2-(Dialkylamino)-4H-1-benzopyran-4-one derivatives modify chloride conductance in CFTR expressing cells

Article abstract of DOI:10.1016/S0014-827X(03)00155-1

Some 2-(diethylamino)-7-hydroxy-4H-1-benzopyran-4-one derivatives, potentially useful as activators of the cystic fibrosis transmembrane conductance regulator (CFTR), were prepared. The synthesized compounds were tested, together with others 2-(dialkylami

Full text of DOI:10.1016/S0014-827X(03)00155-1

Il Farmaco 58 (2003) 961ꢀ970
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2-(Dialkylamino)-4H-1-benzopyran-4-one derivatives modify
chloride conductance in CFTR expressing cells
Mauro Mazzei a,*, Erika Nieddu a, Chiara Folli b, Emanuela Caci b,
Louis V.J. Galietta b
a Dipartimento di Scienze Farmaceutiche,Viale Benedetto XV, 3-16132 Genoa, Italy
b Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini ꢀ
/
Largo Gerolamo Gaslini, 5-16147 Genoa, Italy
Received 11 December 2002; accepted 6 March 2003
Abstract
Some 2-(diethylamino)-7-hydroxy-4H-1-benzopyran-4-one derivatives, potentially useful as activators of the cystic fibrosis
transmembrane conductance regulator (CFTR), were prepared. The synthesized compounds were tested, together with others 2-
(dialkylamino)-7-hydroxybenzopyran-4-one derivatives, by measuring their capacity to modify the kinetics of iodide influx in Fisher
rat thyroid cells expressing wild type CFTR and the halide-sensitive yellow fluorescent protein. Among the tested compounds the
dinitrile derivatives 8 and 9 are endowed with an activity comparable to the reference compound apigenin.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Cystic fibrosis; CFTR; Benzopyran-4-one derivatives
1. Introduction
(CPX, 1) and flavone/isoflavone derivatives (genistein,
2, and apigenin, 3) (Fig. 1).
Cystic fibrosis (CF) is a genetic disease due to an
alteration of a protein named cystic fibrosis transmem-
brane conductance regulator (CFTR) which acts as a
cAMP-regulated Clꢁ channel [1]. This disorder affects
A recent screening of a lead-based chemical library
has identified 7,8-benzoflavones (UCCF-029, 4) as
potent activators of CFTR Clꢁ conductance [4].
Due to our interest in benzopyran-4-one derivatives,
and made curious by the intriguing occurrence of
benzopyran moiety in molecules active on CFTR, we
desired to verify the ability of 2-(diethylamino)-7-
hydroxychromone and its derivatives to modulate the
CFTR-dependent anion transport.
one in 2000ꢀ3000 new-borns and is due to severe
/
impairment of electrolyte and fluid secretion in lung,
pancreas and other organs. Progressive loss of lung
function by bacterial colonization and consequent
inflammation is a major cause of death in CF patients.
Up to date, more than 800 mutations of the gene have
been found. The most common being the deletion of
phenylalanine 508 (DF-508).
Other hydroxybenzopyrans and substituted naphtho-
pyrans will be the object of coming investigations in this
field.
The search for substances potentially useful in the
treatment of the primary defect in CF is still in its
infancy. Nevertheless, some classes of organic com-
pounds have been found that activate native and mutant
CFTR [2,3]. Among the most interesting modifiers of
CFTR channel activity, we must cite purine derivatives
2. Chemistry
In order to amplify the bulk of 2-(dialkylamino)ben-
zopyran-4-ones to test as CFTR modifiers, we selected
the 2-(diethylamino)-7-hydroxychromone 5d as lead
compound to produce and test a reasonable number of
derivatives.
7-Hydroxychromone 5d was treated with N,N-diethy-
laminoethyl chloride in anhydrous potassium carbonate
* Corresponding author.
E-mail address: mazzei@cba.unige.it (M. Mazzei).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00155-1

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M. Mazzei et al. / Il Farmaco 58 (2003) 961ꢀ970
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Fig. 1. Structures of CFTR ion channel modifiers: (1) CPX, (2) Genistein, (3) Apigenin, (4) UCCF-029.
and N,N-dimethylformamide (DMF) to obtain the
N,N-diethylaminoethyl derivative 6. Then, compound
5d was allowed to react with phosphorus pentasulfide in
refluxing pyridine to yield the 4-thiochromone deriva-
tive 7; finally, this derivative was condensed with
malononitrile in the presence of acetic anhydride at
110 8C to give the 4H-chromene derivative 8. The
hydrolysis of this latter in alkaline medium afforded
the corresponding 7-hydroxy derivative 9 (see Scheme
1).
normally, we obtained 5d by demethylation of the
corresponding 7-methoxyderivative [5]. Therefore, re-
sorcinol was treated with an equimolar amount of
bromoalkyl derivative in the presence of anhydrous
potassium carbonate and DMF to yield 3-alkyloxyphe-
nol (10). In turn, 2-(diethylamino) chromones 11 were
prepared by a cyclocondensation reaction between 10
and 3-(diethylamino)-3-oxo-propanoic acid ethyl ester
in the presence of phosphorus oxychloride using 1,2-
dichloroethane (DCE) as solvent, as previously reported
for similar compounds [6,7] (see Scheme 2).
In order to obtain more hydrophobic chains in the
position 7 of 1H-benzopyran-4-ones, instead to alkylate
the 7-hydroxychromone 5d, we preferred to synthesize
Compounds 11 were allowed to react with phospho-
rus pentasulfide in refluxing pyridine to yield the 4-
thiochromone derivatives 12; then these derivatives were
condensed with malononitrile in the presence of acetic
suitable starting phenols 10aꢀ/d. This pathway permits
to avoid the use of the expensive hydriodic acid as,
Scheme 1.

M. Mazzei et al. / Il Farmaco 58 (2003) 961ꢀ
/970
963
Scheme 2.
anhydride at 110 8C to give the 4H-chromene derivatives
13 (see Scheme 2).
fact, the unsymmetrical methylene derivatives sponta-
neously loose the diethylamine yielding the polycyclic
derivatives 17 (see Scheme 4). This behavior was already
observed when, instead of the a-pyrone derivative, 4-
hydroxycoumarin derivatives were used. However, in
the cited case, the intermediate was easily recovered and
the next cyclization occurred under strong conditions
(using boiling acetic acid) [10].
Two 1H-benzopyran-4-ones (5d, 11d) were treated in
acetic anhydride with the Mannich base 14. As pre-
viously reported such reaction affords in good yield the
unsymmetrical methylene derivatives 15 [8,9] (see
Scheme 3).
When two 1H-benzopyran-4-ones (5e, 11b) were
treated with the 3-piperidinomethyl-4-hydroxy-6-
methylpyran-2-one (16) the reaction pattern was mod-
ified in respect to the previously reported Scheme 3. In
Compounds 6, 11, 15, 16 and 17 are white crystals;
compounds 7 and 12 are yellow crystals; compounds 8, 9
and 13 are yellowꢀorange crystals. The structures of all
/
Scheme 3.

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M. Mazzei et al. / Il Farmaco 58 (2003) 961ꢀ970
/
Scheme 4.
synthesized compounds are in agreement with elemental
analyses and spectral data (see Section 3).
CH2), 4.16 (t, 2H, OCH2), 5.40 (s, 1H, H-3), 6.66ꢀ7.09
(m, 2H, H-6, 8), 8.10 (d, 1H, H-5). Anal. C19H28N2O3:
C, H, N.
/
3. Experimental protocols
3.2. 2-(Diethylamino)-7-hydroxy-4H-1-benzopyran-4-
thione (7)
Melting points were determined using a FisherꢀJohns
/
apparatus and are uncorrected. Microanalyses were
carried out on a Carlo Erba 1106 elemental analyzer.
To 1.0 g (4.3 mmol) of 2-(diethylamino)-7-hydroxy-
4H-1-benzopyran-4-one (5d) dissolved in 7 ml of pyri-
dine, 0.8 g of phosphorus pentasulfide were added and
the resulting mixture was refluxed for 1 h. The final
solution was poured onto crushed ice and the brown
solid, which separated out, was stirred for 0.5 h and
filtered. The solid was then crystallized from ethanol
The results of elemental analysis were within 9
/
0.3% of
the theoretical value. H NMR spectra were performed
on a Hitachi PerkinꢀElmer R 600 (60 MHz) spectro-
meter using TMS as internal standard (dꢂ0). IR
spectra were recorded on a PerkinꢀElmer 398 spectro-
photometer.
1
/
/
/
yielding the thiochromone 7. M.p. 131ꢀ132 8C; 91.3%
/
yield. IR (KBr) n (cmꢁ1): 1475, 1557, 1593, 1611, 2951,
1
2974. H NMR (d, CDCl3): 1.39 (t, 6H, CH3), 3.66 (q,
3.1. 2-(Diethylamino)-7-(2-diethylamino)ethyloxy-4H-
1-benzopyran-4-one (6)
4H, Nꢀ
/
CH2), 6.79 (s, 1H, H-3), 7.31ꢀ7.88 (m, 2H, H-6,
/
8), 8.80 (d, 1H, H-5). Anal. C13H15NO2S: C, H, N.
To 1.0 g (4.3 mmol) of 2-(diethylamino)-7-hydroxy-
4H-1-benzopyran-4-one (5d) dissolved in 15 ml of
DMF, 1.25 g of anhydrous potassium carbonate and
0.78 g (4.5 mmol) of diethylaminoethyl chloride were
added and the resulting mixture was heated at 120 8C
for 6 h. The final solution was poured onto crushed ice
and the solid, which separated out, was extracted three
times with chloroform. The pooled organic extracts were
treated with anhydrous sodium sulfate and filtered. The
solid was then crystallized from acetone/light petroleum
3.3. [2-(Diethylamino)-7-acetoxy-4H-chromen-4-
ylidene]malononitrile (8)
A mixture of 1.0 g (4.0 mmol) of thiochromone 7, 0.55
g (8.0 mmol) of malononitrile and 8 ml of freshly
distilled acetic anhydride was heated at 110ꢀ115 8C for
/
1 h cooled and poured onto crushed ice. After stirring
for a 30 min the brown solid which separated out was
collected, washed with water and recrystallized from
ether yielding the chromone 6. M.p. 75ꢀ
yield. IR (KBr) n (cmꢁ1): 1610, 1625, 2881, 2940, 2976.
1H NMR (d, CDCl3): 0.83ꢀ
1.54 (m, 12H, CH3), 2.40ꢀ
3.10 (m, 6H, OCH2CH2N(CH2)2) 3.48 (q, 4H, 2-Nꢀ
/
76 8C; 30.3%
ethanol yielding the 4H-chromene 8. M.p. 136ꢀ137 8C;
/
44.8% yield. IR (KBr) n (cmꢁ1): 1606, 1625, 1767, 2177,
1
/
/
2198, 2937, 2984, 3073. H NMR (CDCl3): 1.33 (t, 6H,
/
Nꢀ
/
CH2CH3), 2.34 (s, 3H, CH3CO), 3.60 (q, 4H, Nꢀ
/

M. Mazzei et al. / Il Farmaco 58 (2003) 961ꢀ
/970
965
CH2CH3), 5.91 (s, 1H, H-3), 6.93ꢀ/7.40 (m, 2H, H-6, 8),
70 8C. After cooling, the organic phase was discarded
8.92 (d, 1H, H-5). Anal. C18H17N3O3: C, H, N.
and the aqueous one was extracted three times with
chloroform. The pooled organic extracts were washed
with water, dried and evaporated under reduced pres-
sure giving dark red oil. The oil was stirred at room
temperature for 2 h with 200 ml of 2 N NaOH and 50 ml
of light petroleum ether. The obtained solid was filtered
off and washed with water. The crude product was
crystallized from ethyl acetate obtaining 11.
3.4. [2-(Diethylamino)-7-hydroxy-4H-chromen-4-
ylidene]malononitrile (9)
A mixture of 0.56 g (2.0 mmol) of 4H-chromene 8 and
2.0 mmol of NaOH in 10 ml of water was heated at
60 8C for 4 h. At the end, the solution was acidified with
6M HCl. The resulting precipitate was filtered off and
crystallized from ethyl acetate yielding the 7-hydroxy
The following products were obtained:
3.6.1. 2-(Diethylamino)-7-n-pentyloxy-4H-1-
benzopyran-4-one (11a)
derivative 9. M.p. 289ꢀ
(cmꢁ1): 1606, 2183, 2201, 2924, 2950, 3188. H NMR
(d, DMSO): 1.21 (t, 6H, CH3), 3.68 (q, 4H, NꢀCH2),
5.68 (s, 1H, H-3), 6.84ꢀ7.04 (m, 2H, H-6, 8), 8.54 (d,
/
290 8C; 32.6% yield. IR (KBr) n
1
M.p. 92ꢀ
1617, 2870, 2936, 2954, 2966. 1H NMR (d, CDCl3):
0.95ꢀ1.61 (m, 15H, NꢀCH2CH3, (CH2)3CH3), 3.47 (q,
4H, NꢀCH2), 4.04 (t, 2H, OCH2), 5.37 (s, 1H, H-3)
6.63ꢀ7.10 (m, 2H, H arom), 8.10 (d, 1H, H arom). Anal.
/
93 8C; 16% yield. IR (KBr) n (cmꢁ1): 1598,
/
/
/
/
1H, H-5). Anal. C15H15N3O2: C, H, N.
/
/
3.5. 3-Alkyloxyphenol (10)
C18H25NO3: C, H, N.
To 4.0 g (0.036 mol) of resorcinol dissolved in 10 ml
of DMF, 8.0 g of anhydrous potassium carbonate and
0.036 mol of suitable bromoalkyl derivative were added.
3.6.2. 2-(Diethylamino)-7-n-octyloxy-4H-1-benzopyran-
4-one (11b)
M.p. 87ꢀ
/
88 8C; 18% yield. IR (KBr) n (cmꢁ1):1601,
The mixture was heated at 110ꢀ115 8C for 5 h under a
/
1
1620, 2863, 2942, 2955, 2972. H NMR (CDCl3): 0.80ꢀ
/
stream of nitrogen. The final mixture was cooled,
poured onto crushed ice (adjusting the pH to 7) and
stirred for 30 min. The aqueous solution was extracted
several times with chloroform. The pooled organic
extracts were counter extracted with 2N NaOH. The
alkaline solution was acidified with 6M HCl and the oil
obtained was distilled in vacuo. The following products
were obtained:
1.92 (m, 21H, Nꢀ
/
CH2CH3, (CH2)6CH3), 3.48 (q, 4H,
NꢀCH2), 4.04 (t, 2H, OCH2), 5.39 (s, 1H, H-3), 6.72ꢀ
/
/
7.04 (m, 2H, H-6, 8), 8.10 (d, 1H, H-5). Anal.
C21H31NO3: C, H, N.
3.6.3. 2-(Diethylamino)-7-n-dodecyloxy-4H-1-
benzopyran-4-one (11c)
M.p. 63ꢀ
1540, 1603, 1625, 2851, 2916. 1H NMR (d, CDCl3):
0.74ꢀ1.60 (m, 29H, NꢀCH2CH3, (CH2)10CH3), 3.46 (q,
4H, NꢀCH2), 3.98 (t, 2H, OCH2), 5.40 (s, 1H, H-3),
6.44ꢀ7.03 (m, 2H, H-6, 8), 8.12 (d, 1H, H-5). Anal.
/
64 8C; 15% yield. IR (KBr) n (cmꢁ1): 1472,
3-n-Pentyloxyphenol (10a) (b.p. 140 8C/5 mm Hg)
[11]
3-n Octyloxyphenol (10b) (b.p. 170 8C/5 mm Hg) [11]
3-n-Dodecyloxyphenol (10c) (b.p. 180 8C/3 mm Hg)
[12]
/
/
/
/
C25H39NO3: C, H, N.
3-n-Hexadecyloxyphenol (10d) (b.p. 210 8C/3 mm
Hg) [12]
3.6.4. 2-(Diethylamino)-7-n-hexadecyloxy-4H-1-
benzopyran-4-one (11d)
3.6. 2-(Diethylamino)-7-alkyloxy-4H-1-benzopyran-4-
one (11)
M.p. 82ꢀ
1540, 1605, 1630, 2851, 2916. 1H NMR (d, CDCl3):
0.80ꢀ1.75 (m, 37H, NꢀCH2CH3, (CH2)14CH3), 3.45 (q,
4H, NꢀCH2), 4.07 (t, 2H, OCH2), 5.40 (s, 1H, H-3),
6.35ꢀ6.90 (m, 2H, H-6, 8), 8.05 (d, 1H, H-5). Anal.
/
83 8C; 15% yield. IR (KBr) n (cmꢁ1): 1473,
/
/
In an ice cooled flask, protected from moisture with a
calcium chloride drying tube, 7.0 ml (78.0 mmol) of
phosphorus oxychloride were added drop wise with
stirring to 10.30 g (55.0 mmol) of 3-(diethylamino)-3-
oxo-propanoic acid ethyl ester [5]. After the addition,
the mixture was removed from the ice bath and
maintained at room temperature for 0.5 h. To the
resulting yellow mixture, a solution of 9.56 g (50.0
mmol) of 3-alkyloxyphenol (10) in 40 ml of DCE was
slowly added with stirring. The reaction mixture was
then heated for 5 h at reflux. After cooling, a solution of
68 g of sodium acetate trihydrate in 200 ml of water was
added and the mixture was then heated for 1.5 h at
/
/
C29H47NO3: C, H, N.
3.7. 2-(Diethylamino)-7-n-alkyloxy-4H-1-benzopyran-
4-thiones (12)
To 3.3 mmol of 2-(diethylamino)-7-alkyloxy-4H-1-
benzopyran-4-one 11 dissolved in 7 ml of pyridine, 0.8 g
of phosphorus pentasulfide were added and the resulting
mixture was refluxed for 1 h. The final solution was
poured onto crushed ice and the brown solid, which
separated out, was stirred for 0.5 h and filtered. The

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M. Mazzei et al. / Il Farmaco 58 (2003) 961ꢀ970
/
solid was then crystallized from ethanol yielding yellow
crystals.
The following products were obtained:
H-3), 6.67ꢀ6.90 (m, 2H, H-6, 8), 8.78 (d, 1H, H-5).
Anal. C21H25N3O2: C, H, N.
/
3.8.2. [2-(Diethylamino)-7-n-octyloxy-4H-chromen-4-
ylidene]malononitrile (13b)
3.7.1. 2-(Diethylamino)-7-n-pentyloxy-4H-1-
benzopyran-4-thione (12a)
M.p. 92ꢀ
1593, 1627, 2182, 2197, 2852, 2923. 1H NMR (d,
CDCl3): 0.85ꢀ1.70 (m, 21H, NꢀCH2CH3,
(CH2)6CH3), 3.58 (q, 4H, NꢀCH2), 4.09 (t, 2H,
OCH2), 5.80 (s, 1H, H-3), 6.73ꢀ7.00 (m, 2H, H-6, 8),
/
93 8C; 45.2% yield. IR (KBr) n (cmꢁ1):
M.p. 135ꢀ
1622, 2857, 2938, 2955, 2969 1H NMR (d, CDCl3):
0.95ꢀ1.61 (m, 15H, NꢀCH2CH3, (CH2)3CH3), 3.54 (q,
4H, NꢀCH2), 4.04 (t, 2H, OCH2), 6.63ꢀ7.10 (m, 3H, H-
/
136 8C; 73.5% yield. IR (KBr) n (cmꢁ1):
/
/
/
/
/
/
/
/
3, 6, 8), 8.72 (d, 1H, H-5). Anal. C18H25NO2S: C, H, N.
8.78 (d, 1H, H-5). Anal. C24H31N3O2: C, H, N.
3.7.2. 2-(Diethylamino)-7-n-octyloxy-4H-1-benzopyran-
4-thione (12b)
3.9. 8-[2?-(Diethylamino)-7?-alkyloxychromon-3?-
yl]methyl-7-acetoxy-4-methylcoumarin (15)
M.p. 144ꢀ
/
145 8C; 71.2% yield. IR (KBr) n (cmꢁ1):
1479,1557,1594,1620, 2851, 2921, 2939, 2980. H NMR
1
In a 100-ml flask, protected from moisture with a
calcium chloride tube, 1.0 mmol of 2-(dialkylamino)-
benzopyran-4-one (5d or 11d) was dissolved in 5 ml of
freshly distilled acetic anhydride. Then 1.0 mmol of
Mannich base 14 [8] was added, and the mixture was
heated at 95 8C for 1.5 h, with stirring. At the end, the
cooled solution was poured onto crushed ice and water,
and stirred for 2 h obtaining a solid residue. The solid
was collected by filtration, washed with water and
crystallized from ethyl acetate.
(d, CDCl3): 0.92ꢀ
(CH2)6CH3), 3.54 (q, 4H, Nꢀ
OCH2), 6.70ꢀ7.12 (m, 3H, H-3, 6, 8), 8.65 (d, 1H, H-
/
1.87 (m, 21H, Nꢀ
/
CH2CH3,
/
CH2), 4.06 (t, 2H,
/
5). Anal. C21H31NO2S: C, H, N.
3.7.3. 2-(Diethylamino)-7-n-dodecyloxy-4H-1-
benzopyran-4-thione (12c)
M.p. 142ꢀ
1620, 2848, 2921, 2980. 1H NMR (d, CF3COOD): 0.93ꢀ
1.68 (m, 29H, NꢀCH2CH3, (CH2)10CH3), 3.79 (q, 4H,
NꢀCH2), 4.24 (t, 2H, OCH2), 6.82ꢀ7.48 (m, 3H, H-3, 6,
/
143 8C; 67.9% yield. IR (KBr) n (cmꢁ1):
/
/
The following products were obtained:
/
/
8), 7.83 (d, 1H, H-5). Anal. C25H39NO2S: C, H, N.
3.9.1. 8-[2?-(Diethylamino)-7?-acetoxychromon-3?-
yl]methyl-7-acetoxy-4-methylcoumarin (15a)
3.7.4. 2-(Diethylamino)-7-n-hexadecyloxy-4H-1-
benzopyran-4-thione (12d)
M.p. 209ꢀ
1596, 1613, 1725, 1760, 2933, 2972. 1H NMR (d,
CDCl3): 0.94 (t, 6H, NꢀCH2CH3), 2.10 (s, 3H, 7-
OCOCH3), 2.26 (s, 3H, 7?-OCOCH3), 2.40 (s, 3H, 4-
CH3), 3.30 (q, 4H, NꢀCH2CH3), 4.15 (s, 2H, CH2
bridge), 6.27 (s, 1H, H-3), 6.86ꢀ7.56 (m, 4H, H-5, 6, 6?,
/
210 8C; 74.5% yield. IR (KBr) n (cmꢁ1):
M.p. 134ꢀ
1479, 1555, 1593, 1620, 2848, 2922. 1H NMR (d,
CF3COOD): 0.97ꢀ1.76 (m, 37H, NꢀCH2CH3,
(CH2)14CH3), 3.75ꢀ4.50 (m, 6H, NꢀCH2, OCH2),
6.80ꢀ7.40 (m, 3H, H-3, 6, 8), 8.00 (d, 1H, H-5). Anal.
/
135 8C; 46.3% yield. IR (KBr) n (cmꢁ1):
/
/
/
/
/
/
/
/
8?), 8.21 (d, 1H, H-5?). Anal. C28H27NO8: C, H, N.
C29H47NO2S: C, H, N.
3.9.2. 8-[2?-(Diethylamino)-7?-n-hexadecylchromon-3?-
yl]methyl-7-acetoxy-4-methylcoumarin (15b)
3.8. [2-(Diethylamino)-7-alkyloxy-4H-chromen-4-
ylidene]malononitriles (13)
M.p. 120ꢀ
1537, 1582, 1603, 1733, 1770, 2849, 2920, 2956. 1H
NMR (d, CDCl3): 0.94ꢀ1.62 (m, 37H, NꢀCH2CH3,
(CH2)14CH3), 2.08 (s, 3H, CH3CO), 2.41 (s, 3H, 4-CH3),
3.23 (q, 4H, NꢀCH2), 4.05 (t, 2H, OCH2), 4.20 (s, 2H,
CH2 bridge), 6.29 (s, 1H, H-3), 6.70ꢀ7.50 (m, 4H, H-5,
/
121 8C; 23.6% yield. IR (KBr) n (cmꢁ1):
A mixture of 4.0 mmol of thiochromones 12, 0.55 g
/
/
(8.0 mmol) of malononitrile and 8 ml of freshly distilled
115 8C for 1 h,
acetic anhydride was heated at 110ꢀ
/
/
cooled and poured onto crushed ice. After stirring for a
30 min the brown solid which separated out was
collected, washed with water and recrystallized from
ethanol yielding orange crystals.
/
6, 6?, 8?), 8.12 (d, 1H, H-5?). Anal. C42H57NO7: C, H, N.
3.10. 4-Hydroxy-6-methyl-3-piperidinomethyl-2-pyrone
(16)
The following products were obtained:
3.8.1. [2-(Diethylamino)-7-n-pentyloxy-4H-chromen-4-
ylidene]malononitrile (13a)
To 20.0 mmol of 4-hydroxy-6-methyl-2-pyrone dis-
solved in 50 ml of ethanol, 20.0 mmol of piperidine and
2.0 ml of 40% formaldehyde were added. The resulting
mixture was refluxed for 6 h. After cooling, the solvent
was evaporated under reduced pressure. The pale yellow
oil obtained was treated with cool acetone, leaving a
M.p. 141ꢀ
/
142 8C; 33.8% yield. IR (KBr) n (cmꢁ1):
1597, 1628, 2179, 2196, 2875, 2944, 2962. H NMR (d,
1
CDCl3): 0.88ꢀ
3.56 (q, 4H, Nꢀ
/
1.65 (m, 15H, Nꢀ
/
CH2CH3, (CH2)3CH3),
CH2), 4.07 (t, 2H, OCH2), 5.80 (s, 1H,
/

M. Mazzei et al. / Il Farmaco 58 (2003) 961ꢀ
/970
967
white solid which was crystallized from ethanol obtain-
ing the Mannich base 16, m.p. 165ꢀ166 8C, 74.8% yield.
IR (KBr) n (cmꢁ1): 1525, 1677, 1705, 2940, 2992. H
NMR (d, CF3COOD): 1.60ꢀ1.21 (m, 6H, bꢃg-piper-
idine CH2), 2.39 (s, 3H, CH3), 3.42ꢀ3.88 (m, 4H, a-
resting conditions, 12 s after addition of 165 ml of a
modified PBS solution in which NaCl was replaced with
NaI. Data were sampled at 5 Hz and stored on
computer disk. CFTR activity was estimated by sub-
tracting the background, normalizing the data for the
initial fluorescence in PBS, and fitting the fluorescence
curve after Iꢁ addition with a single exponential
function. Maximal slope, which is proportional to
d[Iꢁ]/dt, was then calculated using the Igor software
/
1
/
/
/
piperidine CH2), 4.27 (s, 2H, CH2 bridge), 6.46 (s, 1H,
H-5). Anal. C12H17NO3: C, H, N.
3.11. 1,11,12 H-3-methyl-8-alkyloxy-2,5,6-
trioxanaphthacen-1,11-dione (17)
With the same procedure used for the synthesis of
compounds 15, starting from 5e and the Mannich base
16 and after crystallization from acetone, the following
product was obtained:
3.11.1. 1,11,12H-3-methyl-8-methoxy-2,5,6-
trioxanaphthacen-1,11-dione (17a)
M.p. 180ꢀ
/
181 8C; 32.5% yield. IR (KBr) n (cmꢁ1):
1617, 1637, 1670, 1725, 2845, 2943, 3076. H NMR (d,
CF3COOD): 2.48 (s, 3H, CH3), 3.52 (s, 2H, CH2
1
bridge), 4.03 (s, 3H, OCH3), 6.50 (s, 1H, H-4), 7.00ꢀ
/
7.35 (m, 2H, H-7, 9) 7.96 (d, 1H, H-10). Anal.
C17H12O6: C, H, N.
With the same procedure used for the synthesis of
compounds 15, starting from 11b and the Mannich base
16 and after crystallization from ethanol, the following
product was obtained:
3.11.2. 1,11,12H-3-methyl- 7-n-octyloxy-2,5,6-
trioxanaphthacen-1,11-dione (17b)
M.p. 254ꢀ
/
255 8C; 24.2% yield. IR (KBr) n (cmꢁ1):
1570, 1640, 1670, 1705, 2853, 2920, 3430. H NMR (d,
1
CDCl3): 0.89 (t, 3H, CH3), 1.10ꢀ/1.95 (m, 15H, CH2),
3.82ꢀ4.20 (m, 4H, OCH2 and CH2 bridge), 6.42ꢀ
/
/
6.68
(m, 3H, H arom), 7.13 (d, 1H, H arom). Anal.
C24H26O6: C, H, N.
4. CFTR assays
CFTR activity was assessed by using the fluorescent
method described previously [4,13]. Briefly, cells coex-
pressing wild type CFTR and the halide-sensitive yellow
fluorescent protein YFP-H148Q were plated in 96-well
microplates. After 48 h, cells were washed and incubated
in 50 ml PBS with or without test compounds or apigenin
at 10 mM. After 15 min, cell fluorescence was measured
in a Galaxy plate reader (BMG) equipped with high
Fig. 2. CFTR activation by 2-(dialkylamino)-7-hydroxy-4H-1-benzo-
pyran-4-one derivatives. (A) Representative traces showing the time
course of cell fluorescence measured before and after addition of I-
solution in plate reader measurements. Cells were incubated with
physiological saline solution alone or with 10 mM of apigenin or
quality filters for the yellow fluorescent protein (5009
/
10
nm for excitation; 535915 nm for emission) and two
/
dinitrile derivatives 8 and 9. (B) Doseꢀresponse relationships for active
/
compounds. Each point (mean of three experiments) reports the Iꢁ
influx as a function of drug concentration. Data are fitted with Hill
equation. (C) Representative short-circuit current experiment showing
CFTR activation by compound 8 added at the indicated concentra-
tions in the apical chamber. Forskolin (fsk) and glibenclamide (glib)
were added at 0.5 and 400 mM, respectively.
syringe pumps for liquid addition. The assay for each
well consisted in: (1) 22 s before fluorescence reading:
addition of 10 ml of PBS containing forskolin to give a
final submaximal concentration of 0.5 mM; (2) contin-
uous measurement of cell fluorescence for 14 s: 2 s in

968
M. Mazzei et al. / Il Farmaco 58 (2003) 961ꢀ970
/
Table 1
a
Compound
NR2
R?
Rƒ
Rƒ?
Activity
Reference
5a
5b
5c
5d
5e
5f
NMe2
NHEt
OH
H
H
T
I
[5]
OCH3
OH
OH
H
H
[14]
[15]
[5]
NHEt
NEt2
H
H
I
H
H
I
NEt2
NEt2
OCH3
CH3
H
H
I
[5]
H
OH
H
I
[15]
[16]
[17]
[5]
5g
5h
5I
5j
NEt2
NEt2
OCH2CH3
O(CH2)2CH3
OCH3
H
CHO
H
T
I
H
Pyrrolidinyl
Pyrrolidinyl
Piperidinyl
Piperidinyl
Piperidinyl
N(CH2CH2OCH3)2
NEt2
H
H
I
H
OH
H
T
I
[18]
[5]
5k
5l
OCH3
OH
H
H
H
I
[5]
5m
5n
5o
5p
H
H
OH
H
I
[18]
[17]
[16]
[19]
OCH3
OCH2CH3
O(CH2)4CN
H
b
I
H
T
T
NEt2
H
H
T, toxic; I, inactive.
a
In respect to apigenin (100%).
Morpholinomethyl.
b
(WaveMetrics, Inc.). Doseꢀ
active compounds and apigenin using different concen-
trations in the 1ꢀ50 mM range. Active compounds were
/responses were assessed for
/
confirmed by measuring the short-circuit current across
confluent monolayers of FRT cells expressing CFTR
plated on Snapwell permeable supports (Corning
Costar) as previously described [13].
Table 2
5. Results and discussion
The benzopyrans utilized in this pharmacological
a
screening are shown in Tables 1ꢀ
compounds previously synthesized (5aꢀ
synthesized benzopyran derivatives are instead depicted
in Tables 2 and 3 (6ꢀ9, 11ꢀ13, 15, 17).
/
3. Table 1 shows
Compound
X
R
Activity
/
q). Newly
6
O
CH2CH2NEt2
H
CH3CO
I
7
S
C(CN)2
I
A (79%)
/
/
8
Activity of compounds was determined in the plate
reader after prestimulation of CFTR with a low con-
centration of forskolin. Under these conditions, the
reference compound apigenin (10 mM) caused a sig-
nificant increase in CFTR activity as expected (Fig. 2A).
Most of compounds were inactive (the slope was
indistinguishable from that of cells incubated with PBS
alone) or toxic (as evident from the detachment of cells
from the well bottom during the assay upon addition of
solutions). However, compounds 8 and 9 elicited a
CFTR activity which was 79 and 102%, respectively,
of that shown by apigenin at the same concentration
9
C(CN)2
O
H
A (102%)
I
11a
11b
11c
11d
12a
12b
12c
12d
13a
13b
(CH2)4CH3
(CH2)7CH3
(CH2)11CH3
(CH2)15CH3
(CH2)4CH3
(CH2)7CH3
(CH2)11CH3
(CH2)15CH3
(CH2)4CH3
(CH2)7CH3
O
T
O
NS
NS
NS
NS
NS
NS
I
O
S
S
S
S
C(CN)2
C(CN)2
I
A, active; T, toxic; I, inactive; NS, insoluble.
a
In respect to apigenin (100%).

M. Mazzei et al. / Il Farmaco 58 (2003) 961ꢀ
/970
969
Table 3
Compound
R
Activitya
Comp
R
Activity
15a
15b
CH3CO
CH3(CH2)15
NS
NS
17a
17b
CH3
(CH2)7CH3
NS
I
a
In respect to apigenin (100%). I, inactive; NS, insoluble.
(Fig. 2A). Doseꢀ
/
responses were performed in triplicate
confirmed from the fact that the 7-hydroxychromones
5a, 5c, 5d, 5l and 7-hydroxythiochromone 7 are inactive.
Next research will be focused to prove the action of
dinitrile group on dihydroxychromones and naphoth-
pyrans.
In conclusion, we have identified a new class of CFTR
openers. The enlargement of spectrum of CFTR active
compounds is of extreme importance to understand the
molecular mechanisms underlying channel regulation
and gating. Comparison between various CFTR activa-
tors could lead to the identification of consensus
structures that could in turn provide information about
binding site(s). This will be helpful for synthesis of new
effective drugs for the pharmacological correction of CF
basic defect.
and resulting data were fitted with a Hill equation to
obtain information on maximal effect and affinity for
each active compound. Apigenin caused an estimated
maximal Iꢁ influx of 0.75 mM/s with a half effective
concentration of 12.6 mM. Compounds 8 and 9 elicited a
maximal effect smaller than apigenin (around 0.5 mM/s)
whereas the half effective concentration was comparable
or lower (10.2 and 7.9 mM, respectively; Fig. 2B).
The ability of new compounds to induce electrogenic
activity of CFTR was confirmed in using chamber
experiments [13]. CFTR was partially stimulated with
forskolin 0.5 mM, as in the plate reader measurements,
and subsequently test compounds or apigenin were
added at increasing concentrations. Compounds 8 and
9, as well as apigenin, elicited a dose-dependent increase
of short-circuit current which was blocked by the CFTR
inhibitor glibenclamide (Fig. 2C).
Acknowledgements
Though previous studies demonstrated the biological
relevance of 2-(dialkylamino)chromones [5ꢀ
/
8,14ꢀ18],
/
This work was supported by grants from Italian
M.I.U.R. and Telethon (Project GP0296Y01).
compounds 5 are, in general, devoid of activity on
CFTR. The chromones and thiochromones 11 and 12
bearing a long alkyloxy chain are insoluble and, there-
fore, we cannot express a clear conclusion on their value
in this context. The same negative circumstance occurs
for more complex derivatives as 15 and 17. On the other
hand, dinitrile derivatives 8 and 9 seem interesting
compounds in activating CFTR conductance. Their
activity is comparable to that of apigenin and our
interest in these new compounds lies in the presence of
the dinitrile group. In fact, as long as we can see, this
group is not present in substances endowed with this
type of activity. It is significant to note that the presence
of a long alkyloxy group in position 7 of the benzopyran
moiety, as in compounds 13, causes loss of activity even
though the dinitrile substituent is present. As it is likely
that the acetyl derivative 8 could be easily hydrolyzed in
vivo, the presence of the hydroxy group in position 7,
when in the molecule the dinitrile substituent is present,
seems to have great importance. This consideration is
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