Synthesis of 14C-labeled 4-hydroxyindole as an intermediate for the preparation of (S)-2-14-12-13-(indol-2-[14C]-4-yloxy)-2-hydroxypropylaminol -2-methylpropyl]- phenoxylpyridine-5-carboxamide (LY368842-[indole-14C]) glyco

Article abstract of DOI:10.1002/jlcr.631

Synthesis of 4-hydroxyindole labeled with ¹⁴C at the 2-position was accomplished based on the vicarious nucleophilic substitution reaction of benzyl-protected 3-nitrophenol with p-chlorophenoxyacetonitrile-[1-¹⁴C]. This was followed

Full text of DOI:10.1002/jlcr.631

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 1143–1152.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.631
Research Article
14
Synthesis of C-labeled 4-hydroxyindole
as an intermediate for the preparation of
1
4
(
2
S)-2-[4-[2-[3-(indol-2-[ C]-4-yloxy)-
-hydroxypropylamino]-2-methylpropyl]-
phenoxy]pyridine-5-carboxamide
14
(
LY368842-[indole- C]) glycolate
Boris A. Czeskis*, Dean K. Clodfelter and William J. Wheeler
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285,
USA
Summary
1
4
Synthesis of 4-hydroxyindole labeled with
C at the 2-position was
accomplished based on the vicarious nucleophilic substitution reaction of
1
4
benzyl-protected 3-nitrophenol with p-chlorophenoxyacetonitrile-[1- C]. This
was followed by the reductive cyclization of o-nitrocyanomethyl derivative by
1
palladium catalyzed hydrogenation. p-Chlorophenoxyacetonitrile-[1- C] was
4
prepared from commercially available p-chlorophenoxymethyl chloride and
1
sodium cyanide-[ C]. 4-Hydroxyindole-[2- C] was used for the synthesis of
4
14
1
4
C-labeled b adrenergic agonist LY368842. Copyright # 2002 John Wiley &
3
Sons, Ltd.
Key Words: 4-hydroxyindole; b3 adrenergic agonist; LY368842 glycolate;
carbon-14-labeled
*Correspondence to: B. A. Czeskis, Lilly Research Laboratories, Eli Lilly and Company,
Indianapolis, IN 46285, USA. E-mail: czeskis@lilly.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 3 June 2002
Revised 26 June 2002
Accepted 26 June 2002

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B. A. CZESKIS ET AL.
Introduction
4
-Hydroxyindole is a key intermediate in the synthesis of a number
3
1
physiologically active compounds including pindolol and psilocin.
2
14
We were interested in the preparation of C-labeled 4-hydroxyindole to
be used in the synthesis of radiolabeled b adrenergic agonist LY368842,
3
4
a compound with a potential for the treatment of obesity and diabetes.
There are different methods of the synthesis of 4-hydroxyindole
presented in literature. Among them are electrochemical couplings of
5
,3-cyclohexadione with ethyl vinyl ether, and 2,6-dinitrotoluene with
1
6
7
formaldehyde, the Leimgruber-Batcho procedure, and palladium
8
catalyzed N-heteroannulation of styrenes. However, most of these
methods are not suitable for the preparation of the radiolabeled
compound. We report herein an efficient way for the synthesis of 4-
1
4
hydroxyindole-[2- C] which was used for the preparation of b
3
adrenergic agonist LY368842.
Results and discussion
14
Our initial attempt to prepare C-labeled 4-hydroxyindole consisted of
the attractive possibility of allylic bromination followed by cyanation of
6
-substituted 2-nitrotoluenes. The resulting o-nitrosubstituted benzyl
cyanides can be converted into 4-substituted indole by reductive
9
cyclization.
We found that the results of bromination of 2-nitrotoluenes were
dependent on the nature of the substituent in the 6-position. Thus,
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1143–1152

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4
CARBON-14 LABELLED LY368842-[INDOLE- C] GLYCOLATE
1145
hydroxy and aminotoluenes formed aromatic bromination products
only, whereas 2,6-dinitrotoluene was selectively converted into the
desired bromomethyl derivative. The next step of cyanation, however,
proved to be problematic because the starting benzyl bromide reacted
with initially formed benzyl cyanide faster than with sodium cyanide.
This resulted in preferable formation of the dimer (Scheme 1). Lowering
the pH of the reaction with trifluoroacetic acid, previously used for the
1
0
preparation of other 2-nitrobenzyl cyanides, gave a mixture of the
above compounds and trifluoroacetyl derivatives.
Scheme 1.
Another way to obtain 2-nitrobenzyl cyanides is the introduction of a
cyanomethyl group into nitrobenzenes using vicarious nucleophilic
substitution of an ortho-hydrogen with the anion generated from
1
1
p-chlorophenoxyacetonitrile.
obtained by the reaction of p-chlorophenol with chloroacetonitrile,
p-Chlorophenoxyacetonitrile can be
2
1
14
an expensive C-labeled material. We found that p-chlorophenoxyace-
1
tonitrile-[1- C] (2) can be prepared from commercially available p-
4
1
chlorophenoxymethyl chloride (1) and sodium cyanide-[ C] in acetone.
4
1
3
Surprisingly, the addition of sodium iodide lowers the yield of 2 by
leading to the partial formation of p-chlorophenol. This was the only
product formed when the reaction was conducted in dimethylsulfoxide.
Alkylation of benzyl-protected 3-nitrophenol (3) (prepared straightfor-
wardly from 3-nitrophenol, benzyl bromide and potassium carbonate in
acetone) with the anion derived from nitrile 2 in the presence of
potassium tert-butoxide in dimethylformamide, afforded 2-(2-benzy-
Copyright # 2002 John Wiley & Sons, Ltd.
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B. A. CZESKIS ET AL.
14
loxy-6-nitrophenyl)acetonitrile-[1- C] (4). Reductive cyclization of 4
and simultaneous deprotection of the hydroxy-group, using hydrogena-
1
1b
tion on palladium catalyst in the presence of acetic acid,
proceeded
14
smoothly to give 4-hydroxyindole-[2- C] (5) in good yield (Scheme 2).
14
The synthesis of LY368842-[indole- C] glycolate from 4-hydroxyin-
1
dole-[2- C] (5) was accomplished based on the previously developed
4
4
procedure. Thus, reaction of 5 with (S)-glycidyl nosylate (6) in the
presence of potassium carbonate gave (S)-glycidyloxyindole (7), which
was coupled with amine 8 to afford the target radiolabeled compound
14
LY368842-[indole- C], and subsequently its glycolate (Scheme 3).
Scheme 2.
The method described above allowed us to obtain LY368842-
1
indole- C] glycolate with 98.4% radiochemical purity and a specific
4
[
activity of 60.4 mCi/mg in six steps from commercially available p-
1
4
chlorophenoxymethyl chloride and sodium cyanide-[ C] in total 20%
chemical and 17.4% radiochemical yields.
Experimental
14
The sodium cyanide-[ C] was purchased from American Radiolabeled
Chemicals Inc. The NMR spectra were obtained on a Varian
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 1143–1152

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CARBON-14 LABELLED LY368842-[INDOLE- C] GLYCOLATE
1147
Scheme 3.
1
13
MercuryÀ400 at 400 ( H) and 100 ( C) MHz. Chemical shifts are
reported in parts per million (ppm) downfield from tetramethylsilane.
1
4
Flash chromatography was performed as described by Still et al.,
using silica gel 60 (230–400 mesh). Unless otherwise noted, HPLC was
conducted on a Hitachi instrument with UV detection at 230 nm; a
Zorbax C8 column (4.6 mm  25 cm); isocratic elution with a mobile
phase consisting of 50% aqueous 0.5% monobasic ammonium
phosphate and 50% acetonitrile at a flow rate of 1 ml/min. TLC was
performed on precoated plates of silica gel 60 F254
.
1
4
2
-(4-Chlorophenoxy)acetonitrile-[1- C], 2
To a solution of a,4-dichloroanisole (1) (685 mg, 3.87 mmol) in acetone
1
4
(
4 ml) was added sodium cyanide-[ C] (150 mCi, 55 mCi/mmol,
2
3
.73 mmol). The reaction mixture was heated at 60–658C (bath) for
0 min whereupon sodium cyanide (56 mg, 1.14 mmol) was added, and
the mixture was further heated at the same temperature for 5.5 h. After
cooling to room temperature the mixture was evaporated under
vacuum. The residue was diluted with water (3 ml) and extracted with
ethyl ether (30 ml). The extract was washed with saturated aqueous
sodium chloride (2 Â 5 ml). The combined aqueous layer was re-
extracted with ethyl ether (10 ml). The combined organic extract was
dried over sodium sulfate, and evaporated under vacuum. The residue
was subjected to flash chromatography, eluting with ethyl acetate/
hexanes (15:85) to give nitrile 2 (428 mg, 65%) as a colorless oil. TLC:
Copyright # 2002 John Wiley & Sons, Ltd.
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148
B. A. CZESKIS ET AL.
R = 0.39 (20% of ethyl acetate in hexanes). The compound co-eluted
f
with an authentic sample of 2-(4-chlorophenoxy)acetonitrile using the
above TLC conditions. For non-radioactive compound (prepared
1
in model experiment): H-NMR (CDCl , d, ppm): 4.71 (s, 2 H), 6.88
3
(d, J = 8.8 Hz, 2 H), 7.27 (d, J = 8.8 Hz, 2 H).
O-Benzyl-3-nitrophenol, 3
A mixture of 3-nitrophenol (4.0 g, 28.8 mmol), benzyl bromide (3.8 ml,
3
1.9 mmol), and potassium carbonate (12.0 g, 86.8 mmol) in acetone
(55 ml) was stirred at room temperature for 16 h, and evaporated under
vacuum. The residue was diluted with water (20 ml) and extracted with
ethyl ether (50 ml). The extract was washed with saturated aqueous
sodium chloride (2 Â 10 ml), dried over sodium sulfate, and evaporated
under vacuum. The residue was subjected to flash chromatography,
eluting with ethyl acetate/hexanes (10:90) to give benzyl ether 3 (6.29 g,
9
5%) as a light yellow solid. TLC: R = 0.40 (10% of ethyl acetate in
f
1
hexanes). H-NMR (CDCl , d, ppm): 5.10 (s, 2 H), 7.22–7.42 (m, 7 H),
3
7
.77–7.81 (m, 2 H).
1
-(2-Benzyloxy-6-nitrophenyl)acetonitrile-[1- C], 4
4
2
To a solution of potassium tert-butoxide (597 mg, 5.32 mmol) in
dimethylformamide (5 ml) at 0–58C (ice bath) was added a solution of
phenoxyacetonitrile 2 (428 mg, 2.53 mmol) and nitroarene 3 (610 mg,
2
.66 mmol) in dimethylformamide (2.3 ml). The dark blue reaction
mixture was stirred for 1 h in an ice bath, poured onto a mixture of
aqueous hydrochloric acid (1N, 11 ml) and ice (ca. 2 g), and extracted
with ethyl acetate (20 ml  3). The extract was washed with saturated
aqueous sodium chloride (2 Â 10 ml), and the combined aqueous layer
was re-extracted with ethyl acetate (15 ml). The combined organic
extract was dried over sodium sulfate, and evaporated under vacuum.
The residue was subjected to flash chromatography, eluting with ethyl
acetate/hexanes (30:70) to give arylacetonitrile-4 (459 mg, 67%) as a
light yellow solid. TLC: R = 0.34 (30% of ethyl acetate in hexanes).
f
The compound co-eluted with an authentic sample of 2-(2-benzyloxy-6-
nitrophenyl)acetonitrile by HPLC and TLC under the above conditions.
11b
For non-radioactive compound
(prepared in model experiment):
1
H-NMR (CDCl , d, ppm): 4.00 (s, 2 H), 5.19 (s, 2 H), 7.21–7.44
3
(m, 7 H), 7.62 (dd, J = 8.3 and 1.0 Hz, 1 H).
Copyright # 2002 John Wiley & Sons, Ltd.
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CARBON-14 LABELLED LY368842-[INDOLE- C] GLYCOLATE
1149
1
4
4
-Hydroxyindole-[2- C], 5
To a solution of nitrophenylacetonitrile 4 (459 mg, 1.7 mmol) in a
mixture of ethanol (9.0 ml), acetic acid (1.4 ml) and ethyl acetate (2.5 ml)
was added 10% palladium on carbon (46 mg). The resulting suspension
was evacuated and re-filled with hydrogen gas 3 times, followed by
stirring under an atmosphere of hydrogen (balloon) for 18 h. The
reaction mixture was diluted with ethyl acetate (15 ml), filtered through
Hyflo, and evaporated under vacuum. The residue was subjected to
flash chromatography, eluting with ethyl acetate/hexanes (30:70) to give
1
4
4
-hydroxyindole-[2- C] (5) (177 mg, 77%) as a light yellow solid. TLC:
R = 0.32 (30% of ethyl acetate in hexanes). The compound co-eluted
f
with an authentic sample of 4-hydroxyindole by HPLC and TLC under
the above conditions. For non-radioactive compound (prepared in
1
model experiment): H-NMR (CDCl , d, ppm): 4.97 (br. s, 1 H), 6.48
3
(dd, J = 7.3 and 1.0 Hz, 1 H), 6.56 (m, 1 H), 6.95–7.03 (m, 2 H), 7.11
(m, 1 H), 8.13 (br. s, 1 H).
1
4
(
S)-4-(2,3-Epoxypropyloxy)indole-[2- C], 7
14
To a solution of 4-hydroxyindole-[2- C] (5) (177 mg, 1.31 mmol) in
acetone (12 ml) were added nosylate 6 (400 mg, 1.54 mmol) and
potassium carbonate (530 mg, 3.83 mmol). The reaction mixture was
heated at 60–658C (bath) for 20 h, diluted with ethyl acetate (15 ml),
filtered through Hyflo, and evaporated under vacuum. The residue
was subjected to flash chromatography, eluting with ethyl acetate/
hexanes (30:70) to give epoxide 7 (186 mg, 74%) as a white solid. TLC:
R = 0.43 (30% of ethyl acetate in hexanes). The compound co-eluted
f
with an authentic sample of (S)-4-(2,3-epoxypropyloxy)indole by HPLC
and TLC under the above conditions. For non-radioactive compound
1
(
prepared in model experiment): H-NMR (CDCl , d, ppm): 2.78 (dd, J
3
=
4.9 and 2.9 Hz, 1 H), 2.90 (m, 1 H), 3.42 (m, 1 H), 4.12 (dd, J = 11.2
and 5.6 Hz, 1 H), 4.31 (dd, J = 11.2 and 3.4 Hz, 1 H), 6.49 (m, 1 H), 6.65
m, 1 H), 6.97–7.11 (m, 3 H), 8.14 (br. s, 1 H).
(
14
(
methylpropyl]phenoxy]pyridine-5-carboxamide,
S)-2-[4-[2-[3-(Indol-2-[ C]-4-yloxy)-2-hydroxypropylamino]-2-
1
4
(
LY368842-[indole- C])
To a mixture of epoxide 7 (186 mg, 0.97 mmol) in 2-propanol (9 ml) was
added 2-[4-(2-amino-2-methylpropyl)phenoxy]pyridine-5-carboxamide
Copyright # 2002 John Wiley & Sons, Ltd.
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150
B. A. CZESKIS ET AL.
4
8) (330 mg, 1.16 mmol) in one portion. The reaction mixture was
(
heated at 80–838C for 16 h and evaporated under vacuum. The
residue was subjected to flash chromatography, eluting with one
column volume of chloroform/methanol (90:10) following by
three
column
volumes
of
chloroform/methanol/ammonium
1
hydroxide (90:10:1) to give LY368842-[indole- C] (378 mg, 82%)
4
as a pale solid. TLC: R = 0.31 (chloroform/methanol/ammonium
f
hydroxide 90:10:1). The compound co-eluted with an authentic
sample of LY368842 by HPLC and TLC under the above conditions.
For non-radioactive compound (prepared in model experiment):
1
H-NMR (CD OD, d, ppm): 1.10 (d, J = 3.4 Hz, 6 H), 2.75 (d, J =
3
5
.4 Hz, 2 H), 2.85 (m, 1 H), 2.99 (m, 1 H), 4.09 (m, 3 H), 6.45 (dd, J =
4
.9 and 3.4 Hz, 1 H), 6.49 (d, J = 3.4 Hz, 1 H), 6.88–6.94 (m, 5 H), 7.05
(d, J = 2.9 Hz, 1 H), 7.23 (d, J = 8.8 Hz, 2 H), 8.18 (dd, J = 8.8 and
2
.4 Hz, 1 H), 8.57 (d, J = 2.4 Hz).
14
(
methylpropyl]phenoxy]pyridine-5-carboxamide glycolate,
S)-2-[4-[2-[3-(Indol-2-[ C]-4-yloxy)-2-hydroxypropylamino]-2-
1
4
(
LY368842-[indole- C]) glycolate
1
4
To a solution of free base LY368842-[indole- C] (215 mg, 0.45 mmol) in
ethanol (1.4 ml) was added a solution of glycolic acid (35 mg,
0
3
.46 mmol) in ethanol (1.0 ml). The reaction mixture was stirred for
0 min at room temperature and evaporated under vacuum. The residue
was triturated with ethyl ether (3 Â 10 ml), and the supernatant liquid
was decanted each time. After drying under vacuum, LY368842-
1
4
indole- C] glycolate (246 mg, 99%) was obtained as a white solid. The
[
radiochemical purity was determined to be 98.4% by radio-HPLC:
Zorbax SB-Phenyl column (4.6 Â 250 mm); flow rate: 1.0 ml/min;
column temperature: 408C; detection: UV 260 nm; mobile phase:
methanol/0.1% trifluoroacetic acid. The specific activity of the product
was 60.4 mCi/mg. The compound co-eluted with an authentic sample of
LY368842 glycolate by HPLC under the above conditions. For non-
1
radioactive compound (prepared in model experiment): H-NMR
(
(
DMSO-d , d, ppm): 1.01 (d, J = 3.4 Hz, 6 H), 2.70 (s, 2 H), 2.82
m, 1 H), 2.95 (m, 1 H), 3.72 (s, 2 H), 4.01 (m, 3 H), 6.38–6.43 (m, 2 H),
6
6.88–6.99 (m, 5 H), 7.14 (m, 1 H), 7.18 (d, J = 8.8 Hz, 2 H), 7.42 (s, 1 H),
7.97 (s, 1 H), 8.18 (dd, J = 8.3 and 2.4 Hz, 1 H), 8.56 (d, J = 2.4 Hz,
1 H), 11.00 (s, 1 H).
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CARBON-14 LABELLED LY368842-[INDOLE- C] GLYCOLATE
1151
Acknowledgements
The authors are grateful to Dr Mary Peters of Chemical Process
Research and Development for providing a sample of amine 8 as well as
the authentic non-radioactive samples of epoxide 7 and final material
LY368842 glycolate.
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