Synthesis of 4-thiophen-2′-yl-1,4-dihydropyridines as potentiators of the CFTR chloride channel

Article abstract of DOI:10.1016/j.bmc.2009.10.028

The gating of the CFTR chloride channel is altered by a group of mutations that cause cystic fibrosis. This gating defect may be corrected by small molecules called potentiators. Some 1,4-dihydropyridine (DHP) derivatives, bearing a thiophen-2-yl and a furanyl ring at the 4-position of the nucleus, were prepared and tested as CFTR potentiators. In particular, we evaluated the ability of novel DHPs to enhance the activity of the rescued ΔF508-CFTR as measured with a functional assay based on the halide-sensitive yellow fluorescent protein. Most DHPs showed an effect comparable to or better than that of the reference compound genistein. The potency was instead significantly improved, with some compounds, such as 3g, 3h, 3n, 4a, 4b, and 4d, having a half effective concentration in the submicromolar range. CoMFA analysis gave helpful suggestions to improve the activity of DHPs.

Full text of DOI:10.1016/j.bmc.2009.10.028

Bioorganic & Medicinal Chemistry 17 (2009) 7894–7903
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 4-thiophen-20-yl-1,4-dihydropyridines as potentiators of the CFTR
chloride channel
Francesca Cateni a, Marina Zacchigna a, Nicoletta Pedemonte b, Luis J. V. Galietta b, Marco T. Mazzei c,
Paola Fossa d, Michele Giampieri d, Mauro Mazzei d,
*
a Department of Pharmaceutical Sciences, Piazzale Europa 1, 34127 Trieste, Italy
b Molecular Genetics Laboratory, Giannina Gaslini Institute, Largo Gerolamo Gaslini 5, 16147 Genova, Italy
c Department of Experimental Medicine, Viale Benedetto XV 1, 16132 Genova, Italy
d Department of Pharmaceutical Sciences, Viale Benedetto XV 3, 16132 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The gating of the CFTR chloride channel is altered by a group of mutations that cause cystic fibrosis. This
gating defect may be corrected by small molecules called potentiators. Some 1,4-dihydropyridine (DHP)
derivatives, bearing a thiophen-2-yl and a furanyl ring at the 4-position of the nucleus, were prepared
and tested as CFTR potentiators. In particular, we evaluated the ability of novel DHPs to enhance the
Received 30 June 2009
Revised 14 October 2009
Accepted 15 October 2009
Available online 20 October 2009
activity of the rescued
DF508-CFTR as measured with a functional assay based on the halide-sensitive
yellow fluorescent protein. Most DHPs showed an effect comparable to or better than that of the refer-
ence compound genistein. The potency was instead significantly improved, with some compounds, such
as 3g, 3h, 3n, 4a, 4b, and 4d, having a half effective concentration in the submicromolar range. CoMFA
analysis gave helpful suggestions to improve the activity of DHPs.
Keywords:
Cystic fibrosis
CFTR
Dihydropyridine
Potentiators
CoMFA
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
CFTR processing defect (the protein being trapped and degraded at
the endoplasmic [ER] level) and a decrease of its channel activity.6–8
About 55,000 people in Europe and the US are affected by cystic
fibrosis (CF), an autosomal recessive disease caused by mutations
in the cystic fibrosis transmembrane conductance regulator (CFTR)
gene.1,2 CF is characterized by abnormalities of chloride transport
in many organs (pancreas, lungs, intestine, etc.). In particular, the
defective chloride transport in the lungs causes airway surface
dehydration with the production of an abnormally thick, sticky
mucus that helps to initiate a cycle of lung inflammation and infec-
tion. This fact ultimately leads to respiratory failure in most
patients.
The increasing knowledge regarding CFTR function and struc-
ture is helping to discover small molecules able to correct the CF
basic defect, that is, the defective chloride transport. These mole-
cules may be the basis for a specific pharmacotherapy of CF.3,4
In this regard, the search for novel drugs to treat CF has to take
into account the large number of mutations affecting the gene and
the variety of molecular mechanisms through which these muta-
tions cause the functional defect. For instance, deletion of phenyl-
Other mutations, like G551D and G1349D, produce only a gating
defect. Compounds able to deliver the F508-CFTR from the ER
to plasma membrane are called ‘correctors’ whereas compound
able to increase the channel activity are called ‘potentiators’. Gen-
erally, correctors and potentiators have been identified by high-
throughput screening (HTS) of large compound libraries.9–12
D
CF patients homozygote for
DF508 mutation do not have the
D
F508-CFTR in the plasma membrane but, in vitro, the mutant
protein may be helped to escape the endoplasmic reticulum and
reach the plasma membrane by incubating the cells at low temper-
ature (27 °C).13 Under this condition, the ‘rescued’
DF508-CFTR
protein respond to potentiators with a significant increase in activ-
ity and, therefore, in anion transport.
The treatment of CF patients carrying the
DF508 mutation prob-
ably requires two drugs: a corrector to transport the CFTR from the
ER to the plasma membrane, and a potentiator to improve the gat-
ing activity.
In this regard, we have found that the 1,4-dihydropyridines
(DHPs) used to treat hypertension are acting as potentiators and
so are able to stimulate the activity of some CFTR mutants (in par-
alanine at position 508 (DF508), the most frequent mutation
(occurring in more than 50–70% of patients),5 causes both a severe
ticular, G551D, G1349D and rescued
D
F508-CFTR).14,15 Antihyper-
tensive DHPs act by blocking L-type voltage-dependent Ca2+
channels (L-VDCC) and therefore cause the relaxation of arterial
* Corresponding author. Tel.: +39 010 3538371; fax: +39 010 3538358.
E-mail address: mauro.mazzei@unige.it (M. Mazzei).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.028

F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
7895
smooth muscle cells.16 We have obtained evidence that mutant
CFTR is activated by DHPs through a mechanism not involving
the modulation of Ca2+ channels, but by a probable direct interac-
tion with the CFTR protein itself.14
Finally, the CoMFA investigation on the synthesized compounds
gives some helpful suggestions to ameliorate the design of new
potentially active DHPs for a possible CF therapy.
A more recent study, has shown the possibility to generate
DHPs with enhanced selectivity towards CFTR relative to calcium
channels.15 To further elucidate the structure–activity relation-
ships of DHPs as CFTR potentiators, we have now analyzed novel
DHP derivatives in which the phenyl substituent in position 4 is re-
placed by heterocycles like thiophene and furan. The new DHP
derivatives have shown a significant efficacy, comparable to the
reference compound genistein, and improved potency. The activity
on Ca2+ channels was in general similar to that one of felodipine,
with some important differences.
2. Chemistry
The synthesis of the symmetrical dihydropyridines 3a–n was
accomplished by the Hantzsh method17 as shown in Scheme 1,
starting from the corresponding aldehydes 1a–g and the appropri-
ate acetoacetate 2a–d in the presence of ammonia.
For the synthesis of asymmetrical DHPs we slightly changed the
above method to avoid the formation of symmetrical DHPs. In this
regard, the synthesis begins by reacting the aldehyde with the first
acetoacetate. When the condensation reaction is over, the second
R3
R2
X
R1
CH3
R2
R1
RO2C
H3C
CO2R
C
O
a
2
CH2
CO2R
+
R3
N
H
CH3
CHO
X
1a-g
3a-n
2a-d
R2
H
R1
H
R
X
S
R3
NO2
3a
3b
Me
Me
S
S
S
S
O
S
S
S
H
Me
H
Me
H
Me
Me
H
H
H
H
H
H
H
H
H
Me
H
Me
Me
Et
3c
3d
3e
3f
H
H
Me
H
H
Me
3g
3h
3i
X
R1
R2
R3
CH2-CH=CH2
CH2Ph
CH2-CH=CH2
S
H
H
H
H
NO2
Me
1a
1b
NO2
S
S
S
S
H
H
H
H
NO2
CH2Ph
3j
3k
3l
1c
1d
1e
1f
S
S
O
S
Me
H
H
H
H
H
H
H
Me
H
Ph
Ph
H
H
H
H
CH2-CH=CH2
CH2Ph
S SCH2CO2Me
3m
H
Ph
CH2-CH=CH2
1g
CH2Ph
S SCH2CO2Me H
H
3n
S SCH2CO2Me
H
H
R
2a Me
Et
2c CH2-CH=CH2
CH2Ph
2b
2d
Scheme 1. Reagents and conditions: (a) NH3, MeOH, reflux, 6 h.

7896
F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
acetoacetate is added and, finally, ammonia is added, as shown in
Scheme 2.
Compounds 3a–l and 4a,b are white or pale yellow crystals.
Compounds 3m,n and 4c,d are oils. All structures were confirmed
by spectral data and elemental analyses.
removed, the mixture was allowed to stir at room temperature
for 15 min and then refluxed for 6 h. After cooling, the solvent
was evaporated and the crude reaction material obtained was puri-
fied by chromatography on silica gel with petrol ether/AcOEt (7:3).
3.1.1. 1,4-Dihydro-2,6-dimethyl-4-(50-nitro-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid dimethyl ester (3a)
3. Experimental protocols
Prepared from 5 mmol of 1a and 12 mmol of methyl acetoace-
tate 2a. The crude reaction material obtained was purified by chro-
matography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3a as pale yellow microcrystalline precipitate: mp 155–
156 °C; yield 70%.
Nuclear magnetic resonance spectra were recorded with a Var-
ian Gemini 200 MHz spectrometer. 13C NMR: 90.5 MHz, Gemini
200 spectrometer. NMR spectra were obtained by using CDCl3 as
solvent; chemical shifts are expressed as d units (ppm) relative
to tetramethylsilane (TMS) as internal standard. The abbreviations
s, d, t, m and sb refer to singlet, doublet, triplet, multiplet and sin-
glet broad signal, respectively. The EI-MS spectra were measured
with a VG-ZAB 2F spectrometer. The ionizing energy was 70 eV
in all cases and compounds were introduced by direct insertion.
Elemental analyses were carried out on a Carlo Erba model 1016
analyzer. The column chromatography was performed by using sil-
ica gel (Kieselgel 60, 230–400 Mesh, 60 Å Merck). TLC: Kieselgel 60
F254 (20 ꢀ 20 cm; 0.2 mm, Merck). Melting points were determined
on a Büchi 510 micromelting point apparatus and are uncorrected.
Reagents used were AR grade and all solvents for synthesis, extrac-
tion and column chromatography were distilled and dried before
use.
1H NMR (CDCl3): d = 2.40 ppm (s br; 6H, 2CH3-2,6), 3.78 (s br;
6H, 2COOCH3), 5.35 (s; 1H, H-4), 5.90 (s br; 1H, NH), 6.80 (d; 1H,
J = 4.15 Hz, H-30), 7.77 (d; 1H, J = 4.17 Hz, H-40). 13C NMR (CDCl3):
d = 19.6 ppm (2CH3-2,6), 35.4 (C-4), 51.0 (COOCH3), 102.3 (C-3, C-
5), 123.0 (C-30), 128.9 (C-40), 145.8 (C-2, C-6), 148.5 (C-20), 150.0
(C-50), 165.9 (2COO). EI-MS (70 eV): m/z = 352 (M, 20%)+, 337
(Mꢁ15, 18%)+, 321 (Mꢁ31, 22%)+, 293 (Mꢁ59, 100%)+. Anal. Calcd
for C15H16O6N2S: C, 50.91; H, 4.54; N, 7.95; S, 9.09. Found: C,
50.91; H, 4.39; N, 8.02; S, 9.15.
3.1.2. 1,4-Dihydro-2,6-dimethyl-4-(50-methyl-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid dimethyl ester (3b)
Prepared from 5 mmol of 1b and 12 mmol of methyl acetoace-
tate 2a. The crude reaction material obtained was purified by chro-
matography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3b as pale yellow microcrystalline precipitate: mp 179–
180 °C; yield 75%.
3.1. General procedure for the synthesis of symmetrical
1,4-dihydropyridines (3a–n)
1H NMR (CDCl3): d = 2.30 ppm (s; 3H, CH3-50), 2.40 (s br; 6H,
2CH3-2,6), 3.79 (s br; 6H, 2COOCH3), 5.34 (s; 1H, H-4), 5.89 (s br;
1H, NH), 6.62 (d; 1H, J = 4.5 Hz, H-30), 6.63 (d; 1H, J = 4.51 Hz, H-
To an ice-cooled solution of 5 mmol of aldehyde 1a–g and
12 mmol of acetoacetate 2a–d in 15 ml of dry methanol were
added dropwise 0.7 ml of concd ammonia. The ice bath was
O
CH3
O
CH3
O
R
NH3
O
O
R"
R'
H
O
Second acetoacetate
i-PrOH
O
S
1c, g
First acetoacetate
S
R
COOR"
R'OOC
H3C
N
H
CH3
4a-d
4a: R= CH3, R’= allyl, R”= ethyl
4b: R = CH3, R’= benzyl, R”= ethyl
4c: R = SCH2COOCH3, R’ = allyl, R” = ethyl
4d: R = SCH2COOCH3, R’ = benzyl, R” = ethyl
Scheme 2.

F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
7897
13
40). C NMR (CDCl3): d = 14.2 ppm (CH3-50), 19.4 (2CH3-2,6), 33.4
compound 3f as pale yellow microcrystalline precipitate: mp 180–
181 °C. yield 70%.
(C-4), 51.2 (COOCH3), 104.0 (C-3, C-5), 127.3 (C-30, C-40), 137.0
(C-50), 137.5 (C-20), 146.1 (C-2, C-6), 166.2 (2COO). EI-MS (70 eV):
m/z = 321 (M, 28%)+, 306 (Mꢁ15, 21%)+, 290 (Mꢁ31, 14%)+, 262
(Mꢁ59, 93%)+, 224 (MꢁC5H5S, 100%)+, 165 (MꢁC5H5Sꢁ59, 9%)+.
Anal. Calcd for C16H19O4NS: C, 59.81; H, 5.92; N, 4.36; S, 9.97.
Found: C, 59.73; H, 5.90; N, 4.29; S, 9.81.
1H NMR (CDCl3): d = 2.29 ppm (s; 3H, CH3-50), 2.41 (s br; 6H,
2CH3-2,6), 3.79 (s br; 6H, 2COOCH3), 5.33 (s; 1H, H-4), 5.88 (s br;
1H, NH), 5.93 (d; 1H, J = 4.9 Hz, H-30), 6.00 (d; 1H, J = 4.92 Hz, H-
13
40). C NMR (CDCl3): d = 14.3 ppm (CH3-50), 19.4 (2CH3-2,6), 33.4
(C-4), 51.4 (COOCH3), 104.2 (C-3, C-5), 109.4 (C-30, C-40), 149.8
(C-50), 150.0 (C-20), 146.3 (C-2, C-6), 166.3 (2COO). EI-MS (70 eV):
m/z = 305 (M, 40%)+, 290 (Mꢁ15, 30%)+, 274 (Mꢁ31, 100%)+. Anal.
Calcd for C16H19O5N: C, 62.95; H, 6.23; N, 4.59. Found: C, 62.78;
H, 6.11; N, 4.66.
3.1.3. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid dimethyl ester (3c)
Prepared from 5 mmol of 1c and 12 mmol of methyl acetoace-
tate 2a. The crude reaction material obtained was purified by chro-
matography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3c as pale yellow microcrystalline precipitate: mp 216–
217 °C; yield 75%.
3.1.7. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid diallyl ester (3g)
Prepared from 5 mmol of 1c and 12 mmol of allyl acetoacetate
2c. The crude reaction material obtained was purified by chroma-
tography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3g as pale yellow microcrystalline precipitate: mp
82–83 °C; yield 75%.
1H NMR (CDCl3): d = 2.26 ppm (s; 3H, CH3-30), 2.35 (s br; 6H,
2CH3-2,6), 3.35 (s br; 6H, 2COOCH3), 5.33 (s; 1H, H-4), 5.80 (s br;
1H, NH), 6.61 (d; 1H, J = 5.04 Hz, H-40), 6.91 (d; 1H, J = 5.05 Hz,
13
H-50). C NMR (CDCl3): d = 13.6 ppm (CH3-30), 19.4 (2CH3-2,6),
32.8 (C-4), 51.0 (COOCH3), 104.1 (C-3, C-5), 124.3 (C-50), 124.8
(C-40), 132.4 (C-30), 143.8 (C-20), 146.1 (C-2, C-6), 167.6 (2COO).
EI-MS (70 eV): m/z = 321 (M, 28%)+, 306 (Mꢁ15, 21%)+, 290
(Mꢁ31, 14%)+, 262 (Mꢁ59, 93%)+, 224 (MꢁC5H5S, 100%)+, 165
(MꢁC5H5Sꢁ59, 9%)+. Anal. Calcd for C16H19O4NS: C, 59.81; H,
5.92; N, 4.36; S, 9.97. Found: C, 59.69; H, 6.01; N, 4.47; S, 10.02.
1H NMR (CDCl3): d = 2.22 ppm (s; 3H, CH3-30), 2.38 (s br; 6H,
2CH3-2,6), 4.60 (d; 4H, 2CH2CH@CH2), 5.21 (s; 2H, 2CH2CH@CH2),
5.22 (s; 2H, 2CH2CH@CH2), 5.40 (s; 1H, H-4), 5.89 (m; 2H,
2CH2CH@CH2), 6.40 (s br; 1H, NH), 6.60 (d; 1H, J = 5.04 Hz, H-40),
6.95 (d; 1H, J = 5.05 Hz, H-50). 13C NMR (CDCl3): d = 14.1 ppm
(CH3-30), 19.5 (2CH3-2,6), 35.5 (C-4), 64.9 (CH2CH@CH2), 101.8
(C-3, C-5), 117.8 (CH2CH@CH2), 124.1 (C-50), 124.8 (C-40), 132.1
(CH2CH@CH2), 133.1 (C-30), 136.2 (C-20), 145.8 (C-2, C-6), 165.9
(COO). EI-MS (70 eV): m/z = 373 (M, 10%)+, 358 (Mꢁ15, 9%)+, 331
(MꢁC3H6, 50%)+, 288 (MꢁC4H6O2, 53%)+, 203 (Mꢁ2C4H6O2,
100%)+. Anal. Calcd for C20H23O4NS: C, 64.34; H, 6.16; N, 3.75; S,
8.58. Found: C, 64.21; H, 6.09; N, 3.83; S, 8.67.
3.1.4. 1,4-Dihydro-2,6-dimethyl-4-(thiophen-20-yl)-pyridine-
3,5-dicarboxylic acid dimethyl ester (3d)
Prepared from 5 mmol of 1d and 12 mmol of methyl acetoace-
tate 2a. The crude reaction material obtained was purified by chro-
matography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3d as pale yellow microcrystalline precipitate: mp 189–
190 °C; yield 77%.
3.1.8. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid dibenzyl ester (3h)
Prepared from 5 mmol of 1c and 12 mmol of benzyl acetoace-
tate 2d. The crude reaction material obtained was purified by chro-
matography on silica gel with hexane/AcOEt (7:3) to give the
compound 3h as pale yellow microcrystalline precipitate: mp
109–110 °C; yield 77%.
1H NMR (CDCl3): d = 2.40 ppm (s br; 6H, 2CH3-2,6), 3.60 (s br;
6H, 2COOCH3), 5.34 (s; 1H, H-4), 5.80 (s br; 1H, NH), 6.60 (d; 1H,
J = 5.0 Hz, H-30), 6.76 (m; 1H, H-40), 7.01 (d; 1H, J = 5.2 Hz, H-50).
13C NMR (CDCl3): d = 19.3 ppm (2CH3-2,6), 32.8 (C-4), 51.3
(COOCH3), 104.2 (C-3, C-5), 124.2 (C-50), 126.8 (C-40), 126.4 (C-
30), 140.1 (C-20), 146.4 (C-2, C-6), 167.3 (2COO). EI-MS (70 eV): m/
z = 307 (M, 32%)+, 292 (Mꢁ15, 23%)+, 276 (Mꢁ31, 17%)+, 248
(Mꢁ59, 100%)+. Anal. Calcd for C15H17O4NS: C, 58.63; H, 18.06; N,
4.56; S, 10.42. Found: C, 58.49; H, 17.97; N, 4.61; S, 10.53.
1H NMR (CDCl3): d = 2.00 ppm (s; 3H, CH3-30), 2.38 (s br; 6H,
2CH3-2,6), 5.18 (s; 4H, CH2Ph), 5.41 (s; 1H, H-4), 6.17 (s br; 1H,
NH), 6.60 (d; 1H, J = 5.04 Hz, H-40), 6.97 (d; 1H, J = 5.05 Hz, H-50),
7.20–7.34 (m; 10H, arom.H). 13C NMR (CDCl3): d = 13.2 ppm
(CH3-30), 18.9 (2CH3-2,6), 32.2 (C-4), 65.0 (CH2Ph), 103.4 (C-3, C-
5), 121.2 (C-50), 122.3 (C-40), 127.0–128.5 (C-arom.), 131.9 (C-30),
135.8 (C-20), 143.6 (C-1”), 145.4 (C-2, C-6), 166.4 (COO). EI-MS
(70 eV): m/z = 473 (M, 10%)+, 382 (MꢁC7H7, 18%)+, 338 (MꢁC8H7O2,
43%)+, 232 (MꢁC8H7O2ꢁC7H7ꢁCH3, 72%)+, 91 (C7H7, 100%)+. Anal.
Calcd for C28H27O4NS: C, 71.03; H, 5.71; N, 2.96; S, 6.76. Found:
C, 70.91; H, 5.60; N, 3.03; S, 6.84.
3.1.5. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid diethyl ester (3e)
Prepared from 5 mmol of 1c and 12 mmol of ethyl acetoacetate
2b. The crude reaction material obtained was purified by chroma-
tography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3e as pale yellow microcrystalline precipitate: mp
135–137 °C; yield 74%.
1H NMR (CDCl3): d = 1.25 ppm (t; 6H, J = 7.5 Hz, 2CH3CH2), 2.20
(s; 3H, CH3-30), 2.38 (s br; 6H, 2CH3-2,6), 4.15 (m; 4H, CH3CH2-3,5),
5.40 (s; 1H, H-4), 6.15 (s br; 1H, NH), 6.63 (d; 1H, J = 5.04 Hz, H-40),
6.95 (d; 1H, J = 5.05 Hz, H-50). 13C NMR (CDCl3): d = 13.9 ppm (CH3-
30), 14.0 (2CH3CH2–), 19.3 (2CH3-2,6), 32.7 (C-4), 64.5 (2CH2CH3),
103.8 (C-3, C-5), 124.4 (C-50), 124.9 (C-40), 132.6 (C-30), 135.4 (C-
20), 146.0 (C-2, C-6), 168.9 (2COO). EI-MS (70 eV): m/z = 349 (M,
31%)+, 334 (Mꢁ15, 14%)+, 304 (MꢁC2H5O, 100%)+. Anal. Calcd for
C18H23O4NS: C, 61.89; H, 6.59; N, 4.01; S, 9.17. Found: C, 61.77;
H, 6.48; N, 4.11; S, 9.28.
3.1.9. 1,4-Dihydro-2,6-dimethyl-4-(50-nitro-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid diallyl ester (3i)
Prepared from 5 mmol of 1a and 12 mmol of allyl acetoacetate
2c. The crude reaction material obtained was purified by chroma-
tography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3i as red microcrystalline precipitate: mp 124–125 °C;
yield 72%.
1H NMR (CDCl3): d = 2.40 ppm (s br; 6H, 2CH3-2,6), 4.62 (d; 4H,
2CH2CH@CH2), 5.23 (s; 2H, 2CH2CH@CH2), 5.24 (s; 2H,
2CH2CH@CH2), 5.40 (s; 1H, H-4), 5.90 (m; 2H, 2CH2CH@CH2),
6.20 (s br; 1H, NH), 6.83 (d; 1H, J = 4.15 Hz, H-30), 7.78 (d; 1H,
J = 4.17 Hz, H-40). 13C NMR (CDCl3): d = 19.5 ppm (2CH3-2,6), 35.5
(C-4), 64.9 (2CH2CH@CH2), 101.8 (C-3, C-5), 117.8 (2CH2CH@CH2),
123.0 (C-30), 128.9 (C-40), 132.1 (2CH2CH@CH2), 145.8 (C-20), 146.4
(C-2, C-6), 149.6 (C-50), 165.9 (COO). EI-MS (70 eV): m/z = 404 (M,
3.1.6. 1,4-Dihydro-2,6-dimethyl-4-(50-methyl-furan-20-yl)-
pyridine-3,5-dicarboxylic acid dimethyl ester (3f)
Prepared from 5 mmol of 1e and 12 mmol of methyl acetoace-
tate 2a. The crude reaction material obtained was purified by chro-
matography on silica gel with petrol ether/AcOEt (7:3) to give the

7898
F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
37%)+, 363 (MꢁC3H5, 76%)+, 347 (MꢁC3H5O, 18%)+, 319
(MꢁC4H5O2, 92%)+, 276 (MꢁC4H2NO2S, 100%)+. Anal. Calcd for
C19H20O6N2S: C, 56.43; H, 4.95; N, 6.93; S, 7.92. Found: C, 56.29;
H, 4.78; N, 6.99; S, 8.01.
(40 mmol) and dry toluene (10 ml) as described in the literature.18
The crude product was distilled to give 1.76 g (yield 76%) of 3-bro-
mo-2-(2-dioxolanyl) thiophene. 1H NMR (CDCl3): d = 4.0–4.17 ppm
(m; 4H), 6.14 (s; 1H), 6.96 (d; H-5, J = 5.50 Hz), 7.30 (d; 1H, J
= 5.50 Hz). EI-MS (70 eV): m/z = 235 (M, 45%)+.
3-Bromo-2-(2-dioxolanyl) thiophene (1.1 g; 0.005 mmol) in THF
was treated with n-Butyllithium and sulphur at ꢁ100 °C to give 1.3 g
of crude product which was used in the next step without purifica-
tion. Methyl 3-[2-(2-dioxolanyl) thiophene-3-yl]-2-thioacetate
(1.17 g, 0.005 mol) was dissolved in acetone (10 mL), and p-toluene-
sulfonic acid hydrate (0.01 g; 0.068 mmol) was added and stirred
over 1 h. The usual work up gave 0.5 g of crude product which was
purified by chromatography on silica gel with hexane/ether (7:3)
to give 0.45 g of 1g (yield 41%). 1H NMR (CDCl3): d = 3.70 ppm (s;
1H, SCH2), 3.77 (s; 3H, COOCH3), 7.07 (s; 2H, CH@CH), 9.83 (s; 1H,
CHO). EI-MS (70 eV): m/z = 216 (M, 37%)+.
3.1.10. 1,4-Dihydro-2,6-dimethyl-4-(50-nitro-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid dibenzyl ester (3j)
Prepared from 5 mmol of 1a and 12 mmol of benzyl acetoace-
tate 2d. The crude reaction material obtained was purified by chro-
matography on silica gel with hexane/AcOEt (7:3) to give the
compound 3j as pale yellow microcrystalline precipitate: mp
117–118 °C; yield 70%.
1H NMR (CDCl3): d = 2.35 ppm (s br; 6H, 2CH3-2,6), 5.20 (s; 4H,
CH2Ph), 5.40 (s; 1H, H-4), 6.15 (s br; 1H, NH), 6.82 (d; 1H,
J = 4.14 Hz, H-30), 7.20–7.34 (m; 10H, H-arom.), 7.77 (d; 1H,
J = 4.16 Hz, H-40). 13C NMR (CDCl3): d = 18.7 ppm (2CH3-2,6), 32.4
(C-4), 65.2 (CH2Ph), 103.2 (C-3, C-5), 123.4 (C-30), 127.0–128.5
(C-arom.), 128.8 (C-40), 145.7 (C-20), 146.4 (C-2, C-6), 149.5 (C-
50),166.2 (COO). EI-MS (70 eV): m/z = 504 (M, 23%)+, 413 (MꢁC7H7,
100%)+, 376 (MꢁC4H2NO2S, 83%)+. Anal. Calcd for C27H24O6N2S: C,
64.28; H, 4.76; N, 5.55; S, 6.35. Found: C, 64.07; H, 4.54; N, 5.68;
S, 6.47.
3.1.14. 1,4-Dihydro-2,6-dimethyl-4-(3’-methyl 2-thioacetate-
thiophen-20-yl)-pyridine-3,5-dicarboxylic acid diallyl ester (3m)
Prepared from 5 mmol of 1g and 12 mmol of allyl acetoacetate
2c. The crude reaction material obtained was purified by chroma-
tography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3m as an oil: yield 63%.
3.1.11. 1,4-Dihydro-2,6-dimethyl-4-(40-phenyl-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid diallyl ester (3k)
Prepared from 5 mmol of 1f and 12 mmol of allyl acetoacetate
2c. The crude reaction material obtained was purified by chroma-
tography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3k as white microcrystalline precipitate: mp 117–
118 °C; yield 68%.
1H NMR (CDCl3): d = 2.39 ppm (s br; 6H, 2CH3-2,6), 3.48 (s; 1H,
SCH2), 3.68 (s; 3H, COOCH3), 4.60 (d; 4H, 2CH2CH@CH2), 5.22 (s;
2H, 2CH2CH@CH2), 5.23 (s; 2H, 2CH2CH@CH2), 5.40 (s; 1H, H-4),
5.89 (m; 2H, 2CH2CH@CH2), 6.20 (s br; 1H, NH), 6.79 (m; 2H, H-
30, H-40). 13C NMR (CDCl3): d = 19.4 ppm (2CH3-2,6), 34.9 (C-4),
40.5 (CH2S), 51.3 (COOCH3), 65.1 (2CH2CH@CH2), 102.7 (C-3, C-
5), 117.4 (2CH2CH@CH2), 123.7 (C-50), 128.3 (C-40), 131.6 (C-30),
133.4 (C-20), 136.4 (2CH2CH@CH2), 146.4 (C-2, C-6), 166.2, 171.3
(COO). EI-MS (70 eV): m/z = 463 (M, 12%)+, 422 (MꢁC3H5, 92%)+,
378 (MꢁC4H5O2, 100%)+, 276 (MꢁC7H7S2O2, 67%)+. Anal. Calcd for
C22H25O6NS2: C, 57.02; H, 5.39; N, 3.02; S, 13.8. Found: C, 56.89;
H, 5.26; N, 3.11; S, 14.07.
1H NMR (CDCl3): d = 2.40 ppm (s br; 6H, 2CH3-2,6), 4.61 (d; 4H,
2CH2CH@CH2), 5.22 (s; 2H, 2CH2CH@CH2), 5.23 (s; 2H,
2CH2CH@CH2), 5.40 (s; 1H, H-4), 5.89 (m; 2H, 2CH2CH@CH2),
6.20 (s br; 1H, NH), 7.10 (m; 1H, H-30), 7.20–7.51 (m; 5H, H-arom),
7.50 (m; 1H, H-50). 13C NMR (CDCl3): d = 19.4 ppm (2CH3-2,6), 35.6
(C-4), 65.0 (2CH2CH@CH2), 102.0 (C-3, C-5), 117.6 (2CH2CH@CH2),
120.2 (C-50), 123.4 (C-30), 132.5 (2CH2CH@CH2), 127.0–129.3 (C-
arom.), 136.7 (C-100), 143.0 (C-20), 143.2 (C-40), 146.3 (C-2, C-6),
166.2 (COO). EI-MS (70 eV): m/z = 435 (M, 22%)+, 394 (MꢁC3H5,
90%)+, 378 (MꢁC3H5O, 14%)+, 350 (MꢁC4H5O2, 100%)+, 336
(MꢁC4H5O2ꢁCH3, 10%)+, 276 (MꢁC10H7S, 43%)+. Anal. Calcd for
C25H25O4NS: C, 68.96; H, 5.75; N, 3.22; S, 7.36. Found: C, 68.71;
H, 5.60; N, 3.31; S, 7.40.
3.1.15. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl 2-thioacetate-
thiophen-20-yl)-pyridine-3,5-dicarboxylic acid dibenzyl ester
(3n)
Prepared from 5 mmol of 1g and 12 mmol of benzyl acetoace-
tate 2d. The crude reaction material obtained was purified by chro-
matography on silica gel with petrol ether/AcOEt (7:3) to give the
compound 3n as an oil: yield 71%.
1H NMR (CDCl3): d = 2.40 ppm (s br; 6H, 2CH3-2,6), 3.49 (s; 1H,
SCH2), 3.69 (s; 3H, COOCH3), 4.95 (s; 4H, CH2Ph), 5.22 (s; 1H, H-4),
6.10 (s br; 1H, NH), 6.78 (m; 2H, H-40, H-50), 7.17–7.30 (m; 10H, H-
arom.). 13C NMR (CDCl3): d = 19.5 ppm (2CH3-2,6), 34.5 (C-4), 40.6
(CH2S), 51.2 (COOCH3), 65.7 (CH2Ph), 103.3 (C-3, C-5), 124.6 (C-50),
128.3 (C-40), 127.2–129.4 (C-arom.), 133.1 (C-30), 133.6 (C-20),
140.6 (C-100), 146.3 (C-2, C-6), 166.2, 171.3 (COO). EI-MS (70 eV):
m/z = 563 (M, 29%)+, 472 (MꢁC7H7, 72%)+, 428 (MꢁC8H7O2,
100%)+, 376 (MꢁC7H7S2O2, 51%)+. Anal. Calcd for C30H29O6NS2: C,
63.94; H, 5.15; N, 2.48; S, 11.37. Found: C, 63.81; H, 5.06; N,
2.54; S, 11.42.
3.1.12. 1,4-Dihydro-2,6-dimethyl-4-(40-phenyl-thiophen-20-yl)-
pyridine-3,5-dicarboxylic acid dibenzyl ester (3l)
Prepared from 5 mmol of 1f and 12 mmol of benzyl acetoace-
tate 2d. The crude reaction material obtained was purified by chro-
matography on silica gel with hexane/AcOEt (7:3) to give the
compound 3l as white microcrystalline precipitate: mp 148–
150 °C; yield 73%.
1H NMR (CDCl3): d = 2.38 ppm (s br; 6H, 2CH3-2,6), 4.92 (s; 4H,
CH2Ph), 5.20 (s; 1H, H-4), 6.00 (s br; 1H, NH), 6.98 (m; 1H, H-30),
7.19–7.50 (m; 15H, H-arom., H-50). 13C NMR (CDCl3): d =
18.8 ppm (2CH3-2,6), 32.5 (C-4), 65.5 (CH2Ph), 103.1 (C-3, C-5),
120.1 (C-50), 123.5 (C-30), 127.2–129.3 (C-arom.), 137.3, 140.9,
143.0 (C-20), 143.1 (C-40), 146.5 (C-2, C-6), 166.2 (COO). EI-MS
(70 eV): m/z = 535 (M, 24%)+, 444 (MꢁC7H7, 100%)+, 400
(MꢁC8H7O2, 43%)+, 376 (MꢁC10H7S, 75%)+. Anal. Calcd for
C33H29O4NS: C, 7.40; H, 5.42; N, 2.62; S, 5.98. Found: C, 7.32; H,
5.36; N, 2.78; S, 6.03.
3.2. General procedure for the synthesis of asymmetrical 1,4-
dihydropyridines (4a–d)
To an ice-cooled solution of 5 mmol of an aldehyde (A) in 15 ml
of isopropanol, 5 mmol of the first acetoacetate (B) were added and
the mixture is refluxed for 3 h. Then 3 mmol of the second acetoac-
etate (C) is added and the mixture is refluxed for 3 h. At the end,
1 ml of concd ammonia is added and the mixture is refluxed for
1 h. After cooling, the solvent is evaporated under reduced pres-
sure and the crude reaction material obtained is purified by col-
umn chromatography on silica gel with toluene/AcOEt (9:1).
3.1.13. Synthesis of Methyl 3-[2-(2-formylthiophene-3-yl)]-2-
thioacetate (1g)
Pyridinium tosylate (0.4 mmol) was added to a mixture of 3-
bromo-thiophene-2-carboxaldehyde (9.8 mmol), ethylene glycol

F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
7899
3.2.1. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl-thiophen-20-yl)-
pyridine-3-carboxylic acid allyl ester, 5- carboxylic acid ethyl
ester (4a)
13C NMR (CDCl3): d = 13.6 ppm (CH3CH2–), 18.8 (CH3-2), 18.9
(CH3-6), 33.6 (C-4), 40.5 (CH2S), 51.2 (COOCH3), 59.5 (CH2CH3),
64.9 (CH2Ph), 102.3 (C-3), 102.4 (C-5), 125.6 (C-50), 126.8 (C-40),
127.0–129.4 (C-arom.), 133.2 (C-30), 135.9 (C-20), 141.2 (C-100),
146.5 (C-2), 146.9 (C-6), 166.3, 166.5, 171.5 (COO). Anal. Calcd
for C25H27O6NS2: C, 59.86; H, 5.43; N, 2.79; S, 12.78. Found: C,
60.08, H, 5.38, N, 2.85; S, 12.86.
Prepared from 1c (A), ethyl acetoacetate (B) and allyl acetoace-
tate (C). Crystallized from cyclohexane; mp: 157–158 °C; yield: 32%.
IR (KBr)
m
(cmꢁ1): 3313, 2983, 2927, 1697, 1645, 1487, 1206.
1H NMR (d, CDCl3): 1.25 ppm (t, 3H, CH2CH3), 2.21–2.39 (m, 9H,
CH3-30 + CH3-2 + CH3-6), 4.10 (q, 2H, CH2CH3), 4.57 (d, 2H,
CH2CH@CH2), 5.04–5.14 (m, 2H, CH2CH@CH2), 5.35 (s, 1H, H-4),
5.79–5.93 (m, 2H, NH + CH2CH@CH2), 6.60 (d, 1H, H-40), 6.91 (d,
1H, H-50).
4. Methods
4.1. CFTR assays
4.1.1. Cell culture
13C NMR (CDCl3): d = 13.4 ppm (CH3-30), 13.7 (CH3CH2), 18.8
(CH3-2), 18.9 (CH3-6), 32.5 (C-4), 59.5 (CH2CH3), 63.9 (CH2CH@CH2),
103.3 (C-3), 103.7 (C-5), 116.7 (CH2CH@CH2), 120.8 (C-50), 128.3 (C-
40), 131.5 (CH2CH@CH2), 131.5 (C-30), 132.1 (C-20), 143.5 (C-2), 145.5
(C-6), 166.2, 166.6 (COO). Anal. Calcd for C19H23O4NS: C, 63.14; H,
6.41; N, 3.88; S, 8.87. Found: C, 63.38; H, 6.35; N, 3.96; S, 8.76.
Fischer rat thyroid (FRT) cells, stable transfected with
DF508-
CFTR and the halide-sensitive yellow fluorescent protein YFP-
H148Q/I152L19 were cultured in Coon0s modified Ham0s F-12 med-
ium supplemented with 10% fetal calf serum, 2 mM L-glutamine,
100 U/ml penicillin, and 100 g/ml streptomycin. For fluorescence
assays of CFTR activity, cells were plated (100,000 cells/well) on
clear-bottomed 96-well microplates (Corning Life Sciences, Acton,
MA).
H9C2 cells, a cell line derived from rat heart tissue with endog-
enous expression of L-type voltage-dependent Ca2+ channels (L-
VDCCs),20 were grown in Dulbecco0s modified Eagle0s medium/
Ham0s F-12 medium supplemented with 10% fetal calf serum,
3.2.2. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl-thiophen-20-yl)-
pyridine-3-carboxylic acid benzyl ester, 5- carboxylic acid ethyl
ester(4b)
Prepared from 1c (A), ethyl acetoacetate (B) and benzyl acetoac-
etate (C). Crystallized from cyclohexane; mp: 186–187 °C; yield:
35%.
IR (KBr)
m
(cmꢁ1): 3018, 2953, 2930, 1691, 1617, 1470, 1221.
1H NMR (CDCl3): d = 1.23 (t, 3H, CH2CH3), 2.21–2.38 (m, 9H,
CH3-30 + CH3-2 + CH3-6), 4.11 (q, 2H, CH2CH3), 5.05–5.19 (m, 3H,
CH2Ph + H-4), 5.87 (s, 1H, NH), 6.57 (d, 1H, H-40), 6.91 (d, 1H, H-
50), 7.13–7.26 (m, 5H, H-arom).
2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomy-
cin. For fluorescence assays of L-VDCC activity, cells were plated
(100,000 cells/well) on clear-bottomed 96-well microplates. After
24 h, cells received fresh medium containing all-trans-retinol
(10 nM) to increase L-VDCC expression.21 The functional assay
was performed after additional 24 h.
1H NMR (CDCl3): d = 2.00 ppm (s; 3H, CH3-30), 2.38 (s br; 6H,
2CH3-2,6), 5.18 (s; 4H, CH2Ph), 5.41 (s; 1H, H-4), 6.17 (s br; 1H,
NH), 6.60 (d; 1H, J = 5.04 Hz, H-40), 6.97 (d; 1H, J = 5.05 Hz, H-50),
7.20–7.34 (m; 10H, arom.H). 13C NMR (CDCl3): d = 13.2 ppm
(CH3-30), 13.6 (CH3CH2–), 18.7 (CH3-2), 18.9 (CH3-6), 32.1 (C-4),
59.5. Anal. Calcd for C23H25O4NS: C, 67.13; H, 6.12; N, 3.40; S,
7.79. Found: C, 66.90; H, 6.14; N, 3.46; S, 7.70.
4.1.2. Samples
The synthesized compounds 3a–n and 4a–d were dissolved in
dimethyl sulfoxide and for each compound 10 mM stock solutions
were prepared.
3.2.3. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl 2-thioacetate-
thiophen-20-yl)-pyridine-3-carboxylic acid allyl ester, 5-
carboxylic acid ethyl ester (4c)
Prepared from 1g (A), ethyl acetoacetate (B) and allyl acetoace-
tate (C). Pale yellow oil; yield: 33%.
Secondary plates were prepared for screening at 1 mM concen-
tration in dimethyl sulfoxide using a Biomek 2000 liquid handling
workstation (Beckman Coulter, Fullerton, CA). All plates were
stored at ꢁ70 °C.
m
(cmꢁ1): 3020, 2955, 2934, 17634, 1690, 1471, 1225.
4.1.3. Fluorescence assay for CFTR activity
IR (KBr)
1H NMR (CDCl3): d = 1.26 ppm (t, 3H, CH2CH3), 1.97–2.08 (m, 6H,
CH3-2 + CH3-6), 3.33 (s, 1H, SCH2), 3.67 (s, 3H, COOCH3), 4.18 (q, 2H,
CH2CH3), 4.61 (d, 2H, CH2CH@CH2), 5.08–5.25 (m, 2H, CH2CH@CH2),
5.38 (s, 1H, H-4), 5.79–6.05 (m, 2H, NH + CH2CH@CH2), 6.87 (d, 1H,
H-50), 7.12–7.26 (m, 5H, H-arom).
Measurements of CFTR activity were carried out on FRT cells
expressing
plating on microplates. Before the assay,
D
F508-CFTR and the halide-sensitive YFP 48 h after
F508-CFTR-expressing
D
cells were incubated at 27 °C for 20–24 h to allow targeting of
the mutant protein to the plasma membrane.
13C NMR (CDCl3): d = 13.6 ppm (CH3CH2–), 18.7 (CH3-2), 18.9
(CH3-6), 33.4 (C-4), 40.3 (CH2S), 51.3 (COOCH3), 59.1 (CH2CH3),
64.8 (CH2CH@CH2), 102.1 (C-3), 102.4 (C-5), 117.7 (CH2CH@CH2),
125.6 (C-50), 126.3 (C-40), 133.5 (C-30), 135.7 (C-20), 136.5
(CH2CH@CH2), 146.3 (C-2), 146.9 (C-6), 166.2, 166.7, 171.4 (COO).
Anal. Calcd for C21H25O6NS2: C, 55.86; H, 5.58; N, 3.10; S, 14.20.
Found: C, 55.97; H, 5.68; N, 3.03; S, 14.32.
At the time of assay, cells were washed with PBS (containing
137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4,
1 mM CaCl2, and 0.5 mM MgCl2) and stimulated for 30 min with
20 lM forskolin (to maximally enhance intracellular cAMP levels
and allow CFTR phosphorylation) and test compounds at the de-
sired concentration. Then, cells were transferred to a microplate
reader (FluoStar Galaxy; BMG Labtech GmbH, Offenburg, Ger-
many) for CFTR activity determination. The plate reader was
equipped with excitation (HQ500/20X: 500 10 nm) and emission
(HQ535/30M: 535 15 nm) filters for YFP (Chroma Technology
Corp., Brattleboro, VT). Each assay consisted of a continuous 14-s
fluorescence reading: 2 s before and 12 s after injection of an io-
dide-containing solution (PBS with chloride replaced by iodide; fi-
nal iodide concentration in the well: 100 mM). Data were
normalized to the initial background-subtracted fluorescence. To
determine iodide influx rate, the final 10 s of the data for each well
were fitted with an exponential function to extrapolate the initial
slope (dF/dt).
3.2.4. 1,4-Dihydro-2,6-dimethyl-4-(30-methyl 2-thioacetate-
thiophen-20-yl)-pyridine-3-carboxylic acid benzyl ester, 5-
carboxylic acid ethyl ester (4d)
Prepared from 1g (A), ethyl acetoacetate (B) and benzyl acetoac-
etate (C). Pale yellow oil; yield: 36%.
IR (KBr)
m
(cmꢁ1): 3019, 2955, 2929, 1735, 1693, 1470, 1225.
1H NMR (CDCl3): d = 1.23 (t, 3H, CH2CH3), 2.24–2.38 (m, 6H,
CH3-2 + CH3-6), 3.47 (s, 1H, SCH2), 3.69 (s, 3H, COOCH3), 4.15 (q,
2H, CH2CH3), 5.04–5.23 (m, 3H, CH2Ph + H-4), 5.95 (s, 1H, NH),
6.87 (s, 1H, H-50), 7.16–7.38 (m, 10H, H-arom).

7900
F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
Table 1
Kd values for DHPs 3 and 4
4.1.4. Fluorescence assay for VDCC activity
Cells were incubated with a loading solution containing the cal-
cium-sensitive fluorescent probe Fluo-4 AM (4 M), glucose
l
Compound
Kd (lM)
(10 mM) and probenecid (2 mM). After 1 h, cells were washed with
phosphate-buffered saline and then incubated for 20 min in the
presence of test compounds or known VDCC modulators (proben-
ecid and glucose were maintained in the solution). Then, cells were
transferred to the microplate reader (485 nm excitation; 520 nm
emission). Each assay consisted of a continuous 8-s fluorescence
reading, after which a solution containing high-potassium (PBS
with Na+ replaced by K+) was injected into the well to depolarize
the cell membrane and activate VDCCs. The fluorescence was con-
tinuously monitored for additional 27 s. The activity of VDCCs was
determined as the increase in Fluo-4 fluorescence upon injection,
normalized to the initial background-subtracted fluorescence.
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
4a
4b
4c
8.3 2.1
6.0 0.8
3.1 0.3
9.5 1.2
5.7 1.2
6.0 1.8
1.0 0.3
0.7 0.1
3.8 0.6
13.1 0.7
1.5 0.4
16.5 2.2
1.3 0.3
0.3 0.1
0.3 0.1
0.5 0.2
1.9 0.5
0.3 0.1
16.7 0.1
4.2. 3D-QSAR analysis
4d
Genistein
4.2.1. CoMFA procedure
Comparative Molecular Field Analysis (CoMFA) method is a
widely used 3D-QSAR technique to relate the biological activity
of a series of molecules with their steric and electrostatic fields
which are calculated placing the aligned molecules, one by one,
in a 3D cubic lattice with 2 Å grid spacing. The column-filtering
threshold value in this study was set to 2.0 kcal/mol, to improve
the signal-noise ratio. A methyl probe with a +1 charge is used to
calculate steric and electrostatic fields, represented by van der
Waals potential and coulombic term, respectively. A 30 kcal/mol
energy cut-off was applied to avoid infinity of energy values inside
the molecule. Regression analyses were performed applying the
partial least squares (PLS) algorithm in SYBYL; the combination of
the steric and electrostatic fields were used as structural descrip-
tors to evaluate their correlation with the biological activity (ex-
pressed as pKd) data. PLS with cross-validation, performed using
five cancellation groups was used to identify the optimal number
of components to be used in the subsequent analyses. The final
CoMFA model was generated using non-cross-validated PLS analy-
sis. To further assess the statistical confidence and robustness of
derived model, a 100-cycle bootstrap analysis was performed.
The alignment procedure was carried out using the Molecular
Operating Environment (MOE; version 2006.08) suite. CoMFA
About the substituents on thiophene ring of DHPs, the presence
of substituents at the 30 position seems to favour the activity on
CFTR. Thus, the 30-methyl 2-thioacetate derivatives 3m and 3n
show an outstanding efficacy and also good results are obtained
by the 30-methyl derivatives 3g and 3h (the only exception to this
trend is 3c). The phenyl derivatives at the position 40 (3k and 3l)
show a good activity on CFTR. On the other hand, compounds with
substituents at the position 50 (3a and 3b) are less effective. Also
the furanyl isoster 3f presents a modest degree of efficacy. Never-
theless, this compound may deserve a further development (see
later).
In general, not only the kind of substituents of the thiophen-2-
yl ring is important for potency on CFTR, but also the esterification
of the carboxyl groups with allyl and benzyl, instead of methyl and
ethyl, shows improved properties with the exception of the com-
pounds 3i, 3j and 3l.
The synthesized DHPs 3 and 4 were also tested in the calcium
channel assay and compared to felodipine (see Fig. 2). The sym-
metrical DHPs 3a–n had different levels of activity on Ca2+ chan-
nels. In particular, 3f bearing a furanyl substituent at the C-4
position of the dihydropyridine moiety, was the weakest Ca2+
was performed using SYBYL.
22 All calculations were carried out using
a PC, with operative system windows XP and an SGI O2 Silicon
Graphics.
genistein
4d
4c
5. Results
4b
4a
3n
3m
3l
FRT cells expressing
DF508-CFTR were incubated at 27 °C to
rescue the mutant protein from the ER, thus improving its target-
ing to the plasma membrane. Subsequent stimulation of cells with
forskolin plus genistein evoked a threefold increase in halide trans-
port relative to cells treated with forskolin alone. Most DHPs elic-
3k
3j
3i
ited a similar increase in
DF508-CFTR activity. We determined
3h
3g
3f
3e
3d
3c
dose–response relationships for symmetrical (3a–n) and asym-
metrical (4a–d) DHPs and derived apparent Kd values and maximal
effect by fitting the data with a Hill equation (Table 1).
The affinity calculated for our DHPs was high for compounds 3g,
3h, 3n, 4a, 4b, and 4d, with Kd values in the 0.3–1.0 lM range. The
3b
3a
other compounds showed different degrees of affinity. Compounds
3j and 3l were the least potent, the corresponding Kd values being
close to that of genistein (ꢂ16
lM).
0
10 20 30 40 50
CFTR activity
From the data reported in Figure 1 it is possible to see that the
efficacy of symmetrical DHPs 3 is, in general, better than the refer-
ence compound genistein, whereas the asymmetrical DHPs 4 pres-
ent a CFTR activity less than genistein.
Figure 1. Compound efficacy on
response relationships of YFP fluorescence experiments (mean SD, n = 3).
D
F508-CFTR. The efficacy is obtained from dose–

F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
7901
blocker. Also the compounds 3b and 3d had reduced activity on
calcium channels. On the contrary, the behaviour of asymmetrical
DHPs 4a–d on Ca2+ channel activity is quite similar to that of the
reference compound felodipine.
Fig. 1), the asymmetrical DHPs 4a–d are less active than the refer-
ence compound genistein, whereas the symmetrical DHPs 3a–n
present an activity comparable or better than genistein. As the effi-
cacy of DHPs 3 is confined in a narrow range, it causes difficulties
to give an order of importance to the substituents of the DHPs
present in this work, however we can affirm that, regarding the
substituents at positions 3 and 5 of these DHPs, carboxybenzyl
and carboxyallyl esters are more active than carboxymethyl and
carboxyethyl esters. Regarding the substituents on the thiophene
ring, remarkable results are attained when the 30-methyl 2-thio-
acetate group is present: indeed, compounds 3m and 3n (Table 1
and Fig. 1) have both a good affinity and a notable efficacy.
6. Discussion
6.1. Activity of 1,4-dihydropyridines on CFTR
Identification of selective potentiators of the CFTR Clꢁ channel
is important to develop effective drugs for the treatment of the ba-
sic defect in cystic fibrosis patients. In particular, potentiators may
be useful to enhance the activity of the
DF508 mutant, once the
6.2. Activity of 1,4-dihydropyridines on Ca2+ channels
trafficking defect is corrected, at least partially, by suitable phar-
macological chaperones.23,24 In 2005, we showed that DHPs pos-
sess a notable activity in potentiating the conductance in cells
carrying CFTR gating mutants as G551D or when the cells with
The lack of activity on Ca2+ channels is a very important charac-
teristic in order to avoid negative consequences on the cardiovas-
cular system in CF patients. As already observed in previous
work,15 the DHPs potentially useful for CF are often endowed of
a VDCC block. Now, the only derivatives that may represent an
exception to this propensity are 3d, 3f and 3l. These three com-
pounds each presents at position 3 and 5 a carboxymethyl group,
but this fact does not represent a selectiveness because other
Ca2+ channel blocking compounds (e.g., 3a, 3c) also possess this
group. As the substitutions on esters by allyl and benzyl groups
give a great VDCC inhibition (the only partial exception being 3l),
we can conclude that a carboxymethyl group could be useful in
lowering the Ca2+ blocking activity. Also the substitutions on the
thiophene ring do not seem to have a great significance in modify-
ing the activity of DHPs on Ca2+ channels. Interestingly, the only
compound without activity on Ca2+ channels is the isosteric furanyl
derivative 3f, having a relatively good activity on CFTR. This fact
deserves further investigation: indeed, it will be very interesting
to examine the behaviour of other furan derivatives possessing
more interesting groups at the ester level (as benzyl or allyl) or
on the furan ring as the 30-methyl-2-thioacetate group, to test if
it will be possible maximize the activity on CFTR maintaining a
low VDCC inhibition.
D
F508-CFTR were rescued by incubation at low temperature.11 In
this way more than 300 DHPs with a phenyl group in position 4
were evaluated in order to investigate the structure–activity
relationship.
4-Thiophen-2-yl-1,4-dihydropyridines, the object of the present
work, represent an extension of our knowledge on this topic. In
general, the behaviour of the present DHPs are similar to the pre-
vious ones, supporting the idea that the activity of such com-
pounds is unbound from the quality of the substituent present in
position 4 (also the isosteric furanyl derivative 3f has a certain de-
gree of activity). Regarding the affinity of our DHPs (see Table 1)
we can say that both compounds 3 and 4 present Kd values better
than the reference compound genistein, but activity is distributed
in a large array. In this regard, the asymmetrical derivatives 4a–d,
tested as racemates, showed better affinity with Kd values ranging
from 0.3 to 1.9 lM and for this reason constitute a sub-class
deserving a future wider study. Symmetrical DHPs 3a–n present
Kd values spreading in a broad range in comparison to compounds
4. In particular, the derivative 3n possesses the lowest Kd value of
this group (0.3 lM), and such value is identical to that of 4a and 4d.
Moreover, it must be noted that DHPs 3n and 4d have similar sub-
stituents, the only difference being the presence at position 3 of a
carboxybenzyl ester in 3n and the presence at position 3 of a car-
boxyethyl ester in 4d. Regarding the efficacy of our DHPs (see
6.3. CoMFA analysis
In order to give further support to the biological results ob-
tained and to better address the future synthesis, a preliminary
quantitative evaluation of the structure–activity relationship study
inside this series of CFTR chloride channel potentiators has been
performed. This in silico investigation is important because the
true binding site of potentiators in CFTR is unclear. Some studies
indicate that genistein binds CFTR at nucleotide binding domains
(NBDs) level and inside NBDs three or four binding sites, at differ-
ent degree of affinity, are proposed.25,26 Compounds 3a–n and 4a–
d were manually divided into a training set (3b–h, 3j–m, 4a–d) for
model generation and into a test set (3a, 3i, 3n) for model valida-
tion. The analysis was developed using CoMFA steric and electro-
static fields as independent variables and activity (expressed as
pKd) as the dependent one.
genistein
4d
4c
4b
4a
3n
3m
3l
3k
3j
3i
3h
3g
3f
CoMFA steric and electrostatic fields effect on the target prop-
erty can be viewed as 3D coefficient contour plots, thus they could
be helpful to identify important regions where any change in these
fields may affect the biological activity. The final CoMFA model
(Tables 2–4) was generated using non-cross-validated PLS analysis
with the optimum number of components (4) to give an r2ncv 0.93,
Standard Error of Estimate, SEE = 0.165, steric contribution = 0.486
and electrostatic contribution = 0.514. The results of the CoMFA
approach qualitatively points out that the activity of DHPs 3a–n,
4a–d is almost equally affected by the steric and electronic proper-
ties of the ligands. More in detail, the steric contour map shows
3e
3d
3c
3b
3a
0
20 40 60 80 100
VDCC inhibition (%)
Figure 2. Compound activity on VDCC. Activity on VDCC is reported as the
percentage of block obtained at a single concentration (10 lM) on H9C2 cells
(mean SD, n = 3).

7902
F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
Table 2
Summary of CoMFA results
No. of compounds
Opt. No. components
Cross-validated r2
Std. error of estimate
Non cross-validated rn2cv
F values
Steric contribution
Electrostatic contribution
Bootstrap r2
18
4
0.716
0.165
0.930
136.565
0.486
0.514
0.861
0.212
0.973
Std. Error of estimate(bootstrap
r2)
a
r2pred
a
Correlation coefficient for the test set.
favourable interaction polyhedra (green region) in the area around
the 1,4-dihydropyridine ring and around one of the two substitu-
ents at positions 3 and 5, thus suggesting the possibility of a
non-symmetric substitution. In addition, a large yellow polyhe-
dron (unfavourable interaction) underlines the importance of a
small substituent on positions 2 and 6 in order to avoid negative
interactions with a bulky moiety at position 3 and 5. Moreover, a
second yellow area is detected around position 4 of the dihydro-
pyridine ring (Fig. 3).
Figure 3. Contour maps of CoMFA steric regions (green, favoured; yellow,
disfavoured) are shown around the studied compounds.
As concern the electrostatic maps, all the compounds display a
red region, decoding for a favourable electrostatic interaction, be-
hind the substituent on position 4 of the dihydropyrine ring, while
two blue regions (unfavourable interactions) suggest the impor-
tance to limit the polar portion of substituents on position 3 and
5 to the carbonyl group directly linked to the scaffold (Fig. 4).
7. Conclusions
4-Thiophen-20-yl-1,4-dihydropyridines 3 and 4 were found to
act as valuable potentiators of rescued
DF508-CFTR. In this regard,
the activity of DHPs 3 and 4 is consistent with the activity of the 4-
phenyl-1,4-dihydropyridines already tested.14,15 About the activity
on Ca2+ channels, our data show that it is difficult to separate a
Table 3
Experimental and predicted binding affinities of the training set molecules
Compound
pKd exp
pKd pred
3b
3c
3d
3e
3f
3g
3h
3j
5.22
5.51
5.02
5.24
5.22
6.00
6.15
4.88
5.82
4.80
5.89
6.52
6.30
5.72
6.52
5.23
5.35
5.10
5.54
5.19
6.21
6.34
4.95
6.00
4.89
5.94
6.50
6.69
5.66
6.61
3k
3l
Figure 4. Contour maps of CoMFA electrostatic regions are shown around the
studied compounds. Blue regions are favourable for more positively charged
groups; red regions are favourable for less positively charged groups.
3m
4a
4b
4c
4d
positive action on CFTR from a block of Ca2+ channels; that said,
there is the chance that a single DHP may not cause a significant
Ca2+ channel block, while maintainig a notable degree on CFTR
activation. The CoMFA analysis may help in improving the activity
of these potentiators as, even if the number of the tested com-
pounds is small, it gives some interesting suggestions for further
research. In particular, it must be stressed that the CoMFA indica-
tions about the asymmetry of the molecule and the changes at the
position 4 of the DHP ring join the structure of our set of DHPs to
the compound Vx-770, a potential drug now in pharmacological
trial (phase III) as potentiator for CF.27 Vx-770 is a quinolin-4-
Table 4
Experimental and predicted binding affinities of the test set molecules
Compound
pKd exp
pKd pred
3a
3i
3n
5.08
5.42
6.52
5.92
5.42
6.39

F. Cateni et al. / Bioorg. Med. Chem. 17 (2009) 7894–7903
7903
one derivative substituted at position 3 with a carboxyanilide;28
therefore, Vx-770 has tight structural relation with our asymmetric
DHPs. We believe these observations deserve further development.
Panchenko, V.; Rader, J.; Singh, A.; Stack, J. H.; Tung, R.; Grootenhuis, P. D.;
Negulescu, P. Am. J. Physiol. Lung Cell. Mol. Physiol. 2006, 290, L1117.
13. Wang, X.; Koulov, A. V.; Kellner, W. A.; Riordan, J. R.; Balch, W. E. Traffic 2008, 9,
1878.
14. Pedemonte, N.; Diena, T.; Caci, E.; Nieddu, E.; Mazzei, M.; Ravazzolo, R.;
Zegarra-Moran, O.; Galietta, L. J. V. Mol. Pharmacol. 2005, 68, 1736.
15. Pedemonte, N.; Boido, D.; Moran, O.; Giampieri, M.; Mazzei, M.; Ravazzolo, R.;
Galietta, L. J. V. Mol. Pharmacol. 2007, 72, 197.
Acknowledgements
This work was supported by the Italian Cystic Fibrosis Research
Foundation (Grants FFC#3/2006 and FFC#3/2007) with the contri-
bution of ‘La Bottega delle Donne—Montebelluna’ and ‘Associazi-
one Siciliana per la lotta alla Fibrosi Cistica’.
16. Harrold, M. W. Angiotensin Converting Enzyme Inhibitors, Antagonists and
Calcium Blockers. In Foye0s Principles of Medicinal Chemistry; Williams, D. A.,
Lemke, T. L., Eds., 5th ed.; Lippincott Williams & Wilkins: Philadelphia, 2002;
pp 551–556.
17. Falsone, G.; De Nardo, M. M.; Cateni, F.; Kukowetz, W. R.; Holzmann, S. Pharm.
Pharmacol. Lett. 1997, 7, 21.
18. Prugh, J. D.; Hartman, G. D.; Mallorga, P. J.; McKeever, B. M.; Michelson, S. R.;
Murcko, M. A.; Schwam, H.; Smith, R. L.; Sondey, J. M.; Springer, J. P.; Sugrue, M.
F. J. Med. Chem. 1991, 34, 1805.
19. Galietta, L. J. V.; Haggie, P. M.; Verkman, A. S. FEBS Lett. 2001, 499, 220.
20. Hescheler, J.; Meyer, R.; Plant, S.; Krautwurst, D.; Rosenthal, W.; Schultz, G. Circ.
Res. 1991, 69, 1476.
21. Ménard, C.; Pupier, S.; Mornet, D.; Kitzmann, M.; Nargeot, J.; Lory, P. J. Biol.
Chem. 1999, 274, 29063.
22. SYBYL 7.2, Tripos Inc. 1699 South Hanley Road, St. Louis, Missouri, 63144, USA.
23. Wang, Y.; Loo, T. W.; Bartlett, M. C.; Clarke, D. M. J. Biol. Chem. 2007, 16,
33247.
24. Yu, G. J.; Yoo, C. L.; Lodewyk, M. W.; Meng, L.; El-Idreesy, T. T.; Fettinger, J. C.;
Tantillo, D. J.; Verkman, A. S.; Kurth, M. J. J. Med. Chem. 2008, 51, 6044.
25. Moran, O.; Galietta, L. J. V.; Zegarra-Moran, O. Cell Mol. Life Sci. 2005, 62, 446.
26. Huang, S.-Y.; Bolser, D.; Liu, H.-Y.; Hwang, T.-C.; Zou, X. J. Mol. Graphics Modell.
2009, 27, 822.
References and notes
1. Balfour-Lynn, I. M. J. R. Soc. Med. 1999, 92, 23.
2. Ratjen, F.; Döring, G. Lancet 2003, 361, 681.
3. Cuthbert, A. W. J. R. Soc. Med. 2006, 99, 30.
4. Riordan, J. R. Annu. Rev. Biochem. 2008, 77, 701.
5. Bobadilla, J. L.; Macek, M.; Fine, J. P.; Farell, P. M. Hum. Mutat. 2002, 19, 575.
6. Dalemans, W.; Barbry, P.; Champigny, G.; Jallat, S.; Dott, K.; Dreyer, D.; Crystal,
R. G.; Pavirani, A.; Lecocq, J. P.; Lazdunski, M. Nature 1991, 354, 526.
7. Denning, G. M.; Anderson, M. P.; Amara, J. F.; Marshall, J.; Smith, A. E.; Welsh,
M. J. Nature 1992, 358, 761.
8. Kopito, R. R. Physiol. Rev. 1999, 79, 167.
9. Ma, T.; Vetrivel, L.; Yang, H.; Pedemonte, N.; Zegarra-Moran, O.; Galietta, L. J. V.;
Verkman, A. S. J. Biol. Chem. 2002, 277, 37235.
10. Yang, H.; Shelat, A. A.; Guy, R. K.; Gopinath, V. S.; Ma, T.; Du, K.; Lukacs, G. L.;
Taddei, A.; Folli, C.; Pedemonte, N. J. Biol. Chem. 2003, 278, 35079.
11. Pedemonte, N.; Lukacs, G. L.; Du, K.; Caci, E.; Zegarra-Moran, O.; Galietta, L. J.
V.; Verkman, A. S. J. Clin. Invest. 2005, 115, 2564.
12. Van Goor, F.; Straley, K. S.; Cao, D.; Gonzalez, J.; Hadida, S.; Hazlewood, A.;
Joubran, J.; Knapp, T.; Makings, L. R.; Miller, M.; Neuberger, T.; Olson, E.;
27. www.cff.org/research/DrugDevelopmentPipeline/.
28. Grootenhuis, P. D. J.; Ruah, S. H.; Zhou, J.; Hazlewood, A.; Binch, H. Prodrugs of
modulators of ABC transporters, United States Patent Application
20090105272, April 23, 2009.