Discovery of Icenticaftor (QBW251), a Cystic Fibrosis Transmembrane Conductance Regulator Potentiator with Clinical Efficacy in Cystic Fibrosis and Chronic Obstructive Pulmonary Disease

Article abstract of DOI:10.1021/acs.jmedchem.1c00343

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel are established as the primary causative factor in the devastating lung disease cystic fibrosis (CF). More recently, cigarette smoke exposure has been shown to be asso

Full text of DOI:10.1021/acs.jmedchem.1c00343

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Drug Annotation
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Discovery of Icenticaftor (QBW251), a Cystic Fibrosis
Transmembrane Conductance Regulator Potentiator with Clinical
Efficacy in Cystic Fibrosis and Chronic Obstructive Pulmonary
Disease
Darren Le Grand, Martin Gosling, Urs Baettig, Parmjit Bahra, Kamlesh Bala, Cara Brocklehurst,
Emma Budd, Rebecca Butler, Atwood K. Cheung, Hedaythul Choudhury, Stephen P. Collingwood,
Brian Cox, Henry Danahay, Lee Edwards, Brian Everatt, Ulrike Glaenzel, Anne-Lise Glotin,
Paul Groot-Kormelink, Edward Hall, Julia Hatto, Catherine Howsham, Glyn Hughes, Anna King,
Julia Koehler, Swarupa Kulkarni, Megan Lightfoot, Ian Nicholls, Christopher Page, Giles Pergl-Wilson,
Mariana Oana Popa, Richard Robinson, David Rowlands, Tom Sharp, Matthew Spendiff, Emily Stanley,
Oliver Steward, Roger J. Taylor, Pamela Tranter, Trixie Wagner, Hazel Watson, Gareth Williams,
Penny Wright, Alice Young, and David A. Sandham*
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ABSTRACT: Mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR) ion channel are established as the
primary causative factor in the devastating lung disease cystic
fibrosis (CF). More recently, cigarette smoke exposure has been
shown to be associated with dysfunctional airway epithelial ion
transport, suggesting a role for CFTR in the pathogenesis of
chronic obstructive pulmonary disease (COPD). Here, the
identification and characterization of a high throughput screening
hit 6 as a potentiator of mutant human F508del and wild-type CFTR channels is reported. The design, synthesis, and biological
evaluation of compounds 7−33 to establish structure−activity relationships of the scaffold are described, leading to the identification
of clinical development compound icenticaftor (QBW251) 33, which has subsequently progressed to deliver two positive clinical
proofs of concept in patients with CF and COPD and is now being further developed as a novel therapeutic approach for COPD
patients.
amount of protein at the membrane is unaffected, but its open
probability (Po) is reduced, also resulting in a reduced channel
gating.4 Thus, to address the underlying causes of CF, two
distinct CFTR modulators are required: correctors to increase
CFTR levels at the plasma membrane and potentiators to enable
effective opening of the channel.4
Significant progress in the CFTR modulator field was initiated
by Vertex with the 2011 registration of the potentiator ivacaftor
15 (Figure 1) for patients with the G551D mutation followed by
the 2015 approval of the fixed dose combination of ivacaftor
with the corrector lumacaftor (Orkambi) for homozygous
INTRODUCTION
■
Cystic fibrosis (CF) is the most prevalent life-threatening
Mendelian disorder in Caucasian populations.1 CF arises from
mutations of the gene for the cystic fibrosis transmembrane
conductance regulator (CFTR) protein.2 The CFTR ion
channel orchestrates gating of chloride and bicarbonate ions
across epithelial cell membranes in various tissues, including the
lung, pancreas, intestine, reproductive tract, and sweat glands.
While CF is a systemic disorder, the primary mortality derives
from reduced CFTR activity in the airways. Subsequent
acidification3 and dehydration leads to accumulation of a
viscous mucus layer, occluding the airways and trapping
bacteria, leading to infections, reduced lung function, and
ultimately, respiratory failure. The most common CFTR
mutation, F508del (Class II, found in 90% of CF patients),
impairs folding of the CFTR protein (a Class II trafficking
defect), resulting in a reduced amount of channel present at the
plasma membrane. With the G551D mutation (class III), the
Received: February 24, 2021
Published: May 24, 2021
https://doi.org/10.1021/acs.jmedchem.1c00343
© 2021 American Chemical Society
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Drug Annotation
Figure 1. Selected reported CFTR potentiators 1−5 and high throughput screening hit 6.
F508del CF patients.6 Most recently, the addition of a second
site CFTR corrector agent to a potentiator−corrector
combination was shown to give improvement in lung function
in both hetero- and homozygous F508del CF patients, leading to
the 2019 US registration of the ivacaftor−tezacaftor−elexacaftor
(Trikafta) fixed dose combination.7 Further CFTR potentia-
tor−corrector combinations are in development from AbbVie,
with the clinical stage potentiators GLPG1837 28 and
GLPG2451 39 (Figure 1) disclosed, and the broader field of
CFTR potentiator discovery has also been recently reviewed.10
Chronic obstructive pulmonary disease (COPD) is antici-
pated to shortly become the third leading cause of death
globally.11 COPD is characterized by persistent airflow
obstruction12 with cigarette smoke exposure recognized as the
primary risk factor.13 Airflow limitation is associated with all
COPD patients; however, the disease is heterogeneous, with
variable phenotypes ranging from chronic bronchitis (CB) to
emphysema.13 Small airway disease exhibits increased numbers
of goblet cells and mucus plugging with associated smooth
muscle hyperplasia, airway fibrosis, and increased inflamma-
tion.14,15 Excess mucus secretion is believed to play an important
role in COPD pathogenesis16,17 and is associated with
progression of the disease.18−21
Smoking-induced acquired dysregulation of CFTR may
contribute to COPD pathogenesis. In vitro, cigarette smoke
condensate induced CFTR dysfunction results in reduction of
airway surface liquid (ASL) and decreased mucociliary transport
(MCT).22−25 In clinical studies, cigarette smokers and COPD
patients show reduced CFTR function both in upper and lower
airways as well as systemically, which is associated with CB.26−29
Collectively, the importance of mucus hypersecretion, chronic
airway inflammation, and recurrent infections in both COPD
and CF suggests that acquired CFTR dysfunction may
contribute to the pathophysiology of COPD, particularly in
COPD patients with CB.
reduces the risk of exacerbations in COPD patients with CB)
suggests that CFTR activation may contribute to the drug’s
mechanism of action via phosphorylation of the CFTR
regulatory domain and partial restoration of CFTR-dependent
ion transport in cigarette smoke-exposed epithelial cells.30 Prior
to the clinical data in COPD reported herein (vide infra), only
one small pilot study of two weeks in duration with ivacaftor in
COPD patients revealed nonstatistically significant improve-
ments in sweat chloride, nasal potential difference, and symptom
scores compared with placebo.31
Here, the identification and characterization of a high
throughput screening hit 6 as a potentiator of human F508del
and wild-type CFTR channels is reported. The design, synthesis,
and biological evaluation of compounds 7−33 to establish
structure−activity relationships (SAR) of the scaffold are
described, leading to the identification of clinical development
compound icenticaftor (QBW251) 33, which has subsequently
progressed to deliver two positive clinical proofs of concept in
patients with CF and COPD and is now being further developed
as a novel therapeutic approach for COPD.
RESULTS AND DISCUSSION
■
At the outset of CFTR modulator drug discovery activities at
Novartis in the mid-2000s, CF was envisaged as the primary
indication, and a screening strategy based around the most
prevalent CFTR F508del mutation was devised with the known
CFTR potentiators genistein 432 and PG-01 533 (Figure 1)
initially used as reference compounds. A high throughput screen
(approximately 1 million compounds) was conducted using
CFTR F508del stably expressed in human CHO-K1 cells,
determining changes in membrane potential due to CFTR-
mediated ion efflux in response to the adenylate cyclase activator
forskolin. A fluorophore dye partitions across the cell membrane
according to the resting potential; upon efflux of Cl− mediated
by CFTR (and consequent depolarization of the cells), the dye
accumulates inside the cell, where it binds to intracellular
proteins, and the increased fluorescence signal is measured using
a fluorimetric imaging plate reader (FLIPR).34 The assay was
also temperature corrected to facilitate membrane insertion of
the channel.35 This FLIPR assay proved to be a robust and cost-
Preclinical data with the CFTR potentiator ivacaftor indicates
restoration of CFTR-dependent ion transport in cigarette
smoke-exposed epithelial cells and improvement of the
downstream effects of epithelial function.25 Preclinical data
with the phosphodiesterase 4 inhibitor roflumilast (which
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Drug Annotation
effective primary screen which identified multiple hits that were
further characterized via automated 384 well planar patch clamp
electrophysiology (IonWorks Quattro), using the same CFTR
F508del CHO cell line.
A further, higher fidelity, lower throughput characterization of
ion transport properties was also performed, measuring changes
in short circuit current (ΔIsc) in Ussings chambers with CFTR
F508del expressed in Fisher Rat Thyroid (FRT) cells, in parallel
with the same determination on wild-type (WT) CFTR
expressed in FRT cells. An acceptable correlation between
electrophysiology and ion transport assays was found on a test
set of validated hit compounds (Figure 2), providing confidence
for use of the Quattro assay as the primary screen for subsequent
compound optimization.
solubility measurements <0.001 g/L and equilibrium dialysis
experiments using rat plasma confirming a high level of plasma
protein binding at 99.5%. High in vitro clearance in the rat
microsomal assay was confirmed in vivo, with a short intravenous
mean residence time (MRT) of less than 15 min, reflecting a
moderate level of plasma clearance combined with a small
volume of distribution. The high levels of nonspecific binding to
plasma proteins reduced the scope to achieve unbound drug
concentrations in plasma consistent with the free drug
hypothesis without substantially increasing the affinity for the
target or the dose administered. Despite this unfavorable
biopharmaceutical profile, the overall lipophilic ligand efficiency
(LLE)37 of 3.9 (Table 2) based on the Quattro assay potency,
combined with the low molecular weight represented an
acceptable starting point for hit-to-lead chemistry. These
considerations prompted exploration of opportunities to achieve
the desired drug exposure metric through a combination of
increasing binding affinity for CFTR and reducing lipophilicity.
Such an approach by definition focused on increasing the LLE of
the compound class while minimizing liver microsomal
clearance. Furthermore, in the absence of an available animal
PK−PD or disease model for CF, maximization of free drug
exposure was set as a key objective for identification of a
development candidate.
To establish the minimum pharmacophore for CFTR
potentiator activity and identify a lead compound with optimal
ligand efficiency, the scaffold was simplified to identify the key
groups required for binding site recognition. Initially retaining
the methyl ester, the furan ring of 6 with its known potential for
reactive metabolite formation38 was first targeted (Table 3).
Excising the furan to afford the 4-trifluoromethyl substituted
anthranilate ester 7 gave a drop in potency of 23-fold but with
similar efficacy to 6. Further pruning of the scaffold to the
unsubstituted anthranilate 8 abolished all activity, and
exploration of a further range of substituents at the 5-position
gave either inactive or minimally active compounds (data not
shown), leading to retention of the trifluoromethyl group in all
later analogues. The potencies of the iodo synthetic
intermediate 9 and 5- and 6-membered aromatic heterocycles
10 and 11 derived from it, together with the saturated
heterocycle 12, established from the outset the broad tolerance
of this position to a range of substituents. There was little
improvement in rat microsomal clearance with these early
analogues, although solubility was markedly improved with the
heterocycles 10−12. As anticipated, solubility was also
improved by insertion of a nitrogen atom to give the picolinate
analogue 13, at the expense of >50 fold potency loss.
Figure 2. Correlation of automated electrophysiology (Quattro) with
ion transport (FRT) assay potency for F508del CFTR.
With this paradigm, the anthranilate derivative 6 was
identified as one of a number of validated hits with superior
potency to genistein and PG-01 (Table 1) in F508del CFTR and
evidence for potentiation of WT CFTR channels.
Additional profiling to evaluate the general pharmacology of
compound 6 showed it to be selective for the potentiation of
CFTR against a broad panel of 46 G-protein coupled receptor,
enzyme, transporter, and ion channels (including hERG) as well
as 20 kinase targets with no observed inhibition >50% at 10 μM
concentration (Supporting Information Tables S2 and S3). In
addition, a negative result was obtained in a mini-Ames test, in
line with the reported nonmutagenicity of other anthranilates.36
Initial assessment of the biopharmaceutical properties of
compound 6 (Table 2) showed the impact of the high
lipophilicity on solubility and free fraction, with thermodynamic
While the ester group of 6 itself was hydrolytically stable in
plasma, the corresponding acid 14 was readily prepared by basic
hydrolysis and found be >50 fold less potent than 6 (Table 4).
To avoid routine screening of new compounds for plasma
stability, replacement of the ester group with amides was next
studied (Table 4). With an emphasis on identifying compounds
Table 1. CFTR Assay Characterization of HTS Hit 6 in Comparison to Genistein 4 and PG-01 5
CFTR F508del CHO membrane
CFTR F508del CHO Quattro
CFTR F508del FRT ion transport
CFTR WT FRT ion transport
a
b
b
b
b
compound
potential EC50 (μM) (% efficacy )
EC50 (μM) (% efficacy )
EC50 (μM) (% efficacy )
EC50 (μM) (% efficacy )
4
5
6
8.20 (83)
0.39 (100)
0.92 (154)
8.48 (88)
1.25 (100)
0.078 (93)
6.17 (54)
0.260 (100)
0.180 (100)
14.2 μM (138)
0.99 μM (100)
30 μM (206)
c
a
b
c
Data is mean of n ≥ 2. Efficacy relative to Emax of PG-01 set at 100%. Efficacy at a single concentration of 30 μM.
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Table 2. Physicochemical, Biopharmaceutical, and Pharmacokinetic Properties of Compound 6
c
rat in vivo PK
a
b
solubility pH 6.8
(g/L)
PAMPA permeability logPe
rat Clint
(μL/min/mg)
rat PPB
(%)
Cl
Vdss
(L/kg)
MRT
(hr)
Fpo
(%)
MW clogP LLE
285 3.2 3.9
pH 6.8
(mL/min/kg)
<0.001
−4.5
351
99.5
30
c
0.4
0.2
15
a
b
Parallel artificial membrane permeability assay. Plasma protein binding determined by equilibrium dialysis. I.v. dose 0.5 mg/kg, p.o. dose 5 mg/
kg (suspension), vehicle details in Table S4 (Supporting Information).
Table 3. Initial Optimization of HTS Hit 6 Retaining the Methyl Ester
a
b
c
compound
R1
R2
X
dF508 Quattro EC50 (μM) (% efficacy ) rat Clint (μL/min/mg) solubility pH 6.8 (g/L)
LLE
6
7
8
9
10
11
12
13
CF3
CF3
H
CF3
CF3
CF3
CF3
CF3
2-furan
H
H
I
2-oxazole
3-pyridine
( )-2-tetrahydrofuran
2-furan
CH
CH
CH
CH
CH
CH
CH
N
0.078 (98)
3.78 (72)
>30 (0)
0.362 (104)
0.240 (92)
0.329 (100)
0.462 (90)
5.31 (72)
351
nd
nd
342
239
217
185
>700
<0.001
<0.001
nd
0.002
0.036
0.056
0.006
0.011
3.9
2.9
<2.6
3.5
3.8
3.2
3.3
2.4
d
a
b
c
Data is mean of n ≥ 2 determinations. Efficacy relative to PG-01 maximal effect. Microsomal clearance with NADPH cofactor representing CYP
d
mediated metabolism. nd: not determined.
Table 4. Replacements for the Methyl Ester of HTS Hit 6
a
c
dF508 Quattro EC50 (μM)
rat Clint
solubility pH 6.8
(g/L)
b
compound
R1
R2
X
(% efficacy )
(μL/min/mg)
LLE
6
OMe
OH
NHMe
NMe2
NHMe
NH(CH2)2CF3
( )-NHCH2CH(OH)Me
(S)-NHCH2CH(OH)(CF3)
2-furan
2-furan
2-furan
2-furan
Br
Br
Br
Br
CH
CH
CH
CH
N
N
N
N
N
0.078 (98)
5.83 (63)
1.86 (83)
351
201
205
nd
100
55.8
37.7
23
<0.001
nd
0.013
nd
nd
0.049
0.12
0.290
0.177
3.9
2.4
3.1
<1.9
4.5
4.5
5.0
5.2
5.6
d
14
15
16
17
18
19
20
21
>30 (37)
0.250 (97)
0.040 (132)
0.070 (79)
0.023 (102)
(R)-NHCH2CH(OH)(CF3) Br
0.009 (100)
37.1
a
b
c
Data are mean of n ≥ 2 determinations. Efficacy relative to PG-01 maximal effect. Microsomal clearance with NADPH cofactor representing
d
CYP mediated metabolism. nd: not determined.
Table 5. Rat in Vivo Pharmacokinetic and in Vitro Protein Binding Profiles in Plasma of Selected Compounds
Fpo
(%)
AUClast
(nmol/h/L)
Cmax
(nM)
C
max (nM)
CL
Vdss
(L/kg)
MRT
(h)
PPB
(%, human)
a
b
b
compound
[unbound]
(mL/min/kg)
PPB (%, rat)
c
21
22
29
33
32
114
75
652
7739
5929
271
634
595
nd
25
11
53
17
15
5
3.2
6.5
6.4
3.0
1.0
6.4
7.2
nd
nd
97.5
98.6
96.9
98.1
90.3
d
90
20 635
1364
132
10.2
92.3
a
b
Doses i.v. 1 mg/kg (except 21 0.5 mg/kg) and p.o. 3 mg/kg (suspension); vehicle details in Table S4 (Supporting Information). Plasma protein
c
d
binding determined by equilibrium dialysis. nd: not determined. Determined with [14C]-33 (synthesis to be reported separately).
having improved lipophilic ligand efficiency, simple amide
derivatives of both the anthranilate and picolinate scaffolds were
explored. Synthesis of the N-methyl and N-dimethyl amide
derivatives 15 and 16 clearly indicated a preference for the
secondary amide 15. The retention of some activity in the iodo
ester analogue 9 (vide supra) led to consideration of bromine as a
less reactive alternative, together with the combination of ester
replacement and the potentially improved properties of the
picolinate core. Thus, a key advance was made with 6-bromo
picolinate N-methyl amide 17, suggesting divergence from the
SAR of the anthranilate scaffold. With these data available, a
library of commercially available primary amines was employed
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Table 6. Lead Optimization of 21 to Incorporate a Tertiary Alcohol
a
b
c
Data are mean of n ≥ 2 determinations. Efficacy relative to PG-01 maximal effect. Microsomal clearance with NADPH cofactor representing
d
CYP mediated metabolism. nd: not determined.
Figure 3. In vivo metabolism of compound 22 in the rat.
to elaborate 17, resulting in identification of the first sub-100 nM
potency potentiators 18−20, with some improvement in
microsomal clearance compared to earlier compounds. Re-
placement of the methyl group of 19 with trifluoromethyl in
enantiomers 20 and 21 gave a 10-fold improvement in activity
over the N-methyl amide 17 and a further reduction in the
microsomal clearance, with the (R) enantiomer 21 being
marginally more active.
clearance in vitro, 21 showed a high clearance rate in vivo that
limited its oral bioavailability and overall exposure with a short
mean residence time of 1 h. Although metabolism of 21 was not
characterized, it was speculated that the secondary alcohol could
be cleared through direct glucuronidation of the hydroxyl group,
limiting the compounds systemic exposure. In addition, possible
metabolic oxidation of the secondary alcohol to the trifluor-
omethyl ketone derivative was considered, which might then
undergo reversible covalent interactions with nucleophilic
amino acid side chains that could potentially lead to
The oral pharmacokinetic profile of 21 was established in
Sprague−Dawley rats (Table 5). Despite the reduced intrinsic
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Table 7. Final Optimization of Advanced Lead 22
a
b
c
d
compound
R
stereochemistry
dF508 Quattro EC50 (μM) (% efficacy )
rat Clint (μL/min/mg)
solubility pH 6.8 (g/L)
LLE
22
26
27
Br
H
Me
Et
Et
(S)
0.015 (107)
0.229 (78)
0.240 (99)
0.008 (100)
0.043 (101)
0.009 (113)
0.276 (91)
0.111 (95)
0.020 (89)
0.046 (93)
0.015 (116)
0.047 (96)
5.3
0.140
nd
nd
0.091
nd
0.082
0.013
nd
0.165
nd
5.3
4.7
4.5
5.4
4.2
5.5
4.0
4.3
4.9
4.5
5.5
5.2
e
( )
( )
ent-1
ent-2
(S)
ent-1
ent-2
ent-1
ent-2
(S)
<25
73.8
85.6
nd
7.38
<25
nd
102
114
39
28a
28b
29
30a
30b
31a
31b
32
CF3
MeNH
MeNH
Me2N
Me2N
EtO
0.06
0.237
33
MeO
(S)
11
a
b
ent-1 and ent-2 denote single enantiomers of undetermined stereochemistry reported in order of preparative chiral SFC elution. Data are mean of
c
d
n ≥ 2 determinations. Efficacy relative to PG-01 maximal effect. Microsomal clearance with NADPH cofactor representing CYP mediated
metabolism. nd: not determined.
e
Table 8. Further CFTR Ion Transport Characterization of 22 and Icenticafor 33 in Comparison to Ivacaftor 1
c
c
WT primary HBEC EC50
d
CFTR F508del FRT EC50
CFTR G551D FRT EC50
CFTR WT FRT EC50
CF F508del primary HBEC
a
b
b
b
d
compound
(μM) (% efficacy)
(μM) (% efficacy)
(μM) (% efficacy)
EC50 (μM) (% efficacy)
(μM) (% efficacy)
1
22
33
0.049 0.019 (83)
0.018 0.006 (106)
0.079 0.052 (92)
0.131 0.073 (107)
0.150 0.044 (67)
0.497 0.198 (40)
0.070 0.020 (122)
0.043 0.041 (140)
0.113 0.045 (79)
0.068 0.026 (100)
0.025 0.012 (113)
0.039 0.022 (84)
0.034 0.017 (100)
nd
0.072 0.045 (87)
e
a
b
c
d
e
Data is mean of n ≥ 3 determinations SD. Efficacy relative to PG-01 set at 100%. n = 3 donors. Efficacy relative to ivacaftor set at 100%. Not
determined.
idiosyncratic toxicity.39 To address these concerns, a tertiary
alcohol was introduced to hinder access to the hydroxy group
while at the same time prevent its oxidation to the ketone (Table
6).
cannulated from Sprague−Dawley rats dosed intravenously with
1 mg/kg of compound 22. Contrary to in vitro findings, LC-MS
analysis of the bile collected over 24 h indicated the presence of
glutathione adducts M8 and M9 caused by displacement of the
bromine (Figure 3).
In this case, the (S)-enantiomer 22 (absolute configuration
confirmed via single crystal X-ray crystallography analysis,
Supporting Information Figure S1) was slightly more potent and
also showed lower microsomal clearance than the (R)-
enantiomer 23, with the rat pharmacokinetic profile of 22
(Table 5) translating into reduced in vivo clearance and
improved oral exposure, together with a higher free fraction in
both rat and human plasma. The introduction of an asymmetric
carbon in compounds 20−23 without a large enantiomeric
preference for CFTR potentiator activity led to exploration of
alternative achiral analogues. The 2-hydroxy-2-methylpropyl
amide 24 demonstrated a fivefold reduction in potency,
indicating a requirement for the more lipophilic trifluoromethyl
group at this position. While replacing both methyl groups with
trifluoromethyls provided the potent derivative 25, the higher
lipophilicity and poor solubility (pH 6.8 buffer 0.003 g/L) was
inconsistent with the goal of improving lipophilic efficiency and
biopharmaceutical properties.
Mindful of the potential for nucleophilic aromatic substitution
of the 6-bromo group of compound 22 in vivo, leading to
covalent adduct formation and the potential for idiosyncratic
toxicity,39 in vitro incubation studies with rat and human liver
microsomes in the presence of NADPH and glutathione (GSH)
to search for evidence of adduct formation were conducted.
After incubation in both rat and human liver microsomes for 1 h,
no evidence of direct GSH adduct formation was detected by
LC-MS (data not shown). In a follow up in vivo study, bile was
Further optimization was thus focused on replacement of the
6-bromo moiety (Table 7; compounds 28, 30, and 31 were
separated by preparative chiral SFC with enantiomer absolute
configurations not determined). Consistent with the early
analogue 7, potency was reduced on deletion of bromine in 26.
While methyl analogue 27 showed a similar potency reduction,
ethyl analogue 28a delivered comparable potency to 22 at the
expense of higher clearance. The corresponding trifluoromethyl
analogue 29 gave an increase in potency, combined with a
favorable in vitro clearance profile which translated in vivo to low
clearance and good exposure in a rat PK study, although this was
associated with a decrease in unbound fraction compared to 22
(Table 5). Replacing the bromine with N-methyl, compound 30,
and N,N-dimethylamino, compound 31, gave a stepwise
increase in potency with single enantiomer 31a having similar
activity to compound 22, confirming that a hydrophobic
interaction at this position was governing affinity for the
channel, in preference to modulation of the electronic properties
of the aromatic ring. Concordant with this hypothesis, the
ethoxy analogue 32 further improved the potency, but again at
the expense of metabolic stability. The methoxy substituted
compound 33, while not as potent as the trifluoromethyl
substituted compound 29, also provided an attractive
replacement for the bromine group, showing good metabolic
stability. Follow up in vivo pharmacokinetic profiling in the rat
(Table 5) indicated lower clearance, higher exposure, and a
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longer MRT in comparison to compounds 22 and 29 and
notably an enhanced free fraction in both rat and human plasma
compared to trifluoromethyl analogue 29.
Compounds 22 and 33 were next further profiled for their
CFTR potentiator driven ion transport effects in Ussings
chamber experiments, in comparison to ivacaftor/VX770 1 as a
reference standard (Table 8).
a two-week exploratory toxicology study in rats. Compound 22
was dosed at 10 and 100 mg/kg, with animals in the 100 mg/kg
group requiring euthanization after 2 days’ dosing. The
compound was tolerated at the lower dose for the duration of
the study, where mild decreases in all leukocyte subsets were
observed, together with bone marrow hypocellularity, corre-
sponding to a No Observable Adverse Effect Level (NOAEL) of
<10 mg/kg. In contrast, compound 33 was tolerated at 100 mg/
kg, and a NOAEL of 10 mg/kg was identified, with comparable
exposures of both compounds determined in toxicokinetic
analyses (Supporting Information Table S5). It is not known
whether the in vivo GSH adduct formation found for 22 (vide
supra) contributed directly to the observed toxicology of the
compound. With these data in hand, 33 was then prioritized as a
development candidate. In higher species in vivo PK studies
(Table 9), increased clearance compared to the rat was found. In
vivo clearance in the dog was close to liver blood flow in the dog
and approximately 50% of liver blood flow in the monkey.43
Following further GLP toxicology studies in rats and
monkeys, 33 was advanced into a healthy volunteer Phase I
study,44 where the compound was found to be safe and well-
tolerated up to 750 mg bid in a multiple ascending dose study. In
a proof of concept (PoC) clinical study for CF, a cohort of
patients with CFTR Class III and IV mutations (S549N, R117H,
D1152H, R334W, R352Q, and R347H) where channel is
present at the membrane was treated with 33 for 14 days at 150
and 450 mg bid doses. The study results showed evidence of
CFTR target engagement through a reduction in sweat chloride
as well as clinically meaningful improvement in lung function in
comparison to placebo. In contrast, a cohort of patients with
F508del mutation treated with 33 (450 mg bid) showed no
reduction in sweat chloride or improvement in lung function,
consistent with prior clinical data with ivacaftor in subjects with
the same mutation45 and confirming that combination with
CFTR correctors is required for this patient group.
First, using F508del, G551D (as an example of a Class III
gating mutation) or WT CFTR expressed in FRT cells, the
effects of compounds on ΔIsc in the presence of submaximal
forskolin stimulation were determined. For the F508del studies,
data are reported from temperature corrected experiments. As
shown in Table 5, comparable potencies and efficacies were
found for all three compounds against WT and F508del CFTR,
whereas ivacaftor was more efficacious against G551D CFTR.
Next, studies were conducted using the most physiologically
relevant available assay with cultured primary human bronchial
epithelial cells (HBEC) obtained from healthy (i.e. WT) donors
and temperature corrected homozygous F508del CF HBECs. In
this assay, amiloride was initially applied to block the ENaC
current.40 Secondly, potentiation of ΔIsc was measured in
response to forskolin, which stimulates adenylate cyclase,
leading to protein kinase A mediated CFTR activation.41
Thirdly, increasing concentrations of the test compound were
added; then, at the end of the experiment, the CFTR mediated
current was blocked by CFTR inhibitor 172.42 A representative
raw data trace for WT CFTR is shown in Figure 4.
In parallel with the CF clinical study, a further PoC study was
also initiated in moderate to severe COPD patients with chronic
bronchitis, who were treated with 33 at a dose of 300 mg bid for
28 days, on top of standard of care.46 In comparison to placebo,
33 showed significant improvements in lung function, together
with a reduction in sweat chloride. The systemic anti-
inflammatory effect of 33 was supported by the observation of
a significant reduction in the qualified prognostic COPD
biomarker fibrinogen,47 and an exploratory analysis indicated a
trend to reduction in sputum bacterial colonization, which may
arise from increased mucociliary clearance. Following the
COPD PoC data, compound 33 (Novartis code NVP-
QBW251; INN icenticaftor) was progressed into a Phase 2b
dose range finding study in moderate-to-severe COPD patients
with chronic bronchitis, which is currently ongoing.48
Figure 4. Representative raw data trace from a WT CFTR HBEC ion
transport experiment.
Given the equivalent potencies of 22 and 33 in CFTR HBEC
assays and similar, clean off-target profiles in a broad safety
pharmacology assay panel, both compounds were advanced into
Table 9. Higher Species in Vivo Pharmacokinetic Characterization of 33
d
c
species
Fpo (%) AUClast (nmol/h/L)
C
max (nM)
Cmax free (nM) Clplasma (mL/min/kg) Clblood (mL/min/kg) Vdss (L/kg) MRT (h) PPB (%)
a
dog
monkey
146
26
8670
6780
1952
2303
102
276
29
18
41
20.2
7.5
3.5
4.4
3.3
94.8
88.0
b
a
b
c
d
I.v. dose 0.46 mg/kg; p.o. dose 3 mg/kg. I.v. dose 0.1 mg/kg; p.o. dose 10 mg/kg. Determined by equilibrium dialysis with [14C]-33. Derived
from blood/plasma ratios of 0.708 and 0.89 for dog and monkey, respectively.
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Drug Annotation
a
Scheme 1. Synthesis of Compounds 6, 10−12, 15, and 16
a
Reagents and conditions; (i) Ag2SO4, I2, MeOH, rt, 18%; (ii) 6: tri-n-butyl-2-furylstannane, Pd(PPh3)4, 1,4-dioxane, reflux, 62%; 10: 2-(tri-n-
butylstannyl)oxazole, Pd(PPh3)4, 1,4-dioxane, reflux, 34%; 11: [Pd(dppf)Cl2]2, 3-pyridineboronic acid, Na2CO3, DME, 100°C, μW, 65%; 12:
compound 6, ethanol, H-Cube (RaNi CatCart), 70°C, 70 bar, 25%; (iii) 2M NaOH, ethanol, reflux 1 h; 99%; (iv) pyridine/MeCN, triphosgene/
DCM, reflux, 71%; (v) 15: methylamine (40% aq), THF, RT, 45%; 16: dimethylamine, DMF, RT, 13%.
a
Scheme 2. Synthesis of Compounds 17−26
a
Reagents and conditions: (i) POCl3, DMF, 110°C, 85%; (ii) CuCN, NMP, 175°C, 42%; (iii) SnCl2, EtOH, reflux, 45%; (iv) Conc HCl, reflux,
89%; (v) SOCl2, MeOH, reflux, 89%; (vi) aq NaOH, MeOH, RT, 91−94%; (vii) primary amine, HATU, N-methylmorpholine, DMF, RT, 36−
90%; (viii) Br2, AcOH/H2SO4, RT, 67%; (ix) tri-n-butyl-2-furylstannane, Pd(PPh3)4, 1,4-dioxane, reflux, 57%.
Synthesis. Synthetic routes to compounds 6−33 are
depicted in Schemes 1−5. Anthranilate compounds 6 and
10−12 were synthesized according to Scheme 1, with
compound 8 being commercially available. Silver catalyzed
iodination of commercially available 4-trifluoromethyl anthra-
nilate ester 7 afforded the iodo compound 9, which readily
reacted with boronate or trialkyltin derived heterocycles under
Suzuki or Stille cross coupling conditions to afford the
corresponding arylated derivatives 6 and 10−12. Saponification
of the ester in 6 followed by triphosgene treatment afforded an
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Drug Annotation
a
Scheme 3. Synthesis of Compound 41
a
Reagents and conditions: (i) MeNO2, aq LiOH, MgSO4, cetyltrimethylammonium chloride RT, 95%; (ii) H2, Pd/C, then HCl-EtOH, 97%; (iii)
(+)-tartartic acid, EtOH-H2O, RT, 23% (97.4% ee); (iv) SCX-2 ion exchange cartridge, MeOH-NH3, RT, 80%.
a
Scheme 4. Synthesis of Compounds 27−29
a
Reagents and conditions: (i) MeB(OH)2, Pd(dppf)Cl2, K3PO4, toluene−water, μW 150°C, 43%; (ii) 41, HATU, Et3N, CH2Cl2, RT, 70%; (iii)
2,5-hexanedione, TsOH, toluene, reflux, 73%; (iv) EtB(OH)2, Pd(dppf)Cl2, Cs2CO3, THF−water, μW 150°C, 22%; (v) NaOH, aq THF, RT,
54%; (vi) 41, HATU, Et3N, NMP, RT, 19%; (vii) NH2OH·HCl, Et3N, EtOH−water, reflux, 21% then chiral HPLC; (viii) TMS-CF3, KF, CuI,
DMF-NMP μW, 100°C, 33%; (ix) NH2OH·HCl, Et3N, EtOH−water, reflux, 73%; (x) (S)-41, HATU, DIPEA, DMF, RT, 75%.
oxazinedione intermediate, from which amides 15 and 16 were
prepared by ring opening with the appropriate amine (Scheme
1).
genation of the nitro group in 40 over palladium on charcoal
afforded the desired amine in sufficient purity for the subsequent
HATU coupling reactions. The (S)-enantiomer of 41 was
obtained in 97.4% ee via resolution of the (+)-tartrate salt, with
the absolute configuration confirmed by X-ray crystallography of
the free base (Supporting Information Figure S2).
For further substitution at the picolinamide 6-position to
initially access the 6-methyl analogue 27, one pot Suzuki
coupling and ester hydrolysis delivered intermediate 42, which
was readily coupled under HATU conditions (Scheme 4).
Protection of the amino group as the 2,5-dimethylpyrrole
afforded the versatile intermediate 43, which allowed access to
6-ethyl analogue 28 via Suzuki coupling as well as the 6-
trifluoromethyl analogue 29 through treatment with Ruppert’s
reagent in moderate yield (Scheme 4). Intermediate 43 also
served as a precursor to the 6-amino analogues 30 and 31 via
nucleophilic displacement of bromine (Scheme 5). Ester
hydrolysis of 43 in the presence of methanolic NaOH led to
concomitant replacement of bromine by methoxy to afford
intermediate 49, which was transformed to the 6-ethoxy
The picolinamide analogue 13 was synthesized from
commercially available 3-nitro-5-(trifluoromethyl)pyridin-2-
one according to Scheme 2. Chlorination of the pyridone with
phosphorus oxychloride followed by copper mediated displace-
ment of the chlorine with cyanide and tin chloride reduction of
the nitro group afforded the 2-cyanopyridine 36, which was
readily hydrolyzed with concentrated hydrochloric acid to the 3-
amino picolinic acid 37. Esterification followed by selective
bromination at the 6-position of the pyridine ring allowed
installation of the furan group by palladium catalyzed Stille cross
coupling. The 6-bromo-3-amino picolinamide derivatives 17−
25 were synthesized from the bromo amino picolinic ester 39 by
HATU coupling from intermediate chemistry also described in
Scheme 2. The same amide coupling conditions afforded 26 in
51% yield from 37. The racemic amino alcohol 41 was
synthesized by a Henry reaction between nitromethane and
methyltrifluoromethyl ketone shown in Scheme 3. Hydro-
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Drug Annotation
a
Scheme 5. Synthesis of Compounds 30−33
a
Reagents and conditions: (i) aq NaOH, THF, RT, 97%; (ii) aq Me2NH or MeNH2, THF, RT, 56−92%; (iii) 41, HATU, Et3N, NMP, RT, 52−
78%; (iv) NH2OH·HCl, Et3N, EtOH−water, reflux, then chiral HPLC, 34−36%; (v) aq NaOH, MeOH, 60°C, 97%; (vi) cat H2SO4, MeOH,
reflux, 75%; (vii) TMSCl, KI, MeCN, reflux, 54%; (viii) EtOH, DEAD, Ph3P, dioxane, RT, 61%; (ix) aq NaOH, THF, reflux, 26%; (x) (S)-41,
HATU, DIPEA, DMF, RT, 89%; (xi) NH2OH·HCl, Et3N, EtOH−water, reflux, 37−53%; (xii) (S)-41, HATU, DIPEA, NMP, RT, 59%.
Scheme 6. Process Chemistry Route to Key Intermediate 51
intermediate 50 through a sequence of esterification, demethy-
lation, and Mitsonobu reaction with ethanol, leading to
compound 32. Finally, compound 33 could be accessed directly
from picolinic acid intermediate 51. Subsequent to this
discovery phase synthesis, alternative scaled up preparations of
33 have been reported,49 including the truncated process
chemistry route shown in Scheme 6, which delivered the key
intermediate 51 in 72% yield from 5-bromo-2-methoxy-3-
(trifluoromethyl)pyridine.
absence of any available in vivo PK−PD or disease models for
CF, an optimization strategy focused on improving LLE and
maximizing free drug exposure was employed. With this
approach, the scaffold was evolved to the advanced candidates
22 and 33, which both showed potent potentiation of CFTR in
primary HBECs from either CF or non-CF patient donors,
comparable to the reference potentiator ivacaftor 1. The
potential for reactivity of the 6-bromopyridine moiety in 22
presented a concern, with human and rat in vitro and rat in vivo
metabolite identification experiments providing contrasting
results on GSH adduct formation. To further derisk compound
progression, an exploratory 14-day rat toxicology study was
conducted, which clearly indicated a superior profile for 33
CONCLUSION AND PERSPECTIVE
■
High throughput screening identified the anthranilate hit 6 as
novel potentiator of delF508 and WT CFTR channels. In the
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Drug Annotation
with 32 mM NaHCO3, 10% v/v fetal bovine serum, 2 mM L-glutamine,
100 U/mL penicillin, 100 μg/mL streptomycin, and 30 μg/mL
hygromycin B as the growth medium. For Ussing chamber experiments,
the cells were grown as polarized epithelia on Snapwell permeable
support inserts (500 000 cells/insert in growth medium) and cultured
for 7 to 9 days. The inserts were fed with fresh Coon’s modified F-12
growth medium every 48 h and 24 h prior to Ussing chamber
experiments. To increase the delF508 CFTR protein expression at the
cell surface, plates were incubated at 27 °C for 48 h before performing
an Ussing chamber experiment. The FRT epithelial cells were used as
monolayer cultures on permeable supports. Cl− current was measured
using the short circuit current technique (Isc) under an imposed
basolateral to apical Cl− gradient in Ussing chambers. Ussing chamber
studies were likewise conducted at 27 °C, whereas for WT CFTR and
G551D CFTR cells, all protocols were conducted at 37 °C. Under these
conditions, the effects of cumulative additions of test compounds on
delF508 CFTR, G551D, or WT CFTR currents could be quantitated
with both potency and efficacy end points. Compounds were added to
both the apical and basolateral sides subsequent to addition of forskolin
at concentrations corresponding to the EC20 value for WT CFTR (370
nM) and the EC50 values for F508del and G551D CFTR (both 20 μM).
Efficacy of compounds was compared to a known potentiator such as
PG-01 or ivacaftor, and concentration−response curves were fitted
using nonlinear regression fit, sigmoidal dose response with variable
slope using GraphPad Prism.
CFTR Ion Transport Assays with Primary HBEC. HBEC were
expanded and cryopreserved as previously described.50 On thawing,
cells were further expanded using the same method as described above.
When at 90% confluency, cells were seeded onto collagen coated
polyester 12 mm inserts in differentiation media containing 50%
DMEM in BEGM with the same supplements as above but without
triiodothyronine and a final retinoic acid concentration of 50 nM. Cells
were maintained submerged for the first 7 days in culture. After 7 days
submerged, the cells were exposed to air liquid interface (ALI) for the
remainder of the culture period. Cells were used between days 14 and
28 after establishment of the ALI. At all stages of culture, cells were
maintained at 37 °C in 5% CO2. Collagen coated snapwell inserts
(prepared as described in the Supporting Information) were mounted
in vertical diffusion chambers bathed with continuously gassed
physiological salt solution (5% CO2 in O2; pH 7.4) maintained at 37
°C. Cells were voltage clamped to 0 mV, and transepithelial resistance
(RT) was measured by applying a 1 mV pulse at 30-s intervals and
calculating RT by Ohm’s law. Changes in ISC in response to amiloride,
forskolin, CFTR potentiators, and blockers were recorded. Compound
additions were made by removal of 0.5 mL of bathing solution and
replacement of the solution containing the test compound to produce
the stated final bath concentration. Amiloride was added apically (10
μM) at the start of all studies to determine the amiloride-sensitive ISC.
Following amiloride addition, forskolin at a concentration correspond-
ing to the EC20 for WT CFTR (100 nM) or the EC50 for F508del CFTR
(20 μM) was added to both the apical and basolateral chambers,
followed by cumulative concentrations of test compounds. At the end of
the experiment, CFTRinh172 (30 μM apical) was added to inhibit all
CFTR mediated anion transport. Efficacy of compounds was compared
to the known potentiator ivacaftor, and concentration−response curves
were fitted using nonlinear regression fit, sigmoidal dose response with
variable slope using GraphPad Prism.
compared to 22. Based on this differentiation, 33 was advanced
into the clinic and shown to be safe and well-tolerated in healthy
volunteers. An initial clinical PoC as evidenced by improvement
in lung function was obtained in CF patients with Class III and
IV CFTR mutations. This was followed by a second clinical
study in COPD patients with chronic bronchitis, which showed
significant improvement in lung function as well as evidence of
reduction in a qualified systemic COPD inflammatory
biomarker. Compound 33, now known as icenticaftor, is in
ongoing clinical development for COPD with chronic
bronchitis.
EXPERIMENTAL SECTION
■
CFTR Membrane Potential Assay. Chinese hamster ovary
(CHO) cells stably expressing the delF508-CFTR channel were
maintained at 37 °C in 5% v/v CO2 at 100% humidity in Modified
Eagles medium (MEM) supplemented with 8% v/v fetal calf serum, 100
μg/mL methotrexate, and 100 U/mL penicillin/streptomycin. Cells
were grown in 225 cm2 tissue culture flasks. For membrane potential
assays, cells were seeded into 96-well plates at 40 000 cells per well,
allowed to adhere, and then maintained at 26 °C for 48 h to facilitate
channel insertion. The membrane potential screening assay utilized a
low chloride ion containing extracellular solution (∼5 mM) combined
with a double addition protocol. The first addition was of buffer with or
without test compounds followed 5 min later by an addition of forskolin
(1−20 μM): this protocol favors maximum chloride efflux in response
to delF508-CFTR activation. The delF508-CFTR mediated chloride
ion efflux led to a membrane depolarization which was optically
monitored by FLIPR, and efficacy of compounds was compared to a
known potentiator such as PG-01. Low chloride ion extracellular
solution was prepared as follows: 120 mM Na-gluconate, 1.2 mM
CaCl2, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 10.0 mM D-
glucose, 20.0 mM HEPES, pH 7.4 with NaOH. The FLIPR dye
(Molecular Devices) was made up pursuant to the manufacturer’s
instructions in extracellular solution at 10× final concentration and
stored as 1 mL aliquots at −20 °C. Concentration−response curves
were fitted using nonlinear regression fit, sigmoidal dose response with
variable slope using GraphPad Prism.
CFTR Ion Works Quattro Assay. CHO cells stably expressing the
delF508-CFTR channel were maintained at 37 °C in 5% v/v CO2 at
100% humidity in D-MEM supplemented with 10% (v/v) FCS, 100 U/
mL penicillin/streptomycin, 1% (v/v) NEAA, 1 mg/mL Zeocin, and
500 μg/mL Hygromycin B. For experiments, cells were grown in 225
cm2 tissue culture flasks until near confluence and then cultured at 26
°C for 48−72 h to facilitate channel insertion. Cells were removed from
the flask and resuspended in either extracellular recording solution for
immediate experimentation or alternatively in growth medium
supplemented with 10% v/v DMSO and frozen to −80 °C as 1−2
mL aliquots for use at a later date. Cells, at a density of 1.5−3 million per
mL, were placed on the Quattro system and added to the planar patch
array, and seals were allowed to establish for 5−10 min. After assessing
seal resistances (commonly >50 MΩ), whole-cell access was obtained
by perforation with 100 μg/mL amphotericin B. Baseline currents were
measured by a precompound scan obtained by application of a voltage
ramp from −100 to +100 mV. This was followed by addition of either
buffer or test compound diluted in the extracellular solution
supplemented with 20 μM forskolin to each of the 384 wells of the
planar patch array. After the incubation step (5−20 min), the
postcompound currents were measured again by application of a
voltage ramp from −100 to +100 mV. The difference in currents
between the pre- and postcompound scans defined the efficacy of
CFTR potentiation. The efficacies of compounds were compared to a
known potentiator such as PG-01. Concentration−response curves
were fitted using nonlinear regression fit, sigmoidal dose response with
variable slope using GraphPad Prism
Pharmacokinetic Studies and in Vitro Profiling Assays. All
animal experiments reported were conducted in compliance with local
institutional guidelines at NIBR Basel, East Hanover and Horsham. In
vivo PK study details, plasma protein binding assays, and safety profiling
panel targets are described in the Supporting Information available
online. Microsomal clearance and PAMPA permeability assays have
been reported previously.51
Chemistry. The purity of all final compounds was ≥95%. 1H NMR
spectra were measured at 400 MHz; 13C NMR spectra were measured
100 MHz, and 19F NMR spectra were measured 376 MHz on a Bruker
AV400 NMR spectrometer. Low resolution mass spectra (LRMS) were
determined by LCMS recorded on an Agilent 1100 LC system with a
Waters Xterra MS C18 4.6 × 100 5 μM column, eluting with 5−95% 10
CFTR Ion Transport Assays with FRT Cells. FRT cells stably
expressing delF508-CFTR, G551D-CFTR, or WT-CFTR were
cultured on plastic in Coon’s modified F-12 medium supplemented
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mM aqueous ammonium bicarbonate in acetonitrile over 2.5 min with
negative ion electrospray ionization or 5−95% water +0.1% TFA in
acetonitrile with positive ion electrospray ionization. [MH]+ refers to
monoisotopic molecular mass. Gas chromatographs (GC) were
recorded on an Agilent 7890A instrument with a gradient of 25 °C/
min from 40 to 220 °C then 40 °C/min to 280 °C. Flash
chromatography was conducted with prepacked silica columns using
a Teledyne ISCO CombiFlash system. Microwave reactions were
conducted in a Biotage Emrys Personal Chemistry Optimizer
Microwave Synthesizer. Microanalysis for carbon, hydrogen, and
nitrogen was carried out on a LECO CHNS-932 analyzer using
ethylenediaminetetraacetic acid as the primary calibration standard.
Methyl 2-Amino-5-(furan-2-yl)-4-(trifluoromethyl)benzoate
(6). A solution of 2-amino-4-(trifluoromethyl)benzoic acid (3 g, 14.62
mmol) and concentrated H2SO4 (3.9 mL, 73.1 mmol) in MeOH (50
mL) was heated at reflux overnight. Additional concentrated H2SO4
(3.9 mL, 73.1 mmol) was added, and reflux continued for a further 48 h.
The MeOH was evaporated; the residue was added to a stirred solution
of saturated NaHCO3 sufficient to neutralize residual acid, and the
resulting solution was extracted into EtOAc. The organic phase was
washed with saturated brine and dried (MgSO4) and the solvent was
evaporated to afford methyl 2-amino-4-(trifluoromethyl)benzoate 7 as
pale yellow crystals (2.54 g, 75%). LRMS C9H8F3NO2 requires M+
219.05, found [MH]+ 220.1. 1H NMR (DMSO-d6) 3.82 (3H s), 6.78
(1H dd J = 8.4, 1.6), 7.80 (2H br s), 7.14 (1H d J = 0.9), 7.88 (1H d J =
8.3).
7.8, 4.8), 7.65 (1H s), 7.71 (1H d J = 7.8), 8.47 (1H s), 8.59 (1H dd J =
4.8, 1.3).
(
)-Methyl 2-Amino-5-(tetrahydrofuran-2-yl)-4-
(trifluoromethyl)benzoate (12). A solution of methyl 2-amino-5-
(furan-2-yl)-4-(trifluoromethyl)benzoate 6 (0.100 g, 0.351 mmol) in
EtOH (10 mL) was hydrogenated over Raney Nickel (70 °C/70 psi)
for 15 h using an H-Cube flow reactor. The reaction mixture was
evaporated onto silica gel and purified by flash chromatography eluting
with 100% isohexanes-40% isohexanes/60% EtOAc gradient to afford
methyl 2-amino-5-(tetrahydrofuran-2-yl)-4-(trifluoromethyl)benzoate
12 as a white solid (25 mg, 25%). LRMS C13H14F3NO3 requires M+
289.09, found [MH]+ 290.2. 1H NMR (DMSO-d6) 1.45 (1H m), 1.88
(2H m), 2.16 (1H m), 3.71 (1H dd J = 14.9, 7.5), 3.78 (3H s), 4.00 (1H
dd J = 14.4, 7.0), 4.79 (1H t J = 6.7), 6.86 (2H br s), 7.10 (1H s), 7.93
(1H s).
Methyl 3-Amino-6-(furan-2-yl)-5-(trifluoromethyl)picolinate
(13). DMF (1.414 mL, 18.26 mmol) was added dropwise to POCl3
(25.5 mL, 274 mmol) at RT, followed by 3-nitro-5-(trifluoromethyl)-
pyridin-2-ol (9.5 g, 45.7 mmol) portionwise, and the reaction mixture
was heated at 110 °C for 45 min. The reaction was cooled to RT and
quenched by addition to water with vigorous stirring. The resultant
suspension was extracted with EtOAc and the organic phase was dried
(MgSO4) and evaporated to afford 2-chloro-3-nitro-5-
1
(trifluoromethyl)pyridine 34 as a pale yellow oil (8.79 g, 83%). H
NMR (DMSO-d6) 9.16 (1H s), 9.30 (1H s).
CuCN (3.56 g, 39.7 mmol) was added to a solution of 2-chloro-3-
nitro-5-(trifluoromethyl)pyridine 34 (9.0 g, 39.7 mmol) in NMP (17
mL) and was heated to 175 °C for 40 min. After cooling to RT, the
reaction mixture was partitioned between water/EtOAc and the
biphasic mixture was filtered through Celite and washed with EtOAc.
The organic phase was dried (MgSO4), evaporated and purified by flash
chromatography (3:1 isohexanes-EtOAc elution). Recrystallization
from 3:1 isohexanes-EtOAc afforded 3-nitro-5-(trifluoromethyl)-
Ag2SO4 (2.134 g, 6.84 mmol) was added to a solution of methyl 2-
amino-4-(trifluoromethyl)benzoate 7 (1.5 g, 6.84 mmol) followed by
iodine (1.737 g, 6.84 mmol). The mixture was stirred at RT for 3 h and
filtered, and the filtrate was evaporated onto silica gel prior to
purification by flash chromatography eluting with 3:1 isohexanes-
EtOAc to afford methyl 2-amino-5-iodo-4-(trifluoromethyl)benzoate 9
as an orange solid (0.435 g, 18%). LRMS C9H7F3INO2 requires M+
344.95, found M+ 345.0. 1H NMR (DMSO-d6) 3.85 (3H s), 7.08 (2H
br s), 7.30 (1H s), 8.23 (1H s).
1
picolinonitrile 35 (3.65 g, 42%) as a white crystalline solid. H NMR
(DMSO-d6) 9.32 (1H s), 9.64 (1H s).
Pd(Ph3P)4 (17 mg, 0.01 mmol) was added to a degassed solution of
tri-n-butyl-2-furylstannane (0.136 mL, 0.48 mmol) and methyl 2-
amino-5-iodo-4-(trifluoromethyl)benzoate 9 (0.1 g, 0.28 mmol) in
dioxane (3 mL). The reaction was heated at reflux for 5 h and then
filtered and evaporated onto silica gel. Purification by flash
chromatography eluting with 5:1 isohexanes-EtOAc followed by
isohexane trituration afforded methyl 2-amino-5-(furan-2-yl)-4-
(trifluoromethyl)benzoate 6 as a beige solid (51 mg, 62%). LRMS
SnCl2 (14.41 g, 76 mmol) was added to a solution of 3-nitro-5-
(trifluoromethyl)picolinonitrile 35 (3.3 g, 15.20 mmol) in EtOH (30
mL), and the reaction was heated at reflux for 3 h. The solvent was
evaporated, and the residue was taken up in EtOAc and then washed
with saturated NaHCO3. The washings were diluted with water and
back extracted with EtOAc and the combined organic phases were dried
(MgSO4) then evaporated to afford crude 3-amino-5-
(trifluoromethyl)picolinonitrile 36 (2.57 g, 45%) as a yellow solid,
which was taken into conc HCl (13.4 mL, 134 mmol) and heated to
reflux for 4 h. The solvent was evaporated to afford 3-amino-5-
(trifluoromethyl)picolinic acid hydrochloride 37 (3.02 g, 89%) as a
yellow solid. 1H NMR (DMSO-d6) 7.21 (1H s), 7.35 (1H s), 7.47 (1H
s), 7.70 (1H s), 8.21 (1H s).
Thionyl chloride (1.083 mL, 14.84 mmol) was added to a solution of
3-amino-5-(trifluoromethyl)picolinic acid hydrochloride 37 (3.0 g,
12.37 mmol) in MeOH (25 mL), and the reaction mixture heated to
reflux. Two further portions of thionyl chloride (1.083 mL, 14.84
mmol) were added after 16 and 24 h, and after a total of 56 h, the
solvent was evaporated. The residue was partitioned between saturated
NaHCO3 and EtOAc, then the organic phase was dried (MgSO4) and
evaporated. Purification via flash chromatography (3:1 isohexanes-
EtOAc elution) afforded methyl 3-amino-5-(trifluoromethyl)picolinate
38 (1.75 g, 64%) as a yellow solid. 1H NMR (DMSO-d6) 3.85 (3H s),
7.01 (2H br s), 7.6 (1H d J = 1.3), 8.13 (1H d J = 1.4).
A suspension of methyl 3-amino-5-(trifluoromethyl)picolinate 38
(1.0 g, 4.54 mmol) and conc H2SO4 (0.484 mL, 9.08 mmol) in water
was briefly heated to 100 °C (heat gun) to aid dissolution and then
cooled to RT. A solution of bromine (0.234 mL, 4.54 mmol) in acetic
acid (3.12 mL, 54.5 mmol) was added dropwise, and the resultant
suspension was stirred for 2 h. The solid was collected by filtration,
washed with water, dried and recrystallized from hot MeOH to afford
methyl 3-amino-6-bromo-5-(trifluoromethyl)picolinate 39 (0.905 g,
68%) as yellow needles. 1H NMR (DMSO-d6) 3.86 (3H s), 7.15 (1H br
s), 7.77 (1H s).
1
C13H10F3NO3 requires M+ 285.06, found [MH]+ 286.2. H NMR
(CDCl3) 3.94 (3H s), 6.02 (2H br s), 6.48 (1H dd J = 3.4, 1.9), 6.53
(1H d J = 3.4), 7.06 (1H s), 7.51 (1H d J = 1.7), 8.21 (1H s).
Methyl 2-Amino-5-(oxazol-2-yl)-4-(trifluoromethyl)-
benzoate (10). Pd(Ph3P)4 (17 mg, 0.01 mmol) was added to a
degassed solution of tri-n-butyl-2-oxazolylstannane (0.091 mL, 0.44
mmol) and methyl 2-amino-5-iodo-4-(trifluoromethyl)benzoate 9 (0.1
g, 0.28 mmol) in dioxane (4 mL). The reaction was heated at reflux for 5
h and then filtered and evaporated onto silica gel. Purification by flash
chromatography eluting with 9:1 isohexanes-EtOAc followed by
isohexane trituration afforded methyl 2-amino-5-(oxazol-2-yl)-4-
(trifluoromethyl)benzoate 10 (28 mg, 34%) as a pale yellow solid.
1
LRMS C12H9F3N2O3 requires M+ 286.06, found [MH]+ 287.0. H
NMR (DMSO-d6) 3.86 (3H s), 7.40 (1H br s), 7.43 (2H overlapping d
J = 8), 8.18 (1H s), 8.34 (1H s).
Methyl 2-Amino-5-(pyridin-3-yl)-4-(trifluoromethyl)-
benzoate (11). Methyl 2-amino-5-iodo-4-(trifluoromethyl)benzoate
9 (0.100 g, 0.290 mmol) followed by Pd(dppf)2Cl2 (24 mg, 0.029
mmol) were added to a solution of 3-pyridineboronic acid (53 mg,
0.435 mmol) and Na2CO3 (0.154 g, 1.449 mmol) in DME (5 mL) in a
microwave vial. Additional DME (5 mL) was added, and then the vial
was capped and irradiated at 100 °C for 1 h. The reaction mixture was
evaporated onto silica gel and purified by flash chromatography eluting
with 3:1 isohexanes-EtOAc to afford methyl 2-amino-5-(pyridin-3-yl)-
4-(trifluoromethyl)benzoate 11 as a white solid (64 mg, 75%). LRMS
1
C14H11F3N2O2 requires M+ 296.08, found [MH]+ 297.1. H NMR
(DMSO-d6) 3.81 (3H s), 7.11 (2H br s), 7.34 (1H s), 7.45 (1H dd J =
7252
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
Pd(Ph3P)4 (39 mg, 0.033 mmol) was added to a solution of methyl
3-amino-6-bromo-5-(trifluoromethyl)picolinate 39 (0.100 g, 0.334
mmol) and tri-n-butyl-2-furylstannane (0.105 mL, 0.334 mmol) in
dioxane (5 mL), and the reaction was heated at reflux for 5 h. The
reaction was evaporated onto silica gel, and purification by flash
chromatography eluting with 4:1 isohexanes-EtOAc followed by hot
isohexane trituration afforded methyl 3-amino-6-(furan-2-yl)-5-
(trifluoromethyl)picolinate 13 (55 mg, 57%) as a pale yellow solid.
N-Methylmorpholine (0.104 mL, 1.053 mmol) was added to a
solution of 3-amino-6-bromo-5-(trifluoromethyl)picolinic acid (0.150
g, 0.526 mmol) and HATU (0.200 g, 0.526 mmol) in DMF (2.5 mL).
The mixture was stirred at RT for 5 min prior to addition of
methylamine (2 M THF, 0.526 mL, 1.053 mmol). After standing at RT
for 48 h, the reaction was poured into water and extracted twice with
EtOAc. The combined organic extracts were dried (MgSO4) and
evaporated. Purification by flash chromatography (0−100% EtOAc-
isohexanes gradient elution) afforded 3-amino-6-bromo-N-methyl-5-
(trifluoromethyl)picolinamide 17 (0.103 g, 66%) as a white solid.
1
LRMS C12H9F3N2O3 requires M+ 286.06, found [MH]+ 287.1. H
NMR (DMSO-d6) 3.87 (3H s), 6.61 (1H dd J = 3.2, 1.7), 6.76 (1H d J =
3.2), 7.75 (1H s), 7.80 (1H s)
1
LRMS C8H7BrF3N3O requires M+ 296.97, found [MH]+ 298.0. H
2-Amino-5-(furan-2-yl)-4-(trifluoromethyl)benzoic acid (14).
A solution of methyl 2-amino-5-(furan-2-yl)-4-(trifluoromethyl)-
benzoate 6 (2.28 g, 7.99 mmol) and 4 M NaOH (4 mL, 15.98
mmol) in EtOH (25 mL) was stirred at RT for 16 h. The solvent was
evaporated, and the residue was diluted with water and then neutralized
with 0.1 M HCl. The resultant precipitate was collected by filtration and
dried to afford 2-amino-5-(furan-2-yl)-4-(trifluoromethyl)benzoic acid
14 (1.99 g, 84%) as a yellow solid. LRMS C11H7F3N2O3 requires M+
271.05, found [MH]+ 272.2. 1H NMR (CDCl3) 6.02 (2H br s), 6.49
(1H dd, J = 3.4, 1.9), 6.55 (1H d J = 3.4), 7.08 (1H s), 7.51 (1H d J =
1.8), 8.28 (1H s), 12.00−9.41 (1H br s).
NMR (DMSO-d6) 2.78 (3H d J = 4.7), 7.26 (2H br s), 7.67 (1H s), 8.47
(1H br d J = 4.7).
3-Amino-6-bromo-5-(trifluoromethyl)-N-(3,3,3-
trifluoropropyl)picolinamide (18). Prepared with the same
procedure used for 17, using 3,3,3-trifluoropropylamine and isolated
as a yellow oil (0.119 g, 89%). LRMS C10H8BrF6N3O requires M+
378.98, found [MH]+ 380.1. 1H NMR (DMSO-d6) 2.56 (2H m), 3.51
(2H q J = 6.8), 7.28 (2H br s), 7.70 (1H s), 8.69 (1H t J = 6.0).
(
)-3-Amino-6-bromo-N-(2-hydroxypropyl)-5-
(trifluoromethyl)picolinamide (19). Prepared with the same
procedure used for 17, using ( )-1-amino-2-propanol and isolated as
an off-white solid (0.140 g, 80%). LRMS C10H11BrF3N3O2 requires M+
341.00, found [MH]+ 342.0. 1H NMR (DMSO-d6) 1.05 (3H d J = 6.3),
3.14 (1H m), 3.14 (1H m), 3.29 (1H m), 3.78 (1H m), 4.84 (1H d J =
4.8), 7.28 (2H br s), 7.69 (1H s), 8.30 (1H t J = 5.9).
2-Amino-5-(furan-2-yl)-N-methyl-4-(trifluoromethyl)-
benzamide (15). A solution of triphosgene (43 mg, 0.147 mmol) in
DCM (1 mL) was added to a solution of 2-amino-5-(furan-2-yl)-4-
(trifluoromethyl)benzoic acid 14 (0.100 g, 0.368 mmol) and pyridine
(59 μL, 0.736 mmol) in MeCN (2 mL). The reaction was heated to
reflux for 30 min and cooled to RT, and the resultant solid was collected
by filtration, taken into DCM, and washed with water. The organic
phase was dried (MgSO4) and evaporated to afford 6-(furan-2-yl)-7-
(trifluoromethyl)-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (89 mg,
(S)-3-Amino-6-bromo-N-(3,3,3-trifluoro-2-hydroxypropyl)-
5-(trifluoromethyl)picolinamide (20). Prepared with the same
procedure used for 17, starting from (S)-3-amino-1,1,1-trifluoro-2-
propanol, (50 mg, 36%). LRMS C10H8BrF6N3O2M+ requires 394.97,
found [MH]+ 396.0. 1H NMR (DMSO-d6) 3.40 (1H m), 3.58 (1H m),
4.24 (1H m), 6.50 (1H br s), 7.29 (2H br s), 7.71 (1H s), 8.56 (1H br
s).
1
81%) as a light brown solid. H NMR (DMSO-d6) 6.67 (1H dd J =
3.4, 1.8), 6.88 (1H d J = 3.4), 7.56 (1H s), 7.89 (1H d J = 1.2), 8.18 (1H
s), 12.07 (1H s).
(R)-3-Amino-6-bromo-N-(3,3,3-trifluoro-2-hydroxypropyl)-
5-(trifluoromethyl)picolinamide (21). Prepared with the same
procedure used for 17, starting from ( )-3-amino-1,1,1-trifluoro-2-
propanol, with separation of the enantiomers via preparative chiral SFC
(Chiralpak AD-H column, 12% [2-propanol +0.1% DEA]/88% CO2
elution), (85 mg, 15%) retention time 3.1 min, >98% ee (vs retention
time 2.2 min for 20). LRMS C10H8BrF6N3O2M+ requires 394.97, found
[MH]+ 396.0. 1H NMR (DMSO-d6) 3.40 (1H m), 3.58 (1H m), 4.24
(1H m), 6.50 (1H br s), 7.29 (2H br s), 7.71 (1H s), 8.56 (1H br s).
(S)-3-Amino-6-bromo-N-(3,3,3-trifluoro-2-hydroxy-2-meth-
ylpropyl)-5-(trifluoromethyl)picolinamide (22) and (R)-3-
Amino-6-bromo-N-(3,3,3-trifluoro-2-hydroxy-2-methylprop-
yl)-5-(trifluoromethyl)picolinamide (23). LiOH (11.7 g, 0.279
mol) was dissolved in distilled water (1 L), and the solution was cooled
to 10 °C. Nitromethane (170 g, 128 mL, 27.9 mol) was added followed
by cetyltrimethylammonium chloride (120 g, 0.377 mol) and
magnesium sulfate (77 g, 0.558 mol). The cloudy suspension was
maintained at 5 °C in an ice bath while trifluoroacetone (500 g, 400 mL,
4.462 mol) was added dropwise. The internal temperature rose to 15 °C
and upon cooling, the reaction mixture was stirred at 0 °C for 3 h. The
reaction mixture was diluted with TBME (1 L) and the phases
separated. The organic phase was extracted with brine (2 × 500 mL)
and the aqueous phase was washed with TBME (500 mL). To the
combined organic phases was added Biotage supercation exchange
resin (500 g, to remove the cetyltrimethylammonium chloride) and the
mixture was stirred at RT over the weekend. The suspension was
filtered and washed with TBME (2 L). The mother liquor was
concentrated to give the product as a yellow liquid (625 g). NMR
showed remaining cetyltrimethylammonium chloride, so the product
was dissolved in TBME (1 L) and scavenged again with Biotage
supercation exchange resin (500 g) overnight at RT. The suspension
was filtered and washed with TBME (2 L). The mother liquor was
concentrated to afford 1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol 40
(457 g, 95%) as a pale yellow oil. 1H NMR (CDCl3) 1.57 (3H s), 4.56
(1H d J = 12.5), 4.73 (1H d J = 12.5).
Methylamine (40% aq; 26 μL, 0.336 mmol) was added to a solution
of 6-(furan-2-yl)-7-(trifluoromethyl)-2H-benzo[d][1,3]oxazine-
2,4(1H)-dione (50 mg, 0.168 mmol) in THF (2 mL). The reaction
was stirred at RT for 1.5 h, and then additional methylamine (40% aq;
52 μL, 0.672 mmol) was added. After a further 20 min, the reaction
mixture was evaporated onto silica gel and purified by flash
chromatography (3:1 isohexanes-EtOAc elution) to afford 2-amino-
5-(furan-2-yl)-N-methyl-4-(trifluoromethyl)benzamide 15 (21 mg,
44%) as a white solid. LRMS C13H11F3N2O2 requires M+ 284.08,
found [MH]+ 285.2 1H NMR (DMSO-d6) 2.75 (3H d J = 4.5), 6.56
(2H s), 6.93 (2H s), 7.19 (1H s), 7.73 (1H s), 7.77 (1H s), 8.51 (1H br
s).
2-Amino-5-(furan-2-yl)-N,N-dimethyl-4-(trifluoromethyl)-
benzamide (16). A solution of 2-amino-5-(furan-2-yl)-4-
(trifluoromethyl)benzoic acid 14 (27 mg, 0.1 mmol) and triphosgene
(10 mg, 0.34 mmol) in THF (1 mL) was shaken for 1 h, and the solvent
was evaporated. The residue was triturated with Et2O and taken into
DMF (1 mL). Dimethylamine (13 μL, 0.2 mmol) was added, and the
reaction was shaken for 16 h. The crude product was purified by elution
through a solid phase extraction cartridge (PE-AX SCX2) to afford 2-
amino-5-(furan-2-yl)-N,N-dimethyl-4-(trifluoromethyl)benzamide 16
(4 mg, 13%) as a white solid. LRMS C14H13F3N2O2 requires M+ 298.09,
found [MH+] 299.0. 1H NMR (CDCl3) 3.1 (6H s), 4.63 (2H br s), 6.48
(1H dd J = 3.4, 1.8), 6.54 (1H d J = 3.3), 7.08 (1H s), 7.28 (1H s), 7.49
(1H d J = 1.6).
3-Amino-6-bromo-N-methyl-5-(trifluoromethyl)-
picolinamide (17). Four molar aq NaOH (14.04 mL, 28 mmol) was
added to a suspension of methyl 3-amino-5-(trifluoromethyl)picolinate
38 (1.4 g, 4.68 mmol) in MeOH (15 mL) and the reaction was stirred at
RT for 16 h. The solvent was evaporated, the residue was taken into
water, acidified with 5 M HCl, and extracted twice with EtOAc. The
combined organic phases were washed with water and brine and then
dried (MgSO4). Evaporation and trituration with Et2O afforded 3-
amino-6-bromo-5-(trifluoromethyl)picolinic acid (1.25 g, 94%) as a
yellow solid. 1H NMR (DMSO-d6) 7.17 (2H br s), 7.74 (1H s) 13.24
(1H br s).
A suspension of 10% Pd/C (2.4 g) and 1,1,1-trifluoro-2-methyl-3-
nitropropan-2-ol 40 (24.29 g, 140 mmol) in EtOH (250 mL) was
7253
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J. Med. Chem. 2021, 64, 7241−7260

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
hydrogenated at 5 bar pressure for 2 h at RT. The reaction mixture was
filtered through Celite, evaporated and taken into MeOH (50 mL).
HCl (1.25 M EtOH solution; 112 mL, 140 mmol) was added. The
solution was stirred at RT for 1 h and evaporated to afford ( )-3-
amino-1,1,1-trifluoro-2-methylpropan-2-ol 41 hydrochloride (22.72 g,
72%) as a white solid. 1H NMR (DMSO-d6) 1.40 (3H s), 2.99 (2H s),
6.84 (1H br s), 8.34 (3H br s).
hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide 23 (0.123
g, 66%, >98%ee), retention time 4.3 min. LRMS C11H10BrF6N3O2
1
requires M+ 408.99, found [MH]+ 410.1. H NMR (DMSO-d6) 1.24
(3H s), 3.45 (1H dd J = 13.8. 5.7), 3.68 (1H dd J = 13.8, 7.4), 6.29 (1H
s), 7.30 (1H br s), 7.73 (1H s), 8.31 (1H t J = 6.5).
3-Amino-6-bromo-N-(2-hydroxy-2-methylpropyl)-5-
(trifluoromethyl)picolinamide (24). Prepared with the same
procedure used for 17, starting from 1-amino-2-methylpropan-2-ol
and isolated as a yellow solid (0.300 g, 75%). LRMS C11H13BrF3N3O2
( )-3-Amino-1,1,1-trifluoro-2-methylpropan-2-ol (prepared as the
free base by upscaling the above procedure and direct evaporation of
the MeOH solution; 40 g, 280 mmol) and L-(+)-tartaric acid (42.0 g,
280 mmol) were dissolved in EtOH + 4% water (1398 mL), warmed to
65 °C over 30 min, and then left standing at RT for 16 h. The resultant
solid was collected by filtration, dried, taken into EtOH + 4% water
(384 mL), warmed to 80 °C, and then left standing at RT for 16 h. The
resultant solid was collected by filtration, dried and taken into EtOH +
4% water (314 mL), warmed to 80 °C for 2 h and then left standing at
RT for 16 h. The white solid was collected by filtration and dried to
afford (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol 41 (+)-tartrate
salt (18.4 g, 23%) 1H NMR (D2O) 1.31 (3H s), 3.05 (1H d J = 14.4),
3.19 (1H d J = 14.4), 4.35 (2H s). The absolute configuration was
determined by X-ray crystallographic analysis of the free base (vide
infra). The enantiomeric purity was determined via conversion to the
Mosher’s amide: tartrate salt 41 (1 mg) was dissolved in DCM (1 mL)
and then DIPEA (6 μL) was added with sonication followed by (S)-α-
methoxy-α-trifluoromethylphenylacetyl chloride (6 μL), and the
mixture was analyzed directly by GC indicating 97.4% ee.
1
requires M+ 355.01, found [MH]+ 356.1. H NMR (DMSO-d6) 1.10
(6H s), 3.24 (2H d J = 6.2), 4.70 (1H s), 7.30 (2H br s), 7.71 (1H s),
8.14 (1H t J = 6.1).
3-Amino-6-bromo-N-(3,3,3-trifluoro-2-hydroxy-2-
(trifluoromethyl)propyl)-5-(trifluoromethyl)picolinamide (25).
2,2-Bis(trifluoromethyl)oxirane (0.500 g, 2.78 mmol) was added
dropwise to a stirred mixture of 35% aq ammonia (1 mL, 18.1 mol) in
Et2O (1 mL). The biphasic reaction was stirred at RT for 3 h; the layers
were separated and the aqueous phase was extracted twice with Et2O.
The combined organic phases were dried (MgSO4) and evaporated to
afford 2-(aminomethyl)-1,1,1,3,3,3-hexafluoropropan-2-ol as a white
solid (0.355 g, 65%). 1H NMR (DMSO-d6) 3.03 (2H s), 3.15 (1H s),
4.23 (2H br s).
2-(Aminomethyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (0.355 g, 1.80
mmol) was added to a solution of 3-amino-6-bromo-5-
(trifluoromethyl)picolinic acid (0.428 g, 1.50 mmol) in NMP (6 mL)
followed by HATU (0.571 g, 1.50 mmol). Triethylamine (0.209 mL,
1.50 mmol) was added; the reaction was stirred at RT for 30 min and
then diluted with EtOAc and washed with 0.1 M aq NaOH, water, and
brine. The organic phase was dried (MgSO4), evaporated and purified
by flash chromatography 0−20% EtOAc-isohexane gradient to afford 3-
amino-6-bromo-N-(3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)-
propyl)-5-(trifluoromethyl)picolinamide 25 as a pale yellow solid
(0.530 g, 76%). LRMS C11H7BrF9N3O2 requires M+ 462.96, found
[MH]+ 464.0. 1H NMR (DMSO-d6) 3.99 (2H d J = 6.5), 7.31 (2H br
s), 7.74 (1H s), 8.31 (1H s), 8.49 (1H s).
(S)-3-Amino-1,1,1-trifluoro-2-methylpropan-2-ol 41 (+)-tartrate
salt (15.5 g, 52.9 mmol) was dissolved in MeOH (400 mL) and eluted
through a column packed with SCX-2 ion-exchange resin (Isolute Si-
propylsulfonic acid) with DCM/MeOH (9:1) until no more tartaric
acid was detected in the eluent. The eluent was switched to NH3-
MeOH (9:1), and the combined eluent was evaporated to afford (S)-3-
amino-1,1,1-trifluoro-2-methylpropan-2-ol 41 as a white crystalline
solid (6.34 g, 70%). The absolute configuration was determined by
1
single crystal X-ray analysis (Supporting Information Figure S2). H
NMR (DMSO-d6) 1.21 (3H s), 1.45 (2H br s), 2.56 (1H d J = 13.4),
2.72 (1H d J = 13.4), 5.70 (1H br s).
( )-3-Amino-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-
5-(trifluoromethyl)picolinamide (26). 2 M aq NaOH (2 M NaOH
(0.182 mL, 4.54 mmol) was added to a solution of methyl 3-amino-5-
(trifluoromethyl)picolinate 38 (0.998 g, 4.53 mmol) in MeOH (20
mL), and the reaction was stirred at RT for 30 min, then poured into
water and adjusted to pH 1 with 1 M HCl. The product was extracted
with EtOAc and the organic phase was washed with brine, dried
(MgSO4), and evaporated to afford 3-amino-5-(trifluoromethyl)-
picolinic acid 37 as an orange solid (0.850 g, 91%). 1H NMR
(DMSO-d6) 7.08 (2H br s), 7.58 (1H d J = 1.5), 8.12 (1H d J = 1.5),
12.90 (1H br s).
HCl (1.5 M solution in MeOH, 30 mL) was added to a solution of
(S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol 41 (5 g, 34.9 mmol)
in MeOH (30 mL) and the mixture was stirred at RT for 30 min. The
solvent was evaporated, and the residue was recrystallized from MeCN
to afford (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol 41 hydro-
chloride as a white solid (4.73 g, 75%). 1H NMR (DMSO-d6) 1.19 (3H
s), 2.54 (1H d J = 13.4), 2.71 (1H d J = 13.4), 3.30 (3H br s), 5.80 (1H
br s).
N-Methylmorpholine (0.413 mL, 4.18 mmol) was added to a
solution of 3-amino-6-bromo-5-(trifluoromethyl)picolinic acid (0.397
g, 1.39 mmol), ( )-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol hy-
drochloride 41 (0.250 g, 1.39 mmol), and HATU (0.529 g, 1.39 mmol
in DMF (10 mL). The reaction was stirred at RT for 3 h, poured into
ice−water and extracted with EtOAc. The organic phase was washed
with saturated aqueous NH4Cl, dried (MgSO4) and evaporated.
Purification by flash column chromatography (30%-EtOAc-isohexanes
elution) afforded racemic 3-amino-6-bromo-N-(3,3,3-trifluoro-2-hy-
droxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide (0.373 g,
65%), which was separated by preparative chiral SFC (Chiralpak OJ-
H column, eluting with 12% [isopropanol +0.1% DEA]/88% CO2) to
afford (S)-3-amino-6-bromo-N-(3,3,3-trifluoro-2-hydroxy-2-methyl-
propyl)-5-(trifluoromethyl)picolinamide 22 (0.119 g, 64%, >98% ee)
as a beige solid, retention time 3.8 min with absolute stereochemistry
confirmed by X-ray crystallography (Supporting Information, Figure
S2). LRMS C11H10BrF6N3O2 requires M+ 408.99, found [MH]+ 410.2.
Elemental analysis requires C, 32.22%; H, 2.46%; N, 10.25% found C,
32.37 0.00%; H, 2.43 0.00%; N, 10.27 0.04. 1H NMR (DMSO-
d6) 1.24 (3H s), 3.45 (1H dd J = 13.8. 5.7), 3.68 (1H dd J = 13.8, 7.4),
6.29 (1H s), 7.30 (1H br s), 7.73 (1H s), 8.31 (1H t J = 6.5). 13C NMR
(DMSO-d6) 18.93 (q), 42.21 (t), 72.28 (s JF = 26.6), 117.69 (s), 121.91
(s JF = 273.2), 126.32 (s JF = 285.8), 126.41 (d JF = 5.2), 128.37 (s JF =
32.0), 129.62 (s), 145.31 (s), 165.28 (s). 19F NMR (DMSO-d6) −62.73
(s), −80.49 (s) and (R)-3-amino-6-bromo-N-(3,3,3-trifluoro-2-
3-Amino-1,1,1-trifluoro-2-methylpropan-2-ol (99 mg, 0.694 mmol)
was added to a solution of 3-amino-5-(trifluoromethyl)picolinic acid
(0.130 g, 0.631 mmol) in NMP (2 mL), followed by HATU (0.240 g,
0.631 mmol) and then dropwise addition of triethylamine (88 μL, 0.631
mmol). The reaction was stirred at RT for 16 h, then partitioned
between 1 M aq NaOH and EtOAc. The organic phase was washed with
brine, dried (MgSO4) and evaporated. Purification by flash
chromatography (0−80% EtOAc-isohexanes gradient elution) afforded
(
)-3-amino-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-
(trifluoromethyl)picolinamide 26 as a yellow oil (0.108 g, 52%). LRMS
1
C11H11F6N3O2 requires M+ 331.08, found [MH]+ 332.2. H NMR
(DMSO-d6) 1.25 (3H s), 3.48 (1 H dd J = 13.8 7.3), 3.65 (1H dd J =
13.8 7.3), 6.27 (1H s), 7.21 (2H br s), 7.56 (1H d J = 1.5), 8.11 (1H d J
= 1.5), 8.12 (1H t J = 6.4).
( )-3-Amino-6-methyl-N-(3,3,3-trifluoro-2-hydroxy-2-meth-
ylpropyl)-5-(trifluoromethyl)picolinamide (27). A suspension of
methyl 3-amino-6-bromo-5-(trifluoromethyl)picolinate 39 (1.249 g,
4.18 mmol), methylboronic acid (0.250 g, 4.18 mmol), PdCl2(dppf)
(0.153 g, 0.209 mmol), and K3PO4 (2.66 g, 12.53 mmol) in toluene (10
mL) and water (5 mL) was heated in a sealed vial with microwave
irradiation at 150 °C for 30 min. The reaction mixture was diluted with
water and toluene and filtered and the phases separated. The aqueous
phase was adjusted to pH 3 by addition of 2 M HCl and extracted twice
with EtOAc. The combined organic phases were dried (MgSO4),
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Drug Annotation
evaporated and purified by flash chromatography (0−20% MeOH-
DCM gradient elution) to afford 3-amino-6-methyl-5-
(trifluoromethyl)picolinic acid 42 as a yellow oil (0.379 mg, 43%).
LRMS C8H7F3N2O2 requires M+ 220.05, found [MH]+ 221.0.
Triethylamine (0.190 mL, 1.363 mmol) was added to a solution of 3-
amino-6-methyl-5-(trifluoromethyl)picolinic acid 42 (0.150 g, 0.681
mmol) in DCM (5 mL), followed by ( )-3-amino-1,1,1-trifluoro-2-
methylpropan-2-ol hydrochloride 41 (0.122 g, 0.681 mmol) and
HATU (0.285 g, 0.749 mmol). The reaction was stirred for 30 min at
RT and then diluted with water. The phases were separated, and the
organic phase was dried (MgSO4), evaporated and purified by flash
chromatography (0−100% EtOAc-isohexanes gradient elution) to
afford ( )-3-amino-6-methyl-N-(3,3,3-trifluoro-2-hydroxy-2-methyl-
propyl)-5-(trifluoromethyl)picolinamide 27 as a yellow solid (0.175
g, 70%). LRMS C12H13F6N3O2 requires M+ 345.09, found [MH]+
346.1. 1H NMR (CDCl3) 1.43 (3H s), 2.58 (3H s), 3.68 (1H dd J =
14.5, 7.1), 3.78 (1H dd J = 14., 6.2), 4.57 (1H s), 5.99 (2H br s), 8.53
(1H s), 7.30 (1H s).
(ent-1)-3-Amino-6-ethyl-N-(3,3,3-trifluoro-2-hydroxy-2-
methylpropyl)-5-(trifluoromethyl)picolinamide (28a) and (ent-
2)-3-Amino-6-ethyl-N-(3,3,3-trifluoro-2-hydroxy-2-methyl-
propyl)-5-(trifluoromethyl)picolinamide (28b). Acetylacetone
(7.06 mL, 60.2 mmol) was added to a suspension of methyl 3-amino-
6-bromo-5-(trifluoromethyl)picolinate 39 (15 g, 50.2 mmol) in toluene
(60 mL), followed by p-toluenesulfonic acid (0.954 g, 5.02 mmol). The
suspension was heated at reflux with a Dean−Stark trap for 5 h. After
cooling, the reaction mixture was partitioned between EtOAc and
saturated NaHCO3, and the combined organic phases were washed
with brine, dried (MgSO4) and evaporated. Purification by flash
chromatography (0−80% EtOAc-isohexane gradient elution) afforded
methyl 6-bromo-3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-(trifluoromethyl)-
picolinate 43 as a yellow solid (14.9 g, 79%). 1H NMR (DMSO-d6) 1.90
(6H s), 3.68 (3H s), 5.89 (2H s), 8.51 (1H s).
One molar aq Cs2CO3 (1.67 mL, 1.67 mmol) was added to a
suspension of methyl 6-bromo-3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-
(trifluoromethyl)picolinate 43 (0.923 g, 2.47 mmol), ethylboronic
acid (0.181 g, 2.447 mmol), and PdCl2(dppf) (0.200 g, 0.245 mmol) in
THF (5 mL). The reaction was heated in a sealed vial with microwave
irradiation at 150 °C for 15 min. The reaction mixture was filtered
through Celite and washed with EtOAc, and the filtrate was washed
with water, brine, dried (MgSO4) and evaporated. Flash chromatog-
raphy (0−80% EtOAc-isohexane gradient elution) afforded an
inseparable mixture of 43 and methyl 3-(2,5-dimethyl-1H-pyrrol-1-
yl)-6-ethyl-5-(trifluoromethyl)picolinate 44 in approximately 1:1 ratio
based on UV absorbance (0.492 g, ∼22%). LRMS C16H17F3N2O2
requires M+ 326.12, found [MH]+ 327.2.
The above mixture (0.492 g, approximately 1.508 mmol) was taken
into THF (10 mL) and 2 M aq NaOH (66 μL, 1.508 mmol), and the
reaction mixture was stirred at RT for 1 h. Water was added, and the
solution was adjusted to pH 4 with 1 M HCl and extracted with EtOAc.
The combined organic phases were washed with brine, dried (MgSO4)
and evaporated to afford a brown viscous oil (0.482 g) corresponding to
15% by UV peak area 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-ethyl-5-
(trifluoromethyl)picolinic acid. LRMS C15H15F3N2O2 requires M+
312.11, found [MH]+313.2.
hydrochloride (0.232 g, 3.34 mmol). The mixture was heated at reflux
for 10 h and then partitioned between 1 M HCl and EtOAc. The
organic phase was washed with brine, dried (MgSO4) and evaporated.
The crude product was purified by mass-directed preparative LCMS to
afford ( )-3-amino-6-ethyl-N-(3,3,3-trifluoro-2-hydroxy-2-methyl-
propyl)-5-(trifluoromethyl)picolinamide as a pale yellow solid (23
mg, 19%). The racemic mixture was separated by preparative chiral
SFC (Chiralcel OD-H column eluting with 15% 2-propanol +0.1% v/v
diethylamine/85% CO2) to afford (ent-1)-3-amino-6-ethyl-N-(3,3,3-
trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide
28a as an off-white solid (5 mg), retention time 4.03 min. LRMS
1
C13H15F6N3O2 requires M+ 359.11, found [MH]+ 360.2. H NMR
(DMSO-d6) 1.24 (3H t J = 7.4), 1.26 (3H s), 2.75 (2H q J = 7.3), 3.48
(1H dd J = 13.8, 5.6), 3.66 (1H dd J = 13.8, 7.2), 6.32 (1H s), 6.97 (2H
br s), 7.56 (1H s), 8.50 (1H t J = 6.3) and (ent-2)-3-amino-6-ethyl-N-
(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)-
picolinamide 28b as an off-white solid (4 mg), retention time 4.59 min.
1
LRMS C13H15F6N3O2 requires M+ 359.11, found [MH]+ 360.2. H
NMR (DMSO-d6) 1.24 (3H t J = 7.4), 1.26 (3H s), 2.75 (2H q J = 7.3),
3.48 (1H dd J = 13.8, 5.6), 3.66 (1H dd J = 13.8, 7.2), 6.32 (1H s), 6.97
(2H br s), 7.56 (1H s), 8.50 (1H t J = 6.3).
(S)-3-Amino-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-
5,6-bis(trifluoromethyl)picolinamide (29). Nine microwave re-
actions were conducted in batch format as follows: (trifluoromethyl)-
trimethylsilane (2.49 mL, 15.91 mmol) was added to a mixture of
potassium fluoride (0.462 g, 7.95 mmol), copper(I) iodide (1.52 g, 7.95
mmol) and methyl 6-bromo-3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-
(trifluoromethyl)picolinate 43 (2.0 g, 5.30 mmol) in a microwave
vial. The reaction was irradiated at 100 °C for 3 h, and then the 9 crude
reaction mixtures were combined for work up by adding cautiously to 5
M aqueous ammonia and extracting three times with EtOAc. The
combined organic phases were further washed sequentially with 5 M
aqueous ammonia, 1 M HCl, saturated NaHCO3 then brine and dried
(MgSO4). Following evaporation, flash chromatography (0−10%
EtOAc gradient elution) gave a brown oil which partly crystallized on
standing. The supernatant oil was decanted and discarded to afford
methyl 3-(2,5-dimethyl-1H-pyrrol-1-yl)-5,6-bis(trifluoromethyl)-
picolinate 46 as yellow crystals (5.7 g, 33%). LRMS C15H12F6N2O2
1
requires M+ 366.08, found [MH]+ 367.0. H NMR (DMSO-d6) 1.84
(6H s), 3.63 (3H s), 5.80 (2H s), 8.74 (1H s).
One molar NaOH (31 mL 31.1 mmol) was added to a solution of
methyl 3-(2,5-dimethyl-1H-pyrrol-1-yl)-5,6-bis(trifluoromethyl)-
picolinate 46 (5.70 g, 15.6 mmol) in MeOH (90 mL). The reaction
was stirred at RT for 1 h, and the MeOH was evaporated. The residue
was adjusted to pH 3 with 1 M HCl and then extracted three times with
EtOAc. The combined organic phases were washed with brine, dried
(MgSO4) and evaporated to afford 3-(2,5-dimethyl-1H-pyrrol-1-yl)-
5,6-bis(trifluoromethyl)picolinic acid containing 1 equiv EtOAc as a
brown oil (2.96 g, 54%). LRMS C14H10F6N2O2 requires M+ 352.06,
found [MH]+ 353.0. 1H NMR (DMSO-d6) 1.94 (6H s) 5.86 (2H s),
8.71 (1H s), 14.3 (1H br s).
Hydroxylamine hydrochloride (5.84 g, 84.0 mmol) was added to a
solution of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-5,6-bis(trifluoromethyl)-
picolinic acid (2.96 g, 8.40 mmol) in 2:1 EtOH-water (30 mL),
followed by triethylamine (2.34 mL, 16.8 mmol). The reaction was
heated at 100 °C for 3 h, and the solvent was evaporated. The residue
was diluted with water and extracted three times with EtOAc. The
combined organic phases were washed with brine, dried (MgSO4) and
evaporated. The crude product was purified first by normal phase
column chromatography (75−100% EtOAc-isohexanes gradient
elution) and then by reverse phase chromatography (C18 column;
10−95% MeCN-water gradient elution, each containing 1% HCl) to
afford 3-amino-5,6-bis(trifluoromethyl)picolinic acid hydrochloride 47
as a yellow solid (1.95 g, 73%). LRMS C8H4F6N2O2 requires M+
274.02, found [MH]+ 275.2. 1H NMR (DMSO-d6) 7.59 (2H br s), 7.80
(1H s), 13.30 (1H br s).
The above crude material (0.482 g) was taken into NMP (2 mL).
( )-3-Amino-1,1,1-trifluoro-2-methylpropan-2-ol hydrochloride 41
(0.243 g, 1.698 mmol) was added, followed by HATU (0.646 g,
1.698 mmol) and triethylamine (0.473 mL, 3.40 mmol). The reaction
was stirred at RT for 16 h and then partitioned between EtOAc and 1 M
NaOH. The organic phase was washed with brine, dried (MgSO4),
evaporated and partially purified by mass-directed preparative LCMS to
afford 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-ethyl-N-(3,3,3-trifluoro-2-hy-
droxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide 45 as a brown
viscous oil in 67% purity by UV peak area (0.146 g, approx 21%). LRMS
C13H15F6N3O2 requires M+ 453.19, found M+ 453.3
Triethylamine (93 μL, 0.668 mmol) was added to 3-(2,5-dimethyl-
1H-pyrrol-1-yl)-6-ethyl-N-(3,3,3-trifluoro-2-hydroxy-2-methylprop-
yl)-5-(trifluoromethyl)picolinamide 45 (0.146 g, 0.334 mmol) in
EtOH (5 mL) and water (5 mL), followed by hydroxylamine
(S)-3-Amino-1,1,1-trifluoro-2-methylpropan-2-ol hydrochloride 41
(1 g, 5.57 mmol) was added to a solution of 3-amino-5,6-
bis(trifluoromethyl)picolinic acid hydrochloride 47 (1.5 g, 5.47
mmol) in DMF (61 mL) followed by HATU (2.497 g, 6.57 mmol),
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Drug Annotation
and then diispropylethyamine (3.81 mL, 22.28 mmol) was added in
∼0.5 mL portions every 5−10 min over a total of 1 h. The reaction
mixture was added to water and extracted with EtOAc, and the organic
phase was washed with 1 M HCl, 2 M NaOH, water, and brine and
dried (MgSO4). Evaporation and flash chromatography (0−30%
EtOAc-isohexanes gradient elution) afforded an oil, which was taken
into hot DCM and isohexane was added until the solution became
turbid. On cooling, filtration afforded (S)-3-amino-N-(3,3,3-trifluoro-
2-hydroxy-2-methylpropyl)-5,6-bis(trifluoromethyl)picolinamide 29
as awhite solid (1.67 g, 75%). LRMS C12H10F9N3O2 requires M+
399.06, found [MH]+ 400.1. Elemental analysis requires C, 36.10%; H,
2.52%; N, 10.53% found C, 36.20 0.02%; H, 2.49 0.06%; N, 10.56
0.05%. 1H NMR (DMSO-d6) 1.25 (3H s), 3.49 (1H dd J = 14.0, 5.6),
3.67 (1H dd J = 13.8 7.3), 6.33 (1H s), 7.79 (1H s), 7.80 (2H br s), 8.31
(1H t J = 6.4). 13C NMR (DMSO-d6) 18.95 (q), 42.21 (t), 72.14 (s JF =
27), 129.87 (s JF = 272), 121.97 (s JF = 272), 124.28 (d JF = 6), 26.19 (s
JF = 36), 126.32 (s JF = 287), 126.34 (s JF = 34), 127.85 (s), 147.78 (s),
165.22 (s). 19F NMR (DMSO-d6) −59.81(q J = 11), −61.92 (q J = 11),
80.49 (s).
found [MH]+ 361.2. 1H NMR (DMSO-d6) 1.27 (3H s), 2.82 (3H d J =
4.5), 3.45 (1H dd J = 13.8, 5.6), 3.64 (1H dd J = 13.8, 7.3), 5.90 (1H q J
= 4.6), 6.22 (2H br s), 6.32 (1H s), 7.48 (1H s), 8.50 (1H t J = 6.4) and
(ent-2)-3-amino-6-(methylamino)-N-(3,3,3-trifluoro-2-hydroxy-2-
methylpropyl)-5-(trifluoromethyl)picolinamide 30b as a colorless oil
(24 mg, 34%), retention time 4.01 min. LRMS C12H14F6N4O2 requires
M+ 360.1, found [MH]+ 361.2. 1H NMR (DMSO-d6) 1.27 (3H s), 2.82
(3H d J = 4.5), 3.45 (1H dd J = 13.8, 5.6), 3.64 (1H dd J = 13.8, 7.3),
5.90 (1H q J = 4.6), 6.22 (2H br s), 6.32 (1H s), 7.48 (1H s), 8.50 (1H t
J = 6.4).
(ent-1)-3-Amino-6-(dimethylamino)-N-(3,3,3-trifluoro-2-hy-
droxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide (31a)
and (ent-2)-3-Amino-6-(dimethylamino)-N-(3,3,3-trifluoro-2-
hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide
(31b). Dimethylamine (0.209 mL, 1.652 mmol) was added to solution
of 6-bromo-3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-(trifluoromethyl)-
picolinic acid 48 (0.300 g, 0.826 mmol), and the reaction was stirred
at RT for 72 h and then partitioned between 0.5 M HCl and EtOAc.
The organic phase was washed with brine, dried (MgSO4) and
evaporated to afford crude 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-(dime-
thylamino)-5-(trifluoromethyl)picolinic acid as an orange oil (0.151 g,
56%) LRMS C15H16F3N3O2 requires M+ 327.12, found [MH]+ 328.2.
HATU (0.193 g, 0.507 mmol) was added to a solution of 3-(2,5-
dimethyl-1H-pyrrol-1-yl)-6-(dimethylamino)-5-(trifluoromethyl)-
picolinic acid (0.151 g, 0.461 mmol) and ( )-3-amino-1,1,1-trifluoro-
2-methylpropan-2-ol hydrochloride 41 (73 mg, 0.507 mmol) in NMP
(1 mL) followed by triethylamine (0.129 mL, 0.923 mmol). The
reaction was stirred at RT for 16 h and then partitioned between EtOAc
and 1 M NaOH. The organic phase was washed with brine, dried
(MgSO4) and evaporated to afford crude 3-(2,5-dimethyl-1H-pyrrol-1-
yl)-6-(dimethylamino)-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-
5-(trifluoromethyl)picolinamide as an orange oil (0.165 g, 78%) LRMS
C19H22F6N4O2 requires M+ 452.16, found [MH]+ 453.3.
(ent-1)-3-Amino-6-(methylamino)-N-(3,3,3-trifluoro-2-hy-
droxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide (30a)
and (ent-2)-3-Amino-6-(methylamino)-N-(3,3,3-trifluoro-2-hy-
droxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide (30b).
Two molar aq NaOH (2.52 mL, 5.04 mmol) was added to a solution of
methyl 6-bromo-3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-(trifluoromethyl)-
picolinate 43 (1.9 g, 1.504 mmol) in THF (10 mL). The reaction was
stirred at RT for 1 h, then poured into water, adjusted to pH4 with 1 M
HCl and extracted twice with EtOAc. The combined organic phases
were washed with brine, dried (MgSO4), and evaporated to afford crude
6-bromo-3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-(trifluoromethyl)picolinic
acid 48 as an orange solid (1.78 g, 97%). LRMS C13H10BrF3N2O2
1
requires M+ 361.99, found [MH]+ 363.1. H NMR (DMSO-d6) 1.92
(6H s), 5.74 (2H s), 8.14 (1H s), 14.3 (1H br s).
Methylamine (2 M THF, 0.826 mL, 1.652 mmol) was added to a
solution of 6-bromo-3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-
(trifluoromethyl)picolinic acid 48 (0.300 g, 0.826 mmol) in THF (1
mL). The reaction was stirred at RT for 10 days, then partitioned
between 0.5 M HCl and EtOAc. The organic phase was washed with
brine, dried (MgSO4), and evaporated to afford crude 3-(2,5-dimethyl-
1H-pyrrol-1-yl)-6-(methylamino)-5-(trifluoromethyl)picolinic acid as
a red oil (0.240 g, 92%). LRMS C14H14F3N3O2 requires M+ 313.10,
found [MH]+ 314.2. 1H NMR (DMSO-d6) 1.89 (6H s), 2.92 (3H d J =
4.3), 5.72 (2H s), 7.03 (1H q J = 4.2), 7.72 (1H s), 13.3 (1H br s).
HATU (0.320 g, 0.843 mmol) was added to a solution of 3-(2,5-
dimethyl-1H-pyrrol-1-yl)-6-(methylamino)-5-(trifluoromethyl)-
picolinic acid (0.240 g, 0.766 mmol) and ( )-3-amino-1,1,1-trifluoro-
2-methylpropan-2-ol hydrochloride 41 (0.121 g, 0.843 mmol) in NMP
(1 mL) followed by triethylamine (0.214 mL, 1.532 mmol). The
reaction was stirred at RT for 16 h then partitioned between EtOAc and
1 M NaOH. The organic phase was washed with brine, dried (MgSO4)
and evaporated to afford crude 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-
(methylamino)-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-
(trifluoromethyl)picolinamide (0.174 g, 52%). LCMS C18H20F6N4O2
requires M+ 438.15, found [MH]+ 439.3.
Hydroxylamine hydrochloride (0.269 g, 3.88 mmol) was added to a
solution of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-(methylamino)-N-
(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)-
picolinamide (0.170 g, 0.388 mmol) in EtOH (5 mL) and water (3
mL), followed by triethylamine (0.108 mL, 0.776 mmol). The mixture
was heated at reflux for 10 h and stood at RT for 16 h. The reaction was
partitioned between 1 M HCl/EtOAc and the organic phase was
washed with brine, dried (MgSO4), and evaporated. Flash chromatog-
raphy (0−60% EtOAc-isohexanes gradient elution) afforded ( )-3-
amino-6-(methylamino)-N-(3,3,3-trifluoro-2-hydroxy-2-methylprop-
yl)-5-(trifluoromethyl)picolinamide which was separated by prepara-
tive chiral SFC (Chiralpak OJ-H column, eluting with 10%−50%
[MeOH + 0.1% diethylamine]-CO2 gradient to afford (ent-1)-3-amino-
6-(methylamino)-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-
(trifluoromethyl)picolinamide 30a as a colorless oil (25 mg, 36%),
retention time 3.22 min. LRMS C12H14F6N4O2 requires M+ 360.1,
Hydroxylamine hydrochloride (0.253 g, 3.65 mmol) was added to a
solution of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-(dimethylamino)-N-
(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)-
picolinamide (0.165 g, 0.365 mmol) in EtOH (10 mL) and water (5
mL), followed by triethylamine (0.102 mL, 0.729 mmol). The mixture
was heated at reflux for 6 h. The reaction was partitioned between 1 M
HCl/EtOAc, and the organic phase was washed with brine, dried
(MgSO4), and evaporated. Flash chromatography (0−60% EtOAc-
isohexane gradient elution afforded 3-amino-6-(dimethylamino)-N-
(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)-
picolinamide, which was separated by preparative chiral SFC (Chiralcel
OD-H column; 12% [2-propanol +0.1% DEA]/88% CO2 elution) to
afford (ent-1)-3-amino-6-(dimethylamino)-N-(3,3,3-trifluoro-2-hy-
droxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide 31a as a col-
orless glassy solid (25 mg, 36%), retention time 4.09 min. LRMS
1
C13H16F6N4O2 requires M+ 374.12, found [MH]+ 375.3. H NMR
(DMSO-d6) 1.26 (3H s), 2.67 (6H s), 3.48 (1H dd J = 13.8, 5.6), 3.64
(1H dd J = 13.8, 7.2), 6.32 (1H s), 6.79 (2H br s), 7.60 (1H s), 8.31 (1H
t J = 6.4) and (ent-2)-3-amino-6-(dimethylamino)-N-(3,3,3-trifluoro-2-
hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide 31b as a
colorless glassy solid (25 mg, 36%), retention time 4.74 min. LRMS
1
C13H16F6N4O2 requires M+ 374.12, found [MH]+ 375.3. H NMR
(DMSO-d6) 1.26 (3H s), 2.67 (6H s), 3.48 (1H dd J = 13.8, 5.6), 3.64
(1H dd J = 13.8, 7.2), 6.32 (1H s), 6.79 (2H br s), 7.60 (1H s), 8.31 (1H
t J = 6.4).
(S)-3-Amino-6-ethoxy-N-(3,3,3-trifluoro-2-hydroxy-2-meth-
ylpropyl)-5-(trifluoromethyl)picolinamide (32). Two molar
NaOH (135 mL, 270 mmol) was added to a solution of methyl 6-
bromo-3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-(trifluoromethyl)picolinate
43 (34 g, 90 mmol) in MeOH (460 mL), and the reaction was heated at
90 °C for 3 h. The solvent was evaporated, and the residue was adjusted
to pH 1 with 2 M HCl and extracted twice with EtOAc. The combined
organic phases were washed with brine, dried (MgSO4), and evaporated
to afford 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-methoxy-5-
(trifluoromethyl)picolinic acid 49 as a reddish-brown solid (28.2 g,
100%). LRMS C14H13F3N2O3 requires M+ 314.09, found [MH]+ 315.2.
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
1H NMR (DMSO-d6) 1.90 (6H s), 4.07 (3H s), 5.77 (2H s), 8.19 (1H
s), 13.8 (1H br s).
3.65 (1H dd J = 13.6, 7.2), 4.38 (2H q J = 7.0), 6.29 (1H s), 6.67 (1H s),
7.67 (1H s) 8.26 (1H t J = 6.2).
(S)-3-Amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-
methylpropyl)-5-(trifluoromethyl)picolinamide (33). Triethyl-
amine (37.5 mL, 269 mmol) was added to 3-(2,5-dimethyl-1H-pyrrol-
1-yl)-6-methoxy-5-(trifluoromethyl)picolinic acid 49 (28.2 g, 90
mmol) in EtOH (540 mL) and water (270 mL) followed by
hydroxylamine hydrochloride (62.4 g, 897 mmol). The reaction was
heated at 100 °C for 5 h and then partitioned between EtOAc (800 mL)
and 1 M HCl (800 mL). The aqueous phase was extracted with EtOAc
(800 mL) and the combined organic phases were washed with brine,
dried (MgSO4 and evaporated. The foregoing procedure was repeated
analogously starting from 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-methoxy-
5-(trifluoromethyl)picolinic acid (75.7 g, 241 mmol) and the crude
products from both reactions were combined and recrystallized from
isohexanes with hot filtration to afford 3-amino-6-methoxy-5-
(trifluoromethyl)picolinic 51 acid in 3 crops as an orange/yellow
solid (41.59 g, 53%). LRMS C8H7F3N2O3 requires M+ 236.04, found
[MH]+ 237.1. 1H NMR (DMSO-d6) 3.90 (3H s), 7.68 (1H s), 7.80−
9.20 (3H br s).
DIPEA (62.4 g, 897 mmol) was added to a solution of 3-amino-6-
methoxy-5-(trifluoromethyl)picolinic acid 51 (22.0 g, 93 mmol) and
(S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol 41 hydrochloride
(16.73 g, 93 mmol) in NMP (800 mL), followed by HATU (42.5 g,
112 mmol). The reaction was stirred for 1 h at RT, added to EtOAc (4
L) and then washed with 1 M NaOH (2 × 2 L), water (2 L) and brine (2
L) and dried (MgSO4) and evaporated to afford a brown oil. The
foregoing procedure was repeated analogously starting from 3-amino-6-
methoxy-5-(trifluoromethyl)picolinic acid 51 (14.0 g, 59.3 mmol), and
the crude products from both reactions were combined and initially
purified by flash chromatography (5−100% EtOAc-isohexane gradient
elution). The resultant solid was dissolved in the minimum volume of
hot DCM, which on cooling reprecipitated. Filtration and drying
afforded (S)-3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-
methylpropyl)-5-(trifluoromethyl)picolinamide 33 as a white solid
(33.6 g, 59%). LRMS C12H13F6N3O3 requires M+ 361.08, found [MH]+
362.2. Elemental analysis requires C, 39.90%; H, 3.63%; N, 11.63%
found C, 40.22 0.06%; H, 3.68 0.11%; N, 11.76 0.04%. 1H NMR
(DMSO-d6) 1.26 (3H s), 3.46 (1H dd J = 13.3, 5.6), 3.66 (1H dd J =
13.7, 7.3), 3.92 (3H s), 6.29 (1H s), 6.69 (2H br s), 7.68 (1H s), 8.30
(1H t J = 6.4). 13C NMR (DMSO-d6) 18.95 (q), 42.19 (t), 53.56 (q),
72.27 (s JF = 26.8), 116.07 (s JF = 32.3), 122.40 (s JF = 272.1), 124.85
(s), 126.43 (s JF = 287.1), 128.29 (d JF = 5.2), 141.0 (s), 148.51 (s),
166.3 (s). 19F NMR (DMSO-d6) −62.71 (s), −80.46 (s).
Conc H2SO4 (42 μL, 0.795 mmol) was added to a solution of 3-(2,5-
dimethyl-1H-pyrrol-1-yl)-6-methoxy-5-(trifluoromethyl)picolinic acid
49 (500 mg, 1.591 mmol) in MeOH (16 mL). The reaction was heated
at reflux for 20 h, and then the solvent was evaporated. The residue was
adjusted to pH 7 with sat. NaHCO3 and extracted with EtOAc. The
organic phase was washed with brine, dried (MgSO4), and purified by
flash chromatography (0−10% EtOAc-isohexane gradient) to afford
methyl 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-methoxy-5-
(trifluoromethyl)picolinate as an off-white solid (0.391 g, 75%).
1
LRMS C15H15F3N2O3 requires M+ 328.10, found [MH]+ 329.2. H
NMR (DMSO-d6) 1.89 (6H s), 3.64 (3H s), 4.07 (3H s), 5.80 (2H s),
8.29 (1H s).
KI (0.589 g, 3.55 mmol) was added to a solution of methyl 3-(2,5-
dimethyl-1H-pyrrol-1-yl)-6-methoxy-5-(trifluoromethyl)picolinate
(0.291 g, 0.886 mmol) in MeCN (9 mL) followed by TMSCl (0.453
mL, 3.54 mmol). The reaction was heated at reflux for 5 h, and then the
solvent was evaporated. The residue was taken into EtOAc, washed
with water then brine and dried (MgSO4). Evaporation and partial
purification by flash chromatography (0−30% EtOAc-isohexanes
gradient elution) afforded crude methyl 3-(2,5-dimethyl-1H-pyrrol-1-
yl)-6-hydroxy-5-(trifluoromethyl)picolinate as a white solid (0.176 g,
54%). LRMS C14H13F3N2O3 requires M+ 314.09, found [MH]+ 315.4.
Triphenylphosphine (0.250 g, 0.952 mmol) was added to a solution
of methyl 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-hydroxy-5-
(trifluoromethyl)picolinate (0.176 g, 0.476 mmol) in dioxane (5
mL), followed by EtOH (0.6 mL, 10.28 mmol) and dropwise addition
of DEAD (0.151 mL, 0.952 mmol). The reaction was stirred at RT for 2
h and the solvent was evaporated. Purification by flash chromatography
(0−10% EtOAc-isohexanes gradient elution) afforded ethyl 3-(2,5-
dimethyl-1H-pyrrol-1-yl)-6-ethoxy-5-(trifluoromethyl)picolinate 50 as
a colorless viscous oil (0.100 g, 61%). LRMS C16H17F3N2O3 requires
M+ 342.12, found [MH]+ 343.2. 1H NMR (DMSO-d6) 1.38 (3H t J =
7.1), 1.88 (6H s), 3.63 (3H s), 4.52 (2H q J = 7.1), 5.79 (2H s), 8.27
(1H s).
Two molar NaOH (0.613 mL, 1.226 mmol) was added to a solution
of ethyl 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-ethoxy-5-(trifluoromethyl)-
picolinate 50 (0.140 g, 0.409 mmol) in THF (2 mL). The reaction was
heated at reflux for 6 h and the solvent was evaporated. The residue was
acidified to pH 1 with 5 M HCl and extracted with EtOAc. The organic
phase was washed with brine, dried (MgSO4) and evaporated to afford
crude 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-ethoxy-5-(trifluoromethyl)-
picolinic acid as a yellow oil (35 mg, 26%). LRMS C15H15F3N2O3
requires M+ 328.10, found [MH]+ 329.2.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00343.
■
HATU (49 mg, 0.128 mmol) was added to a solution of 3-(2,5-
dimethyl-1H-pyrrol-1-yl)-6-ethoxy-5-(trifluoromethyl)picolinic acid
(35 mg, 0.107 mmol) and (S)-3-amino-1,1,1-trifluoro-2-methylpro-
pan-2-ol hydrochloride 41 (19 mg, 0.107 mmol) in DMF (1 mL)
followed by DIPEA (56 μL, 0.320 mmol). The reaction was stirred at
RT for 3 h then partitioned between EtOAc and water. The organic
phase was washed with 1 M HCl, 2 M NaOH, brine and then dried
(MgSO4). Evaporation afforded crude 3-(2,5-dimethyl-1H-pyrrol-1-
yl)-6-ethoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-
(trifluoromethyl)picolinamide as a yellow oil (43 mg, 89%). LRMS
C19H21F6N3O3 requires M+ 453.15, found [MH]+ 454.3.
sı
Molecular formula strings for final compounds (CSV)
X-ray crystallographic data for compounds 22 and 41; list
of off-target safety profiling assays; CFTR HBEC ion
transport assay additional experimental information;
experimental procedures for in vitro and in vivo
pharmacokinetic and metabolite ID studies together
with in vitro plasma protein binding studies; experimental
details and toxicokinetic data for exploratory toxicology
studies on compounds 22 and 33; characterization data
on final compounds 6−33 (PDF)
Hydroxylamine hydrochloride (66 mg, 0.948 mmol) was added to a
solution of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-6-ethoxy-N-(3,3,3-tri-
fluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide
(43 mg, 0.095 mmol) in EtOH (0.6 mL) and water (0.3 mL), followed
by triethylamine (40 μL, 0.285 mmol), and the mixture was heated at
reflux for 5 h. The reaction was partitioned between water/EtOAc then
organic phase was washed with brine, dried (MgSO4) and evaporated.
Purification by preparative HPLC (C18 column, 50−95% [MeCN +
0.1% DEA] − [water +0.1% DEA] gradient) afforded (S)-3-amino-6-
ethoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-
(trifluoromethyl)picolinamide 32 as a white solid (13 mg, 37%). LRMS
AUTHOR INFORMATION
Corresponding Author
■
David A. Sandham − Novartis Institutes for Biomedical
Research, Cambridge, Massachusetts 02139, United States;
orcid.org/0000-0003-4523-9931;
1
C13H15F6N3O3 requires M+ 375.10, found [MH]+ 376.1. H NMR
(DMSO-d6) 1.25 (3H s), 1.32 (3H t J = 7.0), 3.46 (1H dd J = 13.7, 5.5),
Email: david.sandham@novartis.com
7257
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J. Med. Chem. 2021, 64, 7241−7260

Journal of Medicinal Chemistry
Authors
pubs.acs.org/jmc
Drug Annotation
Christopher Page − Novartis Institutes for Biomedical
Research, Horsham Research Center, Horsham RH12 5AB,
U.K.
Giles Pergl-Wilson − Novartis Institutes for Biomedical
Research, Horsham Research Center, Horsham RH12 5AB,
U.K.
Mariana Oana Popa − Novartis Institutes for Biomedical
Research, Horsham Research Center, Horsham RH12 5AB,
U.K.
Darren Le Grand − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Martin Gosling − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Urs Baettig − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Parmjit Bahra − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Kamlesh Bala − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Cara Brocklehurst − Novartis Institutes for Biomedical
Research, Basel CH 4002, Switzerland; orcid.org/0000-
0002-0484-5540
Emma Budd − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Rebecca Butler − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Atwood K. Cheung − Novartis Institutes for Biomedical
Research, Cambridge, Massachusetts 02139, United States;
orcid.org/0000-0002-2210-6362
Hedaythul Choudhury − Novartis Institutes for Biomedical
Research, Horsham Research Center, Horsham RH12 5AB,
U.K.
Stephen P. Collingwood − Novartis Institutes for Biomedical
Research, Horsham Research Center, Horsham RH12 5AB,
U.K.
Brian Cox − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.;
orcid.org/0000-0002-5980-9707
Henry Danahay − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Lee Edwards − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.;
orcid.org/0000-0002-5645-746X
Richard Robinson − Novartis Institutes for Biomedical
Research, Cambridge, Massachusetts 02139, United States;
orcid.org/0000-0002-7209-3424
David Rowlands − Novartis Institutes for Biomedical Research,
Cambridge, Massachusetts 02139, United States
Tom Sharp − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Matthew Spendiff − Novartis Institutes for Biomedical
Research, Horsham Research Center, Horsham RH12 5AB,
U.K.
Emily Stanley − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Oliver Steward − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Roger J. Taylor − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Pamela Tranter − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Trixie Wagner − Novartis Institutes for Biomedical Research,
Basel CH 4002, Switzerland
Hazel Watson − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Gareth Williams − Novartis Institutes for Biomedical Research,
Basel CH 4002, Switzerland
Penny Wright − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Alice Young − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Brian Everatt − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Ulrike Glaenzel − Novartis Institutes for Biomedical Research,
Basel CH 4002, Switzerland
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00343
Anne-Lise Glotin − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Paul Groot-Kormelink − Novartis Institutes for Biomedical
Research, Basel CH 4002, Switzerland
Edward Hall − Novartis Institutes for Biomedical Research,
Cambridge, Massachusetts 02139, United States
Julia Hatto − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Catherine Howsham − Novartis Institutes for Biomedical
Research, Horsham Research Center, Horsham RH12 5AB,
U.K.
Glyn Hughes − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Anna King − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Julia Koehler − Novartis Institutes for Biomedical Research,
Basel CH 4002, Switzerland
Swarupa Kulkarni − Novartis Institutes for Biomedical
Research, East Hanover, New Jersey 07936, United States
Megan Lightfoot − Novartis Institutes for Biomedical Research,
Horsham Research Center, Horsham RH12 5AB, U.K.
Ian Nicholls − Novartis Institutes for Biomedical Research,
Basel CH 4002, Switzerland
Notes
The authors declare the following competing financial
interest(s): All authors are former or current employees of
Novartis.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the following individuals:
Dr. Hanna Harant (NIBR Vienna) for generation of the FRT
cell lines, Prof Jack Riordan (University of North Carolina
Chapel Hill) for provision of the F508del CFTR CHO cell line
used for the Quattro assay and Prof Scott Randell (University of
North Carolina Chapel Hill) for provision of HBEC.
ABBREVIATIONS USED
■
ALI, air liquid interface; ASL, airway surface liquid; BEGM,
bronchial epithelial cell growth medium; CB, chronic bronchitis;
CHO, Chinese hamster ovary; COPD, chronic obstructive
pulmonary disease; DEA, diethylamine; FLIPR, fluorimetric
imaging plate reader; HATU, (1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
fluorophosphate; LLE, lipophilic ligand efficiency; MCC,
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J. Med. Chem. 2021, 64, 7241−7260

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
(16) McDonough, J. E.; Yuan, R.; Suzuki, M.; Seyednejad, N.; Elliott,
W. M.; Sanchez, P. G.; Wright, A. C.; Gefter, W. B.; Litzky, L.; Coxson,
H. O.; Pare, P. D.; Sin, D. D.; Pierce, R. A.; Woods, J. C.; McWilliams, A.
M.; Mayo, J. R.; Lam, S. C.; Cooper, J. D.; Hogg, J. C. Small-airway
obstruction and emphysema in chronic obstructive pulmonary disease.
N. Engl. J. Med. 2011, 365, 1567−1575.
mucociliary clearance; MRT, mean residence time; PoC, proof
of concept; SFC, supercritical fluid chromatography
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