An efficient large-scale synthesis of 1H-indazole-[3-14C] carboxylic acid

Article abstract of DOI:10.1002/jlcr.1396

An efficient large-scale carbon-14 synthesis of 1H-indazole-[3- ¹⁴C]carboxylic acid starting from [¹⁴C]potassium cyanide is reported. Key transformations encountered during the synthesis include aromatic nucleophilic substitution of

Full text of DOI:10.1002/jlcr.1396

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 675–678.
Published online 8 June 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1396
JLCR
Research Article
An efficient large-scale synthesis of 1H-indazole-
[3-14C]carboxylic acid
RICHARD V. COELHO JR. and KLAAS SCHILDKNEGT*
Pfizer Global Research & Development, Eastern Point Road, Groton, CT 06340, USA
Received 2 April 2007; Revised 17 April 2007; Accepted 7 May 2007
Abstract: An efficient large-scale carbon-14 synthesis of 1H-indazole-[3-14C]carboxylic acid starting from [14C]po-
tassium cyanide is reported. Key transformations encountered during the synthesis include aromatic nucleophilic
substitution of 2-nitrofluorobenzene by ethyl [14C]cyanoacetate, a mild decarbethoxylation and an aniline
nitrosation/cyclization. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: carbon-14; 1H-indazole-3-carboxylic acid; ethyl cyanoacetate
Introduction
perform cyanide displacement on a benzyl bromide
precursor. However, this chemistry proved largely
inefficient for preparing 3b when demonstrated
with a single equivalent of KCN under a variety of
reaction conditions. The benzylic carbon of 3b is
sufficiently acidic (facilitated by the ortho nitro function)
to undergo further alkylation with available benzyl
bromide and predominantly form over-alkylated
byproducts.2
We required ꢀ200 mCi of 1H-indazole-[3-14C]carboxylic
acid (1a) for use as a labeled substructure of a Pfizer drug
development candidate. Of numerous established non-
isotope-labeled indazole synthesis strategies, we chose to
exploit a nitrosation/cyclization approach to 1H-inda-
zole-3-carboxylic acid reported by Yoshida et al.1 for
carbon-14 labeling because we anticipated it would
support the introduction of the carboxylic acid function
as a (labeled) nitrile. The ability of this approach to
efficiently deliver 200 mCi of 1a hinged on the develop-
ment of a succinct and high yielding radiosynthesis of
2-(2-nitrophenyl)-[1-14C]acetonitrile (3a) from K14CN.
This result prompted us to investigate an alternative
approach to 3a that utilized ethyl [14C]cyanoacetate to
install the required carbon-14 nitrile as a labeled
acetonitrile anion equivalent. Bromoacetic acid (4)
was deprotonated with aqueous NaOH and the
resulting anion was heated at reflux with 986 mCi
(52 mCi/mmol, ꢀ1 eq) of K14CN (Scheme 2). After 3 h
the reaction was cooled in an ice bath acidified with
conc. HCl and concentrated to a residue. This residue
was extracted with EtOH, treated with a catalytic
Results and Discussion
Our strategy for preparing 1a (Scheme 1) necessitated
the development of an efficient large-scale synthesis of
3a. Our initial design for accessing 3 was to simply
Scheme 1
*Correspondence to: Klaas Schildknegt, Pfizer Global Research
Development, Eastern Point Road, Groton, CT 06340, USA.
E-mail: klaas.schildknegt@pfizer.com
&
Copyright # 2007 John Wiley & Sons, Ltd.

676 R. V. COELHO JR. AND K. SCHILDKNEGT
Scheme 2
amount of H2SO4 and heated at reflux. When
the esterification was deemed complete by radio-thin
larger chromatography (TLC) the product was
isolated by extraction to afford 801 mCi (81%)
of radiochemically pure 5. Ethyl cyanoacetate has
been shown to efficiently undergo aromatic nucleophi-
lic substitutions on suitably functionalized aryl
fluorides.3 In the present case, 5 (801 mCi) was treated
with K2CO3 and a slight excess of 2-fluoronitrobenzene
in hot DMF to cleanly afford 783 mCi (98%) of
nitrile ester 6. To arrive at the desired substituted
acetonitrile, decarbethoxylation was required without
concurrent nitrile hydrolysis. Premature nitrile hydro-
lysis would result in a symmetrical 12C/14C diacid
intermediate and subsequent loss of label and specific
activity reduction. Krapcho decarboxylation condi-
tions4 effectively performed this operation, converting
6 to 3a in good yield. Benzyl nitrile 3a (759 mCi) was
refluxed in concentrated HCl and acetic acid to effect
nitrile hydrolysis, affording crude carboxylic acid
which was esterified with ethanol to afford 710 mCi
(94%) of 96% radiochemically pure nitroester 7 after
Experimental
Materials and methods
K14CN (52 mCi/mmol) was purchased from PerkinEl-
mer Life & Analytical Sciences. All remaining reagents
and solvents were purchased from Sigma-Aldrich
Corporation and used as received. Radioactivity mea-
surements were performed on
a Packard Tricarb
2900TR liquid scintillation analyzer using Beckman–
Coulter Ready Safe liquid scintillation cocktail. TLC
plates were analyzed on a Bioscan AR-2000 linear
analyzer. Radio-high-performance liquid chromatogra-
phy (HPLC) was performed using an Agilent 1100
Series HPLC system equipped with an Agilent 1100
Series diode array detector and an IN/US Systems B-
RAM Model 2 radioactivity detector. Luna C18 3 mm,
50 ꢁ 4.6 mm, 210 nm, 100% 98:2 10 mM aqueous
formic acid:AcN for 1 min, then linear gradient to 80%
2:98 10 mM aqueous formic acid AcN by 8 min, then
return to 100% 98:2 10 mM aqueous formic acid:AcN
by 8.10 min, 1 mL/min flow rate, HPLC solvent :
Beckman–Coulter Ready Safe liquid scintillation cock-
tail (1:3).
extractive workup. Hydrogenation of
7 delivered
aminoester 8 and this was treated with t-butylnitrite/
acetic anhydride to form a transient nitroso intermedi-
ate 2a that underwent rapid intramolecular cyclization
to indazole 9 (539 mCi, 76% yield from 7). Finally,
215 mCi of 9 was subjected to alkaline hydrolysis to
yield 203 mCi (94%) of 1H-indazole-[3-14C]carboxylic
acid (1a) void of any chemical and radiochemical
impurities.
Ethyl [14C]cyanoacetate (5)
K14CN (986 mCi, 19.0 mmol, 52 mCi/mmol) was added
to a solution of bromoacetic acid (2.90 g, 20.9 mmol) in
20 mL of 1 M aqueous NaOH and the resulting solution
was heated at 1008C for 3 h. The reaction was then
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 675–678
DOI: 10.1002.jlcr

AN EFFICIENT LARGE-SCALE SYNTHESIS OF 1H-INDAZOLE-[3-14C]CARBOXYLIC ACID 677
cooled in an ice bath, treated dropwise with 1.5 mL of
residue. The residue was dissolved in 45 mL of ethanol
and 1 mL of H2SO4 and heated at reflux for 36 h. The
reaction solution was then concentrated to approxi-
mately 5 mL total volume by rotary evaporation and
then partitioned between saturated aqueous NaHCO3
and EtOAc. The organic layer was washed with brine,
dried over anhydrous Na2SO4 and concentrated to
dryness to yield 710 mCi of 7 that was 96% radio-
chemically pure by radio-TLC (Rf ¼ 0:7, EtOAc:hexanes
40:60) versus an authentic product standard.
concentrated aqueous HCl and evaporated to a solid
residue. The residue was suspended in 50 mL of EtOH
and filtered through a fritted glass funnel. The filtrate
was treated with 1 mL of H2SO4 and heated at reflux for
7 h, at which time the esterification was shown to be
complete by radio-TLC (EtOAc:hexanes 20:80) versus
an authentic product standard. The reaction solution
was concentrated to ꢀ5 mL by rotary evaporation,
treated with 50 mL of ice-cold saturated aqueous
NaHCO3 and extracted with 50 mL of EtOAc. The
organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated to dry-
ness to afford 801 mCi of 5 that was >99% radio-
chemically pure by radio-TLC (Rf ¼ 0:2, EtOAc:hexanes
20:80).
Ethyl 2-(2-aminophenyl)-[1-14C]acetate (8)
A nitrogen purged solution of ethyl 2-(2-nitrophenyl)-
[1-14C]acetate (7) (710 mCi, 13.6 mmol, 52 mCi/mmol)
in 20 mL of EtOH and 3 mL of IPA was treated with
ꢀ30 mg of 10% Pd/C and stirred under a balloon of
hydrogen for 14 h. The reaction mixture was filtered
and the filtrate was concentrated to dryness to yield
642 mCi of 8 that was 94% radiochemically pure by
radio-HPLC and co-eluted with an authentic product
standard.
Ethyl [2-14C]cyano-2-(2-nitrophenyl)acetate (6)
2-Nitrofluorobenzene (2.40 g, 17.0 mmol) and K2CO3
(3.19 g, 23.0 mmol) were added to a solution of ethyl
[
14C]cyanoacetate (5) (801 mCi, 15.4 mmol, 52 mCi/
mmol) in 22 mL of DMSO. The resulting suspension
was heated at 1008C for 4 h, at which time the reaction
was deemed complete by radio-TLC (EtOAc:hexanes
20:80) versus an authentic product standard. The
reaction was cooled to room temperature, diluted with
75 mL of EtOAc and washed with 50 mL of 1 N aqueous
HCl, followed by brine. The organic layer was dried
over anhydrous Na2SO4, filtered and concentrated
to dryness to afford 783 mCi of 6 that was 95%
radiochemically pure by radio-TLC (Rf ¼ 0:3, EtOAc:
hexanes 20:80).
Ethyl N-acetyl-1H-indazole-[3-14C]carboxylate (9)
A solution of ethyl 2-(2-aminophenyl)-[1-14C]acetate (8)
(642 mCi, 12.3 mmol, 52 mCi/mmol) in 14 mL of to-
luene was treated with 4 mL of acetic anhydride and
heated to 908C. t-Butyl nitrite (1.9 mL) was added
dropwise to the solution over 90 min. The reaction was
then cooled to room temperature, concentrated to
dryness and the resulting residue was dissolved in
CH2Cl2 and washed with 5% aqueous K2CO3. The
organic layer was dried over anhydrous Na2SO4 and
concentrated to yield 539 mCi of 9 that was 94%
radiochemically pure by radio-HPLC and co-eluted
with an authentic product standard.
2-(2-Nitrophenyl)-[1-14C]acetonitrile (3a)
NaCl (0.23 g, 3.9 mmol) was added to a solution of ethyl
2-[14C]cyano-2-(2-nitrophenyl)acetate (6) (783 mCi,
15.1 mmol, 52 mCi/mmol) and water (2.3 g, 41.4 mmol)
in 23 mL of DMSO. The reaction was heated at 1408C
for 6 h and then cooled to room temperature and
partitioned between 50 mL of EtOAc and 50 mL of
brine. The organic layer was dried over anhydrous
Na2SO4 and concentrated to dryness to yield 759 mCi
of 3a that was 93% radiochemically pure by radio-
HPLC and co-eluted with an authentic product stan-
dard.
1H-Indazole-[3-14C]carboxylic acid (1a)
Ethyl N-acetyl-1H-indazole-3-[14C]carboxylate (9) (215 mCi,
4.1 mmol, 52 mCi/mmol) was taken up in 10 mL of
water and treated with 0.50 g of NaOH pellets. The
resulting mixture was heated at reflux for 3 h at which
time HPLC indicated that the hydrolysis was complete
versus an authentic product standard. The reaction
was cooled to room temperature and acidified with 20%
aqueous HCl, causing solids to precipitate. The mixture
was cooled to 08C and product solids were collected by
filtration, rinsing with water, to afford 0.64 g (203 mCi)
of 1a that was of >99% chemical and radiochemical
purity by radio-HPLC and co-eluted with an authentic
product standard.
Ethyl 2-(2-nitrophenyl)-[1-14C]acetate (7)
A solution of 2-(2-nitrophenyl)-[1-14C]acetonitrile (3a)
(759 mCi, 14.6 mmol, 52 mCi/mmol) in 6 mL of con-
centrated HCl, 1 mL of acetic acid and 1 mL of water
was heated at reflux for 6 h and then concentrated to a
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 675–678
DOI: 10.1002.jlcr

678 R. V. COELHO JR. AND K. SCHILDKNEGT
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 675–678
DOI: 10.1002.jlcr