4,8-Dimethylcoumarin Inhibitors of Intestinal Anion Exchanger slc26a3 (Downregulated in Adenoma) for Anti-Absorptive Therapy of Constipation

Article abstract of DOI:10.1021/acs.jmedchem.9b01192

The chloride/bicarbonate exchanger SLC26A3 (downregulated in adenoma) is expressed mainly in colonic epithelium, where it dehydrates the stool by facilitating the final step of chloride and fluid absorption. SLC26A3 inhibition has predicted efficacy in various types of constipation including that associated with cystic fibrosis. We previously identified, by high-throughput screening, 4,8-dimethylcoumarin inhibitors of murine slc26a3 with IC₅₀ down to a150 nM. Here, we synthesized a focused library of forty-three 4,8-dimethylcoumarin analogues. Structure-activity studies revealed the requirement of 4,8-dimethylcoumarin-3-acetic acid for activity. The most potent inhibitors were produced by replacements at C7, including 3-iodo- (4az) and 3-trifluoromethyl- (4be), with IC₅₀ of 40 and 25 nM, respectively. Pharmacokinetics in mice showed predicted therapeutic concentrations of 4az for >72 h following a single 10 mg/kg oral dose. 4az at 10 mg/kg fully normalized stool water content in a loperamide-induced mouse model of constipation. The favorable inhibition potency, selectivity within the SLC26 family, and pharmacological properties of 4az support its further preclinical development.

Full text of DOI:10.1021/acs.jmedchem.9b01192

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Article
4,8-Dimethylcoumarin inhibitors of intestinal anion exchanger slc26a3 (down-
regulated in adenoma, DRA) for anti-absorptive therapy of constipation
Sujin Lee, Onur Cil, Peter Haggie, and Alan S Verkman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01192 • Publication Date (Web): 07 Aug 2019
Downloaded from pubs.acs.org on August 8, 2019
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Journal of Medicinal Chemistry
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4,8-Dimethylcoumarin inhibitors of intestinal anion exchanger
slc26a3 (down-regulated in adenoma, DRA) for anti-absorptive
therapy of constipation
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Sujin Lee1, Onur Cil1,2, Peter M. Haggie1 and Alan S. Verkman*,1,3
Departments of 1 Medicine, 2 Pediatrics and 3 Physiology, University of California, San Francisco CA 94143-0521, USA
KEYWORDS: Constipation, cystic fibrosis, SLC26A3, DRA
ABSTRACT: The chloride/bicarbonate exchanger SLC26A3 (down-regulated in adenoma, DRA) is expressed mainly in colonic
epithelium where it dehydrates the stool by facilitating the final step of chloride and fluid absorption. SLC26A3 inhibition has
predicted efficacy in various types of constipation including that associated with cystic fibrosis. We previously identified, by high-
throughput screening, 4,8-dimethylcoumarin inhibitors of murine slc26a3 with IC50 down to ~150 nM. Here, we synthesized a focused
library of forty-three 4,8-dimethylcoumarin analogs. Structure-activity studies revealed the requirement of 4,8-dimethylcoumarin-3-
acetic acid for activity. The most potent inhibitors were produced by replacements at C7, including 3-iodo- (4az) and 3-
trifluoromethyl- (4be), with IC50 of 40 nM and 25 nM, respectively. Pharmacokinetics in mice showed predicted therapeutic
concentrations of 4az for >72 h following a single 10 mg/kg oral dose. 4az at 10 mg/kg fully normalized stool water content in a
loperamide-induced mouse model of constipation. The favorable inhibition potency, selectivity within the SLC26 family and
pharmacological properties of 4az support its further preclinical development.
INTRODUCTION
Constipation is a common clinical problem affecting ~15% of the US population,1 with at least 3-fold greater prevalence in cystic
fibrosis (CF) due to impaired function of the pro-secretory chloride channel CFTR in the intestine.2 Currently available treatments for
chronic constipation include dietary and lifestyle changes, over-the-counter medications such as osmotic and stimulant laxatives, and
FDA-approved prescription drugs that stimulate intestinal fluid secretion.3-4 The approved pro-secretory drugs lubiprostone,
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linaclotide and plecanatide activate CFTR and perhaps apical membrane chloride channels indirectly, and showed limited efficacy in
clinical trials.4 These drugs are unlikely to be effective in CF because they primarily rely on functional CFTR. We recently identified
activators of wild-type CFTR with pro-secretory action that showed greater efficacy than lubiprostone and linaclotide in mouse models
of constipation.5-6 The CFTR activators, as expected, were not effective in CF mice lacking functional CFTR. An alternative approach
for increasing stool hydration in constipation is inhibition of intestinal fluid absorption. Tenapanor, an inhibitor of the sodium-
hydrogen exchanger 3 (NHE3) in small intestine and proximal colon,7 recently completed a Phase 3 clinical trial for constipation
predominant irritable bowel syndrome (IBS-C), showing limited efficacy,4 perhaps due to intact fluid absorption in more distal parts
of the intestine. There remains an unmet need for more efficacious anti-constipation drugs with alternative mechanisms of action for
the general population and particularly for CF subjects.
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The SLC26A3 protein, originally named down-regulated in adenoma (DRA), is a chloride/anion exchanger expressed most strongly
at the luminal plasma membrane of intestinal epithelial cells in colon.8-10 On the basis of the finding that loss-of-function mutations
in SLC26A3 in humans cause chloride-losing diarrhea,11 as does slc26a3 knockout in mice,12 SLC26A3 inhibition, by reducing colonic
fluid absorption and thus blocking terminal step of stool dehydration, is predicted to be effective as an anti-absorptive therapy for all
forms of constipation, including that associated with cystic fibrosis.
Using a cell-based high-throughput screen, we recently identified 4,8-dimethylcoumarin inhibitors of the slc26a3 anion exchanger.13
The most potent compound was 4,8-dimethyl-7-(m-bromobenzyloxy)-coumarin-3-acetic acid 4ba, which reversibly inhibited
SLC26A3-mediated anion transport with IC50 ~150 nM and showed efficacy in a mouse model of constipation. Here, following an
initial analysis of 176 commercially available analogs of the originally identified inhibitors, we synthesized a focused library of forty-
three 4,8-dimethylcoumarin analogs, which included a compound that inhibited slc26a3 selectively with IC50 of ~25 nM and had
favorable pharmacological properties and efficacy in an experimental mouse model of constipation.
Scheme 1. Synthesis of 4,8-dimethylcoumarin analogsa
8
O
O
O
O
O
O
7
MeO2C
MeO2C
HO
O
O
b
c or d
HO
OH
R
R
a
R-Br
3
CO2Me
CO2H
CO2Me
6
4
5
O
2
4aa-4bj
3aa-3bj
aReagents: (a) H2SO4, MeOH, 86%. (b) K2CO3, acetone, reflux, 62-99%. (c) NaOH, MeOH, 75 oC, 19-96%. (d) LiOH, MeCN, rt, 28% for
4aq, 27% for 4ar.
RESULTS
Preliminary structure-activity relationship (SAR) analysis. Initially, 176 commercially available coumarin analogs of our
originally reported inhibitor 4ba (DRAinh-A250) were tested.13 SAR revealed the requirement of methyl substitution at C4 and C8,
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Journal of Medicinal Chemistry
and an acetic acid moiety at C3, with C7 substituents producing a wide range of activity. Of 80 compounds purchased with different
C7 substituents (representative compounds shown in Figure S1), 7-hydroxy- (S1), 7-methyl- (S2) and 7-methoxy-coumarin (S3) were
inactive, as were styrenyloxy- (S11), naphthylmethyloxy- (S14), isoprenoxy- (S13), and several annulated coumarins and analogs
containing an additional fused ring. Based on the initial SAR, compounds having different C7 substitutions were synthesized, as well
as compounds with acetic acid replacements.
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Synthesis. Scheme 1 shows the synthesis of 4aa-4bj. Pechmann reaction of 2-methyl resorcinol with dimethyl acetylsuccinate in the
presence of sulfuric acid afforded 4,8-dimethyl-7-hydroxycoumarin ester 2 in 86% yield. O-Alkylation of 2 with alkyl bromides (3aa-
3aj) or aryl methylbromides (3ak-3ap, 3at-3bj) gave substituted benzyloxy-4,8-dimethylcoumarins 3aa-3bj in 28-98% yields, which
were hydrolyzed with 1N sodium hydroxide in methanol to give 4aa-4bj in 52-96% yields. For the benzenesulfonyl analogs 3aq and
3ar, substitution of 2 with m-Cl-, or m-CH3-benzenesulfonyl chloride with potassium carbonate give 3aq and 3ar in 68 and 71%
yields, respectively. Hydrolysis of esters 3aq and 3ar was accomplished with lithium hydroxide in acetonitrile to afford 4aq and 4ar.
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Scheme 2 shows the synthesis of formic acid derivatives 8aa-8ad. Methyl 8-methyl-7-hydroxybenzopyranone-3-carboxyl-ate 6a was
prepared by condensation of 2,4-dihydroxy-3-methylbenzaldehyde 5 with dimethyl malonate under sulfuric acid condition.
Alkylation of 6a with substituted benzyl bromide afforded 7aa and 7ab, which were hydrolyzed to give 8aa and 8ab. Aminolysis of
7aa with ammonia gas in THF gave amide analog 8ac. Hydroxamic acid analog 8ad was synthesized by reaction of ester 7aa with
hydroxylamine under basic conditions. For the propionic acid analogs, Pechmann type reaction with 2-methyl resorcinol and
diethylacetyl glutarate under acidic conditions afforded 6b. O-Alkylation of 6b with bromo- or iodobenzyl bromide gave 7ba-7bb
which upon hydrolysis under basic conditions gave 8ba-8bb.
Scheme 2. Synthesis of 8-methylcoumarin carboxylate analogsa.
HO
OH
HO
O
O
a
CO2Me
CO2Me
H
CO2Me
O
H
O
O
O
O
O
O
5
d or e or f
6a
c
X
X
R
Br
R1
R1
O
R2
R2
HO
O
O
EtO2C
b
HO
OH
8aa
8ab
8ac
8ad
8ba
8bb
X = Br, R1 = CO2H, R2 = H
X = I, R1 = CO2H, R2 = H
X = Br, R1 = CONH2, R2 = H
X = Br, R1 = CONHOH, R2= H
X = Br, R1 = CH2CH2CO2H, R2 = CH3
X = I, R1 = CH2CH2CO2H, R2 = CH3
7aa
7ab
7ba
7bb
X = Br, R1 = CO2Me, R2 = H
X = I, R1 = CO2Me, R2 = H
CO2Et
X = Br. R1 = CH2CH2CO2Et, R2 = CH3
X= I, R1 = CH2CH2CO2Et, R2 = CH3
CO2Et
2
6b
aReagents: (a) H2SO4, MeOH, 57%. (b) H2SO4, EtOH, 81%. (c) K2CO3, acetone, reflux, 86-93%. (d) NaOH, MeOH, 75 oC, 44-94%,
for 8aa-8bb. (e) NH3 in THF, 66% for 8ac. (f) NH2OH-H2O, NaOMe, MeOH, 54% for 8ad.
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SAR analysis for inhibition of slc26a3-mediated chloride/iodide exchange. All synthesized analogs were tested for inhibition of
slc26a3-mediated chloride/iodide exchange using a cell-based kinetic assay in which fluorescence was measured in Fischer rat thyroid
cells expressing (murine) slc26a3 and a yellow fluorescent protein halide sensor (YFP) following extracellular addition of iodide.8
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Table 1 summarizes slc26a3 inhibition data for compounds with different C7 substituents.
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Slc26a3 inhibition was compared for analogs containing various alkyl, allyl, heterocyclic, benzenesulfonyl, and substituted benzyl
groups at C7. Propyl (4ad) or allyl (4aa) analogs showed no inhibition at 5 µM. Inhibition with micromolar IC50 was seen with
elongated alkyl chains, including butoxy-, pentoxy- and hexyloxy- analogs (4ae-4ag). Analogs containing cyclic alkyl chains (4ah-
4aj) showed mildly reduced inhibition compared to linear alkyl analogs (4ad-4ag). To investigate heterocyclic analogs, substituted
pyridines, pyrrol and 5-trifluoromethylfuran were studied. Pyridine (4ak-4an) and pyrrol (4ap) analogs were inactive at 5 µM, with
only the 5-trifluoromethylfuran analog (4ao) showing strong activity with IC50 of 0.3 µM, similar to that of original compound
DRAinh-A250 (4ba). To investigate benzyl analogs, o-, m- and p-halobenzyl bromides were studied (4at-4bc). p-Substituted
halobenzyl analogs (4at-4av) showed no inhibition at 5 µM, whereas o-substituted halobenzyl analogs (4aw-4ay) showed weak
inhibition, with IC50 of 4.5 µM for o-iodobenzyl (4aw), 10 µM for o-bromobenzyl (4ax), and 18 µM for o-chlorobenzyl (4ay) analogs.
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Notably, m-substituted halobenzyl analogs showed strong inhibition, with IC50 of 0.04 µM for iodobenzyl 4az. With the knowledge
that meta-substitution increased inhibition activity, we studied m-nitro 4bd, m-trifluoromethyl 4be, and m-cyclopropyl 4bf analogs.
m-Nitrobenzyl 4bd was less potent (IC50 0.5 µM) than 4bb, and m-cyclopropylbenzyl 4bf was more potent (0.07 µM), with m-
trifluoromethylbenzyl 4be having the greatest potency with IC50 of 0.025 µM. For comparison, the commercially available m-
methylbenzyl analog 4bk had IC50 of 0.5 µM. The 3,5-dimethylbenzyl analog 4bg had much reduced potency compared to its mono-
substituted analog 4bk, and 3,5-bistrifluoromethylbenzyl analog 4bh (IC50 0.3 µM) was much less potent than monosubstituted 4az.
Additional F-substitutions (4bi, 4bj) containing m-trifluoromethylbenzyl mildly reduced inhibition compared to 4az, with additional
m-substitution (4bj) showing greater inhibition than o-substitution (4bi). m-Trifluoromethylphenethyl analog 4as showed no
inhibition at 5 µM. Benzenesulfonyl analogs 4aq and 4ar showed no inhibition at 5 µM.
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Journal of Medicinal Chemistry
Figure 1. Concentration dependent slc26a3-mediated anion exchange inhibition data. (A) Inhibition of slc26a3-mediated Cl- / I- exchange
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by indicated compounds (mean ± S.E.M., n = 3). (B) Inhibition of slc26a3-mediated Cl- / HCO3 exchange (mean ± S.E.M., n = 12–28
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individual cell regions analyzed from 3 or more separate replicates). Curves are data fits to single-site inhibition model.
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Table 1. Inhibition of DRA (slc26a3-mediated Cl–/I– exchange) by 4aa-4bl.
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O
O
O
R
O
7
3
OH
4
Isolated
Yield (%)
60
Isolated
Yield (%)
84
Compound
R
IC50 (µM)a
Compound
R
IC50 (µM)a
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
4ai
Allyl-
>5
4at
4-Iodobenzyl
4-Bromobenzyl
4-Chlorobenzyl
2-Iodobenzyl
>5
>5
CH3OCH2CH2-
HO2CCH2CH2-
CH3CH2CH2-
CH3(CH2)2CH2-
CH3(CH2)3CH2-
CH3(CH2)4CH2-
(CH2)4CH-
19
66
76
76
96
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85
76
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>5
4au
4av
4aw
4ax
4ay
4az
4ba
4bb
4bc
75
79
68
59
63
56
60
73
62
>5
>5
>5
4.5±1.3
10±2.5
18±3
1.5±0.3
1.0±0.4
1.5±0.4
4.5±2.0
3.4±0.8
1.8±0.5
2-Bromobenzyl
2-Chlorobenzyl
3-Iodobenzyl
0.04±0.003
0.15±0.02
1.0±0.5
0.9±0.4
3-Bromobenzyl
3-Chlorobenyzl
3-Fluorobenzyl
(CH2)3CHCH2-
(CH2)4CHCH2-
4aj
N
4ak
4al
23
28
>5
>5
4bd
4be
3-Nitrobenzyl
3-(CF3)-benzyl
52
90
0.5±0.1
N
0.025±0.01
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3-cyclopropyl-
benzyl
N
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4
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7
8
4am
4an
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45
>5
>5
4bf
88
84
0.07±0.003
3,5-dimethyl-
benzyl
4bg
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0.3±0.1
0.1±0.1
0.06±0.05
HO
N
9
F3C
3,5-bis(CF3)-
benzyl
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O
4ao
4ap
4aq
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4bh
4bi
4bj
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2-F, 5-(CF3)-
benzyl
N
>5
3-F, 5-(CF3)-
benzyl
O
S
>5
O
O
S
4ar
4as
27
88
>5
>5
4bkb
3-CH3-benzyl
-
0.5±0.2
Cl
O
4bl
34
>5
F3C
N
CF3
a) Data representative of 3 replicates. b) Purchased from ChemDiv.
Next, the acetic acid portion on C3 was investigated. Preliminary data showed that acetic acid had much greater inhibition potency
than its corresponding ester, and that benzyl or alkyl in place of acetic acid reduced inhibition. To determine the best acid substituents
on C3, we tested formic acid and propionic acid analogs with Br- or I-substituted 7-oxybenzyl-8-dimethylcoumarin. As listed in Table
2, replacement of acetic acid on C3 with formic acid (8aa-8ab) or propionic acid (8ba-8bb) reduced inhibition. Interestingly,
hydroxamic acid 8ad had IC50 of 0.45 µM, whereas its corresponding acid 8aa was inactive. To compare with 4ba, the corresponding
amide of 8aa, namely 8ae, showed much reduced inhibition, supporting the importance of the acetic acid moiety for inhibition. To
investigate inhibitor selectivity, 4be and 4az were chosen for further studies.
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Table 2. Inhibition of DRA (slc26a3-mediated Cl–/I– exchange) by 8aa-8ae, 8ba-8bb.
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O
O
O
X
R1
R2
Compound
X
R1
R2
Isolated Yield (%)
60
IC50 (µM)a
0.15±0.02
4ba
Br
CH2CO2H
Me
H
8aa
8ab
8ac
8ad
8ae
8ba
8bb
Br
I
CO2H
44
62
66
34
57
89
94
>5
>5
CO2H
H
Br
Br
Br
Br
I
CONH2
H
>5
CONHOH
CH2CONH2
CH2CH2CO2H
CH2CH2CO2H
H
0.45±0.1
3.3±0.4
>5
Me
Me
Me
>5
a) Data representative of 3 replicates.
Inhibition of DRA (slc26a3)-mediated chloride / bicarbonate exchange. The concentration-dependence measurements reported
in Table 1 and 2 were done for inhibition of slc26a3-mediated chloride/iodide exchange. Fig. 1A shows representative data obtained
using the YFP quenching assay, which fitted well to a single-site inhibition model with IC50 of 0.040 and 0.025 µM for 4az and 4be,
respectively. For chloride / bicarbonate exchange, which is the physiologically relevant transport process in colon, cytoplasmic pH
was measured in slc26a3-expressing FRT cells using the pH-sensitive fluorescent indicator BCECF.9 BCECF-labeled cells were
incubated with a chloride- and bicarbonate containing solution, after which a chloride-free, bicarbonate-containing solution was
added. The rate of cytoplasmic alkalinization was inhibited by 4az and 4be with IC50 of 0.035 M and 0.023 M, respectively (Fig.
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1B). In control experiments, no change in cytoplasmic pH was observed in cells not expressing slc26a3, or in experiments where
bicarbonate was replaced with chloride in exchange solutions (not shown).
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Figure 2. Selectivity of slc26a3 inhibitors. Original data curves (A) and summary (B) of relative inhibition of slc26a3 homologs by 4az and
4be (mean ± S.E.M., n = 3). Differences not significant.
DRA inhibition selectivity of 4az and 4be. To study selectivity, anion exchange assays were done on related solute carrier 26 family
members, including (murine) slc26a4 (pendrin; ~48 % amino acid identity to slc26a3), (murine) slc26a6 (PAT-1; ~38 % amino acid
identity), and (human) SLC26A9 (~39 % amino acid identity). Chloride/iodide exchange was not significantly inhibited by 10 µM
4az or 4be in cells expressing each of these solute carrier family 26 members, with original fluorescence time course data shown in
Fig. 2A and summary data in Fig. 2B. Based on the inhibition activity, aqueous solubility and preliminary in vitro microsomal stability
test (data not shown), 4be and 4az were chosen for further studies including in vivo efficacy.
Pharmacokinetics in mice. Fig. 3 shows the serum concentrations of 4ba, 4be and 4az after single dose (5 mg/kg for 4ba and 4be,
10 mg/kg for 4az) oral or intraperitoneal administration at zero time. 4ba had levels well above the IC50 determined in vitro for at
least 24 h after single dose oral or intraperitoneal administration. 4be had high serum levels initially, but disappeared relatively rapidly
with little compound remaining at 16 h. 4az had predicted therapeutic concentrations for several days after single dose oral or
intraperitoneal administration, with serum concentration of 4az of approximately 1 µM even at 72 h. The deduced pharmacokinetic
parameters for 4ba, 4be and 4az are given in Table 3. Computed oral bioavailability values were (mean ± S.E.M.): 54 ± 6.5% (4ba),
16 ± 7% (4be) and 39 ± 4% (4az).
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Figure 3. Pharmacokinetics. Serum concentrations of 4ba, 4be and 4az in mice after single dose intraperitoneal or oral administration
at 5 mg/kg (4ba and 4be) or 10 mg/kg (4az). Mean ± S.E.M., 3 mice per group. ip: intraperitoneal, po: per oral.
Table 3. Pharmacokinetic parameters of slc26a3 inhibitors.
Compound
Route
Oral
IP
Cmax (µM)
9.4 ± 1.8
Tmax (h)
5.8 ± 5.1
1.5 ± 0.5
0.8 ± 0.2
0.8 ± 0.2
6.5 ± 4.8
0.5 ± 0
AUC (µmol*h/L)
124 ± 15
4ba
24.3 ± 3.1
12.9 ± 3.7
70.2 ± 5.0
4.7 ± 0.5
231 ± 33
Oral
IP
69 ± 28
4be
4az
418 ± 78
Oral
IP
130 ± 12
29.1 ± 2.3
331 ± 42
Efficacy in a mouse model of constipation. Opioids such as loperamide slow gut motility14 and are commonly used to induce
constipation in animal models as they have direct relevance to opioid-induced constipation, one of the most common types of
constipation. Fig. 4A shows the experimental model in which slc26a3 inhibitor was administered orally 1 hour prior to intraperitoneal
injection of loperamide, with stool collected over the following 3 hours. As shown in Fig. 4B, 4be administration partially prevented
constipation in loperamide-treated mice as quantified by improved 3-hour stool weight, pellet number and water content. The efficacy
of 4be was similar to that found previously for original inhibitor 4ba.13 Notably, 4az administration fully normalized stool water
content in loperamide-treated mice (Fig. 4C), even at a low dose of 3 mg/kg. In control mice not treated with loperamide, 4az had no
significant effect on stool parameters compared to vehicle treatment.
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Figure 4. Inhibitor efficacy in a loperamide-induced constipation model in mice. A. Experimental protocol. B. 4be at 10 mg/kg improved 3-
hour stool weight, pellet number and water content in loperamide-treated mice (mean ± S.E.M., 4-6 mice per group). C. Dose-dependence
of 4az (in mg/kg) effect on 3-hour stool weight, pellet number and water content in loperamide-treated mice (5-8 mice per group, comparisons
made with one-way analysis of variance with post-hoc Newman Keuls multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns:
not significant (p ≥ 0.05). ip: intraperitoneal, po: per oral).
DISCUSSION AND CONCLUSION
The goal of this study was to advance 4,8-dimethylcoumarin inhibitors of the slc26a3 anion exchange protein for anti-absorptive
therapy of constipation. SAR analysis of commercially available 4,8-dimethylcoumarin analogs as well as a focused library of
synthesized compounds identified compounds with ~10-fold improved potency compared with reference compound 4ba, with good
pharmacokinetic properties and efficacy in a mouse model of constipation when administered orally. A low oral dose (1 mg/kg) of
4az produced greater than 50% normalization of stool hydration in loperamide-treated mice, suggesting effective compound
accumulation in enterocytes in colon. It is not possible, however, to directly relate serum concentrations to efficacy in constipation
because we do not know the concentration of free compound in serum, the intracellular compound concentration, and whether the
compound accumulates in enterocytes by delivery from the luminal or basolateral surfaces of the colonic epithelium. We speculate,
though do not know with certainty, that the 4,8-dimethylcoumarins act from the cytoplasmic surface of slc26a3 because they were
effective when administered intraperitoneally (data not shown) and their onset of action was not immediate in cell cultures in vitro.13
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Depending on their physicochemical properties, slc26a3 inhibitors could accumulate at their target site, the colonic epithelium, by
delivery from the luminal membrane with minimal systemic absorption, or by absorption in small intestine and delivery from the
basolateral membrane.
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Compounds 4be, 4az and 4ba have favorable drug-like properties according to the Lipinski’s rule of 5,15 with molecular mass less
than 500 Da (406, 464, and 416, respectively), tPSA of 72.8, pKa of 3.70, cLogP of 4.6-4.8, and multiple hydrogen bond acceptors
and donors. In general, coumarins are polyphenolic derivatives found in plants that contain a benzopyranone moiety. They are
considered as phytoalexins with diverse cytoprotective and modulatory activities including antimicrobial, antiviral, anticancer, anti-
inflammatory, antioxidant and central nervous system activities.16 Prior studies have provided information on the coumarin
substituents (C3, 4, 7 and 8) involved in the various biological activities. For example, 3-acetic acid coumarin analogs have MRSA
antibacterial activity,17 DNA gyrase inhibition,18 ATPase inhibition,19 antituberculosis activity,20 and inhibition of NF-kB/DNA
interaction.21 3-carboxamide coumarin analogs inhibit monoamine oxidase.22, 23 The most widely used coumarin-containing drug is
warfarin.24 Warfarin and its analogs contain a hydroxyl group at C4, in which its replacement by sulfhydryl, halogen or hydrogen
abolished anticoagulant activity.25 The compounds reported herein are not expected to have anticoagulant activity because they do
not contain a 4-hydroxy group. For the C7 substituent on coumarin, 6,7-annulated coumarins were reported as inhibitors of NF-
kB/DNA interaction,26, 27 and C7 biaryl coumarin analogs as antibacterials.28
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This study builds on our original identification, by high-throughput screening, of the first selective inhibitors of anion exchanger
SLC26A3.13 Compounds 4be and 4az synthesized and characterized herein fully inhibited slc26a3-mediated anion exchange with
IC50 down to 25 nM. 4az gave sustained therapeutic concentration in serum for over 72 h after single-dose oral or intraperitoneal
administration, and fully normalized stool hydration in a loperamide mouse model of constipation. Having favorable inhibition
potency in vitro and in vivo, selectivity within the SLC26 family, and pharmacokinetics, 4az is suitable candidate for further
preclinical development.
EXPERIMENTAL DETAILS
General synthesis procedure.
All chemicals and solvents were used as purchased and used without further purification. Reaction progress was monitored by TLC
1
using pre-coated silica gel plates with fluorescence at 254 nm and ethylacetate/n-hexane solvent system. H and 13C NMR spectra
were obtained on a Bruker Avance 300 instrument using CDCl3, CD3OD, trifluoroacetic acid-d, acetic acid-d4, or DMSO-d6 as
solvent. Chemical shifts are given in parts per million (ppm). HRMS was performed using a hybrid quadrupole Orbitrap mass
analyzer, QExactive (Thermo, Bremen, Germany) with an electrospray ionization (ESI) source. The mass resolution was set as 70000
at m/z 200, and mass accuracy >3 ppm. Purity of all final compounds was 95% or higher as determined by high performance liquid
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chromatography (HPLC) using UV absorbance at 254 nm. HPLC was done on an Xterra MS C18 column (2.1 mm × 100 mm, 3.5
1
2
µm) with 0.2 mL/min water/acetonitrile (containing 0.1% formic acid), 25 min linear gradient, 5−95% acetonitrile.
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Methyl 2-(7-hydroxy-4,8-dimethyl-2-oxo-2H-chromen-3-yl) acetate (2).
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To a solution of 2-methylresorcinol (5.0 g, 40 mmol) and dimethyl 2-acetylsuccinate (7.6 g, 40 mmol) in absolute methanol (40 mL)
was added 20 mL of sulfuric acid at 0 °C for 1 h. The reaction mixture was stirred at room temperature for 24 h and then the mixture
was poured into water. The resulting precipitate was collected by filtration, washed with water, and dried at 50 °C under vacuum
overnight to give an off-white solid (9.0 g, 86% yield) of the desired product. 1H NMR (300 MHz, CDCl3) δ 7.14 (d, 1H, J = 8.2 Hz),
6.65 (d, 1H, J = 8.2 Hz), 3.81 (s, 3H), 3.74 (s, 2H), 2.32 (s, 3H), 2.21 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 172.7, 162.3, 157.3,
151.7, 150.1, 122.5, 114.9, 113.1, 111.8, 111.5, 52.6, 32.7, 15.3, 7.9; LRMS: 263 (M + H+).
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Methyl 2-(7-((3-bromobenzyl)oxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl)acetate (3ba)
A mixture of 2 (200 mg, 0.8 mmol), bromobenzyl bromide (270 mg, 1.1 mmol), and potassium carbonate (210 mg, 1.5 mmol) in
acetone (10 mL) was heated at reflux overnight. The reaction mixture was cooled to rt and poured into ice water. The resulting
precipitate was collected by filtration, washed with methanol, and dried to give 170 mg (96%) of the product as white solid. 1H NMR
(300 MHz, CDCl3) δ 7.61 (brs, 1H), 7.51-7.48 (m, 1H), 7.46 (d, 1H, J = 8.9 Hz), 7.39 (d, 1H, J = 8.0 Hz), 7.29 (t, 1H, J = 7.8 Hz),
6.87 (d, 1H, J = 8.9 Hz), 3.75 (brs, 2H), 3.73 (s, 3H), 2.39 (brs, 6H); 13C NMR (75 MHz, CDCl3) δ 170.9, 161.8, 158.6, 151.6, 149.0,
138.8, 131.2, 130.2, 130.0, 125.5, 122.76, 122.72, 116.5, 114.5, 114.4, 108.1, 69.5, 52.2, 32.7, 15.3, 8.4; LRMS: 431 (M + H+).
2-(7-((3-Bromobenzyl)oxy)-4,8-dimethyl-2-oxo-2H-chromen-3-yl)acetic acid (4ba)
To a solution of methyl ester 3ba (160 mg, 0.4 mmol) in methanol (8 mL) was added NaOH (25 mg, 0.6 mmol) in water (2 mL) and
refluxed for 1 h. The mixture was cooled to room temperature, diluted with water, and acidified with 1N HCl. The resulting precipitate
was collected by filtration, washed with water and dried. Final product was recrystallized in methanol to give the product as a white
powder (105 mg, 68%). 1H NMR (300 MHz, DMSO-d6) δ 7.70 (brs, 1H), 7.65 (d, 1H, J = 9.0 Hz), 7.55 (d, 1H, J = 8.4 Hz), 7.51 (d,
1H, J = 7.9 Hz), 7.41 (d, 1H, J = 7.9 Hz), 7.12 (d, 1H, J = 9.0 Hz), 5.29 (s, 2H), 3.51 (s, 2H), 2.34 (s, 3H), 2.26 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ 172.0, 161.4, 158.4, 151.1, 149.1, 140.1, 131.2, 130.4, 126.7, 124.0, 122.2, 117.8, 114.4, 112.9, 109.2, 69.3, 52.2,
15.5, 8.5; HRMS for C20H17BrO5: calcd, 417.0332; found, 417.0330 (M + H+).
Mice
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Mice were bred at the University of California San Francisco (UCSF) Animal Facility. Animal protocols were approved by the UCSF
Institutional Animal Care and Use Committee.
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Pharmacokinetics
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Pharmacokinetics of 4ba, 4be and 4az were determined in mice. Following single-dose intraperitoneal or oral administration of 4ba
(5 mg/kg, in saline with 5% DMSO and 10% Kolliphor HS 15), 4be (5 mg/kg, same vehicle as 4ba) or 4az (10 mg/kg, in PBS with
5% DMSO and 20% (2- hydroxypropyl)-β-cyclodextrin), blood samples were collected via retroorbital puncture at specified time
points and serum was separated by centrifugation at 4000 g for 20 min at 24 °C. Compound concentrations were measured in serum
using LC/MS. Oral bioavailability was calculated by using area under the curve (AUC) for oral and ip routes as AUC (oral) / AUC
(ip).
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Loperamide constipation model
Female CD1 mice (age 8–10 weeks) were administered loperamide (0.3 mg/kg, intraperitoneally, in 5% ethanol in PBS, 0.1 mg/mL
final concentration) to produce constipation or vehicle in control mice. 4be (5 mg/kg), 4az (1, 3 or 10 mg/kg, in PBS with 5% DMSO
and 20% (2-hydroxypropyl)-β-cyclodextrin) or vehicle were administered by oral gavage 1 h before loperamide. After loperamide
injection, mice were placed individually in metabolic cages with free access to food and water. Stool samples were collected for 3 h,
and total stool weight and number of fecal pellets were quantified. To measure stool water content, stool samples were dried at 80°
C for 24 h and water content was calculated as [wet weight-dry weight]/wet weight. 29, 30
Anion exchange assays
Assay of (murine) slc26a3-mediated chloride/iodide exchange was done as described8 in which FRT rat thyroid epithelial cells
expressing slc26a3 and halide-sensitive EYFP-H148Q/I152L/F46L were incubated in phosphate buffered saline (PBS) prior to
addition of iodide-substituted PBS (140 mM NaCl substituted with NaI). The rate of fluorescence quenching was determined as a
quantitative measure of chloride/iodide exchange. Measurement of chloride/bicarbonate exchange was done as described14 in which
intracellular pH in FRT cells expressing slc26a3 was measured using the pH-sensitive probe BCECF in a bicarbonate-containing
buffer following chloride replacement by gluconate. Transporter selectivity was studied as described13 in which FRT cells containing
slc26a transporters were preincubated with 0 or 10 µM 4az or 4be for 10 min prior to anion exchange assay.
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website.
Structures of commercial slc26a3 inhibitors, and synthesis details and spectroscopic characterization of new molecules (PDF)
Molecular strings and data for all final inhibitor candidates (CSV).
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AUTHOR INFORMATION
Corresponding Author
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* Alan S. Verkman, M.D., Ph.D., 1246 Health Sciences East Tower, University of California San Francisco, CA 94143-0521, U.S.A.; Phone
415-476-8530; Fax 415-665-3847; E-mail Alan.Verkman@ucsf.edu.
Author Contributions
SL performed synthesis and compound analysis, some transport assays, and LC/MS for pharmacokinetic analysis. OC performed murine
constipation and pharmacokinetic studies. PMH performed some transport assays. All authors designed experiments, analyzed data and wrote
the manuscript. OC, PMH and ASV developed the original concept for this study.
Funding Sources
This work was supported by grants from the NIH (DK72517, DK099803, DK101373), University of California Drug Discovery Consortium
and the UCSF Clinical & Translational Science Institute Catalyst Program.
ACKNOWLEDGMENTS
The authors thank Joseph-Anthony Tan for assistance with transport assays and Amber A. Rivera for assistance with cell culture. The authors
thank to Dr. Robert Yen of the SFSU Mass Spectrometry Facility for HRMS measurements, funded by National Science foundation grant
CHE-1228656.
ABBREVIATIONS
DRA, down-regulated in adenoma; SAR, structure-activity relationship; AUC, area under the curve; ip, interperitoneal; po, per oral;
slc26a3, solute carrier 26 family member a3; YFP, yellow fluorescent protein halide sensor; BCECF, 2',7'-Bis(carboxy ethyl)-5(6)-
carboxyfluorescein; FRT, Fischer rat thyroid epithelial cells.
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