Biological evaluation of bishydroxymethyl-substituted cage dimeric 1,4-dihydropyridines as a novel class of P-glycoprotein modulating agents in cancer cells

Article abstract of DOI:10.1021/jm058046w

A series of N-substituted cage dimeric 1,4-dihydropyridines 3a-e was evaluated as inhibitors of membrane efflux pump P-glycoprotein (P-gp) in multidrug resistant (mdr) cancer cells. Structure-activity relationships (SAR) and cytotoxic properties are discu

Full text of DOI:10.1021/jm058046w

2838
J. Med. Chem. 2006, 49, 2838-2840
Brief Articles
Biological Evaluation of Bishydroxymethyl-Substituted Cage Dimeric 1,4-Dihydropyridines as a
Novel Class of P-Glycoprotein Modulating Agents in Cancer Cells
Martin Richter,† Jo´sef Molna´r,‡ and Andreas Hilgeroth*,†
Institute of Pharmaceutical Chemistry, Martin-Luther-UniVersity Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany, and
UniVersity of Szeged, Faculty of Medicine, Department of Medical Microbiology, Do´m te´r 10, 6720 Szeged, Hungary
ReceiVed August 15, 2005
A series of N-substituted cage dimeric 1,4-dihydropyridines 3a-e was evaluated as inhibitors of membrane
efflux pump P-glycoprotein (P-gp) in multidrug resistant (mdr) cancer cells. Structure-activity relationships
(SAR) and cytotoxic properties are discussed. Effective concentrations for overcoming mdr have been
demonstrated in competition studies with the P-gp substrate epirubicin.
Scheme 1a
Introduction
Multidrug resistance (mdr) is a main problem in cancer
treatment. Membrane efflux pumps such as P-glycoprotein (P-
gp) have been identified as causative agents which partly
transport cytostatic agents out of the cells before they reach the
cytosol.1,2 With a broad substrate specifity, P-gp was found to
transport even novel cytostatics such as tyrosine kinase inhibitor
imatinib or monoclonal antibody gemtuzumab ozagamicin.3,4
Intense efforts to overcome mdr by influencing transporter
expressions via signal transduction pathways or by direct
transcriptional control have not been successful in clinical trials.5
The most hopeful alternative to combate mdr is the inhibition
of the transporter activity itself. Almost all modulators of mdr
have been derived from known drugs such as verapamil with
structural modifications which only occasionally led to effective
modulators in concentration ranges below their drug-like
pharmacological activities.6 Moreover, most inhibiting agents
proved to be transporter substrates themselves, thus requiring
high applied concentrations for effectively overcoming mdr.7,8
We report a novel series of bishydroxymethyl-substituted cage
dimeric 1,4-dihydropyridines 3a-e which prove to be effective
and superior P-gp inhibitors compared to verapamil, without
showing additional pharmacological activities, as do the struc-
turally related tetrabishydroxymethyl-substituted compounds
which have been demonstrated to act as HIV-protease inhibi-
tors.9 Moreover, the comparison of the cytotoxic properties
indicates that they are practically not P-gp substrates and thus
form an innovative class of new mdr modulators for further
development efforts.
a Reagents and conditions: (i) CuI (5%), PhMgCl, THF, RT; (ii) hν, 25
°C, THF; (iii) LiAlH4, -8 °C, THF.
Chemistry. The alcoholic target compounds 3a-e (Scheme
1) have all been prepared from the ester group-substituted cage
dimers 2a-e by reduction with lithium aluminum hydride in
THF at low temperatures (-8 °C), in effective reaction times
of a few hours and overall yields of about 70%. N-Alkyl cage
dimers 2a,b have been prepared by a solution dimerization
reaction of monomeric 1,4-dihydropyridines 1a,b under UV-
excitation of the dihydropyridine chromophore with λmax ) 346
nm (a) and 348 nm (b). Monomeric 1,4-dihydropyridines 1a,b
were prepared by a regioselective 4-phenylation of the N-
alkylpyridinium salt starting compounds by the use of equimolar
amounts of phenylmagnesium chloride and catalytic traces of
copper(I) iodide.
Results and Discussion
P-gp inhibiting properties have been evaluated by comparing
the concentration-dependent uptakes of P-gp specific fluorescent
substrate rhodamine 123 in both mdr1-gene transfected and
parental mouse T-lymphoma cell lines as fluorescence activity
* To whom correspondence should be addressed. Tel: 49-345-55-25168.
Fax: 49-345-55-27026. E-mail: hilgeroth@pharmazie.uni-halle.de.
† Martin-Luther-University Halle-Wittenberg.
‡ University of Szeged.
10.1021/jm058046w CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/07/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2839
Table 1. P-gp Inhibition, Cytotoxicity and MDR Overcoming Properties of Cage Dimeric 1,4-Dihydropyridines in Tumor Cell Lines
FARa cytotoxicitya overcoming MDR,a IC50 [µM]
at 30 µM
at 3 µM
max.
IC50 (µM)
Par, IC50 (µM)
MDR1, IC50 (µM)
modulator
>22.5
epirubicin
3a
3b
3c
3d
3e
4
1.2 ( 0.03
24.5 ( 0.5
1.1 ( 0.04
6.8 ( 0.3
28.1 ( 1.2
0.7 ( 0.03
34.3 ( 0.8
26.5 ( 0.5
21.6 ( 0.3
33.0 ( 0.3
30.8 ( 1.3
7.3 ( 0.3d
35
27
22b
33
32
-
17.6 ( 0.4
<1
7.1 ( 0.3
11.9 ( 0.8
n.dc
2.5 ( 0.1
12.7 ( 0.5
7.5 ( 0.3
13.3 ( 0.5
n.dc
3.2 ( 0.2
21.0 ( 0.9
10 ( 0.6
1.3 ( 0.5
n.dc
>10
<5
6.5 ( 0.06
3.8 ( 0.5
6.1 ( 0.1
<1
n.dc
7.2 ( 0.07
5.6 ( 0.1
n.dc
a Mean of two determinations. b saturation. c n.d., not determined. d Determined at the clinically relevant concentration of 11 µM with saturation effects.
ratios (FAR). Within the N-acyloxy derivatives 3c-e, an
increase in activity is observed at low concentration from methyl
to tert-butyl and finally phenyl substitution. With increased
lipophilicity of the varied substituents at the nitrogen atom,
the phenyl-substituted compound 3e finally reaches a more
than 15-fold higher ratio in comparison to verapamil (4) as a
standard with a FAR value of 7.27 (11 µM) (Table 1). At higher
concentrations we reached saturation effects of the P-gp
inhibitions with ratios of about 30 for the lipophilic tert-butyl
and phenyloxycarbonyl substitution.
Within the N-alkyl derivatives 3a,b, benzylic substitution
leads to a more than 15-fold higher activity than does methyl
substitution and both compounds reach final P-gp inhibition
saturation effects at higher concentrations. Again, the more
lipophilic substituent is found superior at 3 µM.
Comparing equivalents of both N-acyloxy and N-alkyl
compound series in each case, the additional acyl functions may
play a role in hydrogen bonding to the P-gp binding region of
these compounds. These additional functions lead to a lowered
activity in comparison of compounds 3a and 3c while they
increase activity in comparison of compounds 3b and 3e. From
these results it may be concluded that both the differing
lipophilicity as well as hydrogen bonding patterns will play a
complementary role in the biological activity within this class
of highly active compounds.
The IC50 values for the reduction of fluorescence uptake for
compounds of similar comparable P-gp saturation effects
(3a,b,d,e) with FAR values of about 32, indicate the lowest
activity for methyl substitution and highest biological activities
for benzyl- and the phenyloxycarbonyl-substituted derivatives
b and e. The structure-activity relationships stress the impor-
tance of lipophilic substitution patterns in effective mdr modula-
tors.10,11
The cytotoxicity of the comparable compounds 3a,b,d,e have
been determined in both mdr1-gene transfected and parental
mouse T-lymphoma cell lines to ascertain whether any potential
cytotoxic properties in the parental cell line are reduced in the
mdr1 subline by P-gp transport efflux of the compounds out of
the cells. Such a reduction of compound cytotoxicity suggests
unfavorable P-gp substrate properties so that higher concentra-
tions are necessary to achieve the same cytotoxic effects.8
Some cytotoxicity has been found for compound 3d in the
parental cell line, while the cytotoxicity of compounds b and e
are reduced. When both the parental and the mdr1 transfected
cell lines are compared, interestingly, only the phenoxycarbonyl-
substituted derivative e showed differences in cytotoxicity, while
for the other three derivatives a, b, and d, almost identical IC50
values are found. Only derivative 3e of this novel series of mdr
modulators exhibited some P-gp substrate properties with
reduced cytotoxic activity in the mdr1 transfected cell line
caused by the efflux from this mdr resistant cell line as a P-gp
substrate.
tion rates compared to those mdr1 modulators which are being
effluxed from the cells as P-gp substrates themselves and thus
require higher unfavorable application rates.8 We also investi-
gated the concentrations to overcome mdr by comparing the
cytotoxicity in mouse T lymphoma parental and mdr1 subline
using combinations of each fixed modulator and cytotoxic P-gp-
substrate epirubicin concentrations, respectively, in competition
with varying epirubicin and modulator concentrations, respec-
tively.
In the study, with varying epirubicin concentrations at each
fixed modulator concentration we found the lowest cytotoxicities
for the mdr1 subline compared to the parental cell line at the
lowest modulator concentrations. These lowest cytotoxicities
result from the concentration-dependent low P-gp inhibiting
properties. We found subsequent increased cytotoxicities in the
mdr1 subline due to increased intracellular epirubicin levels
caused by increased P-gp inhibition of increasing modulator
concentrations and thus increased cytotoxicity of epirubicin.
Increased cytotoxicity was partly found also in the parental cell
line due to some increasing cytotoxic effects of the modulators
themselves.
With mainly reduced IC50 values of cytotoxicity in the mdr1
subline we observed an overcoming of mdr for compounds
3b,d,e with equal concentrations in the low range of 5.6 and
7.2 µM in both cell lines but not for the poor mdr modulator
3a (Table 1).
Using each fixed epirubicin concentration in combination with
varied modulator concentrations we observed certain differences
in the potential of epirubicin to overcome mdr with equal
epirubicin concentrations in both cell lines. While the use of
compound 3d did not overcome mdr within the epirubicin
concentration range up to 10 µM, the benzyl- and phenyloxy-
carbonyl-substituted derivatives 3b and 3e were successful
competitors that overcame mdr at an epirubicin concentration
of 1.3 µM, with compound 3b as the best derivative.
Conclusion
We report a novel class of mdr modulators which exhibit
effective P-gp inhibitory properties significantly superior to that
of verapamil. The most active compounds are lipophilic
substituted N-benzyl and -phenyloxycarbonyl derivatives 3b and
3e. Some P-gp substrate properties have been suggested only
for the N-phenyloxycarbonyl compound 3e. Competition studies
with cytotoxic P-gp substrate epirubicin indicated the overcom-
ing of mdr in comparison of the cell line at concentrations below
cytotoxic ranges of the most effective mdr-modulating concen-
trations of the compounds themselves. The N-benzyl compound
exhibited the highest activity and practically no P-gp substrate
properties. Therefore, it could be a promising candidate for
further clinical studies and structural improvements as a novel
lead compound for the overcoming of mdr.
Experimental Section
This is an important finding with respect to a potential clinical
application of these compounds which allows lowered applica-
General. Commercial reagents were used without further
purification. 1H NMR (500 MHz) spectra were recorded using

2840 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Brief Articles
tetramethylsilane as an internal standard. TLC was performed on
E. Merck 5554 silica gel plates. Mass spectra were measured with
an AMD 402 and Finigan-LCQ Classic mass spectrometer,
respectively. Elemental analysis was performed using a Leco
CHNS-932 apparatus.
sorbance of the formazane was corrected by the absorbance of the
cell control and the compounds. Plotting of each compound
concentration and corrected UV absorbance led to a sigmoidal curve
from which the IC50 values could be derived. For the determination
of the mdr overcoming concentrations, cytotoxicity was determined
as described by combining each fixed concentration of epirubicin
within a range of 0.02 to 10 µM and modulator within a range of
0.13 (3d) to 64.5 (3e) µM, respectively, and varying concentrations
of modulator and epirubicin in each of both cell lines. Plotting of
each fixed concentration and resulting IC50 values of cytotoxicity
for the varied concentration of the other combined compounds
resulted in sigmoid-like curves with an intersection point of the
resulting concentration for overcoming of mdr.
The synthesis of cage compounds 2c-e has been reported in ref
12.
General Procedure for the Formation of 4-Phenyl-1,4-
dihydropyridines 1a,b from N-Alkylpyridinium Salts.13 The
N-alkylpyridinium salt (1 equiv, 10 mmol) was suspended in dry
THF (100 mL) and anhydrous methyl sulfide (27 mL). After
addition of copper(I) iodide (0.095 g, 0.5 mmol), a solution of
phenylmagnesium chloride (1 M) in a mixture of THF (10 mL, 10
mmol) was added dropwise. After 2 h of stirring at room
temperature, the solution was treated with an aqueous solution of
ammonium chloride (60 mL, 113 mmol) and then extracted with
diethyl ether (150 mL). The ether phase was washed with 20 mL
each of a 20% mixture of ammonia/ammonium chloride (1/1),
water, twice with hydrochloric acid (10%), water, and finally
saturated aqueous sodium chloride following the described proce-
dure in ref 12. After final removal of the dried ether, the oily residue
was solved in ethanol from which the product crystallized.
General Procedure for the Formation of N-Alkyl Cage
Compounds (2a,b). N-Alkyl-1,4-dihydropyridine 1 (1 equiv, 2.18
mmol) was dissolved in dry THF (40 mL) with stirring. The solution
was irradiated in a borosilicate flask with an Ultra Vitalux lamp
from a distance of 60 cm at 25 °C. After eight weeks, the cage
compound 2 precipitated from the solution and the reaction was
carried out until no more precipitate was formed. The compound
was collected by filtration and dried.
General Procedure for the Formation of Alcoholic Target
Cage Compounds (3a-e). Cage compound 2 (1 equiv, 0.04 mmol)
was dissolved in dry THF (20 mL). The solution was cooled to
-8 °C, and then a solution of lithium aluminum hydride (0.04 mL,
0.04 mmol) in THF (1 M) was added. After 2 h, the reaction mixture
was kept overnight at -8 °C and the next day hydrolyzed with 1
mL of cold water at 0 °C. The solution was then extracted with 20
mL of chloroform three times. The combined extracts were then
dried over sodium sulfate. After filtration, the organic phase was
removed in a vacuum and the remaining oil was dissolved in
chloroform/methanol/water from which 3 crystallized after dropwise
addition each of diethyl ether and petroleum ether.
Fluorescence Uptake Assay. Cells from L5178Y mouse T-cell
lymphoma parental cell line and from its multidrug resistant subline
L5178YvMDR which was a gift from the National Cancer Institute
(NCI) were adjusted to a concentration of 2 × 106/mL in serum-
free McCoys 5A medium and distributed in 0.5 mL aliquots to
eppendorf centrifuge tubes. The test compounds were added from
stock solutions (1.0 mg/mL) and incubated for 10 min at room
temperature. Then rhodamine 123 with 5.2 µM as a final concentra-
tion was added and incubation continued for 20 min at 37 °C. After
being washed twice with phosphate-buffered saline (PBS), the
samples were resuspended in PBS for analysis. The fluorescence
of 1 × 104 cells was measured by flow cytometry. Fluorescence
activity ratios (FAR) have been calculated from fluorescence uptake
relationships of each treated and untreated control cell line. Exact
IC50 values have been determined from the resulting sigmoidal
curves obtained by plotting each concentration and corresponding
FAR value.
MTT Cytotoxicity Assay. Cells were cultured for 48 h under
5% CO2 atmosphere and 37 °C. Then 10 µL of an MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution
with a final concentration of 0.5 mg/mL was added, and incubation
was continued for additional 4 h at 37 °C. Precipitated formazane
crystals were resolved under addition of 100 µL of SDS solution
(10% in 0.01 M HCl), and then samples were left standing overnight
under the described conditions. Concentration-dependent UV ab-
Acknowledgment. The work was financially supported
within the framework of the COST B16 Action of the EU, the
BMBF, and the DFG.
Supporting Information Available: Detailed spectroscopic and
purity data of the compounds. Sigmoidal curves of the fluorescence
uptake assays are shown. This material is available free of charge
via the Internet at http://pubs.acs.org.
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