Synthesis and evaluation of a new class of nifedipine analogs with T-type calcium channel blocking activity

Article abstract of DOI:10.1124/mol.61.3.649

We have synthesized a novel series of 18 dialkyl 1,4-dihydro-4-(2′alkoxy-6′-pentadecylphenyl)-2,6-dimethyl-3,5 pyddine dicarboxylates from anacardic acid, a natural compound found in cashew nut shells, and investigated their blocking action on L- and T-type calcium channels transiently expressed in tSA-201 cells. The IC₅₀ values for L-type calcium channel block obtained with the series ranged from 1 to ～40 μM, with higher affinities being favored by increasing the size of the alkoxy group on the 4-phenyl ring and ester substituent in the 3,5 positions. A detailed analysis of the strongest L-type channel blocker of the series (PPK-12) revealed that block was poorly reversible and mediated an apparent speeding of the time course of inactivation. Moreover, in the presence of PPK-12, the midpoint of the steady state inactivation curve was shifted by 20 mV toward more hyperpolarized potentials, resulting in an increase in blocking efficacy at more depolarized holding potentials. Surprisingly, PPK-12 blocked T- and L-type calcium channels with similar affinities. One of the weakest L-type channel inhibitors (PPK-5) exhibited a T-type channel affinity that was similar to that seen with PPK-12, resulting in a 40-fold selectivity of PPK-5 for T- over L-type channels. Thus, dialkyl 1,4-dihydro-4-(2′alkoxy-6′-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine dicarboxylates may serve as excellent candidates for the development of T-type calcium-channel specific blockers.

Full text of DOI:10.1124/mol.61.3.649

0026-895X/02/6103-649–658$3.00
MOLECULAR PHARMACOLOGY
Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 61:649–658, 2002
Vol. 61, No. 3
1339/964059
Printed in U.S.A.
Synthesis and Evaluation of a New Class of Nifedipine Analogs
with T-Type Calcium Channel Blocking Activity
P. PHANI KUMAR, STEPHANIE C. STOTZ, R. PARAMASHIVAPPA, AARON M. BEEDLE, GERALD W. ZAMPONI, and
A. SRINIVASA RAO
Vittal Mallya Scientific Research Foundation, Bangalore, India (P.P.K., R.P., A.S.R.); and Department of Physiology and Biophysics, University
of Calgary, Calgary, Alberta, Canada (S.C.S., A.M.B., G.W.Z.)
Received September 10; 2001; accepted November 21, 2001
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
We have synthesized a novel series of 18 dialkyl 1,4-dihydro-4- presence of PPK-12, the midpoint of the steady state inactivation
(2Јalkoxy-6Ј-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine dicar- curve was shifted by 20 mV toward more hyperpolarized poten-
boxylates from anacardic acid, a natural compound found in tials, resulting in an increase in blocking efficacy at more depolar-
cashew nut shells, and investigated their blocking action on L- and ized holding potentials. Surprisingly, PPK-12 blocked T- and L-
T-type calcium channels transiently expressed in tSA-201 cells. type calcium channels with similar affinities. One of the weakest
The IC50 values for L-type calcium channel block obtained with L-type channel inhibitors (PPK-5) exhibited a T-type channel af-
the series ranged from 1 to ϳ40 ␮M, with higher affinities being finity that was similar to that seen with PPK-12, resulting in a
favored by increasing the size of the alkoxy group on the 4-phenyl 40-fold selectivity of PPK-5 for T- over L-type channels. Thus,
ring and ester substituent in the 3,5 positions. A detailed analysis dialkyl 1,4-dihydro-4-(2Јalkoxy-6Ј-pentadecylphenyl)-2,6-dimeth-
of the strongest L-type channel blocker of the series (PPK-12) yl-3,5 pyridine dicarboxylates may serve as excellent candidates
revealed that block was poorly reversible and mediated an appar- for the development of T-type calcium-channel specific blockers.
ent speeding of the time course of inactivation. Moreover, in the
Voltage-gated calcium channels are important regulators activate and can be divided further into N-, P/Q-, R- and
of calcium influx in a number of cell types. Calcium entry L-types based on their pharmacological properties (for re-
through these channels activates a plethora of intracellular view, see Stea et al., 1995; Zamponi, 1997). Molecular cloning
events, from the broad stimulation of gene expression, calci-
um-dependent second messenger cascades, and cell prolifer-
ation to the specific release of neurotransmitter within the
nervous system and contraction in smooth and cardiac mus-
cle (Tsien et al., 1991; Wheeler et al., 1994; Dunlap et al.,
1995). A number of different types of calcium channels have
been identified in native tissues and divided based on their
biophysical profiles into low-voltage–activated (LVA) and
high-voltage–activated (HVA) channels (Nowycky et al.,
1985; Tsien et al., 1991). LVA channels first activate at
relatively hyperpolarized potentials and rapidly inactivate
(Akaike et al., 1989; Takahashi et al., 1991). By contrast,
HVA channels require stronger membrane depolarizations to
has revealed that HVA channels are heteromultimers com-
posed of a pore-forming ␣1 subunit plus ancillary ␣2-␦, ␤, and
possibly ␥ subunits (Pragnell et al., 1994; Klugbauer et al.,
1999, 2000; for review, see Catterall, 2000), whereas LVA
channels seem to contain only the ␣1 subunit (Lacinova et al.,
2000). So far, 10 different types of calcium channel ␣1 sub-
units have been identified and shown to encode the previ-
ously identified native calcium channel isoforms. Expression
studies show that alternative splicing of ␣1A generates both
P- and Q-type Ca2ϩ channels (Bourinet et al., 1999); ␣1B
encodes N-type channels (Dubel et al., 1992); ␣1C, ␣1D, and
␣1F are L-type channels (Williams et al., 1992; Bech-Hansen
et al., 1998); ␣1G, ␣1H, and ␣1I form T-type channels (i.e.,
McRory et al., 2001); ␣1E may encode R-type channels (Soong
et al., 1993; Tottene et al., 1996); and ␣1S encodes the skeletal
muscle L-type channel isoform (Tanabe et al., 1987).
Dihydropyridine (DHP) antagonists of L-type calcium
channels are widely used therapeutics in the treatment of
hypertension, angina, arrhythmias, congestive heart failure,
cardiomyopathy, atherosclerosis, and cerebral and periph-
eral vascular disorders (Janis and Triggle, 1990). The ability
of DHPs to both block and enhance native L-type calcium
This work was supported in part by an operating grant from the Heart and
Stroke Foundation of Alberta and the Northwest Territories to G.W.Z. G.W.Z.
holds faculty scholarships from the Canadian Institutes of Health Research
(CIHR), the Alberta Heritage Foundation for Medical Research (AHFMR), and
the EJLB Foundation. S.C.S. holds studentship awards from the CIHR and the
AHFMR; A.M.B. holds studentships from the AHFMR, the Natural Sciences
and Engineering Research Council of Canada, and the Neuroscience Canada
Foundation; P.P.K. holds a junior research fellowship from the Council of
Scientific and Industrial Research (India). P.P.K. and S.C.S. contributed
equally to this study.
ABBREVIATIONS: LVA, low-voltage–activated; HVA, high-voltage–activated; DHP, dihydropyridine.
649

650
Kumar et al.
currents has been well documented (Bean, 1984; Bechem and
Hoffmann, 1993; Bangalore et al., 1994; Peterson and Cat-
terall, 1995). Although they are considered to be selective
inhibitors of the L-type, a number of reports have suggested
that native T-type channels may also show sensitivity to
certain commonly used DHPs (Akaike et al., 1989; Romanin
et al., 1992; Formenti et al., 1993; Santi et al., 1996). In
Materials and Methods
Chemistry. Anacardic acid was isolated from cashew nut shell
liquid by a novel method reported by Paramashivappa et al. (2001).
As illustrated in Fig. 1, the ene mixture of anacardic acid obtained by
this method was hydrogenated using Pd/C to obtain saturated anac-
ardic acid (Fig. 1, scheme 1.1), which after alkylation with dimethyl
or diethyl sulfate using potassium carbonate gave the dialkylated
contrast, for cloned calcium channels, only ␣1S, ␣1C, and ␣1D derivative (Fig. 1, scheme 1.2). Di isopropyl anacardic acid was
obtained by using isopropyl bromide (Fig. 1 , scheme 2). Dialkylated
anacardic acid (Fig. 1, scheme 1.2) was reduced to alcohol (Fig. 1,
scheme 1.3) using lithium aluminum hydride and oxidized to corre-
sponding aldehyde (Fig. 1, scheme 1.4) using pyridinium chloro chro-
mate. The aldehyde is then converted to 1,4-dihydropyridine using
the modified Hantzsch procedure. All the 1,4-dihydro pyridines were
converted to pyridine derivatives (Yadav et al., 2000; Fig. 1, scheme
3) for further characterization of the compounds.
have been shown to be sensitive to the most commonly used
DHPs (Williams et al., 1992; Tomlinson et al., 1993; Grabner
et al., 1996; Berjukow et al., 2000; Koschak et al., 2001).
None of the remaining calcium channel subtypes cloned from
brain exhibit significant DHP sensitivity; however, informa-
tion on DHP block of cloned T-type calcium channels is still
sparse.
All of the compounds were characterized by IR, 1H-NMR, 13C-
Here, we report the calcium channel blocking action of a
novel series of DHP derivatives [dialkyl 1,4-dihydro-4- NMR, and electrospray mass spectrometry. The melting points for
all of the compounds were recorded on an electrically heated melting
point apparatus and are uncorrected. IR spectra were recorded on a
Galaxy 4020 FT-IR instrument (Mattson, Madison, WI) as KBr discs.
NMR (1H and 13C) spectra were recorded on a DPX200 (Bruker,
Fallenden, Switzerland; 40 MHz for 13C and 200 MHz for 1H), Fou-
rier transform-NMR in CDCl3 using tetramethylsilane as an inter-
nal standard. In the case of 1H NMR, typically 500 scans were
accumulated. All signals were referenced to tetramethylsilane to
within Ϯ0.01 ppm. Typically 1000–2000 scans were accumulated for
the proton noise decoupled 13C NMR spectra. TLC was done using
(2Јalkoxy-6Ј-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine di-
carboxylates] derived from anacardic acid, a phenolic constit-
uent present in cashew nut shell liquid (Paul and
Yeddanapalli, 1956). Our data show that these novel DHPs
have the propensity to mediate high-affinity inhibition of
cloned L-type calcium channels. However, more interest-
ingly, these compounds also effectively block T-type calcium
channels, in some cases with a substantial unparalleled se-
lectivity over the L-type channel.
Fig. 1. Synthesis of the series of dialkyl 1,4-dihydro-4-(2Јalkoxy-6Ј-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine dicarboxylates from anacardic acid as
described in detail under Materials and Methods section. In scheme 1, the reagents used in each step were as follows: (CH3)2SO4/K2CO3, acetone, reflux
3 h (a); LiAlH4, Tetrahydrofuran, reflux 3 h (b); PCC, dichloromethane, rt. 3 h (c); CH3COCH2COOR1/piperidine/acetic acid, n-butanol, rt. 3 h (d); and
(CH3)(NH2)C ϭ CH(COOR2), n-butanol, reflux 30 h (e).

DHP Block of T-type Channels
651
precoated silicagel GF254 plates (Merck, Darmstadt, Germany) with 5% CO2 for 1 to 3 days before recording. The cDNA constructs
hexane/EtOAc/acetic acid (7:3:0.1) as the developing solvent and encoding calcium channel ␣1C, ␣2-␦, and ␤1b subunits were kindly
visualized by UV at 254 and 360 nm. Electrospray mass spectra were
recorded on VG QUATTRO II from Micromass, UK.
donated by Dr. Terry Snutch, and the “b” splice variant of the human
construct was isolated as we have described elsewhere (A. M.
␣
1G
Preparation of Dialkyl 1,4-Dihydro-4-(2؅alkoxy-6؅-pentadecyl- Beedle, J. Hamid, and G. W. Zamponi, submitted). The electrophys-
phenyl)-2,6-dimethyl-3,5 Pyridine Dicarboxylates. 2-Ethoxy-6- iological and biophysical properties of our human ␣1G clone (i.e.,
pentadecyl-2-benzaldehyde (3 g, 8.3 mmol) and ethylacetoacetate (1.08 half-activation potential of Ϫ47 mV, half-inactivation potential of
g, 8.3 mmol) were dissolved in n-butanol (20 ml). Acetic acid (0.5 g, 8.3 Ϫ79 mV in 2 mM barium) closely parallel those described for the “b”
mmol) and piperidine (0.7 g, 8.3 mmol) were added and stirred at room splice variant by Monteil et al. (2000).
temperature for 3–4 h. Ethyl-3-amino crotonate (1.1 g, 8.3 mmol) was
Electrophysiological Recordings. Expressed calcium channels
then added and refluxed for 30 h. 2-n-Butanol was distilled off under were screened at room temperature for macroscopic currents using
vacuum and product was purified by column chromatography using an Axopatch 200B amplifier (Axon Instruments, Union City, CA)
silicagel (100–200 mesh) with hexane/EtoAc (94:6) solvent system to linked to a personal computer equipped with pCLAMP version 6.0.
give dialkyl 1,4-dihydro-4-(2Јalkoxy-6Ј-pentadecylphenyl)-2,6-dimethyl- Patch pipettes (Sutter borosilicate glass, BF150–86-15), pulled using
3,5 pyridine dicarboxylates (PPK4) as white powder (0.69 g, 14.2%).
a microelectrode puller (P87; Sutter Instruments, Novato, CA) to a
Cell Culture and Transfections. The human embryonic kidney typical resistance of ϳ4 M⍀ and fire-polished with a Narashige
tSA-201 cell line was used to transiently express cloned rat ␣1C and microforge, were used for whole-cell patch clamp recordings. Typi-
human ␣1G) calcium channels. The cells were grown in standard cally, series resistance and capacitance were compensated by at least
Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) sup- 85% to minimize contamination of records by voltage errors caused
plemented with 10% fetal bovine serum, and 0.5 mg/ml penicillin by insufficient voltage clamp. The internal pipette solution consisted
streptomycin. Once the cells were at 90% confluence, they were split of 108 mM cesium methanesulfonate, 4 mM MgCl2, 9 mM EGTA, and
with trypsin-EGTA, and plated on glass coverslips at 10% confluence 9 mM HEPES, pH 7.2 with cesium hydroxide. The external bath
12 h before transfection. Immediately before transfection, fresh me- solution consisted of 5 mM BaCl2, 1 mM MgCl2, 10 mM HEPES, 40
dia was given to the cells. A standard calcium phosphate protocol mM tetraethylammonium chloride, 10 mM glucose, and 90 mM CsCl,
was used to transiently transfect the cells with cDNA constructs pH 7.2 with tetraethylammonium hydroxide. The DHP derivatives
encoding for the calcium channel ␣1 subunit (␣1C or ␣1G) and the were solubilized in dimethyl sulfoxide to a concentration of 100 mM.
green fluorescent protein EGFP (7 and 4 ␮g of DNA, respectively). From the stock, the compounds were diluted to the appropriate
DNA constructs encoding ancillary ␤1b and ␣2-␦ subunits were added concentration in external solution. The final concentration of di-
to the ␣1C mix (7 ␮g of each DNA). After a 12-h incubation at 36°C, methyl sulfoxide in the recording solution was kept below 1:1000,
the cells were washed with fresh DMEM and allowed to recover for
which did not affect calcium channel activity. External solutions
an additional 12 h. Subsequently, the cells were incubated at 28°C in were applied to cells expressing calcium channels through a sole-
TABLE 1
Nomenclature, chemical structures, and chemical properties of the series of compounds examined in this study
Note that PPK-17 and -18 do not belong to the dihydropyridine class.
Sample Name
R
R1
R2
Melting Point
PPK-1
PPK-2
PPK-3
PPK-4
PPK-5
PPK-6
PPK-7
PPK-8
PPK-9
PPK-10
PPK-11
PPK-12
PPK-13
PPK-14
PPK-15
PPK-16
PPK-17
PPK-18
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
CH3
CH2CH3
CH3
CH2CH3
CH3
CH3
CH2CH3
CH3
CH2CH3
CH(CH3)2
CH3
CH2CH3
CH(CH3)2
CH3
CH2CH3
CH(CH3)2
CH2CH3
CH2CH3
52–54°C
42–44°C
CH2CH3
CH2CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH3
36–38°C
65–68°C
Viscous liquid
Viscous liquid
Viscous liquid
Viscous liquid
Viscous liquid
Viscous liquid
Viscous liquid
Viscous liquid
48–50°C
CH2CH3
CH2CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH2CH3
CH2CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH3
Viscous liquid
Viscous liquid
Viscous liquid
48–50°C
CH2CH3
Low melting solid

652
Kumar et al.
noid-driven perfusion system. In our experiments, rundown of ␣1C holding potential on drug affinity, the holding potential was changed
and ␣1G currents was typically minimal (Ͻ10% over the course of a to either Ϫ60 mV or Ϫ40 mV and the duration of the test potential
typical experiment), and the few cells that exhibited significant was reduced to 30 ms and 15 ms, respectively. Steady-state inacti-
rundown were discarded. To minimize the possibility of contamina- vation curves were obtained in the presence and absence of com-
tion from rundown during prolonged drug applications, cells were pounds by providing conditioning pulses between Ϫ100 mV and ϩ 20
repeatedly pulsed in the presence of 5 mM barium control solution
until a stable baseline was obtained. Only after currents had reached currents at a standard potential of 10 mV (␣1C) or Ϫ20 mV (␣1G).
equilibrium were the compounds bath-applied at the concentrations Data Analysis. All data were analyzed using Clampfit (Axon
mV in 10 mV increments for 5 s and measuring the corresponding
detailed in the figures. The time course of the effects of the various Instruments) and Sigmaplot 4.0 (Jandel Scientific, Costa Madre,
PPK-compounds was determined at a test potential of 0 mV (␣1C) or CA). Steady-state inactivation curves were fit with the Boltzmann
Ϫ20 mV (␣1G) applied every 10 s for 200 ms from a holding potential equation to obtain half inactivation potentials. The dose-response
of Ϫ80 mV. After the drug effect had stabilized, the cells were curves were fit with the Hill equation to determine the IC50 values of
washed with control external solution for up to 10 min to determine the compounds. The data are given as mean Ϯ S.E. and the numbers
whether any effects were reversible. To determine the effect of the in parentheses reflect the number of experiments per group. Stu-
dent’s t tests were carried out to ascertain the statistical significance
of the results (P Ͻ 0.05).
Results
Structure Activity Relationship of the Dialkyl 1,4-
Dihydro-4-(2؅alkoxy-6؅-pentadecylphenyl)-2,6-dimeth-
yl-3,5 Pyridine Dicarboxylates. Anacardic acid is a phe-
nolic constituent present in cashew nut shell liquid (Paul and
Yeddanapalli, 1956) and is reported to exhibit antibacterial
(Kubo et al., 1993a), antitumor (Kubo et al., 1993b) and
antiacne (Kubo et al., 1994a) properties. It is also known to
inhibit such medicinally important enzymes as prostaglan-
din synthase (Grazzini et al., 1991), lipoxygenase (Shobha et
al., 1994), and tyrosinase (Kubo et al., 1994b). We have used
saturated anacardic acid (2-hydroxy-6-pentadecyl benzoic
acid) to prepare a series of 18 novel dialkyl 1,4-dihydro-4-
(2Јalkoxy-6Ј-pentadecylphenyl)-2,6-dimethyl-3,5 pyridine di-
carboxylates (termed PPK-1 through 18; Table 1). Because
these derivatives are structurally related to DHP compounds
of the nifedipine class, we investigated their abilities to block
transiently expressed L-type calcium channels via whole-cell
patch-clamp recordings. In these experiments, cells were
held at Ϫ80 mV to approximate the membrane potential of
native excitable cells and to isolate tonic block from any
Fig. 2. Inhibitory effects of the PPK series on L-type (␣1C ϩ␣2- ␦ ϩ ␤1b
)
calcium channels transiently expressed in tSA-201 cells. Whole-cell cur-
rents were recorded in 5 mM external barium by stepping from a holding
potential of Ϫ80 mV to a test depolarization of 0 mV. The degree of block
was determined from the percentage of peak current inhibition obtained
in the presence of each of the compounds. The bars reflect means, error
bars are S.D., and numbers in parentheses indicate the number of exper-
iments.
Fig. 3. Effects of PPK-12 on
transiently expressed L-type
channels at a holding potential
of Ϫ80 mV. A, representative
time course of PPK-12 block of
transiently expressed L-type cal-
cium channels. Symbols reflect
peak current amplitudes in the
presence and absence of 10 ␮M
PPK-12. B, representative cur-
rent records obtained before and
after application of PPK-12.
Note the inhibition of peak cur-
rent amplitude and the speeding
of the rate of current decay. C,
ensemble dose-response curve
obtained from 10 experiments.
The data were fitted with the
Hill equation (solid line). The pa-
rameters obtained from the fit
were as follows; IC50 ϭ 0.54 ␮M,
nHϭ0.95). Error bars reflect S.E.
and the symbols are means of 7
to 10 independent determina-
tions of drug effect.

DHP Block of T-type Channels
653
Fig. 4. Dependence of PPK-12 block of L-type calcium channel on holding potential. A, ensemble steady-state inactivation curves obtained in the
absence (E) and presence (Ⅺ) of 1 ␮M PPK-12. The data were fitted with the Boltzmann equation (solid lines). The half inactivation potentials obtained
from the fits were Ϫ36.5 mV and Ϫ56.8 mV in the absence and the presence of 1 ␮M PPK-12, respectively. In each case, steady-state inactivation
curves in the absence and presence of PPK-12 were recorded from the same cell for a total of 10 cells. Error bars are S.E. B, dependence of
half-inactivation potential on the concentration of PPK-12. The asterisks indicate statistical significance relative to control (P Ͻ 0.05; Student’s t test).
C, dependence of the blocking effects of 300 nM PPK-12 on the holding potential. Note the significant increase in the degree of block (P Ͻ 0.05) at more
depolarized membrane potentials. The asterisks indicate significance relative to the data obtained at Ϫ80 mV (p Ͻ 0.05).
putative inactivation state dependent effects. As seen in Fig. records, significant block already occurs at 300 nM concen-
2, under these conditions, the application of a standard con- trations, whereas block is greater than half-maximal at 1 ␮M
centration for each compound (10 ␮M) results in varying PPK-12. Upon inspection of the current records, a significant
degrees of inhibition of ␣1C ϩ ␣2- ␦ ϩ ␤1b calcium channels speeding of the macroscopic time constant for current inac-
transiently expressed in tSA-201 cells ranging from about tivation becomes apparent. This could reflect additional open
25% (i.e., PPK-2) to virtually complete block (PPK-12). Ex- channel block developing during the time course of the mem-
amination of the R group structures of these compounds brane depolarization or, alternatively, a drug-induced inac-
reveals a moderate structure activity relationship: increasing tivation of the currents similar to what has been proposed to
the size of alkoxy group on 4-phenyl ring and ester substitu- occur for certain DHPs (Berjukow et al., 2000). Irrespective of
ent in the 3,5 positions increases the efficacy of these com- the mechanism, this speeding would serve to further depress
pounds, where the role of the C15 alkyl chain is to ensure calcium influx during prolonged membrane depolarization.
perpendicularity between 4-aryl and dihydropyridine rings. Figure 3C displays a complete dose-response curve for
Moreover, modification of the dihydropyridine ring of deriv- PPK-12 block obtained at a holding potential of Ϫ80 mV. The
atives with inherent symmetry to nonsymmetric analogs data are nicely described by the Michaelis-Menten equation,
seems to result in more potent block, as suggested by earlier yielding an apparent IC50 of 540 nM. For comparison, the
work (Rovnyak et al., 1992) on nifedipine analogs. Hence, IC50 obtained for nifedipine block under identical conditions
dialkyl 1,4-dihydro-4-(2Јalkoxy-6Ј-pentadecylphenyl)-2,6-di- was 170 nM (see Fig. 6), indicating that PPK-12 is only about
methyl-3,5 pyridine dicarboxylates can yield effective block- 3-fold less effective in inhibiting L-type currents compared
ers of L-type calcium channels with SAR in both the phenyl with established DHP compounds.
and DHP rings.
The blocking affinities of a number of DHPs are strongly
Analysis of PPK-12 Block of L-Type Calcium Chan- increased at more depolarized membrane potentials (i.e., Sun
nels. Figs. 3 and 4 more precisely characterize the effects of and Triggle, 1995). This strong inactivated state dependence
the most efficient blocker identified in the initial screen, of blocking action is typically reflected at the whole-cell levels
PPK-12. Figure 3A illustrates a typical time course of the as a hyperpolarizing shift in the midpoint of the steady state
effects of PPK-12 on transiently expressed L-type calcium inactivation curve. Figure 4 illustrates just such a property
channels. Block developed rapidly, but was not reversible for PPK-12. In the presence of 1 ␮M PPK-12, the steady-state
after washout, indicating either a tight interaction with the inactivation curve was shifted about 20 mV into the hyper-
blocking site, or accumulation of the compounds in the polarizing direction (Fig. 4A). This effect was observed at
plasma membrane. Figure 3B displays current records ob- concentrations as low as 300 nM PPK-12 and would predict
tained with transiently expressed L-type calcium channels in significant additional current inhibition as the membrane
the presence and the absence of PPK-12. As seen from the became more depolarized. This is shown quantitatively in

654
Kumar et al.
Fig. 5. Effects of PPK-12 on tran-
siently expressed T-type calcium
channels. A, representative time
course of peak current block in-
duced by 10 ␮M PPK-12. Note that
the inhibition is virtually complete
and not reversible after washout.
B, effect of 3 ␮M PPK-12 on whole-
cell current records elicited by
stepping from Ϫ80 mV to a test
potential of Ϫ20 mV. C, dose de-
pendence of the effects of PPK-12
on transiently expressed T-type
calcium channels. Data from 12 ex-
periments are included in the fig-
ure. Symbols reflect means from 8
to 12 individual determinations,
the solid line is a fit according to
the Hill equation (IC50 ϭ 1.65 ␮M,
nHϭ1.88), error bars reflect S.E. D,
comparison of the half-inactivation
potentials obtained in the absence
and presence of 1 ␮M PPK-12. The
asterisk indicates statistical sig-
nificance (P Ͻ 0.005, paired Stu-
dent’s t test).
Fig. 4C, where the degree of block inducing a fixed concen-
The experiments shown in Fig. 5 raise the intriguing pos-
tration of PPK-12 is examined at different holding potentials. sibility that if the drug structural requirements underlying
As seen from the figure, the degree of PPK-12 block increases block of T- and L-type calcium channels were to be distinct,
significantly such that an inhibition of greater than 50% is then members of the dialkyl 1,4-dihydro-4-(2Јalkoxy-6Ј-pen-
obtained in the presence of 300 nM PPK-12. Hence, PPK-12 tadecylphenyl)-2,6-dimethyl-3,5 pyridine dicarboxylate se-
block of L-type calcium channels can occur with submicromo- ries may offer the potential to yield a selective T-type calcium
lar affinity.
channel inhibitor. To examine this possibility, we tested the
PPK-12 and PPK-5 Block T-Type Calcium Channels. actions of one of the least effective L-type calcium channel
In native cells, a number of DHPs have been shown to exert blockers of the series (PPK-5) on transiently expressed T-
a low-affinity block of T-type calcium channels. To determine type calcium channels and compared the selectivity of this
whether this occurred for dialkyl 1,4-dihydro-4-(2Јalkoxy-6Ј- compound relative to that seen with nifedipine and PPK-12.
pentadecylphenyl)-2,6-dimethyl-3,5 pyridine dicarboxylates, Figure 6, A and B, illustrates block of T-type calcium chan-
we examined the effects of PPK-12 on ␣1G (Cav 3.1) T-type nels by PPK-5. As seen from the figure, the application of a 3
calcium channels transiently expressed in tSA-201 cells. Ap- ␮M concentration of this compound mediated a robust inhi-
plication of 3 ␮M PPK-12 mediated a dramatic reduction in bition of T-type channels that, similar to the action of PPK-
␣1G peak current amplitude without any additional speeding 12, could not be reversed with washes. The dose dependence
of the macroscopic time course of inactivation that was typ- of the PPK-5 effects indicates an IC50 of 1.14 ␮M with a Hill
ically irreversible after washout (Figs. 5, A and B). Interest- coefficient of 2.06 (Fig. 6C), the latter of which may be reflec-
ingly, the dose dependence of PPK-12 block of ␣1G was sur- tive of additional effects on steady-state inactivation, like the
prisingly steep (nHϭ1.88; Fig. 5C). Given the negative ones seen with PPK-12. Hence, despite the dramatic reduc-
inactivation range of T-type calcium channels and our stan- tion in L-type calcium channel blocking efficacy seen with
dard holding potential of Ϫ80 mV, a drug induced hyperpo- PPK-5 compared with PPK-12, the abilities of PPK-5 and
larizing shift in the midpoint of the steady-state inactivation PPK-12 to inhibit T-type calcium channels are remarkably
curve could result in additional inhibition, thus increasing similar. The relative selectivities of PPK-5 and PPK-12 for L-
the slope of the dose-response curve. To examine this possi- and T-type calcium channels are illustrated in Fig. 6D in
bility, we assessed the effects of PPK-12 on half-inactivation comparison with nifedipine. Whereas nifedipine blocked L-
potential. As seen in Fig. 5D, 1 ␮M PPK-12 resulted in a type channels more effectively than T-type channels by al-
small (ϳ5 mV) but statistically significant (p Ͻ 0.05, paired most 3 orders of magnitude, PPK-12 blocked both channel
t test) hyperpolarizing shift in the midpoint of the steady types with comparable affinities, and PPK-5 exhibited a 40-
state inactivation curve (Fig. 5D). Although the magnitude of fold selectivity for T-type over L-type channels. Thus, L-type
this effect did not approach that seen with the L-type calcium and T-type calcium channels require distinct drug structural
channel isoform, it may contribute to the shape of the dose- requirements for effective DHP block.
response curve shown in Fig. 5C. Overall, these data indicate
that at hyperpolarized membrane potentials, PPK-12 blocks overall structural similarity between our series of compounds
L- and T-type calcium channels with almost equal efficacy. and nifedipine, it is possible that nifedipine may bind to
Nifedipine Antagonizes PPK-5 Action. In view of the

DHP Block of T-type Channels
655
T-type calcium channels without significantly affecting cur- dimethyl-3,5 pyridine dicarboxylates starting from saturated
rent activity. To examine this possibility, we compared the
time course of development of PPK-5 block with and without
prior application of 30 ␮M nifedipine. Figure 7 shows that
although channels that were exposed to nifedipine could still
be completely blocked by 3 ␮M PPK-5, the time constant for
development of PPK-5 block was slowed 2-fold [from 70.6 Ϯ
11.6 s (n ϭ 5) to 138.9 Ϯ 12.6 s (n ϭ 4), p Ͻ 0.05]. The
simplest explanation for this effect is that nifedipine may
indeed occupy part of the PPK-5 binding site without actually
blocking current activity and that for PPK-5 to mediate its
blocking effects, nifedipine may have to first dissociate from
the channel. If so, these would suggest that T-type calcium
channels contain a binding pocket for the DHP pharmacoph-
ore, but that channel block requires the presence of specific R
groups on the DHP molecule.
anacardic acid (2-hydroxy-6-pentadecyl benzoic acid). These
compounds are structurally related to nifedipine (see Table
1), and it is thus not surprising that they are able to inhibit
L-type calcium channel activity, albeit to a somewhat lesser
extent compared with nifedipine per se. The most efficacious
L-type channel blocker of the series, PPK-12, showed several
similarities to nifedipine action: first, like nifedipine, PPK-12
accelerated the time course of current decay (Lee and Tsien,
1983), which may reflect either a true effect on inactivation of
the channel (Berjukow et al., 2000) or additional open chan-
nel block that develops during the test depolarization. Sec-
ond, like nifedipine and several other DHPs (Lee and Tsien,
1983; Shen et al., 2000), PPK-12 mediated a robust shift in
the midpoint of the steady-state inactivation curve toward
more hyperpolarized potentials, indicative of strong inacti-
vated channel block. Unlike nifedipine, however, the action
of PPK-12 or any of the other derivatives examined, could not
be reversed during the time course of a typical experiment. In
view of evidence that DHPs must at least partially partition
into the plasma membrane to mediate L-type channel block
(Kass et al., 1991; Strubing et al., 1993; Bangalore et al.,
1994), it is possible that the added hydrophobicity arising
from the pentadecyl side chain results in accumulation of
these compounds in the plasma membrane, thus precluding
rapid reversal of the blocking action.
Discussion
Comparison with Previous Work. Of the DHP blockers,
dialkyl 1,4-dihydro-4-aryl-2,6-dimethyl-3,5-pyridine dicar-
boxylates of the nifedipine class have been found to offer
longer bioavailability and greater tissue selectivity (Gold-
mann and Stoltefuss, 1991). The superior blocking efficacy of
this class of compounds containing bulky ortho substituents
on the 4-aryl ring has been attributed to a forced perpendic-
ular orientation between 4-aryl ring and 1,4-dihydropyridine
ring (Loev et al., 1974). Although 4-aryl(2Ј,6Ј-di substitut-
ed)1,4 DHPs were predicted to possess high affinity for the
L-type calcium channel (Loev et al., 1974; Coburn et al.,
1988), these derivatives received less attention because of
low product yield obtained even with modified Hantzsch syn-
thesis (Loev et al., 1974). For this study, we prepared a series
In our experiments, nifedipine only weakly inhibited T-
type calcium channel activity. This is consistent with a re-
cent report by Lacinova et al. (2000), who reported a 14%
inhibition of ␣1G current activity by 10 ␮M nifedipine. These
authors also found a weak dependence of nifedipine blocking
affinity on holding potential. Although we did not examine
of dialkyl 1,4-dihydro-4-(2Јalkoxy-6Ј-pentadecylphenyl)-2,6- this property for nifedipine, PPK-12 mediated a small but
Fig. 6. Block and selectivity
of PPK-5 for T-type calcium
channels. A, representative
time course of block of T-
type calcium channel by 3
␮M PPK-5. Note the lack of
reversibility. B, current
records obtained in the ab-
sence and presence of
PPK-5 under the same con-
ditions as those in Fig. 5B.
C, dose dependence of
PPK-5 action on transiently
expressed T-type channels
obtained from 11 different
cells. The experimental con-
ditions were the same as in
Fig. 5C, the IC50 and Hill
coefficient obtained from
the fit were 1.14 ␮M and
2.06, respectively. D, rela-
tive selectivities of nifedi-
pine, PPK-12, and PPK-5
among L- and T-type cal-
cium channels. Note that
the IC50 values shown on
the ordinate are on a loga-
rithmic scale. Error bars
are S.E., numbers in paren-
theses reflect the number of
cells used to calculate the
means.

656
Kumar et al.
statistically significant leftward shift in the position of the site on the ␣1G subunit. Moreover, it will be interesting to
steady-state inactivation curve toward more hyperpolarizing examine the abilities of these compounds to inhibit other
potentials, which is qualitatively consistent with the results T-type calcium channel isoforms such as ␣1H and ␣1I (i.e.,
of Lacinova et al. (2000).
Mechanism of T-Type Calcium Channel Block. Per- is conserved across all members of the T-type channel family.
haps the most striking result from our study is the strong Yet qualitative features of this site can be inferred from the
McRory et al., 2001) to determine whether this site of action
inhibition of T-type calcium channel activity by PPK-5 and observation that nifedipine, although on its own being able to
PPK-12. Moreover, the 40-fold selectivity of PPK-5 for T-type mediate only a small reduction in the peak current amplitude
over L-type calcium channels is particularly interesting, al- at 30 ␮M concentrations, antagonized PPK-5 block of ␣1G
though one must be aware that this value was obtained at a channels. If this concentration of nifedipine were to act on
holding potential of Ϫ80 mV, where little inactivated channel only a fraction of T-type channels in a given cell, then the
block of the L-type isoform is expected. A large (ϳ20 mV) remaining (nifedipine-free) channels would be expected to
hyperpolarizing shift in the midpoint of the voltage depen- respond normally to the application of PPK-5. Instead, the
dence of L-type channel inactivation (such as that observed time course of development of PPK-5 block was slowed dra-
with PPK-12, Fig. 4A) would be expected to reduce the degree matically in channels pretreated with nifedipine. It is un-
of selectivity for T-type channels at very depolarized holding likely that this effect is caused by a perfusion artifact, be-
potentials; at membrane potentials more negative than Ϫ40 cause our microperfusion system achieves complete solution
mV, however, significant T-type channel selectivity would exchanges in less than 1 s and is thus much faster than the
remain (for example, the apparent blocking affinity for time course of development of block observed in our experi-
PPK-12 increased by less than 5-fold when the holding po- ments. We also do not expect the formation of inactive nifed-
tential was switched from Ϫ80 mV to Ϫ40 mV).
ipine-PPK-5 complexes, because PPK-5 was not applied as
The DHP receptor site in the L-type calcium channel com- part of a nifedipine-PPK-5 mixture. Thus, we conclude that
prises as few as nine single amino acid residues in the do- nifedipine is able to bind to a DHP interaction site on the
main IIIS5, IIIS6, and IVS6 regions of the ␣1C subunit (Ito et T-type calcium channel molecule without significantly inhib-
al., 1997; Yamaguchi et al., 2000; Wappl et al., 2001). These iting current flux. By doing so, nifedipine might perhaps
residues are not conserved in T-type calcium channels, sug- allosterically reduce the affinity of the channel for PPK-5
gesting that the DHP blocking site on the latter channels is acting at a distinct site. However, given the structural ho-
of a fundamentally different nature. This is also consistent mology between nifedipine and PPK-5, we favor a model in
with our observation that PPK-12 mediated much more pro- which the two compounds compete for the same site, but
nounced effects on the steady-state inactivation behavior of because of its bulkier substituents, PPK-5 is able to effec-
L-type channels compared with the T type. Without molecu- tively block channel activity whereas nifedipine is not. For
lar biological or biochemical studies, however, it is difficult PPK-5 to be able to occupy the blocking site, nifedipine would
even to speculate on a possible location of the DHP blocking then have to first dissociate from the channel, thereby ac-
Fig. 7. Slowing of the develop-
ment of PPK-5 block of T-type
calcium channels by prior
application of nifedipine. A,
whole-cell current records ob-
tained from ␣1G calcium chan-
nels with (right) and without
(left) prior application of 30
␮M nifedipine (for 3 min), mea-
sured 90 s after application of 3
␮M PPK-5. The currents ob-
tained in the absence of PPK-5
were normalized relative to
each other to facilitate compar-
ison of the PPK-5 effect. Note
that in the absence of pretreat-
ment with nifedipine, a 90-s
application of PPK-5 mediates
65% block, whereas the same
exposure in cells pretreated
with nifedipine results in
a
much smaller degree of block
(ϳ20%), and longer exposure
times are required achieve a
comparable level of inhibition
(in this example, 170 s is re-
quired for 3 ␮M PPK-5 to block
70% of the current). B, quanti-
tative comparison of the effects
illustrated in A. Note that ni-
fedipine
dramatically
in-
creases the time constant for
development of PPK-5 block. A
total of five experiments is in-
cluded in the figure.

DHP Block of T-type Channels
657
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We thank Dr. Terry Snutch (The University of British Columbia,
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