Phenylhydrazones as Correctors of a Mutant Cystic Fibrosis Transmembrane Conductance Regulator

Article abstract of DOI:10.1002/ardp.201500352

The phenylhydrazone RDR-1 is endowed with moderate activity as F508del-CFTR corrector; nevertheless, its simple structure enables stimulating developments in this class of correctors. Therefore, we synthesized a number of phenylhydrazones 3 by reacting phenylhydrazine derivatives 1 with furfural derivatives 2. By the same reaction, also the pyridine derivatives 4, the thiophene derivatives 5, and the hydrazides 6 and 7 were prepared. All compounds were tested as F508del-CFTR correctors in the cystic fibrosis (CF) bronchial epithelial cell line CFBE41o-, using corr-4a and VX-809 as controls. Some of the tested compounds emerged as interesting F508del-CFTR correctors at 20 μM (3c) and 2 μM (5d). 3c and 5d administered together with VX-809 produced a satisfactory additivity of action. When the structure of 5d was overlapped with RDR-1 and five other established correctors, a shared central design was clearly visible. This fact may be of interest in the search for new F508del-CFTR correctors. Starting from RDR-1, a known F508del-CFTR corrector, some phenylhydrazones were synthesized. Two new compounds (3c and 5d) showed encouraging activity and, when administered together with the corrector VX-809, gave an additivity of action. Overlapping the molecular design of 5d with RDR-1 and other known correctors revealed a common structural moiety, which may help in the search for new drugs against cystic fibrosis.

Full text of DOI:10.1002/ardp.201500352

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Full Paper
Phenylhydrazones as Correctors of a Mutant Cystic Fibrosis
Transmembrane Conductance Regulator
Erika Nieddu1, Benedetta Pollarolo1, Marco T. Mazzei1, Maria Anzaldi1, Silvia Schenone1,
Nicoletta Pedemonte2, Luis J. V. Galietta2, and Mauro Mazzei1
1
Department of Pharmacy, University of Genova, Genova, Italy
Laboratory of Molecular Genetics, Giannina Gaslini Institute, Genova, Italy
2
The phenylhydrazone RDR-1 is endowed with moderate activity as F508del-CFTR corrector; neverthe-
less, its simple structure enables stimulating developments in this class of correctors. Therefore, we
synthesized a number of phenylhydrazones 3 by reacting phenylhydrazine derivatives 1 with furfural
derivatives 2. By the same reaction, also the pyridine derivatives 4, the thiophene derivatives 5, and
the hydrazides 6 and 7 were prepared. All compounds were tested as F508del-CFTR correctors in the
cystic fibrosis (CF) bronchial epithelial cell line CFBE41o-, using corr-4a and VX-809 as controls. Some
of the tested compounds emerged as interesting F508del-CFTR correctors at 20 mM (3c) and 2 mM
(5d). 3c and 5d administered together with VX-809 produced a satisfactory additivity of action. When
the structure of 5d was overlapped with RDR-1 and five other established correctors, a shared central
design was clearly visible. This fact may be of interest in the search for new F508del-CFTR correctors.
Keywords: CFTR / Correctors / Cystic fibrosis / Ion channels / Phenylhydrazones
Received: September 25, 2015; Revised: November 23, 2015; Accepted: November 26, 2015
DOI 10.1002/ardp.201500352
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
disease occurs in very severe form when the CFTR is lacking the
phenylalanine in position 508. This mutation is present in ca.
70% of CF homozygote patients. In this case, the mutant CFTR
Introduction
Cystic fibrosis (CF) is a genetic disease due to mutations
affecting the gene coding for the protein CFTR (cystic fibrosis
transmembrane conductance regulator). CFTR is a channel for
anions (particularly, for chloride) located in the apical
membrane of epithelial cells. When CFTR is missing or heavily
damaged, electrolyte and fluid secretion from the epithelial
cells to the lumen of many organs (lung, pancreas, intestine,
etc.) are hampered. In addition, the mucus coating the lungs
becomes more viscous and is transformed into a favorable
place for the growth of bacterial infections [1, 2].
(F508del-CFTR) remains entrapped in the endoplasmic reticu-
lum (ER) where a prolonged association with the chaperone
HSC70 conveys the F508del-CFTR toward the degradation via
the ubiquitin/proteasome pathway. Therefore, only a mar-
ginal amount of the mutant CFTR reaches the plasma
membrane and patients present a severe form of CF [3].
Since 1991, researchers know that F508del-CFTR may be
rescued in vitro by culturing cells at low temperature (27–
30°C) [4]. In fact, under these conditions, the cellular quality
control system allows the trafficking of the F508del-CFTR from
the ER to the plasma membrane [5]. Unfortunately, F508del-
CFTRalsopresentsagatingdefectwhenitsucceedsingettingto
the plasma membrane [6]. Furthermore, a third issue arises
from the fact that the F508del-CFTR in the membrane is
subjected to a rapid recycle leading to a fast lysosomial
degradation; therefore, the F508del-CFTR also presents a
stability defect [7]. Although the F508del-CFTR shows multiple
faults, pharmacological correction of trafficking defect, is
feasible [8]. Since 1996, researchers have found that some
Although the CFTR gene is subjected to many mutations
(more than 1900 mutations have been identified so far), the
Correspondence: Prof. Mauro Mazzei, Department of Pharmacy,
University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy.
E-mail: mauro.mazzei@unige.it
Fax: þ39 010 3538358
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chemicals may partially rescue the mutant protein obtaining
a result similar to the use of low temperature [9, 10]. Later
on, such chemicals were called correctors. By the time, the
number of the correctors has been growing. At present,
such class is constituted of several compounds recently
reviewed [8, 11]. The therapeutic use of correctors has, until
now, been hampered by their toxicity, as a high dosage is
required to rescue the F508del-CFTR. Moreover, their mecha-
nism of action is still unclear. For instance, some of them
could bind to CFTR itself [12–15], while others could act
favorablyonthecomplexsystemofproteins(namely,molecular
chaperones, phosphodiesterases, kinases, and PARPs), helping
in the folding and trafficking of F508del-CFTR [16–19].
Clearly, the more interesting correctors are those binding
the F508del-CFTR itself with respect to correctors favoring the
partial inhibition of proteins essential for the cell-life, for
example, chaperones. Among the more recent correctors, our
interest was pointed at the phenylhydrazone RDR-1. RDR-1 is
a medium level corrector and its mechanism of action is still
not clear, especially if it binds or not NBD1 [14, 20].
Nevertheless, this molecule possesses a quite simple structure,
and therefore it is easy to extend the synthesis to other
phenylhydrazones and investigate their activity on F508del-
CFTR rescue. Interestingly, the synthesis of RDR-1 derivatives
also leads to investigate in more detail on a common
molecular design we have already identified [21] for some
different correctors, including RDR-1. This fact may be of
interest in the search of new F508del-CFTR correctors.
Results and discussion
Chemistry
As depicted in Scheme 1, phenylhydrazones 3a–l were easily
prepared by reacting phenylhydrazines
1 with furfural
derivatives 2 in ethanol. Then, by the same reaction, the
pyridine derivatives (4a and 4b), the thiophene derivatives
(5a–e), the benzohydrazides (6a–c), and the isonicotinohydra-
zides (7a–c) were prepared. The formulas of the synthesized
compounds are shown in Table 1.
All compounds are very colored crystals. The proposed
structures were confirmed by spectral data and elemental
analyses (see Experimental section).
Scheme 1. Synthesis of phenylhydrazones 3, 4, 5, 6, and 7.
potentiator. Indeed, F508del-CFTR has also a partial gating
defect as well that requires treatment with a potentiator to
generate full anion transport.
Data were normalized with respect to control dimethyl
sulfoxide (DMSO): compound activity in Table 1 shows how
many times the compound is more efficient than DMSO. In
parallel experiments, the selective blocker CFTRinh-172 [25]
was added to the solution containing forskolin plus genistein
to demonstrate that the activity was CFTR-dependent.
From the compounds presented in Table 1, it is possible to
draw some interesting considerations. Looking at the general
formula of Fig. 1, which encompasses only 15 derivatives in
Table 1, the best results in the activity are found when the A
substituent is Br or CH3O. About the C substituent, we can
conclude that the presence of the nitro group exerts
indubitably a positive effect for the F508del-CFTR correction:
in fact, some of the better correctors presented in Table 1 (3c,
3f, 3l, and RDR-1) benefit from such substitution.
CFTR corrector activity
Table 1 reports the effectiveness of compounds at both 20 and
2 mM. Activity was calculated as variation of fluorescence
during time due to influx of iodide through CFTR and
quenching of YFP fluorescence (fluorescence quenching rate,
FQR). Two known correctors (corr-4a and VX-809) were used
as reference compounds at 10 and 1 mM, respectively [22–24].
Although corr-4a has a moderate activity in CFBE41o- cells at
10 mM, it was not tested at higher concentration (i.e., at
20 mM) because of signs of cellular toxicity. After incubation
with correctors, the functional assay evaluating F508del-CFTR
in the membrane was done in the presence of genistein as a
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Table 1. Evaluation of compounds on CFBE41o- F508del-CFTR cells.a)
Activity (FQR with respect to DMSO)
Compound
Formula
20 mM
2 mM
RDR-1b)
1.3 ꢀ 0.1
1.3 ꢀ 0.2
3a
3b
3c
0.7 ꢀ 0.1
1.0 ꢀ 0.2
1.8 ꢀ 0.1
1.0 ꢀ 0.1
1.1 ꢀ 0.1
1.0 ꢀ 0.1
1.2 ꢀ 0.2
0.9 ꢀ 0.1
3d
3e
1.0 ꢀ 0.2
1.0 ꢀ 0.1
3f
3g
3h
3i
1.2 ꢀ 0.1
1.0 ꢀ 0.1
1.2 ꢀ 0.1
1.1 ꢀ 0.2
1.1 ꢀ 0.1
1.0 ꢀ 0.1
1.1 ꢀ 0.2
1.1 ꢀ 0.1
1.1 ꢀ 0.1
1.2 ꢀ 0.2
3j
(Continued)
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Table 1. (Continued)
Activity (FQR with respect to DMSO)
Compound
3k
Formula
20 mM
2 mM
0.9 ꢀ 0.2
1.1 ꢀ 0.1
3l
1.4 ꢀ 0.1
1.1 ꢀ 0.1
1.2 ꢀ 0.1
1.0 ꢀ 0.1
4a
4b
1.1 ꢀ 0.2
1.0 ꢀ 0.2
5a
5b
5c
1.5 ꢀ 0.1
0.9 ꢀ 0.1
1.1 ꢀ 0.1
1.1 ꢀ 0.1
0.9 ꢀ 0.2
1.1 ꢀ 0.1
5d
5e
0.6 ꢀ 0.1
0.7 ꢀ 0.2
1.5 ꢀ 0.1
1.1 ꢀ 0.1
6a
6b
0.5 ꢀ 0.1
0.9 ꢀ 0.2
0.9 ꢀ 0.2
1.0 ꢀ 0.1
(Continued)
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Table 1. (Continued)
Activity (FQR with respect to DMSO)
Compound
6c
Formula
20 mM
2 mM
1.1 ꢀ 0.1
1.0 ꢀ 0.1
7a
7b
7c
0.8 ꢀ 0.1
0.9 ꢀ 0.1
0.8 ꢀ 0.1
1.0 ꢀ 0.1
1.0 ꢀ 0.1
0.8 ꢀ 0.1
corr-4a (10 mM)
1.4 ꢀ 0.1
2.9 ꢀ 0.1
VX-809 (1 mM)
a) Activity values are the mean ꢀ SEM of five to ten experiments.
b) RDR-1 was synthesized by the same general procedure described in the Experimental section “General procedure for the
synthesis of the phenylhydrazones 3, 4, 5, 6, and 7” (mp 192–194°C).
The two derivatives 3b and 5b bearing B ¼ CH3 are inactive;
therefore, the presence of NH seems to be essential for the
searched correction activity.
Ten derivatives (3d, 3e, 4, 6, and 7) do not match the
formula of Fig. 1. All the six compounds coming from
isoniazid (7a–c) and benzhydrazide (6a–c) are toxic or
ineffective, the two chloro derivatives 3d and 3e are
inactive and the two hydrazinopyridine derivatives 4a
and 4b are endowed with a very low activity at 20 mM:
it is clear that the insertion of a carbonyl group between
the aromatic ring and the NH does not attain good
results.
Wanting to suggest some remarks from the data here
presented and referring to Fig. 1, we can deduce that a good
corrector coming from these RDR-1-like derivatives should
possess the methoxy or bromo substituent in the A position,
the hydrogen in the B position, and the nitro group in the C
position. We do not have sufficient data to establish the
nature of the heteroatom
X since we could take in
consideration only a small number of thiophene derivatives,
although the presence of sulfur seems to provide a slightly
better activity together with a major toxicity in respect to
furan derivatives.
Figure 1. General structure of phenyhydrazones 3a–c, 3f–l, and
5a–e.
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Table 2. Evaluation of compounds 3c and 5d in associa-
tion with VX-809 on CFBE41o- F508del-CFTR cells.a)
As 5d seems to be interesting at low concentration, we tested
its behavior at 0.1, 0.5, and 10.0 mM obtaining activity values
(as defined in Tables 1 and 2) of 1.1, 1.3, and 1.7, respectively.
Therefore, also taking in account the values from Table 1, for
5d it was possible to draw the dose–response graph of Fig. 2.
The drop of activity at 20 mM may be ascribed to sign of
toxicity revealed in CFBE41o- cells.
The results described above allow us to enlarge a study
done in a previous paper in which we concentrated attention
to a structural design present in some F508del-CFTR correc-
tors [21]. Briefly, in that study we compared six correctors,
each of them being the lead of a greater family: besides the
already cited RDR-1 and corr-4a (see Table 1), we took in
account a locked bithiazole [26], a pyrazine derivative
patented by Novartis [27], a pyrazolothiazole [28], and a
thiazole derivative (EM-2) [29] (see Fig. 3).
Overlapping the six molecules by Chimera and MOE
software [30], we could highlight a shared structural design,
from which we drew the general formula depicted in Fig. 4. In
this figure, great prominence was given to the presence of N
(nitrogen) and X (sulfur or oxygen) whereas the other parts of
the structure (the presence of one or two cycles, the presence
or the absence of Y, the size of Ar and R) may be highly
variable.
Compound
Activity (FQR with
respect to DMSO)
VX-809 (1 mM) þ 3c (20 mM)
VX-809 (1 mM) þ 3c (2 mM)
VX-809 (1 mM) þ 5d (20 mM)
VX-809 (1 mM) þ 5d (2 mM)
3.6 ꢀ 0.1
3.3 ꢀ 0.1
3.0 ꢀ 0.1
4.1 ꢀ 0.1
a) Activity values are the mean ꢀ SEM of four experiments.
Two compounds in our series emerge as potential F508del-
CFTR correctors: 3c with an interesting activity at 20 mM and
5d, with a good activity at 2 mM. Therefore, we tested 3c and
5d together with VX-809 to search any positive effect, which
should be derived by such association. The results are shown in
Table 2, from which it is possible to see an additivity of action
between our compounds and VX-809.
In our point of view, N and X in this spatial geometry may
represent a signature for this type of F508del-CFTR correction.
On these grounds, in the present paper we try to validate
our previous indications searching new substituents able to
improve the activity of one among the above correctors
(namely, RDR-1) introducing some modifications in various
parts of the parent compound and also a more significant
change at the level of the heteroatom X (see Fig. 4). This first
synthetic approach was performed using commercial
reagents: consequently, the set of the synthesized compounds
is quite limited in the series of thiophene derivatives (X ¼ S)
with respect to the series of furan derivatives (X ¼ O). Thus,
the thiophene derivatives do not present any substitution on
Figure 2. Dose–response graph of 5d.
Figure 3. Structures of some F508del-CFTR correctors.
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need the availability of several drugs with different chemical
structures and different targets, possibly given in association
with minimizing toxicity and tolerance: in this regard, the
results presented in Table 2 show, at low concentration, a
satisfactory additivity of action (especially for 5d), which could
be of value in succeeding association therapy.
Figure 4. General formula of RDR-1-like correctors.
Moreover, it must be stressed that all compounds displayed
here start from cheap raw materials, rely on a very simple
synthetic method, and have excellent yield. The possibility to
obtain correctors in a cheap and efficient way is very
interesting for this research and for clinical application, too.
We think that future research in CF drugs will be directed to
the finding and assessment of clusters of F508del-CFTR
modulators (correctors plus potentiators). Although phenyl-
hydrazones present a low activity as correctors with respect to
VX-809, we think that this class of compounds deserves further
study since by modifying and refining the terminal endings of
the basic chemical scaffold, more active compounds could be
found. Eventually, the results attained in the present paper
confirm that the molecular design outlined in Fig. 4 may be
useful in the search of new F508del-CFTR correctors.
Figure 5. Superimposition of seven correctors: RDR-1 (black),
corr-4a (green), locked bithiazole (blue), pyrazolothiazole
(magenta), EM-2 (red), pyrazine derivative (yellow), and
phenylhydrazone 5d (light blue).
the phenyl inserted in the position 5 of the heterocycle (see
Fig. 4, X ¼ S, R ¼ C6H5).
Experimental
Chemistry
Considering the difference between 5d and RDR-1 at the
level of the heteroatom (S instead of O) in the pentacyclic
ring, we were induced to amplify the figure depicted in our
previous paper [21]: in Fig. 5, the superimposition of seven
correctors can be seen.
All reagents and solvents were purchased from Sigma–Aldrich
(St. Louis, MO, USA). Melting points were determined using
an electrothermal apparatus and are uncorrected. Micro-
analyses 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 ¼ 0). IR spectra were recorded on a
Perkin–Elmer 398 spectrophotometer.
Taking into account the similarity, in the central part, of the
correctors displayed in Fig. 5, we can draw some observations.
Firstly, it is important to stress that VX-809 does not match the
correctors of Fig. 5 and it is likely, for this very active CF
investigational drug, to think a binding site in the F508del-
CFTR different from those of the correctors depicted in Fig. 5.
Secondly, the existence of the molecular structure outlined in
Fig. 4 cannot be regarded as a necessary/sufficient condition
to have an F508del-CFTR correction, but may only represent
an interesting support in the search of new correctors. Thirdly,
the presence of furan or thiophene may drive the molecules to
different targets in the mutant CFTR. Fourthly, 5d shows at
20 mM an evident sign of toxicity, at least in CFBE41o- cells (see
Fig. 2): this behavior delineates a correspondence between 5d
and corr-4a [22].
Eventually, our working assumption is, for 5d, a mode of
action similar to corr-4a, whereas the furan derivatives (for
instance, 3c) could follow the same pathway of RDR-1. This
conclusion is in agreement with already published results in
which it was proposed for RDR-1, corr-4a, and VX-809
different targets in F508del-CFTR [31].
Regardless of the action mechanism, we judged interesting
to test the behavior of our best phenylhydrazones (3c and 5d)
when administered together with VX-809: this fact may be
very interesting for CF patients who must take the drugs for
their entire life. Indeed, the long-term cure of a CF patient will
Compounds used in biological testing possess purity of no
less than 98% as determined by elemental analysis (accuracy
ꢀ0.4%) and mass spectrometry.
General procedure for the synthesis of the
phenylhydrazones 3, 4, 5, 6, and 7
In a 100-mL flask, to a solution of 2.3 mmoles of aldheyde A in
10 mL of ethanol, a solution of 2.3 mmoles of the suitable
phenylhydrazine (or hydrazide) B in ethanol was added. The
resulting mixture was refluxed for 1 h. After cooling, a red/
orange precipitate separated out. Then, the solid was filtered
off and crystallized from a suitable solvent. The following
compounds were obtained (see also the Supporting Informa-
tion providing the InChI codes).
1-Phenyl-2-[(5-phenylfuran-2-yl)methylene]hydrazine (3a)
A:5-Phenylfurfural(0.40 g). B:Phenylhydrazine (0.25 g). Yellow
crystals from toluene; yield: 48%; mp 144–146°C. 1H NMR
(CDCl3): d ¼ 6.70–6.80 (m, 3H, H-2,4,6 phenylhydrazine), 6.84 (d,
1H, J ¼ 3.6, H-3 furan), 6.89–7.01 (m, 2H, H-3,5 phenylhydra-
zine), 7.12–7.60 (m, 4H, H-4 furan, H-3,4,5 phenylfuran), 7.64 (s,
–
1H, CH–),7.76(d,2H,J ¼ 8.6,H-2,6phenylfuran).IR(KBr):1266,
–
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1510, 1604, 3317 cmꢁ1. Anal. calcd. for C17H14N2O: C, 77.84; H,
5.38; N, 10.68, found: C, 79.17; H, 5.73; N, 10.52.
1-(4-Chlorophenyl)-2-{[5-(4-nitrophenyl)furan-2-yl]-
methylene}hydrazine (3f)
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: 4-Chlorophenylhydra-
zine (0.41 g). Violet crystals from cyclohexane/ether; yield:
83%; mp 172–174°C. 1H NMR (CDCl3): d ¼ 6.69 (d, 1H, J ¼ 3.2,
H-3 furan), 6.93 (d, 1H, J ¼ 3.2, H-4 furan), 7.07 (d, 2H, J ¼ 8.6,
H-2,6 phenylhydrazine), 7.21 (d, 2H, J ¼ 8.6, H-3,5 phenyl-
1-Methyl-2-{[5-(4-nitrophenyl)furan-2-yl]methylene}-1-
phenylhydrazine (3b)
A: 5-(4-Nitrophenyl)furfural (0.49 g). B: 1-Methyl-1-phenylhy-
drazine (0.28 g). Redpurple crystals from ethylacetate/ethanol;
yield: 80%; mp 158–160°C. 1H NMR (DMSO-d6): d ¼ 3.45 (s, 3H,
N–CH3), 6.88 (d, 1H, J ¼ 3.6, H-3 furan), 6.95 (t, 1H, J ¼ 7.2, H-4
phenylhydrazine), 7.30–7.47 (m, 5H, H-4 furan, H-2,3,5,6
–
hydrazine), 7.69 (s, 1H, CH–), 7.82 (d, 2H, J ¼ 8.8, H-2,6, 4-
–
nitrophenyl), 8.24 (d, 2H, J ¼ 8.8, H-3,5, 4-nitrophenyl). IR
(KBr): 1328, 1506, 1575, 3320 cmꢁ1
. Anal. calcd. for
–
phenylhydrazine), 7.67 (s, 1H, CH), 7.99 (d, 2H, J ¼ 9.0, H-2,6,
C
17H12ClN3O3: C, 59.75; H, 3.54; Cl, 10.37; N, 12,30, found:
–
4-nitrophenyl), 8.31 (d, 2H, J ¼ 9.0, H-3,5, 4-nitrophenyl). IR
(KBr): 1331, 1497, 1598 cmꢁ1. Anal. calcd. for C18H15N3O3: C,
67.28; H, 4.71; N, 13.08, found: C, 67.14; H, 4.85; N, 13.11.
C, 60.01; H, 3.56; N, 12.32.
4-(2-[(5-Phenylfuran-2-yl)methylene]hydrazinyl)-
benzonitrile (3g)
1-(4-Bromophenyl)-2-{[5-(4-nitrophenyl)furan-2-yl]-
methylene}hydrazine (3c)
A: 5-Phenylfurfural (0.40 g). B: 4-Cyanophenylhydrazine
hydrochloride (0.39 g). Yellow crystals from ethyl acetate;
yield: 83%; mp 230–232°C. 1H NMR (DMSO-d6): d ¼ 6.92 (d, 1H,
J ¼ 3.6, H-3 furan), 7.19–7.40 (m, 3H, H-3,5 benzonitrile, H-4
furan), 7.34 (t, 1H, J ¼ 7.2, H-4 phenylfuran), 7.46 (d, 2H,
J ¼ 7.8, H-2,6 benzonitrile), 7.65 (dd, 2H, J ¼ 8.7, J ¼ 7.2, H-3,5,
phenylfuran), 7.80 (d, 2H, J ¼ 8.7, H-2,6 phenylfuran), 7.90 (s,
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: 4-Bromophenylhydra-
zine (0.51 g). Red crystals from toluene; yield: 85%; mp 174–
176°C. 1H NMR (DMSO-d6): d ¼ 6.93 (d, 1H, J ¼ 3.6, H-3 furan),
7.05 (d, 2H, J ¼ 8.8, H-2,6 phenylhydrazine), 7.36–7.47 (m, 3H,
–
H-4 furan, H-3,5 phenylhydrazine), 7.85 (s, 1H, CH–), 8.01 (d,
–
1H, CH–). IR (KBr): 1309, 1539, 1603, 2209, 3289 cmꢁ1. Anal.
–
–
2H, J ¼ 9.0, H-2,6, 4-nitrophenyl), 8.32 (d, 2H, J ¼ 9.0, H-3,5, 4-
nitrophenyl), 10.76 (s, 1H, NH). 13C NMR (CDCl3): d ¼ 110.1 ppm
(C-3 furan), 113.2 (C-4 furan), 113.4 (C-4 phenylhydrazine),
113.6 (2C-2,6 phenylhydrazine), 122.9 (2C-3,5 nitrophenyl),
123.3 (2C-2,6 nitrophenyl), 123.5 (2C-3,5 phenylhydrazine),
123,8 (C-2 furan), 131.1 (C-1 phenylhydrazine), 131.2 (C-1
calcd. for C18H13N3O: C, 75.25; H, 4.56; N, 14.63, found: C,
75.54; H, 4.71; N, 14.73.
4-(2-[(5-(4-Chlorophenyl)furan-2-yl)methylene]-
hydrazinyl)benzonitrile (3h)
–
nitrophenyl), 149.3 (C-4 nitrophenyl), 150.9 ( CH–), 151.4 (C-5
A: 5-(4-Chlorophenyl)furfural (0.48 g). B: 4-Cyanophenylhy-
drazine hydrochloride (0.39 g). Pale yellow crystals from ethyl
acetate; yield: 83%; mp 226–228°C. 1H NMR (DMSO-d6):
d ¼ 6.93 (d, 1H, J ¼ 3.6, H-3 furan), 7.09–7.21 (m, 3H, H-3,5
benzonitrile, H-4 furan), 7.54 (d, 2H, J ¼ 8.6, H-2,6 benzoni-
trile), 7.65 (d, 2H, J ¼ 8.7, H-3,5 4-chlorophenyl), 7.81 (d, 2H,
–
furan). IR (KBr): 1329, 1510, 1586, 3320 cmꢁ1. Anal. calcd. for
C
17H12N3BrO3: C, 52.87; H, 3.13; Br, 20.69; N, 10.88, found: C,
53.16; H, 3.33; N, 10.78.
1-(2-Chlorophenyl)-2-{[5-(4-nitrophenyl)furan-2-yl]-
methylene}hydrazine (3d)
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: 2-Chlorophenylhydra-
zine (0.41 g). Red purple crystals from cyclohexane/ether;
yield: 85%; mp 180–181°C. 1H NMR (CDCl3): d ¼ 6.80–7.09 (m,
3H, H-3,4 furan, H-6 phenylhydrazine), 7.22–7.45 (m, 3H, H-
–
J ¼ 8.7, H-2,6, 4-chlorophenyl), 7.90 (s, 1H, CH–), 11.04 (s, 1H,
–
NH). IR (KBr): 1318, 1520, 1606, 2207, 3281 cmꢁ1. Anal. calcd.
for C18H12ClN3O: C, 67.19; H, 3.76; Cl, 11.02; N, 13.06, found: C,
67.23; H, 4.01; N, 12.92.
–
3,4,5 phenylhydrazine), 7.67 (s, 1H, CH), 7.94 (d, 2H, J ¼ 7.2,
4-{2-[(5-(4-Nitrophenyl)furan-2-yl)methylene]hydrazinyl}-
benzonitrile (3i)
–
H-2,6 4-nitrophenyl), 8.35 (d, 2H, J ¼ 7.2, H-3,5 4-nitrophenyl).
IR (KBr): 1330, 1510, 1580, 3317 cmꢁ1. Anal. calcd. for
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: 4-Cyanophenylhydra-
zine hydrochloride (0.39 g). Red crystals from toluene; yield:
73%; mp 246–248°C. 1H NMR (DMSO-d6): d ¼ 7.02 (d, 1H,
J ¼ 3.6, H-3 furan), 7.18 (d, 2H, J ¼ 8.4, H-3,5 benzonitrile), 7.46
(d, 1H, J ¼ 3.6, H-4 furan), 7.67 (d, 2H, J ¼ 8.4, H-2,6
C
17H12ClN3O3: C, 59.75; H, 3.54; Cl, 10.37; N, 12.30, found:
C, 59.97; H, 3.,52; N.12.55.
1-(3-Chlorophenyl)-2-{[5-(4-nitrophenyl)furan-2-yl]-
methylene}hydrazine (3e)
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: 3-Chlorophenylhydra-
zine (0.41 g). Violet crystals from cyclohexane/ether; yield:
81%; mp 161–163°C. 1H NMR (CDCl3): d ¼ 6.77 (d, 1H, J ¼ 3.6, H-
3 furan), 6.87–7.10 (m, 3H, H-4 furan, H-2,6 phenylhydrazine),
–
benzonitrile), 7.93 (s, 1H, CH–), 8.02 (d, 2H, J ¼ 8.7, H-2,6,
–
4-nitrophenyl), 8.32 (d, 2H, J ¼ 8.7, H-3,5, 4-nitrophenyl), 11.17
(s, 1H, NH). IR (KBr): 1336, 1512, 1608, 2209, 3272 cmꢁ1. Anal.
calcd. for C18H12N4O3: C, 65.06; H, 3.64; N, 16.86, found: C,
65.02; H, 3.90; N, 16.73.
–
7.20–7.38 (m, 2H, H-4,5 phenylhydrazine), 7.66 (s, 1H, CH–),
–
7.87 (d, 2H, J ¼ 8.4, H-2,6, 4-nitrophenyl), 8.29 (d, 2H, J ¼ 8.4, H-
1-{[5-(4-Chlorophenyl)furan-2-yl]methylene}-2-[4-
(trifluoromethyl)phenyl]hydrazine (3j)
3,5, 4-nitrophenyl). IR (KBr): 1330, 1512, 1570, 3333 cmꢁ1
.
Anal. calcd. for C17H12ClN3O3: C, 59.75; H, 3.54; Cl, 10.37; N,
12.30, found: C, 59.78; H, 3.98; N, 12.58.
A: 5-(4-Chlorophenyl)furfural (0.48 g). B: 4-(Trifluoro-
methyl)phenylhydrazine (0.41 g). Yellow brownish crystals
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119

RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2016, 349, 112–123
E. Nieddu et al.
Archiv der Pharmazie
from cyclohexane; yield: 71%; mp 122–124°C. 1H NMR
(CDCl3): d ¼ 6.68–7.35 (m, 4H, H-3,4 furan, H 2,6 phenyl-
hydrazine), 7.40–7.72 (m, 7H, H-2,3,5,6 4-chlorophenyl, H-
1-Phenyl-2-[(5-phenylthiophen-2-yl)methylene]-
hydrazine (5a)
A: 5-Phenylthiophene-2-carboxaldehyde (0.43 g). B: Phenyl-
hydrazine (0.25 g). Yellow crystals from ethanol; yield: 79%;
mp 166–168°C. 1H NMR (CDCl3): d ¼ 6.82–7.23 (m, 3H, H-4
phenylhydrazine, H-3,4 thiophene), 7.26–7.50 (m, 8H, H-
–
3,5 phenylhydrazine, CH). IR (KBr): 1319, 1521, 1615,
–
3324 cmꢁ1. Anal. calcd. for C18H12ClF3N2O: C, 59.27; H,
3.32; Cl, 9.72; F, 15.63; N, 7.68, found: C, 59.32; H, 2.92; N,
7.44.
–
2,3,5,6 phenylhydrazine, CH–, H-3,4,5 phenylthiophene),
–
7.67 (d, 2H, J ¼ 7.6, H-2,6 phenylthiophene). IR (KBr): 1493,
1602, 3312 cmꢁ1. Anal. calcd. for C17H14N2S: C, 73.35; H,
5.07; N, 10.06; S, 11.52; found: C, 73.06; H, 5.15; N, 9.93; S,
11.24.
1-{[5-(4-Nitrophenyl)furan-2-yl]methylene}-2-[4-
(trifluoromethyl)phenyl]hydrazine (3k)
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: 4-(Trifluoromethyl)-
phenylhydrazine (0.41 g). Red purple crystals from toluene;
yield: 64%; mp 188–190°C. 1H NMR (DMSO-d6): d ¼ 6.97 (d, 1H,
J ¼ 3.8, H-3 furan), 7.22 (d, 2H, J ¼ 8.4, H-2,6 phenylhydrazine),
7.43 (d, 1H, J ¼ 3.8, H-4 furan), 7.58 (d, 2H, J ¼ 8.4, H-3,5
1-Methyl-1-phenyl-2-[(5-phenylthiophen-2-yl)methylene]-
hydrazine (5b)
A: 5-Phenylthiophene-2-carboxaldehyde (0.43 g). B: 1-Methyl-
1-phenylhydrazine (0.28 g). Yellow crystals from ethanol;
yield: 87%; mp 114–116°C. 1H NMR (CDCl3): d ¼ 3.46 (s, 3H,
N–CH3), 7.04 (t, 1H, J ¼ 7.4, H-4 phenylhydrazine), 7.15 (d, 1H,
J ¼ 3.2, H-3 thiophene), 7.24–7.57 (m, 8H, H-4 thiophene, H-
2,3,5,6 phenylhydrazine, H-3,4,5 phenylthiophene), 7.69
–
phenylhydrazine), 7.90 (s, 1H, CH–), 8.00 (d, 2H, J ¼ 9.1, H-2,6,
–
4-nitrophenyl), 8.30 (d, 2H, J ¼ 9.1, H-3,5, 4-nitrophenyl), 11.03
(s, 1H, NH). IR (KBr): 1327, 1505, 1597, 3307 cmꢁ1. Anal. calcd.
for C18H12F3N3O3: C, 57.61; H, 3.22; F, 15.19; N, 11.20, found: C,
57.70; H, 3.49; N, 10.93.
–
(d, 2H, J ¼ 8.4, H-2,6 phenylthiophene), 7.89 (s, 1H, CH). IR
–
1-(4-Methoxyphenyl)-2-{[5-(4-nitrophenyl)furan-2-yl]-
methylene}hydrazine (3l)
(KBr): 1371, 1500, 1595 cmꢁ1. Anal. calcd. for C18H16N2S: C,
73.94; H, 5.52; N, 9.58; S, 10.97, found: C, 73.60; H, 5.41; N,
9.69; S, 10.63.
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: 4-Methoxyphenylhy-
drazine (0.40 g). Yellow brownish crystals from cyclohexane/
ether; yield: 53%; mp 168–170°C. 1H NMR (CDCl3): d ¼ 3.84 (s,
3H, CH3), 6.77 (d, 1H, J ¼ 3.2, H-3 furan), 6.85–7.20 (m, 5H, H-4
furan, H-2,3,5,6 phenylhydrazine), 7.84 (d, 2H, J ¼ 9.0, H-2,6, 4-
1-(4-Bromophenyl)-2-[(5-phenylthiophen-2-yl)methylene]-
hydrazine (5c)
A: 5-Phenylthiophene-2-carboxaldehyde (0.43 g). B: 4-Bromo-
phenylhydrazine (0.51 g). Yellow brownish crystals from
toluene; yield: 57%; mp 163–165°C. 1H NMR (CDCl3): d ¼ 6.92–
7.17 (m, 3H, H-3 thiophene and H-2,6 phenylhydrazine), 7.26
(d, 1H, J ¼ 3.8, H-4 thiophene), 7.34–7.58 (m, 5H, H-3,5
phenylhydrazine, H-3,4,5 phenylthiophene), 7.67 (d, 2H,
–
–
nitrophenyl), 8.23–8.40 (m, 3H, CH–, H-3,5, 4-nitrophenyl). IR
–
(KBr): 1319, 1506, 1594, 3322 cmꢁ1
C
.
Anal. calcd. for
18H15N3O4: C, 64.09; H, 4.48; N, 12.46, found: C, 64.00; H,
4.70; N, 12.31.
2-{2-[(5-(4-Chlorophenyl)furan-2-yl)methylene]-
hydrazinyl}pyridine (4a)
J ¼ 6.8, H-2,6, phenylthiophene), 7.86 (s, 1H,
CH–).
–
13C NMR (CDCl3): d ¼ 111.0 (C-4 phenylhydrazine), 113.3 (2C-
2,6 phenylhydrazine), 122.1 (2C-2,6 phenyl), 124.6 (2C-3,5
phenylthiophene), 126.7 (C-2 thiophene), 126.8 (C-4 phenyl-
thiophene), 127.9 (C-3 thiophene), 131.0 (2C-3,5 phenyl-
hydrazine), 131.8 (C-1 phenylthiophene), 133.0 (C-1
thiophene), 138.2 (C-4 thiophene), 142.3 (C-1 phenylhydra-
A: 5-(4-Chlorophenyl)furfural (0.48 g). B: 2-Hydrazinopyridine
(0.25 g). Yellow crystals from ethanol; yield: 74%; mp 211–
213°C. 1H NMR (DMSO-d6): d ¼ 7.00–7.14 (m, 1H, H-5 pyridine),
7.20–7.39 (m, 3H, H-3,4 furan, H-3 pyridine), 7.57 (d, 2H,
J ¼ 8.6, H-3,5 4-chlorophenyl), 7.91 (d, 2H, J ¼ 8.6, H-2,6 4-
chlorophenyl), 7.99–8.10 (m, 1H, H-4 pyridine), 8.14 (d, 1H,
ꢁ1
–
zine), 143.8 ( CH–). IR (KBr): 1252, 1491, 1593, 3312 cm
.
–
–
J ¼ 6.4, H-6 pyridine), 8.25 (s, 1H, CH–), 13.08 (s, 1H, NH). IR
Anal. calcd. for C17H13BrN2S: C, 57.15; H, 3.67; Br, 22.37; N,
7.84; S, 8.98, found: C, 57.30; H, 3,77; N, 7.48.
–
(KBr): 1474, 1606, 1653, 3376 cmꢁ1
. Anal. calcd. for
C
16H12ClN3O: C, 64.54; H, 4.06; Cl, 11.91; N, 14.11, found: C,
64.35; H, 3.98; N, 14.23.
1-(4-Methoxyphenyl)-2-[(5-phenylthiophen-2-yl)-
methylene]hydrazine (5d)
2-{2-[(5-(4-Nitrophenyl)furan-2-yl)methylene]hydrazinyl}-
pyridine (4b)
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: 2-Hydrazinopyridine
(0.25 g). Orange crystals from ethanol; yield: 76%; mp 242–
244°C. 1H NMR (DMSO-d6): d ¼ 7.08–7.20 (m, 1H, H-3 furan, H-5
pyridine), 7.37 (d, 1H, J ¼ 9.0, H-3 pyridine), 7.53 (d, 1H, J ¼ 3.4,
H-4 furan), 8.01–8.42 (m, 7H, H-4,6 pyridine, H-3,5, 4-nitro-
A: 5-Phenylthiophene-2-carboxaldehyde (0.43 g). B: 4-
Methoxyphenylhydrazine (0.40 g). Orange brownish crystals
from toluene; yield: 63%; mp 146–148°C. 1H NMR (CDCl3):
d ¼ 3.80 (s, 3H, CH3O), 6.81–6.98 (m, 3H, H-3 thiophene and H-
2,6 phenylhydrazine), 7.25 (d, 1H, J ¼ 3.8, H-4 thiophene),
7.30–7.55 (m, 5H, H-3,5 phenylhydrazine, H-3,4,5 phenyl-
thiophene), 7.60–7.73 (m, 3H, H-2,6, phenylthiophene and
phenyl, CH–), 13.60 (s, 1H, NH). IR (KBr): 1329, 1506, 1595,
CH–). IR (KBr): 1236, 1505, 1520, 3311 cmꢁ1. Anal. calcd. for
–
–
–
–
1650 cmꢁ1. Anal. calcd. for C16H12N4O3: C, 62.33; H, 3.92; N,
18.17, found: C, 62.10; H, 4.13; N, 18.02.
C18H16N2OS: C, 70.10; H, 5.23; N, 9.08; S,10.40, found: C, 74.22;
H, 5.05; N, 8.85; S, 9.43.
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120

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Arch. Pharm. Chem. Life Sci. 2016, 349, 112–123
Phenylhydrazones as CFTR Correctors
Archiv der Pharmazie
1-[(5-Phenylthiophen-2-yl)methylene]-2-(4-
trifluoromethyl)phenyl)hydrazine (5e)
A: 5-Phenylthiophene-2-carboxaldehyde (0.43 g). B: 4-(Tri-
1H NMR (DMSO-d6): d ¼ 7.15 (d, 1H, J ¼ 3.7, H-3 furan), 7.22 (d,
1H, J ¼ 3.7, H-4 furan), 7.56 (d, 2H, J ¼ 8.6, H-3,5 4-chloro-
phenyl), 7.68–7.92 (m, 4H, H-2,6, 4-chlorophenyl and H-3,5,
–
fluoromethyl)phenylhydrazine (0.41 g). Yellow crystals from
pyridine), 8.40 (s, 1H, CH–), 8.81 (d, 2H, J ¼ 5.6, H-2,6
–
1
ethanol; yield: 89%; mp 166–167°C. H NMR (CDCl3): d ¼ 6.95
pyridine), 12.09 (s, 1H, NH). IR (KBr): 1291, 1549, 1658,
3189 cmꢁ1. Anal. calcd. for C17H12ClN3O2: C, 62.68; H, 3.71; Cl,
10.88; N, 12.90, found: C, 63.22; H, 4.47; N, 12.73.
(d, 1H, J ¼ 3.8, H-3 thiophene), 7.16 (d, 2H, J ¼ 7.8, H-2,6
phenylhydrazine), 7.27 (d, 1H, J ¼ 3.8, H-4 thiophene), 7.35–
7.60 (m, 5H, H-3,5 phenylhydrazine, H-3,4,5 phenylthio-
phene), 7.68 (d, 2H, J ¼ 7.0, H-2,6, phenylthiophene), 7.91
N0-{[5-(4-Nitrophenyl)furan-2-yl]methylene}pyridine-4-
carbohydrazide (7b)
(s, 1H, CH–). IR (KBr): 1334, 1536, 1616, 3318 cmꢁ1. Anal.
–
–
calcd. for C18H13F3N2S: C, 62.42; H, 3.78; F, 16.46; N, 8.09; S,
9.26; found: C, 62.63; H, 3,61; N, 7.90; S, 9.03.
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: Isoniazid (0.32 g).
Yellow crystals from toluene; yield: 45%; mp 207–208°C.
1H NMR (DMSO-d6): d ¼ 7.22 (d, 1H, J ¼ 3.7, H-3 furan), 7.50 (d,
1H, J ¼ 3.7, H-4 furan), 7.84 (d, 2H, J ¼ 4.8, H-3,5 pyridine), 8.05
(d, 2H, J ¼ 8.7, H-2,6, 4-nitrophenyl), 8.33 (d, 2H, J ¼ 8.7, H-3,5,
N’-{[5-(4-Chlorophenyl)furan-2-yl]methylene)-
benzohydrazide (6a)
A: 5-(4-Chlorophenyl)furfural (0.48 g). B: Benzhydrazide
(0.31 g). Pale yellow crystals from toluene; yield: 60%; mp
218–220°C. 1H NMR (CDCl3): d ¼ 6.69 (d, 1H, J ¼ 3.4, H-3 furan),
7.80–7.97 (m, 2H, H-4 furan, H-4 benzoyl), 7.25–7.70 (m, 6H, H-
3,5 benzoyl, H-2,3,5,6 4-chlorophenyl), 7.83–8.08 (m, 3H, H-
–
4-nitrophenyl), 8.43 (s, 1H, CH–), 8.81 (d, 2H, J ¼ 4.8, H-2,6
–
pyridine), 12.17 (s, 1H, NH). 13C NMR (DMSO-d6): d ¼ 111.7 (C-3
furan), 112.0 (2C-3,5 pyridine), 116.4 (C-4 furan), 121.0 (C-3
nitrophenyl), 124.0 (C-5 nitrophenyl), 124.2 (C-2 nitrophenyl),
125.4 (C-6 nitrophenyl), 134.6 (C-1 nitrophenyl), 137.6 (C-2
furan), 139.8 (C-4 pyridine), 145.9 (C-4 nitrophenyl), 149.9 (C-2
–
2,6, benzoyl, CH–), 11.40 (s, 1H, NH). IR (KBr): 1281, 1564,
–
1652, 3022 cmꢁ1. Anal. calcd. for C18H13ClN2O2: C, 66.57; H,
4.03; N, 8.63, found: C, 66.21; H, 4.08; N, 8.66.
pyridine), 150.2 (C-6 pyridine), 152.3 ( CH–), 161.2 (C-5 furan),
–
–
178.5 (C O). IR (KBr): 1344, 1516, 1685, 3117 cmꢁ1. Anal. calcd.
–
–
for C17H12N4O4: C, 60.71; H, 3.60; N, 16.66, found: C, 60.96; H,
3.88; N, 16.36.
N0-{[5-(4-Nitrophenyl)furan-2-yl]methylene}-
benzohydrazide (6b)
A: 5-(4-Nitrophenyl)furfural (0.50 g). B: Benzhydrazide
(0.31 g). Yellow crystals from toluene; yield: 90%; mp 261–
263°C. 1H NMR (DMSO-d6): d ¼ 7.18 (d, 1H, J ¼ 3.8, H-3 furan),
7.48–7.63 (m, 4H, H-4 furan, H-3,4,5 benzoyl), 7.94 (d, 2H,
J ¼ 7.6, H-2,6 benzoyl), 8.06 (d, 2H, J ¼ 8.9, H-2,6, 4-nitro-
phenyl), 8.34 (d, 2H, J ¼ 8.9, H-3,5 4-nitrophenyl), 8.47 (s, 1H,
N0-[(5-Phenylthiophen-2-yl)methylene]pyridine-4-
carbohydrazide (7c)
A: 5-Phenylthiophene-2-carboxaldehyde (0.43 g). B: Isoniazid
(0.32 g). Pale yellow crystals from toluene; yield: 12%; mp
240–242°C. 1H NMR (DMSO-d6): d ¼ 7.31–7.62 (m, 5H, H-3,4
furan, H-3,4,5 phenyl), 7.74–7.90 (m, 4H, H-2,6 phenyl,
–
–
–
CH–), 11.98 (s, 1H, NH).
H-3,5, pyridine), 8.67 (s, 1H, CH–), 8.81 (d, 2H, J ¼ 4.4,
–
13C NMR (DMSO-d6): d ¼ 112.0 ppm (C-3 furan), 115.8 (C-4
furan), 124.1 (2C-3,5 nitrophenyl), 124.2 (2C-2,6 benzoyl),
127.2 (2C-2,6 nitrophenyl), 128.1 (C-4 benzoyl), 131.5 (C-1
benzoyl), 132.8 (C-3 benzoyl), 134.7 (C-5 benzoyl), 136.6 (C-2
H-2,6 pyridine), 12.09 (s, 1H, NH). IR (KBr): 1302, 1569,
1643, 2978 cmꢁ1. Anal. calcd. for C18H17N3OS: C, 66.85; H,
5.30; N, 12.99; S, 9.91, found: C, 66.69; H, 5.36; N, 13.29; S,
10.29.
–
furan), 145.9 (2C-1,4 nitrophenyl), 151.0 ( CH–), 152.0 (C-5
–
ꢁ1
–
–
Biology
Cell culture
furan), 162.8 (C O). IR (KBr): 1332, 1517, 1659, 3095 cm
.
Anal. calcd. for C18H13N3O4: C, 64.47; H, 3.91; N, 12.53, found:
C, 64.22; H, 4.23; N, 12.23.
The CF bronchial epithelial cell line CFBE41o-, with stable
expression of F508del-CFTR, was obtained by Dr. J. P.
Clancy [32]. The cell line was subsequently transfected
with the halide-sensitive yellow fluorescent protein (HS-
YFP) YFP-H148Q/I152L [33]. The culture medium was mini-
mum essential medium (MEM), supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and
100 mg/mL streptomycin.
N0-{[5-Phenylthiophen-2-yl]methylene)benzohydrazide (6c)
A: 5-Phenylthiophene-2-carboxaldehyde (0.43 g). B: Benzhy-
drazide (0.31 g). Pale yellow crystals from toluene; yield: 22%;
mp 247–248°C. 1H NMR (DMSO-d6): d ¼ 7.29–7.60 (m, 8H, H-3,4
thiophene, H-3,4,5 phenylthiophene, H-3,4,5 benzoyl), 7.76 (d,
2H, J ¼ 7.6, H-2,6 phenylthiophene), 7.92 (d, 2H, J ¼ 7.8, H-2,6
–
benzoyl), 8.67 (s, 1H, CH–), 11.95 (s, 1H, NH). IR (KBr): 1288,
For fluorescence assays of CFTR activity, CFBE41o- cells were
plated (50000 cells/well) on clear-bottomed 96-well black
microplates (Corning Life Sciences, Acton, MA).
–
1565, 1638, 2995 cmꢁ1. Anal. calcd. for C18H14N2OS: C, 70.56; H,
4.61; N, 9.14; S, 10.47, found: C, 70.24; H, 4.91; N, 8.94; S, 10.79.
N0-{[5-(4-Chlorophenyl)furan-2-yl]methylene}pyridine-4-
carbohydrazide (7a)
A: 5-(4-Chlorophenyl)furfural (0.48 g). B: Isoniazid (0.32 g).
Fluorescence assay for CFTR activity
Measurements of CFTR activity were carried out on
CFBE41o-cells expressing mutant CFTR and the HS-YFP
48 h after plating on microplates. Twenty-four hours after
Pale yellow crystals from toluene; yield: 51%; mp 248–250°C.
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plating, the cells were incubated with test compounds at
37°C for 20–24 h. At the time of assay, the 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 forskolin (20 mM) in the
presence of genistein (50 mM). Then the cells were
transferred to a microplate reader (FluoStar Galaxy; BMG
Labtech GmbH, Offenburg, Germany) for CFTR activity
determination. The plate reader was equipped with high
quality excitation (HQ500/20X: 500 ꢀ 10 nm) and emission
(HQ535/30M: 535 ꢀ 15 nm) filters for YFP (Chroma Technol-
ogy Corp., Brattleboro, VT). Each assay consisted of a
continuous 14-s fluorescence reading with 2 s before and
12 s after injection of an iodide-containing solution (PBS
with Clꢁ replaced by I–; final I– concentration in the well,
100 mM). In parallel experiments, the selective blocker
CFTRinh-172 [25] was added to the activating cocktail
containing forskolin plus genistein. The data were normal-
ized to the initial background-subtracted fluorescence. To
determine fluorescence quenching rate (FQR) associated
with Iꢁ influx, the final 11 s of the data for each well were
fitted with an exponential function to extrapolate initial
slope (dF/dt).
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This work was financially supported by Italian Cystic Fibrosis
Foundation (Project FFC#3/2013). The funders had no role in
study design, data collection and analysis, decision to publish,
or preparation of the paper.
The authors have declared no conflicts of interest.
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