Imidazo[2,1-b]thiazole system: A scaffold endowing dihydropyridines with selective cardiodepressant activity

Article abstract of DOI:10.1021/jm070681+

The synthesis, characterization, and functional in vitro assays in cardiac tissues and smooth muscle (vascular and nonvascular) of a number of 4-imidazo[2,1-b]thiazole-1,4-dihydropyridines are reported. The binding properties for the novel compounds have been investigated and the interaction with the binding site common to other aryl-dihydropyridines has been demonstrated. Interestingly, the novel 4-aryl-dihydropyridines are L-type calcium channel blockers with a peculiar pharmacological behavior. Indeed, the imidazo[2,1-b]thiazole system is found to confer to the dihydropyridine scaffold an inotropic and/or chronotropic cardiovascular activity with a high selectivity toward the nonvascular tissue. Finally, molecular modeling studies were undertaken for the most representative compounds with the aim of describing the binding properties of the new ligands at molecular level and to rationalize the found structure-activity relationship data. Due to the observed pharmacological behavior of our compounds, they might be promising agents for the treatment of specific cardiovascular pathologies such as cardiac hypertrophy and ischemia.

Full text of DOI:10.1021/jm070681+

1592
J. Med. Chem. 2008, 51, 1592–1600
Imidazo[2,1-b]thiazole System: A Scaffold Endowing Dihydropyridines with Selective
Cardiodepressant Activity
Roberta Budriesi,*,† Pierfranco Ioan,† Alessandra Locatelli,*,† Sandro Cosconati,‡ Alberto Leoni,† Maria P. Ugenti,†
Aldo Andreani,† Rosanna Di Toro,§ Andrea Bedini,§ Santi Spampinato,§ Luciana Marinelli,‡ Ettore Novellino,‡ and
Alberto Chiarini†
Dipartimento di Scienze Farmaceutiche, UniVersità degli Studi di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Dipartimento di Chimica
Farmaceutica e Tossicologica, UniVersità degli Studi di Napoli “Federico II”, Via Montesano 49, 80131 Napoli, Italy, and Dipartimento di
Farmacologia, UniVersità degli Studi di Bologna, Via Irnerio 48, 40126 Bologna, Italy
ReceiVed June 12, 2007
The synthesis, characterization, and functional in vitro assays in cardiac tissues and smooth muscle (vascular
and nonvascular) of a number of 4-imidazo[2,1-b]thiazole-1,4-dihydropyridines are reported. The binding
properties for the novel compounds have been investigated and the interaction with the binding site common
to other aryl-dihydropyridines has been demonstrated. Interestingly, the novel 4-aryl-dihydropyridines are
L-type calcium channel blockers with a peculiar pharmacological behavior. Indeed, the imidazo[2,1-b]thiazole
system is found to confer to the dihydropyridine scaffold an inotropic and/or chronotropic cardiovascular
activity with a high selectivity toward the nonvascular tissue. Finally, molecular modeling studies were
undertaken for the most representative compounds with the aim of describing the binding properties of the
new ligands at molecular level and to rationalize the found structure–activity relationship data. Due to the
observed pharmacological behavior of our compounds, they might be promising agents for the treatment of
specific cardiovascular pathologies such as cardiac hypertrophy and ischemia.
Chart 1
Introduction
Voltage-gated Ca2+ channels are heteromultimeric trans-
membrane proteins that are made of four or five subunits (R1,
R2, δ, ꢀ, γ). Among them, the main R1 subunit allows the
selective passage of Ca2+ ions into excitable cells and controls
the voltage sensitivity and the gating mechanism.1 These
proteins have a crucial role in a broad range of cellular
processes, such as neurotransmitter release, second messenger
cascades, cardiac excitation and contraction, and gene regulation
supporting learning and memory.2 Due to their important
functions, voltage-gated Ca2+ channels have been extensively
studied and, to date, different drugs are available that are known
to interact with them. Among the others, L-type calcium channel
(LTCCa) blockers have gained a critical role in the treatment
of different cardiovascular pathologies. The above-mentioned
drugs can be divided in four structurally different classes: 1,4-
dihydropyridines (1,4-DHPs), such as Nifedipine, phenylalkyl-
amines (PAAs), such as Verapamil, 1,5-benzothiazepines (BTZs),
such as (+)-cis-Diltiazem, and pyrrolidineethanamine, such as
Bepridil (Chart 1).
the different tissue-selectivity has to be ascribed to the different
topographic localization of their binding sites of the channel. It
has been demonstrated that these drugs bind at distinct sites
within the R1 subunit of the L-type Ca2+ channel (CaV1) and
they noncompetitively affect each other’s binding.3 Moreover,
it can also be argued that the targeted subclass of L-type channel
and the state-dependent interactions of LTCC blockers with the
protein might have a role in determining tissue selectivity. In
addition to the above-described factors, small structural variation
on the 1,4-DHPs scaffold led to significant differences in voltage
sensitivity of binding, which has a direct influence on the
vascular/cardiac selectivity. Interestingly, in our earlier works
we demonstrated that compounds such as fluodipine displayed
a high cardiac selectivity.4 The differential tissue-selectivity
displayed by LTCC blockers has allowed their employment as
“specific probes” for the pharmacological and structural char-
acterization of the CaV1 channel.5 To date, several R1 subunits
Each class of LTCC blockers exhibits distinct characteristics
in their cardiovascular profiles in mediating antihypertensive,
antianginal and antiarrhythmic activities.3 In particular, Vera-
pamil and Diltiazem are essentially nonselective and 1,4-DHPs
usually display a vascular selectivity. One obvious reason for
* To whom corresondence should be addressed. Tel.: +39-051-
2099737(R.B.); +39-051-2099712(A.L.). Fax: +39-051-2099721 (R.B.);
+39-051-2099721 (A.L.). E-mail: roberta.budriesi@unibo.it (R.B.); alessandra.
locatelli@unibo.it (A.L.).
†
Dipartimento di Scienze Farmaceutiche, Università degli Studi di
Bologna.
‡
Università degli Studi di Napoli “Federico II”.
Dipartimento di Farmacologia, Università degli Studi di Bologna.
§
a Abbreviations: LTCC, L-type calcium channel; 1,4-DHPs, 1,4-dihy-
dropyridines; PAAs, phenylalkylamines; BTZs, 1,5-benzothiazepines;
GPILSM, guinea-pig ileum longitudinal smooth muscle.
10.1021/jm070681+ CCC: $40.75
2008 American Chemical Society
Published on Web 02/28/2008

Imidazo[2,1-b]thiazole-1,4-DHPs as Cardiac Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1593
Scheme 1a
have been described and the sequence relationship for these
channel subtypes has also been disclosed.6 Recently, the R1
subunits have been classified in R1S found in skeletal muscle,7
R
1C mainly cardiac,8 R1D in neurons and heart,9 and R1F retinal
not yet functionally expressed.10 The classification of the L-type
voltage dependent calcium channel family nomenclature was
recently revised accordingly by Ertel et al. in Cav1.1 (R1S) found
in skeletal muscle; Cav1.2 (R1C) found in cardiac, smooth, and
endocrine tissues; Cav1.3 (R1D) found in brain, ear, and
endocrine tissues; Cav1.4 (R1F) found on retina, respectively.1,6
Unfortunately, to date, a comprehensive description of the
sensitivity of the different channel subtypes toward 1,4-DHPs
is still unavailable.11 Indeed, there are subtype-specific differ-
ences in ligand recognition properties for the LTCC; these
differences are extremely common for a large number of
receptor types and subtypes specific agonists and antagonists.
From this point of view, the availability of tissue-selective LTCC
blockers could facilitate an accurate description of the phar-
macological profile of these channels. On the other hand, tissue
selective LTCC blockers might also have an enhanced thera-
peutic utility in specific cardiovascular pathologies. In this
context, it is worth noting that the 1,4-DHP nucleus appears to
be an interesting structure interacting with a wide variety of
channel and receptors and is an example of a “privileged
structure”: a core structure that by appropriate molecular
decoration can be directed to diverse pharmacological tissues.12
The term “privileged structure” introduced by Evans et al.13
prompts that adequate modification of such a structure could
be a valid route in the search for new LTCC blockers with well-
defined channel subtype selectivity. Interestingly, in a previous
work promising results were obtained by testing a series of
different 4-imidazothiazole-DHPs for their antiarrhythmic, ino-
tropic, and chronotropic properties.14 Therefore, in the present
paper starting from these encouraging results we designed and
synthesized a small library of imidazo[2,1-b]thiazole derivatives
(Chart 1) with the aim of identifying new tissue selective 1,4-
DHPs. In particular, taking into account that the double bond
at the position 2,3 is necessary for the activity and the 2-methyl
derivatives showed antiarhythmic but not cardiodepressant
activity,14 to define the structure–activity relationship we
performed the synthesis of new analogues bearing: unsubstituted
at the 2,3-position; a chlorine or a small aliphatic group (methyl,
ethyl, prophyl) at the position 2; a methyl at the position 3. In
addition, based on reported results14 to investigate the influence
of the substitution at the position 6, we consider the following
feature as suitable pharmacophoric groups: chlorine, methyl,
trifluoromethyl, unsubstituted/substituted phenyl ring. Moreover,
molecular modeling studies were undertaken with the aim of
describing the binding properties of the new ligands at molecular
level and to rationalize the structure–activity relationship (SAR)
data could, in turn, prospectively guide the design of novel
analogues endowed of the same pharmacological properties.
a Reagents and conditions: (I) (a) acetone reflux; (b) hydrochloridric acid
2 N reflux; (II) phosphorus oxychloride, N,N-dimethylformamide reflux;
(III) methyl acetoacetate, ammonia solution 30%, isopropyl alcohol. For
R, R1, and R2, see Table 1.
chronotropic effects on guinea pig isolated left atria driven at 1
Hz and on right atria spontaneously beating, respectively. The
vasorelaxant activity was tested on K+-depolarized (80 mM)
aortic strips. Details of which have been reported in Supporting
Information. For some selected compounds, the investigation
was extended on determination of the relaxant activity on
nonvascular smooth muscle: K+-depolarized (80 mM) guinea-
pig ileum longitudinal smooth muscle (GPILSM). According
to Langendorff, the guinea-pig isolated perfused heart was used
to assay the whole cardiac activity of compound 5Cb and of
the reference Nifedipine. Compounds were checked at increasing
concentrations to evaluate changes in left ventricular pressure
(inotropic activity), heart rate (chronotropic activity), coronary
perfusion pressure (coronary activity), and electrocardiogram
(ECG) signal.
Data were analyzed using Student’s t-test and are presented
as mean ( S.E.M.16 Because the drugs were added in cumula-
tive manner, the difference between the control and the
experimental values at each concentration were tested for a P
value <0.05. The potency of drugs defined as EC50, EC30, and
IC50 was evaluated from log concentration–response curves (Probit
analysis using Litchfield and Wilcoxon16 or GraphPad Prism
software17,18) in the appropriate pharmacological preparations.
Binding Experiments. The compounds were screened for
their affinity for calcium channels from guinea pig heart
ventricles. Binding site was determined by using a competitive
radiometric receptor binding assays. PN200-110, (+)-[5-methyl-
3H] was used to label 1,4-DHPs binding site. Binding affinities
were expressed as Ki and IC50 values (mean ( S.E.M.).19 Data
were analyzed with Student’s t-test. The criterion for significance
was a P value of <0.001.
Displacement binding assays were also carried out on intact
HEK-293 cells transfected with either the rabbit Cav1.2a (R1c-
a) or the rabbit Cav1.2b (R1c-b) coding plasmid. Plasmids were
a generous gift by Dr. Andrea Welling. PN200-110, (+)-[5-
methyl-3H] was used to label 1,4-DHPs binding sites. Binding
affinities were expressed as IC50 values (mean ( S.E.M.).19
Chemistry
1,4-DHPs 5, reported in Scheme 1, were synthesized by
means of the well-known Hantzsch reaction15 with the appropri-
ate aldehydes 4, obtained in turn by means of the Vilsmeier
method on the corresponding imidazo[2,1-b]thiazoles 3. The
bicyclic heterocycles were obtained by cyclocondensation using
the appropriate 2-aminothiazoles 1 and bromoketones 2.
Results and Discussion
A-Functional Studies. A small library of 1,4-DHPs bearing
in C-4 a different substituted imidazo[2,1-b]thiazole system was
designed and synthesized (5Ac-5Gq, Chart 1). All compounds
were tested for their cardiovascular profile on guinea pig left
atrium driven at 1 Hz and on spontaneously beating right atrium
to evaluate their inotropic and chronotropic effects, respectively,
Pharmacology
Functional Assays. All compounds were checked at increas-
ing doses to evaluate the percent decrease of inotropic and

1594 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Table 1. New 1,4-Dihydropyridines 5Ac-5Gpa
Budriesi et al.
cmpd
R
R1
R2 (a-r)
formula
MW
yield %
Mp, °C (purif.)
5Ac
5Ae
5Af
5Ag
5Ah
5Ai
5Aj
5Ak
5Al
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
H
CF3
2-(CF3)C6H4
3-(CF3)C6H4
C18H16F3N3O4S
C23H20F3N3O4S
C23H20F3N3O4S
C23H20F3N3O5S
C23H20F3N3O5S
C23H23N3O5S
C23H23N3O5S
C24H25N3O6S
C24H25N3O6S
C24H25N3O6S
C24H25N3O6S
C25H27N3O7S
C24H24N4O8S
C24H24N4O8S
C24H24N4O8S
C17H18ClN3O4S
C16H15Cl2N3O4S
C22H20ClN3O4S
C24H24ClN3O6S
C24H23ClN4O8S
C24H23ClN4O8S
C18H21N3O4S
C17H18ClN3O4S
C18H18F3N3O4S
C25H27N3O6S
C25H26N4O8S
C25H26N4O8S
C17H18ClN3O4S
C23H23N3O4S
C18H20ClN3O4S
C23H25N3O4S
C26H29N3O6S
C27H30N4O8S
427.4010
491.4867
491.4867
507.4861
507.4861
453.5147
453.5147
483.5408
483.5408
483.5408
483.5408
513.5669
528.5385
528.5385
528.5385
395.8637
416.2818
457.9335
517.9857
562.9833
562.9833
375.4455
395.8637
429.4169
497.5675
542.5652
542.5652
395.8637
437.5153
409.8903
439.5312
511.5942
570.6185
10
40
15
12
10
10
10
14
10
10
8
10
10
20
15
15
16
18
10
10
15
24
10
18
11
10
10
10
8
222–224 (A)
223–227 (A)
94 (A)
2-(OCF3)C6H4
3-(OCF3)C6H4
2-(OCH3)C6H4
3-(OCH3)C6H4
2,5-(OCH3)2C6H3
3,5-(OCH3)2C6H3
3,4-(OCH3)2C6H3
2,4-(OCH3)2C6H3
3,4,5-(OCH3)3C6H2
6-(NO2)2,5-(OCH3)2C6H2
4-(NO2)2,5-(OCH3)2C6H2
6-(NO2)3,4-(OCH3)2C6H2
CH3
215–216 (D)
171–172 (D)
226–228 (B)
217–220 (B)
220–222 (A)
218–220 (B)
180–182 (D)
189–191 (G)
194–196 (E)
195–197 (C)
196–198 (C)
200–202 (E)
225–228 (B)
230–232 (B)
200–203 (A)
226–228 (B)
219–221 (B)
228–231 (B)
210–213 (B)
231–235 (E)
270–275 (E)
217–222 (D)
99–105 (A)
200–203 (E)
215–219 (F)
201–204 (B)
220–223 (F)
192–194 (A)
210–211 (B)
244–248 (B)
5Am
5An
5Ao
5Ap
5Aq
5Ar
5Ba
5Bb
5Bd
5Bk
5Bp
5Bq
5Ca
5Cb
5Cc
5Ck
5Cp
5Cq
5Db
5Dd
5Eb
5Ed
5Fk
5Gp
Cl
C6H5
2,5-(OCH3)2C6H3
6-(NO2)2,5-(OCH3)2C6H2
4-(NO2)2,5-(OCH3)2C6H2
CH3
Cl
CF3
2,5-(OCH3)2C6H3
6-(NO2)2,5-(OCH3)2C6H2
4-(NO2)-2,5-(OCH3)2C6H2
Cl
C6H5
Cl
H
CH3
CH3
C2H5
C3H7
15
6
22
19
C6H5
2,5-(OCH3)2C6H3
6-(NO2)2,5-(OCH3)2C6H2
H
a Purified by (A) flash chromatography, eluent acetone-petroleum ether, 40–60 °C from 1.9 to 4.6, v/v, to afford an oil that was crystallized from
acetone-petroleum ether, 40–60 °C; (B) flash chromatography, eluent acetone-petroleum ether, 40–60 °C from 1.9 to 4.6, v/v, to afford an oil that was
crystallized from acetone; (C) flash chromatography, eluent acetone-petroleum ether, 40–60 °C from 1.9 to 4.6, v/v, to afford an oil that was crystallized
from methanol-acetone; (D) flash chromatography, eluent acetone-petroleum ether, 40–60 °C from 1.9 to 4.6, v/v, to afford an oil that was triturated with
ethanol-hexane, 2:8 v/v; (E) flash chromatography, eluent acetone-petroleum ether, 40–60 °C from 1.9 to 4.6, v/v; (F) flash chromatography, eluent
acetone-diethyl ether, 0.5:9.5 v/v; (G) flash chromatography, eluent diethyl ether, to afford an oil that was triturate with acetone.
and on K+-depolarized (80 mM) guinea pig aortic strips to
assess their vasorelaxant activity. Data are collected in Table 2
using Nifedipine as the reference drug. All the synthesized
compounds showed a marked selectivity for the cardiac over
vascular tissue (Table 2) and, for the tested ones, a selective
nonvascular over vascular activity (Table 3). As a matter of
fact, none of them possesses a vasorelaxant activity on the K+-
depolarized aortic strips greater than 50% (Table 2), whereas
they showed a relaxation activity on K+-depolarized guinea-
pig ileum longitudinal smooth muscle between 51 and 97%
(Table 3). In the series, only three (5Ao, 5Ca, and 5Cc) of 33
compounds synthesized displayed a very low intrinsic activity
on all considered parameters.
A significant group of compounds (11 of 33) were endowed
of negative inotropic selectivity: 5Ae, 5Ag, 5Ak, 5Ap, 5Ba,
5Bb, 5Cb, 5Cp, 5Db, 5Eb, and 5Gp with potency between
0.039 and 1.96 µM. Among them, eight compounds (5Ae, 5Ag,
5Ba, 5Bb, 5Cb, 5Cp, 5Eb, and 5Gp) were more potent than
the reference Nifedipine [EC50 ) 0.026 µM (c.l. 0.018–0.036)];
in particular, 5Gp was found to be the most potent as a negative
inotropic agent [EC50 ) 0.039 µM (c.l. 0.030–0.051)] even if
its efficacy resulted to be lower if compared with that of
Nifedipine.
this group, only two compounds 5Aj and 5Fk were revealed to
have weak negative chronotropic selectivity. Among these
compounds, 5Cq and 5Aq are the most potent and selective
negative inotropic agents [EC50 ) 0.026 µM (c.l. 0.018–0.036);
EC50 ) 0.054 µM (c.l. 0.036–0.079), respectively]. Compounds
5Cq and 5Aq are 10- and 5-fold more potent than Nifedipine,
respectively, as negative inotropic agents; moreover, they
showed a good negative chronotropic/inotropic ratio (81 and
73, respectively). Both 5Aj and 5Fk showed a slight negative
chronotropic activity with an inotropic/chronotropic ratio of 3.
It is well-known that calcium entry blockers, such as Nifedipine,
have relevant inhibitory effects on nonvascular smooth muscle.20
Deeper investigation tested the relaxant activity of some selected
compounds on K+ depolarized (80 mM; GPILSM). Data are
collected in Table 3. For all tested compounds, we observed an
increased activity in nonvascular with respect to vascular smooth
muscle, even if they are, on the whole, less potent than the
reference Nifedipine. Compounds 5Ak, 5Ap, and 5Gp, selective
negative inotropic compounds, are inactive on guinea pig aortic
strips, whereas the potency on nonvascular smooth muscle are
in a 11–13 µM range [IC50 ) 12.35 µM (c.l. 8.56–17.80); IC50
) 11.04 µM (c.l. 8.18–14.90); IC50 ) 12.81 µM (c.l.
10.00–16.41), respectively]. Compound 5Ao acted selectively
on nonvascular smooth muscle with a significant potency [EC50
) 0.55 µM (0.43–0.69)]. Negative chronotropic selective
compound 5Al [EC30 ) 0.86 µM (c.l. 0.653–0.99)] is 36-fold
less potent then Nifedipine, relaxing guinea pig longitudinal
smooth muscle. Compound 5Cq revealed an interesting activity
because it was active on left and right atria and on nonvascular
As shown in Figure 1, most of the tested compounds (5Ac,
5Af, 5Ah, 5Ai, 5Aj, 5Am, 5An, 5Aq, 5Ar, 5Bd, 5Bk, 5 Bq,
5Ck, 5Cq, 5Dd, 5Ed, and 5Fk) showed both negative inotropic
and chronotropic properties with different selectivity. In par-
ticular, different from the reference Nifedipine, they largely
showed a negative inotropic over chronotropic selectivity. Inside

Imidazo[2,1-b]thiazole-1,4-DHPs as Cardiac Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1595
Table 2. Cardiovascular Activity of Compounds 5Ac-5Gp
% decrease (M ( SEM)
EC50 of inotropic negative activity EC30 of chronotropic negative activity vasorelaxant activity
negative inotropic negative chronotropic
95% conf lim
95% conf lim
activityd
(M ( SEM)
c
c
cmpd
activitya
activityb
EC50 (µM)
(×10-6
)
EC30 (µM)
(×10-6
)
5Ac
5Ae
5Af
5Ag
5Ah
5Ai
5Aj
5Ak
5Al
90 ( 3.8f
56 ( 2.4g
89 ( 3.3h
67 ( 2.0i
86 ( 0.7h
54 ( 1.1
90 ( 3.7
92 ( 3.4h
26 ( 1.3
61 ( 2.7
71 ( 2.8h
42 ( 0.2g
71 ( 0.9f
54 ( 3.4i
91 ( 1.4h
63 ( 4.7h
77 ( 2.9h
58 ( 4.1
55 ( 2.3
42 ( 2.1g
58 ( 3.2f
43 ( 3.3j
63 ( 2.7g
46 ( 1.3g
78 ( 0.9h
71 ( 0.1i
75 ( 3.6i
79 ( 3.1g
59 ( 4.2
73 ( 3.4i
76 ( 3.4
72 ( 3.6h
55 ( 1.6h
97 ( 2f
54 ( 2.7g
25 ( 1.8
78 ( 2.5
47 ( 1.4
69 ( 4.3i
95 ( 3.7l
94 ( 2.0i
31 ( 2.1
90 ( 0.5i
80 ( 1.9h
55 ( 2.5
25 ( 1.5i
12 ( 0.9l
67 ( 2.3
90 ( 0.8h
30 ( 2.4
37 ( 3.5
87 ( 3.4
84 ( 2.7l
64 ( 3.6l
90 ( 2.4i
33 ( 2.6h
22 ( 0.7
36 ( 1.7
58 ( 3.4
47 ( 1.2l
87 ( 5.3
39 ( 1.5
88 ( 3.6i
29 ( 1.9i
94 ( 2.4i
92 ( 2.7
25 ( 1.3i
85 ( 4.2k
0.071
0.093
0.31
0.075
0.83
1.97
1.90
0.44
0.021–0.14
0.068–0.12
0.22–0.42
0.050–0.093
0.60–1.04
1.71–2.31
1.65–2.27
0.29–0.65
0.57
0.22–0.87
34 ( 2.1i
24 ( 1.4
27 ( 1.3h
47 ( 1.8
26 ( 1.3
28 ( 1.7
48 ( 1.7f
24 ( 1.4
40 ( 2.4i
15 ( 0.9h
42 ( 3.1
44 ( 3.7
17 ( 0.9
35 ( 2.9h
35 ( 1.7
24 ( 1.8h
34 ( 2.9h
22 ( 1.3h
11 ( 1.0
32 ( 2.2
19 ( 1.4f
38 ( 1.5
19 ( 1.3h
28 ( 1.5h
15 ( 0.9
27 ( 1.9
32 ( 2.5
47 ( 1.1h
30 ( 1.6h
29 ( 1.7f
12 ( 1.1
29 ( 2.4h
5 ( 0.3
1.90
1.75–2.03
1.18
1.99
0.55
0.83–1.36
1.65–2.24
0.34–0.78
0.86
1.44
5.88
0.65–0.99
1.39–1.77
4.93–6.12
5Am
5An
5Ao
5Ap
5Aq
5Ar
5Ba
5Bb
5Bd
5Bk
5Bp
5Bq
5Ca
5Cb
5Cc
5Ck
5Cp
5Cq
5Db
5Dd
5Eb
5Ed
5Fk
5Gp
Nife
2.64
1.24
2.03–3.01
0.93–1.48
0.36
0.054
1.34
0.13
0.18
1.96
0.90
0.25–0.51
0.036–0.079
0.94–1.88
0.079–0.20
0.13–0.23
1.67–2.35
0.56–1.44
3.97
6.97
3.45–4.18
6.03–7.24
2.14
0.36
11.16
0.31
1.78–2.43
0.095–0.58
8.71–13.43
0.27–0.62
0.36
0.22–0.59
0.056
0.041–0.076
1.43
1.02–1.94
0.063–0.14
0.018–0.036
0.08–0.15
0.55–1.21
0.056–0.11
0.43–0.81
1.85–2.43
0.030–0.051
0.19–0.36
1.36
2.11
0.64
1.03–1.75
1.86–2.35
0.23–0.91
0.093
0.026
0.103
0.83
0.081
0.59
2.24
0.039
0.26
0.33
0.73
0.25–0.56
0.52–0.97
0.025
0.019–0.031
82 ( 1.3g
a Decrease in developed tension in isolated guinea-pig left atrium at 10-4 M, expressed as percent changes from the control (n ) 5–6). The left atria were
driven at 1 Hz. 10-4 M gave the maximum effect for most compounds. b Decrease in atrial rate on guinea-pig spontaneously beating isolated right atrium
at 10-5 M, expressed as percent changes from the control (n ) 7–8). Pretreatment heart rate ranged from 165 to 190 beats/min. 10-5 M gave the maximum
effect for most compounds. c Calculated from log concentration–response curves (Probit analysis by Litchfield and Wilcoxon with n ) 6–7).16 When the
maximum effect was <50%, the EC50 ino., EC30 chrono., IC50 values were not calculated. d Percent inhibition of calcium-induced contraction on K+-
depolarized guinea-pig aortic strip at 10-4 M (n ) 5–6). 10-4 M gave the maximum effect for most compounds. e IC50 ) 0.009 µM (c.l. 0.003–0.02). f At
the 10-5 M. g At the 10-6 M. h At the 5 × 10-5 M. i At the 5 × 10-6 M. j At the 5 × 10-7 M. k At the 10-7 M. l At the 10-4 M.
Table 3. Relaxant Activity of Some Compounds and Nifedipine on
K+-Depolarized Guinea Pig Ileum Longitudinal Smooth Muscle
b
cmpd activitya (M ( SEM) IC50 (µM) 95% conf lim (×10-6
)
5Ae
5Ag
5Aj
5Ak
5Al
92 ( 1.5
81 ( 3.2c
51 ( 2.4d
51 ( 0.6
89 ( 3.5e
64 ( 2.6f
86 ( 5.2g
97 ( 1.4
93 ( 0.5
94 ( 3.5f
89 ( 2.4g
70 ( 0.36h
1.66
1.95
0.083
12.35
0.055
0.55
11.04
2.56
2.06
1.36–2.05
1.54–2.47
0.066–0.103
8.56–17.80
0.043–0.070
0.43–0.69
5Ao
5Ap
5Aq
5Cb
5Cq
5Gp
Nif
8.18–14.90
1.90–3.11
1.55–2.73
0.19–0.29
10.00–16.41
0.0011–0.0022
0.24
12.81
0.0015
a Percent inhibition of calcium-induced contraction on K+-depolarized
(80 mM) guinea-pig longitudinal smooth muscle at 10-5 M. The 10-5
M
concentration gave the maximum effect for most compounds. b Calculated
from log concentration–response curves (Probit analysis according to
Litchfield and Wilcoxon16 with n ) 6–8). When the maximum effect was
<50%, the IC50 values were not calculated. c At 5 × 10-6 M. d At 10-7
M. e At 5 × 10-7 M. f At 10-6 M. g At 10-4 M. h At 5 × 10-9 M.
Figure 1. Correlation between negative chronotropic and inotropic
potencies (pEC50) for some of the new synthesized 1,4-DHPs. (O)
Compounds with selective negative inotropic potency. (b) Compounds
with selective negative chronotropic potency. (9) Compounds without
selectivity between negative inotropic and chronotropic activities.
smooth muscle with a good selectivity between the three
parameters. The peculiar cardiovascular profile of 5Cb was more
deeply investigated by comparing the negative inotropic and
chronotropic activities, coronary relaxation, and electrocardio-
gram (ECG) effects with those of Nifedipine on isolated
perfused heart according to Langendorff (Table 4). In perfused
Langendorff models, 1,4-DHPs produce ECG alterations such
as prolongation of ventricular repolarization time (QT interval),
and at high concentrations, they might prolong the atrioven-

1596 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Budriesi et al.
Table 4. Cardiac Parameters of 5Cb and Nifedipine Evaluated in the
Isolated Perfused Guinea Pig Spontaneously Beating Heart
functional studies. As shown in Figure 2a, 5Al that is endowed
with a selective negative chronotropic activity causes a con-
centration-dependent inhibition of PN200-110, (+)-[5-methyl-
3H] binding with pKi of 6.51, whereas nifedipine pKi was 8.83.
On the contrary, 5Ac (pKi ) 5.35) and 5Cq (pKi < 5), which
possess both negative inotropic and chronotropic activity, or
5Ag (pKi ) 6.8) and 5Cb (pKi < 5), which show a selective
negative inotropic activity, interact with the 1,4-DHPs binding
site with low affinity (Figure 2b). It is worth noting that the
discrepancy between functional and binding studies are not
unusual and several explanations for this peculiarity are possible:
the existence of high and low affinity sites for 1,4-DHPs and
their ability to bind other receptors; the affinity of the 1,4-DHPs
for the binding site is dependent upon the conformation of the
receptor, which is itself dependent on membrane potential; each
1,4-DHP may have unique effects on calcium channel gating.23
In addition, some 1,4-DHPs have demonstrated activity at a
variety of potassium and sodium channels.24 Of particular
cmpd
% changea
Nif
+(dP/dt)maxb
-96 ( 3g
-60 ( 2.3g
-36 ( 3.9h
+66 ( 2h
+37 ( 2g
-35 ( 1.5
+6 ( 0.5
+14 ( 0.9
+61 ( 3.1
-16 ( 1.1
HRc
CPPd
PRe
QTf
5Cb
+(dP/dt)max
HR
CPP
PR
QT
a Percentage change of the basal value at the highest concentration tested
(10 µM). Each value corresponds to the mean ( SEM (GraphPad Prism
Software,17,18 n ) 4–6). b +(dP/dt)max: maximal rate of the rise in left
ventricular pressure. c HR: heart rate calculated from ECG signal. d CPP:
coronary perfusion pressure. e PR: atrio-ventricular conduction time. f QT:
ventricular repolarization time. g At the 10-8 M. h At the 10-6 M.
interest are 1,4-dihydropyridines that are activators of K+
ATP
channels, where they are more active than at Ca2+ channels.25
1,4-DHPs also display activity at several G-protein-coupled
receptors and this is of interest because in several systems
significant selectivity of action occurs. Thus, the combination
of some of these factors could contribute to explain some
differences between radioligand binding and functional studies
and will stimulate further investigation to establish the activity
and/or selectivity toward other channels and receptors.
To further examine whether compounds 5Ac, 5Al, and 5Cq
bind directly to the R1c subunit or allosterically modulate it,
their effects on PN200-110, (+)-[5-methyl-3H] specific binding
were evaluated in intact HEK-293 cells stably expressing the
cardiac R1c-a or the vascular R1c-b subunit.26 No detectable
specific binding of PN200-110, (+)-[5-methyl-3H] was observed
in non-transfected HEK-293 cells (data not shown). In trans-
fected cells, the nonspecific binding of PN200-110, (+)-[5-
methyl-3H] was approximately 10–20% of the total binding.
Displacement of PN200-110, (+)-[5-methyl-3H] specific
binding by nifedipine, 5Ac, 5Al, and 5Cq in transfected HEK-
293 is shown in Figure 3. In agreement with literature data,26
nifedipine exhibited a lower affinity for the recombinant R1c-a
(IC50 ) 45 nM; Figure 3a) than R1c-b (IC50 ) 3.6 nM; Figure
3b) subunit. Similarly to nifedipine, compound 5Al exhibited a
lower affinity for the recombinant R1c-a (IC50 ) 265 nM; Figure
3a) than for the R1c-b subunit (IC50 ) 1.45 nM; Figure 3b). On
the contrary, compound 5Ac, which behaved as a weak
competitor in displacement binding assays carried out on guinea
pig cardiac membranes (Figure 2b), did not affect PN200-110,
(+)-[5-methyl-3H] specific binding in intact HEK-293 express-
ing either R1c-a or R1c-b subunit (Figure 3). Therefore, this
behavior is suggestive of an allosteric rather than directly
competitive binding of the compound 5Ac to the R1c cardiac
subunit occurring in guinea pig ventricular membranes.
Compound 5Cq behaved as a weak competitor for PN200-
110, (+)-[5-methyl-3H] specific binding both in ventricular
membranes (Figure 2b) and in HEK-293 cells expressing the
R1c-a subunit (Figure 3a), whereas it did not displace PN200-
110, (+)-[5-methyl-3H] in HEK-293 cells expressing the R1c-b
subunit (Figure 3b). This latter compound showed a profile of
cardiac versus vascular selectivity, which has been previously
reported for other 1,4-dihidropiridine substitutes.27
Figure 2. Effect of Nifedipine (4) and 5Al (b) (a) and 5Ac (9), 5Ag
(2), 5Cb ()), and 5Cq (O) (b) on PN 200-110, (+)-[5-methyl-3H]
binding on membranes prepared from guinea pig ventricular myocar-
dium. Points are the mean from three independent experiments (S.E.M.
smaller than the symbols).
tricular conduction time (PR interval).21,22 In our model,
Nifedipine showed to prolong the PR interval only at the highest
tested concentrations. This behavior might be related to the
depression of the heart rate (HR) and to first-degree atrio-
ventricular blocks that occasionally occur. In the same way,
5Cb prolonged PR interval but slightly reduced QT interval,
avoiding arising of the long QT syndrome.
B-Binding Assays. For some selected compounds, binding
assays on guinea pig ventricular membranes were carried out
to establish their binding affinity to the 1,4-DHP-sensitive site.
PN200-110, (+)-[5-methyl-3H] (0.5nM) was incubated with
increasing concentrations (0.1-50 µM) of the compounds as
described in Supporting Information. Data presented in Figure
2, showed the effects of 5Al (a) and 5Ac, 5Ag, 5Cb, 5Cq (b)
on PN200-110, (+)-[5-methyl-3H] binding. Compounds affect
the binding of PN200-110, (+)-[5-methyl-3H], with different
affinities which could be related to their different selectivity on
C-Computational Studies. To get major insights on the
molecular basis of the interactions between LTCC and the new
4-aryl-1,4-DHPs, docking simulations of the most active and
structurally diverse compounds (5Cb, 5Cq, and 5Ag) were

Imidazo[2,1-b]thiazole-1,4-DHPs as Cardiac Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1597
Figure 3. Effect of nifedipine (4), 5Al (b), 5Ac (9), and 5Cq (O) on
PN200-110, (+)-[5-methyl-3H] specific binding in HEK-293 cells
expressing the R1c-a (a) or R1c-b (b) subunit. Points are the mean from
three independent experiments (S.E.M. smaller than the symbols).
carried out using the published three-dimensional structure of
LTCC.28 Docking experiments were conducted employing the
new version of AutoDock (AutoDock4) software.29 The latest
version of this software implements a new force field that, using
an improved thermodynamic model of the binding process,
allows inclusion of intramolecular terms in the estimated free
energy. Moreover, the new force field includes a full desolvation
model that contains terms for all atom types, including the
favorable energetics of desolvating carbon atoms as well as the
unfavorable energetics of desolvating polar and charged atoms.
This force field also incorporates an improved model of
directionality in hydrogen bonds, now predicting the proper
alignment of groups with multiple hydrogen bonds. This
program was employed to perform 100 independent docking
runs for the selected compounds, which usually converged to a
small number of different clusters (“clusters” of results differing
by less than 1.5 Å rmsd). Even if the predicted free energy of
binding associated with each solution should be used as a
criterion for the choice of the “best” posing, herein the
preference for one solution has been also governed by its
consistency with experimental data (mutagenesis experiments
and SARs). In the following section, a brief description of the
calculated binding modes of the selected 1,4-DHPs into LTCC
is given together with the rationalization of the new SARs data.
Hereafter, to distinguish between the two sides of the 1,4-DHP
ring, as suggested by Goldmann et al.,30 the preferred conforma-
tion of 1,4-DHP moiety will be regarded as a flattered boat with
C4 as the bow, the axial aryl ring as the bowsprit, and the N1
atom as the stern. Accordingly, the two sides of the 1,4-DHP
ring will be referred as the port side (left) and the starboard
side (right).30 Docking of 5Cb placed dihydropyridine ring in
the cleft formed by IIIS6, IIIS5, and IVS6 segments in
consonance with experimental evidences (photoaffinity labeling,
construction of chimeric channels, and mutagenesis experiments)
suggesting that they contain all the residues critical for DHP
Figure 4. Docked structures of 5Cb (a), 5Cq (b), and 5Ag (c) in the
three-dimensional structure of L-type Ca2+ channel. DHPs are displayed
as orange sticks, and key interacting residues are shown in cyan.
Hydrogen bonds as represented with dashed yellow lines. The figure
was created by using PYMOL software.32
binding.31 Moreover, the plane of the ligand 1,4-DHP ring is
parallel to the pore axis, the ligand NH group faces the IIIS5
segment, the starboard side of the heterocyclic ring points
upward, and the plane of the imidazo[2,1-b]thiazole system is
perpendicular to the pore axis. Such a binding pose allows the
ligand to establish numerous interactions with the protein (Figure
4a).32

1598 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Budriesi et al.
In particular, the N1 hydrogen atom of the 1,4-DHP hetero-
cyclic ring H-bonds with the carbonyl oxygen of Q1060 side
chain in IIIS5. This is in consonance with SAR studies reported
for other 1,4-DHPs compounds which indicate that the N1
hydrogen atom has a key role in the binding of 1,4-DHPs to
LTCC.30 Moreover, mutagenesis data clearly demonstrated that
Q1070 contributes to the binding of 1,4-DHPs.31 As depicted
in Figure 4a,32 both the carbonyl and the ester oxygens on the
starboard side of the dihydropyridine ring form two H-bonds
with the S1131 and Y1169 side chains. The involvement of
Y1169 in a H-bond with the carbonyl oxygen of the starboard
side esters of the most common 1,4-DHPs is in agreement with
mutagenesis data. In fact, when mutating Y1169 to A (S)-
isradipine resulted to be 25-fold less active on the resulting
mutant.33 Moreover, in the Y1169F mutant (S)-isradipine
demonstrated to be 12.4-fold less active if compared with the
wild-type channel.33 This demonstrates the involvement of
Y1169 hydroxyl group in a H-bond with the ligand in agreement
with the proposed binding pose. The involvement of S1131 in
the binding of 1,4-DHPs was demonstrated by Yamaguchi et
al., who reported that when mutating S1131 to A the IC50 value
of (S)-nitrendipine was 39.4 times higher than that of rbCII (rat
brain Ca2+ channel R1C subunit type II).34 Differently from other
known 1,4-DHPs, 5Cb features the presence of an imidazo[2,1-
b]thiazole system instead of a phenyl ring. This system perfectly
fits into the channel 1,4-DHP binding cavity and establishes
with it favorable interactions. Most precisely, the aromatic
system is well oriented to form a T-shaped interaction with
Y1508, which is predicted to be reinforced by the electron-
withdrawing effect of the chlorine atom in R2 (see Table 1).
The involvement of Y1508 in the binding of Ca2+-antagonists
1,4-DHPs was experimentally proven by mutagenesis studies.
In fact, replacement of this residue to A has large effects on
1,4-DHP activities, with the KD for 1,4-DHP binding in
Y1508A mutant increased by 6.1-fold.35 In addition, the methyl
group in R takes favorable hydrophobic contacts with M1509.
Indeed, the importance of an electron-withdrawing group in R2
and a lipophilic one in R is further demonstrated by biological
data. In fact, the structurally related compounds 5Ac displayed
comparable inotropic activities (Table 2), whereas the different
chemical properties of the above-mentioned groups in 5Ba, 5Bb,
and 5Db results in a slightly lower activity. Docking calculations
conducted on 5Cq resulted in a binding pose similar to that
found for 5Cb (Figure 4b).32 Although, differently to 5Cb, in
5Cq the imidazo[2,1-b]thiazole system is substituted in R2 with
a phenyl ring that establishes additional π-stacking interactions
with Y1508 side chain. Moreover, the above cited interaction
is further reinforced by the presence of the electron-withdrawing
nitro group in para-position on the phenyl ring in R2. In 5Cq
this phenyl ring is also substituted in ortho-position by a
methoxy group, which with its ether oxygen H-bonds with
T1132 side chain. On the contrary, the other ortho-methoxy
group on the port side of the molecule engages hydrophobic
interactions with M1177 side chain. Also, in this case, the above-
cited residue has been confirmed to be critical for 1,4-DHP
binding.33 The presence of supplementary interactions with
LTCC might explain why 5Cq resulted to have a higher
inotropic potency if compared with 5Cb. Docking results on
5Cq also allow to infer the reasons for the different inotropic
potencies recorded for its structural congeners. In fact, good
negative inotropic potencies, ranging from 0.026 to 0.36 µM,
were found for structurally related compounds 5Ap, 5Aq, 5Bq,
5Cp, 5Cq, and 5Gp, which feature the same kind of substitu-
tions on the phenyl ring in R2 but in different positions, still
Figure 5. Synopsis of the SAR findings: the red groups for the best
inotropic potency, the blue groups for the best chronotropic potency,
the magenta groups for the best inotropic selectivity, and the green
groups for the best chronotropic selectivity.
allowing the interaction with the above cited residues. It remains
unknown the anomalous of 5Bp (weak negative inotropic agent)
and the low negative inotropic potency of 5Ar. On the other
hand, the absence of the electron-withdrawing nitro group on
the phenyl ring could explain why compounds such as 5Ai, 5Aj,
5Am, 5An, 5Bk, 5Ck, and 5Fk are endowed of lower inotropic
potency (range from 0.90 to 2.64 µM). Docking on 5Ag
converged toward a well-defined solution in which the DHP
ring fits in the cleft formed by IIIS6, IIIS5, and IVS6 segments
similarly to what found for the above-described compounds.
Nevertheless, if compared with 5Cq, the presence of a single
ortho-substitution in 5Ag allows this ring to be nearly parallel
to the imidazo[2,1-b]thiazole moiety (Figure 4c). This different
orientation allows the R2 aromatic portion to establish a
T-shaped interaction with Y1508 and an off-centered parallel
displaced π-π interaction with F1133, which are strengthened
by the trifluoromethoxy group that provides an electron-
withdrawing effect on the aromatic ring. Moreover, the same
group also H-bonds through its fluorine and oxygen atoms with
T1132 side chain. The proposed binding mode is in accordance
with the SARs presented herein. In fact, when the trifluo-
romethoxy group is moved from the ortho- to the meta-position
(5Ah) lower inotropic potency was found. This might be
rationalized by the absence of the H-bond interactions with
T1132 side chain. Indeed, when the trifluorometoxy is substi-
tuted by a trifluoromethyl group (5Ae), the same interactions
were detected (data not shown), which explains why similar
potencies were recorded in functional studies. In the same way,
if the H-bond accepting group is moved from the ortho- to the
meta-position (5Af), lower inotropic activities were recorded
due to the loss of a polar interaction with T1132.
Conclusions
In this paper we report the in Vitro biological characterization
of a new set of 1,4-DHPs. In summary, it is notable the lack of
vasorelaxant activity of new derivatives of 1,4-DHPs in which
the phenyl ring on the C-4 position was replaced with imi-
dazo[2,1-b]thiazole. As a matter of fact, in a previous work we
underlined this for some 1,4-DHPs bearing different heterocyclic
systems.4 A number of structure–activity relationship trends have
been identified and rationalized through molecular modeling
studies. We found that the group in position 6 (R2) is
determining to influence the cardiovascular activity (Figure 5).
Nevertheless, the weak affinity to calcium channel measured
by binding assay on the new 4-aryl-DHPs showed a peculiar
cardiovascular activity profile. Morel et al. comparing some
well-known 1,4-DHPs in cells expressing the cardiac Cav1.2a
and smooth muscle Cav1.2b subunits demonstrated that these
subunits are different in sensitivity to 1,4-DHPs.26 There are

Imidazo[2,1-b]thiazole-1,4-DHPs as Cardiac Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1599
several explanations for selective action of 1,4-DHPs toward a
tissue: the existence of various types and heterogeneous
distribution of voltage-operated Ca2+ channels,36 discovery of
splicing variants of R1 subunits,37 actions other than calcium
channel blocking,38 frequency and voltage-dependency of the
action,39 and physicochemical properties of drugs40 are some
descriptors for this effect.
We used HEK-293 cells transfected either with cDNA
encoding for the R1c-a or R1c-b subunit of the LTCC to ascertain
that some of these novel compounds, such as 5Ac, may
allosterically modulate binding affinity of PN200-110, (+)-[5-
methyl-3H] to the LTCC.
Compounds that selectively act on cardiac tissue with a
negative inotropic and/or chronotropic effect and devoid of
effects on vascular tissues might be used in the treatment of
many cardiovascular dysfunctions, such as cardiac hypertrophy
and ischemia. Although attempts have been made to improve
the prognosis of ischemia-evoked cardiac failure with second-
generation calcium antagonists, this could be an interesting
approach.41 In fact, the most common agents used to treat the
above cited pathologies are the ꢀ1 adrenoreceptor antagonists,
which most of the time are not devoid of side effects due to the
lack of ꢀ1/ꢀ2-adrenoreceptor selectivity. In conclusion, the
encouraging and interesting pharmacological profile of the new
imidazo[2,1-b]thiazole 1,4-DHPs certainly deserves further
studies that are currently in progress to identify new tissue-
selective calcium antagonists useful to treat common cardio-
vascular pathologies such as cardiac hypertrophy and ischemia.
vascular subunits. Supported by grants from M.I.U.R.: PRIN-
2003 “Design, synthesis and biological evaluation of new
cardiovascular drugs” and from the University of Bologna.
Supporting Information Available: Chemistry, analytical data,
functional assays, receptor binding studies, and computational
methods. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) (a) Catterall, W. A.; Striessnig, J.; Snutch, T. P.; Perez-Reyes, E.
International Union of Pharmacology. XL. Compendium of voltage-
gated ion channels. Calcium channels. Pharmacol. ReV. 2003, 55, 579–
581. (b) Catterall, W. A.; Perez-Reyes, E.; Terrance, P.; Striessnig, J.
International Union of Pharmacology. XLVIII. Nomenclature and
structure-function relationships of voltage-gated calcium channels.
Pharmacol. ReV. 2005, 57, 411–425.
(2) (a) Heilbrunn, L. V. An Outline of General Physiology, 1st, 2nd, and
3rd editions; Saunders Co.: Philadelphia, PA, 1952; pp 1937–1943.
(b) Carafoli, E. Calcium signaling: A tale for all seasons. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 1115–1122. (c) Berridge, M. J.; Lipp, P.;
Bootman, M. D. The versatility and universality of calcium signaling.
Nat. ReV. Mol. Biol. 2000, 1, 11–21. (d) Verkhratsky, A.; Landfield,
P. W.; Thibault, O. Ca2+ and neuronal pathology. Eur. J. Pharmacol.
2002, 447, 115–296.
(3) Triggle, D. J. Calcium-channel drugs: Structure-function relationships
and selectivity of action. J. CardioVasc. Pharmacol. 1991, 18, S1–6.
(4) (a) Budriesi, R.; Aicardi, G.; Campana, G.; Spampinato, S.; Zaza, A.;
Bisi, A.; Rampa, A.; Valenti, P.; Chiarini, A. Selective cardiodepressant
activity of fluodipine, a fluorenone 1,4-dihydropyridine derivative. Eur.
J. Pharmacol. 1998, 359, 161–170. (b) Budriesi, R.; Rampa, A.; Bisi,
A.; Fabbri, G.; Chiarini, A.; Valenti, P. Structure-activity relationship
studies of xanthone and fluorenone-1,4-dihydropyridine-5-phospho-
nates. Arzneim.-Forsch. 1996, 46, 374–377.
(5) Spedding, M.; Paoletti, R. Classification of calcium channels and the
sites of action of drugs modifying channel function. Pharmacol. ReV.
1992, 44, 363–376.
(6) Ertel, E. A.; Campbell, K. P.; Harpold, M. M.; Hofmann, F.; Mori,
Y.; Perez-Reyes, E.; Schwartz, A.; Snutch, T. P.; Tanabe, T.;
Birnbaumer, L.; Tsien, R. W.; Catterall, W. A. Nomenclature of
voltage-gated calcium channel. Neuron 2000, 25, 533–535.
(7) Tanabe, T.; Takeshima, H.; Kikami, A.; Flockerzi, V.; Takahashi, H.;
Kangawa, K.; Kojima, M.; Matsuo, H.; Hirose, T.; Numa, S. Primary
structure of the receptor for calcium blockers from skeletal muscle.
Nature 1987, 328, 313–318.
(8) Mikami, A.; Imoto, K.; Tanabe, T.; Niidome, T.; More, Y.; Takeshima,
H.; Narumiya, S.; Numa, S. Primary structure and functional expression
of the cardiac dihydropyridine-sensitive calcium channel. Nature 1989,
340, 230–233.
Experimental Section
A. Chemistry. The melting points are uncorrected. Analyses
(C, H, N) were within (0.4% of the theoretical values (see
Supporting Information, Table S3). TLC was performed on Bak-
erflex plates (Silica gel IB2-F); the eluent was a mixture of
petroleum ether 60–80 °C/acetone in various proportions. Kieselgel
60 (Merck) was used for flash chromatography. The IR spectra were
recorded in nujol on a Nicolet Avatar 320 E.S.P.; νmax is expressed
in cm-1. The 1H NMR spectra were recorded on a Varian Gemini
(300 MHz); the chemical shift (referenced to solvent signal) is
expressed in δ (ppm) and J in Hz (see Supporting Information,
Table S2). All solvents and reagents, unless otherwise stated, were
supplied by Aldrich Chemical Company Ltd. and were used as
supplied.
(9) Williams, M. E.; Feldman, D. H.; McCue, A. F.; Brenner, R.;
Velicelebi, G.; Ellis, S. B.; Harpold, M. M. Structure and functional
expression of R1, R2, and ꢀ subunits of a novel human neuronal calcium
channel subtype. Neuron 1992, 8, 71–84.
For a new starting compound, please see Table S1 in Supporting
Information.
(10) Strom, T. M.; Nyakatura, G.; Apfelstedt Sylla, E.; Hellebrand, H.;
Lorenz, B.; Weber, B. H.; Wurtz, K.; Gutwillinger, N.; Ruther, K.;
Drescher, B.; Sauer, C.; Zrenner, E.; Meitinger, T.; Rosenthal, A.;
Meindl, A. An L-type calcium-channel gene mutated in incomplete
X-linked congenital stationary night blindness. Nat. Genet. 1998, 19,
260–263.
(11) Triggle, D. J. The 1,4-Dihydropyridines nucleus: A pharmacophoric
template Part 1. Actions at ion channels. Mini.-ReV. Med. Chem. 2003,
3, 215–223.
General Procedure for the Synthesis of the Dihydropy-
ridines 5Ac-5Gr. Methylacetoacetate (2 mM) and 30% NH4OH
(4 mM) were added to a stirred solution of the appropriate aldehyde
4 (1 mM) dissolved in isopropyl alcohol (50 mL). The reaction
mixture was refluxed for 1–4 days (according to a TLC test acetone/
petroleum ether 55–85 °C, 1:9 v/v, 2:8 v/v) and added of
methylacetoacetate (4 mM) and 30% NH4OH (2 mM) every 12 h.
After cooling, the mixture was evaporated to dryness under reduced
pressure. All the derivatives were purified by flash chromatography.
See Table 1 for eluent and yield.
B. Functional Studies. For details, please see Supporting
Information, section S11.
C. Receptor Binding Studies. For details, please see Supporting
Information, section S14.
(12) Triggle, D. J. L-Type calcium channel. Curr. Pharm. Des. 2006, 12,
443–457.
(13) Evans, B. E.; Rittle, K. E.; Block, M. G.; Di Pardo, R. M.; Friedinger,
R. M.; Whitter, W. L.; Lindell, G. F.; Veber, D. F.; Anderson, P. S.;
Chang, R. S. L.; Lotti, V. J.; Cerino, D. J.; Chen, T. B.; Kling, P. J.;
Kunkel, K. A.; Springer, J. P.; Hirshfield, J. Methods for drug
discovery: Development of potent, selective, orally effective chole-
cystokinin antagonists. J. Med. Chem. 1988, 31, 2235–2246.
(14) Andreani, A.; Leoni, A.; Rambaldi, M.; Locatelli, A.; Bossa, R.; Galatulas,
I.; Chiericozzi, M.; Bissoli, M. Dihydropyridines bearing an imidazo[2,1-
b]thiazole system. Eur. J. Med. Chem. 1997, 32, 151–157.
(15) Phillips, A. P. Hantzsch’s pyridine synthesis. J. Am. Chem. Soc. 1949,
71, 4003–4007.
D. Computational Methods. For details, please see Supporting
Information, section S15.
Acknowledgment. The authors are grateful to Prof. A. J.
Olson (The Scripps Research Institute) for providing a beta
version of the software AutoDock4 and to Prof. A. Welling
(Institut für Pharmakologie and Toxicologie der Technischen
Universität Mñchen) for providing cloned R1-cardiac and
(16) Tallarida, R. J.; Murray, R. B. Manual of Pharmacologic Calculations
with Computer Programs, 2nd ed.; Springer-Verlag: New York, 1987.
(17) Motulsky, H.; Christopoulos, A. Fitting Models to Biological Data
Using Linear and Non Linear Regression, 2003, www.GraphPad.com.

1600 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Budriesi et al.
(18) Motulsky H. Statistic Guide: Statistical Analysis for Laboratory and
Clinical Research: A Pratical Guide to Curve Fitting, 2003, www.
GraphPad.com.
(30) Goldmann, S.; Stoltefuss, J. 1,4-Dihydropyridines: effects of chirality
and conformation on the calcium antagonist and calcium agonist
activities. Angew. Chem., Int. Ed. Engl. 1991, 30, 1559–1578.
(31) Hockerman, G. H.; Johnson, B. D.; Scheuer, T.; Catterall, W. A.
Molecular determinants of high-affinity phenylalkylamine block of
L-type calcium channels. J. Biol. Chem. 1995, 270, 22119–22122.
(32) DeLano, L. The PyMOL Molecular Graphics System, 2002, http://
www.pymol.org.
(33) Peterson, B. Z.; Johnson, B. D.; Hockerman, G. H.; Acheson, M.;
Scheuer, T.; Catterall, W. A. Analysis of the dihydropyridine receptor
site of L-type calcium channels by alanine-scanning mutagenesis.
J. Biol. Chem. 1997, 272, 18752–18758.
(19) GraphPad, version 3.0; GraphPad Software, Inc.: San Diego, CA.
(20) Bolger, G. T.; Genco, P.; Klockowski, R.; Luchowski, E.; Siegel, H.;
Janis, R. A.; Triggle, A. M.; Triggle, D. J. Characterization of binding
of the Ca+2 channel antagonist, [3H]nitrendipine, to guinea-pig ileal
smooth muscle. J. Pharmacol. Exp. Ther. 1983, 225, 291–309.
(21) Nielsen-Kudsk, F.; Askholt, J.; Hyldgard Jensen, P. A comparative
study of the pharmacodynamic effects of nimodipine and nifedipine
in the isolated spontaneously beating rabbit heart. Acta Pharmacol.
Toxicol. 1983, 52, 105–111.
(34) Yamaguchi, S.; Zhorov, B. S.; Yoshioka, K.; Nagao, T.; Ichijo, H.;
Adachi-Akahane, S. Key roles of Phe1112 and Ser1115 in the pore-
forming IIIS5-S6 linker of L-type Ca2+ channel alpha-1C subunit
(Cav1.2) in binding of dihydropyridines and action of Ca2+ channel
agonists. Mol. Pharmacol. 2003, 64, 235–248.
(35) (a) Mitterdorfer, J.; Wang, Z.; Sinnegger, M. J.; Hering, S.; Striessnig,
J.; Grabner, M.; Glossmann, H. Two amino acid residues in the IIIS5
segment of L-type calcium channels differentially contribute to 1,4-
dihydropyridine sensitivity. J. Biol. Chem. 1996, 271, 30330–30335.
(b) Mitterdorfer, J.; Wang, Z.; Sinnegger, M. J.; Hering, S.; Striessnig,
J.; Grabner, M.; Glossmann, H. Two amino acid residues in the IIIS5
segment of L-type calcium channels differentially contribute to 1,4-
dihydropyridine sensitivity. J. Biol. Chem. 1996, 271, 30330–30335.
(36) Hermsmeyer, K. Role of T channels in cardiovascular function.
Cardiology 1998, 89 (Suppl. 1), 2–9.
(37) Welling, A.; Ludwig, A.; Zimmer, S.; Klugbauer, N.; Flockerzi, V.;
Hofmann, F. Alternatively spliced IS6 segments of the alpha 1C gene
determine the tissue-specific dihydropyridine sensitivity of cardiac and
vascular smooth muscle L-type Ca2+ channels. Circ. Res. 1997, 81,
526–532.
(38) Godfraind, T. Calcium antagonists and vasodilatation. Pharmacol.
Ther. 1994, 64, 37–75.
(39) (a) Triggle, D. J. Calcium, calcium channels, and calcium channel
antagonists. Can. J. Physiol. Pharmacol. 1990, 68, 1474–1481. (b)
Gurney, A. M. Mechanisms of drug-induced vasodilatation. J. Pharm.
Pharmacol. 1994, 46, 242–251.
(40) (a) Hattori, Y.; Fukao, M.; Houzen, H.; Qi, F.; Yamada, Y.; Kanno,
M. In vitro assessment for vascular selectivity of a new dihydropyridine
derivative, NB-818. Eur. J. Pharmacol. 1996, 301, 99–106. (b)
Hockerman, G. H.; Peterson, B. Z.; Johnson, B. D.; Caterall, W. A.
Molecular determinants of drug binding and action on L-type calcium
channels. Annu. ReV. Pharmacol. Toxicol. 1997, 37, 361–396.
(41) Singh, N. B.; Nademanee, K.; Baky, S. H. Calcium Antagonists:
Clinical use in the treatment of arrhythmias. Drugs 1983, 25, 125–153.
(22) Askholt, J.; Nielsen-Kudsk, F. Felodipine, pharmacodynamics and
pharmacokinetics in the isolated rabbit heart. Acta Pharmacol. Toxicol.
1986, 59, 17–26.
(23) (a) Hamilton, S. L.; Yatani, A.; Brush, K.; Schwartz, A.; Brown, A.
A comparison between the binding and electrophysiological effects
of dihydropyridines on cardiac membranes. Mol. Pharmacol. 1987,
31, 221–231. (b) Schwartz, A.; Triggle, D. J. Cellular action of calcium
channel blocking drugs. Annu. ReV. Med. 1984, 35, 325–329.
(24) Triggle, D. J. Mechanisms of action of calcium antagonists In Calcium
Antagonists in Clinical Medicine; Epstein, M. Ed.; Hanley and Belfus:
Philadelphia, PA, 2002; pp 1–32..
(25) (a) Frank, C. A.; Forst, J. M.; Harris, R. J.; Kau, S. T.; Li, J. H.;
Ohmmachb, C. J.; Smith, R. W.; Trainor, D. A.; Trivedi, S.
Dihydropyridine KATP potassium channel openers. Bioorg. Med. Chem.
Lett. 1993, 3, 2725–2726. (b) Trivedi, S.; Stetz, S. L.; Potter-Lee, L.;
McConville, M.; Li, J. H.; Empfield, J.; Ohnmacht, C. J.; Russell, K.;
Brown, F. J.; Trainor, D. A.; Kau, S. T. K-channel opening activity
of ZD6169 and its analogs: effect on 86Rb efflux and 3H-P1075
binding in bladder smooth muscle. Pharmacology 1995, 50, 388–397.
(26) Morel, N.; Buryi, V.; Feron, O.; Gomez, J. P.; Christen, M. O.;
Godfraind, T. The action of calcium channel blockers on recombinant
L-type calcium channel alpha1-subunits. Br. J. Pharmacol. 1998, 125,
1005–1012.
(27) Alker, D.; Campbell, S. F.; Cross, P. E.; Burges, R. A.; Carter, A. J.;
Gardiner, D. G. Long-acting dihydropyridine calcium antagonists. 4.
Synthesis and structure-activity relationships for a series of basic and
nonbasic derivatives of 2-[(2-aminoethoxy)methyl]-1,4-dihydropyridine
calcium antagonists. J. Med. Chem. 1990, 33, 585–591.
(28) Cosconati, S.; Marinelli, L.; Lavecchia, A.; Novellino, E. Character-
izing the 1,4-dihydropyridines binding interactions in the L-Type Ca2+
channel: Model construction and docking calculations. J. Med. Chem.
2007, 50, 1504–1513.
(29) Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. A semiempirical
free energy force field with charge-based desolvation. J. Comput.
Chem. 2007, 28, 1145–1152.
JM070681+