Palladium-catalyzed hydroxycarbonylation as the key step in the synthesis of a carbon-14 labeled Maxi-K channel blocker

Article abstract of DOI:10.1002/jlcr.1755

Carbon-14 labeled sodium formate was utilized for the preparation of a radiolabeled tracer to support the development of a Maxi-K channel blocker. Key to the process was a palladium-catalyzed hydroxycarbonylation of a bromoarene for installation of the radiolabel. Copyright

Full text of DOI:10.1002/jlcr.1755

Research Article
Received 19 October 2009,
Revised 20 January 2010,
Accepted 25 February 2010
Published online 25 April 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1755
Palladium-catalyzed hydroxycarbonylation as
the key step in the synthesis of a carbon-14
labeled Maxi-K channel blocker
Ã
Joseph P. Simeone,a Matthew P. Braun,a Luping Liu,b and
Swaminathan R. Natarajanb
Carbon-14 labeled sodium formate was utilized for the preparation of a radiolabeled tracer to support the development of
a Maxi-K channel blocker. Key to the process was a palladium-catalyzed hydroxycarbonylation of a bromoarene for
installation of the radiolabel.
Keywords: Maxi-K channel; carbon-14; palladium-catalyzed hydroxycarbonylation; [14C]carbon monoxide; carbonylation
intermediate, 6-methoxy-1H-indazole-3-carbonitrile (2),3 was trea-
ted with 4-bromophenylmagnesium iodide, formed in situ from
Introduction
The movement of ions across cell membranes is a process that is
controlled by the action of specialized pore-forming proteins called
ion channels. The high-conductance, calcium-activated potassium
(Maxi-K) channels are one such group of proteins that are present in
many cell types throughout the body including epithelial, neuronal,
endocrine, and smooth muscle tissue cells.1 Maxi-K channel
modulators have been reported for the treatment of glaucoma,2
a disease characterized by increased intraocular pressure due to an
imbalance between the production and the release of aqueous
humor from the anterior section of the eye. In support of a
medicinal chemistry program aimed at developing a Maxi-K
channel blocker, the preparation of a carbon-14 labeled version
of indazole (1) was required for drug metabolism studies.3 Herein,
we report the synthesis of this compound as well as a simple and
efficient palladium-catalyzed hydroxycarbonylation utilizing carbon-
14 sodium formate as a radiolabeled carbon monoxide source.
isopropylmagnesium chloride and 1-bromo-4-iodobenzene,
resulting in ketone (3). Alkylation of the basic nitrogen with
1-bromopinacolone in the presence of potassium carbonate
provided (4), which would serve as the starting point for the
introduction of the radiolabel, which was to be placed at the
methylene carbon adjacent to the phosphate of compound (1).
The synthesis of carbon-14 labeled carbonyl derivatives from
aryl halides can be achieved via palladium-catalyzed carbonyla-
tion; however, this process is limited by the efficiency in which
one can generate and trap carbon-14 carbon monoxide.4 Elmore
has reported a procedure for the generation of carbon-14
carbon monoxide from the dehydration of formic acid with
sulfuric acid and has applied this to the synthesis of carbon-14
labeled carbonyl derivatives via palladium catalysis.5 This
method results in moderate-to-high radiochemical yields and
can be extended to the preparation of esters, amides, aldehydes,
and ketones.
In the absence of high carbon monoxide or hydrogen
pressure, reductive carbonylation often requires the slow
addition of a reducing agent such as Bu3SnH6 or Et3SiH7
in order to minimize arene side products resulting from
hydrodehalogenation. An alternative method first introduced
by Okano utilizes sodium formate as a reducing agent at
atmospheric carbon monoxide pressure.8 We sought to
combine the Elmore procedure for carbon-14 carbon monoxide
O
MeO
N
N
O
O
HO
PO
HO
aDepartment of Process Research, Merck Research Laboratories, P.O. Box 2000,
Rahway, NJ 07065-0900, USA
1
bDepartment of Medicinal Chemistry, Merck Research Laboratories, P.O. Box
2000, Rahway, NJ 07065-0900, USA
Results and discussion
*Correspondence to: Joseph P. Simeone, Department of Process Research, Merck
Research Laboratories, P.O. Box 2000, Rahway, NJ 07065-0900, USA.
Our strategy for carbon-14 incorporation required the synthesis of
a suitable unlabeled precursor (Scheme 1). The medicinal chemistry E-mail: joe_simeone@merck.com
J. Label Compd. Radiopharm 2010, 53 511–516
Copyright r 2010 John Wiley & Sons, Ltd.

J. P. Simeone et al.
H
MeO
N
N
H
MeO
N
1. iPrMgCl, 4-iodo-bromobenzene,
-40°C to r.t.
N
O
CN
2. HCl (aq), MeOH, 91%
Br
2
3
O
MeO
N
N
K2CO3, 1-bromopinacolone
O
DMF, 80°C, 2hrs, 85%
Br
4
Scheme 1. Synthesis of radiolabeling precursor.
generation, with the use of sodium formate as a reducing agent, in reaction conditions as shown in Table 1. The Cacchi procedure
an attempt to prepare a carbon-14 labeled aldehyde (Scheme 2). uses three equivalents of the formate salt as the optimal reaction
We envisioned that this compound could be converted to the conditions. We first sought to reduce the amount of formate, since
benzylic alcohol via selective reduction in the presence of both this is the source of the radiolabel. Using 1.4equiv. of carbon-14
ketone functionalities.
sodium formate (entry 1) we were able to obtain a 23% chemical
Radiolabeled carbon monoxide was generated using 2.1 equiv. yield and a 16% radiochemical yield of the carboxylic acid (6);
of carbon-14 sodium formate and trapped into a separate flask however, none of the aldehyde was detected. With 2.3equiv. of
containing the catalyst, solvent, and 1.5 equiv. of unlabeled formate (entry 2), a 2:1 ratio of (6) to (5) was obtained in a
sodium formate as the reducing agent. Unfortunately, we were moderate yield. The use of polymethylhydrosiloxane (PMHS)12 as a
only able to obtain the desired aldehyde (5) in 10% yield. hydride source (entry 3) completely inhibited the formation of
The major product of the reaction was the carboxylic acid (6), radiolabeled products. However, incorporation of Et3SiH (entry 4)
which was isolated in 15% yield, with the remainder being resulted in selective formation of the aldehyde, albeit in low yield,
unreacted bromide and a trace amount of the arene resulting from with the major product arising from hydrodehalogenation of the
hydrodehalogenation. As expected, we were able to selectively bromide. Interestingly, complete selectivity for the carboxylic acid
reduce the aldehyde in the presence of both ketones by heating (6) was achieved with the highest yield by removing the amine
with sodium triacetoxyborohydride in benzene to obtain the base from the reaction. This is in contrast to Cacchi’s report in
benzylic alcohol (7). However, we required further synthetic steps which the presence of the base was determined to give the
to obtain our target compound and this led us to pursue a more highest yields of carboxylic acid products.
efficient method for the introduction of the radiolabel.
With an efficient procedure to make the carboxylic acid (6) in
We considered using the slow addition of Bu3SnH to obtain hand, we reconsidered our synthetic plan and searched for a
the aldehyde as reported by Elmore5, but we were discouraged method to selectively reduce the carboxylic acid in the presence
by the need to use a syringe pump in addition to the double of both ketones. We were encouraged by the report from Taddei
flask apparatus required for the gas generation. There has been which details the reduction of aryl carboxylic acids to alcohols
considerable interest in the recent literature for the develop- with hydrogen gas through the intermediacy of [1,3,5]triazine
ment of safer and environmentally benign carbonylation esters.13 We therefore treated compound (6) with 2-chloro-4,6-
procedures, which do not use gaseous carbon monoxide.9 dimethoxytriazine and NMM in dimethoxyethane to form the
One such procedure reported by Cacchi uses acetic anhydride activated ester, which was subsequently hydrogenated over
and lithium formate as a carbon monoxide source, presumably 10% Pd/C (Scheme 3). Using this method, we were able to
through the formation and decomposition of acetic formic obtain the desired alcohol (7) in 55% radiochemical yield, with
anhydride. This one pot procedure has been used to generate the major byproduct (25% rcy) resulting from ketone reduction.
aryl carboxylic acids from aryl and vinyl halides and triflates.10 A number of modifications of this procedure were attempted,
In addition, preformed acetic formic anhydride has been used in but all failed to increase the selectivity of the reaction. However,
the presence of Et3SiH to prepare aryl aldehydes.11 We set out to due to the efficiency of the hydroxycarbonylation for the
explore whether the Cacchi method was amenable to the formation of the carboxylic acid, this method still resulted in
preparation of carbon-14 labeled carboxylic acid derivatives, by greater than a 2-fold increase in radiochemical yield over the
applying it to the synthesis of our target compound.
aldehyde route. The remainder of the synthesis was completed
By using readily available carbon-14 labeled sodium formate as by converting alcohol (7) to the phosphate (8) via treatment
a surrogate for lithium formate, we explored a number of different with di-tert-butyl-N,N-diethylphosphoramidate in the presence
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 511–516

J. P. Simeone et al.
O
MeO
N
N
Pd(PPh3)2Cl2, HCOO-Na+
DMF, 90 C, 16h
[
14C]HCOO-Na+, H2O + H2SO4
[
14C]CO
°
O
Br
4
O
O
MeO
MeO
N
N
N
N
NaBH(OAc)3, AcOH
benzene, 80 C, 80%(80%)
from compound 5
O
°
O
H214C
X
OH
5, X=14C(O)H, 10%(5%)
6, X=14COOH, 15%(7%)
7
radiochemical yield in ( )
Scheme 2. Synthesis of carbon-14 labeled benzylic alcohol intermediate.
Table 1. Comparison of hydroxycarbonylation conditions
O
MeO
N
O
O
MeO
MeO
N
N
N
N
N
1) Ac2O, [14C]HCOO-Na+, DMF
2) Pd(PPh3)2Cl2, LiCl, DMF
O
O
O
90°C, 16h
H14
C
O
Br
HOO14
C
6
5
1) 1.4 eq. [14C]HCOO-Na+, 2 eq. (i-Pr)2NEt
2) 2.3 eq. [14C]HCOO-Na+, 2 eq. (i-Pr)2NEt
3) 2.3 eq. [14C]HCOO-Na+, 2 eq. (i-Pr)2NEt, 1.4 eq. PMHS
4) 2.3 eq. [14C]HCOO-Na+, 2 eq. (i-Pr)2NEt, 1 eq. Et3SiH
5) 2.3 eq. [14C]HCOO-Na+
0 %
20%(10%)
0%
20%(9%)
0%
23%(16%)
46%(23%)
0%
0%
72%(32%)
Radiochemical yield in parentheses.
of tetrazole, followed by in situ oxidation with tert-butylhydro- labeled Maxi-K channel blocker. Furthermore, through careful
peroxide. Removal of the tert-butyl esters with HCl in ethyl control of the reaction conditions we were able to obtain
acetate, followed by HPLC purification, resulted in the target complete selectivity for the aryl carboxylic acid over the aryl
radiotracer (9).
aldehyde with moderate radiochemical yields. This allowed us to
In summary, we have successfully applied a one-pot hydro- obtain our target compound via a slight modification of our
xycarbonylation procedure to the synthesis of a carbon-14 original route. We envision the one-pot hydroxycarbonylation
J. Label Compd. Radiopharm 2010, 53 511–516
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

J. P. Simeone et al.
O
MeO
N
O
MeO
N
N
1) 2-chloro-4,6-dimethoxy triazine,
NMM, DME, 0 C to r.t., 16hrs
1) di-t-butyl-N,N-diethyl phosphoramidate,
1-H-tetrazole, CH2Cl2
N
°
O
2) H2, 10% Pd/C, 1hr, (55%)
2) t-butylhydroperoxide, 0°C to r.t
O
H214C
OH
HOO14
C
7
6
O
O
N
MeO
MeO
N
N
N
HCl, EtOAc
(42%) from 7
O
O
H214C
H214C
O
O
O
P
Ot-Bu
O P
OH
OH
Ot-Bu
radiochemical yield in ( )
8
9
Scheme 3. Completing the synthesis of the carbon-14 labeled Maxi-K channel blocker.
protocol to have broad utility in radiosynthesis laboratories due (4-Bromophenyl)(6-methoxy-1H-indazol-3-yl)methanone (3)
to its operational simplicity.
To a suspension of 1-bromo-4-iodobenzene (1.7 g, 6.0 mmol) in
THF (3 mL) at À401C was added dropwise a 2 M solution of
isopropylmagnesium chloride (3.0 mL, 6 mmol) in THF. After 4 h
at that same temperature, a solution of 6-Methoxy-1H-indazole-
Experimental
Materials and methods
3-carbonitrile 2 (519 mg, 3.0 mmol) in THF (4.5 mL) was added
and the mixture was stirred for 15 min and then allowed to
warm to room temperature, at which time 2 N HCl (7.5 mL)
was added and stirring was continued for an additional 2 h.
The reaction mixture was extracted with methylene chloride
(3 Â 75 mL). The organic extracts were combined, washed with
water (1 Â 50 mL), and dried over sodium sulfate. Evaporation
under reduced pressure yielded a yellow solid (907 mg, 91%).
1H NMR (400 MHz, CDCl3) d 10.30 (1H, broad s), 8.30 (1H, d,
J = 8.89 Hz), 8.22–8.19 (2H, m), 7.68–7.64 (2H, m), 7.04 (1H, dd,
J = 8.90, 2.14 Hz), 6.92 (1H, d, J = 2.11 Hz), 3.93 (3H, s). MS (ESI1)
[M1H]1 330.9.
All reagents and solvents were purchased from commercial
sources and used without further purification. Sodium
[14C]formate was purchased from Amersham Radiochemicals.
6-Methoxy-1H-indazole-3-carbonitrile 2 was prepared by Merck
Medicinal Chemistry according to reference 3. Radioactivity was
measured with a Packard Tri-Carb 1000TR Liquid Scintillation
Analyzer. 1H NMR spectra (400 MHz) were measured at 400 MHz
on a Varian Inova Spectrometer. Chemical shifts are reported as
d parts per million (ppm) downfield from tetramethylsilane.
An Agilent series 1100 HPLC system coupled to a Packard
Radiomatic 525TR Flow Scintillation Analyzer was used to
monitor reactions and check for purity. The following conditions
were used unless otherwise noted: Zorbax XDB C8 column,
4.6 Â 150 mm, 5% acetonitrile:H2O (0.1% TFA) to 100% acetoni-
trile, 15 min linear gradient, 1 mL/min. Preparative HPLC was
performed using Varian Pro Star Model 210 pumps coupled to a
Shimadzu SPD-10A UV/Vis detector. LC/MS and specific activity
measurements were obtained by electrospray ionization using
an Agilent 1100 Series LC/MSD. After injection, samples were
loaded onto a 4.6 Â 150 mm Zorbax XDB C8 column and eluted
directly into the mass spectrometer using a linear gradient of
acetonitrile in water with ammonium formate as a mobile phase
modifier. Specific activity was calculated by comparison of the
ratio of carbon-14/carbon-12 for the tracer against the
unlabeled reference.
1-{3-[(4-Bromophenyl)carbonyl]-6-methoxy-1H-indazol-1-yl}-
3,3-dimethylbutan-2-one (4)
To a solution of 3 (741 mg, 2.23 mmol) in DMF (7 mL) was added
potassium carbonate (754 mg, 5.45 mmol) and 1-bromopinaco-
lone (647 mL, 4.80 mmol) and the mixture was heated at 801C for
4 h. The reaction was quenched with water (50 mL) and
extracted into diethyl ether (3 Â 50 mL). The organic extracts
were combined, washed with water (1 Â 50 mL), and dried over
sodium sulfate. Evaporation under reduced pressure yielded a
light yellow residue, which was triturated with warm diethyl
ether (100 mL) and aged at 01C for 1 h. The solids were collected
on a filter funnel and dried in a vacuum pistol overnight to give
a white solid (816 mg, 85%). 1H NMR (400 MHz, CDCl3) d 8.28 (1H,
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J. Label Compd. Radiopharm 2010, 53 511–516

J. P. Simeone et al.
d, J = 8.90 Hz), 8.18–8.14 (2H, m), 7.64–7.60 (2H, m), 7.01 (1H, dd, (1H, d, J = 8.93Hz), 5.87 (2H, s), 4.81 (2H, s), 4.02 (3H, s), 1.33 (9H, s).
J = 8.91, 2.08 Hz), 6.52 (1H, d, J = 2.06 Hz), 5.39 (3H, s), 3.86 (3H, s), MS (ESI1) [M1H]1 383.0.
1.34 (9H, s). MS (ESI1) [M1H]1 429.0.
4-{[1-(3,3-Dimethyl-2-oxobutyl)-6-methoxy-1H-indazol-3-yl]-
carbonyl}(formyl-14C)benzaldehyde (5) via in situ formation
4-{[1-(3,3-Dimethyl-2-oxobutyl)-6-methoxy-1H-indazol-3-yl]-
of [14C] carbon monoxide
carbonyl}(formyl-14C)benzaldehyde (5) and 4-{[1-(3,3-
dimethyl-2-oxobutyl)-6-methoxy-1H-indazol-3-yl]carbonyl}
(carboxy-14C)benzoic acid (6) by Elmore method with
sodium formate as a reducing agent
In a 0.5–2 mL Biotage microwave vial was placed sodium
[14C]formate (17 mg, 0.242 mmol, 13.3 mCi, 55 mCi/mmol), DMF
(1 mL), and N,N-diisopropylethylamine (36 mL, 0.210 mmol).
A rubber septum was placed on the vial and the mixture was
purged with argon gas. Acetic anhydride (20 mL, 0.210 mmol)
was added, the septum was quickly removed, and a crimp cap
was placed on the vial. The mixture was stirred at room
temperature for 1 h. In a separate vial was placed 4 (45 mg,
0.105 mmol), Pd(PPh3)2Cl2 (3.5 mg, 0.005 mmol), lithium chloride
(9.0 mg, 0.221 mmol), DMF (500 mL), and triethylsilane (16 mL,
0.105 mmol). A rubber septum was placed on the vial and the
mixture was purged with argon gas. An argon-filled balloon was
inserted through the septum of the vial containing the sodium
[14C]formate mixture and the substrate/catalyst mixture was
added via syringe. The crimp cap was quickly removed and
replaced with a new one and the mixture was heated at 901C
overnight. The reaction mixture was cooled to room tempera-
ture, diluted with water (2 mL), and extracted with ethyl acetate
(3 Â 5 mL). The organic extracts were combined, washed with
water (1 Â 5 mL), and dried over sodium sulfate. Evaporation
under reduced pressure yielded a residue, which was dissolved
in acetonitrile (1 mL) and subjected to preparative chromato-
graphy (Phenomenex Luna C18(2) column, 21.2 Â 250 mm, 40%
acetonitrile:H2O (0.1% TFA) to 70% acetonitrile, 30 min linear
gradient, 20 mL/min, 1 mL injection). 1.2 mCi (9% rcy) of 5 was
obtained, which had a radiochemical purity of 99.5% by HPLC.
1H NMR (400 MHz, CDCl3) d 10.13 (1H, s), 8.39–8.35 (3H, m), 8.00
(2H, d, J = 8.09 Hz), 7.13 (1H, d, J = 8.92 Hz), 5.86 (2H, s), 4.02
(3H, s), 1.33 (9H, s). MS (ESI1) [M1H]1 381.0.
In a 3-neck 50 mL round-bottomed flask connected to a vacuum
manifold was added sodium [14C]formate (65 mg, 0.90 mmol,
49.5 mCi, 55 mCi/mmol) and water (2 mL). The flask was fitted
with a dropping funnel capped with a rubber septum and a 901
bent adapter attached to a 10 mL recovery flask containing
4 (185 mg, 0.430 mmol), sodium formate (44 mg, 0.645 mmol),
Pd(PPh3)2Cl2 (15 mg, 0.022 mmol), and DMF (1 mL). The entire
system was evacuated to 0.01 mm Hg and concentrated sulfuric
acid (5 mL) was added via the dropping funnel. The 3-neck
flask was heated at 701C for 1 h at which time it was removed
from the oil bath, the recovery flask was placed inside, and
the temperature was increased to 901C and held for 16 h. The
reaction mixture was cooled to room temperature and the
vacuum was released. Water (2 mL) and 1 N NaOH (2 mL) were
added and the mixture was extracted with ethyl acetate
(3 Â 5 mL). The combined extracts had a total radioactivity of
3.8 mCi (5% rcy) by liquid scintillation counting. After drying
over sodium sulfate and evaporation under reduced pressure,
the resulting brown residue containing 5, which had a radio-
chemical purity of 66.7% by HPLC (tR = 13.0 min), was taken on
directly to the next step. The alkaline aqueous layer was then
adjusted to pH = 6 with concentrated hydrochloric acid and
extracted with ethyl acetate (3 Â 5 mL). The organic extracts
were combined and dried over sodium sulfate. Evaporation
under reduced pressure yielded a residue that was dissolved in
2:1 methanol:DMSO (3 mL) and subjected to preparative
chromatography
(Phenomenex
Luna
C18(2)
column,
21.2 Â 250 mm, 35–65% acetonitrile:H2O (0.1% TFA), 30 min
linear gradient, 20 mL/min, 3 Â 1 mL injections). 4.0 mCi (7%
rcy) of 6 was obtained, which had a radiochemical purity of
89.8% by HPLC (tR = 11.8 min). 1H NMR (400 MHz, CD3OD) d 8.28
(2H, d, J = 8.04 Hz), 8.19 (1H, d, J = 8.91 Hz), 8.13 (2H, d,
J = 8.08 Hz), 7.01 (1H, dd, J = 9.00, 1.40 Hz), 6.91 (1H, d,
J = 1.40 Hz), 5.68 (2H, s), 3.88 (3H, s), 1.33 (9H, s). MS (ESI1)
[M1H]1 397.5.
4-{[1-(3,3-Dimethyl-2-oxobutyl)-6-methoxy-1H-indazol-3-yl]-
carbonyl}(carboxy-14C)benzoic acid (6) via in situ formation
of [14C] carbon monoxide
In a 0.5–2 mL Biotage microwave vial was placed sodium
[14C]formate (23.2 mg, 0.329 mmol, 18.2 mCi, 55 mCi/mmol) and
DMF (1 mL). A rubber septum was placed on the vial and the
mixture was purged with argon gas. Acetic anhydride (28.2 mL,
0.298 mmol) was added, the septum was quickly removed, and a
crimp cap was placed on the vial. The mixture was stirred at
room temperature for one hour. In a separate vial was placed
1-[3-({4-[Hydroxy(14C)methyl]phenyl}carbonyl)-6-methoxy-
1H-indazol-1-yl]-3,3-dimethylbutan-2-one (7) from (5)
4
(64 mg, 0.149 mmol), Pd(PPh3)2Cl2 (5.3 mg, 0.008 mmol),
lithium chloride (13.3 mg 0.313 mmol), and DMF (1 mL). A rubber
septum was placed on the vial and the mixture was purged with
argon gas. An argon filled balloon was then inserted through
the septum of the vial containing the sodium [14C]formate
mixture and the substrate/catalyst mixture was added via
syringe. The crimp cap was quickly removed and replaced with
a new one and the mixture was heated at 901C overnight. The
reaction mixture was cooled to room temperature and
evaporated under reduced pressure. The residue was parti-
tioned between 1 N NaOH (4 mL) and ethyl acetate (5 mL).
The alkaline aqueous layer was adjusted to pH = 6 with
concentrated hydrochloric acid and extracted with ethyl
acetate (3 Â 5 mL). The combined extracts had a total radio-
activity of 6.7 mCi (32% rcy) by liquid scintillation counting.
To the crude residue containing 5 (2.5 mCi, 0.045 mmol) was
added benzene (2 mL), acetic acid (10 mL, 0.175 mmol), and
sodium triacetoxyborohydride (23 mg, 0.108 mmol). The mixture
was purged with nitrogen gas and heated at reflux for 1 h.
The reaction was diluted with acetonitrile (2 mL) and passed
through a 0.45 mM filter. Evaporation under reduced pressure
yielded a residue, which was dissolved in acetonitrile (1.5 mL)
and subjected to preparative chromatography (Phenomenex
Luna C18(2) column, 21.2 Â 250 mm, 40% acetonitrile:H2O (0.1%
TFA) to 70% acetonitrile, 30 min linear gradient, 20 mL/min,
1 Â 1.5 mL injections). 2.0 mCi (80% rcy) of 7 was obtained,
which had
(tR = 11.5 min). 1H NMR (400 MHz, CDCl3)
J = 8.92 Hz), 8.26 (2H, d, J = 8.07 Hz), 7.54–7.47 (3H, m), 7.11
a
radiochemical purity of 99.9% by HPLC
d
8.34 (1H, d,
J. Label Compd. Radiopharm 2010, 53 511–516
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J. P. Simeone et al.
After drying over sodium sulfate and evaporation under reduced
pressure, the resulting residue containing 6, which had a
radiochemical purity of 88.4% by HPLC, was taken on directly to
the next step. H NMR and MS data were consistent with that
given for the sample prepared by the alternate method
described above.
(4-{[1-(3,3-Dimethyl-2-oxobutyl)-6-methoxy-1H-indazol-3-yl]-
carbonyl}phenyl)(14C)methyl dihydrogen phosphate (9)
1
The ethyl acetate solution containing 8 was evaporated under
reduced pressure and diluted with a solution of saturated HCl in
ethyl acetate (2 mL). The solution was stirred at room temperature
for 2 h, at which time it was concentrated to dryness, dissolved in
methanol (2 mL), and subjected to preparative chromatography
(Phenomenex Synergi Max RP column, 21.2 Â 250 mm, 25%
acetonitrile:H2O (0.1% HClO4) to 55% acetonitrile, 30 min linear
gradient, 20 mL/min, 2 Â 1 mL injections). 2.5 mCi (42% rcy from 7)
of 9 was obtained, which had a radiochemical purity of 99.2% by
HPLC (Phenomenex Luna C8 column, 4.6 Â 250 mm, 60% acetoni-
trile:H2O (0.1% HClO4), tR = 12.5 min, 1 mL/min) and a specific
activity of 53.1 mCi/mmol. 1H NMR (400MHz, CD3OD) d 8.25 (2H, d,
J= 7.50 Hz), 8.18 (1H, d, J= 7.50 Hz), 7.60 (2H, d, J= 7.60 Hz), 7.00
(1H, d, 7.50 Hz), 6.90 (1H, broad s), 5.65 (2H, s), 5.10 (2H, broad s),
3.90 (3H, S), 1.35 (9H, s). MS (ESI1) [M1H]1 463.0.
1-[3-({4-[Hydroxy(14C)methyl]phenyl}carbonyl)-6-methoxy-
1H-indazol-1-yl]-3,3-dimethylbutan-2-one (7) from (6)
To
a solution of 2-chloro-4,6-dimethoxytriazine (28.7 mg,
0.163 mmol) in DMF (1 mL) at 01C was added 4-methylmorpho-
line (18.8 mL, 0.171 mmol). A precipitate immediately formed.
The mixture was removed via syringe and added to a
suspension of 6 (7.2 mCi, 0.131 mmol) in dimethoxyethane
(2 mL) at 01C. The reaction was warmed to room temperature
and stirred for 16 h at which time it was passed through a
0.45 mM filter. The filter was rinsed with additional dimethox-
yethane (2 mL) and the filtrate was transferred to a 15 mL
recovery flask. The flask was purged with nitrogen and
10 wt% palladium on carbon (10 mg, 0.009 mmol) was added.
The mixture was then stirred under a balloon of hydrogen for
1 h. The catalyst was removed by filtration and the filtrate was
evaporated under reduced pressure. The residue was dissolved
in acetonitrile (3 mL) and subjected to preparative chromato-
graphy (Phenomenex Luna C18(2) column, 21.2 Â 250 mm,
40% acetonitrile:H2O (0.1% TFA) to 70% acetonitrile, 30 min
linear gradient, 20 mL/min, 2 Â 1.5 mL injections). 4.0 mCi (55%
rcy) of 7 was obtained, which had a radiochemical purity of
Acknowledgements
We thank Mr Herb Jenkins of Merck Analytical Research for
analytical support and Dr Jeffrey Hale of Merck Medicinal
Chemistry for helpful discussions related to phosphate chemistry.
References
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1
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Di-tert-butyl(4-{[1-(3,3-dimethyl-2-oxobutyl)-6-methoxy-1H-
indazol-3-yl]carbonyl}phenyl)(14C)methyl phosphate (8)
To a solution of 7 (5.9 mCi, 0.111 mmol) in dichloromethane
(3 mL) under an argon atmosphere was added a solution of
tetrazole in acetonitrile (740 mL, 0.45 M, 0.333 mmol) followed by
di-tert-butyl N,N-diethylphosphoramidite (59 mL, 0.211 mmol).
The reaction was stirred for 30 min at room temperature and
cooled to 01C, at which time tert-butyl hydroperoxide in
decane (111 mL, 5 M, 0.555 mmol) was added. After 30 min, a
solution of 1 M sodium sulfite (250 mL) was added and
stirring was continued for a further 5 min. The mixture was
diluted with ethyl acetate (10 mL) and washed with brine
(5 mL). The organic layer, which had a total radioactivity of
5.5 mCi by liquid scintillation counting and a radiochemical
purity of 88.2% by HPLC (tR = 14.3 min), was taken on directly to
the next step.
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J. Label Compd. Radiopharm 2010, 53 511–516