Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation

Article abstract of DOI:10.1016/j.trsl.2016.10.003

Constipation is a common condition for which current treatments can have limited efficacy. By high-throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride chan

Full text of DOI:10.1016/j.trsl.2016.10.003

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ORIGINAL ARTICLE
Phenylquinoxalinone CFTR activator as potential
prosecretory therapy for constipation
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ONUR CIL, PUAY-WAH PHUAN, JUNG-HO SON, JIE S. ZHU, COLTON K. KU,
NILOUFAR AKHAVAN TABIB, ANDREW P. TEUTHORN, LORETTA FERRERA,
NICHOLAS C. ZACHOS, RUXIAN LIN, LUIS J. V. GALIETTA, MARK DONOWITZ, MARK J. KURTH,
and A. S. VERKMAN
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SAN FRANCISCO, AND DAVIS, CALIF; GENOVA, ITALY; AND BALTIMORE, MD
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Constipation is a common condition for which current treatments can have limited
efficacy. By high-throughput screening, we recently identified a phenylquinoxali-
none activator of the cystic fibrosis transmembrane conductance regulator (CFTR)
chloride channel that stimulated intestinal fluid secretion and normalized stool out-
put in a mouse model of opioid-induced constipation (Cil et al Cell Mol Gastroenterol
Hepatol 2:317-327, 2016). Here, we report phenylquinoxalinone structure-activity
analysis, mechanism of action, animal efficacy data in acute and chronic models
of constipation, and functional data in ex vivo primary cultured human enterocytes.
Structure-activity analysis was done on 175 phenylquinoxalinone analogs, including
15 synthesized compounds. The most potent compound, CFTRact-J027, activated
CFTR with EC50 ꢀ 200 nM, with patch-clamp analysis showing a linear CFTR current-
voltage relationship with direct CFTR activation. CFTRact-J027 corrected reduced
stool output and hydration in a mouse model of acute constipation produced by sco-
polamine and in a chronically constipated mouse strain (C3H/HeJ). Direct compar-
ison with the approved prosecretory drugs lubiprostone and linaclotide showed
substantially greater intestinal fluid secretion with CFTRact-J027, as well as greater ef-
ficacy in a constipation model. As evidence to support efficacy in human constipa-
tion, CFTRact-J027 increased transepithelial fluid transport in enteroids generated
from normal human small intestine. Also, CFTRact-J027 was rapidly metabolized
in vitro in human hepatic microsomes, suggesting minimal systemic exposure
upon oral administration. These data establish structure-activity and mechanistic
data for phenylquinoxalinone CFTR activators, and support their potential efficacy
in human constipation. (Translational Research ;-:1–13)
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Abbreviations: CFTR ¼ cystic fibrosis transmembrane conductance regulator; CIC ¼ chronic idi-
opathic constipation; OIC ¼ opioid-induced constipation; IBS-C ¼ constipation-predominant
irritable bowel syndrome; ppm ¼ parts per million; PBS ¼ phosphate-buffered saline; LC/MS ¼
liquid chromatography mass spectrometry; Ednrb ¼ endothelin receptor B
From the Departments of Medicine and Physiology, University of
California San Francisco, San Francisco, Calif; Department of
Chemistry, University of California, Davis, Calif; Istituto Giannina
Gaslini, Genova, Italy; Departments of Physiology and Medicine,
Gastroenterology Division, Johns Hopkins University School of
Medicine, Baltimore, Md.
Reprint requests: Alan S. Verkman, University of California, 1246
Health Sciences East Tower, San Francisco, CA 94143-0521;
e-mail: Alan.Verkman@ucsf.edu.
1931-5244/$ - see front matter
Ó 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.trsl.2016.10.003
Submitted for publication June 14, 2016; revision submitted
September 9, 2016; accepted for publication October 6, 2016.
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We recently introduced the idea of prosecretory ther-
apy for constipation by direct activation of the cystic fib-
rosis transmembrane conductance regulator Cl2
channel, based on the idea that increasing intestinal fluid
secretion would increase stool hydration and thereby ac-
celerate intestinal transit. Intestinal fluid secretion in-
volves active Cl2 secretion across the enterocyte
epithelium, which is controlled by apical membrane
Cl2 channels, including CFTR and perhaps Ca21-acti-
vated Cl2 channels. CFTR is a compelling target for
constipation therapy as its overactivation by bacterial
enterotoxins in cholera and Traveler’s diarrhea (entero-
toxigenic E. coli) produces marked intestinal fluid se-
cretion.10,11 We recently reported that the
phenylquinoxalinone CFTRact-J027, a small-molecule
CFTR activator identified by high-throughput screen-
ing, increased intestinal fluid secretion and normalized
stool output, stool water content, and intestinal transit
in a mouse model of acute constipation produced by lo-
peramide.12 The compound had no effect in CFTR-
deficient mice, showed no toxicity, and had minimal
systemic exposure following oral administration be-
cause of rapid hepatic metabolism.
AT A GLANCE COMMENTARY
Cil O, et al.
Background
Constipation is a common problem. Despite 2
FDA-approved drugs that promote intestinal fluid
secretion, lubiprostone and linacotide, there re-
mains an unmet need for drugs with better efficacy
and side-effect profile. The cystic fibrosis trans-
membrane conductance regulator (CFTR) is a ma-
jor prosecretory intestinal chloride channel, and
hence a compelling target to promote stool hydra-
tion in constipation.
Translational Significance
Our results support the application of a phenylqui-
noxalinone CFTR activator as first-in-class ther-
apy of opioid and nonopioid constipation. The
compound activates CFTR by direct binding, stim-
ulates intestinal fluid secretion, and, when admin-
istered orally, normalizes stool output and
hydration in experimental animal models of consti-
pation.
Here, for development of phenylquinoxalinones for
therapy of constipation, we studied compound
structure-activity relationships, mechanism of action
by patch-clamp analysis, and animal efficacy in acute
and chronic rodent models of constipation. In addition,
functional studies were done in human enterocytes to
support their potential utility for treatment of constipa-
tion in humans.
INTRODUCTION
Constipation is a common clinical problem affecting
15% of the U.S. population, with annual health care
costs estimated at ꢀ7 billion dollars of which more
than 800 million dollars is spent on laxatives.1 The
most frequent types of constipation include chronic idi-
opathic constipation (CIC), opioid-induced constipation
(OIC), and constipation-predominant irritable bowel
syndrome (IBS-C). Current treatment options include
dietary modification and over-the-counter laxatives in-
cluding agents that increase stool bulk, soften stool, cre-
ate an osmotic load, or stimulate intestinal contraction.2
There are 3 FDA-approved prescription drugs for treat-
ment of various types of constipation: linaclotide, a pep-
tide agonist of the guanylate cyclase C receptor that acts
by inhibiting visceral pain, stimulating intestinal motil-
ity, and increasing intestinal secretion3; lubiprostone, a
prostaglandin E analog that is thought to activate the en-
terocyte ClC-2 channel and perhaps cystic fibrosis
transmembrane conductance regulator (CFTR)4,5; and
naloxegol, a peripherally acting m-opioid receptor
antagonist.6 These FDA-approved drugs generally
showed efficacy in 40%–50% of patients in different
clinical trials albeit with a baseline 25%–35% patient
response to placebo.7-9 Despite the wide range of
therapeutic options, there is a continued need for safe
and effective drugs to treat constipation.
MATERIALS AND METHODS
General chemistry procedures. All chemicals were
purchased from commercial suppliers and used without
further purification. Commercial analogs were pur-
chased from ChemDiv (San Diego, Calif). All other
chemicals were purchased from Sigma-Aldrich (St.
Louis, Mo) unless otherwise stated. Analytical thin
layer chromatography was carried out on precoated
plates (silica gel 60 F254, 250-mm thickness) and
visualized with UV light. Flash chromatography was
ꢀ
performed using 60 A, 32–63 mm silica gel (Fisher
Scientific, Waltham, Mass). Concentration in vacuo
refers to rotary evaporation under reduced pressure.
1H NMR spectra were recorded at 400, 600, or
800 MHz at ambient temperature with acetone-d6,
DMSO-d6, or CDCl3 as solvents. 13C NMR spectra
were recorded at 100, 150, or 200 MHz at ambient
temperature. Chemical shifts are reported in parts per
million (ppm) relative to the residual solvent peak.
High-resolution mass spectra were acquired on an
LTQ Orbitrap XL mass spectrometer equipped with an
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electrospray ionization source (ThermoFisher, San Jose,
Calif), operating in the positive ion mode. Samples were
introduced into the source via loop injection at a flow
rate of 200 mL/min in a solvent system of 1:1
acetonitrile/water with 0.1% formic acid. Mass spectra
were acquired using Xcalibur, version 2.0.7 SP1
(ThermoFinnigan, San Jose, Calif). The spectra were
externally calibrated using the standard calibration
mixture and then calibrated internally to ,2 ppm with
the lock mass tool. Analytical data are reported in
Supplemental Data.
was neutralized with 1-M NaOH solution. The product
4 was extracted with ethyl acetate or dichloromethane
and purified by flash column chromatography.
Synthesis of CFTRact-J133, J142–144 (Path II). Path II/RXN
5: N-(R3-substituted-2-(3-oxo-3,4-dihydroquinoxalin-2-yl)
phenyl)acetamide (5). A solution of 1-acetyl-
(R3-substituted)indoline-2,3-dione (1 mmol) and
o-phenylenediamine (1 mmol) in toluene (10 mL) was
heated at 120ꢁC overnight. The resulting tan
precipitate of 5 was collected by filtration, washed
sequentially with toluene and hexane, and then used in
the next step without further purification.
Synthesis of CFTRact-J102-J105, J109, and J134–J141
(Path I). Path I/RXN 1: N-benzyl-R2-substituted-2-
nitroanilines (1). A stirred solution of R2-substituted-2-
Path II/RXN 6: N-(2-(4-R1-substituted-benzyl-3-oxo-3,4-
dihydroquinoxalin-2-yl)-R3-substituted-phenyl)acetamide
(6). A solution of 5 (0.5 mmol), R1-substituted benzyl
nitroaniline (4 mmol) and benzyl bromide (5 mmol) in
water (8 mL) was sealed in a thick-wall glass tube
(10 mL) and heated at 110ꢁC overnight. The reaction
mixture was cooled to room temperature, diluted with
ethyl acetate, and solid sodium bicarbonate (4 mmol)
was added. The resulting mixture was washed with
water and the organic layer was dried over MgSO4.
After filtration, the organic layer was concentrated in
vacuo, and the final product was purified by silica gel
column chromatography to yield brightly colored
nitroaniline 1.
bromide (0.6 mmol) and K2CO3 (1 mmol) in DMF
(20 mL) was stirred overnight at room temperature.
The reaction mixture was diluted with water and ex-
tracted with ethyl acetate or dichloromethane. The or-
ganic layer was washed sequentially with water and
brine and then dried over MgSO4. Filtration and re-
moval of solvent yielded 6 as a tan product that was
washed with ethanol and used in the deacylation reac-
tion described above without further purification.
Cell culture. Fischer rat thyroid (FRT) cells stably co-
expressing human wild-type CFTR and the halide-
sensitive yellow fluorescent protein -H148Q were as
described.13 Cells were cultured on plastic in Coon’s-
modified Ham’s F12 medium supplemented with 10%
fetal bovine serum, 2-mM L-glutamine, 100 units/mL
penicillin, and 100 mg/mL streptomycin. For
platereader assays, cells were plated in black 96-well
microplates (Corning Costar Corp., New York, N.Y.)
at a density of 20,000 cells per well and assayed 24–
48 hours after plating.
Path
I/RXN
2:
N1-benzyl-R2-substituted-1,2-
diaminobenzene (2). Nitroaniline 1 (5 mmol) was dis-
solved in ethanol (ꢀ100 mL; requires warming) and,
after cooling to room temperature, Zn (50 mmol) and
a 4-M HCl (4 mL) were added to the solution. The mix-
ture was stirred until brightly colored 1 was consumed at
which point the solution was filtered through a pad of
Celite and the solvent was removed in vacuo. The resi-
due was dissolved in ethyl acetate and the mixture was
neutralized with 1-M NaOH. The ethyl acetate solution
was washed with water, dried over MgSO4, filtered, and
concentrated in vacuo. The product was purified by
silica gel column chromatography to yield 2 as a dark-
colored viscous oil.
Plate reader assay of CFTR activity. CFTR activity was
assayed as described.13 Briefly, cells were washed 3
times with phosphate-buffered saline (PBS) and then
incubated for 10 minutes with 60 mL of PBS-
containing test compounds (at 10 mM) and a low
concentration of forskolin (125 nM). I2 influx was
measured in a plate reader by recording fluorescence
continuously (200 ms per point) for 2 seconds
(baseline) and then for 12 seconds after rapid
(,1 second) addition of 165 mL of PBS in which 137-
mM Cl2 was replaced by I2. The initial rate of I2
influx was computed using exponential regression.
Path I/RXN 3: N-(2-(4-benzyl-R2-substituted-3-oxo-3,4-
dihydroquinoxalin-2-yl)-R3-substituted-phenyl)acetamide
(3). A solution of 2 (0.5 mmol) and 1-acetyl-(R3-substi-
tuted)indoline-2,3-dione (0.5 mmol) in glacial acetic
acid (20 mL) was heated at 90ꢁC overnight. Upon cool-
ing to room temperature, the solvent was removed in va-
cuo and the product was washed with ethanol and
filtered to yield 3, which was used without further puri-
fication.
Path I/RXN 4: 3-(2-amino-R3-substituted-phenyl)-1-
Short-circuit current measurement. Short-circuit cur-
rent was measured in FRT cells stably expressing hu-
man wild-type CFTR cultured on porous filters as
described.14 The basolateral solution contained (in
mM): 120 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10
glucose, 25 NaHCO3, and 5 HEPES (pH 7.4, 37ꢁC).
benzyl-R2-substituted-quinoxalin-2(1H)-one (4). To a sol-
ution of 3 (0.2 mmol) in methanol (125 mL) was added
4-M HCl (2.5 mL) and the resulting mixture was heated
at 80ꢁC overnight. On cooling to room temperature, the
solvent was removed in vacuo and the reaction mixture
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In the apical solution, 60-mM NaCl was replaced by Na
gluconate, CaCl2 was increased to 2 mM, and the
basolateral membrane was permeabilized with 250 mg/
mL amphotericin B.
Animals were housed in communal cages in a tempera-
ture- and humidity-controlled environment with 12-
hour light/dark cycle and provided standard rodent
chow and water ad libitum. Wild-type female CD1
mice were bred in the UCSF Laboratory Animal
Resource Center. Female C3H/HeJ mice, which have
a spontaneous mutation in TLR4 (Tlr4lps-d), and their
control background C3H/HeOuJ mice were purchased
from Jackson Laboratories (Bar Harbor, Maine).
Patch-clamp experiments. Whole-cell and inside-out
membrane currents were recorded in FRT cells stably
expressing human wild-type CFTR. For whole-cell
experiments, the extracellular (bath) solution
contained (in mM): 150 NaCl, 1 CaCl2, 1 MgCl2, 10
glucose, 10 mannitol, 10 Na-HEPES (pH 7.4). The
pipette (intracellular) solution contained (in mM): 120
CsCl, 10 TEA-Cl, 0.5 EGTA, 1 MgCl2, 10 Cs-
HEPES, 40 mannitol, and 1 ATP (7.4). For inside-out
patch-clamp experiments, the pipette solution
contained (in mM): 150 N-methyl-D-glucamine
chloride, 3 CaCl2, 2 MgCl2, and 10 Na-Hepes (pH
7.3). The bath solution contained (in mM): 150 N-
methyl-D-glucamine chloride, 2 MgCl2, 10 EGTA, 10
Na-Hepes, 1 ATP (pH 7.3), and 125-nM catalytic
Constipation models in mice. CD1 mice (age 8–
10 weeks) were administered loperamide (0.3 mg/kg)
or scopolamine (0.5 mg/kg) intraperitoneally (ip) to in-
duce constipation. CFTRact-J027 (10 mg/kg, in saline
containing 5% DMSO and 10% Kolliphor HS 15) was
given ip or orally (po) either 1 hour before, at the
same time, or 1 hour after loperamide/scopolamine in
different experiments. Control mice were treated with
vehicle only. Some mice were treated orally with lubi-
prostone (0.5 mg/kg) or linaclotide (0.5 mg/kg, Toronto
Research Chemicals Inc, Toronto, Ontario, Canada). At
designated time points, mice were placed individually
in metabolic cages with food and water provided ad li-
bitum. Stool samples were collected for 3 hours, and to-
tal stool weight and number of fecal pellets were
determined. Stool samples were dried at 80ꢁC for
24 hours and stool water content was calculated as
[wet weight 2 dry weight]/wet weight.
The efficacy of orally administered CFTRact-J027
(10 mg/kg) was also tested in a genetically constipated
mouse strain15 (C3H/HeJ, age 8–11 weeks) and their
wild-type counterparts (C3H/HeOuJ, age 8–11 weeks).
Mice were placed in metabolic cages after CFTRact-
J027 or vehicle administration at zero time and stool
was collected for 4 hours. Stool weight, pellet number,
and water content were determined as described above.
Whole-gut transit time was measured to assess gut motil-
ity, in which mice treated with CFTRact-J027 (10 mg/kg,
ip) or vehicle at zero time were given with 100-mL blue
marker (5% Evans blue and 5% gum Arabic) orally.
The time of blue dye appearance in stool was determined.
All experiments in C3H/HeJ and C3H/HeOuJ mice were
done in paired animals to minimize variability.
subunit of protein kinase
A (PKA) (Promega,
Sunnyvale, Calif). Pipette electrical resistance for both
whole-cell and inside-out experiments was 3–5 MU.
The protocol for stimulation consisted of 600-ms
voltage steps from 2100 to 1100 mV in 20-mV
increments starting from a holding potential of
260 mV. The interval between steps was 4 seconds.
Membrane currents were filtered at 1 kHz and
digitized at 5 kHz. Data were analyzed using the Igor
software (Wavemetrics, Portland, Ore) with custom
software kindly provided by Dr Oscar Moran.
In vitro gastric acid and metabolic stability. To study
gastric acid stability, CFTRact-J027 (10 mM) was dis-
solved in simulated gastric fluid (0.2 NaCl, 0.7% HCl,
pH 2) and incubated for 3 hours at 37ꢁC. To study
in vitro metabolic stability, CFTRact-J027 (5 mM) was
incubated for specified times at 37ꢁC with human liver
microsomes (1-mg protein/mL; Sigma-Aldrich) in
potassium phosphate buffer (100 mM) containing
1-mM NADPH. After specified incubation periods, the
mixtures were chilled on ice, and 0.5 mL of ice-cold
ethyl acetate was added. Samples were centrifuged for
15 minutes at 3000 rpm, the supernatants evaporated
to dryness, and the residues were dissolved in 100-mL
mobile phase (acetonitrile:water, 3:1) and assayed by
liquid chromatography mass spectrometry (LC/MS).
The solvent system consisted of a linear gradient from
5% to 95% acetonitrile over 16 minutes (0.2 mL/min
Closed-loop model of intestinal fluid secretion. Mice
were given access to 5% dextrose water but not solid
food for 24 hours before experiments. Mice were anes-
thetized with isoflurane and body temperature was
maintained during surgery at 36ꢁC–38ꢁC using a heat-
ing pad. A small abdominal incision was made to ex-
pose the small intestine, and closed mid-jejunal loops
(length 2–3 cm) were isolated by sutures. Loops were
injected with 100-mL vehicle alone or 100-mg
CFTRact-J027, lubiprostone, or linaclotide in vehicle.
The abdominal incision was closed with sutures, and
flow). Mass spectra were acquired on
a mass
spectrometer (Waters 2695 and Micromass ZQ,
Milford, Mass) using electrospray (1) ionization, mass
ranging from 100 to 1500 Da, cone voltage 40 V.
Animals. Animal experiments were approved by
UCSF Institutional Animal Care and Use Committee.
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mice were allowed to recover from anesthesia. Intestinal
loops were removed at 90 minutes and loop length and
weight were measured to quantify fluid secretion.
Swelling measurements in human enteroids. Tissues
from human subjects were obtained under approval of
the Johns Hopkins University School of Medicine Insti-
tutional Review Board (protocol NA_00038329). Duo-
denal and jejunal biopsy specimens were obtained from
adults during routine endoscopy at Johns Hopkins Hos-
pital. Crypt isolation, enteroid propagation, and culture
were as described.16 For swelling measurements,
enteroids were seeded in 35-mm dishes with bottom
coverglass with 1.5-mL media. On the day of the
experiment, the media was replaced with 3-mL
Advanced DMEM/F12, and enteroids were incubated
with 1-mM calcein green-acetoxymethyl ester for
1 hour at 37ꢁC to label cytoplasm. Relative enteroid
volume following addition of specified concentrations
of forskolin was measured using a laser scanning
confocal microscope (Fluoview FV10i-LIV; Olympus)
at 37ꢁC, 5% CO2, and 95% relative humidity. In some
studies, CFTRact-J027 was added 10 minutes prior to
forskolin. Images were acquired every 10 minutes and
analyzed with MetaMorph version 7.7 software
(Olympus) to quantify enteroid areas.
information from commercial compounds, synthesis of
targeted analogs was undertaken.
A total of 2 routes were developed to synthesize the
phenylquinoxalinone analogs (Fig 1, A). The Path I
route begins with N-benzylation of the appropriate 2-
nitroaniline (/ 1) and subsequent nitro group reduc-
tion to give 1,2-diamino analog 2. Condensation of
this diamine with the appropriate N-acylisatin in acetic
acid delivers quinoxalin-2(1H)-one 3; attempts to effect
this transformation with nonacylated isatins usually
produced the desired product in low yield. Finally,
acid-catalyzed deacylation of 3 delivered the quinoxa-
lin-2(1H)-one 4. Path II allowed for late-stage introduc-
tion of the N-benzyl moiety, but requires the use of a
symmetrical 1,2-diamine (here, 1,2-diaminobenzene)
so as to avoid regioisomer formation in quinoxalin-
2(1H)-one 5. N1-benzylation with the appropriate ben-
zyl bromide delivered quinoxalin-2(1H)-one 6 and de-
acylation gave the quinoxalin-2(1H)-one 4.
CFTR activity was measured using a plate reader as-
say of iodide influx following extracellular addition of
iodide in FRT cells stably expressing human wild-type
CFTR and a yellow fluorescent protein fluorescent hal-
ide sensor. Fig 1, B shows the concentration-
dependence data of selected compounds, with EC50 val-
ues for all synthesized compounds given in Table I.
Fig 1, C summarizes structure-activity results for the
synthesized compounds reported in Table I (compounds
with J1## designations). In general, having a substitu-
tion group (R1) on the benzyl group, especially in the
Statistical analysis. Experiments with 2 groups were
analyzed with Student’s t-test; when there are 3 or
more groups, analysis was done with one-way analysis
of variance and post-hoc Newman-Keuls multiple
comparisons test. P , 0.05 was taken as statistically
significant.
para- or ortho-positions, reduced activity (CFTRact
-
J102, -J103, -J104, -J143, -J144). Acetylation of the
amino-group on CFTRact-J027, giving CFTRact-J109,
also reduced activity. Analogs with halide substitution
at the 6-position (R2) of the quinoxalinone core ring
(CFTRact-J105, -J135, -J136) gave similar or slightly re-
duced activity compared with CFTRact-J027, and mod-
ifying R2 to a methyl substituent was tolerated
(CFTRact-J141). Changing the 5-nitro on the phenyl
ring of the phenylquinoxalinone to a 5-fluoro, 5-
bromo, or 5-iodo slightly reduced activity (for example,
compare CFTRact-J134 with -J027), while shifting to
the 6-position (compare CFTRact-J134 with -J142)
greatly reduced activity. CFTRact-J135, the most potent
synthesized analog, fully activated CFTR (Fig 1, D), as
the cAMP agonist forskolin produced minimal further
increase in current, with EC50 ꢀ300 nM, which was
comparable to CFTRact-J027. As none of the commer-
cial and synthesized analogs were significantly more
potent than CFTRact-J027, which was already shown
to have favorable pharmacological properties and effi-
cacy in mice,12 subsequent studies were done with
CFTRact-J027.
RESULTS
Synthesis
and
structure-activity
analysis
of
phenylquinoxalinone CFTR activators. Limited structure-
activity relationship information emerged from analysis
of 160 commercially available phenylquinoxalinone
analogs, as most had quite different structures or
multiple substituent modifications in relation to
CFTRact-J027. Table I reports CFTR activity of selected
commercial analogs most closely related to CFTRact-
J027 (J051 - J062). The benzyl (R1) substituent on the
phenylquinoxalinone appeared to be important, as
unsubstituted (CFTRact-J051), methyl (CFTRact-J052),
and phenylacetyl (CFTRact-J058) greatly reduced
activity. Limited substitution (R2 position) on the
quinoxalinone ring showed that a nitro group (CFTRact-
J054) was tolerated. Substituent changes on the phenyl
ring also strongly modulated activity; for example,
changing R3 and R4 substituents to N-benzyl and bromo
(CFTRact-J056) or N-acetyl and unsubstituted (CFTRact-
J057) greatly reduced activity. Because of the limited
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643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
Table I. Cystic fibrosis transmembrane conductance regulator (CFTR) activation by phenylquinoxalinone
analogs
R3
R4
R2
N
N
O
R1
Compound
R1
R2
R3
R4
EC50 (mM)
CFTRact-J027
CFTRact-J051
CFTRact-J052
CFTRact-J053
CFTRact-J054
CFTRact-J055
CFTRact-J056
CFTRact-J057
CFTRact-J058
CFTRact-J059
CFTRact-J060
CFTRact-J061
CFTRact-J062
CFTRact-J102
CFTRact-J103
CFTRact-J104
CFTRact-J109
CFTRact-J105
CFTRact-J135
CFTRact-J136
CFTRact-J133
CFTRact-J134
CFTRact-J140
CFTRact-J141
CFTRact-J143
CFTRact-J144
CFTRact-J142
CFTRact-J139
Bn
H
CH3
CH3
CH3
CH3
Bn
Bn
H
H
H
NH2
NH2
NH2
NH2
NH2
NHMe
NHBn
NHAc
NHAc
NHAc
NHAc
NHAc
H
NH2
NH2
NH2
NHAc
NH2
NH2
NH2
NH2
NH2
5-NO2
5-NO2
5-NO2
H
5-NO2
5-NO2
5-Br
H
H
H
5-CH3
H
5-NHAc
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-F
5-Br
5-F
5-I
5-I
5-I
0.2
.25
ꢀ10
.25
ꢀ10
.25
ꢀ15
ꢀ15
ꢀ20
ꢀ19
.25
ꢀ20
.25
.25
1.2
H
NO2
NO2
H
H
H
H
H
H
H
H
H
H
H
Phenylacetyl
3-BrBn
Bn
Phenethyl
n-propyl
4-BrBn
3-BrBn
2-BrBn
Bn
7.3
1.5
Bn
Bn
Bn
Bn
Bn
Bn
Bn
F
0.53
0.11
0.26
0.79
0.31
0.70
0.47
.25
.25
1.5
Cl
Br
H
H
CH3
CH3
H
H
H
NH2
NH2
NH2
NH2
NH2
NH2
2-NO2Bn
2-CNBn
Bn
6-Br
6-Br
Bn
CH3
0.65
Approximate EC50 of commercial analogs (CFTRact-J051–J062) deduced from 2-concentration point analysis. EC50 of synthesized compounds
measured from full dose-response study.
Patch-clamp electrophysiology shows direct CFTR
activation by CFTRact-J027. Patch-clamp was done to in-
strongly increased CFTR activity (Fig 2, D). CFTRact
-
J027, thus activates CFTR by a direct binding
mechanism, which is consistent with prior data
showing that CFTRact-J027 does not elevate cellular
cAMP concentration.12
CFTRact-J027 efficacy in a scopolamine-induced mouse
model of acute constipation. We previously showed
CFTRact-J027 efficacy in a loperamide-induced mouse
model of constipation.12 To show efficacy in an
alternative, nonopioid mouse model of acute
constipation, CFTRact-J027 was administered orally
1 hour prior to scopolamine. Fig 3, A shows
normalization of stool parameters by CFTRact-J027 in
the scopolamine-treated mice. CFTRact-J027 was also
vestigate the mechanism of CFTR activation by
CFTRact-J027. In whole-cell recordings, CFTR was
partially activated using a low concentration of
forskolin (150 nM) to give ꢀ25% maximal
stimulation. Addition of 1-mM CFTRact-J027 in the
extracellular condition further increased CFTR
activity by more than 4-fold (Fig 2A–C). The currents
activated by forskolin and CFTRact-J027 changed
linearly with applied voltage and were blocked by 10-
mM CFTRinh-172. CFTR activation was also measured
under cell-free conditions using the inside-out patch-
clamp configuration using large pipette tips to obtain
macropatches containing multiple CFTR channels.
After inducing phosphorylation with submaximal ATP
and the catalytic subunit of PKA, CFTRact-J027
effective
after development of constipation in both loperamide
when
administered
intraperitonally
and scopolamine models (Fig 3, B). However,
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Fig 1. Synthesis and structure-activity analysis of phenylquinoxalinone CFTR activators. A, General synthetic
scheme. B, Concentration-dependent activation of CFTR by selected phenylquinoxalinones in FRT cells express-
ing wild-type CFTR (mean 6 S.E.M., n 5 3). Dashed line indicates response to 125-nM forskolin. C, Structural
determinants of phenylquinoxalinone activation of wild-type CFTR. D, Short-circuit current measurement in FRT
cells expressing wild-type CFTR cells showing responses to indicated concentrations of forskolin (fsk), CFTRact
J135, and CFTRinh-172 (representative of 3 experiments).
-
CFTRact-J027 was not effective when given orally after
development of constipation (Fig 3, C), which is not
unexpected as both scopolamine and loperamide delay
gastric emptying and intestinal transit, likely
preventing CFTRact-J027 delivery to its site of action.
affect stool parameters or whole-gut transit time in the
wild-type control mice.
CFTRact-J027 is stable in gastric acid and rapidly
metabolized by human hepatic microsomes. Incubation
of 10-mM CFTRact-J027 in simulated gastric fluid (pH
2) at 37ꢁC for 3 hours showed no compound degradation
(Fig 5, A). In vitro metabolic stability measurements in
human hepatic microsomes revealed rapid compound
metabolism (elimination half-life ꢀ35 minutes), with
only 34% of the compound remaining at 60 minutes
(Fig 5, B).
CFTRact-J027 efficacy in a genetic mouse model of
chronic constipation. CFTRact-J027 was also tested in
a genetic mouse model (C3H/HeJ) of chronic constipa-
tion. C3H/HeJ mice have a spontaneous mutation in
Toll-like receptor 4 gene (Tlr4lps-d), which causes
impaired interactions between gut microbiota and
enteric neurons resulting in reduced enteric neuronal
survival with delayed intestinal transit and decreased
stool output.15 We found that C3H/HeJ mice have
ꢀ30% reduced stool output compared with control
mice in a matched background (C3H/HeOuJ), with
remarkably decreased stool water content and
prolonged whole-gut transit time. Oral administration
of 10 mg/kg CFTRact-J027 increased 4-hours stool
weight, pellet number, and water content in C3H/HeJ
mice to the level of wild-type controls (Fig 4, A).
CFTRact-J027 normalized the increased whole-gut
transit time in C3H/HeJ mice (Fig 4, B), but did not
Greater efficacy of CFTRact-J027 compared with
lubiprostone and linaclotide in increasing intestinal
secretion and normalizing stool parameters in
constipation. The efficacy of CFTRact-J027 in increas-
ing intestinal secretion was compared with that of the
approved prosecretory drugs lubiprostone and linaclo-
tide. In a first set of studies, intestinal fluid accumulation
was measured in a closed intestinal loop model. Closed
mid-jejunal loops were injected with CFTRact-J027 or
test drug, and fluid accumulation was quantified after
90 minutes. Significantly greater intestinal fluid
accumulation was produced by CFTRact-J027
compared to equal doses of lubiprostone or linaclotide
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Fig 2. Patch-clamp analysis of CFTR activation by CFTRact-J027. A, Representative whole-cell patch-clamp in
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FRT cells expressing human wild-type CFTR. Each panel shows superimposed membrane currents elicited at vol-
tages between 2100 and 1 100 mV (with 20 mV steps). Cells were exposed to a submaximal concentration of
forskolin (fsk, 150 nM) with and then to CFTRact-J027 (1 mM) followed by CFTRinh-172 (10 mM). B, Current-
voltage relationships from the experiment in A. C, Membrane conductance deduced from experiments as in B
(mean 6 S.E.M., 4 experiments). D, Currents measured in inside-out patch-clamp experiment. CFTR was acti-
vated by submaximal ATP and catalytic subunit of protein kinase A (PKA), followed by CFTRact-J027 (1 mM).
The voltage stimulation protocol was the same used for whole-cell experiments. Data representative of 3 sets
of experiments.
Q4
(Fig 6, A). In a second set of studies, drug efficacy was
compared in a mouse model of scopolamine-induced
constipation. CFTRact-J027 was more effective in
increasing stool output, pellet number, and water
content compared with supramaximal (250–500 fold
greater than human mg/kg dose) doses of lubiprostone
and linaclotide (Fig 6, B).
responses were measured in enteroids generated from
normal human duodenum and jejunum. The enteroids
comprise a sealed epithelial layer of enterocytes with
CFTR expressed at the inner, luminal membrane, in
which CFTR activation produces a swelling response
over tens of minutes.16,17 Enteroids generated from
human duodenum and jejunum showed a slow
swelling response to 0.1-mM or 0.2-mM forskolin
(Fig 7), which was increased by pretreatment for
10 minutes with CFTRact-J027, with greatest effect
seen for enteroids from jejunum. The response
CFTRact-J027 increases fluid secretion in enteroids
generated from normal human duodenum and
jejunum. To study CFTRact-J027 efficacy in a model
system of direct relevance to human intestine, swelling
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Fig 3. CFTRact-J027 efficacy in mouse models of acute constipation. A, Experimental protocol (left) and 3-hour
stool weight, pellet number, and water content in mice treated with CFTRact-J027 (10 mg/kg, po) or vehicle 1 hour
before scopolamine (0.5 mg/kg, ip) (mean 6 S.E.M., 4 mice per group). B, Experimental protocol (left) and 3-hour
stool weight and pellet number in mice treated with CFTRact-J027 (10 mg/kg, ip) or vehicle 1 hour after scopol-
amine (0.5 mg/kg, ip) or loperamide (0.3 mg/kg, ip) (mean 6 S.E.M., 4 mice per group). C, Experimental protocol
(left) and 3-hour stool weight and pellet number in mice treated with CFTRact-J027 (10 mg/kg, po) or vehicle
1 hour after scopolamine (0.5 mg/kg, ip) or loperamide (0.3 mg/kg, ip) (mean 6 S.E.M., 4 mice per group).
One-way analysis of variance was used for A and B, Student’s t-test was used for C, *P , 0.05, **P , 0.01,
***P , 0.001, ns: not significant.
produced by CFTRact-J027 was approximately 50% of
that produced by maximal (5 mM) forskolin.
colon,18-20 we used C3H/HeJ mice here because
they manifest chronic constipation but with a milder
phenotype and normal survival,15 which make them
more relevant to nonlife-threatening CIC and
IBS-C. We found that CFTRact-J027 was effective
in both pharmacological and genetic models of con-
stipation, which supports its potential use in acute
and various forms of chronic constipation in humans
including CIC, IBS-C, and OIC.
DISCUSSION
CFTRact-J027 showed efficacy in rodent models of
constipation induced by opioid and nonopioid anti-
motility agents, as well as in chronically constipated
C3H/HeJ mice. Opioid and nonopioid antimotility
agents have been widely used to test laxatives in ro-
dents, as these models are technically simple and in-
formative in short-term studies, and the opioid
models have high relevance to OIC in humans. How-
ever, they may have limited relevance to human CIC
and IBS-C, which are chronic conditions. Genetic
models of chronic constipation have more relevance
to human CIC and IBS-C due to their chronic pheno-
type, and do not require pharmacological interven-
tions targeting intestinal motility. Although there
are genetically constipated models of Hirschprung’s
disease with spontaneous mutations in endothelin re-
ceptor B (Ednrb) and targeted mutation in EDNRB
A modular approach was developed for efficient syn-
thesis of phenylquinoxalinone analogs using substituted
nitro-anilines, benzyl bromides, and isatins. The most
potent phenylquinoxalinones have favorable drug-like
properties, including the presence of multiple hydrogen
bond acceptors, average molecular weight of ꢀ400 Da,
aLogP of ꢀ4.0 and topological polar surface areas of
ꢀ2 21,22
ꢀ80 A .
Patch-clamp studies indicated direct activation of
CFTR by CFTRact-J027. CFTR activation by
CFTRact-J027 required a low level of phosphorylation,
as produced by submaximal forskolin in whole-cell re-
cordings and ATP/catalytic subunit of PKA in inside-
out patches, and by a low concentration of forskolin in
ligand endothelin
3 that manifest aganglionic
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Fig 4. CFTRact-J027 reverses constipation in chronically constipated C3H/HeJ mice. A, 4-hour stool weight, pel-
let number, and water content (left) and percent change in these parameters after CFTRact-J027 (10 mg/kg, po) or
vehicle treatment (center) in C3H/HeJ and C3H/HeOuJ mice (mean 6 S.E.M., 10 mice per group). Experimental
protocol is on top right. B, Whole-gut transit time in C3H/HeJ and C3H/HeOuJ mice treated with CFTRact-J027
(10 mg/kg, ip) or vehicle at zero time (mean 6 S.E.M., 5 mice per group). Student’s t-test, *P , 0.05, **P , 0.01,
***P , 0.001, ns: not significant. All experiments were done in paired animals.
enteroid swelling measurements. Basal CFTR phos-
phorylation is required as well for other CFTR activa-
tors, including the clinically approved compound VX-
770, a potentiator of some mutant CFTRs causing cystic
fibrosis.23 The current-voltage relationship in cells
stimulated with CFTRact-J027 was linear, as expected
for CFTR-mediated function. CFTR activation by
CFTRact-J027 in inside-out patches indicates a direct
activation mechanism, likely at a site on the cytoplasmic
domain of CFTR. Further studies are needed by define
the precise binding site of CFTRact-J027 on CFTR,
which are likely to be quite challenging because of the
large size of CFTR and its complex gating mechanism.
Indeed, the site of action of clinically approved poten-
tiators and correctors of mutant CFTRs causing cystic
fibrosis is not known.
greater efficacy of CFTRact-J027 may be due to
greater intestinal secretion. Lubiprostone and linaclo-
tide are thought to act largely by inducing intestinal
fluid secretion, though by different mechanisms.
These compounds activate intestinal secretory path-
ways indirectly by increasing cellular cyclic nucleo-
tide levels through their actions on prostanoid and
guanylate cyclase C receptors, respectively.3,5 The
non-selective activation of cyclic nucleotide path-
ways may be responsible in part for the side effects
of these drugs. Compared with lubiprostone and lina-
clotide, direct-acting CFTR activators have a defined
mechanism of action targeting a single prosecretory
ion transporter, with less likelihood of side effects
due to absence of global cyclic nucleotide eleva-
tion.24 Also, targeting CFTR directly rather than up-
stream receptor or signaling pathways is less likely
to induce tachyphylaxis. Whether the greater thera-
peutic action of CFTRact-J027 compared with lubi-
prostone or linaclotide seen here in mice will
translate to humans must await clinical trial data.
We found greater effect of CFTRact-J027 compared
with lubiprostone or linaclotide in stimulating fluid
secretion in mouse intestine, which translated to
greater efficacy in increasing stool output and hydra-
tion in a scopolamine model of constipation. The
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Fig 5. CFTRact-J027 is rapidly metabolized by human liver microsomes and is gastric acid-stable. A, In vitro
metabolic stability of CFTRact-J027 assayed in human liver microsomes in the presence of NADPH after in-
cubation for specified times, with representative chromatograms on the left. B, Stability of CFTRact-J027 in
simulated gastric fluid (pH 2) after 3 hours incubation, with representative chromatograms on the left
(mean 6 S.E.M., n 5 3).
The preclinical results here support the testing of
CFTRact-J027 or alternative CFTR-targeted activa-
tors in constipation in humans. We previously re-
ported an ED50 of 0.5 mg/kg for orally administered
CFTRact-J027 in a loperamide model in mice, which
translates to a dose of 35 mg for a 70-kg human.
The duration of effect for CFTRact-J027 is at least
3–4 hours in mice, suggesting that a once-a-day ad-
ministration may be adequate for treatment of consti-
pation in humans. Pharmacology studies in mice12
and the rapid metabolism found here with human hep-
atic microsomes (t1/2 35 minutes) predict minimal
systemic exposure following oral administration of
CFTRact-J027 because of rapid hepatic metabolism
probably by a first-pass mechanism, though formal
pharmacokinetics measurements will be needed in
humans. The minimal systemic exposure is expected
to limit the extraintestinal off-target effects of
Fig 6. Relative efficacy of CFTRact-J027 with lubiprostone and linaclotide in increasing intestinal fluid se-
cretion and stool output. A, Intestinal fluid secretion was measured in closed midjejunal loops in mice. Loops
were injected with 100-mL vehicle or 100-mg CFTRact-J027, lubiprostone, or linaclotide. Loop weight/length
was measured at 90 minutes (mean 6 S.E.M., 4–8 loops per group, representative photos on right). B, 3-hours
stool weight, pellet number, and water content in mice orally treated with CFTRact-J027 (10 mg/kg), lubipro-
stone (0.5 mg/kg), linaclotide (0.5 mg/kg), or vehicle in a scopolamine model of constipation as done in Fig 3,
A (mean 6 S.E.M., 4 mice per group). One-way analysis of variance, *P , 0.05, **P , 0.01, and
***P , 0.001.
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Fig 7. CFTRact-J027 produces swelling in enteroids generated from human small intestine. Percentage increase in
Q6
enteroid area, as deduced by confocal fluorescence microscopy of calcein-stained enteroids, relative to initial area.
Data shown for enteroids from human jejunum (left) and duodenum (right). Mean 6 S.E.M., n $ 10 enteroids for
each condition.
CFTRact-J027, as evidenced by our prior toxicity
studies in mice showing no effect of chronic oral
high-dose CFTRact-J027 on blood counts, serum
chemistries, and lung water content.12 A potential
side effect of any laxative is diarrhea, which might oc-
cur with CFTRact-J027 in a dose-dependent manner
and require appropriate dose adjustment.
from the Cystic Fibrosis Foundation, a Catalyst grant from
funds awarded to the UCSF CTSI (UL1 TR000004), a
Gates Foundation Grand Challenges Grant, and grants
from Ministero della Salute (Ricerca Corrente: Cinque Q3
per mille).
REFERENCES
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small molecule increases intestinal fluid secretion and is
efficacious orally in mouse models of acute and chronic
constipation. CFTRact-J027 induced enterocyte fluid se-
cretion in human enteroids and showed rapid metabo-
lism in human hepatic microsomes, supporting its
utility for human constipation.
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ACKNOWLEDGMENTS
Conflicts of Interest: O. Cil and A. S. Verkman are
named inventors on provisional patent filings, with
rights owned by the University of California, San Fran-
cisco. The other authors have nothing to declare.
This work was supported by grants DK099803,
DK72517, DK101373, DK35124, EB00415, EY13574,
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Appendix
Analytical data for synthesized 1-benzyl-3-
phenylquinoxalin-2(1H)-one analogs
3-(2-Amino-5-nitrophenyl)-1-(2-bromobenzyl)qui-
1H
noxalin-2(1H)-one
(CFTRact-J104).
NMR
(800 MHz, DMSO-d6) d 9.11 (d, J 5 2.8 Hz, 1H),
8.05 (dd, J 5 9.2, 2.8 Hz, 1H), 8.02 (dd, J 5 8.0,
1.5 Hz, 1H), 7.83 (s, 2H), 7.79–7.69 (m, 1H), 7.58
(ddd, J 5 8.6, 7.2, 1.5 Hz, 1H), 7.43 (ddd, J 5 8.2,
7.2, 1.2 Hz, 1H), 7.32–7.22 (m, 2H), 7.19 (dd,
J 5 8.5, 1.2 Hz, 1H), 6.98–6.88 (m, 2H), 5.48 (s, 2H).
13C NMR (201 MHz, DMSO) d 155.00, 154.50,
153.41, 135.34, 134.37, 133.25, 132.81, 132.31,
131.32, 130.08, 129.80, 129.53, 128.56, 127.68,
126.89, 124.32, 122.41, 116.10, 115.96, 115.00,
3-(2-Amino-5-nitrophenyl)-1-(4-bromobenzyl)qui-
(CFTRact-J102). 1H
noxalin-2(1H)-one
NMR
(800 MHz, DMSO-d6) d 9.13 (d, J 5 2.8 Hz, 1H),
8.05 (dd, J 5 9.2, 2.8 Hz, 1H), 7.97 (dd, J 5 7.9,
1.5 Hz, 1H), 7.80 (s, 2H), 7.57 (ddd, J 5 8.6, 7.2,
1.5 Hz, 1H), 7.54–7.48 (m, 2H), 7.44 (dd, J 5 8.5,
1.2 Hz, 1H), 7.40 (ddd, J 5 8.2, 7.2, 1.2 Hz, 1H), 7.34
(d, J 5 8.5 Hz, 2H), 6.91 (d, J 5 9.2 Hz, 1H), 5.55 (s,
2H). 13C NMR (201 MHz, DMSO) d 154.99, 154.61,
153.42, 135.85, 135.34, 132.70, 132.27, 131.97,
131.12, 130.03, 129.76, 129.53, 126.87, 124.20,
120.88, 116.16, 115.95, 115.33, 45.47. HRMS
47.07. HRMS [C21H15BrN4O3
451.0406/found 451.0401.
1
H]1: calcd
[C21H15BrN4O3
451.0417.
1
H]1: calcd 451.0406/found
3-(2-Amino-5-nitrophenyl)-1-(3-bromobenzyl)quinoxa-
lin-2(1H)-one (CFTRact-J103). 1H NMR (600 MHz,
DMSO-d6) d 9.09 (d, J 5 2.8 Hz, 1H), 8.03 (dd,
J 5 9.2, 2.8 Hz, 1H), 7.95 (dd, J 5 7.9, 1.5 Hz, 1H),
7.77 (s, 2H), 7.62 (s, 1H), 7.56 (ddd, J 5 8.6, 7.2,
1.6 Hz, 1H), 7.48–7.40 (m, 2H), 7.38 (dd, J 5 15.2,
1.1 Hz, 1H), 7.32 (dt, J 5 7.8, 1.3 Hz, 1H), 7.27 (t,
J 5 7.8 Hz, 1H), 6.88 (d, J 5 9.2 Hz, 1H), 5.54 (s, 2H).
13C NMR (151 MHz, dmso) d 154.98, 154.64, 153.50,
139.18, 135.42, 132.76, 132.29, 131.21, 131.08, 130.71,
130.18, 129.99, 129.49, 126.79, 126.42, 124.16, 122.40,
3-(2-Amino-5-nitrophenyl)-1-benzyl-6-fluoroquin
oxalin-2(1H)-one (CFTRact-J105). NMR
1H
(600 MHz, DMSO-d6) d 9.24 (d, J 5 2.7 Hz, 1H),
8.02 (dd, J 5 9.2, 2.8 Hz, 1H), 7.91 (s, 2H), 7.86 (d,
J 5 8.9 Hz, 1H), 7.50–7.37 (m, 2H), 7.32 (s, 4H),
7.24 (t, J 5 6.8 Hz, 1H), 6.90 (d, J 5 9.2 Hz, 1H),
5.55 (s, 2H). 13C NMR (151 MHz, dmso) d 158.44 (d,
JC-F 5 242 Hz), 157.64, 155.15, 154.42, 154.34,
136.13, 132.77 (br), 129.74, 129.63, 129.11, 127.77,
127.33, 126.95, 118.53 (d, JC-F 5 22 Hz), 116.92 (br),
116.22, 115.46, 115.01 (d, JC-F 5 21 Hz), 46.25.
HRMS [C21H15FN4O3 1 H]1: calcd 391.1206/found
391.1206.
116.24,
115.92,
115.24,
45.54.
HRMS
[C21H15BrN4O3 1 H]1: calcd 451.0406/found 451.0404.
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3-(2-Amino-5-bromophenyl)-1-benzylquinoxalin-
2(1H)-one (CFTRact-J134). 1H NMR (600 MHz,
Chloroform-d) d 8.43 (d, J 5 2.4 Hz, 1H), 7.87–7.79
(m, 1H), 7.54–7.42 (m, 1H), 7.36–7.26 (m, 7H), 6.68
(d, J 5 8.6 Hz, 2H), 5.60 (s, 2H), 5.57 (s, 2H). 13C
NMR (101 MHz, CDCl3) d 154.62, 153.89, 146.69,
135.14, 134.20, 134.00, 132.43, 132.33, 130.45,
129.75, 128.99, 127.78, 126.93, 123.97, 120.52,
N-(2-(4-Benzyl-3-oxo-3,4-dihydroquinoxalin-2-yl)-
4-nitrophenyl)-acetamide (CFTRact-J109). 1H NMR
(800 MHz, DMSO-d6) d 10.27 (s, 1H), 8.57 (d,
J 5 2.8 Hz, 1H), 8.21 (d, J 5 9.2 Hz, 1H), 7.94 (dd,
J 5 7.9, 1.5 Hz, 1H), 7.61 (ddd, J 5 8.7, 7.1, 1.6 Hz,
1H), 7.51–7.45 (m, 1H), 7.45–7.39 (m, 3H), 7.35 (q,
J 5 7.7, 7.0 Hz, 3H), 7.28 (t, J 5 7.4 Hz, 1H), 5.55
(s, 2H), 2.01 (s, 3H). 13C NMR (201 MHz, DMSO) d
169.26, 154.72, 154.30, 143.54, 142.57, 136.23,
133.47, 133.14, 131.44, 130.30, 129.04, 128.13,
127.79, 127.47, 127.16, 125.50, 124.15, 122.90,
115.55, 45.80, 24.42. HRMS [C23H18N4O4 1 H]1:
calcd 415.1401/found 415.1389.
118.78, 114.46,
[C21H16BrN3O 1
406.0548.
108.76,
46.47.
HRMS
H]1: calcd 406.0555/found
3-(2-Amino-5-nitrophenyl)-1-benzyl-6-chloroquino
xalin-2(1H)-one (CFTRact-J135). NMR
1H
3-(2-Amino-5-fluorophenyl)-1-benzylquinoxalin-
2(1H)-one (CFTRact-J133). 1H NMR (600 MHz,
Chloroform-d) d 8.05 (dd, J 5 10.7, 3.0 Hz, 1H), 7.85
(dd, J 5 7.9, 1.5 Hz, 1H), 7.45 (td, J 5 8.6, 7.9,
1.6 Hz, 1H), 7.38–7.25 (m, 7H), 6.99 (ddd, J 5 8.7,
7.5, 3.0 Hz, 1H), 6.73 (dd, J 5 8.9, 4.8 Hz, 1H), 5.57
(s, 2H), 5.42 (s, 2H). 13C NMR (151 MHz, cdcl3) d
154.90 (d, JC-F 5 234 Hz), 154.60, 143.97, 135.22,
132.48, 132.37, 130.30, 129.73, 128.92, 127.71,
126.95, 123.84, 119.61 (br), 118.57 (d, JC-F 5 23 Hz),
118.04, 117.99, 117.79 (d, JC-F 5 24 Hz), 114.39,
46.41. HRMS [C21H16FN3O 1 H]1: calcd 346.1356/
found 346.1371.
(600 MHz, DMSO-d6) d 9.25 (d, J 5 2.7 Hz, 1H),
8.09 (d, J 5 2.4 Hz, 1H), 8.02 (dd, J 5 9.3, 2.8 Hz,
1H), 7.93 (s, 2H), 7.56 (dd, J 5 9.1, 2.4 Hz, 1H), 7.42
(d, J 5 9.1 Hz, 1H), 7.31 (d, J 5 4.4 Hz, 4H), 7.25
(td, J 5 5.3, 2.7 Hz, 1H), 6.90 (d, J 5 9.3 Hz, 1H),
5.54 (s, 2H). 13C NMR (151 MHz, dmso) d 155.18,
154.39, 154.32, 136.01, 135.37, 132.85, 131.71,
130.47, 129.77, 129.11, 128.83, 128.01, 127.79,
127.30, 126.98, 117.05, 116.27, 115.26, 46.21. HRMS
[C21H15ClN4O3
407.0908.
1
H]1: calcd 407.0911/found
FLA 5.4.0 DTD ꢀ TRSL1105_proof ꢀ 31 October 2016 ꢀ 8:26 pm ꢀ ce

Translational Research
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3-(2-Amino-5-fluorophenyl)-1-benzyl-6-methylquin
1H
3-(2-Amino-5-nitrophenyl)-1-benzyl-6-bromoquin
oxalin-2(1H)-one (CFTRact-J136). NMR
1H
oxalin-2(1H)-one
(CFTRact-J140).
NMR
(600 MHz, Chloroform-d) d 8.04 (dd, J 5 10.7,
3.0 Hz, 1H), 7.67–7.63 (m, 1H), 7.35–7.29 (m, 2H),
7.31–7.23 (m, 4H), 7.19 (d, J 5 8.6 Hz, 1H), 6.98
(ddd, J 5 8.8, 7.6, 3.0 Hz, 1H), 6.72 (dd, J 5 8.9,
4.9 Hz, 1H), 5.55 (s, 2H), 5.41 (s, 2H), 2.42 (s, 3H).
13C NMR (101 MHz, CDCl3) d 154.94 (d, JC-
5 235 Hz), 154.55, 154.08, 143.91, 135.32, 133.82,
132.33, 131.69, 130.26, 129.55, 128.94, 127.71,
126.98, 119.85 (d, JC-F 5 7 Hz), 118.50 (d, JC-
(600 MHz, DMSO-d6) d 9.23 (d, J 5 2.8 Hz, 1H),
8.25 (d, J 5 2.3 Hz, 1H), 8.03 (dd, J 5 9.3, 2.7 Hz,
1H), 7.90 (s, 2H), 7.68 (dd, J 5 9.0, 2.4 Hz, 1H), 7.37
(d, J 5 9.1 Hz, 2H), 7.31 (d, J 5 4.4 Hz, 4H), 6.91
(d, J 5 9.3 Hz, 1H), 5.54 (s, 2H). 13C NMR
(101 MHz, DMSO) d 155.20, 154.44, 154.32, 136.03,
135.26, 133.23, 132.12, 131.88, 129.83, 129.15,
127.82, 127.31, 127.06, 117.80, 117.39, 116.26,
F
115.74,
[C21H15BrN4O3
451.0398.
115.30,
1
46.14.
HRMS
H]1: calcd 451.0406/found
5 23 Hz), 118.05 (d, JC-F 5 7 Hz), 117.82 (d, JC-
F
5
24 Hz), 114.20, 46.39, 20.64. HRMS
F
[C22H18FN3O 1 H]1: calcd 360.1512/found 360.1505.
3-(2-Amino-6-bromophenyl)-1-benzyl-6-methylquin
oxalin-2(1H)-one (CFTRact-J139). NMR
1H
3-(2-Amino-5-iodophenyl)-1-benzyl-6-methylquin
1H
(800 MHz, Chloroform-d) d 8.24 (d, J 5 8.6 Hz, 1H),
7.65 (dd, J 5 2.1, 1.0 Hz, 1H), 7.37–7.32 (m, 2H),
7.32–7.28 (m, 4H), 7.20 (d, J 5 8.5 Hz, 1H), 6.98 (d,
J 5 2.0 Hz, 1H), 6.93 (dd, J 5 8.6, 2.0 Hz, 1H), 5.78
(s, 2H), 5.57 (s, 2H), 2.45 (d, J 5 0.7 Hz, 3H). 13C
NMR (201 MHz, CDCl3) d 154.65, 154.20, 148.91,
135.34, 133.79, 133.44, 132.25, 131.49, 130.12,
129.35, 128.93, 127.70, 126.96, 125.39, 120.03,
119.46, 117.67, 114.19, 46.41, 20.65. HRMS
oxalin-2(1H)-one
(CFTRact-J141).
NMR
(600 MHz, Chloroform-d) d 8.20 (dd, J 5 7.9, 1.5 Hz,
1H), 7.65 (s, 1H), 7.34–7.26 (m, 4H), 7.28–7.24 (m,
3H), 7.27–7.20 (m, 1H), 7.18 (dd, J 5 8.6, 1.2 Hz,
1H), 6.85–6.77 (m, 1H), 6.57 (dd, J 5 8.6, 0.9 Hz,
1H), 5.55 (s, 2H), 2.42 (s, 3H). 13C NMR (101 MHz,
CDCl3) d 154.52, 140.03, 139.54, 135.48, 135.25,
133.84, 133.63, 131.97, 131.27, 129.50, 128.93,
128.90, 127.68, 127.01, 126.90, 119.25, 117.29,
114.18, 46.36, 20.64. HRMS [C22H18IN3O 1 H]1:
calcd 468.0573/found 468.0567.
[C22H18BrN3O
420.0704.
1
H]1: calcd 420.0711/found
FLA 5.4.0 DTD ꢀ TRSL1105_proof ꢀ 31 October 2016 ꢀ 8:26 pm ꢀ ce

Translational Research
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(m, 1H), 7.89 (dq, J 5 8.1, 1.7 Hz, 1H), 7.52–7.39
(m, 4H), 7.41–7.33 (m, 1H), 7.04 (dq, J 5 8.4,
1.5 Hz, 1H), 6.95–6.87 (m, 1H), 6.57 (dd, J 5 8.7,
1.4 Hz, 1H), 5.95 (s, 2H), 5.75 (s, 2H). 13C NMR
(151 MHz, cdcl3) d 154.47, 153.57, 147.50, 140.08,
139.82, 134.17, 132.26, 132.11, 130.64, 130.61,
129.84, 129.75, 128.53, 127.10, 125.78, 124.32,
119.23, 117.09, 113.89, 113.79, 44.61. HRMS
[C21H15IN4O3 1 H]1: calcd 499.0267/found 499.0251.
3-(2-Amino-6-bromophenyl)-1-benzylquinoxalin-
2(1H)-one (CFTRact-J142). 1H NMR (600 MHz,
Chloroform-d) d 8.21 (d, J 5 8.6 Hz, 1H), 7.82 (dd,
J 5 8.0, 1.5 Hz, 1H), 7.46–7.41 (m, 1H), 7.36–7.21
(m, 7H), 6.96–6.88 (m, 2H), 5.77 (s, 2H), 5.56 (s,
2H). 13C NMR (101 MHz, CDCl3) d 154.73, 154.28,
148.95, 135.22, 133.44, 132.32, 130.22, 129.56,
128.97, 128.89, 127.76, 126.96, 125.53, 123.93,
120.03, 119.48, 117.46, 114.44, 46.47. HRMS
[C21H16BrN3O
406.0550.
1
H]1: calcd 406.0555/found
3-(2-Amino-5-iodophenyl)-1-(2-cyanobenzyl)qui-
noxalin-2(1H)-one
(CFTRact-J144).
1H
NMR
(600 MHz, Chloroform-d) 1H NMR (600 MHz, Chloro-
form-d) d 8.59 (s, 1H), 8.23 (d, J 5 6 Hz, 1H), 7.87 (ddd,
J 5 8.1, 3.9, 1.5 Hz, 1H), 7.81 (dd, J 5 8.0, 1.5 Hz, 1H),
7.53–7.42 (m, 2H), 7.37 (dq, J 5 23.7, 8.3, 7.8 Hz, 2H),
7.28–7.19 (m, 1H), 7.09 (dq, J 5 24.8, 8.7, 7.9 Hz, 2H),
6.90–6.77 (m, 1H), 6.57 (d, J 5 8.6 Hz, 1H), 5.77 (d,
J 5 2.2 Hz, 2H). 13C NMR (151 MHz, cdcl3) d
154.56, 153.56, 147.43, 140.04, 139.81, 138.68,
133.49, 133.22, 132.28, 131.88, 130.76, 130.65,
129.87, 128.75, 128.20, 126.78, 124.31, 119.23,
3-(2-Amino-5-iodophenyl)-1-(2-nitrobenzyl)quinox-
1
alin-2(1H)-one (CFTRact-J143). H NMR (600 MHz,
Chloroform-d) d 8.59 (t, J 5 1.9 Hz, 1H), 8.27–8.19
116.89,
113.84,
111.25,
44.59.
HRMS
[C22H15IN4O 1 H]1: calcd 479.0369/found 479.0362.
FLA 5.4.0 DTD ꢀ TRSL1105_proof ꢀ 31 October 2016 ꢀ 8:26 pm ꢀ ce