Dihydropyridines as inhibitors of capacitative calcium entry in leukemic HL-60 cells

Article abstract of DOI:10.1016/S0006-2952(02)01488-0

A series of 1,4-dihydropyridines (DHPs) were investigated as inhibitors of capacitative calcium influx through store-operated calcium (SOC) channels. Such channels activate after ATP-elicited release of inositol trisphosphate (IP₃)-sensitive calcium stores in leukemia HL-60 cells. The most potent DHPs were those containing a 4-phenyl group with an electron-withdrawing substituent, such as m- or p-nitro- or m-trifluoromethyl (IC₅₀ values: 3-6μM). Benzyl esters, corresponding to the usual ethyl/methyl esters of the DHPs developed as L-type calcium channel blockers, retained potency at SOC channels, as did N-substituted DHPs. N-Methylation reduced by orders of magnitude the potency at L-type channels resulting in DHPs nearly equipotent at SOC and L-type channels. DHPs with N-ethyl, N-allyl, and N-propargyl groups also had similar potencies at SOC and L-type channels. Replacement of the usual 6-methyl group of DHPs with larger groups, such as cyclobutyl or phenyl, eliminated activity at the SOC channels; such DHPs instead elicited formation of inositol phosphates and release of IP₃-sensitive calcium stores. Other DHPs also caused a release of calcium stores, but usually at significantly higher concentrations than those required for the inhibition of capacitative calcium influx. Certain DHPs appeared to cause an incomplete blockade of SOC channel-dependent elevations of calcium, suggesting the presence of more than one class of such channels in HL-60 cells. N-Methylnitrendipine (IC₅₀ 2.6μM, MRS 1844) and N-propargylnifrendipine (IC₅₀ 1.7μM, MRS 1845) represent possible lead compounds for the development of selective SOC channel inhibitors.

Full text of DOI:10.1016/S0006-2952(02)01488-0

Biochemical Pharmacology 65 (2003) 329±338
Dihydropyridines as inhibitors of capacitative calcium entry in
leukemic HL-60 cells
Jacquie L. Harper, Carol S. Camerini-Otero, An-Hu Li, Soon-Ai Kim,
Kenneth A. Jacobson, John W. Daly*
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 8, Rm. 1A17,
National Institutes of Health, Bethesda, MD 20892, USA
Received 25 April 2002; accepted 13 June 2002
Abstract
A series of 1,4-dihydropyridines (DHPs) were investigated as inhibitors of capacitative calcium in¯ux through store-operated calcium
(SOC) channels. Such channels activate after ATP-elicited release of inositol trisphosphate (IP3)-sensitive calcium stores in leukemia HL-
60 cells. The most potent DHPs were those containing a 4-phenyl group with an electron-withdrawing substituent, such as m- or p-nitro- or
m-tri¯uoromethyl (IC50 values: 3±6 mM). Benzyl esters, corresponding to the usual ethyl/methyl esters of the DHPs developed as L-type
calcium channel blockers, retained potency at SOC channels, as did N-substituted DHPs. N-Methylation reduced by orders of magnitude
the potency at L-type channels resulting in DHPs nearly equipotent at SOC and L-type channels. DHPs with N-ethyl, N-allyl, and N-
propargyl groups also had similar potencies at SOC and L-type channels. Replacement of the usual 6-methyl group of DHPs with larger
groups, such as cyclobutyl or phenyl, eliminated activity at the SOC channels; such DHPs instead elicited formation of inositol phosphates
and release of IP3-sensitive calcium stores. Other DHPs also caused a release of calcium stores, but usually at signi®cantly higher
concentrations than those required for the inhibition of capacitative calcium in¯ux. Certain DHPs appeared to cause an incomplete
blockade of SOC channel-dependent elevations of calcium, suggesting the presence of more than one class of such channels in HL-60
cells. N-Methylnitrendipine (IC 2.6 mM, MRS1844) and N-propargylnifrendipine (IC 1.7 mM, MRS1845) represent possible lead
50
50
compounds for the development of selective SOC channel inhibitors.
Published by Elsevier Science Inc.
Keywords: Calcium channels; Dihydropyridines; Capacitative calcium in¯ux; Inositol trisphosphate; Store-operated calcium channels
1. Introduction
blockade, or modulation of such channels. A variety of
structurally different compounds have been reported to
cause some degree of SOC channel blockade [4±9], but
there is no clear pattern regarding the structural require-
ments for the blockade of SOC channels, nor insights into
the mechanisms involved. Many of the putative blockers of
SOC channels also cause the release of intracellular cal-
cium, rendering them unsatisfactory as speci®c probes for
SOC channels [5,7,9,10].
DHPs are known as a class of potent and selective
inhibitors of voltage-dependent L-type calcium channels
[11]. Other classes of voltage-sensitive calcium channels,
however, can be inhibited by certain DHPs [12]. In addi-
tion, certain structurally novel DHPs have proven to be
highly selective as antagonists of A3-adenosine receptors
[13±15]. DHPs, such as nicardipine, nitrendipine (6), and
nifedipine (8), have been reported to cause inhibition of
capacitative calcium in¯ux in HL-60 and U937 cells,
Cell function is regulated in a wide variety of cells by
changes in cytosolic free calcium. In non-excitable cells,
changes in intracellular calcium are often associated with
receptor-mediated release of calcium from intracellular
calcium stores and subsequent capacitative calcium entry
due to the activation of SOC channels in the plasma
membrane. The activation of SOC channels appears to
be linked to the ®lling state of the calcium stores, but
the pathways of activation and inactivation remain unclear
[1±3]. A major problem in studying SOC channels has
been the lack of selective probes for detection, activation,
* Corresponding author. Tel.: ‡1-301-496-4024; fax: ‡1-301-402-0008.
E-mail address: jdaly@nih.gov (J.W. Daly).
Abbreviations: DHPs, 1,4-dihydropyridines; IP3, inositol trisphosphate;
and SOC, store-operated calcium.
0006-2952/02/$ ± see front matter. Published by Elsevier Science Inc.
PII: S 0 0 0 6 - 2 9 5 2 ( 0 2 ) 0 1 4 8 8 - 0

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J.L. Harper et al. / Biochemical Pharmacology 65 (2003) 329±338
which lack L-type calcium channels, but high micromolar
concentrations are required [10,16]. Certain p-substituted
4-phenyl DHPs inhibit a capacitative calcium current in
skeletal muscle cells [17], while nifedipine (8), which is an
o-nitro-substituted 4-phenyl DHP, enhances the current in
muscle cells [18].
All other materials were obtained from standard commer-
cial sources.
2.2. Synthesis
The syntheses of DHPs 1±5, 7, 13±15, and 29 have been
reported previously [13,14]. The DHPs 9±12 and 16±28
were prepared in a similar manner using the Hantzsch
condensation reaction (Fig. 1, see below). Reduction of the
nitro group of DHP 7 to the amine gave DHP 10, which was
then acetylated to give DHP 12. N-Methylation with
methyl iodide of 7 and 20 gave DHPs 30 and 31, respec-
tively. Alkylation of nitrendipine (6) with methyl iodide,
ethyl iodide, allyl bromide, propargyl bromide, or benzyl
chloride gave DHPs 32, 33, 34, 35, and 36, respectively
(Fig. 1). The yield for 32 was nearly quantitative. For
structures, see Table 1.
A series of DHPs have now been assayed with HL-60
cells, which lack L-type calcium channels, to provide
insight into the structural requirements for inhibition of
the SOC channels by DHPs in this cell line. The IP3-
sensitive calcium stores were depleted in the HL-60 cells
using the receptor agonist ATP to initiate the formation of
IP3, the release of intracellular calcium, and the subsequent
activation of the SOC channels [10]. The inhibition of SOC
channels by the DHPs was determined by monitoring the
effects after ATP on intracellular calcium levels using a
¯uorescent calcium probe. Several DHPs also were tested
in GH4C1 cells, labeled with a ¯uorescent calcium probe,
for effects on calcium in¯ux through voltage-sensitive L-
type calcium channels to ascertain if there was any corre-
lation between inhibition of L-type calcium channels and
SOC channels by the DHPs.
Equimolar amounts (0.5 to 1.0 mmol) of the appropriate
b-enaminoester, aldehyde, and b-ketoester were dissolved
in 2±5 mL of absolute ethanol. The mixture was sealed in a
Pyrex tube and heated with stirring to 908 overnight. After
cooling to room temperature, the solvent was evaporated
and the product was puri®ed by preparative thin-layer
chromatography (silica 60; 1000 mm; Analtech) with pet-
roleum ether±ethyl acetate. The ratio of solvents varied for
different compounds from 1:1 to 4:1. The DHPs were
characterized by chromatographic analysis, nuclear mag-
netic resonance spectra, mass spectrometry, and, in most
cases, C, H, N analysis. Proton nuclear magnetic resonance
spectroscopy was performed on a Varian GEMINI-300
spectrometer, and all spectra were obtained in CDCl3.
Chemical-ionization (CI) mass spectrometry was per-
formed with a Finnigan 4600 mass spectrometer, and
electron-impact (EI) mass spectrometry with a VG7070F
mass spectrometer at 6 kV. Elemental analysis was per-
formed by Galbraith Laboratories, Inc. The yields were as
follows: 9, 77%; 10, 83%; 12, 71%; 16, 36%; 17, 76%; 18,
41%; 19, 92%; 20, 55%; 21, 49%; 22, 45%; 23, 20%; 24,
12%; 25, 48%; 26, 89%; 27, 58%; 28, 54%; 30, 22%; 31,
74%; 32, 95%; 33, 21%; 34, 22%; 35, 22%; and 36, 16%.
S-Ethyl 3-oxothiobutyrate, S-ethyl 3-oxothiovalerate,
benzyl 3-oxo-3-cyclopropylpropionate, and benzyl 3-
oxo-3-cyclobutylpropionate were prepared as described
[13]. 2-Hydroxyethyl acetoacetate and p-methoxybenzyl
acetoacetate were prepared, respectively, by heating
2,2,6-trimethyl-4H-1,3-dioxin-4-one and either ethylene
glycol or p-methoxybenzyl alcohol in toluene for 4 hr.
2. Materials and methods
2.1. Materials
Nitrendipine (6), nifedipine (8), diltiazem, and methox-
yverapamil (D-600) were obtained from Research Bio-
chemicals International (RBI). Dibutyryl cyclic AMP was
obtained from the Sigma Chemical Co., and ATP from
Fluka. Roswell Park Memorial Institute (RPMI) 1640
medium, F10 nutrient mixture (Ham), fetal bovine serum,
horse serum, L-glutamine, trypsin-EDTA, and penicillin/
streptomycin (10,000 units/mL of penicillin G sodium and
10,000 mg/mL of streptomycin sulfate) were purchased
from Gibco BRL, Life Technologies. Fluo3-AM, ¯uo4-
AM, and fura2-AM were from Molecular Probes, Inc.
[3H]myo-Inositol was obtained from NEN Life Science
Products. 3-Nitrobenzaldehyde, 3-tri¯uoromethylbenzal-
dehyde, ethyl 3-aminocrotonate, ethyl acetoacetate, 3-ami-
nocrotonitrile, 2-methoxyethyl acetoacetate, benzyl
acetoacetate, p-nitrobenzyl acetoacetate, ethyl 4,4,4-tri-
¯uoroacetoacetate, methyl acetoacetate, ethyl propionyla-
cetate, iodomethane, zinc, ammonium acetate, and sodium
hydride were purchased from the Aldrich Chemical Co.
Fig. 1. General procedure for synthesis of 1,4-dihydropyridines and N-alkylation.

J.L. Harper et al. / Biochemical Pharmacology 65 (2003) 329±338
331
Table 1
Inhibition of capacitative calcium in¯ux through SOC channels in HL-60 cells by 1,4-dihydropyridines
a
b
Imax (%)
Compound
R1
R2
R3
R4
R5
R6
IC
50
(mM)
1
2
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOMe
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOCH2Ph
COOEt
COOMe
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOMe
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
Ph
COOMe
COOMe
COOMe
COOEt
Me
40 Æ 8
15 Æ 6
NIc
75 Æ 6
50 Æ 6
NI
H
CH=CH±Ph
CBC±Ph
Me
3
H
Me
Me
4
H
2-Thienyl
4-NO2±Ph
3-NO2±Ph
3-NO2±Ph
2-NO2±Ph
3,5-(NO2)2±Ph
3-NH2±Ph
3-CF3±Ph
3-(NHCOMe)±Ph
4-OMe±Ph
2-CF3±Ph
3-OMe-4-OH±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
Me
NI
NI
5
H
COOEt
Me
6.5 Æ 2.8
3.3 Æ 1.2
5.0 Æ 2.1
16 Æ 2
7
70 Æ 10
80 Æ 6
90 Æ 3
75 Æ 3
95
6d
7
H
COOMe
COOEt
Me
H
Me
Me
8e
9
H
COOMe
COOEt
H
Me
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
H
COOEt
Me
NI
NI
H
COOEt
COOEt
Me
Me
2.6 Æ 0.6
NI
55 Æ 2
NI
H
H
COOMe
COOMe
COOMe
COSEt
Me
36 Æ 3
35 Æ 4
25
75 Æ 5
75 Æ 4
75
H
Me
Me
H
H
Me
8
70
H
CN
Me
NI
NI
H
COO(CH2)2OH
COO(CH2)2OMe
COOCH2Ph
COOCH2Ph
COOCH2-(4-NO2)±Ph
COOMe
COOEt
Me
Me
30
80
NDf
H
>100
1.8 Æ 0.1
3.0 Æ 1.5
NI
H
Me
95 Æ 5
97 Æ 2
NI
H
Me
H
Me
Me
H
8.0 Æ 4.0
10
50 Æ 5
55
H
Et
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
COSEt
COOEt
Et
15
NI
75
NI
H
CF3
Cyclopropyl
Cyclobutyl
Ph
H
COOCH2Ph
COOCH2Ph
COOEt
NI
NI
H
NI
NI
H
NI
NI
Me
Me
Me
Et
Allyl
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
COOMe
COOEt
Me
2.8 Æ 0.3
2.0 Æ 0.8
2.6 Æ 1.1
6
88 Æ 4
72 Æ 11
84 Æ 12
75
Me
COOMe
COOMe
COOMe
COOMe
COOMe
Me
Me
Me
6.8 Æ 1.8
1.7 Æ 1.3
28
69 Æ 7
78 Æ 12
51
Propargyl Me
Benzyl Me
Me
Me
a The IC values represent the mean concentration Æ SEM (N ˆ 3±6) required for 50% of the maximum inhibition observed for each compound, as
50
shown in Fig. 3A±C. Single values are the average of two determinations.
b Imax represents the maximum inhibition Æ SEM by the DHP of the sustained calcium levels elicited by 10 mM ATP.
c NI: no inhibition observed at concentrations up to 100 mM.
d Nitrendipine.
e Nifedipine.
f NDˆnot determined.
3,5-Dinitrobenzaldehyde was prepared by pyridinium
chlorochromate oxidation of 3,5-dinitrobenzyl alcohol.
and 2 mM L-glutamine. The HL-60 cells were differen-
tiated in supplemented RPMI 1640 medium containing
500 mM dibutyryl cyclic AMP for 48 hr prior to each
experiment. The GH4C1 cells were grown in F10 nutrient
mixture supplemented with 15% horse serum, 2.5% fetal
bovine serum, 100 units/mL of penicillin, and 100 mg/mL
of streptomycin. Prior to an experiment, the GH4C1 cells
were removed from the cell culture surface with trypsin-
EDTA (2 mL, 2 min) and suspended in 20 mL of F10
nutrient mixture.
2.3. Cell culture
Human leukemic HL-60 cells and rat pituitary GH4C1
cells were from the American Type Culture Collection.
The HL-60 cells were grown in suspension in RPMI 1640
medium supplemented with 10% fetal bovine serum,
100 units/mL of penicillin, 100 mg/mL of streptomycin,

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J.L. Harper et al. / Biochemical Pharmacology 65 (2003) 329±338
2.4. Calcium assay
vol.). The columns were washed with 10 mL of water,
followed by 10 mL of borate/formate buffer (5 mM sodium
borate, 60 mM sodium formate), and then the [3H]inositol
phosphates were eluted with 5 mL of elution buffer (0.1 M
formic acid, 0.2 M ammonium formate). Hydro¯uor (2:1,
v/v) was added to the eluent, and the radioactivity was
measured using a Beckman LS6500 Multipurpose Scin-
tillation Counter. The anion exchange columns were regen-
erated with 0.1 M formic acid, 3 M ammonium formate.
Aliquots (10±20 mL) of suspended cells were subjected
to low speed centrifugation (200 g, 258; HL-60 cells for
10 min, GH4C1 cells for 3 min), and the supernatants were
removed. The packed HL-60 cells were resuspended in 10±
20 mL of buffered medium containing 1 part supplemented
RPMI 1640 medium and 1 part Krebs±Ringer HEPES
buffer (KRH: 125 mM NaCl, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 2 mM CaCl2, 6 mM glucose, 25 mM HEPES, pH
7.4). The GH4C1 cells were resuspended in buffered saline
solution (BSS: 118 mM NaCl; 4.6 mM KCl; 10 mM glu-
cose; 20 mM HEPES; pH 7.4). To the cell suspension was
added either 10 mg ¯uo3-AM for HL-60 cells and 10 mg
fura2-AM or 30 mg ¯uo4-AM for GH4C1 cells in 10 mL
dimethyl sulfoxide. In some experiments, fura2-AM was
used for HL-60 cells. After 30±40 min in the dark at 258,
the cell suspension was centrifuged as described above and
the supernatant removed. The packed cells were resus-
pended in either KRH or BSS to give a ®nal cell count of
approximately 106/mL. A 2-mL aliquot of the dye-loaded
cells was transferred into a cuvette and stirred with a
magnetic stirrer bar throughout the entire experiment. In
most experiments with HL-60 cells, the ATP (10 mM) was
added, followed in 2 min by the DHP in dimethyl sulf-
oxide. For experiments with GH4C1 cells, 40 mM KCl was
added followed in 3 min by the DHP in dimethyl sulfoxide.
Intracellular calcium levels were monitored by ¯uorescent
spectroscopy using a PTI Deltascan Fluorescence Spectro-
photometer (Photon Technology International) set at
506 nm (excitation) and 526 nm (emission) for cells loaded
with ¯uo3 and at 494 nm (excitation) and 516 nm (emis-
sion) for cells loaded with ¯uo4. For cells loaded with
fura2, the spectrophotometer was set at 380 and 340 nm for
excitation and at 500 nm for emission. Either the ratio is
reported or the levels of intracellular calcium were calcu-
lated as described [19].
3. Results and discussion
Although a variety of SOC channel inhibitors are now
known, there has been little investigation into the structural
requirements necessary for SOC channel inhibition, and no
potent (sub-micromolar) or selective inhibitors are avail-
able. Imidazoles, such as SKF 96365 and miconazole [5±
7], tricyclics, such as tri¯uoperazine [7], and DHPs, such as
nitrendipine [10], represent distinct structural classes of
SOC channel inhibitors. Most imidazoles and tricyclics, in
addition to blocking SOC channels, cause the release of
intracellular calcium and in some cases in¯ux of calcium
[5,7,9,10]. The DHPs in the present study represent a
preliminary investigation into the structural requirements
for SOC channel inhibition by the DHPs. A typical set of
concentration-dependent inhibitions for N-methylnitrendi-
pine (32, MRS1844) is shown in Fig. 2. Representative
composite inhibition curves are shown for several DHPs in
panels A±C of Fig. 3. The inhibitory effects of the DHPs
with respect to SOC channels are summarized in Table 1.
DHP 1 with a phenyl group at the 4-position of the DHP
ring showed much greater potency than the 4-phenethynyl
DHP 3 or the 4-thienyl DHP 4, both of which were inactive
at 100 mM (Table 1). The 4-styryl DHP 2 showed some-
what greater potency than 1, but was less ef®cacious. The
introduction of a variety of substituents on the phenyl ring
resulted in a wide range of activities. DHPs 5±7, 9, and 11
with an electron-withdrawing substituent(s), such as nitro
or tri¯uoromethyl in the m- or p-position, were more potent
than DHPs 8 and 14 with an o-nitro or o-tri¯uoromethyl
substituent. DHPs 10, 13, and 15 with electron-donating
substituents were also less potent. DHP 7 with a m-nitro
group was signi®cantly more ef®cacious than DHP 11 with
a m-tri¯uoromethyl group, but both had similar potencies.
Further DHPs, namely 16±28, with a m-nitrophenyl group
at the 4-position were synthesized.
2.5. Inositol phosphate assay
[3H]Inositol (2 mCi/mL) and dibutyryl cyclic AMP were
added to the HL-60 cells 48 hr prior to each experiment.
The differentiated [3H]inositol-labeled HL-60 cells were
then centrifuged (10 min, 200 g, 258) and resuspended in
supplemented RPMI medium to give a ®nal cell concen-
tration of 106/mL. Aliquots (1 mL) of cells were incubated
after the addition of LiCl (®nal concentration, 10 mM) at
room temperature for 20 min. The agents were added at the
appropriate concentrations, and the cells were incubated
further for 10 min at room temperature. The cells were
centrifuged (10 min, 200 g, 258), and the supernatant was
removed. The cell pellet was treated with 750 mL of ice-
cold formic acid (20 mM), and the suspension was left at 48
for 30 min. The suspensions were neutralized with 60 mM
NH4OH and loaded onto anion exchange columns (Bio-
Rad AG-1-X8 Resin, 100±200 mesh, formate form, 1 mL
DHPs 20 and 21, which are benzyl esters, had inhibitory
properties similar to those of the comparable methyl/ethyl
esters 6 and 7. Exchanging an ester in 7 for a thioester in 16
had little effect on the potency, but the thioester was less
ef®cacious. DHPs with a nitrile (17) or with polar groups in
the ester moiety (18, 19) were much less potent.
DHPs having a methyl group on the ring nitrogen, as
in 30±32, were among the most potent inhibitors of
SOC channels. A series of N-substituted derivatives of

J.L. Harper et al. / Biochemical Pharmacology 65 (2003) 329±338
333
Fig. 2. Concentration-dependent inhibition by N-methylnitrendipine (32, MRS1844) of SOC channels in HL-60 cells. Intracellular calcium levels of calcium
were elevated by 10 mM ATP (first arrow) followed by DHP 32 (second arrow). (A) 0.3 mM; (B) 1 mM; (C) 3 mM; (D) 10 mM; (E) 30 mM. A representative
experiment is shown. Cells were loaded with fura2-AM. The basal levels of intracellular calcium were calculated to be about 100±150 nM (see Fig. 4).
nitrendipine (6) were synthesized. N-Methylnitrendipine
(32, MRS1844) and the N-propargyl (35, MRS1845)
analog were more potent than the N-ethyl (33) or N-allyl
(34) derivatives, while the N-benzyl (36) analog was
signi®cantly less potent than the other N-substituted DHPs.
DHPs with a cyclopropyl (27), cyclobutyl (28), or phenyl
(29) substituent at the 6-position were inactive at SOC
channels; all caused an increase in levels of intracellular
calcium whether added before or after ATP (Fig. 4A).
DHPs 28 and 29 were the most active with half-maximal

334
J.L. Harper et al. / Biochemical Pharmacology 65 (2003) 329±338
Fig. 3. Concentration-dependent inhibition by DHPs of SOC channels in HL-60 cells. Intracellular calcium levels were first elevated with 10 mM ATP,
followed by the addition of increasing concentrations of the following agents: (A) 1, 5, nitrendipine (6), and nifedipine (8). (B) 11, 13, and 21. (C) 32 and 35.
Values are means Æ SEM (N ˆ 3). (D) Structures. Curves fitted with GraphPad Prism (GraphPad Software).
stimulations at 5 mM. An increase in calcium levels was
also observed with another SOC channel-inactive DHP (3)
containing a 4-phenethynyl substituent and a methyl group
at the 6-position, but a 10-fold higher concentration was
required. The elevation of intracellular calcium elicited by
3 occurred in both the presence and absence of extracellular
calcium (Fig. 4B and D). The elevation of intracellular
calcium levels by such DHPs would appear to be associated
with the generation of inositol phosphates (Fig. 5A), which
would cause the release of calcium from IP3-sensitive
stores. The DHP-induced generation of inositol phosphates
appeared to be additive with the ATP-induced generation of

J.L. Harper et al. / Biochemical Pharmacology 65 (2003) 329±338
335
Fig. 4. Elevation of intracellular calcium levels by certain DHPs in HL-60 cells. (A) Effect of the addition of 10 mM DHP 29 after the elevation of
intracellular calcium levels by 10 mM ATP in the presence of external calcium. (B) Effect of the addition of 10 mM DHP 29 before the elevation of
intracellular calcium levels by ATP in the presence of external calcium. (C) Effect of the addition of 10 mM DHP 29 after the elevation of intracellular
calcium levels by ATP in the absence of external calcium. (D) Effect of 10 mM DHP 29 before the elevation of intracellular calcium levels by ATP in the
absence of external calcium. External calcium was removed by the addition of 2 mM EGTA to the cell suspensions 2 min prior to the addition of the agents.
Similar effects were obtained for 28, while higher concentrations of 3 and 27 were required for similar effects.
inositol phosphates (Fig. 5B). The DHPs that were the more
potent SOC channel inhibitors, such as nitrendipine (6), and
compounds 7, 11, 20, 21, 30±32, and 35 did not cause a
signi®cant generation of inositol phosphates at the concen-
trations required for the inhibition of SOC channels
(Fig. 5A). At concentrations of 30 mM or greater, most
of the DHPs caused an elevation of intracellular calcium
levels (data not shown).
Certain generalities as to structure±activity relationships
for DHPs as blockers of SOC channels in HL-60 cells are
possible. With regard to the aryl R4-substituent (see
Table 1), an electron-withdrawing group at the m-position
of the phenyl ring (5, 9, 11, 20, 21) or the p-position (5)
enhanced activity relative to that of the DHP with an
unsubstituted phenyl ring (1). The nature of the ester
moieties (COOMe, COOEt, COSEt, COOCH2Ph) at R3
and R5 (6, 7, 16, 20, 21, 23) had no or only modest effects
on activity. However, certain larger ester moieties (18, 19,
22) or a nitrile moiety (17) were not tolerated. Virtually all
the DHPs had methyl groups at R2 and R6. Replacement of
such methyl groups with ethyl groups (24) had little effect,
but replacement of one methyl with CF3, cycloalkyl, or
phenyl (26±29) eliminated activity. N-Methylation at the
R1-position of DNPs (6 ! 32, 7 ! 31) slightly increased
potency. Nitrendipines with N-ethyl, N-allyl, or N-propar-
gyl substituents (33±35) all were potent, while an N-benzyl
(36) was poorly tolerated. Most of the DHPs caused at least
a 70% apparent inhibition of the SOC channel-dependent

336
J.L. Harper et al. / Biochemical Pharmacology 65 (2003) 329±338
Fig. 5. Effect of DHPs on the generation of [3H]inositol phosphates in HL-60 cells. (A) [3H]Inositol-labeled HL-60 cells were incubated with 10 mM DHP,
and the accumulation of [3H]inositol phosphates was determined as described in ``Section 2.'' (B) Effect of 10 mM DHP 3 or 29 in combination with 10 mM
ATP on the accumulation of [3H]inositol phosphates. The generation of [3H]inositol phosphates by 10 mM ATP alone is set arbitrarily at 100%. Values are
means Æ SEM (N ˆ 3).
elevation of intracellular calcium (Table 1). However, a
few DHPs (2, 11, 23, 24, 36) caused only a maximal
apparent inhibition of 50±65%. It is possible that more
than one class of SOC channel exists in HL-60 cells and
that one subclass is poorly inhibited by certain DHPs.
Some of the DHPs alone caused signi®cant elevation of
intracellular calcium at concentrations greater than 10 mM
(data not shown), and this might result in an apparently less
than complete inhibition of SOC channels by such DHPs.
It has been suggested that the inhibitory effect of the
DHPs at SOC channels might re¯ect structural similarities
between the voltage-dependent L-type calcium channels
and the voltage-independent SOC channels [16]. Thirteen
structurally diverse DHPs were tested for inhibition of L-
type calcium channels in GH4C1 cells (Table 2). All of the
DHPs caused inhibition of L-type calcium channels. In
most instances, inhibition was observed at much lower
concentrations (1100- to 3000-fold) than what was
required for inhibition of SOC channels (Table 2). There
were some striking exceptions, namely the styryl DHP 2,
which appeared only 15-fold more potent at the L-type
channels, and the p-methoxyphenyl DHP 13, which was
only 12-fold more potent, and the N-substituted DHPs (30,
32±35), which had greatly reduced potencies as inhibitors
of L-type calcium channels. Such N-substituted DHPs each
had similar potencies at L-type channels and SOC chan-
nels. The present results indicate that DHP sites on the two
channels are quite different with respect to tolerance of
certain substituents at R4 and to N-substitution. The L-type
calcium channels are also inhibited by benzothiazepines,
such as diltiazem, and by phenylalkylamines, such as
methoxyverapamil [11], neither of which caused inhibition
of SOC channels in HL-60 cells at concentrations up to
100 mM ([5], and data not shown). Certain of the DHPs
caused a rapid but only a partial reduction of the intracel-
lular calcium in¯ux through L-type calcium channels,
suggestive of more than one subtype of such channels in
GH4C1 cells. There has been a report of both ``fast'' and
``slow'' effects of DHPs on calcium channels in such
pituitary tumor cells [20].
In conclusion, the classical DHPs, such as nitrendipine
(6) and nifedipine (8), while being SOC channel blockers,
are many fold more potent as blockers of L-type calcium
channels. The N-substituted DHPs, exempli®ed by N-
methylnitrendipine (32, MRS1844), represent lead com-
pounds for the development of selective SOC channel
blockers. However, the present N-substituted DHPs (31±
35) are not selective for SOC channels, since, although
potency at L-type channels is reduced, it is not abolished,
and the DHPs (31±35) now have comparable potencies at
the two classes of calcium channels. The N-substituted
DHPs do not elicit increases in intracellular calcium at
concentrations required to block SOC channels in HL-60
cells. Release of calcium by the classical, widely used

J.L. Harper et al. / Biochemical Pharmacology 65 (2003) 329±338
337
Table 2
Inhibition of calcium in¯ux through voltage-dependent L-type calcium channels in GH4C1 cells by DHPs: comparison to inhibition of SOC channels in HL-
60 cells
Compound
R1
R2
R3
R4
Ph
R5
R6
L-type channel
SOC
Ratio
c
channel IC (mM) SOC/L-type
50
a
Imax (%)
b
IC (mM)
50
1
2
6d
7
8e
13
20
21
30
32
33
34
35
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
COOEt
COOEt
COOEt
COOEt
COOMe
COOEt
COOEt
COOMe
Me
Me
Me
Me
Me
Me
54
0.01
1
40 Æ 8
15 Æ 6
4000
15
H
CH=CH±Ph COOMe
40
H
3-NO2±Ph
3-NO2±Ph
2-NO2±Ph
4-OMe±Ph
3-NO2±Ph
COOMe
COOEt
92 Æ 4
77
0.003 Æ 0.001
0.003
3.3 Æ 1.2
5.0 Æ 2.1
16 Æ 2
1100
1600
1600
12
H
H
COOMe
COOMe
85 Æ 3
60
0.01 Æ 0.005
3
H
36 Æ 3
H
COOCH2Ph Me
COOCH2Ph Me
55
0.001
1.8 Æ 0.1
3.0 Æ 1.5
2.8 Æ 0.3
2.6 Æ 1.1
6
1800
3000
H
COOCH2Ph 3-NO2±Ph
60
70
0.001
2
Me
Me
Et
Allyl
COOMe
COOEt
COOEt
COOEt
COOEt
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
3-NO2±Ph
COOMe
COOMe
COOMe
COOMe
COOMe
Me
Me
Me
Me
Me
1.4
86 Æ 7
82 Æ 4
72 Æ 5
86 Æ 7
1.3 Æ 0.3
2.3 Æ 0.4
3.8 Æ 1.8
2.1 Æ 0.2
2.0
2.6
1.8
0.8
6.8 Æ 1.8
Propargyl Me
1.7 Æ 1.3
a Imax represents the maximum mean inhibition Æ SEM (N ˆ 3) by the DHP of the elevation of calcium levels elicited by depolarization of the cell
membrane with 40 mM KCl. Single values are the average of two determinations.
b The IC values represent the mean concentration Æ SEM (N ˆ 3) required for 50% of the maximum inhibition observed for each compound. Single
50
values are the average of two determinations.
c The IC values are from Table 1.
50
d Nitrendipine.
e Nifedipine.
[8] Guse AH, de Wit C, Klokow T, Schweitzer K, Mayr GW. Unique
properties of the capacitative Ca2‡-entry antagonist LU 52396: its
imidazole blockers of SOC channels, such as SKF 96365,
miconazole, and clotrimazole, has represented a major
disadvantage to their use [5,7,9,10]. Thus, the N-substi-
inhibitory activity depends on the activation state of the cells. Cell
Calcium 1997;22:91±7.
tuted DHPs appear to represent at present the only class of
SOC channel blockers without the undesirable side-effect
of releasing intracellular calcium. Further structural mod-
i®cation of N-substituted DHPs hopefully will result in
even more potent and selective SOC channel blockers.
[9] Harper JL, Daly JW. Effect of calmidazolium analogs on calcium
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[10] Harper JL, Shin Y, Daly JW. Loperamide: a positive modulator for
store-operated calcium channels? Proc Natl Acad Sci USA 1997;94:
14912±7.
[11] Triggle DJ. Calcium channel drugs: structure-function relationships
and selectivity of action. J Cardiovasc Pharmacol 1991;18(Suppl 10):
S1±6.
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