Potent, metabolically stable benzopyrimido-pyrrolo-oxazine-dione (BPO) CFTR inhibitors for polycystic kidney disease

Article abstract of DOI:10.1021/jm200505e

We previously reported the discovery of pyrimido-pyrrolo-quinoxalinedione (PPQ) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in an organ culture model of polycystic kidney disease (PKD) (J. Med. Chem. 2009, 52, 6447-6455). Here, we report related benzopyrimido-pyrrolo-oxazinedione (BPO) CFTR inhibitors. To establish structure-activity relationships and select lead compound(s) with improved potency, metabolic stability, and aqueous solubility compared to the most potent prior compound 8 (PPQ-102, IC₅₀ ～ 90 nM), we synthesized 16 PPQ analogues and 11 BPO analogues. The analogues were efficiently synthesized in 5-6 steps and 11-61% overall yield. Modification of 8 by bromine substitution at the 5-position of the furan ring, replacement of the secondary amine with an ether bridge, and carboxylation, gave 6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9, 10-tetrahydro-6H-benzo[b]pyrimido [4′,5′:3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid 42 (BPO-27), which fully inhibited CFTR with IC₅₀ ～ 8 nM and, compared to 8, had >10-fold greater metabolic stability and much greater polarity/aqueous solubility. In an embryonic kidney culture model of PKD, 42 prevented cyst growth with IC ₅₀ ～ 100 nM. Benzopyrimido-pyrrolo-oxazinediones such as 42 are potential development candidates for antisecretory therapy of PKD.

Full text of DOI:10.1021/jm200505e

ARTICLE
pubs.acs.org/jmc
Potent, Metabolically Stable Benzopyrimido-pyrrolo-oxazine-dione
(BPO) CFTR Inhibitors for Polycystic Kidney Disease
David S. Snyder,†,‡ Lukmanee Tradtrantip,† Chenjuan Yao,† Mark J. Kurth,‡ and A. S. Verkman*,†
†Departments of Medicine and Physiology, University of California, San Francisco, California 94143-0521, United States
‡Department of Chemistry, University of California, Davis, California 95616, United States
S Supporting Information
b
ABSTRACT: We previously reported the discovery of pyrimi-
do-pyrrolo-quinoxalinedione (PPQ) inhibitors of the cystic
fibrosis transmembrane conductance regulator (CFTR) chlor-
ide channel and showed their efficacy in an organ culture
model of polycystic kidney disease (PKD) (J. Med. Chem.
2009, 52, 6447ꢀ6455). Here, we report related benzopyrimido-
pyrrolo-oxazinedione (BPO) CFTR inhibitors. To establish
structureꢀactivity relationships and select lead compound(s)
with improved potency, metabolic stability, and aqueous solubility compared to the most potent prior compound 8 (PPQ-102,
IC50 ∼ 90 nM), we synthesized 16 PPQ analogues and 11 BPO analogues. The analogues were efficiently synthesized in 5ꢀ6 steps
and 11ꢀ61% overall yield. Modification of 8 by bromine substitution at the 5-position of the furan ring, replacement of the
secondary amine with an ether bridge, and carboxylation, gave 6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-
tetrahydro-6H-benzo[b]pyrimido [40,50:3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid 42 (BPO-27), which fully inhibited
CFTR with IC50 ∼ 8 nM and, compared to 8, had >10-fold greater metabolic stability and much greater polarity/aqueous solubility.
In an embryonic kidney culture model of PKD, 42 prevented cyst growth with IC50 ∼ 100 nM. Benzopyrimido-pyrrolo-
oxazinediones such as 42 are potential development candidates for antisecretory therapy of PKD.
’ INTRODUCTION
the CFTR pore to block CFTR chloride conductance.20ꢀ22
CFTRinh-172 has been used widely as a research tool to study
CFTR function in cell culture, ex vivo tissues, and animal models.
CFTRinh-172 inhibits CFTR, with IC50 in the range of
300ꢀ3000 nM, depending on cell type and membrane potential,
and was shown to have low toxicity and metabolic stability with
primarily renal excretion.21,23 The tetrazolo-substituted thiazoli-
dinone Tetrazolo-172 (Figure 1) has improved water solubility
compared to CFTRinh-172,24 reducing cyst expansion in organ
culture and mouse models of PKD.25 A second class of small-
molecule CFTR inhibitors, the glycine hydrazides, such as GlyH-
101 (Figure 1), block CFTR at an external site within the CFTR
pore.26 Membrane-impermeant, nonabsorbable conjugates of
glycine hydrazides with polyethylene glycols (Figure 1)27,28
and lectins29 inhibit CFTR with IC50 down to 50ꢀ100 nM
whenadded atthe mucosal cellsurfaceand wereeffective in rodent
models of secretory diarrheas such as cholera. An absorbable
glycine hydrazide, phenyl-GlyH-101 (Figure 1), reduced cyst
growth in PKD.25
Polycystic kidney disease (PKD) is one of the most common
human genetic diseases and a major cause of chronic renal
insufficiency requiring dialysis and kidney transplantation.1 Cyst
growth in autosomal dominant polycystic kidney disease
(ADPKD) involves progressive fluid accumulation.2,3 Fluid secre-
tion into the cyst lumen requires chloride secretion by the cystic
fibrosis transmembrane conductance regulator (CFTR) protein,4,5
a cAMP-regulated chloride channel in which loss-of-function
mutations cause the genetic disease cystic fibrosis.6 CFTR is
expressed strongly in epithelial cells lining cysts in ADPKD.7
Cystic fibrosis (CFTR-deficient) mice are resistant to cyst forma-
tion, and CFTR inhibitors block cyst formation in cell/organ
cultureandin vivo models.8,9 Inrarefamilies affected with ADPKD
and cystic fibrosis, individuals with both ADPKD and CF have less
severe renal disease than those with ADPKD only.10ꢀ12 Cyst
expansion in ADPKD also requires cyst epithelial cell proliferation
involving mTor signaling,13,14 which is the basis of several “anti-
proliferative” therapies under development.15ꢀ18 “Antisecretory”
(CFTR inhibition) therapy is predicted to complement antipro-
liferative therapy or be effective alone in life-long treatment
of ADPKD.
Additional small molecule screening yielded pyrimido-pyrro-
lo-quinoxalinedione (PPQ) compounds, which were the first
uncharged, and thus membrane-potential insensitive, CFTR
inhibitors.30 7,9-Dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,
We identified several classes of small-molecule CFTR inhibi-
tors by high-throughput screening (reviewed in ref 19). Thiazo-
lidinone-class CFTR inhibitors such as CFTRinh-172 (Figure 1)
act on the cytoplasmic side of the plasma membrane at a site near
Received: April 25, 2011
Published: June 27, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structures of CFTR inhibitors.
Figure 2. Metabolism of compound 8 in hepatic microsomes. (A) LC/MS showing disappearance over 30 min during incubation with microsomes in
the presence of NADPH. (B) Appearance of metabolites at +14 and +16 Da. (C) Schematic of potential sites of metabolism.
6-dihydro-pyrimido-[40,50-3,4]pyrrolo[1,2-a]quinoxaline-8,10-
(7H,9H)-dione (PPQ-102, Figure 1) 8 inhibited CFTR chloride
conductance with IC50 ∼ 90 nM by a mechanism involving
stabilization of the channel closed-state. PPQ analog 8 prevented
cyst expansion and reduced the size of preformed cysts in an
embryonic kidney organ culture model of PKD. However, as
described below, 8 has poor metabolic stability, precluding animal
testing, as well as low polarity (clogP 4.92) and hence low aqueous
solubility. To improve compound stability, water solubility, and
CFTR inhibition potency, herein we synthesized and characterized
a focused library of 27 PPQ and BPO analogues. The most potent
compound, 42 (BPO-27) had an IC50 ∼ 8 nM for CFTR
inhibition, more than 10-fold improved metabolic stability and
much greater polarity (clogP 1.76) compared to 8, andwaseffective
in preventing renal cyst expansion in a PKD model.
’ RESULTS
We reported 8 as a small-molecule inhibitor of CFTR chloride
conductance with efficacy in preventing and reversing cyst
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Scheme 1. Synthesis of PPQ Analoguesa
a Reagents: (a) NaOH, H2O, dimethylsulfate, rt, 3 days; (b) benzoyl chloride, ZnCl2, chlorobenzene, reflux; (c) Br2, CH2Cl2, cat. H2O, reflux; (d) 4-R2-
1,2-phenylenediamine, EtOH, reflux; (e) X d O, R2 = H, 4-R5-5-R6-furfural, 1,2-dichloroethane, TsOH, reflux; X d S, 5-methylthiophene carbaldehyde,
1,2-dichloroethane, TsOH, reflux; X d O, R2 = NO2, 5-bromo-2-furaldehyde CHCl3, TFA reflux; (f) R4 = CH3SO2ꢀ, methanesulfonyl chloride, DCM,
Et3N; R4 = ClCH2COꢀ, chloroacetyl chloride, DCM, Et3N; R4 = CH3COꢀ, acetic anhydride, DMAP, 100 °C; R4 = NO, t-butyl nitrite, DCM; (g) N-
methyl-1,2-phenylenediamine, EtOH, reflux; (h) 5-methylfurfural, 1,2-dichloroethane, TsOH, reflux.
formation in an organ culture model of PKD.30 Preliminary
analysis of the metabolic stability and aqueous solubility of 8,
done in preparation for in vivo testing in mouse models of PKD,
indicated poor metabolic stability and low water solubility (<2
μM in albumin-free saline). In vitro metabolic stability was
determined by compound incubation with hepatic microsomes
at 37 °C for specified times in the absence vs presence of
NADPH, following by LC/MS analysis. Figure 2A shows loss
of 8 in hepatic microsomes in the presence of NADPH, with
∼60% disappearance in 30 min. No loss of 8 was seen in the
absence of NADPH (data not shown). PPQ analog 8 was
undetectable in serum, kidney, and urine at 30ꢀ60 min after
intravenous bolus administration of 300 μg 8 in mice using an
LC/MS assay with sensitivity better than 100 nM (data not
shown). Although the precise metabolic fate of 8 is not known,
structural considerations and the presence of prominent meta-
bolites at +14 and +16 Da (Figure 2B) suggested possible
oxidation, aromatization, and hydroxylation (Figure 2C). To
improve on the drug-like properties of 8, a series analogues was
synthesized and tested.
Scheme 1, 6-methyluracil 1 was exhaustively alkylated using
dimethyl sulfate to give 1,3,6-trimethyluracil 2 in 98% yield.
1,3,6-Trimethyluracil 2 was subject to FriedelꢀCrafts acylation
utilizing benzoyl chloride and anhydrous zinc chloride to give
ketone 3 in 66% yield. Bromination of ketone 3 gave 4 in
quantitative yield. At the first point of diversification, 4 was
reacted with substituted 1,2-phenylenediamines (2 equiv) to give
pyrroles 5ꢀ7 in 97% (R2 = H), 89% (R2 = NO2), and 83% (R4 =
Me) yield. Pyrroles 5ꢀ7 were condensed with the appropriately
substituted furfural or thiophene carbaldehyde using catalytic
acid to give 13ꢀ23 and 38 with yields of 57ꢀ98%. PPQ analog 8
was obtained on a gram scale in 83% yield. Amide analogues were
synthesized from 8 and acid halides or anhydrides to give 9ꢀ11,
in 73%, 80% and 79% yield, respectively. The nitrosamine 12 was
synthesized from 8 and t-butyl nitrite in 79% yield.
Scheme 2 shows the synthesis of benzoxazine BPO (YdO)
compounds. Ketone 24 (R1 = Me) was synthesized from 2 by
FriedelꢀCrafts acylation utilizing m-tolyl chloride in 26% yield,
and subsequently brominated to give 25 in 93% yield. The
condensation of substituted 2-aminophenols with 4 and 25 in
ethanol gave pyrroles 26ꢀ30 in 96% (R1 = CH3, R2 = R3 = H),
96% (R1 = R2 = R3 = H), 94% (R1 = R3 = H, R2 = NO2), 88%
(R1 = H, R2 = Cl, R3 = NO2), and 98% (R1 = R3 = H, R2 =
COOEt) yield, respectively. Pyrroles 26ꢀ30 were then con-
densed with substituted furfurals using catalytic acid at 150 °C to
give 31ꢀ37 and 39ꢀ41 in yields of 43ꢀ84%. BPO analog 40 was
PPQ and BPO Analogue Synthesis. Scheme 1 shows the
synthesis of dihydroquinoxoline PPQ (YdN) compounds and
Scheme 2 of benzoxazine BPO (YdO) compounds. Table 1
shows structures and CFTR inhibition data for all synthesized
analogues. Our initial efforts focused on improving the synthesis
of 8, as the original synthesis had low yield.30 As shown in
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Scheme 2. Synthesis of BPO Analoguesa
a Reagents: (a) R1 = H, benzoyl chloride, ZnCl2, chlorobenzene, reflux; R1 = Me, m-tolyl chloride, ZnCl2, chlorobenzene, reflux; (b) Br2, CH2Cl2, cat. H2O,
reflux; (c) 2-amino-3-R2-4-R3-phenol, EtOH, reflux; (d) 5-R6-furfual, TFA, CHCl3, or 1,2-dichloroethane, 150 °C; (e) KOH, THF, H2O, HCl workup.
saponified utilizing KOH in THF and water to give 42 in 83%
yield after acid workup.
basic hydrazine only yielded 8. To maintain basicity and prevent
aromatization, we tried to synthesize the N-methyl analogue 13 via
reductive alkylation using formaldehyde but were unsuccessful.
Fortunately, the N-methyl precursor 7 was obtained in good yield
from N-methyl-1,2-phenylenediamine and 4. Pyrrole 7 was then
used to synthesize 13, which was weakly active, and so further
derivitization at the secondary amine was abandoned.
Characterization of PPQ and BPO Analogues. Initial com-
pound testing for CFTR inhibition was done using a plate-reader
assay in which iodide influx was measured in FRT cells coex-
pressing human wild-type CFTR and the iodide-sensing yellow
fluorescent protein YFP-H148Q/I152L. Figure 3A (top) shows
representative fluorescence data for inhibition of CFTR-
mediated iodide influx by one of the synthesized PPQ analogues,
showing reduced negative slope after iodide addition with
increasing inhibitor concentration. Figure 3A (bottom) shows
concentrationꢀinhibition data for selected compounds, with
IC50 values for all compounds listed in Table 1. Though relative
IC50 values are useful for comparisons, absolute IC50 values from
the plate-reader assay are approximate, generally underestimat-
ing compound potency because of assay nonlinearities, pH-
dependent YFP fluorescence, the use of iodide instead of
chloride, and compound dilution at the start of the assay.31
Quantitative concentrationꢀinhibition data for the most potent
compounds was obtained by analysis of short-circuit current,
which represents a definitive electrophysiological measure of
compound potency. Current was measured in CFTR-expressing
FRT cells in which the basolateral membrane was permeabilized
and in the presence of a transepithelial chloride concentration
gradient, so that current is a linear measure of CFTR function.
CFTR was activated by forskolin, followed by serial additions of
increasing concentrations of test compounds. The representative
short-circuit current measurement in Figure 3B shows increased
CFTR chloride conductance following addition of the cAMP
agonist forskolin, which was reduced in a concentration-depen-
dent manner by a PPQ inhibitor, with complete inhibition at high
inhibitor concentration.
Re-examination of SAR data obtained from ∼350 commer-
cially available analogues (as reported in ref 30 and unpublished
data) suggested that the 5-position of the furan ring in 8 was
privileged. Therefore, compounds 14ꢀ23 were synthesized to
probe the steric and electronic requirements for activity of the
furyl moiety. Substituting bromine at the 5-position of the furyl
ring yielded 18, which was substantially more stable than 8
and had similar CFTR inhibition potency (Figure 3B,C). To
further probe oxidative aromatization as a metabolic pathway,
the secondary amine in 18 was replaced by oxygen forming
an ether bridge, which is unable to undergo oxidation. The
resulting benzoxazine 31 had similar CFTR inhibition potency
(Figure 3A) and better stability than 8. Synthesis of 32ꢀ33
confirmed the CFTR inhibition activity (Table 1) and improved
stability (Figure 3C) imparted by bromine and illustrated the
synergy between the 5-bromofuran moiety and the ether bridge.
Previous SAR data30 showed that an m-tolyl moiety (R1 = Me)
increased CFTR inhibition potency. Unfortunately, synthesis of
34ꢀ35 revealed that the m-tolyl moiety reduced CFTR inhibition
in the BPO series. To increase the polarity and hence the aqueous
solubility of 31, a nitro group was introduced at R2. The resulting
compound, 36, had substantially greater CFTR inhibition potency
(Figure 4A) with excellent metabolic stability in hepatic micro-
somes (Figure 4B). BPO analogs 37 and 38 showed that the
increased CFTR inhibition activity conferred by the nitro sub-
stituent at R2 was independent of both the 5-bromo substituent
and the ether bridge.
Our initial synthesis efforts focused on preventing aromatiza-
tion, which was initially achieved by derivatizing the secondary
amine of 8 to give analogues 9ꢀ13. The amides 9ꢀ11 were,
however, substantially less active than 8. As the loss of the basic
amine coincided with a loss in activity, we synthesized nitrosa-
mine 12, but our attempts to reduce the nitrosamine to a strongly
To further increase compound polarity, we replaced the nitro
functionality at R2 in 36 with a carboxyl moiety to give 42.
Compound 42 was found the most potent CFTR inhibitor to date
with IC50 ∼ 8 nM (Figure 4A) and had excellent stability in
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Table 1. CFTR Inhibition by PPQ and BPO Analogues
compd
X
Y
R1
R2
R3
R4
R5
R6
IC50 (μM)a
IC50 (μM)b
8
O
O
O
O
O
O
N
N
N
N
N
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
0.25
0.1
9
H
H
H
H
H
CH3SO2
ClCH2CO2
CH3CO2
NO
inactive
inactive
inactive
2
10
11
12
13
Me
1
14
15
16
17
18
19
20
21
22
23
O
S
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
Et
0.3
Me
Me
Cl
1.7
O
O
O
O
O
O
O
O
1.7
0.15
Br
0.09
0.05
0.1
I
0.17
CF3
Ph
0.35
inactive
inactive
inactive
CH2OH
ꢀ(CH)2ꢀ
ꢀ(CH)2ꢀ
31
32
33
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
Br
I
0.2
0.09
0.5
Me
0.15
34
35
O
O
O
O
Me
Me
H
H
H
H
H
H
Cl
0.37
0.6
Me
36
37
38
39
O
O
O
O
O
O
N
O
H
H
H
H
NO2
NO2
NO2
Cl
H
H
H
H
H
Br
0.12
0.1
0.025
0.025
H
Me
Br
H
H
0.17
0.17
NO2
Me
40
41
42
O
O
O
O
O
O
H
H
H
COOEt
COOEt
COOH
H
H
H
H
H
H
Br
0.05
0.08
0.04
0.025
0.008
Me
Br
a By microplate reader assay. b By short-circuit current assay.
hepatic microsomes with <5% compound loss in 30 min
(Figure 4B). At physiological pH, 42 is deprotonated and thus
substantially more polar than 8 (clogP 1.76 vs 4.92), with solubility
∼17 μM in a pH 7.4 aqueous phosphate buffer. BPO analog 42 was
synthesized by hydrolysis of the ethyl ester 40, which also strongly
inhibited CFTR, and could potentially serve as a pro-drug of 42.
Inhibition of Cyst Growth in a Kidney Organ Culture
Model of PKD. An established embryonic ex vivo kidney organ
culture model of PKD was used to test the efficacy of 42 in
reducing cAMP agonist-induced renal cystogenesis. Figure 5A
shows progressive renal cyst formation and growth in 8-Br-
cAMP agonist-treated cultures, as seen by transmitted light
microscopy. Kidney growth without cyst formation was seen in
the absence of 8-Br-cAMP. Cyst growth in 8-Br-cAMP-treated
kidneys was remarkably reduced by inclusion of 42 in the culture
medium. As quantified by percentage area occupied by cysts, 42
inhibited cyst growth with IC50 ∼ 100 nM (Figure 5B), much
better than that of >500 nM measured for 8.30
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Figure 3. Characterization of PPQ analogues. (A) Fluorescence plate-reader assay of CFTR inhibition. FRT cells expressing human wild-type CFTR
and iodide-sensing YFP fluorescent dye were incubated with test compound and CFTR agonists and then subjected to an inwardly directed iodide
gradient. (top) Representative data showing kinetics of fluorescence decrease following iodide addition (causing YFP fluorescence quenching) in the
absence of cAMP agonists and in the presence of cAMP agonists and indicated concentrations of 18. (bottom) Summary of concentrationꢀinhibition
data for indicated compounds (SE n = 4). See Table 1 for IC50 values. (B) Short-circuit current analysis of CFTR inhibition in CFTR-expressing FRT
cells in the presence of a transepithelial chloride gradient and basolateral membrane permeabilization. Where indicated, forskolin (20 μM) was added to
activate CFTR chlorideconductance, followingbyindicated concentrations of 18. (C) (top) LS/MS analysis showingdisappearanceof18and 32 in hepatic
microsomes in the presence of NADPH. (bottom) Summary of kinetics of compound disappearance (SEM, n = 3). Data for 8 shown for comparison.
as well as greater polarity and potency for inhibition of CFTR
chloride conductance in vitro (∼10-fold) and renal cystogenesis
ex vivo (>5-fold). SAR analysis suggested that the greater stability
of 42 over 8 is the consequence of the 5-Br substituted furan and
the ether bridge, which largely prevent hydroxylation and
aromatization modifications. The greatly improved water solu-
bility of 42 over 8 is a consequence of the carboxylic acid
substituent, which is charged at physiological pH. The carboxylic
acid addition also, unexpectedly, improved CFTR inhibition
potency. The high CFTR inhibition potency of the esterified
form of 42, 40, suggests its possible use as a pro-drug for efficient
intestinal absorption and cell accumulation following de-ester-
ification by ubiquitous intracellular esterases.
Several noteworthy observations were made in synthesizing
the PPQ analogues. In an experiment intended to improve yield,
Figure 4. BPO CFTR inhibitors with high potency, metabolic stability,
bromination of ketone 3 was conducted under strict anhydrous
and water solubility. (A) Short-circuit current analysis showing CFTR
inhibition by 36 and 42. (B) Compound stability in hepatic microsomes
(1 equiv), but after several hours TLC showed little product.
in the presence of NADPH.
conditions. Ketone 3 was refluxed in CH2Cl2 and bromine
On a whim, several drops of wet CH2Cl2 were added to the
reaction, which resulted in remarkably rapid discharge of the
bromine color and evolution of fuming HBr gas. After several
’ DISCUSSION AND CONCLUSIONS
minutes, TLC indicated a near quantitative yield of 4, which spon-
taneously crystallized whendriedinvacuo. When4 was condensed
The best compound emerging from this study, 42, had
substantially improved metabolic stability (by >10-fold) over 8,
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Figure 5. Compound 42 reduces renal cytogenesis. (A) Transmission light micrographs of E13.5 embryonic kidneys cultured for indicated days without
or in the presence of 100 μM 8-Br-cAMP, and with 0, 0.1, or 1 μM 42. (B) Summary of percentage cyst areas at 5 days in cultures (SEM, n = 4ꢀ6,
* P < 0.001.
with N-methyl-1,2-phenylenediamine, the more reactive second-
ary amine can undergo alkylation by displacement of the readily
accessible bromide in 4. The high regioselectivity evident in the
83% yield of 5 suggests that the formation of the pyrrole ring in the
initial reaction in is one of imine formation rather than alkylation
despite the crowded reaction center.
complementary data on CFTR function in the absence of
compensatory phenomena that can occur in transgenic animal
models.
In conclusion, the synthesis and evaluation of a rationally
designed series of PPQ analogues produced compounds with
much improved metabolic stability, CFTR inhibitory potency,
and aqueous solubility compared with reference compound 8.
Compound 42 and related analogues are thus suitable for in vivo
testing of efficacy and represent potential development candi-
dates for antisecretory therapy of PKD.
ADPKD is a major health care burden with a prevalence of 1 in
500ꢀ1000 individuals.1 There is no approved drug at present to
slow the progression of renal disease in PKD. As mentioned in
the Introduction, there is compelling rationale and experimental
proof-of-concept for CFTR inhibitor antisecretory therapy for
PKD. An alternative antisecretory therapy, vasopressin V2 antag-
onism, isinclinical trials forPKD and is based onthe ideathatcysts
in PKD often express V2 receptors, which, when stimulated by
antidiuretic hormone, elevate cytoplasmic cAMP and activate both
CFTR chloride conductance and mTOR signaling.15,32 Our lab
introduced an alternative, V2 receptor-independent strategy to
reduce cAMP involving small-molecule phosphodiesterase activa-
tors, which were shown to cyst growth in an in vitro PKD
model.33 Pure antiproliferative therapies, most based on the
central role of mTOR signaling in proliferation of cyst-lining
epithelial cells, are also in clinical trials as well as renin-angiotensin
inhibitors and statins.16ꢀ18 Whether antiproliferative or antisecre-
tory therapy alone, or in combination, will slow the progression of
kidney destruction in PKD awaits human clinical trials.
Compound 42, with IC50 ∼ 8 nM, is substantially more potent
than existing CFTR inhibitors. In addition to their potential
utility for antisecretory therapy of PKD, potent small-molecule
CFTR inhibitors have potential efficacy in the therapy of
secretory diarrheas and as research tools in defining CFTR-
dependent cellular and physiological processes. Secretory diar-
rheas caused by enterotoxins, such as cholera and travelers’
diarrhea (enteropathogenic E. coli), require functional CFTR
for primary chloride secretion into the intestinal lumen, which
secondarily drives sodium and water secretion.34,35 Testing of 42
and related PPQ analogues is needed to establish their efficacy in
animal models of secretory diarrheas, as has been done for
thiazolidinone36 and glycine hydrazide28,29 CFTR inhibitors.
CFTR inhibitors are also useful to create the CF phenotype in
ex vivo human and animal tissues, as has been done, for example,
in studies of the role of CFTR in airway submucosal gland fluid
secretion.37 Finally, although mouse, pig, and ferret models of
CFTR gene deletion exist, pharmacological creation of the
CFTR phenotype in animals by CFTR inhibitors might provide
’ EXPERIMENTAL SECTION
Cell Culture and Platereader Assay of CFTR Inhibition.
Fischer rat thyroid (FRT) cells coexpressing human wild-type CFTR
and the halide indicator YFP-H148Q were cultured in 96-well black-
walled microplates (Corning Costar) at a density of 20000 cells per well
in Coon’s modified F12 medium containing 10% fetal bovine serum,
2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.
CFTR chloride conductance was assayed at 48 h after plating on a
FluoStar fluorescence plate-reader (BMG Lab Technologies) as
described.21 Each well was washed 3 times with PBS, leaving 60 μL of
PBS. Test compounds were added and incubated with the cells for
45 min. Then, 5 μL of a CFTR-activating cocktail (10 μM forskolin,
100 μM IBMX, 20 μM apigenin in PBS) was added. After 15 min, each
well was assayed for iodide influx by recording fluorescence continu-
ously (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.
Short-Circuit Current. Snapwell inserts containing CFTR-expres-
sing FRT cells were mounted in an Ussing chamber. The hemichambers
contained 5 mL of buffer containing 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, as described.27 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.
Liquid Chromatography/Mass Spectrometry. Compounds
(each 5 μM) were incubated for specified times at 37 °C with rat
liver microsomes (1 mg protein/mL; Sigma-Aldrich, St. Louis, MO) in
potassium phosphate buffer (100 mM) containing NADPH (0 or 1 mM).
The mixture was then chilled on ice, and 0.5 mL of ice-cold ethyl acetate
was added. Samples were centrifuged for 15 min at 3000 rpm, and the
supernatant was evaporated to dryness under nitrogen. The residue was
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Journal of Medicinal Chemistry
ARTICLE
dissolved in 150 μL mobile phase (acetonitrile:water 3:1, containing 0.1%
formic acid) for LC/MS analysis. Reverse-phase HPLC separations were
carried out using a Waters C18 column (2.1 mm ꢁ 100 mm, 3.5 mm
particle size) equipped with a solvent delivery system (Waters model 2690,
Milford, MA). The solvent system consisted of a linear gradient from 5 to
95% acetonitrile run over 16 min (0.2 mL/min flow rate). Mass spectra
were acquired on an Alliance HT 2790 + ZQ mass spectrometer using
negativeion detection, scanning from 150 to 1500 Da. The electrospray ion
source parameters were: capillary voltage 3.5 kV (positive ion mode), cone
voltage 37 V, source temperature 120 °C, desolvation temperature 250 °C,
cone gas flow 25 L/h, and dessolvation gas flow 350 L/h.
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.30 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 streptomycin. Kidneys were maintained in a
37 °C humidified CO2 incubator for up to 8 days. Culture medium
containing 100 μM 8-Br-cAMP, with or without test compound, was
replaced (in the lower chamber) every 12 h. Kidneys were photographed
using a Nikon inverted microscope (Nikon TE 2000-S) equipped with
2ꢁ 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.
was added 2 (5.00 g, 32.4 mmol), anhydrous zinc chloride40 (freshly
dried, 4.45 g, 32.6 mmol), dry chlorobenzene (20 mL), and benzoyl
chloride (freshly distilled, 4 mL, 4.84 g, 34.4 mmol). The reaction was
refluxed in an oil bath equipped with an air lock and vigorously stirred for
3 h. The reaction was allowed to cool and the condenser arranged for
distillation. Water (40 mL) was added dropwise at first and then with
increasing speed. Chlorobenzene (30 mL) and water were distilled off,
and the mixture was cooled on ice. Diethyl ether (30 mL) was added
while stirring, causing a precipitate to form. The precipitate was collected
by filtration and recrystallized from 2-propanol (50 mL) to yield 3a
(5.53 g, 66%); mp 143.2ꢀ143.7 °C. 1H NMR (600 MHz, CD3CN) δ
8.74 (d, J = 7.1 Hz, 2H), 8.46 (t, J = 6.0 Hz, 1H), 8.37ꢀ8.29 (m, 2H),
4.25 (s, 3H), 4.07 (s, 3H), 2.99 (s, 3H). 13C NMR (151 MHz, CD3CN)
δ 195.6, 162.2, 153.7, 153.2, 139.2, 134.9, 130.5, 130.0, 113.4, 32.8, 28.6,
18.3. MS (ES+) (m/z): [M + 1]+ calculated for C14H15N2O3, 259.28,
found 259.11.
5-Benzoyl-6-(bromomethyl)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione
(4).39 In a 50 mL round-bottom flask, 3 (1.00 g, 38.7 mmol) was
dissolved in CCl4 (5 mL) and CH2Cl2 (4 mL) at 50 °C. Bromine
(200 μL, 0.621 g, 38.8 mmol) was mixed with CCl4 (5 mL) and CH2Cl2
(5 mL) in an addition funnel and added dropwise to the solution of 3
such that the brown color disappeared between drops. The last few
drops caused the reaction to remain brown. The reaction was then
brought to reflux for 10 min before the brown color was discharged by
the addition of a few drops of acetone. The reaction was refluxed for
30 min to remove HBr. The reaction was quantitative by TLC, and the
product crystallized when concentrated in vacuo to yield 4 (1.305 g,
100%). The product was recrystallized from 2-propanol as colorless
needles; mp 171.0ꢀ171.7 °C. 1H NMR (600 MHz, CD2Cl2) δ
7.88ꢀ7.80 (m, 2H), 7.65ꢀ7.60 (m, 1H), 7.52ꢀ7.45 (m, 2H), 4.24
(s, 2H), 3.59 (s, 3H), 3.31 (s, 3H). 13C NMR (151 MHz, CD2Cl2) δ
192.9, 160.9, 152.0, 149.6, 137.8, 134.5, 129.8, 129.2, 114.8, 32.0, 28.6,
23.7. MS (ES+) (m/z): [M + 1]+ calculated for C14H14BrN2O3, 338.18,
found 338.81.
Ethyl 3-(1,3-Dimethyl-2,4-dioxo-5-phenyl-3,4-dihydro-1H-pyrrolo-
[3,4-d]pyrimidin-6(2H)-yl)-4-hydroxybenzoate (30). In a 100 mL
round-bottom flask, ethyl 3-amino-4-hydroxybezoate (1.10 g, 6.07 mmol)
and 4 (1.00 g, 2.98 mmol) were refluxed in ethanol (50 mL). After 2 h,
the condenser was rearranged for distillation, and ethanol (25 mL)
was distilled off. The resulting solution was slowly poured into a
vigorously stirred solution of ice-cold water (200 mL) and citric acid
(50 mg), giving a pink solid precipitate. The mixture was stirred for 10
min, and then the solid was collected by filtration and rinsed with cold
water to give 30 (1.23 g, 98.5%) after drying; mp 129.2ꢀ130 °C.
1H NMR (600 MHz, CD2Cl2) δ 8.58 (s, 1H), 7.89 (dd, J = 2.1 Hz, 8.7,
1H), 7.67 (d, J = 2.1 Hz, 1H), 7.36ꢀ7.29 (m, 2H), 7.28ꢀ7.18 (m, 3H),
6.97 (d, J = 8.7 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 3.27 (s, 3H), 3.17
(s, 3H), 1.30 (t, J = 7.1 Hz, 3H). 13C NMR (151 MHz, CD2Cl2)
δ 165.4, 159.7, 156.8, 151.9, 136.1, 132.1, 130.9, 130.7, 129.5, 129.0,
128.8, 127.8, 125.7, 122.9, 117.7, 104.7, 103.1, 61.2, 32.0, 28.1, 14.2. MS
(ES+) (m/z): [M + 1]+ calculated for C23H22N3O5, 420.16, found 420.13.
Ethyl 6-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-
7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [40,50:3,4]pyrrolo[1,2-d]-
[1,4]oxazine-2-carboxylate (40). Pyrrole 30 (500 mg, 1.19 mmol),
5-bromofurfural (240 mg, 1.37 mmol), chloroform (7 mL), TFA (10 μL,
14.8 mg, 130 μmol), and 3 Å molecular sieves (2.0 g, 8ꢀ12 mesh beads)
were sealed in an Emrys 10ꢀ20 mL process vial and submerged to the
level of solvent in an oil bath at 150 °C. The reaction was stirred for
24 min and then removed from the oil bath. Once the internal pressure
dropped the reaction vial was rapidly cooled in water. After cooling, the
reaction was filtered through Celite into a 50 mL recovery flask and then
dried in vacuo. The residue was dissolved in a minimum amount of
CH2Cl2 and quickly diluted with warm ethanol (25 mL). Fine crystals
began to form immediately. The mixture was then placed on a rotary
Synthesis Procedures. NMR spectra (1H at 600 MHz; 13C at
150 MHz) were obtained in methylene chloride (CD2Cl2), chloroform
(CDCl3), acetonitrile (CD3CN), or dimethyl sulfoxide (DMSO-d6)
using a 600 MHz Varian spectrometer. Chemical shifts are expressed in
parts per million relative to the solvent. NMR spectra for PPQ and BPO
compounds were acquired at ꢀ20 °C due to excessive broadening of the
11-phenyl protons at ambient temperature. NMR spectra for the
intermediates were obtained at ambient temperature. Mass spectro-
metry was done using a Waters LC/MS instrument with MS, electro-
spray (+) ionization, mass ranging from 100 to 900 Da, 20 V cone
voltage; LC, Xterra MS C18 column (2.1 mm ꢁ 50 mm ꢁ 3.5 μm),
0.2 mL/min water/acetonitrile (containing 0.1% TFA). Purity was
judged by the peak area percentage of the UV absorbance signal.
Compound purities by RP-HPLC were >98%. Flash chromatography
was done using EM silica gel (230ꢀ400 mesh), and thin-layer chroma-
tography was done on EMD silica gel 60 F254 plates (Darmstadt,
Germany). Melting points are uncorrected. To follow is the synthesis
procedure for 42. See Supporting Information for synthesis of all PPQ
and BPO compounds, and analytical data.
1,3,6-Trimethyl-1H,3H-pyrimidine-2,4-dione (2).38 In a 250 mL
round-bottom flask, 6-methyluracil (1; 15.0 g, 119 mmol) and NaOH
(9.55 g, 239 mmol) were dissolved in water (150 mL) at low heat. The
solution was brought to 25 °C in an ice bath and maintained at 25 °C
while dimethyl sulfate (23 mL, 30.59 g, 243 mmol) was added dropwise
over 20 min with vigorous stirring. After 22 h, the reaction mixture
contained a white precipitate and had pH 9. NaOH (5.0 g, 125 mmol)
was added to make the solution homogeneous, and an ice bath was used
to maintain a temperature of 25 °C while dimethyl sulfate (12 mL,
15.96 g, 127 mmol) was added dropwise over 10 min. The reaction was
stirred for 72 h, then NaOH (2 g, 50 mmol) was added and the reaction
extracted with CHCl3 (5 ꢁ 50 mL). The chloroform extracts were
pooled, dried over Na2SO4, and concentrated in vacuo to yield 2 (18 g,
98% yield); mp 114ꢀ115 °C. 1H NMR (600 MHz, CDCl3) δ 5.67 (s,
1H), 3.45 (s, 3H), 3.35 (s, 3H), 2.29 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 162.6, 152.5, 151.8, 101.2, 31.9, 28.1, 20.6. MS (ES+) (m/z):
[M + 1]+ calculated for C7H11N2O2, 155.17, found 155.14.
5-Benzoyl-1,3,6-trimethylpyrimidine-2,4(1H,3H)-dione (3).39 In a
100 mL round-bottom flask equipped with a condenser and an air lock
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Journal of Medicinal Chemistry
ARTICLE
evaporator, and the CH2Cl2 was removed to increase the quantity of
crystals. The mixture was then chilled, filtered, and the crystals rinsed
with cold ethanol to give 40 (0.500 g, 76.4%) as fine white needle-like
crystals. No mp (slow decomposition). 1H NMR (600 MHz, CD2Cl2) δ
7.81 (d, J = 7.7 Hz, 1H), 7.68 (dd, J = 1.9 Hz, 8.4, 1H), 7.63 (t, J = 7.5 Hz,
1H), 7.52 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.23 (d, J = 1.8 Hz,
1H), 7.09 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 7.7 Hz, 1H), 6.86 (s, 1H), 6.14
(d, J = 3.4 Hz, 1H), 5.98 (d, J = 2.9 Hz, 1H), 4.11 (dq, J = 7.2 Hz, 10.7,
1H), 4.00 (dq, J = 7.1 Hz, 10.7, 1H), 3.48 (s, 3H), 3.26 (s, 3H), 1.14
(t, J = 7.1 Hz, 3H). 13C NMR (151 MHz, CD2Cl2) δ 165.0, 159.3, 151.9,
151.8, 149.1, 131.8, 131.3, 130.0, 129.8, 129.7, 128.8, 128.8, 128.3, 125.4,
124.7, 124.5, 124.4, 121.6, 119.6, 114.9, 112.4, 105.9, 105.7, 68.4, 61.3,
32.4, 28.0, 14.2. MS (ES+) (m/z): [M + 1]+ calculated for C28H23-
BrN3O6, 576.08, found 576.04.
Development Program and Drug Discovery grants from the
Cystic Fibrosis Foundation.
’ ABBREVIATIONS USED
ADPKD, autosomal dominant polycystic kidney disease; BPO,
benzopyrimido-pyrrolo-oxazinedione; CFTR, cystic fibrosis
transmembrane conductance regulator; CPT-cAMP, chlorophe-
nylthio-cAMP; IBMX, isobutylmethylxanthine; PKD, polycystic
kidney disease; PPQ, pyrimido-pyrrolo-quinoxalinedione; YFP,
yellow fluorescent protein
’ REFERENCES
6-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-
tetrahydro-6H-benzo[b]pyrimido [40,50:3,4]pyrrolo[1,2-d][1,4]oxazine-
2-carboxylic Acid (42). In a 500 mL round-bottom flask equipped with
stir bar, 40 (1.00 g, 1.73 mmol) was dissolved in THF (100 mL) with
gentle heat and then allowed to cool. A mixture of water (70 mL) and
KOH (768 mg, 13.7 mmol) was quickly added to the vigorously stirred
solution, which formed a white suspension. After 3 days, the mixture was
a homogeneous yellow solution free of 40 as determined by LC/MS.
The THF was removed using a rotary evaporator and warm water bath,
leaving behind a viscous aqueous solution. The solution was made
strongly acidic to litmus using 1% aq HCl while stirring vigorously with a
glass rod. The resulting gel was shaken with EtOAc (125 mL) and then
quickly poured into a 1 L separatory funnel, where a precipitate formed
in the organic layer. The 500 mL flask was rinsed with additional EtOAc
(125 mL). Additional EtOAc (400 mL) was added until all solids
dissolved. After settling, the lower yellow aqueous layer was discarded.
The EtOAc layer was washed with brine, dried over Na2SO4, and dried
on a rotary evaporator. The resulting slightly yellow amorphous powder
was loosened by swirling with CH2Cl2 (15 mL) and then diluted with
diethyl ether (15 mL). The solids were collected by filtration and rinsed
with CH2Cl2:Et2O (1:1) to give 42 (791 mg, 83.2%) as a white solid. No
mp (slow decomposition). 1H NMR (600 MHz, 91% CD2Cl2, 9%
DMSO-d6) δ 12.30 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.63 (dd, J = 1.9 Hz,
8.4, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.29 (t, J =
7.5 Hz, 1H), 7.17 (d, J = 1.7 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 7.01
(d, J = 7.7 Hz, 1H), 6.89 (s, 1H), 6.12 (d, J = 3.4 Hz, 1H), 5.96 (d, J = 3.3
Hz, 1H), 3.44 (s, 3H), 3.21 (s, 3H). 13C NMR (151 MHz, 91% CD2Cl2,
9% DMSO-d6) δ 166.7, 159.1, 151.9, 151.6, 149.0, 131.8, 131.0, 129.7,
129.7, 129.5, 128.6, 128.5, 128.5, 125.3, 125.2, 124.4, 124.2, 121.9, 119.4,
114.8, 112.3, 106.0, 105.7, 68.2, 32.3, 27.8. MS (ES+) (m/z): [M + 1]+
calculated for C26H19BrN3O6, 548.05, found 548.00.
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’ ASSOCIATED CONTENT
S
Supporting Information. Detailed synthesis procedures
b
and NMR spectra for all compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: 415-4768530. Fax: 415-6653847. E-mail: alan.verkman@
ucsf.edu. Webstie: http://www.ucsf.edu/verklab. Address:
University of California, San Francisco, Box 0521, HSE 1246,
513 Parnassus Avenue, San Francisco, CA 94143-0521.
’ ACKNOWLEDGMENT
This work was supported by NIH grants DK86125, DK72517,
HL73856, EB00415, DK35124, and EY13574 and Research
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