Carbon-14 radiosynthesis of 4-(5-chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl) -6-(trifluoromethyl)-[4-14C]quinolin-2(1H)-one (XEN-D0401), a novel BK channel activator

Article abstract of DOI:10.1002/jlcr.1740

A method has been developed for the carbon-14 radiosynthesis of [ ¹⁴C]XEN-D0401, a 4-(2-hydroxyphenyl)quinolin-2(1H)-one derivative. The radiosynthetic route involves a series of ortho-lithiations directed by both 2-methoxymethyl (MOM) and pivaloyl protecting groups. This is demonstrated in the first key radiochemical step between the reaction of 5-chloro-2- methoxymethoxy-[carboxyl-¹⁴C]benzoic acid methyl ester [ ¹⁴C]-3 and the ortho-lithiated intermediate generated from 2,2-dimethyl-N-(4-trifluoromethylphenyl)-propionamide (2) to afford the protected benzophenone [¹⁴C]-4. The other key radiochemical step utilizes a variation of the Friedlaender quinoline synthesis, which involves a base catalysed, one-pot condensation process between (2-amino-5- trifluoromethyl-phenyl)-(5-chloro-2-methoxymethoxy-phenyl)-[¹⁴C] methanone [¹⁴C]-5 and c-butyrolactone to give MOM-protected [ ¹⁴C]-6. On MOM deprotection, [¹⁴C]XEN-D0401 was isolated with a radiochemical purity of >97%, with a specific activity of 55 mCi/mmol from seven radiochemical steps, starting from barium [¹⁴C]carbonate in a radiochemical yield of 10.5%. Copyright

Full text of DOI:10.1002/jlcr.1740

Research Article
Received 13 October 2009,
Revised 4 December 2009,
Accepted 8 December 2009
Published online 25 January 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1740
Carbon-14 radiosynthesis of 4-(5-chloro-
2-hydroxyphenyl)-3-(2-hydroxyethyl)-6-
(trifluoromethyl)-[4-14C]quinolin-2(1H)-one
(XEN-D0401), A novel BK channel activatory
Ã
Sean L. Kitson,a Stuart Jones,a William Watters,a Fiona Chan,b and
David Madgeb
A method has been developed for the carbon-14 radiosynthesis of [14C]XEN-D0401, a 4-(2-hydroxyphenyl)quinolin-2(1H)-
one derivative. The radiosynthetic route involves a series of ortho-lithiations directed by both 2-methoxymethyl (MOM) and
pivaloyl protecting groups. This is demonstrated in the first key radiochemical step between the reaction of 5-chloro-2-
methoxymethoxy-[carboxyl-14C]benzoic acid methyl ester [14C]-3 and the ortho-lithiated intermediate generated from 2,2-
dimethyl-N-(4-trifluoromethylphenyl)-propionamide (2) to afford the protected benzophenone [14C]-4. The other key
¨
radiochemical step utilizes a variation of the Friedlander quinoline synthesis, which involves a base catalysed, one-pot
condensation process between (2-amino-5-trifluoromethyl-phenyl)-(5-chloro-2-methoxymethoxy-phenyl)-[14C]methanone
[
14C]-5 and c-butyrolactone to give MOM-protected [14C]-6. On MOM deprotection, [14C]XEN-D0401 was isolated with a
radiochemical purity of 497%, with a specific activity of 55 mCi/mmol from seven radiochemical steps, starting from
barium [14C]carbonate in a radiochemical yield of 10.5%.
Keywords: 3-substituted-4-arylquinolin-2(1H)-one; carbon-14; [14C]XEN-D0401; BK channel activator; overactive bladder (OAB);
BMS-223131
The retro-synthetic analysis for the incorporation of a single
carbon-14 label at position 4 on the substituted quinolin-2(1H)-
one ring to give [14C]XEN-D0401 is shown in Scheme 1. The
Introduction
The drug candidate 4-(5-chloro-2-hydroxyphenyl)-3-(2-hydro-
disconnection of the amide bond in the 3-substituted-
4-arylquinolin-2(1H)-one derivative of [14C]XEN-D0401 gives
the retro-target (A) with the aliphatic primary hydroxyl
protected. Further disconnection provides the target benzo-
phenone synthon (B), which can be synthesized from the
building blocks (D) and (E). The key retro-synthetic step is the
formation of the retro-target (A), which in turn can be
synthesized from the condensation of ‘synthons’ (B) with (C).
The carbon-14 radiolabel can be incorporated into (E) from
reacting synthon (F) with [14C]carbon dioxide.
xyethyl)-6-(trifluoromethyl)-quinolin-2(1H)-one, XEN-D0401, shown
in Figure 1 has found application in the management of disease
states arising from the dysfunction of cellular membrane polariza-
tion and conductance.1 XEN-D0401 contains a 3-substituted-4-
arylquinolin-2(1H)-one derivative and is a novel, selective small
molecule activator to open the large-conductance calcium-
activated potassium channel, known as the BK channel.2 Com-
pounds with this heterocyclic scaffold have also been used as
inhibitors of acyl-coenzyme A and cholesterol acyltransferase.1 This
drug candidate is in Phase I development for the treatment of
overactive bladder (OAB).3 Currently, it is estimated that 200 million
people worldwide suffer from this condition.4 Treatments using
anti-muscarinic-based drugs (which operate on the muscarinic
acetylcholine receptors) have poor tolerability profiles and therefore
new drugs for the management of OAB are needed.
The target [14C]XEN-D0401 was prepared in seven radio-
chemical steps starting from barium [14C]carbonate. This included
aIsotope Chemistry Laboratories, Almac Sciences, Almac House, 20 Seagoe
Industrial Estate, Craigavon, Northern Ireland, BT63 5QD, UK
This paper describes a method for the radiosynthesis of
[14C]XEN-D0401, having a single carbon-14 label (denoted by
bXention Limited, Iconix Park, London Road, Pampisford, Cambridge CB22 3EG,
UK
Ã
)
in the quinolin-2(1H)-one ring with a specific activity greater
than 50 mCi/mmol, as illustrated in Figure 2. This strategy is
based on a viable route published by Wang et al.5 and a
personal communication with Crispino et al. to produce multi-
kilogram quantities of the drug candidate BMS-223131 for the
treatment of male erectile dysfunction.6
*Correspondence to: Sean L. Kitson, Isotope Chemistry Laboratories, Almac
Sciences, Almac House, 20 Seagoe Industrial Estate, Craigavon, Northern Ireland,
BT63 5QD, UK.
E-mail: sean.kitson@almacgroup.com
yPresented at the 18th International Isotopes Society (UK Group) Symposium.
J. Label Compd. Radiopharm 2010, 53 140–146
Copyright r 2010 John Wiley & Sons, Ltd.

S. L. Kitson et al.
underwent azeotropic-drying and the resultant oil was then
taken up in acetone to react with methyl iodide in the presence
of potassium carbonate.
3-substituted-4-arylquinolin-2(1H )-one scaffold
H
N
O
On analysis of the reaction mixture by normal phase radio-
TLC, formation of a major impurity was observed. This was
isolated in 47% radiochemical yield and identified by proton-
NMR spectroscopy as 5-chloro-2-hydroxy-[carboxyl-14C]benzoic
acid methyl ester [14C]-10. The reason for this low radiochemical
yield was during the methylation procedure, the MOM protect-
ing group had been cleaved and resulted in O-methylation to
F3C
OH
OH
pKa ~7.9
Cl
Figure 1. XEN-D0401.
give
[ [
14C]-11. Consequently, the substrate 14C]-10 was
successfully re-protected with MOMCl in 97% radiochemical
yield by generating the phenoxide derivative using sodium
hydride in anhydrous dimethylformamide. The labelled sub-
strate [14C]-3 was isolated with a total activity of 86 mCi with a
radiochemical yield of 35% from barium [14C]carbonate.
H
N
O
MeO
O
carbon-14 label
F3C
OH
O
O
OH
A key step in the radiosynthesis of [14C]XEN-D0401 was the
[
14C]-4. This
Cl
formation of the protected benzophenone
Cl
14C]XEN-D0401
unsymmetrical benzophenone derivative was made from react-
ing the ortho-lithiated intermediate with [14C]-3 as illustrated in
Scheme 2. This reaction was trialed and optimized several times
due to the poor yields of [14C]-4 from the incomplete
lithium–halogen exchange on pivaloyl protected (2). On further
investigation an acceptable yield of 71% of the benzophenone
derivative, [14C]-4, was achieved after purification.
[
[
14C]-3
Figure 2. Position of carbon-14 label.
the intermediate 5-chloro-2-methoxymethoxy-[carboxyl-14C]benzoic
acid methyl ester [14C]-3, shown in Scheme 2, which was prepared
using in-house carboxylation radiochemistry and is outlined in
Scheme 3.
The other challenging step was the de-protection of the
pivaloyl group under basic conditions. It was found that the best
procedure was to add all the sodium hydroxide in ethanol
(25 wt%) in a single batch at the start of the reaction. After
heating the reaction at reflux for 1 h, analysis of the reaction
mixture, using normal phase radio-TLC, showed formation of
Results and discussion
The starting material 2,2-dimethyl-N-(4-trifluoromethylphenyl)-
propionamide (2) was easily prepared in 83% isolated yield from
the N-acylation of 4-trifluoromethylphenylamine (1) using
pivaloyl chloride, as illustrated in Scheme 2.7
[
and radiochemical purity of 90.2%.
14C]-5 which was isolated with a radiochemical yield of 68%
The most common route to quinoline derivatives utilizes the
¨
Directed ortho-lithiation on the substrate (2) was achieved
with 2.5 equivalents of a solution of n-butyl lithium in
tetrahydrofuran at ꢀ781C. Progress of the reaction was
monitored by quenching an aliquot of the reaction mixture in
D2O. Reaction completion was established when the proton-
NMR spectrum in d6-DMSO of the quenched reaction mixture
showed replacement of the symmetrical AB quartet group of
signals, integrating for four aryl ring protons, with a series of
multiplets with an ABX splitting pattern, integrating for only 3
protons.
The introduction of the single carbon-14 label was achieved
via the substrate [14C]-3, which was prepared using in-house
carboxylation radiochemistry, as outlined in Scheme 3. The
carbon-14-labelled material [14C]-3 was synthesized from the
commercially available 4-chloro-2-bromophenol (7) and proved
capricious in terms of yield.
Friedlander ‘quinoline’ synthesis, which involves the condensa-
tion of aromatic ortho-amino aldehydes/ketones with an
adjacent methylene aldehyde or ketone. This reaction step has
been further explored (Crispino et al., personal communication)
to synthesize 4-aryl-quinolin-2(1H)-one derivatives. The limiting
¨
9
factor in using the Friedlander strategy is the requirement of an
activating group, which must be adjacent to an ester or
carboxylic acid group. This condition is conveniently met by
employing a lactone in the condensation with 2-aminobenzo-
phenone derivatives. It has been demonstrated (Crispino et al.,
personal communication) using a one-pot procedure that the
required 4-aryl-quinolin-2(1H)-one can be produced by reacting
g-butyrolactone with the unlabelled substrate (5). The advan-
tage of using g-butyrolactone is that it immediately gives the
required side chain (2-hydroxyethyl) in the correct position on
the 4-aryl-quinolin-2(1H)-one ring.
First the conversion from [14C]-5 into [14C]-6 was not
successful due to the formation of [14C]-5 hydrate. The
proton-NMR spectrum of the [14C]-5 hydrate gave the new
resonance signals at 8.44 and 5.59–5.60 ppm. During unlabelled
synthesis, the cyclized product was prepared successfully
because the resultant unlabelled solid (5) was filtered off and
dried under high vacuum at 401C to remove any residual water.
For the cyclization step to be efficient, it was important to dry
Phenol (7) was protected using chloromethyl methyl ether
(MOMCl) to give the MOM ether (8).8 Subsequent lithium–
halogen exchange preferentially at the bromo atom, followed
by carboxylation of the lithiated intermediate, with [14C]carbon
dioxide, was carried out to give benzoic acid [14C]-9, after an
aqueous basic quench and mild acidic work-up with 5%
aqueous citric acid. [14C]Carbon dioxide used for the carboxyla-
tion was generated in situ by the action of concentrated
sulphuric acid on 250 mCi of barium [14C]carbonate (specific
activity 450 mCi/mmol) using high vacuum procedures. The
[14C]carbon dioxide generated was dried and stored in a glass
bulb containing P2O5 until required. The crude product [14C]-9
[
14C]-5 prior to the reaction. Unfortunately, in the radiochemical
synthesis the solid [14C]-5 was filtered off on a sinter funnel, and
the solid dissolved in tetrahydrofuran and concentrated using
rotary evaporator to obtain oil. The oil was azeotropically dried
J. Label Compd. Radiopharm 2010, 53 140–146
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

S. L. Kitson et al.
H
NH2
CO2H
N
O
quinolin-2(1H)-one
F3C
OH
F3C
OP1
OH
OH
Cl
14C]-XEN-D0401
Cl
(A)
[
condensation
NH2
HO
O
P1, P2 = Protecting groups
* = Carbon-14 label
O
F3C
*
+
OH
OP1
Cl
(C)
(B)
14CO2
coupling
P2
+
RO
O
+
X
*
carboxylation
Cl
N-protection
NH2
NHP2
OH
OH
+
F3C
F3C
Cl
(D)
(E)
(1)
Scheme 1. Retro-synthetic pathway of [14C]XEN-D0401.
(F)
several times from tetrahydrofuran but residual water may still measured by analytical HPLC, was 497% with a gravimetric
be present. The ‘semi-dried’ oil that was then subsequently used specific activity of 55 mCi/mmol. The 500 MHz proton NMR-
in the cyclization step and consequently did not give the desired spectrum of [14C]XEN-D0401 was consistent with the molecular
product [14C]-6 but only starting material.
structure.3 One feature of interest in the proton-NMR spectrum
For the radiochemistry, success was finally achieved by prior, of [14C]XEN-D0401 was that multiplets at d 2.75 and 2.71 were
rigorous drying of [14C]-5 to ensure the absence of any residual assigned to the diastereotopic methylene protons (CH2CH2OH)
water. This was accomplished by azeotropic drying from toluene, on the 2-hydroxyethyl moiety, an interpretation consistent with
followed by pentane. Analysis of the proton-NMR spectrum of the the reported atropisomerism of XEN-D0401.3–10
rigorously dried [14C]-5 was consistent with the unlabelled
Experimental procedures
material and showed the absence of the proton-NMR signals at
d 8.44 and 5.59–5.60 that were present in the wet product.
The anion at the amino moiety of the benzophenone
Materials and methods
derivative [14C]-5 was successfully achieved by using lithium
bis(trimethylsilyl)amide (LiHMDS) in tetrahydrofuran. The gener-
ated anion was reacted with a solution of g-butyrolactone in
tetrahydrofuran and resulted in the formation of the cyclized
MOM protected [14C]-6 after aqueous work-up, with a radio-
chemical purity of 78.8%. The proposed mechanism for the
reaction of [14C]-5 with g-butyrolactone is analogous to the one
already described by Crispino et al.5
The final step in the radiosynthesis of [14C]XEN-D0401 was the
cleavage of the MOM protecting group using concentrated
hydrochloric acid in propan-2-ol at elevated temperature. The
crude [14C]XEN-D0401 was isolated and purified by flash column
chromatography on silica gel to give [14C]XEN-D0401. Radio-TLC
analysis established the radiochemical purity at 96%. Crystal-
lization from a mixture of ethanol and water gave [14C]XEN-
D0401 as an off white solid. The radiochemical purity, as
The custom synthesis of [14C]XEN-D0401 and all associated
radioactive experiments were carried out in the Isotope
Chemistry Laboratories at Almac Sciences in specially designed
fume hoods dedicated to carbon-14 only. Reactions were
performed under an inert atmosphere using filtered nitrogen
gas unless specified differently. Chemical reagents and solvents
used in the synthetic procedures were obtained from various
chemical suppliers without further purification. Barium [14C]car-
bonate was purchased from a commercial supplier with specific
activity 450 mCi/mmol. High-purity HPLC solvents were used.
Intermediates and products synthesized were characterized
based on proton-NMR spectra and recorded on a Bruker DPX
spectrometer operating at 500 MHz spectrometer. Chemical
shifts were recorded in ppm (d) from an internal tetramethylsi-
lane standard in either d6-DMSO or CDCl3 and coupling
constants (J) are reported in Hertz. The NMR peak patterns are
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 140–146

S. L. Kitson et al.
O
O
O
Li
Cl
N
NH2
HN
Li
n-BuLi/Hexane, THF
-78 °C
NaOH
pH 7.5-8.0
CF3
0 °C, 1h
CF3
CF3
(2)
(1)
'ortho-lithiated
intermediate'
MeO
O
O
O
O
NH
O
O
O
[14C]-3
NaOH / Ethanol
1h / reflux
Cl
THF
-78 °C
CF3
O
Cl
[14C]-4
H
N
O
O
NH2
O
O
O
O
F3C
OH
O
LiHMDS / THF
-20 °C to -10 °C, 5.5h
addn. H2O, 19h, rt
CF3
Cl
Cl
[14C]-5
[14C]-6
H
N
O
IPA / HCl
F3C
OH
OH
40-45 °C, 2h
Cl
[14C]XEN-D0401
Scheme 2. Radiosynthetic route to [14C]XEN-D0401.
reported as broad (br), singlets (s), doublets (d), triplets (t), trifluoroacetic acid in water/acetonitrile (90:10), solvent B, 0.1%
quartets (q), septets (sep), and multiplets (m). Silica gel (200–400 trifluoroacetic acid in water/acetonitrile (10:90), gradient,
˚
mesh, 60 A) was used for flash column chromatography. TLC 0–100% B over 0–30 min; flow rate, 1.0 mL/min; injection volume
plates (pre-coated glass plates with silica gel 60 F254, 0.25 mm 10 mL; monitored at 230 nm wavelength. HPLC analyses are
thickness) were used to monitor reactions and developed using described as area%.
the Cyclone Storage Phosphor System. Final products were
dried in a high vacuum desiccator over phosphorous pentoxide.
Carbon-14 radiosynthesis
The radiochemical yield was determined by liquid scintillation
counting using the Perkin–Elmer Tricarb 2900 Scintillation
Analyzer. Purity of the final compound was further analysed
using an Agilent 1100 HPLC system with UV and radiochem-
Synthesis of 2,2-dimethyl-N-(4-trifluoromethylphenyl)-propiona-
mide (2)
A 500 mL three-necked round bottom flask was charged with
water (60 mL), acetone (30 mL) and (1) (15.2 g, 94.3 mmol). The
ical detection;
a Symmetry C18 analytical HPLC column
3.9 mm ꢁ 150 mm, 5 mm. T = ambient; eluents, solvent A, 0.1%
J. Label Compd. Radiopharm 2010, 53 140–146
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

S. L. Kitson et al.
Br
Br
Li
n-BuLi/Hexane
OH
1. NaH / DMF
2. MOM-Cl
O
O
O
O
THF
-78 oC, 20 mins
Cl
Cl
Cl
(7)
(8)
lithiated intermediate
c H2SO4
-196 °C to rt
Ba14CO3
14CO2
MeO
O
MeO
O
HO
O
OMe
K2CO3 / Acetone
OH
O
O
+
CH3I
°C
Cl
Cl
Cl
40
[14C]-11
[14C]-10
[14C]-9
Isolate [14C]-10
1. NaH / DMF
2. MOM-Cl
MeO
O
O
O
Cl
[14C]-3
Scheme 3. Radiosynthetic route to labelled starting material [14C]-3.
reaction mixture was cooled to 01C and pivaloyl chloride (13.1 g, ortho-lithiated product was connected to the high vacuum
108 mmol) was added dropwise via a pressure equalized manifold via an H-piece and cooled down to –1961C using a
dropping funnel with concurrent addition of aqueous sodium liquid nitrogen Dewar. The [14C]carbon dioxide was generated
hydroxide (1M), so as to maintain the pH in the range of from the action of concentrated sulphuric acid on barium
7.5–8.0 using a pH probe. The reaction mixture was left stirring [14C]carbonate (250 mCi, Specific Activity 450 mCi/mmol) on
for 1 h at 01C, filtered off and the solid washed with water the high vacuum manifold and dried over P2O5. The [14C]carbon
(3 ꢁ 100 mL) and water/acetone (4:1, 4 ꢁ 100 mL). The dioxide was transferred to the flask containing the ortho-
white solid was dried in the high vacuum oven to constant lithiated product and the reaction mixture was allowed to warm
weight to yield (2) (19.3 g, 83%): 1H-NMR (CDCl3, 500 MHz) to ambient temperature. During this process the mixture went
d = 7.66 (m, 2H, J = 10.0 Hz), 7.57 (m, 2H, J = 10.0 Hz), 3.93 (s, 1H), from orange to straw coloured. The reaction mixture was
1.33 (s, 9H).
allowed to stir for 1 h and then re-cooled to –1961C and purged
with nitrogen. This allowed for the addition of aqueous sodium
hydroxide (1.0 M, 10 mL) and the quenched mixture was allowed
to warm to ambient temperature overnight. The non-acidic
organics were extracted into dichloromethane (5 ꢁ 10 mL) and
the aqueous phase was made acidic to pHꢂ3–4 using aqueous
citric acid (5%, 100 mL). The crude product was extracted into
dichloromethane (2 ꢁ 10 mL), washed with brine, dried over
magnesium sulphate, filtered and counted to give a total activity
of 191 mCi and radiochemical yield of 76%. The dried organic
layer was concentrated on the rotary evaporator to give the oil
Synthesis of 5-chloro-2-methoxymethoxy[carboxyl-14C]benzoic
acid [14C]-9
A two-necked 100 mL round bottom flask was charged with (8)
(1.68 g, 6.70 mmol) and anhydrous tetrahydrofuran (9 mL) under
nitrogen. The resultant solution was cooled down to ꢀ781C and
a solution of n-butyl lithium in hexane (2.0 M, 3.3 mL, 6.6 mmol)
was added over 5 min via syringe. On complete addition, the
reaction mixture was allowed to stir at ꢀ781C for 20 min to
give the ortho-lithiated product. The flask containing the
1
product [14C]-9: H-NMR (CDCl3, 500 MHz) d = 10.69 (s, 1H), 8.04
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 140–146

S. L. Kitson et al.
(d, 1H, J = 3.0 Hz), 7.47 (dd, 1H, J = 11.5, 3.0 Hz), 7.25 (d, 1H, reaction mixture was quenched with D2O and concentrated in
J = 11.5 Hz), 5.34 (s, 2H), 3.54 (s, 3H).
vacuo. The residue was analysed by proton NMR spectroscopy
to confirm that ortho-lithiation had taken place.
Synthesis of 5-chloro-2-methoxymethoxy-[carboxyl-14C]benzoic
acid methyl ester [14C]-3
The reaction mixture was cooled to –781C and a solution of
[
14C]-3 (86 mCi) in anhydrous tetrahydrofuran (2 mL) was added
over 5 min. On complete addition, the reaction was left stirring
at –781C for 20 min. The mixture was warmed to 0–51C and left
stirring under nitrogen. An aliquot of the reaction mixture was
quenched with a mixture of tert-butyl methyl ether and water.
The organic phase was analysed by radio-TLC and showed the
formation of [14C]-4. The reaction mixture was quenched with
water (5 mL), followed by the addition of tert-butyl methyl ether
(5 mL). The organic components were extracted into tert-
butyl methyl ether (2 ꢁ 50 mL) and concentrated in vacuo to
afford an oil. The oil was dissolved in tetrahydrofuran
(2 ꢁ 50 mL) and each time concentrated in vacuo to give a
straw coloured oil. The oil was taken up in a mixture of
dichloromethane/hexane and the active solution applied to a
column of silica gel, which was neutralized with triethylamine.
The product was eluted off first with hexane, hexane/
dichloromethane (9:1), hexane/dichloromethane (8:2) and finally
with hexane/dichloromethane (7:3). The product fractions were
collected, combined and concentrated in vacuo to give a solid.
The solid was dissolved in tetrahydrofuran and counted to give
a total activity of 60.7 mCi and radiochemical yield of 71%.
Activity from a previous batch of 8.4 mCi was combined with the
60.7 mCi batch to give a total activity of 69.1 mCi. Radio-TLC
analysis on normal phase silica of the combined batches gave a
radiochemical purity of 88.7%. Proton-NMR spectroscopic
analysis was consistent with the titled compound: 1H-NMR
(CDCl3, 500 MHz) d = 11.85 (bs, NH), 8.98 (d, 1H, J = 9.0 Hz), 7.78
(d, 1H, J = 9.0 Hz), 7.71 (s, 1H), 7.45 (dd, 1H, J = 9.0, 2.5 Hz), 7.35 (d,
1H, J = 2.5 Hz), 7.23 (d, 1H, J = 9.0 Hz), 5.02 (s, 2H), 3.28 (s, 3H),
1.39 (s, 9H).
A 250 mL round bottom flask containing [14C]-9 (191 mCi) was
taken up in anhydrous acetone (200 mL). To the resultant
solution was added anhydrous potassium carbonate (2.36 g,
17.1 mmol, 5.0 eqv) and methyl iodide (0.318 mL, 5.11 mmol, 1.5
eqv). The mixture was heated to 401C and left stirring for 18.5 h.
Radio-TLC analysis on normal phase silica gel showed that the
reaction under these conditions produced very little product.
Another aliquot of methyl iodide (2.0 mL) was added and the
mixture was left stirring at 401C for 2.3 h. Radio-TLC analysis
showed two products, without further progress of the reaction.
The reaction mixture was filtered and the filtrate concentrated
on the rotary evaporator to give a residue. Proton-NMR
spectroscopic analysis indicated that the MOM protecting group
was cleaved and the dominate product was [14C]-10. The
[14C]methyl ester was isolated on a column of normal phase
silica gel, eluting the product off with a mixture of hexane/
dichloromethane (4:1) to give 89.1 mCi. Radio-TLC analysis, on
normal phase silica gel, eluting with diethyl ether/hexane/
triethylamine (100:900:2) showed a radiochemical purity of
99.5%: Rf = 0.42 (silica gel, n-hexane/diethylether/triethylamine,
1
400:100:1); H-NMR (CDCl3, 500 MHz) d = 10.67 (s, 1H), 7.82 (m,
1H), 7.40 (m, 1H), 7.42 (m, 1H), 3.97 (s, 3H).
The substrate, [14C]-10 (89.1 mCi), was taken up in anhydrous
N,N-dimethylformide (5.0 mL) and the resultant solution was
cooled down to 01C. Sodium hydride (60% dispersion in mineral
oil, 67.2 mg, 1.68 mmol, 1.05 eqv) was then added under
nitrogen, followed by the addition of MOMCl (0.128 mL,
1.68 mmol, 1.05 eqv). The reaction mixture was left stirring for
20 min at 01C, followed by 20 min at ambient temperature.
Radio-TLC analysis on normal phase silica gel, eluting with
hexane/diethyl ether/triethylamine (400:100:1), gave a radio-
chemical purity of 98.4%. The reaction mixture was diluted with
dichloromethane (20 mL) and the organic phase was washed
with water (3 ꢁ 30 mL) and brine (1 ꢁ 50 mL). The product layer
was concentrated on the rotary evaporator and dried azeo-
tropically with tetrahydrofuran (3 ꢁ 100 mL) to give a colourless
oil. The oil was taken up in a mixture of diethyl ether and hexane
and applied to a column of silica gel and the product was eluted
with a mixture of diethyl ether/hexane. The product fractions
were combined and counted to give a total activity of (86 mCi).
The solution was concentrated on the rotary evaporator to give
Synthesis of (2-amino-5-trifluoromethyl-phenyl)-(5-chloro-2-meth-
oxymethoxy-phenyl)-[14C]methanone [14C]-5
A 250 mL round bottom flask was charged with [14C]-4
(69.1 mCi), ethanol (30 mL) and aqueous sodium hydroxide (25
wt%, 6 mL). The resultant yellow solution was heated to reflux
for 1 h, then allowed to cool to ambient temperature. Radio-TLC
analysis on normal phase silica of the reaction mixture showed
product that co-eluted with an authentic sample and gave a
radiochemical purity of 80%. The reaction mixture was
quenched with water (70 mL) over 10 min and resulted in an
off white slurry. The slurry was left stirring at 0–51C for 2 h, then
filtered off and washed with water until the pH of the filtrate
was 6. The solid was taken up in tetrahydrofuran (250 mL) and
counted to give a total activity of 60.6 mCi. Radio-TLC analysis
on normal phase silica of the active solution gave a radio-
chemical purity of 90.2%. The solution was concentrated on the
rotary evaporator to give a yellow oil. Analysis using proton-
NMR spectroscopy indicated that the crude product may be a
hydrate: 1H-NMR (CDCl3, 500 MHz)d = 8.44 (bs), 7.51–7.53 (m, 1H),
7.46–7.48 (m, 1H), 7.38–7.40 (m, 1H), 7.27–7.27 (m, 1H), 7.19–7.20
(m, 1H), 6.75–6.77 (m, 1H), 6.71 (bs, NH), 5.59–5.60 (m) 5.05 (s,
2H), 3.32 (s, 3H). The crude product was dried by azeotropic
distillation from toluene (12 ꢁ 20 mL) and then from hexane
(2 ꢁ 20 mL) to give ketone [14C]-5 (46.8 mCi) and radiochemical
yield of 68%. Radio-TLC Rf = 0.41 (silica gel, dichloromethane/
[
14C]-3 as an oil: Rf = 0.15 (silica gel, n-hexane/diethylether/
triethylamine, 400:100:1); 1H-NMR (d6-DMSO, 500 MHz) d = 7.65
(m, 1H), 7.57 (m, 1H), 7.25 (m, 1H), 5.24 (s, 2H), 3.82 (s, 3H), 3.39
(s, 3H).
Synthesis of N-[2-(5-chloro-2-methoxymethoxy-[14C-carbonyl]ben-
zoyl)-4-trifluoromethyl-phenyl]-2,2-dimethyl-propionamide [14C]-4
A 50 mL, three-necked round bottom flask was charged with (2)
(414 mg, 1.69 mmol, 1.1 eqv) and anhydrous tetrahydrofuran
(5 mL) under nitrogen. The solution was cooled to –781C and
held for 10 min. A solution of n-butyl lithium in hexane (2.19 M,
3.84 mmol, 1.75 mL, 2.5 eqv) was then added to the cold
solution over 1 min and the reaction mixture was stirred at
–781C for a further 10 min, followed by stirring for 4.5 h at 0–51C.
Before quenching the slurry with water, an aliquot of the
J. Label Compd. Radiopharm 2010, 53 140–146
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

S. L. Kitson et al.
1
methanol 9:1); H-NMR (CDCl3, 500 MHz) d = 7.51–7.53 (m, 1H), point of precipitation. Then on heating gave a solution which
7.46–7.48 (m, 1H), 7.39–7.40 (m, 1H), 7.26–7.27 (m, 1H), 7.19–7.20 was allowed to cool to ambient temperature over 18 h. The
(m, 1H), 6.75–6.77 (m, 1H), 6.70 (bs, NH) 5.05 (s, 2H), 3.32 (s, 3H). resultant crystals were filtered under vacuum and washed with
water/ethanol (9:1, 3 ꢁ 2 mL). The solid was dried in the high
Synthesis of 4-(5-chloro-2-methoxymethoxy-phenyl)-3-(2-hydro- vacuum dessicator to constant weight to give [14C]XEN-D0401
xyethyl)-6-trifluoromethyl-[4-14C]-quinolin-2(1H)-one [14C]-6
(207.4mg, 142.4 mCi/mg, 29.5mCi) which co-eluted with an
authentic unlabelled sample: radiochemical purity (HPLC):
A 100 mL round bottom flask containing the white solid [14C]-5
97.1%; specific activity 55mCi/mmol; radio-TLC Rf = 0.32 (silica
(46.8 mCi) was dissolved in tetrahydrofuran (7 mL) and gave a
gel, dichloromethane/methanol 9:1) radiochemical purity: 97.3%;
total activity of 46.8 mCi. The resultant straw coloured solution
1H-NMR (d4MeOH, 500 MHz) s = 7.72 (dm, 1H, J = 3.4 Hz), 7.51
was cooled to –201C and a solution of lithium bis(trimethylsi-
(d, 1H, J = 3.4 Hz), 7.40 (dd, 1H, J = 3.1, 0.9 Hz), 7.24 (m, 1H), 7.16
lyl)amide in tetrahydrofuran (1 M, 9 mL) was added slowly over
(d, 1H, J = 0.9 Hz), 7.01 (d, 1H, J = 3.1 Hz), 3.66 (dd, 2H, J = 7.5,
5 min to give an orange solution. The solution was allowed to
7.5 Hz), 2.75 (dt, 1H, J = 12.5, 7.5 Hz), 2.71 (dt, 1H, J = 12.5, 7.5 Hz).
warm to –101C over 20 min then a solution of g-butyrolactone
(0.48 mL) in tetrahydrofuran (4 mL) was added slowly over
10 min. On complete addition the yellow/orange solution was
Conclusion
allowed to warm to ambient and left to stir for 5.5 h. The
At Almac Sciences we have utilized a route to the radiosynthesis
of [14C]XEN-D0401, developing the chemistry from (Crispino
reaction mixture was quenched with water (10 mL) and left
stirring at ambient for 19 h. tert-Butyl methyl ether (30 mL) was
added and the organics were extracted into tert-butyl methyl
a single carbon-14 radiolabel in the quinolin-2(1H)-one scaffold.
et al., personal communication) which allowed incorporation of
The main features to highlight in this radiosynthesis of [14C]XEN-
D0401 are the formation of methyl ester [14C]-3 and the
base catalysed, one-pot condensation process involving the
MOM-protected benzophenone [14C]-5 with g-butyrolactone to
give the cyclized MOM-protected [14C]-6. Other features to this
radiosynthesis are the ortho-lithiation reactions and removal of
MOM and pivaloyl protecting groups at the various stages in the
radiochemical synthesis to release [14C]XEN-D0401. The synth-
esis was completed in seven radiochemical steps and gave an
overall radiochemical yield of 10.5% from barium [14C]carbonate.
ether (2 ꢁ 50 mL), washed with brine (50 mL) and water (50 mL).
The combined organic phases were concentrated on the rotary
evaporator to give a yellow oil, which was dried using
azeotropic distillation from toluene (6 ꢁ 20 mL), followed by
pentane (6 ꢁ 20 mL) to give a yellow solid. Proton NMR
spectroscopic analysis of the solid was consistent with the
structure of the titled compound. The radiochemical purity was
established as 78.8% by radio-TLC on normal phase silica gel:
TLC Rf = 0.41 (silica gel, dichloromethane/methanol 9:1); 1H-NMR
(d6DMSO, 500 MHz) d = 12.27 (bs, NH), 7.78 (dm, 1H, J = 8.3 Hz),
7.57 (dd, 1H, J = 9.5, 3.2 Hz), 7.52 (d, 1H, J = 8.3 Hz), 7.39 (d, 1H,
J = 3.2 Hz), 7.34 (d, 1H, J = 9.5 Hz), 7.02–7.02 (m, 1H), 5.17 (d, 1H,
J = 7.3 Hz), 5.05 (d, 1H, J = 7.3 Hz), 4.56 (m, OH), 3.38–3.45 (m, 2H),
3.06 (s, 3H), 2.52–2.63 (m, 1H), 2.38–2.44 (m, 1H).
Acknowledgements
Almac Sciences and the authors (S. L. K., S. J., W. W.) thank
Xention Limited for their sponsorship in this radiochemistry
project. The authors also thank the Almac Analytical Chemistry
Group and Dr Mathilde Gilles for final product release.
Synthesis of 4-(5-chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl)-6-
trifluoro-methyl-2(1H)-quinolinone [14C]XEN-D0401
A 250 mL round bottom flask was charged with the [14C]-6 and
propan-2-ol (20 mL). To the resultant yellow solution was added
dropwise concentrated hydrochloric acid (36%, 5 mL). The
mixture was heated to 40–451C and after 2 h radio-TLC on
normal phase silica gel showed the formation of product with a
radiochemical purity of 86.7%. The reaction mixture was allowed
to cool to ambient temperature and quenched with ethyl
acetate (100 mL), followed by the addition of water (50 mL). The
lower aqueous layer was separated and the organic layer
washed with water (3 ꢁ 100 mL) and concentrated on the rotary
evaporator to give an oil. The oil was taken up in toluene
(3 ꢁ 20 mL), followed by pentane (3 ꢁ 20 mL) and each time
concentrated on the rotary evaporator to give a yellow solid.
The solid was dissolved in a mixture of dichloromethane/
methanol and the active solution applied to a column of silica.
The product was eluted off with dichloromethane/methanol
(95:5) and the product fractions combined and analysed by
radio-TLC on normal phase silica gel to give a radiochemical
purity of 97.8%, with a total activity of 33 mCi. The solution was
concentrated on the rotary evaporator to give an off white solid.
Radio-TLC on normal phase silica gel of the solid gave a
radiochemical purity of 96.6%. The solid was taken up in hot
absolute ethanol (5 mL) and water was added dropwise until the
References
[1] E. S. Hamanaka, 4-aryl-3-(heteroarylureido)-1,2-dihydro-2-oxo-
quinoline derivatives as antihypercholesterolemic and antiather-
osclerotic agents, US 5,565,472, 1996. Available from: www.
freepatentsonline.com/5565472.html.
[2] P. Hewawasam, J. E. Starrett, S. G. Swartz, 4-aryl-3-aminoquinoline-2-
one derivatives as potassium channel modulators, US 5,972,961,
1999. Available from: www.freepatentsonline.com/5972961.html.
[3] Xention Limited, Investigational Medicinal Product Dossier
XEN-D0401-CL-02 IMPD: XEN-D0401 Phase Ib Clinical Studies,
Cambridge, UK, 2007.
[4] Xention Limited, Fact Sheet-Overactive Bladder, Available from:
http://www.xention.com/wp-content/uploads/Fact-sheet-Overactive-
bladder.pdf, 2009 [Friday 11 September 2009].
[5] J. Wang, R. P. Discordia, G. A. Crispino, J. Li, J. A. Grosso,
R. Polniaszek, V. C. Truc, Tetrahedron Lett. 2003, 44, 4271–4273.
[6] D. V. Sivarao, K. Newberry, S. Langdon, A. V. Lee, P. Hewawasam,
M. J. Plym, L. Signor, R. Myers, N. J. Lodge, J. Pharmacol. Exp. Ther.
2005, 313, 840–847.
[7] E. L. Elliott, S. M. Bushell, M. Cavero, B. Tolan, T. R. Kelly, Org. Lett.
2005, 7, 2449–2451.
´
[8] P. J. Kocienski, Protecting Groups, 3rd ed, Thieme, New York, 2005.
[9] J. I. Ubeda, M. Villacampa, C. Avendano, Synthesis 1998, 1176–1180.
[10] V. M. Vrudhula, B. Dasgupta, J. Qian-Cutrone, E. S. Kozlowski,
C. G. Boissard, S. I. Dworetzky, D. Wu, Q. Gao, R. Kimura,
V. K. Gribkoff, J. E. Starrett, J. Med. Chem. 2007, 50, 1050–1057.
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 140–146