethyl iodopyridines 1a–c, respectively, at À40 °C in dried
THF under stirring for 1 h (Scheme 1). Each 1.5 equiv of the result-
2.2. Biological evaluation and structure–activity relationships
(SAR) of the P-gp inhibition
R
R
The P-gp inhibition of our target compounds has been carried
out by the determination of the uptaken amount of the fluorescent
P-gp substrate rhodamine 123 in both a mouse T lymphoma cell
line without P-gp and another one with overexpressing P-gp. The
later cell line has been derived fom the non-P-gp expressing cell
line after retroviral transfection with the human mdr1 gene and a
consequent cell culturing in a colchicine-containing medium to se-
lect only the P-gp expressing cells which survive the colchicine-
culturing by the extracellular colchicine efflux. Thus both cell lines
exclusively differ in the expression of human P-gp so that a differ-
ence in the uptake of the fluorescent rhodamine 123 can be di-
rectly reasoned with the expression of this efflux pump. By the
way the use of different P-gp inhibitor concentrations in pretreated
cells caused differences in the fluorescence uptake so that the inhi-
bition of the efflux pump activity increased the fluorescence up-
take in the P-gp expressing cell line.
i
I
MgCl
R2
1a, R = Cl
b, R = Br
c, R = CF3
R3
R4
O
O
O
OR1
OR
OR
ii
iii
+
Br -
N
N
N
2a, R= Me
b, R= Et
3a, R= Me
b, R= Et
4a - l
The P-gp inhibition-characterizing size is the fluorescence activ-
ity ratio named as FAR value which was calculated by dividing the
Scheme 1. Reagents and conditions: (i) (CH3)2CHMgCl, THF, À40 °C, 1 h; (ii) BnBr,
(CH3)2CHOH, rt, 18 h; (iii) ArylMgBr, Cu(I)I, LiCl, THF, À20 °C—rt.

168
C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
6a, R1 = H, R2 = H, R3 = H
b, R1 = OMe, R2 = H, R3 = H
c, R1 = H, R2 = H, R3 = OMe
O
NH2
d, R1 = H, R2 = H, R3 = Me
R3
R1
OR1
e, R1= -CH=CH-CH=CH-R2
N
R2
R3 = H
O
R4
R2
iv
OR
R3
7a, R1 = Me, R2 = H, R3 = H, R4 = H
b, R1 = Me, R2 = OMe, R3 = H, R4 = H
c, R1 = Me, R2 = H, R3 = H, R4 = OMe
d, R1 = Me, R2 = H, R3 = H, R4 = Me
e, R1 = Me, R2 = -CH=CH-CH=CH-R3
R4 =H
5a, R = Me
b, R = Et
f, R1 = Et, R2 = H, R3 = H, R4 = H
g, R1 = Me, R2 = OMe, R3 = H, R4 = H
h, R1 = Me, R2 = H, R3 = H, R4 = OMe
i, R1 = Me, R2 = H, R3 = H, R4 = Me
j, R1 = Me, R2 = -CH=CH-CH=CH-R3
R4 =H
OC2H5
OC2H5
R1
R2
R1
O
H
v
R2
I
9a, R1= R2 = H
8a, R1= R2 = H
b, R1 = -CH=CH-CH=CH-R2
b, R1 = -CH=CH-CH=CH-R2
O
vi
OR1
7b-e, 7g-h
+
9a
R2
N
R2
R3
R4
R3
10a - h
R4
O
vi
OR1
7a, f
+
9b
N
10i, j
Scheme 2. Reagents and conditions: (iv) THF, rt, 5 h; (v), (C4H9)4NAc, K2CO3, KCl, Pd(II)AC2, DMF, 90 °C, 2–3 h;(VI) Na2SO4, Fe(III)Cl3 Â 6 H2O, CH2Cl2, 40 °C, 12 h.
fluorescence uptake in the P-gp expressing subline after inhibitor
addition through the fluorescence uptake in the non-P-gp express-
ing subline after inhibitor addition. Each used values have been re-
lated to the untreated control values without inhibitor before final
division. Verapamil has been used as established standard for
in vitro assays because of its reliable high in vitro activity. This
was confirmed in comparison with tariquidar which proved to be
only 1.2-fold more active than verapamil in our assay system at
Verapamil for comparison was not active at the lower inhibitor
concentration. The activity data of the 3-bromophenyl derivative
4b were similar to those of the 3-chlorophenyl compound 4a.
The 3-trifluorophenyl compound 4c resulted in the best activity
with a FAR value of 1.53 at the higher concentration. Nifedipine
as used 1,4-dihydropyridine reference showed a poorer activity
with a FAR value of only 1.0.
The change of the 3-methyl ester in compounds 4d–f each led
to main increases in the P-gp inhibiting properties, namely at the
higher inhibitor concentration. All 3-ethyl ester derivatives were
slightly better inhibitors than the 3-methyl ester derivatives with
each improved FAR values at the lower concentration. The FAR va-
lue of the 3-chlorophenyl 3-ethyl ester 4d at the higher concentra-
tion almost duplicated if compared to that of the 3-methyl ester
compound 4a and in the case of the 3-bromophenyl 3-ethyl ester
4e more than the twofold FAR value with 2.76 was reached.
We then introduced a methoxy function into the 4-aryl residue
by the use of the corresponding available Grignard reagents. Meth-
oxy functions are known to serve as hydrogen bond acceptor func-
the used inhibitor concentration of 10 lM.
In our earlier N-acyloxy 1,4-dihydropyridines we varied the
substitution patterns in the 4-aryl residue by introducing methoxy
functions into both the 2- and the 4-position of the aryl residue. In
our present series of N-benzyl derivatives we varied all positions of
the 4-aryl residue starting with the introduction of synthetically
accessible 3-halogen substituents. We characterized the introduc-
tion of the halogen substituents chloro, bromo and, finally, trifluo-
romethyl within the compounds 4a–f under additional variation of
the 3-ester substituents of the molecular scaffold as will be dis-
cussed. We preferred 3-ester derivatives because 3-acetyl deriva-
tives were less active within the series of N-acyloxy compounds.4
The 3-chlorophenyl derivative 4a showed P-gp inhibiting activ-
tions in P-gp inhibitors.11,12 The introduction of
a methoxy
function into the 2-position of the 4-phenyl residue resulted in im-
proved activity of compound 4h at both the lower and the higher
concentration if compared to the activity data of the 3-halogen
ities at both the lower concentration of 1
lM and the higher
concentration of 10 M with FAR values >1 (Table 1).
l

C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
169
Table 1
of the aromatic N-benzyl residue increased the activity at the high-
er concentration.
Target compound (4a–l and 10a–j) activities in the concentration dependent
inhibition of P-gp as determined FAR values for each three measurements
With the 4-methoxy function of 10b being more favourable
than the 2-methoxy we found a similar tendency as we observed
for the methoxy positioning in the 4-aryl substituted compounds.
The replacement of the 4-methoxy with a 4-methyl group further
increased the activity. At the higher concentration the FAR value of
7.80 for derivative 10c made much more than the double value of
verapamil.
Within the 3-ethyl ester compound series 10d–f we found the
same substituent effects than those observed for the 3-methyl es-
ters. By the way almost all resulting activities were better, espe-
cially at the higher inhibitor concentration. The 2-methoxy
derivative 10d was more active than the corresponding 3-methyl
ester at the higher concentration. The 4-methoxy compound 10e
showed an increased FAR value if compared to the 2-methoxy com-
pound 10d and to the 4-methoxy 3-methyl ester derivative 10b at
the higher concentration. The replacement of the 4-methoxy by a
4-methyl group led to the most active derivative 10f so far with
a threefold increased FAR value at the lower concentration if com-
pared to our verapamil standard.
Compd
Residues
FAR-Values
P-gp inhibition
10 lM
R1
R2
R3
R4
1
lM
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
verapamil
Me
Me
Me
Et
Et
Et
Me
Me
Me
Et
Et
Et
Me
Me
Me
Et
Et
Et
Me
Et
Me
Et
H
H
H
H
H
H
Cl
Br
CF3
Cl
Br
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
1.12 0.15
1.05 0.08
1.05 0.23
1.25 0.06
1.27 0.07
1.17 0.07
1.11 0.03
1.13 0.09
0.85 0.04
1.34 0.22
1.18 0.13
1.52 0.03
1.82 0.07
1.50 0.19
1.72 0.26
1.72 0.27
1.32 0.09
2.54 0.23
1.19 0.27
1.28 0.11
1.22 0.09
1.43 0.05
0.89 0.21
1.39 0.16
1.22 0.43
1.53 0.19
2.67 0.17
2.76 0.40
2.33 0.27
1.88 0.27
1.76 0.40
2.95 0.24
2.54 0.95
2.38 0.21
3.13 0.33
3.97 0.47
4.13 1.12
7.80 1.48
4.64 1.05
4.89 0.86
7.80 1.42
2.19 0.63
2.74 0.61
2.35 0.39
3.45 0.56
2.94 0.78
Me
H
OMe
Me
H
OMe
OMe
H
H
OMe
H
H
OMe
Me
H
OMe
Me
H
H
OMe
H
H
As the methyl group effects on increasing activity turned out to
be significant in the group of the varied aryl substituents of our
compound series of both 3-methyl and 3-ethyl esters we extended
our aromatic substitution patterns with the introduction of
aryl-annelated residues. We started with the replacement of the
–CH@CH–CH@CH–
–CH@CH–CH@CH–
H
H
H
–CH@CH–CH@CH
–CH@CH–CH@CH–
4-phenyl residue of our 1,4-dihydropyridine scaffold with
a
1-naphthyl residue. Derivative 10i showed increased activity if
compared to the 4-tolyl derivative 4g at both inhibitor concentra-
tions. At the lower concentration it is the most active inhibitor of
the 4-phenyl varied compound series with the 3-methyl ester.
Again the corresponding 3-ethyl ester compound 10j is more active
at both concentrations and also the most active compound of the 4-
aryl 3-ethyl ester series. Thus, the additional benzo-annelation led
to compounds with highest activitis of all 4-aryl varied derivatives.
We have also been interested to investigate the influence of a
benzo-annelation to the phenyl ring of the N-benzyl substituent.
The resulting 3-methyl ester derivative 10g almost reached the
activity of the 4-(1-naphthyl) substituted compound 10i. However,
if compared to the excellent activity data of the N-(4-methylben-
zyl) substituted compound 10c the activity was found mainly
reduced. The same tendency was observed for the corresponding
3-ethyl ester compound 10h that was again more active than
the 3-methyl ester compound 10g but less active than the
N-(4-methylbenzyl) substituted compound 10f.
Summarising our novel structure-activity relationship (SAR) re-
sults we can conclude that variations of substitution patterns in
both the 4-phenyl and the N-benzyl residue show similar tenden-
cies. As far as evaluated the 2- and 4-substitutions are most favour-
able if substituted with single substituents. Most active derivatives
have been given in the series of the N-benzyl substituted
compounds. In all cases of our investigated compounds the ethyl
esters tended to be more potent than the methyl ester derivatives.
substituted derivatives of the 3-methyl ester series. A movement of
the methoxy function into the 4-position of the 4-phenyl residue
again improved the activity at the higher concentration with a
FAR value of 2.95 of derivative 4i. The replacement of the
4-methoxy function with a 4-methyl group led to decreases in
the activity of compound 4g at the higher concentration. However,
the activity of 4g was again better than that of the 3-halogenphe-
nyl substituted compounds of this 3-methyl ester series. The
4-position seems to be most favourable for the introduction of
possible substituents either with size-enlarging effects in the case
of methyl or methoxy groups or as potential hydrogen bond
acceptor functions.
The same tendency of the effects of introduced 4-phenyl
substituents was observed for the compounds of the 3-ethyl ester
series 4j–l. The 2-methoxyphenyl derivative 4k showed similar
activities than the 3-halogen substituted compounds. The move-
ment of the methoxy function into the 4-position mainly increased
activity up to a FAR value of 3.13 of compound 4l at the higher
inhibitor concentration which meant a higher activity than that
of verapamil. The replacement of the 4-methoxy with a 4-methyl
group lowered this activity but the 4-methylphenyl compound 4j
was more active than the 2-methoxyphenyl derivative 4k. Again
the 4-phenyl ring substitution was the most attractive one for
the introduction of possible substituents.
We then varied the substituents in the N-benzyl residue
concentrating in the most effective methoxy and methyl substitu-
tions as evaluated in the 4-phenyl residue of derivatives 4a–l.
Again we varied the 3-ester substituent with both methyl and
ethyl ester functions.
We started with the introduction of the methoxy function into
the 2-position of the phenyl ring of the N-benzyl substituent of the
3-methyl ester compound. Derivative 10a showed increased
activity if compared to all compounds of the 4-phenyl substituted
series 4a–l at both tested inhibitor concentrations reaching the
highest FAR values so far. The FAR value at the lower concentration
made almost twice as much than the FAR value of verapamil. A
movement of the methoxy function from the 2- to the 4-position
2.3. MDR reversal studies
The observed compound effectiveness in inhibiting the P-gp
activity in our in vitro assay encouraged to further studies. So we
investigated the ability of our compounds to reverse the MDR in
anticancer drug studies. The undertaken studies with rhodamine
123 were essential to characterize the SAR of the P-gp inhibition.
However, the proof-of-concept would be a concentration depen-
dent inhibition profile of selected compounds to really reverse
the MDR of a cytostatic drug.
We used our model system of both mouse T lymphoma cell
lines to further investigate the cell-profiled inhibitors in this

170
C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
system for their activity to reverse MDR which meant that a re-
duced cytostatic drug activity in the P-gp overexpressing cell line
had to be restored ideally reaching the same toxicity which was
found for the cytostatic drug in the non-P-gp expressing cell line.
We chose the anthracycline drug daunorubicin for our antican-
cer studies. Daunorubicin is an established anticancer drug with
well known P-gp substrate properties.11 First we investigated the
cytotoxic effect of the drug in both cell lines, the P-gp expressing
and the non-expressing mouse T lymphoma cell line. The cytotoxic
effects were determined in the MTT assay in which viable cells are
characterized by the activity of mitochondrial dehydrogenases of
reducing the MTT reagent to formazan which is determined UV
spectroscopically after cell lysis.
side effect by developing liposomal applications.19,20 Moreover,
anthracyclines are effected by the MDR phenomenon as known
P-gp substrates so that such liposomal applications were encour-
aged by hopes to reduce the P-gp mediated resistance phenome-
non.21,22 Our encouraging P-gp inhibitor studies led to extended
investigations including a liposomal daunorubicin application for
consideration.
We used DaunoXomeÒ as liposomal daunorubicin application
and determined its toxicity, again in both mouse T lymphoma cell
lines with and without P-gp expression. The resulting IC50 values
are displayed in Figure 3.
We found different IC50 values with 0.3
lM in the non-P-gp
expressing cell line and 2.32 M in the P-gp expressing cell line.
l
The concentration dependent daunorubicin toxicity has been
determined and sigmoidal curves resulted which are displayed in
Figure 2.
Both IC50 values are reduced if compared to those of the non-
liposomal daunorubicin. Interestingly, also the liposomal daunoru-
bicin turned out to have P-gp substrate properties although the
cytotoxic effect is stronger in the P-gp expressing cell line if
compared to the application of the non-liposomal daunorubicin.
We decided to investigate the potential of two of our P-gp
inhibitors to reverse this determined MDR phenomenon of the
liposomal daunorubicin application. We uniformly used two rele-
vant concentrations in our SAR studies discussed above to charac-
terize the activity of the P-gp inhibitors. For two of our best
inhibitors 10c and 10e we found increased FAR values of 17.6
(10c) and 11.8 (10e) at higher concentrations which were tested
to investigate possible saturation effects of the P-gp inhibition at
The calculated corresponding IC50 values of the daunorubicin
toxicity derived from the cell viability made 0.73 0.03
lM for
the non-P-gp expressing cell line and 7.73 0.86 M for the P-gp
l
expressing cell line which meant a loss of the anticancer drug
activity by a factor of more than 10 caused by the P-gp dependent
efflux of the anticancer drug out of the P-gp expressing cell line.
We used one of our best inhibitors 10f to investigate the effects
of restoring the anticancer drug activity. We started with a concen-
tration of 2.5
the IC50 value of the daunorubicin toxicity with a value of
3.33 0.36 M which meant a more than 50% restoration of the
cytotoxic anticancer drug effect. We then used 5 M and, finally,
10 M of compound 10f. The resulting sigmoidal curves are shown
lM of 10f. We observed a more than 50% reduction of
l
the chosen concentration of 10
first investigated MDR reverser showed saturation effects at
10 M. However this concentration led to a complete reversal of
lM. Compound 10f tested as our
l
l
l
in Figure 2. The shift of the curves towards the curve of the dauno-
rubicin toxicity in the non-P-gp expressing cell line indicates the
further restoration of the anticancer drug toxicity. A further re-
the MDR phenomenon as demonstrated, so that it was a com-
pletely sufficient concentration. With the potential to show en-
hanced MDR reversal effects at higher concentrations than 10 lM
duced IC50 value of 1.96 0.15
P-gp inhibitor application if compared to the IC50 value at the
2.5 M application. At the concentration 10 M an almost com-
plete restoration of the daunorubicin toxicity was observed with
a resulting IC50 value of 0.96 0.13 M which meant an anticancer
drug activity similar to that in the non-P-gp expressing cell line.
As anthracyclines show certain cardiotoxicity in anticancer
drug therapies attempts have been made to reduce that undesired
l
M was calculated for the 5
l
M
we selected both compounds 10c and 10e for the liopsomal
daunorubicin studies.
l
l
At the lowest application concentration of 2.5 lM both inhibi-
tors showed a main decrease in the IC50 values of the determined
liposomal daunorubicin toxicity. Compound 10c with the higher
P-gp inhibiting activity than 10e at the lower concentration of
l
1
l
M showed better MDR reversing properties than 10e with a
more than 60% restoration of the MDR sensitivity at this lower
concentration of 2.5 M and a resulting IC50 value of 0.88 M for
the liposomal daunorubicin toxicity.
The IC50 values further decreased at a concentration of 5
and finally led to a complete reversal of the MDR phenomenon at
a concentration of 10 M with almost identical IC50 values than
l
l
l
M
l
in the non-P-gp expressing cell line. So also the observed resistance
against liposomal daunorubicin could be successfully reversed by
our P-gp inhibitors. Interestingly, also the little weaker inhibitor
100
50
0
3
2.32
2.5
2
1.5
1.05
0.88
0.72
1
0.5
0
0.69
0.35
10
0.32
10
0.3
P
MDR
2.5
5
2.5
5
-2
-1
0
1
2
3
log c daunorubicin in µmol/L
daunorubicin
10c
10e
Figure 2. Concentration dependent cell viability profiles (a) with incubated
daunorubicin in the mouse T lymphoma cell line without P-gp (left curve d) and
in the mouse T lymphoma cell line with overexpressing P-gp (right curve d) and (b)
Figure 3. IC50 values of liposomal daunorubicin (DaunoXomeÒ) toxicity (a) in the
non-P-gp expressing mouse T lymphoma cell line (P) and the P-gp overexpressing
mouse T-lymphoma cell line (MDR) shown in the left columns, after preincubation
with P-gp inhibitors 10c (middle columns) and 10e (right columns) at each
with preincubated P-gp inhibitor 10f concentrations of 2.5
j), of 5 M (middle curve withN) and of 10 M (middle curve with ꢀ) each in the P-
gp overexpressing mouse T lymphoma cell line, number (n) of experiences n = 3.
lM (middle curve with
l
l
increasing concentrations of 2.5, 5 and 10
lM in the P-gp overexpressing mouse T
lymphoma cell line, number (n) of experiences n = 3.

C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
171
10e was able to completely reverse the MDR phenomenon similar
3. Conclusions
to the more potent P-gp inhibitor 10c.
Finally we investigated the cytotoxic effects of our proved MDR
reversing compounds in our cellular assay system. We determined
the cell viability with increasing compound concentrations up to
Early pyridine-2-ones with P-gp inhibiting activities partly in
the range of verpamil were further developed by removing the car-
bonyl function from the molecular scaffold with a resulting 1,4-
dihydropyridine structure. A complex structure-activity discussion
was carried out by structural variations of the 4-phenyl, the
N-benzyl and the 3-ester function.
80
l
M (Fig. 4).
All compounds showed complete MDR reversing activity at
M. Moreover, their potential to reverse MDR has been demon-
M. Most
active compounds 10c and 10e were completely nontoxic at the
concentration 2.5 M with cell viability data of 95% for 10c and
10
l
strated already at the lowest used concentration of 2.5
l
4-Aryl-1,4-dihydropyridines are known to adopt
a boat
conformation of the 1,4-dihydropyridine ring which has been
demonstrated to flip.24,25 However, if the ring was substituted in
the 3-position the free rotability of the 4-aryl residue was hin-
dered. So taking these facts in account, one may wonder whether
the exact positioning of a substituent either in the 4-phenyl or
the N-benzyl residue may be important with respect to the biolog-
ical activity. Interestingly, similar tendencies of the aromatic
substituent effects were observed for both the 4-phenyl and the
N-benzyl residues. The 4-position of both aromatic residues was
favoured for both a methyl and a methoxy group. The activity data
of all of such N-benzyl substituted derivatives were much better
than the activities of those compounds with the aromatic substit-
utents at the 4-phenyl residue. The replacement of the 4-phenyl
with a 4-(1-naphthyl) residue was of favour for the biological
activity. However, a replacement of the N-benzyl substituent with
the naphthyl residue was less favourable. All ethyl ester com-
pounds were better inhibitors than the corresponding methyl ester
compounds, so that the slightly extended alkyl side chain of the
3-ester substituent seemed to be significantly important for the
l
of 97% for 10e in the non-P-gp expressing cell line. Both com-
pounds were also nontoxic in the P-gp overexpressing cell line
although compound 10c showed a partly reduced cell viability of
81 %. However, refering to literature such a minimally reduced cell
viability >80% under compound application qualifies a compound
as nontoxic. At the concentration of 10 lM the cell viability of com-
pound 10c was already about 75% in both cell lines. Compound 10e
maintained practically nontoxic with cell viabilities of 85% in the P-
gp overexpressing and 100% in the non-expressing cell line. Com-
pound 10f as our third MDR reversing compound showed similar
cell viability results after application than compound 10c. How-
ever, the cell viability at the concentration of 10 lM proved to be
slightly better with values of about 78% in both cell lines. So we
can finally state that a compound toxicity which may contribute
to the toxic effects of daunorubicin in our final reversal assay can
almost be excluded.
120
100
80
60
40
20
0
120
100
80
60
40
20
0
1
2,5
5
10
20
40
80
160
1
2,5
5
10
20
40
80
160
concentration [µmol/L]
10c
concentration [µmol/L]
10c
120
100
80
60
40
20
0
120
100
80
60
40
20
0
1
2,5
5
10
20
40
80
160
1
2,5
5
10
20
40
80
160
concentration [µmol/L]
10e
concentration [µmol/L]
10e
120
100
80
60
40
20
0
120
100
80
60
40
20
0
1
2,5
5
10
20
40
80
160
1
2,5
5
10
20
40
80
160
concentration [µM]
10f
concentration [µM]
10f
Figure 4. Concentration dependent cell viability profiles of respective compounds 10c, 10e and 10f in both cell lines with the results of the non-P-gp expressing cell line
displayed on the left site and those of the P-gp overexpressing cell line displayed on the right site.

172
C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
molecular properties of the P-gp inhibition. Similar observed sub-
stituent effects both in the 4-phenyl and the N-benzyl residue
may indicate that the inhibitor may show a flexible interaction
with the P-gp protein binding site which is supported by the con-
formational flexibility of such 1,4-dihydropyridines as cited.
The MDR reversal studies of various anticancer drug applica-
tions were carried out with the most active P-gp inhibitors. We
found increased cytotoxic effects for the liposomal daunorubicin
application in our P-gp-expressing cell line if compared to the
application of the sole daunorubicin. So the liposomal application
was more effective but still it was affected by the MDR phenome-
non as demonstrated.
Low concentrations of all our best P-gp inhibitors led to a main
restoration of more than 50% of the anticancer drug activity of both
the liposomal and the sole daunorubicin application. At higher
concentrations the restoration was complete and the MDR was
overcome even by the compound which showed a saturation effect
of the P-gp inhibition at this concentration.
4.1.2. General procedure for the preparation of the 4-phenyl
substituted 1,4-dihydropyridines (4a–l).
One equivalent of the nicotinic acid ester salts 3a,b were sus-
pended in dried THF under addition of 0.2 equiv of lithium chloride
and 0.1 equiv of copper(I) iodide. One and a half equivalent of the
Grignard reagent were added dropwise at À20 °C. In the case of the
3-halogen substituted derivatives the Grignard reagent was freshly
prepared from one equivalent of the 3-halogen substituted iodo-
benzol and 1.2 equiv of isopropyl magnesium chloride in dried
THF at À40 °C by stirring under argon atmosphere for 1 h. After
addition of the grignard reagent stirring continued at rt until no
more starting compound was detectable by tlc. Then the remaining
excess of reagent was hydrolysed by the addition of an ammonium
chloride solution in water (20%). Extraction with portions of
diethyl ether followed. The unified organic layer was then ex-
tracted with an ammonium chloride solution in water (20%), with
a concentrated solution of ammonia in water (1:1), with water, a
hydrochloric acid solution in water (10%), water and, finally, with
a saturated sodium chloride solution in water. Then the organic
layer was dried over sodium sulfate, filtered and evaporated. The
remaining oil was purified by column chromatography using a
mixture of dichloromethane and heptane (90:10).
So the best P-gp inhibiting compounds convinced as MDR rever-
sal agents thus giving hope for coming studies of further improving
activity by combined aromatic substitutions.
4. Experimental
4.1. Chemistry
4.1.2.1. Methyl 1-benzyl-4-(3-chlorophenyl)-1,4-dihydropyri-
dine-3-carboxylate (4a).
Yellow oil; yield 72%; MS (ESI) m/z
340 (M+H+, 100%). 1H NMR (CDCl3) d 3.57 (s, 3H, CH3), 4.43 (s,
2H, N–CH2), 4.51 (d, J = 4.7 Hz, 1H, 4-H), 4.86 (dd, J = 7.8, 4.7 Hz,
1H, 5-H), 5.93 (d, J = 7.8 Hz, 1H, 6-H), 7.10–7.17 (m, 4H, C6ClH4),
7.25–7.39 (m, 6H, 2-H, CH2–C6H5). 13C NMR (CDCl3) d 43.5 (d, C-
4), 52.3 (q, COOCH3), 54.8 (t, N–CH2), 109.8 (s, C-3), 112.0 (d, C-
5), 125.8 (d, C4-4-Ph), 126.8 (d, C-4–N–CH2–Ph), 126.9 (d, C-2, –
6-N–CH2–Ph), 127.1 (d, C-6–4-Ph), 128.6 (d, C-3, -5-N–CH2–Ph),
128.8 (d, C-2-4-Ph), 129.3 (d, C-6), 130.1 (d, C-5-4-Ph), 134.2 (s,
C-3–4-Ph), 141.6 (s, C-1–N–CH2–Ph), 143.6 (s, C-1-4-Ph), 144.6
(d, C-2), 167.2 (s, CO). Anal. Calcd for C20H18ClNO2: C, 70.84; H,
5.35; N, 4.13. Found: C, 70.55; H, 5.23; N, 3.95.
All the chemical agents used were either synthesized or have
been commercially available. Melting points were determined
using a Boetius melting desk microscope and are uncorrected. 1H
NMR and 13C NMR spectra were recorded on a Varian Gemini
2000 at 400 and 100 MHz, respectively. Chemical shifts are re-
ported in ppm units with tetramethylsilane as internal reference
standard. Mass spectra were recorded on an AMD 402 mass spec-
trometer named AMD INTEGRA (EI masses) or on a Finnigan LCQ
classic (ESI masses). Elemental analyses (C, H, N) were carried
out with a Leco analyzer apparatus (CHN-932) and the results were
within 0.4% of the theoretical values.
4.1.2.2. Methyl 1-benzyl-4-(3-bromophenyl)-1,4-dihydropyri-
dine-3-carboxylate (4b).
Yellow oil; yield 65%; MS (ESI) m/z
4.1.1. General procedure for the preparation of the N-benzyl
pyridinium bromides (3a, b).
384 (M+H+, 100%). 1H NMR (CDCl3) d 3.57 (s, 3H, CH3), 4.43 (s,
2H, N–CH2), 4.49 (d, J = 4.7 Hz, 1H, 4-H), 4.86 (dd, J = 7.7, 4.7 Hz,
1H, 5-H), 5.91 (d, J = 7.7 Hz, 1H, 6-H), 7.01–7.19 (m, 4H, C6BrH4),
7.28–7.49 (m, 6H, 2-H, CH2-C6H5).
One equivalent of the respective nicotinic acid ester 2a, b was
dissolved in isopropanol and one and a half equivalent of benzyl
bromide were added dropwise under stirring. Stirring continued
for 18 h at rt. Then the solution was extracted with diethyl ether
and finally evaporated. The remaining product was washed with
diethyl ether and kept in vacuum in an exsicator.
13C NMR (CDCl3) d 43.3 (d, C-4), 52.2 (q, COOCH3), 54.9 (t, N–
CH2), 109.9 (s, C-3), 112.5 (d, C-5), 123.0 (s, C-3–4-Ph), 126.7 (d,
C-4–N–CH2–Ph), 126.9 (d, C-2, –6-N–CH2–Ph), 128.0 (d, C-6–4-
Ph), 128.5 (d, C-3, –5-N–CH2–Ph), 128.6 (d, C4–4-Ph), 129.1 (d, C-
6), 130.9 (d, C-5–4-Ph), 133.8 (d, C-2–4-Ph), 141.5 (s, C-1–N–
CH2–Ph), 144.4 (s, C-1–4-Ph), 144.7 (d, C-2), 167.1 (s, CO). Anal.
Calcd for C20H18BrNO2: C, 62.71; H, 4.74; N, 3.66. Found: C,
62.67; H, 4.70; N, 3.41.
4.1.1.1.
(3a).
1-Benzyl-3-methoxycarbonyl-pyridinium
Bright-pink semi-solid product; yield 95%; MS (EI) m/z
bromide
(cation) 228 (M+, 100%), 137 (M+-Bn, 50%), 91 (Bn, 80%).
1H NMR (acetone-d6) d 4.00 (s, 3H, CH3), 6.48 (s, 2H, N–CH2),
7.42–7.86 (m, 5H, CH2–C6H5), 8.40 (t, J = 6.8 Hz, 1H, 5-H), 9.07 (d,
J = 6.8 Hz, 1H, 4-H), 9.94 (s, 1H, 2-H), 10.12 (d, J = 6.8 Hz, 1H, 6-
H). Anal. Calcd for C14H14NO2Br: C, 54.17; H, 4.58; N, 4.55. Found:
C, 53.99; H, 4.47; N, 4.38.
4.1.2.3. Methyl 1-benzyl-4-(3-trifluorophenyl)-1,4-dihydropyri-
dine-3-carboxylate (4c).
Yellow oil; yield 70%; MS (ESI) m/z
374 (M+H+, 100%). 1H NMR (CDCl3) d 3.56 (s, 3H, CH3), 4.44 (s,
2H, N–CH2), 4.60 (d, J = 4.7 Hz, 1H, 4-H), 4.88 (dd, J = 7.7, 4.7 Hz,
1H, 5-H), 5.95 (d, J = 7.7 Hz, 1H, 6-H), 7.19–7.53 (m, 10H, 2-H,
C6CF3H4,CH2–C6H5). 13C NMR (CDCl3) d 44.3 (d, C-4), 52.3 (q,
COOCH3), 54.8 (t, N–CH2), 109.8 (s, C-3), 112.5 (d, C-5), 122.1 (d,
C4–4-Ph), 124.5 (s, CF3), 126.6 (d, C-4–N–CH2–Ph), 126.7 (d, C-2,
-6-N–CH2–Ph), 127.0 (d, C-2–4-Ph), 128.6 (d, C-3, –5-N–CH2–Ph),
129.1 (d, C-6), 129.2 (d, C-5–4-Ph), 130.9 (s, C-3-4-Ph), 132.3 (d,
C-6-4-Ph), 141.7 (s, C-1-N–CH2–Ph), 142.5 (s, C-1-4-Ph), 144.5 (d,
C-2), 167.3 (s, CO). Anal. Calcd for C21H18F3NO2: C, 67.60; H,
3.75; N, 3.66. Found: C, 67.42; H, 3.54; N, 3.39.
4.1.1.2.
(3b).
1-Benzyl-3-ethoxycarbonyl-pyridinium
White-yellow semi-solid product; yield 90%; MS (EI)
bromide
m/z (cation) 242 (M+, 100%), 151 (M+-Bn, 40%), 91 (Bn, 85%). 1H
NMR (acetone-d6) d 1.40 (t, J = 7.2 Hz, 3H, CH2–CH3), 4.48 (q,
J = 7.2 Hz, 2H, CH2–CH3), 6.46 (s, 2H, N–CH2), 7.44–7.85 (m, 5H,
CH2–C6H5), 8.40 (t, J = 6.8 Hz, 1H, 5-H), 9.09 (d, J = 6.8 Hz, 1H, 4-
H), 9.97 (s, 1H, 2-H), 10.04 (d, J = 6.8 Hz, 1H, 6-H). Anal. Calcd for
C15H16NO2Br: C, 55.92; H, 5.01; N, 4.35. Found: C, 55.77; H, 4.88;
N, 4.32.

C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
173
4.1.2.4. Ethyl 1-benzyl-4-(3-chlorophenyl)-1,4-dihydropyridine-
3-carboxylate (4d). Yellow oil; yield 62%; MS (ESI) m/z 354
(s, 3H, COOCH3), 3.81 (s, 3H, C6H4–OCH3), 4.55 (s, 2H, N–CH2),
4.88 (d, J = 4.8 Hz, 1H, 4-H), 4.94 (dd, J = 7.7, 4.8 Hz, 1H, 5-H),
5.94 (d, J = 7.7 Hz, 1H, 6-H), 7.82–7.10 (m, 4H, C6H4–OCH3), 7.26–
7.38 (m, 5H, CH2–C6H5), 7.57 (s, 1H, 2-H). 13C NMR (CDCl3) d 38.1
(d, C-4), 52.3 (q, COOCH3), 54.8 (t, N–CH2), 56.1 (q, C-2–Ph–
OCH3), 108.9 (s, C-3), 111.8 (d, C-5), 114.2 (d, C-3–4–Ph), 121.0
(d, C-5–4-Ph), 121.4 (s, C-1–4-Ph), 126.7 (d, C-4–N–CH2–Ph),
126.8 (d, C4–4-Ph), 126.9 (d, C-2, –6-N–CH2–Ph), 128.4 (d, C-3, –
5-N–CH2–Ph), 129.1 (d, C-6), 130.0 (d, C-6–4-Ph), 141.6 (s, C-1–
N–CH2–Ph), 144.6 (d, C-2), 158.6 (s, C-2–4-Ph), 167.2 (s, CO). Anal.
Calcd for C21H21NO3: C, 75.20; H, 6.31; N, 4.18. Found: C, 74.94; H,
6.61; N, 4.06.
(M+H+, 100%). 1H NMR (CDCl3) d 1.13 (ABX3, J = 7.1 Hz, 3H, CH2–
CH3), 3.95–4.10 (ABX3, J = 10.3, 7.1 Hz, 2H, CH2–CH3), 4.43 (s, 2H,
N–CH2), 4.51 (d, J = 4.7 Hz, 1H, 4-H), 4.86 (dd, J = 7.8, 4.7 Hz, 1H,
5-H), 5.91 (d, J = 7.8 Hz, 1H, 6-H), 7.10–7.19 (m, 4H, C6ClH4),
7.26–7.39 (m, 6H, 2-H, CH2–C6H5). 13C NMR (CDCl3) d 14.2 (q,
COOCH2CH3), 43.5 (d, C-4), 54.6 (t, N–CH2), 61.7 (t, COOCH2CH3),
108.0 (s, C-3), 112.4 (d, C-5), 125.6 (d, C4-4-Ph), 126.3 (d, C-4–N–
CH2–Ph), 126.5 (d, C-2,–6-N–CH2–Ph), 127.1 (d, C-6–4-Ph), 128.6
(d, C-3,–5-N–CH2–Ph), 128.7 (d, C-2–4-Ph), 129.5 (d, C-6), 130.1
(d, C-5–4-Ph), 134.2 (s, C-3–4-Ph), 141.6 (s, C-1–N–CH2–Ph),
143.6 (s, C-1–4-Ph), 144.6 (d, C-2), 167.2 (s, CO). Anal. Calcd for
C21H20ClNO2: C, 71.43; H, 5.71; N, 3.97. Found: C, 71.22; H, 5.67;
4.1.2.9. Methyl 1-benzyl-4-(4-methoxyphenyl)-1,4-dihydropyri-
N, 3.92.
dine-3-carboxylate (4i).
Yellow semi-solid product; yield
75%; MS (ESI) m/z 358 (M+Na+, 100%). 1H NMR (acetone-d6) d
3.47 (s, 3H, COOCH3), 3.72 (s, 3H, C6H4–OCH3), 4.38 (d, J = 4.9 Hz,
1H, 4-H), 4.58 (s, 2H, N–CH2), 4.85 (dd, J = 7.7, 4.9 Hz, 1H, 5-H),
6.10 (d, J = 7.7 Hz, 1H, 6-H), 6.77–7.11 (m, 4H, C6H4–OCH3), 7.29–
7.51 (m, 6H, 2-H, CH2–C6H5). 13C NMR (CDCl3) d 44.0 (d, C-4),
52.3 (q, COOCH3), 54.7 (t, N–CH2), 55.8 (q, C-4–Ph–OCH3), 109.4
(s, C-3), 112.3 (d, C-5), 114.2 (d, C-3, –5–4-Ph), 126.8 (d, C-4–N–
CH2–Ph), 126.9 (d, C-2, –6–N–CH2–Ph), 128.4 (d, C-3, –5-N–
CH2––Ph), 129.3 (d, C-6), 130.0 (d, C-2, –6–4-Ph), 134.5 (s, C-1–
4-Ph), 141.6 (s, C-1–N–CH2–Ph), 144.6 (d, C-2), 157.6 (s, C4–4-
Ph), 167.2 (s, CO). Anal. Calcd for C21H21NO3: C, 75.20; H, 6.31; N,
4.18. Found: C, 74.87; H, 6.51; N, 4.10.
4.1.2.5. Ethyl 1-benzyl-4-(3-bromophenyl)-1,4-dihydropyridine-
3-carboxylate (4e).
Yellow oil; yield 68%; MS (ESI) m/z 398
(M+H+, 100%). 1H NMR (CDCl3) d 1.13 (ABX3, J = 7.1 Hz, 3H, CH2–
CH3), 3.95-4.09 (ABX3, J = 10.3, 7.1 Hz, 2H, CH2–CH3), 4.43 (s, 2H,
N–CH2), 4.49 (d, J = 4.7 Hz, 1H, 4-H), 4.86 (dd, J = 7.7, 4.7 Hz, 1H,
5-H), 5.91 (d, J = 7.7 Hz, 1H, 6-H), 7.01–7.19 (m, 4H, C6BrH4),
7.28–7.48 (m, 6H, 2-H, CH2–C6H5). 13C NMR (CDCl3) d 14.3 (q,
COOCH2CH3), 43.1 (d, C-4), 54.6 (t, N–CH2), 61.9 (t, COOCH2CH3),
108.3 (s, C-3), 112.2 (d, C-5), 123.4 (s, C-3–4-Ph), 126.7 (d, C-4–
N–CH2–Ph), 126.9 (d, C-2, –6-N–CH2–Ph), 128.8 (d, C-6–4-Ph),
128.9 (d, C-3, –5-N–CH2–Ph), 128.9 (d, C4–4-Ph), 129.2 (d, C-6),
130.7 (d, C-5–4-Ph), 133.6 (d, C-2–4-Ph), 141.6 (s, C-1–N–CH2–
Ph), 144.8 (s, C-1-4-Ph), 146.1 (d, C-2), 167.2 (s, CO). Anal. Calcd
for C21H20BrNO2: C, 63.52; H, 5.08; N, 3.53. Found: C, 63.42; H,
5.18; N, 3.63.
4.1.2.10. Ethyl 1-benzyl-4-(4-tolyl)-1,4-dihydropyridine-3-car-
boxylate (4j).
Yellow oil; yield 68%; MS (ESI) m/z 334
(M+H+, 100%). 1H NMR (acetone-d6) d 1.08 (ABX3, J = 7.1 Hz, 3H,
CH2–CH3), 2.24 (s, 3H, C6H4–CH3), 3.88–3.99 (ABX3, J = 10.9,
7.1 Hz, 2H, CH2–CH3), 4.43 (d, J = 4.9 Hz, 1H, 4-H), 4.55 (s, 2H, N–
CH2), 4.85 (dd, J = 7.7, 4.9 Hz, 1H, 5-H), 5.94 (d, J = 7.7 Hz, 1H, 6-
H), 7.01–7.11 (m, 4H, C6H4–CH3), 7.30–7.42 (m, 6H, 2-H, CH2–
C6H5). 13C NMR (CDCl3) d 14.2 (q, COOCH2CH3), 24.2 (q, C-4–Ph–
CH3), 44.3 (d, C-4), 54.8 (t, N–CH2), 61.7 (t, COOCH2CH3), 108.0 (s,
C-3), 111.7 (d, C-5), 126.8 (d, C-4–N–CH2–Ph), 126.8 (d, C-2, –6-
N–CH2–Ph), 128.6 (d, C-3, –5-N–CH2–Ph), 128.9 (d, C-2, –6–4-Ph),
129.3 (d, C-6), 129.4 (d, C-3, –5–4-Ph), 139.1 (s, C-1–4-Ph), 135.4
(s, C4–4-Ph), 141.7 (s, C-1–N–CH2–Ph), 146.1 (d, C-2), 167.2 (s,
CO). Anal. Calcd for C22H23NO2: C, 79.31; H, 6.96; N, 4.20. Found:
C, 79.11; H, 6.75; N, 4.11.
4.1.2.6. Ethyl 1-benzyl-4-(3-trifluorophenyl)-1,4-dihydropyri-
dine-3-carboxylate (4f).
Yellow oil; yield 71%; MS (ESI) m/z
410 (M+Na+, 100%). 1H NMR (CDCl3) d 1.13 (ABX3, J = 7.1 Hz, 3H,
CH2–CH3), 3.95–4.09 (ABX3, J = 10.3, 7.1 Hz, 2H, CH2–CH3), 4.44 (s,
2H, N–CH2), 4.60 (d, J = 4.7 Hz, 1H, 4-H), 4.87 (dd, J = 7.7, 4.7 Hz,
1H, 5-H), 5.94 (d, J = 7.7 Hz, 1H, 6-H), 7.26–7.53 (m, 10H, 2-H,
C6CF3H4, CH2–C6H5). 13C NMR (CDCl3) d 14.2 (q, COOCH2CH3),
44.1 (d, C-4), 54.8 (t, N–CH2), 61.8 (t, COOCH2CH3), 108.2 (s, C-3),
112.1 (d, C-5), 122.6 (d, C4–4-Ph), 124.0 (s, CF3), 126.6 (d, C-4–
N–CH2–Ph), 126.8 (d, C-2, –6-N–CH2–Ph), 127.8 (d, C-2–4-Ph),
128.9 (d, C-3, –5-N–CH2–Ph), 129.3 (d, C-6), 129.7 (d, C-5–4-Ph),
130.9 (s, C-3–4-Ph), 132.1 (d, C-6–4-Ph), 141.6 (s, C-1–N–CH2–
Ph), 142.4 (s, C-1–4-Ph), 146.6 (d, C-2), 167.4 (s, CO). Anal. Calcd
for C22H20F3NO2: C, 68.26; H, 5.21; N, 3.62. Found: C, 68.16; H,
5.20; N, 3.54.
4.1.2.11. Ethyl 1-benzyl-4-(2-methoxyphenyl)-1,4-dihydropyri-
dine-3-carboxylate (4k).
Yellow semi-solid product; yield
61%; MS (ESI) m/z 350 (M+H+, 100%). 1H NMR (acetone-d6) d 1.06
(ABX3, J = 7.1 Hz, 3H, CH2–CH3), 3.81 (s, 3H, C6H4–OCH3), 3.94–
4.04 (ABX3, J = 10.7, 7.1 Hz, 2H, CH2–CH3), 4.39 (s, 2H, N–CH2),
4.98–5.25 (m, 2H, 4-H, 5-H), 5.75 (d, J = 6.9 Hz, 1H, 6-H), 6.80–
7.35 (m, 9H, C6H4–OCH3, CH2–C6H5), 7.47 (s, 1H, 2-H).
4.1.2.7. Methyl 1-benzyl-4-(4-tolyl)-1,4-dihydropyridine-3-car-
boxylate (4g).
Yellow semi-solid product; yield 68%; MS (ESI)
m/z 320 (M+H+, 100%). 1H NMR (acetone-d6) d 2.23 (s, 3H, C6H4–
CH3), 3.48 (s, 3H, COOCH3), 4.41 (d, J = 4.9 Hz, 1H, 4-H), 4.59 (s,
2H, N-CH2), 4.86 (dd, J = 7.7, 4.9 Hz, 1H, 5-H), 6.11 (d, J = 7.7 Hz,
1H, 6-H), 7.03–7.09 (m, 4H, C6H4–CH3), 7.30–7.41 (m, 6H, 2-H,
CH2–C6H5). 13C NMR (CDCl3) d 24.3 (q, C-4–Ph-CH3), 44.0 (d, C-4),
52.3 (q, COOCH3), 53.9 (t, N–CH2), 108.7 (s, C-3), 111.9 (d, C-5),
126.5 (d, C-4–N–CH2–Ph), 126.6 (d, C-2, –6-N–CH2–Ph), 128.6 (d,
C-3, –5-N–CH2–Ph), 128.9 (d, C-6, –2–4-Ph), 129.1 (d, C-6), 129.2
(d, C-5, –3–4-Ph), 135.4 (s, C4–4-Ph), 139.2 (s, C-1–4-Ph), 141.9
(s, C-1–N–CH2–Ph), 144.6 (d, C-2), 167.2 (s, CO). Anal. Calcd for
13C NMR (CDCl3) d 14.2 (q, COOCH2CH3), 38.1 (d, C-4), 54.6 (t,
N–CH2), 56.4 (q, C-2–Ph–OCH3), 61.8 (t, COOCH2CH3), 108.0 (s, C-
3), 111.9 (d, C-5), 114.4 (d, C-3–4-Ph), 121.0 (s, C-1–4-Ph), 121.6
(d, C-5–4-Ph), 126.6 (d, C4–4-Ph), 126.7 (d, C-4–N–CH2–Ph),
126.9 (d, C-2, –6–N-CH2–Ph), 128.6 (d, C-3, –5–N–CH2–Ph), 129.3
(d, C-6), 130.3 (d, C-2–4-Ph), 141.5 (s, C-1–N-CH2–Ph), 146.1 (d,
C-2), 158.8 (s, C-2–4-Ph), 167.3 (s, CO). Anal. Calcd for
C22H23NO3: C, 75.62; H, 6.63; N, 4.01. Found: C, 75.61; H, 6.74;
N, 3.71.
C21H21NO2: C, 79.04; H, 6.63; N, 4.39. Found: C, 78.87; H, 6.46;
N, 4.21.
4.1.2.12. Ethyl 1-benzyl-4-(4-methoxyphenyl)-1,4-dihydropyri-
dine-3-carboxylate (4l).
Yellow oil; yield 72%; MS (ESI) m/z
4.1.2.8. Methyl 1-benzyl-4-(2-methoxyphenyl)-1,4-dihydropyri-
350 (M+H+, 100%). 1H NMR (acetone-d6) d 1.10 (ABX3, J = 6.9 Hz,
3H, CH2–CH3), 3.52–3.57 (ABX3, J = 13.9, 6.9 Hz, 2H, CH2–CH3),
3.77 (s, 3H, C6H4–OCH3), 4.38 (d, J = 4.9 Hz, 1H, 4-H), 4.59 (s, 2H,
dine-3-carboxylate (4h).
Yellow semi-solid product; yield
73%; MS (ESI) m/z 336 (M+H+, 100%). 1H NMR (acetone-d6) d 3.46

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C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
N–CH2), 4.86 (dd, J = 7.7, 4.9 Hz, 1H, 5-H), 6.11 (d, J = 7.7 Hz, 1H, 6-
H), 6.76-7.12 (m, 4H, C6H4–OCH3), 7.27–7.42 (m, 6H, 2-H, CH2–
C6H5). 13C NMR (CDCl3) d 14.0 (q, COOCH2CH3), 44.2 (d, C-4), 54.8
(t, N–CH2), 55.9 (q, C-4–Ph–OCH3), 61.7 (t, COOCH2CH3), 109.0 (s,
C-3), 112.0 (d, C-5), 114.6 (d, C-3, –5–4-Ph), 126.8 (d, C-4–N–
CH2–Ph), 126.9 (d, C-2, –6-N–CH2–Ph), 128.4 (d, C-3, –5-N–CH2–
Ph), 129.1 (d, C-6), 131.0 (d, C-2,–6–4-Ph), 134.9 (s, C-1–4-Ph),
141.3 (s, C-1–N–CH2–Ph), 146.6 (d, C-2), 158.3 (s, C4–4-Ph),
167.1 (s, CO).Anal. Calcd for C22H23NO3: C, 75.62; H, 6.63; N,
4.01. Found: C, 75.33; H, 6.47; N, 3.83.
7.55 (m, 8H, E/Z–C10H7), 7.61 (dd, J = 13.3, 6.9 Hz, 1H, E–2-H), 7.78–
7.93 (m, 6H, E/Z–C10H7), 8.21 (br s, 2H, NH). Anal. Calcd for
C15H15NO2: C, 74.72; H, 6.27; N, 5.81. Found: C, 74.55; H, 6.23;
N, 5.67.
4.1.3.6. E/Z-Ethyl 3-(benzylamino)acrylat (7f).
Yellow oil;
yield 90%; MS (ESI) m/z 205 (M+H+, 100%). 1H NMR (CDCl3) d
1.24–1.26 (m, 6H, E/Z–CH2–CH3), 4.06–4.12 (ABX3, J = 14.3,
7.2 Hz, 4H, E/Z–CH2–CH3), 4.20 (d, J = 5.2 Hz, 2H, E–N–CH2), 4.34
(d, J = 6.0 Hz, 2H, Z–N–CH2), 4.53 (d, J = 8.1 Hz, 1H, Z–1-H), 4.82
(d, J = 13.2 Hz, 1H, E–1-H), 6.67 (dd, J = 13.2, 8.1 Hz, 1H, Z–2-H),
7.28–7.34 (m, 10H, C6H5), 7.57 (dd, J = 13.2, 8.1 Hz, 1H, E-2-H),
8.11 (br s, 2H, NH). Anal. Calcd for C12H15NO2: C, 70.16; H, 7.37;
N, 6.83. Found: C, 69.95; H, 7.24; N, 6.72.
4.1.3. General procedure for the preparation of the b-
enaminocarbonyl compounds (7a–j)
One equivalent of the respective propiolic acid ester 5a, b and
one equivalent of the benzylamine compound 6a–d were stirred
in THF for 5 h at rt.23 Then the solvent was removed in vacuum
and the resulting yellow oil of compounds 7a–j was used without
further purification.
4.1.3.7.
(7g).
E/Z-Ethyl
3-(2-methoxybenzylamino)acrylat
Yellow oil; yield 91%; MS (ESI) m/z 236 (M+H+, 100%).
1H NMR (CDCl3) d 1.21–1.25 (m, 6H, E/Z–CH2–CH3), 3.82 (s, 3H,
Z–C6H4–OCH3), 3.83 (s, 3H, E–C6H4–OCH3), 4.05–4.12 (m, 4H, E/
Z–CH2–CH3), 4.20 (d, J = 5.7 Hz, 2H, Z–N–CH2), 4.30 (d, J = 6.2 Hz,
2H, E–N–CH2), 4.46 (d, J = 8.0 Hz, 1H, Z–1-H), 4.80 (d, J = 13.2 Hz,
1H, E–1-H), 6.68 (dd, J = 13.1, 8.0 Hz, 1H, Z–2-H), 6.83–7.27 (m,
8H, C6H4–OCH3), 7.53 (dd, J = 13.2, 8.4 Hz, 1H, E–2-H), 8.07 (br s,
2H, NH). Anal. Calcd for C13H17NO3: C, 66.41; H, 7.29; N, 5.96.
Found: C, 66.25; H, 7.23; N, 5.77.
4.1.3.1. E/Z-Methyl 3-(benzylamino)acrylat (7a).
Yellow oil;
yield 98%; MS (ESI) m/z 214 (M+Na+, 100%). 1H NMR (CDCl3) d 3.63
(s, 3H, Z–CH3), 3.64 (s, 3H, E–CH3), 4.21 (d, J = 5.2 Hz, 2H, E–CH2),
4.34 (d, J = 6.0 Hz, 2H, Z–CH2), 4.54 (d, J = 8.0 Hz, 1H, Z–1-H), 4.82
(d, J = 13.2 Hz, 1H, E–1-H), 6.67 (dd, J = 12.8, 8.0 Hz, 1H, Z–2-H),
7.22–7.34 (m, 10H, C6H5), 7.57 (dd, J = 13.2, 8.1 Hz, 1H, E–2-H),
8.10 (br s, 2H, NH). Anal. Calcd for C11H13NO2: C, 69.13; H, 6.86;
N, 7.33. Found: C, 68.86; H, 6.75; N, 7.24.
4.1.3.8.
(7h).
E/Z-Ethyl
3-(4-methoxybenzylamino)acrylat
Yellow oil; yield 88%; MS (ESI) m/z 236 (M+H+, 100%).
4.1.3.2.
(7b).
E/Z-Methyl
3-(2-methoxybenzylamino)acrylat
1H NMR (CDCl3) d 1.23–1.26 (m, 6H, E/Z–CH2–CH3), 3.78 (s, 3H,
Z–C6H4–OCH3), 3.87 (s, 3H, E–C6H4–OCH3), 4.06–4.14 (m, 6H, E/
Z–CH2–CH3, E–N–CH2), 4.27 (d, J = 5.8 Hz, 2H, Z–N–CH2), 4.51 (d,
J = 8.1 Hz, 1H, Z–1-H), 4.82 (d, J = 13.3 Hz, 1H, E–1-H), 6.66 (dd,
J = 13.2, 8.1 Hz, 1H, Z–2-H), 6.84–7.19 (m, 8H, C6H4–OCH3), 7.55
(dd, J = 13.3, 7.8 Hz, 1H, E–2-H), 8.05 (br s, 2H, NH). Anal. Calcd
for C13H17NO3: C, 66.41; H, 7.29; N, 5.96. Found: C, 66.05; H,
7.10; N, 5.88.
Yellow oil; yield 95%; MS (ESI) m/z 222 (M+H+, 100%).
1H NMR (CDCl3) d 3.61 (s, 3H, Z–COOCH3), 3.63 (s, 3H; E–COOCH3),
3.82 (s, 3H, Z–C6H4–OCH3), 3.83 (s, 3H, E–C6H4–OCH3), 4.20 (d,
J = 5.7 Hz, 2H, Z–CH2), 4.30 (d, J = 6.2 Hz, 2H, E–CH2), 4.47 (d,
J = 8.0 Hz, 1H, Z–1-H), 4.81 (d, J = 13.2 Hz, 1H, E–1-H), 6.69 (dd,
J = 13.1, 8.0 Hz, 1H, Z–2-H), 6.83–7.27 (m, 8H, C6H4–OCH3), 7.53
(dd, J = 13.2, 8.5 Hz, 1H, E–2-H), 8.07 (br s, 2H, NH). Anal. Calcd
for C12H15NO3: C, 65.18; H, 6.84; N, 6.33. Found: C, 64.97; H,
6.63; N, 6.25.
4.1.3.9. E/Z-Ethyl 3-(4-Methylbenzylamino)acrylat (7i).
Yel-
low oil; yield 83%; MS (ESI) m/z 220 (M+H+, 100%). 1H NMR (CDCl3)
d 1.16-1.20 (m, 6H, E/Z–CH2–CH3), 2.26 (s, 3H, Z-C6H4–CH3), 2.27 (s,
3H, E-C6H4–CH3), 4.00–4.09 (m, 6H, E/Z–CH2–CH3, E–N–CH2), 4.22
(d, J = 5.9 Hz, 2H, Z–N–CH2), 4.44 (d, J = 8.1 Hz, 1H, Z–1-H), 4.72
(d, J = 13.2 Hz, 1H, E–1-H), 6.59 (dd, J = 13.1, 8.1 Hz, 1H, Z–2-H),
7.07–7.18 (m, 8H, C6H4–CH3), 7.55 (dd, J = 13.2, 7.9 Hz, 1H, E–2-
H), 8.03 (br s, 2H, NH). Anal. Calcd for C13H17NO2: C, 71.26; H,
7.82; N, 6.39. Found: C, 70.95; H, 7.69; N, 6.27.
4.1.3.3.
(7c).
E/Z-Methyl
3-(4-methoxybenzylamino)acrylat
Yellow oil; yield 98%; MS (ESI) m/z 222 (M+H+, 100%).
1H NMR (CDCl3) d 3.62 (s, 3H, Z–COOCH3), 3.64 (s, 3H; E–COOCH3),
3.77 (s, 3H, Z–C6H4–OCH3), 3.78 (s, 3H, E–C6H4–OCH3), 4.12 (d,
J = 5.1 Hz, 2H, Z–CH2), 4.27 (d, J = 5.9 Hz, 2H, E–CH2), 4.52 (d,
J = 8.0 Hz, 1H, Z–1-H), 4.82 (d, J = 13.2 Hz, 1H, E–1-H), 6.66 (dd,
J = 13.1, 8.0 Hz, 1H, Z–2-H), 6.83–7.18 (m, 8H, C6H4–OCH3), 7.53
(dd, J = 13.2, 8.0 Hz, 1H, E–2-H), 8.04 (br s, 2H, NH). Anal. Calcd
for C12H15NO3: C, 65.18; H, 6.84; N, 6.33. Found: C, 65.08; H,
6.75; N, 6.30.
4.1.3.10. E/Z-Ethyl 3-(1-naphthylylamino)acrylat (7j).
Yel-
low oil; yield 98%; MS (ESI) m/z 256 (M+H+, 100%). 1H NMR (CDCl3)
d 1.22–1.28 (m, 6H, E/Z–CH2–CH3), 4.06–4.17 (m, 4H, E/Z–CH2–
CH3), 4.55 (d, J = 8.1 Hz, 1H, Z–1-H), 4.60 (br s, 2H, Z–N–CH2),
4.80 (d, J = 5.7 Hz, 2H, E–N–CH2), 4.95 (d, J = 13.3 Hz, 1H, E–1-H),
6.72 (dd, J = 13.1, 8.1 Hz, 1H, Z–2-H), 7.41–7.55 (m, 8H, E/Z–
4.1.3.4.
(7d).
E/Z-Methyl
3-(4-methylbenzylamino)acrylat
Yellow oil; yield 93%; MS (ESI) m/z 205 (M+H+, 100%).
1H NMR (CDCl3) d 2.26 (s, 3H, Z–C6H4–CH3), 2.27 (s, 3H, E–C6H4–
CH3), 3.57 (s, 3H, Z–COOCH3), 3.59 (s, 3H; E–COOCH3), 4.10 (d,
J = 5.3 Hz, 2H, E–CH2), 4.24 (d, J = 5.9 Hz, 2H, Z–CH2), 4.47 (d,
J = 7.9 Hz, 1H, Z–1-H), 4.76 (d, J = 13.2 Hz, 1H, E–1-H), 6.61 (dd,
J = 13.2, 8.0 Hz, 1H, Z–2-H), 7.07–7.18 (m, 8H, C6H4–CH3), 7.53
(dd, J = 13.2, 8.0 Hz, 1H, E–2-H), 8.01 (br s, 2H, NH). Anal. Calcd
for C12H15NO2: C, 70.27; H, 7.37; N, 6.83. Found: C, 69.95; H,
7.18; N, 6.75.
C10H7), 7.62 (dd, J = 13.3, 6.9 Hz, 1H, E–2-H), 7.77–7.93 (m, 6H, E/
Z–C10H7), 8.21 (br s, 2H, NH). Anal. Calcd for C16H17NO2: C, 75.33;
H, 6.72; N, 5.49. Found: C, 74.99; H, 6.65; N, 5.38.
4.1.4. General procedure for the preparation of the cinnamyl
aldehydes (9a,b).
One equivalent of the corresponding aryl iodide 8a, b was dis-
solved in DMF and then 3 equiv of acrolein diethyl acetale, one
equivalent of tetrabutyl ammonium acetate, one and a half equiv-
alents of potassium carbonate and, finally, 1 equiv of potassium
chloride were added together with 0.03 equiv of palladium(II) ace-
tate. The mixture was stirred for 2–3 h at 90 °C. After cooling to rt
2 M hydrochloric acid was added and stirring continued for 10 min.
4.1.3.5. E/Z-Methyl 3-(1-naphthylamino)acrylat (7e).
Yel-
low oil; yield 94%; MS (ESI) m/z 242 (M+H+, 100%). 1H NMR (CDCl3)
d 3.63 (s, 3H, Z–CH3), 3.64 (s, 3H; E–CH3), 4.56 (d, J = 8.1 Hz, 1H, Z–
1-H), 4.60 (br s, 2H, Z–CH2), 4.80 (d, J = 5.7 Hz, 2H, E–CH2), 4.94 (d,
J = 13.3 Hz, 1H, E–1-H), 6.73 (dd, J = 13.1, 8.1 Hz, 1H, Z–2-H), 7.41–

C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
175
Then the mixture was extracted with diethyl ether for several
times and the unified organic layer was dried over sodium sulfate,
filtered and evaporated in vacuum. The remaining oily product was
purfied by column chromatography using an eluent mixture of
hexane and acetic acid ethyl ester (90/10).
CH2– Ph-CH3), 44.1 (d, C-4), 52.3 (q, COOCH3), 54.0 (t, N–CH2),
109.8 (s, C-3), 112.3 (d, C-5), 125.0 (d, C-4–4-Ph), 126.8 (d, C-2, –
6-N–CH2-Ph), 128.7 (d, C-3, –5–4-Ph), 129.0 (d, C-2, –6–4-Ph),
129.2 (d, C-6), 128.8 (d, C-3, –5-N–CH2–Ph), 136.4 (s, C4–N–CH2–
Ph), 138.5 (s, C-1–N–CH2–Ph), 142.1 (s, C-1–4-Ph), 145.3 (d, C-2),
167.2 (s, CO). Anal. Calcd for C21H21NO2: C, 79.02; H, 6.63; N,
4.39. Found: C, 78.89; H, 6.45; N, 4.25.
4.1.4.1. Cinnamyl aldehyde (9a).
Yellow oil; yield 75%; MS
(ESI) m/z 132 (M+H+, 100%). 1H NMR (CDCl3) d 6.68 (dd, J = 16.0,
7.4 Hz, 1H, 2-H), 7.23–7.44 (m, 6H, 3-H, C6H5), 9.69 (d, J = 7.4 Hz,
1H, 1-H). Anal. Calcd for C9H8O: C, 82.20; H, 6.13. Found: C,
81.90; H, 5.95.
4.1.5.4. Ethyl 1,4-dihydro-1-(2-methoxybenzyl)-4-phenylpyri-
dine-3-carboxylate (10d).
Yellow oil; yield 89%; MS (ESI)
m/z 350 (M+H+, 100%). 1H NMR (CDCl3) d 1.07 (ABX3, J = 7.0 Hz,
3H, CH2–CH3), 3.78 (s. 3H, C6H4–OCH3), 3.49–4.03 (m, 2H, CH2–
CH3), 4.20–4.35 (m, 1H, 4-H), 4.41–4.49 (m, 3H, 5-H, N–CH2),
6.21–6.38 (m, 1H, 6-H), 6.83–7.72 (m, 10H, 2-H, C6H5, C6H4–
OCH3). 13C NMR (CDCl3) d 14.2 (q, COOCH2CH3), 43.8 (d, C-4),
49.2 (t, N–CH2), 56.3 (q, C-2–N–CH2– Ph–OCH3), 61.7 (t,
COOCH2CH3), 109.4 (s, C-3), 112.0 (d, C-5), 114.4 (d, C-5– N–
CH2–Ph), 121.2 (d, C-5–N–CH2–Ph), 128.0 (s, C-1–N–CH2–Ph),
125.8 (d, C-4–4-Ph), 127.4 (d, C4–N-CH2–Ph), 129.3 (d, C-2, –6–4-
Ph), 129.1 (d, C-3, –5–4-Ph), 129.3 (d, C-6), 127.4 (d, C-6–N–CH2–
Ph), 142.2 (s, C-1–4-Ph), 144.9 (d, C-2), 156.7 (s, C-2–N–CH2–Ph),
167.1 (s, CO). Anal. Calcd for C22H23NO3: C, 75.68; H, 6.64; N,
4.01. Found: C, 75.44; H, 6.53; N, 3.88.
4.1.4.2. 3-Naphthylacrylaldehyde (9b).
Yellow oil; yield 90%;
MS (ESI) m/z 183 (M+H+, 100%). 1H NMR (CDCl3) d 6.84 (dd, J = 15.7,
7.6 Hz, 1H, 2-H), 7.49–8.16 (m, 7H, C10H7), 8.29 (d, J = 15.7 Hz, 1H,
3-H), 9.83 (d, J = 7.6 Hz, 1H, 1-H). Anal. Calcd for C13H10O: C, 85.79;
H, 6.37. Found: C, 85.55; H, 6.25.
4.1.5. General procedure for the preparation of the N-benzyl and
4-naphthyl substituted 1.4-dihydropyridines (10a–j)
One equivalent of the corresponding cinnamyl aldehyde 9a, b
and one equivalent of the corresponding b-enaminocarbonyl com-
pound 7a–j were dissolved in dichloromethane. After addition of
three equivalents of sodium sulfate and 0.05 equiv of iron(II) chlo-
ride hexahydrate (5%) the solution was stirred at 40 °C for 12 h.
After both starting products disappeared as monitored by tlc the
solution was filtered and the solvent was removed in vacuum.
The remaining oil was purified by MPLC using cyclohexane, t-
butylmethylether and dichloromethane in a mixture of 80/15/5.
4.1.5.5. Ethyl 1,4-dihydro-1-(4-methoxybenzyl)-4-phenylpyri-
dine-3-carboxylate (10e).
Yellow oil; yield 71%; MS (ESI) m/
z 350 (M+H+, 100%). 1H NMR (CDCl3) d 1.10 (ABX3, J = 7.0 Hz, 3H,
CH2–CH3), 3.79 (s. 3H, C6H4–OCH3), 3.97–4.03 (ABX3, J = 14.3,
7.0 Hz, 2H, CH2–CH3), 4.35 (s, 2H, N-CH2), 4.50 (d, J = 4.9 Hz, 1H,
4-H), 4.88 (dd, J = 7.7, 4.9 Hz, 1H, 5-H), 5.88 (d, J = 7.7 Hz, 1H, 6-
H), 6.89–7.49 (m, 10H, 2-H, C6H5, C6H4–OCH3). 13C NMR (CDCl3) d
14.5 (q, COOCH2CH3), 39.8 (d, C-4), 55.1 (t, N-CH2), 55.8 (q, C-4–
N–CH2–Ph–OCH3), 62.1 (t, COOCH2CH3), 108.0 (s, C-3), 112.0 (d,
C-5), 114.5 (d, C-3, –5-N–CH2–Ph), 125.9 (d, C-4–4-Ph), 127.8 (d,
C-2, –6-N–CH2–Ph), 129.0 (d, C-2, –6–4-Ph), 129.1 (d, C-3, –5–4-
Ph), 129.3 (d, C-6), 134.1 (s, C-1–N–CH2–Ph), 142.2 (s, C-1–4-Ph),
146.1 (d, C-2), 158.6 (s, C4–N–CH2–Ph), 167.2 (s, CO). Anal. Calcd
for C22H23NO3: C, 75.68; H, 6.64; N, 4.01. Found: C, 75.38; H,
6.54; N, 4.03.
4.1.5.1. Methyl 1,4-dihydro-1-(2-methoxybenzyl)-4-phenylpyri-
dine-3-carboxylate (10a).
Yellow oil; yield 85%; MS (ESI) m/z
336 (M+H+, 100%). 1H NMR (CDCl3) d 3.81 (s, 3H, COOCH3), 3.94 (s.
3H, C6H4–OCH3), 4.05–4.08 (m, 1H, 4-H), 4.09–4.15 (m, 2H, N–CH2),
4.22–4.35 (m, 1H, 5-H), 6.23–6.33 (m, 1H, 6-H), 6.83–7.71 (m, 10H,
2-H, C6H5, C6H4–OCH3).
13C NMR (CDCl3) d 44.0 (d, C-4), 52.2 (q, COOCH3), 48.9 (t, N–
CH2), 56.1 (q, C-2–N–CH2– Ph–OCH3), 109.8 (s, C-3), 111.9 (d, C-
5), 114.1 (d, C-3–N–CH2–Ph), 120.9 (d, C-5–N–CH2–Ph), 125.8 (d,
C-4–4-Ph), 127.3 (s, C-1–N–CH2–Ph), 127.8 (d, C4–N–CH2–Ph),
127.9 (d, C-6–N–CH2–Ph), 128.7 (d, C-3, –5–4-Ph), 129.0 (d, C-2,
–6–4-Ph), 129.3 (d, C-6), 142.2 (s, C-1–4- Ph), 144.5 (d, C-2),
156.5 (s, C-2–N–CH2–Ph), 167.2 (s, CO). Anal. Calcd for
4.1.5.6. Ethyl 1,4-Dihydro-1-(4-methylbenzyl)-4-phenylpyri-
dine-3-carboxylate (10f).
Yellow oil; yield 73%; MS (ESI) m/
z 334 (M+H+, 100%). 1H NMR (CDCl3) d 1.13 (ABX3, J = 7.0 Hz, 3H,
CH2–CH3), 2.25 (s, 3H, C6H4–CH3), 3.97–4.03 (ABX3, J = 14.2,
7.0 Hz, 2H, CH2–CH3), 4.13–4.23 (m, 1H, 4-H), 4.25–4.44 (m, 1H,
5-H), 4.51 (s, 2H, N–CH2), 5.77-5.90 (m, 1H, 6-H), 6.73–7.59 (m,
9H, C6H5, C6H4–CH3), 7.63 (s, 1H, 2-H). 13C NMR (CDCl3) d 14.5
(q, COOCH2CH3), 24.2 (q, C-4–N–CH2–Ph–CH3), 44.0 (d, C-4), 54.4
(t, N–CH2), 62.0 (t, COOCH2CH3), 109.4 (s, C-3), 112.0 (d, C-5),
125.5 (d, C-4–4-Ph), 128.0 (d, C-3, –5-N–CH2–Ph), 129.3 (d, C-2, –
6–4-Ph), 128.7 (d, C-3, –5–4-Ph), 129.0 (d, C-6), 126.4 (d, C-2, –6-
N–CH2–Ph), 136.7 (s, C4–N–CH2–Ph), 138.9 (s, C-1–N–CH2–Ph),
142.4 (s, C-1–4-Ph), 145.9 (d, C-2), 167.1 (s, CO). Anal. Calcd for
C21H21NO3: C, 75.26; H, 6.32; N, 4.18. Found: C, 75.21; H, 6.28;
N, 4.04.
4.1.5.2. Methyl 1,4-dihydro-1-(4-methoxybenzyl)-4-phenylpyri-
dine-3-carboxylate (10b).
Yellow oil; yield 80%; MS (ESI) m/z
336 (M+H+, 100%). 1H NMR (CDCl3) d 3.33 (s, 3H, COOCH3), 3.80 (s.
3H, C6H4–OCH3), 4.35 (s, 2H, N–CH2), 4.50 (d, J = 4.8 Hz, 1H, 4-H),
4.89 (dd, J = 7.7, 4.8 Hz, 1H, 5-H), 5.88 (d, J = 7.8 Hz, 1H, 6-H),
6.90–7.49 (m, 10H, 2-H, C6H5, C6H4–OCH3). 13C NMR (CDCl3) d
44.2 (d, C-4), 52.4 (q, COOCH3), 54.8 (t, N–CH2), 55.8 (q, C-4–N–
CH2– Ph–OCH3), 109.5 (s, C-3), 112.5 (d, C-5), 114.3 (d, C-3, –5-
N–CH2–Ph), 125.5 (d, C-4–4-Ph), 127.6 (d, C-2, –6-N–CH2–Ph),
128.3 (d, C-3, –5–4-Ph), 129.0 (d, C-6), 129.4 (d, C-2, –6–4–Ph),
133.9 (s, C-1–N–CH2–Ph), 142.0 (s, C-1–4-Ph), 144.1 (d, C-2),
158.6 (s, C4–N–CH2–Ph), 167.1 (s, CO). Anal. Calcd for C21H21NO3:
C, 75.26; H, 6.32; N, 4.18. Found: C, 75.21; H, 6.28; N, 4.04.
C22H23NO2: C, 79.31; H, 6.96; N, 4.20. Found: C, 79.25; H, 6.76;
N, 4.15.
4.1.5.7. Methyl 1,4-dihydro-1-(1-naphthylmethyl)-4-phenylpyr-
idine-3-carboxylate (10g).
Yellow crystals; mp 135-137 °C;
yield 87%; MS (ESI) m/z 356 (M+H+, 100%). 1H NMR (CDCl3) d
3.54 (s, 3H, CH3), 4.55 (d, J = 4.7 Hz, 1H, 4-H), 4.89–4.93 (m. 3H,
5-H, N–CH2), 5.94 (d, J = 7.8 Hz, 1H, 6-H), 7.11–7.91 (m, 13H, 2-H,
C6H5, C10H7). 13C NMR (CDCl3) d 45.1 (d, C-4), 53.1 (q, COOCH3),
53.1 (t, N–CH2), 108.8 (s, C-3), 113.2 (d, C-5), 124.2 (d, C-2, –8-N–
CH2–Nph), 125.0 (d, C-4–4-Ph), 125.6 (d, C-6–N–CH2–Nph), 125.8
(d, C7–N–CH2–Nph), 126.5 (d, C-4–N–CH2–Nph), 126.9 (d, C-3-N-
CH2-Nph), 128.6 (d, C-5-N–CH2–Nph), 129.1 (d, C-3, –5–4-Ph),
4.1.5.3. Methyl 1,4-dihydro-1-(4-methylbenzyl)-4-phenylpyri-
dine-3-carboxylate (10c).
Yellow oil; yield 77%; MS (ESI) m/
z 320 (M+H+, 100%). 1H NMR (CDCl3) d 2.25 (s, 3H, C6H4–CH3),
3.48 (s, 3H, COOCH3), 4.24-4.38 (m, 1H, 4-H), 4.42-4.46 (m, 3H,
5-H, N-CH2), 5.78-5.95 (m, 1H, 6-H), 6.81–7.49 (m, 9H, C6H5,
C6H4–OCH3), 7.63 (s, 1H, 2-H). 13C NMR (CDCl3) d 24.3 (q, C-4–N–

176
C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
129.6 (d, C-6), 129.8 (d, C-2, –6–4-Ph), 132.6 (s, C-8a–N–CH2–Nph),
133.5 (s, C-4a–N–CH2–Nph), 133.8 (s, C-1–N–CH2–Nph), 142.1 (s,
C-1–4-Ph), 145.3 (d, C-2), 167.2 (s, CO). Anal. Calcd for
4.3. P-gp inhibition assay
Cells of both cell lines were taken in an adjusted concentration
of 1 x 106 cells per mL medium and resuspended in serum free Mc
Coys 5A medium. 0.5 mL Aliquots were filled into Eppendorf cen-
trifuge tubes. The test compounds taken from prepared stock solu-
C24H21NO2: C, 81.16; H, 5.96; N, 3.94. Found: C, 81.05; H, 6.04;
N, 3.94.
4.1.5.8. Ethyl 1,4.dihydro-1-(1-naphthylmethyl)-4-phenylpyri-
tions in DMSO (1.0 mg/mL) were added in a volume of 5
10 min of inhibitor preincubation at rt the P-gp substrate rhoda-
mine 123 was added using 5 L of a 0.5 mM solution in water.
lL. After
dine-3-carboxylate (10h).
Yellow crystals; mp 145–148 °C;
yield 84%; MS (ESI) m/z 370 (M+H+, 100%). 1H NMR (CDCl3) d
1.09 (ABX3, J = 7.1 Hz, 3H, CH2–CH3), 3.93–4.07 (ABX3, J = 10.6,
7.1 Hz, 2H, CH2–CH3), 4.55 (d, J = 4.8 Hz, 1H, 4-H), 4.89–4.92 (m,
3H, 5-H, N–CH2), 5.93 (d, J = 7.9 Hz, 1H, 6-H), 7.11–7.92 (m, 13H,
2-H, C6H5, C10H7). 13C NMR (CDCl3) d 14.0 (q, COOCH2CH3), 44.8
(d, C-4), 53.3 (t, N–CH2), 61.7 (t, COOCH2CH3), 108.0 (s, C-3),
112.2 (d, C-5), 124.5 (d, C-2, –8-N–CH2–Nph), 125.5 (d, C-6–N–
CH2–Nph), 125.6 (d, C-4–4-Ph), 125.9 (d, C7–N–CH2–Nph), 126.3
(d, C-4–N–CH2–Nph), 126.7 (d, C-3–N–CH2–Nph), 128.4 (d, C-5–
N–CH2–Nph), 129.1 (d, C-6), 129.3 (d, C-3, –5-4–Ph), 129.4 (d, C-
2, –6-4–Ph), 132.3 (s, C-8a–N–CH2–Nph), 133.4 (s, C-4a–N–CH2–
Nph), 133.9 (s, C-1–N–CH2–Nph), 142.3 (s, C-1–4-Ph), 145.8 (d,
C-2), 167.1 (s, CO). Anal. Calcd for C25H23NO3: C, 81.27; H, 6.27;
N, 3.79. Found: C, 80.95; H, 6.11; N, 3.76.
l
Incubation was continued for 40 min at 37 °C. After that the cells
were centrifuged and washed twice with phosphate-buffered sal-
ine (PBS). Then they were resuspended in PBS for measurement.
The non-inhibitor containing cells were treated in the same way
as the inhibitor preincubated cells. The fluorescence uptake of rho-
damine 123 within a number of 1 Â 104 counted cells was deter-
mined by flow cytometry using a Becton Dickinson FACScan flow
cytometer. The fluorescence activity ratio (FAR) value was calcu-
lated from the quotient of the determined fluorescence uptake ra-
tios of the P-gp expressing cell line and the non-P-gp expressing
parental cell line. Both ratios have been corrected by division with
the fluorescence determined in the inhibitor untreated control cell
lines.
4.1.5.9. Methyl 1-benzyl-1,4-dihydro-4-(1-naphthyl)pyridine-3-
4.4. MDR reversal studies
carboxylate (10i).
Yellow crystals; mp 125–127 °C; yield
85%; MS (ESI) m/z 356 (M+H+, 100%). 1H NMR (CDCl3) d 3.47 (s,
3H, CH3), 4.45 (s, 3H, N–CH2), 5.04 (dd, J = 7.8, 4.7 Hz, 1H, 5-H),
5.37 (d, J = 4.7 Hz, 1H, 4-H), 5.80 (d, J = 7.8 Hz, 1H, 6-H), 7.26-8.24
(m, 13H, 2-H, C6H5, C10H7). 13C NMR (CDCl3) d 42.3 (d, C-4), 53.2
(q, COOCH3), 54.8 (t, N–CH2), 109.2 (s, C-3), 113.3 (d, C-5), 124.1
(d, C-2, –8–4-Nph), 125.6 (d, C-6–4-Nph), 125.9 (d, C7–4-Nph),
126.4 (d, C-4–4-Nph), 126.6 (d, C-3–4-Nph), 126.8 (d, C-4–N–
CH2–Ph), 126.9 (d, C-2, –6-N–CH2–), 128.5 (d, C-5–4-Nph), 128.6
(d, C-3, –5-N–CH2–Ph), 129.6 (d, C-6), 132.7 (s, C-8a–4-Nph),
133.5 (s, C-4a–4-Nph), 134.0 (s, C-1–4-Nph), 141.6 (s, C-1-N–
CH2–Ph), 145.3 (d, C-2), 167.2 (s, CO). Anal. Calcd for C24H21NO2:
C, 81.16; H, 5.96; N, 3.94. Found: C, 81.10; H, 6.30; N, 3.78.
Cells of both cell lines were corrected to a cell number of
5 Â 105 cells per mL. Daunorubicin both as pure compound and
as liposomal DaunoXomeÒ preparation was added from stock solu-
tions reaching a final DMSO concentration of 1%. The daunorubicin
has been used in concentrations of 0.01 lM to 10 lM. Then 100 lL
of the cell and daunorubicin containing suspension were divided
into 96-well plates and additionally supplemented with the P-gp
inhibitor in the respective concentration. Cells were incubated
for 24 h at 37 °C under the carbon dioxide containing atmosphere
(5%). Then 10
nyltetrazolium bromide) solution (5 mg/mL) in PBS were added to
each well. After additional four hours of incubation 100 L of lysis
lL of a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
l
buffer containing 50 g of sodium dodecyl sulfate (SDS), 125 mL of
acetic acid and 375 mL of DMSO each per liter were added and cells
were shaken for 30 min for a complete cell lysis. Each plate was
measured spectrophotometrically with a microplate reader Polar-
Star Galaxy at 560 nm. The experiments were repeated in triplicate
and the IC50 values were determined from the mean percent
growth inhibitions.
4.1.5.10. Ethyl 1-benzyl-1,4-dihydro-4-(1-naphthyl)pyridine-3-
carboxylate (10j).
Yellow oil; yield 79%; MS (ESI) m/z 370
(M+H+, 100%). 1H NMR (CDCl3) d 0.93 (ABX3, J = 7.0 Hz, 3H, CH2–
CH3), 3.90–4.05 (m, ABX3, J = 13.6, 7.0 Hz, 3H, 4-H, CH2–CH3),
4.45 (s, 2H, N–CH2), 5.02 (dd, J = 7.7, 5.0 Hz, 1H, 5-H), 5.80 (d,
J = 7.7 Hz, 1H, 6-H), 7.18–8.26 (m, 13H, 2-H, C6H5, C10H7). 13C
NMR (CDCl3) d 14.2 (q, COOCH2CH3), 42.3 (d, C-4), 61.5 (q,
COOCH2CH2), 54.6 (t, N–CH2), 109.1 (s, C-3), 112.8 (d, C-5), 124.5
(d, C-2, –8–4-Nph), 126.5 (d, C-4–N–CH2–Ph), 125.6 (d, C–6–4-
Nph), 125.7 (d, C8–4-Nph), 126.4 (d, C-4–4-Nph), 126.6 (d, C-3–
4-Nph), 126.9 (d, C-2, –6-N–CH2–Ph), 128.5 (d, C-5–4-Nph), 128.6
(d, C-3, –5-N–CH2–Ph), 129.6 (d, C-6), 132.7 (s, C-8a–4-Nph),
133.5 (s, C-4a–4-Nph), 134.0 (s, C-1–4-Nph), 141.6 (s, C-1-N–
CH2–Ph), 146.0 (d, C-2), 167.2 (s, CO). Anal. Calcd for C25H23NO2:
C, 81.33; H, 6.28; N, 3.79. Found: C, 81.28; H, 6.24; N, 3.67.
4.5. Cytotoxicity studies
Cells, cell numbers and both incubation as well as workup pro-
cedure were exactly the same as used for the determination of the
daunorubicin toxicity except the use of the respective 1,4-dihydro-
pyridines instead of the daunorubicin. The used concentration
range of the 1,4-dihydropyridines varied from 1 lM up to 80 lM
as the final concentration. The spectrophotometrical analysis fol-
lowed the described protocol. The experiments were repeated in
triplicate.
4.2. Cell culture
Both cell lines the mouse T-lymphoma parental cell line L5178Y
and the P-gp expressing subline L5178Y mdr which resulted from
retrovirus-mediated gene transfection26,27 were cultured at 37 °C
under carbon dioxide containing atmosphere (5%) in McCoys 5A
Acknowledgements
The authors gratefully acknowledge the financial support of
their work by the DFG (Deutsche Forschungsgemeinschaft)
within the projects HI 687/5-1 and HI 687/5-3 to Christiane
Baumert, Sören Krawczyk and Andreas Hilgeroth and by the EU
within the project 1124856 to Marc Hemmer and Andreas
Hilgeroth.
medium containing 10% of fetal calf serum, L-glutamine (2 mM)
and 5 mL of a gentamicin solution (5 mg/mL). The medium used
for culturing the P-gp expressing subline L5178Y has been addi-
tionally supplemented with colchicine (60 ng/mL) to ensure a
stable P-gp expression.

C. Baumert et al. / Bioorg. Med. Chem. 21 (2013) 166–177
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