4′-Methyl-4,5′-bithiazole-based correctors of defective ΔF508-CFTR cellular processing

Article abstract of DOI:10.1016/j.bmcl.2008.03.037

The synthesis and ΔF508-CFTR corrector activity of a 148-member methylbithiazole-based library are reported. Synthetic routes were devised and optimized to generate methylbithiazole analogs in four steps. Corrector potency and efficacy were assayed using epithelial cells expressing human ΔF508-CFTR. These structure-activity data establish that the bithiazole substructure plays a critical function; eight novel methylbithiazole correctors were identified with low micromolar potencies.

Full text of DOI:10.1016/j.bmcl.2008.03.037

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2610–2614
40-Methyl-4,50-bithiazole-based correctors of defective
DF508-CFTR cellular processing
Choong Leol Yoo,a Gui Jun Yu,a Baoxue Yang,b Lori I. Robins,a
A. S. Verkmanb and Mark J. Kurtha,*
aDepartment of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA
bDepartments of Medicine and Physiology, University of California, San Francisco, CA 94143, USA
Received 24 December 2007; accepted 11 March 2008
Available online 16 March 2008
Abstract—The synthesis and DF508-CFTR corrector activity of a 148-member methylbithiazole-based library are reported. Syn-
thetic routes were devised and optimized to generate methylbithiazole analogs in four steps. Corrector potency and efficacy were
assayed using epithelial cells expressing human DF508-CFTR. These structure–activity data establish that the bithiazole substruc-
ture plays a critical function; eight novel methylbithiazole correctors were identified with low micromolar potencies.
Ó 2008 Elsevier Ltd. All rights reserved.
Cystic fibrosis (CF), a lethal genetic disease afflicting
ꢀ0.04% of white individuals,1 results in chronic lung
infections because mutant cystic fibrosis transmembrane
conductance regulator (CFTR) protein fails to confer
chloride permeability to epithelial cells in lung and other
tissues.2 DF508-CFTR, the most common CF mutation
(present in at least 1 allele in ꢀ90% of CF patients),1
contains a single amino acid deletion of phenylalanine
508, which causes the nascent protein to be retained in
the endoplasmic reticulum and rapidly degraded.3 When
allowed to reach the cell plasma membrane by low-tem-
perature (27 °C) rescue, DF508-CFTR can function as a
cAMP-activated chloride channel, but with significantly
decreased activity compared with WT-CFTR.4 While
programs aimed at the discovery of small-molecule effec-
tors of defective DF508-CFTR folding and cellular pro-
cessing (e.g., correctors) and channel gating (e.g.,
potentiators) have identified a number of DF508-CFTR
potentiators,5 the discovery of effective correctors is a
substantially greater challenge as protein folding and
trafficking are complex processes involving multiple cel-
lular targets, some of which may be cell type-specific. As
previously reported, we identified several small-molecule
DF508-CFTR correctors [aminoarylthiazoles (e.g., 1
and 2), quinazolinylaminopyrimidinones (e.g., 3), and
40-methyl-4,50-bithiazole (e.g., 4); Fig. 1] by screening a
structurally diverse set of 150,000 compounds.6 Func-
tional and biochemical analyses established methylbi-
thiazoles as particularly promising for further
development based on their efficacy in human DF508-
CFTR airway epithelial cells and their CFTR-specific-
ity. Herein, we report the preparation and screening of
148 new methylbithiazole analogs aimed at establishing
initial SAR data for this lead class of correctors.
To verify our initial screening result as well as to develop
an effective route to methylbithiazoles, we began with a
resynthesis of corrector 4. This work commenced by treat-
ing 3-chloropentane-2,4-dione with thiourea (Scheme 1)
under reflux in absolute ethanol; 5-acetyl-2-amino-4-
MeO
H3C
H
N
N
S
H3C
N
N
N
NH
NH
S
S
1
2
4
Cl
H
OH
O
N
S
S
HN
N
OMe
N
N
N
N
N
H
S
N
N
N
Keywords: DF508-CFTR correctors; Bithiazole; Structure–activity;
Small-molecule library.
H
OMe
3
*
Corresponding author. Tel.: +1 530 752 8192; fax: +1 530 752
8995; e-mail: mjkurth@ucdavis.edu
Figure 1. Correctors of defective human DF508-CFTR.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.037

C. L. Yoo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2610–2614
2611
tion resolved this problem, presumably because this
strongly acidic medium promotes enolization. Finally,
bromoacetyl thiazole 8 (obtained from 6) was treated with
thiourea 9, in turn prepared by the reaction of 5-chloro-2-
methoxyaniline with ammonium thiocyanate, in refluxing
ethanol to deliver corrector 4 in excellent yield. Impor-
tantly, no chromatic purifications were required through-
out the four-step process outlined in Scheme 2.
O
O
S
O
Δ
EtOH,
12 h
N
+
H2N
NH2
N
S
H2N
Cl
5
O
O
S
N
H
conditions
6
The concentration–activity data for resynthesized meth-
ylbithiazole 4 are shown in Figure 2 where the dashed
line indicates the level of activity with low-temperature
(27 °C) rescue which is used as a positive control and
reference. Activation of DF508-CFTR was confirmed
for each of the compounds by showing no activity on
non-transfected FRT cells and near-complete inhibition
of the increased iodide influx by the thiazolidinone
CFTRinh-172 at 10 lM (data not shown).7
Scheme 1. Aminothiazole N-acylation problem. Reagents and condi-
tions: (1) Et3N (1.2 equiv), C6H5COCl, DCM, 0 °C to rt, overnight, 5
is recovered; (2) Et3N (1.2 equiv), C6H5COCl, DCM, 0 °C to rt,
overnight, except 5 was pre-washed with 10% aq NaOH, results in
complex mixture; (3) Et3N (1.2 equiv), C6H5COCl, toluene, reflux,
overnight, 5 is recovered + trace of 6; (4) i-Pr2NEt (15 equiv),
C6H5COCl (10 equiv), DCM, rt, overnight, 5 is consumed, mono-
and bis-benzoylation products.
methylthiazole (5) was obtained in 90% yield. Surpris-
ingly, attempts to N-acylate the C2-amino moiety in 5
with benzoyl chloride under various bases, solvents, and
temperature conditions failed or delivered the targeted
N-acyl aminothiazole 6 in low yield. While perhaps sur-
mountable in any particular case, the malfunction of
5 ! 6 is a consequence of the 5-acetyl moiety reducing
the nucleophilicity of the C2-amino group of 5. Since
the plan was to ultimately diversify with a spectrum of
acid chlorides, this problematic reaction caused us to eval-
uate the inverse of these two reactions—that is, use ben-
zoyl isothiocyanate as the starting material in place of
thiourea (Scheme 2). Passing ammonia gas through a
dichloromethane solution of benzoyl isothiocyanate
delivered N-carbamothioylbenzamide (7) which then re-
acted with 3-chloropentane-2,4-dione to afford 6 in good
yield. a-Bromination of the acetyl group of 6 proved to be
quite challenging with the recovery of unreacted 6 being
the principle issue [Br3 on Amberlite A-26 resin, CHCl3,
rt, 24 h/no reaction; Br2 in AcOH, rt, 24 h/>50% recov-
ered 6; NBS, CHCl3, reflux, 24 h/>50% recovered 6].
Pyridinium tribromide in a 30% HBr in acetic acid solu-
To establish SAR information, it was decided to begin
by making a small collection of analogs of 4 in which
the N-(40-methyl-4,50-bithiazol-20-yl)benzamide moiety
was held constant while the aniline moiety was varied.
As outlined in Scheme 3, each of these derivatives was
80
27 oC
60
-
d[I ]
40
20
0
dt
(μM/s)
0.1
1
10
[methylbithiazole 4] (μM)
Figure 2. Concentration–activity analysis of methylbithiazole
(mean SE, n = 4).
4
CH3
O
step 1
step 2
Δ
EtOH,
N
O
O
O
S
NH3, DCM
S
N
N=C=S
N
H
NH2
0 o
C → rt
H
O
Br
Cl
O
7
Ar
N
8
H2
CH3
S
NH4SCN
CH3
step 3
Ar EtOH, Δ
O
H2
N
N
H
N
1.5 M aq. HCl, Δ
O
N
O
pyrH+ Br3
-
O
S
N
N
H
S
30% HBr in HOAc
rt
H
Br
CH3
held
constant
6
8
N
S
O
Cl
Ar
CH3
2'
S
N
2
N
H
N
H
R1
step 4
N
S
O
Δ
EtOH,
10
S
N
N
H
N
H
a
: Ar = p-MeC6H4
Cl
b
: Ar = p-MeOC6H4
S
OMe
c: Ar = p-FC6H4
H2
N
N
H
4
R2
d
: Ar = m-FC6H4
OMe
e
: Ar = m-MeOC6H4
f: Ar = p-ClC6H4
9
NH4SCN
1.5 M aq. HCl, Δ
Cl
g
h
: Ar = m-Cl(o-MeO)C6H3
: Ar = o-Cl(m-Cl)C6H3
H2
N
OMe
Scheme 2. Synthesis of corrector 4.
Scheme 3. Aniline derivatives of the methylbithiazole corrector.

2612
C. L. Yoo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2610–2614
A
B
*
80
60
40
20
0
80
*
*
60
*
*
*
-
-
d[I ]
dt
μM/s
d[I ]
dt
μM/s
*
*
40
20
0
*
*
*
*
*
10 a
b
c
d
e
f
g
h
C 4a
10 a
b
c
d
e
f
g
h
C 4a
bithiazole analogs (10 μM)
bithiazole analogs (25 μM)
Figure 3. Maximal iodide influx in DF508-CFTR-expressing FRT cells incubated at 37 °C for aniline-based methylbithiazole correctors 10a–h (see
Scheme 3) compared with 4a (C, negative DMSO vehicle control) (mean SE, n = 4, *P < 0.05 tested by Student’s test comparing with C).
available in one step by condensation of the appropriate
arylthiourea with 8.
N
S
O
R2
S
N
R1
N
H
N
H
The corrector efficacies of methylbithiazoles 10a–h are
summarized in Figure 3. Although all eight of these
methylbithiazoles were less effective correctors of
DF508-CFTR than the initial hit compound 4, we were
encouraged to find that three (10f, 10g, and10h) were
moderately effective. Indeed, compounds 10g and 10h
showed comparable corrector activity to 4 at both 10
and 25 lM concentrations. Additionally, this small set
of compounds established that placing an electron with-
drawing fluorine group on the aniline moiety (e.g., 10c
and 10d) caused a significant decrease in activity and this
decrease was independent of where the fluoride was lo-
cated (10c and 10d). This small initial set of compounds
established that the peripheral modification of the meth-
ylbithiazole core modulates corrector activity and, as
such, a broader study of the amide and aniline substruc-
tures of methylbithiazole 4 was undertaken.
11Aa-11Dj
R1
R2
Cl
CH3
//
\\
\\
\\
Cl
CH3
CH3
Cl
a
c
A
b
Cl
Cl
\\
f
Cl
Cl
//
\\
\\
Cl
OMe
OMe
B
d
e
OMe
//
\\
\\
\\
i
OMe
OMe
C
g
h
With these data for bithiazoles 10a–h in hand, a 40-
member second library set (11Aa–11Dj; Fig. 4) was pre-
pared by reagent-based modification of the protocol
outlined in Scheme 2 (e.g., the isothiocyanate employed
in step 1 incorporate aryl moieties A–D and the N-aryl
thioureas used in step 4 incorporated aryl moieties a–
j). Based on the results with 10c and 10d, fluorine was
precluded from the R2 aryls.
O2N
//
\\
j
OEt
D
Figure 4. Library set two bithiazoles.
its C5 acyl moiety. In addition, adding the R1 diversity
input in the last step was expected to provide consider-
able time- and labor-saving benefits, particularly with
regards to product purification.
While library set two was under preparation, a third li-
brary set was targeted in which R1 diversity (Scheme 2)
was expanded to include non-aryl substituents. Scheme
2 chemistry afforded high yielding reactions and re-
quired no chromatographic purifications, but R1 diver-
sity in the bithiazoles accessible by this chemistry was
limited in that few isothiocyanates are commercially
available. Since previous attempts at benzoylation
5 ! 6 (Scheme 1) were problematic, it was decided to
construct library set three by assembly of the bithiazole
first with subsequent N-acylation (Scheme 4). It was
postulated that the C20-amino group in the planned 40-
methyl-N-aryl-4,50-bithiazole-2,20-diamine intermediate
(e.g., 14a–j) would be considerably more nucleophilic
than the corresponding amine in aminothiazole 5 with
Bromination of 5 using pyridinium tribromide plus 30%
HBr in acetic acid afforded 12 in 50% yield. While the
isolated product yield of this reaction was significantly
lower than the nearly quantitative bromination 6 ! 8,
the cause of this modest yield was the water solubility
of the ammonium salt of 12. Aromatic thioureas 13a–j
were prepared by bubbling ammonia gas through the
commercially available aromatic isothiocyanates in
dichloromethane. After simply evaporating the dichlo-
romethane, pure aromatic thioureas 13a–j were obtained

C. L. Yoo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2610–2614
2613
CH3
Cl
CH3
-
N
pyrH+ Br3
EtOH,
Δ
O
5
O
N
S
H2N
S
S
30% HBr in HOAc
rt
C2 moiety
S
N
R2
Br
N
H
N
H
12
C2' moiety
H2N
N
OMe
H
4
13a-j
NH3, DCM
μ
μ
Vmax ( M/s) Kd ( M)
122 +9 0.9
bithiazole
CH3
S=C=N
R2
0 o
C → rt
CH3
μ
μ
Vmax ( M/s) Kd ( M)
N
S
R2
O
S
N
N
H
H2N
CH3
CH3
O
N
S
14a-j
R1
Cl
N
S
O
S
N
Et3N, CHCl3
N
N
H
R2
H
S
N
R1
N
H
N
H
0 o
11Ca
CH3
102 + 7
112 + 6
127 + 8
97 + 8
1.1
1.1
0.7
0.8
1.1
C → rt
Cl
15Ea-15Nj
OMe
OMe
R1 diversity*
O
N
S
\\
\\
\\
\\
\\
S
N
N
H
N
H
H
F
G
E
11Ci
Cl
Cl
CH3
O
\\
\\
\\
\\
O
O
N
S
L
K
J
I
S
N
N
N
H
H
\\
OMe
OEt
15Jf
CH3
M
N
N
S
Scheme 4. Synthesis of library set three bithiazoles.
S
N
N
H
N
H
in high yield. Bromothiazole 12 was subsequently re-
fluxed in ethanol with thioureas 13a–j to afford C20-
aminobithiazoles 14a–j in 60–80% yields. These com-
pounds were contaminated by the modest quantities of
impurities which proved difficult to remove by flash col-
umn chromatography as product and impurity Rf values
were similar. Consequently, the crude C20-aminobithiaz-
oles were used in the next step without purification. As
hoped, acylation of the C20-amino moiety of 14a–j with
various acid chlorides in the presence of triethylamine in
chloroform afforded library set three bithiazoles 15Ea–
15Nj (Scheme 4). LC–MS analysis of this last reaction
generally showed 40–60% reaction completion. Addi-
tional reaction time or the addition of more acid chlo-
ride made product purification more difficult due to a
corresponding increase in side products. Upon work-
up, the products were purified by prep-HPLC and again
analyzed by LC–MS. All showed 90% or better purity as
well as the correct molecular ion peak.
15Jj
CH3
Cl
O
N
S
S
N
N
N
H
H
11Cd
CH3
126 + 11
Cl
Cl
O
N
S
S
N
N
N
H
H
11Dd
119 + 7
113 + 9
114 + 8
0.8
1.1
1.1
O2N
CH3
O
N
S
S
N
N
H
N
H
OMe
15Jh
CH3
OMe
O
N
S
S
N
N
H
N
H
OMe
15Ni
The Kd/Vmax data for the eight most active methylbi-
thiazoles from 11Aa–11Dj and 15Ea–15Nj series are
shown in Figure 5. It is interesting to note that six out
of the eight best methylbithiazole share either a 4-chlo-
robenzamide (11Ca, 11Cd, 11Ci) or a pivalamide
(15Jf, 15Jh, 15Jj) at the C20-position and five out of
these eight share either o-methoxy or o-ethoxy aromatic
amines (11Ci, 15Jf, 15Jh, 15Jj, 15Ni) at the C2-position.
Figure 5. Kd/Vmax data for the most active bithiazoles in library sets
two and three.
substituent showed DF508-CFTR corrector activity.
Nevertheless, among the bithiazoles screened (including
the original hit 4), new bithiazole 15Jf is the most effec-
tive corrector as judged by both Vmax and Kd data.
When there is a p-substituted aromatic amide at the C20-
position (11Ca, 11Cd, 11Ci, 11Dd), p-substituted aro-
matic amines at the C2-position have elevated activity
(11Ca, 11Cd, 11Dd). It is also interesting that bithiazole
15Ni, which presents a relatively long alkyl amide chain,
expressed comparable corrector activity. None of the
methylbithiazoles incorporating a 2,4-dichloroaniline
DF508-CFTR corrector activity measurements were
made in this study as described previously6 using FRT
epithelial cells stably co-expressing human DF508-CFTR
and the sensitive halide-sensing green fluorescent protein
YFP-H148Q/I152L.8 Cells were grown at 37 °C (90%

2614
C. L. Yoo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2610–2614
humidity; 5% CO2) for 24 h and then incubated for 20 h
with 50 ll of medium containing the test compounds.
At the time of the assay, cells were washed with PBS
and then incubated with PBS containing forskolin
(20 lM) and genistein (50 lM) for 20 min. Measurements
were carried out using FLUOstar fluorescence plate read-
ers (Optima; BMG LABTECH GmbH), each equipped
with 500 10 nm excitation and 535 15 nm emission
filters (Chroma Technology Corp.). Each well was as-
sayed individually for iodide influx by recording fluores-
cence continuously (200 ms per point) for 2 s (baseline)
and then for 12 s after rapid (<1 s) addition of 165 ll
PBS in which 137 mM chloride was replaced by iodide.
The iodide influx rate was computed by fitting the final
11.5 s of the data to an exponential for the extrapolation
of initial slope and normalized for the background-sub-
tracted initial fluorescence. All experiments contained
negative controls (DMSO vehicle) and positive controls
(methylbithiazole 4).
Foundation [CHE-0614756 as well as CHE-0443516
and CHE-9808183 (NMR spectrometers)] for their gen-
erous financial support.
References and notes
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In conclusion, we have developed two versatile synthetic
routes for the reliable preparation of bithiazole deriva-
tives. Screening data for the 148 bithiazoles provides
valuable structural activity information. In particular,
the information gathered from bithiazoles 11Ca, 11Cd,
11Ci, 11Dd, 15Jf, 15Jh, 15Jj, and 15Ni is guiding our
ongoing efforts to further refine and improve the
DF508-CFTR corrector activity of bithiazoles.
6. Pedemonte, N.; Lukacs, G. L.; Du, K.; Caci, E.; Zegarra-
Moran, O.; Galietta, L. J. V.; Verkman, A. S. J. Clin.
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Acknowledgments
The authors thank the National Institutes of Health
(DK072517 and GM076151) and the National Science
8. Galietta, L. J.; Haggie, P. M.; Verkman, A. S. FEBS Lett.
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