Cystic fibrosis: A new target for 4-imidazo[2,1-b]thiazole-1, 4-dihydropyridines

Article abstract of DOI:10.1021/jm200199r

The pharmacology of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel has attracted significant interest in recent years with the aim to search for rational new therapies for diseases caused by CFTR malfunction. Mutatio

Full text of DOI:10.1021/jm200199r

ARTICLE
pubs.acs.org/jmc
Cystic Fibrosis: A New Target for 4-Imidazo[2,1-b]thiazole-1,
4-dihydropyridines
Roberta Budriesi,*,† Pierfranco Ioan,† Alberto Leoni,† Nicoletta Pedemonte,‡ Alessandra Locatelli,†
Matteo Micucci,† Alberto Chiarini,† and Luis J. V. Galietta*,‡
†Dipartimento di Scienze Farmaceutiche, Universitꢀa degli Studi di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
‡Laboratorio di Genetica Molecolare, Istituto Giovannina Gaslini, Largo Gerolamo Gaslini 5, 16147 Genova, Italy
S Supporting Information
b
ABSTRACT: The pharmacology of the cystic fibrosis trans-
membrane conductance regulator (CFTR) Clꢀ channel has
attracted significant interest in recent years with the aim to
search for rational new therapies for diseases caused by CFTR
malfunction. Mutations that abolish the function of CFTR
cause the life-threatening genetic disease cystic fibrosis (CF).
The most common cause of CF is the deletion of phenylalanine
508 (ΔF508) in the CFTR chloride channel. Felodipine,
nifedipine, and other antihypertensive 1,4-dihydropyridines
(1,4-DHPs) that block L-type Ca2þ channels are also effective
potentiators of CFTR gating, able to correct the defective
activity of ΔF508 and other CFTR mutants (Mol. Pharmacol. 2005, 68, 1736). For this purpose, we evaluated the ability of the
previously and newly synthesized 4-imidazo[2,1-b]thiazoles-1,4-dihydropyridines without vascular activity and inotropic and/or
chronotropic cardiac effects (J. Med. Chem. 2008, 51, 1592) to enhance the activity of ΔF508-CFTR. Our studies indicate
compounds 17, 18, 20, 21, 38, and 39 as 1,4-DHPs with an interesting profile of activity.
’ INTRODUCTION
defect is the most severe consequence of the mutation, consisting
of the inability of the CFTR protein to exit the endoplasmic
reticulum. The latter defect appears instead as a partial response
to the cAMP activating stimulus. The two defects can be sep-
arately treated by pharmacological compounds called correctors
and potentiators, respectively.6ꢀ9 Besides ΔF508, other CF
mutations are also sensitive to potentiators.10
Cystic fibrosis (CF), the most common channelopathy, is
caused by mutations in the cystic fibrosis transmembrane con-
ductance regulator (CFTR) gene.1 The CFTR protein works as a
cAMP-activated plasma membrane Clꢀ channel. As a matter of
fact, in CF, the lack of CFTR Clꢀ channel function in the apical
membranes of many epithelial cells prevents the normal secre-
tion of Clꢀ onto the epithelial surface. There is also a loss of
control of Naþ channels, resulting in increased fluid reabsorp-
tion. Consequently, there is a production of a thick and dehy-
drated mucus that is responsible for many of the most important
manifestations of the disease in the respiratory tract. In particular,
the impairment of mucociliary clearance in the airways, due to
altered ion/fluid transport, favors the colonization of the airway
surface by antibiotic-resistant bacteria. The presence of bacteria
triggers in turn a severe inflammatory response that irreversibly
damages the lung.2ꢀ4 The CFTR channel contains 12 transmem-
brane (TM) domains, with TM1, TM6 and TM12 contributing
to the formation of the ion pore. In addition, there are two
domains that bind cytoplasmic nucleotides (NBD1 and NBD2)
and a regulatory domain. The channel is regulated by binding of
ATP to NBDs and by the cAMP-dependent phosphorylation of
serine residues in the regulatory domain. Deletion of phenylala-
nine (ΔF508) in NBD1 occurs in nearly 70% of patients with
CF.5 ΔF508 causes two types of problems: an impaired traffick-
ing of the protein and a defect in channel activity. The former
Chart 1
Antihypertensive 1,4-dihydropyridines (1,4-DHPs) are a well-
known class of drugs that block voltage-dependent Ca2þ chan-
nels. Many DHPs are also effective potentiators of CFTR gating,
able to correct the defective activity of ΔF508 and other CFTR
mutants.8 Activation of mutant CFTR by 1,4-DHPs occurs
Received: February 22, 2011
Published: April 28, 2011
r
2011 American Chemical Society
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through a mechanism that does not involve the inhibition of
Ca2þ channels but possibly a direct interaction with the CFTR
protein itself.9 Therefore, 1,4-DHPs represent an interesting
family of compounds for the development of effective drugs
for CF therapy. Indeed, they have been extensively studied, and a
large amount of information is available on their medicinal
chemistry properties.11 However, optimization of potency for
CFTR versus Ca2þ channels is required to minimize possible
side effects. In fact, felodipine, nifedipine, and other 1,4-DHPs
used as drugs activate CFTR at concentrations that are consider-
ably higher than those effective on Ca2þ channels. A better
understanding of structural features required for CFTR acti-
vation may help in the development of more selective and
effective DHP-based potentiators.7ꢀ9 In a recent study, we
have published 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.12 The binding properties for the novel
compounds have been investigated, and the interaction with
the binding site common to other 4-aryl-1,4-DHPs has been
demonstrated. Interestingly, the novel 4-aryldihydropyridines
are L-type calcium channel blockers with a peculiar pharma-
cological behavior. Indeed, the imidazo[2,1-b]thiazole system
is found to confer to the 1,4-dihydropyridine scaffold an
inotropic and/or chronotropic cardiovascular activity with a
high selectivity toward the nonvascular tissue. With this
information in mind, we designed and synthesized new
imidazo[2,1-b]thiazole derivatives 16ꢀ26, and in an attempt
to get major insights on structural features required for
specific CFTR activation, we screened a set of newly
(16ꢀ26) and previously synthesized 1,4-DHPs (27ꢀ47)
(Chart 1) using cell-based assays to determine their activity
as potentiator on ΔF508-CFTR. Moreover, we report func-
tional studies to define the cardiovascular activity profile, the
relaxant activity on Kþ-depolarized guinea pig ileum long-
itudinal smooth muscle, and the bronchodilatation effect on
guinea pig trachea with the aim to evaluate potency for CFTR
vs L-type calcium channels (LTCCs). In our analysis of
cardiac and smooth muscle function, we have also included
1,4-DHPs [48 (DHP-256), 49 (DHP-194), 50 (DHP-229)]13
as reference compounds previously reported as CFTR poten-
tiators with reduced potency on Ca2þ channels. Our results
show that modifications of the 1,4-DHP scaffold, particularly
in the 4-phenyl ring at C4, may lead to compounds with highly
improved selectivity as CFTR potentiators.
’ CHEMISTRY
As shown in Scheme 1, the syntheses of the compounds
16ꢀ26 (Table 1) were accomplished starting from the bicyclic
heterocycles obtained by cyclocondensation of the appropriate
2-aminothiazoles 1 and 2 and bromoketones 3 and 4. Imidazo-
[2,1-b]thiazoles 5ꢀ7 gave the corresponding aldehydes 8ꢀ10 by
means of the Vilsmeier method. 1,4-DHPs 16ꢀ26 were synthe-
sized by means of the well-known Hantzsch reaction with the
appropriate aldehydes 8ꢀ15.12,14ꢀ16 All final products were
confirmed with infrared spectra and 1H nuclear magnetic
resonance.
’ PHARMACOLOGY
CFTR Studies. The activity of 1,4-DHPs on ΔF508-CFTR was
tested in FRT cells coexpressing the mutant CFTR protein and
the halide-sensitive yellow fluorescent protein (HS-YFP) as
previously described.8,13 Briefly, cells were previously incubated
at 27 °C for 24 h to rescue the mutant protein from the
endoplasmic reticulum8 and then acutely stimulated for 30ꢀ40
min with forskolin (20 μM) in the presence and absence of 1,4-
DHPs at three different concentrations (0.2, 2, and 20 μM).
CFTR activity was finally evaluated with a microplate reader by
continuously measuring the fluorescence in each well before and
after the addition of an Iꢀ rich solution (final concentration of
100 mM), as previously described.8,13 Iꢀ influx through the
CFTR channel causes YFP quenching. Therefore, the rate of
fluorescence quenching reflects the extent of CFTR activity.
Accordingly, the readings from each well were normalized for the
initial fluorescence value and the fluorescence decay following Iꢀ
addition was fitted with an exponential function to derive the
maximal quenching rate (dF/dt). The quenching rate (QR) was
plotted against compound concentrations. The resulting doseꢀ
response relationships were then fitted with the Hill equation to
estimate potency and maximal effect.
Functional Studies on Cardiac and Smooth Muscle Func-
tion. The pharmacological profile of compounds was derived in
guinea pig isolated left and right atria to evaluate their inotropic
and chronotropic effects, respectively, and in Kþ-depolarized
(80 mM) guinea pig vascular (aortic strips) and nonvascular
[ileum longitudinal smooth muscle (GPILSM)] to assess cal-
cium antagonist activity. Compounds were checked at increasing
doses to evaluate the percent decrease of developed tension in
isolated left atrium driven at 1 Hz (negative inotropic activity),
Scheme 1
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Table 1. 1,4-Dihydropyridines 16ꢀ47
of drugs defined as EC50 and IC50 was evaluated from log
concentrationꢀresponse curves (Probit analysis using Litchfield
and Wilcoxon17 or GraphPad Prism18,19software) in the appro-
priate pharmacological preparations.
’ RESULTS
A. CFTR Study. FRT cells expressing ΔF508-CFTR were
incubated at 27 °C for 24 h to rescue the mutant protein from ER,
thus improving its targeting to the plasma membrane. Subse-
quently the cells were stimulated with forskolin (20 μM) in the
presence and absence of 1,4-DHPs at different concentrations.
Under these conditions, the cAMP activating pathway is satu-
rated by the high forskolin concentration. Therefore, any in-
crease in ΔF508-CFTR activity caused by a compound above the
level reached with forskolin alone can be attributed to a
potentiator-like activity. We tested 4-imidazo[2,1-b]thiazole-
1,4-dihydropyridines as well as 48, 49, and 50, identified as
CFTR-selective potentiators from a previous study.13 We also
included genistein as a non-1,4-DHP potentiator. Nifedipine, our
reference compound, shows a maximal activity of 58 and Kd =
2.9 ( 0.4 μM. Compounds 48 [Kd = 0.37 ( 0.07 μM], 49 [Kd =
0.17 ( 0.03 μM], and 50 [Kd = 0.40 ( 0.05 μM] show efficacy
comparable to that of nifedipine but, as reported previously,13
with greatly improved potency. In particular, 49 has 17-fold
increased potency relative to nifedipine. Among 4-imidazo[2,1-b]-
thiazole-1,4-dihydropyridines, compounds 23, 24, 30, 32, 35, 37,
40ꢀ47 are inactive. In contrast, as shown in Figure 1, com-
pounds 17, 20, 21, 38, and 39 effectively induce CFTR activity,
with a maximal effect elicited at 20 μM comparable to that of
nifedipine and 48ꢀ50. However, the 4-imidazo[2,1-b]thiazole-
1,4-dihydropyridines appear significantly less potent given the
small activity induced at 0.2 μM. There are compounds such as
18 with apparently high potency [Kd = 0.18 ( 0.03] but with
partial activity (Emax = 42) (Table 2).
B. Functional Study in Cardiovascular System. All com-
pounds were tested for their cardiovascular profile in guinea pig
left atrium driven at 1 Hz and in spontaneously beating right
atrium to evaluate their negative inotropic and/or chronotropic
effects, respectively, and in 80 mM Kþ-depolarizing guinea pig
aortic strips to assess their vasorelaxant activity. Data are
collected in Table 3 for 16ꢀ26 and in Table S4 (Supporting
Information) for 27ꢀ47, using nifedipine, genistein, and 48ꢀ50
as reference compounds. Nifedipine is active on all cardiovas-
cular parameters considered with selectivity for vascular smooth
muscle [IC50 = 0.009 μM (CL 0.003ꢀ0.02)]. In contrast, 48ꢀ50
have no negative chronotropic and vascular effects. These three
compounds have selective negative inotropic activity with po-
tency higher than those of nifedipine; in particular the potency of
49 is EC50 = 0.0022 μM (CL 0.0014ꢀ0.0036).
compd
xꢀy
R
R1
16
HCdCH
HCdCH
HCdCH
CF3
C2H5
CH3
17
4-(CF3)-C6H4
4-(CF3)-C6H4
18
C2H5
19
H3CCdCCH3 4-(CF3)-C6H4
CH2CHdCH2
CH3
20
HCdCH
4-(OCF3)-C6H4
21
HCdCH
4-(OCF3)-C6H4
CH2CHdCH2
C2H5
CH3
22
H3CCdCH
H2CꢀCH2
HCdCH
C6H5
23
C6H5
24
2,5-(OCH3)-C6H3
CH2CHdCH2
C2H5
C2H5
CH3
25
H3CCdCH
HCdCH
Cl
26
2,3,4-Cl3C6H2
27a
28a
29a
30a
31a
32a
33a
34a
35a
36a
37a
38a
39a
40a
41a
42a
43a
44a
45a
46a
47a
HCdCH
CF3
CF3
CH3
CH3
Cl
H3CCdCH
ClCdCH
H3CCdCH
ClCdCH
H3CCdCH
HCdCCH3
CH3
CH3
CH3
CH3
Cl
CH3
Cl
CH3
H3CCdCCH3 Cl
CH3
HCdCH
HCdCH
HCdCH
HCdCH
HCdCH
H3CCdCH
ClCdCH
H5C2CdCH
HCdCH
ClCdCH
ClCdCH
HCdCH
HCdCH
2-(OCH3)C6H4
CH3
3-(OCH3)C6H4
2-(OCF3)C6H4
CH3
CH3
3-(OCF3)C6H4
CH3
3-(CF3)C6H4
CH3
2,5-(OCH3)2C6H3
2,5-(OCH3)2C6H3
2,5-(OCH3)2C6H3
3,4-(OCH3)2C6H3
3,4,5-(OCH3)3C6H2
CH3
CH
CH3
CH3
CH3
4-(NO2)-2,5-(OCH3)2C6H2 CH3
6-(NO2)-2,5-(OCH3)2C6H2 CH3
6-(NO2)-3,4-(OCH3)2C6H2 CH3
a Reference 12.
the percent decrease in atrial rate in spontaneously beating right
atrium (negative chronotropic activity), and the percent inhibi-
tion of calcium-induced contraction in Kþ-depolarized aortic
strips and GPILSM (vascular and nonvascular relaxant activity,
respectively). These details are reported in the Supporting
Information.
For some selected compounds (see Table 5) the characteriza-
tion was extended to guinea pig tracheal ring preparations
precontracted with carbachol (1 μM) to evaluate the induced
inhibition.
In isolated vascular smooth muscle depolarized with 80 mM
Kþ, genistein is inactive. In cardiac isolated tissues genistein
induced negative inotropic and chronotropic activities with
about 1588-fold selectivity for negative inotropic potency
[EC50 = 0.028 μM (CL 0.021ꢀ0.037)] respective to negative
chronotropic potency [EC50 = 44.48 μM (CL 34.98ꢀ56.61)]. As
previously described, genistein up to 10 μM induced positive
inotropic activity.20
All other compounds, except 20, are devoid of vascular effects.
The potency of 20 was not significantly different from that of
nifedipine [IC50 = 0.016 μM (CL 0012ꢀ0025), IC50 = 0.009 μM
Data were analyzed using Student’s t-test and are presented as
the mean ( SEM.17 Since the drugs were added in a cumulative
manner, the difference between the control and the experimental
values at each concentration was tested for P < 0.05. The potency
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Figure 1. Compound efficacy on ΔF508-CFTR. The efficacy is obtained from doseꢀresponse relationships of YFP fluorescence experiments (mean (
SEM, n = 3).
(CL 0003ꢀ0020), respectively] even if the intrinsic activity is
significantly lower.
Compounds 28, 30, and 44 are also devoid of cardiac effects.
In fact, their intrinsic activity on cardiac parameters is less than
50% for the inotropic and 40% for the chronotropic effect at the
highest concentration studied.
(CL 14.19ꢀ24.16) respectively] even though 286 and 446 times
less than that of nifedipine.
C. Functional Study in Guinea Pig Longitudinal Smooth
Muscle. It is well-known that calcium entry blockers such as
nifedipine have relevant inhibitory effects on nonvascular smooth
muscle.21 All compounds were also tested in Kþ-depolarized
(80 mM) guinea pig ileum longitudinal smooth muscle to
evaluate both the effects on nonvascular smooth muscle and
the selective action by comparison with the cardiovascular
system. These data are collected in Table 4. For compounds
32, 36, 37, 44, and 46, these data are reported in the Supporting
Information (Table S5). As previously mentioned, among the
cardiovascular system, nifedipine showed a clear-cut vascular
potency and selectivity [EC50 = 0.009 μM (CL 0.003ꢀ0.02)].
However, with regard to the nonvascular smooth muscle, the
potency of nifedipine increases 6 times if compared to that measured
in aortic strips [EC50 = 0.0015 μM (CL 0.0011ꢀ0.0022)].
All three 1,4-DHPs previously described (48ꢀ50)13 are able
to relax the intestinal smooth muscle but with a potency
significantly lower than that of nifedipine. In the same prepara-
tion genistein induced relaxation with intrinsic activity similar
to that of 48 but with lower potency [IC50 = 9.95 μM (CL 8.01ꢀ
12.35); IC50 = 0.0015 μM (CL 0.0011ꢀ0.0022); respectively].
All the 1,4-DHPs bearing a imidazo[2,1-b]thiazole system at
C4 are able to relax the guinea pig longitudinal smooth muscle of
ileum. They show an increased activity in nonvascular smooth
muscle with respect to vascular smooth muscle with the excep-
tion of 18, which has a potency comparable to that of nifedipine
[IC50 = 0.00088 μM (CL 0.00021ꢀ0.0017); IC50 = 0.0015 μM
A significant group of compounds (11 of 32) expressed
negative inotropic selectivity: 16, 17, 24, 25, 29, 31ꢀ34, 37,
and 46, with potency between 0.039 and 1.96 μM. Among them,
nine compounds (17, 25, 29, 31ꢀ34, 37, and 46) were more
potent than nifedipine [EC50 = 0.26 μM (CL 0.19ꢀ0.36)].
Derivatives 17, 29, 31, and 33 have negative inotropic effect
about 2.6 times greater than that of nifedipine. However, all these
compounds have no intrinsic activity that exceeds 80%. There-
fore, the intrinsic activity is significantly lower than that of
nifedipine considering the concentration needed to obtain the
maximal intrinsic activity with the exception of 16 and 17.
Most of the tested compounds (18, 19, 21, 23, 26, 27, 35, 36,
38ꢀ43, and 47) showed both negative inotropic and chrono-
tropic properties with different selectivity. In particular, unlike
the reference nifedipine, none of these compounds showed
negative chronotropic selectivity. Compounds 19, 23, 27, 39,
and 47, different from the reference nifedipine, showed a negative
inotropic over chronotropic selectivity. In particular in 19, 23, 39,
and 47 are predominantly negative inotropic agents with a selec-
tively ratio of about 33, 22, 12, and 9, respectively. Derivatives 35, 38,
and 40 (Table S4) have only a weak negative inotropic selectivity.
Only compounds 22 and 45 have negative chronotropic selec-
tivity [EC50 = 7.15 μM (CL 4.30ꢀ10.01) and EC50 = 18.52 μM
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Table 2. Activity of 1,4-DHPs and Genistein on
minor effects on cardiovascular parameters were also tested on
isolated tracheal rings to measure bronchodilatation (data of
selected compounds and references are collected in Table 5
with those of reference compounds). The compounds studied
have a different potency profile in guinea pig trachea smooth
muscle.
49 has a strong selectivity for the trachea compared to
nifedipine. Indeed, 49 is 163 times more potent than nifedipine.
48 and 50 have intrinsic activity e30%, while genistein has
intrinsic activity lover then 20%.
The new 1,4-DHPs studied showed a panel of various
activities. Among the compounds studied, 19ꢀ21, 26, 30, 37,
39, and 45 have intrinsic activity less than 50%. 17 and 18 have
the same intrinsic activity of nifedipine, while 38 reaches the
same intrinsic activity at a concentration 10 times higher.
However, the calculated potencies for 17, 18, and 38 were not
significantly different from that of nifedipine [EC50 = 0.061 μM
ΔF508-CFTRa
compd
Emax (msꢀ1
)
nH
mean Kd ( SEM (μM)
16
60
0.66
2.15
1.69
1.14
1.8
19.4 ( 2.4
3.2 ( 0.5
17
64
18
42
0.18 ( 0.03
0.32 ( 0.06
3.5 ( 0.7
19
42
20
64
21
60
1.09
0.93
4.8 ( 0.7
22
60
14.8 ( 1.9
23
inactiveb
inactiveb
60
24
25
0.86
0.44
1.89
0.88
1.55
8.4 ( 0.8
3.2 ( 0.6
28.3 ( 2.5
12.0 ( 1.6
42.9 ( 4.9
26
46
27
60
(CL 0.043ꢀ0.084), EC50 = 0.13 μM (CL 0.10ꢀ0.17), EC50
=
28
60
0.058 μM (CL 0.026ꢀ0.099), and EC50 = 0.096 μM (CL
0.070ꢀ0.13), respectively]. The relaxant potencies of 16, 25,
and 31 are lower than that of nifedipine (11-, 24-, and 38-fold,
respectively).
29
60
inactiveb
30
31
60
1.04
6.8 ( 1.0
32
inactiveb
60
Interestingly, compound 28, which has no effect on cardio-
vascular parameters, relaxes both intestinal and tracheal smooth
muscles.
33
0.82
1.32
21.4 ( 2.4
10.9 ( 1.8
34
60
inactiveb
35
36
60
1.5
34.2 ( 4.4
’ DISCUSSION
37
inactiveb
64
Our study had the main objective of identifying 1,4-DHPs
with optimal activity as potentiators of the CFTR Clꢀ channel
and negligible effects on voltage-dependent Ca2þ channels
(VDCCs). In a previous paper, it was shown that these two
activities might be separated by modifying the substituents of 1,4-
DHP scaffold.13 Indeed a cell-based functional assay approach
was used to identify compounds, such as 48ꢀ50, with high
potency and efficacy on mutant CFTR and dramatically reduced
activity on VDCCs.13 In the present study, we have proved these
results by testing these compounds on physiologically relevant
functions associated with Ca2þ channel activity in heart, aorta
smooth muscle strips, and ileum. Our results in aortic strips
(Figure 2) demonstrate that the CFTR-selective 1,4-DHPs pre-
viously identified13 indeed have low potency and efficacy, com-
pared to an antihypertensive drug such as nifedipine, on the
contractility of smooth muscle of blood vessels. These com-
pounds, namely, 48ꢀ50, also have negligible negative chrono-
tropic activity on the guinea pig right atrium. In contrast, these
1,4-DHPs are similar to nifedipine as negative inotropic agents
and also as relaxant agents on intestinal smooth muscle (Tables 3
and 4). These results may reflect a different expression of Ca2þ
channel subtypes in tissues used for functional assays. It is known
that the activities of the cardiac calcium channel blockers are
related to their interaction with the R1-subunits of VDCCs. In
particular, the negative inotropic effect is due to interaction with
the Cav1.2 isoform while the negative chronotropic effect seems
to be related to interaction with the Cav1.3 isoform.22 Further-
more, effects of Ca2þ channel blockers such as nifedipine on
vascular and nonvascular smooth muscle cells appear to be linked
to the interaction with the Cav1.2 isoform.23 Moreover, the
discovery that CFTR is expressed not only in epithelial cells but
also in the smooth muscle, where it may modulate contractility,
complicates the interpretation of results.24 To address this point,
we have also tested a classical non-1,4-DHP CFTR potentiator
38
0.81
1.01
1.4 ( 0.1
2.8 ( 0.4
39
65
40
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
58
41
42
43
44
45
46
47
nifedipine
48
2.58
0.97
3.58
1.18
1.3
2.9 ( 0.4
62
0.37 ( 0.07
0.17 ( 0.03
0.40 ( 0.05
9.4 ( 1.1
49
56
50
59
genistein
61
a The table reports the activity of compounds on ΔF508-CFTR
measured as Iꢀ influx (HS-YFP quenching rate). Doseꢀresponse
relationships were generated, and the data were fitted with the Hill
equation to obtain maximal effect on quenching rate (Emax), Hill
coefficient (nH), and apparent dissociation constant (Kd). For com-
pounds having low potency (i.e. 16, 22, 27, 29, 33, and 36) the fit was
done with a forced Emax value of 60. Data were generated from three
independent determinations. b Inactive up to 20 μM.
(CL 0.0011ꢀ0.0022); respectively]. All other compounds are on
the whole less potent than the reference nifedipine. In particular,
compounds 17, 20, and 21 are about 3 times less potent than
nifedipine, whereas 16, 25, 26, 28, 36, 38, 39, and 45 are about 65
times less potent. Compound 28 showed a selective activity for
the GPILSM, since it was ineffective on the cardiovascular
parameters studied.
D. Functional Study in Trachea. Compounds showing an
interesting activity profile as CFTR potentiators combined with
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Table 3. Cardiovascular Activity of Tested Compounds
% decrease (mean ( SEM)
EC50 of inotropic negative activity
EC50 of chronotropic negative activity
95% CL
c
negative inotropic negative chronotropic
EC50
95% CL
mean vasorelaxant
activitydactivity ( SEM
compd
16
activitya
activityb
(μM)
(ꢁ10-6)
EC50 c (μM)
(ꢁ10-6)
93 ( 2.4
96 ( 3.7f
90 ( 2.8g
93 ( 2.2i
89 ( 2.2
88 ( 0.1
44 ( 1.3f
78 ( 2.2
81 ( 2.2l
56 ( 1.7g
90 ( 2.4e
97 ( 2.0f
80 ( 2.3e
71 ( 1.1h
83 ( 1.4e
71 ( 0.3l
40 ( 1.4e
33 ( 0.5g
64 ( 1.1h
52 ( 0.7
33 ( 1.6k
77 ( 1.6k
84 ( 1.6
90 ( 1.4k
32 ( 1.1e
36 ( 2.1l
92 ( 2.7
85 ( 4.2g
38 ( 0.4l
5 ( 0.3
1.07
0.12
0.033
0.27
2.33
0.73
0.76ꢀ1.49
0.082ꢀ0.17
0.026ꢀ0.044
0.21ꢀ0.36
1.90ꢀ2.66
0.51ꢀ1.01
48 ( 0.9
36 ( 2.1h
6 ( 0.1
17
18
0.15
8.96
0.071ꢀ0.67
7.65ꢀ10.50
19
32 ( 1.9
60 ( 2.4e,j
31 ( 2.8e
5 ( 0.1f
20
21
1.97
7.15
8.63
1.01ꢀ3.54
4.30ꢀ10.01
5.93ꢀ10.25
22
23
0.39
0.26ꢀ0.61
46 ( 3.1
31 ( 1.3
8 ( 0.2
24
0.34
0.23ꢀ0.48
25
0.031
0.43
0.024ꢀ0.039
0.31ꢀ0.57
26
0.61
0.41ꢀ0.89
0.031ꢀ0.051
17 ( 0.9
82 ( 1.3e,m
17 ( 0.3f
12 ( 0.3f
1 ( 0.01f
22 ( 0.7i
nifedipine
48
0.26
0.19ꢀ0.36
0.039
0.029
0.0022
0.034
0.028
0.021ꢀ0.040
0.0014ꢀ0.0036
0.025ꢀ0.046
0.021ꢀ0.037
49
50
6 ( 0.4
68 ( 2.1k
genistein
44.48
34.95ꢀ56.61
a Decrease in developed tension on isolated guinea pig left atrium at 10ꢀ4 M, expressed as percent changes from the control (n = 5ꢀ6). The left atria were
driven at 1 Hz. b Decrease on atrial rate on guinea pig spontaneously beating isolated right atrium at 10ꢀ5 M, expressed as percent changes from the
control (n = 7ꢀ8). Pretreatment heart rate ranged from 165 to 190 beats/min. c Calculated from log concentrationꢀresponse curves (Probit analysis by
Litchfield and Wilcoxon17 with n = 6ꢀ7). When the maximum effect was <50%, the EC50 inotropic, EC50 chronotropic, and IC50 values were not
calculated. d Percent inhibition of calcium-induced contraction on Kþ-depolarized guinea pig aortic strip at 10ꢀ4 M (n = 5ꢀ6). e At 10ꢀ6 M. f At 10ꢀ5 M.
g At 10ꢀ7 M. h At 5 ꢁ 10ꢀ7 M. At 5 ꢁ 10ꢀ5 M. IC50 = 0.016 μM (CL 0.012ꢀ0.025). k At 10ꢀ4 M. At 5 ꢁ 10ꢀ6 M. m IC50 = 0.009 μM (CL
i
j
l
0.003ꢀ0.020).
Figure 2. Evaluation of selected compounds on CFTR, negative inotropic activity (INO), negative chronotropic activity (CHRONO), vasorelaxation
of guinea pig aortic strips (AORTA), ileum longitudinal smooth muscle (GPILSM), and tracheal smooth muscle (TRACHEA). Data are normalized for
the most effective compound (100%) in each assay.
such as genistein. The finding that genistein has, at least in part,
a profile similar to that of CFTR-selective 1,4-DHPs (i.e., low
activity on aorta and high relaxant effect on ileum) suggests the
possibility that CFTR contributes to smooth muscle function in
some organs (Figure 2).
Here we have tested 4-imidazo[2,1-b]thiazole-1,4-dihydro-
pyridines as a possible new class of CFTR potentiators. Some
of these compounds are indeed highly effective on ΔF508-CFTR
and have a low activity on vascular smooth muscle cells similar to
48ꢀ50. However, they have a relatively low potency as CFTR
potentiators (Kd in the micromolar range).
The evaluation of the screening tests on CFTR, on cardiac
tissue, and on vascular and nonvascular tissues for identification
of the most promising molecules of the small library of the
imidazo[2,1-b]thiazole-1,4-DHP allows us to define some emer-
ging relationships.
As previously demonstrated, imidazo[2,1-b]thiazole substitu-
ents at C4 are able to change not only the cardiovascular
parameters12 but also the CFTR activity (Figure 2). From a
quantitative standpoint, compounds bearing a 3- or 4-trifluor-
omethylphenyl group or a 3- or 4-trifluoromethoxyphenyl group
at the 6-position of the imidazo[2,1-b]thiazole (17, 18, 20, 21,
38, 39) are the most effective on ΔF508-CFTR. A shift of the
trifluoromethoxyphenyl group at position 2 of the phenyl ring 37
or a replacement of the trifluoromethoxyphenyl group with a
methoxy group (35, 36) has a detrimental effect on ΔF508-
CFTR. Also the introduction of more than one methoxy group or
a nitro group on the phenyl ring drastically decreases activity on
ΔF508-CFTR (24, 40ꢀ47). All compounds with a trifluoro-
methyl group or a methyl group or chlorine in position 6 are less
CFTR-selective (16, 25, 27ꢀ34). The 2- and 3-positions of the
imidazo[2,1-b]thiazole ring moderately affect the ΔF508-CFTR
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Journal of Medicinal Chemistry
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Table 4. Relaxant Activity of Tested Compounds on
Kþ-Depolarized Guinea Pig Ileum Longitudinal Smooth
Muscle
Table 5. Bronchodilator Effect of Same Compounds on
Guinea Pig Trachea
compd
mean activitya ( SEM
IC50 b (μM)
95% CL (ꢁ10-6)
compd
mean activitya ( SEM
IC50 b (μM)
95% CL (ꢁ10-6)
16
77 ( 1.7c
67 ( 1.3
57 ( 2.4
33 ( 1.1
26 ( 2.1
38 ( 2.2
92 ( 1.4
40 ( 2.1
64 ( 1.6d
27 ( 1.3
87 ( 1.5c
47 ( 1.1
64 ( 2.5c
19 ( 0.7d
35 ( 2.2c
62 ( 2.3
30 ( 2.6
68 ( 2.2
29 ( 1.7
14 ( 1.1
1.11
0.061
0.13
0.87ꢀ1.23
0.043ꢀ0.084
0.10ꢀ0.17
16
90 ( 1.4c
69 ( 3.6d
83 ( 1.3e
72 ( 2.3
73 ( 3.2d
90 ( 3.4f
83 ( 2.4
80 ( 1.1
96 ( 2.5g
94 ( 1.4g
77 ( 0.2
70 ( 2.3h
92 ( 1.4
96 ( 3.3g
70 ( 1.3i
60 ( 1.4
97 ( 0.6g
78 ( 1.2
88 ( 2.2g
76 ( 1.3f
96 ( 1.0j
74 ( 1.4i
91 ( 2.3k
92 ( 1.1i
90 ( 2.3
92 ( 2.2j
86 ( 1.3k
70 ( 0.36e
83 ( 2.6i
52 ( 0.6i
68 ( 1.5i
84 ( 1.6k
0.014
0.0046
0.00088
0.46
0.010ꢀ0.018
0.0036ꢀ0.0058
0.00021ꢀ0.0017
0.36ꢀ0.61
0.0025ꢀ0.0042
0.0038ꢀ0.0073
0.19ꢀ0.32
0.20ꢀ0.33
0.24ꢀ0.42
17
17
18
18
19
19
20
20
0.0033
0.0053
0.25
21
21
25
2.34
1.89ꢀ2.89
22
26
23
0.26
28
0.031
3.66
0.023ꢀ0.041
1.21ꢀ4.74
24
0.32
30
25
0.057
0.038
0.21
0.043ꢀ0.076
0.029ꢀ0.050
0.14ꢀ0.31
31
26
37
27
38
0.058
0.026ꢀ0.099
28
0.095
0.55
0.072ꢀ0.13
0.19ꢀ0.93
39
29
45
30
8.83
5.53ꢀ10.41
0.55ꢀ0.84
nifedipine
48
0.096
0.070ꢀ0.13
31
0.68
33
0.51
0.39ꢀ0.62
49
0.00059
0.00042ꢀ0.00083
34
0.32
0.24ꢀ0.41
50
35
0.86
0.64ꢀ1.02
genistein
a Percent inhibition of carbachol (1 μM) induced contraction on guinea
pig trachea at 10ꢀ5 M. The 10ꢀ5 M concentration gave the maximum
effect for most compounds. b Calculated from log concentrationꢀ
response curves (Probit analysis by Litchfield and Wilcoxon17 with
n = 6ꢀ7). When the maximum effect was <50%, the IC50 values were not
calculated. c At 10ꢀ4 M. d At 10ꢀ7 M.
38
0.018
0.036
4.89
0.010ꢀ0.097
0.028ꢀ0.046
3.74ꢀ6.40
39
40
41
1.14
0.73ꢀ1.85
42
2.16
1.03ꢀ2.94
43
0.45
0.10ꢀ0.91
45
0.074
11.43
0.0015
0.23
0.057ꢀ0.097
8.78ꢀ14.87
0.0011ꢀ0.0022
0.17ꢀ0.31
’ CONCLUSIONS
47
Our study confirms the possibility in the development of
1,4-DHPs with increased activity and selectivity as potentiators
of mutant CFTR and, at the same time, reduced activity as
inhibitors of voltage-dependent Ca2þ channels of the cardiovas-
cular system and smooth muscle of nonvascular tissues (see
Figure 3). This is highly desirable for developing drugs able to
correct the basic defect in cystic fibrosis while having minimal side
effects. However, the 1,4-DHPs evaluated in this study still keep
some activity in nonepithelial cells. Thismay be caused by residual
activity on some types of Ca2þ channels and/or expression of
CFTR in cardiac and smooth muscle cells. To further minimize
undesired effects, it is possible to generate novel 1,4-DHPs
based on SAR obtained from the present and previous studies.
In general the results suggest that the core imidazo[2,1-b]thiazole
is confirmed as a suitable heterocycle to explore new potentiators
of mutant CFTR. Of further interest is the effect of the imidazo-
[2,1-b]thiazole substitution pattern on the profile of biological
action. CFTR-selective potentiators might be better achieved by
a phenyl ring with appropriate substituents at the 6-position of the
imidazo[2,1-b]thiazole, while the substituents at the 2-position
can influence cardiovascular parameters to the same extent. In
addition, systemic effects may be further minimized by consider-
ing delivery of 1,4-DHP potentiators via aerosol.
nifedipine
48
49
1.85
1.31ꢀ2.03
50
0.94
0.64ꢀ1.38
genistein
9.95
8.01ꢀ12.35
a Percent inhibition of calcium-induced contraction on Kþ-depolarized
(80 mM) guinea pig longitudinal smooth muscle at 10ꢀ6 M. b Calculated
from log concentrationꢀresponse curves (Probit analysis by Litchfield
and Wilcoxon17 with n = 6ꢀ7). When the maximum effect was <50%,
the IC50 values were not calculated. c At 10ꢀ7 M. d At 10ꢀ8 M. e At 5 ꢁ
10ꢀ9 M. f At 5 ꢁ 10ꢀ8 M. g At 5 ꢁ 10ꢀ6 M. h At 3 ꢁ 10ꢀ7 M. i At 10ꢀ5 M.
j At 5 ꢁ 10ꢀ7 M. k At 5 ꢁ 10ꢀ5 M.
activity more than the cardiovascular profile. In both cases, the
aromaticity of the system must be kept as demonstrated by
compound 23 which is inactive in the assays. A comparison of
the ΔF508-CFTR activities of compounds 27, 28, 33, and 34
seems to indicate that a methyl in position 2 of the heterocycle
can positively influence the effect. This influence is greater if the
L-type calcium channel blocker activity is considered (27 vs 28).
The results in the functional assay point out that esterification of
the carboxylic functions in positions 3 and 5 of the 1,4-DHP ring
with bulky and more hydrophobic groups such as allylic or ethyl
groups decreases potency on CFTR while two methyl groups
are better for CFTR potentiators13 (see compounds 21 and 18 vs
20 and 17). On the contrary, the introduction of ester, ethyl, or
allyl chains enhances L-type calcium channel blocker activity.
’ EXPERIMENTAL SECTION
A. Chemistry. All the compounds prepared have a purity of at
least 95% as determined by combustion analysis. The melting points are
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Journal of Medicinal Chemistry
ARTICLE
Figure 3. (a) Comparison of potencies of different biological parameters for compounds with the best, interesting profile. (b) Reference compounds.
Data are expressed as ꢀlog potency ( CL.
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 60ꢀ80 °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.;
(1H, d, th, J = 4.6), 9.06 (1H, s, NH, ex D2O). Mp = 185ꢀ190 °C. MW =
443.4435. Anal. (C19H20F3N3O4S) C, H, N.
Dimethyl 2,6-Dimethyl-4-(6-(4-(trifluoromethyl)phenyl)imidazo-
[2,1-b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (17). 25%
yield. 1H NMR: 2.20 (6H, s, CH3), 3.18 (6H, s, COOCH3), 5,72 (1H, s,
py), 7.27 (1H, m, th), 7.33 (1H, m, th), 7.79 (2H, d, ar, J= 5.2), 8.09 (2H, d,
ar, J = 5.2), 9.13 (1H, s, NH, ex D2O). Mp = 227. MW = 491.1126. Anal.
(C23H20F3N3O4S) C, H, N.
Diethyl 2,6-Dimethyl-4-(6-(4-(trifluoromethyl)phenyl)imidazo[2,1-
b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (18). 30% yield.
1H NMR: 0.81 (6H, t, COOCH2CH3, J = 7.1), 2.15 (6H, s, CH3), 3.75
(4H, m, COOCH2CH3), 5.65 (1H, s, py), 7.32 (1H, d, th, J = 4.4), 7.43
(1H, d, th, J = 4.4), 7.70 (2H, d, ar, J = 8.4), 8.04 (2H, d, ar, J = 8.4), 8.98
(1H, s, NH, ex D2O). Mp = 175ꢀ180. MW = 519.5400. Anal.
(C25H24F3N3O4S) C, H, N.
Diallyl 4-(2,3-Dimethyl-6-(4-(trifluoromethyl)phenyl)imidazo[2,1-
b]thiazol-5-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (19).
10% yield. 1H NMR: 1.88 (6H, s, CH3), 2.31 (3H, s, CH3), 2.64 (3H, s,
CH3), 4.31 (2H, dd, J = 5.45, J = 13.65, CH2CHdCH2), 4.50 (2H, dd,
J = 5.45, J = 13.65, CH2CHdCH2), 4.88 (2H, dd, J = 1.55, J = 17.2,
CH2CHdCH2), 5.00 (2H, dd, J = 1.55, J = 10.5, CH2CH=CH2), 5,43 (1H,
s, py), 5.58 (2H, m, CH2CHdCH2), 7.30 (2H, d, ar, J = 8.2), 7.68 (2H, d, ar,
J = 8.2), 8.18 (1H, s, NH, ex D2O). Mp = 165ꢀ170. MW = 571.6149. Anal.
(C29H28F3N3O4S) C, H, N.
Dimethyl 2,6-Dimethyl-4-(6-(4-(trifluoromethoxy)phenyl)imidazo-
[2,1-b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (20).20% yield.
1H NMR: 2.20 (6H, s, CH3), 3.21 (6H, s, COOCH3), 5.67 (1H, s, py),
7.27 (1H, d, th, J = 3.1),7.32 (1H, d, th, J = 3.1), 7.42 (2H, d, ar, J = 5.7), 7.96
(2H, d, ar, J = 5.7), 9.08 (1H, s, NH, ex D2O). Mp = 217. MW = 507.1075.
Anal. (C23H20F3N3O5S) C, H, N.
1
Rmax is expressed in cmꢀ1. The H 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. Abbreviations are as follows:
th = thiazole, im = imidazole, ar = aromatic, py = pyridine, ex = H linked
to N that exchanged with D2O.
All solvents and reagents, unless otherwise stated, were supplied by
Sigma-Aldrich Chemical Co. Ltd. and were used as supplied. For a new
starting compound, see Table S1 in Supporting Information.
General Procedure for the Synthesis of the 1,4-Dihydropyridines
16ꢀ26. Methyl acetoacetate or ethyl acetoacetate or allyl acetoacetate
(2 mM) and 30% NH4OH (4 mM) were added to a stirred solution of
the appropriate aldehyde 8ꢀ15 (1 mM) dissolved in isopropyl alcohol
(50 mL). The reaction mixture was refluxed for 1ꢀ4 days (according to a
TLC test acetone/petroleum ether 55ꢀ85 °C, 1:9 v/v, 2:8 v/v), and to it
were added methyl acetoacetate or ethyl acetoacetate or allyl acetoace-
tate (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 40ꢀ60 °C from 1.9 to 4.6, v/v) to provide the pure
DHPs as a pale yellow syrup. The resulting oily residue was diluted with
cold acetone to afford a white solid collected by filtration for 16, 19,
21ꢀ26. For 18 the oily residue was diluted with diethyl ether. For 17 and
20 resulting oils were taken with acetoneꢀpetroleum ether 1/1 v/v to
obtain a white solid collected by filtration.
Diethyl 2,6-Dimethyl-4-(6-(trifluoromethyl)imidazo[2,1-b]thiazol-5-
yl)-1,4-dihydropyridine-3,5-dicarboxylate (16). 25% yield. 1H NMR:
0.96 (6H, t, COOCH2CH3, J = 7.1), 2.23 (6H, s, CH3), 3.90 (4H, q,
COOCH2CH3, J = 7.1), 5.52 (1H, s, py), 7.44 (1H, d, th, J = 4.6), 7.63
Diallyl 2,6-Dimethyl-4-(6-(4-(trifluoromethoxy)phenyl)imidazo[2,1-
b]thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (21). 10% yield.
1H NMR: 2.16 (6H, s, CH3), 4.15 (2H, dd, J = 5.55, J = 13.5,
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Journal of Medicinal Chemistry
ARTICLE
CH2CHdCH2), 4.33 (2H, dd, J = 5.55, J = 13.5, CH2CHdCH2), 4.91
(2H, dd, J = 1.5, J = 10.35, CH2CHdCH2), 4.98 (2H, dd, J = 1.5, J = 10.35,
CH2CHdCH2), 5.54 (2H, m, CH2CHdCH2), 5.62 (1H, s, py), 7.28 (1H,
d, th, J = 4.35), 7.36 (2H, d, ar, J = 8.7), 7.38 (1H, d, th, J = 4.35), 7.86 (2H,
d, ar, J = 8.7), 9.00 (1H, s, NH, ex D2O). Oil. MW = 559.1388. Anal.
(C27H24F3N3O5S) C, H, N.
Diethyl 2,6-Dimethyl-4-(2-methyl-6-phenylimidazo[2,1-b]thiazol-5-
yl)-1,4-dihydropyridine-3,5-dicarboxylate (22). 20% yield. 1H NMR:
0.85 (6H, t, COOCH2CH3, J = 7.05), 2.12 (6H, s, CH3), 2.42 (3H, s,
CH3), 3.71 (2H, q, COOCH2CH3, J = 7.05), 3.81 (2H, q, COOCH2CH3,
J = 7.05), 5.54 (1H, s, py), 7.25 (3H, m, th þ ar), 7.36 (1H, t, ar, J = 6.95),
7.72 (2H, d, ar, J = 6.95), 8.80 (1H, s, NH, ex D2O). Mp = 205. MW =
465.1722. Anal. (C25H27N3O4S) C, H, N.
Dimethyl 2,6-Dimethyl-4-(6-phenyl-2,3-dihydroimidazo[2,1-b]thiazol-
5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (23). 30% yield. 1H NMR:
2.16 (6H, s, CH3), 3.23 (6H, s, COOCH3), 3.89 (2H, d, thn, J =
6.2), 3.98 (2H, d, thn, J = 6.2), 5.44 (1H, s, py), 7.23 (1H, t, ar, J =
7.3),7.35 (2H, t, ar, J = 7.3), 7.70 (2H, d, ar, J = 7.3), 8.82 (1H, s,
NH, ex D2O). Mp = 245. MW = 424.4966. Anal. (C22H22N3O4S)
C, H, N.
’ ACKNOWLEDGMENT
This work was supported by grants from University of
Bologna, Italy, from Telethon Italy (Grant GGP10026), from
Fondazione Italiana per la Fibrosi Cistica (Grant FFC2/2009),
and from the Cystic Fibrosis Foundation Therapeutics. The
authors thank Alessandro Casoni for animal care.
’ ABBREVIATIONS USED
CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane con-
ductance regulator; TM, transmembrane; LTCC, L-type calcium
channel; 1,4-DHP, 1,4-dihydropyridine; GPILSM, guinea pig
ileum longitudinal smooth muscle; NMR, nuclear magnetic
resonance; IR, infrared; DMSO, dimethylsulfoxide; DMF, N,N-
dimethylformamide; SEM, standard error of the mean; ER, en-
doplasmic reticulum; CL, confidence limit
’ REFERENCES
(1) Riordan, J. R.; Rommens, J. M.; Kerem, B.; Alon, N.; Rozmahel,
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tion of the cystic fibrosis gene: cloning and characterization of com-
plementary DNA. Science 1989, 245, 1066–1973.
(2) Wilschanski, M.; Durie, P. R. Patterns of GI disease in adulthood
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Diallyl 4-(6-(2,5-Dimethoxyphenyl)imidazo[2,1-b]thiazol-5-yl)-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (24). 15% yield.
1H NMR: 1.92 (6H, s, CH3), 3.46 (3H, s, OCH3), 3.69 (3H, s, OCH3),
4.35 (2H, dd, J = 5.15, J = 13.95, CH2CHdCH2), 4.48 (2H, dd, J =
5.15, J = 13.95, CH2CHdCH2), 4.86 (2H, dd, J = 1.4, J = 17.2,
CH2CHdCH2), 4.98 (2H, dd, J = 1.4, J = 10.6, CH2CHdCH2), 5.09
(1H, s, py), 5.69 (2H, m, CH2CHdCH2), 6.44 (1H, s, ar), 6.86 (1H, s,
ar), 7.11 (1H, d, th, J = 4.6), 7.74 (1H, d, th, J = 4.6), 8.14 (1H, s, NH,
ex D2O). Mp = 200ꢀ205. MW = 535.6157. Anal. (C28H29N3O6S)
C, H, N.
(4) Mitchell, I.; Corey, M.; Woenne, R.; Krastins, I. R.; Levison, H.
Bronchial hyperreactivity in cystic fibrosis and asthma. J. Pediatr. 1978,
93, 744–748.
Diethyl 4-(6-Chloro-2-methylimidazo[2,1-b]thiazol-5-yl)-2,6-di-
methyl-1,4-dihydropyridine-3,5-dicarboxylate (25). 75% yield.
1HNMR: 0.99 (6H, t, COOCH2CH3, J = 7.2), 2.25 (6H, s, CH3),
2.42 (3H, s, CH3), 3.90 (4H, m, COOCH2CH3), 5.12 (1H, s, py), 7.58
(1H, s, th), 9.04 (1H, s, NH, ex D2O). Mp = 175. MW = 423.1019. Anal.
(C19H22ClN3O4S) C, H, N.
Diethyl 2,6-Dimethyl-4-(6-(2,3,4-trichlorophenyl)imidazo[2,1-b]-
thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (26). 20% yield.
1H NMR: 0.93 (6H, t, COOCH2CH3, J = 7.1), 1.94 (6H, s, CH3), 3.91
(4H, m, COOCH2CH3), 5.07 (1H, s, py), 7.05 (1H, d, ar, J = 8.4), 7.26
(1H, d, th, J = 4.4), 7.66 (1H, d, ar, J = 8.4), 7.95 (1H, d, th, J = 4.4), 8.21
(1H, s, NH, ex D2O). Mp = 230ꢀ235. MW = 554.8765. Anal.
(C24H22Cl3N3O4S) C, H, N.
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B. CFTR Assays. For details, see Supporting Information, sec-
tion S10.
C. Functional Studies. For details, see Supporting Information,
section S11.
’ ASSOCIATED CONTENT
S
Supporting Information.
Chemistry details; analytical
b
data for compounds 5ꢀ26; IR data for compounds 16ꢀ26;
CFTR and functional assays; functional data for compounds
previously described12 (cardiovascular data for compounds
27ꢀ47 and relaxant activity on GPLSM for compounds 32,
36, 37, 44, and 46). This material is available free of charge via the
Internet at http://pubs.acs.org.
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Antagonists and Calcium Blockers. In Foye’s Principles of Medicinal
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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
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’ AUTHOR INFORMATION
Corresponding Author
*For R.B.: phone, þ39-051-2099737; fax, þ39-051-2099721;
e-mail, roberta.budriesi@unibo.it. For L.J.V.G.: phone, þ39-
010-5636801; fax, þ39-010-3779797; e-mail, galietta@unige.it.
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