Stereoselective behavior of the functional diltiazem analogue 1-[(4-chlorophenyl)sulfonyl]-2-(2-thienyl)pyrrolidine, a new L-type calcium channel blocker

Article abstract of DOI:10.1021/jm9008696

We studied the stereoselective behavior of 1-[(4-chlorophenyl)sulfonyl]-2- (2-thienyl)pyrrolidine, a recently described blocker of cardiovascular L-type calcium channels that binds to the diltiazem site. Given the stereocenter at C-2 of the pyrrolidine ri

Full text of DOI:10.1021/jm9008696

J. Med. Chem. 2009, 52, 6637–6648 6637
DOI: 10.1021/jm9008696
Stereoselective Behavior of the Functional Diltiazem Analogue
1-[(4-Chlorophenyl)sulfonyl]-2-(2-thienyl)pyrrolidine, a New L-Type Calcium Channel Blocker
Emanuele Carosati,† Roberta Budriesi,*,‡ Pierfranco Ioan,*,‡ Gabriele Cruciani,† Fabio Fusi,§ Maria Frosini,§
Simona Saponara,§ Francesco Gasparrini, Alessia Ciogli, Claudio Villani, Philip J. Stephens,^ Frank J. Devlin,^
Domenico Spinelli,# and Alberto Chiarini‡
†
‡
Dipartimento di Chimica, Universita degli Studi di Perugia, Via Elce di Sotto 10, 06123 Perugia, Italy, Dipartimento di Scienze Farmaceutiche,
ꢀ
Universita degli Studi di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Dipartimento di Neuroscienze, Universita degli Studi di Siena, Via A.
§
ꢀ
Moro 2, 53100 Siena, Italy, Dipartimento di Chimica e Tecnologie del Farmaco, Universita “La Sapienza”, Piazzale A. Moro 5, 00185 Roma,
ꢀ
ꢀ
Italy, ^Department of Chemistry, University of Southern California, Los Angeles, California 90089-0482, and #Dipartimento di Chimica
ꢀ
“G. Ciamician”, Universita degli Studi di Bologna, Via Selmi 2, 40126 Bologna, Italy
Received June 15, 2009
We studied the stereoselective behavior of 1-[(4-chlorophenyl)sulfonyl]-2-(2-thienyl)pyrrolidine, a
recently described blocker of cardiovascular L-type calcium channels that binds to the diltiazem site.
Given the stereocenter at C-2 of the pyrrolidine ring, the two enantiomers were separated by chiral
HPLC and, using VCD in conjunction with DFT calculations of chiroptical properties, the absolute
configuration was assigned as R-(þ)/S-(-). For both forms, functional, electrophysiological, and
binding properties were studied and the three-dimensional superimpositions of the two enantiomers
over diltiazem were obtained in silico. The significant differences observed for the two enantiomers well
agreed with the experimental data, and molecular regions were hypothesized as responsible for the
cardiac stereoselectivity and vascular stereospecificity.
Introduction
molecules with LTCC-blocking activity. In this context, we
first optimized the inotropic activity of a series of thiazinoox-
adiazolones,2,3 then recently developed a ligand-based virtual
screening (LBVS) procedure that provided some interesting
hits2 that are structurally different from the starting com-
pound. The R-(-) form of 8-(4-bromophenyl)-8-ethoxy-
5-methyl-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3-one (2, in
previous papers named 5b)4 (Chart 1) was the reference
compound used in the cited LBVS procedure.4 It was used as
reference because of its selective and relevant negative inotro-
pic activity, enantiospecificity, and binding to the diltiazem site
of LTCCs. With the LBVS only 20 molecules were selected and
The search for new chemotypes for any pharmacological
target requires the high- or low-throughput analysis of large
private or commercial libraries, followed by biological testing.
Today an ever increasing interest exists in computational
strategies for enhancing the enrichment capability of such
preliminary screening, in other words for finding molecules
with the desired pharmacological activity. These are usually
referred to as hits. For this reason both industrial and aca-
demic research laboratories routinely use cost- and time-saving
in silico approaches, such as virtual screening, to search
through large chemical databases1 with the aim of finding
new chemotypes and/or to identify new chemical groups
related to the desired biological activity in order to design
more potent and selective molecules.
Chart 1
Our group is involved in the research field of L-type calcium
channels (LTCCsa) blockers, with a special interest in the
diltiazem (1) binding site. The synergic application of many
different techniques is directed toward the discovery of new
*To whom correspondence should be addressed. For R.B.: phone,
þ39-051-2099737; fax, þ39-051-2099721; e-mail, roberta.budriesi@
unibo.it. For P.I.: phone, þ39-051-2099737; fax, þ39-051-2099721;
e-mail, pierfranco.ioan3@unibo.it.
aAbbreviations: AC, absolute configuration; B3PW91/TZ2P, meth-
od to calculate the vibrational circular dichroism spectra; BTZ, ben-
zothiazepine; CD, circular dichroism; DFT, density functional theory;
DTZ, diltiazem; ECD, electronic circular dichroism; FLAP, fingerprint
for ligands and proteins; GPILSM, guinea-pig ileum longitudinal
smooth muscle; HPLC, high performance liquid chromatography;
LBVS, ligand based virtual screening; LCS, low Ca2þ solution; LTCC,
L-type calcium channel; MIF, molecular interaction fields; OR, optical
rotation; PSS, physiological salt solution; rmsd, root mean square
deviation; SAR, structure-activity relationship; VCD, vibrational cir-
cular dichroism.
r
2009 American Chemical Society
Published on Web 10/15/2009
pubs.acs.org/jmc

6638 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Carosati et al.
Table 1. Functional Activity of Tested Compounds
cardiac activity
% decrease (M ( SEM)
negative negative
inotropic chronotropic
EC50 of inotropic negative activity
vasorelaxant activity
95% conf
GPILSM relaxant activity
95% conf
lim
(ꢀ10-6)
c
c
c
EC50
(μM)
95% conf
lim (ꢀ10-6)
activityd
IC50
lim
(ꢀ10-6)
activitye
IC50
compd
activitya
activityb
(M ( SEM) (μM)
(M ( SEM) (μM)
1f
R-(-)-2
78 ( 3.5
94 ( 5.6g
16 ( 0.8
4 ( 0.2k
5 ( 0.4k
5 ( 0.3k
0.79
0.07
0.31
1.33
0.31
0.70-0.85
0.04-0.08
0.22-0.42
1.04-1.70
0.24-0.39
88 ( 2.3
11 ( 0.7k
74 ( 2.1
57 ( 1.6
54 ( 3.1j
2.6
2.2-3.1
98 ( 1.5h
3 ( 0.2k
90 ( 0.7
83 ( 2.6i
83 ( 1.7
0.11 0.085-0.13
58 ( 3.4h
(R,S)-(()-3 84 ( 1.6g
5.62 4.46-7.08
25.1 15.3-32.0
17.7 14.3-21.7
1.18 0.84-1.67
14.77 11.1-19.7
4.32 3.65-5.11
4
92 ( 0.9
5
73 ( 1.4h
a Activity: decrease in developed tension in isolated guinea pig left atrium at 10-5 M, expressed as percent changes from the control (n = 4-6). The left
atria were driven at 1 Hz. The 10-5 M concentration gave the maximum effect for most compounds. b Activity: decrease in atrial rate in guinea pig
spontaneously beating isolated right atria at 10-4 M, expressed as percent changes from the control (n = 6-8). Pretreatment heart rate ranged from 170
to 195 beats/min. The 10-4 M concentration gave the maximum effect for most compounds. c Calculated from log concentration-response curves
(Probit analysis according to Litchfield and Wilcoxon with n = 6-8).10 When the maximum effect was <50%, the EC50 inotropic, EC30 chronotropic, and IC50
values were not calculated. d Activity: percent inhibition of calcium-induced contraction in 80 mM Kþ-depolarized guinea pig aortic strip at 10-4 M. The 10-4
M
concentration gave the maximum effect for most compounds. e Percent inhibition of calcium-induced contraction in 80 mM Kþ-depolarized guinea pig
longitudinal smooth muscle (GPILSM) at 10-5 M. The 10-5 M concentration gave the maximum effect for most compounds. f EC30 = 0.07 (confidence limit
0.064-0.075). g At 5 ꢀ 10-6 M. h At 10-6 M. i At 5 ꢀ 10-5 M. j At 5 ꢀ 10-4 M. k P < 0.05.
biologically tested, and seven hits were found to be very
promising for their LTCCs negative inotropic activity; further-
more, few vasorelaxant compounds were found as well.
On one hand, the negative inotropic activity refers to the
decrease of myocardial contractility, and compounds with
such activity have clinical application to decreasing the car-
diac workload in conditions such as hypertrophic obstructive
cardiomyopathy.5 A vasorelaxant compound, on the other
hand, is a drugthatrelaxes the smoothmuscleinblood vessels,
thus being useful in pathologies such as hypertension.6
Interestingly, the selective negative inotropic versus chron-
otropic and vasorelaxant activity, observed for the reference
compound 2, was found in many but not all screened hits.
In particular, three other compounds were shown to
exhibit vasorelaxant properties; in addition to (()-1-[(4-
chlorophenyl)sulfonyl]-2-(2-thienyl)pyrrolidine, ((()-3, in a
previous paper named as (()-M8),4 the most interesting
among the three,2 there were 1-[(4-chlorophenyl)sulfonyl]-
2-(methylthio)-1,4,5,6-tetrahydropyrimidine (4, previously
named as M4)4 and N-allyl-4-chloro-N-phenylbenzenesulfo-
namide (5, previously named as B3).4
The further investigation of some of the hits found by virtual
screening is inserted within our framework of analysis7 and
search for new chemotypes.4,8 The present study was carried
out to explore the chemical space around the thiazinooxadia-
zolone and benzothiazepine (BTZ) scaffolds4,7 and to obtain
information on variations of structural features responsible for
LTCC blocking activities. In particular, these three com-
pounds (3, 4, and 5) possess a common p-chlorobenzensulfo-
namidic scaffold that is differently substituted at the nitrogen,
which for 4 is part of a six-membered ring (the tetrahydropyr-
imidin-1-yl residue), for 5 is bisubstituted with phenyl and allyl
groups, and for (()-3 is part of a five-membered ring (the
pyrrolidin-1-yl residue). Considering the chiral center in the
pyrrolidine ring of 3, the two enantiomers were considered
separately,9 and a stereoselective behavior was observed.
circular dichroism (VCD) in conjunction with density func-
tional theory (DFT) calculations of their predicted chiroptical
properties. Then functional, electrophysiological, and binding
properties were studied for both R and S enantiomers.
Functional Characterization and Binding Profile. The effect
of compounds 4 and 5 on the longitudinal smooth muscle
was measured as detailed in the Experimental Section. The
functional data of compounds 4 and 5, expressed as intrinsic
activity and potency values on 1 Hz driven left and sponta-
neously beating right atria and in 80 mM KCl depolarized
vascular and nonvascular smooth muscle, are reported in
Table 1 with those of references 1 and R-(-)-2 as well as of
compound (()-3.
Compounds (()-3, 4, and 5 revealed intrinsic negative
inotropic activity without chronotropic effect. In particular,
compounds (()-3 and 5 were equipotent, weaker than R-(-)-2
but 2.5-fold more potent than 1, while compound 4 was less
effective. Concerning the activity on smooth muscles, com-
pounds (()-3, 4, and 5 showed profiles similar to that of
diltiazem, all being more potent on nonvascular than on
vascular smooth muscles even if less selective than 1. Com-
pound (()-3, which is the most potent in both tissues followed
by 5 and 4, had vasorelaxant potency 2.2 times lower than 1,
whereas compounds 4 and 5 were 9.7 and 6.8 times less potent
than 1, respectively.
Binding assays with [3H]diltiazem on rat cardiomyocytes
were carried out for compounds 4 and 5, while for (()-3
binding data have been already published.4 Only compound
4 displayed a weak interaction with the BTZ-binding site,
producing a maximum inhibition of [3H]diltiazem binding
of about 50% at 100 μM (Figure 1). In contrast, compound
5 stimulated [3H]diltiazem binding in a concentration-
dependent fashion over the concentration range 10 nM to
100 μM. Thus, compound 5 might interact with a distinct site
allosterically linked to the BTZ receptor. This hypothesis is
in agreement with the stimulation profile of diltiazem bind-
ing produced by dihydropyridines and phenylalkylamines in
purified porcine cardiac sarcolemmal membrane vesicles
reported by Garcia et al.11 Because of its different binding
profile, compound 5 might be separately investigated in
detail, and only compounds 3 and 4 would be further
compared with 1.
Results
Overview. For comprehensive comparison with the refer-
ences 1 ((þ)-cis-diltiazem) and R-(-)-2, the pharmacological
profile of 4 and 5 was completed by measuring their effect on
the longitudinal smooth muscle. For compound 3, the two
enantiomers were separated by chiral HPLC, and their
absolute configurations were determined using vibrational
Determination of Absolute Configuration of Compound 3.
To elucidate the biological activity of (()-3 and to assign the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6639
Figure 1. Effects of 4 (a) and 5 (b) on [3H]diltiazem binding.
absolute configuration (AC) of its enantiomers, (()-3 was
first separated by chiral HPLC. Then the AC of each
enantiomer was determined using VCD in tandem with
calculations of the predicted spectra.12 The principle behind
this powerful method for the determination of AC is deter-
mining one or more chiroptical properties of an enantiomer
experimentally and comparing the spectrum/spectra with
that/those predicted by DFT calculations for one AC. This
technique is becoming very popular in recent years,13-20 and
it was already used to determine the AC of the above-
mentioned chiral oxadiazol-3-one 2 by a concerted compar-
ison of DFT calculations of its VCD spectrum, electronic
circular dichroism (ECD) spectrum, and optical rotation
(OR) to experimental VCD, ECD, and OR data.21
Figure 2. HPLC traces for the analysis of (a) (()-3, (b) (-)-3, and
(c) (þ)-3 monitored by UV and CD at 254 nM. Bottom right: ECD
spectra of the individual enantiomers isolated by semipreparative
HPLC.
First, the pharmacologically active (()-3 was resolved by
semipreparative enantioselective HPLC on a chiral station-
ary phase. Chiralpak IA was chosen as the chiral stationary
phase for these purposes. The chiral selector tris-3,5-di-
methylphenylcarbamate derivative of amylose immobilized
on silica allows the use of a broad range of organic solvents in
which the analyte is readily soluble and shows a good level of
enantioselectivity and high loading ability.22,23
The pair of enantiomers was resolved with a selectivity
value of R = 1.22 (k1 = 1.33) using hexane/isopropanol/
dichloromethane (85/10/5, v/v/v) as eluent. In the semipre-
parative scale-up, each stereoisomer (11 mg, process yield
85%) was obtained with an enantiomeric excess of 99%
(Figure 2). The elution order as monitored online by circular
dichroism (CD) detection at 254 nm and off-line by polari-
metric measurements on the isolated enantiomers was the
(-)-enantiomer first ([R]2D5 -179.53 (c 0.60, CH3CN)) and
the (þ)-enantiomer second ([R]2D5 þ173.48 (c 0.64, CH3CN)).
Additional information is provided in the Experimental
Section.
Then the AC of 3 was determined by comparison of
experimental and predicted VCD spectra. The VCD spectra
of 0.06 M CDCl3 solutions of (þ)-3 and (-)-3 were measured
using cell path lengths of 236 and 546 μm; to minimize
artefacts, the baselines for the spectra were the VCD spectra
of racemic 3. The resulting VCD spectrum of (þ)-3 is shown
in Figure 3, together with the IR absorption spectrum of
(()-3. The spectra are quite dense because of the large size
and conformational flexibility of 3.
Given the conformational flexibility of 3, its conforma-
tional analysis was also quite complicated, since there are
many possible conformations. According to DFT calcula-
tions, by use of the B3PW91 functional and the TZ2P basis
set, there are 10 conformations with energy values within 3
kcal/mol of the lowest free-energy conformation. The free
energy value of each conformation was calculated in order to
evaluate the relative population of each conformation at
room temperature using Boltzmann statistics. The relative
free energies and the resulting room temperature popula-
tions are listed in Table 2. The prediction of the IR and VCD
spectra of 3 requires calculations of the spectra of the 10
conformers and the calculation of the conformationally
averaged spectra, using the calculated populations.
Finally, the B3PW91/TZ2P conformationally averaged
VCD spectra of the R and S ACs of 3 were compared to
the experimental VCD of (þ)-3, as shown in Figure 4. The
R-3 VCD spectrum is in much better agreement with the
experimental VCD spectrum than the S-3 VCD spectrum.
The assignment of 16 bands is indicated using the letters a-p.
As a result, the AC of 3 was assigned as R-(þ)/S-(-).
Determination of the LTCCs Blocking Properties of the
Two Enantiomers of 3. A. Functional Study. The functional
profile observed for the two enantiomers was significantly
different. Data for the S-(-) and R-(þ) forms of 3 are listed
in Table 3 with those of (()-3 taken here as reference. Both
enantiomers had negative inotropic activity, R-(þ) being
twice as potent as S-(-). Also, for the relaxant effect on
guinea pig longitudinal smooth muscle, the R-(þ) form was
twice as potent as S-(-). In contrast, the vasorelaxant
property was uniquely shown by S-(-)-3, with a potency
comparable to that of (()-3.
Finally, for comprehensive comparison with the three
above-mentioned compounds, the pharmacological profiles
of the enantiomers of 3 are graphically shown in Figure 5;
(()-3, 4, and 5 had relaxant properties in guinea pig aortic
strips (orange bars) and ileum longitudinal smooth muscle
(fuchsia-colored bars) while showing the same selectivity
profile. For all three, the inotropic activity (blue bars) was

6640 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Carosati et al.
Figure 4. Comparison of the conformationally averaged B3PW91/
TZ2P VCD spectra of the R and S configurations of 3 to the
experimental VCD spectrum of (þ)-3. The peaks used to assign
the AC are labeled a-p.
Figure 3. (a) IR absorption spectrum of racemic 3 in CDCl3: 0.05 M,
path length 546 μm. (b) Half-difference, 1/2[Δε(þ) - Δε(-)],
VCD spectrum of 3 in CDCl3: 0.06 M. For the frequency ranges
1650-1479, 1431-1390, 1321-1200, 1072-955, and 852-800 cm-1
the VCD was measured with a 546 μm path length cell. For the
frequency ranges 1479-1431, 1390-1321, 1200-1072, and 864-
852 cm-1 the VCD was measured with a 236 μm path length cell.
shown in Figure 5. Diltiazem (1) was the less selective
compound because of its activity in all tissues; conversely,
R-(-)-2 was the most selective one, since it showed only a
negative inotropic activity. Compounds (()-3 and 4 had the
same biological profile, similar to that of diltiazem although
they lacked of chronotropic activity (green bar). Finally, it is
evident in the figure that the profiles of the two enantiomers of
3 are different, with S-(-)-3 being responsible for the vaso-
relaxant activity (orange bar) found in the racemate (()-3.
B. Binding Study. Binding assays were carried out in rat
cardiomyocytes to establish the binding site of the two
enantiomers of 3. [3H]Diltiazem (5 nM) was incubated with
increasing concentrations (0.01 nM to 100 μM) of the
compounds as described in the Experimental Section.
As reported in Figure 6, S-(-)-3 and R-(þ)-3 interacted
with the BTZ-binding site, producing a reproducible
inhibition of [3H]diltiazem binding that reached the
maximum of about 50% already at 1 μM. The findings
presented in this study strongly suggest that both enantio-
mers of 3 equally inhibited LTCCs activity by interacting at
the BTZ-binding site in the cardiac R1 subunit of the
LTCCs.
Table 2. B3PW91/TZ2P Conformations, Free Energy Differences, and
Populations of 3
conformer
4G (kcal)
P (%)a
a
b
c
d
e
f
0.00
0.22
0.49
0.65
0.70
0.76
1.43
1.75
2.39
2.79
31.1
21.5
13.6
10.4
9.6
8.6
2.8
g
h
i
1.6
0.6
j
0.3
a Populations based on G values; T = 298 K.
the main effect, with the same potency registered for 5 and
(()-3. The latter was also the most potent compound con-
cerning the relaxant properties on both aorta and ileum. In
particular, while the negative inotropic effect was common
to both enantiomers, the vascular action of 3 was only
observed with the S-(-) form.
Finally, for comprehensive comparison, the potencies of
all tested compounds on left driven atria, spontaneous beat-
ing right atria, Kþ-depolarized aortic strips, and guinea pig
ileum logitudinal smooth muscle (GPILSM) are graphically
C. Electrophysiological Study. In addition, the effect of
(()-3, S-(-)-3, and R-(þ)-3 on L-type barium currents
[IBa(L)] recorded in vascular myocytes was assessed in order
to provide direct evidence of their calcium antagonistic
activity. The racemic mixture of 3 and both enantiomers
individually inhibited peak IBa(L) in a concentration-depen-
dent manner, with IC50 values of 23.5 ( 2.0 μM ((()-3,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6641
Table 3. Functional Activity of Tested Compounds
cardiac activity
% decrease (M ( SEM)
EC50 of inotropic negative activity
vasorelaxant activity
95% conf
GPILSM relaxant activity
95% conf
negative
inotropic
activitya
negative
chronotropic
activityb
95% conf
lim
(ꢀ10-6)
c
c
c
EC50
(μM)
activityd
IC50
lim
(ꢀ10-6)
activitye
IC50
lim
(ꢀ10-6)
compd
(M ( SEM) (μM)
(M ( SEM) (μM)
(()-3
84 ( 1.6
4 ( 0.2h
7 ( 0.3h
10 ( 0.7h
0.31
0.45
0.23
0.22-0.42
0.35-0.64
0.13-0.31
74 ( 2.1
83 ( 4.5
24 ( 2.3
5.62
5.16
4.46-7.08
3.33-6.99
90 ( 0.7f
69 ( 4.7
94 ( 1.1
1.18
2.06
1.07
0.84-1.67
1.68-5.53
0.88-1.29
S-(-)-3 96 ( 1.7f
R-(þ)-3 98 ( 1.1g
a Activity: decrease in developed tension in isolated guinea pig left atrium at 5 ꢀ 10-6 M, expressed as percent changes from the control (n = 4-6). The
left atria were driven at 1 Hz. The 5ꢀ10-6 M concentration gave the maximum effect for most compounds. b Activity: decrease in atrial rate in guinea pig
spontaneously beating isolated right atria at 5 ꢀ 10-5 M, expressed as percent changes from the control (n = 6-8). Pretreatment heart rate ranged from
170 to 195 beats/min. The 5ꢀ10-5 M concentration gave the maximumeffect for most compounds. c Calculated from log concentration-response curves
(Probit analysis according to Litchfield and Wilcoxon with n = 6-8).10 When the maximum effect was <50%, the IC50 value was not calculated. d Activity:
percent inhibition of calcium-induced contraction in 80 mM Kþ-depolarized guinea pig aortic strip at 10-4 M. The 10-4 M concentration gave the maximum effect
for most compounds. e Percent inhibition of calcium-induced contraction in 80 mM Kþ-depolarized GPILSM at 5 ꢀ 10-6 M. The 5 ꢀ 10-6 M concentration gave
the maximum effect for most compounds. f At 10-5 M. g At 10-4 M. h P < 0.05.
Figure 7. Effects of 3 and its enantiomers on IBa(L) (5 mM Ba2þ
,
0 mV, Vh = -80 mV). Each point is the mean ( SEM (n = 4-7).
Where error bars are not shown, these are covered by the point itself.
Figure 5. Negative inotropic and negative chronotropic potencies
reported together with relaxant potency in 80 mM Kþ-depolarized
guinea pig aortic strips and ileum longitudinal smooth muscle
(GPILSM) reported for the reference compounds 1 and R-(-)-2,
for (()-3 and its S-(-) and R-(þ) enantiomers, and for compound 4.
features are important or detrimental for biological activity.
Thus, a common 3D-orientation was searched for 1, 3, and 4
with a molecular modeling study by means of the program
FLAP,24 which is a recently developed algorithm suited to
describe ligands on the basis of a common reference frame-
work. Compound 1 was taken as reference (“template
molecule”), whereas the R- and S-enantiomers of 3 and
compound 4 constituted the so-called ligand molecules.
Their best-possible FLAP-based orientations with respect
to 1 were selected and are discussed in the next section in
comparison with the experimental data. It is noteworthy that
the final aim of this ligand-based application was not to
claim for the compounds presented here a definitive orienta-
tion within the LTCC diltiazem binding site but to discuss
one of their possible orientations with respect to 1 in order to
facilitate the understanding of the relationship between
molecular structure and experimental data. For a better
comprehension of the 3D-superimpositions obtained, the
core concepts of the program FLAP are described below
while more details are given in the Experimental Section.
The program FLAP aligns two molecules one over the
other and quantifies the similarity between them. The
“ligand molecule” is aligned over the “template molecule”,
which is the reference, on the basis of their pharmacophores.
A FLAP pharmacophore is a combination of four hotspots
(molecular atoms): each pharmacophore of the “ligand” is
searched over the “template” pharmacophores. For each
match, which is obtained when four hotspots of the “ligand”
molecule are found to fit over four hotspots of the
Figure 6. Binding curves for the (a) S-(-) and (b) R-(þ) enantio-
mers of 3.
n = 5), 22.2 ( 5.1 μM (S-(-)-3, n = 4), and 27.1 ( 8.5 μM
(R-(þ)-3, n = 3). Hence, comparable profiles for the two
enantiomers and the racemic mixture emerged from this
study (Figure 7).
D. Molecular Modeling Study. One of the main focuses
of in silico procedures applied to drug discovery processes is
to provide hypotheses for understanding which molecular

6642 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Carosati et al.
Figure 8. Scheme for the FLAP procedure that compares the “ligand” molecule R-(þ)-3 with the “template” molecule 1. Nitrogen atoms are
represented in blue, oxygen atoms in red, sulfur atoms in orange. Carbon atoms of 1 and R-(þ)-3 are differently colored (1 = green, R-(þ)-3 =
magenta). Hydrogen atoms are not reported for clarity. Nodes of the GRID MIF, reported at the energy level of -3.5 kcal/mol for the probe
OH2 and -0.8 kcal/mol for the probe DRY, are differently colored: orange (DRY) and blue (OH2) for 1 and yellow (DRY) and cyan (OH2) for
R-(þ)-3. One match between two quadruplets of hotspots is reported as example. The figure was prepared using PyMol.26
“template”, a potentially favorable superimposition is de-
tected. This iterative process continues until all the “tem-
plate” pharmacophores are combined in all possible ways
with all the “ligand” pharmacophores. Each time, a score
associated with the 3D-superimposition of the two molecules
and with their common pharmacophores quantifies the 3D
similarity. At the end of the process, only the best score
obtained, which corresponds to the best superimposition of
each “ligand” to the “template”, is used to assess the 3D-
similarity between the two molecules.
The FLAP 3D-similarity is an estimation of how the
molecular interaction fields (MIF) calculated for the two
molecules with the GRID force field25 are intersected. The
MIF encode into energy values the information of hydro-
phobic and hydrogen bonding interactions of the molecule
with a virtual receptor that is schematized by the GRID
probes. The best intersection of the MIF of two molecules
represents the interaction with a virtual receptor that the two
molecules potentially have in common.
The non-negligible flexibility of the channel and of its
ligands would affect any computational study of the chan-
nel-ligand interaction, and for the same compound several
possible orientations within the channel could be hypothe-
sized. Therefore, in this study a conformational sampling
guaranteed an adequate treatment of molecular flexibility.
For the “template” 1 the conformational analysis was carried
out on-the-fly by FLAP, producing 10 different conforma-
tions and repeating for each conformation the entire align-
ment procedure. Also, “ligand” molecules were subjected to
a fast conformational analysis. Since the FLAP method has
been created for virtual screening projects in order to run
over libraries of thousands of compounds, the conforma-
tional analysis of FLAP was carried out on-the-fly to span
reasonably the conformational space, a reasonable number

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6643
of conformations (up to 100) were generated for each “ligand
molecule”, and each conformer was treated individually. At
the end of the process, only the best superimposition of each
“ligand” to the “template” was memorized. A flowchart of
the procedure is schematized in Figure 8.
Discussion
To date, the absence of crystallographic structures of
calcium channels is likely responsible for the prevalence of
ligand-based versus structure-based studies. In particular,
the BTZ-binding site of LTCCs is the least characterized.
Only recently, Tikhonov and Zhorov27 have published a
homology model of the pore-forming subunit (R1) of
LTCC, using ligand-docking computational experiments
to elaborate a 3D model of the BTZ binding site. Their
model, rationalizing various experimental observations
reported in the literature,28,29 proposes two binding modes
for BTZs depending on the number of calcium ions in the
selectivity-filter region. In one mode, the ammonium group
of BTZ is attracted to the outer pore; this binding mode
would prevail when the negative charges at the selectivity-
filter glutamates in the outer pore are not compensated by
calcium ions. In the other mode, the carbonyl oxygen of
BTZ could directly interact with a calcium ion chelated
by selectivity-filter glutamates. This binding mode would
prevail when the negative charges in the selectivity-filter
glutamates are instead neutralized by calcium ions. In this
case, the ammonium group is not directly involved. Then
for molecules without such an ammonium group a similar
binding mode could be hypothesized as well.
Figure 9. 3D-Superimpositions over 1 (a) obtained for 4 (b), R-(þ)-
3 (c), and S-(-)-3 (d) with the program FLAP. The figure was
prepared using PyMol.26
Given the binding profiles reported above for the R-(þ) and
S-(-) enantiomers of 3 and for compound 4 and given the
common vasorelaxant properties of 1, (()-3, and 4, it would
be interesting to provide binding mode hypotheses for
R-(þ)-3, S-(-)-3, and 4 in comparison with 1. What makes
it more interesting is that compounds 3 and 4 possess neither
the ammonium group of 1 nor an alternative basic group with
pKa in the range of diltiazem structural analogues, i.e., 7-9.
In order to hypothesize the binding orientations, one might
use the 3D-superimpositions obtained with FLAP for 4,
R-(þ)-3, and S-(-)-3 over 1, reported in Figure 9. In parti-
cular, it is worth investigating the “driving motifs” of such
molecular superimpositions; many polar oxygen atoms (red
atoms in Figure 9) and polar groups lie in a central polar
region, while a common orientation of the p-substituted
phenyl rings canbenoted for all the compounds. In particular,
the 4-chlorophenyl ring of 4 is twisted when compared to the
other corresponding phenyl rings and also the sulfonyl group
of the sulfonamides is differently oriented for 4 with respect to
R-(þ)-3 and S-(-)-3.
Figure 10. Chemical features hypothesized as responsible for the
modulation of negative inotropic activity (highlighted in cyan
squares), for vasorelaxant effect (red circles), and for both effects
(purple). The 3D-superimpositions are also reported. The figure was
prepared using PyMol.26
with the sulfonyl group aligned over the carbonyl oxygen of
the thiazepine ring and the p-substituted phenyl ring slightly
twisted. The lower inotropic and vasorelaxant activities of4 as
well as its low affinity toward the diltiazem binding site could
be related to this “less precise alignment”. The tetrahydropyr-
imidine ring, aligned over the thiazepine moiety, forces the
methylmercapto group to lie over the N,N-dimethylami-
noethyl side chain of 1. Also the 2-thienyl group of S-(-)-3
lies over the hydrophobic side chain of1, while for R-(þ)-3 the
same ring is better aligned over the benzo-fused ring of 1.
These differences could help to rationalize the stereoselective
behavior of 3.
On the basis of this in silico superimposition, obtained over
the 3D structures of 1 and limited to this set of functional
diltiazem analogues, it could be hypothesized that one region
is specific for the modulation of the vasorelaxant activity, one
region is specific for the modulation of the negative inotropic
activity, and a third region, composed of a phenyl ring and a
polar area, is common to compounds endowed with both
cardiac and vascular properties, as highlighted in Figure 10.
These considerations agree to some extent with the classical
structure-activity relationships (SAR) of structural analo-
gues of 1. The importance of the basic center in the N,N-
dimethylaminoethyl side chain of 1 and the importance of its
two aromatic rings about 7 A apart are well-known. In
particular, modifications to the aliphatic chain modulated
A better understanding could arise from analyzing the
hydrophilic MIF in Figure 8. Two large regions are evident,
which are generated by the oxygen atoms of the carboxylate
group and by the carbonyl oxygen from the thiazepine moiety
of 1 (blue MIF in Figure 8) and by the oxygen atoms of the
sulfonyl group of R-(þ)-3 (cyan MIF in Figure 8). As
described above, the molecular superimposition proposed is
the result of the best MIF intersection for the ligand and
template molecules. Thus, for both enantiomers of 3, the
sulfonyl group is aligned to the carboxylate of 1 (see
Figure 9) whereas for compound 4 the best compromise of
molecular shape fitted over 1 and MIF alignment (over the
MIF of 1) is obtained for the orientation proposed in Figure 9,

6644 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Carosati et al.
the vasorelaxant activity30 whereas substituents on the con-
densed phenyl ring enhanced the potency.31 A comprehensive
analysis of the SARs of 1 and its structural and functional
analogues is given in refs 7 and 32.
1-[(4-Chlorophenyl)sulfonyl]-2-(2-thienyl)pyrrolidine ((()-3). 1H
NMR (400 MHz, CD3OD) δ 1.76-2.10 (m, 4H), 3.38-3.46
(m, 1H), 3.50-3.60 (m, 1H), 5.17 (dd, 1H, J = 2.80 Hz, J =
6.86 Hz), 6.93 (dd, 1H, 2J = 3.51 Hz, 3J = 5.06 Hz), 6.97-7.00
2
3
(m, 1H), 7.26 (dd, 1H, J = 1.25 Hz, J = 5.06), 7.54-7.59
(m, 2H), 7.73-7.78 (m, 2H) ppm. HPLC: 254 nm, tR =12.39 min,
purity >99%. MS (ESI): m/z = 328 [M þ H]þ.
Conclusion
1-[(4-Chlorophenyl)sulfonyl]-2-(methylthio)-1,4,5,6-tetrahydro-
pyrimidine (4). 1H NMR (400 MHz, CD3OD) δ 1.80 (quin, 2H,
3J = 5.90 Hz), 2.22 (s, 3H), 3.46 (t, 2H, 3J = 5.90 Hz), 3.85 (t,
2H, 3J = 5.90 Hz), 7.64 (d, 2H, Jortho = 8.50 Hz), 7.97 (d,
2H, Jortho = 8.50 Hz) ppm. HPLC: 254 nm, tR= 10.05 min,
purity >99%. MS (ESI): m/z = 305 [M þ H]þ.
For the new cardiovascular LTCCs blocker 1-[(4-
chlorophenyl)sulfonyl]-2-(2-thienyl)pyrrolidine (3), found by
virtual screening, a comparison of experimental and calcu-
lated VCD spectra permitted the unambiguous assignment of
the absolute configuration as R-(þ)/S-(-), and a broad
investigation of the antagonist activity of both enantiomers
of 3 involving functional, electrophysiology, and binding
studies. The functional profile on guinea pig was studied by
assessing the negative inotropic and chronotropic action on
left and right atria. The vasorelaxant activity was assessed on
high Kþ-depolarized aortic strips and ileum longitudinal
smooth muscle. The effect on IBa(L) recorded in rat vascular
myocytes was assessed via the patch-clamp technique. The
affinity to and the direct interaction with the cardiac LTCCs
was verified using binding experiments carried out on rat
cardiomyocytes with [3H]diltiazem.
As a result of such a large analysis, stereoselective behavior
for the negative inotropic activity and stereospecific behavior
for the vasorelaxant activity were observed, whereas compar-
able binding and electrophysiological profiles were observed
for the two enantiomers. While R-(þ)-3 was the more potent
negative inotropic enantiomer, the vasorelaxant profile was
exclusively assigned to S-(-)-3.
In conclusion, with the results presented here, compound 3
can be considered as a new and valid pharmacological tool to
characterize the BTZ site in the cardiac R1 subunit of the
LTCC. Interestingly, all these data are in agreement with the
in silico 3D-superimpositions obtained with the algorithm of
the program FLAP specifically designed for ligand-based
studies and used here for modeling some functional analogues
of diltiazem. Combining this multidisciplinary research with
the in silico procedure, the 3D orientations obtained led us to
hypothesize relationships of molecular moieties to biological
properties. In prospective, these results could pave the way to
the design of new molecules as selective calcium modulators.
Preliminary studies are currently in progress in this direction
and will be published in due course.
1
N-Allyl-4-chloro-N-phenylbenzenesulfonamide (5). H NMR
3
(400 MHz, CD3OD) δ 4.26 (d, 2H, J = 6.20 Hz), 5.06 (dd,
1H, 2J = 1.0 Hz, 3J = 11.2 Hz), 5.12 (dd, 1H, 2J = 1.0 Hz, 3J =
17.1 Hz), 5.70-5.82 (m, 1H), 7.05-7.10 (m, 2H), 7.28-7.35
(m,3H), 7.57 (d, 2H, Jortho = 8.90 Hz), 7.59 (d, 2H, Jortho =
8.90 Hz) ppm. HPLC: 254 nm, tR =13.09 min, purity >99%.
MS (ESI): m/z = 308 [M þ H]þ.
B. Details for Enantiomers Separation and Absolute Config-
uration Determination. Experimental Data. The analytical
HPLC resolution of (()-3 was performed on an analytical
chromatograph equipped with a Rheodyne model 7725i 20 μL
loop injector, a PU-1580-CO2 and PU-980 Jasco HPLC pumps,
a UV detector Jasco-975, and a circular dichroism detector
Jasco 995-CD. Chromatographic data were collected and pro-
cessed using Borwin software (Jasco Europe, Italy). The chemi-
cal purity of (()-3 was checked on an analytical chromatograph
equipped with a Rheodyne model 7725i 20 μL loop injector, a
Waters 1525 HPLC pump, and a UV Waters 2487 detector.
Chromatographic data were collected and processed using
Empower2 (Waters, Milford, MA). The semipreparative HPLC
resolution of (()-3 was performed on a Waters chromatograph
(Waters Associates) equipped with a Rheodyne model 7012 500 μL
loop injector and a UV SpectraMonitor 4100 and a refractive index
Waters R401 detectors. The ECD spectra of (þ)- and (-)-3 in
acetonitrile were recorded on a Jasco J710 spectropolarimeter.
IR spectra of (þ)-3, (-)-3, and (()-3 (potassium bromide dis-
persion) were recorded on a Jasco 430 FT-IR spectrometer.
Specific rotations at 589 nm of (þ)-3 and (-)-3 dissolved in
acetonitrile were recorded on a Jasco P-1030 polarimeter.
Methods. All HPLC-grade solvents and reagents were pur-
chased from Sigma-Aldrich, and the mobile phases were filtered
before use over 0.45 μm filters. The chemical purity check of
(()-3 was performed by gradient RP-HPLC using a C18 Luna
Phenomenex column (250 mm ꢀ 4.6 mm i.d.) and gradient
elution from (A) H2O/ACN (90/10) and (B) ACN/H2O (90/10)
at a flow rate of 1 mL/min. The gradient elution started from
A/B 40/60 (3 min hold) to B 100% in 15 min. The chromato-
graphic profiles were acquired at 25 ꢀC, using UV detection at
254 nm. The analytical HPLC resolution of (()-3 was carried
out on a Chiralpak-IA column (250 mm ꢀ 4.6 mm i.d.) using
hexane/isopropanol/dichloromethane, 85/10/5, as eluent, deliv-
ered at a flow rate of 1 mL/min. The column temperature
was set at 25 ꢀC. UV detection was carried out at 254 nm. The
enantiomers of (()-3 were resolved by semipreparative HPLC
using a Chiralpak-IA column (250 mm ꢀ 10 mm i.d.) with
hexane/isopropanol/dichloromethane, 85/10/5, as eluent deliv-
ered at a flow rate of 4 mL/min. The column temperature was set
at 25 ꢀC. UV detection was carried out at 254 nm. Racemic 3 was
dissolved in the mobile phase (c = 30.3 mg/mL), and for each
run, an amount of 100 μL of solution, corresponding to 3 mg of
racemic 3, was injected.
Experimental Section
Purity of compounds was >99%, as determined by HPLC (see
below, section A).
A. Purity of Tested Compounds. Starting materials (()-3 and 4
were purchased from Maybridge Chemicals,33 and compound 5
was from Key Organics Ltd.34 HPLC-grade water and acetoni-
trile were purchased from Carlo Erba (Carlo Erba Reagents,
Rodano, Italy). HPLC system consisted of a Waters 2695
(Waters, Milford, MA) chromatograph equipped with auto-
matic injector and column heater and coupled with a model 996
PDA detector (Waters, Milford, MA). Thermo Finnigan LCQ
Deca XP Plus ion trap mass spectrometer equipped with an ESI
ion source was used for mass detection. Purity of compounds
was checked by RP-HPLC using a Phenomenex Luna C18
column (250 mm ꢀ 4.6 mm i.d.). Gradient elution was obtained
with solvents A (H2O/ACN, 90/10) and B (ACN/H2O, 90/10)
starting from 60% B and reaching 100% B in 15 min at a flow
rate of 1 mL/min. Eluents were 0.45 μm filtered before use. PDA
data were acquired in the 200-400 nm range, and the 254 nm
signal was extracted. Solutions of each sample in acetonitrile
(c = 10-5 M) were employed for MS flow injection analysis.
Determination of Absolute Configuration. The experimental
IR and VCD spectra of the enantiomers and racemate of 3 were
measured using CDCl3 solutions in cells of path lengths 236 and
546 μm using a Thermo Nicolet Nexus 670 FTIR instrument
and a Bomem/BioTools ChiralIR Fourier transform VCD
instrument. Conformational analysis of 3 was carried out using

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6645
the MMFF94 molecular mechanics force field and the SPAR-
TAN 02 program.35 Conformations obtained were reoptimized
using DFT and the Gaussian 03 program.36 Harmonic vibra-
tional frequencies were then calculated for all conformations to
verify their stability and to predict their relative free energies.
Harmonic dipole and rotational strengths were simultaneously
calculated, and IR and VCD spectra were then obtained using
Lorentzian bandshapes (γ = 4 cm-1). Comparison of the DFT
calculated VCD spectra for both enantiomers of 3 to the
experimental VCD spectrum of (þ)-3 enables the absolute
configuration of 3 to be reliably determined.12
C. Details for Functional Experiments. Functional Studies.
The selected compounds were assayed on guinea pig left and
right atria for negative inotropic and chronotropic action,
respectively, while activity on vascular and nonvascular smooth
muscle was assessed on high Kþ-depolarized guinea pig aortic
strips and ileum longitudinal smooth muscle. In particular,
compounds were checked at increasing concentrations to eval-
uate the percent decrease of developed tension on isolated left
atrium driven at 1 Hz (negative inotropic activity), the percent
decrease in atrial rate on spontaneously beating right atrium
(negative chronotropic activity), and the percent inhibition of
calcium-induced contraction in 80 mM Kþ-depolarized aortic
strips (vasorelaxant activity) or ileum longitudinal smooth
muscle (GPILSM relaxant activity).3 The functional activity
determined was expressed as efficacy and potency.
equilibrated with 95% O2-5% CO2 gas at pH 7.4. The vessel
was cleaned of connective tissue. Two helicoidal strips (10 mm ꢀ
1 mm) were cut from each aorta beginning from the end most
proximal to the heart. Vascular strips were then tied with
surgical thread (6-0) and suspended in a jacketed organ bath
(15 mL) containing aerated PSS at 35 ꢀC. Aortic strips were
secured at one end to Plexiglas hooks and connected via the
surgical thread to a force displacement transducer (FT 0.3,
Grass Instruments Corporation, Quincy, MA) for monitoring
changes in isometric contraction. Aortic strips were subjected to
a resting tension of 1 g. The intestine was excised above the
ileocecal junction and the mesenteric tissue removed. The long-
itudinal smooth muscle was gently separated from the ileum
portion, and segments of 2 cm length were mounted at one end
to Plexiglas hooks under a resting tension of 300-400 mg. Strips
were secured at the other end to a force displacement transducer
(FT 0.3, Grass Instruments Corporation, Quincy, MA) for
monitoring changes in isometric contraction and washed every
20 min with fresh PSS for 1 h. After the equilibration period,
guinea pig aortic strips were contracted by washing in PSS
containing 80 mM KCl (equimolar substitution of Kþ for Naþ).
When the contraction reached a plateau (about 45 min), various
concentrations of the compounds (0.1, 0.5, 1, 5, 10, 50, 100, and
500 μM) were added cumulatively to the bath, allowing for any
relaxation to obtain an equilibrated level of force. Addition of
the drug vehicle (DMSO for all compounds) had no appreciable
effect on Kþ-induced contraction.
Statistical Analysis. Data were analyzed using Student’s t test
and are presented as the mean ( SEM.10 Since the drugs were
added in cumulative manner, significant differences (P < 0.05)
between control and experimental values at each concentration
were indicated by asterisks. The potency of drugs defined as
EC50, EC30, and IC50 was evaluated from log concentra-
tion-response curves (Probit analysis using Litchfield and
Wilcoxon10 or GraphPad Prism software37) in the appropriate
pharmacological preparations.
D. Details for Binding Experiments. Cardiomyocytes Isola-
tion. This investigation conforms to the Guide for the Care and
Use of Laboratory Animals published by the U.S. National
Institutes of Health (NIH Publication No. 85-23, revised 1996),
and the animal protocols used were reviewed and approved by
In Vitro Screening. Isolated Guinea Pig Left and Right Atria
Preparations. Female guinea pigs (300-400 g) were sacrificed by
cervical dislocation. After thoracotomy, the heart was immedi-
ately removed and washed by perfusion through the aorta with
oxygenated Tyrode solution of the following composition
(mM): NaCl, 136.9; KCl, 5.4; CaCl2, 2.5; MgCl2, 1.0; NaH2-
PO4 H2O, 0.4; NaHCO3, 11.9; glucose, 5.5. The physiological
3
salt solution (PSS) was buffered to pH 7.4 by saturation with
95% O2-5% CO2 gas, and the temperature was maintained at
37 ꢀC. Isolated guinea pig heart preparations were used, i.e.,
spontaneously beating right atria and left atria driven at 1 Hz.
For each preparation, the entire left and right atria were
dissected from the ventricles, cleaned of excess tissue, and hung
vertically in a 15 mL organ bath containing the PSS continu-
ously bubbled with 95% O2-5% CO2 gas at 35 ꢀC, pH 7.4. The
contractile activity was recorded isometrically by means of force
transducer (FT 0.3, Grass Instruments, Quincy, MA) using
Power Lab software (ADInstruments Pty Ltd., Castle Hill,
Australia). The left atria were stimulated by rectangular pulses
of 0.6-0.8 ms duration and about 50% threshold voltage
through two platinum contact electrodes in the lower holding
clamp (Grass S88 stimulator, Quincy, MA). The right atrium
was in spontaneous activity. After the tissue was beating for
several minutes, a length-tension curve was determined, and
the muscle length was fixed at that eliciting 90% of maximum
contractile force observed at the optimal length. A stabilization
period of 45-60 min was allowed before the atria were used to
test compounds. During the equilibration period, the bathing
solution was changed every 15 min and the threshold voltage
was ascertained for the left atria. Atrial muscle preparations
were used to examine the inotropic and chronotropic activity of
the compounds (0.1, 0.5, 1, 5, 10, 50, and 100 μM) first dissolved
in DMSO and then diluted with PSS. According to this proce-
dure, the concentration of DMSO in the bath solution never
exceeded 0.3%, a concentration that did not produce significant
inotropic and chronotropic effects. During the construction of
cumulative concentration-response curves, the next higher
concentration of the compound was added only after the
response to the previous one had reached a steady state.
ꢀ
the Animal Care and Ethics Committee of the Universita degli
Studi di Siena, Italy. Single cardiac myocytes (CM) were isolated
from male Wistar rats (body weight 376 ( 12 g, n = 19, Charles
River Italia, Calco, Italy), injected with 500 U heparin/100 g
body weight, i.p., anesthetized with a mixture of Ketavet (30 mg
kg-1 ketamine; Gellini, Italy) and Xilor (8 mg kg-1 xylazine;
Bayer, Germany), decapitated, and bled.38 After thoracotomy,
the heart was rapidly removed, mounted on a micro-Langen-
dorff apparatus, and perfused for 20 min at 37 ꢀC with a
nominally Ca2þ-free solution (low Ca2þ solution, LCS) of the
following composition (mM): NaCl, 120; KCl, 10; HEPES, 10;
glucose, 10; MgCl2, 1.2; KH2PO4, 1.2; sodium pyruvate, 5;
taurine, 20; pH 7.2, equilibrated with 95% O2-5% CO2. The
solution was then quickly changed to LCS complemented with
0.9 mg/mL collagenase type I (Sigma Chimica, Milan, Italy),
0.05 mg/mL dispase I (Roche Gmbh, Penzbeg, Germany), and
1.5 mg/mL bovine serum albumin acid-free (Sigma Chimica,
Milan, Italy) for 10-15 min. When the heart was soft, perfusion
was stopped and the tissue was chopped into small pieces and
gently stirred in fresh LCS at room temperature. The cardio-
myocytes that appeared in the supernatant were purified by
centrifugation (5 min at 800g) and frozen at -80 ꢀC until use.
Pooled cells, derived from at least two animals, were used for
each binding experiment. Protein concentrations were estimated
by the method of Bradford39 with BSA as standard.
Guinea Pig Aortic Strips and Ileum Longitudinal Smooth
Muscle (GPILSM). The thoracic aorta and ileum were removed
and placed in Tyrode solution (PSS) of the following composi-
tion (mM): NaCl, 118; KCl, 4.75; CaCl2, 2.54; MgSO4, 1.20;
KH2PO4, 1.19; NaHCO3, 25; glucose, 11. This solution was
[3H]Diltiazem Binding Assays. [3H]Diltiazem binding assays
were performed according to earlier methods40,41 with some
modifications. Aliquots of thawed rat CM (200 μg) were in-
cubated with ligands in 50 mM Tris buffer (pH 7.4) at 25 ꢀC for

6646 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Carosati et al.
90 min in a final volume of 0.2 mL. In homologous competition
curves, [3H]diltiazem (specific activity 85.5 Ci/mmol) was pre-
sent at 20 nM in tubes containing increasing concentrations of
unlabeled diltiazem (5 nM to 100 μM) and at 0.1-20 nM in
tubes without unlabeled ligand. In heterologous competition
curves, a fixed amount of the tracer (5 nM) was displaced by
increasing concentrations of several unlabeled ligands (0.1 nM
to 100 μM). The incubation was terminated by a rapid filtration
on Whatman GF/B glass fiber filters (presoaked for at least 1 h
in 0.5% polyethyleneimmine) and washed three times with 3 mL
of ice-cold wash buffer. The filters were then placed in scintilla-
tion vials. An amount of 5 mL of scintillation liquid was added
and the radioactivity measured in a LS5000CE β-counter
(Beckman Instruments, Palo Alto, CA). Nonspecific binding
was defined by means of 100 μM unlabeled diltiazem. All the
experiments were always run in triplicate.
Data Analysis. All the data, presented as mean ( SEM, were
reported by plotting specific binding (% of control) versus log of
the inhibitor concentration (M) and fitted with a nonlinear
(sigmoidal) analysis.
E. Details for Electrophysiological Experiments. Tail Main
Artery Dissection. This investigation conforms to the Guide for
the Care and Use of Laboratory Animals published by the U.S.
National Institutes of Health (NIH Publication No. 85-23,
revised 1996), and the animal protocols used were reviewed
and approved by the Animal Care and Ethics Committee of the
5 mM Ba2þ-containing recording solution. Current was elicited
with 250 ms clamp pulses (0.067 Hz) to 0 mV from a Vh of -80 mV
until a stable current response was achieved (usually 6-9 min
after the whole-cell configuration had been obtained). IBa(L) did
not run down during the following 20-30 min under these
conditions.44 Kþ currents were blocked with 30 mM tetraethy-
lammonium in the external solution and Csþ in the internal
solution (see below). Current values were corrected for leakage
using 10 μM nifedipine, which was proved to block completely
I
Ba(L). Following control measurements, each cell was exposed
to cumulative concentration of a drug by flushing through the
experimental chamber recording solution containing the drug.
Compounds (()-3, S-(-)-3, and R-(þ)-3, dissolved directly in
DMSO, were diluted at least 1000 times prior to use. The
resulting concentrations of DMSO (below 0.1%, v/v) failed to
alter the response of the preparations. Acquisition and analysis
of data were accomplished by using pClamp 9.2.1.8 software
and GraphPad Prism version 5.01.33 Data are reported as the
mean ( SEM. n is the number of cells analyzed (indicated in
parentheses), isolated from at least three animals. The IC50
values were calculated from log concentration-response curves.
Solutions for IBa(L) Recordings. The Ca2þ-free external solu-
tion contained the following (mM): NaCl, 110; KCl, 5.6;
HEPES, 10; glucose, 20; taurine, 20; MgCl2, 1.2; sodium pyr-
uvate, 5; pH 7.4. The Ca2þ-free recording solution contained the
following (in mM): NaCl, 130; KCl, 5.6; HEPES, 10; glucose,
20; MgCl2, 1.2; sodium pyruvate, 5; pH 7.4. The internal
solution (pCa 8.4) consisted of the following (mM): CsCl, 100;
HEPES, 10; EGTA, 11; MgCl2, 2; CaCl2, 1; Na-pyruvate, 5;
succinic acid, 5; oxalacetic acid, 5; Na2-ATP, 3; phosphocrea-
tine, 5; pH was adjusted to 7.4 with CsOH. The osmolarity of
recording solution (320 mosmol) and that of the internal solu-
tion (290 mosmol) were checked with an osmometer (Osmostat
OM 6020, Menarini Diagnostics, Italy).
ꢀ
Universita degli Studi di Siena, Italy (31.01.2006). Male Wistar
rats (300-400 g, Charles River Italia, Calco, Italy) were an-
esthetized with a mixture of Ketavet (30 mg kg-1 ketamine;
Gellini, Italy) and Xilor (8 mg kg-1 xylazine; Bayer, Germany),
decapitated, and bled. Tail was immediately removed, cleaned
of skin, and placed in 0.1 mM Ca2þ external solution (see below
for composition). The tail main artery was dissected free,
removing the connective tissue.
Cell Isolation Procedure for IBa(L) Recordings. Smooth muscle
cells were freshly isolated from the tail main artery incubated at
37 ꢀC in 2 mL of 0.1 mM Ca2þ external solution (see below)
containing 1 mg/mL collagenase (type XI), 1 mg/mL soybean
trypsin inhibitor, and 1 mg/mL bovine serum albumin and
gently bubbled with a 95% O2-5% CO2 gas mixture, as
previously described.42 Cells exhibited an ellipsoid form
(10-15 μm in width, 35-55 μm in length) and were continuously
superfused with recording solution containing 0.1 mM Ca2þ
using a peristaltic pump (LKB 2132, Bromma, Sweden), at a
F. Computational methods. Details for the 3D-Superimposi-
tion Procedure. The program FLAP24 (fingerprint for ligands
and proteins) was originally conceived as a fast algorithm for
performing virtual screening by means of a fingerprint-type
description of both ligands and protein binding sites. It was
designed to describe molecules and protein structures in terms of
a common reference framework. This framework can be built
from information obtained from the active site of the protein of
interest, in which case the resulting study is called structure-
based study, or from ligands that are known to be active for a
certain biological target, in which case the resulting study is
called a ligand-based study. In ligand-based virtual screening,
compounds from virtual libraries are ranked according to their
similarity toward a given reference molecule, called a template.24
Given the 3D structure of a molecule, with the GRID force
field25 the interaction energy between the molecule and a probe
is calculated at different points in space. As a result, one has the
so-called molecular interaction fields (MIF): the hydrophobic
and hydrogen bonding interactions of the molecule with a
virtual receptor are schematized by the probes and encoded into
energy values assigned to the nodes of a grid built around each
molecule. MIF are of central importance in the execution of the
FLAP algorithm since, given two molecules, their MIF are used
by FLAP to align one over the other. The first step of the FLAP
procedure is the creation of a database: all the molecules
imported will be referred to as ligand molecules. The ligand
molecules are subjected to a fast conformational analysis, a
reasonable number of conformations are generated, and each
conformer is individually treated (in this application, up to 100
conformations were generated by FLAP). Some representative
fields are extracted from the MIF of the individual elements
entered and stored in the database. Then a molecular template is
imported, the corresponding MIF are calculated with the pro-
gram GRID, and again some representative fields are selected
from the MIF. The pairwise similarity is calculated on the
basis of MIF superimposition which, in turn, is obtained by
flow rate of 400 μL min-1
.
Whole-Cell Patch-Clamp Recordings. The conventional
whole-cell patch-clamp method43 was employed to voltage-
clamp smooth muscle cells. Recording electrodes were pulled
from borosilicate glass capillaries (WPI, Berlin, Germany) and
fire-polished to obtain a pipet resistance of 2-5 MΩ when filled
with internal solution (see below). A low-noise, high-perfor-
mance Axopatch 200B (Molecular Devices Corporation, CA)
patch-clamp amplifier driven by a personal computer (pClamp
9.2.1.8 software; Molecular Devices Corporation, CA) in con-
junction with an A/D, D/A board (DigiData 1200 A/B series
interface, Molecular Devices Corporation, CA) was used to
generate and apply voltage pulses to the clamped cells and record
the corresponding membrane currents. At the beginning of each
experiment, the junction potential between the pipette and bath
solutions was electronically adjusted to zero. Cell break-in
was accomplished by gentle suction at a holding potential (Vh) of
-50 mV. Then Vh was set to -80 mV. Micropipette seals had to
be GΩ in nature with leak currents less than 0.25 pA mV-1
.
Current signals, after compensation for whole-cell capacitance
and series resistance (70-80%), were low-pass-filtered at 1 kHz
and digitized at 3 kHz prior to being stored on the computer hard
disk. Electrophysiological responses were tested at room tem-
perature (20-22 ꢀC) only in those cells that were phase dense.
IBa(L) Recordings. Cells used in this study expressed LTCCs
but not T-type Ca2þ channels.44 IBa(L) was always recorded in

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6647
combining quadruplets of hotspots that could be either molec-
ular atoms or points from the MIF. The first case was used:
FLAP classifies all the heavy atoms of ligand molecules as
hydrophobic, hydrogen bond donating or accepting, and posi-
tively or negatively charged. Then FLAP describes each mole-
cule as a set of quadruplets, derived by combining all the
hotspots, four by four, in an exhaustive way. Each quadruplet
is defined by the character of its composing hotspots, by their six
interatomic distances, measured in angstroms and binned in 1 A
intervals, and by an additional flag for the chirality of the
quadruplet. The huge information obtained is then documented
in a fingerprint mode where the absence or presence of all
quadruplets corresponds to 0 or 1, respectively. For computa-
tional reasons, only present quadruplets are stored in such a way
that facilitates further searches and uses. The information
obtained for the template molecule is stored in a virtual bit
string, making future computations much easier and quicker to
complete and compare. The next step in the FLAP process
would be finding the matches of the four hotspots of the
individual ligands to those of the template. When four hotspots
of the ligand molecule are found to fit over four hotspots of the
template framework, a potentially favorable superimposition
has been detected. Of course, the process is iterative, and it will
continue until all the template quadruplets are combined in all
possible ways with all ligand quadruplets. Each time, a score
associated with the two molecules and their common quadruplet
of hotspots quantifies the overlap of the two MIF: a score is
assigned to the superimposition in order to evaluate its “good-
ness” of MIF-overlapping, using the MIF of the two molecules
produced with the GRID probes DRY and OH2. At the end of
the process, only the best superimposition of each ligand to each
template is memorized. Some details of the FLAP calculations
are briefly listed: (1) the tolerance between pairwise atoms (from
template and ligand molecules) was set to 1 A. (2) The con-
formers are produced by FLAP on-the-fly with a routine that
randomly modifies the molecule and adds a new conformation
to the set only when it differs from the ones already existing. The
difference is calculated by using rmsd (root-mean-square
deviation) criteria, using 0.1 as threshold for considering two
conformations enough diverse to be treated individually. In this
way, the FLAP conformational sampling guarantees an ade-
quate treatment of molecular flexibility in order to span as much
as possible the conformational space. For the template 1 the
conformational analysis was carried out on-the-fly producing 10
different conformations and repeating for each conformation
the entire procedure described, whereas up to 100 conformers
were generated for each ligand molecule, and the best results
obtained among these was the result assigned to the compound.
(3) GRID probes DRY and OH2 was used to describe the
template and each ligand molecule. In addition, the GRID
probe H is used for describing the molecular shape. (4) The
molecular atoms were used as hotspots. (5) The level of speed
was set to 75%.
rotational strengths. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) McGregor, M. J.; Luo, Z.; Jiang, X. Virtual Screening in Drug
Discovery. In Drug Discovery Research. New Frontiers in the Post-
Genomic Era; Huang, Z., Ed.; Wiley-VCH: Weinheim, Germany, 2007;
pp 63-88.
(2) Budriesi, R.; Cosimelli, B.; Ioan, P.; Lanza, C. Z.; Spinelli, D.;
Chiarini, A. CardiovascularCharacterizationof[1,4]Thiazino[3,4-c]-
[1,2,4]oxadiazol-1-one-derivatives: Selective Myocardial Calcium
Channel Modulators. J. Med. Chem. 2002, 45, 3475–3481.
(3) Budriesi, R.; Carosati, E.; Chiarini, A.; Cosimelli, B.; Cruciani, G.;
Ioan, P.; Spinelli, D.; Spisani, R. A New Class of Selective
Myocardial Calcium Channel Modulators. 2. The Role of the
Acetal Chain in Oxadiazol-3-one Derivatives. J. Med. Chem.
2005, 48, 2445–2456.
(4) Carosati, E.; Cruciani, G.; Chiarini, A.; Budriesi, R.; Ioan, P.;
Spisani, R.; Spinelli, D.; Cosimelli, B.; Fusi, F.; Frosini, M.;
Matucci, R.; Gasparrini, F.; Ciogli, A.; Stephens, P. J.; Devlin,
F. J. New Calcium Channel Antagonists Discovered by a Multi-
disciplinary Approach. J. Med. Chem. 2006, 49, 5206–5216.
(5) Maron, B. J. Hypertrophic Cardiomyopathy:
A Systematic
Review. JAMA, J. Am. Med. Assoc. 2002, 287, 1308–1320.
(6) Varon, J. Treatment of Acute Severe Hypertension: Current and
Newer Agents. Drugs 2008, 68, 283–97.
(7) Budriesi, R.; Cosimelli, B.; Ioan, P.; Carosati, E.; Ugenti, M. P.;
Spisani, R. Diltiazem Analogues: The Last Ten Years on Structure
Activity Relationships. Curr. Med. Chem. 2007, 14, 279–287.
(8) Carosati, E.; Budriesi, R.; Ioan, P.; Ugenti, M. P.; Frosini, M.;
Fusi, F.; Corda, G.; Cosimelli, B.; Spinelli, D.; Chiarini, A.;
Cruciani, G. Discovery of Novel and Cardioselective Diltiazem-
like Calcium Channel Blockers via Virtual Screening. J. Med.
Chem. 2008, 51, 5552–5565.
(9) Agrawal, Y. K.; Bhatt, H. G.; Raval, H. G.; Oza, P. M.; Gogoi, P. J.
Chirality, a New Era of Therapeutics. Mini-Rew Med. Chem. 2007,
7, 451–460.
(10) Tallarida, R. J.; Murray, R. B. Manual of Pharmacologic Calcula-
tions with Computer Programs, 2nd ed.; Springer-Verlag: New York,
1987.
(11) Garcia, M. L.; King, V. F.; Siegl, P. K.; Reuben, J. P.; Kaczor-
owski, G. J. Binding of Ca2þ Entry Blockers to Cardiac Sarco-
lemmal Membrane Vesicles. Characterization of Diltiazem-
Binding Sites and Their Interaction with Dihydropyridine and
Aralkylamine Receptors. J. Biol. Chem. 1986, 261, 8146–8157.
(12) Stephens, P. J.; Devlin, F. J.; Pan, J. J. The Determination of the
Absolute Configuration of Chiral Molecules Using Vibrational
Circular Dichroism (VCD) Spectroscopy. Chirality 2008, 20, 643–
663.
(13) Stephens, P. J.; Pan, J. J.; Devlin, F. J.; Cheeseman, J. R. Determi-
nation of the Absolute Configurations of Natural Products Using
TDDFT Optical Rotation Calculations: The Iridoid Oruwacin. J.
Nat. Prod. 2008, 71, 285–288.
(14) Krohn, K.; Gehle, D.; Dey, S. K.; Nahar, N.; Mosihuzzaman, M.;
Sultana, N.; Sohrab, M. H.; Stephens, P. J.; Pan, J. J.; Sasse, F.
Prismatomerin, a New Iridoid from Prismatomeris tetrandra.
Structure Elucidation, Determination of Absolute Configuration
and Cytotoxicity. J. Nat. Prod. 2007, 70, 1339–1343.
(15) Stephens, P. J.; Pan, J. J.; Krohn, K. Determination of the Absolute
Configurations of Pharmacological Natural Products via Density
Functional Theory Calculations of Vibrational Circular Dichro-
ism: The New Cytotoxic Iridoid Prismatomerin. J. Org. Chem.
2007, 72, 7641–7649.
Acknowledgment. Financial support was provided by
grants from Universities of Bologna, Perugia, Siena, and
€
(16) Stephens, P. J.; Devlin, F. J.; Schurch, S.; Hulliger, J. Determina-
tion of the Absolute Configuration of Chiral Molecules via Density
Functional Theory Calculations of Vibrational Circular Dichroism
and Optical Rotation: The Chiral Alkane D3-anti-trans-anti-trans-
anti-trans-Perhydrotriphenylene. Theor. Chem. Acc. 2008, 119, 19–
28.
ꢀ
Roma and from Ministero dell’Istruzione, dell’Universita e
della Ricerca (Grants PRIN 2005034305_001, PRIN
20078J9L2A_005, and FIRB RBPR05NWWC_003). We
are also grateful for financial support from the U.S. National
Science Foundation (to P.J.S., Grants CHE-0209957 and
CHE-0614577). We also thank the USC High Performance
Computing and Communication (HPCC) facility for compu-
ter time.
(17) Stephens, P. J.; Devlin, F. J.; Villani, C.; Gasparrini, F.; Mortera, S.
L. Determination of the Absolute Configurations of Chiral Orga-
nometallic Complexes via Density Functional Theory Calculations
of Their Vibrational Circular Dichroism Spectra: The Chiral
Chromium Tricarbonyl Complex of N-Pivaloyl-tetrahydroquino-
line. Inorg. Chim. Acta 2008, 361, 987–999.
ꢁ
(18) Stephens, P. J.; Pan, J. J.; Devlin, F. J.; Urbanova, M.; Julinek, O.;
ꢁ ꢂ
Hajı
´
cek, J. Determination of the Absolute Configurations of
Supporting Information Available: The B3PW91/TZ2P struc-
tures and geometries of the conformers a-g of R-(þ)-3 (with
room-temperature equilibrium populations of >2%) together
with their DFT calculated frequencies, dipole strengths, and
Natural Products via Density Functional Theory Calculations of
Vibrational Circular Dichroism, Electronic Circular Dichroism
and Optical Rotation: The Isoschizozygane Alkaloids Isoschizoga-
line and Isoschizogamine. Chirality 2008, 20, 454–470.

6648 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Carosati et al.
(19) Drabowicz, J.; Zajac, A.; Lyzwa, P.; Stephens, P. J.; Pan, J. J.;
Devlin, F. J. Determination of the Absolute Configurations of
Isotopically Chiral Molecules Using Vibrational Circular Dichro-
ism (VCD) Spectroscopy: The Isotopically Chiral Sulfoxide Per-
deuteriophenyl-phenyl-sulfoxide. Tetrahedron: Asymmetry 2008,
19, 288–294.
Halogen-Substituted 1,5-Benzothiazepine Derivatives and Their Vaso-
dilating and Hypotensive Activities. J. Med. Chem. 1991, 34, 675–
687.
(31) Schleifer, K. J.; Tot, E. Molecular Modeling Study of Diltiazem
Mimics at L-Type Calcium Channels. Pharm. Res. 1999, 16, 1506–
1513.
(32) Budriesi, R.; Ioan, P.; Carosati, E.; Cruciani, G.; Zhorov, B. S.;
Chiarini, A. Ligands of Diltiazem Binding Site: An Overview of
Some Chemotypes. Mini-Rev. Med. Chem., in press.
ꢀ
(20) Figadere, B.; Devlin, F. J.; Millar, J. G.; Stephens, P. J. Determina-
tion of the Absolute Configuration of the Sex Pheromone of the
Obscure Mealybug by Vibrational Circular Dichroism Analysis.
Chem. Commun. 2008, 9, 1106–1108.
(33) http://www.maybridge.com.
(21) Stephens, P. J.; Devlin, F. J.; Gasparrini, F.; Ciogli, A.; Spinelli, D.;
Cosimelli, B. Determination of the Absolute Configuration of a
Chiral Oxadiazol-3-one Calcium Channel Blocker, Resolved Using
Chiral Chromatography, via Concerted Density Functional The-
ory Calculations of Its Vibrational Circular Dichroism, Electronic
Circular Dichroism, and Optical Rotation. J. Org. Chem. 2007, 72,
4707–4715.
(34) http://www.keyorganics.ltd.uk.
(35) Spartan 02; Wavefunction Inc.: Irvine, CA.
(36) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.;
Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi,
J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;
Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.;
Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.;
Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H.
P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Strat-
mann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.;
Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck,
A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.
Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong,
M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03; Gaussian, Inc.:
Pittsburgh, PA, 2004.
(22) Zhang, T.; Kientzy, C.; Franco, P.; Ohnishi, A.; Kagamihara, Y.;
Kurosawa, H. Solvent Versatility of Immobilized 3,5-Dimethyl-
phenylcarbamate of Amylose in Enantiomeric Separations by
HPLC. J. Chromatogr., A 2005, 1075, 65–75.
ꢀ
(23) Cere, V.; Peri, F.; Pollicino, S.; Ricci, A.; Devlin, F. J.; Stephens, P.
J.; Gasparrini, F.; Rompietti, R.; Villani, C. Synthesis, Chromato-
graphic Separation, Vibrational Circular Dichroism Spectroscopy,
and ab Initio DFT Studies of Chiral Thiepane Tetraol Derivatives.
J. Org. Chem. 2005, 70, 664–669.
(24) Baroni, M.; Cruciani, G.; Sciabola, S.; Perruccio, F.; Mason, J. S. A
Common Reference Framework for Analyzing/Comparing Pro-
teins and Ligands. Fingerprints for Ligands and Proteins (FLAP):
Theory and Application. J. Chem. Inf. Model. 2007, 47, 279–294.
(25) (a) Goodford, P. J. A Computational Procedure for Determining
Energetically Favorable Binding Sites on Biologically Important
Macromolecules. J. Med. Chem. 1985, 28, 849–857. (b) Carosati, E.;
Sciabola, S.; Cruciani, G. Hydrogen Bonding Interactions of Covalently
Bonded Fluorine Atoms: From Crystallographic Data to a New Angular
Function in the GRID Force Field. J. Med. Chem. 2004, 47, 5114–
5125. (c) GRID, version 22 for Linux; Molecular Discovery Ltd.
(215 Marsh Road, HA5 5NE Pinner, Middlesex, U.K.); http://www.
moldiscovery.com.
(37) GraphPad Prism, versions 3.02, 4.03, and 5.01; GraphPad Software
Inc.: La Jolla, CA; http://www.graphpad.com.
(38) Matucci, R.; Ottaviani, M. F.; Barbieri, M.; Cerbai, E.; Mugelli, A.
Protective Effect of Darodipine, a Calcium Antagonist, on Rat
Cardiomyocytes against Oxygen Radical-Mediated Injury. Br. J.
Pharmacol. 1997, 122, 1353–1360.
(39) Bradford, M. M. A Rapid and Sensitive Method for the Quantita-
tion of Microgram Quantities of Protein Utilizing the Principle of
Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254.
(40) Wei, X. Y.; Rutledge, A.; Triggle, D. J. Voltage-Dependent Bind-
ing of 1,4-Dihydropyridine Ca2þ Channel Antagonists and Acti-
vators in Cultured Neonatal Rat Ventricular Myocytes. Mol.
Pharmacol. 1989, 35, 541–552.
(26) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano
Scientific LLC: Palo Alto, CA, 2008; http://www.pymol.org.
(27) Tikhonov, D. B.; Zhorov, B. S. Benzothiazepines in L-Type
Calcium Channel: Insights from Molecular Modelling. J. Biol.
Chem. 2008, 283, 17594–17604.
(28) Hering, S.; Savchenko, A.; Strubing, C.; Lakitsch, M.; Striessnig, J.
(41) Zobrist, R. H.; Mecca, T. E. [3H]TA-3090, a Selective Benzothia-
zepine-Type Calcium Channel Receptor Antagonist: In Vitro
Characterization. J. Pharmacol. Exp. Ther. 1990, 253, 461–465.
(42) Fusi, F.; Saponara, S.; Gagov, H.; Sgaragli, G. P. 2,5-Di-t-butyl-
1,4-benzohydroquinone (BHQ) Inhibits Vascular L-Type Ca2þ
Channel via Superoxide Anion Generation. Br. J. Pharmacol.
2001, 133, 988–996.
€
Extracellular Localization of the Benzothiazepine Binding Domain
of L-Type Ca2þ Channels. Mol. Pharmacol. 1993, 43, 820–826.
(29) Dilmac, N.; Hilliard, N.; Hockerman, G. H. Molecular Determi-
nants of Ca2þ Potentiation of Diltiazem Block and Ca2þ-Depen-
dent Inactivation in the Pore Region of Cav1.2. Mol. Pharmacol.
2003, 64, 491–501.
(30) (a) Inoue, H.; Konda, M.; Hashiyama, T.; Otsuka, H.; Watanabe,
A.; Gaino, M.; Takahashi, K.; Date, T.; Okamura, K.; Takeda, M.;
Narita, H.; Murata, S.; Odawara, A.; Sasaki, H.; Nagao, T.
Synthesis and Biological Evaluation of Alkyl, Alkoxy, Alkylthio,
or Amino-Substituted 2,3-Dihydro-1,5-benzothiazepin-4(5H)-
ones. Chem. Pharm. Bull. 1997, 45, 1008–1026. (b) Inoue, H.; Konda,
M.; Hashiyama, T.; Otsuka, H.; Takahashi, K.; Gaino, M.; Date, T.; Aoe,
K.; Takeda, M.; Murata, S.; Narita, H.; Nagao, T. Synthesis of
(43) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J.
Improved Patch-Clamp Techniques for High-Resolution Current
Recording from Cells and Cell-Free Membrane Patches. Pflugers
Arch. 1981, 391, 85–100.
(44) Petkov, G. V.; Fusi, F.; Saponara, S.; Gagov, H.; Sgaragli, G. P.;
Boev, K. K. Characterization of plasma membrane calcium cur-
rents in freshly isolated smooth muscle cells from rat tail main
artery. Acta Physiol. Scand. 2001, 173, 257–265.