Structure-activity relationships of cyanoquinolines with corrector-potentiator activity in Δf508 cystic fibrosis transmembrane conductance regulator protein

Article abstract of DOI:10.1021/jm201372q

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The most common CF-causing mutation, ΔF508-CFTR, produces CFTR loss-of-function by impairing its cellular targeting to the plas

Full text of DOI:10.1021/jm201372q

Article
pubs.acs.org/jmc
Structure−Activity Relationships of Cyanoquinolines with Corrector−
Potentiator Activity in ΔF508 Cystic Fibrosis Transmembrane
Conductance Regulator Protein
John M. Knapp,† Alex B. Wood,† Puay-Wah Phuan,‡ Michael W. Lodewyk,† Dean J. Tantillo,†
A. S. Verkman,‡ and Mark J. Kurth*,†
†Department of Chemistry, University of California, Davis, California 95616, United States
‡Departments of Medicine and Physiology, University of California, San Francisco, California 94143-0521, United States
S
* Supporting Information
ABSTRACT: Cystic fibrosis (CF) is caused by mutations in the cystic
fibrosis transmembrane conductance regulator (CFTR) chloride
channel. The most common CF-causing mutation, ΔF508-CFTR,
produces CFTR loss-of-function by impairing its cellular targeting to the
plasma membrane and its chloride channel gating. We recently identified
cyanoquinolines with both corrector (“Co”, normalizing ΔF508-CFTR
targeting) and potentiator (“Po”, normalizing ΔF508-CFTR channel
gating) activities. Here, we synthesized and characterized 24 targeted cyanoquinoline analogues to elucidate the conformational
requirements for corrector and potentiator activities. Compounds with potentiator-only, corrector-only, and dual potentiator−
corrector activities were found. Molecular modeling studies (conformational search ⇒ force-field lowest energy assessment ⇒
geometry optimization) suggest that (1) a flexible tether and (2) a relatively short bridge between the cyanoquinoline and
arylamide moieties are important cyanoquinoline-based CoPo features. Further, these CoPo’s may adopt two distinct π-stacking
conformations to elicit corrector and potentiator activities.
nylacetamide, PG01}7g and 4 {N-(2,4-di-tert-butyl-5-hydrox-
INTRODUCTION
Cystic Fibrosis (CF) is a genetic disorder caused by mutations
■
yphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide, VX-
770};7h Figure 1b). Recently, we identified a dual-acting
in the cystic fibrosis transmembrane conductance regulator
(CFTR) protein,1 which is expressed in epithelia in lung,
cyanoquinoline having independent corrector and potentiator
pancreas, intestine, testis, and other tissues.2 While there are
many CF-causing mutations, the most common CFTR
activities (5, {N-(2-((3-cyano-5,7-dimethylquinolin-2-yl)-
amino)ethyl)-3-methoxybenzamide, CoPo-22};8 Figure 1c).
Such compounds would be advantageous over CF treatments
mutation is a deletion of the phenylalanine residue at position
requiring separate corrector and potentiator administration.8
508 (ΔF508-CFTR). The ΔF508 mutation causes folding and
conformation defects resulting in its retention in the
Moreover, the development costs for a single drug that treats
endoplasmic reticulum and accelerated degradation.3,4 Addi-
both defects would be significantly lower than development
costs for a two-drug paradigm.
tionally, any ΔF508-CFTR that reaches the cell plasma
membrane is poorly functional as a chloride channel.2
Cyanoquinoline 5 contains three distinct subunits consisting
of a cyanoquinoline core and arylamide moieties linked by a
flexible tether (Figure 1). We postulated that this tether allows
5 to adopt two active conformations, an active corrector
conformation and an active potentiator conformation, and that
altering this tether would modulate CoPo activities by
Consequently, the ΔF508-CFTR mutation results in greatly
reduced cell membrane chloride permeability.
An ideal small molecule treatment of CF would fully restore
ΔF508-CFTR structure to the native wild type state, resulting
in normalization of its cellular processing and chloride channel
function.5 Such compounds have not been identified. Instead,
restricting its conformations. The two active conformations of
5 could allow binding to two separate targets (mode 1), two
separate compounds, called “correctors”, which partially rescue
ΔF508-CFTR cellular misprocessing, and “potentiators”, which
distinct binding sites on the same target (mode 2), or two
different forms of a single binding site on ΔF508-CFTR that
change shape during protein transport from the endoplasmic
reticulum (ER) to the cell plasma membrane (mode 3, Figure
2). Of these, mode 3 appears to be the most likely on a
statistical basis; i.e., one molecule addressing one binding site
partially restore ΔF508-CFTR chloride channel function, have
been identified (Figure1). We and others have identified a
variety of correctors6 (e.g., 1 {N-(2-((5-chloro-2-
methoxyphenyl)amino)-4′-methyl-[4,5′-bithiazol]-2′-yl)-
benzamide, corr-4a}6d and 2 {4-(cyclohexyloxy)-2-(1-(4-((4-
methoxyphenyl)sulfonyl)piperazin-1-yl)ethyl)quinazoline,
VRT-325};6h Figure 1a) and potentiators7 (e.g., 3 {2-(2-(1H-
indol-3-yl)-N-methylacetamido)-N-(4-isopropylphenyl)-2-phe-
Received: October 11, 2011
Published: January 3, 2012
© 2012 American Chemical Society
1242
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
Figure 1. Structures of (a) correctors 1 and 2, (b) potentiators 3 and 4, and (c) cyanoquinoline 5 with structural subunits identified.
Scheme 1. Synthesis of Chlorocyanoquinoline Intermediate
a
5
Figure 2. Possible CoPo conformational adaptation during the ER ⇒
cell membrane transition.
a
that changes shape as it matures is statistically less challenging
than one molecule addressing two distinct binding sites (modes
1 and 2). The goal of the work reported here was to examine
how the tethering moiety in 5 influences its ability to afford
both corrector (Co) and potentiator (Po) activities and to
assess the feasibility of mode 3 by computational analysis of the
proclivities of 5 and tethered analogues for particular
conformations.
Reagents: (a) acetic anhydride, THF; (b) POCl3, DMF; (c)
NH2OH·HCl, Et3N, EtOH; (d) SOCl2, benzene.
Acylation of 6 with acetic anhydride gave 7 in near quantitative
yield, and subsequent Vilsmeier−Haack12 type reaction gave
chloroquinoline carboxaldehyde 8. Neutralization, filtration,
and drying produced pure 8 in 95% yield. Aldehyde 8 was then
condensed with hydroxylamine to yield oxime 9, which was
directly converted to nitrile 10 by dehydration with SOCl2 in
refluxing benzene (93% yield over two steps).
Aromatic substitution of 10 with various diamines allowed
for ready diversification of the tethering subunit as depicted in
Scheme 2. The tethers examined were derived from 1,2-
diaminoethane (the tether in original screening hit 5), 1,3-
CHEMISTRY
■
This study examines the role of the tether and, secondarily, the
arylamide subunits on CoPo activities. Our tether-focused
strategy is depicted in Figure 3 where the cyanoquinoline
a
Scheme 2. Tether Diversification
Figure 3. Tether-focused CoPo library diversification strategy.
moiety is fixed while the tether and arylamide moieties are
varied. Indeed, quinolines hold privileged heterocycle status9
and have received much attention as antibacterial agents10 and
cancer therapeutics.11 Their biological importance has led to
the development of several strategies to construct this aromatic
ring system.
a
Reagents: (e) 1,2-diaminoethylene, dioxane, reflux; (f) 1,3-
The CoPo cyanoquinoline core was synthesized starting
from commercially available 3,5-dimethylaniline 6 (Scheme 1).
diaminopropylene, dioxane, reflux; (g) piperazine, dioxane, reflux.
1243
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
Figure 4. Cyanoquinoline-based CoPo arylamide diversity.
diaminopropane (a longer and more flexible tether), and 1,4-
piperazine (a more rigid tether). All three diamino tethers gave
their intended products (10 → 11−13) in good yields (61−
62%).
Further diversification of the distal primary or secondary
amine in 11−13 was accomplished by EDC-mediated aryl acid
coupling (Figure 4). Heteroaromatic amides were not evaluated
in our initial screening study. So, as a preliminary test of the
effect of heterocycles on CoPo potency, a range of pyridyl
acids, an array of arylmethoxy acids (similar to that of initial hit
5), and one methoxypyridyl acid were employed in the
acylation of 11−13. The specific aryl acids used in this coupling
step are depicted in Figure 4.
each bond between the aryl moieties was given 2-fold rotational
freedom (sp2−sp2 bonds) or 3-fold rotational freedom (sp2−sp3
and sp3−sp3 bonds) and atoms in rings were given 3-fold
puckering mobility. This procedure resulted in 36 candidate
conformations for 5 and 42 candidate conformations for 26,
each within 10 kcal/mol of the lowest energy conformation, as
assessed by the MMFF94 force field.14 These candidates were
then subjected to further geometry optimization using the
M06-2X/6-31+G(d,p)15 density functional theory method as
implemented in the Gaussian '09 software suite.16 After this
refinement, five conformers within 3 kcal/mol of the lowest
energy conformer of 5 and 18 conformers within 3 kcal/mol of
the lowest energy conformer of 26 (four within 2 kcal/mol)
were found, as assessed by computed electronic energies with
solvent (water) modeled by the SMD continuum solvation
model17 (via single-point calculations). Finally, select con-
formers described below were further refined with full solvent
optimization and frequency calculations in order to include
entropy contributions to the computed relative free energies.
Atomic coordinates and computed energies for all structures
can be found in the Supporting Information. As described
below, constrained calculations were also performed on some
conformers.
PHARMACOLOGY
■
ΔF508-CFTR correctors were previously identified by screen-
ing of a large collection of diverse small molecules.6d Our
screening assay utilized Fischer rat thyroid (FRT) epithelial
cells coexpressing ΔF508-CFTR and the yellow fluorescent
protein (YFP) halide indicator YFP-H148Q/I152L at 37 °C in
a 96-well-plate format. Test compounds at 10 μM (and
negative/positive controls) were added to each well for 18−24
h. ΔF508-CFTR-facilitated iodide influx was determined from
the kinetics of decreasing YFP fluorescence following addition
of extracellular iodide in the presence of the potentiators
genistein and forskolin. Active compounds from the primary
high-throughput screen were verified and then counterscreened
for ΔF508-CFTR potentiator activity. For the potentiator assay
ΔF508-CFTR was low-temperature rescued by incubating FRT
cells coexpressing ΔF508-CFTR and YFP at 27 °C for 24 h.
Test compounds at 25 μM (and negative/positive control)
were added to each well for 10 min in the presence of forskolin.
Similar to the corrector assay, ΔF508-CFTR-facilitated iodide
influx was determined from the kinetics of decreasing YFP
fluorescence following addition of extracellular iodide.
RESULTS AND DISCUSSION
■
Corrector and potentiator activities for the 24 cyanoquinolines
synthesized in this study were assayed separately in ΔF508-
CFTR expressing FRT epithelial cells. Table 1 lists corrector
(Co) and potentiator (Po) activities as EC50 and Vmax from
concentration-dependence studies. EC50 is a measure of
compound potency and Vmax is a measure of compound
efficacy in increasing cell chloride permeability. Figure 5 shows
representative concentration-dependence data for the corrector
and potentiator plate reader assays from which EC50 and Vmax
values were deduced from concentration-dependence data by
nonlinear regression as described in the Experimental Section
(plate reader assay). Figure 6 shows representative short-circuit
assays of corrector and potentiator activities of compound 15,
compared to 5, in which apical membrane chloride current was
measured in the ΔF508-CFTR expressing FRT cells after
COMPUTATIONS
■
For cyanoquinolines 5 and 26, a two-step conformational
search was performed. First, candidate conformers were
identified via a systematic search in Spartan ’1013 in which
1244
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
Table 1. Corrector and Potentiator Activities, Determined in Triplicate, of CoPo Analogues As Measured by Fluorescence
a
Plate−Reader Assay
a
Concentration−response curves were determined for eight compound concentrations (maximum 50 μM for potentiator assay; 25 μM for corrector
assay). EC50 ( SE) and Vmax were computed as described in the Experimental Section (plate reader assay).
Figure 5. Concentration-dependence of corrector (left) and potentiator (right) activities of indicated compounds in FRT cells expressing ΔF508-
CFTR measured by iodide (I−) influx. See Table 1 for summary of deduced EC50 and Vmax
.
1245
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
Two notable observations from these data are that
constraining the diamino tether in a piperazine ring abolished
corrector activity but, conversely, resulted in excellent
potentiator activity (25−27), better than that of 5. One
interpretation of these findings is that that the binding site
evolves during the endoplasmic reticulum ⇒ cell membrane
transition (mode 3 discussed above; see Figure 2) and that
piperazine-constrained cyanoquinolines cannot adequately
adapt to the ΔF508-CFTR corrector-binding site when the
protein is in the endoplasmic reticulum. Consequently,
transport to the cell membrane does not occur with these
piperazine-constrained cyanoquinolines. Conversely, an in-
crease in mobility of the chain by lengthening (ethylene →
propylene) results in CoPo’s (32−33, 35, 36) with comparable,
albeit, lower corrector and potentiator activity compared to that
of 5. This increased tether chain length causes increased
flexibility and different conformational preferences, which in
turn results in a reduced probability of these CoPo’s adopting
an active conformation. Thus, the propylene-based tether is
slightly less active compared to the ethylene-based tether.
If this model is correct, then the action of cyanoquinoline-
based CoPo’s likely involves a slight, but important, conforma-
tional change allowing them to adapt to a single binding site
that matures as it progresses from the ER to the cell membrane.
Indeed, the juxtaposition of Co- and Po-activities with 5 versus
only Po-activity with 25−27 implies that, in this cyanoquino-
line-based series, the CoPo must be able to access a
conformation not precluded by the constraints associated
with tethering. In addition, this conformation, which is active
for Po-activity, must differ from that associated with Co-
activity, implying that 5 must also be able to readily access a
conformation that is not accessible by 25−27. The active
conformation enabling potentiation is satisfied by (indeed,
enabled by) piperazine-constrained cyanoquinolines (e.g., 25−
27 are all better potentiators than 5). Taken together, the
tether-differentiated SAR results delineated in Table 1 suggest
that (1) a flexible tether and (2) a relatively short bridge
between the cyanoquinoline and arylamide moieties are vital for
these cyanoquinoline-based CoPo’s to adopt the two distinct
conformations required for dual Co- and Po-activities.
Figure 6. Short-circuit current measurement of ΔF508-CFTR chloride
conductance. Measurements of corrector and potentiator activities of
15 and 5 were done in the presence of an apical-to-basolateral chloride
gradient in basolateral membrane-permeabilized cells. Added com-
pounds included forskolin (fsk, 20 μM), genistein (gen, 50 μM), and
CFTRinh-172 (10 μM).
basolateral membrane permeabilization and in the presence of a
transepithelial chloride gradient. The corrector assay was done
by incubation of cells for 18−24 h with test compound at 37
°C, followed by addition of forskolin and the potentiator
genistein. The potentiator assay was done in low temperature-
rescued cells by addition of forskolin followed by test
compound. Short-circuit data as in Figure 6 confirmed the
main findings obtained using the fluorescence plate reader
assay.
On the basis of these function studies, 12 compounds in this
series were identified as having both corrector and potentiator
activities (14, 15, 17, 18, 20, 21, 23, 24, 32, 33, 35, and 36), 8
compounds were identified as having potentiator-only activity
(16, 19, 25−28, 31, and 34), and 1 compound was identified as
having corrector-only activity (22). For compounds having
both corrector and potentiator activities, cyanoquinoline 5 is
the most active (corrector/potentiator values of 2.2 μM/5.9
μM): the corrector activity EC50 values for cyanoquinolines 15,
17, and 18 were 3.0, 2.7, and 7.3 μM, respectively, and the
potentiator activity EC50 values were 4.1, 13, and 10 μM,
respectively. The maximal efficacies, Vmax, of cyanoquinoline 5
for corrector and potentiator actions are comparable with those
of 1 and genistein, respectively.
Are there energetically accessible conformations of 5 and
25−27 that meet these requirements? One such pair of
conformers (there may well be others, but these conformers
illustrate the point) located by our conformational search
calculations are shown in Figure 7, a conformation possibly
associated with the Co-activity of 5 at left and a conformation
possibly associated with Po-activity for 26 (the molecule in the
Figure 7. (a) Possible “Co”-active conformer of 5. (b) Possible “Po”-active conformer of 26. Two views of each structure are shown. In the left-hand
presentation of each structure, the viewing angle is perpendicular to the C−C bond of the tether (downward double-arm arrow); this view
emphasizes the differing dihedral angles between the C−N bonds. In the right-hand presentation of each structure, the orientation is systematized by
viewing down this same C−C bond (↓). Selected through-space distances are shown in Å.
1246
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
25−27 series that has the same aryl group as 5) at right. First,
note that both of these conformers are folded to allow for some
degree of π-stacking between the quinoline and methoxyphenyl
groups, an effect that is perhaps maximized in water. The
conformer shown for 26 is predicted to be the lowest energy
conformer in water for this molecule (despite the twist-boat
conformation of its piperazine core), while that for 5 is
predicted to be the seventh-lowest conformer in water, only
approximately 3 kcal/mol higher in energy than the lowest
energy conformer. Here we focus on a low energy conformer of
5 that could possibly mimic that shown for 26 by undergoing
relatively minor conformational changes; the lowest energy
conformers predicted for 5 (see Supporting Information),
although still displaying π-stacking interactions, do not orient
their NH groups in such a way as to allow for such a
conformational exchange to occur. Although strong binding to
a target can overcome energy penalties associated with
nonoptimal conformations of ligands, if the conformer shown
for 26 does correspond to the Po-active conformation, no
energy penalty associated with changing this conformation
from the predominant conformation in water will need to be
paid.18 Since 5 is also active, however, a 26-like conformation
would need to be energetically accessible. Altering the
conformation of 5 such that the quinoline and methoxyphenyl
groups are in the same relative orientation as in 26 (and
allowing the rest of the molecule to relax; see Supporting
Information for details) is predicted to be accompanied by an
increase in energy of approximately 4 kcal/mol (likely the result
of not only disrupting π-stacking but also disrupting the gauche
conformation of the N−C−C−N substructure, which is
preferred on both steric and orbital-based grounds over other
staggered conformations;19 note that this distorted conforma-
tion is predicted to be approximately 8 kcal/mol above the
lowest energy conformation of 5 in water). Thus, 5 could adopt
the shape of 26 if this energy penalty is not enough to cause
dissociation or is counterbalanced by favorable interactions
with a binding site cavity complementary to 26. We see no
reason why this relatively small change to the conformation of
5 could not occur within a binding site (i.e., without
dissociation) that switches between a Co- and a Po-form,
each complementary to one or the other arrangement of the
quinoline and methoxyphenyl groups shown in Figure 7a and
Figure 7b. Although this model is speculative, it should be
testable through synthesis of, and activity assays on, quinolines
with further conformational constraints.
potentiator activity, while most other modifications giving
better corrector or better potentiator activity did so at the
expense of the other. These data suggest that dual CoPo
activity is not a general feature of cyanoquinolines but is rather
dependent on the particular tether and/or arylamide
substituent.
CONCLUSION
■
This work provides structure−activity insight around the
unique cyanoquinoline-based CoPo scaffold, which has dual,
independent ΔF508-CFTR corrector and potentiator activities.
The most active diamine tether was shown to be 1,2-
diaminoethane-based compared to more constrained (piper-
azine) and more flexible (1,3-diaminopropylene) counterparts.
Additionally, variations of the arylamide moiety can tune the
corrector and/or potentiator activities. This work has led to the
hypothesis that cyanoquinoline-based CoPo’s have dual activity
if they can conformationally adapt to a single binding site that
evolves during the endoplasmic reticulum ⇒ cell membrane
transition. While the target site(s) of CoPo’s remain unknown,
this work provides a working hypothesis regarding the corrector
and potentiator structural features of the reported cyanoquino-
line-based CoPo scaffold.
EXPERIMENTAL SECTION
■
General Experimental. All purchased starting materials and
reagents were used without further purification. Product purification
was performed either on an automated flash chromatography system
(Combiflash by Teledyne, 35 min of elution with linear gradient from
100% hexane to 100% EtOAc solvent) with silica gel columns or on an
HPLC system [Waters, 15 mL/min flow rate, linear gradient elution
with 0.1% TFA-containing H2O/MeCN from 5% to 95% MeCN in 20
min, Xterra Prep MS C18 OBD column (19 mm × 100 mm), and dual
wavelength absorbance detector]. NMR spectra (1H at 400 MHz, 13C
at 100 MHz) were recorded in CDCl3 solvent on a Varian 600.
Chemical shifts are expressed in parts per million relative to solvent.
Coupling constants are expressed in units of hertz (Hz). Splitting
patterns are designated as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), and bs (broad singlet). LC/MS (Waters
Micromass ZQ) specifications are as follows: electrospray (+)
ionization, mass ranging from 100 to 900 Da, 20 V cone voltage.
For LC, the specifcations are Xterra MS C18 column (2.1 mm × 50
mm × 3.5 μm), 0.2 mL/min water/acetonitrile (containing 0.1%
TFA), 30 min linear gradient 0−100% acetonitrile. The LC/MS UV
detector is a diode array with 200−400 nm wavelength range. Purity is
based on integration of peak area as a percentage of the UV diode
array signals. Compound purities are determined by RP-HPLC
(≥95%).
Additional structure−activity insight was derived from
examination of the arylamide moiety. Substitution at the
ortho position of the arylamide generally gave worse corrector
activity but better potentiator activity (e.g., CoPo’s 14, 16, 25,
28, and 34). The best corrector activity was seen with an
ethylene tether employing a methoxy substitution at either the
meta (5) or para (CoPo 15) position of the arylamide, and the
greatest corrector activity was obtained by placing a methoxy
substituent at both (CoPo 21); note that electron-donating
methoxy groups may well enhance the preference for π-stacked
conformations. A similar trend can be seen with the propylene
tether, as CoPo’s 32 and 33 are superior correctors compared
to 31. In general, hydrogen bond accepting groups at the meta
and para positions afforded the best corrector activity as
exemplified in the propylene diamine series with CoPo’s 31−
36. Methoxy substitution proved to be superior to nitrogen
heterocyclic amides in corrector activity. A hybrid pyridyl-
methoxyamide (CoPo 23) maintained corrector and improved
ΔF508-CFTR Corrector and Potentiator Activity Assays.
-
Plate Reader Assay. FRT cells stably coexpressing human ΔF508-
CFTR and the halide-sensing fluorescent protein YFP-H148Q/I152L
were used as described previously.6d For corrector assay, cells were
grown at 37 °C for 24 h and then incubated for 16−20 h with 200 μL
of medium containing the test compound. At the time of the assay,
cells were washed with PBS and then incubated with PBS containing
forskolin (20 μM) and genistein (50 μM) for 20 min. For potentiator
assay, cells were grown at 37 °C for 18−24 h and then for 18−24 h at
27 °C. At the time of the assay, cells were washed with PBS and then
incubated for 10 min with PBS (50 μL) containing forskolin (20 μM)
and test compound (0−50 μM final concentration). Measurements
were carried out using FLUOstar fluorescence plate readers (Optima,
BMG LABTECH Gmbh), equipped with 500 10 nm excitation and
535 15 nm emission filters. Each well was assayed individually for I−
influx by recording fluorescence for 2 s (baseline) and then for 12 s
after rapid addition of 165 μL of PBS in which 137 mM Cl− was
replaced by I−. I− influx rate was computed by exponential regression.
All experiments contained negative control (DMSO vehicle) and
1247
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
positive controls (potentiator assay, genistein; corrector assay, 1). EC50
and Vmax, and their associated uncertainties, were determined by four-
parameter logistic nonlinear regression from concentration−activity
data using GraphPad Prism, version 5.01. Initial iodide influx rates
were fitted to the Hill equation: activity (initial slope) = Ao + VmaxCiH/
(CiH + IC50H), where Ci is compound concentration, H is coefficient,
and Ao is background signal.
5,7-Dimethyl-2-(piperazin-1-yl)quinoline-3-carbonitrile (13).
Orange solid. Yield: 62%. 1H NMR (600 MHz, CDCl3) δ 8.35 (d, J =
4.1, 1H), 7.35 (s, 1H), 6.95 (s, 1H), 3.60 (m, 4H), 3.02 (m, 4H), 2.49
(s, 3H), 2.40 (s, 3H), 1.87 (s, 1H); 13C NMR (150 MHz, CDCl3) δ
158.00, 149.29, 143.62, 142.63, 134.79, 127.89, 125.21, 120.08, 118.47,
97.37, 50.30, 46.16, 22.23, 18.47. ESI-MS m/z [M + H]+: 267.28.
Purity: 99%.
Short-Circuit Current Measurements. ΔF508-CFTR-expressing
FRT cells were cultured on Snapwell inserts for 7−9 days. For
corrector assay, test compounds were incubated with cells for 18−24 h
at 37 °C prior to measurements. For potentiator assay, the FRT cells
were incubated for 18−24 h at 27 °C prior to measurements. Short-
circuit current was recorded using established procedures.6d
General Procedure for Amide Coupling Reactions. EDC
hydrochloride (0.192 g, 1 mmol), aryl acid (1 mmol), and
triethylamine (0.14 mL, 2.5 mmol) were dissolved in dry DCM (10
mL). The mixture was stirred at room temperature for 30 min.
Carbonitrile (1 mmol) dissolved in dry DCM (5 mL) was added
dropwise to the solution, and the mixture was stirred for 18 h. The
mixture was diluted with DCM (50 mL) and washed with 1 M
NaHSO4 (aq, 2 × 100 mL). Organics were dried over Na2SO4, filtered,
and the solvent was removed under reduced pressure. The crude
product was purified by flash chromatography. Characterization data
can be found in the Supporting Information for all final compounds.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-2-
N-(3,5-Dimethylphenyl)acetamide (7). Acetic anhydride (2.84
mL, 30 mmol) was dissolved in dry THF (10 mL), purged with N2,
and brought to 0 °C. 3,5-Dimethylaniline (1.25 mL, 10 mmol) was
added dropwise, and upon completion of the addition, the mixture was
allowed to warm to room temperature and stirred an additional hour.
The reaction solution was then poured over ice, and 1 M NaOH (aq)
was added to adjust the pH to 12−14. The precipitate was collected by
filtration, dissolved in DCM, and dried over Na2SO4. The drying agent
was removed by filtration and the solvent was removed under reduced
pressure to afford pure product in 99% yield as a white solid.12
2-Chloro-5,7-dimethylquinoline-3-carbaldehyde (8). Phos-
phorus oxychloride (6.52 mL, 70 mmol) and dry DMF (1.94 mL,
25 mmol) were refluxed for 2 h under a N2 atmosphere. Acetamide 2
(1.632 g, 10 mmol) was then added to the reaction solution as a solid,
and the mixture was stirred at room temperature for an additional 3 h.
The reaction solution was poured slowly over ice, diluted with water
(200 mL), and carefully neutralized with solid K2CO3. The precipitate
was then collected by filtration, dissolved in chloroform, and dried
over Na2SO4. The drying agent was removed by filtration and the
solvent was removed under reduced pressure to afford pure product in
95% yield as an orange solid.12
1
methoxybenzamide (14). Yellow solid. Yield 72%. H NMR (600
MHz, CDCl3) δ 8.31 (s, 1H), 8.24−8.17 (m, 2H), 7.41 (t, J = 7.4,
1H), 7.30 (s, 1H), 7.06 (t, J = 7.4, 1H), 6.96−6.88 (m, 2H), 5.85 (s,
1H), 3.90−3.78 (m, 9H), 2.52 (s, 3H), 2.41 (s, 3H); 13C NMR (150
MHz, CDCl3) δ 166.69, 157.65, 154.31, 143.81, 140.49, 135.15,
133.00, 132.45, 131.07, 128.98, 126.76, 124.66, 121.49, 119.08, 117.09,
111.45, 94.37, 56.13, 42.05, 39.91, 22.31, 18.82. ESI-MS m/z [M +
H]+: 375.18. Purity: 98%.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-4-
1
methoxybenzamide (15). Yellow solid. Yield: 72%. H NMR (600
MHz, CDCl3) δ 8.26 (s, 1H), 7.87 (s, 1H), 7.61 (d, J = 8.0, 2H), 7.24
(s, 1H), 6.90 (s, 1H), 6.67 (d, J = 8.7, 2H), 5.80 (s, 1H), 3.83 (q, J =
5.2, 3H), 3.70 (s, 3H), 3.67 (q, J = 5.2, 2H), 2.46 (s, 3H), 2.37 (s, 3H);
13C NMR (150 MHz, CDCl3) δ 167.82, 162.18, 155.18, 149.70,
144.14, 140.93, 135.45, 132.48, 129.16, 127.12, 124.50, 119.39, 116.87,
113.66, 94.49, 55.43, 43.09, 41.29, 22.15, 18.47. ESI-MS m/z [M +
H]+: 375.18. Purity: 98%.
2-Chloro-5,7-dimethylquinoline-3-carbonitrile (10). Aldehyde
8 (1.095 g, 5 mmol), hydroxylamine hydrochloride (0.365 g, 5.25
mmol), and triethylamine (1.00 mL, 7 mmol) were combined in
ethanol (50 mL). The solution was refluxed for 3 h, and then the
ethanol was removed under reduced pressure. Aqueous HCl (1M, 100
mL) was added to the crude material, and product was extracted with
DCM (100 mL). The organic layer was separated and dried over
Na2SO4. The drying agent was filtered and solvent was removed under
reduced pressure. The crude product 4 was then dissolved in dry
benzene (50 mL). Thionyl chloride (0.73 mL, 10 mmol) was added
dropwise to the solution, and the mixture was refluxed under a N2
atmosphere for 4 h. The solution was allowed to cool to room
temperature. Then the benzene and excess thionyl chloride were
removed under reduced pressure to afford pure product 5 in 93% yield
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-
1
picolinamide (16). Yellow solid. Yield: 60%. H NMR (600 MHz,
CDCl3) δ 8.87 (s, 1H), 8.43−8.41 (m, 1H), 8.32 (s, 1H), 8.20 (d, J =
7.8, 1H), 7.81 (td, J = 7.7, 1.7, 1H), 7.49 (s, 1H), 7.40−7.35 (m, 1H),
6.95 (s, 1H), 5.78 (s, 1H), 3.91 (dd, J = 11.3, 5.7, 2H), 3.81 (dd, J =
11.2, 5.6, 2H), 2.53 (s, 3H), 2.47 (s, 3H); 13C NMR (150 MHz,
CDCl3) δ 165.40, 154.49, 150.13, 148.14, 143.59, 140.55, 137.44,
135.08, 126.82, 126.28, 125.02, 122.49, 119.24, 117.07, 94.17, 41.66,
40.86, 22.30, 18.53. ESI-MS m/z [M + H]+: 346.16. Purity: 96%.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-
1
nicotinamide (17). Yellow solid. Yield: 66%. H NMR (600 MHz,
CDCl3) δ 8.93 (s, 1H), 8.66 (d, J = 4.6, 1H), 8.38 (s, 1H), 8.34 (s,
1H), 8.00 (d, J = 7.8, 1H), 7.24−7.20 (m, 2H), 7.00 (s, 1H), 5.79 (s,
1H), 3.94 (q, J = 5.6, 2H), 3.77 (q, J = 4.3, 2H), 2.56 (s, 3H), 2.43 (s,
3H); 13C NMR (150 MHz, CDCl3) δ 166.28, 155.17, 152.18, 148.59,
144.58, 141.05, 135.54, 135.18, 130.46, 127.41, 124.24, 123.35, 119.45,
116.77, 94.37, 43.57, 41.25, 22.26, 18.55. ESI-MS m/z [M + H]+:
346.16. Purity: 97%.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-
isonicotinamide (18). Yellow solid. Yield: 69%. 1H NMR (600 MHz,
CDCl3) δ 8.53−8.46 (m, 3H), 8.33 (s, 1H), 7.45 (d, J = 4.5, 2H), 7.17
(s, 1H), 6.96 (s, 1H), 5.71 (s, 1H), 3.88 (q, J = 5.4, 2H), 3.68 (q, J =
5.0, 2H), 2.51 (s, 3H), 2.38 (s, 3H); 13C NMR (151 MHz, CDCl3) δ
166.03, 155.37, 150.49, 144.68, 141.89, 141.18, 135.74, 127.49, 124.18,
121.36, 119.54, 116.72, 113.58, 94.48, 77.43, 77.22, 77.01, 44.14,
41.00, 22.27, 18.56. ESI-MS m/z [M + H]+: 346.16. Purity: 98%.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-
benzamide (19). Yellow solid. Yield: 84%. 1H NMR (600 MHz,
CDCl3) δ 8.35 (s, 1H), 8.14 (s, 1H), 7.71 (d, J = 8.0, 2H), 7.41 (t, J =
7.4, 1H), 7.28 (s, 1H), 7.26 (t, J = 5.5, 1H), 6.98 (s, 1H), 5.82 (t, J =
5.5, 1H), 3.91 (q, J = 5.7, 2H), 3.75 (q, J = 4.6, 2H), 2.54 (s, 3H), 2.43
(s, 3H); 13C NMR (151 MHz, CDCl3) δ 168.16, 155.19, 149.55,
144.15, 140.96, 135.46, 131.40, 130.30, 128.49, 127.40, 127.15, 124.46,
1
as a light brown solid. H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H),
7.62 (s, 1H), 7.33 (s, 1H), 2.66 (s, 3H), 2.54 (s, 3H).
General Procedure for Aromatic Substitution of 5 with
Diamine Tether (11−13). Carbonitrile 5 (1.083 g, 5 mmol) and
diamine (15 mmol) were refluxed in dioxane (50 mL) until judged
complete by TLC. The mixture was allowed to cool to room
temperature, and dioxane was removed under reduced pressure. The
crude product was suspended in NH4Cl (1 M, aq) and collected by
filtration. The solids were washed with diethyl ether and allowed to
dry on a filter paper under vacuum. Yields ranged from 40% to 80%.
2-((2-Aminoethyl)amino)-5,7-dimethylquinoline-3-carboni-
trile (11). 1H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 7.32 (s, 1H),
6.92 (s, 1H), 5.64 (t, J = 4.8, 1H), 3.65 (q, J = 5.7, 2H), 3.01 (t, J = 6.0,
2H), 2.52 (s, 3H), 2.42 (s, 3H).8
2-(3-Aminopropylamino)-5,7-dimethylquinoline-3-carboni-
1
trile (12). Yellow solid. Yield: 61%. H NMR (600 MHz, CDCl3) δ
8.30 (s, 1H), 7.32 (s, 1H), 6.91 (s, 1H), 6.18 (s, 1H), 3.72 (q, J = 6.2,
2H), 2.89 (t, J = 6.2, 2H), 2.52 (s, 3H), 2.42 (s, 3H), 1.82 (quint, J =
6.4, 2H), 1.64 (s, 2H); 13C NMR (150 MHz, CDCl3) δ 154.56,
150.37, 143.73, 140.41, 135.13, 126.48, 124.52, 118.89, 117.41, 94.14,
40.40, 40.05, 32.52, 22.20, 18.53. ESI-MS m/z [M + H]+: 255.33.
Purity: 99%.
1248
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
119.40, 116.89, 94.37, 43.40, 41.25, 22.24, 18.60. ESI-MS m/z [M +
H]+: 345.12. Purity: 99%.
NMR (150 MHz, CDCl3) δ 170.48, 161.10, 157.75, 149.16, 144.02,
142.69, 134.98, 129.43, 128.49, 127.82, 125.38, 120.51, 118.18, 114.00,
97.72, 55.62, 49.33, 29.91, 22.29, 21.60, 18.86, 18.53. ESI-MS m/z [M
+ H]+: 401.13. Purity: 93%.
5,7-Dimethyl-2-(4-picolinoylpiperazin-1-yl)quinoline-3-car-
bonitrile (28). Yellow Solid. Yield: 40%. 1H NMR (600 MHz,
CDCl3) δ 8.62 (d, J = 4.8, 1H), 8.48 (s, 1H), 7.83 (t, J = 8.4, 1H), 7.71
(d, J = 7.8, 1H), 7.43 (s, 1H), 7.38 (dd, J = 5.2, 7.2, 1H), 7.07 (s, 1H),
3.85 (m, 8H), 2.58 (s, 3H), 2.47 (s, 3H); 13C NMR (150 MHz,
CDCl3) δ 167.89, 157.64, 154.06, 149.20, 148.56, 143.99, 142.74,
137.36, 134.94, 128.41, 125.38, 124.86, 124.28, 120.46, 118.25, 97.55,
49.58, 48.82, 47.23, 42.46, 22.28, 18.53. ESI-MS m/z [M + H]+:
360.24. Purity: 99%.
5,7-Dimethyl-2-(4-nicotinoylpiperazin-1-yl)quinoline-3-car-
bonitrile (29). Yellow solid. Yield: 49%. 1H NMR (600 MHz, CDCl3)
δ 8.73 (s, 1H), 8.70 (d, J = 3.7, 1H), 8.50 (s, 1H), 7.81 (d, J = 7.8,
1H), 7.45 (s, 1H), 7.40 (dd, J = 4.9, 7.8, 1H), 7.09 (s, 1H), 4.02 (s,
2H), 3.70 (m, 6H), 2.59 (s, 3H), 2.48 (s, 3H); 13C NMR (150 MHz,
CDCl3) δ 168.14, 157.66, 151.16, 149.09, 148.25, 144.13, 142.70,
135.29, 135.01, 131.64, 128.66, 125.42, 123.73, 120.62, 118.07, 97.80,
49.42, 49.15, 47.79, 42.35, 22.29, 18.54. ESI-MS m/z [M + H]+:
360.31. Purity: 99%.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-2,4-
dimethoxybenzamide (20). Yellow solid. Yield: 52%. 1H NMR
(600 MHz, CDCl3) δ 8.28 (s, 1H), 8.18 (d, J = 8.7, 1H), 8.04 (s, 1H),
7.29 (s, 1H), 6.91 (s, 1H), 6.57 (dd, J = 8.8, 2.2, 1H), 6.40 (d, J = 2.1,
1H), 5.87 (s, 1H), 3.86−3.76 (m, 10H), 2.51 (s, 3H), 2.40 (s, 3H);
13C NMR (150 MHz, CDCl3) δ 166.26, 163.58, 159.00, 154.31,
150.21, 143.68, 140.37, 135.08, 134.17, 126.67, 124.71, 119.05, 117.09,
114.68, 105.41, 98.69, 94.18, 56.06, 55.70, 42.11, 39.57, 22.21, 18.52.
ESI-MS m/z [M + H]+: 405.15. Purity: 98%.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-3,4-
dimethoxybenzamide (21). Yellow solid. Yield: 41%. 1H NMR
(600 MHz, CDCl3) δ 8.37 (s, 1H), 7.89 (s, 1H), 7.36 (d, J = 1.8, 1H),
7.29 (s, 1H), 7.18 (dd, J = 8.3, 1.9, 1H), 6.99 (s, 1H), 6.65 (d, J = 8.3,
1H), 5.78 (s, 1H), 3.92 (q, J = 5.8, 2H), 3.86 (s, 3H), 3.78 (s, 3H),
3.75 (q, J = 5.3, 2H), 2.56 (s, 3H), 2.43 (s, 3H); 13C NMR (150 MHz,
CDCl3) δ 168.07, 155.20, 151.72, 149.68, 149.04, 144.18, 140.94,
135.52, 127.57, 127.11, 124.58, 119.87, 119.40, 116.89, 110.89, 110.24,
94.37, 56.11, 56.09, 43.14, 41.37, 22.25, 18.59. ESI-MS m/z [M + H]+:
405.15. Purity: 98%.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-
1
pyrazine-2-carboxamide (22). Yellow solid. Yield: 38%. H NMR
2-(4-Isonicotinoylpiperazin-1-yl)-5,7-dimethylquinoline-3-
1
carbonitrile (30). Yellow solid. Yield: 50%. H NMR (600 MHz,
(600 MHz, CDCl3) δ 9.36 (s, 1H), 8.94 (s, 1H), 8.66−8.65 (m, 1H),
8.32 (s, 1H), 8.32−8.30 (m, 1H), 7.48 (s, 1H), 6.96 (s, 1H), 5.76 (s,
1H), 3.92 (q, J = 5.0, 2H), 3.80 (q, J = 5.2, 2H), 2.53 (s, 3H), 2.47 (s,
3H); 13C NMR (150 MHz, CDCl3) δ 163.98, 154.64, 149.96, 147.22,
144.85, 144.53, 143.61, 142.56, 140.67, 135.19, 126.93, 124.93, 119.32,
116.98, 94.15, 41.66, 41.17, 22.35, 18.53. ESI-MS m/z [M + H]+:
376.12. Purity: 97%.
CDCl3) δ 8.74 (d, J = 5.6, 2H), 8.50 (s, 1H), 7.45 (s, 1H), 7.34 (d, J =
5.8, 2H), 7.10 (s, 1H), 4.01 (s, 2H), 3.76 (s, 2H), 3.61 (s, 4H), 2.59 (s,
3H), 2.48 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 168.10, 157.67,
150.62, 149.08, 144.16, 143.40, 142.70, 135.03, 128.71, 125.42, 121.45,
120.65, 118.04, 97.85, 49.45, 49.12, 47.45, 42.08, 22.30, 18.55. ESI-MS
m/z [M + H]+: 360.31. Purity: 99%.
N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-2-
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-5-
methoxypicolinamide (23). Yellow solid. Yield: 78%. 1H NMR
(600 MHz, CDCl3) δ 8.69 (s, 1H), 8.29 (s, 1H), 8.13 (d, J = 8.6, 1H),
8.05 (d, J = 2.8, 1H), 7.47 (s, 1H), 7.22 (dd, J = 8.6, 2.9, 1H), 6.93 (s,
1H), 5.81 (t, J = 5.5, 1H), 3.88 (q, J = 5.5, 2H), 3.85 (s, 3H), 3.78 (q, J
= 5.6, 2H), 2.51 (s, 3H), 2.46 (s, 3H); 13C NMR (150 MHz, CDCl3) δ
165.32, 157.93, 154.51, 150.12, 143.50, 142.88, 140.49, 136.52, 135.06,
126.74, 125.02, 123.58, 120.18, 119.19, 117.08, 94.16, 55.88, 41.73,
40.79, 22.31, 18.52. ESI-MS m/z [M + H]+: 376.12. Purity: 99%.
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-2,6-
dimethoxybenzamide (24). 1H NMR (600 MHz, CDCl3) δ 8.30 (s,
1H), 7.26 (t, J = 4.3, 1H), 7.22 (t, J = 8.4, 1H), 6.98 (s, 1H), 6.90 (s,
1H), 6.49 (d, J = 8.4, 2H), 5.69 (t, J = 5.6, 1H), 3.85 (q, J = 5.6, 2H),
3.74 (q, J = 4.9, 2H), 3.69 (s, 6H), 2.51 (s, 3H), 2.33 (s, 3H); 13C
NMR (150 MHz, CDCl3) δ 166.38, 157.60, 154.45, 149.77, 143.85,
140.52, 135.05, 130.63, 126.84, 124.35, 119.10, 116.95, 116.35, 104.15,
94.19, 56.15, 41.58, 41.35, 22.17, 18.47. ESI-MS m/z [M + H]+:
405.15. Purity: 99%.
1
methoxybenzamide (31). Yellow solid. Yield: 88%. H NMR (600
MHz, CDCl3) δ 8.28 (s, 1H), 8.21 (t, J = 5.3, 1H), 8.18 (dd, J = 1.8,
7.8, 1H), 7.44 (td, J = 1.8, 8.4, 1H), 7.20 (s, 1H), 7.07 (t, J = 7.9, 1H),
6.97 (d, J = 8.2, 1H), 6.89 (s, 1H), 5.76 (t, J = 5.7, 1H), 3.92 (s, 4H),
3.74 (q, J = 6.3, 2H), 3.59 (q, J = 6.2, 2H), 2.51 (s, 3H), 2.36 (s, 3H),
1.98 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 166.01, 157.51, 154.46,
150.24, 143.66, 140.43, 135.16, 132.75, 132.37, 126.51, 124.41, 122.32,
121.44, 118.93, 117.23, 111.40, 94.16, 56.11, 38.60, 37.11, 30.17,
22.25, 18.55. ESI-MS m/z [M + H]+: 389.32. Purity: 99%.
N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-3-
1
methoxybenzamide (32). Yellow solid. Yield: 87%. H NMR (600
MHz, CDCl3) δ 8.30 (s, 1H), 7.66 (t, J = 6.0, 1H), 7.42 (m, 2H), 7.35
(t, J = 7.9, 1H), 7.11 (s, 1H), 7.06 (dd, J = 2.5, 8.1, 1H), 6.90 (s, 1H),
5.65 (t, J = 6.3, 1H), 3.83 (s, 2H), 3.80 (dd, J = 6.3, 12.2, 2H), 3.51
(dd, J = 6.1, 12.0, 2H), 2.51 (s, 3H), 2.30 (s, 3H), 1.92 (m, 2H); 13C
NMR (150 MHz, CDCl3) δ 168.26, 159.97, 154.81, 149.96, 144.07,
140.81, 137.26, 135.35, 129.67, 126.72, 124.17, 119.32, 119.01, 117.62,
117.14, 112.70, 94.10, 55.60, 37.89, 36.37, 30.43, 30.42, 22.12, 18.54.
ESI-MS m/z [M + H]+: 389.32. Purity: 99%.
2-(4-(2-Methoxybenzoyl)piperazin-1-yl)-5,7-dimethylquino-
1
line-3-carbonitrile (25). Yellow solid. Yield: 40%. H NMR (600
MHz, CDCl3) δ 8.47 (s, 1H), 7.44 (s, 1H), 7.37 (t, J = 7.9, 1H), 7.28
(m, 1H), 7.07 (s, 1H), 7.01 (t, J = 7.4, 1H), 6.94 (d, J = 8.3, 1H), 4.03
(m, 2H), 3.85 (s, 3H), 3.76 (dd, J = 6.4, 10.8, 2H), 3.54 (m, 4H), 2.58
(s, 3H), 2.47 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 168.23, 157.75,
155.51, 149.20, 144.02, 142.73, 134.97, 130.82, 128.44, 128.29, 125.72,
125.36, 121.24, 120.47, 118.22, 111.13, 97.67, 55.80, 49.62, 48.95,
46.90, 41.69, 22.29, 18.54. ESI-MS m/z [M + H]+: 401.13. Purity:
93%.
N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-4-
1
methoxybenzamide (33). Yellow solid. Yield: 81%. H NMR (600
MHz, CDCl3) δ 8.30 (s, 1H), 7.83 (d, J = 8.6, 2H), 7.50 (s, 1H), 7.20
(s, 1H), 6.92 (m, 3H), 5.77 (s, 1H), 3.84 (s, 3H), 3.80 (dd, J = 5.9,
11.8, 2H), 3.51 (dd, J = 6.1, 11.9, 2H), 2.51 (s, 3H), 2.34 (s, 3H), 1.93
(m, 2H); 13C NMR (150 MHz, CDCl3) δ 167.96, 162.21, 154.61,
135.47, 130.31, 129.31, 128.52, 127.87, 127.25, 126.78, 124.01, 118.85,
117.05, 113.91, 94.27, 55.61, 38.14, 36.46, 30.32, 22.22, 18.55. ESI-MS
m/z [M + H]+: 389.32. Purity: 99%.
2-(4-(3-Methoxybenzoyl)piperazin-1-yl)-5,7-dimethylquino-
1
line-3-carbonitrile (26). Yellow solid. Yield: 50%. H NMR (600
N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-
1
MHz, CDCl3) δ 8.49 (s, 1H), 7.44 (s, 1H), 7.33 (td, J = 1.4, 7.4, 1H),
7.08 (s, 1H), 6.98 (m, 3H), 3.83 (s, 3H), 3.76 (m, 8H), 2.58 (s, 3H),
2.47 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 170.51, 159.92, 157.76,
149.15, 144.05, 142.69, 137.10, 134.99, 129.90, 128.54, 125.40, 120.55,
119.30, 118.14, 115.93, 112.64, 97.77, 55.59, 49.59, 49.18, 47.71,
42.20, 29.91, 22.29, 18.54. ESI-MS m/z [M + H]+: 401.26. Purity:
99%.
picolinamide (34). Yellow solid. Yield: 42%. H NMR (600 MHz,
CDCl3) δ 8.72 (s, 1H), 8.58 (d, J = 4.2, 1H), 8.27 (s, 1H), 8.18 (d, J =
7.8, 1H), 7.80 (td, J = 1.7, 7.7, 1H), 7.59 (s, 1H), 7.39 (ddd, J = 1.1,
4.7, 7.5, 1H), 6.87 (s, 1H), 5.56 (t, J = 5.9, 1H), 3.70 (q, J = 6.2, 2H),
3.51 (q, J = 6.4, 2H), 2.48 (s, 3H), 2.36 (s, 3H), 1.89 (quint, J = 6.2,
2H); 13C NMR (150 MHz, CDCl3) δ 167.44, 164.92, 154.65, 150.40,
148.15, 143.70, 140.54, 137.55, 135.13, 126.65, 126.32, 124.84, 122.67,
119.08, 117.24, 94.09, 38.25, 36.50, 30.60, 22.33, 18.55. ESI-MS m/z
[M + H]+: 372.24. Purity: 99%.
2-(4-(4-Methoxybenzoyl)piperazin-1-yl)-5,7-dimethylquino-
1
line-3-carbonitrile (27). Yellow solid. Yield: 45%. H NMR (600
MHz, CDCl3) δ 8.47 (s, 1H), 7.44 (m, 3H), 7.07 (s, 1H), 6.94 (d, J =
N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-
8.8, 2H), 3.84 (s, 8H), 3.83 (m, 8H), 2.57 (s, 3H), 2.47 (s, 3H); 13C
nicotinamide (35). Yellow solid. Yield: 40%. H NMR (600 MHz,
1
1249
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
CDCl3) δ 9.09 (s, 1H), 8.77 (d, J = 6.3, 1H), 8.34 (s, 1H), 8.15 (d, J =
7.9, 1H), 7.70 (s, 2H), 7.41 (dd, J = 4.8, 7.8, 1H), 7.03 (s, 1H), 6.92 (s,
1H), 5.51 (t, J = 6.4, 1H), 3.82 (s, 2H), 3.53 (s, 2H), 2.53 (s, 3H), 2.31
(s, 3H), 1.94 (quint, J=5.5, 2H); 13C NMR (150 MHz, CDCl3) δ
166.37, 154.83, 152.30, 149.84, 148.46, 144.32, 140.91, 135.50, 135.49,
131.46, 126.91, 124.03, 123.64, 119.11, 117.03, 94.19, 37.89, 36.43,
30.41, 22.18, 18.53. ESI-MS m/z [M + H]+: 372.24. Purity: 99%.
N-(3-(3-Cyano-5,7-dimethylquinolin-2-ylamino)propyl)-
isonicotinamide (36). Yellow solid. Yield: 42%. 1H NMR (600 MHz,
CDCl3) δ 8.70 (d, J = 4.4, 2H), 8.28 (s, 1H), 7.73 (s, 1H), 7.61 (dd, J
= 1.6, 4.3, 2H), 6.92 (s, 1H), 6.87 (s, 1H), 5.43 (d, J = 6.0, 1H), 3.76
(q, J = 6.3, 2H), 3.46 (q, J = 5.8, 2H), 2.47 (s, 3H), 2.23 (s, 3H), 1.87
(m, 2H); 13C NMR (150 MHz, CDCl3) δ 166.36, 154.88, 150.74,
149.78, 144.33, 142.93, 140.96, 135.62, 126.97, 123.86, 121.50, 119.15,
116.95, 94.26, 37.84, 36.40, 30.40, 22.20, 18.54. ESI-MS m/z [M +
H]+: 372.24. Purity: 99%.
1999, 79, S23−S45. (c) Zeiher, B. G.; Eichwald, E.; Zabner, J.; Smith,
J. J.; Puga, A. P.; McCray, P. B. Jr.; Capecchi, M. R.; Welsh, M. J.;
Thomas, K. R. A mouse model for the ΔF508 allele of cystic fibrosis. J.
Clin. Invest. 1995, 96, 2051−2064.
(3) (a) Bobadilla, J. L.; Macek, M; Fine, J. P.; Farrell, P. M. Cystic
fibrosis: a worldwide analysis of CFTR mutations-correlation with
incidence data and application to screening. Hum. Mutat. 2002, 19,
575−606. (b) Sharma, M.; Benharouga, M.; Hu, W.; Lukacs, G. L.
Conformational and temperature-sensitive stability defects of the
ΔF508 cystic fibrosis transmembrane conductance regulator in post-
endoplasmic reticulum compartments. J. Biol. Chem. 2001, 276, 8942−
8950. (c) Thibodeau, P. H.; Richardson, J. M.; Wang, W.; Millen, L.;
Watson, J.; Mendoza, J. L.; Du, K.; Fischman, S.; Senderowitz, H.;
Lukacs, G. L.; Kirk, K.; Thomas, P. J. The cystic fibrosis-causing
mutation ΔF508 affects multiple steps in cystic fibrosis trans-
membrane conductance regulator biogenesis. J. Biol. Chem. 2010,
285, 35825−35835.
(4) (a) Skach, W. R. Defects in processing and trafficking of the
cystic fibrosis transmembrane conductance regulator. Kidney Int. 2000,
57, 825−831. (b) Denning, G. M.; Anderson, M. P.; Amara, J. F.;
Marshall, J.; Smith, A. E.; Welsh, M. J. Processing of mutant cystic
fibrosis transmembrane conductance regulator is temperature-
sensitive. Nature 1992, 358, 761−764. (c) Lukacs, G. L.; Mohamed,
A.; Kartner, N.; Chang, X.-B.; Riordan, J. R.; Grinstein, S.
Conformational maturation of CFTR but not its mutant counterpart
(ΔF508) occurs in the endoplasmic reticulum and requires ATP.
EMBO J. 1994, 13, 6076−6086. (d) Kopito, R. R. Biosynthesis and
degradation of CFTR. Physiol. Rev. 1999, 79, S167−S173. (e) Du, K.;
Sharma, M.; Lukacs, G. L. The ΔF508 cystic fibrosis mutation impairs
domain−domain interactions and arrests post-translational folding of
CFTR. Nat. Struct. Mol. Biol. 2005, 12, 17−25.
ASSOCIATED CONTENT
* Supporting Information
■
S
ΔF508-CFTR corrector and potentiator activity assay exper-
imentals; experimental details and compound characterization
data for compounds 7, 8, 10, and 14−36; constrained
calculation for 5; coordinates for computed conformers of 5;
and coordinates for computed conformers of 26. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 530-554-2145. Fax: 530-752-8995. E-mail: mjkurth@
ucdavis.edu.
■
(5) Drug discovery reviews: (a) Riordan, J. R. CFTR function and
prospects for therapy. Annu. Rev. Biochem. 2008, 77, 701−726.
(b) Verkman, A. S.; Galietta, L. J. Chloride channels as drug targets.
Nat. Rev. Drug Discovery 2009, 8, 153−171.
ACKNOWLEDGMENTS
■
The authors thank the Tara K. Telford Fund for Cystic Fibrosis
Research at the University of California, Davis, the National
Institutes of Health (Grants DK072517, GM089153, and
HL073856), and the National Science Foundation (Grant
CHE-0910870; Grants CHE-0910870, CHE-0443516, CHE-
0449845, and CHE-9808183 supporting NMR spectrometers;
Grant CHE-030089 supporting calculations).
(6) (a) Yoo, C. L.; Yu, G. J.; Yang, B.; Robins, L. I.; Verkman, A. S.;
Kurth, M. J. 4′-Methyl-4,5′-bithiazole-based correctors of defective
ΔF508-CFTR cellular processing. Bioorg. Med. Chem. Lett. 2008, 18,
2610−2614. (b) Yu, G. J.; Yoo, C. L.; Yang, B.; Lodewyk, M. W.;
Meng, L.; El-Idreesy, T. T.; Fettinger, J. C.; Tantillo, D. J.; Verkman,
A. S.; Kurth, M. J. Potent s-cis-Locked Bithiazole Correctors of ΔF508
Cystic Fibrosis Transmembrane Conductance Regulator Cellular
Processing for Cystic Fibrosis Therapy. J. Med. Chem. 2008, 51,
6044−6054. (c) Ye, L.; Knapp, J. M.; Sangwung, P.; Fettinger, J. C.;
Verkman, A. S.; Kurth, M. J. Pyrazolylthiazole as ΔF508-Cystic
Fibrosis Transmembrane Conductance Regulator Correctors with
Improved Hydrophilicity Compared to Bithiazoles. J. Med. Chem.
2010, 53, 3772−3781. (d) Pedemonte, N.; Lukacs, G. L.; Du, K.; Caci,
E.; Zegarra-Moran, O.; Galietta, L. J.; Verkman, A. S. Small-molecule
correctors of defective ΔF508-CFTR cellular processing identified by
high-throughput screening. J. Clin. Invest. 2005, 115, 2564−2571.
(e) Noel, S.; Wilke, M.; Bot, A. G.; De Jonge, H. R.; Becq, F. Parallel
improvement of sodium and chloride transport defects by miglustat
(n-butyldeoxynojyrimicin) in cystic fibrosis epithelial cells. J.
Pharmacol. Exp. Ther. 2008, 325, 1016−1023. (f) Van Goor, F.;
Hadida, S.; Grootenhuis, P. D.; Stack, J. H.; Burton, B.; Olson, E.;
Wine, J.; Frizzell, R. A.; Ashlock, M.; Negulescu, P. Rescue of the
protein folding defect in cystic fibrosis in vitro by the investigational
small molecule, VX-809. J. Cystic Fibrosis 2010, 9, S14. (g) Pedemonte,
N.; Tomati, V.; Sondo, E.; Caci, E.; Millo, E.; Armirotti, A.; Damonte,
G.; Zegarra-Moran, O.; Galietta, L. J. Dual activity of amino-
arylthiazoles on the trafficking and gating defects of the cystic fibrosis
transmembrane conductance regulator chloride channel caused by
cystic fibrosis mutations. J. Biol. Chem. 2011, 286, 15215−15226.
(h) Wang, Y.; Loo, T. W.; Bartlett, M. C.; Clarke, D. M. Modulating
the folding of P-glycoprotein and cystic fibrosis transmembrane
conductance regulator truncation mutants with pharmacological
chaperones. Mol. Pharmacol. 2007, 71, 751−758.
ABBREVIATIONS USED
■
CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane
conductance regulator; ΔF508-CFTR, deletion of phenyl-
alanine-508 in cystic fibrosis transmembrane conductance
regulator; CoPo, corrector and potentiator; THF, tetrahydro-
furan; DMF, dimethylformamide; DCM, dichloromethane;
EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride; LCMS, liquid chromatography−mass spectrometry
REFERENCES
■
(1) (a) Dalemans, W.; Barbry, P.; Champigny, G.; Jallat, S.; Dott, K.;
Dreyer, D.; Crystal, R. G.; Pavirani, A.; Lecocq, J. P.; Lazdunski, M.
Altered chloride ion channel kinetics associated with ΔF508 cystic
fibrosis mutation. Nature 1991, 354, 526−528. (b) Snouwaert, J. N.;
Brigman, K. K.; Latour, A. M.; Malouf, N. N.; Boucher, R. C.;
Smithies, O.; Koller, B. H. An animal model for cystic fibrosis made by
gene targeting. Science 1992, 257, 1083−1088. (c) Boucher, R. C.
Cystic fibrosis: a disease of vulnerability to airway surface dehydration.
Trends Mol. Med. 2007, 13, 231−240. (d) Okiyoneda, T.; Barriere, H.;
Bagdany, M.; Rabeh, W. M.; Du, K.; Hohfeld, J.; Young, J. C.; Lukacs,
G. L. Peripheral protein quality control removes unfolded CFTR from
the plasma membrane. Science 2010, 329, 805−810.
(2) (a) Pilewski, J. M.; Frizzell, R. A. Role of CFTR in airway disease.
Phys. Rev. 1999, 79, S215−S255. (b) Sheppard, D. N.; Welsh, M. J.
Structure and function of the CFTR chloride channel. Physiol. Rev.
1250
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251

Journal of Medicinal Chemistry
Article
(7) (a) Pedemonte, N.; Boido, D.; Moran, O.; Giampieri, M.; Mazzei,
M.; Ravazzolo, R.; Galietta, L. J. Structure−activity relationship of 1,4-
dihydropyridines as potentiators of the cystic fibrosis transmembrane
conductance regulator chloride channel. Mol. Pharmacol. 2007, 72,
197−207. (b) Pedemonte, N.; Sonawane, N. D.; Taddei, A.; Hu, J.;
Zegarra-Moran, O.; Suen, Y. F.; Robins, L. I.; Dicus, C. W.;
Willenbring, D.; Nantz, M. H.; Kurth, M. J.; Galietta, L. J.;
Verkman, A. S. Phenylglycine and sulfonamide correctors of defective
ΔF508 and G551D cystic fibrosis transmembrane conductance
regulator chloride-channel gating. Mol. Pharmacol. 2005, 67, 1797−
1807. (c) Yang, H.; Shelat, A. A.; Guy, R. K.; Gopinath, V. S.; Ma, T.;
Du, K.; Lukacs, G. L.; Taddei, A.; Folli, C.; Pedemonte, N.; Galietta, L.
J.; Verkman, A. S. Nanomolar affinity small molecule correctors of
defective ΔF508-CFTR chloride channel gating. J. Biol. Chem. 2003,
278, 35079−35085. (d) Van Goor, F.; Straley, K. S.; Cao, D.;
Gonzalez, J.; Hadida, S.; Hazlewood, A.; Joubran, J.; Knapp, T.;
Makings, L. R.; Miller, M.; Neuberger, T.; Olson, E.; Panchenko, V.;
Rader, J.; Singh, A.; Stack, J. H.; Tung, R.; Grootenhuis, P. D.;
Negulescu, P. Rescue of DeltaF508-CFTR trafficking and gating in
human cystic fibrosis airway primary cultures by small molecules. Am.
J. Physiol.: Lung Cell. Mol. Physiol. 2006, 290, L1117−L1130. (e) Van
Goor, F.; Hadida, S.; Grootenhuis, P. D.; Burton, B.; Cao, D.;
Neuberger, T.; Turnbull, A.; Singh, A.; Joubran, J.; Hazlewood, A.;
Zhou, J.; McCartney, I.; Arumugam, V.; Decker, C.; Yang, J.; Young,
C.; Olson, E. R.; Wine, J. J.; Frizzell, R. A.; Ashlock, M.; Negulescu, P.
Rescue of CF airway epithelial cell function in vitro by a CFTR
potentiator, VX-770. Proc. Natl Acad. Sci. U.S.A. 2009, 106, 18825−
18830. (f) Accurso, F. J.; Rowe, S. M.; Clancy, J. P.; Boyle, M. P.;
Dunitz, J. M.; Durie, P. R.; Sagel, S. D.; Hornick, D. B.; Konstan, M.
W.; Donaldson, S. H.; Moss, R. B.; Pilewski, J. M.; Rubenstein, R. C.;
Uluer, A. Z.; Aitken, M. L.; Freedman, S. D.; Rose, L. M.; Mayer-
Hamblett, N.; Dong, Q.; Zha, J.; Stone, A. J.; Olson, E. R.; Ordonez, C.
L.; Campbell, P. W.; Ashlock, M. A.; Ramsey, B. W. Effect of VX-770
in persons with cystic fibrosis and the G551D-CFTR mutation. N.
Engl. J. Med. 2010, 363, 1991−2003. (g) Mills, A. D.; Yoo, C.; Butler, J.
D.; Yang, B.; Verkman, A. S.; Kurth, M. J. Design and synthesis of a
hybrid potentiator−corrector agonist of the cystic fibrosis mutant
protein DeltaF508-CFTR. Bioorg. Med. Chem. Lett. 2010, 20, 87−91.
(h) Jones, A. M.; Helm, J. M. Emerging treatments in cystic fibrosis.
Drugs 2009, 69, 1903−1910.
covalent interactions, excited states, and transition elements: two new
functionals and systematic testing of four M06-class functionals and 12
other functionals. Theor. Chem. Acc. 2008, 120, 215−241.
(16) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
̈
Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;
Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09,
revision B.01; Gaussian, Inc., Wallingford CT, 2009.
(17) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. Universal
solvation model based on solute electron density and a continuum
model of the solvent defined by the bulk dielectric constant and
atomic surface tensions. J. Phys. Chem. B 2009, 113, 6378−6396.
(18) Since conformations preferred in water are often substantially
different from bioactive conformations, the energy penalties associated
with changing between them can contribute significantly to energies of
binding.
(19) Chang, Y.-P.; Su, T.-M.; Li, T.-W.; Chao, I. Intramolecular
hydrogen bonding, gauche interactions, and thermodynamic functions
of 1,2-ethanediamine, 1,2-ethanediol, and 2-aminoethanol: a global
conformational analysis. J. Phys. Chem. A 1997, 101, 6107−6117.
(8) Phuan, P.-W.; Yang, B.; Knapp, J. M.; Wood, A. B.; Lukacs, G. L.;
Kurth, M. J.; Verkman, A. S. Cyanoquinolines with independent
corrector and potentiator activities restore ΔF508-CFTR chloride
channel function in cystic fibrosis. Mol. Pharmacol. 2011, 80, 683−693.
(9) Solomon, V. R.; Lee, H. Quinoline as a privileged scaffold in
cancer drug discovery. Curr. Med. Chem. 2011, 18, 1488−508.
(10) Massarani, E.; Nardi, D.; Pozzi, R.; Degen, L.; Magistretti, M. J.
8-Hydroxyquinoline derivatives. Synthesis and biological evaluation of
arylglyoxal N-7-amino-5-substituted 8-hydroxyquinoline hemiacetals
and 5-phenylglyoxylidenamino-8-hydroxyquinolines. J. Med. Chem.
1970, 13, 380−383.
(11) Heiniger, B.; Gakhar, G.; Prasain, K.; Hua, D. H.; Nguyen, T. A.
Second-generation substituted quinolines as anticancer drugs for
breast cancer. Anticancer Res. 2010, 30, 3927−3932.
(12) (a) Fischer, O.; Muller, A.; Vilsmeier, A. Action of phosphorus
oxychloride upon methyl- and ethylacetanilide. Synthesis of γ-
chloroisoquinocyanines. J. Prakt. Chem. 1925, 109, 69−87. (b) Vils-
meier, A.; Haack, A. Action of phosphorus halides on alkylformani-
lides. A new method for the preparation of secondary and tertiary p-
alkylaminonobenzaldehydes. Ber. 1927, 60B, 119−122. (c) Moussaoui,
F.; Belfaitah, A.; Debache, A.; Rhouati, S. Synthesis and character-
ization of some new aryl quinolyl α,β-unsaturated ketones. J. Soc. Alger.
Chim. 2002, 12, 71−78.
(13) Spartan ’10; Wavefunction, Inc.: Irvine, CA.
(14) Halgren, T. A. Merck molecular force field. I. Basis, form, scope,
parameterization, and performance of MMFF94. J. Comput. Chem.
1996, 17, 490−519.
(15) Zhao, Y.; Truhlar, D. G. The M06 suite of density functionals
for main group thermochemistry, thermochemical kinetics, non-
1251
dx.doi.org/10.1021/jm201372q | J. Med. Chem. 2012, 55, 1242−1251