Discovery of N-(3-Carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-lH-pyrazole-5-carboxamide (GLPG1837), a Novel Potentiator Which Can Open Class III Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels to a High Extent

Article abstract of DOI:10.1021/acs.jmedchem.7b01288

Cystic fibrosis (CF) is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). With the discovery of Ivacaftor and Orkambi, it has been shown that CFTR function can be partially restored by administering one or more small molecules. These molecules aim at either enhancing the amount of CFTR on the cell surface (correctors) or at improving the gating function of the CFTR channel (potentiators). Here we describe the discovery of a novel potentiator GLPG1837, which shows enhanced efficacy on CFTR mutants harboring class III mutations compared to Ivacaftor, the first marketed potentiator. The optimization of potency, efficacy, and pharmacokinetic profile will be described.

Full text of DOI:10.1021/acs.jmedchem.7b01288

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Drug Annotation
Discovery of N-(3-carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-
thieno[2,3-c]pyran-2-yl)-lH-pyrazole-5-carboxamide (GLPG1837), a
Novel Potentiator which can open Class III Mutant Cystic Fibrosis
Transmembrane Conductance Regulator (CFTR) Channels to a high extent.
Steven Emiel Van der Plas, Hans Kelgtermans, Tom De Munck, Sébastien Laurent Xavier Martina,
Sébastien Dropsit, Evelyne Quinton, Ann De Blieck, Caroline Joannesse, Linda Tomaskovic, Mia
Jans, Thierry Christophe, Ellen Van der Aar, Monica Borgonovi, Luc Nelles, Maarten Gees, Pieter
FW Stouten, Jan Van Der Schueren, Oscar Mammoliti, Katja Conrath, and Martin J. Andrews
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01288 • Publication Date (Web): 17 Nov 2017
Downloaded from http://pubs.acs.org on November 17, 2017
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Discovery of N-(3-carbamoyl-5,5,7,7-tetramethyl-
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,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-lH-
pyrazole-5-carboxamide (GLPG1837), a Novel
Potentiator which can open Class III Mutant Cystic
Fibrosis Transmembrane Conductance Regulator
(CFTR) Channels to a high extent.
Steven E. Van der Plas†*, Hans Kelgtermans†, Tom De Munck†, Sébastien L. X. Martina†, Sébastien
║
Dropsit†, Evelyne Quinton†, Ann De Blieck†§, Caroline Joannesse†, Linda Tomaskovic , Mia Jans†,
Thierry Christophe†, Ellen van der Aar†, Monica Borgonovi‡, Luc Nelles †, Maarten Gees†, Pieter
Stouten†, Jan Van Der Schueren†, Oscar Mammoliti†, Katja Conrath†*, Marꢀn Andrews†
†Galapagos NV, Generaal De Wiꢀelaan L11 A3, 2800 Mechelen, Belgium
‡
Galapagos SASU, 102 avenue Gaston Roussel, 93230 Romainville, France
║
Fidelta Ltd., Prilaz baruna Filipovića 29, Zagreb, HR-10000, Croatia
KEYWORDS
Cystic fibrosis, CFTR, potentiator
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ABSTRACT
Cystic fibrosis (CF) is caused by mutations in the gene for the cystic fibrosis transmembrane conductance
regulator (CFTR). With the discovery of Ivacaftor and Orkambi, it has been shown that CFTR function can
be partially restored by administering one or more small molecules. These molecules aim at either
enhancing the amount of CFTR on the cell surface (correctors) or at improving the gating function of the
CFTR channel (potentiators). Here we describe the discovery of a novel potentiator GLPG1837 which
shows enhanced efficacy on CFTR mutants harboring Class III mutations compared to Ivacaftor, the first
marketed potentiator. The optimization of potency, efficacy and pharmacokinetic profile will be
described.
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INTRODUCTION
Cystic fibrosis (CF) is the most common life-threatening autosomal recessive disorder in Caucasian
1
populations, affecting approximately 70,000 patients worldwide. It is caused by mutations of the gene
2
for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The ion channel CFTR is
involved in the gating of chloride ions across epithelial cell membranes in various tissues including the
lung, pancreas, intestine, reproductive tract and sweat duct. Although CF is a systemic disease, the most
severe effects come from the reduced chloride secretion in the airways. Subsequent lung surface
dehydration leads to the buildup of a thick mucus layer, which clogs the airways and traps bacteria
leading to infections, extensive lung damage and eventually, respiratory failure. Most treatments like
antibiotics and mucus-clearing agents are symptomatic and hence there is a great need for disease
3
modifying agents.
4
More than 2,000 mutations of the CFTR gene are known. These CFTR defects have been grouped in 6
5
classes based on their defective mechanisms. The most common mutation, F508del, belongs to the
class II trafficking defects, where folding of the CFTR protein is impaired, resulting in a reduction of the
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amount of ion channel present on the cell surface. With the G551D mutation (class III), the amount of
6
protein is not affected but its open probability (P ) is reduced, resulting as well in a reduced channel
o
gating. To address the underlying causes of cystic fibrosis, two biomolecular activities are required,
namely correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective
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opening of the CFTR channel. For patients with the F508del mutation, a combination of correctors and
potentiator will be necessary to restore CFTR function. For patients with the G551D mutation, a
potentiator alone has been shown to be effective. In 2012, 1 (Ivacaftor) was approved as a monotherapy
7
for patients who carry the G551D mutation and has recently been approved for additional mutations
8
influenced by a potentiator alone. This was followed in 2015 by the approval of Orkambi, a combination
of Ivacaftor and the corrector 3-[6-[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropanecarbonyl]amino]-
9
3
-methyl-2-pyridyl]benzoic acid (VX-809), for cystic fibrosis patients with 2 copies of the F508del allele.
This treatment results in only minor benefit to the patients and thus there is a demand for more
10
effective therapies to further improve clinical benefit for CF patients with the F508del mutation.
RESULTS AND DISCUSSION
To check for compounds which can restore chloride transport mediated by the CFTR ion channel, a
CFTR dependent yellow fluorescent protein (YFP) halide assay was developed. A bronchial epithelial cell
line derived from a CF patient (CFBE41o- cells) was used to overexpress F508del CFTR and yellow
11
fluorescent protein. In order to have enough F508del CFTR on the membrane, cells were incubated at
2
7°C. Alternatively, activity on different class III and IV mutants was measured in HEK293 cells,
expressing the respective mutant CFTR and YFP. The CFTR activity is proportional to the quenching of
the YFP related fluorescence and the % activation is a measure for the extent or efficacy of channel
opening.
The search for novel potentiators started by performing a screen on 589 commercial compounds
which were selected based on similarity around existing potentiators (Figure 1. Overview potentiators).
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From this screen, the most active hits were found around compound 2 (P5, Figure 1), a potentiator
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described by Verkmann et al. Relative to Ivacaftor, the hit showed good starting potencies on F508del
CFTR but failed to restore the chloride transport through G551D CFTR (Figure 2). In terms of molecular
properties, the compound was perceived as a low molecular weight starting point, albeit having a
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calculated lipophilicity XLogP3 >4. In line with the lipophilic nature of the compound, it showed high
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unbound clearance in the rat liver microsomal stability assay.
OH
OH
O
O
S
O
O
O
O
O
O
N
N
N
H
H
HO
O
N
H
N
H
OH
Genistein
SF-01
1
Ivacaftor (VX-770)
H N
O
2
O
NH
N
N
Cl
NH
H
O
N
N
S
H
O
OH
2
P5
PG-01
VRT-532
15
Figure 1. Overview potentiators. Genistein has been used as a general tool compound. Compounds
sulfonamide-01 (SF-01), phenylglycine-01 (PG-01) and 2 were described by Verkmann et al. to have
16,12
17
potentiating activites.
Phenyl-pyrazole (VRT-532) has been disclosed by Vertex Pharmaceuticals.
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In terms of chemical exploration, 2 was considered as an interesting starting point. The core was readily
built up by a Gewald reaction (Scheme 2), allowing exploration of different scaffolds by simply varying
the starting ketone. Keeping the high rat microsomal clearance in mind, initial exploration focused at
installing a hetero-atom into the cyclohexyl ring to improve the unbound clearance.
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OH
H N
2
O
O
O
Cl
NH
N
S
H
O
N
H
1
2
Ivacaftor
F508del: 326nM, 71%
G551D: >10ꢀM, 18%
MW/xLogP/PSA: 335/4.1/100
Clint,unb,ꢀsome,rat (L/h/kg): 84
F508del: 126nM, 100%
G551D: 1300nM, 50%
MW/xLogP/PSA: 393/5.59/78
Figure 2. Comparison of 2 with Ivacaftor.
Introduction of a basic nitrogen resulted in the loss of activity (Table 1, 4). The ether analogue 5 showed
activity comparable to 3 but with an improved polarity. Further potency improvements were made by
introducing steric bulk next to the oxygen atom (6, 7 and 8). Compound 6 was the least potent while 7
and 8 had similar potencies and efficacies on the F508del mutant. On G551D CFTR however, 8 was more
efficacious in opening the ion channel as represented by the higher % activation. Therefore, the
tetramethyl-tetrahdyropyran (THP) scaffold was chosen for as the starting point for further exploration.
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Table 1. Activity in YFP halide assay of selected compounds.
H N
H N
2
2
O
O
NH
S
NH
S
S
0
1
2
3
4
5
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0
O
O
HO
A
B
Cmpd
R
Amide
group
XLogP3
3.8
F508del
EC , % activity
G551D
EC , % activity
50
50
3
4
5
A
A
A
652nM, 72%
NA, 3%
-
-
-
2.1
HN
O
2.3
778nM, 74%
6
7
B
B
2.7
3.0
13nM, 93%
4nM, 98%
490nM, 51%
236nM, 36%
O
O
8
B
3.3
4nM, 111%
162nM, 52%
O
Using the novel tetramethyl-THP scaffold, the amide SAR was more thoroughly explored. Although 8
had good potency and efficacy on the 2 tested CFTR mutants, the phenolic moiety was considered as an
issue because of the risk of phase II metabolism. A broad exploration was undertaken, but in general it
appeared that groups containing a proton donor were consistently more active. Table 2 summarizes the
SAR collected by targeting groups containing an acid proton, mimicking the phenol group of 8. Close
isosters like 9 and 10 showed a >40 fold loss in potency. Installing small heterocycles with an acidic
proton like the triazole 11 showed an even greater loss in potency. The pyrazole 12 also was much less
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active, but its isomer 13 appeared to very active on the F508del mutant. The elongated compound 14
had similar activity to 12, showing that the position of the proton donor and acceptor as present in 13 is
crucial for good activity.
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1
1
1
1
1
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2
2
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Table 2. Activity in YFP halide assay on F508del CFTR of selected amides.
H N
2
O
NH
O
S
R
O
Cmpd
9
R
F508del
Cmpd
R
F508del
EC (nM)
EC (nM)
50
50
O
190
147
15
16
101
NH
HO
N
NH
10
>10,000
HN
S
O
O
O
H
N
1
1
1
1
2
3
467
125
3
17
18
19
20
23
16
15
75
N
HO
HO
N
NH
N
H
N
N
HO
H
N
14
136
N
HO
3
3 18
In effort to increase the fraction sp ( fsp ), lactams and saturated alcohols were explored to see
whether the H-bonding pattern present in 8 and 13 could be mimicked. In this case, a big difference in
potency between the 2 lactam isomers 15 and 16 was observed. Nevertheless, 15 still showed a > 30
fold loss in potency compared to 13. More successfully, alcohols 17, 18 and 19 showed good potencies
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<
25 nM. In contrast to the sharp difference in potency between 12, 13 and 14, it seemed that the
position of the alcohol was less crucial.
From this amide exploration, 2 subseries emerged as being very promising, namely the pyrazoles and
alcohols. These were further expanded and profiled against both the F508del and the G551D CFTR
mutant.
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Figure 3. YFP halide assay profiling of the alcohol and pyrazole subseries on the F508del and G551D
CFTR. Green dots represent pyrazoles and yellow dots alcohols. A) shows the potencies against F508del
and G551D CFTR. B) shows the correlation between the potency and efficacy on the G551D CFTR for
both subseries.
For both subseries, it was possible to get to compounds with potencies <10 nM on the F508del CFTR.
For the F508del mutation, the % activation, a measure for the extent or efficacy of channel opening, was
between 90-110% (data not shown) for most compounds. Although there was a good correlation
between the potencies for F508del and G551D, most of the compounds were in general less potent for
the G551D than for the F508del CFTR. This is consistent with the erosion in potency of Ivacaftor for
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F508del vs G551D CFTR (own data in Figure 2 and reference 6c). Whereas it was difficult to discriminate
the 2 subseries based on their F508del profile, the pyrazole subseries consistently showed to be more
potent and more effective in opening the G551D CFTR channel (Figure 3). Indeed, it looked as if the
alcohol subseries was less effective in opening the G551D CFTR. This is represented best with alcohol 23
in Table 3. Although the compound had a sub-nanomolar potency on F508del, it was only 981 nM on
G551D, reflecting almost a 2000 fold loss in potency. In addition, the % activation of 23, but also the
other alcohols 19, 21 and 22, was significantly lower than most pyrazoles. Also, the chirality of the
alcohols had a strong impact on the potency. The ‘S’ enantiomer 21 is 47 and 18 times more potent on
F508del and G551D CFTR respectively than its ‘R’ isomer.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
0
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9
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9
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8
9
0
Starting from pyrazole 13, the SAR around the heterocycle was further explored. In general,
substitution on the pyrazole either on the 3 or 4 position, did improve the potency for G551D CFTR 2 to
4 fold (24, 25 and 26). For F508del CFTR, no benefit was obtained with these substitutions. However,
improving the potency did coincide with a reduction in efficacy. Compound 26 was the most striking
example with an efficacy reduced to 49%. These changes did not influence potency on F508del CFTR.
Double substitution as in the case of 28 resulted in a similar profile as the mono-substituted pyrazoles.
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Summary of the SAR around the alcohols and pyrazoles.
H N
2
O
NH
O
S
R
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
F508del
G551D
F508del
G551D
Cmpd
R
EC50
,
EC50
,
Cmpd
R
EC50
,
EC50,
%
activity
% activity
% activity
% activity
H
N
15 nM,
05%
1,320 nM,
57%
3 nM,
105%
339 nM,
103%
1
2
9
1
13
25
N
N
1
HO
H
N
2
.7 nM,
5%
421 nM,
50%
2 nM,
96%
123 nM,
83%
9
OH
4
H
N
1
27 nM,
95%
7,777 nM,
31%
3 nM,
107%
130 nM,
49%
N
2
2
26
3
OH
CF3
H
N
0
.54 nM,
02%
981 nM,
58%
13 nM,
81%
590 nM,
73%
N
N
23
27
28
1
3
OH
H
N
H
N
2 nM,
96%
81 nM,
77%
11 nM,
92%
199 nM,
68%
2
4
N
4
Cl
3
Cl 4
In the end, 13 showed to have the most interesting profile, having an EC of 3 nM and 339 nM on
50
F508del and G551D CFTR respectively. Upon benchmarking 13 against Ivacaftor, which had an efficacy of
0% (Figure 2) for G551D CFTR, it seemed that 13 was twice as efficacious as Ivacaftor in the YFP halide
5
19
G551D assay. This observation was confirmed in a Trans-epithelial Clamp Circuit (TECC) assay using
primary patient cells. The increased conductivity of the G551D CFTR channel by compound 13 was
nearly 200% compared that of Ivacaftor (Figure 4). The obtained potency of 13 was 181 nM. This is the
first time, to our knowledge, that a novel potentiator is reported that shows an improved potency and
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efficacy in G551D primary human bronchial epithelial (HBE) cells compared to the current standard of
care Ivacaftor. A detailed mechanistic study has been published recently, showing that 13 increases the
20
P0 of CFTR .
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
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4
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5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. G551D/F508del HBE cells treated with Ivacaftor and 13.
This feature of improved conductivity was further investigated on the class III gating mutations G178R,
S549N and the class IV conductance mutation R117H For these mutant CFTR forms, it has been shown
8,21
that the addition of Ivacaftor can restore CFTR function. . In many (but not all) cases the tetramethyl-
THP potentiators were able to improve the defect to a greater extent when compared to Ivacaftor
(
Figure 5). Compound 13 was on all mutations more potent and showed a higher efficacy when
compared to Ivacaftor. The extent of the increased efficacy varies between the different mutations
158% for G178R, 143% for S549N and 119% for R117H).
(
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0
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9
0
1
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3
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Progression of potency (Y-axis) and efficacy (% activation to control, X-axis) of potentiator
series on various CFTR mutants (A. G178R; B. S549N, C. R117H) in YFP halide assay. Top right corner of
each panel shows dose response curve of 13 (black) and Ivacaftor (grey).
Compound 13 had an attractive in vitro ADME profile, showing low Clint,unb in both microsomal and
hepatocytes stability assays, good permeability and no off target inhibition of CYPs and the hERG
channel (Table 4). On the other hand, the thermodynamic solubility was low at neutral and acidic pH,
but this did not limit the bioavailability (F%) at the 5 mg/kg dose. Moreover, the pharmacokinetic profile
of 13 was attractive, showing a low Cl,unb and good F% in both rat and dog (Table 5).
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Table 4. Physicio-chemical and in vitro ADME parameters for 13.
MW/XLogP/PSA/Fsp3
348 / 1.8 / 138 / 0.44
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
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1
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0
Clint,unb, µsome (L/h/kg) (r,h)
Clint,unb, hep (L/h/kg) (r,h)
1.7 / 1.1
5.7 / 1.8
CYP inhibition (1A2, 2C19, 2C9, 2D6, 3A4)
>100 for all tested
CYPs
(
EC , µM)
50
hERG (manual Patch Clamp, EC µM)
>33
50
Thermodynamic ASOL (pH 7.4, 3.0) (µM)
MDCK
8.6 / 11
11 / 2.6
-
6
-1
(
A2B cm.10 sec , Efflux ratio)
Table 5. Pharmacokinetic properties of 13 in rat and dog. IV parameters were determined using a 1
mg/kg dose. F% was determined after a PO dose of 5 mg/kg.
Cl
Cl,unb
Vd
T1/2
(h)
F%
(L/h/kg) (L/h/kg)
(L/kg)
Rat PK
Dog PK
1.92
0.32
24
4.8
1.4
1.84
3.0
67
6.4
>100
However, due to the low thermodynamic solubility, the observed exposures at 100mg/kg and 300
mg/kg in rat were lower than expected (data not shown). The calculated lipophilicity of 13 was 1.8,
indicating that the low solubility was not originating from the lack of polarity and related poor solvation
22
(
‘grease ball molecules’). On the contrary, with a melting point of >300°C, 13 could be considered as a
‘
brick dust’ molecule. The crystal lattice seemed to be stabilized by strong inter-molecular hydrogen
bonds originating from the carboxamide and the pyrazole ring (Figure 6Error! Reference source not
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2
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2
2
3
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6
found.). Both groups are a donor-acceptor pair in terms of hydrogen bonding, allowing efficient
hydrogen bonding with a donor-acceptor
0
1
2
3
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9
0
1
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3
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9
0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. X ray structure of 13. Sulfur atoms are coloured yellow, oxygens red, nitrogens blue, carbons
grey and hydrogen white.
pair from another molecule. The X ray analysis also showed that there was extensive intra-molecular
bonding. Not surprisingly, the 2 amides in the molecule were locked up by an internal hydrogen bond.
Additionally, the carbonyl of the amide ortho on the thiophene ring, was held planar by an intra-
molecular interaction where the carbonyl electrons were interacting with the electrophilic sulfur atom
23
of the thiophene ring. This explained why a compound with high PSA value of 138, still had very good
permeability as measured in the MDCK assay (Table 4). Due to the intra-molecular interactions, one
hydrogen bond donor and 2 hydrogen bond acceptors are shielded, lowering the effective PSA.
In order to improve the solubility, a phosphate prodrug strategy was applied (Scheme 1). This approach
is well documented and has proven to improve solubility and exposure for a range of compounds
24
administered by either an intravenous (IV) or oral (PO) route. The phosphate prodrug is selectively
25
cleaved in the gut by alkaline phosphatases, located at the brush border of the epithelial cells. Upon
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26
cleavage, formaldehyde and the parent compound are released. In addition of improving the solubility,
the phosphate prodrug has the advantage of only having the parental compound systemically exposed.
In all PK experiments performed with the prodrug 29, the prodrug could never be detected in the
plasma.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Phosphate prodrug 29 and its mechanism of cleavage
Na+
ꢁO
Oꢁ
P
O
Na+
H N
H N
2
2
O
O
O
Phosphatase enzymes
gut, apical brush border
O
H
N
+
NH
N
NH
N
N
O
O
S
S
H
H
O
O
29
13
When comparing the exposure of 13 via its prodrug 29, it seemed that at 5 mg/kg, there was no
difference in obtained Cmax and AUC. This implies, that at the lower dose, the low solubility of 13 is not
limiting its exposure. On the other hand, an increase in both Cmax and AUC was observed when dosing
3
00mg/kg, showing that the phosphate prodrug strategy was effective at addressing the low solubility
and low F% at higher doses.
In a parallel effort, various solid state formulations were explored and in the end, a nanosuspension of
13 was developed leading to similar results as obtained with 29 (data not shown).
Table 6. Pharmacokinetic properties of 13 in rat at higher doses. Comparison of the AUCs of 13 obtained
after administration of the parental compound directly, or the prodrug 29 is given.
1
3
13 via prodrug 29
Rat PK (5mg/kg)
3
31/1,540
741/1,562
Cmax (ng/ml)/ AUC (ng*hr/ml))
Rat PK (300mg/kg)
4,674/68,170
12,378/192,264
Cmax (ng/ml)/ AUC (ng*hr/ml))
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6
With an improved formulation in hand, 13 was nominated as GLPG1837 and selected for pre-clinical
safety evaluation after which it was progressed into a phase I study to evaluate its PK and safety in
humans. Compound 13 was generally safe and well tolerated in healthy volunteers, with single doses up
to 2000 mg and 14-day dosing up to 800mg b.i.d. The efficacy was explored in 2 phase II studies the
SAPHIRA I and SAPHIRA II, for which data will be reported soon.
0
1
2
3
4
5
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8
9
0
1
2
3
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8
9
0
1
2
3
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8
9
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0
1
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0
CHEMISTRY
a
Scheme 2. Synthesis of the main scaffold 31 by a Gewald reaction
H N
2
O
O
O
O
a)
N
b)
+
+
S8
NH2
H N
O
2
S
O
Phorone
3
1
3
0
a
Reagents and conditions: (a) 1M HCl, 40°C, 6 days, 45%; (b) S8, diethylamine, EtOH, 40°C, 18h, 35%.
The core 31 was synthesized by a multi-component Gewald reaction (Scheme 2) in which the ketone
2
7
3
0 reacts with sulfur and cyanoacetamide in the presence of a base. Although 30 was commercially
available, the first large quantities were prepared by stirring the readily available Phorone at 40°C in
acidified water.
Derivatisation of the scaffold 31 was done by amide bond formation. It was found that the
28
Mukaiyama reagent (2-chloro-1-methylpyridinium iodide, CMPI ) gave the most consistent results,
allowing the coupling of great variety of different amides (Scheme 3).
a
Scheme 3. Synthesis of pyrazole derivatives using the Mukaiyama reagent
H N
H N
2
2
O
O
O
H
a)
N
H
+
HO
NH
N
NH2
N
N
O
O
S
S
R'
O
3
1
R'
a
Reagents and conditions: (a) 2-chloro-1-methylpyridinium iodide, DMAP, TEA, DCM, 50°C, 2h.
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For the synthesis of the alcohols, generally the alcohol function needed to be masked to guarantee a
successful amide coupling. One option was to couple the corresponding ketone or ester, followed by
reduction (Scheme 4). Alternatively, the alcohol was first protected, before engaging into the amide
coupling. This is exemplified for compound 21 in Scheme 5. Here, an acetate protecting group was used
which could be subsequently removed by hydrolysis.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 4. Synthesis of alcohol 19
H N
2
H N
H N
2
2
O
O
O
HO
O
+
a)
b)
NH
NH2
NH
O
O
O
O
S
S
3
S
O
1
O
O
O
OH
32
O
19
a
Reagents and conditions: (a) 2-chloro-1-methylpyridinium iodide, DMAP, TEA, DCM, 50°C, 2h; (b)
LiBH , dry THF, 0° -> rT, 32%.
4
a
Scheme 5. Synthesis of alcohol 21
O
O
a)
HO
HO
H
2
N
2
H N
O
O
OH
O
O
3
3
b)
c)
NH
NH
O
O
S
S
O
O
O
OH
2
H N
O
34
O
21
NH
2
O
S
31
a
Reagents and conditions: (a) ethanoly chloride, pyridine, DCM, 0°C - rT (b) 2-chloro-1-
methylpyridinium iodide, DMAP, TEA, DCM, 40°C, 16h (c) LiOH.H O, THF, water, 40°C, 16h.
2
For the synthesis of the phosphate prodrug 29, the di-tert-butyl chloromethyl phosphate group was
introduced on the pyrazole ester by alkylation (Scheme 6). This gave rise to two regio-isomers that could
be separated by column chromatography. Hydrolysis with LiOH gave the corresponding acid that was
coupled to 31 using the Mukaiyama reagent. Once coupled, the tBu protecting groups were removed
under very mild conditions.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 6. Synthesis of the phosphate prodrug 29 .
O
O
O
P
O OtBu
O P
O
O O
P
a)
OtBu
H
N
N
O
+
N
O
O
O
+
Cl
O
O
OtBu
N
O
OtBu
N
O
N
35
36
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b)
Na+
Oꢁ
OtBu
P
OtBu
O
O
Oꢁ
Na+
H N
P
H N
2
2
O
O
O
O O
P
d)
c)
OtBu
N
NH
O
N
NH
O
N
HO
O
N
O
N
OtBu
S
N
S
2
9
38
37
a
Reagents and conditions: (a) Cs CO , DMA, 55°C, 35% after column chromatography; (b) LiOH (1 M),
THF/MeOH/H2O, 45°C, 99%; (c) 31, Mukaiyama reagent, DMAP, Et N, DCM, 45°C, 99%; (d)
2
3
3
acetone/water, 45°C, 51%.
CONCLUSION
Starting from the described potentiator 2 a novel potentiator series was discovered using the tetra-
methyl-THP scaffold. In contrast to 2, the novel series was able to efficiently potentiate G551D CFTR.
During lead optimization, two subseries (alcohols and pyrazoles) were thoroughly explored. Upon
benchmarking against Ivacaftor, it seemed that the pyrazole subseries consistently showed an improved
restoration of the channel activity of G551D CFTR. In the end, the best efficacy was obtained with the
unsubstituted pyrazole 13. The enhanced efficacy was confirmed with a Tecc experiment on primary
human G551D/F508del bronchial epithelial cells. Here, 13 not only induced a current which was almost
2
00% compared to Ivacaftor, but was in the same time also more potent. Hence, 13 represents a novel
potentiator with improved potency and efficacy compared to the current standard of care, Ivacaftor.
Phase II evaluation of 13 in patients recently finished and data will be reported soon.
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EXPERIMENTAL SECTION
Yellow Fluorescent Protein (YFP) halide assays
CFBe41o- cells were cultured in Eagle’s minimal essential medium (MEM) (Life Technologies)
supplemented with 10 % fetal bovine serum (FBS), 1 % penicillin/streptomycin, 1 % L-glutamine and 500
µg/ml hygromycin B. The cells were grown on culture flasks coated with 0.01 % bovine serum albumin
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
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0
1
2
3
4
5
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9
0
1
2
3
4
5
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9
0
1
2
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0
1
2
3
4
5
6
7
8
9
0
(
BSA) (Sigma), 30 µg/ml Purecol (Nutacon) and 0.001 % human fibronectin (Sigma). CFBe41o- cells were
transduced with adenoviruses containing F508del CFTR and YFP (H148Q/I152L/ F47L). HEK293 cells were
cultured in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 10% FBS
and 1 % penicillin/streptomycin. HEK293 cells were transfected with plasmids containing G551D, G178R,
,
S549N, R117H, CFTR and YFP (H148Q/I152L/F47L). Directly after transfection, the HEK293 cells were
seeded in black 96-well plates coated with Poly-D-lysine at a density of 70,000 cells per well. The next
day, cells were incubated for 24h at 27°C (CFBE41o-) or 37°C (HEK293). Then, cells were treated for 10
min with 10 µM forskolin and the desired concentration of potentiator at room temperature. The YFP
fluorescence was recorded during 7 s (CFBE41o-) or 2 min (HEK293), starting immediately before
addition of NaI buffer (CFBE41o-: 137 mM NaI, 2.7 mM KI, 1.7 mM KH2PO4, 10.1 mM Na2HPO4, 5 mM
D-glucose, HEK923: (375 mM NaI, 7.5 mM KI, 1.76 mM KH2PO4, 10.1 mM Na2HPO4, 13.75 mM glucose)
to the wells, using a fluorescence reader. The capacity of potentiators to increase CFTR channel function
was expressed as 1-(fluorescence after NaI addition (F) / fluorescence before NaI addition (F0)).
TECC experiments
Trans-epithelial clamp circuit (TECC) recordings were performed using the TECC instrument developed
and sold by EP Design (Bertem, Belgium). During the recording,the epithelia were bathed in a NaCl-
Ringer solution (120 mM NaCl, 20 mM HEPES, 1.2 mM CaCl2, 1.2 mM MgCl2, 0.8 mM KH2PO4, 0.8 mM
K2HPO4, 5 mM glucose, pH 7.4) on both the basolateral and the apical side and kept at 37°C. Apical
amiloride was used to inhibit the endogenous epithelium sodium channel (ENaC) currents (100 µM)
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while forskolin (10 µM) was applied on both the apical and basolateral sides to stimulate CFTR. For acute
potentiator experiments, compounds were added on both sides to test their potential for increasing
CFTR gating. Measurements were done during a 20-minute timeframe with recordings every 2 minutes.
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The transepithelial potential (PD) and transepithelial resistance (R ) were measured in an open circuit
t
and transformed to I using Ohm’s law. The increase in I (Δ I) was used as a measure for the increased
eq
eq
CFTR activity. EC50 values were generated by measuring impact of different concentrations of
compound on I in primary cells. For this purpose each transwell was treated with a different
eq
compound concentration. CFTRinh-172 was used at 10 µM to assess the specificity of the tested
compounds. Each concentration of compound was tested in duplicate or triplicate, and average and
standard error of the mean (SEM) were calculated.
Chemistry
All reagents were of commercial grade and were used as received without further purification, unless
otherwise stated. Commercially available anhydrous solvents were used for reactions conducted under
inert atmosphere. Reagent grade solvents were used in all other cases, unless otherwise specified.
Column chromatography was performed on silica gel 60 (35-70 µm). Thin layer chromatography was
1
carried out using pre-coated silica gel F-254 plates (thickness 0.25 mm). H NMR spectra were recorded
on a Bruker DPX 400 NMR spectrometer (400 MHz) or a Bruker Advance 300 NMR spectrometer (300
1
MHz). Chemical shifts (δ) for H NMR spectra are reported in parts per million (ppm) the appropriate
residual solvent peak, i.e. CHCl (δ 7.27), as internal reference. Multiplicities are given as singlet (s),
3
doublet (d), triplet (t), quartet (q), quintuplet (quin), multiplet (m) and broad (br). Microwave heating
was performed with a Biotage Initiator.
Ultra-violet and electrospray MS spectra were obtained on a Waters Acquity H-Class UPLCwhich was
coupled to a Waters Mass detector QDA or ZQ2000 and an Acquity PDA Detector (210-400nm). All final
compounds reported were analyzed using one of these analytical methods and were at least 95% pure.
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Columns used: Waters Acquity UPLC BEH C18 1.7µm, 2.1mm ID x 50mm L or Waters Acquity UPLC CSH
C18 2.1x50mm 1.7 µm. The methods are using MeCN/H O gradients (H O contains either 0.1% TFA or 15
2
2
mM NH3).
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Racemic mixtures were separated on an Agilent HP1100 system with UV detection. Column used:
Chiralpak 1A (10x250 mm, 5µm). Solvents used: iPrOH and tBME. Alternatively, separation was done
using a SFC2 system. Column used: Lux Cellulose-4. Solvents used: CO2 and MeOH. Enantiomeric purity
was determined on an Agilent HP1100 system with UV detection. Column used: Chiralpak IA (4.6x250
mm, 5µm). Solvents used: iPrOH and tBME.
N-(3-carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-5-
carboxamide (13). Compound 31 (15 g, 59 mmol) and 2H-pyrazole-3-carboxylic acid (9.9 g, 88 mmol)
were suspended in DCM (250 mL). Next, Mukaiyama reagent (2-chloro-1-methylpyridinium iodide) (18.1
g, 71 mmol), TEA (24.7 mL, 177 mmol) and DMAP (3.6 g, 29 mmol) were added. The reaction mixture
was stirred at 40°C overnight and then cooled. The mixture was evaporated and the obtained crude was
suspended in a 1 M HCl solution. After stirring for 10 min, the suspension was filtered and obtained
precipitate was isolated. This precipitate was re-suspended in a 0.1 M citric acid solution. Again,
filtration gave a precipitate. A third trituration was done using ether as a solvent to give a precipitate
after filtration. Finally, the precipitate (13.6 g) was suspended in EtOH (816 mL) and heated at reflux. To
this suspension, 65 mL of DMF was added and a clear solution was obtained. The solution was
concentrated to 275 mL and cooled at 0°C. A suspension was obtained, the solid was separated by
1
filtration, and the cake was dried affording the desired product. H NMR δ (ppm)(DMSO-d6) : 13.55 (1 H,
s br), 12.53 (1 H, s br), 7.94 (1 H, s br), 7.54 (1 H, s br), 7.10 (1 H, s br), 6.79 (1 H, s), 2.73 (2 H, s), 1.45 (6
+
H, s), 1.20 (6 H, s), 1.45 (6 H, s). LC−MS: m/z = 371 [M + Na] .
2
-(1-(hydroxymethyl)cyclopropanecarboxamido)-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-
c]pyran-3-carboxamide (19). Compound 31 (2 g, 7.9 mmol) was mixed with dimethyl 1,1-
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cyclopropanedicarboxylate (CAS: 6914-71-2, 1.33 g, 10.2 mmol), DMAP (480 mg, 3.9 mmol), Mukaiyama
reagent (2.4 g, 9.4 mmol) and triethylamine (3.3 mL, 24 mmol) in DCM (25 mL) under N . The mixture
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was stirred at 40°C overnight, after which the mixture was evaporated. A yellow precipitate was
obtained that was suspended in water. Subsequent filtration gave a yellow powder. This was re-
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suspended in 0.1 M HCl , followed by filtration to give an off-white solid. The obtained solid was
aq
dissolved in DCM and dried over MgSO . In the end, 3.4 g (113%) of compound 32 was obtained which
4
was used as such. LC-MS: m/z = 381 [M+H].
Compound 32 (100 mg, 0.26 mmol) was weighed together with LiBH (17 mg, 0.79 mmol) in a flask.
4
The flask is put in an ice bath and dry THF (2 mL) was slowly added. After stirring for 5 min, the ice bath
was removed and the mixture was stirred at room temperature. Reaction was subsequently quenched
with 2 M HCl, diluted with water and extracted with EtOAc. The organic phase was evaporated and the
1
obtained residue was purified by preparative chromatography, yielding 29 mg white powder (32%). H
NMR δ (ppm)(400 MHz,CDCl ): 12.42 (s, 1H), 5.74 (br. s., 2H), 3.81 (d, J = 4.9 Hz, 2H), 3.02 (t, J = 5.0 Hz,
3
1
+
H), 2.64 (s, 2H), 1.49 (s, 6H), 1.32 - 1.40 (m, 2H), 1.27 (s, 6H), 0.84 - 0.92 (m, 2H). LC−MS: m/z = 353 [M
H].
2
-[[(2S)-2-hydroxy-3,3-dimethyl-butanoyl]amino]-5,5,7,7-tetramethyl-4H-thieno[2,3-c]pyran-3-
carboxamide (21). To a solution of (2S)-2-hydroxy-3,3-dimethyl-butanoic acid (0.76 g, 5.8 mmol) in
dichloromethane (20 ml) were added successively pyridine (1 ml, 11.5 mmol) and ethanoyl chloride
(
drop wise, 821 µl, 11.5 mmol) at 0°C. The reaction mixture was stirred 1hour at room temperature. The
mixture was diluted with water followed by extraction with dichloromethane. The combined organic
layers were washed with water and brine, dried over Na SO , filtered and concentrated in vacuo to
2
4
afford crude (2S)-2-acetoxy-3,3-dimethyl-butanoic acid (33) which was used as such in the next step. LC-
MS: m/z= 175.1 [M+H].
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Compound 31 (1.5 g, 5.8 mmol) was added to a solution of 33 (1.5 g, 5.8 mmol), 2-chloro-1-
methylpyridinium iodide (2.2 g, 8.7 mmol), triethylamine (2.4 mL, 17.4 mmol) and 4-
dimethylaminopyridine (350 mg, 2.9 mmol) in dichloromethane (40 mL). The resulting reaction mixture
was heated at 40°C for 16 h. After cooling to room temperature, 10% citric acid aqueous solution was
added and the reaction mixture was extracted with dichloromethane. The combined organic layers were
washed with water and brine, dried over Na SO , filtered and concentrated in vacuo. to afford crude
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[(1S)-1-[(3-carbamoyl-5,5,7,7-tetramethyl-4H-thieno[2,3-c]pyran-2-yl)carbamoyl]-2,2-dimethyl-propyl]
acetate (34) which was used as such in the next step. LC-MS: m/z= 411.2 [M+H].
Lithiumhydroxide monohydrate (486 mg, 11.6 mmol) was added to a solution of crude 34 (2.36 g, 5.8
mmol) in THF (15 ml) and water (3 ml). The reaction mixture was stirred for 16h at 40°C. Organic solvent
was removed under reduced pressure and the remaining solution was acidified to pH = 1 with 1 N HClaq
solution. The resulting mixture was extracted with DCM. The combined organic layers were washed with
water and brine, dried over Na SO , filtered and concentrated in vacuo to afford crude 2-[[(2S)-2-
2
4
hydroxy-3,3-dimethyl-butanoyl]amino]-5,5,7,7-tetramethyl-4H-thieno[2,3-c]pyran-3-carboxamide (21).
Crude mixture was purified by column chromatography on silica gel (eluens: petroleum ether/ethyl
acetate: 75/25) and subsequent crystallisation from EtOH to afford pure title compound (398 mg, 18%).
LC-MS: m/z= 369.1 [M+H]. ¹H NMR δ (ppm)(DMSO-d6): 11.98 (s, 1H), 7.49 (br.s, 1H), 6.93 (br.s, 1H), 6.19
(
d, J = 5.2 Hz , 1H), 3.73 (d, J = 5.2 Hz, 1H), 2.70 (d, 2H), 1.43 (s, 6H), 1.19 (d, J = 8.8 Hz, 6H), 0.93 (s, 9H).
Disodium phosphoric acid mono-[5-(3-carbamoyl-5,5,7,7-tetramethyl-4,7-dihydro-5H-thieno[2,3-
c]pyran-2-ylcarbamoyl)-pyrazol-1-ylmethyl] ester (29). To a solution of phosphoric acid di-tert-butyl
ester chloromethyl ester (46.2 g, 179 mmol) in DMA (425 mL) under N , 1H-pyrazole-3-carboxylic acid
2
ethyl ester (25 g, 179 mmol) and Cs CO (73 g, 223 mmol) were added (portion wise) at room
2
3
temperature. The resulting turbid mixture was stirred at 55°C under N overnight. Once reaction was
2
finished (determined by LC-MS), the mixture was allowed to reach room temperature, added to water
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(
800 mL) and extracted with Et2O (~5x 200 mL). The combined organic fractions were washed with
water/brine (~4x 500 mL), dried over Na SO and concentrated in vacuo. The two regioisomers 35 and
2
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6 were separated by means of silica chromatography (columns were pre-treated with Et N in
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petroleum ether to avoid deprotection of the tert-butyl groups) with a gradient (100% petroleum ether
to 50% EtOAc in petroleum ether), yielding the targeted isomer 36 (17 g, 26%) which was identified via
1
NOESY experiments (see SI). H NMR (400 MHz, DMSO-d6) δ (ppm) 8.03 (d, J = 2.5 Hz, 1H), 6.81 (s, 1H),
5
.90 (d, J = 11.6 Hz, 2H), 4.28 (q, J = 7.1 Hz, 2H), 1.37 (s, 18H), 1.28 (t, J = 7.1 Hz, 3H). LC-MS: m/z = 363
[M+H].
To a solution of 2-(di-tert-butoxy-phosphoryloxymethyl)-2H-pyrazole-3-carboxylic acid ethyl ester 36
18.6 g, 51.3 mmol) in THF/MeOH/H O (270 mL, 54 mL, 54 mL), LiOH (1.29 g, 53.9 mmol) was added.
(
2
The resulting mixture was stirred at 45°C under N for 2 hours. After cooling down, mixture diluted with
2
water and acidified to pH=3-4 (HCl, 2M). Aqueous phase was extracted with Et O whilst maintaining pH
2
at 3-4. The combined organic fractions were dried over Na SO and concentrated in vacuo. Compound
2
4
3
7 was obtained as a white solid (17 g, 99%) and used as such in the next reaction. LC-MS: m/z = 335
[
M+H].
To a solution of 37 in CH Cl (350 mL), 2-chloro-1-methylpyridinium iodide (17.7 g, 69.3 mmol), 4-
2
2
dimethylaminopyridine (7.06 g, 57.8 mmol) and Et N (12.1 mL, 13.1 mmol) were added at room
3
temperature. The resulting suspension was stirred for 10 minutes at room temperature, after which 31
was added and finally heated at reflux for 24 hours under N . After completion, reaction was cooled
2
down, added to water and extracted with CH Cl . Combined organic fractions were washed with HCl
2
2
(
0.1M, 2x), dried over Na SO and concentrated in vacuo, yielding 38 as a dark red viscous oil which was
2 4
used as such. LC-MS: m/z = 571 [M+H].
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To a solution of 38 (26.5 g, 46.4 mmol) in acetone (280 mL), water (280 mL) was added. The resulting
mixture was stirred at 45°C in a RB flask under N2 for 24 h, after which the mixture was alkalized with
NaOH (5M) to pH=~10. The aqueous mixture was washed with Et O twice, acidified to pH=~2 (HCl, 2M)
2
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and finally extracted with EtOAc. During extraction a precipitate was formed which was collected via
filtration and combined with the concentrated organic fractions. The resulting solid was taken up again
in water (100 mL), filtered and rinsed with water, Et O and acetone, yielding the title product as a white
2
1
solid (10.8 g, 51%). H NMR (400 MHz, DMSO-d6) δ (ppm) 12.65 (s, 1H), 7.78 (d, J = 2.0 Hz, 1H), 7.68 (s,
1H), 7.22 (s, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.18 (d, J = 8.3 Hz, 2H), 2.75 (s, 2H), 1.47 (s, 6H), 1.22 (s, 6H). LC-
MS: m/z = 459 [M+H].
2
,2,6,6-tetramethyltetrahydro-4H-pyran-4-one (30). Phorone or 2,6-dimethyl-2,5-heptadien-4-one
(
CAS: 504-20-1, 750 g) was mixed with an aqueous 1 M HCl solution (8 L) and the obtained emulsion is
stirred at 40°C for 6 days. The water phase is extracted with DCM, and the organic phase is concentrated
1
and purified by distillation to afford the desired product as an oil (448.8 g, 45%). H NMR (CDCl ):2.42 (s,
3
.
3
4
H), 1.39 (s, 12H). GC-MS: m/z= 141 [M-CH ].
2-Amino-5,5,7,7-tetramethyl-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylic acid amide (31). To a
flask containing 30 (32.3 g, 206.8 mmol), cyanoacetamide (17.4 g, 208 mmol) and sulfur (6.0 g, 186.1
mmol) were stirred in EtOH (85 mL). Diethylamine (23.5 mL, 227.4 mmol) was added and the reaction
was stirred at 40°C overnight. The reaction was diluted with water (200 mL) and partially concentrated
by evaporation causing the precipitation of a solid that is separated by filtration. The cake is then
1
washed with water (2 x 150 mL) to afford the desired product (18.4 g, 35%). H NMR (DMSO-d6): 6.80 (s,
+
1
H), 6.51 (br. s., 1H), 2.49 (s, 1H), 1.25 (s, 3H), 1.10 (s, 3H). LC-MS: m/z= 238 [M-H 0]
2
ASSOCIATED CONTENT
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Preparation and characterization for additional final compounds, NOESY analysis of 35 and 36 are
available at http://pubs.acs.org.
AUTHOR INFORMATION
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Corresponding Authors
*
Steven E. Van der Plas: phone, +3215342900; e-mail: steven.vanderplas@glpg.com
*Katja Conrath: phone, +3215342900; e-mail: katja.conrath@glpg.com
REFERENCES
1
https://www.cff.org/What-is-CF/About-Cystic-Fibrosis, last accessed on Nov 13, 2017.
2
(
a) Lubamba, B.; Dhooghe, B.; Noel, S.; Leal, T. Cystic fibrosis: insight into CFTR pathophysiology and
pharmacotherapy. Clin Biochem. 2012, 15, 1132-1144. (b) Hanrahan, J. W.; Sampson, H. M.; Thomas, D.
Y. Novel pharmacological strategies to treat cystic fibrosis. Trends Pharmacol Sci. 2013, 34, 119-125. (c)
Merk, D.; Schubert-Zsilavecz, M. Repairing mutated proteins--development of small molecules targeting
defects in the cystic fibrosis transmembrane conductance regulator. Expert Opin Drug Discov. 2013, 8,
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91-708. (d) Riordan J.R.; Rommens J.M.; Kerem R.S.; Alon N.; Rozma-Hel R.; Grzel- czak Z.; Zielenski J.;
Lok S.; Plavsic N.; Chou J.L.; Drumm M.L.; Iannuzzi C.; Collins F.S.; Tsui L.C. Identification of the cystic
fibrosis gene: cloning and characterization of complementary DNA. Science 1989, 245, 1066– 1073.
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(
a) Schmidt, B.; Haaf, J.; Leal, T.; Noel, S. Cystic fibrosis transmembrane conductance regulator
modulators in cystic fibrosis: current perspectives. Clin. Pharmacol. Adv. Appl. 2016, 8, 127–140. (b)
Lopes-Pacheco, M. CFTR modulators: shedding light on precision medicine for cystic fibrosis. Front.
Pharmacol. 2016, 7, 1–20.
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http://www.genet.sickkids.on.ca/Home.html, last accessed on Nov 13, 2017.
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Amaral, M. D.; Farinha, C. M. Rescuing mutant CFTR: a multi-task approach to a better outcome in
treating cystic fibrosis. Curr. Pharm. Des. 2013, 19 (19), 3497–3508.
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(a) Hadida, S.; Van Goor, F.; Zhou, J.; Arumugam, V.; McCartney, J.; Hazlewood, A.; Decker, C.;
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Van Goor, F.; Hadida, S.; Grootenhuis, P. D. J.; Burton, B.; Stack, J. H.; Straley, K. S.; Decker, C. J.; Miller,
M.; McCartney, J.; Olson, E. R.; Wine, J. J.; Frizzell, R. A.; Ashlock, M.; Negulescu, P. A. Correction of the
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F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc. Natl. Acad. Sci.
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Wainwright, C.E.; Elborn, J.S.; Ramsey, B.W.; Marigowda, G.; Huang, X.; Cipolli, M.; Colombo, C.;
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XlogP3 is a program for the prediction of the octanol/water partition coefficients of organic
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coefficients, J.Chem.Inf.Comput.Sci. 1997, 37, 615-621.
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To obtain meaningful drug concentrations for the prediction of intrinsic clearance, non-specific
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fraction unbound in hepatocyte incubations (Fu,inc). (a) Obach, R.S. The importance of nonspecific
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implications for in vitro-in vivo correlations. Drug. Metab. Dispos., 1996, 24, 1047-1049. (b) Gertz, M.;
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Hwang, T.C.; Wang, F.; Yang, I.C.; Reenstra, W.W Genistein potentiates wild-type and delta F508-CFTR
channel activity. Am. J. Physiol., 1997, 273, C988-C998.
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Pedemonte, N.; Sonawane, N. D.; Taddei, A.; Hu, J.; Zegarra-moran, O.; Suen, Y. F.; Robins, L. I.; Dicus,
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Van Goor, F.; Straley, K. S.; Cao, D.; Gonzalez, J.; Hadida, S.; Hazlewood, A.; Joubran, J.; Knapp, T.;
Makings, L. R.; Miller, M.; Neuberger, T.; Olson, E.; Panchenko, V.; Rader, J.; Singh, A.; Stack, J. H.; Tung,
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The TECC instrument is developed and sold by EP Design (Bertem, Belgium), and measures the
potential difference (PD) and transmembrane resistance (R ) over epithelial cells and these values are
t
used to calculate the apparent short circuit current (I ) using Ohms law. The I can be interpreted as
eq
eq
the charge flow per time when the tissue is short circuited i.e. the transepithelial voltage V is clamped
t
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