Asymmetric 4-Aryl-1,4-dihydropyridines Potentiate Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Article abstract of DOI:10.1002/cmdc.201200311

Some of the genetic mutations that cause cystic fibrosis (CF) impair the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- ion channel. This defect can be corrected with pharmacological tools (potentiators) that belong to various chemical families, including the 1,4-dihydropyridines (DHPs). A small set of asymmetric 4-aryl-DHPs was synthesized, and each racemic couple was tested in a functional assay carried out on cells expressing the G1349D, ΔF508, and G551D mutants. The most active racemates were subjected to chiral separation by HPLC, and the pure enantiomers were tested to evaluate any gains in activity. Although three enantiomers demonstrated high potency (K_d values less than 0.09, 0.1, and 0.5μM in G1349D, ΔF508, and G551D, respectively), in general, the screening of pure enantiomers did not produce a great diversity in potency values. It is probable that the degree of DHP asymmetry considered in our analysis is still insufficient with respect to that allowed in a putative DHP binding site in CFTR, so that the site could equally accommodate both enantiomers.

Full text of DOI:10.1002/cmdc.201200311

MED
DOI: 10.1002/cmdc.201200311
Asymmetric 4-Aryl-1,4-dihydropyridines Potentiate Mutant
Cystic Fibrosis Transmembrane Conductance Regulator
(CFTR)
Michele Giampieri,[a] Nicolas Vanthuyne,[b] Erika Nieddu,[a] Marco T. Mazzei,[a] Maria Anzaldi,[a]
Nicoletta Pedemonte,[c] Luis J. V. Galietta,[c] Christian Roussel,[b] and Mauro Mazzei*[a]
Some of the genetic mutations that cause cystic fibrosis (CF)
impair the gating of the cystic fibrosis transmembrane conduc-
tance regulator (CFTR) ClÀ ion channel. This defect can be cor-
rected with pharmacological tools (potentiators) that belong
to various chemical families, including the 1,4-dihydropyridines
(DHPs). A small set of asymmetric 4-aryl-DHPs was synthesized,
and each racemic couple was tested in a functional assay car-
ried out on cells expressing the G1349D, DF508, and G551D
mutants. The most active racemates were subjected to chiral
separation by HPLC, and the pure enantiomers were tested to
evaluate any gains in activity. Although three enantiomers
demonstrated high potency (Kd values less than 0.09, 0.1, and
0.5 mm in G1349D, DF508, and G551D, respectively), in general,
the screening of pure enantiomers did not produce a great di-
versity in potency values. It is probable that the degree of DHP
asymmetry considered in our analysis is still insufficient with
respect to that allowed in a putative DHP binding site in CFTR,
so that the site could equally accommodate both enantiomers.
Introduction
Cystic fibrosis (CF) is a genetic pathology caused by mutations
in the cAMP-activated cystic fibrosis transmembrane conduc-
tance regulator (CFTR) ClÀ ion channel. Patients mainly suffer
from pulmonary infection and inflammation, pancreatic insuffi-
ciency, and male sterility.[1,2] CFTR is a very complex plasma
membrane protein composed of 1480 amino acids arranged in
two transmembrane domains, each containing six a-helices,
two nucleotide binding domains (NBD1 and NBD2), and a regu-
latory domain.[3] At this point, more than 1900 mutations have
been discovered, with the deletion of the phenylalanine resi-
due at position 508 (DF508 CFTR) being the most common
and one of the most severe mutations in CF patients (http://
www.genet.sickkids.on.ca/cftr/app).
membrane stability.[14] These defects can be corrected by the
same potentiators that are effective on G551D and G1349D
mutations.[15]
The precise mechanism of action of potentiators is still un-
clear. Mutations of class III allow the normal trafficking of the
CFTR protein to the plasma membrane, but impair channel
gating by delaying channel opening and decreasing the stabili-
ty of the open channel conformation.[8,9] Potentiators such as
genistein,[16] benzoflavones,[17] phenylglycines,[18] and others are
thought to have one or more binding sites at the NBD1/NBD2
level, the occupancy of which by the potentiator molecule
leads to partial restoration of channel activity. In this regard,
many theoretical studies have been performed, particularly
The severity of disease for each CF patient depends largely
on the specific set of mutations that affect the CFTR gene. De-
pending on the type of molecular defect, researchers have or-
ganized the CFTR mutations into five classes: lack of protein
synthesis (class I), CFTR protein maturation defect (class II),
channel gating defect (class III), decrease in single channel con-
ductance (class IV), and decreased CFTR synthesis (class V).[4–6]
Compounds able to repair class II CFTR mutations (among
which the most relevant is DF508) are defined as “correctors”,
whereas compounds able to ameliorate the gating defect in
class III mutations (which include G551D and G1349D) are de-
fined as “potentiators”.[7–10] Among these latter potential thera-
peutics, great interest has been placed on VX-770,[11,12] a 4-qui-
nolone derivative very recently approved by the USFDA and
marketed as Kalydeco (http://www.cff.org/treatments/Pipeline).
Notably, when cells with the DF508 mutation are incubated at
low temperature, the mutant protein may escape from degra-
dation and reach the plasma membrane.[13] However, the “res-
cued DF508 CFTR” has decreased channel activity and low
[a] Dr. M. Giampieri, Dr. E. Nieddu, Dr. M. T. Mazzei, Dr. M. Anzaldi,
Prof. M. Mazzei
Department of Pharmacy, University of Genova
Viale Benedetto XV, 3, 16132 Genova (Italy)
E-mail: mauro.mazzei@unige.it
[b] Dr. N. Vanthuyne, Prof. C. Roussel
Aix-Marseille University, ISM2-UMR-CNRS 7313, Chirosciences
Avenue Escadrille Normandie-Niꢀmen, 13397 Marseille Cedex 20 (France)
[c] Dr. N. Pedemonte, Dr. L. J. V. Galietta
Laboratory of Molecular Genetics, Giannina Gaslini Institute
Largo Gerolamo Gaslini, 5, 16147 Genova (Italy)
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M. Mazzei et al.
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using genistein as a ligand, showing some putative binding
sites on NBDs. However, to shed more conclusive light on this
difficult and important aspect, additional refinements are nec-
essary.[19–21]
The present work deals with the search for new CF potentia-
tors. Some years ago, in order to find chemical substances
useful in treating CF, the anti-hypertensive 4-phenyl-1,4-dihy-
dropyridines (DHPs) nifedipine, felodipine, and nitrendipine
were identified as compounds able to stimulate the activity of
class III CFTR mutants.[22,23] Unfortunately, CFTR channel activa-
tion only becomes apparent at doses higher than those re-
quired for the anti-hypertensive activity of DHPs; such activity
should be avoided in CF patients.
Recently, the challenge to obtain suitable DHPs endowed
with sufficient channel potency, yet free of anti-hypertensive
activity, led to the evaluation of 4-thiophenyl-1,4-dihydropyri-
dines[24] and 4-imidazo[2,1-b]thiazole-1,4-dihydropyridines[25] as
sound and effective potentiators. In particular, in the evalua-
tion of 4-thiophenyl derivatives, we found that more favorable
activity was present in some asymmetric DHPs bearing differ-
ent substituents at the ester level (namely, at positions 3 and 5
of the 1,4-dihydropyridine ring). Among those compounds,
a good activity value (Kd =0.3 mm) was observed for the deriva-
Scheme 1. Synthesis of asymmetric DHPs 4a–e (see the Experimental Sec-
tion for specific reaction conditions).
tive
1,4-dihydro-2,6-dimethyl-4-(3’-methylthiophen-2’-yl)pyri-
dine-3-carboxylic acid allyl ester, 5-carboxylic acid ethyl ester.
Figure 1. 4-(Naphth-2’-yl)-1,4-dihydropyridines 4 f–i.
1e (or 1a, 1b), acetoacetate 2a (or 2b, 2d), and aminocroto-
nate 3, DHPs 4j–l were synthesized (Scheme 2).
In this work, we set out to evaluate the activity of 4-aryl-
DHPs with dissimilar substituents at ester level toward class III
mutant CFTR forms. For this purpose, we synthesized a small
set of asymmetric DHPs and tested their biological activity ini-
tially as racemates; then, the more active DHPs were submit-
ted to chiral chromatography to determine whether the activi-
ty could be assigned to a single enantiomer.
Despite the careful addition of reagents, HPLC analyses of
compounds 4a–l also showed the presence of symmetric de-
rivatives, formed as by-products through an unavoidable
double reaction of the acetoacetates on a single aldehyde and
transesterification reaction. Therefore, the need for several
crystallization steps in the purification of asymmetric solid
DHPs (and chromatographic purifications for oils) explains the
low yields of final products. Compounds 4a–l are white or
pale-yellow crystals or pale-yellow oils. The proposed struc-
tures were confirmed by spectral data and elemental analyses.
Chemistry
The synthesis of the asymmetric dihydropyridines 4a–l was
carried out by a modified Hantzsh method. DHPs 4a–e were
synthesized in three steps to minimize the formation of sym-
metric DHPs. Therefore, as shown in Scheme 1, the aromatic al-
dehydes 1a (or 1b, 1c) were treated with an equimolar
amount of the appropriate acetoacetate 2a (or 2b) in isopro-
panol. Upon completion of the first condensation reaction, an-
other equimolar amount of the second acetoacetate 2c (or
2d) was added. After this reaction was completed, ammonia
was finally added. Following the same procedure, starting from
2-naphthaldehyde 1d and 2a (or 2b) as the first acetoacetate
and 2c (or 2d, 2e) as the second acetoacetate, DHPs 4 f–
i were obtained (Figure 1). Finally, by using aromatic aldehydes
Scheme 2. Synthesis of asymmetric DHPs 4j–l (see the Experimental Section
for specific reaction conditions).
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4-Aryl-DHPs Potentiate Mutant CFTR
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Results
Biological results of racemates
Compounds 4a–l were first tested on Fischer rat thyroid (FRT)
cells expressing the G1349D mutation to evaluate, by means
of the iodide influx assay, whether the synthesized DHPs could
ameliorate the gating defect of mutant CFTR. As shown in
Table 1, all the 4-phenyl-DHPs 4a–e bearing two different car-
Table 1. Evaluation of G1349D CFTR gating effect by compounds 4a–
l tested as racemates.[a]
Compd
Kd [mm]
Emax [msÀ1
]
4a
4b
4c
4d
4e
4 f
4g
4h
4i
0.29Æ0.13
0.65Æ0.15
0.072Æ0.035
0.060Æ0.017
0.087Æ0.025
1.30Æ0.30
0.39Æ0.12
0.53Æ0.15
2.94Æ0.40
4.70Æ0.80
10.00Æ1.60
3.90Æ0.60
0.35Æ0.06
119Æ6.3
115Æ7.5
117Æ6.7
102Æ6.9
109Æ7.7
112Æ8.4
121Æ9.6
116Æ8.3
113Æ7.8
115Æ9.1
112Æ7.2
114Æ7.4
123Æ8.9
4j
4k
4l
felodipine
[a] Kd and Emax values are the mean ÆSEM of n=5–10 experiments.
Figure 2. Functional evaluation of asymmetric DHP 4c and 4d. A) Represen-
tative traces from the YFP assay. FRT cells co-expressing DF508 CFTR and the
halide-sensitive YFP were stimulated with forskolin (20 mm) alone or with in-
dicated DHPs (2 mm). Addition of iodide-rich buffer caused YFP fluorescence
quenching at a rate proportional to CFTR activity. The y-axis scale bar repre-
sents the fraction of normalized cell fluorescence. B) Representative short-
circuit current recording from FRT cells expressing DF508 CFTR. Following
stimulation with forskolin and with increasing concentrations of compound
4d, total CFTR current was blocked with CFTR inhibitor-172 (inh-172, 10 mm).
boxyalkyl esters elicited significant effects, whereas 4-naphthyl-
DHPs 4 f–i are endowed with moderate activity. Asymmetric
DHPs with a carboxyalkyl ester and a cyano group (4j–l) are
weakly active.
Five asymmetric DHPs, namely 4a, 4c, 4d, 4e, and 4g, the
Kd values of which are all <0.4 mm (Table 1), were also tested
on DF508 and G551D mutations (Table 2), and interesting re-
Chiral separation of asymmetric DHPs
The above compounds 4a, 4c, 4d, 4e, and 4g were subjected
to enantiomeric separation by chiral HPLC. For each com-
pound the stationary phase and the eluent composition were
optimized to give the best results in peak separation. The sta-
tionary phases and eluents used (H=hexane, I=isopropanol)
are listed in Table 3 along with retention times (tR), retention
factors (k), enantioselectivity (a), and resolution (Rs). The re-
ported sign of the enantiomers is the sign of the optical rota-
tion at l 589 nm in chloroform. All experimental details are
given in the Experimental Section.
Table 2. Evaluation of rescued DF508 and G551D CFTR gating effects by
selected compounds tested as racemates.[a]
Rescued DF508
Kd [mm]
G551D
Compd
Emax [msÀ1
]
Kd [mm]
Emax [msÀ1
]
4a
4c
4d
4e
4g
0.67Æ0.18
0.067Æ0.025
0.17Æ0.09
0.27Æ0.11
0.51Æ0.11
0.88Æ0.16
25Æ1.4
24Æ1.7
23Æ1.5
22Æ2.0
26Æ2.2
29Æ1.9
1.10Æ0.20
0.60Æ0.19
0.62Æ0.15
0.19Æ0.05
1.80Æ0.30
22.60Æ2.20
19Æ1.3
18Æ1.4
22Æ1.9
16Æ1.2
21Æ2.0
21Æ1.7
Chiral separation of the above-indicated DHPs gave good re-
sults. For instance, Figure 3 shows chromatograms from the
chiral separation of 4c, together with determination of enan-
tiomeric purity of the separated peaks obtained after semi-
preparative chromatography. Enantiomeric excesses (%ee)
were measured by integration of the UV signal at l 254 nm
and confirmed by the circular dichroism (CD) signal.
felodipine
[a] Kd and Emax values are the mean ÆSEM of n=5–10 experiments.
sults relative to felodipine were observed, especially in terms
of potency (Figure 2A). The values of efficacy between the
new DHPs and felodipine are similar. The results obtained with
the YFP assay were also confirmed with short-circuit current re-
cordings (Figure 2B). Application of asymmetric DHPs elicited
dose-dependent increases in the CFTR-dependent ClÀ current.
With samples (~20 mg) of the enantiomers (+)-4a, (À)-4a,
(+)-4c, (À)-4c, (+)-4d, (À)-4d, (+)-4e, (À)-4e, (+)-4g, and
(À)-4g available, the biological tests on FRT cells were repeat-
ed. Table 4 presents the results of these five pairs of enantio-
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Discussion
Table 3. Chromatographic data for selected DHPs.
[b]
[c]
Enantiomer
Column
Eluent[a]
H/I 7:3
H/I 9:1
H/I 9:1
H/I 9:1
H/I 7:3
tR1/tR2
k1/k2
a[d]
Rs[e]
1.16
2.53
2.78
1.21
1.26
ee [%]
Previous works that describe
a potential pharmacotherapy for
CF stress the importance of
small molecules that are able to
improve the gating and/or traf-
ficking of mutant CFTR.[26–28] For
class III mutations, it was demon-
strated that the 4-phenyl-DHPs
act as very effective potentiators,
as these compounds are able to
restore the correct gating pro-
cess.[22,23] Moreover, potentiators
are also thought to be very im-
portant therapeutic agents for
DF508 CFTR, because this
mutant also has a gating defect
in addition to impaired traffick-
ing.[29]
(À)-4a
(+)-4a
(+)-4c
(À)-4c
(+)-4d
(À)-4d
(À)-4e
(+)-4e
(À)-4g
(+)-4g
13.3
15.0
20.7
25.3
23.4
29.6
11.2
12.3
7.9
3.36
3.90
96
95
94
95
99
98
97
96
99
98
(S,S)-Whelk-O1
(S,S)-Whelk-O1
(S,S)-Whelk-O1
Chiralpak IC
1.45
1.26
1.31
1.15
1.18
12.63
15.64
14.38
18.47
2.60
2.98
4.17
4.72
(S,S)-Whelk-O1
8.7
[a] H=hexane; I=iPrOH. [b] Retention time ratio. [c] Retention factor ratio. [d] Enantioselectivity. [e] Resolution.
Table 4. Evaluation of G1349D, rescued DF508, and G551D CFTR gating effects by selected DHPs tested as
pure enantiomers.[a]
G1349D
Rescued DF508
Kd [mm]
Emax [msÀ1
G551D
Compd
Kd [mm]
Emax [msÀ1
]
]
Kd [mm]
Emax [msÀ1
]
To improve our knowledge of
DHPs as CFTR potentiators, we
synthesized a small set of asym-
metric DHPs (4a–l). Such DHPs
were prepared with commercial
starting materials, and so they
do not present great asymmetry
because at the ester level we
can provide only a small differ-
ence between R’ and R’’ groups
(Scheme 1 and Figure 1); the
highest degree of asymmetry is
(+)-4a
(À)-4a
(+)-4c
(À)-4c
(+)-4d
(À)-4d
(+)-4e
(À)-4e
(+)-4g
(À)-4g
0.362Æ0.125
0.210Æ0.085
0.088Æ0.055
0.036Æ0.025
0.046Æ0.021
0.216Æ0.031
0.080Æ0.055
0.158Æ0.045
0.559Æ0.155
0.313Æ0.075
126Æ9.8
121Æ8.0
123Æ8.3
120Æ7.8
97Æ6.7
1.0Æ0.3
29Æ2.2
28Æ1.9
28Æ2.3
27Æ2.0
28Æ2.4
25Æ2.1
30Æ2.5
29Æ2.3
28Æ2.0
30Æ2.5
1.8Æ0.40
0.6Æ0.20
1.1Æ0.30
18Æ1.6
18Æ1.5
18Æ1.1
18Æ1.0
23Æ1.9
19Æ1.6
17Æ1.6
16Æ1.1
19Æ1.2
21Æ1.8
0.5Æ0.2
0.16Æ0.02
0.082Æ0.011
0.061Æ0.022
0.187Æ0.025
0.09Æ0.2
0.20Æ0.04
0.46Æ0.10
0.77Æ0.12
0.14Æ0.04
0.24Æ0.08
2.3Æ0.5
109Æ8.1
130Æ10.2
125Æ11.3
121Æ9.4
122Æ7.2
0.25Æ0.03
1.4Æ0.3
0.48Æ0.17
1.5Æ0.4
[a] Kd and Emax values are the mean ÆSEM of n=5–10 experiments.
mers; compounds (À)-4a, (À)-4c, (+)-4d, (+)-4e, and (À)-4g
generally show a slightly higher activity than their enantiomer-
ic counterparts in all cell lines. In particular, the Kd ratios of the
above values range from 1.7 to 4.7 for G1349D, from 2.0 to 3.1
for rescued DF508, and from 1.5 to 5.5 for G551D.
in derivatives 4c and 4i, where R’ and R’’ are methyl and
benzyl groups, respectively. A greater degree of asymmetry
would require more complex syntheses, and so may be
a future development in this topic.
Figure 3. Enantiomeric HPLC separation of 4c detected by UV and CD: A) analytical separation of 4c; B) enantiomeric excess after semi-preparative separation
of (+)-4c; C) enantiomeric excess after semi-preparative separation of (À)-4c.
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4-Aryl-DHPs Potentiate Mutant CFTR
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Looking at the structure–activity relationship of the synthe-
sized derivatives, DHPs 4a–e emerged as interesting potentia-
tors when tested as racemates, suggesting the relevance of
esters at positions 3 and 5, with a phenyl group present at po-
sition 4.[22–24] In particular, compounds 4c, 4d, and 4e show ac-
tivity at concentrations <0.1 mm in the G1349D cell line, giving
evidence, in this small series, for the importance of a branched
substituent at 4’. The presence of a benzyl substituent in the
ester group also seems to be valuable for this type of activity.
On the other hand, 4-naphthyl derivatives 4 f–i showed moder-
ate activity (only 4g has a Kd value lower than 0.4 mm in the
G1349D cell line). Cyano derivatives 4j–l were weakly active
(Table 1), thus confirming that both esters (at positions 3 and
5) are mandatory for high activity.
bulky alkyl groups at the 4’-position of the 4-phenyl substitu-
ent is sufficient to render such derivatives inefficient as calcium
blockers.[30,31] Because our most active derivatives (À)-4c and
(+)-4d have an isopropyl group at 4’, and (+)-4e bears an iso-
propyloxy group at 4’, we hypothesize that these compounds
possess negligible anti-hypertensive activity.
Conclusions
The synthesis and biological evaluation of some asymmetric
DHPs led to racemates endowed, in some cases, with signifi-
cant activity as CFTR potentiators. The more interesting race-
mates were subjected to enantiomeric separation: for specific
pure enantiomers (À)-4c, (+)-4d, and (+)-4e an increase in
potency was observed. Unfortunately, such increased potency
for the enantiomerically pure derivatives is modest relative to
the starting racemates; the maximum expected increase in ac-
tivity would be twice the activity of the racemate if the activity
were isolated to a single enantiomer. For this reason, the in-
vestment in terms of work and cost for the separation is very
high and demanding in view of the obtained results. It is likely
that a more advantageous use of enantiomers would be realiz-
ed by using derivatives with a greater degree of asymmetry
than that of our derivatives. In fact, the most potent DHPs,
namely (À)-4c, (+)-4d, and (+)-4e, exhibit the greatest asym-
metry in our derivatives, because at the ester level a benzyl
group and a short alkyl group are present. This hypothesis is
consistent with the observation that VX-770 may also be struc-
turally considered as a 1,4-dihydropyridine with a broad asym-
metry. For highly asymmetric DHPs, the synthetic procedures
could be more elaborate and complex. The generation of
highly asymmetric DHPs may be guided by molecular model-
ing provided that a suitable binding site for DHPs in mutant
CFTR is identified.
Compounds that showed the best values in the G1349D cell
line (Kd <0.4 mm) were also tested against rescued DF508 and
G551D mutations, demonstrating high potency (Table 2). For
example, 4c, 4d, and 4e have Kd values less than 0.3 and
0.7 mm in rescued DF508 and G551D cell lines, respectively.
The above compounds show greater potency in all cell lines
relative to other DHPs and the reference compound felodipine
(Tables 1 and 2). Interestingly, we found that the compounds
appear to be more active toward G1349D than toward the
DF508 mutant. This is not surprising, as these two mutations
affect different NBDs (NBD1 for DF508 and NBD2 for G1349D);
therefore, they may affect the potentiator binding site(s) differ-
ently (possibly localized at the NBD1/NBD2 interface). More-
over, emphasis must be placed on the potency values of 4c,
4d, and 4e with respect to felodipine in the G551D cell line:
in particular, 4e shows a gain in potency of about two orders
of magnitude (Table 2).
To increase the specificity of the binding, the asymmetric
DHPs 4a, 4c, 4d, 4e, and 4g were subjected to enantiomeric
separation by HPLC using chiral stationary phases. The separa-
tion of each compound was initiated by an analytical study to
obtain good peak resolution; this was then followed by scaled-
up separation with a semi-preparative column. The enantio- Experimental Section
meric excess of each collected peak was evaluated by UV and
Chemistry
CD spectroscopy. As evident in Figure 3 and in the Experimen-
Melting points were determined using an Electrothermal apparatus
tal Section, all peaks presented an enantiomeric excess
between 94 and 99%.
and are uncorrected. Microanalyses were carried out on a Carlo
Erba 1106 elemental analyzer. 1H NMR and 13C NMR spectra were
performed on a Varian Gemini 200 (200 MHz) spectrometer using
TMS as internal standard (d=0). IR spectra were recorded on a Per-
kinElmer 398 spectrophotometer. GC–MS analyses were performed
on an HP 6890-5973 spectrometer; GC parameters: injector tem-
perature 2508C; HP5 poly(methylphenylsiloxane) column 30 m,
0.25 mm, 0.25 mm; temperature profile: from 100 to 3008C;
108CminÀ1; MS parameters mode SCAN 40–600 Da. Compounds
used in biological testing possess purity of no less than 98% as de-
termined by elemental analysis (accuracy Æ0.4%) and mass spec-
trometry.
The biological tests on FRT cells were repeated for each sep-
arated enantiomer, and the results listed in Table 4 indicate
a clear, although not dramatic, increase in activity for com-
pounds (À)-4a, (À)-4c, (+)-4d, (+)-4e, and (À)-4g with re-
spect to the enantiomeric counterparts in all cell lines. In par-
ticular, compounds (À)-4c and (+)-4d showed Kd values lower
than 0.050 and 0.09 mm in G1349D and rescued DF508, respec-
tively, whereas compound (+)-4e had Kd <0.15 mm in G551D.
On the whole, (À)-4c, (+)-4d, and (+)-4e can be classified
among the most active derivatives of this series and also
among the best potentiators identified so far.
General procedure for the synthesis of asymmetric 1,4-dihy-
dropyridines (4a–i).
Commercial DHPs have anti-hypertensive activity due to the
blockade of Ca2+ channels, but such activity must be avoided
in CF patients. In the present work, we did not test the activity
of our DHPs on Ca2+ channels on the grounds that previous
research with DHPs has demonstrated that the presence of
To an ice-cooled solution of an aldehyde A (5 mmol) in iPrOH
(15 mL), the first acetoacetate B (5 mmol) was added, and the mix-
ture was held at reflux for 3 h. The second acetoacetate C
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M. Mazzei et al.
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(5 mmol) was then added, and the mixture was again held at
reflux for 3 h. At the end, NH3 (concd, 1 mL) was added, and the
mixture was held at reflux for 1 h. After cooling, the solvent was
evaporated under reduced pressure, and the crude reaction materi-
al obtained was crystallized from suitable solvent or purified by
column chromatography on silica gel with toluene/EtOAc (9:1).
(C5), 126.2 (2C-3’,5’), 127.5 (2C-2’’,6’’), 127.7 (C4’’), 128.2 (2C-3’’,5’’),
128.9 (2C-2’,6’), 135.1 (C1’), 140.8 (C1’’), 145.7 (C4’), 145.9 (C2),
146.3 (C6), 166.2, 166.9 ppm (COO); IR (KBr): n˜ =3440, 2963, 2872,
1690, 1618, 1470, 1229 ppm; MS: m/z=433 (100); Anal. calcd for
C27H31NO4: C 74.80, H 7.21, N 3.23, found: C 75.03, H 7.29, N 3.16.
1,4-Dihydro-2,6-dimethyl-4-(3’,4’-diisopropoxyphenyl)pyridine-3-
carboxylic acid benzyl ester, 5- carboxylic acid ethyl ester (4e):
(A): 3,4-diisopropyloxybenzaldehyde 1c, (B): ethyl acetoacetate 2b,
(C): benzyl acetoacetate 2d; pale yellow oil; yield: 29%, 0.73 g;
1H NMR (CDCl3): d=1.04–1.35 (m, 15H, CH(CH3)2 +CH2CH3), 2.24 (s,
3H, CH3-2), 2.25 (s, 3H, CH3-6), 4.02 (q, J=7.0, 2H, CH2CH3), 4.12-
4.38 (m, 2H, CH(CH3)2), 4.90–5.07 (m, 3H, CH2Ph+H-4), 5.79 (s, 1H,
NH), 6.29–7.31 (m, 8H, H-arom); 13C NMR (CDCl3): d=13.8 ppm
(CH3CH2), 18.8 (CH3-2), 18.9 (CH3-6), 22.4 (CH3CHCH3 in 3’), 22.6
(CH3CHCH3 in 4’), 32.3 (C4), 59.9 (CH3CH2), 70.7 (CH3CHCH3 in 3’),
70.8 (CH3CHCH3 in 4’), 72.5 (CH2Ph), 101.7 (C3), 102.3 (C5), 114.7
(C5’), 115.5 (C2’), 121.4 (C6’), 129.9 (C1’), 127.5 (2C-2’’,6’’), 127.7
(C4’’), 128.2 (2C-3’’,5’’), 140.8 (C1’’), 141.5 (C4’), 144.4 (C3’), 145.7
(C2), 146.6 (C6), 166.5, 166.8 ppm (COO); IR (KBr): n˜ =3440, 2980,
2874, 1690, 1618, 1500, 1227 cmÀ1; MS: m/z=507 (100); Anal.
calcd for C30H37NO6: C 70.98, H 7.35, N 2.76, found: C 71.26, H 7.30,
N 2.84.
1,4-Dihydro-2,6-dimethyl-4-(4’-bromophenyl)pyridine-3-carbox-
ylic acid allyl ester, 5-carboxylic acid methyl ester (4a): (A): 4-
bromobenzaldehyde 1a, (B): methyl acetoacetate 2a, (C): allyl ace-
1
toacetate 2c; pale yellow oil; yield: 39%, 0.79 g; H NMR (CDCl3):
d=2.24 (s, 3H, CH3-2), 2.26 (s, 3H, CH3-6), 3.55 (s, 3H, OCH3), 4.06
(d, J=5.4, 2H, CH2CH=CH2) 4.51 (d, J=9.6, 2H, CH2CH=CH2), 4.84-
5.11 (m, 2H, CH2CH=CH2 +H-4), 5.61 (s, 1H, NH), 7.01–7.34 ppm (m,
4H, H-arom); 13C NMR (CDCl3): d=18.7 ppm (CH3-2), 18.9 (CH3-6),
32.2 (C4), 51.2 (COOCH3), 70.3 (CH2CH=CH2), 102.4 (C3), 103.1 (C5),
116.2 (CH2-CH=CH2), 120.1 (C4’), 131.4 (2C-2’,6’), 131.7 (2C-3’,5’),
136.7 (C1’), 145.8 (C2), 146.5 (C6), 166.1, 166.7 ppm (COO); IR (KBr):
n˜ =3440, 2951, 2843, 1697, 1619, 1471, 1217 cmÀ1; MS: m/z: 407
(100); Anal. calcd for C19H20BrNO4: C 56.17, H 4.96, N 3.45, Br 19.67,
found: C 56.40, H 4.89, N 3.48, Br 19.81.
1,4-Dihydro-2,6-dimethyl-4-(4’-bromophenyl)pyridine-3-carbox-
ylic acid allyl ester, 5-carboxylic acid ethyl ester (4b): (A): 4-bro-
mobenzaldehyde 1a, (B): ethyl acetoacetate 2b, (C): allyl acetoace-
tate 2c; pale yellow solid; mp: 131–1328C; yield: 33%, 0.69 g; crys-
1,4-Dihydro-2,6-dimethyl-4-(naphth-2’-yl)pyridine-3-carboxylic
acid ethyl ester, 5-carboxylic acid isopropyl ester (4 f): (A): 2-
naphthaldehyde 1d, (B): ethyl acetoacetate 2b, (C): isopropyl ace-
toacetate 2e; white solid; mp: 154–1558C; yield: 27%, 0.53 g; crys-
tallized from cyclohexane; 1H NMR (CDCl3): d=0.96–1.24 (m, 9H,
CH2CH3 +CH(CH3)2), 2.26 (s, 3H, CH3-2), 2.28 (s, 3H, CH3-6), 4.00 (q,
J=7.2, 2H, CH2CH3), 4.77–4.93 (m, 1H, CH(CH3)2), 5.08 (s, 1H, H-4),
5.70 (s, 1H, NH), 7.28-7.74 (m, 7H, H-arom); 13C NMR (CDCl3): d=
13.5 ppm (CH3CH2), 18.6 (CH3-2), 18.8 (CH3-6), 22.0 (2C-CH3CHCH3),
32.2 (C4), 59.6 (CH3CH2), 69.0 (CH3CHCH3), 101.6 (C3), 101.9 (C5),
124.2 (C6’), 125.7 (C5’), 126.7 (C2’), 127.1 (C9’), 127.4 (C4’), 127.4
(C7’), 127.7 (C10’), 131.6 (C8’), 133.5 (C3’), 135.2 (C1’), 145.9 (C2),
146.7 (C6), 166.3, 166.7 (COO); IR (KBr): n˜ =3358, 2982, 2906, 1695,
1679, 1484, 1265; MS: m/z=393 (100); Anal. calcd for C24H27NO4: C
73.26, H 6.92, N 3.56, found: C 73.02, H 7.00, N 3.48.
1
tallized from acetone/cyclohexane; H NMR (CDCl3): d=1.14 (t, J=
7.2, 3H, CH2CH3), 2.24 (s, 3H, CH3-2), 2.25 (s, 3H, CH3-6), 4.01 (q, J=
7.2, 2H, CH2CH3), 4.47 (d, J=5.0, 2H, CH2CH=CH2), 4.83-5.18 (m,
3H, CH2CH=CH2 +H-4) 5.69-5.93 (m, 2H, CH2CH=CH2 +NH), 7.06–
7.27 ppm (m, 4H, H-arom); 13C NMR (CDCl3): d=13.7 ppm
(CH3CH2), 18.7 (CH3-2), 18.9 (CH3-6), 32.2 (C4), 59.5 (CH3CH2), 70.3
(CH2CH=CH2), 102.4 (C3), 103.1 (C5), 116.2 (CH2-CH=CH2), 120.1
(C4’), 131.4 (2C-2’,6’), 131.7 (2C-3’,5’), 136.7 (C1’), 145.8 (C2), 146.5
(C6), 166.1, 166.7 ppm (COO); IR (KBr): n˜ =3357, 2984, 2893, 1695,
1651, 1485, 1210 cmÀ1
; MS: m/z: 419 (100); Anal. calcd for
C20H22BrNO4: C 57.15, H 5.28, N 3.33, Br 19.01, found: C 56.88, H
5.38, N 3.25, Br 18.84.
1,4-Dihydro-2,6-dimethyl-4-(4’-isopropylphenyl)pyridine-3-car-
boxylic acid benzyl ester, 5- carboxylic acid methyl ester (4c):
(A): 4-isopropylbenzaldehyde 1b, (B): methyl acetoacetate 2a, (C):
benzyl acetoacetate 2d; pale yellow oil; yield: 31%, 0.65 g;
1H NMR (CDCl3): d=1.11-1.22 (m, 6H, CH(CH3)2), 2.30 (s, 3H, CH3-2),
2.33 (s, 3H, CH3-6), 2.77–2.94 (m, 1H, CH(CH3)2), 3.57 (s, 3H, OCH3),
4.90-5.07 (m, 3H, CH2Ph+H-4), 5.79 (s, 1H, NH), 6.83–7.26 ppm (m,
9H, H-arom); 13C NMR (CDCl3): d=18.8 (CH3-2), 18.9 (CH3-6), 24.4
(CH3CHCH3), 31.7 (CH3CHCH3), 32.1 (C4), 50.8 (COOCH3), 72.6
(CH2Ph), 102.5 (C3), 103.0 (C5), 126.2 (2C-3’,5’), 127.3 (2C-2’’,6’’),
127.8 (C4’’), 128.2 (2C-3’’,5’’), 128.9 (2C-2’,6’), 135.1 (C1’), 140.8
(C1’’), 145.7 (C4’), 145.9 (C2), 146.3 (C6), 166.2, 166.9 ppm (COO); IR
(KBr): n˜ =3440, 2961, 2871, 1692, 1617, 1470, 1227 cmÀ1; MS: m/z=
419 (100); Anal. calcd for C26H29NO4: C 74.44, H 6.97, N 3.34, found:
C 74.17, H 7.06, N 3.44.
1,4-Dihydro-2,6-dimethyl-4-(naphth-2’-yl)pyridine-3-carboxylic
acid allyl ester, 5-carboxylic acid ethyl ester (4g): (A): 2-naphthal-
dehyde 1d, (B): ethyl acetoacetate 2b, (C): allyl acetoacetate 2c;
white solid; mp: 130–1318C; yield: 30%, 0.58 g; crystallized from
acetone/cyclohexane; 1H NMR (CDCl3): d=1.15 (t, J=7.0, 3H,
CH2CH3), 2.30 (s, 3H, CH3-2), 2.31 (s, 3H, CH3-6), 3.98 (q, J=7.0, 2H,
CH2CH3), 4.47 (d, J=5.4, 2H, CH2CH=CH2), 5.01–5.22 (m, 3H,
CH2CH=CH2 +H-4), 5.75-5.92 (m, 2H, NH+CH2CH=CH2), 7.23–7.75
(m, 7H, H-arom); 13C NMR (CDCl3): d=13.5 ppm (CH3CH2), 18.6
(CH3-2), 18.8 (CH3-6), 32.2 (C4), 59.6 (CH3CH2), 70.3 (CH2CH=CH2),
101.8 (C3), 102.4 (C5), 115.1 (CH2CH=CH2), 124.2 (C6’), 125.6 (C5’),
126.7 (C2’), 127.1 (C9’), 127.4 (C4’), 127.5 (C7’), 127.7 (C10’), 131.6
(C8’), 133.5 (C3’), 135.2 (C1’), 137.5 (CH2CH=CH2), 145.9 (C2), 146.7
(C6), 166.3, 166.7 ppm (COO); IR (KBr): n˜ =3340, 2968, 2849, 1701,
1652, 1485, 1210 cmÀ1
; MS: m/z=391 (100); Anal. calcd for
1,4-Dihydro-2,6-dimethyl-4-(4’-isopropylphenyl)pyridine-3-car-
boxylic acid benzyl ester, 5-carboxylic acid ethyl ester (4d): (A):
4-isopropylbenzaldehyde 1b, (B): ethyl acetoacetate 2b, (C): benzyl
acetoacetate 2d; pale yellow oil; yield: 42%, 0.91 g; 1H NMR
(CDCl3): d=1.04–1.23 (m, 9H, CH(CH3)2 +CH2CH3), 2.29 (s, 3H, CH3-
2), 2.31 (s, 3H, CH3-6), 2.78–2.94 (m, 1H, CH(CH3)2), 4.03 (q, J=7.0,
2H, CH2CH3), 4.85–5.09 (m, 3H, CH2Ph+H-4), 5.69 (s, 1H, NH), 6.90–
7.07 ppm (m, 9H, H-arom); 13C NMR (CDCl3): d=13.7 ppm
(CH3CH2), 18.8 (CH3-2), 18.9 (CH3-6), 24.4 (CH3CHCH3), 31.7
(CH3CHCH3), 32.1 (C4), 59.8 (CH3CH2), 72.6 (CH2Ph), 102.5 (C3), 103.0
C24H25NO4: C 73.64, H 6.44, N 3.58, found: C 73.87, H 6.41, N 3.49.
1,4-Dihydro-2,6-dimethyl-4-(naphth-2’-yl)pyridine-3-carboxylic
acid benzyl ester, 5-carboxylic acid ethyl ester (4h): (A): 2-naph-
thaldehyde 1d, (B): ethyl acetoacetate 2b, (C): benzyl acetoacetate
1
2d; pale yellow oil; yield: 31%, 0.68 g; H NMR (CDCl3): d=1.14 (t,
J=7.0, 3H, CH2CH3), 2.28 (s, 3H, CH3-2), 2.30 (s, 3H, CH3-6), 4.01 (m,
2H, CH2CH3), 4.96-5.17 (m, 3H, CH2Ph+H-4), 5.66 (s, 1H, NH), 7.04–
7.76 (m, 12H, H arom); 13C NMR (CDCl3): d=13.5 ppm (CH3CH2),
18.6 (CH3-2), 18.8 (CH3-6), 32.2 (C4), 59.6 (CH3CH2), 72.6 (CH2Ph),
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4-Aryl-DHPs Potentiate Mutant CFTR
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101.6 (C3), 101.9 (C5), 124.2 (C6’), 125.6 (C5’), 126.7 (C2’), 127.1
(C9’), 127.3 (2C-2’’,6’’), 127.5 (2C-4’,4’’), 127.7 (C7’), 127.9 (C10’),
128.2 (2C-3’’,5’’), 131.6 (C8’), 133.5 (C3’), 135.2 (C1’), 140.8 (C1’’),
145.9 (C2), 146.7 (C6), 166.3, 166.7 ppm (COO); IR (KBr): n˜ =3401,
2931, 2891, 1692, 1619, 1471, 1227 cmÀ1; MS: m/z=441 (100);
Anal. calcd for C28H27NO4: C 76.17, H 6.16, N 3.17, found: C 75.91, H
6.23, N 3.23.
1H, NH), 6.97–7.17 ppm (m, 4H, H-arom); 13C NMR (CDCl3): d=13.6
(CH3CH2), 18.3 (CH3-6), 18.7 (CH3-2), 24.4 (CH3CHCH3), 31.5 (C4), 31.7
(CH3CHCH3), 59.6 (CH3CH2), 78.2 (C5), 101.9 (C3), 117.2 (CN), 126.2
(2C-3’,5’), 128.9 (2C-2’,6’), 134.9 (C1’), 145.7 (C4’), 145.8 (C2), 149.9
(C6), 166.4 ppm (COO); IR (KBr): n˜ =3022, 2963, 2203, 1693, 1665,
1480, 1211 cmÀ1; MS: m/z=324 (100); Anal. calcd for C20H24N2O2: C
74.04, H 7.46, N 8.64, found: C 74.29, H 7.55, N 8.52.
1,4-Dihydro-2,6-dimethyl-4-(naphth-2’-yl)pyridine-3-carboxylic
acid benzyl ester, 5-carboxylic acid methyl ester (4i): (A): 2-naph-
thaldehyde 1d, (B): methyl acetoacetate 2a, (C): benzyl acetoace-
tate 2d; pale yellow oil; yield: 30%, 0.64 g; 1H NMR (CDCl3): d=
2.29 (s, 3H, CH3-2), 2.30 (s, 3H, CH3-6), 3.61 (s, 3H, OCH3), 4.91-5.17
(m, 3H, CH2Ph+H-4), 5.68 (s, 1H, NH), 7.04–7.78 ppm (m, 12H, H-
arom); 13C NMR (CDCl3): d=18.6 ppm (CH3-2), 18.8 (CH3-6), 32.2
(C4), 50.9 (COOCH3), 72.6 (CH2Ph), 101.6 (C3), 101.9 (C5), 124.8 (C6’),
125.7 (C5’), 126.7 (C2’), 127.1 (C9’), 127.3 (2C-2’’,6’’), 127.5 (2C-4’,4’’),
127.7 (C7’), 127.9 (C10’), 128.2 (2C-3’’,5’’), 131.6 (C8’), 133.5 (C3’),
135.2 (C1’), 140.8 (C1’’), 145.9 (C2), 146.7 (C6), 166.3, 166.7 ppm
Chiral separation
Compounds 4a, 4c, 4d, 4e, and 4g were subjected to chiral sepa-
ration with the aim of obtaining 10–20 mg of each separated
enantiomer to be tested as a potentiator for mutant CFTR. For this
reason, samples were initially subjected to analytical separation
and then to semi-preparative separation. In this regard, samples
were injected several (~50) times until the desired amount of
enantiomers was obtained. The reported sign of the enantiomers
is the sign of the optical rotation at l 589 nm in CHCl3. Retention
times (tR) are given in min. The HPLC unit used was a Merck
D-7000 System Manager with vacuum pump Merck-Lachrom
L-7100 and oven Merck-Lachrom L-7360, UV detector Merck-Lach-
rom L-7400, CD detector CD-1595. Hexane and iPrOH (HPLC grade)
were degassed and filtered (0.45 mm) before use.
(COO); IR (KBr): n˜ =3440, 2950, 2929, 1694, 1618, 1471, 1216 cmÀ1
;
MS: m/z=427 (100); Anal. calcd for C27H25NO4: C 75.86, H 5.89, N
3.28, found: C 76.09, H 6.01, N 3.21.
General procedure for the synthesis of asymmetric 1,4-dihy-
dropyridines (4j–l).
Separation of 4a: Analytical: column: (S,S)-Whelk-O1 (250ꢁ
4.6 mm); mobile phase: hexane/iPrOH 7:3; flow: 1 mLminÀ1; UV de-
To an ice-cooled solution of aldehyde A (5 mmol) in iPrOH (15 mL),
acetoacetate B (5 mmol) was added, and the mixture was held at
reflux for 3 h. Aminocrotonate 3 (5 mmol) was then added, and
the mixture was held at reflux again for 3 h. After cooling, the sol-
vent was evaporated under reduced pressure, and the crude reac-
tion material obtained was crystallized from toluene.
tector set at l 254 nm. (À)-4a: tR1 =13.3, k1 =3.36; (+)-4a: tR2
=
15.0, k2 =3.90, a=1.45, Rs=1.16. Semi-preparative: column: (S,S)-
Whelk-O1 (250ꢁ10 mm); mobile phase: hexane/iPrOH 7:3; flow:
5 mLminÀ1; UV detector set at l 254 nm. Samples of the first enan-
tiomer were collected from 12.0 to 13.5 min. Samples of the
second enantiomer were collected from 14.5 to 17.0 min. First
enantiomer (À)-4a: 96% ee; a2D5 =À18.4 (CHCl3, c=1.58). Second
enantiomer (+)-4a: 95% ee; a2D5 = +19.2 (CHCl3, c=1.15).
1,4-Dihydro-2,6-dimethyl-4-(4’-bromophenyl)-5-cyanopyridine-3-
carboxylic acid methyl ester (4j): (A): 4-bromobenzaldehyde 1a,
(B): methyl acetoacetate 2a; pale yellow solid; mp: 203–2048C;
Separation of 4c: Analytical: column: (S,S)-Whelk-O1 (250ꢁ
4.6 mm); mobile phase: hexane/iPrOH 9:1; flow: 2 mLminÀ1; UV de-
1
yield: 19%, 0.33 g; H NMR (CDCl3): d=2.00 (s, 3H, CH3-2), 2.30 (s,
3H, CH3-6), 3.85 (s, 3H, OCH3), 4.53 (s, 1H, H-4), 5.78 (s, 1H, NH),
6.92–7.38 ppm (m, 4H, H-arom); 13C NMR (CDCl3): d=18.3 (CH3-6),
18.7 (CH3-2), 31.5 (C4), 51.3 (COOCH3), 78.2 (C5), 101.9 (C3), 117.2
(CN), 120.1 (C4’), 131.4 (2C-2’,6’), 131.7 (2C-3’,5’), 136.7 (C1’), 145.8
(C2), 149.9 (C6), 166.4 ppm (COO); IR (KBr): n˜ =3316, 2955, 2196,
1686, 1659, 1500, 1252 cmÀ1; MS: m/z=346 (100); Anal. calcd for
C16H15BrN2O2: C 55.35, H 4.35, N 8.07, Br 23.01, found: C 55.54, H
4.24, N 8.16, Br 22.85.
tector set at l 254 nm. (+)-4c: tR1 =20.7, k1 =12.63; (À)-4c: tR2
=
25.3, k2 =15.64, a=1.26, Rs=2.53. Semi-preparative: column: (S,S)-
Whelk-O1 (250ꢁ4.6 mm); mobile phase: hexane/iPrOH 9:1; flow:
2 mLminÀ1; UV detector set at l 254 nm. Samples of the first enan-
tiomer were collected from 19.5 to 22.0 min. Samples of the
second enantiomer were collected from 25.5 to 30.0 min. First
enantiomer (+)-4c: 94% ee; a2D5 = +12.7 (CHCl3, c=0.70). Second
enantiomer (À)-4c: 95% ee; a2D5 =À12.1 (CHCl3, c=0.73).
1,4-Dihydro-2,6-dimethyl-4-(2’,3’-dichlorophenyl)-5-cyanopyri-
dine-3-carboxylic acid benzyl ester (4k): (A): 2,3-dichlorobenzal-
dehyde 1e, (B): benzyl acetoacetate 2d; white solid; mp: 169–
Separation of 4d: Analytical: column: (S,S)-Whelk-O1 (250ꢁ
4.6 mm); mobile phase: hexane/iPrOH 9:1; flow: 2 mLminÀ1; UV de-
tector set at l 254 nm. (+)-4d: tR1 =23.4, k1 =14.38; (À)-4d: tR2
=
1
1708C; yield: 48%, 0.99 g; H NMR (CDCl3): d=2.00 (s, 3H, CH3-2),
25.3, k2 =18.47, a=1.31, Rs=2.78. Semi-preparative: column: (S,S)-
Whelk-O1 (250ꢁ4.6 mm); mobile phase: hexane/iPrOH 9:1; flow:
2 mLminÀ1; UV detector set at l 254 nm. Samples of the first enan-
tiomer were collected from 22.0 to 25.0 min. Samples of the
second enantiomer were collected from 25.5 to 33 min. First enan-
tiomer (+)-4d: 99% ee; a2D5 = +16.7 (CHCl3, c=1.0). Second enan-
tiomer (À)-4d: 98% ee; a2D5 =À17.0 (CHCl3, c=1.0).
2.29 (s, 3H, CH3-6), 4.96-5.25 (m, 3H, CH2Ph+H-4), 5.95 (s, 1H, NH),
7.02–7.17 (m, 8H, H-arom); 13C NMR (CDCl3): d=18.3 ppm (CH3-6),
18.7 (CH3-2), 25.8 (C4), 72.6 (CH2Ph), 78.2 (C5), 102.8 (C3), 117.2
(CN), 127.5 (3C-4’,2’’,6’’), 127.7 (C4’’), 127.9 (C5’), 128.2 (3C-6’,3’’,5’’),
134.1 (C3’), 134.9 (C2’), 139.5 (C1’), 140.8 (C1’’), 145.8 (C2), 149.9
(C6), 166.2 (COO); IR (KBr): n˜ =3314, 2984, 2202, 1682, 1659, 1501,
1249 cmÀ1; MS: m/z=412 (100); Anal. calcd for C22H18Cl2N2O2: C
63.93, H 4.39, N 6.78, Cl 17.16, found: C 64.22, H 4.33, N 6.87.
Separation of 4e: Analytical: column: Chiralpak IC (250ꢁ4.6 mm);
mobile phase: hexane/iPrOH 9:1; flow: 1 mLminÀ1; UV detector set
at l 254 nm. (À)-4e: tR1 =11.2, k1 =2.60; (+)-4e: tR2 =12.3, k2 =
2.98, a=1.15, Rs=1.21. Semi-preparative: column: Chiralpak IC
1,4-Dihydro-2,6-dimethyl-4-(4’-isopropylphenyl)-5-cyanopyri-
dine-3-carboxylic acid ethyl ester (4l): (A): 4-isopropylbenzalde-
hyde 1b, (B): ethyl acetoacetate 2b; white solid; mp: 82–838C;
(250ꢁ4.6 mm); mobile phase: hexane/iPrOH 9:1; flow: 1 mLminÀ1
;
1
yield: 40%, 0.65 g; H NMR (CDCl3): d=0.93-1.28 (m, 9H, CH2CH3 +
UV detector set at l 254 nm. Samples of the first enantiomer were
collected from 10.0 to 11.5 min. Samples of the second enantiomer
were collected from 12.5 to 14.0 min. First enantiomer (À)-4e:
CH(CH3)2), 1.98 (s, 3H, CH3-2), 2.26 (s, 3H, CH3-6), 2.67-2.88 (m, 1H,
CH(CH3)2), 3.96 (q, J=7.0, 2H, CH2CH3), 4.48 (s, 1H, H-4), 5.93 (s,
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M. Mazzei et al.
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97% ee; a2D5 =À18.7 (CHCl3, c=1.0). Second enantiomer (+)-4e:
in Ussing-chamber-like systems in the presence of a ClÀ gradient
(65 mm apical/130 mm basolateral). The trans-epithelial electrical
potential difference was clamped at zero with a DVC-1000 voltage-
clamp amplifier (World Precision Instruments) via Ag/AgCl electro-
des. The resulting short-circuit current was digitized with an
analog/digital converter (PowerLab 4/25, ADInstruments).
96% ee; a2D5 = +14.5 (CHCl3, c=1.1).
Separation of 4g: Analytical: column: (S,S)-Whelk-O1 (250ꢁ
4.6 mm); mobile phase: hexane/iPrOH 7:3; flow: 2 mLminÀ1; UV de-
tector set at l 254 nm. (À)-4g: tR1 =7.9, k1 =4.17; (+)-4g: tR2 =8.7,
k2 =4.72, a=1.18, Rs=1.26. Semi-preparative: column: (S,S)-Whelk-
O1 (250ꢁ10 mm); mobile phase: hexane/iPrOH 9:1; flow:
5 mLminÀ1; UV detector set at l 254 nm. Samples of the first enan-
tiomer were collected from 7.0 to 8.5 min. Samples of the second
enantiomer were collected from 9.0 to 10.5 min. First enantiomer
(À)-4g: 99% ee; a2D5 =À24.7 (CHCl3, c=1.2). Second enantiomer
(+)-4g: 98% ee; aD25 = +24.2 (CHCl3, c=1.0).
Data analysis: Data for all experiments are presented as representa-
tive traces or as mean values ÆSEM. Statistical analyses were per-
formed with Student’s t test for unpaired data. Values for Emax and
Kd were obtained by fitting dose–response data with the Hill equa-
tion. The activity elicited by forskolin (20 mm) alone was subtracted
from the Emax value. Felodipine properties are reported for compar-
ison.
Biology
Acknowledgements
CFTR assays
This work was supported by grants (FFC#2/2009) from the Italian
Cystic Fibrosis Foundation with contribution from the FFC Dele-
gation of Vicenza (Italy). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation
of the manuscript. The authors declare no competing personal
and financial interests.
Cell culture: Fischer rat thyroid (FRT) cells, stably transfected with
G1349D, G551D, or DF508 CFTR and the halide-sensitive yellow flu-
orescent protein YFP-H148Q/I152L[32] were cultured in Coon’s
modified Ham’s F-12 medium supplemented with 10% fetal calf
serum, l-glutamine (2 mm), penicillin (100 UmLÀ1), and streptomy-
cin (100 mgmLÀ1). For fluorescence assays of CFTR activity, cells
were plated (105 cells per well) on clear-bottomed 96-well micro-
plates (Corning Life Sciences, Acton, MA, USA).
Keywords: asymmetric
1,4-dihydropyridines
·
chiral
Samples: Synthesized compounds 4a–l were dissolved in DMSO,
and for each compound a stock solution (10 mm) was prepared.
Secondary plates were prepared for screening at 1 mm in DMSO
using a Biomek 2000 liquid handling workstation (Beckman Coult-
er, Fullerton, CA, USA). All plates were stored at À708C.
separation cystic fibrosis transmembrane conductance
·
regulator (CFTR) · potentiators
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Fluorescence assay for CFTR activity: Measurements of CFTR activity
were carried out on FRT cells expressing G1349D, G551D, or DF508
CFTR and the halide-sensitive YFP 48 h after plating on micro-
plates. The 96-well microplates containing FRT cells expressing
DF508 CFTR and the halide-sensitive YFP were incubated at 278C
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(137 mm), KCl (2.7 mm), Na2HPO4 (8.1 mm), KH2PO4 (1.5 mm), CaCl2
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4-Aryl-DHPs Potentiate Mutant CFTR
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Published online on && &&, 0000
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FULL PAPERS
M. Giampieri, N. Vanthuyne, E. Nieddu,
M. T. Mazzei, M. Anzaldi, N. Pedemonte,
L. J. V. Galietta, C. Roussel, M. Mazzei*
The racemate for a cure: 1,4-Dihydro-
pyridines (DHPs) are already known as
potential drugs for cystic fibrosis caused
by class III mutations. Asymmetric DHPs
were synthesized and tested as race-
mates on Fischer rat thyroid cells ex-
pressing three mutations. Some DHPs
are effective potentiators at the nano-
molar level. The most active DHPs (e.g.,
4c) were subjected to chiral separation
and retested, but the resulting differ-
ence in activity between enantiomers
was modest.
&& – &&
Asymmetric 4-Aryl-1,4-
dihydropyridines Potentiate Mutant
Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR)
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These are not the final page numbers!