Constrained bithiazoles: Small molecule correctors of defective δf508-CFTR protein trafficking

Article abstract of DOI:10.1021/jm5007885

Conformationally constrained bithiazoles were previously found to have improved efficacy over nonconstrained bithiazoles for correction of defective cellular processing of the δF508 mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. In this study, two sets of constrained bithiazoles were designed, synthesized, and tested in vitro using δF508-CFTR expressing epithelial cells. The SAR data demonstrated that modulating the constraining ring size between 7-versus 8-membered in these constrained bithiazole correctors did not significantly enhance their potency (IC₅₀), but strongly affected maximum efficacy (V_max), with constrained bithiazoles 9e and 10c increasing V_max by 1.5-fold compared to benchmark bithiazole corr4a. The data suggest that the 7-and 8-membered constrained ring bithiazoles are similar in their ability to accommodate the requisite geometric constraints during protein binding.

Full text of DOI:10.1021/jm5007885

Article
pubs.acs.org/jmc
Open Access on 07/16/2015
Constrained Bithiazoles: Small Molecule Correctors of Defective
ΔF508−CFTR Protein Trafficking
Keith C. Coffman,† Huy H. Nguyen,† Puay-Wah Phuan,‡ Brandi M. Hudson,† Gui J. Yu,†
Alex L. Bagdasarian,† Deanna Montgomery,† Michael W. Lodewyk,† Baoxue Yang,‡ Choong L. Yoo,†
A. S. Verkman,‡ Dean J. Tantillo,† and Mark J. Kurth*,†
†Department of Chemistry, University of California, One Shields Avenue, Davis, California 95616, United States
‡Department of Medicine and Physiology, University of California, 513 Parnassus Avenue, San Francisco, California 94143, United
States
S
* Supporting Information
ABSTRACT: Conformationally constrained bithiazoles were
previously found to have improved efficacy over noncon-
strained bithiazoles for correction of defective cellular
processing of the ΔF508 mutant cystic fibrosis transmembrane
conductance regulator (CFTR) protein. In this study, two sets
of constrained bithiazoles were designed, synthesized, and
tested in vitro using ΔF508−CFTR expressing epithelial cells.
The SAR data demonstrated that modulating the constraining
ring size between 7- versus 8-membered in these constrained bithiazole correctors did not significantly enhance their potency
(IC50), but strongly affected maximum efficacy (Vmax), with constrained bithiazoles 9e and 10c increasing Vmax by 1.5-fold
compared to benchmark bithiazole corr4a. The data suggest that the 7- and 8-membered constrained ring bithiazoles are similar
in their ability to accommodate the requisite geometric constraints during protein binding.
INTRODUCTION
■
Cystic fibrosis (CF) is a recessive genetic disease found
primarily in Caucasians.1 Of the ∼2000 known mutations in the
CF gene, deletion of phenylalanine at position 508 (ΔF508) in
the cystic fibrosis transmembrane conductance regulator
(CFTR) protein is the most common mutation, present in at
least one allele in ∼90% of patients.2−4 Due to this deletion, the
ΔF508−CFTR protein is misfolded, retained at the endoplas-
mic reticulum, and rapidly degraded.4 CFTR protein is a cyclic
Figure 1. Constrained bithiazole corrector 9a, lead corrector corr15jf,
and benchmark corrector corr4a.
AMP-regulated chloride channel expressed in the plasma
membrane of secretory epithelia in the airways, intestines,
pancreas, testes, and exocrine glands, as well as some
nonepithelial cell types.4,5 CF patients suffer from chronic
studies showed that the conformation of the bithiazole core
lung infections leading to deterioration of lung function, which
is the main cause of morbidity and mortality.4 Currently, there
is only one drug (potentiator) that targets mutant CFTR
(KalydecoTM; VX-770), but it is only helpful to the small
number of patients with the G551D mutation (4.3% of CF
patients) and some other gating mutations.3,6
structure is an important determinant of corrector activity.
Corrector 9a (Figure 1), with its constrained bithiazole
substructure compared to lead bithiazole corr15jf, was
identified in these previous studies.8
The present study was designed to explore the constrained
bithiazole core further by (1) defining the optimum size of the
ring constraining the bithiazole core and (2) refining the
peripheral groups in these constrained correctors using in vitro
evaluation with ΔF508−CFTR FRT epithelial cells. It is hoped
that refined bithiazole structural features may lead to useful
insight into the mechanism of small molecule ΔF508−CFTR
protein binding and interactions.8,9
Correctors, first recognized by Verkman et al.,5a are believed
to address the misfolding and trafficking of defective ΔF508−
CFTR protein and are being investigated by several groups.6
The investigational correctors VX-809 and VX-661 are in
clinical trials.7 However, the limited understanding of how
small molecules modulate the trafficking of ΔF508−CFTR
creates a challenge in efforts to discover effective corrector drug
candidates. We previously reported that bithiazoles (cf., corr4a
and corr15jf, Figure 1) can correct ΔF508−CFTR, and these
Received: May 20, 2014
Published: July 16, 2014
© 2014 American Chemical Society
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Constrained bithiazoles are of particular interest because the
bithiazole moiety is found in some natural products.10 Also,
bithiazole 9a, with its constrained conformational advantage
over freely rotating bithiazoles corr4a and corr15jf, can be
expected to have a more favorable entropy of binding in
formation of the corrector-bound-CFTR complex.11 Herein, we
report the synthesis and biological evaluation of a set of novel
constrained bithiazoles.
dione, the second bromination/thiazole formation sequence
failed to deliver the targeted bithiazole because SN2 reaction on
the neopentyl bromide failed under all conditions attempted
(for example, using AgBF6 to activate the C−Br bond).
Consequently, the only constrained bithiazole 1m = 1 evaluated
in vitro (vide supra) was aromatized analogue 3.
We next directed our attention to the preparation of
constrained bithiazoles 9 (1m = 2; 7-membered constraining
ring) and 10 (1m = 3; 8-membered constraining ring). This
series contained constrained corrector 9a plus several amide
(e.g., “R” in 9a−f/10a−f; Scheme 2a) and aniline (e.g., “Ar” in
9g−j/10g−j; Scheme 2b) analogues. These various constrained
bithiazoles were synthesized by modification of our previously
published procedures8,12 starting from 1,3-cycloheptadione
[prepared from (cyclopent-1-en-1-yloxy)trimethylsilane as out-
lined in Scheme 1a]8 and 1,3-cyclooctadione (prepared from
diethyl heptanedioate as outlined in Scheme 1b).13
CHEMISTRY
■
On the basis of our previous bithiazole studies,8,12 we were
interested in exploring different constraining ring sizes in the
bithiazole core substructure. Indeed, as the ring size increases
or decreases, the bithiazole substructure becomes more or less
rigid, and the interatomic distances and dihedral angles
between the thiazole rings modulate. To fully probe these
structural features, the constraining carbocycle (red highlight in
1; Figure 2) would, ideally, range from five to eight-membered
rings (i.e., m = 0−3 in generalized bithiazole 1).
Scheme 1. (a) Synthesis of 1,3-Cycloheptadione. (b)
Synthesis of 1,3-Cyclooctadione
Figure 2. Target analogues (1) of constrained bithiazole corrector 9a.
For 1m = 0 (5-membered constrained bithiazole 2; Figure 3), a
variety of routes were explored without success. For instance, α-
With the requisite diones in hand, α-bromination [CHCl3/
H2O (1:1) for 1,3-cycloheptadione; CCl4/H2O (1:1) for 1,3-
cyclooctadione] by the dropwise addition of bromine at 0 °C
followed by treatment of the crude 2-bromo-1,3-dione with
thiourea in ethanol overnight delivered thiazoles 5 and 6 in
38% and 51% yield, respectively, over two steps. As a result of
the difficulty in workup, thiazole 6 required basification (sat.
NH4OH to pH 8−9) before purification followed by
reacidification (HBr in HOAc) ahead of the second
bromination. Following a second α-bromination by dropwise
addition of bromine to 5 or 6 in HOAc, the resulting α-
bromoketone was refluxed in EtOH overnight with 1-(5-chloro-
2-methoxyphenyl)thiourea to yield 7a and 8a in 79% and 65%
yield, respectively. Bithiazoles 7a and 8a were stirred in DCM
and Et3N at 60 °C overnight with various acid chlorides to give
the final constrained bithiazoles 9a−f and 10a−f. Constrained
bithiazoles 9g−j and 10g−j, wherein the amide moiety was
maintained as a pivalamide and the aniline aryl moiety was
varied, were prepared in similar fashion, except that different
aryl substituted thioureas were used to prepare 7g−j and 8g−j.
Figure 3. Constrained bithiazole analogues 2−4.
bromination of 1,3-cyclopentanedione followed by displace-
ment with thiourea cleanly gave 2-amino-4H-cyclopenta[d]-
thiazol-6(5H)-one. The introduction of the second thiazole
ring by a second α-bromination and subsequent condensation
with a substituted thiourea failed, even at elevated temperatures
in a microwave reactor, to give the targeted bithiazole 2.
Presumably the 5,5,5-ring system in 2 is too strained to form
under these conditions. In contrast, the first and second
1
thiazole-forming steps for 1m = (6-membered constraining
ring) proceeded smoothly, but during the final N-acylation step,
the central cyclohexadiene ring spontaneously aromatized
(even at −4 °C) to give 3 (Figure 3). To avoid this
aromatization, we employed 5,5-dimethyl-1,3-cyclohexanedione
and found that the first bromination/thiazole formation
sequence worked well. However, as with 1,3-cyclopentane-
ΔF508−CFTR CORRECTION BIOASSAY
■
The 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.5,8,12 Correctors at various
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concentrations (and negative/positive controls) were added to
each well and incubated for 18−24 h. ΔF508−CFTR-facilitated
iodide influx was determined from the kinetics of YFP-H148Q/
I152L quenching in response to iodide addition in cells treated
with a cAMP agonist forskolin and the potentiator genistein.
Table 1. Corrector Activities of Amide-Varied Constrained
Bithiazoles 7a/8a and 9a−f/10a−f Relative to Bithiazole
corr4a
a
corr4a: Vmax = 475 μM/s, IC50 = 6.0 μM
9a−f; n = 1
10a−f; n = 2
RESULTS AND DISCUSSION
Vmax
IC50
Vmax
IC50
■
compound
(μM/s)
(μM)
compound
(μM/s)
(μM)
Prior bithiazole-based structure−activity relationship studies
supported the premise that the conformation about the
bithiazole moiety is an important determinant of ΔF508−
CFTR corrector activity.8 Although bithiazoles that are free to
rotate around the thiazole−thiazole tethering C,C-bond can
accommodate many conformations, we went on to demonstrate
that constrained bithiazoleswhere the bithiazole moiety was
locked into an s-cis conformation (see Figure 1)led to
improved corrector activity.8 With this backdrop, the objectives
of this investigation were 3-fold: (i) correlate corrector activity
with bithiazole S−C−C−N (see corr4a in Figure 1) dihedral
angle; (ii) correlate corrector activity with amide R-group (see
corr4a in Figure 1) structural features; and (iii) correlate
corrector activity with aniline Ar (see corr4a in Figure 1)
structural features.
Our inability to construct the 7H-cyclopenta[1,2-d:3,4-
d′]bis(thiazole) analogue of 1 (m = 0) coupled with the fact
that the 4,5-dihydrobenzo[1,2-d:3,4-d′]bis(thiazole) analogue
of 1 (m = 1) spontaneously aromatizes to 3 limited the probe
of lower value S−C−C−N dihedral angle to 0°. Moreover, fully
aromatized bithiazole 3 proved to be toxic, killing the ΔF508−
CFTR transfected FRT cells employed in the assay. These
outcomes evolved objective (i) of this study to a direct
comparison of constrained bithiazoles 9a (1; m = 2) and 10a
(1; m = 3) to benchmark bithiazole corrector corr4a (these
three correctors each has the aniline Ar = 5-chloro-2-
methoxyphenyl). The IC50 values of 9a and 10a (2.3 and 4.1
μM, respectively) were improved relative to corr4a (6.0 μM),
and the 8-membered constrained corrector 10a was found to
have a higher Vmax value (Table 1; 512 μM/s) than either the 7-
7a
9a
9b
9c
9d
9e
9f
351
336
473
329
447
678
505
11.0
2.3
8.9
5.8
6.6
8.8
4.8
8a
592
512
247
717
286
481
467
9.2
4.1
4.5
8.7
2.9
8.3
6.6
10a
10b
10c
10d
10e
10f
a
Corrector activity of benchmark bithiazole corr4a.
data8,12 suggested that the electron-withdrawing groups (EWG)
on the phenyl ring, like fluorine, reduce corrector activity,
whereas electron-donating groups (EDG) on the phenyl ring,
like methoxy, improve corrector activity. However, we noted
that the aryls substituted simultaneously with EWG and EDG
like -Cl and -OMe generally were among the better correctors.
Also, LogP is an important feature in drug design, and we
reasoned that N-alkylated aniline or pyridine moieties could
form ammonium salts to facilitate hydrophilicity. Therefore, we
set out to prepare constrained bithiazole analogues 9a,g−j and
10a,g−j to probe these two issues. As outlined in Table 2, all of
Table 2. Corrector Activities of Aniline-Varied Constrained
Bithiazoles 9a/10a and 9g−j/10g−j Relative to Bithiazole
corr4a
a
corr4a: Vmax = 475 μM/s, IC50 = 6.0 μM
9a,g−j n = 1
10a,g−j n = 2
Vmax
(μM/s)
IC50
(μM)
Vmax
(μM/s)
IC50
(μM)
compound
compound
9a
9g
9h
9i
336
366
509
336
485
2.3
6.9
7.2
2.2
3.6
10a
10g
10h
10i
512
198
280
291
243
4.1
2.1
5.2
5.2
0.9
membered analogue 9a (Table 1; 336 μM/s) or corr4a (Vmax
475 μM/s).
=
With comparable IC50 values for 9a, 10a, and corr4a and a
higher Vmax value for 10a, we were encouraged to continue the
exploration of these 7- and 8-membered constrained bithiazole
analogues. On the basis of previous research into the bithiazole
class of compounds,8,12 we knew that the amide moiety of the
bithiazole had improved activity as a pivalamide group
compared to a benzamide. A series of six pivalamide-like
constrained bithiazoles (Scheme 2a; 9b−e and 10b−e) as well
as four pivalamide-unlike constrained bithiazoles (Scheme 2a;
ethyl propanoates 9e/10e and isoxazoles 9f/10f)all with 5-
chloro-2-methoxyphenyl as the aniline moietywere prepared
and evaluated for ΔF508−CFTR corrector activity. Although
the IC50 values for this set of amides did not reveal any
significant increase or decrease in activity relative to corr4a,
there were significantly improved Vmax values for 9e (678 μM/
s) and 10c (717 μM/s) relative to corr4a (475 μM/s).
Importantly, these increases in Vmax are indicative of better
chloride cell permeability in the rescued ΔF508−CFTR
protein. Finally, although their IC50 values were highest in
the Table 1 series, we were surprised to find that free amine
analogues 7a and 8a were modest correctors with Vmax values
approximately similar to amides 9a−f and 10a−f.
9j
10j
a
Corrector activity of benchmark bithiazole corr4a.
the constrained bithiazoles in this series were active correctors.
Of the set, 7-membered bithiazole 9i and 8-membered
bithiazoles 10g and 10j had the lowest IC50 values. Only 7-
membered constrained bithiazoles 9h and 9j afforded a
significant Vmax improvement relative to 9a.
In an effort to understand the relationship between ring size
and activity, a search of possible conformations of each of the
bithiazole analogues (including full optimizations with density
functional theory) was carried out. Figure 4 shows the two
lowest energy conformations of bithiazoles 3, 9a, and 10a. Not
surprisingly, more conformations of 9a and 10a are accessible
compared to 3. Constrained bithiazoles 9a and 10a are not
forced into a flat U-shaped conformation (i.e., a central dihedral
angle of 0°), which is perhaps related to the cytotoxicity of 3
(although this may also be a result of increased unsaturation).
Because the size of the fused ring (7-membered vs 8-
membered) does not significantly affect activity, it seems that
Our next objective was to explore modification of the aniline
aryl moiety in these constrained bithiazoles. Previous SAR
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Figure 4. Lowest energy conformers of 3, 9a, and 10a. Geometries and energies were computed with M06-2X/6-31+G(d,p) (selected distances are
shown in Å; dihedral angles are shown for the central S−C−C−N substructure; see the Computational Methods section in Supporting Information
for values and details). Top structures are the lowest energy conformers, whereas bottom structures are the second lowest energy conformers; free
energies for the latter relative to the former (0.0 kcal/mol) are shown in kcal/mol.
the target active site can accommodate nonpolar moieties of
varying size and shape.
that the 7- and 8-membered ring sizes studied accommodate
the requisite flexibility in the bithiazole core during protein
binding.
On the basis of these structural insights, future discovery on
constrained bithiazoles could address how structural manipu-
lation of the 7- and 8-membered constraining ring might
improve compound potency, pharmacokinetics, and pharma-
codynamics. Two broadly cast “constrained-X” options (9x and
10x) are depicted in Figure 4. Although 7-membered analogue
9x is appealing from a symmetry perspective, Scheme 2
chemistry may be complicated by regiochemical issues imposed
by the nonsymmetrical starting 1,3-dicarbonyl. The 8-
membered analogue 10x, which could be prepared from a
symmetrical 1,3-dicarbonyl starting material, would avoid these
complications. Finally, in addition to various constrained-X
modifications, manipulation of the peripheral groups (i.e., the
amide “R” and aniline “Ar” moieties; see Scheme 2) in
constrained-X analogues 9x and 10x may prove insightful.
EXPERIMENTAL SECTION
■
General Procedures. All chemicals were purchased from
commercial suppliers and used without further purification. Analytical
thin layer chromatography was carried out on precoated plates (silica
gel 60 F254, 250 μm thickness) and visualized with UV light. Flash
chromatography was performed using 60 Å, 32−63 μm silica gel
(Scientific Adsorbents). Concentration in vacuo refers to rotary
evaporation under reduced pressure. The chemical purity of all
compounds was determined by HPLC and HRMS or LC−MS and
1
confirmed to be ≥95%. H NMR spectra were recorded at 300, 400,
600, or 800 MHz at ambient temperature with acetone-d6, DMSO-d6,
CDCl3, CD3CN, or CD3OD as solvents. 13C NMR spectra were
recorded at 75, 100, 150, or 200 MHz at ambient temperature with
acetone-d6, DMSO-d6, CDCl3, CD3CN, or D3OD as solvents.
Chemical shifts are reported in parts per million (ppm) relative to
the residual solvent peak. Infrared spectra were recorded on an ATI-
FTIR spectrometer. The specifications of the Waters LC/MS are as
follows: electrospray (+) ionization, mass range 100−1500 Da, 20 V
cone voltage, and Xterra MS C18 column (2.1 mm × 50 mm × 3.5
μm), 0.2 mL/min; eluents were water/0.1% HCOOH and MeCN/
0.1% HCOOH in gradient, and Waters 996 PDA. Preparative HPLC
specifications are as follows: 15 mL/min flow rate, Xterra Prep MS
C18 OBD column (19 mm × 100 mm); eluents were water/0.1%
HCOOH and MeCN/0.1% HCOOH in gradient, and dual wavelength
absorbance detector. High-resolution mass spectra were acquired on
an LTQ Orbitrap XL mass spectrometer equipped with an
electrospray ionization source (ThermoFisher, San Jose, CA),
operating in the positive ion mode. Samples were introduced into
the source via loop injection at a flow rate of 200 μL/min, in a solvent
CONCLUSIONS
■
Twenty-three analogues of constrained bithiazole 9a were
designed and synthesized to probe how the constrained
bithiazole core affects the rescue and chloride channel function
of ΔF508−CFTR. We found that modulating the constraining
ring size (7- vs 8-membered) in these constrained bithiazole
correctors did not significantly increase or decrease the potency
(IC50). However, the cell chloride permeability (Vmax) of
corrected ΔF508−CFTR was significantly influenced by
bithiazole peripheral groups; most notably, bithiazoles 9e and
10c had Vmax 200 μM/s higher than benchmark bithiazole
corr4a while maintaining comparable IC50 values. This suggests
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then for 12 s after rapid (<1 s) addition of 165 μL of PBS in which 137
mM Cl− was replaced by I−. I− influx rate was computed by fitting the
final 11.5 s of the data to an exponential for extrapolation of initial
slope and normalizing for background-subtracted initial fluorescence.
All experiments contained negative control (DMSO vehicle) and
positive control [N-(2-(5-chloro-2-methoxyphenylamino)-4′-methyl-
4,5′-bithiazol-2′-yl)benzamide]. EC50 and Vmax 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) = A0 +
VmaxCiH/(CiH + IC50H), where Ci is compound concentration, H is
coefficient, and A0 is background signal.
Scheme 2. (a) Synthesis of Constrained Bithiazole Amide
Analogues 9a−f and 10a−f. (b) Synthesis of Constrained
Bithiazole Aniline Analogues of 9g−j and 10g−j
Computational Methods: All calculations were performed using
Gaussian 09.14 Conformational analyses were performed using the
Spartan10 software suite.15 Geometries were optimized at the M06-
2X/6-31+G(d,p)16,17 level. The identities of all minima were verified
by frequency analysis. Structural drawings were produced using
CYLView.18
Synthesis of Constrained Bithiazole Analogues. N8-(5-
Chloro-2-methoxyphenyl)-5,6-dihydro-4H-cyclohepta[1,2-d:3,4-d′]-
bis(thiazole)-2,8-diamine (7a). 1,3-Cycloheptadione (1.6 g, 12.7
mmol) was dissolved in CHCl3 (21 mL) and H2O (21 mL). The
biphasic mixture was stirred in an ice bath, and bromine (2.24 g, 12.0
mmol) was added dropwise via addition funnel. The mixture was
stirred for 1 h in an ice bath, then extracted with DCM (3 × 10 mL),
dried over Na2SO4, and concentrated under reduced pressure to yield
a yellow oil, 2-bromo-1,3-cycloheptadione (3.1 g crude material). This
oil was dissolved in EtOH (28 mL), thiourea was added, stirred at
room temperature overnight, concentrated, and recrystallized from
DCM/hexane (decanted cloudy white solution from yellow oil that
separated during addition of hexane and stored in freezer) to yield
colorless crystals of 2-amino-4,5,6,7-tetrahydro-8H-cyclohepta[d]-
thiazol-8-one (1.83 g, 38% yield, two steps). The yellow oil can be
purified via column chromatography (100% EtOAc) to obtain
additional 2-amino-4,5,6,7-tetrahydro-8H-cyclohepta[d]thiazol-8-one.
1
These crystals were carried forward with only a crude H and 13C
NMR for characterization, which was in agreement with previously
published results.8 To 2-amino-4,5,6,7-tetrahydro-8H-cyclohepta[d]-
thiazol-8-one (0.5 g, 1.9 mmol) in HOAc (18 mL) was added bromine
(0.34 g, 2.1 mmol) dropwise and stirred at rt for 30 min. The solid was
filtered off, rinsed with cold acetone, and dried to yield 2-amino-7-
bromo-4,5,6,7-tetrahydro-8H-cyclohepta[d]thiazol-8-one as an off-
white solid (0.48 g, 74%). To 2-amino-7-bromo-4,5,6,7-tetrahydro-
8H-cyclohepta[d]thiazol-8-one (0.48 g, 1.4 mmol) in EtOH (10 mL)
was added 1-(5-chloro-2-methoxyphenyl)thiourea (0.32 g, 1.5 mmol)
and refluxed overnight. The reaction mixture was concentrated and
recrystallized from EtOH to yield 7a as an off-white powdery solid
system of 1:1 acetonitrile/water with 0.1% formic acid. Mass spectra
were acquired using Xcalibur, version 2.0.7 SP1 (ThermoFinnigan).
The spectra were externally calibrated using the standard calibration
mixture and then calibrated internally to <2 ppm with the lock mass
tool.
Reagent Preparations. 1,3-Cycloheptadione was prepared
following a literature procedure, and spectral data were in agreement
with literature data.8 1,3-Cyclooctadione was prepared following a
literature procedure, and spectral data were in agreement with
literature data.13 Substituted thioureas were also prepared following
literature procedures, and spectral data were in agreement with
literature data.8
1
(0.42 g, 79%). H NMR (600 MHz, CDCl3): δ 9.83 (s, 1H), 9.11 (s,
2H), 8.47 (d, J = 2.5 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H) 6.97 (dd, J =
8.8, 2.5 Hz, 1H), 3.85 (s, 3H), 2.92 (t, J = 5.5 Hz, 2H), 2.89 (t, J = 5.7
Hz, 2H), 2.05−1.98 (m, 2H). HRMS (m/z): [M + H]+ calcd for
C16H15ClN4OS2, 379.0449; found, 379.0468.
ΔF508−CFTR Corrector Activity Assay. The assay was
performed using FRT epithelial cells stably coexpressing human
ΔF508−CFTR, and the high-sensitivity halide-sensing fluorescent
protein YFP-H148Q/I152L were used as described previously.5,8,12
Cells were grown at 37 °C (95% air/5% CO2) for 24 h and then
incubated for 16−20 h with 50 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. Measurements were carried out using a M1000
plate reader (Tecan Trading AG, Switzerland) equipped with a
monochromator (excitation: 500 10 nm, emission: 535 10 nm).
Each well was assayed individually for I− influx by recording
fluorescence continuously (200 ms per point) for 2 s (baseline) and
N-(8-((5-Chloro-2-methoxyphenyl)amino)-5,6-dihydro-4H-
cyclohepta[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (9a). Bithia-
zole 7a (20.0 mg, 0.053 mmol, 1.0 equiv) was dissolved in dry THF
(1 mL). Triethylamine (18.4 μL, 0.132 mmol, 2.5 equiv) and pivaloyl
chloride (10.1 μL, 0.0688 mmol, 1.3 equiv) were added sequentially.
The reaction mixture was purged with nitrogen, heated to 60 °C, and
stirred overnight. Reaction progress was monitored by TLC in 50/50
ethyl acetate/hexanes. The reaction mixture was concentrated, and the
product was isolated by HPLC as a white solid (12.5 mg, 51%). mp
225−229 °C. IR (neat): vmax 2966, 2929, 2856, 1678, 1597, 1533,
1244, 1124, 1024 cm−1. 1H NMR (600 MHz, CDCl3): δ 8.24 (s, 1H),
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8.07 (d, J = 2.4 Hz, 1H), 7.57 (s, 1H), 6.92 (dd, 2.4 and 8.4 Hz, 1H),
6.78 (d, J = 8.4 Hz, 1H), 3.90 (s, 3H), 3.06 (at, 5.7 Hz, 2H), 2.98 (at,
5.6 Hz, 2H), 2.17−2.13 (m, 2H), 1.34 (s, 9H). 13C NMR (150 MHz,
CDCl3): δ 184.2, 176.2, 158.9, 157.0, 145.5, 145.0, 138.6, 130.8, 126.3,
120.8, 120.1, 115.9, 110.6, 56.0, 39.2, 32.0, 27.2, 27.1, 22.6. HRMS
(m/z): [M + H]+ calcd for C21H23ClN4O2S2, 463.1024; found,
463.1021.
1.54 (m, 1H), 1.26 (d, J = 7.0 Hz, 3H), 0.96 (t, J = 7.4 Hz, 3H). 13C
NMR (150 MHz, CDCl3): δ 175.1, 166.5, 159.3, 158.0, 145.6, 137.9,
130.6, 126.3, 122.2, 121.0, 120.8, 116.1, 110.7, 56.0, 38.7, 35.5, 32.3,
31.0, 26.8, 22.3, 19.0. HRMS (m/z): [M + H]+ calcd for
C21H23ClN4O2S2, 463.1024; found, 463.1024.
Methyl 4-((8-((5-chloro-2-methoxyphenyl)amino)-5,6-dihydro-
4H-cyclohepta[1,2-d:3,4-d′]bis(thiazole)-2-yl)amino)-4-oxobuta-
noate (9e). The procedure for 9a was followed, except 3-
(carbomethoxy)-propionyl chloride (28.7 μL, 0.233 mmol, 2.2
equiv) was substituted, yielding a yellow solid (7.9 mg, 15%). 1H
NMR (600 MHz, CDCl3): δ 8.07 (d, J = 2.3 Hz, 1H), 7.56 (s, 1H),
6.91 (dd, J = 8.6, 2.3 Hz, 1H), 6.77 (d, J = 8.6, 1H), 3.89 (s, 3H), 3.71
(s, 3H), 3.09 (at, J = 5.5 Hz, 2H), 2.98 (at, J = 5.5 Hz, 2H), 2.81−2.73
(m, 2H), 2.17−2.12 (m, 2H). 13C NMR (150 MHz, CDCl3): δ 173.1,
168.7, 159.0, 155.4, 145.7, 138.5, 130.8, 126.3, 123.1, 120.8, 120.2,
115.9, 113.1, 110.6, 56.0, 52.1, 32.6, 31.0, 28.9, 27.2, 22.6. HRMS (m/
z): [M + H]+ calcd for C21H21ClN4O4S2, 493.0766, found: 493.0758.
N-(8-((5-Chloro-2-methoxyphenyl)amino)-5,6-dihydro-4H-
c y c l o h e p t a [ 1 , 2 - d : 3 , 4 - d ′ ] b i s ( t h i a z o l e ) - 2 - y l ) -
cyclopropanecarboxamide (9b). The procedure for 9a was followed,
except cyclopropanecarbonyl chloride (0.14 mmol, 2.6 equiv)
(prepared freshly from cyclopropane carboxylic acid and oxalyl
chloride 1:1.2 eq in 50 μL of DCM and a drop of DMF) was
substituted, yielding a yellow solid (12 mg, 51%). 1H NMR (600 MHz,
CDCl3): δ 8.05 (d, J = 2.3 Hz, 1H), 7.57 (s, 1H), 6.91 (dd, J = 8.6, 2.3
Hz, 1H), 6.77 (d, J = 8.6, 1H), 3.89 (s, 3H), 3.09 (at, J = 5.5 Hz, 2H),
2.98 (at, J = 5.5 Hz, 2H), 2.16−2.12 (m, 2H), 1.70−1.64 (m, 1H),
1.23−1.20 (m, 2H), 0.98−0.95 (m, 2H). 13C NMR (150 MHz,
CDCl3): δ 171.4, 159.1, 156.8, 145.6, 143.5, 138.1, 130.7, 126.3, 122.7,
120.9, 120.6, 116.0, 110.7, 56.0, 31.9, 29.7, 27.0, 22.4, 15.1, 9.3. HRMS
(m/z): [M + H]+ calcd for C20H19ClN4O2S2, 447.0711; found,
447.0708.
N-(8-((5-Chloro-2-methoxyphenyl)amino)-5,6-dihydro-4H-
cyclohepta[1,2-d:3,4-d′]bis(thiazole)-2-yl)isoxazole-5-carboxamide
(9f). The procedure for 9a was followed, except isoxazole 5-carbonyl
chloride (6.64 μL, 0.069 mmol, 1.3 equiv) was substituted, yielding a
bright yellow solid (11.7 mg, 47%). mp 115−120 °C. IR (neat): vmax
1
2924, 1597, 1531, 1415, 1294, 1244, 1124, 1020, 750 cm−1. H NMR
N-(8-((5-Chloro-2-methoxyphenyl)amino)-5,6-dihydro-4H-
cyclohepta[1,2-d:3,4-d′]bis(thiazole)-2-yl)isobutyramide (9c). The
procedure for 9a was followed, except isobutyryl chloride (7.2 μL,
0.069 mmol, 1.3 equiv) was substituted, yielding an orange solid (5.9
mg, 25%). mp 208−215 °C. IR (neat): vmax 2964, 2924, 1597, 1531,
1417, 1268, 1248, 1126, 1026, 796 cm−1. 1H NMR (600 MHz,
CDCl3): δ 8.21 (s, 1H), 8.06 (d, J = 2.5 Hz, 1H), 7.59 (s, 1H), 6.92
(dd, J = 8.6, 2.5 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 3.90 (s, 3H), 3.04
(at, J = 5.7 Hz, 2H), 2.98 (at, J = 5.6 Hz, 2H), 2.73 (h, J = 6.9 Hz, 1H),
2.18−2.12 (m, 2H), 1.29 (d, J = 6.9 Hz, 6H). 13C NMR (150 MHz,
CDCl3): δ 175.1, 166.5, 159.3, 158.0, 145.6, 137.9, 130.6, 126.3, 122.2,
121.0, 120.7, 116.1, 110.7, 56.0, 35.5, 31.0, 26.8, 22.3, 19.0. HRMS
(m/z): [M + H]+ calcd for C20H21ClN4O2S2, 449.0867; found,
449.0870.
(400 MHz, DMSO-d6): δ 9.72 (s, 1H), 8.78 (d, J = 2.0 Hz, 1H), 8.61
(d, J = 2.4 Hz, 1H), 8.11 (s, 1H), 7.36 (s, 1H), 7.0 (d, J = 8.7 Hz, 1 H),
6.96 (dd, J = 8.6, 2.5 Hz, 1H), 3.85 (s, 3H), 3.04 (at, J = 5.6 Hz, 2H),
2.94 (at, J = 5.5 Hz, 2H), 2.05−2.00 (m, 2H). HRMS (m/z): [M +
H]+ calcd C20H16ClN5O3S2, 474.0.456; found, 474.0462.
2-Amino-5,6,7,8-tetrahydrocycloocta[d]thiazol-9(4H)-one (A). To
a heterogeneous mixture of cyclooctane-1,3-dione (2.08 g, 14.8 mmol)
in CCl4/water (1:1, 50 mL) was added a solution of Br2 (2.61g, 0.84
mL, 16.3 mmol) in CCl4 (25 mL) dropwise at 0 °C. The mixture was
stirred at 0 °C for 1 h and extracted with DCM. The organic layer was
collected, and DCM was removed under reduced pressure to afford 2-
bromocyclooctane-1,3-dione as a yellow oil which was used without
further purification. To a solution of 2-bromocyclooctane-1,3-dione in
anhydrous EtOH (35 mL) was added thiourea (1.24 g, 16.3 mmol).
The mixture was stirred at room temperature overnight. EtOH was
removed under reduced pressure, and the resulting light brown residue
was redissolved in water (50 mL), basified with concentrated NH4OH
solution until pH = 8−9, and extracted with EtOAc (3 × 50 mL). The
combined organics were dried over anhydrous Na2SO4 and
concentrated to give the crude product which was triturated with
CHCl3 to afford A as a yellow solid (1.47 g, 51% yield over two steps.
1H NMR (600 MHz, DMSO-d6): δ 7.88 (s, 2H), 3.01 (t, J = 7.0 Hz,
2H), 2.78 (t, J = 7.0 Hz, 2H), 1.73−1.55 (m, 4H), 1.45−1.33 (m, 2H).
N-(8-((5-Chloro-2-methoxyphenyl)amino)-5,6-dihydro-4H-
cyclohepta[1,2-d:3,4-d′]bis(thiazole)-2-yl)-2-methylpentanamide
(9d). The procedure for 9a was followed, except 2-methylbutyryl
chloride (8.54 μL, 0.069 mmol, 1.3 equiv) was substituted, yielding a
yellow solid (11.8 mg, 48%). mp 138−144 °C. IR (neat): vmax 3207,
2031, 2011, 1969, 1531, 1261, 1178, 1090, 1016, 897, 798, 644 cm-1.
1H NMR (600 MHz, CDCl3): δ 8.22 (s, 1H), 8.05 (d, J = 2.4 Hz, 1
H), 7.57 (s, 1H), 6.92 (dd, J = 8.6, 2.5 Hz, 1H), 6.78 (d, J = 8.7 Hz,
1H), 3.90 (s, 3H), 3.05 (at, J = 5.7 Hz, 2H), 2.99 (at, J = 5.6 Hz, 2H),
2.52−2.46 (m, 1H), 2.17−2.13 (m, 2H), 1.84−1.76 (m, 1H), 1.61−
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13C NMR (150 MHz, DMSO-d6): δ 191.1, 171.6, 159.9, 125.4, 39.6,
30.0, 24.0, 23.1, 22.6. HRMS (m/z): [M + H]+ calcd C9H12N2OS,
197.0749; found, 197.0749.
cyclopropanecarboxamide (10b). The procedure for 10a was
followed, except cyclopropane carbonyl chloride (83 μL, 0.917
mmol, 1.1 equiv) was substituted, yielding a yellow solid (0.0359 g,
2-Amino-8-bromo-5,6,7,8-tetrahydrocycloocta[d]thiazol-9(4H)-
one dihydrobromide salt (B). Compound A (0.70 g, 3.59 mmol)
dissolved in glacial acetic acid (18 mL) was treated dropwise with a
solution of HBr in HOAc (18 mL, 33 wt % in acetic acid). The
mixture was stirred at room temperature for 30 min. Br2 (0.573 g,
0.184 mL, 3.59 mmol) was added dropwise. The reaction mixture was
stirred at room temperature for 1 h. Removing solvent under reduced
pressure yielded the crude product which was triturated with DCM to
afford B as a tan solid (1.08 g, 70%). 1H NMR (600 MHz, DMSO-d6):
δ 10.26 (s, 1H), 9.35 (s, 3H), 5.84 (dd, J = 11.9, 7.2 Hz, 1H), 3.42−
3.26 (m, 1H), 2.97 (dd, J = 15.1, 6.5 Hz, 1H), 2.23 (dtd, J = 13.5, 7.2,
3.3 Hz, 1H), 2.05 (t, J = 12.6 Hz, 1H), 1.75 (dd, J = 13.5, 6.5 Hz, 1H),
1.62 (dd, J = 9.7, 5.5 Hz, 2H), 1.42−1.24 (m, 1H). 13C NMR (150
MHz, DMSO-d6): δ 184.5, 170.0, 150.0, 121.0, 55.7, 35.6, 27.1, 23.5,
22.4. HRMS (m/z): [M + H]+ calcd for C9H11BrN2OS, 274.9854;
found, 274.9867.
1
9%). H NMR (600 MHz, CDCl3): δ 11.15 (s, 1H), 8.04 (d, J = 2.2
Hz, 1H), 7.80 (s, 1H), 6.89 (dt, J = 8.6, 2.2 Hz, 1H), 6.75 (dd, J = 8.6,
2.2 Hz, 1H), 3.85 (d, J = 3.4 Hz, 3H), 2.90−2.77 (m, 4H), 1.78 (s,
4H), 1.61 (tt, J = 8.1, 4.6 Hz, 1H), 1.20−1.15 (m, 2H), 0.90 (dt, J =
7.2, 3.4 Hz, 2H). 13C NMR (150 MHz, CDCl3): δ 171.8, 159.7, 158.4,
146.7, 146.0, 139.0, 130.9, 126.2, 122.6, 121.2, 120.6, 116.4, 110.8,
56.1, 29.9, 27.1, 24.8, 24.4, 15.0, 9.2. HRMS (m/z): [M + H]+ calcd
for C21H21ClN4O2S2, 461.0873; found, 461.0865.
N-(9-((5-Chloro-2-methoxyphenyl)amino)-4,5,6,7-tetrahydrocy-
cloocta [1,2-d:3,4-d′]bis(thiazole)-2-yl)isobutyramide (10c). The
procedure for 10a was followed, except isobutyryl chloride (87 μL,
0.830 mmol, 1.2 equiv) was substituted, yielding a yellow solid (0.132
g, 41%). 1H NMR (600 MHz, CDCl3): δ 9.56 (s, 1H), 8.04 (d, J = 2.5
Hz, 1H), 7.81 (s, 1H), 6.94−6.83 (m, 1H), 6.75 (d, J = 8.6 Hz, 1H),
3.84 (s, 3H), 2.90−2.78 (m, 4H), 2.59 (hept, J = 6.9 Hz, 1H), 1.84−
1.74 (m, 4H), 1.23 (d, J = 6.9 Hz, 6H). 13C NMR (150 MHz, CDCl3):
δ 174.5, 159.7, 157.6, 146.9, 145.9, 139.0, 130.9, 126.2, 122.6, 121.1,
120.8, 116.4, 110.8, 56.1, 35.6, 30.0, 27.1, 24.7, 24.4, 19.3. HRMS (m/
z): [M + H]+ calcd for C21H23ClN4O2S2, 463.1029; found, 463.1021.
N9-(5-Chloro-2-methoxyphenyl)-4,5,6,7-tetrahydrocycloocta[1,2-
d:3,4-d′]bis(thiazole)-2,9-diamine (8a). A suspension of B (0.451 g,
1.03 mmol, 1.0 equiv) and 1-(5-chloro-2-methoxyphenyl)thiourea
(0.224 g, 1.03 mmol, 1.0 equiv) in anhydrous EtOH (10 mL) was
heated at reflux for 24 h. EtOH was removed under reduced pressure,
and the residue was redissolved in water (10 mL), basified with 1 M
NaOH (50 mL), and extracted with EtOAc (2 × 50 mL). The
combined organics were dried over anhydrous Na2SO4, concentrated,
and purified by column chromatography (100% EtOAc, Rf = 0.20) to
1
afford 8a as a light brown solid (0.266 g, 65%). H NMR (600 MHz,
CDCl3): δ 8.05 (d, J = 2.3 Hz, 1H), 7.67 (s, 1H), 6.88 (dd, J = 8.6, 2.3
Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 6.12 (s, 2H), 3.85 (s, 3H), 2.79 (t, J
= 10.5 Hz, 4H), 1.78 (dt, J = 10.5, 5.2 Hz, 4H). 13C NMR (150 MHz,
CDCl3): δ 167.3, 159.5, 145.8, 138.8, 130.9, 126.2, 123.2, 121.9, 121.0,
116.3, 115.4, 110.8, 56.1, 29.5, 27.3, 24.5, 23.5. HRMS (m/z): [M +
H]+ calcd for C17H17ClN4OS2, 393.0611; found, 393.0593.
N-(9-((5-Chloro-2-methoxyphenyl)amino)-4,5,6,7-
tetrahydrocycloocta[1,2-d:3,4-d′]bis(thiazole)-2-yl)-2-methylbuta-
namide (10d). The procedure for 10a was followed, except 2-
methylbutyryl chloride (85 μL, 0.683 mmol, 1.1 equiv) was
substituted, yielding a light yellow solid (0.10 g, 34%). 1H NMR
(600 MHz, CDCl3): δ 9.72 (s, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.85 (s,
1H), 6.88 (dd, J = 8.6, 2.4 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 3.84 (s,
3H), 3.04−2.61 (m, 4H), 2.35 (sextet, J = 7.0 Hz, 1H), 1.89−1.66 (m,
5H), 1.50 (dp, J = 14.0, 7.0 Hz, 1H), 1.20 (d, J = 7.0 Hz, 3H), 0.89 (t,
J = 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 174.1, 159.7, 157.5,
146.9, 146.0, 139.0, 130.9, 126.2, 122.6, 121.2, 120.8, 116.4, 110.8,
56.1, 42.8, 30.0, 27.2, 27.1, 24.7, 24.3, 17.1, 11.8. HRMS (m/z): [M +
H]+ calcd for C22H25ClN4O2S2, 477.1186; found, 477.1176.
N-(9-((5-Chloro-2-methoxyphenyl)amino)-4,5,6,7-tetrahydrocy-
cloocta-[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (10a). To a sol-
ution of 8a (0.266 g, 0.676 mmol, 1.0 equiv) in anhydrous DCM (7
mL) was added TEA (188 μL, 1.35 mmol, 2.0 equiv). Pivaloyl chloride
(92 μL, 0.743 mmol, 1.1 equiv) was then added in one portion. The
reaction mixture was purged with nitrogen and stirred at 60 °C
overnight at which time LC-MS indicated the completion of reaction.
DCM was removed in vacuo, and the resulting solid was purified by
1
HPLC to yield 10a as a yellow solid (0.183 g, 57%). H NMR (600
MHz, CDCl3): δ 9.46 (s, 1H), 8.07 (d, J = 2.5 Hz, 1H), 7.64 (s, 1H),
6.97−6.84 (m, 1H), 6.77 (dd, J = 8.6, 2.5 Hz, 1H), 3.88 (d, J = 3.0 Hz,
3H), 2.93−2.77 (m, 4H), 1.81 (m, 4H), 1.34 (s, 9H). 13C NMR (150
MHz, CDCl3): δ 175.8, 159.4, 157.3, 146.6, 145.7, 139.0, 130.8, 126.3,
122.8, 120.9, 120.9, 116.1, 110.7, 56.1, 39.2, 29.9, 27.3, 27.0, 24.7, 24.3.
HRMS (m/z): [M + H]+ calcd for C22H25ClN4O2S2, 477.1186; found,
477.1176.
N-(9-((5-Chloro-2-methoxyphenyl)amino)-4,5,6,7-
tetrahydrocycloocta[1,2-d:3,4-d′]bis(thiazole)-2-yl)-
Methyl 4-((9-((5-chloro-2-methoxyphenyl)amino)-4,5,6,7
tetrahydrocycloocta[1,2-d:3,4-d′]bis(thiazole)-2-yl)amino)-4-oxo-
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butanoate (10e). The procedure for 10a was followed, except methyl
4-chloro-4-oxobutyrate (76 μL, 0.618 mmol, 1.1 equiv) was
1
substituted, yielding a light yellow solid (0.121 g, 42%). H NMR
(600 MHz, CDCl3): δ 10.63 (s, 1H), 8.05 (d, J = 2.5 Hz, 1H), 7.91 (s,
1H), 6.85 (dd, J = 8.6, 2.5 Hz, 1H), 6.72 (d, J = 8.6 Hz, 1H), 3.80 (s,
3H), 3.65 (s, 3H), 2.85 (d, J = 7.0 Hz, 2H), 2.80−2.72 (m, 6H), 1.80−
1.70 (m, 4H). 13C NMR (150 MHz, CDCl3): δ 173.1, 169.5, 159.7,
157.4, 147.1, 145.9, 138.9, 130.8, 126.0, 122.4, 121.0, 120.6, 116.4,
110.7, 56.0, 52.0, 30.7, 29.9, 28.7, 27.0, 24.5, 24.2. HRMS (m/z): [M +
H]+ calcd for C22H23ClN4O4S2, 507.0927; found, 507.0918.
N-(8-((3-(Dimethylamino)phenyl)amino)-5,6-dihydro-4H-
cyclohepta[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (10i). The pro-
cedures for 7a and 9a were followed, except 1-(2-(dimethylamino)-
phenyl)thiourea (0.195 g, 1.1 mmol, 1.1 equiv) was substituted,
yielding a yellow solid (0.010g, 10%). mp 111−112 °C. 1H NMR (600
MHz, CDCl3): δ 9.09 (s, 1H), 7.21 (s, 1H), 7.18 (t, J = 8.2 Hz, 1H),
7.00 (s, 1H), 6.59 (d, J = 8.2, 1H), 6.45 (d, J = 8.2, 1H), 3.07 (t, J = 5.7
Hz, 2H), 3.01 (s, 3H), 2.95 (t, J = 5.7 Hz, 2H), 2.16−2.12 (m, 2H),
1.32 (s, 9H). 13C NMR (150 MHz, CDCl3): δ 175.8, 161.1, 155.8,
151.6, 145.3, 141.5, 138.4, 130.0, 123.3, 119.5, 107.5, 106.5, 102.5,
40.9, 39.3, 32.6, 29.9, 27.5, 22.9. HRMS (m/z): [M + H]+ calcd for
C22H27N5OS2, 442.1730; found, 442.1725.
N-(9-((5-Chloro-2-methoxyphenyl)amino)-4,5,6,7-tetrahydrocy-
clo-octa[1,2-d:3,4-d′]bis(thiazole)-2-yl)isoxazole-5-carboxamide
(10f). The procedure for 10a was followed, except isoxazole-5-
carbonyl chloride (76 μL, 0.787 mmol, 1.1 equiv) was substituted,
yielding a yellow solid (0.0422 g, 12%). 1H NMR (600 MHz, CDCl3):
δ 9.55 (s, 1H), 8.39 (d, J = 1.7 Hz, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.76
(s, 1H), 7.15−7.11 (m, 1H), 6.90 (dd, J = 8.6, 2.4 Hz, 1H), 6.77 (d, J =
8.6 Hz, 1H), 3.87 (s, 3H), 2.97−2.75 (m, 4H), 1.88−1.75 (m, 4H).
13C NMR (150 MHz, CDCl3): δ 161.6, 159.8, 156.9, 153.7, 151.3,
N-(8-((2-Methoxy-5-nitrophenyl)amino)-5,6-dihydro-4H-
cyclohepta[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (10j). The pro-
cedures for 7a and 9a were followed, except 1-(2-methoxy-5-
nitrophenyl)thiourea (0.227 g, 1.1 mmol, 1.1 equiv) was substituted,
yielding a yellow solid (0.006 g, 6%). 1H NMR (600 MHz, CDCl3): δ
10.60 (s, 1H), 8.89 (s, 1H), 7.94 (d, J = 9.3, 1H), 7.62 (s, 1H), 6.94 (d,
J = 9.3, 1H), 3.97 (s, 3H), 3.13−2.94 (m, 4H), 2.25−2.05 (m, 2H),
1.25 (s, 9H). 13C NMR (150 MHz, DMSO): δ 176.1, 159.1, 155.7,
152.4, 146.2, 140.8, 137.5, 130.4, 121.1, 120.5, 117.4, 111.1, 110.1,
56.6, 38.7, 32.3, 26.7, 26.1, 22.3. HRMS (m/z): [M + H]+ calcd for
C21H23N5O4S2, 474.1264; found, 474.1253.
146.4, 145.9, 138.5, 130.8, 126.3, 123.0, 121.9, 121.2, 116.3, 110.9,
108.3, 56.1, 29.7, 27.0, 24.6, 24.0. HRMS (m/z): [M + H]+ calcd for
C21H18ClN5O3S2, 488.0618; found, 488.0609.
N-(8-((5-Chloro-2-(dimethylamino)phenyl)amino)-5,6-dihydro-
4H-cyclo-hepta[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (9g). The
procedures for 7a and 9a were followed, except 1-(5-chloro-2-
(dimethylamino)-phenyl)thiourea (0.229 g, 1.1 mmol, 1.1 equiv) was
1
substituted, yielding a yellow solid (0.015 g, 15%). H NMR (400
N-(9-((5-Chloro-2-(dimethylamino)phenyl)amino)-4,5,6,7-tetra-
hydrocycloocta-[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (10g).
The procedures for 8a and 10a was followed, except 1-(5-chloro-2-
(dimethylamino)-phenyl)thiourea (0.260 g, 1.13 mmol, 1.0 equiv) was
MHz, CDCl3): δ 8.28 (s, 1H), 7.99 (d, J = 2.3, 1H), 7.07 (d, J = 8.4,
1H), 6.91 (dd, J = 2.3, 8.4, 1H), 3.18−2.85 (m, 4H), 2.64 (s, 6H), 2.15
(s, 2H), 1.35 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 184.3, 176.2,
159.2, 156.9, 145.3, 140.2, 138.8, 136.7, 130.8, 122.8, 121.4, 120.7,
115.6, 44.9, 39.3, 38.7, 32.3, 27.5, 22.8. HRMS (m/z): [M + H]+ calcd
for C22H27ClN5OS2, 476.1340; found, 476.1338.
1
substituted, yielding a tan solid (0.146 g, 53%). H NMR (600 MHz,
CDCl3): δ 8.92 (s, 1H), 8.37 (s, 1H), 8.02 (d, J = 2.3 Hz, 1H), 7.02 (d,
J = 8.4 Hz, 1H), 6.84 (dd, J = 8.4, 2.3 Hz, 1H), 2.87−2.72 (m, 4H),
2.56 (s, 6H), 1.82−1.67 (m, 4H), 1.27 (s, 9H). 13C NMR (150 MHz,
CDCl3): δ 175.6, 159.3, 156.9, 147.3, 140.0, 139.1, 136.5, 130.4, 122.4,
121.2, 121.1, 120.8, 115.6, 44.7, 39.0, 30.0, 27.2, 26.8, 24.7, 24.3.
HRMS (m/z): [M + H]+ calcd for C23H28ClN5OS, 490.1502; found,
490.1491.
N-(8-((3-Methylpyridin-2-yl)amino)-5,6-dihydro-4H-cyclohepta-
[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (10h). The procedures for
7a and 9a were followed, except 1-(3-methylpyridin-2-yl)thiourea
(0.167 g, 1.1 mmol, 1.1 equiv) was substituted, yielding a yellow solid
(0.024 g, 26%). mp 154−155 °C. 1H NMR (400 MHz, CDCl3): δ 8.85
(br s, 1H), 8.16 (d, J = 4.0, 1H), 7.97 (br s, 1H), 7.37 (d, J = 7.1, 1H),
6.78 (dd, J = 5.1, 7.2, 1H), 3.13−2.93 (m, 4H), 2.27 (s, 3H), 2.14−
2.10 (m, 2H), 1.30 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 175.6,
156.5, 155.2, 149.7, 145.9, 144.4, 138.3, 137.0, 123.5, 123.3, 117.8,
116.5, 39.3, 33.1, 27.5, 27.0, 23.0, 16.8. HRMS (m/z): [M + H]+ calcd
for C20H23N5OS2, 414.1417; found, 414.1404.
N-(9-((3-Methylpyridin-2-yl)amino)-4,5,6,7-tetrahydrocycloocta-
[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (10h). The procedure for
8a and 10a was followed, except 1-(3-methylpyridin-2-yl)thiourea
(0.171 g, 1.02 mmol, 1.0 equiv) was substituted, yielding a yellow solid
(0.0845 g, 40%). 1H NMR (600 MHz, CDCl3): δ 8.97 (s, 1H), 8.45−
7.82 (m, 2H), 7.35 (d, J = 7.2 Hz, 1H), 6.92−6.59 (m, 1H), 2.81 (s,
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Article
4H), 2.21 (s, 3H), 1.76 (s, 4H), 1.26 (s, 9H). 13C NMR (150 MHz,
CDCl3): δ 175.5, 156.7, 156.6, 149.6, 147.1, 144.1, 138.1, 137.1, 125.3,
120.9, 117.7, 116.3, 39.0, 30.1, 27.3, 27.2, 24.4, 24.3, 16.7. HRMS (m/
z): [M + H]+ calcd for C21H25N5OS2, 428.1579; found, 428.1570.
ACKNOWLEDGMENTS
■
We thank Professor Makhluf J. Haddadin (American University
of Beirut, Beirut, Lebanon) for helpful insights and Kelli
Gottlieb for assistance collecting HRMS data. The authors
thank the Tara K. Telford Fund for Cystic Fibrosis Research at
UC Davis, the National Institutes of Health (DK072517 and
GM076151), the National Science Foundation (CHE-0614756,
CHE-0443516, CHE-0449845, CHE-9808183 − NMR spec-
trometers; CHE-030089−computer time from the Pittsburgh
Supercomputer Center), and the Petroleum Research Fund of
the American Chemical Society (grant 52801-ND4) for their
generous support. This work used the Extreme Science and
Engineering Discovery Environment (XSEDE), which is
supported by the National Science Foundation [ACI-1053575].
N-(9-((3-(Dimeth ylamino)phenyl)amino)-4, 5, 6, 7-
tetrahydrocycloocta[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide (10i).
The procedure for 8a and 10a was followed, except 1-(3-
(dimethylamino)phenyl)thiourea (0.197 g, 1.01 mmol, 1.0 equiv)
1
was substituted, yielding a tan solid (0.0817 g, 22%). H NMR (600
ABBREVIATIONS:
MHz, CDCl3): δ 8.92 (s, 1H), 8.26 (s, 1H), 7.12 (t, J = 8.1 Hz, 1H),
6.77 (t, J = 2.1 Hz, 1H), 6.60 (dd, J = 7.8, 1.9 Hz, 1H), 6.39 (d, J = 8.3
Hz, 1H), 2.92 (s, 6H), 2.90−2.67 (m, 4H), 1.88−1.69 (m, 4H), 1.26
(s, 9H). 13C NMR (150 MHz, CDCl3): δ 175.6, 161.8, 157.0, 151.5,
146.9, 141.5, 138.5, 129.8, 121.2, 121.1, 107.2, 106.5, 102.4, 40.6, 39.0,
30.0, 27.2, 26.8, 24.7, 24.5. HRMS (m/z): [M + H]+ calcd for
C23H29N5OS2, 456.1892; found, 456.1884.
■
cystic fibrosis transmembrane conductance regulator, (CFTR);
cystic fibrosis, (CF); Fischer rat thyroid, (FRT); yellow
fluorescent protein, (YFP); electron-withdrawing group,
(EWG); electron-donating group, (EDG)
REFERENCES
■
(1) Bobadilla, J. L.; Macek, M., Jr.; 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.
(2) CF mutation database: genet.sickkids.on.ca/cftr/StatisticsPage.
html (accessed 5/2/2014).
(3) Cystic Fibrosis Foundation Patient Registry 2012 Annual Data
Report: http://www.cff.org/livingwithcf/qualityimprovement/
patientregistryreport/ (accessed 5/2/2014).
(4) (a) Amaral, M. D.; Kunzelmann, K. Molecular targeting of CFTR
as a therapeutic approach to cystic fibrosis. Trends Pharmacol. Sci.
2007, 28, 334−341. (b) Amaral, M. D. CFTR and chaperones:
processing and degradation. J. Mol. Neurosci. 2004, 23, 41−48.
(c) Amaral, M. D. Therapy through chaperones: sense or anti-sense?
Cystic fibrosis as a model disease. J. Inherit. Metab. Dis. 2005, 29, 477−
487.
(5) (a) Pedemonte, N.; Lukacs, G. L.; Du, K.; Caci, E.; Zegarra-
Moran, O.; Galietta, L. J. V.; Verkman, A. S. Small-molecule correctors
of defective Delta F508-CFTR cellular processing identified by high-
throughput screening. J. Clin. Invest. 2005, 115, 2564−2571.
(b) Verkman, A. S.; Galietta, L. J. Chloride Channels as Drug Targets.
Nat. Rev. Drug Discovery 2009, 8, 153−171. (c) Lukacs, G. L.;
Verkman, A. S. CFTR: Folding, Misfolding and Correcting the
DeltaF508 Conformational Defect. Trends Mol. Med. 2012, 18, 81−91.
(d) Okiyoneda, T.; Veit, G.; Dekkers, J. F.; Bagdany, M.; Soya, N.; Xu,
H.; Roldan, A.; Verkman, A. S.; Kurth, M.; Simon, A.; Hegedus, T.;
Beekman, J. M.; Lukacs, G. L. Mechanism-based corrector
combination restores DeltaF508-CFTR folding and function. Nat.
Chem. Biol. 2013, 9, 444−454.
N - ( 9- ( ( 2 - M e th ox y - 5- n i t ro p h e n y l ) a m i n o) - 4 , 5 , 6 , 7 -
tetrahydrocycloocta[1,2-d:3,4-d′]bis(thiazole)-2-yl)pivalamide
(10j). The procedure for 8a and 10a was followed, except 1-(2-
methoxy-5-nitrophenyl)thiourea (0.229 g, 1.01 mmol, 1.0 equiv) was
1
substituted, yielding a yellow solid (0.0997 g, 34%). H NMR (600
MHz, CDCl3): δ 9.04 (d, J = 2.7 Hz, 1H), 8.90 (s, 1H), 7.77 (dd, J =
9.0, 2.7 Hz, 2H), 6.84 (d, J = 9.0 Hz, 1H), 3.94 (s, 3H), 2.90−2.72 (m,
4H), 1.86−1.67 (m, 4H), 1.28 (s, 9H). 13C NMR (150 MHz, CDCl3):
δ 175.6, 158.6, 157.0, 151.5, 147.2, 141.7, 139.2, 130.1, 122.9, 120.8,
117.6, 110.9, 108.9, 56.5, 39.1, 29.9, 27.2, 26.8, 24.6, 24.3. HRMS (m/
z): [M + H]+ calcd for C22H25N5O4S2, 488.1426; found, 488.1415.
(6) (a) Wang, Y.; Wrennall, J. A.; Cai, Z.; Li, H.; Sheppard, D. N.
Understanding how cystic fibrosis mutations disrupt CFTR function:
From single molecules to animal models. Int. J. Biochem. Cell B 2014,
52, 47−57. (b) Ramsey, B. W.; Davies, J.; McElvaney, N. G.; Tullis, E.;
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra as well as a corrector activity figure
for compounds 9a, 10a, 9e, and 10c; HRMS data summary for
all compounds; and computational data. This material is
available free of charge via the Internet at http://pubs.acs.org.
Bell, S. C.; Dre
̌
vínek, P.; Griese, M.; McKone, E. F.; Wainwright, C. E.;
Konstan, M. W.; Moss, R.; Ratjen, F.; Sermet-Gaudelus, I.; Rowe, S.
M.; Dong, Q.; Rodriguez, S.; Yen, K.; Ordonez, C.; Elborn, J. S. A
̃
CFTR potentiator in patients with cystic fibrosis and the G551D
mutation. N. Engl. J. Med. 2011, 365, 1663−1672. (c) Van Goor, F.;
Yu, H.; Burton, B.; Hoffman, B. J. Effect of ivacaftor on CFTR forms
with missense mutations associated with defects in protein processing
or function. J. Cyst. Fibros. 2014, 13, 29−36.
(7) (a) Deeks, E. D. Ivacaftor: A Review of Its Use in Patients with
Cystic Fibrosis. Drugs 2013, 73, 1595. (b) Kopeikin, Z.; Yuksek, Z.;
Yang, H.-Y.; Bompadre, S. G. Combined effects of VX-770 and VX-
AUTHOR INFORMATION
Corresponding Author
*E-mail: mjkurth@ucdavis.edu. Fax: (530) 752-8995. Tel.:
(530) 554-2145.
■
Notes
The authors declare no competing financial interest.
6737
dx.doi.org/10.1021/jm5007885 | J. Med. Chem. 2014, 57, 6729−6738

Journal of Medicinal Chemistry
Article
809 on several functional abnormalities of F508del-CFTR channels. J.
Cystic Fibrosis 2014, DOI: 10.1016/j.jcf.2014.04.003.
(8) 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.
(9) Vistoli, G.; Pedretti, A.; Testa, B. Assessing drug-likeness-what are
we missing? Drug Discovery Today 2008, 13, 285−294.
(10) Du, L.; Chen, M.; Zhang, Y.; Shen, B. BlmIII and BlmIV
Nonribosomal Peptide Synthetase-Catalyzed Biosynthesis of the
Bleomycin Bithiazole Moiety Involving Both in Cis and in Trans
Aminoacylation. Biochemistry 2003, 42, 9731−9740.
(11) Dinner, A. R.; Sali, A.; Smith, L. J.; Dobson, C. M.; Karplus, M.
Understanding protein folding via free-energy surfaces from theory
and experiment. Trends Biochem. Sci. 2000, 25, 331−339.
(12) 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
DF508-CFTR cellular processing. Bioorg. Med. Chem. Lett. 2008, 18,
2610−2614.
(13) Pirrung, M. C.; Webste, N. J. G. Mechanism of Intramolecular
Photocycloadditons of Cyclooctenones. J. Org. Chem. 1987, 52, 3603−
3613.
(14) Frisch, M. J.; et al. Gaussian 09, revision B.01; Gaussian, Inc.,
Wallingford, CT, 2009 (full reference in Supporting Information).
(15) Shao, Y.; et al. Phys. Chem. Chem. Phys. 2006, 8, 3172−3191
(full reference in Supporting Information).
(16) (a) Becke, A. D. Density−functional thermochemistry. III. The
role of exact exchange. J. Chem. Phys. 1993, 98, 5648−5652. (b) Becke,
A. D. A new mixing of Hartree−Fock and local-density-functional
theories. J. Chem. Phys. 1993, 98, 1372−1377. (c) Lee, C.; Yang, W.;
Parr, R. G. Development of the Colle−Salvetti correlation-energy
formula into a functional of the electron density. Phys. Rev. B 1988, 37,
785−789. (d) Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch,
M. J. Ab initio calculation of vibrational absorption and circular
dichroism spectra using density functional force fields. J. Phys. Chem.
1994, 98, 11623−11627.
(17) (a) Zhao, Y.; Truhlar, D. G. The M06 suite of density
functionals for main group thermochemistry, thermochemical kinetics,
noncovalent 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.
(b) Zhao, Y.; Truhlar, D. G. Density Functionals with Broad
Applicability in Chemistry. Acc. Chem. Res. 2008, 41, 157−167.
́
(18) CYLview, 1.0b; Legault, C. Y. Universite de Sherbrooke, 2009
(http://www.cylview.org).
6738
dx.doi.org/10.1021/jm5007885 | J. Med. Chem. 2014, 57, 6729−6738