Nanomolar potency pyrimido-pyrrolo-quinoxalinedione CFTR inhibitor reduces cyst size in a polycystic kidney disease model

Article abstract of DOI:10.1021/jm9009873

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are predicted to slow cyst enlargement in polycystic kidney disease and reduce intestinal fluid loss in secretory diarrheas. Screening of ～110000 small synthetic and natural compounds for inhibition of halide influx in CFTR-expressing epithelial cells yielded a new class of pyrimido-pyrrolo-quinoxalinedione (PPQ) CFTR inhibitors. Testing of 347 analogues established structure-activity relationships. The most potent compound, 7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido- [4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione, PPQ-102, completely inhibited CFTR chloride current with IC₅₀ ～90 nM. The PPQs, unlike prior CFTR inhibitors, are uncharged at physiological pH, and therefore not subject to membrane potential-dependent cellular partitioning or block efficiency. Patch-clamp analysis confirmed voltage-independent CFTR inhibition by PPQ-102 and showed stabilization of the channel closed state. PPQ-102 prevented cyst expansion and reduced the size of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease. PPQ-102 is the most potent CFTR inhibitor identified to date. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9009873

J. Med. Chem. 2009, 52, 6447–6455 6447
DOI: 10.1021/jm9009873
Nanomolar Potency Pyrimido-pyrrolo-quinoxalinedione CFTR Inhibitor Reduces Cyst Size in a
Polycystic Kidney Disease Model
Lukmanee Tradtrantip, N. D. Sonawane, Wan Namkung, and A. S. Verkman*
Departments of Medicine and Physiology, University of California, San Francisco, California 94143-0521
Received July 3, 2009
Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are
predicted to slow cyst enlargement in polycystic kidney disease and reduce intestinal fluid loss in
secretory diarrheas. Screening of ∼110000 small synthetic and natural compounds for inhibition of
halide influx in CFTR-expressing epithelial cells yielded a new class of pyrimido-pyrrolo-quinoxaline-
dione (PPQ) CFTR inhibitors. Testing of 347 analogues established structure-activity relationships.
The most potent compound, 7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido-
[40,50-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione, PPQ-102, completely inhibited CFTR chlor-
ide current with IC50 ∼90 nM. The PPQs, unlike prior CFTR inhibitors, are uncharged at physiological
pH, and therefore not subject to membrane potential-dependent cellular partitioning or block
efficiency. Patch-clamp analysis confirmed voltage-independent CFTR inhibition by PPQ-102 and
showed stabilization of the channel closed state. PPQ-102 prevented cyst expansion and reduced the size
of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease. PPQ-102 is the
most potent CFTR inhibitor identified to date.
Introduction
CFTRinh-172 (Figure 1A) acts from the cytoplasmic side of
the plasma membrane to block CFTR chloride conductance
with IC50 ∼ 0.3-3 μM depending on cell type and membrane
potential.16 Patch-clamp analysis indicated a voltage-inde-
pendent channel block mechanism in which CFTRinh-172
stabilizes the channel closed state;17 CFTR mutagenesis sug-
gested CFTRinh-172 interaction at arginine-347 located near
the cytoplasmic entrance of the CFTR pore.18 CFTRinh-172
has low toxicity, undergoes renal excretion with minimal
metabolism, and accumulates in the intestine by enterohepatic
recirculation.19 A second compound class, the glycine hydrazides
(GlyH-101, Figure 1A), inhibit CFTR with IC50 ∼ 5 μM.20
Patch-clamp analysis showed inward rectifying chloride cur-
rent following GlyH-101 application with rapid channel
flicker, indicating an external pore occlusion mechanism.
Nonabsorbable conjugates of glycine hydrazides with poly-
ethylene glycols21,22 and lectins4 inhibited CFTR when added
at the mucosal surface and had improved potency compared
to GlyH-101. Cell culture and animal models provided proof-
of-concept for the potential utility of thiazolidinones
and glycine hydrazides in secretory diarrheas3,16,19-22 and
PKD.23
Although CFTRinh-172 has been used extensively to
block CFTR chloride conductance in cells and tissues, its
low aqueous solubility is a potential concern, as is its
membrane-potential dependent partitioning across cell
membranes, which reduces its potency in cells because of
their interior negative membrane potential. Thiazolidinone
analogues with improved water solubility were synthe-
sized,24 although they had reduced CFTR inhibition po-
tency compared to CFTRinh-172 and retained the negative
charge that reduces their accumulation in cytoplasm.
The glycine hydrazides, including their macromolecular
The cystic fibrosis transmembrane conductance regulator
(CFTRa) gene encodes a cAMP-regulated chloride channel
expressed in epithelial cells in the airways, intestine, testis, and
other tissues.1,2 Mutations in CFTR cause the hereditary
disease cystic fibrosis. CFTR is excessively active in entero-
toxin-mediated secretory diarrheas such as cholera and tra-
veler’s diarrhea. CFTR inhibitors are predicted to reduce
intestinal fluid loss in secretory diarrheas,3-5 as well as slow
renal cyst expansion in polycystic kidney disease (PKD),
where fluid accumulation in renal cysts is CFTR-dependent.6-13
There is interest in the identification of CFTR inhibitors for these
clinical indications and as research tools to study disease patho-
genesis in cystic fibrosis by blocking CFTR function in ex vivo
human tissues and animal models.
Older CFTR inhibitors, including glibenclamide, dipheny-
lamine-2-carboxylate (DPC), and 5-nitro-2-(3-phenylpropyl-
amino)benzoate (NPPB) (Figure 1A) are nonselective in their
action and have low potency. One study reported strong
CFTR inhibition by R-aminoazaheterocyclic-methylglyoxal
adducts,14 although CFTR inhibition was not subsequently
confirmed.15 We identified two classes of improved CFTR
inhibitors by high-throughput screening. The thiazolidinone
*To whom correspondence should be addressed. Phone: 415-
4768530. Fax: 415-6653847. E-mail: alan.verkman@ucsf.edu. Website:
http://www.ucsf.edu/verklab. Address: University of California, San
Francisco, Box 0521, HSE 1246, 513 Parnassus Avenue, San Francisco,
CA 94143-0521.
a Abbreviations: ADPKD, autosomal dominant polycystic kidney
disease; CFTR, cystic fibrosis transmembrane conductance regulator;
CPT-cAMP, chlorophenylthio-cAMP; IBMX, isobutylmethylxanthine,
PKD, polycystic kidney disease; PPQ, pyrimido-pyrrolo-quinoxaline-
dione; YFP, yellow fluorescent protein.
r
2009 American Chemical Society
Published on Web 09/28/2009
pubs.acs.org/jmc

6448 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Tradtrantip et al.
Figure 1. Discovery of pyrimido-pyrrolo-quinoxalinedione (PPQ) CFTR inhibitors. (A) Chemical structures of CFTR inhibitors. Shown are
the original CFTR inhibitors DPC, NPPB, and glibenclamide, the thiazolidinone CFTRinh-172, the glycine hydrazide GlyH-101, and new
compound PPQ-102. (B) Representative screening data. Cell-based screening done in 96-well plates containing FRT cells expressing human
CFTR and the YFP halide sensor YFP-H148Q/I152L. CFTR was maximally stimulated by an agonist mixture and CFTR-mediated iodide
influx was measured as YFP fluorescence quenching. Fluorescence data from individual wells shown in the absence of agonists and in the
presence of agonists for the negative control (DMSO vehicle alone), positive control (10 μM CFTRinh-172), and test compounds (at 25 μM).
conjugates, also suffer from reduced CFTR inhibition
potency at physiological interior-negative membrane po-
tentials, but for a different reason. Glycine hydrazides
produce strongly inwardly rectifying CFTR currents, with
reduced inhibition potency at interior-negative membrane
potential as a consequence of their electrostatic expulsion
from the CFTR pore.20,22 Here, we screened synthetic and
natural small molecules to identify new classes of CFTR
inhibitors. We report here the discovery, structure-activity
analysis, and characterization of pyrimido-pyrrolo-quinox-
alinediones (PPQs), which are the first uncharged, and thus
membrane-potential insensitive, CFTR inhibitors, and are
the most potent small-molecule CFTR inhibitors identified
to date.
Figure 1B shows examples of YFP fluorescence data in
negative control (vehicle-only) and positive control (10 μM
CFTRinh-172) wells, and wells containing test compounds,
showing data for two active compounds. Of 54 compounds
giving >50% CFTR inhibition at 25 μM, rescreening and
electrophysiological measurements indicated three com-
pounds with >50% CFTR inhibition at 5 μM. Several active
compounds were related to previously identified CFTR in-
hibitors, and a new class of pyrimido-pyrrolo-quinoxaline-
dione (PPQ) compounds was identified. The structure of the
PPQ analogue with greatest CFTR inhibition potency, PPQ-
102, is shown in Figure 1A. The PPQs are not related
structurally to known CFTR or ion channel inhibitors, and
unlike prior CFTR inhibitors they are uncharged at physio-
logical pH.
Results
Structure-activity analysis was done to identify the most
potent PPQ-class CFTR inhibitors for further characteriza-
tion and biological testing. Of 347 commercially available
PPQ analogues screened using the fluorescence platereader
assay, 54 compounds inhibited CFTR-mediated iodide influx
by>50%at 25μM (structuresandactivity datafor analogues
provided in Supporting Information). Table 1 summarizes
CFTR inhibition data for some compounds. Apparent IC50
values obtained using the fluorescence platereader assay are
semiquantitative because of the dilution procedure used but
useful for comparative purposes. Figure 2A summarizes the
structural determinants for CFTR inhibition deduced from
the functional data. Analogues containing a 5-methyl furanyl
moiety (PPQ-101 to PPQ-105) showed greater inhibition
potencies than unsubstituted furanyl and thiophene analogues
(group 1, Table 1). Compounds containing other heterocycles
Screening of collections of synthetic and natural com-
pounds was carried out to identify new classes of CFTR
inhibitors with improved properties over known inhibitors.
A cell-based fluorescence assay was used in which CFTR
inhibitors wereidentified by reduced iodide influx in FRTcells
coexpressinghuman CFTR and a YFP halidesensor, inwhich
CFTR was maximally activated by a mixture of agonists
having different activating mechanisms. Inhibition of iodide
influx was seen as reduced YFP fluorescence quenching in
response to rapid iodide addition to each well of 96-well
plates. On the basis ofprior knowledge of the small percentage
of active CFTR inhibitors identified from screening of ran-
dom compounds, primary screening was done at 25 μM
concentration with test compounds preincubated for 15 min
prior to assay.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6449
Table 1. CFTR Inhibition by PPQ Analoguesa
a IC50app is an apparent IC50 determined from concentration-inhibition data from the fluorescence platereader assay.
such as 1H-benzimidazole-2-yl and chromenone in place of
furan were inactive. Analogues containing phenyls, PPQ-201
to PPQ-203, PPQ-209, and PPQ-210 (group 2, Table 1) were
mildly less potent than the furan analogues PPQ-101 and
PPQ-102. All active analogues contained a phenyl ring
(substituted or unsubstituted) at the 2-position of pyrrole.
Methyl substituents on this phenyl ring increased CFTR
inhibition potency, as seen in PPQ-101, 102, 103, and PPQ-
215, 202, 213. Aromatization of PPQ-102 to PPQ-102b
(Figure 2B), which removes the stereocenter resulting in a
planar structure, abolished CFTR inhibition activity.
The most potent CFTR inhibitor confirmed by electrophy-
siological testing, PPQ-102, was resynthesized, confirmed,
and further characterized. Synthesis of PPQ-102 was achieved
in six steps (Figure 2B). Commercially available 6-methylur-
acil 1 was methylated using dimethylsulfate to produce 1,3,6-
trimethyluracil 2, which upon Friedel-Crafts acylation using
zinc chloride as a catalyst yielded 5-benzoyl-1,3,6-trimethyl-
pyrimidine-2,4(1H,3H)-dione 3 as a white powder. Bromina-
tion of 3 followed by reaction with N-(2-aminophenyl)-
acetamide generated amino-protected intermediate 5. The
acetamido function of 5 was hydrolyzed, and the resultant
intermediate 6 was cyclocondensed with 5-methylfurfural to
yield yellowish-white racemic PPQ-102. Aromatic com-
pound PPQ-102b, which lacks a stereocenter, was prepared
from PPQ-102 by oxidation with potassium permanganate.
Short-circuit current analysis of CFTR chloride conduc-
tance was done to study compound potency, reversibility, and
specificity. Figure 3A (left) shows PPQ-102 inhibition of
chloride current in CFTR-expressing FRT cells following
CFTR stimulation by the cAMP agonist CPT-cAMP. Mea-
surements were done in the presence of a transepithelial
chloride gradient and following basolateral membrane per-
meabilization with amphotericin B, so that measured current
is a direct, quantitative measure of CFTR chloride conduc-
tance. PPQ-102 inhibition of CFTR was approximately 100%
at higher concentrations, with IC50 ∼90 nM (Figure 3A,
right). Inhibition occurred over several minutes at low PPQ-
102 concentrations, suggesting an intracellular site of action.
Inhibition was reversible, as seen by complete restoration of
CFTR chloride current after 30 min incubation with 2 μM
PPQ-102 followed by 10 min washout (data not shown).
Figure 3B shows PPQ-102 inhibition of CFTR chloride
current following CFTR activation by apigenin, a flavone-
type CFTR agonist that acts by direct CFTR binding, and
IBMX, a phosphodiesterase inhibitor that also binds directly
to CFTR. ThemildlyreducedPPQ-102 potency inresponseto
these agonists, compared to a pure cAMP agonist (CPT-cAMP)
that activates CFTR by a physiological phosphorylation
mechanism, is consistent with PPQ-102 action at nucleotide
binding domain(s) on the intracellular CFTR surface.
Figure 3C shows PPQ-102 inhibition of short-circuit current
in (nonpermeabilized) human intestinal (T84) and bronchial
cells following maximal CFTR activation by forskolin and
IBMX. CFTR inhibition was near 100% at 10 μM PPQ-102
with IC50 well below 1 μM.
PPQ-102 did not inhibit calcium-activated chloride chan-
nels or cellular cAMP production. Figure 3D shows little
inhibition of UTP-induced chloride currents in cystic fibrosis
human bronchial cells by 10 or 20 μM PPQ-102. Figure 3E

6450 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Tradtrantip et al.
markedly, as seen by the less frequent channel openings
(Figure 4B, left). Channel open probability (Po) was reduced
from 0.50 ( 0.04 to 0.14 ( 0.03. Mean channel open time did
not significantly change, but mean channel closed time was
greatly increased (Figure 4B, right). These results suggest that
PPQ-102inhibits CFTR by analtered channel gatingmechan-
ism, with stabilization of the channel closed state.
PPQ-102 was tested in an embryonic kidney culture model
of polycystic kidney disease. Kidneys were removed from day
13.5 embryonic mice and maintained in organ culture where
they continue to grow. Whereas kidneys do not form cysts
under control conditions as seen by transmission light micro-
scopy, multiple cysts form and progressively enlarge when the
culture medium was supplemented with the CFTR agonist
8-Br-cAMP (Figure 5A, left). Inclusion of PPQ-102 in the
culture medium did not affect kidney growth but remarkably
reduced the number and size of renal cysts formed in the 8-Br-
cAMP-containing medium. Figure 5A (right) summarizes the
percentage area occupied by cysts from studies done on many
kidneys, showing ∼60% inhibition of cyst formation by
0.5 μM PPQ-102 and near complete absence of cysts at 2.5
and 5 μM PPQ-102. In control studies in which 2.5 μM PPQ-
102 was removed after 3 days in organ culture, cysts rapidly
enlarged in the continued presence of 8-Br-cAMP (data not
shown), indicating that the inhibition effect of PPQ-102 is
reversible. Figure 5B shows representative hematoxylin and
eosin-stained paraffin sections of control and 8-Br-cAMP-
treated kidneys cultured for 4 days in the presence of indicated
concentrations of PPQ-102. In agreement with the transmis-
sion light micrographs of intact kidneys, PPQ-102 remarkably
reduced cyst size.
The ability of PPQ-102 to reduce fluid accumulation in
preformed cysts was tested by adding PPQ-102 to the 8-Br-
cAMP-containing medium after kidneys were cultured for
3 days in the presence of 8-Br-cAMP. Figure 5C shows
remarkable reduction in cyst size over 1 and 2 days after
inclusion of PPQ-102 in the culture medium.
Figure 2. Structure-activity analysis and synthesis of PPQ-102.
(A) Summary of structure-activity analysis, showing the structural
determinants for PPQ-102 inhibition of CFTR chloride conduc-
tance. (B) Synthesis of PPQ-102 and PPQ-102b. Reagents and
conditions: (a) Me2SO4, NaOH, 40 °C, 4 h, 43%; (b) PhCOCl,
ZnCl2, toluene, reflux, 6 h, 28%; (c) Br2, CHCl3, rt, 2 h, 57%;
(d) N-(2-aminophenyl)acetamide, microwave, 170 °C, 1 h, 51%;
(e) HCl, reflux, 6 h, 67%; (f) 5-Me-furan-2-carbaldehyde, 170 °C,
10 min, 43%; (g) KMnO4, Me2CO, 1 h, 40%.
Discussion and Conclusions
This paper reports the discovery and characterization of
PPQ CFTR inhibitors, which are the most potent CFTR
inhibitors identified to date and the first uncharged CFTR
inhibitors. Prior CFTR inhibitors, and most chloride channel
inhibitors in general, are negatively charged, which may be
required for their competition with chloride for binding to key
positively charged amino acids in the channel pores.25 As
predicted for an uncharged inhibitor and confirmed by patch-
clamp analysis, CFTR inhibition by PPQ-102 is voltage-
independent, which, as explained in the Introduction, is
advantageous to maintain CFTR inhibition potency in inter-
ior-negative cells. Single-channel analysis indicated that PPQ-
102 stabilizes the CFTR channel closed state, which together
with its neutral charge and relatively slow time course of
inhibition suggests action at a site on the cytoplasmic-facing
surface of CFTR distinct from its pore.
The most potent PPQ CFTR inhibitor identified by SAR
analysis, PPQ-102, inhibited CFTR chloride conductance
with IC50 ∼ 90 nM. Screening of PPQ analogues revealed
many active compounds having a wide range of potencies. In
nonpermeabilized T84 and bronchial epithelial cells, which
have a strong interior-negative membrane potential, the
IC50 for CFTR inhibition by PPQ-102 of ,1 μM was
substantially better than that of 3-5 μM measured previously
shows no significant effect of 10 μM PPQ-102 on basal or
forskolin-stimulated cAMP production.
Whole-cell membrane current was measured by patch-
clamp in CFTR-expressing FRT cells (Figure 4A, left).
Stimulation by 10 μM forskolin produced a membrane cur-
rent of 172 ( 39 pA/pF (n = 4) at þ100 mV (total membrane
capacitance 13 ( 1 pF). PPQ-102 at 0.5 μM gave ∼65%
inhibition of CFTR chloride current. Figure 4A (right) shows
an approximately linear current-voltage relationship for
CFTR, as found previously.1,2 The CFTR current-voltage
relationship remained linear after PPQ-102 addition, indicat-
ing a voltage-independent block mechanism, as expected for
an uncharged inhibitor. Cell-attached patch recordings were
done to examine single-channel CFTR function (Figure 4B).
Addition of 10 μM forskolin and 100 μM IBMX to the bath
resulted in CFTR channel opening. CFTR unitary conduc-
tance was 7 pS at þ80 mV. Application of 1 μM PPQ-102 did
not change unitary conductance but reduced channel activity

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6451
Figure 3. CFTR inhibition by PPQ-102. (A) Apical membrane current measured in CFTR-expressing FRT cells in the presence of a
transepithelial chloride gradient and with amphotericin B permeabilization of the basolateral membrane. CFTR was activated by CPT-cAMP
(100 μM), with increasing concentrations of PPQ-102 added as shown. (left) Original recording; (right) dose-response (SE n = 4). (B)
Measurements as in (A), but with apigenin (100 μM) or IBMX (100 μM) as agonists. Representative of three sets of experiments. (C) Short-
circuit current measured in T84 (left) and human bronchial airway epithelial cells (right). CFTR was maximally activated by 10 μM forskolin
and 100 μM IBMX (“forsk”). Current in the absence of inhibitor indicated as “control”. (D) Calcium-activated chloride channels were
activated by UTP (100 μM) in cystic fibrosis (CFTR-deficient) human bronchial epithelial cells, with PPQ-102 added as indicated. ENaC was
inhibited by amiloride (10 μM). (E) Cellular cAMP assayed in CHO-K1 cells under basal conditions and after 20 μM forskolin (SE n=4,
differences with PPQ-102 not significant).
for CFTRinh-172 and GlyH-101.16,20 The SAR analysis, as
reported in Table 1 and summarized in Figure 2A, showed
that greatest CFTR inhibition activity required a 5-methyl
furan ring, 3-methylphenyl moiety, and the pyrimido[40,50-
3,4]pyrrolo[1,2-a]quinoxaline template. The furan moiety in
many of the active compounds could be replaced by the
relatively stable phenyl moiety with minimal loss of activity.
Abolishing the chiral center in PPQ-102 by oxidation resulted
in loss of inhibition activity. SAR analysis also indicated the
need for a phenyl ring at the 2-position of pyrrole.
In conclusion, screening of >100000 chemically diverse
small molecules yielded a novel class of potent, uncharged
CFTR chloride channel inhibitors that were effective in
reducing cyst size in an organ culture model of PKD. The
PPQ inhibitors are likely to be effective as well in reducing
intestinal fluid accumulation in enterotoxin-mediated secre-
tory diarrheas, although we did not study their antidiarrheal
properties because existing malonic acid hydrazide-type
CFTR inhibitors have the advantage over PPQs of being
nonabsorbed and active extracellularly. The high potency of
PPQs should also make them useful, as CFTRinh-172 has
been, in characterizing CFTR function in cellular and in vivo
models.
Multiple lines of evidence support the application of CFTR
inhibitors to slow cyst growth in autosomal dominant PKD
(ADPKD): (a) CFTR is expressed strongly in epithelial cells
lining cysts in ADPKD, (b) cystic fibrosis (CFTR-deficient)
mice are resistant to cyst formation, (c) CFTR inhibitors
block cyst formation in cell/organ culture and in vivo models,
and (d) in some families affected with ADPKD and cystic
fibrosis, individuals with both ADPKD and CF have less
severe renal disease than those with ADPKD only.9,11 We
used here an established neonatal kidney organ culture model
to test the efficacy of PPQ-102 to reduce cyst volume. Intact
kidney models to study cystogenesis, although technically
more difficult than cell culture models such as MDCK cells
grown in collagen gels, are superior in recapitulating native
kidney anatomy and cellular phenotype.26 We found >60%
reduction in the size of developing cysts in kidneys cultured in
medium containing 0.5 μM PPQ-102, and near complete
absence of cysts at 2.5 μM PPQ-102, demonstrating substan-
tially greater potency of PPQ-102 compared to the best
thiazolidinone and glycine hydrazide CFTR inhibitors tested
previously.23 The shrinking of preformed cysts by PPQ-102
supports the notion that renal cystogenesis involves a balance
between active fluid secretion into and absorption from the
cyst lumen. The efficacy of PPQ inhibitors to prevent and
reverse cyst formation in vivo will require testing in PKD
animal models and, ultimately, in clinical trials.
Experimental Section
Cell Lines and Compounds. Fischer rat thyroid (FRT) cells
coexpressing human wildtype CFTR and the halide indicator
YFP-H148Q were generated as described.16 Cells were plated in
96-well black-walled microplates (Corning Costar) at a density
of 20000 cells per well in Coon’s modified F12 medium supple-
mented with 5% fetal calf serum, 2 mM L-glutamine, 100 U/mL
penicillin, and 100 μg/mL streptomycin. Assays were done at
48 h after plating when cells were just confluent. Some measure-
ments were made on T84 human intestinal epithelial cells and on
primary cultures of human bronchial epithelial cells, obtained
and grown as described.3,27 The compound collections used for
screening included: ∼105000 synthetic small molecules from
ChemDiv (San Diego, CA) and Asinex (San Diego, CA), and
∼7500 purified natural compounds from Analyticon (Potsdam,
Germany), Timtek (Newark, NJ), and Biomol (Plymouth Meet-
ing, PA). Compounds were maintained as DMSO stock solu-
tions. Structure-activity analysis was done on analogues
purchased from ChemDiv and Asinex.
Screening Procedures. Assays were done using an automated
screening platform (Beckman) equipped with FluoStar fluores-
cence plate readers (BMG Lab Technologies) as described.16
Each well of a 96-well plate was washed three times in PBS (300 μL/
wash), leaving 50 μL of PBS. Ten μL of a CFTR-activating

6452 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Tradtrantip et al.
Figure 4. Patch-clamp analysis of PPQ-102 inhibition of CFTR. (A) (left) Whole-cell currents measured in CFTR-expressing FRT cells
recorded at a holding potential at 0 mV, and pulsing to voltages between (100 mV in steps of 20 mV in the absence and presence of 500 nM
PPQ-102. CFTR was stimulated by 10 μM forskolin. (right) Current/voltage (I/V) plot of mean currents. Representative of four sets of
measurements. (B) (left) Single channel recordings in the cell-attached configuration. CFTR was activated by 10 μM forskolin and 100 μM
IBMX. Pipette potential was þ80 mV. (right) Effect of 1 μM PPQ-102 on CFTR channel open probability (Po), mean channel open time, and
mean channel closed time (SE, n = 3-4, * P < 0.01). O, open; C, closed.
cocktail (5 μM forskolin, 100 μM IBMX, 25 μM apigenin in
PBS) was added, and after 5 min test compounds (0.5 μL of
1 mM DMSO solution) were added to each well at 25 μM final
concentration. After 15 min, 96-well plates were transferred to a
plate reader for fluorescence assay. Each well was assayed
individually for CFTR-mediated iodide influx by recording
fluorescence continuously (200 ms per point) for 2 s (baseline)
and then for 10 s after rapid addition of 160 μL of isosmolar PBS
in which 137 mM chloride was replaced by iodide. The initial
rate of iodide influx was computed from fluorescence data by
nonlinear regression. Each plate contained negative (DMSO vehicle)
and positive (10 μM CFTRinh-172) controls.
hemichambers were filled with 5 mL of 75 mM NaCl and 75 mM
Na gluconate (apical) and 150 mM NaCl (basolateral) (pH 7.3),
and the basolateral membrane was permeabilized with 250 μg/
mL amphotericin B. For bronchial epithelial cells and T84 cells,
both hemichambers contained a Krebs-bicarbonate solution.
Hemichambers were continuously bubbled with air (FRT cells)
or 5% CO2 in air (bronchial and T84 cells) and maintained at
37 °C. Short-circuit current was recorded continuously using a
DVC-1000 voltage clamp (World Precision Instruments) using
Ag/AgCl electrodes and 3 M KCl agar bridges.
Patch-Clamp Analysis. Whole cell recordings were done on
FRT cells stably expressing human wild-type CFTR. The pipet
solution contained 140 mM N-methyl D-glucamine chloride
(NMDG-Cl), 5 mM EGTA, 1 mM MgCl2, 1 mM Tris-ATP,
and 10 mM HEPES (pH 7.2), and the bath solution contained
Short-Circuit Current. Snapwell inserts containing CFTR-
expressing FRT cells, T84 cells, or human bronchial epithelial
cells were mounted in Ussing chambers. For FRT cells, the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6453
Figure 5. PPQ-102 prevents and reverses renal cyst expansion in an embryonic kidney organ culture model of PKD. E13.5 embryonic kidneys
were maintained in organ culture in defined medium. (A) Inhibition of cyst formation. (left) Transmission light micrographs of kidneys in
culture. As indicated, the culture medium contained 0 or 100 μM 8-Br-cAMP and/or 0, 0.5, or 5 μM PPQ-102. (right) Summary of cyst volumes
after 4 days in culture shown as the fractional kidney area occupied by cysts (SE, 6-8 kidneys, * P < 0.001 compared to þ8-Br-cAMP, 0 μM
PPQ-102). (B) Hematoxylin and eosin-staining of kidney paraffin sections after 4 days in culture in the presence of 0 or 100 μM 8-Br-cAMP and
indicated concentrations of PPQ-102. Representative of studies on three kidneys for each condition. (C) Reversal of preformed renal cysts.
(left) Transmission light micrographs of kidneys cultured in 8-Br-cAMP for 3 days, with 5 μM PPQ-102 added at day 3 (two kidneys shown per
condition). Micrographs at the right show kidneys at day 5 that were not exposed to PPQ-102. (right) Summary of cyst volumes at day 5 (SE, six
kidneys, * P < 0.001).
140 mM N-methyl D-glucamine chloride, 1 mM CaCl2, 1 mM
MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). Experi-
ments were done at room temperature (22-25 °C). Pipettes were
pulled from borosilicate glass and had resistances of 3-5 MΩ
after fire polishing. Seal resistances were typically between 3 and
10 GΩ. After establishing the whole-cell configuration, CFTR
was activated by adding forskolin and 3-isobutyl-1-methyl-
xanthine (IBMX). Whole cell currents were elicited by applying
hyperpolarizing and depolarizing voltage pulses from a holding
potential of 0 mV to potentials between -100 and þ100 mV in
steps of 20 mV. Current was filtered at 5 kHz and digitized and
analyzed using an AxoScope 10.0 system and a Digidata 1440A
AC/DC converter. The single channel characteristics of CFTR
were analyzed in the cell-attached configuration using fire-
polished pipettes with a resistance of 10-15 MΩ. The pipet
solution contained (in mM): 140 NMDG-Cl, 1 CaCl2, 1 MgCl2,
5 glucose, and 10 HEPES (pH 7.4), and the bath solution
contained 140 KCl, 1 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES
(pH 7.4). Recordings were performed at room temperature
using an Axopatch-200B (Axon Instruments). The voltage and
current data were low-pass filtered at 1 kHz and stored for later
analysis. Single channel data were digitally filtered at 25 Hz and
analyzed using Clampfit 10.0 software (Axon Instruments).
Embryonic Organ Culture Model of PKD. Mouse embryos
were obtained at embryonic day 13.5 (E13.5). Metanephroi were
dissected and placed on transparent Falcon 0.4 mm diameter
porous cell culture inserts as described.23 To the culture inserts
was added DMEM/Ham’s F-12 nutrient medium supplemented
with 2 mM L-glutamine, 10 mM HEPES, 5 μg/mL insulin, 5 μg/
mL transferrin, 2.8 nM selenium, 25 ng/mL prostaglandin E,
32 pg/mL T3, 250 U/mL penicillin, and 250 μg/mL streptomy-
cin. Kidneys were maintained in a 37 °C humidified CO2

6454 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Tradtrantip et al.
incubator for up to 8 days. Culture medium containing 100 μM
8-Br-cAMP, with or without CFTR inhibitor, was replaced
(in the lower chamber) every 12 h. In some studies, CFTR
inhibitor was added at 3 days after 8-Br-cAMP to test its efficacy
in reversing preformed cysts. Kidneys were photographed using
a Nikon inverted microscope (Nikon TE 2000-S) equipped with
2ꢀ objective lens, 520 nm bandpass filter, and high-resolution
CCD camera. Percentage cyst area was calculated as total cyst
area divided by total kidney area.
67%) as a white precipitate; mp > 300 °C. 1H NMR (DMSO-d6):
δ7.32-7.26 (m, 2H), 7.23-7.15 (m, 3H), 7.00 (t, 1H, J= 7.32 Hz),
6.87 (s, 1H), 6.80 (d, 1H, J=7.69), 6.70 (d, 1H, J=8.06 Hz), 6.41
(t,1H,J=7.32Hz),5.00(s,2H),3.29(s,3H),3.16(s,3H).MS(ESþ)
(m/z): [M þ 1]þ calculated for C20H19N4O2, 347.39, found 347.10.
7,9-Dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-
pyrimido[40,50-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione
(PPQ-102). A mixture of 5-methylfuran-2-carbaldehyde (32 mg,
29 μL, 0.29 mmol), compound 6 (101 mg, 0.29 mmol), and
ethanol (1 mL) were heated in a microwave reactor at 170 °C
for 10 min. A white product was isolated, washed, and recrys-
tallized from ethanol to afford51mg of PPQ-102 (42% yield); mp
Synthesis Procedures. 1H and 13C NMR spectra were obtained
in deuterated dimethyl sulfoxide (DMSO-d6) using a 400-MHz
Varian spectrometer referenced to DMSO. Mass spectrometry
was done using a Waters LC/MS system (Alliance HT 2790þZQ,
HPLC: Waters model 2690, Milford, MA). Flash chromatogra-
phy was done using EM silica gel (230-400 mesh), and thin-layer
chromatography was done on Merk silica gel 60 F254 plates
(Darmstadt, Germany). Microwave reactions were performed in
a Biotage Initiator (0.5-2 mL vials) with target temperature
reached within 30 s at ∼55 W. Melting points are uncorrected.
Purity to >98% was confirmed by LCMS.
1
>300 °C. H NMR (DMSO-d6): δ 7.41 (broad m, 4H), 6.95
(d, 2H, J=8.42 Hz), 6.90-6.83 (m, 2H), 6.29 (d, 2 H, J=2.93 Hz),
6.08 (d, 1H, J=2.19 Hz), 5.80 (d, 1H, J=2.93 Hz), 5.69 (d, 1H, J=
2.93 Hz), 3.50 (s, 3H), 3.12 (s, 3H), 2.11 (s, 3H). 13C NMR
(DMSO): 159.2, 153.1, 151.9, 151.9, 139.2, 131.5, 129.6, 129.4,
128.8, 126.9, 124.3, 122.8, 120.6, 118.2, 117.6, 111.9, 108.9, 107.1,
105.2, 47.8, 32.3, 28.2, 13.9. HRMS (ESþ) (m/z): [M þ 1]þ
calculated for C26H23N4O3, 439.1765, found, 439.1771.
1,3,6-Trimethyl-1H,3H-pyrimidine-2,4-dione (2).27 Dimethyl-
sulfate (106 g, 80 mL, 844 mmol) was added dropwise to a
solution of 2,4-dihydroxy-6-methylpyrimidine (30 g, 238 mmol)
in 280 mL of 4 N NaOH at 40 °C. After stirring for 4 h at 40 °C,
the reaction mixture was neutralized with careful addition of
acetic acid and extracted three times with 100 mL of ethyl
acetate. Combined organics were dried over MgSO4 and con-
centrated in vacuo to yield a white solid. Recrystallization from
ethanol yielded 2 (15.8 g, 43%); mp 113-114 °C. 1H NMR
(DMSO-d6): δ 5.58 (s, 1H), 3.26 (s, 3H), 3.09 (s, 3H,), 2.19
(s, 3H,). MS (ESþ) (m/z): [M þ 1]þ calculated for C7H11N2O2,
155.17, found 155.93. This compound matched the analytical
data as reported.27
5-Benzoyl-1,3,6-trimethylpyrimidine-2,4(1H,3H)-dione (3). A
mixture of 1,3,6-trimethyl-compound 2 (12.3 g, 80 mmol),
benzoyl chloride (11.5 g, 9.5 mL, 82 mmol), and anhydrous zinc
chloride (10.8 g, 79 mmol) in toluene (100 mL) was heated to
reflux for 6 h. The mixture was poured over ice (200 g), and the
separated toluene layer was concentrated in vacuo. The crude
residue was purified by flash chromatography to yield 3 (5.8 g,
28%); mp 132-134 °C. MS (ESþ) (m/z): [M þ 1]þ calculated for
C14H15N2O3, 259.28, found 259.09. This compound matched
analytical data as reported.28
N-(2-(1,3-Dimethyl-2,4-dioxo-5-phenyl-3,4-dihydro-1H-pyrr-
olo[3,4-d]pyrimidin-6(2H)-yl)phenyl)-5-methylfuran-2-carboxa-
mide (PPQ-102b). To a solution of PPQ-102 (12 mg, 27 μmol in
2 mL acetone) was added dropwise a saturated solution of
potassium permanganate (13 mg, 80 μmol, 200 μL). The reac-
tion mixture was stirred for 1 h at room temperature and filtered.
The residue was processed by standard methods,30 and the
acetone solution was evaporated to yield PPQ-102b (5 mg,
40%). MS (ESþ) (m/z): [M þ 1]þ calculated for C26H21N4O3,
437.47, found, 437.12.
Acknowledgment. Supported by NIH grants DK86125,
HL73856, DK72517, EB00415, HL59198, DK35124 and
EY13574, and Research Development Program and Drug
Discovery grants from the Cystic Fibrosis Foundation.
Supporting Information Available: Analytical data for synthe-
sized compounds and list of PPQ analogues tested and their
CFTR inhibition activities. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
5-Benzoyl-6-(bromomethyl)-1,3-dimethylpyrimidine-2,4(1H,-
3H)-dione (4).28,29 To a solution of 3 (2.61 g, 10.1 mmol) in
chloroform (13 mL) was added bromine (1.62 g, 0.52 mL,
20.3 mmol in 3 mL chloroform) dropwise over 30 min at room
temperature. The reaction mixture was further stirred for 1 min
at room temperature before concentrated in vacuo. The crude
reaction mixture was purified by flash chromatography to yield
4 (1.96 g, 57%); mp 164-167 °C. MS (ESþ) (m/z): [M þ 1]þ
calculated for C14H14BrN2O3, 338.18, found 337.15, 338.93.
This compound matched analytical data as reported.28,29
N-(2-(1,3-dimethyl-2,4-dioxo-5-phenyl-3,4-dihydro-1H-pyrr-
olo[3,4-d]pyrimidin-6(2H)-yl)phenyl)acetamide (5). A mixture of
N-(2-aminophenyl)acetamide (315 mg, 2.1 mmol), bromo-com-
pound 4 (680 mg, 2 mmol), triethylamine (200 mg, 280 μL,
2 mmol), and ethanol (2 mL) was microwave-heated at 170 °C
for 1 h (2-5 mL vial, pressure 13 bar). The shiny white crystal-
line mass was filtered, washed, and recrystallized from ethanol
to give 5 (392 mg, 51% yield); mp > 250 °C. 1H NMR (DMSO-
d6): δ 9.17 (s, 1H), 7.69 (d, 1H, pyrrole CH, J = 8.06 Hz),
7.33-7.14 (m, 6H), 7.10-6.94 (m, 3H), 3.31 (s, 3H), 3.17 (s, 3H),
1.87 (s, 3H). MS (ESþ) (m/z): [M þ 1]þ calculated for
C22H21N4O3, 389.43, found 389.19.
(1) Sheppard, D. N.; Welsh, M. J. Structure and function of the CFTR
chloride channel. Physiol. Rev. 1999, 79, S23–45.
(2) Gadsby, D. C.; Vergani, P.; Csanady, L. The ABC protein turned
ꢀ
chloride channel whose failure causes cystic fibrosis. Nature 2006,
440, 477–483.
(3) Thiagarajah, J.; Broadbent, T.; Hsieh, E.; Verkman, A. S. Prevention
of toxin-induced intestinal ion and fluid secretion by a small-
molecule CFTR inhibitor. Gastroenterology 2004, 126, 511–519.
(4) Sonawane, N.; Zhao, D.; Zegarra-Moran, O.; Galietta, L. J. V.;
Verkman, A. S. Lectin conjugates as potent, nonabsorbable CFTR
inhibitors for reducing intestinal fluid secretion in cholera. Gastro-
enterology 2007, 132, 1234–1244.
(5) Kunzelmann, K.; Mall, M. Electrolyte transport in the mammalian
colon: mechanisms and implications for disease. Physiol. Rev. 2002,
82, 245–289.
(6) Hanaoka, K.; Guggino, W. B. cAMP regulates cell proliferation
and cyst formation in autosomal polycystic kidney disease cells.
J. Am. Soc. Nephrol. 2000, 11, 1179–1187.
(7) Brill, S. R.; Ross, K. E.; Davidow, C. J.; Ye, M.; Grantham, J. J.;
Caplan, M. J. Immunolocalization of ion transport proteins in
human autosomal dominant polycystic kidney epithelial cells.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10206–10211.
(8) Torres, V. E.; Harris, P. C. Mechanisms of disease: autosomal
dominant and recessive polycystic kidney diseases. Nat. Clin.
Pract. Nephrol. 2006, 2, 40–55.
(9) O’Sullivan, D. A.; Torres, V. E.; Gabow, P. A.; Thibodeau, S. N.;
King, B. F.; Bergstralh, E. J. Cystic fibrosis and the phenotypic
expression of autosomal dominant polycystic kidney disease. Am.
J. Kidney Dis. 1998, 32, 976–983.
6-(2-Aminophenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]-
pyrimidine-2,4(3H,6H)-dione (6). Acetamide compound 5 (200 mg,
0.5 mmol) was refluxed in hydrochloric acid (4 N, 10 mL) for 6 h.
The reaction mixture was evaporated under vacuum, and the
residue was dissolved in water and neutralized to give 6 (120 mg,
(10) Xu, N.; Glockner, J. F.; Rossetti, S.; Babovich-Vuksanovic, D.;
Harris, P. C.; Torres, V. E. Autosomal dominant polycystic kidney

Article
disease coexisting with cystic fibrosis. J. Nephrol. 2006, 19, 529–
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6455
(20) Muanprasat, C.; Sonawane, N. D.; Salinas, D.; Taddei, A.;
Galietta, L. J. V.; Verkman, A. S. Discovery of glycine hydrazide
pore-occluding CFTR inhibitors: mechanism, structure-activity
analysis and in vivo efficacy. J. Gen. Physiol. 2004, 124, 125–137.
(21) Sonawane, N. D.;Hu, J.;Muanprasat, C.;Verkman, A. S. Luminally-
active, nonabsorbable CFTR inhibitors as potential therapy to reduce
intestinal fluid losses in cholera. FASEB J. 2006, 20, 130–132.
(22) Sonawane, N.;Zhao, D.; Zegarra-Mora, O;Galietta, L. J.;Verkman,
A. S. Nanomolar CFTR inhibition by pore-occluding divalent
polyethylene glycol-malonic acid hydrazides. Chem. Biol. 2008,
15, 718–728.
(23) Yang, B.; Sonawane, N. D.; Zhao, D.; Somlo, S.; Verkman, A. S.
Small-molecule CFTR inhibitors slow cyst growth in polycystic
kidney disease. J. Am. Soc. Nephrol. 2008, 19, 1300–1310.
(24) Sonawane, N.; Verkman, A. S. Thiazolidinone CFTR inhibitors
with improved water solubility identified by structure-activity
analysis. Bioorg. Med. Chem. 2008, 16, 8187–8195.
534.
(11) Cotton, C. U.; Avner, E. D. PKD and CF: an interesting family
provides insight into the molecular pathophysiology of polycystic
kidney disease. Am. J. Kidney Dis. 1998, 32, 1081–1083.
(12) Davidow, C. J.; Maser, R. L.; Rome, L. A.; Calvet, J. P.; Gran-
tham, J. J. The cystic fibrosis transmembrane conductance reg-
ulator mediates transepithelial fluid secretion by human autosomal
dominant polycystic kidney disease epithelium in vitro. Kidney Int.
1996, 50, 208–218.
(13) Li, H.; Findlay, I. A.; Sheppard, D. N. The relationship between
cell proliferation, Cl- secretion, and renal cyst growth: a study
using CFTR inhibitors. Kidney Int. 2004, 66, 1926–1938.
(14) Routaboul, C.; Norez, C.; Melin, P.; Molina, M. C.; Boucherle, B.;
Bossard, F.; Noel, S.; Robert, R.; Gauthier, C.; Becq, F.; Decout,
J. L. Discovery of r-aminoazaheterocycle-methylglyoxal adducts
as a new class of high-affinity inhibitors of cystic fibrosis trans-
membrane conductance regulator chloride channels. J. Pharmacol.
Exp. Ther. 2007, 322, 1023–1035.
(25) Verkman, A. S.; Galietta, L. J. Chloride channels as drug targets.
Nat. Rev. Drug Discovery 2009, 8, 153–171.
(15) Sonawane, N. D.; Zegarra-Moran, O.; Namkung, W.; Galietta, L.
J. V.; Verkman, A. S. r-Aminoazaheterocyclic-methylglyoxyl ad-
ducts do not inhibit cystic fibrosis transmembrane conductance
chloride channel activity. J. Pharm. Exp. Ther. 2008, 325, 529–535.
(16) Ma, T.; Thiagarajah, J. R.; Yang, H.; Sonawane, N. D.; Folli, C.;
Galietta, L. J.; Verkman, A. S. Thiazolidinone CFTR inhibitor
identified by high-throughput screening blocks cholera-toxin in-
duced intestinal fluid secretion. J. Clin. Invest. 2002, 110, 1651–
1658.
(17) Taddei, A.; Folli, C.; Zegarra-Moran, O.; Fanen, P.; Verkman,
A. S.; Galietta, L. J. Altered channel gating mechanism for CFTR
inhibition by a high-affinity thiazolidinone blocker. FEBS Lett.
2004, 558, 52–56.
(26) Magenheimer, B. S.; St. John, P. L.; Isom, K. S.; Abrahamson,
D. R.; De Lisle, R. C.; Wallace, D. P.; Maser, R. L.; Grantham,
J. J.; Calvet, J. P. Early embryonic renal tubules of wild-type and
polycystic kidney disease kidneys respond to cAMP stimulation
wiþth cystic fibrosis transmembrane conductance regulator/Naþ,
K ,2Cl- co-transporter-dependent cystic dilation. J. Am. Soc.
Nephrol. 2006, 17, 3424–3437.
(27) Azas, N.; Rathelot, P.; Djekou, S.; Delmas, F.; Gellis, A.;
Di Giorgio, C.; Vanelle, P.; Timon-David, P. Antiparasitic activity
of highly conjugated pyrimidine-2,4-dione derivatives. Farmaco
2003, 58, 1263–1270.
(28) Tsupak, E. B.; Shevchenko, M. A.; Pozharskii, A. F.; Tkachenko,
Y. N. Pyrollopyrimidines. 5. Interaction of 6-amino-1,3-
dimethylpyrrolo[3,4-d]-pyrimidin-2,4(1H,3H)-diones with 1,3-di-
ketones. Khim. Geterotsikl. Soedin. 2003, 7, 1096–1102.
(29) Tsupak, E. B.; Shevchenko, M. A. [3,4]-annulated pyrroles
1. Polynuclear heterocyclic systems based on pyrrolo[3,4-d]pyrimi-
dine-2,4-dione. Russ. Chem. Bull. 2006, 55, 2265–2270.
(18) Caci, E.; Caputo, A.; Hinzpeter, A.; Arous, N.; Fanen, P.; Sona-
wane, N. D.; Verkman, A. S.; Ravazzolo, R.; Zegarra-Moran, O.;
Galietta, L. J. V. Evidence for direct CFTR inhibition by CFTRinh
-
172 based on arginine 347 mutagenesis. Biochem. J. 2008, 413, 135–
142.
(19) Sonawane, N. D.; Muanprasat, C.; Nagatani, R.; Song, Y.;
Verkman, A. S. In vivo pharmacology and antidiarrheal efficacy
of a thiazolidinone CFTR inhibitor in rodents. J. Pharm. Sci. 2005,
94, 134–143.
(30) Oels, R.; Storer, R.; Young, D. W. Reinvestigation of synthesis of
3-dimethylallyl-4-hydroxy-2-quinolones;novel route to tetracyc-
lic heteroaromatic-compounds. J. Chem. Soc., Perkin Trans. 1977,
23, 2546–2551.