A new 9-alkyladenine-cyclic methylglyoxal diadduct activates wt- and F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2014.06.028

Cystic fibrosis transmembrane conductance regulator (CFTR) is the main chloride channel present in the apical membrane of epithelial cells and the F508 deletion (F508del-CFTR) in the CF gene is the most common cystic fibrosis-causing mutation. In the sear

Full text of DOI:10.1016/j.ejmech.2014.06.028

European Journal of Medicinal Chemistry 83 (2014) 455e465
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A new 9-alkyladenine-cyclic methylglyoxal diadduct activates wt- and
F508del-cystic fibrosis transmembrane conductance regulator (CFTR)
in vitro and in vivo
,
,
Benjamin Boucherle a 1, Johanna Bertrand b 1, Bruno Maurin a, Brice-Loïc Renard a,
b
b
b
a, *
ꢀ a
ꢀ ꢀ
ꢀ
Antoine Fortune , Brice Tremblier , Frederic Becq , Caroline Norez , Jean-Luc Decout
Universite Grenoble Alpes, Joseph Fourier/CNRS, UMR 5063, Departement de Pharmacochimie Moleculaire, 470 rue de la Chimie, F-38041 Grenoble, France
a
ꢀ
ꢀ
ꢀ
b
ꢀ
Universite de Poitiers/CNRS, Laboratoire Signalisation et Transports Ioniques Membranaires, 1 rue Georges Bonnet, F-86022 Poitiers, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Cystic fibrosis transmembrane conductance regulator (CFTR) is the main chloride channel present in the
apical membrane of epithelial cells and the F508 deletion (F508del-CFTR) in the CF gene is the most
common cystic fibrosis-causing mutation. In the search for a pharmacotherapy of cystic fibrosis caused
by the F508del-CFTR, a bi-therapy could be developed associating a corrector of F508del-CFTR trafficking
and an activator of the channel activity of CFTR. Here, we report on the synthesis of 9-alkyladenine
derivatives analogues of our previously discovered activator of wt-CFTR and F508del-CFTR, GPact-11a,
and the identification of a new activator of these channels, GPact-26a, through various flux assays on
human airway epithelial CF and non-CF cell lines and in vivo measurement of rat salivary secretion. This
study reveals that the possible modifications of the side chain introduced at the N9 position of the main
pharmacophore are highly limited since only an allyl group can replace the propyl side chain present in
GPact-11a to lead to a strong activation of wt-CFTR in CHO cells. Docking simulations of the synthesised
compounds and of four described modulators performed using a 3D model of the wt-type CFTR protein
suggest five possible binding sites located at the interface of the nucleotide binding domains NBD1/
NBD2. However, the docking study did not allow the differentiation between active and non-active
compounds.
Received 8 February 2014
Received in revised form
12 June 2014
Accepted 13 June 2014
Available online 14 June 2014
Keywords:
Cystic fibrosis
F508del-CFTR
Modulators
Activators
Salivary secretion
Docking
Nucleotide binding domains
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
The mutations of the CF gene encoding CFTR cause the genetic
disease cystic fibrosis (CF) [1,4e6] that is the most common lethal
The Cystic Fibrosis Transmembrane conductance Regulator
(CFTR) protein is a cAMP-regulated chloride channel controlling
transepithelial Cle transport and belonging to the ATP-binding
cassette (ABC) superfamily [1e6]. CFTR is the only one of the
numerous members of the ABC superfamily known to function as
an ion channel.
autosomal recessive genetic disease in Caucasians (more than 1900
identified mutations, http://www.genet.sickkids.on.ca/cftr). These
numerous mutations can be classified according to the fate of the
final product into five classes [6]. The F508 deletion (F508del) is the
most common cystic fibrosis-causing mutation that induces
reduced channel function, early degradation and poor trafficking of
modified CFTR protein to the apical membrane of epithelial cells
(Class II) [7]. The protein consists of 1480 amino acid residues
forming (i) two membrane-spanning domains (MSD1 and MSD2)
composed each by six transmembrane helices (TM1 to TM6 and
TM7 to TM12) and by intracellular loops (ICLs), (ii) two cytoplasmic
nucleotide binding domains (NBD1 and NBD2) and (iii) a large
regulatory domain (R). The two NBDs form tightly interacting di-
mers upon ATP-binding at their interface [8]. ATP-binding and
dimerization of the NBDs are coupled to channel opening, whereas
ATP hydrolysis at composite site 2 leads to the channel closure. The
Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conduc-
tance regulator; CHO cells, Chinese hamster ovaries cells; CMGD, cyclic methyl-
glyoxal diadducts; DMTr, 4,40-dimethoxytrityl; ICL, intracellular loop; MG,
methylglyoxal; MSD, membrane-spanning domain; NBD, nucleotide binding
domain; TM, transmembrane helice.
* Corresponding author.
ꢀ
E-mail address: jean-luc.decout@ujf-grenoble.fr (J.-L. Decout).
Equal contributions of these authors.
1
http://dx.doi.org/10.1016/j.ejmech.2014.06.028
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

456
B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
non-canonical ATP-binding site 1 binds nucleotide tightly but is not
hydrolytic, suggesting that this site remains closed throughout the
gating site and that a limited separation of the NBDs occur [8].
As a large polytopic membrane protein containing disordered
regions, intact CFTR has been refractory to efforts to solve a
high-resolution structure using X-ray crystallography [9]. Only
crystal structures of the wild type and mutated NBD1 [10,11] and
NBD2 [12] have been solved, which together with previous
modelling studies of the NBD1:NBD2 heterodimer [13,14] have
provided the first significant insights into the role that the F508
residue would play in the formation of inter-domain contacts. This
role was clearly evidenced by the two first homology models of the
whole NBD-MSD assembly, build on the basis of the crystal struc-
ture of the bacterial Sav1866 [10,11,15], as the F-508 residue was
indeed shown to be located at the interface between NBD1 and ICL4
from MSD2 [16]. These models were assumed to approximate the
open form of the channel, at least in the cytoplasmic domains, as
both NDB are tightly associated, with ATP bound at their interfaces.
Recent approval of a CF protein modulator, ivacaftor (Kalydeco),
in a small group of patients with the G551D mutation has ignited an
explosion of interest for other modulators directed at different
CFTR mutation classes [17e29]. This Class III mutation results in
a channel open probability strongly lower than that of the wild-
type channel. One of the major therapeutic strategy in CF aims at
developing modulators of CFTR activity and, in the case of F508del,
a bi-therapy could be used associating a corrector of F508del-CFTR
trafficking and an activator.
In the search for modulators of CFTR, we have recently identified
GPact-11a (1a) (Fig. 1) as a non-toxic and water-soluble CFTR acti-
vator [30]. We showed that GPact-11a activates F508del-CFTR
when rescued by correctors including miglustat, in nasal,
tracheal, bronchial, pancreatic CF cell lines and in human CF ciliated
epithelial cells freshly dissociated from lung samples (iodide efflux,
fluorescence imaging and patch-clamp recordings). GPact-11a also
stimulates ex vivo the colonic chloride secretion (short-circuit
current measurements) and increases in vivo the salivary secretion
in cftrþ/þ but not in cftrꢀ/ꢀ mice [30]. The effects appeared to be
selective for CFTR since they are inhibited by CFTRinh-172 [31],
GlyH-101 [32], glibenclamide and GPinh-5a (2a) (Fig. 1), an inhib-
itor of CFTR that we have identified previously having a structure
closely related to the structure of GPact-11a [33] (Fig. 1).
GPact-11a and inhibitor GPinh-5a are made of a same heterocyclic
core and differ only from the substituent present at the 9-position
of the heterocyclic ring which are a propyl chain and a 2-
deoxyribosyl substituent, respectively (numbering according to
the usual adenine ring numbering, Fig. 1). In these structures, the
nature of the 9-substituent appears to be essential for modulating
the effects on CFTR and we modified this side chain in the search for
new CFTR modulators through delineation of structureeactivity
relationships.
Here, we report on the synthesis of analogues of GPact-11a and
the identification, in the same family, of a new CFTR-activator
GPact-26a in CHO cells stably expressing wt-CFTR. This com-
pound showed similar in vitro and in vivo effects to those of GPact-
11a and was also able to activate rescued F508del-CFTR in human
airway epithelial CF15 cell line. Since, GPact-26a is able to activate
wt-CFTR, we carried out molecular modelling docking simulations
using the 3D model published by Mornon et al. [11]. In the search
for potential binding sites of discovered activators and inhibitors,
we also report here on these simulations that suggests several
common possible binding sites shared by activators and inhibitors
of the GP-family and located at the key NBD1/NBD2 interface in the
protein. These possible targets remain to be related to the observed
biological effects through site-directed mutagenesis experiments.
2. Results
2.1. Chemistry
GPact-11a has been prepared by condensation of 9-
propyladenine (prepared by alkylation of the nucleic base
adenine) and methylglyoxal (MG). Such a condensation led to four
isomers of cyclic methylglyoxal diadducts (CMGD) isolated as two
racemic mixtures of enantiomers 1a (GPact-11a) and 1b (GPact-
11b) formed in a 60:40 ratio [34].
In a first approach in the delineation of structureeactivity re-
lationships, the chemical modifications in the GPact-11a structure
were designed in order to maintain the hydrophobic character of
the 9-substituent and the propyl side chain was replaced by
different alkyl chains: methyl, ethyl, isopropyl, butyl, allyl, prop-
argyl, benzyl and 3-phenylpropyl groups (Scheme 1). A compound
in which the 3-hydroxypropyl group replaces the propyl chain was
also prepared in order to evaluate the role of the chain lipophilicity
(Scheme 1).
The previously identified activator GPact-11a 1a and inhibitor
GPinh-5a 2a have been obtained by condensation of two molecules
of methylglyoxal (MG) with one molecule of 9-propyladenine and
20-deoxyadenosine, respectively, leading to cyclic methylglyoxal
diadducts (CMGD) [34]. More recently, we also described two 5-
pyrimidinols, obtained from prepared CMGD to 2-aminopyridine
and 1-aminoisoquinoline, that are able to strongly inhibit the ac-
tivity of CFTR channels in wt-CFTR CHO cells [35]. The activator
Fig. 1. Structure of the previously identified activator GPact-11a [30] and inhibitor
GPinh-5a [33] of wt- and F508del-CFTR channels in vitro and in vivo.
Scheme 1. General method used for the preparation of the 9-alkyladenine-cyclic
methylglyoxal diadducts.

B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
457
Previously, we have observed that both mixtures of enantiomers
1a and 1b have close EC50 on wt-CFTR in CHO cells (125I iodide
M, respectively) [30]
efflux assay: EC50 ¼ 2.1 1.3 and 2.9 0.9
m
and thus, in a first approach, the mixtures of major enantiomers a
of the prepared analogues were isolated and evaluated.
The commercially available 9-methyladenine
3 and 9-
ethyladenine 4 were converted to the corresponding CMGD de-
rivatives 13a,b and 14a,b through reaction with MG (Scheme 1).
The diasteroisomeric mixtures of enantiomers 13a and 14a were
isolated by reversed phase chromatography. With the exception of
the 3-hydroxypropyl derivative 21a, the other 9-alkylated CMGD
analogues of GPact-11a 15ae20a were prepared from adenine in
two steps (Scheme 1) (i) alkylation with the corresponding bro-
moalkylating reagent in the presence of NaH in DMF for obtaining
the 9-alkyladenine derivatives 5e10 and (ii) reaction of 5e10 with
MG in excess. The second step led as previously to four isomers as
two racemic mixtures of enantiomers a and b from which the
mixtures a were isolated. The 3-hydroxypropyl derivatives 21a
were obtained in four steps (Scheme 1): (i) preparation of the
alkylating reagent 3-bromo-1-(4,40-dimethoxytrityloxy)propane,
(ii) alkylation of adenine with this reagent to lead to 11, (iii) removal
of the trityl group in the presence of dichloroacetic acid and (iv)
reaction with MG of the deprotected intermediate 12.
Fig. 2. Effect of the synthesised GPact11a analogues (100
ulated by forskolin (Fsk, 1
centage of Fsk induced response, n ¼ 4 for each condition, **p < 0.01.
m
M) on iodide efflux stim-
m
M) in wt-CFTR CHO cells. Data are expressed as a per-
We also tried to synthesise the corresponding CMGD to 7-
methyl- and 7-propyl-adenines which have been isolated as mi-
nor products in the step of adenine alkylation. Unfortunately the
cyclization with MG did not occur probably due to steric hindrance.
2.2. Biological evaluation
A number of methods are currently employed to assess the
functional properties of CFTR channels and their response to
pharmacological potentiators and correction of the defective CFTR
trafficking [36].
First, we examined the effect of the synthesised compounds in
CHO cells stably expressing wt-CFTR through a robotic cell-based
primary screening assay using radioactive iodide efflux measure-
ments. Because the CFTR channel is permeable to iodide, these
measurements are related to the chloride efflux and allow a rapid
detection of CFTR channel activation or inhibition.
Fig. 3. Viability of wt-CFTR CHO cells in the presence of 17a (GPact-26a) (24 h) using
the colorimetric MTT assay. Ctrlþ corresponds to untreated cells and Ctrlꢀ corresponds
to cells private of medium during 24 h.
The slow-response potential-sensitive probe bis-(1,3 diethylth-
iobarbituric acid)trimethine oxonol (DiSBAC2(3)) can be used to
estimate the intracellular chloride concentration change. Increased
chloride concentrations results in additional influx of the anionic
dye and an increase in cell fluorescence. Thus, GPact-26a was
further studied using the single cell fluorescence imaging tech-
nique developed with the oxonol (DiSBAC2(3)) probe applied to the
human tracheal gland epithelial cell line MM39 (Fig. 5A, B) [37].
The synthesised analogues of GPact-11a 13ae21a were evaluated
by this iodide efflux technique at a concentration of 100
mM in the
presence of forskolin (Fsk) at 1 M (Fig. 2). Fsk, a terpene derivative of
m
plant origin, activates adenylate cyclase and leads to an intracellular
increase of cAMP sufficient to activate the cAMP-dependent protein
kinaseA (PKA)withthe consequentactivationof theCFTR. Amongthe
evaluated compounds, only 17a (GPact-26a) revealed a detectable
potentiation of the Fsk effect under the conditions used (Fig. 2).
The cell viability in the presence of 17a was then evaluated in
wt-CFTR CHO cells after 24 h of incubation using the colorimetric
MTT assay (reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium in the mitochondria to purple formazan, Fig. 3).
Since 17a appeared to be non-cytotoxic up to 300 mM concen-
trations, we determined in a second step the half maximal effective
concentration EC50 of this compound. The study of the
concentration-response effect, in the presence of Fsk (1
mM),
showed an EC50 of 7.5 1.7 M (standard error of mean, Fig. 4).
m
In order to investigate the effects of the corresponding isomers
b, the mixture 17b was isolated and the corresponding EC50 was
measured in a similar experiment with CHO cells. The value ob-
tained was EC50 ¼ 2.1 1.2
mM. In regard to the errors calculated
from the concentrations of the prepared solutions of 17a and 17b,
we concluded that there is no major difference of activity between
both mixtures of enantiomers 17a and 17b. Thus, the major isomers
17a (GPact-26a) were further studied.
Fig. 4. Evaluation of 17a (GPact-26a) using the iodide efflux technique in wt-CFTR CHO
cells in the presence of Fsk at 1
mM. The half-maximal effective concentration for
activation (EC50) was calculated after nonlinear regression of the data.

458
B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
Fig. 5. Functional evaluation of 17a (GPact-26a) by DISBAC2 assay [37] in human tracheal gland epithelial cells MM39. (A) Example of mean traces showing potentiation of the
forskolin (Fsk, 1 M)-induced activation of CFTR; (B): activation in the absence of Fsk. Data represent the mean ( SEM) of the relative fluorescence collected (Fx) in arbitrary units
lex ¼ 535 nm, lem ¼ 560 nm). CFTRinh-172 at 1 M concentration was used to inhibit the CFTR activity.
m
(
m
Fig. 6. Evaluation of 17a (GPact-26a) using the iodide (125I) efflux technique in miglustat-corrected CF15 cells in the presence of Forskolin (Fsk) at 10
mM. (A): Concentration-
response time-dependent curves were plotted by measuring the rate of iodide efflux (minꢀ1); (B): The half-maximal effective concentration for activation (EC50) was calculated
after nonlinear regression of the data.
Fig. 7. Evaluation of 17a (GPact-26a) on rat salivary secretion collected in response to subcutaneous injection. Basal salivary secretion was inhibited by atropine (1 mM) prior to
injection of a saline solution containing atropine (1 mM) mixed with isoprenaline (Iso, 10
act26a alone (30 M). (A): The average of salivary secretion was determined in dividing the weight of collected saliva by the time required for collection in min and by the weight of
the rat in g; (B): The total saliva secretion was determined by addition of all the samples of saliva collected during the experiment.
mM) alone or the mixture isoprenaline þ GP-act26a (10 and 30
mM, respectively) or GP-
m

B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
459
We observed that GPact-26a (17a) increases the fluorescence in
the presence of Fsk at 1 M confirming its potentiating effect in
GPinh-5a and 13ae21a) in comparison to the previously
described genistein, MBP-104 and IBMX activators [30,39] and
CFTRinh-172 inhibitor [31,33], we performed in silico docking sim-
ulations using the homology model of the wt-CFTR outward-facing
conformation, made on the basis of the crystal structure of Sav1866
in an outward-facing conformation [11]. This model is assumed to
approximate the open form of the channel, at least in the cyto-
plasmic domains, as the two NDB are tightly associated, with ATP
bound at their interfaces. In the first step of simulation, we
explored the ability that the selected compounds bind to the
cytoplasmic part of the protein excluding thereby the trans-
membrane helices (TMs) of the membrane spanning domains
(MSDs). Both the nucleotide binding domains NBDs (NBD1: resi-
dues 386e649, NBD2: 1204e1446) and the intracellular loops
(ICLs) of the MSDs constituting the interface between NBDs and
MSDs (ICL1: 155e189, ICL2: 255e289, ICL3: 947e980, ICL4:
1048e1082) were conserved. In regard to the large size of the
corresponding docking box, the most appropriate software VINA
was used in the first step of the docking study with the forty-eight
selected compounds performed in the presence of nucleotide (ADP)
in both NBDs (Scheme 2).
The first docking calculations showed that all compounds can
bind in five different cavities (Fig. 8, Table 1). Two cavities (C1 and
C2) are located at the entrance of the nucleotide binding sites
occupied by nucleotides. The third identified water-accessible
pocket (C3) is located at the opposite face of NBDseICLs contact
area. The two last cavities (C4, C5) are inner pockets located close to
the canonical and non-canonical nucleotide-binding sites, respec-
tively, and delimited in part by ICLs (ICL1,2 and ICL2,3, respectively).
All these identified cavities appeared to be located at the nucleotide
binding domains interface (NBD1/NBD2).
In the second step of the simulation (Scheme 2), accurate
dockings were performed using the VINA, Autodock and GOLD
[40,41] software in five small boxes, each ones including one
different cavity previously identified. For a same cavity, the three
docking software gave similar homogeneous scores with low
standard deviations for the affinity of all compounds (data not
shown, see in Table 1 the average affinities or scores and the
standard deviations calculated for the 48 compounds studied).
Thus, the scores obtained for a same cavity appeared to be close for
all the GP-family compounds including their four isomers. There
was no significant difference between active and non-active com-
pounds including the known modulators. With Autodock and VINA,
better homogeneous energies or scores were obtained for the
binding of all compounds in the C4 and C5 cavities. With GOLD, the
scores in the large inner cavity C4 close to non-canonical ATP
binding site jumped about 20% (Table 1) suggesting that cavity C4
could be a preferential possible binding site of GPact-11a and
GPact-26a.
m
MM39 cells. This observed Fsk/GPact-26a-induced response was
fully inhibited by the previously reported inhibitor CFTRinh-172
[31](Fig. 5A). Clearly, these results confirm the wt-CFTR potenti-
ating effect of GPact-26a in human epithelial cells. Moreover, in the
absence of Fsk, the fluorescent signal was also increased by GPact-
26a alone and fully inhibited by addition of the CFTRinh-172 sug-
gesting that GPact-26a (17a) is an activator of CFTR and not only a
CFTR potentiator in the presence of Fsk (Fig. 5B). The same results
have been obtained previously with GPact-11a (1a) [30].
To study the effect of GPact-26a on F508del-CFTR, we deter-
mined its effect on miglustat-corrected human airway epithelial
cell line CF15 by the iodide efflux technique. This compound
induced a concentration-dependent potentiation of iodide efflux
(Fig. 6A) with an EC50 ¼ 104
10 M as shown in Fig. 6B.
Measurement of salivary secretion (weight of saliva) is a good
2 mM in the presence of forskolin at
m
non-invasive technique to study fluid and electrolyte secretion
in vivo [30,38]. Saliva is secreted in response to cholinergic and
adrenergic agonists by the coordinated action and regulation of
multiple water and ion transporters and ionic channels. Fluid and
electrolyte transport is driven by transepithelial Clꢀ movement. The
opening of Clꢀ channels in the apical membrane of salivary gland
acinar cells initiates the fluid secretion process, whereas the acti-
vation of Clꢀ channels in both the apical and the basolateral
membranes of ductal cells is thought to be necessary for NaCl re-
absorption. Different classes of Clꢀ channels have been identified
in salivary cells, activated by intracellular Ca2þ, gated by cAMP, and
activated following changes in membrane potential and cell vol-
ume. Importantly, only adrenergic response is impaired in most CF
exocrine glands, because the secretory portion of CF glands failed to
respond to b-adrenergic agonists.
In order to evaluate the effect of GPact-26a in vivo, we studied
the CFTR-dependent salivary secretion in rat after sub-cutaneous
injection of atropine to block the cholinergic dependent secretion
(Fig. 7A and B). First, we determined the secretory capacity of saliva
by stimulating the salivary glands with GPact-26a alone. GPact-26a
at 30
the contrary, isoprenaline, a b1- and b2-adrenoreceptor agonist, at
10
M, stimulated the salivary secretion (N ¼ 9). Addition of GPact-
26a to isoprenaline (30 and 10 M, respectively) increased the
m
M by itself did not increase the salivary secretion (N ¼ 5). On
m
m
collected saliva weights (Fig. 7A and B). Clearly, GPact-26a poten-
tiates the isoprenaline-induced wt-CFTR-dependent salivary
secretion in rat (N ¼ 10) as observed previously in mice with GPact-
11a [30].
2.3. Molecular modelling
Additional docking simulations were performed in each nucle-
otide binding sites after removal of ADP. In the corresponding
In order to delineate potential target sites of the compounds of
the GP-family (forty-four compounds: four isomers of GPact-11a,
Scheme 2. Method of in silico docking used for the delineation of potential targeted sites by the compounds of the GP-family (forty-four compounds: four isomers of GPact-11a,
GPinh-5a and 13ae21a) and previously described activators, genistein, MBP-104 and IBMX, and inhibitor CFTRinh-172 with the CFTR homology model of the CFTR outward-facing
conformation (closed channel) developed by Mornon et al. [11] and the VINA, Autodock and GOLD software. TMs: Transmembrane helices.

460
B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
Fig. 8. 3D structure representation of wt-CFTR. The five cavities identified in the presence of ADP (shown as sticks) are shown through coloured volumes: C1, NBD1/NBD2 interface,
slate blue; C2, NBD1/NBD2 interface, red; C3, NBD1/NBD2 interface, violet; C4, NBD1/NBD2/ICL1/ICL4 interface, cyan; C5, NBD1/NBD2/ICL2/ICL3 interface, yellow. (A): MSDs: green,
NBD1: light blue, NBD2 not drawn; (B): 90ꢁ rotation about the x axis relative to (A); MSDs not drawn, NBD2: salmon surface. Pictures made with Pymol [50]. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
boxes, the main binding solutions obtained for all selected com-
pounds partly overlap the ADP binding sites and the corresponding
scores appeared to be similar to those obtained with the C1eC3 and
C5 sites.
of the hydrophobic group introduced at the 9-position is a key
parameter in the activating effect and (ii) this group should be
located in a small hydrophobic site of CFTR in which the position of
the terminal methyl (GPact-11a) or methylene group (GPact-26a) is
crucial for an activation.
The molecular docking study of forty-eight compounds
including the GP-family compounds and four known modulators
(genistein, MBP-104, IBMX, CFTRinh-172) was performed using the
homology model of the wt-CFTR outward-facing conformation and
the VINA, GOLD and Autodock software. These three software
pointed out five main possible binding sites common to the GP
compounds and to the four reported modulators studied. These five
identified cavities are located at the NBD1/NBD2 interface that is a
key feature in the CFTR opening. Accurate dockings in the corre-
sponding boxes did not allow the discrimination between the af-
finities for a same cavity of the active and non-active compounds.
Cavity 4 (NBD1/NBD2/ICL1/ICL4 interface) gave, with the three
software used, the better average scores calculated from the scores
of the forty eight compounds docked. However, the scores obtained
for each compound are high enough to consider a possible binding
in the five identified cavities.
These results and the lack of discrimination between active and
non-active compounds in the docking study point out the limits of
such an in silico approach to explain dynamic effects. The observed
drastic changes in CFTR activation by modifications of the length
and the steric hindrance of the 9-side chain attached to the adenine
core cannot be explained at the molecular level through the
docking simulations performed. Site-directed mutagenesis experi-
ments especially in cavity 4 are underway in order to determine the
CFTR-activating binding cavity of GPact-11a and GPact-26a and to
progress in the design of more active activators.
3. Discussion and conclusion
In the search for new CFTR modulators, the analogues
(13ae21a) of GPact-11a (1a) in which the 9-propyl side chain is
modified, were synthesised. Among these analogues, only GPact-
26a (17a) and the corresponding isomers GPact-26b (17b)
appeared to be able to strongly activate wt-CFTR at similar con-
centrations in CHO cells. GPact-26a also activates wt-CFTR in hu-
man epithelial MM39 and F508del-CFTR rescued by miglustat in
human CF ciliated epithelial cells. GPact-26a was also able to acti-
vate wt-CFTR-dependent salivary secretion in rat. It is thus a water-
soluble wt-CFTR and F508-del activator in several airway epithelial
cell lines as observed in vitro (iodide efflux and fluorescence im-
aging) and in vivo (salivary secretion) techniques. GPact-26a
showed similar activation properties to those of GPact-11a and,
thus, is a good candidate in combination with F508del-CFTR cor-
rectors for further developments towards a cystic fibrosis therapy.
From the results obtained with nine analogues of our first lead
GPact-11a, it appeared that the possible modifications of the
pharmacophoric core at the 9-position are highly limited to the
replacement of the 9-propyl chain by a 9-allyl group incorporating
the same number of carbon atoms. Introduction at the N9 position
of shorter and longer side chains led to the loss of activation of wt-
type CFTR in CHO cells. Since the presence of 9-isopropyl (16a) and
9-propargyl (19a) side groups made of three carbon atoms also led
to a lack of activity, it can be concluded that (i) the steric hindrance
Table 1
Location of the identified binding cavities C1eC5 (NBD: Nucleotide Binding Domain, ICL: intracellular loops) and corresponding average binding energy or fitness score
calculated using the VINA, Autodock and GOLD software for the forty-eight compounds studied and standard deviation.
Cavity
C1
C2
C3
C4
C5
Location
Colour in Fig. 8
NBD1/NBD2
slate blue
NBD1/NBD2
red
NBD1/NBD2
violet
NBD1/NBD2/ICL1/ICL4
cyan
NBD1/NBD2/ICL2/ICL3
yellow
Average binding energy and standard deviation
ꢀ7.9 0.7
ꢀ8.1 0.7
ꢀ7.6 0.5
ꢀ8.9 0.6
ꢀ8.5 0.6
(VINA) kcal molꢀ1
Average binding energy and standard deviation
(Autodock) kcal molꢀ1
ꢀ7.7 1.4
ꢀ7.7 1.2
ꢀ6.9 1.3
ꢀ8.6 1.2
ꢀ8.3 1.4
Average fitness score and standard deviation (GOLD)
56
5
57
7
55
7
65
7
54
7

B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
461
4. Experimental
4.1.1.4. Synthesis of 9-allyladenine [43]7. After evaporation under
reduced pressure, the residue was dissolved in 100 mL of aqueous
NaOH (1 M) and extracted by 4 ꢂ 100 mL of AcOEt. Organic layers
are combined, dried and evaporated under reduced pressure. The
residue was chromatographed on silica gel eluting with DCM-
4.1. Chemistry
All starting materials were commercially available research-
grade chemicals and used without further purification. Reactions
were monitored by analytical TLC on silica gel (Alugram Sil G/
MeOH giving 9-allyladenine
23.20 mmol, 63%) obtained from 5.00 g (37.04 mmol) of adenine. 1H
NMR (400 MHz, D2O): 7.83 (1H, s, CHAr), 7.82 (1H, s, CHAr),
7 as a white solid (4.06 g,
UV254) from MachereyeNagel with fluorescent indicator UV254
.
d
Melting points were determined in open glass capillaries using a
Büchi 510 apparatus and are reported uncorrected. LRMS were
achieved with a NERMAG spectrometer for the FAB, DCI and EI
techniques and with a ZQ Waters for the ESI. HRMS and elemental
analysis were obtained from the Mass Spectrometry Service,
CRMPO, at the University of Rennes I, France, using a Micro-Tof-Q-II
Micromass Zabspec-Tof spectrometer for ESI and a Varian Mat311
spectrometer for EI technique, and a Microanalyseur Flash EA1112
CHNS/O Thermo Electron.
5.89e5.81 (1H, m, CH), 5.11 (1H, d, J ¼ 10.4 Hz, CH2), 4.88 (1H, d,
J ¼ 17.2 Hz, CH2), 4.54 (2H, dd, J ¼ 1.2 and 4.4 Hz, CH2), 13C NMR
(100 MHz, D2O): d 154.8 (CIV), 151.8 (CHAr), 147.9 (CIV), 141.7 (CHAr),
131.5 (CH), 117.9 (CH2), 117.6 (CIV), 45.7 (CH2), LRMS (FABþ, glycerol)
calcd for C8H10N5: [M þ H]þ 176, found 176.
4.1.1.5. Synthesis of 9-propargyladenine [44] 8. After evaporation
under reduced pressure, the residue was chromatographed on sil-
ica gel eluting with DCM-MeOH giving 9-propargyladenine 8 as a
white solid (1.39 g, 8.03 mmol, 67%) obtained from 1.62 g
1H and 13C NMR spectra were recorded on a Bruker Avance 400
at 400 MHz and 100 MHz, respectively, using the residual solvent
signal as internal standard. Chemical shifts are reported in ppm
(parts per million) relative to the residual signal of the solvent, and
the signals are described as singlet (s), broad singlet (bs), doublet
(d), triplet (t), doublet of doublet (dd), quartet (q), sextuplet (sext),
septuplet (sept), multiplet (m); coupling constants are reported in
Hertz (Hz). Columns chromatography were performed on silica gel
(MN Kieselgel 60, 0.063e0.2 mm/70e230 mesh, MachereyeNagel)
or on C18 reversed phase (MachereyeNagel polygoprep 60e50 C18).
HPLC analysis was performed with an Agilent 1100 series using a
diode array detector and a C18 reversed-phase column (Nucleodur
(11.99 mmol) of adenine. 1H NMR (400 MHz, MeOD):
d 8.24 (1H, s,
CH), 8.23 (1H, s, CH), 5.08 (2H, d, J ¼ 2.6 Hz, CH2), 3.00 (1H, t,
J ¼ 2.6 Hz, CH), 13C NMR (100 MHz, MeOD):
d 155.9 (CIV), 152.5
(CHAr), 148.8 (CIV), 140.5 (CHAr), 118.6 (CIV), 76.2 (CIV), 74.3 (CH2),
32.3 (CH). LRMS (FABþ, glycerol) calcd for C8H8N5: [M þ H]þ 174,
found 174.
4.1.1.6. Synthesis of 9-benzyladenine [42] 9. After evaporation un-
der reduced pressure, the residue was dissolved in 100 mL of
aqueous NaOH (1 M) and extracted by 4 ꢂ 100 mL of DCM. Organic
layers are combined, dried and evaporated under reduced pressure.
The residue was chromatographed on silica gel eluting with DCM-
C18 ISIS, MachereyeNagel, 5
m
m particle size, 250 mm ꢂ 4.6 mm),
with a mobile phase composed of A ¼ ammonium acetate 0.1 M pH
7 and B ¼ methanol with a gradient: to 0:100 A:B over 30 min,
MeOH giving 9-benzyladenine
14.80 mmol, 40%) obtained from 5.00 g (37.04 mmol) of adenine. 1H
NMR (400 MHz, CDCl3): 8.42 (1H, s, CH), 7.78 (1H, s, CH),
7.43e7.28 (5H, m, CH), 5.64 (2H, Bs, NH2), 5.39 (2H, S, CH2), 13C NMR
(100 MHz, CDCl3): 155.6 (CIV), 153.5 (CHAr), 150.6 (CIV), 140.7
9 as a white solid (3.33 g,
1 mL/min, 10 mL injection, detection at 260 nm.
Purity of the evaluated compounds was determined by
elemental analysis or HPLC analysis and in every case appeared to
be ꢃ95%.
d
d
(CHAr), 135.7 (CIV), 129.3 (CHAr), 128.7 (CHAr), 128.0 (CHAr), 47.5
(CH2), HRMS (ESIþ) calcd for C12H11N5: [M]þ 225.10145 found
225.1015, [MꢀH]þ 224.09362 found 224.0947.
4.1.1. Synthesis of 9-alkyled adenines
4.1.1.1. General procedure used for preparing 9-alkylated adenine.
To a suspension of adenine in dry DMF, NaH (1.1 equiv) was added
under argon. The mixture was stirred at rt for 1 h then the alky-
lating agent (1.05 equiv) was added and the mixture was stirred
until complete reaction.
4.1.1.7. Synthesis of 9-(30-phenylpropyl)adenine [45] 10. After
evaporation under reduced pressure, the residue was crystallized
from propan-2-ol giving 9-(30-phenylpropyl)adenine 10 as a white
solid (5.31 g, 20.96 mmol, 57%) obtained from 5.00 g (37.04 mmol)
4.1.1.2. Synthesis of 9-isopropyladenine [42] 5. After evaporation
under reduced pressure, the residue was chromatographed on sil-
ica gel eluting with DCM-MeOH giving 9-isopropyladenine 5 as a
white solid (457 mg, 2.58 mmol, 26%) obtained from 1.40 g
of adenine. 1H NMR (400 MHz, CDCl3):
d 8.39 (1H, s, CH), 7.76 (1H, s,
CH), 7.36e7.14 (5H, m, CH), 5.63 (2H, Bs, NH2), 4.23 (2H, t, J ¼ 7.2 Hz,
CH2), 2.69 (2H, t, J ¼ 7.2 Hz, CH2), 2.28 (2H, m, CH2), 13C NMR
(100 MHz, CDCl3):
d 155.5 (CIV), 153.1 (CHAr), 150.4 (CIV), 140.7
(10.36 mmol) of adenine. 1H NMR (400 MHz, CDCl3):
d 8.36 (1H, s,
(CHAr), 140.4 (CIV), 128.8 (CHAr), 128.6 (CHAr), 120.0 (CIV), 43.6 (CH2),
32.9 (CH2), 31.4 (CH2), LRMS (ESIþ) calcd for C14H16N5: [M þ H]þ
254, found 254.
CH), 7.88 (1H, s, CH), 5.96 (2H, bs, NH2), 4.85 (1H, m, CH),1.61 (6H, d,
J ¼ 6.8 Hz, CH3), 13C NMR (100 MHz, CDCl3):
d 157.7 (CIV), 152.9
(CHAr), 149.9 (CIV), 138.2 (CHAr), 120.2 (CIV), 47.2 (CH), 22.9 (CH3),
LRMS (DCI, NH3, isobutane): calcd for C8H12N5: [M þ H]þ 178, found
178.
4.1.1.8. Synthesis of 3-bromo-1-(40,400-dimethoxytrityloxy)propane
[46]. To
a
solution of 4,40-dimethoxytrityl chloride (2.44 g,
4.1.1.3. Synthesis of 9-butyladenine [42] 6. After evaporation under
reduced pressure, the residue was crystallized from propan-2-ol
giving 9-butyladenine 6 as a white solid (9.04 g, 47.28 mmol,
64%) obtained from 10.00 g (74.08 mmol) of adenine. 1H NMR
7.19 mmol) in pyridine (5 mL), 3-bromopropanol (1.00 g, 0.66 mL)
was added. The mixture was stirred at rt during 2 h and then
precipitated into MeOH (30 mL). After filtration, the solid was
washed with MeOH to give 3-bromopropoxy-DMTr as a white solid
(1.97 g, 4.46 mmol, 62%), mp: 85e86 ꢁC.1H NMR (400 MHz, CDCl3):
(400 MHz, CDCl3):
NH2), 4.20 (2H, m, CH2), 1.89 (2H, m, CH2), 1.36 (2H, m, CH2), 0.95
(3H, m, CH3), 13C NMR (100 MHz, CDCl3):
155.4 (CIV), 152.9 (CIV),
d 8.37 (1H, s, CH), 7.80 (1H, s, CH), 6.34 (2H, bs,
d
7.63 (2H, d, J ¼ 7.6 Hz, CHAr), 7.51 (4H, d, J ¼ 8.8 Hz, CHAr), 7.42 (1H,
d
t, J ¼ 7.6 Hz, CHAr), 7.36e7.31 (2H, m, CHAr), 6.99e6.96 (4H, d,
J ¼ 8.8 Hz, CHAr), 3.85 (6H, s, CH3), 3.68 (2H, t, J ¼ 6.8 Hz, CH2),
3.40e3.39 (2H, m, CH2), 2.24 (2H, m, CH2), HRMS (EI) calcd for
140.4 (CHAr), 129.0 (CHAr), 119.6 (CIV), 43.7 (CH2), 32.0 (CH2), 19.9
(CH2), 13.5 (CH3), LRMS (FABþ, NBA) calcd for C9H14N5: [M þ H]þ
192, found 192.
C
24H25O739Br: [MþNa]þ 463.0885, found 463.0884.

462
B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
4.1.1.9. Synthesis of 9-[30-(400,4000-dimethoxytrityloxy)propyl]adenine
11. After evaporation under reduced pressure, the residue was
chromatographed on silica gel eluting with DCM-MeOH. After
evaporation under reduced pressure, the residue was crystallized
from propan-2-ol giving 9-[30-(400,4000-dimethoxytrityloxy)propyl]
adenine 11 as a pale yellow solid (1.26 g, 2.54 mmol, 56%) obtained
from 612 mg (4.53 mmol) of adenine, mp: 162e164 ꢁC. 1H NMR
13C NMR (100 MHz, D2O):
d 176.5 (CIV), 146.3 (CIV), 144.4 (CHAr),
142.6 (CHAr), 117.4 (CIV), 89.1 (CIV), 70.5 (CH), 63.2 (CIV), 36.7 (CH2),
25.6 (CH3), 22.0 (CH3), 14.4 (CH3), HRMS (EI) calcd for C13H17N5O4:
[M þ H]þ 308.1359,found 308.1359, elemental analysis calcd for
C13H17N5O4, H2O: C 48.00, H 5.89, N 21.53, found C 47.61, H 5.56, N
21.28.
(400 MHz, CDCl3):
d
8.39 (1H, s, CH), 7.66 (1H, s, CH), 7.46 (2H, d,
4.1.2.4. Synthesis of 9-isopropyladenine adducts 15a. 15a (121 mg,
0.38 mmol, 25%) obtained as a white solid from 250 mg (1.41 mmol)
of 9-isopropyladenine 5 in 40% aqueous solution of MG and 3 mL of
water after 18 h at 50 ꢁC, mp: 144 ꢁC (dec.). 1H NMR (400 MHz,
J ¼ 7.6 Hz, CHAr), 7.36e7.26 (7H, m, CHAr), 6.88 (4H, d, J ¼ 7.6 Hz,
CHAr), 5.88 (2H, bs, NH2), 4.21 (2H, t, J ¼ 6.4 Hz, CH2), 3.85 (6H, s,
CH3), 3.18 (2H, t, J ¼ 6.4 Hz, CH2), 2.12 (2H, t, J ¼ 6.4 Hz, CH2), 13
C
NMR (100 MHz, CDCl3):
d
158.7 (CIV), 155.4 (CIV), 152.7 (CHAr), 150.2
D2O):
(1H, s, CH), 1.61 (3H, s, CH3), 1.58 (3H, s, CH3), 1.07 (6H, d, J ¼ 8.4 Hz,
2 CH3), 13C NMR (100 MHz, D2O):
176.5 (CIV), 146.3 (CIV), 145.1
d 8.77 (1H, s, CH), 8.28 (1H, s, CH), 4.81e4.75 (1H, m, CH), 4.44
(CIV), 145.0 (CIV), 141.1 (CHAr), 136.2 (CIV), 130.1 (CHAr), 128.2 (CHAr),
128.1 (CHAr), 127.1 (CHAr), 119.9 (CIV), 113.3 (CHAr), 86.3 (CIV), 59.9
(CH2), 55.4 (CH3), 41.5 (CH2), 30.3 (CH2), HRMS (EI) calcd for
d
(CIV),142.8 (CHAr), 142.3 (CHAr),117.7 (CIV), 89.0 (CIV), 70.5 (CH), 63.2
(CIV), 48.5 (CH), 25.6 (CH3), 22.1 (CH3), 21.4 (CH3), HRMS (EI) calcd
for C14H19N5O4: [M þ H]þ 344.1335, found 344.1333, elemental
analysis calcd for C14H19N5O4, Na, ½ C3H7OH: C 49.86, H 5.94, N
18.76, found C 49.56, H 6.06, N 18.89.
C
29H30N5O3: [M þ H]þ 496.2349, found 496.2362.
4.1.1.10. Synthesis of 9-(30-hydroxypropyl)adenine [42] 12. A solu-
tion of 11
9-[30-(400,4000-dimethoxytrityloxy)propyl]adenine
(800 mg, 1.61 mmol) in dichloroacetic acid (2% in DCM, 65 mL) was
stirred at rt during 1.5 h. The mixture was then neutralized using a
saturated solution of NaHCO3. The aqueous layer was evaporated
under reduce pressure and the residue was chromatographed on
reverse phase (C18) eluting with water-methanol giving 9-(3-
hydroxypropyl)-adenine 11 as a white solid (305 mg, 1.58 mmol,
4.1.2.5. Synthesis of 9-butyladenine adducts 16a. 16a (432 mg,
1.29 mmol, 11%) obtained as a white solid from 2.50 g (13.07 mmol)
of 9-butyladenine 6 in 40% aqueous solution of MG and 5 mL of
water after 48 h at 50 ꢁC, mp: 140e142 ꢁC (dec.). 1H NMR (400 MHz,
D2O): d 8.84 (1H, s, CH), 8.21 (1H, s, CH), 4.53 (1H, s, CH), 4.25 (2H, t,
96%), mp: 178e179 ꢁC. 1H NMR (400 MHz, MeOD):
d
8.23 (1H, s,
J ¼ 6.8 Hz, CH2), 1.82 (2H, m, CH2), 1.74 (3H, s, CH3), 1.67 (3H, s, CH3),
CH), 8.14 (1H, s, CH), 4.37 (2H, t, J ¼ 7.2 Hz, CH2), 3.57 (2H, t,
1.26 (2H, sext, J ¼ 7.2 Hz, CH2), 0.87 (3H, t, J ¼ 7.2 Hz, CH3), 13C NMR
J ¼ 6.4 Hz, CH2), 2.09 (2H, m, CH2), 13C NMR (100 MHz, MeOD):
(100 MHz, D2O):
d 175.6 (CIV), 146.5 (CIV), 145.4 (CIV), 144.8 (CHAr),
d
155.9 (CIV), 152.2 (CHAr), 149.2 (CIV), 141.5 (CHAr), 57.9 (CH2), 40.4
142.6 (CHAr), 117.4 (CIV), 89.1 (CIV), 70.5 (CH), 63.2 (CIV), 44.2 (CH2),
31.2 (CH2), 25.5 (CH3), 22.0 (CH3), 19.0 (CH2), 12.6 (CH3), HRMS (EI)
(CH2), 32.0 (CH2), HRMS (EI) calcd for C8H11N5O: [M]þ 193.0964,
found 193.0970.
calcd for
C
15H22N5O4: [M
þ
H]þ 336.1672, found 336.1683,
elemental analysis calcd for C15H21N5O4, H2O: C 50.98, H 6.56, N
19.82, found C 51.11, H 6.36, N 19.76.
4.1.2. Preparation of the adenine methylglyoxal diadducts
4.1.2.1. General procedure used for preparing methylglyoxal adducts.
To the commercial concentrated aqueous solution of methylglyoxal
4.1.2.6. Synthesis of 9-allyladenine adducts 17a. 17a (1.23 g,
3.86 mmol, 44%) obtained as a white solid from 1.50 g (8.70 mmol)
of 9-allyladenine in 40% aqueous solution of MG and 5 mL of water
after 12 h at 50 ꢁC, mp: 136e139 ꢁC (dec.). 1H NMR (400 MHz, D2O):
(MG) (40%) (8 equiv), the
a-aminoazaheterocycle was added. In
some cases, the reaction mixture was diluted by addition of water.
Argon was flushed through the solution and the mixture was
heated at 50 ꢁC until complete reaction under argon. After evapo-
ration under reduced pressure, the residue was chromatographed
on Sep-Pak® C18 cartridges (10 g) eluting with H2O and then
H2OeMeOH (95:5) giving the major isomers adducts a except for
compound 17, which for the two isomers were isolated. Com-
pounds 15a, 17a,18a, and 21a were also crystallized from propan-2-
ol.
d
8.81 (1H, s, CH), 8.22 (1H, s, CH), 6.10e6.01 (1H, ddt, J ¼ 17.0, 10.4
and 5.2 Hz, CH), 5.29 (1H, d, J ¼ 10.4 Hz, CH2), 5.07 (1H, d,
J ¼ 17.0 Hz, CH2), 4.86 (2H, d, J ¼ 5.2 Hz, CH2), 4.56 (1H, s, CH),
1.75e1.67 (6H, m, CH3), 13C NMR (100 MHz, D2O):
d 176.8 (CIV),
146.8 (CIV), 145.3 (CIV), 144.8 (CHAr), 142.9 (CHAr), 131.5 (CH), 118.5
(CH2), 117.5 (CIV), 89.1 (CIV), 70.5 (CH), 63.6 (CIV), 46.3 (CH2), 25.9
(CH3), 22.1 (CH3), LRMS (FABþ, glycerol) calcd for C14H18N5O4:
[M þ H]þ 320, found 320, HPLC purity ¼ 96.3%.
4.1.2.2. Synthesis of 9-methyladenine adducts 13a. 13a (245 mg,
0.80 mmol, 28%) obtained as a pale yellow solid from 450 mg
(3.00 mmol) of 9-methyladenine 3 in 40% aqueous solution of MG
and 5 mL of water after 24 h at 50 ꢁC, mp: 102e103 ꢁC (dec.). 1H
4.1.2.7. Synthesis of 9-allyladenine adducts 17b. 17b (601 mg,
1.89 mmol, 22%) obtained as a white solid from 1.50 g (8.70 mmol)
of 9-allyladenine in 40% aqueous solution of MG and 5 mL of water
after 12 h at 50 ꢁC, mp: 143e146 ꢁC (dec.). 1H NMR (400 MHz, D2O):
NMR (400 MHz, D2O):
CH3), 1.99 (3H, s, CH3), 1.54 (3H, s, CH3), 1H NMR (400 MHz, D2O,
50 ꢁC):
8.63 (1H, s, CH), 8.23 (1H, s, CH), 5.04 (1H, s, CH), 3.95 (3H,
s, CH3), 2.22 (3H, s, CH3), 1.82 (3H, s, CH3), 13C NMR (100 MHz, D2O):
177.4 (CIV), 149.4 (CIV), 148.3 (CHAr), 144.0 (CHAr), 118.8 (CIV), 82.1
d 8.31 (1H, s, CH), 8.00 (1H, s, CH), 3.73 (3H, s,
d
8.81 (1H, s, CH), 8.25 (1H, s, CH), 6.11e6.01 (1H, ddt, J ¼ 17.0, 10.4
d
and 5.2 Hz, CH), 5.29 (2H, d, J ¼ 10.4 Hz, CH2), 5.09 (1H, d,
J ¼ 17.0 Hz, CH2), 4.90 (1H, d, J ¼ 5.2 Hz, CH2), 4.21 (1H, s, CH),
d
1.95e1.76 (6H, m, 2 CH3), 13C NMR (100 MHz, DMSO D6):
d 175.2
(CIV), 69.9 (CH), 64.8 (CIV), 30.1 (CH3), 26.7 (CH3), 21.2 (CH3), HRMS
(EI) calcd for C12H15N5O4: [M þ H]þ 294.1202, found 294.1204,
HPLC purity ¼ 96.3%.
(CIV), 152.2 (CIV), 151.3(CIV), 151.1 (CHAr), 146.7 (CIV), 141.1 (CHAr),
131.4 (CH), 118.5 (CH2), 118.0 (CIV), 73.6 (CH), 65.3 (CIV), 46.4 (CH2),
24.0 (CH3), 18.2 (CH3), HRMS (EI) calcd for C14H18N5O4: [M þ H]þ
320.13588 found 320.1361, HPLC purity ¼ 96.1%.
4.1.2.3. Synthesis of 9-ethyladenine adducts 14a. 14a (680 mg,
2.21 mmol, 33%) obtained as a white solid from 1.00 g (6.19 mmol)
of 9-ethyladenine 4 in 40% aqueous solution of MG and 15 mL of
water after 10 h at 50 ꢁC, mp: 139e141 ꢁC (dec.). 1H NMR (400 MHz,
4.1.2.8. Synthesis of 9-propargyladenine adducts 18a. 18a (495 mg,
1.56 mmol, 27%) obtained as a pale yellow solid from 1.00 g
(5.78 mmol) of 9-propargyladenine in 40% aqueous solution of MG
20 mL of water after 24 h at 50 ꢁC, mp: 147 ꢁC (dec.). 1H NMR
D2O):
d 8.75 (1H, s, CH), 8.11 (1H, s, CH), 4.42 (1H, s, CH), 4.14 (2H, m,
CH2), 1.63 (3H, s, CH3), 1.56 (3H, s, CH3), 1.35 (3H, t, J ¼ 7.2 Hz, CH3),
(400 MHz, D2O): d 8.76 (1H, s, CH), 8.23 (1H, s, CH), 4.98 (2H, s, CH2),

B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
463
4.40 (1H, s, CH), 2.82 (1H, s, CH), 1.60 (3H, s, CH3), 1.55 (3H, s, CH3),
13C NMR (100 MHz, D2O):
176.2 (CIV), 146.1 (CIV), 145.1 (CIV), 144.0
collagen IV (50 mg/ml, SIGMA chemicals, USA) and cultured in
d
Dulbecco's minimal essential medium/Ham F-12 (3:1) (Invitrogen
corporation, USA) supplemented with 10% FCS (BIO-Whittaker
Europe, Belgium) and seven growth factors [38]The non CF human
tracheal cell line MM39 was also used and cultures as previously
described [47].
(CHAr), 143.0 (CHAr),143.0 (CIV), 117.5 (CIV), 98.3 (CIV), 75.6 (CH), 70.4
(CH), 63.3 (CIV), 33.7 (CH2), 25.5 (CH3), 22.0 (CH3), HRMS (EI) calcd
for C14H16N5O4 [M þ H]þ 318.1202, found 318.1195, elemental
analysis calcd for C14H15N5O4, Na, ½ C3H7OH: C 50.41, H 4.91, N
18.96, found C 50.69, H 4.82, N 19.06.
4.2.2. Iodide efflux
4.1.2.9. Synthesis of 9-benzyladenine adducts 19a. 19a (140 mg,
0.35 mmol, 14%) obtained as a pale brown solid from 600 mg
(2.67 mmol) of 9-benzyladenine in 40% aqueous solution of MG and
5 mL of water after a week at 50 ꢁC, mp: 170e171 ꢁC (dec.). 1H NMR
CFTR Clꢀ channel activity was assayed on population of cells by
the iodide (125I) efflux in the presence of the compounds [48].
Concentration response curves were determined by measuring
the rate of iodide (125I) efflux with a high-capacity robotic system
(MultiProbe II EXT; PerkinElmer Life and Analytical Sciences,
Courtaboeuf, France) adapted to the determination of iodide efflux.
The protocol of screening was as follows. CHO cells were cultured in
multi-well plates and incubated at 37 ꢁC in Krebs' solution con-
(400 MHz, D2O):
m, 5CH), 5.36 (2H, s, CH2), 4.89 (1H, s, CH), 2.03 (3H, s, CH3), 1.53
(3H, s, CH3), 13C NMR (100 MHz, D2O):
177.5 (CIV), 151.3 (CIV), 147.8
d 8.24 (1H, s, CH), 8.10 (1H, s, CH), 7.30e7.17 (5H,
d
(CIV), 142.5 (CHAr), 135.7 (CIV), 135.5 (CHAr), 129.8 (CHAr), 128.2
(CHAr), 127.2 (CHAr), 119.1 (CIV), 98.1 (CIV), 81.1 (CH), 64.8 (CIV), 47.5
(CH2), 20.2 (CH3), 12.3 (CH3), LRMS (ESIþ, glycerol) calcd for
taining 1
mM KI and 1 m
Ci of Na125I/mL (PerkinElmer Life and
Analytical Sciences, Boston, MA) during 30 min (CHO cells) to
permit the 125I to reach equilibrium. The first three aliquots were
used to establish a stable baseline in cold Krebs' buffer (from t0 to
t2). A medium containing the appropriate compound was then used
for the remaining aliquots from t3 to t8. Residual radioactivity was
extracted at the end of the experiment with a mixture of 0.1 M
NaOH and 0.1% SDS and determined using a gamma counter (Cobra
II; PerkinElmer Life and Analytical Sciences). The fraction of initial
intracellular 125I lost during each time point was determined, and
time-dependent rates (k ¼ peak rate, minꢀ1) of 125I efflux were
calculated from the following equation: k ¼ ln(125It1/125It2)/(t1ꢀt2),
where 125It is the intracellular 125I at time t, and t1 and t2 are suc-
cessive time points. Relative rates were calculated as kpeakꢀkbasal
(minꢀ1), i.e., the maximal value for the time-dependent rate
(kpeak minꢀ1) excluding the third point used to establish the base-
line (kbasal minꢀ1). Concentration-dependent activation curves
were constructed as a percentage of maximal activation (set at
100%) transformed from the calculated relative rates. CFTR-
dependent iodide efflux was stimulated by forskolin.
C
18H20N5O4: [M þ H]þ 370, found 370, HPLC purity ¼ 95.6%.
4.1.2.10. Synthesis of 9-(30-phenylpropyl)adenine adducts 20a.
20a (808 mg, 2.11 mmol, 25%) obtained as a white solid from 2.00 g
(8.36 mmol) of 9-(30-phenylpropyl)adenine in 40% aqueous solu-
tion of MG and 10 mL of water after 48 h at 50 ꢁC, mp: 149 ꢁC (dec.).
1H NMR (400 MHz, D2O):
d 8.11 (1H, s, CH), 7.76 (1H, s, CH),
6.66e6.62 (5H, m, 5CH), 4.80 (1H, s, CH), 4.02 (2H, t, J ¼ 6.6 Hz,
CH2), 2.43 (2H, t, J ¼ 6.6 Hz, CH2), 2.04 (2H, m, CH2), 2.00 (3H, s,
CH3), 1.46 (3H, s, CH3), 13C NMR (100 MHz, D2O):
d 177.0 (CIV), 151.1
(CHAr), 149.6 (CIV), 147.2 (CIV), 142.0 (CHAr), 139.8 (CIV), 127.4 (CHAr),
127.3 (CHAr), 125.2 (CHAr), 118.6 (CIV), 80.1 (CH), 64.4 (CIV), 43.5
(CH2), 31.8 (CH2), 28.7 (CH2), 26.3 (CH3), 20.2 (CH3), LRMS (FABþ,
NBA) calcd for C18H20N5O4: [M þ H]þ 398, found 398, elemental
analysis calcd for C20H23N5O4: C 60.44, H 5.83, N 17.62, found C
60.32, H 5.94, N 17.54.
4.1.2.11. Synthesis of 9-(30-hydroxypropyl)adenine adducts 21a.
21a (283 mg, 0.84 mmol, 65%) obtained as a white solid from
250 mg (1.29 mmol) of 9-(30-hydroxypropyl)adenine in 40%
aqueous solution of MG and 2 mL of water after 48 h at 50 ꢁC, mp:
4.2.3. Cytotoxicity assay for cell survival
Wt-CFTR CHO cells, grown in 96-well plates, were incubated
103e105 ꢁC (dec.). 1H NMR (400 MHz, D2O):
d
8.58 (1H, s, CH), 8.14
24
h
with different concentrations of drugs. Then, 3-[4,5-
bromide (MTT,
(1H, s, CH), 4.72 (1H, s, CH), 4.34 (2H, m, CH2), 3.57 (2H, m, CH2),
dimethylthiazol-2-yl]-2,5diphenyltetrazolium
2.09e2.00 (5H, m, CH2 and CH3), 1.72 (3H, s, CH3), 1H NMR
5 mg/ml) (Sigma, USA) was added to the cell medium during 4 h.
The absorbance at 570 nm and 630 nm was read. Bars values were
calculated from three independent experiments.
(400 MHz, D2O, 45 ꢁC):
d 8.33 (1H, s, CH), 8.13 (1H, s, CH), 4.95 (1H,
s, CH), 4.36 (2H, d, J ¼ 7.2 Hz, CH2), 3.57 (2H, m, CH2), 2.10 (2H, m,
CH2), 1.91 (3H, s, CH3), 1.74 (3H, s, CH3), 13C NMR (100 MHz, D2O):
d
175.6 (CIV), 152.0 (CIV), 147.9 (CHAr), 141.8 (CHAr), 118.8 (CIV), 97.4
4.2.4. Single-cell fluorescence
(CIV), 79.0 (CH), 63.8 (CIV), 58.0 (CH2), 40.8 (CH2), 32.1 (CH2), 25.7
(CH3), 20.9 (CH3), HRMS (EI) calcd for C14H19N5O5: [M þ H]þ
338.1464, found 338.1472,elemental analysis calcd for C14H18N5O5,
Na, ½ H2O, ½ C3H7OH: C 45.31, H 5.51, N 18.22, found C 45.38, H
5.48, N 18.08.
CFTR activity was assayed by single-cell fluorescence imaging
using the potential-sensitive probe, bis(1,3-diethylthiobarbituric
acid)trimethine oxonol (DisBAC2(3)), Molecular Probes, Eugene,
OR; (for more details see Ref. [37]). The results of fluorescence
imaging are presented as transformed data to obtain the percent-
age signal variation (Fx) relative to the time of addition of the
stimulus, according to the equation: Fx ¼ ([FtꢀF0]/F0] ꢂ 100 where
Ft and F0 are the fluorescent values at the time t and at the time of
addition of the stimulus, respectively. For histogram representa-
tion, the values correspond to the level of stable variation of fluo-
rescence induced by each drug.
4.2. Biological evaluations
4.2.1. Cell culture
For this study, we used Chinese Hamster Ovary (CHO) cells,
stably transfected with pNUT vector containing wild-type CFTR
(wt-CFTR CHO). Cells cultured at 37 ꢁC in 5% CO2 were maintained
in
7% FCS (BIO-Whittaker Europe, Belgium), 50 IU penicillin/ml
(Invitrogen corporation, USA), 50 g streptomycin/ml (Invitrogen
corporation, USA) and 100 M methotrexate (SIGMA chemicals,
USA). The human nasal airway epithelial cell line JME/CF15, derived
from a CF patient homozygous for the F508del mutation, was
passaged once a week, plated in flasks coated with human placental
a
MEM with Glutamax (Invitrogen corporation, USA), containing
4.2.5. Salivary secretion assay
All experiments were performed on male Wistar rats
(250e300 g) according to our previous reports [30,38]. Briefly,
quiescent rats were maintained in supine orientation with adhesive
tape. Subcutaneous injection of GP-act26a was performed in the
cheek and saliva was collected with small pieces of Whatman paper
inserted in the mouth near to the previously injected cheek. Every 3
m
m

464
B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
[5] J.A. Tabcharani, X.B. Chang, J.R. Riordan, J.W. Hanrahan, Phosphorylation-
regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene,
Nature 352 (1991) 628e631.
[6] M.J. Welsh, A.E. Smith, Molecular mechanisms of cftr chloride channel
dysfunction in cystic-fibrosis, Cell 73 (1993) 1251e1254.
or 4 min, a piece of paper was removed and replaced with a second.
At the end of the experiment, the net weight of saliva secreted
during the collection period was evaluated. For all experiments,
basal salivary secretion was inhibited by atropine (1 mM, 50
prior to injection of a saline solution containing atropine (1 mM)
mixed with isoprenaline (10 M), GP-act26a (30 M) or a mixture
isoprenaline (10 M). The secretory rate was
M) þ GP-act26a (30
mL)
[7] H.A. Lewis, X. Zhao, C. Wang, J.M. Sauder, I. Rooney, B.W. Noland, D. Lorimer,
M.C. Kearins, K. Conners, B. Condon, P.C. Maloney, W.B. Guggino, J.F. Hunt,
m
m
S. Emtage, Impact of the DF508 mutation in first nucleotide-binding domain of
human cystic fibrosis transmembrane conductance regulator on domain
folding and structure, J. Biol. Chem. 280 (2005) 1346e1353.
m
m
determined by dividing the weight of sample by the time required
for collection in min. The total saliva secretion was determined by
addition of all the samples collected during the experiment. These
data were then normalized by dividing the result by the weight in
grams of the rat.
[8] A. Szollosi, D.R. Muallem, L. Csanady, P. Vergani, Mutant cycles at CFTR's non-
canonical ATP-binding site support little interface separation during gating,
J. Gen. Physiol. 137 (2011) 549e562.
[9] J.L. Mendoza, A. Schmidt, P.J. Thomas, Introduction to section IV: biophysical
methods to approach CFTR structure, in: M.D. Amaral, K. Kunzelmann (Eds.),
Cystic Fibrosis Methods Mol. Biol., Humana Press, 2011, pp. 321e327.
[10] J.P. Mornon, P. Lehn, I. Callebaut, Atomic model of human cystic fibrosis
transmembrane conductance regulator: membrane-spanning domains and
coupling interfaces, Cell. Mol. Life Sci. 65 (2008) 2594e2612.
4.2.6. Data analysis
Data are presented as mean value S.E.M, where n refers to the
number of isolated cell (single cell measurements), cell populations
(iodide efflux and cAMP measurements) and N to the number of
animals. Sets of data were compared with a Student's t test. All
graphs are plotted with GraphPad Prism 5.0 for Windows (Graph-
Pad Software, San Diego, CA). Values of p < 0.05 were considered as
statistically significant: *p < 0.05; **p < 0.01; ***p < 0.001. No sig-
nificant difference was p > 0.05.
[11] J.P. Mornon, P. Lehn, I. Callebaut, Molecular models of the open and closed
states of the whole human CFTR protein, Cell. Mol. Life Sci. 66 (2009)
3469e3486.
[12] S. Atwell, C.G. Brouillette, K. Conners, S. Emtage, T. Gheyi, W.B. Guggino,
J. Hendle, J.F. Hunt, H.A. Lewis, F. Lu, I.I. Protasevich, L.A. Rodgers, R. Romero,
S.R. Wasserman, P.C. Weber, D. Wetmore, F.F. Zhang, X. Zhao, Structures of a
minimal human CFTR first nucleotide-binding domain as a monomer, head-
to-tail homodimer, and pathogenic mutant, Protein Eng. Des. Sel. 23 (2010)
375e384.
[13] I. Callebaut, R. Eudes, J.P. Mornon, P. Lehn, Nucleotide-binding domains of
human cystic fibrosis transmembrane conductance regulator: detailed
sequence analysis and three-dimensional modeling of the heterodimer, Cell.
Mol. Life Sci. 61 (2004) 230e242.
4.3. Molecular modelling
[14] R. Eudes, P. Lehn, C. Ferec, J.P. Mornon, I. Callebaut, Nucleotide binding do-
mains of human CFTR: a structural classification of critical residues and
disease-causing mutations, Cell. Mol. Life Sci. 62 (2005) 2112e2123.
[15] H.A. Lewis, S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart,
R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, J.F. Hunt, M.C. Kearins,
D. Lorimer, P.C. Maloney, K.W. Post, K.R. Rajashankar, M.E. Rutter, J.M. Sauder,
S. Shriver, P.H. Thibodeau, P.J. Thomas, M. Zhang, X. Zhao, S. Emtage, Structure
Docking studies used the modelled structure of human wild
type CFTR [11]. The protein 3D structure was prepared using Protein
€
Preparation Wizard module from Schrodinger [49]. All compounds
were built and their energies minimized with Schrodinger/
MAESTRO [49], using a conjugate gradient method and MMFF94
force field and charges. Exploratory docking was achieved by VINA
on a large box of 60 Å3, exhaustiveness parameter was 12 and
Energy Range was 5 kcal molꢀ1. Accurate dockings were achieved
using GOLD, VINA and Autodock4 docking software with default
parameters, on limited areas selected from former docking. The
genetic algorithms of GOLD, VINA and Autodock performed flexible
dockings with small molecules while keeping the protein structure
rigid. For each compound, 20 poses were generated by VINA and
GOLD, 100 by Autodock then ranked according to their binding
energy or GOLD-PLP fitness scoring function. Molecular surfaces
and docking solutions were analysed with the PYMOL software
[50].
of nucleotide-binding domain
1 of the cystic fibrosis transmembrane
conductance regulator, EMBO J. 23 (2004) 282e293.
[16] A.W. Serohijos, T. Hegedus, A.A. Aleksandrov, L. He, L. Cui, N.V. Dokholyan,
J.R. Riordan, Phenylalanine-508 mediates a cytoplasmic-membrane domain
contact in the CFTR 3D structure crucial to assembly and channel function,
Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 3256e3261.
[17] D.M. Amaral, M.C. Farinha, Rescuing mutant CFTR: a multi-task approach to a
better outcome in treating cystic fibrosis, Curr. Pharm. Des. 19 (2013)
3497e3508.
[18] V. Birault, R. Solari, J. Hanrahan, D.Y. Thomas, Correctors of the basic traf-
ficking defect of the mutant F508del-CFTR that causes cystic fibrosis, Curr.
Opin. Chem. Biol. 17 (2013) 353e360.
[19] R.A. Denny, L.K. Gavrin, E. Saiah, Recent developments in targeting protein
misfolding diseases, Bioorg. Med. Chem. Lett. 23 (2013) 1935e1944.
[20] A.G. Durmowicz, K.A. Witzmann, C.J. Rosebraugh, B.A. Chowdhury, Change in
sweat chloride as a clinical end point in cystic fibrosis clinical trials: the
ivacaftor experience, Chest 143 (2013) 14e18.
[21] I. Fajac, I. Sermet-Gaudelus, Cystic fibrosis: new treatments targeting the CFTR
protein, Rev. Mal. Respir. 30 (2013) 255e261.
[22] C.M. Farinha, P. Matos, M.D. Amaral, Control of cystic fibrosis transmembrane
conductance regulator membrane trafficking: not just from the endoplasmic
reticulum to the Golgi, FEBS J. 280 (2013) 4396e4406.
[23] G. Faure, N. Bakouh, N. Odolczyk, H. Xu, F. Saul, G. Planelles, P. Zielenkiewicz,
G.L. Lukacs, M. Ollero, A. Edelman, Correcting effect of PLA2 subunit of cro-
toxin on mutated CFTR: original perspective to investigate the pharmaco-
therapy of cystic fibrosis, Toxicon 75 (2013) 212.
Acknowledgements
This work was supported by the French Associations “Vaincre la
Mucoviscidose” (BB) and Mucovie (JB) which are gratefully
acknowledged.
[24] L.R. Hoffman, B.W. Ramsey, Cystic fibrosis therapeutics: the road ahead, Chest
J. 143 (2013) 207e213.
Appendix A. Supplementary data
[25] D. Merk, M. Schubert-Zsilavecz, Repairing mutated proteins-development of
small molecules targeting defects in the cystic fibrosis transmembrane
conductance regulator, Expert Opin. Drug. Discov. 8 (2013) 691e708.
[26] S. Monterisi, V. Casavola, M. Zaccolo, Local modulation of cystic fibrosis
conductance regulator: cytoskeleton and compartmentalized cAMP signalling,
Br. J. Pharmacol. 169 (2013) 1e9.
[27] E. Nieddu, B. Pollarolo, L. Merello, S. Schenone, M. Mazzei, F508del-CFTR rescue:
a matter of cell stress response, Curr. Pharm. Des. 19 (2013) 3476e3496.
[28] T. Ong, B.W. Ramsey, Modifying disease in cystic fibrosis: current and future
therapies on the horizon, Curr. Opin. Pulm. Med. 19 (2013) 645e651.
[29] C. Schwarz, Drug therapy for cystic fibrosis, Arzneimitteltherapie 31 (2013)
80e88.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.028.
References
[1] P.B. Davis, M. Drumm, M.W. Konstan, Cystic fibrosis, Am. J. Respir. Crit. Care
Med. 154 (1996) 1229e1256.
[2] D.C. Gadsby, Ion channels versus ion pumps: the principal difference, in
principle, Nat. Rev. Mol. Cell. Biol. 10 (2009) 344e352.
[30] J. Bertrand, B. Boucherle, A. Billet, P. Melin-Heschel, L. Dannhoffer,
C. Vandebrouck, C. Jayle, C. Routaboul, M.C. Molina, J.L. Decout, F. Becq,
C. Norez, Identification of a novel water-soluble activator of wild-type and
F508del CFTR: GPact-11a, Eur. Respir. J. 36 (2010) 311e322.
[31] T.H. Ma, J.R. Thiagarajah, H. Yang, N.D. Sonawane, C. Folli, L.J.V. Galietta,
A.S. Verkman, Thiazolidinone CFTR inhibitor identified by high-throughput
[3] D.C. Gadsby, P. Vergani, L. Csanady, The ABC protein turned chloride channel
whose failure causes cystic fibrosis, Nature 440 (2006) 477e483.
[4] J.R. Riordan, J.M. Rommens, B. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak,
J. Zielenski, S. Lok, N. Plavsic, J.L. Chou, et al., Identification of the cystic fibrosis
gene: cloning and characterization of complementary DNA, Science 245
(1989) 1066e1073.

B. Boucherle et al. / European Journal of Medicinal Chemistry 83 (2014) 455e465
465
screening blocks cholera toxin-induced intestinal fluid secretion, J. Clin.
Invest. 110 (2002) 1651e1658.
[40] Gold-Suite in, CCDC, Cambridge, UK.
[41] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of
docking with new scoring function, efficient optimization, and multi-
[32] C. Muanprasat, N.D. Sonawane, D. Salinas, A. Taddei, L.J.V. Galietta,
A.S. Verkman, Discovery of glycine hydrazide pore-occluding CFTR inhibitors:
mechanism, structure-activity analysis, and in vivo efficacy, J. Gen. Physiol.
124 (2004) 125e137.
[33] C. Routaboul, C. Norez, P. Melin, M.C. Molina, B. Boucherle, F. Bossard, S. Noel,
R. Robert, C. Gauthier, F. Becq, J.L. Decout, Discovery of alpha-
aminoazaheterocycle-methylglyoxal adducts as a new class of high-affinity
inhibitors of cystic fibrosis transmembrane conductance regulator chloride
channels, J. Pharmacol. Exp. Ther. 322 (2007) 1023e1035.
a
threading, J. Comput Chem. 31 (2010) 455e461.
[42] C. Lambertucci, I. Antonini, M. Buccioni, D.D. Ben, D.D. Kachare, R. Volpini, K.-
N. Klotz, G. Cristalli, 8-Bromo-9-alkyl adenine derivatives as tools for devel-
oping new adenosine A2A and A2B receptors ligands, Bioorg. Med. Chem. 17
(2009) 2812e2822.
[43] J. Thibon, L. Latxague, G. Deleris, Synthesis of silicon analogues of acyclonu-
cleotides incorporable in oligonucleotide solid-phase synthesis, J. Org. Chem.
62 (1997) 4635e4642.
[34] C. Routaboul, L. Dumas, I. Gautier-Luneau, J. Vergne, M.C. Maurel, J.L. Decout,
New stereoselective reaction of methylglyoxal with 2-aminopyridine and
adenine derivatives: formation of imino acid-nucleic base derivatives in water
under mild conditions, Chem. Commun. (Camb) (2002) 1114e1115.
[35] B.L. Renard, B. Boucherle, B. Maurin, M.C. Molina, C. Norez, F. Becq, J.L. Decout,
An expeditious access to 5-pyrimidinol derivatives from cyclic methylglyoxal
diadducts, formation of argpyrimidines under physiological conditions and
discovery of new CFTR inhibitors, Eur. J. Med. Chem. 46 (2011) 1935e1941.
[36] O. Moran, O. Zegarra-Moran, On the measurement of the functional properties
of the CFTR, J. Cyst. Fibros. 7 (2008) 483e494.
[37] C. Norez, F. Antigny, S. Noel, C. Vandebrouck, F. Becq, A cystic fibrosis respi-
ratory epithelial cell chronically treated by miglustat acquires a non-cystic
fibrosis-like phenotype, Am. J. Respir. Cell. Mol. Biol. 41 (2009) 217e225.
[38] S. Noel, C. Faveau, C. Norez, C. Rogier, Y. Mettey, F. Becq, Discovery of pyrrolo
[2,3-b]pyrazines derivatives as submicromolar affinity activators of wild type,
G551D, and F508del cystic fibrosis transmembrane conductance regulator
chloride channels, J. Pharmacol. Exp. Ther. 319 (2006) 349e359.
[44] W. Edward Lindsell, C. Murray, P.N. Preston, T.A.J. Woodman, Synthesis of 1,3-
diynes in the purine, pyrimidine, 1,3,5-triazine and acridine series, Tetrahe-
dron 56 (2000) 1233e1245.
[45] V.I. Petrov, A.A. Ozerov, M.S. Novikov, C. Pannecouque, J. Balzarini, E. De
Clercq, 9-(2-Aryloxyethyl) derivatives of adenine e a new class of non-
nucleosidic antiviral agents, Chem. Heterocycl. Compd. 39 (2003)
1218e1226.
[46] M. Nielsen, A.H. Thomsen, E. Clo, F. Kirpekar, K.V. Gothelf, Synthesis of linear
and tripoidal oligo(phenylene ethynylene)-based building blocks for appli-
cation in modular DNA-programmed assembly, J. Org. Chem. 69 (2004)
2240e2250.
[47] M.D. Merten, W. Kammouni, W. Renaud, F. Birg, M.G. Mattei, C. Figarella,
A transformed human tracheal gland cell line, MM-39, that retains serous
secretory functions, Am. J. Respir. Cell. Mol. Biol. 15 (1996) 520e528.
[48] C. Norez, G.D. Heda, T. Jensen, I. Kogan, L.K. Hughes, C. Auzanneau, R. Derand,
L. Bulteau-Pignoux, C. Li, M. Ramjeesingh, H. Li, D.N. Sheppard, C.E. Bear,
J.R. Riordan, F. Becq, Determination of CFTR chloride channel activity and
[39] C. Marivingt-Mounir, C. Norez, R. Derand, L. Bulteau-Pignoux, D. Nguyen-Huy,
B. Viossat, G. Morgant, F. Becq, J.-M. Vierfond, Y. Mettey, Synthesis, SAR,
crystal structure, and biological evaluation of benzoquinoliziniums as acti-
vators of wild-type and mutant cystic fibrosis transmembrane conductance
regulator channels, J. Med. Chem. 47 (2004) 962e972.
pharmacology using radiotracer flux methods, J. Cyst. Fibros.
119e121.
3 (2004)
€
[49] Maestro, in, Schrodinger LLC.
€
[50] PyMOL Molecular Graphics System, in, Schrodinger, LLC.