Fluorinated DF508-CFTR correctors and potentiators for PET imaging

Article abstract of DOI:10.1016/j.bmcl.2011.12.128

¹⁹F-modified bithiazole correctors and phenylglycine potentiators of the DF508-CFTR chloride channel were synthesized and their function assayed in cells expressing human DF508-CFTR and a halide-sensitive fluorescent protein. Fluorine was incorporated into each scaffold using prosthetic groups for future biodistribution imaging studies using positron emission tomography (PET). The DF508-CFTR corrector and potentiator potencies of the fluorinated analogs were comparable to or better than those of the original compounds.

Full text of DOI:10.1016/j.bmcl.2011.12.128

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1602–1605
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorinated
D
F508-CFTR correctors and potentiators for PET imaging
Holly R. Davison a, Danielle M. Solano a, Puay-Wah Phuan b, A.S. Verkman b, Mark J. Kurth a,
⇑
a Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
b Departments of Medicine & Physiology, University of California, San Francisco, Health Science East Tower, Room 1246, 505 Parnassus Avenue, San Francisco, CA 94143, United States
a r t i c l e i n f o
a b s t r a c t
19F-modified bithiazole correctors and phenylglycine potentiators of the
D
F508-CFTR chloride channel
F508-CFTR and a halide-sensi-
tive fluorescent protein. Fluorine was incorporated into each scaffold using prosthetic groups for future
biodistribution imaging studies using positron emission tomography (PET). The F508-CFTR corrector
Article history:
Received 4 December 2011
Revised 23 December 2011
Accepted 27 December 2011
Available online 3 January 2012
were synthesized and their function assayed in cells expressing human
D
D
and potentiator potencies of the fluorinated analogs were comparable to or better than those of the ori-
ginal compounds.
Keywords:
Cystic fibrosis
PET
Ó 2011 Elsevier Ltd. All rights reserved.
Corrector
Potentiator
D
F508-CFTR
Cystic fibrosis (CF) is the most prevalent lethal hereditary dis-
the non-modified compounds could be studied. Also, the limited
penetration of light into tissues precluded non-invasive whole-
body imaging.
ease among Caucasians, affecting approximately one in 2500 indi-
viduals.1 The average life expectancy in CF is about 40 years. The
principal cause of mortality in CF is deterioration of lung function
caused by chronic lung infection.2,3 Cystic fibrosis is caused by
mutations in the cystic fibrosis transmembrane conductance regu-
lator (CFTR) protein, the most common being a deletion of phenyl-
To address these issues, we report here a first step to apply pos-
itron emission tomography (PET) imaging to non-invasively visual-
ize the biodistribution of a bithiazole corrector and a phenylglycine
potentiator.15–17 Analogs suitable for PET imaging have the advan-
tage of minimally modifying the original compound structure
(replacing a H with an 18F),18 which increases the likelihood of
maintaining activity while enabling in vivo monitoring of uptake
and biodistribution. Indeed, imaging modalities can be relevant
at all stages of CF drug development, with potential applications
in animal models, tissue preparations, and human in vivo studies.
Relevant imaging could enlighten at the basic mechanistic level
leading to a better understanding CF pathophysiology, at the level
alanine at position 508 (D
F508).4,5 This mutation causes CFTR
protein misfolding, resulting in retention and rapid degradation
in the endoplasmic reticulum.6–9
Compounds have been synthesized that rescue defective
D
F508-CFTR protein processing (correctors) and defective chloride
channel gating (potentiators).10–12 Bithiazole
1
(corr-4a) and
indolylacetamidophenylacetamide 2 (PG-01) are the benchmark
corrector and potentiator studied here, respectively (Fig. 1). Struc-
ture–activity relationship studies based on corr-4a revealed that
substitution of a 2-chloro-5-(N,N-dimethylamino)phenyl moiety
for the 5-chloro-2-methoxyphenyl group improved water
solubility (3; Fig. 1)13 and that locking the bithiazole system into
an s-cis conformation with a seven-membered ring (3; Fig. 1) im-
proved corrector efficacy.11
We recently reported the synthesis of a fluorescently-labeled
bithiazole corrector 4 (Fig. 1), which enabled the evaluation of its
biodistribution in mice by fluorescence imaging.14 Though this ap-
proach produced information about the in vivo handling of a func-
tional CF corrector, the inclusion of a fluorophore label resulted in
poor compound metabolic stability, limiting the time over which
⇑
Corresponding author.
E-mail address: mjkurth@ucdavis.edu (M.J. Kurth).
Figure 1. DF508-CFTR correctors (1/3/4) and potentiator (2).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.128

H. R. Davison et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1602–1605
1603
of assessing the potential of new correctors or potentiators, at the
effectiveness of treatment level, and/or at the level of measuring
function to better drug delivery.19
PET produces images through the detection of positron emitting
radioisotopes. The most commonly used radionucleotide is
fluorine-18 due to its relatively long half-life (t1/2 = 110 min).
Other elements, such as carbon (11C t1/2 = 20.4 min), nitrogen
(
13N t1/2 = 9.97 min), and oxygen (15O t1/2 = 2.04 min), have much
shorter half-lives. To address the need for new disease-specific
probes, prosthetic groups18 have been developed for the rapid
incorporation of radioisotopes into the specific tracers. A variety of
amine-reactive prosthetic groups have been developed, the most
commonly being
[
18F]N-succinimidlyl-4-fluorobenzoate (5;
Fig. 2).20 Other 18F-acylating agents include the carboxylate reac-
tive [18F]4-fluoroaniline prosthetic group (6; Fig. 2).21 A strategy
that has received considerable attention is the use of click chemis-
try to incorporate small molecular weight 18F-labeled alkynes such
as [18F]5-fluoroalkyne (7; Fig. 2) into azide substituted peptides.22
Copper(I)-catalyzed 1,2,3-triazole formation is fast, chemoselec-
tive, and performed under mild conditions.22
Figure 3. Fluorine-containing corrector and potentiator analogs.
As a first step in enabling CF-relevant PET studies, we report
here the design, synthesis, and
D
F508-CFTR activity of two 19F-la-
beled correctors (8 and 9; Fig. 3) and two 19F-labeled potentiators
(10 and 11; Fig. 3). Since 18F has a 110 min half-life, late-stage
introduction of the fluorinated moiety is essential and, conse-
quently, targets 8–11 were designed with the aforementioned
18F-prosthetic groups in mind.
Targeting fluorobenzoyl analogs 19F-8 and 19F-9 (Scheme 1), the
first synthetic step was the condensation of 3-chloropentane-2,4-
dione with thiourea in refluxing ethanol over 12 h to afford amino-
thiazole 12 in 95% yield.11 The acetyl group of 12 was subsequently
a-brominated with bromine in acetic acid to produce 13 in 86%
yield. Aminothiazole building block 15 was obtained in 79% yield
by the condensation of 13 with 1-(2-chloro-5-dimethylaminophe-
nyl)thiourea (14) in refluxing ethanol. The final step in the synthe-
sis of 19F-bithiazole 8 proved to be more challenging than expected
as numerous attempts to couple it with fluorobenzoic acid by
amide-coupling condensation with HATU, EDC, or CDI proved
unsuccessful. Eventually, bithiazole 15 was acylated with 4-fluor-
obenzoyl chloride (prepared by treatment of 4-fluorobenzoic acid
with oxalyl chloride) in the presence of triethylamine at room tem-
perature (10 min) to afford 19F-8.
Given these difficulties, a new strategy was developed for the
synthesis of fluorinated bithiazole 9. Work began here with the
CDI-mediated coupling of aminothiazole 12 with 4-fluorobenzoic
acid. Thiazole 17 was markedly easier to purify than 8. Bromina-
tion of 17 proved significantly more difficult than 12, requiring
the use of a stronger acidic medium (HBr vs AcOH) with pyridini-
um tribromide to afford 18 (91% yield). The final reaction involved
condensation of 18 with 1-(5-chloro-2-methoxyphenyl)thiourea
(19), delivering 19F-9 in 76% yield.
Scheme 1. Synthesis of fluorinated corrector analogs. Reagents and conditions: (a)
EtOH, reflux; (b) Br2, AcOH; (c) 1-(2-chloro-5-dimethylaminophenyl)thiourea (14),
EtOH, reflux; (d) (i) 4-fluorobenzoic acid, CH2Cl2, oxalyl chloride, (ii) 4-fluor-
obenzoyl chloride (16), CH2Cl2, TEA; (e) 4-fluorobenzoic acid, CDI, DMF, 100 °C; (f)
ꢀ
PyrH+Br3
, 33% HBr in AcOH; (g) 1-(5-chloro-2-methoxyphenyl)thiourea (19),
EtOH, reflux.
Yield and purification difficulties were due to the poor solubility
of aminobithiazole intermediate 15 as well as the corresponding
fluorinated bithazoles 8 and 9. Because [18F]N-succinimidlyl-4-flu-
orobenzoate (18F-SFB) would be used in picomolar quantities for
the final coupling to aminobithiazole intermediates, the reaction
solution would be quite dilute and solubility is not expected to
be a significant issue.
Figure 4. Concentration-dependence of
19F-9 compared to corr-4a.
D
F508-CFTR corrector activity of 19F-8 and
The
using a cell-based fluorescence assay in Fischer rat thyroid (FRT)
cells co-expressing human F508-CFTR and the halide-sensitive
fluorescent protein YFP-H148Q/I152L.11 The concentration-
D
F508-CFTR corrector activity of 19F-8 and 19F-9 was assayed
D
Figure 2. Potential 18F-prosthetics.

1604
H. R. Davison et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1602–1605
Scheme 2. Synthesis of fluorinated potentiator 19F-10 and azido intermediate 23.
Reagents and conditions: (a) CuI, NaN3, -proline, NaOH, DMSO, 60 °C; (b) EDC,
HOBt, DMF, DCM, 0 °C to rt; (c) (i) TFA, DCM, (ii) EDC, DMAP, DCM, DMF, 0 °C to rt.
L
Figure 5. Concentration-dependence of
and 19F-11 compared to genistein.
D
F508-CFTR potentiator activity of 19F-10
The
assayed in FRT cells co-expressing
I152L after rescue of
F508-CFTR by 24 h culture at 27 °C.11,12
Fluorinated phenylglycine analogs had EC50 of 0.09
M (19F-10)
M reported previ-
M for bench-
D
F508-CFTR potentiator activity of 19F-10 and 19F-11 was
F508-CFTR and YFP-H148Q/
dependence data show slightly greater potency of 19F-8 and 19F-9,
compared to benchmark corrector corr-4a, as shown in Figure 4.
D
D
EC50 values (in lM) were: 8 = 1.4; 9 = 1.4; corr-4a = 2.3].
Synthesis of fluoroaniline potentiator 19F-10 (Scheme 2) began
with an EDC-mediated coupling of 4-fluoroaniline to Boc-N-
l
and 1.1 l l
M (19F-11), comparable to that of 0.3
ously for PG-01,10 and much better than that of 7.0
mark potentiator genistein (Fig. 5).
l
methyl-L
-phenylglycine (Scheme 2).10,23 Boc-protected 21, ob-
tained in 61% yield, was then deprotected by treatment with TFA
and subsequent EDC-activated coupling with 3-indole acetic acid
and DMAP gave 19F-10 in 72% yield over two steps.
Synthesis of 4-(3-fluoropropyl)-1H-1,2,3-triazole analog 11
began with the synthesis of 4-azidoaniline via a CuI-catalyzed, pro-
line-promoted coupling reaction24 followed by an EDC-mediated
The retained corrector activity of 19F-8 and 19F-9 and potentia-
tor activity of 19F-10 is likely the consequence of the enhanced
binding interactions of fluorine.29 Indeed, fluorine is found in
approximately 5–15% of drugs that have come to market over
the past 50 years.29 Fluorine has also been shown to improve met-
abolic stability and generally enhance physicochemical proper-
ties,29 features which would be useful in future in vivo studies.
In conclusion, two active fluorinated corrector analogs and two
active fluorinated potentiator analogs were synthesized and the
introduction of a fluorine atom was found to improve potency. Both
fluorinated correctors(19F-8 and 19F-9) and a fluorinated potentiator
condensation of 20 (Scheme 2) with Boc-N-methyl-L-phenylglycine
to afford 22 (20, 74%; and 22, 97%, respectively). Subsequent Boc
removal (TFA) followed by treatment with EDC-activated 3-indole
acetic acid and DMAP afforded azido phenylglycine intermediate
23 (90% yield).
The synthesis of 5-fluoroalkyne for the triazole-forming cyclo-
addition with azido-phenylglycine intermediate 23 proved to be
unsuccessful. Initially, we focused on utilizing diethylaminosulfur
trifluoride (DAST) for the conversion of the alcohol moiety of 4-
pentyne-1-ol into an alkyl fluoride.25 Unfortunately, no fluorinated
alkyne was isolated. Das et al. reported that ionic liquids can be
used in an improved purification procedure of the dehydroxy-fluo-
rination reaction with DAST; again, no desired product was iso-
lated.26 Likewise, a modified procedure employing tosylate 24
(synthesized by the treatment of 4-pentyn-1-ol with p-toluenesul-
fonyl chloride and triethylamine27) and nucleophilic fluoride [KF
plus 18-Crown-6 or Bu4N+Fꢀ(tBuOH)428] was unsuccessful.
With this as a backdrop, tosylate 24 was clicked to azido inter-
mediate 23 in a Huisgen copper-catalyzed 1,3-dipolar cycloaddi-
tion to afford 25 in 85% yield (Scheme 3). Finally, potentiator
analog 19F-11 was obtained by treatment of 25 with Bu4N+Fꢀ(t-
BuOH)4 (26) in 31% yield.
(
19F-10) showed improved or comparable activity to the original
compounds. These active, fluorinated analogs are potentially useful
for non-invasive PET imaging of in vivo compound uptake and bio-
distribution. Given the short half-life of 19F (t1/2 = 110 min), the next
stepen route to PETstudies withthese compoundswill be todevelop
synthetic routes to 18F-8, 19F-9, and 19F-10 where the 19F moiety is
introduced in the last synthetic step. Current efforts are focused on
developing viable and high-yielding routes to 18F-8 from N2-(2-chloro-
5-(dimethylamino)phenyl)-40-methyl-[4,50-bithiazole]-2,20-diamine
(15) + 7 and 18F-10 from (S)-2-(2-(1H-indol-3-yl)-N-methylacet-
amido)-2-phenylacetic acid + 6.
Acknowledgments
The authors thank Professor Julie Sutcliffe and Ms. Robin Cum-
ming (UC Davis, Department of Biomedical Engineering) for helpful
suggestions in selecting prosthetic groups and, for financial sup-
port, the Tara K. Telford Fund for Cystic Fibrosis Research at the
University of California/Davis, the National Institutes of Health
(Grants DK072517 and GM0891583), and the National Science
Foundation [Grants CHE-0910870, CHE-0443516, CHE-0449845,
and CHE-9808183 (NMR spectrometers)].
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.128.
References and notes
Scheme 3. Synthesis of fluorinated potentiator 19F-11. Reagents and conditions: (a)
TsCl, TEA, DCM, 0 °C to rt; (b) Na ascorbate, CuSO4, 23, DCM, tBuOH, H2O; (c) (i)
Bu4N+FꢀꢁH2O, tBuOH, hexane, 90 °C, 30 min forms Bu4N+Fꢀ(tBuOH)4 (26), (ii) 25, 26,
ACN, 70 °C, 1 h.
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