Thiazolidinone CFTR inhibitors with improved water solubility identified by structure-activity analysis

Article abstract of DOI:10.1016/j.bmc.2008.07.044

The thiazolidinone 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR_inh-172) inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel conductance with submicromolar affinity and blocks cholera toxin-induced intestinal fluid secretion. Fifty-eight CFTR_inh-172 analogs were synthesized to identify CFTR inhibitors with improved water solubility, exploring modifications in its two phenyl rings, thiazolidinone core, and core-phenyl connectors. Greatest CFTR inhibition potency was found for 3-CF₃ and polar group-substituted-phenyl rings, and a thiazolidinone core. Two compounds with ～1 μM CFTR inhibition potency and solubility >180 μM (>10-fold more than CFTR_inh-172) were identified: Tetrazolo-172, containing 4-tetrazolophenyl in place of 4-carboxyphenyl, and Oxo-172, containing thiazolidinedione in place of the thiazolidinone core. These water soluble thiazolidinone analogs had low cellular toxicity. The improved water solubility of Tetrazolo- and Oxo-172 make them potential lead candidates for therapy of secretory diarrheas and polycystic kidney disease.

Full text of DOI:10.1016/j.bmc.2008.07.044

Bioorganic & Medicinal Chemistry 16 (2008) 8187–8195
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thiazolidinone CFTR inhibitors with improved water solubility identified
by structure–activity analysis
*
N. D. Sonawane , A. S. Verkman
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, 1246 Health Sciences East Tower, San Francisco, CA 94143-0521, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The thiazolidinone 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazo-
lidinone (CFTRinh-172) inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride
channel conductance with submicromolar affinity and blocks cholera toxin-induced intestinal fluid secre-
tion. Fifty-eight CFTRinh-172 analogs were synthesized to identify CFTR inhibitors with improved water
solubility, exploring modifications in its two phenyl rings, thiazolidinone core, and core-phenyl connec-
tors. Greatest CFTR inhibition potency was found for 3-CF3 and polar group-substituted-phenyl rings, and
Received 5 June 2008
Revised 14 July 2008
Accepted 16 July 2008
Available online 23 July 2008
Keywords:
CFTR
Cystic fibrosis
Diarrhea
SAR
CFTR inhibitors
CFTRinh-172
a thiazolidinone core. Two compounds with ꢀ1
lM CFTR inhibition potency and solubility >180 lM (>10-
fold more than CFTRinh-172) were identified: Tetrazolo-172, containing 4-tetrazolophenyl in place of 4-
carboxyphenyl, and Oxo-172, containing thiazolidinedione in place of the thiazolidinone core. These
water soluble thiazolidinone analogs had low cellular toxicity. The improved water solubility of Tetraz-
olo- and Oxo-172 make them potential lead candidates for therapy of secretory diarrheas and polycystic
kidney disease.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
tive charge at neutral pH, which precipitates in an acid environ-
ment. Also, the concentration of CFTRinh-172 in cytoplasm, where
The cystic fibrosis transmembrane conductance regulator
(CFTR) is a cAMP-regulated chloride channel, which when mutated
can produce the hereditary disease cystic fibrosis. CFTR inhibition
is a potential strategy for therapy of secretory diarrheas1–6 and
polycystic kidney disease,7–9 as well as for pharmacological crea-
tion of the cystic fibrosis phenotype in human and animal tis-
sues.10 We previously discovered by high-throughput screening
the thiazolidione CFTR inhibitor 3-[(3-trifluoromethyl)phenyl]-5-
[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTRinh-172),
which blocked CFTR chloride conductance fully with
it acts, is likely reduced in a Nernstian manner compared with its
extracellular concentration because of the interior-negative mem-
brane potential of its target epithelial cells. We report here a struc-
ture–activity analysis of CFTRinh-172, with the goal of improving
water solubility while maintaining CFTR inhibition potency. We
systematically modified Rings A and C of CFTRinh-172, as well as
the thiazolidinone core (Ring B) and the core-ring linkers (Fig. 1).
CFTR analogs with >10-fold higher water solubility than CFTRinh
-
172 and only mildly reduced potency were synthesized and
characterized.
IC50 < 0.5 l
M.11 Patch-clamp analysis indicated a voltage-indepen-
dent channel block mechanism with prolongation of mean channel
closed time,12 involving interaction at arginine-347 located near
the CFTR cytoplasmic surface.13 In vivo analysis indicated
CFTRinh-172 efficacy in reducing cholera toxin-induced intestinal
fluid secretion in rodent models.11,14,15 CFTRinh-172 was found to
have low toxicity, renal excretion with minimal metabolism, and
2. Results
2.1. Chemistry
A total of 58 CFTRinh-172 analogs were assayed for CFTR inhibi-
tion activity, with the most potent compounds further character-
ized. Of the 58 compounds, several (compounds 5, 6, 9, 14, 15,
17, 20, 21, 25, 26, 27, 29, 33, and 38) were reported previously as
part of other studies from our laboratory.9,11,13,14
Compounds with different substituents on Ring A were synthe-
sized, keeping the remainder of the molecule the same. Commer-
cially available substituted anilines 1 were reacted with carbon
disulfide, followed by reaction with bromoacetate and acidic cycli-
zation to give thiazolidinone intermediate 3 (Route 1, Scheme 1).
intestinal accumulation by enterohepatic recirculation.14 CFTRinh
-
172 has been used extensively to block CFTR chloride channel
function in variety of cell culture, tissue, and in vivo systems.16–23
Limitations of CFTRinh-172 include its low water solubility and
oral bioavailability. CFTRinh-172 is a weak acid with a single nega-
* Corresponding author. Tel.: +1 415 476 8530; fax: +1 415 665 3847.
E-mail address: Nitin.Sonawane@ucsf.edu (N.D. Sonawane).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.044

8188
N. D. Sonawane, A. S. Verkman / Bioorg. Med. Chem. 16 (2008) 8187–8195
though use of toxic compounds (phosgene or thiophosgene) was
required.
Carboxylate modification to various esters and amides was
Ring A Ring B Ring C
H
O
achieved by three reaction routes as shown in Scheme 1. Amide
42 and ester 45 were synthesized by condensation of thiazolidi-
none intermediate 3 with 4-carbamoylbenzaldehyde and ethyl 4-
formylbenzoate, respectively. Alternatively, the carboxyl in
CFTRinh-172 was converted to the acid chloride using thionyl chlo-
ride, followed by reaction with equimolar amounts of compounds
(ammonia, aminoethanol, ethylenediamine, acetoxymethanol) to
yield 42–44 and 46. Reaction involving activation of the carboxy
function by DCC also generated 42–44 and 46 amides (Scheme 1).
Thiazolidinedione 48 was synthesized by condensation of 2,4-
O
N
S
OH
F3C
S
Linker 1 Linker 2
CFTRinh-172
Figure 1. Chemical structure of CFTRinh-172 showing Rings A, B, C and Linkers 1
and 2.
The same reaction route was used for synthesis of analogs contain-
ing a methylene group between Rings A and B (Route 1, Scheme 1).
These intermediates upon Knoevenagel condensation with aro-
matic aldehydes in ethanol under reflux in presence of piperidine
produced analogs 5–41 and 47–49. TLC showed quantitative for-
mation of products. This reaction generates a double bond that
can produce E and Z isomers. Similar analogs are reported to exist
predominantly as Z-isomers.24–29 It is presumed that the analogs
synthesized here are mainly Z-isomers.
Scheme 1 also shows an alternate isothiocyanate route (Route
2) for synthesis of the 2-thiaoxo-4-thiazolidinone ring intermedi-
ate 3. Route 2 was used for the synthesis of 13, 18, 20, 22, and
27. Isothiocyanates 2 were prepared by single step reaction of cor-
responding amino compounds 1 with thiophosgene and reacted
with thioglycolic acid in presence of triethylamine to yield dithio-
carbamate intermediates. This intermediate upon in situ acidificat-
ion (HCl) and reflux generated 2-thioxo-4-thiazolidinones 3. Route
2 was single-pot and increased overall yields compared to Route 1,
thiazolidinedione intermediate
3 (R1, R3 = H, R2 = CF3, Y = S,
Z = O) with 4-carboxybenzaldehyde (Scheme 1). Route 2 was used
for efficient synthesis of corresponding intermediate 3. For synthe-
sis of compounds 50 and 51, maleimide intermediates 4 (R4 = Cl or
H) were prepared by reaction of 3-trifluoromethylaniline with
dichloromaleic anhydride (R4 = Cl) or maleic anhydride (R4 = H)
in refluxing acetic anhydride (Scheme 2). Subsequent reaction with
4-aminobenzoic acid and 4-mercaptobenzoic acid produced com-
pounds 50 and 51 (Scheme 2, dotted line indicate double bond in
50).30–33 Compounds 52–55 were synthesized by reaction of aryl
isothiocyanates with 3 in presence of base DBU at room tempera-
ture (Scheme 1). Reduction of the double bond in CFTRinh-172
using LiBH4 in pyridine24,34 at room temperature gave 56 (Scheme
1).
Thiazole analogs 59 and 60 were synthesized by bromination of
acetophenone 57 in acetic acid at 0 °C for 2 h, followed by reaction
with substituted phenylthiourea in refluxing ethanol (Scheme
3).35,36 Synthesis of thiadiazole compounds 64 and 65 was accom-
O
Z = O, S
g
R2
R1
X
N
z
S
n = 0; R1, R3 = H; R2 = CF3; Y, Z = S
R3
C
N
n
O
F3C
S
H2
f,c
X1
R1, R3, X1, X2, X4 = H;
R2 = CF3; X3 = COOH;
2
O
O
R2
R1
R2
R1
X2
X3
Route 2
h or i, j
g
Y, Z = S
d or e
R2
R3
C
n N
R3
C
n N
Y
l
Y
H2
H2
a,b,c
42, X = NH2; 43, X = NH-C2H4-OH
44, X = NH-C2H4-NH2; 45, X = O-C2H5
46, X = O-CH2-O(C=O)-CH3
R1
X4
z
z
3
Route 1
5-41, 47-49
n = 0
R3
C
NH2
n
H2
n = 0
R2 = CF3
k
R1,R3 = H
Y, Z = S
1
R1,R3,X1,X2,X4 = H
Y, Z = S
X3
O
S
O
F3C
O
O−
+
S
S
O
F3C
O
X2
O
Na
N
H
N
H
N
N
N
S
COOH
S
S
52, X2 = H; X3 = COOH
53, X2 = OH; X3 = COOH
54, X2 = H; X3 = SO3Na
F3C
S
S
S
55
56
Scheme 1. Reagents and conditions: (a) CS2, TEA, EtOAc, rt, 12 h; (b) BrCH2COOH, NaHCO3, rt, 2 h; (c) HCl, reflux, 2 h; (d) COCl2 or CSCl2, TEA, 10 °C, 2 h; (e) Triphosgene,
CHCl3, reflux, 3 h; (f) HSCH2COOH, TEA, 4 h. (g) aldehyde, piperidine/ethanol, reflux, 2 h.; (h) SOCl2, cat. DMF, rt, 2 h; (i) DCC, N-hydroxy-succinimide, rt,; (j) HX-R, pyridine,
0o-rt, 2 h; (k) SCN-(3X2–4X3–Ph) or sodium 5-SCN-furan-2-sulfonate (for 55), DBU, THF, rt, 2 h. (l) LiBH4, pyridine, rt, 12 h.
O
O
R4
R4
R4
W
a
b
COOH
NH2
N
N
F3C
F3C
F3C
O
O
4
50, R4 = Cl, W = NH; 51, R4 = H; W = S
Scheme 2. Reagents and conditions: (a) malic anhydride/dichloromalic anhydride, Ac2O, NaOAc, 80°, 2 h; (b) (4-COOH)–Ph–WH, TEA, THF, rt, 5 h.

N. D. Sonawane, A. S. Verkman / Bioorg. Med. Chem. 16 (2008) 8187–8195
8189
H
N
R2
R1
O
O
a
b
N
Br
S
F3C
F3C
F3C
59, R1 = COOH, R2 = H;
60, R1 = OH, R2 = COOH
57
58
Scheme 3. Reagents and conditions: (a) Br2, AcOH, 0°, rt, 2 h; (b) n-(4-carboxyphenyl)thiourea, AcOH, reflux, 4 h.
O
O
S
H
N
R2
R1
H
N
a,b
c
S
N
Cl
N
H
N
H
N
F3C
F3C
R2
F3C
61
R1
62, R1 = COOH, R2 = H;
63, R1 = OH, R2 = COOH
64, R1 = COOH, R2 = H;
65, R1 = OH, R2 = COOH
Scheme 4. Reagents and conditions: (a) NH2NH2 ÁxH2O, pyridine, rt, 2 h; (b) (4-COOH)–Ph–NCS, TEA, THF, rt, 12 h; (c) H2SO4, rt, 2 h.
R2
N
a
N
N
R1
N3
COOH
+
R2
R1
COOH
66, R1 = H, R2 = CF3;
67, R1 = CF3, R2 = H
68, R1 = H, R2 = CF3;
69, R1 = CF3, R2 = H
Scheme 5. Reagents and conditions: (a) 1 mol% CuSO4, 0.1 eq. sodium ascorbate, H2O/t-BuOH (1:1), rt, 24 h.
plished in three steps. Reaction of the acid hydrazide (prepared
from benzoyl chloride 61 and hydrazine) with substituted pheny-
lisothiocyanates gave thiosemicarbazides 62 and 63 in good yields.
Treatment of 62 and 63 with sulfuric acid produced the 2-amino-
thiadiazoles 64 and 65 (Scheme 4).37
Scheme 5 shows the synthesis of 1,2,3-triazoles. 1,3-Dipolar
cycloaddition38,39 of alkynes 66 and 67 with 4-azidobenzoic acid
produced the 1,2,3-triazoles (compounds 68 and 69) in high yields.
Single spots in TLC indicated that adducts were predominantly 4-
regioisomers.
(17 lM; Table 3). Addition of polar substituents such as hydroxy,
SO3Na or COOH in Ring A, or removal of CF3, produced highly
water-soluble though inactive compounds.
The thiazolidinone core, Ring B, was replaced by thiazolidined-
ione, maleimide, succinimide, thiazole, thiadiazole, and triazole,
while keeping Rings A and C and their substituents the same as
in CFTRinh-172. These heterocycles were selected because of their
resemblance to Ring B, their ease of synthesis, and prior knowledge
of good bioavailability and metabolic and chemical stability.40,41
The 2,4-thiazolidinedione 48 is a close analog of CFTRinh-172 in
which the thioxo group is replaced by an oxo-group (referred to as
Oxo-172). Replacement of 2-thioxo by 2-oxo increased solubility in
saline by ꢀ25-fold, while reducing CFTR inhibition potency 3.6-
fold in short-circuit current assays (Fig. 2B and F; Table 2). Malei-
mide analog 50 had weak activity, though 2,5-pyrrolidinedione 51
2.2. Functional studies
CFTR inhibition potency for all compounds was measured by
short-circuit current analysis, with representative original data
and concentration–inhibition curves for compounds of greatest
interest provided in Figure 2. In the short-circuit current assay,
CFTR was stimulated by the cAMP agonist forskolin, followed by
compound additions at increasing concentration. Table 1 summa-
rizes the effects of Ring A substitutions on CFTR inhibition potency.
CF3 substitution at the 3-position, as in CFTRinh-172, gave greater
inhibition potency than at the 2- or 4-position (compounds 20
and 25).9 Substitution of 3-CF3 by 3-CH3 gave the less active analog
12. Compounds 8 and 11, in which Ring A contained 3-CF3 along
with 4-F substituents, had intermediate activity. Similar substitu-
tions by chloro at 2- or 4-position greatly reduced activity (com-
pounds 24 and 30).
had moderate activity with IC50 ꢀ 7
lM. The Ring B variation in 51
removed the double bond in CFTRinh-172, making it less reactive
for Michael addition. The aminothiazole and aminothiadiazole ana-
logs were inactive, however. Replacement of thiazolidinone by
other heterocycles such as aminothiazoles (59 and 60) gave weakly
active compounds. Replacement of thiazolidinone by aminothi-
adiazoles (64 and 65), and 1,2,3-triazoles (68 and 69) yielded inac-
tive compounds.
C-ring substitutions were carried out in an attempt to increase
compound solubility, and to convert compound net negative
charge at physiological pH to neutral in order to increase com-
pound accumulation in cytoplasm. Analogs were synthesized with
different substitutions on Ring C, keeping Rings A and B, and both
linkers the same as in CFTRinh-172. Substitutions were chosen to
increase compound polarity and H-bond capacity, including car-
boxy, esters, amides, hydroxy, methoxy, and sulfonate.
To improve aqueous solubility, we first tried to disrupt the pla-
nar configuration of Rings A and B, which is predicted to reduce
ring-ring stacking, by adding bulky ortho substituents to Ring A.
Introduction of methyl at the 2-position in
a-Me-172, 18, in-
creased water solubility, but reduced inhibition potency consider-
ably. Figure 2 shows concentration-dependent inhibition of CFTR
Monosubstituted compounds containing 4-COOH (CFTRinh-172)
or 3-COOH, 9, showed greater CFTR inhibition potency than 2-
COOH, 26. Esterification or amidation of 4-COOH in CFTRinh-172
gave inactive compounds 42–46. Compounds 31, 38, 19, and 34
containing mono, di-, or tri-hydroxy functions at Ring C had low
by CFTRinh-172 (A) and
respectively. As determined by LC/MS, the solubility of
in saline was 259 M, substantially greater than that of CFTRinh-172
a
-Me-172 (E) with IC50 0.4 and 8
lM,
a-Me-172
l

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N. D. Sonawane, A. S. Verkman / Bioorg. Med. Chem. 16 (2008) 8187–8195
A
B
CFTRinh-172 (μM)
Oxo-172 (μM)
0.25
forsk
forsk
ap
0.25
0.75
1.75
0.75
I
50
μA
3.75
1.75
3.75
8.75
8.75
20 min
Tetrazolo-172 (μM)
Pyridine-NO-172 (μM)
1.5
C
forsk
D
F
0.25
forsk
3.5
1.25
8
18
33
8.25
83
20 min
E
forsk
CFTRinh-172
Tetrazolo-172
100
75
50
25
0
α-Me-172 (μM)
1
3
α-Me-172
8
Oxo-172
18
Pyridine-NO-172
0.01 0.1
1
10
100
[Compound] (μM)
Figure 2. Short-circuit current measurements of CFTR inhibition. (A) CFTR-mediated apical membrane chloride current measured in FRT cells expressing human wildtype
CFTR after permeabilization of the basolateral membrane in the presence of a chloride gradient (see Section 4). CFTR was activated by 20 M forskolin and indicated
-Me-172, and Pyridine-NO-172. See Table 3 for fitted
l
concentrations of inhibitors were added. (B) Concentration–inhibition data for CFTRinh-172, Tetrazolo-172, Oxo-172,
IC50 values.
a
activity. Based on predicted pKa of >7.5, these compounds are ex-
pected to be neutral at physiological pH. Creation of a negative
charge by addition of 3,4-dibromo electron-withdrawing moieties,
that lower the pKa of 4-OH, resulted in modest inhibition activity
(compounds 14 and 15; Table 1), whereas modification of the ion-
izable 4-OH to 4-OMe gave the inactive compound 39. However,
sulfonic acid derivatives 35 and 37, which carry at physiological
pH single and double negative charges, respectively, were inactive.
Ring C was also replaced by heterocyclic-equivalent ring sys-
tems. Replacement of the phenyl by a pyridyl ring gave the inactive
neutral compound 33; however, N-oxidation of pyridyl nitrogen
gave the first, net-neutral thiazolidinone CFTR inhibitor, pyridine-
stitution increased water solubility 11-fold (Table 3) with IC50 of
0.8 M (Fig. 2F). As shown in Figure 2C, Tetrazolo-172 showed
slower inhibition kinetics than CFTRinh-172 or Oxo-172. Similarly
replacement of carboxylate in 48 by tetrazolo in 49 gave lower
inhibition potency (IC50 17 lM; Table 2).
l
CFTRinh-172 contains a single bond as a Linker 1 that directly
connects lipophilic Ring A to heterocyclic Ring B. We examined
the effect of introduction of a methylene group as a bridge between
Rings A and B. Compound 47 was inactive, however, indicating lit-
tle tolerance of Linker 1 modification.
Because the double bond in Linker 2 is a Michael electrophile,
alternative linkers between Ring B and Ring C were investigated.
First, reduction of double bond linker of CFTRinh-172 gave
compound 56, which was inactive. The double bond reduction dis-
turbes the rigid geometry of CFTRinh-172, which is presumed as
Z-configuration, allowing free rotation of Rings B and C. Further,
replacement of the double bond-methylidyne bridge by thioamide
in 52 and 53, though highly water soluble, were inactive, as were
analogs 54 and 55 containing sulfonic acid substituents (Scheme
1 and Table 2).
NO-172, 17, with IC50
9 lM (Fig. 2D and F). This is a zwitterionic
compound containing a positive charge on the ring nitrogen and
negative charge on the oxygen. Addition of hetero atoms is ex-
pected to increase aqueous solubility by H-bonding and increased
polarity. Pyridine-NO-172 had high aqueous solubility of 264
Similarly, the polar analog 10 containing a 4-carboxymethoxy
group had an IC50 of 2.6 M (Table 1). Moving the carboxymethoxy
lM.
l
group from the 4-position (as in 10) to the 3-postion (as in 36) sub-
stantially reduced CFTR inhibition.
Preliminary cytotoxicity analysis was done for the principle
As another approach to improve water solubility, the 4-COOH in
ring C was replaced by tetrazolo-5-yl, an isoster of carboxy with
delocalized negative charge at physiological pH. The tetrazolo sub-
compounds of interest, Tetrazolo-172, Oxo-172, a-Me-172, and
Pyridine-NO-172, comparing with CFTRinh-172 (Table 3). Com-
pounds were incubated with cell cultures for 48 h and cell viability

N. D. Sonawane, A. S. Verkman / Bioorg. Med. Chem. 16 (2008) 8187–8195
8191
Table 1
Variations in Rings A and C
X1
O
O
X2
N
X
R3
N
S
S
X3
F3C
S
N+
O
R2
X4
R1
S
17: X =
33: X = N
Compound
R3
R2
R1
X1
X2
X3
X4
IC50 (lM)
5 CFTRinh-172
6 Tetrazolo-172
7
8
9
10
11
12
13
14
15
16
H
H
H
F
H
H
F
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CH3
CH3
CF3
CF3
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
H
H
COOH
H
COOH
H
COOH
H
H
Br
Br
COOH
Tetrazolo
OH
COOH
H
O–CH2–COOH
H
COOH
COOH
OH
H
H
H
H
H
H
H
H
H
Br
Br
H
0.4
0.8
1.4
1.5
1.5
2.6
5
5
7
7
8
CH3
H
H
OH
COOH
H
H
17 Pyridine-NO-172
9
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
a-Me-172
H
H
H
CF3
H
CF3
CF3
H
H
H
CH3
H
CF3
H
H
H
H
H
H
H
H
H
COOH
COOH
H
COOH
H
H
OH
H
COOH
COOH
H
H
H
H
H
COOH
H
H
COOH
OH
COOH
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
9
11
12
13
14
14
15
15
15
18
19
20
20
24
25
28
28
29
30
50
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
CF3
OH
Ring A: 3,5-di-CF3–Ph–
Cl
CF3
H
H
COOH
COOH
COOH
H
H
H
H
COOH
OH
CF3
H
CF3
H
H
H
H
H
H
CF3
CF3
H
H
CF3
Ring A: 2-Cl-5-CF3–Ph–
H
CF3
–
CF3
H
H
H
H
H
H
COOH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
SO3Na
H
OH
OCH3
H
H
H
H
H
OH
H
OH
SO3Na
H
SO3Na
OH
OMe
Br
OMe
CONH2
CONHC2H4OH
CONHC2H4NH2
COOCH2CH3
H
H
H
H
H
Br
H
H
H
H
H
H
H
O–CH2–COOH
H
OH
Br
H
H
H
H
H
H
H
H
COOCH2OCOCH3
was determined by crystal violet staining. Compounds at 20
l
M
pounds with greatly increased water solubility compared to
CFTRinh-172. Tetrazolo-172 and Oxo-172 are thus potential candi-
dates for further development.
showed little cytotoxicity, with staining ꢀ90% of that of control
(vehicle-treated cultures). Crystal violet staining was reduced for
Tetrazolo-172 and
a
-Me-172 at 50
l
M. CFTRinh-172 was not stud-
Though the most active thiazolidinones contain a negative
charge at neutral pH, compound Pyridine-NO-172, which has IC50
ied at 50 M because of its limited aqueous solubility.
l
of 9 lM for CFTR inhibition, is zwitterionic and net neutral. To-
gether with analysis of site-directed CFTR mutants,13 the existence
of a moderately potent CFTR inhibitor with net neutral charge indi-
cates that a negative charge is not necessary for CFTR inhibition by
thiazolidinones. Though Pyridine-NO-172 is not a candidate for
further development because of its predicted poor in vivo stability,
it may be possible to identify other net neutral thiazolidinones
3. Discussion
We synthesized and characterized 58 CFTRinh-172 analogs with
the goal to increase water solubility and retain CFTR inhibition po-
tency. As summarized in Figure 3, SAR analysis revealed that com-
pounds containing 3-CF3 on Ring A, thiazolidinone core as Ring B,
and 4-carboxy at Ring C had greatest CFTR inhibition potency. Po-
lar ionizable substituents such as carboxy, hydroxy, tetrazolo,
which created a negative charge on Ring C, gave greatest inhibition
activity, whereas non-polar substituents were required on Ring A.
The thiazolidinone ring (Ring B) could be replaced by thiazolidin-
edione, and the COOH group on Ring C by tetrazolo, yielding com-
with improved accumulation in cytoplasm compared to CFTRinh
-
172.
A recent study from our laboratory identified Tetrazolo-172 as
the best thiazolidinone for inhibition of renal cyst growth in mod-
els of polycystic kidney disease.9 Prevention of cyst formation by
Tetrazolo-172 in an MDCK cell model was substantially better than

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N. D. Sonawane, A. S. Verkman / Bioorg. Med. Chem. 16 (2008) 8187–8195
Table 2
Variations in Ring B and Linkers 1 and 2
O
O
O
F
F
F
F
N
N
S
O
C
N
Cl
S
H2
F
F
OH
O
O
O
F3C
S
N
47
X3
H
48, 49
50
OH
Compound
X3
IC50 (lM)
47
Inactive
1.4
48 Oxo-172
COOH
49
50
(2H)-Tetrazolo-5-yl
17
36
O
S
S
X3
F
F
F
F
F
O
S
O
O−
F
Na+
N
N
N
S
S
X2
F
F
F
O
O
O
O
O
S
N
N
H
52-54
H
S
S
55
51
OH
Compound
X2
X3
IC50
(l
M)
51
52
53
54
55
7
9
11
25
27
H
OH
H
COOH
COOH
SO3Na
O
N
S
H
F
N
F
N
N
O
N
S
F
S
F
F
X2
OH
S
N
H
F
X3
56
F
X3
F
F
59, 60
64, 65
X2
N
N
O
N
OH
R3
R2
68, 69
Compound
X3/R3
X2/R2
IC50
(l
M)
56
59
60
64
65
68
69
27
28
32
Inactive
Inactive
Inactive
Inactive
COOH
OH
COOH
OH
H
CF3
H
COOH
H
COOH
CF3
H
model of pkd1 gene deletion. Whether Tetrazolo-172 or other
small-molecule CFTR inhibitors are effective in human polycystic
kidney disease will require clinical trials. Our recent study also
identified a cell permeable phenyl derivative of the glycine hydra-
zide-type CFTR inhibitor as effectively reducing cyst formation and
growth in in vitro and mouse models of polycystic kidney disease.
In contrast to polycystic kidney disease, which is a life-long con-
dition, therapy of enterotoxin-mediated secretory diarrheas such
as cholera or Traveler’s diarrhea requires short-term therapy of
days or less. Small-molecule CFTR inhibitors are predicted to re-
duce intestinal fluid secretion. Thiazolidinones, as ‘absorbable-
type’ CFTR inhibitors that act from the cytoplasmic surface of CFTR,
are taken up into enterocytes and enter the systemic circulation.
Absorbable-type CFTR inhibitors are expected to resist potential
Table 3
Inhibition potency, solubility, and toxicity of CFTR inhibitors
Compound
IC50
(
l
M)
Solubility
in saline (
% Cell viability
50
lM)
20
lM
lM
CFTRinh-172
Tetrazolo-172
Oxo-172
0.38 0.04
0.76 0.2
1.4 0.2
8.2 0.4
8.7 0.7
17
189
420
259
264
86
87
93
89
not soluble
73
91
72
a-Me-172
Pyridine-NO-172
not determined
by CFTRinh-172. Tetrazolo-172 reduced cyst formation and expan-
sion in an embryonic kidney organ culture model and in a mouse

N. D. Sonawane, A. S. Verkman / Bioorg. Med. Chem. 16 (2008) 8187–8195
8193
Linker 1
Linker 2
Best: Ph-N
Best: C=C
Inactive: Ph-CH2-N Weak: CH-CH2
Ring C
H
O
Ring A
Best:3-CF3
Best: 4-COOH, 4-Tetrazolo,
Good: 2/3-COOH, 3,5-DiBr2-4-OH
Inactive: -(C=O)OR, -(C=O)NHR, 4-NMe2
Good: 2/4-CF3, 4-Cl-3-CF3
O
N
S
Inactive: COOH, SO3Na,OH
OH
F3C
Active: O or S
S
Best: S
Inactive: NH
Ring B
Best: Thiazolidinone
Inactive: Thiazole, thiadiazole
Figure 3. Structure–activity analysis of thiazolidinone CFTR inhibitors.
washout, a theoretical concern of non-absorbable CFTR inhibitors
in which rapid fluid transit through the intestine may dilute and
wash out compounds that weakly associate with targets on the
surface of the intestinal lumen. Our laboratory has developed a
series of non-absorbable CFTR inhibitors that block the CFTR pore
from its external surface,42,43 including macromolecular conju-
gates that stick tightly to the intestinal surface.44 The clinical util-
ity of absorbable vs. non-absorbable CFTR inhibitors in the
treatment of secretory diarrheas will require human clinical trials.
4.2.1. 2-Thioxo-3-[3-(trifluoromethyl)phenyl]-4-thiazolidinone
(intermediate 3; R1 = H, R2 = CF3, R3 = H, n = 0, Y and Z = S)
Mp 177–178 °C; 1H NMR (CDCl3): d 7.72 (d, 1H, phenyl,
J = 7.6 Hz), 7.64 (t, 1H, phenyl, J = 8.0 Hz), 7.48 (s, 1H, phenyl),
7.40 (d, 1H, phenyl, J = 8.0 Hz), d 4.18 (s, 2H, CH2); MS (ES+) (m/
z): [M+1]+ calculated for C10H6F3NOS2, 278.29, found 277.93.
4.2.2. 2-Thioxo-3-[2-methyl-3-(trifluoromethyl)phenyl]-4-thia-
zolidinone
MS (ES+) (m/z): [M+1]+ calculated for C11H8F3NOS2, 292.32,
found 292.02.
4. Experimental methods
4.1. Synthesis procedures
4.3. Synthesis of compounds 5–41, 47, 48
1H nuclear magnetic resonance spectra were obtained in CDCl3
or dimethyl sulfoxide (DMSO)-d6 using a 400-MHz Varian Spec-
trometer referenced to CDCl3 or DMSO. Mass spectrometry was
done on a Waters LC/MS system (Alliance HT 2790+ZQ, HPLC:
Waters model 2690, Milford, MA); HRMS were recorded at mass
spectrometry facility, University of California, Riverside. Flash
chromatography was done using EM silica gel (230–400 mesh),
and thin-layer chromatography was performed on Merk silica gel
60 F254 plates (Darmstadt, Germany).
4.3.1. 4-[[4-Oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]-5-thia-
zolidinylidene]methyl]benzoic acid (5, CFTRinh-172)14
A mixture of 2-thioxo-3-(3-trifluoromethyl phenyl)-4-thiazo-
lidinone 3 (55 mg, 0.2 mmol,15 4-carboxybenzaldehyde (30 mg,
0.2 mmol), and a drop of piperidine in absolute ethanol (0.5 ml)
was refluxed for 2 h. Solvent was evaporated, and the residue
was crystallized from ethanol and further purified by normal phase
flash chromatography to yield 54 mg yellow powder (yield 67%);
mp 182–183 °C; 1H NMR (DMSO-d6): d 13.20 (bs, 1H, COOH, D2O
exchange), 8.07 (d, 2H, carboxyphenyl, J = 8.31 Hz), 7.80–8.00 (m,
5H, trifluoromethyl-phenyl and CH), 7.78 (d, 2H, carboxyphenyl,
J = 8.2 Hz); HRMS (ESIÀ) (m/z): [MÀ1]À calculated for
Intermediates were confirmed by mass spectrometry and 1H
NMR. Full analytical data for representative and the main com-
pounds, including their intermediates, are provided. Melting points
are uncorrected. Weakly active and inactive compounds and their
intermediates were characterized by mass spectral analysis (LC/
MS). Purity was determined by TLC and HPLC. Compounds with
purity >95% were used for CFTR inhibition testing.
C18H9F3NO3S2, 407.9976, found 407.9976.
4.3.2. 5-[[4-(2H-Tetrazol-5-yl)phenyl]methylene]-2-thioxo-3-
[3-(trifluoromethyl)phenyl]-4-thiazolidinone (6, Tetrazolo-
172)9
Mp 216–219 °C; 1H NMR (DMSO-d6): d 11.92 (bs, 1H, tetrazolo-
H, D2O exchange), 8.16 (d, 2H, phenyl, J = 8.3 Hz), 8.02 (s, 1H), 7.92
(s, 1H), 7.87–7.84 (m, 3H, phenyl or trifluoromethyl-phenyl and/or
CH), 7.79–7.76 (m, 2H, trifluoromethyl-phenyl); MS (ESÀ) (m/z):
[MÀ1]À calculated for C18H9F3N5OS2, 432.0201, found 432.0205.
4.2. Synthesis of thiazolidinone intermediate 3
Typical procedure for Route 1:15 An equimolar amount of carbon
disulfide was added dropwise to an ice-cold solution of aniline 1
and triethylamine in ethyl acetate over 30 min (Scheme 1). After
stirring overnight, a yellow dithiocarbamate was isolated by filtra-
tion and reacted with an equimolar amount of aqueous bromoace-
tic acid solution (NaHCO3, pH 8-9). After 2 h, the solution was
acidified (HCl), refluxed, and the resultant precipitate crystallized
from ethanol to yield thiazolidinone intermediate 3. Typical proce-
dure for Route 2: A solution of isothiocyanate 2 (5 mmol, in THF)
was added dropwise to a stirred aqueous solution of thioglycolic
acid (0.347 g, 3.7 mmol) and triethylamine (1.38 ml, 10 mmol).
After 30 min at 0 °C, the reaction mixture was further stirred at
room temperature for 3 h. The reaction mixture was acidified
(HCl), refluxed, and resultant precipitate crystallized from ethanol
to yield thiazolidinone intermediate 3.
4.3.3. 5-(1-Oxido-4-pyridinyl)methylene)-2-thioxo-3-[3-(triflu-
oromethyl)phenyl]-4-thiazolidinone(17, Pyridine-NO-172)13
Mp 209–210 °C (decomp); MS (ES+) (m/z): [M+H]+ calculated
for C16H9F3N2O2S2, 382.39, found 383.01.
4.3.4. 4-[[4-Oxo-2-thioxo-3-[2-methyl-3-(trifluoromethyl)phe-
nyl]-5-thiazolidinylidene]methyl] benzoic acid(18, a-Me-172)
Mp 156–158 °C; 1H NMR (DMSO-d6): d 12.74 (bs, 1H, COOH,
D2O exchange), 8.05 (d, 2H, carboxyphenyl, J = 8.3 Hz), 7.90 (1H,
s, @CHA), 7.85 (d, 1H, J = 7.8 Hz, trifluoromethylphenyl), 7.79 (d,
2H, J = 8.3, carboxyphenyl), 7.74 (d, 1H, J = 7.3 Hz, trifluoromethyl-
phenyl), 7.58 (t, 1H, J = 7.8 Hz, trifluoromethylphenyl), 2.13 (s, 3H,
CH3); HRMS (ESIÀ) (m/z): [MÀ1]À calculated for C19H11F3NO3S2,
422.0132, found 422.0140.
Isothiocyanates and isocyanates 2, if not available commer-
cially, were prepared by reaction of respective amino compounds
1 with phosgene or thiophosgene, following known procedures.29

8194
N. D. Sonawane, A. S. Verkman / Bioorg. Med. Chem. 16 (2008) 8187–8195
4.3.5. 5-(4-Pyridinylmethylene)-2-thioxo-3-[3-(trifluoromethyl)
phenyl]-4-thiazolidinone (33)
4.3.14. 4-[(5-(3-Trifluoromethylphenyl)-1,3,4-thiadiazol-2-yl)
amino]-benzoic acid (64)37
Mp 186–188 °C; 1H NMR (DMSO-d6): d 8.72 (dd, 2H, J = 6.35,
2.93 Hz, pyridine), 7.92 (s, 1H, =CH-), 7.87 (d, 1H, J = 7.32 Hz, tri-
fluoromethylphenyl), 7.81–7.75 (m, 3H, trifluoromethylphenyl),
7.59 (dd, 2H, J = 6.35, 2.93 Hz, pyridine); MS (ES+) (m/z): [M+1]+
calculated for C16H9F3N2OS2, 367.40, found 367.20.
Thiosemicarbazide 62 (2.6 mmol) was added slowly to 10 ml of
concentrated sulfuric acid, stirred for 30 min at room temperature,
and slowly dumped into a stirred ice-water mixture. The precipi-
tated product was purified by flash chromatography to give 64
(yield 77%). 1H NMR (DMSO-d6): d 10.98 (bs, 1H, COOH), 8.14–
8.11 (m, 3H, trifluoromethylphenyl), 7.90 (d, 2H, J = 8.3 Hz, car-
boxyphenyl) 7.84–7.71 (m, 4H, (2H carboxyphenyl, 1H trifluoro-
methylphenyl, NH)); MS (ES+) (m/z): [M+1]+ calculated for
4.3.6. 4-[[4-Oxo-2-thioxo-3-[(3-(trifluoromethyl)phenyl)
methyl]-5-thiazolidinylidene]methyl]benzoic acid (46)
1H NMR (DMSO-d6): d 13.19 (s, 1H, COOH), 8.01 (d, 2H,
J = 8.3 Hz, carboxyphenyl), 7.86 (s, 1H, trifluoromethylphenyl),
7.73–7.70 (m, 3H, carboxyphenyl and trifluoromethylphenyl),
7.63–7.51 (m, 3H, trifluoromethyl and @CHA), 5.29 (s, 2H, CH2);
MS (ES+) (m/z): [M+1]+ calculated for C19H12F3NO3S2, 424.45, found
424.17.
C16H10F3N3O2S, 366.35, found 366.46.
4.3.15. 4-Hydroxy-3-[[5-(3-trifluoromethyl)phenyl-1,3,4-thia-
diazol-2-yl]amino]-benzoic acid (65)
Synthesized by same procedure as for 64. MS (ES+) (m/z):
[M+1]+ calculated for C16H10F3N3O2S, 382.34, found 382.03.
4.3.7. 4-[[3-[3-(trifluoromethyl)phenyl]-2,4-dioxo-5-thiazolidi-
nylidene]methyl]benzoic acid (48, Oxo-172)
4.3.16. 4-[4-[3-(trifluoromethyl)phenyl]-1H-1,2,3-triazol-1-yl]-
benzoic acid (68)38
1-Ethynyl-3-(trifluoromethyl)benzene (0.85 g, 5 mmol) and 4-
azidobenzoic acid (0.815 g, 5 mmol) were suspended in a 1:1 mix-
ture of water and tert-butyl alcohol (10 mL). A freshly prepared
Mp 168–170 °C; 1H NMR (DMSO-d6): d 13.05 (bS, 1H, COOH,
D2O exchange), 8.06–8.04 (d, 2H, carboxyphenyl, J = 8.3 Hz), 8.01
(s, 1H), 7.92 (s, 1H), 7.86–7.84 (m, 1H), 7.78–7.74 (m, 4H); HRMS
(ESIÀ) (m/z): [MÀ1]À calculated for C18H9F3NO4S, 392.0204, found
392.0207.
solution of sodium ascorbate (0.3 mmol, 300
followed by copper (II) sulfate pentahydrate (7.5 mg, 0.03 mmol, in
100 L of water). The mixture was stirred for 24 h at room temper-
lL of 1 M) was added,
l
4.3.8. 4-[[2,5-dioxo-1-[3-(trifluoromethyl)phenyl]-3-pyrrolidinyl]
thio]-benzoic acid (51)
ature. The reaction mixture was diluted with water, and the white
precipitate was collected by filtration, washed, and dried to give
1.53 g (92%) of pure product as an off-white powder. Mp
>250 °C; MS (ES+) (m/z): [M+1]+ calculated for C16H10F3N3O2,
334.28, found 334.09.
MS (ESÀ) (m/z): [MÀ1]À calculated for C18H12F3NO4S, 394.35,
found 394.18.
4.3.9. 4-Oxo-[(3-trifluoromethyl)phenyl]-2-thioxo-N-[4-(carbo-
xy)phenyl]-5-thiazolidinethio carboxamide (52)
MS (ES+) (m/z): [M+1]+ calculated for C18H11F3N2O3S3, 457.49,
found 457.37.
4.3.17. Solubility
As reported,45 a saturated compound solution was prepared by
addition of DMSO stock to phosphate buffered saline (final DMSO
2%) followed by sonication for 5 min at 25 °C and shaking at room
temperature for 1 h. After centrifugation at 15,000 rpm for 1 h, the
supernatant was analyzed by LC/MS with concentration deter-
mined from area under the curve, standardized against calibration
data.
4.3.10. 4-Oxo-[(3-trifluoromethyl)phenyl]-2-thioxo-N-[(4-carbo-
xy-3-hydroxy)phenyl]-5-thiazolidinethiocarboxamide (53)
MS (ES+) (m/z): [M+1]+ calculated for C18H11F3N2O4S3, 473.50,
found 473.23.
Standard curves for each compound were obtained by plotting
area under the curve from chromatograms against inhibitor con-
centration. The concentration range of the standard solutions
4.3.11. 4-[[4-Oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]-5-
thiazolidinyl]methyl]benzoic acid (56)24,34
To a stirred solution of 5 (102 mg, 0.25 mmole) in pyridine
was 1–15 lM (for 5) and 1–100 lM (other compounds). In all
(1 ml), lithium borohydride (1 M solution in THF, 500
ll) was
cases, standard curves prepared were linear with r > 0.998.
added dropwise over 30 min. The reaction mixture was refluxed
(2 h), cooled, and added to an ice-cold solution of hydrochloric acid
(4 N, 5 ml). The resulting material was refluxed for 30 min, filtered
hot, and cooled. The precipitate was dried to yield 56. MS (ES+) (m/
z): [MÀ1]À calculated for C18H12F3NO3S2, 410.42, found 410.13.
4.3.18. Short-circuit current measurements
FRT cells (stably expressing human wildtype CFTR) were cul-
tured on Snapwell filters with 1 cm2 surface area (Corning-Costar)
to resistance >1000
X
cm2 as described.11 Filters were mounted in
an Easymount Chamber System (Physiologic Instruments, San Die-
go). For apical ClÀ current measurements the basolateral hemi-
chamber contained (in mM): 130 NaCl, 2.7 KCl, 1.5 KH2PO4, 1
CaCl2, 0.5 MgCl2, 10 Na–HEPES, 10 glucose (pH 7.3). The basolateral
4.3.12. 3-Trifluoromethyl benzoic acid hydrazide
Hydrazine hydrate (4 equivalents) was added to the stirred
solution of 3-trifluoromethyl benzoyl chloride 61 in pyridine at
0 °C. The reaction mixture was added to the ice-cold water and
the collected precipitate was recrystallized from ethanol (yield
83%). MS (ES+) (m/z): [M+1]+ calculated for C8H7F3N2O, 205.16,
found 205.01.
membrane was permeabilized with amphotericin B (250 lg/ml)
for 30 min. In the apical solution 65 mM NaCl was replaced by so-
dium gluconate, and CaCl2 was increased to 2 mM. Solutions were
bubbled with 95% O2/5% CO2 and maintained at 37 °C. Current was
recorded using a DVC-1000 voltage-clamp (World Precision Instru-
ments) using Ag/AgCl electrodes and 1 M KCl agar bridges.
4.3.13. 3-Trifluoromethyl-2-[[(4-carboxyphenyl)
amino]thioxomethyl]benzoic acid hydrazide (62)
A mixture of 3-trifluoromethyl benzoic acid hydrazide (1 g,
5 mmol, prepared as above) and appropriate carboxyphenylisothi-
ocyanate (5 mmol) in THF was refluxed for 2 h. The solid obtained
after solvent evaporation was purified by recrystallization from
ethanol to give thiosemicarbazide 62 (yield 74%). MS (ES+) (m/z):
[M+1]+ calculated for C16H12F3N3O3S, 384.35, found 384.06.
4.3.19. Cytotoxicity
FRT cells in confluent monolayers were incubated with com-
pounds for 2 days. Cells were washed 3 times, fixed (cytofix,
30 min), and stained with crystal violet (100 ll, 0.5%, 10 min) using
standard procedures.46 Excess crystal violet was removed by wash-
ing and dye was extracted with Sorenson’s buffer (0.1 M sodium

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citrate, 50% ethanol, pH 4.2). Crystal violet was quantified by mea-
surement of absorbance at 650 nm. Percentage crystal violet stain-
ing was determined from test wells measured 8 times, compared
to blanks (wells not containing cells) and vehicle-treated cells.
Acknowledgements
Supported by Grants DK72517, HL73854, EB00415, EY13574,
DK35124, and DK43840 from the National Institutes of Health,
and Drug Discovery and Research Development Program grants
from the Cystic Fibrosis Foundation.
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