Permanently charged chiral 1,4-dihydropyridines: Molecular probes of L- type calcium channels. Synthesis and pharmacological characterization of methyl (ω-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3- nitrophenyl)-3,5-pyridinedicarboxylate iodide

Article abstract of DOI:10.1021/jm000028l

We report the synthesis of the single enantiomers of permanently charged dihydropyridine derivatives (DHPs with alkyl linker lengths of two and eight carbon atoms) and their activities on cardiac and neuronal L-type calcium channels. Permanently charged c

Full text of DOI:10.1021/jm000028l

2906
J . Med. Chem. 2000, 43, 2906-2914
P er m a n en tly Ch a r ged Ch ir a l 1,4-Dih yd r op yr id in es: Molecu la r P r obes of L-Typ e
Ca lciu m Ch a n n els. Syn th esis a n d P h a r m a cologica l Ch a r a cter iza tion of Meth yl
(ω-Tr im eth yla lk yla m m on iu m ) 1,4-Dih yd r o-2,6-d im eth yl-4-(3-n itr op h en yl)-3,5-
p yr id in ed ica r boxyla te Iod id e, Ca lciu m Ch a n n el An ta gon ists
Ravikumar Peri,‡,# Seetharamaiyer Padmanabhan,†,# Aleta Rutledge, Satpal Singh, and David J . Triggle*
Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo,
Buffalo, New York 14260
Received J anuary 24, 2000
We report the synthesis of the single enantiomers of permanently charged dihydropyridine
derivatives (DHPs with alkyl linker lengths of two and eight carbon atoms) and their activities
on cardiac and neuronal L-type calcium channels. Permanently charged chiral 1,4-dihydro-
pyridines and methyl (ω-trimethylalkylammonium) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-
3,5-pyridinedicarboxylate iodides were synthesized in high optical purities from (R)-(-) and
(S)-(+)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-3-pyridinecarboxylic acid,
obtained by resolution of racemic 1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-
3-pyridinecarboxylic acid. Competition binding experiments with radioligand [3H]-(+)-PN200-
110 and the block of whole cell barium currents through L-type calcium channels in GH4C1
cells show that the compounds with the eight-carbon alkyl linker optimally block the L-type
Ca2+ channels, and that the S-enantiomer is more potent than the R-enantiomer.
In tr od u ction
linked, sites on the R1 subunit of the calcium channel.
Molecular characterization of the binding sites of these
drugs is therefore of importance to understand their
mode of action.
L-type Ca2+ channels play a crucial role in many
physiologic functions, including muscle contraction,
neurosecretion, etc. These heteromultimeric proteins are
comprised of R1, â, and R2δ subunits: additionally,
skeletal muscle L-type Ca2+ channels and some neu-
ronal Ca2+ channels may uniquely express a γ subunit
(for review, see ref 1). The pore forming R1 subunit
confers basic functionality to the channel and comprises
four homologous domains (I, II, III, and IV) of six
putative transmembrane segments (S1-S6) and two
short segments SS1-SS2 between segment five and six.
Segments S5 and S6 form the pore lining and intrac-
ellular mouth of the channel, and segments SS1-SS2
form the extracellular mouth of the pore. Three different
gene products and their splice variants code for the R1
subunit of the L-type calcium channel: these are the
DHPs act both as L-type calcium channel activators
and antagonists at the DHP receptor. The differentia-
tion of the activity derives from the nature of the
functional groups substituted on the 1,4-dihydropyridine
moiety, the stereochemistry at C-4, and the membrane
potential (voltage dependence of interaction). The loca-
tion of the DHP binding site within the R1 subunit of
L-type calcium channel has been studied by three
different approaches, including the use of charged DHP
analogues,12-14 photoaffinity labeling studies,15,16 and
molecular biology approaches with chimeric channels
and site directed mutagenesis. Different approaches
have, however, localized several different regions on the
channel as sites for DHP binding. Studies using chi-
meric channels reveal the determinants of DHP binding
in the domain IIIS6 and in domain IV between segments
IVS5 and IVS6 and in segment IVS6.17-19 Site-directed
mutagenesis studies have also identified several amino
acid residues that are critical for DHP binding.20 These
studies indicate that a concerted allosteric action at
different residues is important for DHP binding.
skeletal muscle type R1S,2 the cardiac type R1C,3 and the
neuronal/neuroendocrine type R1D
4,5
.
The L-type Ca2+ channel represents a complex phar-
macophore with sites of interaction for drugs of three
different chemical categoriess1,4-dihydropyridines (DHP)
(e.g., nifedipine), phenylalkylamines (e.g., verapamil),
and the benzothiazepines (e.g., diltiazem).6-11 These
drugs are used in the treatment of cardiovascular
disorders, including hypertension, arrhythmias, angina,
and cerebral and peripheral vascular disorders. They
are known to bind to three different, but allosterically
The DHP binding site in the cardiac L-type calcium
channel has also been characterized using permanently
charged 1,4-dihydropyridine probes with the DHP phar-
macophore linked to a permanently charged quaternary
ammonium ion through a polymethylene chain of vari-
able length. The charged quaternary ammonium ion
anchors either at the cell surface or at a negatively
charged residue in the channel in the aqueous environ-
ment and permits the 1,4-DHP moiety to diffuse into
* Corresponding author address: Office of the Provost, 562 Capen
Hall, State University of NewYork at Buffalo, Buffalo, NY 14260.
‡ Current address: Rammelkamp Center for Education and Re-
search, Room R303, 2500 Metro Health Drive, Cleveland, OH 44109.
† Current address: Cambridge Neuroscience, Inc., One Kendall
Square, Bldg. #700, Cambridge, MA 02139.
# These individuals contributed dominantly and equally to this work.
10.1021/jm000028l CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/07/2000

Permanently Charged Chiral 1,4-Dihydropyridines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2907
F igu r e 2. Scheme of synthesis of optically pure permanently
charged 1,4 DHP enantiomers used in the present study.
F igu r e 1. Structures of 1,4-dihydropyridines (1,4 DHP). (A)
Bay K 8644, (B) PN 202-791, (C) racemic permanently charged
1,4 DHP analogues used in earlier experiments, (D and E)
optically pure permanently charged 1,4 DHP enantiomers used
in the present study.
The present study reports the synthesis of single
enantiomers of permanently charged DHPs. Using these
as probes we (a) verified the depth of DHP binding site
in the R1D subunit of L-type calcium channel in pituitary
GH4C1 cells and membranes, the R1C-type calcium
channels in heart membranes, and the neuronal R1D
-
the lipophilic lipid bilayer to an extent depending on
the length of the polymethylene linker. With compounds
having tether lengths of 2-16 carbon atoms, the eight-
carbon tether had optimal activity.12 Patch clamp stud-
ies in guinea pig ventricular myocytes revealed that
these compounds access the binding site from the
extracellular surface and inhibit the channel activity
depending on the length of the alkyl tether.13 These
studies suggest the DHP binding site to be 11-14 Å
from the extracellular surface of the membrane.12
Neutral DHPs show access to the binding site that is
essentially independent of the length of the carbon
tether. The activity of charged and neutral DHPs on
cardiac R1C channel, and various chimeric channels
having mutations in the putative DHP binding regions
has been reported recently.14 All of these earlier studies
were, however, carried out with racemic nifedipine
analogues on cardiac L-type calcium channels only.
These did not address the effects of permanently
charged DHPs on other L-type calcium channels, nor
did they address the stereoselectivity of interaction.
Stereoselectivity plays an important role in determin-
ing the activity of a number of drugs including
DHPs.8,11,21,22 On the basis of the stereochemistry at C-4,
DHPs can exhibit both qualitative and quantitative
differences in activity. Drugs such as BAY K-8644
[Figure 1A] exhibit qualitative differences in activity;
one enantiomer is an activator of the channel and the
other is an antagonist.23-25 The stereoisomers of some
DHPs such as PN 202-791, on the other hand, show a
more conventional quantitative difference in activity
(100-fold difference in antagonist potencies).24,26-28 It is
therefore of interest to examine the stereoselectivity of
permanently charged DHPs at the binding site.
and R1C-type channels in brain membranes; and (b)
investigated the stereoselectivity of 1,4-DHP action at
the binding site. The permanently charged 1,4-DHPs
[Figure 1] were synthesized as pure enantiomers and
were evaluated for their effect on L-type calcium chan-
nels in GH4C1 cells, cardiac tissue, and neural tissue.
Clonal pituitary GH4C1 cells exhibit both L- and T-type
calcium channels,29,30 heart expresses cardiac R1C-type
calcium channels, and brain expresses two L-type
31
calcium channels, namely the R1D and neuronal R1C
.
Resu lts
Ch em ist r y: Syn t h esis of t h e P er m a n en t ly
Ch a r ged R a cem ic a n d E n a n t iom er ic Dih yd r o-
p yr id in es. Synthesis of the permanently charged ra-
cemic and enantiomeric nifedipine analogues with a
variable carbon chain alkyl tether between the quater-
nary ammonium ion and the DHP is illustrated in the
scheme shown in [Figure 2]. It involves the preparation
of the key intermediate, dihydropyridinecarboxylic acid
(1),32 which was esterified through the acid chloride with
dimethylaminoethanol and ω-dimethylaminooctanol to
give the corresponding esters (() 2a and 2b. Subse-
quent treatment with methyl iodide gave the final
racemic quarternized salts (() 3a and 3b.
Synthesis of chiral compounds of the DHP class can
be achieved either through an asymmetric synthesis33
or through the classical method of resolution of racemic
mixtures. Optically active 1,4-dihydropyridine deriva-
tives have been synthesized from the enantiomers of the
1-ethoxymethylated carboxylic acid followed by removal
of the protecting group.34-36 The absolute configurations
of the carboxylic acid derivatives have been previously

2908 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Peri et al.
determined by X-ray crystallography.34,37 It has been
established that (+)- and (-)- have S- and R-configura-
tions at C-4, respectively. We chose the resolution
protocol of the unprotected racemic acid 132,38 using
quinidine and cinchonidine as chiral bases to give the
carboxylic acids (S)-(+)-1 and (R)-(-)-1 respectively.
The resulting enantiomeric carboxylic acids (S)-(+)-1
and (R)-(-)-1 were separately esterified with both
dimethylaminoethanol and (ω-dimethylamionooctanol)
via the acid chlorides. Subsequent quarternization of
the chiral esters with methyl iodide led to the chiral
permanently charged compounds.
Bioch em ica l Exp er im en ts. Charged DHPs were
evaluated for their ability to compete with [3H]-(+)-
PN200-110 in membrane binding assays and in whole
cell binding assays in normal resting and depolarized
conditions. These compounds were also evaluated for
their ability to block L-type calcium channels in elec-
trophysiological assays.
Bin d in g Stu d ies: Com p etition Bin d in g of [3H]-
(+)-P N200-110 w ith P er m a n en tly Ch a r ged 1,4-
Dih yd r op yr id in e An t a gon ist s (DH P ) in Tissu e
Mem br a n es. Specific binding of [3H]-(+)-PN200-110 in
rat cerebral cortex membranes [Figure 3A], heart
membranes [Figure 3B], and in GH4C1 membranes
[Figure 3C] was inhibited competitively by 1,4-dihydro-
pyridine antagonists. The KI values and the pseudo Hill
coefficients are presented in Table 1. The affinity of
charged DHP antagonists for the receptor depends on
the length of the alkyl linker and is greater for the eight-
carbon alkyl linker relative to the two-carbon alkyl
linker by a 15-170-fold factor depending on the prepa-
ration and the stereochemistry of the DHP. These
results indicate the higher accessibility of the com-
pounds with the longer chain length to the binding site
as opposed to compounds with chain length of two
carbons. These observations parallel those made previ-
ously for binding of charged DHP analogues to ven-
tricular myocytes.12 The binding affinity also exhibits
stereoselectivity and the (4S)(+)3b enantiomer is more
potent than the (4R)-(-)3b enantiomer in all the tissues
with a ratio of 37 in GH4C1 membranes, 29.2 in cerebral
cortex membranes, and 62.2 in heart membranes.
Similarly, the ratios of DHP (4S)-(+)3a and DHP (4R)-
(-)3a were 24.8 times in GH4C1 membranes, 11.2 in
cerebral cortex membranes, and 13.7 in heart mem-
branes. The potency of the racemic compound lies
between the S- and the R-enantiomers and is closer to
the S-enantiomer. The DHP (4S)-(+)3b had the highest
affinity for the heart membranes followed by GH4C1
membranes and cerebral cortex membranes. These
differences are statistically significant at p ) 0.05 in
one-way Anova test. Even though the DHP (4R)-(-)3b
seems to have the highest affinity for GH4C1 membranes
followed by the heart membranes and cerebral cortex
membranes, the results are not statistically significant.
Figure 3D gives a schematic representation of the
dissociation constants for the binding of the charged
DHPs to GH4C1 membranes, rat cerebral cortex mem-
branes, and rat heart membranes. It shows an ap-
proximate 15-170-fold difference between the dissocia-
tion constants of DHPs with eight- and two-carbon
linkers depending on the preparation, and an ap-
proximate 10-60-fold difference between the S- and
F igu r e 3. Competitive inhibition of [3H]-(+)-PN200-110 bind-
ing by permanently charged 1,4-dihydropyridine analogues (A)
to rat cerebral cortex membranes, (B) to rat heart membranes,
(C) to GH4C1 membranes, and (E) to intact GH4C1 cells under
depolarized condition (50 mM K+). (D) Comparison of dissocia-
tion constants (KI) for the charged DHP binding to the three
different membranes. (E) Comparison of dissociation constants
(KI) for the charged DHP binding to intact GH4C1 cells under
depolarized condition (50 mM K+). (n ) 4-6 experiments, error
bars represent SEM).
R-enantiomers. The lower limits of the ratios (15 and
10, respectively) may be obscured by the fact that the
KI values of (4R)-(-)3a are extrapolated values as the
IC50 value was very high and could not be reached in
the actual experiment.
Com p etition Bin d in g of [3H]-(+)-P N200-110 w ith
P er m a n en tly Ch a r ged 1,4-Dih yd r op yr id in e (DHP )
An ta gon ists in Cu ltu r ed GH4C1 Cells u n d er Dep o-
la r ized (50 m M K+) a n d Restin g Con d ition s (5.8
m M K+). Competition binding of [3H]-(+)-PN200-110 by
permanently charged 1,4-dihydropyridine antagonists
in GH4C1 cells was carried out in 5.8 mM K+ and 50
mM K+ buffer to determine the binding properties under
resting and depolarized conditions. The KI values and
the pseudo Hill coefficients are presented in Table 2.
As was described for membrane binding, the affinity of
the charged DHPs used in the experiment depends on
the length of the alkyl linker and the stereochemistry

Permanently Charged Chiral 1,4-Dihydropyridines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2909
Ta ble 1. Comparative Analysis of Inhibition of [3H]-(+)-PN200-110 Binding to Different Membrane Proteins by Permanently
Charged 1,4-Dihydropyridine Analogues
GH4C1
cerebral cortex
KI (nM)
heart
drug
KI (nM)
nH
nH
KI (nM)
nH
(4S)-(+)3b
(4R)-(-)3b
(()3b
(4S)-(+)3a
(4R)-(-)3a
(()3a
11.4 ( 3.9
420 ( 93
1.3 ( 0.2
1.2 ( 0.0
1.5 ( 0.1
0.9 ( 0.1
0.6 ( 0.1
0.9 ( 0.1
24.6 ( 2.0
717.8 ( 75.4
37.2 ( 5.3
1246 ( 293
14013 ( 2743
1372 ( 226
1.6 ( 0.2
1.0 ( 0.0
1.4 ( 0.1
0.9 ( 0.1
0.8 ( 0.0
1.0 ( 0.1
8.0 ( 1.7
1.2 ( 0.1
1.1 ( 0.0
1.1 ( 0.2
1.0 ( 0.1
0.9 ( 0.1
1.0 ( 0.1
497 ( 63.4
99.2 ( 19.2
999 ( 59
16.8 ( 5.7
1940 ( 373
48120 ( 1485
2111 ( 435
13720 ( 95.5
1512 ( 139
Ta ble 2. Comparative Analysis of Inhibition of
[3H]-(+)-PN200-110 Binding to GH4C1 Cells under Depolarized
Conditions by Permanently Charged 1,4-Dihydropyridine
Analoguesa
50 K+
drug
KI (nM)
nH
(4S)-(+)3b
(4R)-(-)3b
(()3b
(4S)-(+)3a
(4R)-(-)3a
(()3a
129.2 ( 27.8
895 ( 336
1.1 ( 0.1
1.0 ( 0.1
1.3 ( 0.1
0.9 ( 0.2
0.5 ( 0.0
1.7 ( 0.7
68.6 ( 3.6
651 ( 343
3988 ( 1752
248.5 ( 50.5
a
The compounds had a very low affinity under resting condi-
tions and no significant dose-dependent inhibition was observed.
However, there was significant inhibition at a concentration of
10 µM.
at C-4. The S-enantiomer has a higher affinity than the
R-enantiomer. Under depolarized conditions the DHP
(4S)-(+)3b was 5 times more potent than the DHP (4S)-
(+)3a and 6.9 times more potent than DHP (4R)-(-)-
3b in inhibiting [3H]-(+)-PN200-110 binding to intact
cells [Figure 3E]. This shows the requirement for
optimal access to the binding site reflected by the length
of the linker, as well as stereochemistry at C-4, to be
important determinants in the binding of DHPs to the
site on receptor. The compounds had a very low affinity
under resting conditions, and no dose response curves
could be constructed in 5.8 mM K+ buffer. However,
under resting conditions (5.8 K+), a concentration of 10-5
M of the DHP with the eight-carbon linker showed a
significant inhibition of [3H]-(+)-PN200-110 binding.
Electr oph ysiologic Stu dies: In h ibition of Bar iu m
Cu r r en ts in Cu ltu r ed GH4C1 Cells by P er m a n en tly
Ch a r ged 1,4-Dih yd r op yr id in e An ta gon ists (DHP ).
The permanently charged DHPs were also evaluated for
their functional activity in an electrophysiological assay.
The currents were elicited by either a 200 or 50 ms test
pulse to +10 mV from a holding potential of -40 mV,
and instantaneous dose response curves (DRC) of the
DHP effects were evaluated after a brief equilibration
of the cell with a given dose of DHP for 30 s. Longer
equilibration times were not used to avoid the interfer-
ence in response due to channel run down. Figure 4A
shows the effect of increasing concentrations of charged
DHPs (4S)-(+)3b, (4R)-(-)3b, and (()3b, respectively,
on the barium currents in GH4C1 cells. Figure 4B shows
the effect of increasing concentrations of charged DHPs
(4S)-(+)3a , (4R)-(-)3a , and (()3a , respectively, on the
barium currents. The DHP with the eight-carbon alkyl
linker is a more potent inhibitor of the channel activity
than the DHP with the two-carbon linker. Hill coef-
ficient signifies the stoichiometry of ligand receptor
interaction in both binding experiments as well as
functional studies. n ) 1 indicates a 1:1 interaction
under equilibrium conditions. The compounds (4S)-(+)-
F igu r e 4. Inhibition of barium currents through L-type
calcium channels in GH4C1 cells by permanently charged 1,4-
dihydropyridine analogues. Currents were elicited by depo-
larizing pulse to +10 mV from a holding potential of -40 mV.
The cell was equilibrated with different concentrations of DHP
for 30 s before the test pulse was applied. DHPs (0, 0.03, 0.1,
0.3, 1, 3 and 10 µM) with an eight-carbon linker (A) and DHPs
(0, 0.1, 1, 3 and 10 µM) with a two-carbon linker (B) were used
in the experiment. The last trace in both (A) and (B) represents
the recovery of currents after DHP wash off in external saline
at -80 mV. The recordings represent the average data from 6
to 7 cells. (C) The dose response curves for charged DHP
inhibition of barium currents. Currents were normalized to
the current before the application of drug.
3b, (4R)-(-)3b, and (()3b having an eight-carbon
linker exhibit a Hill coefficients close to 1, indicating
optimal 1:1 interaction at the DHP binding site. Shallow
numbers of less than 1 for compounds of (4S)-(+)3a ,
(4R)-(-)3a , (()3a indicate that the small tether length
of two carbons is preventing these compounds from
establishing an optimal interaction at the binding site.
It is also evident that both the S and R charged DHP
enantiomers act as neuronal/neuroendocrine L-type
calcium channel antagonists. However, the (4S)-(+)3b
enantiomer is 3.6 times more potent than the (4R)-
(-)3b enantiomer. The relative potencies of these
charged DHP antagonists on barium currents in GH4C1
cells are depicted in Table 3. The racemic compounds
seemed to be slightly more potent than the S-enanti-
omer, but the difference is not statistically significant.
The block was rapid, and no use dependence of block

2910 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Peri et al.
Ta ble 3. Comparative Analysis of Inhibition of Barium
Currents in GH4C1 Cells by Permanently Charged
1,4-Dihydropyridine Analogues
of the (4R)-(-)3b enantiomer as compared with that
of the (4S)-(+)3b enantiomer. I-V experiments were
not done for DHPs with the two-carbon alkyl linker
because they did not show a significant effect on the
currents at 1 µM concentration.
The results from electrophysiology experiments paral-
lel the effects observed in 50 K+ binding experiments.
The compounds with an eight-carbon linker were more
potent than the compounds with the two-carbon linker
and the S-enantiomer is more potent than the R-
enantiomer.
drug
IC50 (µM)
nH
(4S)-(+)3b
(4R)-(-)3b
(()3b
(4S)-(+)3a
(4R)-(-)3a
(()3a
1.4 ( 0.3
5.1 ( 1.3
1.0 ( 0.1
0.8 ( 0.2
0.9 ( 0.1
0.4 ( 0.0
0.3 ( 0.0
0.7 ( 0.2
1.5 ( 0.3
28.1 ( 4.4
270.9 ( 188.3
17.7 ( 3.9
Discu ssion
This study brings forth two important conclusions.
Our data show that the DHP binding domain is in the
membrane and is accessed optimally by charged DHPs
that have an eight-carbon alkyl linker between the
quaternary ammonium ion and the DHP, but not by
DHPs that have a two-carbon alkyl linker. This is in
agreement with previous binding12 and electrophysi-
ological studies13 and supports the hypothesis that
DHPs access the binding site from the extracellular
side.39,40 Our study further strengthens the conclusion
that the DHP binding domain is located within the
membrane, 11-14 Å from the anchor point of the
quaternary trimethylammonium headgroup. The second
conclusion from our study is that the stereochemistry
at C-4 plays an important role in determining the
potency of the charged DHP analogues that were used
to characterize the DHP binding site. The S-enantiomer
is more potent than the R-enantiomer. This is in
agreement with literature reports on studies done with
PN-200-110 and other 1,4-DHP antagonists.27 However,
qualitative differences in terms of antagonist-activator
transitions, as seen with BAY K-8644,23,26 are not seen
in these charged DHPs, and both enantiomers are
channel antagonists. Our results show also that the
charged DHP enantiomers used as probes to character-
ize the binding site exhibit differences in tissue selectiv-
ity. Binding studies on membranes show that the DHPs
used in this study had the highest affinity for the
cardiac R1C-type channels followed by the R1D-type
pituitary channels and the neuronal R1D and R1C chan-
nels [Figure 3D]. The charged DHP probes used in the
study bind to intact GH4C1 cells under depolarized
conditions with high affinity, but not to cells under
resting conditions, indicating that they exhibit voltage-
dependent block, although we did not quantify the
difference in affinities in polarized and depolarized
preparations. The difference in the potency of DHPs
between the GH4C1 membranes and intact GH4C1 cells
under depolarized conditions is probably due to several
factors, including the loss of any biochemical control
(e.g., phosphorylation) in the isolated membrane prepa-
rations. It could also be due to differences in the states
of the channel in the membrane preparation and in the
intact cells under depolarized conditions. The membrane
preparation represents 0 mV membrane potential at
which the channels are more inactivated than in the
intact GH4C1 cells under depolarized condition where
the membrane potential is between -15 and -25 mV.
F igu r e 5. 5. Inhibition of barium currents through L-type
calcium channels in GH4C1 cells. I-V plots of currents elicited
by 200 ms depolarizing steps from a holding potential of -40
mV in the presence and absence of permanently charged 1,4-
DHP with an eight-carbon alkyl linker. Control represents
currents before drug application. The cells were briefly equili-
brated with 1 µM DHP for 30 s, and I-V responses were
measured in the presence of DHP. Recovery represents the
currents elicited from -40 mV after the drug wash out for 1
min at -80 mV. The current traces as well as the I-V plot
represent averages of data from 4 to 9 cells. (A) represents
the current traces and (B) represents the I-V plots (filled
symbols indicate controls and open symbols indicate drug
effect).
was observed. The block of the pituitary L-type calcium
channels by these charged DHPs is reversible, and the
drug is partially washed out when the cells are held at
a hyperpolarizing potential of -80 mV in a stream of
drug free external solution. This is unlike the effect of
charged DHPs on the cardiac L-type calcium channels,
where the block was irreversible.13 Current-voltage
relationships (I-V plots) in the presence of 1 µM of the
DHPs with the eight-carbon linker (Figure 5A and B)
also showed a decrease in the barium currents similar
to the decrease seen in the dose response curve. These
DHPs inhibit the barium currents without significantly
altering the kinetics of the currents. The stereoselec-
tivity of action is observed as a decrease in the potency
Several studies have identified a total of nine L-type
specific and five conserved amino acid residues that
define the DHP binding site in the L-type calcium

Permanently Charged Chiral 1,4-Dihydropyridines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2911
channel.18,41 These are located in the transmembrane
regions IIIS5 (2 L-type residues), IIIS6 (3 L-type resi-
dues), and IVS6 (4 L-type residues). The effects of
racemic charged and neutral DHPs were previously
characterized on cardiac R1Cb chimeric channels.14 It
was shown that mutations in IVS6 (Tyr 1485Ile,
Met1486Phe, Ile1493Leu) lead to a marked decrease in
the effects of neutral DHPs, but not of charged DHPs.
Similarly, mutations in IIIS6 (Ile1175Phe, Ile1178Phe,
Met1183Val) lead to the loss of neutral DHP activity.
The (Thr1061Tyr) point mutation in IIIS5 leads to loss
of neutral as well as charged DHP interaction with
receptor under resting conditions, but only the charged
DHP shows a remarkably higher potency under depo-
larized conditions. Most of these residues are located
within 4-5 turns of the R helical structure. Substitution
of these nine amino acids into the DHP insensitive P/Q-
type R1A subunit, confers DHP sensitivity.42 Neutraliza-
tion of the pore region glutamate residues of the
channels abolished the spacer-chain length dependence
of charged DHPs, suggesting that the quaternary am-
monium ion might anchor at negatively charged gluta-
mate residues in the pore.43
The [R] values of 3a enantiomers (4S)-(+)3a and (4R)-(-)-
3a are in quite good agreement as expected (+72.37 and -71.1,
respectively). However, the [R] values of 3b enantiomers (4S)-
(+)3b and (4R)-(-)-3b are quite different (+3.8 and -7.77,
respectively) though the chiral shift reagent addition NMR
experiment indicated the purity to be >96% ee. The difference
in optical rotation values for the enantiomers may be due to
the accidentally trapped solvents, and this may indeed be the
reason for the disagreement of the combustion analysis values.
P r ep a r a tion of 8-(Dim eth yla m in o)-1-octa n ol Hyd r o-
br om id e. 8-Dimethylamino-1-octanol hydrobromide was pre-
pared from 8-bromooctanol and aqueous dimethylamine fol-
lowing the reported procedure.12
P r ep a r a tion of 1,4-Dih yd r o-5-m eth oxyca r bon yl-2,6-
dim eth yl-4-(3-n itr oph en yl)-3-pyr idin ecar boxylic Acid (1).
Racemic 1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-4-(3-ni-
trophenyl)-3-pyridinecarboxylic acid and the enantiomers were
prepared following the previously reported literature proce-
dure.32
Ester ifica tion of Op tica lly P u r e a n d Ra cem ic DHP
Ca r boxylic Acid s: (4S)-Dim eth yla m in oeth yl Meth yl 1,4-
Dih yd r o-2,6-d im et h yl-4-(3-n it r op h en yl)-3,5-p yr id in ed i-
ca r boxyla te (2a ). A suspension of (4R)-(-)-acid, 1 (400 mg,
1.20 mmol) in CH2Cl2 (20 mL), and DMF (2 mL) was cooled in
an ice bath to 5-10 °C, and oxalyl chloride (0.14 mL, 1.66
mmol) was added and stirred for 2-3 h. To this acid chloride
was added dimethylaminoethanol (0.12 mL, 0.12 mmol) as a
solution in CH2Cl2. The reaction mixture was stirred overnight,
diluted with CH2Cl2 (10 mL), washed with saturated NaHCO3
solution (10 mL) and brine, dried, and concentrated to give
the crude product. Purification by column chromatography
using initially CH2Cl2 and grading to CH2Cl2 containing 5%
MeOH gave the ester, (4S)-2a 336 mg, 69%; mp 114-117 °C;
1H NMR (CDCl3) δ 2.21 (s, 6 H), 2.35 (s, 6 H), 2.50 (t, 2 H),
3.62 (s, 3 H), 4.12 (t, 2 H), 5.09 (s, 1 H), 5.8 (br. s, 1 H), 7.31-
7.39 (m, 1 H), 7.63 (dd, 1 H), 7.98 (dd, 1 H), 8.12 (s, 1 H).
(4S)-(ω-Dim eth yla m in ooctyl) Meth yl 1,4-Dih yd r o-2,6-
dim eth yl-4-(3-n itr oph en yl)-3,5-pyr idin edicar boxylate (2b).
Ester (4S)-2b was prepared by the esterification of (4R)-(-)
acid 1, via the acid chloride using 8-(dimethylamino)-1-octanol
hydrobromide instead of dimethylaminoethanol in the above
procedure. Yield 39%; 1H-NMR (CDCl3) δ 1.15-1.30 (m, 8 H),
1.3-1.5 (m, 2 H), 1.5-1.65 (m, 2 H), 2.15-2.25 (m, 9 H), 2.35-
2.45 (m, 5 H), 3.6 (s, 3 H), 3.9-4.1 (m, 2 H), 5.05 (s, 1 H), 6.15
(s, 1 H), 7.3-7.45 (m, 1 H), 7.65 (dd, 1 H), 8.0 (dd, 1 H), 8.1 (s,
1 H).
In concert with previous studies, the present inves-
tigation therefore suggests that the DHP binding do-
main is in the membrane, at a distance of approximately
1/3 to 2/3 the membrane thickness from the extracellular
surface. The spacer-chain of eight carbons (11-14 Å)
from the point of anchor of charged quaternary am-
monium ion provides optimal block. The stereochemistry
at C-4 is critical to provide the right conformation for
optimal interaction of the charged DHP and the recep-
tor.
Exp er im en ta l Section
Ch em istr y. Melting points were all uncorrected and were
1
determined on a Mel-Temp apparatus. H NMR spectra were
recorded on a Varian Gemini 300 MHz instrument using TMS
as internal standard. Elemental analysis was obtained from
Atlantic Microlab, Norcross, GA. Optical rotations were mea-
sured on a Perkin-Elmer 141 polarimeter using a sodium vapor
lamp.
(4R)-Dim eth yla m in oeth yl Meth yl 1,4-Dih yd r o-2,6-d i-
m eth yl-4-(3-n itr op h en yl)-3,5-p yr id in ed ica r boxyla te (2a ).
1
Yield 68%; mp 124-126 °C; H-NMR (CDCl3) δ 2.22 (s, 6 H),
The optical purity of the series of quaternized compounds
were determined by 1H NMR by the addition of the chiral shift
reagent, tris-[3-(heptafluoropropylhydroxymethylene)-(+)cam-
phorato] europium(III) [(+)-Eu(hfc)3]. In the case of compounds
with dimethylaminooctyl side chain, (4S)-(+)-3b and (4R)-
(-)-3b, the NMR experiments were carried out in CDCl3; and
for those with dimethylaminoethyl side chain, (4S)-(+)-3a and
(4R)-(-)-3a , the optical purity estimation was carried out in
CD3CN. With (4S)-(+)-3b and (4R)-(-)-3b, 1H NMR was
carried out by addition of 20 µL portions of a solution (100 mg
in 1 mL of CDCl3) of the chiral shift reagent to a solution of
(()-3b (7 mg in 0.5 mL of CDCl3). The 1,4-dihydropyridine
C-2 and C-6 methyl resonances at δ 2.39 and 2.44 were each
split into two resonances which appeared at δ 2.56, 2.58 and
2.65, 2.67, respectively. Addition of (+)-Eu(hfc)3 to (4S)-(+)-
3b or (4R)-(-)-3b, as described above, resulted in single
resonances for the C-2 and C-6 methyl groups (ee > 96%). With
(4S)-(+)-3a and (4R)-(-)-3a , 1H NMR measurements were
carried out by addition of 50 µL portions of a solution (100 mg
in 1 mL of CD3CN and filtering the insolubles) of the chiral
shift reagent to a solution of (()-3a (5 mg in 0.5 mL of CD3-
CN). The 1,4-dihydropyridine C2-methyl resonance at δ 2.4
was split into two resonances which appeared at δ 2.48 and
2.62. Addition of (+)-Eu(hfc)3 to (4S)-(+)-3a or (4R)-(-)-3a
as described above, resulted in single resonances for the C-2
and C-6 methyl groups (ee > 96%).
2.34 (s, 6 H), 2.53 (t, 2 H), 3.63 (s, 3 H), 4.12 (t, 2 H), 5.09 (s,
1 H), 6.15 (s, 1 H), 7.33-7.39 (m, 1 H), 7.64 (dd, 1 H), 7.96
(dd, 1 H), 8.09 (s, 1 H).
(4R)-(ω-Dim eth yla m in ooctyl) Meth yl 1,4-Dih yd r o-2,6-
dim eth yl-4-(3-n itr oph en yl)-3,5-pyr idin edicar boxylate (2b).
1
Yield 37%; H-NMR (CDCl3) δ 1.23-1.45 (m, 8 H), 1.5-1.85
(m, 4 H), 2.28 (2s, 6 H), 2.45-2.75 (m, 8 H), 3.63 (s, 3 H), 3.95-
4.2 (m, 2 H), 5.07 (s, 1 H), 6.48 (s, 1 H), 7.35-7.55 (m, 1 H),
7.62 (dd, 1 H), 7.97 (dd, 1 H), 8.09 (s, 1 H).
(()-Dim eth yla m in oeth yl Meth yl 1,4-Dih yd r o-2,6-d i-
m eth yl-4-(3-n itr op h en yl)-3,5-p yr id in ed ica r boxyla te (2a ).
Yield 52%; 1H-NMR (CDCl3) δ 2.22 (s, 6 H), 2.43 (s, 6 H), 2.53
(t, 2 H), 3.63 (s, 3 H), 4.15 (t, 2 H), 5.12 (s, 1 H), 5.9 (s, 1 H),
7.32-7.42 (m, 1 H), 7.65 (dd, 1 H), 8.0 (dd, 1 H), 8.1 (s, 1 H).
(()-(ω-Dim eth yla m in ooctyl) m eth yl 1,4-d ih yd r o-2,6-
dim eth yl-4-(3-n itr oph en yl)-3,5-pyr idin edicar boxylate (2b).
Yield 65%; 1H-NMR (CDCl3) δ 1.05-1.4 (m, 8 H), 1.4-1.6 (m,
2 H), 1.65-1.8 (m, 2 H), 2.15-2.28 (m, 8 H), 2.35 (2s, 6 H),
3.65 (s, 3 H), 4.0 (m, 2 H), 5.1 (s, 1 H), 6.2 (br. s, 1 H), 7.3-
7.42 (m, 1 H), 7.65 (dd, 1 H), 7.95 (dd, 1 H), 8.1 (s, 1 H).
P r ep a r a tion of Qu a ter n ized Sa lts: (4S)-(+)-Dim eth yl-
a m in oeth yl Meth yl 1,4-Dih yd r o-2,6-d im eth yl-4-(3-n itr o-
p h en yl)-3,5-p yr id in ed ica r boxyla te Meth iod id e (3a ). To
a solution of the ester (4S)-2a (336 mg, 0.833 mmol) in acetone
(5 mL) was added methyl iodide (1 mL) while stirring for 2 h.

2912 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Peri et al.
The product separated out from the initially clear reaction
mixture solution. Filtration of the solid and washing with cold
acetone (5 mL) gave the product, (4S)-(+)-3a , 331 mg; Yield
for electrophysiology experiments. The medium was changed
every 2 days. Cells 2-6 days in culture were used for
experiments.
1
73%; mp 235-238 °C (dec); H NMR (CH3OH-d4) δ 2.27 (s, 3
Solu tion s. Normal Tyrode’s solution contained the follow-
ing (in mM): NaCl (132.0), KCl (5.8), MgCl2‚6H2O (1.2), CaCl2‚
2H2O (2.0), HEPES (10.0), and dextrose (5.0) (pH adjusted to
7.4 with NaOH). The external recording solution contained the
following (in mM): N-methyl-D-glucamine (125.0), CsCl (5.0),
HEPES (10.0), MgCl2‚6H2O (1.0), dextrose (5.0), and BaCl2
(20.0). A total of 105 mL of 1 N HCl was added per liter of
solution (pH adjusted to 7.4 with CsOH). The internal record-
ing solution contained the following (in mM): CsCl (60.0),
CaCl2‚2H2O (1.0), MgCl2‚6H2O (1.0), HEPES (10.0), EGTA
(11.0), aspartic acid (50.0), and Na2ATP (5.0) (pH was adjusted
to 7.4 with CsOH). Hank’s buffer (5.8 K+) contained the
following (in mM): NaCl (127.0), KCl (5.36), CaCl2‚2H2O
(1.26), MgCl2‚6H2O (0.98), KH2PO4 (0.44), NaHCO3 (4.16),
NaH2PO4‚7H2O (0.63), dextrose (5.56), and HEPES (20.0) (pH
adjusted to 7.4 with NaOH). Hank’s buffer (50 K+) contained
the following (in mM): NaCl (82.0), KCl (50.0), CaCl2‚2H2O
(1.26), MgCl2‚6H2O (0.98), KH2PO4 (0.44), NaHCO3 (4.16),
NaH2PO4‚7H2O (0.63), dextrose (5.56), and HEPES (20.0 (pH
adjusted to 7.4 with NaOH).
H), 2.37 (s, 3 H), 3.08 (s, 9 H), 3.66 (s, 3 H), 3.68 (t, 2 H), 4.50
(t, 2 H), 5.06 (s, 1 H), 7.42-7.48 (m, 1 H), 7.66 (dd, 1 H), 7.98
(dd, 1 H), 8.05 (s, 1 H) [R]D22 ) +72.37 (c ) 0.7, MeOH); Anal.
(C21H28IN3O6) C, H, N.
(4S)-(+)-(ω-Dim eth yla m in ooctyl) m eth yl 1,4-d ih yd r o-
2,6-d im et h yl-4-(3-n it r op h en yl)-3,5-p yr id in ed ica r b oxy-
la te m eth iod id e (3b). Yield 70%; mp 72-76 °C; 1H NMR
(CDCl3) δ 1.15-1.4 (m, 8 H), 1.45-1.65 (m, 2 H), 1.7-1.85 (m,
2 H), 2.40 (s, 3 H), 2.43 (s, 3 H), 3.52 (s, 9 H), 3.55-3.75 (s &
m, 5 H), 3.98 (m, 1 H), 4.08 (m, 1 H), 5.09 (s, 1 H), 6.43 (s, 1
H), 7.35-7.45 (m, 1 H), 7.65 (dd, 1 H), 7.98 (dd, 1 H), 8.14 (s,
1 H); [R]2D2 ) +3.8 (c ) 0.2, MeOH); Anal. (C27H40IN3O6 3.75
H2O) C, N; H: calcd. 6.82; found 5.98.
(4R)-(-)-Dim eth yla m in oeth yl Meth yl 1,4-Dih yd r o-2,6-
dim eth yl-4-(3-n itr oph en yl)-3,5-pyr idin edicar boxylate Me-
th iod id e (3a ). Yield 96%; mp 234-236 °C (dec); 1H NMR
(CH3OH-d4) δ 2.31 (s, 3 H), 2.41 (s, 3 H), 3.13 (s, 9 H), 3.65 (s,
3 H), 3.73 (t, 2 H), 4.51 (t, 2 H), 5.09 (s, 1 H), 7.48-7.53 (m, 1
H), 7.69 (dd, 1 H), 8.02 (dd, 1 H), 8.09 (s, 1 H); [R]2D2 ) -71.1
(c ) 0.5, MeOH); Anal. C21H28IN3O6 C, H, N.
(4R)-(-)-(ω-Dim eth yla m in ooctyl) Meth yl 1,4-Dih yd r o-
2,6-d im et h yl-4-(3-n it r op h en yl)-3,5-p yr id in ed ica r b oxy-
la te Meth iod id e (3b). Yield 50%; mp 74-77 °C; 1H NMR
(CDCl3) δ 1.17-1.38 (m, 8 H), 1.53-1.63 (m, 2 H), 1.75-1.85
(m, 2 H), 2.39 (s, 3 H), 2.42 (s, 3 H), 3.45 (s, 9 H), 3.55-3.7 (m,
5 H), 3.98 (m, 1 H), 4.08 (m, 1 H), 5.08 (s, 1 H), 6.49 (s, 1 H),
7.35-7.45 (m, 1 H), 7.65 (dd, 1 H), 7.98 (dd, 1 H), 8.15 (s, 1
H); [R]2D2 ) -7.77 (c ) 0.26, MeOH); Anal. (C27H40IN3O6
2H2O) C, N; H: calcd. 6.66; found 6.14.
Electr op h ysiology: Electrodes were pulled from thin-
walled borosilicate glass capillaries with an OD of 1.2 mm (TW-
120, World Precision Instruments, Sarasota, FL). The pipets
were pulled in two stages on a vertical electrode puller (David
Kopf Instruments, Model 750, Tujunga, CA) and had a
resistance of 8-10 MΩ when filled with internal recording
solution.
Whole cell voltage-clamp experiments were carried out using
an Axopatch 200 amplifier (Axon Instruments, Inc. Foster City,
CA). Voltage commands were generated on a Macintosh IIci
computer through a 12-bit digital-to-analog converter using a
MacADIOS II/16 board (GW Instruments, Somerville, MA).
Data were acquired after 16-bit analog-to-digital conversion.
Further analysis was performed with a program written in
the laboratory in Think-C (Symantec Corporation, Cupertino,
CA). Test currents were digitally sampled every 500 µs, except
during the examination of capacitive currents, when they were
sampled every 100 µs. Currents were digitally corrected for
linear leakage with respect to currents obtained at -60 mV
(()-Dim eth yla m in oeth yl Meth yl 1,4-Dih yd r o-2,6-d i-
m eth yl-4-(3-n itr op h en yl)-3,5-p yr id in ed ica r boxyla te Me-
th iod id e (3a ). Yield 63%; mp 212-216 °C; 1H NMR (DMSO-
d6) δ 2.26 (s, 3 H), 2.32 (s, 3 H), 2.99 (s, 9 H), 3.56 (s & t, 5 H),
4.40 (t, 2 H), 4.98 (s, 1 H), 7.54-7.60 (m, 1 H), 7.62 (dd, 1 H),
7.95 (s, 1 H), 7.99 (dd, 1 H), 9.21 (s, 1 H); Anal. (C21H28IN3O6)
C, H, N.
(()-(ω-Dim eth yla m in ooctyl) Meth yl 1,4-Dih yd r o-2,6-
dim eth yl-4-(3-n itr oph en yl)-3,5-pyr idin edicar boxylate Me-
1
from
a holding potential of -40 mV. Current densities,
th iod id e (3b). Yield 77%; mp 73-77 °C; H NMR (CDCl3) δ
expressed as pA/pF, were calculated by dividing the measured
current with the cell capacitance to avoid differences in current
amplitude arising from the differences in cell size. Currents
were filtered at 5 kHz, with a Lowpass Bessel filter. All
voltage-clamp traces represent average data from several
different cells. To generate the current voltage profiles (I-V
plots), current amplitudes at the peak current value were
measured for each voltage command. All data are presented
as mean values ( SEM.
Cells were rinsed with 1 mL of normal Tyrode’s solution
and bathed in 1.5-2.0 mL of the same solution until a cell
was patched and the whole cell configuration established. The
cell was then perfused with external recording solution locally
by a gravity driven perfusion system. Drug solutions were
made to a final concentration in recording solution from a 1
mM stock solution in ethanol. Whole cell barium currents were
elicited by step depolarizations to different potentials from a
holding potential of -40 mV. T-type channels present in these
cells inactivate by holding at -40 mV, and the L-type channels
can be studied selectively. The cell was allowed to equilibrate
for 30 s with each concentration of drug (ranging from 0.01-
10 µM in an increasing order), and currents were elicited at
the end of each equilibration by either a 200 or a 50 ms test
pulse to +10 mV from the holding potential of -40 mV.
Capacitative currents settled rapidly within 2 ms in these cells,
hence the first 3 ms of recording have been removed from the
current traces for clarity.
1.23-1.40 (m, 8 H), 1.5-1.62 (m, 2 H), 1.70-1.95 (m, 2 H),
2.40-2.44 (2s, 6 H), 3.45 (s, 9 H), 3.60-3.75 (m, 5 H), 3.95 (m,
1 H), 4.08 (m, 1 H), 5.08 (s, 1 H), 6.44 (s, 1 H), 7.35-7.48 (m,
1 H), 7.64 (dd, 1 H), 7.98 (dd, 1 H), 8.12 (s, 1 H); Anal. (C27H40
IN3O6 3.75 H2O) C, N; H: calcd. 6.82; found 6.12.
-
Bioch em ica l Exp er im en ts: Ma ter ia ls. Radioactive iso-
tope [3H]-(+)-PN200-110 [isopropyl-4-(2,1,3-benzoxdiazole-4-
yl)-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridine-
carboxylate] was purchased from Dupont-New England Nuclear,
Boston, MA.
F-10 medium and fetal bovine serum were purchased from
Sigma Chemical Co. (St. Louis, MO). Horse serum was
purchased from either Sigma Chemical Co. or from GIBCO
(Grand Island, NY).
Dr u gs. Nifedipine [2,6-dimethyl-3,5-dicarbomethoxy-4-(2-
nitrophenyl)-1,4-dihydropyridine] analogues, with the ester
side chain linked to a permanently charged quaternary am-
monium ion through an alkyl linker chain of either eight or
two carbons ((4S)-(+)3b, (4R)-(-)3b, (()3b, (4S)-(+)3a , (4R)-
(-)3a and (()3a ) were synthesized in our laboratory as
described. All drugs were made up as 1 mM stock solutions in
EtOH, and appropriate dilutions were made from these. Drugs
were stored protected from light.
Meth od s: Cell Cu ltu r e. The rat anterior pituitary cell
line, GH4C1, was obtained from Bayer Inc., West Haven, CT
(Dr. J ane Chisholm). Cells were maintained in a monolayer
culture in Ham’s F-10 media supplemented with 15% horse
serum and 2.5% fetal bovine serum at 37 °C in a humidified
incubator under an atmosphere containing 5% CO2. Cells were
removed from flasks once a week with 0.05% trypsin and were
plated either into flasks or sterile (Corning) 35 mm Petri dishes
Com p etition Bin d in g of 1,4-Dih yd r op yr id in e An ta go-
n ists. Mem br a n e P r ep a r a tion s. The affinities of the series
of charged 1,4-dihydropyridine antagonists were determined
in competition binding experiments using techniques in our

Permanently Charged Chiral 1,4-Dihydropyridines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2913
laboratory described previously.15,16 Experiments were done
on rat cerebral cortex membranes, rat heart membranes, and
GH4C1 membranes, and protein was determined by Bradford’s
method.44
Bin d in g in Mem br a n e P r ep a r a tion s. Competition bind-
ing assays of calcium channel antagonists with [3H]-(+)-
PN200-110 were carried out by methods previously established
in our laboratory.45,46
Bin d in g in In ta ct Cells. Competition binding experiments
were performed on intact GH4C1 cells attached to the culture
dish under resting (5.8 mM K+) as well as depolarizing (50
mM K+) conditions in Hank’s buffer (pH 7.4). These conditions
were similar to those previously established by us for binding
in intact cardiac cells.28
Da ta An a lysis. Data analysis was done using an IBM
personal computer with the program EBDA.47 The concentra-
tion for half-maximal inhibition (IC50) and the dissociation
constant (KI) for the 1,4-DHPs tested were calculated using
the Hill equation
(9) Triggle, D. J . Sites, mechanisms of action, and differentiation
of calcium channel antagonists. Am. J . Hypertension 1991, 4,
422S-429S.
(10) Triggle, D. J . Molecular pharmacology of voltage-gated calcium
channels. Ann. N.Y. Acad. Sci. 1994, 15, 267-281.
(11) Triggle, D. J . Ion channels as pharmacologic receptors: the
chirality of drug interactions. Chirality 1996, 8, 35-38.
(12) Baindur, N.; Rutledge, A.; Triggle, D. J . A homologous series of
permanently charged 1,4-dihydropyridines: novel probes de-
signed to localize drug binding sites on ion channels. J . Med.
Chem. 1993, 36, 3743-3745.
(13) Bangalore, R.; Baindur, N.; Rutledge, A.; Triggle, D. J .; Kass,
R. S. L-type calcium channels: asymmetrical intramembrane
binding domain revealed by variable length, permanently charged
1,4-dihydropyridines. Mol. Pharmacol. 1994, 46, 660-666.
(14) Lacinova, L.; An, R. H.; Xia, J .; Ito, H.; Klugbauer, N.; Triggle,
D.; Hofmann, F.; Kass, R. S. Distinctions in the molecular
determinants of charged and neutral dihydropyridine block of
L-type calcium channels. J . Pharmacol. Exp. Ther. 1999, 289,
1472-1479.
(15) Regulla, S.; Schneider, T.; Nastainczyk, W.; Meyer, H. E.;
Hofmann, F. Identification of the site of interaction of the
dihydropyridine channel blockers nitrendipine and azidopine
with the calcium-channel alpha 1 subunit. EMBO J . 1991, 10,
45-49.
B/Bmax ) 1/(1+ [x/IC50]n)
(16) Kalasz, H.; Watanabe, T.; Yabana, H.; Itagaki, K.; Naito, K.;
Nakayama, H.; Schwartz, A.; Vaghy, P. L. Identification of 1,4-
dihydropyridine binding domains within the primary structure
of the alpha 1 subunit of the skeletal muscle L-type calcium
channel. FEBS Lett. 1993, 331, 177-181.
(17) Tang, S.; Yatani, A.; Bahinski, A.; Mori, Y.; Schwartz, A.
Molecular localization of regions in the L-type calcium channel
critical for dihydropyridine action. Neuron 1993, 11, 1013-1021.
(18) Grabner, M.; Wang, Z.; Hering, S.; Striessnig, J .; Glossmann,
H. Transfer of 1,4-dihydropyridine sensitivity from L-type to
class A (BI) calcium channels. Neuron 1996, 16, 207-218.
(19) Schuster, A.; Lacinova, L.; Klugbauer, N.; Ito, H.; Birnbaumer,
L.; Hofmann, F. The IVS6 segment of the L-type calcium channel
is critical for the action of dihydropyridines and phenylalky-
lamines. EMBO J . 1996, 15, 2365-2370.
(20) Peterson, B. Z.; Tanada, T. N.; Catterall, W. A. Molecular
determinants of high affinity dihydropyridine binding in L-type
calcium channels. J . Biol. Chem. 1996, 271, 5293-5296.
(21) Triggle, D. J . On the other hand: the stereoselectivity of drug
action at ion channels. Chirality 1994, 6, 58-62.
(22) Kwon, Y. W.; Triggle, D. J . Chiral aspects of drug action at ion
channels: a commentary on the stereoselectivity of drug actions
at voltage-gated ion channels with particular reference to
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404.
where B is DPM specific bound in the presence of drug, Bmax
is DPM specific bound in the absence of any drug, x is the
concentration of the drug, IC50 is the concentration of the drug
required to inhibit the specific binding of the radioactive tracer
by 50%, and n is the slope factor (Hill coefficient). n ) 1
represents 1:1 interaction between the ligand and receptor
under equilibrium conditions.
KI ) IC50/(1+[L]/KD)
[L] is the concentration of the radioligand used, and KD is the
dissociation constant of the radioligand.
Ack n ow led gm en t. This work was supported by
grants from NIH (HL16003, GM50779), NSF (MCB-
9604457), and from Bayer Inc. (West Haven, CT). We
thank Dr. Michael Detty of the Department of Med.
Chem. at SUNY Buffalo and Dr. Anthony Williams of
Eastman Kodak, Rochester, NY, for the help with initial
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