Ligand based approach to L-type calcium channel by imidazo[2,1-b]thiazole- 1,4-dihydropyridines: From heart activity to brain affinity

Article abstract of DOI:10.1021/jm301839q

The synthesis, characterization, and functional in vitro assay in cardiac and smooth muscle (vascular and nonvascular) of a series of 4-imidazo[2,1-b] thiazole-1,4-dihydropyridines are reported. To define the calcium blocker nature of the imidazo[2,1-b]thiazole-1,4-DHPs and their selectivity on Ca _v1.2 and Ca_v1.3 isoforms, we performed binding studies on guinea pig atrial and ventricular membranes on intact cells expressing the cloned Ca_v1.2a subunit and on rat brain cortex. To get major insights into the reasons for the affinity for Ca_v1.2 and/or Ca _v1.3, molecular modeling studies were also undertaken. Some physicochemical and pharmacokinetic properties of selected compounds were calculated and compared. All the biological data collected and reported herein allowed us to rationalize the structure-activity relationship of the 4-imidazo[2,1-b]thiazole-1,4-DHPs and to identify which of these enhanced the activity at the central level.

Full text of DOI:10.1021/jm301839q

Article
pubs.acs.org/jmc
Ligand Based Approach to L‑Type Calcium Channel by
Imidazo[2,1‑b]thiazole-1,4-Dihydropyridines: from Heart Activity to
Brain Affinity
Alessandra Locatelli,† Sandro Cosconati,‡ Matteo Micucci,† Alberto Leoni,*,† Marinelli Luciana,§
Andrea Bedini,† Pierfranco Ioan,† Santi Mario Spampinato,† Ettore Novellino,§ Alberto Chiarini,†
and Roberta Budriesi*,†
†Dipartimento di Farmacia e Biotecnologie, Universita
̀
degli Studi di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
‡DiSTABiF, Seconda Universita
̀
di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
§Dipartimento di Chimica Farmaceutica e Tossicologica, Universita
Napoli, Italy
̀
degli Studi di Napoli “Federico II”, Via Montesano 49, 80131
S
* Supporting Information
ABSTRACT: The synthesis, characterization, and functional in vitro assay in cardiac and smooth muscle (vascular and
nonvascular) of a series of 4-imidazo[2,1-b]thiazole-1,4-dihydropyridines are reported. To define the calcium blocker nature of
the imidazo[2,1-b]thiazole-1,4-DHPs and their selectivity on Cav1.2 and Cav1.3 isoforms, we performed binding studies on
guinea pig atrial and ventricular membranes on intact cells expressing the cloned Cav1.2a subunit and on rat brain cortex. To get
major insights into the reasons for the affinity for Cav1.2 and/or Cav1.3, molecular modeling studies were also undertaken. Some
physicochemical and pharmacokinetic properties of selected compounds were calculated and compared. All the biological data
collected and reported herein allowed us to rationalize the structure−activity relationship of the 4-imidazo[2,1-b]thiazole-1,4-
DHPs and to identify which of these enhanced the activity at the central level.
cardiac inotropy and in neurons, Cav1.3 (α1D subunit),
predominantly expressed in neurons and in cardiac pacemaker
cells, and Cav1.4 (α1F subunit), with physiological function as
neurotransmitter release in retinal cells.3 Several studies have
demonstrated that specific drugs, activator or blocker, interact
at discrete receptor sites in accordance with their chemical
heterogeneity which has been extensively reviewed since the
1970s.4,5 LTCC blockers are a group of leading drugs directed
at cardiovascular diseases including hypertension, angina,
peripheral vascular disease, and some arrhythmias.6,7 In
particular, LTCC isoforms Cav1.2 and Cav1.3 are predom-
inantly expressed in neurons and, for this reason, are now
considered viable drug targets to treat central nervous system
(CNS) disease such as Parkinson or pain perception.3,8,9 The
INTRODUCTION
■
Voltage-gated Ca2+ channels (VGCCs) play a critical role in
many physiological functions, from muscle contraction to
neurosecretion in a variety of tissues.1 The 10 members of
VGCCs family, with different role in cellular signal trans-
duction, have been characterized in mammals.2 The calcium
channels are complex proteins of four or five distinct subunits.
The α1 subunit is the largest, and it incorporates the conduction
pore and the majority of the known sites of channel regulation
by drugs. This main site of action is located at α1 subunit
isoforms that belong to three different subfamilies (Cav1, Cav2,
and Cav3). The accessory subunits (α2-δ, β, and γ) fine-tune the
channel function. L-Type Ca2+ channels (LTCCs) are formed
by the Cav1 family, which comprises four isoforms: Cav1.1 (α1S
subunit), predominantly expressed in skeletal muscle, Cav1.2
(α1C subunit), also known as cardiac isoform widely expressed
in cardiovascular system where it regulates vascular tone and
Received: December 13, 2012
© XXXX American Chemical Society
A
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class of ligands with LTCCs higher affinity are 1,4-
dihydropyridines, whose historical lead is Nifedipine. In
cardiovascular systems, Nifedipine is selective for vascular
smooth muscle, allowing its use and that of subsequent 1,4-
DHP’s generations in the treatment of hypertension.10,11 It is
well-known that high doses of 1,4-DHPs, such as Nifedipine,
cause a variety of brain effects such as amelioration of age-
related working memory deficits, anxiolytic and antidepressant-
like actions, inhibition of fear memory extinction, anticonvul-
sant effects, and effects on drug-taking behavior.12 Unfortu-
nately, so far, it is not clear if these effects are mediated by
LTCCs or by other blocking target because of the difficulty in
finding selective LTCC modulators for the brain.12 At the same
time, it should not be underestimated the problem related to
therapeutic use of the LTCC modulators for diseases of the
CNS because of their poor selectivity. Indeed, Nifedipine binds
both Cav1.2 and Cav1.3 isoforms with high affinity. Its low
specificity together with its difficulty in overcoming the blood−
brain barrier are key requirements for use in cardiovascular
therapy but also greater limits.12
Chart 1
1,4-DHPs, known from different decades and spread across
several generations, could still represent a valuable tool for the
characterization of peripheral and central Cav1.2 and Cav1.3
isoforms LTCC. In the framework of our research, we
recently13 described the design, synthesis, and structure−
activity relationship of a small 1,4-DHPs library bearing in
position 4 a variously substituted imidazo[2,1-b]thiazole
system. The substitutions on the heterocycle scaffold, along
with the profile of activity shown on isolated cardiac tissues
streamlined through computational studies and confirmed by
binding studies on isolated cardiomyocytes and HEK-293 cells
transfect with α1c‑a and α1c‑b subunit, made it possible to
correlate the selectivity and potency both with specific
substituents and both with their position on the heterocyclic
system. Herein, to deepen the influence of the chemical
modifications of imidazo[2,1-b]thiazole-1,4-DHPs on the
peripheral and central activity and/or on the selectivity of
calcium channels, we synthesized a new series of compounds
1−13 (Chart 1) using a ligand based approach and taking into
account the previous data13 of the inotropic or chronotropic
effects caused by substituents in the imidazo[2,1-b]thiazole
system. Moreover, we extended the investigation not only to
the functional studies but also to the binding studies to verify
the calcium blocker nature of the imidazo[2,1-b]thiazole-1,4-
DHPs on Cav1.2 and Cav1.3.
Scheme 1
5.96, and finally, the NH gives rise to a broad singlet in the
range 8.01 and 9.13 which exchange by treatment with D2O.
The signals corresponding to the imidazothiazole substituted
moietys were assigned on the basis of previous papers.13,19
PHARMACOLOGY
■
Functional Assays. The pharmacological profile of all
compounds was derived on guinea pig isolated left and right
atria to evaluate their inotropic and/or chronotropic effects,
respectively, and on K+-depolarized (80 mM) guinea pig
vascular (aortic strips) and nonvascular [ileum longitudinal
smooth muscle (GPILSM)] to assess the calcium antagonist
activity. Compounds were checked at increasing doses to
evaluate: (i) the percent decrease of developed tension on
isolated left atrium driven at 1 Hz and on spontaneously
beating right atrium (negative inotropic activity), (ii) the
percent decrease in atrial rate on spontaneously beating right
atrium (negative chronotropic activity), and (iii) the percent
inhibition of calcium-induced contraction on K+-depolarized
aortic strips and GPILSM (vascular and nonvascular relaxant
activity, respectively). Details have been reported in Supporting
Information. Data were analyzed using Student’s t-test and are
presented as mean SEM.20 Because the analyzed compounds
were added in cumulative 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 and IC50 was evaluated from log concentration−response
CHEMISTRY
■
As shown in Scheme 1, the synthesis of the new 4-imidazo[2,1-
b]thiazole-1,4-dihydropyridines 1−13 (Table 1) was accom-
plished by means of the well-known Hantzsch reaction:14 one-
pot condensation of the appropriate β-ketoester, methylaceta-
toacetate, or ethylacetoacetate with the opportune aldehydes
a−m15−18 in a solution of aqueous ammonia and isopropyl
alcohol. After standard purification the 1,4-DHPs were
obtained with a range of 5−25% yield. All the structures of
1
final products were confirmed with infrared and H nuclear
magnetic resonance spectra. The new compounds synthesized
gave 1H NMR spectra in agreement with the assigned
structures and showed common features as regards the 1−4
DHP moiety: the methyl groups in position 2,6 appear as a
singlet in the range 1.91 and 2.23 ppm, the methyl groups of
the ester give a singlet in the range 3.21 and 3.44 ppm, the
singlet corresponding to H-4 is evident in the range 4.99 and
B
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Table 1. 1,4-Dihydropyridines
RESULTS
■
A. Functional Study on Cardiac System. All the newly
starting
compd aldheydes
x−y
R
R1
synthesized (1−13, Chart 1) 1,4-DHPs bearing in C-4 a
differently substituted imidazo[2,1-b]thiazole system were
tested for their cardiovascular profile on guinea pig left atrium
driven at 1 Hz and on a spontaneously beating right atrium to
evaluate their inotropic and chronotropic effects, respectively,
as previously done for the analogues that have already been
published (14−31).13 Data of cardiac activity for all
compounds (1−31) are collected in Table 2 using Nifedipine
as the reference drug. All compounds show interesting
cardiovascular activity profile, except 5 and 31, which are
devoid of activity. As shown in Table 2, most of the tested
compounds (1, 11, 13, 15−18, 20, 24−26, and 28) exhibited
both negative inotropic and chronotropic properties with
b
1
2
a
HCCH
H3CCCH
HCCH
2-Pyr
2-Pyr
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
c
b
b
3
c
3-Pyr
c
4
d
H3CCCH
HCCH
3-Pyr
c
5
e
4-Pyr
c
6
f
g
h
H3CCCH
HCCH
4-Pyr
d
d
7
2-thio
8
H3CCCH
HCCH
2-thio
d
d
d
9
i
j
3-thio
10
11
12
13
H3CCCH
HCCH
3-thio
k
2,4-Cl2-3-thio
2,4-Cl2-3-thio
d
l
H3CCCH
HCCH
e
m
2,5-(OCH3)2-
C6H3
different selectivity. 26 is the most potent of the series [EC50
=
0.59 μM (c.l. 0.43−0.81); EC50 = 0.66 μM (c.l. 0.51−0.85)
negative inotropic and chronotropic potency, respectively]. 1
and 11 have negative inotropic potency 45 and 47 times greater
than the negative chronotropic potency, respectively. Com-
pounds 12, 14, 21−23, 27, and 29−30 possess selective
negative inotropic effect also on the spontaneously beating right
atrium, even though in this case, the potency is lower. 12, 22−
23, and 27 are significantly more potent than Nifedipine as
negative inotropic compounds. Among the compounds studied,
2−4, 6−10, and 19 have only negative chronotropic properties
and they are less potent than Nifedipine.
B. Functional Studies on Guinea Pig Smooth Muscles.
All new 1,4-DHPs derivatives (1−13) were tested on K+-
depolarized (80 mM) guinea pig smooth muscles: aortic strips
and ileum longitudinal smooth muscle to assess their vascular-
or nonvascular-relaxant activity, respectively. The data of the
new compounds (1−13) and of the previously published
compounds (14−31)13 selected for the particular cardiovas-
cular profile are collected in Table 3 using Nifedipine as the
reference drug. All compounds (1−31) had no effect on
vascular smooth muscle. On the contrary, all compounds (1−
31) inhibited K+ induced contraction on guinea pig nonvascular
smooth muscle. Only the compound 9 showed an activity
comparable to that of Nifedipine [EC50 = 0.0023 μM (c.l.
0.0018−0.0030); EC50 = 0.0015 μM (c.l. 0.0011−0.0022);
respectively]. 13, 19, and 20 resulted from 30−50 times less
potent than Nifedipine.
C. Binding Studies on Guinea Pig Atria, Ventricles,
and on Intact Cells Expressing the Cloned Cav1.2a
Subunit. For compounds 1−31, competition binding assays
on guinea pig atrial and ventricular membranes have been
carried out; Nifedipine was employed as a reference compound.
All compounds were first dissolved in DMSO in order to obtain
solutions at 10−2 M concentration. These stock solutions were
diluted with buffer. All compounds at the concentrations used
were soluble in DMSO and in buffer. Addition of the drug
vehicle had no appreciable effect in binding experiments.
Binding assays have been also carried out on intact HEK-293
cells stably expressing the cardiac Cav1.2a subunit of the
LTCC.24 PN200−110, (+)-[5-methyl-3H] (0.5 nM) was
incubated with increasing concentrations (0.001−100 μM) of
all the tested compounds; IC50 and Ki values of all the tested
compounds are reported in Table 4 together with those of
Nifedipine. As expected, the reference compound Nifedipine
significantly bind to LTCC in both atrial and ventricular
membranes as well as in intact HEK-293 cells expressing the
a
14
15
16
17
18
19
HCCH
H3CCCH
ClCCH
HCCH
HCCH
HCCH
2,5-(OCH3)2-
C6H3
CH3
CH3
CH3
CH3
CH3
CH3
a
a
a
a
a
2,5-(OCH3)2-
C6H3
2,5-(OCH3)2-
C6H3
2,4-(OCH3)2-
C6H3
3,4-(OCH3)2-
C6H3
3,5-(OCH3)2-
C6H3
a
a
20
21
HCCH
HCCH
3-(OCH3)-C6H4
CH3
CH3
6-NO2-2,5-
(OCH3)2-C6H2
a
a
a
a
22
23
24
25
H3CCCH
H3C(CH2)2CCH
HCCH
6-NO2-2,5-
(OCH3)2-C6H2
CH3
CH3
CH3
CH3
6-NO2-2,5-
(OCH3)2-C6H2
4-NO2-2,5-
(OCH3)2-C6H2
H3CCCH
4-NO2-2,5-
(OCH3) -C6H2
2
a
a
a
a
a
a
26
27
28
29
30
31
H3CCCCH3
HCCH
C6H5
2-(CF3)-C6H4
(CF3)
Cl
CH3
CH3
CH3
CH3
CH3
CH3
HCCH
H3CCCH
HCCCH3
H3CCCH
Cl
CH3
a
b
c
d
Taken from ref 13. Taken from ref 15. Taken from ref 16. Taken
from ref 17. Taken from ref 18.
e
curves (Probit analysis using Litchfield and Wilcoxon20 or
GraphPad Prism software21,22) in the appropriate pharmaco-
logical preparations.
Binding Studies. Compounds were screened for their
affinity for calcium channels from guinea pig heart ventricles
and atria and rat brain cortex. Binding site was determined by
using a competitive radiometric receptor binding assay.
PN200−110, (+)-[5-methyl-3H] was used to label 1,4-DHPs
binding site. Binding affinity were expressed as Ki and IC50
values (mean SEM).23 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 the rabbit Cav1.2a (α1c‑a
)
encoding plasmid. Cav1.2a plasmid was 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 SEM).23
C
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Journal of Medicinal Chemistry
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Table 2. Cardiac Activity of Compounds 1-31
left atrium
right atrium
negative inotropy
negative inotropy
negative chronotropy
c
d
e
d
f
d
activity
compd (M SEM)
EC50
95% conf lim
(× 10−6
activity
EC50
95% conf lim
(× 10−6
activity
EC50
95% conf lim
(× 10−6
(μM)
)
(M SEM)
(μM)
)
(M SEM)
(μM)
)
g
1
2
58 2.7
42 3.2
25 1.6
18 0.7
46 1.7
44 2.6
46 1.4
40 1.3
47 2.2
12 0.7
74 2.1
60 1.9
93 1.2
92 3.4
78 0.9
55 2.3
71 2.8
61 2.7
26 1.3
90 3.7
71 0.9
71 0.1
55 1.6
54 3.4
75 3.6
76 1.4
56 2.4
90 3.8
63 2.7
79 3.1
43 3.3
97 2.0
0.54
0.36−0.79
71 1.3
72 0.9
98 0.7
24.39
39.91
28.44
12.46
18.50−29.21
32.71−45.72
21.38−36.96
9.67−16.10
h
g
3
h
h
h
i
4
86 1.6
g
5
25 1.2
i
6
93 2.1
7.49
1.30
40.50
1.06
2.30
2.16
3.52−9.91
1.04−1.62
31.62−48.87
0.80−1.41
1.74−3.04
1.83−2.55
j
7
85 1.6
h
8
70 3.4
j
9
87 3.3
10
11
12
13
85 1.8
j
0.046
0.31
1.16
0.44
1.43
0.90
1.24
2.64
0.032−0.064
0.093−0.81
0.85−1.68
0.29−0.65
1.02−1.94
0.56−1.44
0.93−1.48
2.03−3.01
77 1.4
g
82 1.6
85 2.7
0.73
0.56
0.35−1.12
0.38−0.82
22 1.1
g
88 2.1
2.63
2.10−3.28
a
i
i
g
14
31 1.6
a
,
b
b
i
15
16
58 3.4
6.62
0.98
8.60
3.01
1.36
1.36
4.37−10.02
0.73−1.33
7.47−9.90
2.41−3.76
1.08−1.78
0.97−1.70
a
,
84 2.7
a
i
i
17
55 2.5
a
,
b
i
18
80 1.9
a
j
19
90 0.5
a
,
b
j
20
21
1.90
1.65−2.27
0.25−0.51
94 2.0
a
,
b
0.36
87 2.1
80 1.1
1.11
0.31
0.39
0.74−1.63
0.18−0.48
0.25−0.62
12 0.9
47 3.2
a
a
a
a
a
a
j
22
0.093
0.039
0.054
0.026
0.59
0.063−0.14
0.030−0.051
0.036−0.079
0.018−0.036
0.43−0.81
0.068−0.12
0.021−0.14
0.041−0.076
0.08−0.15
h
j
j
i
23
24
25
26
27
75 3.4
25 1.3
g
67 2.3
6.08
3.59
0.66
5.19−7.12
2.71−4.76
0.51−0.85
j
g
87 5.3
j
94 2.4
h
i
j
g
0.093
0.071
0.056
0.10
90 2.5
0.16
0.12−0.22
25 1.8
a
,
,
,
,
b
k
28
54 4.2
0.86
0.74−1.01
a
a
a
b
b
b
k
i
i
29
30
31
95 1.7
1.70
1.89
1.26−2.04
1.32−2.09
22 0.7
h
i
67 1.8
39 1.5
l
i
33 2.6
g
k
Nif
0.26
0.19−0.36
c
85 4.2
0.039
0.031−0.051
a
b
Taken from ref 13. Taken from ref 19. Decrease in developed tension on 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. The 10−4 M concentration gave the maximum effect for most compounds.
Calculated from log concentration−response curves (Probit analysis by Litchfield and Wilcoxon20 with n = 6−7). When the maximum effect was
d
e
<50%, the EC50 ino., EC50 chrono, values were not calculated. Decrease in developed tension on guinea pig spontaneously beating isolated right
atrium at 10−5 M, expressed as percent changes from the control (n = 7−8). The 10−5 M concentration gave the maximum effect for most
f
compounds. Decrease in atrial rate on guinea pig spontaneously beating isolated right atrium at 10−4 M, expressed as percent changes from the
control (n = 7−8). The 10−4 M concentration gave the maximum effect for most compounds. Pretreatment heart rate ranged from 165 to 190 beats/
g
h
i
j
k
l
min. At the 10−5 M. At the 10−6 M. At the 5 × 10−5 M. At the 5 × 10−6 M. At the 10−7 M. At the 5 × 10−7 M.
Cav1.2a subunit, showing similar affinity and potency in all the
three above-mentioned experimental settings. In regard to the
tested compounds, 4 and 14 significantly displace PN200−110,
(+)-[5-methyl-3H] binding to LTCC in atrial membranes but
not in ventricular membranes or in intact HEK-293 stably
expressing the cardiac α subunit. Thus 4 and 14 showed a good
selectivity toward atrial LTCC and an affinity within the
nanomolar range; however, neither 4 nor 14 could displace
more than 60% of PN200−110, (+)-[5-methyl-3H] binding to
atrial LTCC. Compounds 27 and 29 selectively inhibited the
PN200−110, (+)-[5-methyl-3H] binding to atrial LTCC, with a
maximal displacement of PN200−110, (+)-[5-methyl-3H]
greater than 91%. As with 4 and 14, compounds 27 and 29
have similar affinity values. Compound 7, on the contrary,
could not significantly alter PN200−110, (+)-[5-methyl-3H]
binding to LTCC expressed on atrial membranes, but it was
effective in displacing radioligand binding to both ventricular
LTCC and to the cloned alpha subunit, with similar affinity and
potency in both the experimental settings. Nevertheless, 7
could not displace more than 50% of the PN200−110, (+)-[5-
methyl-3H] binding to both ventricular LTCC and the cloned α
subunit. Compound 20 was able to significantly reduce
PN200−110, (+)-[5-methyl-3H] binding to LTCC in both
atrial and ventricular membranes but not in intact HEK-293
expressing the cloned α subunit. These findings suggest that 20
may bind to LTCC subunits other than Cav1.2a. Compound 20
showed affinity values in the nanomolar range and a maximal
displacement of PN200−110, (+)-[5-methyl-3H] of about 92%.
Compounds 9, 13, 18, 19, and 23 significantly displaced
PN200−110, (+)-[5-methyl-3H] binding to LTCC in both
atrial and ventricular membranes as well as in HEK-293 cells
stably expressing Cav1.2 α subunit, with affinity values and
D
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Journal of Medicinal Chemistry
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solutions at 10−2 M concentration. These stock solutions were
diluted with buffer. All compounds at the concentrations used
were soluble in DMSO and in buffer. Addition of the drug
vehicle had no appreciable effect in binding experiments.
PN200−110, (+)-[5-methyl-3H] was incubated with increasing
concentrations (0.001−100 μM) of all the tested compounds;
affinity of 1−31 for rat cortex LTCC (as IC50 and Ki) and their
maximal [3H]PN200−100 displacement are listed in Table 5.
As expected, Nifedipine could significantly compete with
PN200−110, (+)-[5-methyl-3H] for the 1,4-dihydropiridine
binding site on rat cortex LTCC, displaying affinity values
within the nanomolar range and a maximal displacement of
PN200−110, (+)-[5-methyl-3H] greater than 90%. Potency was
in a nanomolar range (EC50 = 0.0061 0.0008). Compound 4
displayed the best affinity toward rat cortex LTCC (Ki = 0.059
μM) and displaced the 70% of PN200−110, (+)-[5-methyl-3H]
binding. On the contrary, compounds 27 and 29−30 had the
worst affinity to rat cortex LTCC, with Ki values of 13.9, 340,
and 34.9 μM, respectively, as their maximal displacement of
PN200−110, (+)-[5-methyl-3H] was lower than 60%. 13−14,
18−20, and 28 significantly bound to the rat cortex LTCC,
showing an affinity ranging from 0.1 to 0.5 μM and a maximal
displacement of PN200−110, (+)-[5-methyl-3H] between 56
and 98%. The remaining 20 compounds (1−3, 5−8, 10−12,
15−17, 21−26, and 31) did not affect PN200−110, (+)-[5-
methyl-3H] binding to calcium channels in rat cortex
membranes, therefore showing no affinity greater than 50%
to cerebral LTCC up to 100 μM concentration.
E. Computational Studies. In a previous study, a
theoretical model of the LTCC Cav1.2 isoform inner pore
was constructed by some of us and used to suggest a viable
binding mode for several 1,4-DHPs endowed with affinity for
this isoform.13,25 In the present inspection, for some of the new
1,4-DHPs the binding to the Cav1.3 isoform of LTCC was also
demonstrated. Therefore, in the attempt of getting major
insights into the reasons for the affinity for both LTCC
isoforms (Cav1.2 and Cav1.3) of these new compounds, we also
constructed a model of the LTCC Cav1.3 starting from the
Cav1.2 one. The sequence alignment (see Supporting
Information) between the two isoforms demonstrated that
the two channels displays a high sequence homology (≈66%
identity).2 Moreover, the residues lining the putative 1,4-DHPs
binding site in the two isoforms are identical, with the
conservative I1173(Cav1.2)/V1139(Cav1.3) mutation being the
only exception. Construction of the two LTTCs allowed for the
docking of the 1,4-DHPs that displayed diverse binding
properties for the two channels. In particular, for this
inspection, we considered compounds 4 and 28, which can
bind only the Cav1.3 and, with strong selectivity, the Cav1.2
isoforms, respectively, and 29, which can bind both isoforms.
These simulations were conducted with the AutoDock4.2
(AD4) software that was previously used to propose reasonable
binding hypothesis for the other 1,4-DHPs.13,25 As happened
for our previous inspections, also in this case the choice of the
“best” posing was also made by considering the consistency
with experimental data (mutagenesis experiments and SARs).
Analysis of docking results revealed that all the three 1,4-DHPs
are able to share the same binding pose (Figure 1) within the
LTCC regardless the considered isoform (Cav1.2 or Cav1.3),
which was somehow expected given the high sequence identity
of the two binding sites (Cav1.2 and Cav1.3). Moreover, the
same interaction pattern has also been proposed by us for
different 1,4-DHPs in our previous papers, demonstrating its
Table 3. Relaxant Activity of Compounds 1−31 on K+-
Depolarized Guinea Pig Vascular and Nonvascular Smooth
Muscle
aorta
ileum
c
c
d
activity
activity
IC50
95% conf lim
(× 10−6
compd
(M SEM)
(M SEM)
(μM)
)
e
1
28 1.3
87 2.4
78 2.1
91 0.4
2.57
2.81
3.55
15.96
0.96
9.69
0.11
1.37
0.0023
3.52
0.18
3.23
0.046
12.35
4.89
1.14
1.21
0.45
0.055
0.083
11.04
1.84
12.81
2.56
0.24
2.96
1.66
0.21
2.06
0.51
8.83
1.97−3.35
2.20−3.57
2.05−6.16
10.93−20.10
0.74−1.06
7.61−12.35
0.086−0.13
1.08−1.72
0.0018−0.0030
2.68−4.61
0.10−0.31
2.39−4.36
0.029−0.071
8.56−17.80
3.74−6.40
0.73−1.85
0.91−1.61
0.10−0.91
0.043−0.070
0.066−0.103
8.18−14.90
1.49−2.26
10.00−16.41
1.90−3.11
0.19−0.29
2.24−3.92
1.36−2.05
0.14−0.31
1.55−2.73
0.39−0.62
5.53−10.41
0.0011−0.0022
2
13 1.2
36 2.4
3
e
e
4
19 0.7
83 2.5
i
5
14 0.5
90 2.1
j
6
4
0.1
97 0.5
f
k
7
23 1.4
0.8
11 0.1
0.2
91 0.3
i
8
9
81 2.3
l
9
85 1.7
10
11
12
13
6
74 2.6
e
e
k
22 1.3
31 3.1
29 1.1
24 1.4
15 0.9
11 1.0
42 3.1
15 0.9
40 2.4
48 1.7
17 0.9
27 1.9
95 2.6
87 1.1
k
85 2.3
a
14
51 0.6
74 1.4
b
15
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
,
b
b
e
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
91 2.3
90 1.7
,
e
g
f
g
90 2.3
k
89 3.5
,
,
b
b
l
51 2.4
j
86 5.2
i
67 3.2
j
5
0.3
89 2.4
35 1.4
32 2.5
12 1.1
24 1.4
34 2.1
19 1.3
47 3.1
38 1.5
82 1.3
97 1.4
g
94 3.5
74 3.8
92 1.5
,
,
,
,
b
b
b
b
e
m
70 2.3
93 0.5
i
97 0.6
70 1.3
g
,
h
n
Nif
70 0.36
0.0015
c
a
b
Taken from ref 13. Taken from ref 19. Percent inhibition of
calcium-induced contraction on K+-depolarized (80 mM) guinea pig
aortic strips (at 10−4 M) and longitudinal smooth muscle (at 10−5 M).
The 10−4 M and the 10−5 M concentration gave the maximum effect
d
for most compounds respectively. Calculated from log concen-
tration−response curves (Probit analysis by Litchfield and Wilcoxon20
with n = 6−7). When the maximum effect was <50%, the IC50 values
e
f
g
were not calculated. At the 5 × 10−5 M. At the 10−5 M. At the 10−6
h
i
j
M. IC50 = 0.009 μM (c.l. 0.003−0.020). At the 5 × 10−6 M. At the
k
l
m
n
10−4 M. At the 5 × 10−7 M. At the 10−7 M. At the 3 × 10−7 M. At
the 5 × 10−9 M.
potency profiles similar to Nifedipine. The remaining
compounds 1−3, 5−6, 8, 10−12, 15−17, 21−22, 24−26,
28, and 30−31 did not significantly affect the PN200−110,
(+)-[5-methyl-3H] binding to calcium channels in atrial and
ventricular membranes as well as in HEK-293 intact cells stably
expressing the cloned Cav1.2a subunit, therefore showing no
affinity greater than 50% to cardiac LTCC up to 100 μM
concentration.
D. Binding Studies on Rat Cortex. Compounds 1−31
have been also assayed in competition binding experiments
carried out on rat cortex membranes; Nifedipine was employed
in the same experiments as a reference compound. All
compounds were first dissolved in DMSO in order to obtain
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also mutations in these paths should be considered.27−29
Unfortunately, in the cases of LTCC ligands, no accurate
information is available about the ligand entrance path.
Nevertheless, it can be postulated that the different chemical
composition as well the extension of the entrance/exit path
could have an influence on the Cav1.2/Cav1.3 selectivity.
According to this theory, 4, with its bulky 3-pyridine
substituent, should only be able to reach the Cav1.3 binding
site while the small and highly electron-withdrawing CF3 group
in 28 should allow this compound to easily reach interact with
the Cav1.2 1,4-DHPs binding site. However, this is a pure
theoretical hypothesis that has to be confirmed by future
experimental data.
Table 5. Rat Cortex Binding Affinity of Compounds 1−31
b
% of max disp
(M SEM)
IC50
SEM
Ki
SEM
c
a
c
compd
(μM)
(μM)
4
70
53
98
56
77
79
90
52
75
60
52
92
3
2
4
5
6
5
4
3
5
4
2
6
0.10 0.005
1.03 0.006
0.24 0.03
0.21 0.01
0.35 0.02
1.21 0.08
0.99 0.05
38.60 1.5
1.65 0.051
1023 24
0.059 0.003
0.74 0.03
0.17 0.02
0.16 0.03
0.11 0.02
0.41 0.09
0.33 0.04
13.90 1.3
0.52 0.020
340 21
9
13
14
18
19
20
27
28
29
30
Nif
106
9
34.7 2.3
F. Physicochemical and Pharmacokinetic Properties
Prediction. Some physicochemical and pharmacokinetic
properties of 4, 28, and 29 were calculated and compared as
a coarse assessment of the drug-like character of our 1,4-DHPs.
For such a purpose, we employed the Qikprop program
0.0061 0.008
0.0019 0.005
a
Compounds 1−3, 5−8, 10−12, 15−17, 21−26, and 31 have not
been put in the table because they have an affinity greater than 50% at
b
100 μM. Percent of maximal [3H]PN200−110 displacement on rat
c
cortex homogenate at 100 μM The concentration of the tested
(Schrodinger. LLC New York). The results are summarized in
̈
compounds that inhibited [3H]PN200−110 binding on rat cortex
homogenate by 50% (IC50) was determined by −log probit analysis
with six concentration of the displacers, each performed in triplicate.
The IC50 values were used to calculate apparent inhibition constant
(Ki) by Prusoff method. Values are the mean SEM of 3−5 separate
experiments performed in triplicate.
Table S1, Supporting Information. Quantitative structure−
activity relationship (QSAR) studies on CNS active compounds
and their derivatives, together with analyses based on CNS
drugs, have suggested the physical and chemical properties that
CNS compounds must possess. They are: molecular weight
(MW) less than 450, ClogP less than 5, number of H-bond
acceptor atoms less than 7, polar surface area (PSA) less than
90 Å, number of rotatable bonds (RB) less or equal to 10.
Thus, for CNS penetration, the physical properties, usually,
have a smaller range than general therapeutics (the latter ranges
are reported in Table S1, Supporting Informations). On the
basis of these premises, all the inspected 1,4-DHPs are
predicted to have a good probability of entering the CNS, as
all its estimated physicochemical parameters fall in the
aforementioned ranges.
correlation with both mutagenesis data and the wide amount of
SARs data available for the 1,4-DHPs.13,25
Thus, it is clear that from docking alone that the different
binding selectivity profile displayed by 4, 28, and 29 cannot be
explained. Obviously, other differences between Cav1.2 and
Cav1.3, not residing in the 1,4-DHPs binding site, should exist
and contribute to the selective recognition of the above-
mentioned compounds. Indeed, it is well-known that ligand
entrance/exit may strongly affect the binding processes, thus
Figure 1. Docked structures of 4 (a,d), 28 (b,e), and 29 (c,f) in the three-dimensional structure of Cav1.2 (a−c) and Cav1.3 (d−f) LTCC. DHPs are
displayed as orange sticks, and key interacting residues are shown in green (Cav1.2) and cyan (Cav1.3). Hydrogen bonds as represented with dashed
yellow lines. The figure was created using the UCSF Chimera software.26
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Ki values of all the tested compounds are reported in Table 4
together with those of Nifedipine. Considering the overall
results of binding assays, we note that a considerable number of
compounds show a weak affinity to calcium channel (intrinsic
activity less than 50%): 1−3, 5, 6, 8, 10−12, 15−17, 21, 22,
24−26, and 31. For the remaining compounds 4, 7, 9, 13, 14,
18−20, 23, and 27−30, considering the percentage of maximal
[3H]PN200−110 displacement and the potency of displace-
ment (IC50 and Ki), a difference of affinity toward 1,4-DHPs
binding site of LTCC isoform emerges. It is interesting to note
that these data are confirmed by functional studies on isolated
organs, reflecting the different distribution of LTCC. In
particular, 14 possess negative inotropic activity [EC50 = 0.44
μM (c.l. 0.29−0.65)] and showed a good selectivity toward
atrial LTCC and an affinity within the nanomolar range; 14
displaces also more than 60% of PN200−110, (+)-[5-
methyl-3H] binding to atrial LTCC (65 3). It is enough to
modify from methyl to ethyl ester of the 1,4-dihydropyridine
core to have a compound more potent but less selective: 13
significantly displaced PN200−110, (+)-[5-methyl-3H] binding
DISCUSSION
■
Novel studies describe Cav1.3 as a determinant of heart rate but
not of ventricular excitation−contraction coupling. The
development of Cav1.3 selective blockers might be an attractive
possibility for managing cardiac ischemia and other coronary
artery diseases without the concomitant negative inotropy due
to Cav1.2 channel inhibition.3 Moreover, a Cav1.3 knockouts
study revealed unexpected physiological and pathophysiological
roles that should prompt development of Cav1.3-selective drugs
with potential therapeutic effects of their pharmacological
inhibition: antidepressant-like effects, neuroprotection of CNS
dopaminergic neurons, and prevention neuronal loss in
Parkinson’s disease.12 Taking into account these emerging
biological results and some data published from us in a previous
paper,13 we designed and synthesized a small library of new 1,4-
DHPs bearing a 4-imidazo[2,1-b]thiazole ring. Using a ligand-
based approach, we tried to determine the SARs of imidazo-
[2,1-b]thiazole ring in binding the two different LTCC
isoforms: Cav1.2 and Cav1.3. The functional studies have
allowed us to delineate the pharmacological profile of all
compounds. We evaluated their inotropic and/or chronotropic
effects on guinea pig isolated left atria driven at 1 Hz and on
spontaneously beating right atria, respectively. By these
preliminary functional studies, first of all we have hypothesized
for some derivatives a Cav1.2 and/or Cav1.3 channel inhibition
knowing that Cav1.2 is expressed in heart tissue and it is
proposed to influence the physiological function of excitation-
contraction coupling while Cav1.3 is expressed in sinoatrial
node, atrioventricular node, heart atria and influences sinoatrial
heart rate control, and atrioventricular conduction.3 To support
the hypothesis that emerged from functional studies, we
performed binding studies on guinea pig atria and ventricular
cells using [3H]PN200−100. Only Ki and IC50 of compounds
having an intrinsic activity greater than 50% are reported in
Table 4. As in the CNS Cav1.2 accounts for approximately 80%
of Cav1 channels, whereas Cav1.3 appears to be restricted to a
much smaller subset of neurons,3 compounds 1−31 have been
assayed in competition binding experiments carried out on rat
membranes cortex so as to investigate the affinity of 1−31 for
rat cortex LTCC (as IC50 and Ki) and their maximal
[3H]PN200−100 displacement. Data are listed in Table 5.
Comparing the results of binding on cardiomyocytes and on rat
cortex, it has been possible to assess the presence of
substituents of the skeleton of imidazo[2,1-b]thiazole able to
discriminate between the central and peripheral LTCC
isoforms. Finally, it is well-known that Cav1.2a is also
distributed in smooth muscle, including blood vessels, intestine,
lung, and uterus and that Cav1.3a is only expressed in vascular
smooth muscle.2,24 Therefore, all new 1,4-DHPs derivatives
(1−13) were tested on K+-depolarized (80 mM) guinea pig
smooth muscles: aortic strips and ileum longitudinal smooth
muscle to assess their vascular- and/or nonvascular-relaxant
activity, respectively. The data of the new compounds (1−13)
together with those of compounds previously published (14−
31)13 selected for the particular cardiovascular profile are
collected in Table 3 using Nifedipine as the reference drug. All
compounds (1−31) had no effect on vascular smooth muscle.
On the contrary, all compounds (1−31) inhibited K+-induced
contraction on guinea pig nonvascular smooth muscle. The
study of the LTCC isoforms selectivity was therefore concluded
by binding assays carried out on intact HEK-293 cells stably
expressing the cardiac Cav1.2a subunit of the LTCC. IC50 and
to LTCC in both atrial and ventricular membranes [IC50
=
0.078 0.009 and IC50 = 1.432 0.051, respectively] as well as
in HEK-293 cells stably expressing Cav1.2a subunit [IC50 = 0.86
0.07], with affinity values and potency profiles similar to
Nifedipine [IC50 = 0.0051 0.0008; IC50 = 0.0057 0.0007;
IC50 = 0.086 0.006; respectively]. 13 possesses both negative
inotropic and chronotropic activities [EC50 = 1.16 μM (c.l.
0.85−1.68), EC50 = 2.63 μM (c.l. 2.10−3.28), respectively]. 14
and 13 significantly bind the rat cortex LTCC, showing an
affinity potency in a μM range (IC50 = 0.21 0.05 and IC50
=
0.24 0.03, respectively) and a maximal displacement of
PN200−110, (+)-[5-methyl-3H] between 56 5 and 98 4%.
The position of the two methoxy groups on the phenyl ring
influences the functional and binding profile. 17 does not affect
PN200−110, (+)-[5-methyl-3H] binding to calcium channels in
rat cortex membranes, therefore showing no affinity greater
than 50% for cerebral LTCCs up to 100 μM concentration. 17
and 18 exhibited both negative inotropic and chronotropic
properties, but in binding studies on guinea pig atria and
ventricular membranes, no specific binding was observed. 19
only has negative chronotropic properties. [Decrease in atrial
rate on guinea pig spontaneously beating isolated right atrium
at 5 × 10−5 M is 90 0.5, EC50 = 1.36 μM (c.l. 1.08−1.78)]. 18
and 19 significantly displace PN200−110, (+)-[5-methyl-3H]
binding to LTCC in both atrial [IC50 = 0.39 0.06 and IC50
0.25 0.01, respectively] and ventricular membranes [IC50
=
=
0.046
0.009 and IC50 = 0.84
0.09, respectively], with
affinity values and potency profiles similar to Nifedipine. 18 and
19 bind the rat cortex LTCC. The elimination of a methoxy
group in 19 to give 20 increases the potency but reduced
selectivity. The introduction of a nitro group in the ortho or
para position on the aromatic ring leads to uninteresting
derivatives: comparing the biological activity of 14 with 21−25,
the potency and selectivity decreases. Only with the
introduction of a short aliphatic chain in position two of the
imidazo[2,1-b]thiazole nucleus (see the structure of 21−23), an
appreciable negative inotropic effect with a weak affinity to
calcium channel (an intrinsic activity about 50%) in cardiac
membranes can be recorded. The lack of the methoxy groups
on the aromatic ring in position 6, 26, or replacement with a
bioisoster moiety as thienyl or pyridyl 1−10 leads to derivatives
in which the negative chronotropic activity is potentiated with
respect to the negative inotropic one, 3−4, 6, 9, 10, and 26.
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Figure 2. Potency of compounds with the best interesting profile and Nifedipine as reference in different biological preparations: isolated tissues
(ino, decrease in developed tension on isolated guinea pig left atrium; chrono, decrease in atrial rate on guinea pig spontaneously beating isolated
right atrium) and in binding assays (atria, displacement on guinea pig atrial membranes; ventr, displacement on guinea pig ventricular membranes;
Cav1.2a, displacement on HEK-293 cells expressing the α1c‑a; cortex, displacement on rat cortex homogenate).
These latter compounds do not exhibit any appreciable affinity
in binding assays, with the exception of 4 and 9. 4 has minimal
structural differences in comparison to other analogues with a
pyridyl in position 6, 1−6, but binds to the rat cortex LTCC,
occurs for other derivatives with a phenyl in position 6 14 and
20.
CONCLUSION
■
showing a IC50 = 0.105
0.005 μM and a maximal
Because Cav1.2 and Cav1.3 channels have diverse and essential
functions (often in the same organ), they can be considered as
potential therapeutic target in individual organs. Indeed, the
development of an isoform-selective activator or blocker could
be therapeutically valuable in a wide range of diseases.
However, despite the fact that all the isoforms can bind 1,4-
DHPs currently used in cardiovascular diseases such as
Nifedipine, because the expression and function of each
isoform is not restricted to one tissue or cell type, it is
necessary to be aware of potential side effects.3 At the
peripheral level, in concert with previous studies13 the present
investigation suggests that 1,4-DHP scaffold bearing at C-4 a
imidazo[2,1-b]thiazole ring is critical to provide compounds
with cardiac selectivity over vascular one. On the basis of their
activity profile in isolated cardiovascular tissues, the substituents
at imidazo[2,1-b]thiazole ring can modulate the activity of
compounds on different heart function influenced by cardiac L-
type calcium channels isoforms Cav1.2 and Cav1.3. In particular
the choice of an appropriate substituent in position 6 in the
heterocyclic nucleus of the imidazo[2,1-b]thiazole allows
obtaining of some 1,4-DHPs with a particular activity probably
due to their binding with Cav1.2 or Cav1.3 with potential
therapeutic effects. An appropriately substituted phenyl ring
with one or two methoxy group as in 20 or in 14 enhances the
negative inotropic activity. Bioisosteric replacement, as for the
pyridine ring (4), leads to a derivative with higher negative
chronotropic potency (Figure 2). All three of these compounds
have different affinities of binding to the channels Cav1.2 and/
or Cav1.3 of the various tissues. The affinities of heteroge-
displacement of PN200−110, (+)-[5-methyl-3H] of 70 3%.
9 is the only one of the series of derivatives with a thienyl group
in 6, 7−8 and 10−12, which has negative chronotropic potency
[EC50 = 1.06 μM (c.l. 0.80−1.41)] and an appreciable binding
in ventricle membranes [IC50 = 2.05
displacement equal to 70 3%]. It is worth noting that, in
binding assays, the more cortex selective compound of the
0.12, maximal
series is 28 (IC50 = 1.65
0.51 μM and a maximal
displacement of PN200−110, (+)-[5-methyl-3H] of 75
5%). This 1,4-DHP exhibited high negative inotropic effect and
potency on the left atrium [90
3.8 at 5 × 10−5
M
concentration with EC50 = 0.071 μM (c.l. 0.021−0.14)] and
selectivity versus negative chronoscopic potency [EC50 = 0.86
μM (c.l. 0.74−1.01)]. Replacement of the trifluoromethyl
moiety in 28 with chlorine, 29 and 30, or a methyl, 31, causes
significant changes in activity. In particular, the methyl group
abrogates the LTCC affinity while the chlorine potentiates
negative inotropic potency on spontaneously beating right atria,
decreases the developed tension on isolated guinea pig left
atrium at 10−5 M = 95
1.7, and induces an appreciable
binding in atrial membranes. When the methyl in position 2 of
the imidazo[2,1-b]thiazole ring is moved to position 3, 30,
binding and functional data show a weak calcium antagonist
activity. Finally, the selectivity of 30 for the calcium channels of
the cortex is lost when the trifluoromethyl group is not bound
directly to the heterocycle nucleus but it is in the ortho position
of a phenyl ring (27). This structural change causes a shift in
the affinity for calcium channels of the atrium membranes as
I
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Dimethyl 2,6-Dimethyl-4-(2-methyl-6-(pyridin-3-yl)imidazo[2,1-
b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (4). Yield
16%. IR: 1699, 1670, 1643, 1210, 1120. 1H NMR: 2.18 (6H, s,
CH3), 2.42 (3H, s, CH3), 3.23 (6H, s, COOCH3), 5.59 (1H, s, dhp),
7.08 (1H, s, th), 7.43 (1H, dd, py-5, J = 7.9, J = 4.9), 8.15 (1H, d, py-6,
J = 7.8), 8.49 (1H, d, py-4, J = 4.9), 8.94 (1H, s, py-2), 8.99 (1H, s,
NH, ex D2O). Melting point = 225 °C. MW = 438.4995. Anal.
(C22H22N4O4S) C, H, N.
neously expressed Cav1.2 and Cav1.3 channels in heart and in
brain is even more evident when the aromatic ring in position 6
is substituted with a chlorine or a trifluoromethyl. Despite the
small diversity in substitution at imidazo[2,1-b]thiazole in 1,4-
DHP ring, 29 has selectivity in cardiovascular parameters
(negative chronotropic) and is able of displacing labeled
PN200−110, (+)-[5-methyl-3H] from brain tissue. It is
interesting to note that compound 28 has both negative
inotropic and negative chronotropic effects and does not
displace the labeled from atrium and ventricle cells, but it is
active at the central level (Figure 2). The results obtained with
these compounds encourages us to further study the 1,4-DHP
bearing imidazo[2,1-b]thiazole scaffold SARs to clarify the
impact of altered channel regulation in brain functions.
Dimethyl 2,6-Dimethyl-4-(6-(pyridin-4-yl)imidazo[2,1-b]thiazol-
5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (5). Yield 10%. IR:
1
1709, 1665, 1600, 1505, 1211. H NMR: 2.23 (6H, s, CH3), 3.21
(6H, s, COOCH3), 5.79 (1H, s, dhp), 7.27 (1H, d, th, J = 4.6), 7.36
(1H, d, th, J = 4.6), 7.89 (2H, d, py-2,6, J = 6.2), 8.60 (2H, d, py-3,5, J
= 6.2), 9.13 (1H, s, NH, ex D2O). Melting point = 243−245 °C. MW=
424.4729. Anal. (C21H20N4O4S) C, H, N.
Dimethyl 2,6-Dimethyl-4-(2-methyl-6-(pyridin-4-yl)imidazo[2,1-
b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (6). Yield
25%. IR 1703, 1672, 1602, 1207, 1115. 1H NMR: 2.23 (6H, s,
CH3), 2.42 (3H, s, CH3), 3.21 (6H, s, COOCH3), 5.75 (1H, s, dhp),
7.01 (1H, s, th), 7.87 (2H, d, py-2,6, J = 6.2), 8.59 (2H, d, py-3,5, J =
6.2), 9.13 (1H, s, NH, ex D2O). Melting point = 240 °C. MW=
437.4995. Anal. (C22H22N4O4S) C, H, N.
EXPERIMENTAL SECTION
■
A. Chemistry. The melting points are uncorrected. Analyses (C, H,
N) were within (0.4% of the theoretical values. TLC was performed on
Bakerflex 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
Dimethyl 2,6-Dimethyl-4-(6-(thiophen-2-yl)imidazo[2,1-b]-
thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (7). Yield 10%.
1
IR 1701, 1634, 1269, 1200, 1093. H NMR: 2.23 (6H, s, CH3), 3.25
1
cm−1. The H NMR spectra were recorded on a Varian Gemini (300
(6H, s, COOCH3), 5.74 (1H, s, dhp), 7.12 (1H, dd, thio-4, J = 5.1, J =
3.6), 7.20 (1H, d, th, J = 4.4), 7.30 (1H, d, th, J = 4.4), 7.48 (1H, d,
thio-5, J = 5.1), 7.55 (1H, d, thio-3, J = 3.6), 9.13 (1H, s, NH, ex D2O).
Melting point = 215−220 °C. MW = 429.5126. Anal. (C20H19N3O4S2)
C, H, N.
MHz); the chemical shift (referenced to solvent signal) is expressed in
δ (ppm) and J in Hz. All solvents and reagents, unless otherwise
stated, were supplied by Aldrich Chemical Co. Ltd. and were used as
supplied.
General Procedure for the Synthesis of the 1,4-Dihydropyridines
1−13. Methylacetoacetate or ethylaacetoacetate (2 mM) and 30%
NH4OH (4 mM) were added to a stirred solution of the appropriate
aldehyde a−m (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
methylacetoacetate or ethylaacetoacetate (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 on silica gel (acetone/petroleum ether 40−60
°C from 1.9 to 4.6 v/v for 1−9, 11, and 13; petroleum ether 40−60
°C/diethyl ether:9/1 v/v for 10 and 12) to provide the pure-DHPs as
pale-yellow syrup. The resulting oily residue was diluted with cold
acetone to afford white solid collected by filtration for 2, 4, 5, and 8.
For 1, 3, 6, 7, 9, 11, and 13, resulting oils were taken with acetone/
petroleum ether 40−60 °C 1/1 v/v and with diethyl ether for 10 and
12 to obtain solid products collect by filtration.
Dimethyl 2,6-Dimethyl-4-(2-methyl-6-(thiophen-2-yl)imidazo-
[2,1-b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (8). Yield
1
10%. IR: 1701, 1620, 1306, 1215, 1102. H NMR: 2.23 (6H, s, CH3),
2.41 (3H, s, CH3), 3.25 (6H, s, COOCH3), 5.69 (1H, s, dhp),
6.97(1H, s, th), 7.10 (1H, dd, thio-4, J = 3.4, J = 2.4), 7.45 (1H, dd,
thio-5, J = 3.4, J = 0.8), 7.51 (1H, d, thio-3, J = 2.4), 9.08 (1H, s, NH,
ex D2O). Melting point = 195 °C. MW= 443.5391. Anal.
(C21H21N3O4S2) C, H, N.
Dimethyl 2,6-Dimethyl-4-(6-(thiophen-3-yl)imidazo[2,1-b]-
thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (9). Yield 10%.
1
IR: 1701, 1620, 1306, 1215, 1102. H NMR: 2.23 (6H, s, CH3), 3.23
(6H, s, COOCH3), 5.72 (1H, s, dhp), 7.18 (1H, d, th, J = 4.4), 7.28
(1H, d, th, J = 4.4), 7.58 (2H, m, thio-4−5), 7.79 (1H, s, thio-2), 9.12
(1H, s, NH, ex D2O). Melting point = 220−223 °C. MW = 429.5126.
Anal. (C20H19N3O4S2) C, H, N.
Dimethyl 2,6-Dimethyl-4-(2-methyl-6-(thiophen-3-yl)imidazo-
[2,1-b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (10).
1
Dimethyl 2,6-Dimethyl-4-(6-(pyridin-2-yl)imidazo[2,1-b]thiazol-
Yield 12%. IR: 1706, 1301, 1204, 1107, 1025. H NMR: 2.22 (6H,
5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (1). Yield 18%. IR:
s, CH3), 2.40 (3H, s, CH3), 3.24 (6H, s, COOCH3), 5.67 (1H, s, dhp),
6.94 (1H, s, th), 7.56 (2H, m, thio-4−5), 7.74 (1H, m, thio-2), 9.03
(1H, s, NH, ex D2O). Melting point = 210 °C. MW = 443.5391. Anal.
(C21H21N3O4S2) C, H, N.
1
1699, 1646, 1514, 1282, 1089. H NMR: 2.16 (6H, s, CH3), 3.22
(6H, s, COOCH3), 5.96 (1H, s, dhp), 7.20 (1H, qd, py-4, J = 4.7, J =
1.1), 7.28 (1H, d, th, J = 4.4), 7.63(1H, d, th, J = 4.4), 7.75 (1H, td, py-
5, J = 7.9, J = 1.1), 7.92 (1H, d, py-6, J = 7.9), 8.56 (1H, dd, py-3, J =
4.7, J = 1.1), 8.95 (1H, s, NH, ex D2O). Melting point = 190 °C. MW
= 424.4729. Anal. (C21H20N4O4S) C, H, N.
Dimethyl 4-(6-(2,5-Dichlorothiophen-3-yl)imidazo[2,1-b]thiazol-
5-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (11). Yield
1
11%. IR: 1705, 1672, 1308, 1213, 1119. H NMR: 2.12 (6H, s, CH3),
Dimethyl 2,6-Dimethyl-4-(2-methyl-6-(pyridin-2-yl)imidazo[2,1-
b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (2). Yield
20%. IR: 1703, 1587, 1270, 1201, 1093. 1H NMR: 2.15 (6H, s,
CH3), 2.45 (3H, s, CH3), 3.23 (6H, s, COOCH3), 5.74 (1H, s, dhp),
7.17 (1H, td, py-4, J = 5.1, J = 1.3), 7.41 (1H, s, th), 7.73 (1H, td, py-5,
J = 7.8, J = 1.3), 7.89 (1H, d, py-6, J = 7.8), 8.54 (1H, dd, py-3, J = 5.1,
J = 1.3), 8.92 (1H, s, NH, ex D2O). Melting point = 245 °C. MW =
438. 4995. Anal. (C22H22N4O4S) C, H, N.
3.34 (6H, s, COOCH3), 5.17 (1H, s, dhp), 6.90 (1H, s, thio), 7.28
(1H, d, th, J = 4.4), 7.79 (1H, d, th, J = 4.4), 8.66 (1H, s, NH, ex
D2O). Melting point = 202 °C. MW = 498.4027. Anal.
(C20H17Cl2N3O4S2) C, H, N.
Dimethyl 4-(6-(2,5-Dichlorothiophen-3-yl)-2-methylimidazo[2,1-
b]thiazol-5-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
1
(12). Yield 10%. IR: 1705, 1670, 1209, 1112, 1020. H NMR: 2.11
(6H, s, CH3), 2.45 (3H, s, CH3), 3.44 (6H, s, COOCH3), 5.10 (1H, s,
dhp), 6.86 (1H, s, thio), 7.55 (1H, s, th), 8.61 (1H, s, NH, ex D2O).
Melting point = 225 °C. MW = 512.4293. Anal. (C21H19Cl2N3O4S2)
C, H, N.
Dimethyl 2,6-Dimethyl-4-(6-(pyridin-3-yl)imidazo[2,1-b]thiazol-
5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (3). Yield 10%. IR:
1
1701, 1649, 1567, 1216, 1122. H NMR: 2.19 (6H, s, CH3), 3.22
(6H, s, COOCH3), 5.64 (1H, s, dhp), 7.31 (1H, d, th, J = 4.6), 7.33
(1H, d, th, J = 4.6), 7.46 (1H, dd, py-5, J = 7.8, J = 5.1), 8.19 (1H, dt,
py-6, J = 7.8, J = 1.7), 8.52 (1H, dd, py-4, J = 5.1, J = 1.7), 8.97 (1H, d,
py-2, J = 1.7), 9.06 (1H, s, NH, ex D2O). Melting point = 209 °C. MW
= 424.4279. Anal. (C21H20N4O4S) C, H, N.
Diethyl 4-(6-(2,5-Dimethoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (13). Yield
10%. IR: 1697, 1666, 1511, 1202, 1124. 1H NMR: 0.94 (6H, t,
COOCH2CH3, J = 7.1), 1.91 (6H, s, CH3), 3.48 (3H, s, OCH3), 3.70
(3H, s, OCH3), 3.89 (4H, q, COOCH2CH3, J = 7.1), 5.05 (1H, s,
J
dx.doi.org/10.1021/jm301839q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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dhp), 6.46 (1H, s, ar-6), 6.85 (1H, s, ar-3,4), 7.17 (1H, d, th, J = 4.6),
7.84 (1H, d, th, J = 4.6), 8.04 (1H, s, NH, ex D2O). Mp = 235−240
°C. MW = 510.5820. Anal. (C26H28N3O6S) C, H, N.
B. Receptor Binding Studies. For details, see Supporting
Information, section S2.
C. Functional Assays. For details, see Supporting Information,
section S4.
D. Computational Methods. For details, see Supporting
Information, section S5.
(9) Sinnegger-Brauns, M. J.; Huber, I. G.; Koschak, A.; Wild, C.;
Obermair, G. J.; Einzinger, U.; Hoda, J. C.; Sartori, S. B.; Striessnig, J.
Expression and 1,4-dihydropyridine-binding properties of brain L-type
calcium channel isoforms. Mol. Pharmacol. 2009, 75, 407−414.
(10) Ioan, P.; Carosati, E.; Micucci, M.; Cruciani, G.; Broccatelli, F.;
Zhorov, B. S.; Chiarini, A.; Budriesi, R. 1,4-Dihydropyridine Scaffold in
Medicinal Chemistry, The Story So Far and Perspectives. Part 1:
Action in Ion Channels and GPCRs. Curr. Med. Chem. 2011, 18,
4901−4922.
(11) Carosati, E.; Ioan, P.; Micucci, M.; Broccatelli, F.; Cruciani, G.;
Zhorov, B. S.; Chiarini, A.; Budriesi, R. 1,4-Dihydropyridine Scaffold in
Medicinal Chemistry, The Story So Far and Perspectives. Part 2:
Action in Other Targets and Antitargets. Curr. Med. Chem. 2012, 19,
4306−4323.
(12) Striessnig, J.; Koschak, A.; Sinnegger-Brauns, M. J.; Hetzenauer,
A.; Nguyen, N. K.; Busquet, P.; Pelster, G.; Singewald, N. Role of
voltage-gated L-type Ca2+ channel isoforms for brain function.
Biochem. Soc. Trans. 2006, 34, 903−909.
(13) Budriesi, R.; Ioan, P.; Leoni, A.; Locatelli, A.; Cosconati, S.;
Ugenti, M. P.; Andreani, A.; Di Toro, R.; Bedini, A.; Spampinato, S.;
Marinelli, L.; Novellino, E.; Chiarini, A. Imidazo[2,1-b]thiazole
System: A Scaffold Endowing Dihydropyridines With Selective
Cardiodepressant Activity. J. Med. Chem. 2008, 51, 1592−1600.
(14) Phillips, A. P. Hantzsch’s pyridine synthesis. J. Am. Chem. Soc.
1949, 71, 4003−4007.
ASSOCIATED CONTENT
■
S
* Supporting Information
Details for functional assays and for receptor binding studies
and for computational methods. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For R.B.: phone, +39-051-2099737; fax, +39-051-2099721; E-
mail, roberta.budriesi@unibo.it. For A.L.: phone, +39-051-
2099714; fax, +39-051-2099721; E-mail, alberto.leoni@unibo.it.
Notes
The authors declare no competing financial interest.
(15) Andreani, A.; Rambaldi, M.; Andreani, F.; Bossa, R.; Galatulas, I.
6-Pyridinylimidazo[2,1-b]thiazoles and Thiazolines as Potential
Cardiotonic Agents. Eur. J. Med. Chem. 1985, 20, 93−94.
(16) Andreani, A.; Burnelli, S.; Granaiola, M.; Leoni, A.; Locatelli, A.;
Morigi, R.; Rambaldi, M.; Varoli, L.; Farruggia, G.; Stefanelli, C.;
Masotti, L.; Kunkel, M. W. Synthesis and Antitumor Activity of
Guanylhydrazones from 6-(2,4-Dichloro-5-nitrophenyl)imidazo[2,1-
b]thiazoles and 6-Pyridilimidazo[2,1-b]thiazoles. J. Med. Chem. 2006,
49, 7897−7901.
(17) Andreani, A.; Burnelli, S.; Granaiola, M.; Leoni, A.; Locatelli, A.;
Morigi, R.; Rambaldi, M.; Varoli, L.; Calonghi, N.; Cappadone, C.;
Farruggia, G.; Zini, M.; Stefanelli, C.; Masotti, L.; Radin, N. S.;
Shoemaker, R. H. New Antitumor Imidazo[2,1-b]thiazole Guanylhy-
drazones and Analogues. J. Med. Chem. 2008, 51, 809−816.
(18) Andreani, A.; Rambaldi, M.; Locatelli, A.; Bossa, R.; Galatulas, I.;
Ninci, M. Synthesis and cardiotonic activity of 2,5-
dimethoxyphenylimidazo[2,1-b]thiazoles. Eur. J. Med. Chem. 1992,
27, 431−433.
ACKNOWLEDGMENTS
■
Supported by grants from University of Bologna. We are
grateful to Istituto Nazionale Per le Ricerche Cardiovascolari
(INRC) for financial support. We thank Alessandro Casoni for
animal care.
ABBREVIATIONS USED
■
CNS, central nervous system; LTCC, L-type calcium channel;
1,4-DHPs, 1,4-dihydropyridines; GPILSM, guinea pig ileum
longitudinal smooth muscle; NMR, nuclear magnetic reso-
nance; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylforma-
mide; SEM, standard error mean; QSAR, quantitative
structure−activity relationship; PSA, polar surface area; RB,
rotatable bonds
(19) Budriesi, R.; Ioan, P.; Leoni, A.; Pedemonte, N.; Locatelli, A.;
Micucci, M.; Chiarini, A.; Galietta, L. J. Cystic Fibrosis: A New Target
for 4-Imidazo[2,1-b]thiazole-1,4-DHPs. J. Med. Chem. 2011, 54,
3885−3094.
REFERENCES
■
(1) Triggle, D. J. L-Type calcium channels. Curr. Pharm. Des. 2006,
12, 443−457.
(2) Catterall, W. A.; Perez-Reyes, E.; Snutch, T. P.; Striessnig, J.
International Union of Pharmacology. XLVIII. Nomenclature and
structure−function relationships of voltage-gated calcium channels.
Pharmacol. Rev. 2005, 57, 411−425.
(3) Zuccotti, A.; Clementi, S.; Reinbothe, T.; Torrente, A.; Vandael,
D. H.; Pirone, A. Structural and functional differences between L-type
calcium channels: crucial issues for future selective targeting. Trends
Pharmacol. Sci. 2011, 6, 366−375.
(4) Fleckenstein, A. Specific pharmacology of calcium in myocar-
dium, cardiac pacemakers, and vascular smooth muscle. Annu. Rev.
Pharmacol. Toxicol. 1977, 17, 149−166.
(5) Striessnig, J.; Grabner, M.; Mitterdorfer, J.; Hering, S.; Sinnegger,
M. J.; Glossmann, H. Structural basis of drug binding to L Ca2+
channels. Trends Pharmacol. Sci. 1998, 19, 108−115.
(6) McDonough, S. I., Ed. Calcium Channels Pharmacology; Kluwer
Academic/Plenum Publishers: New York, 2004.
(7) Triggle, D. J. Calcium channel antagonists: clinical usespast,
present and future. Biochem. Pharmacol. 2007, 74, 1−9.
(8) Hetzenauer, A.; Sinnegger-Brauns, M. J.; Striessnig, J.; Singewald,
N. Brain activation pattern induced by stimulation of L-type Ca2+-
channels: contribution of Ca(V)1.3 and Ca(V)1.2 isoforms. Neuro-
science 2006, 139, 1005−1015.
(20) Tallarida, R. J.; Murray, R. B. Manual of Pharmacologic
Calculations with Computer Programs, 2nd ed.; Springer-Verlag: New
York, 1987.
(21) Motulsky, H., Christopoulos, A. Fitting Models to Biological Data
Using Linear and Nonlinear Regression, 2003, www.graphpad.com.
Accessed on 1/12/2011.
(22) Motulsky, H. J. Prism 5 Statistics Guide; GraphPad Software Inc.:
San Diego, CA, 2007; www.graphpad.com. Accessed on 1/12/2011.
(23) GraphPad, version 3.0; GraphPad Software, Inc.: San Diego,
CA.
(24) Tarabova, B.; Novakova, M.; Lacinova, L. Haloperidol
́ ́ ́ ́
moderately inhibits cardiovascular L-type calcium current. Gen. Physiol.
Biophys. 2009, 28 (3), 249−529.
(25) Cosconati, S.; Marinelli, L.; Lavecchia, A.; Novellino, E.
Characterizing the 1,4-dihydropyridines binding interactions in the
L-Type Ca2+ channel: model construction and docking calculations. J.
Med. Chem. 2007, 50, 1504−1513.
(26) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera−a
visualization system for exploratory research and analysis. J. Comput.
Chem. 2004, 25, 1605−1612.
K
dx.doi.org/10.1021/jm301839q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(27) Limongelli, V.; Marinelli, L.; Cosconati, S.; La Motta, C.; Sartini,
S.; Mugnaini, L.; Da Settimo, F.; Novellino, E.; Parrinello, M.
Sampling protein motion and solvent effect during ligand binding.
Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 1467−1472.
(28) Limongelli, V.; Bonomi, M.; Marinelli, L.; Gervasio, F. L.;
Cavalli, A.; Novellino, E.; Parrinello, M. Molecular basis of
cyclooxygenase enzymes (COXs) selective inhibition. Proc. Natl.
Acad. Sci. U. S. A. 2010, 107, 5411−5416.
(29) Lo Monte, F.; Kramer, T.; Gu, J.; Anumala, U. R.; Marinelli, L.;
La Pietra, V.; Novellino, E.; Franco, B.; Demedts, D.; Van Leuven, F.;
Fuertes, A.; Dominguez, J. M.; Plotkin, B.; Eldar-Finkelman, H.;
Schmidt, B. Identification of Glycogen Synthase Kinase-3 Inhibitors
with a Selective Sting for Glycogen Synthase Kinase-3α. J. Med. Chem.
2012, 55, 4407−4424.
L
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