Synthesis of 14C-labeled and tritiated AMPA potentiator LY450108

Article abstract of DOI:10.1002/jlcr.899

Asymmetric synthesis of AMPA potentiator LY450108-[¹⁴C] containing ¹⁴C-label attached to the chiral center of the molecule, was accomplished based on Evans' chiral oxazolidinone auxiliary method. Diastereoselective methylation of p-n

Full text of DOI:10.1002/jlcr.899

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 85–100.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.899
Research Article
14
Synthesis of C-labeled and tritiated AMPA
potentiator LY450108
Boris A. Czeskis*, Douglas D. O’Bannon, William J. Wheeler and
Dean K. Clodfelter
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
Summary
Asymmetric synthesis of AMPA potentiator LY450108-[14C] containing 14C-label
attached to the chiral center of the molecule, was accomplished based on Evans’ chiral
oxazolidinone auxiliary method. Diastereoselective methylation of p-nitrophenylacetic
acid derivative was used as a key step. The auxiliary was reductively removed, and the
resulting primary alcohol was converted into the corresponding amine. Its
sulfonylation, reduction of the aromatic nitro group, and acylation with 3,5-
difluorobenzoyl chloride led to the final product. The synthesis of tritiated LY450108
is also detailed. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: AMPA potentiator; LY450108; asymmetric synthesis; carbon-14 labeled
Introduction
Glutamic acid is the major excitatory neurotransmitter in the central nervous
system. It interacts with multiple subtypes of excitatory amino acid receptors
like 2-amino-3-(5-methyl-3-hydroxyisoxazol-4-yl)propanoic acid (AMPA)
receptors. Signals are transformed at AMPA receptors through conductance
of sodium and calcium ions into cells upon activation by glutamic acid.
AMPA potentiators are compounds that enhance ion influx through AMPA
receptors. These compounds are important for learning and memory processes
and could be used in the development of pharmacological agents for the
treatment of cognitive disorders including Alzheimer’s disease and schizo-
phrenia.1 In the process of conducting non-clinical ADME studies, tritium and
C-14 labeled LY4501082 were required.
We report herein the asymmetric synthesis of this compound, which allowed
the introduction of the 14C label in a metabolically stable position, next to the
*Correspondence to: B. A. Czeskis, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis,
IN 46285, USA. E-mail: czeskis@lilly.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 27 August 2004
Accepted 9 September 2004

86
B. A. CZESKIS ET AL.
chiral center. In addition, we report on the iridium catalyzed tritiation of
LY450108 using Crabtree’s catalyst.
H
N
O
O
S
O
F
N
H
F
LY450108
Results and discussion
In order to generate the chiral fragment of the molecule we decided to use
Evans’ chiral oxazolidinone auxiliary approach.3 In the first step (Scheme 1),
acylation of the lithium derivative of (R)-(+)-4-benzyl-2-oxazolidinone with a
mixed anhydride generated in situ from p-nitrophenylacetic acid (1) and
pivaloyl chloride, resulted in less than 20% yield of the desired amide 3 both
under standard4 and recently modified5 conditions. Preparation of the acyl
chloride from the acid 1 and oxalyl chloride was even less efficient. The best
reproducible result was obtained when the acid 1 was initially converted to
p-nitrophenylacetyl fluoride (2) using cyanuric fluoride.6
O
O
O
O
O2N
HN
O
Bn
Me3CCOCl,
Et3N
n-BuLi
O
O
N
CO2H
Bn
O
O2N
O2N
3
1
F
O
N
N
HN
, Py
F
O
F
F
N
Bn
n-BuLi
O
O2N
2
Scheme 1.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 85–100

SYNTHESIS OF 14C-
87
The key step was the alkylation of enolate derived from amide 3 with methyl
iodide (Scheme 2). The best experimental conditions in our hands consisted of
the addition of sodium hexamethyldisilazide to a mixture of equimolar
amounts of 3 and methyl iodide. According to HPLC and NMR data only one
stereoisomer 4 was formed, which was evident after a mixture of both isomers
4 and 5 was prepared in a separate experiment from the racemic 2-(4-
nitrophenyl)-propionic acid. The diastereomers 4 and 5 appeared to be
separable on silica gel, and one of them (less polar) corresponded to the above
compound 4, the absolute (R)-stereochemistry of which was proven by the
comparison of optical rotations of the final product LY450108 and authentic
sample of this compound.
In an attempt to increase the yield in the asymmetric alkylation step we
decided to replace the nitro group of 3 with dibenzylamino group in order to
reduce the possibility of decomposition of N-acylated oxazolidinone through
ketene formation under basic conditions.5 The target substrate 9 was prepared
as shown on Scheme 3 by the bis-benzylation of the 4-aminophenylacetic acid
(6), subsequent hydrolysis of ester 7, and acylation of the lithium derivative of
(R)-(+)-4-benzyl-2-oxazolidinone with the acid chloride prepared in situ from
8 and oxalyl chloride.
The dibenzyl derivative 9 unexpectedly appeared to be extremely unstable
under the deprotonation conditions in the presence of sodium hexamethyldi-
silazide. After the addition of methyl iodide, neither starting material nor
desired alkylation product were found in the complex reaction mixture.
Therefore the nitro compound 4 was used in the preparation of the target
molecule LY450108.
O
O
O
O
N
MeI
N
Bn
O
NaHMDS
O2N
Bn
O
O2N
4
3
O
O
1.Me3CCOCl
N
CO2H
4 +
O
Bn
O
2.
O2N
O2N
LiN
O
5
Bn
Scheme 2.
Copyright # 2005 John Wiley & Sons, Ltd.
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88
B. A. CZESKIS ET AL.
CO2Bn
BnBr
CO2H
K2CO3
Bn2N
H2N
7
6
NaOH,
THF/MeOH
O
1. (COCl)2,
DMF, CH2Cl2
O
N
CO2H
O
2.
Bn
O
Bn2N
Bn2N
LiN
O
8
9
Bn
Scheme 3.
After an unsuccessful attempt of direct transamination of the oxazolidinone
4 with ammonium chloride and trimethylaluminum,7 the reductive removal of
the chiral auxiliary was accomplished using sodium borohydride8 to give the
primary alcohol 10 in a good yield. Conversion of 10 to the corresponding
primary amine 13 was performed in two alternative ways. Firstly initial
formation of the azide 11 using Mitsunobu reaction with hydrazoic acid9 or
diphenylphosphoryl azide (DPPA),10 and subsequent chemoselective reduc-
tion of 11 with triphenylphosphine11 or sodium borohydride in the presence of
copper (II) sulfate.12 The second, and more convenient approach, consisted of
the reaction of the alcohol 10 with phthalimide under Mitsunobu conditions
followed by hydrazinolysis9 of the N-substituted imide 12 (Scheme 4).
Conversion of amine 13 into the final product LY450108 was accomplished
in three straightforward steps: sulfonylation with isopropylsulfonyl chloride,
catalytic hydrogenation of nitro compound 14, and acylation of aniline 15
with 3,5-difluorobenzoyl chloride (Scheme 5). The optical rotation of the
compound thus obtained was exactly the same as the rotation of an authentic
sample of LY450108, which was prepared by resolution of a b-methylphe-
nethylamine salt, and subsequent nitration of the aromatic ring.
The synthesis of radiolabeled product LY450108-[14C] (Scheme 6) was
accomplished according to the sequence discussed above. Thus, diastereose-
lective alkylation of N-acyloxazolidinone 3 with methyl iodide-[14C] smoothly
gave methylated product 4a with modest but expected yield. Sodium
borohydride reduction of 4a afforded the primary alcohol 10a, which was
converted by Mitzunobu reaction into the substituted phthalimide 12a.
Primary amine 13a was obtained after treatment of 12a with hydrazine.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 85–100

SYNTHESIS OF 14C-
89
O
O
N
N3
Bn
O
O2N
O2N
4
Ph3P, H2O or
NaBH4, CuSO4
11
HN3 or
DPPA
NaBH4,
THF/H2O
Ph3P,
DEAD
NH2
OH
O2N
O2N
10
13
Phthalimide,
Ph3P, DEAD
NH2NH2
O
N
O
O2N
12
Scheme 4.
Reaction of 13a with isopropylsulfonyl chloride led to sulfonamide 14a, which
was subjected to catalytic hydrogenation to give arylamine 15a. Finally,
acylation of 15a with 3,5-difluorobenzoyl chloride afforded the target
compound LY450108-[14C].
The final product LY450108-[14C] thus obtained had the radiochemical
purity of 99.4% and optical purity 99.8% (according to chiral HPLC). Its
overall radiochemical yield was 17%, which exactly corresponds to the total
chemical yield from methyl iodide-[14C].
LY450108-[3H] was prepared by tritium exchange labeling on LY450108 in
the presence of [(cod)Ir(py)(Pcy3)]PF6 (Crabtree’s catalyst,13 Scheme 7).y The
LY450108-[3H] had a specific activity of 96 Ci/mmol and a radiochemical
purity of 98.8% as determined by HPLC. Analysis of the product by 3H-NMR
y The tritiation of LY450108 was conducted at Amersham Biosciences, Cardiff, Wales, UK using
methodology provided by the authors.
Copyright # 2005 John Wiley & Sons, Ltd.
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90
B. A. CZESKIS ET AL.
H
N
NH2
i-PrSO2Cl
Et3N
SO2
O2N
O2N
14
13
H2, Pd/C
H
N
SO2
H
N
3,5-F2C6H3COCl
Et3N
SO2
HN
F
H2N
O
15
LY450108
F
Scheme 5.
(in CDCl3) showed resonances at d 7.50 and 7.75 ppm indicating tritia in the
3,5, 20 and 60 positions.
Experimental
The methyl iodide-[14C] was purchased from Amersham Pharmacia Biotech.
The Crabtree’s catalyst was obtained from Strem Chemicals. The NMR
spectra were obtained in CDCl3 on a Varian Mercury-400 at 400 (1H) and 100
(13C) MHz. Chemical shifts are reported in parts per million (ppm) downfield
from tetramethylsilane. Optical rotations, IR, MS, and microanalytical data
were provided by the Physical Chemistry Department of the Lilly Research
Laboratories. Flash chromatography was performed on silica gel 60 (230–400
mesh). TLC was conducted on precoated plates of silica gel 60 F254. All 14C-
compounds were identified by TLC comparison with the corresponding non-
radiolabeled isotopomers. The radiochemical purity of the final material
LY450108-[14C] was determined by radio-HPLC: Zorbax SB-phenyl,
150 ꢀ 4.6 mm; mobile phase: 0.05% aqueous trifluoroacetic acid/acetonitrile
3:2; flow rate: 1 ml/min; UV detector wavelength: 225 nm.Its optical purity was
determined by chiral radio-HPLC: Chiracel OD-H 250 ꢀ 4.6 mm; mobile
phase: ethanol/hexane 1:9; flow rate 1 ml/min; UV detector wavelength:
275 nm; column temperature 40 8C. The radiochemical purity of LY450108-
[3H] was determined by radio-HPLC: Spherosorb Phenyl (5m, 4.6 ꢀ 250 mm)
with gradient elution at 1.0 ml/min (Solvent A ¼ 0:1% TFA/water, Solvent
B ¼ 0:1% TFA/CH3CN) from 20% B to 100% B over 30 min and
simultaneous radiochemical and UV (at 225 nm) detection.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 85–100

SYNTHESIS OF 14C-
91
O
O
14CH3
O
O
N
14CH3I
N
Bn
O
NaHMDS
O2N
Bn
O
34%
O2N
3
4a
NaBH4,
THF/H2O
87%
O
14CH3
14CH3
Phthalimide,
Ph3P, DEAD
OH
N
90%
O
O2N
O2N
10a
12a
NH2NH2
14CH3
94%
14CH3
H
NH2
N
i-PrSO2Cl
SO2
Et3N
85%
O2N
O2N
13a
14a
14CH3
H2, Pd/C
84%
H
N
14CH3
SO2
H
N
3,5-F2C6H3COCl
Et3N
SO2
HN
F
H2N
O
94%
15a
LY450108-[14C]
F
Scheme 6.
4-Nitrophenylacetyl fluoride, 2
To a solution of 1 (4.29 g, 23.7 mmol) and pyridine (2.6 ml, 32 mmol) in
acetonitrile (40 ml) was added cyanuric fluoride (2.0 ml, 23.7 mmol) dropwise.
The reaction mixture was stirred at room temperature for 3.5 h, and
evaporated under vacuum. The residual solid was triturated with ethyl ether
(50–60 ml) and filtered. The filtrate was washed with ice water (ꢁ5 ml), dried
over magnesium sulfate, and evaporated under vacuum to give 2 (4.183 g,
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92
B. A. CZESKIS ET AL.
H
N
O
S
O2
F
N
H
LY450108
F
T2/CH2Cl2
[(cod)Ir(py)(PCy3)]PF6
H
N
3H
3H
O
S
O2
F
N
H
3H
3H
F
LY450108-[3H]
Scheme 7.
1
96%) as a light yellow solid. H-NMR (d, ppm): 3.95 (s, 2 H), 7.49 (d,
J ¼ 8:3 Hz, 2 H), 8.25 (d, J ¼ 8:3 Hz, 2 H).
4R-Benzyl-3-(4-nitrophenylacetyl)-2-oxazolidinone, 3
To a solution of (R)-(+)-4-benzyl-2-oxazolidinone (1.88 g, 10.61 mmol) and
triphenylmethane (ꢁ20 mg) in tetrahydrofuran (22 ml) at ꢂ788C was added n-
butyllithium (1.6 M in hexane) until yellowish color persisted (ca. 7 ml). After
10 minutes a solution of 2 (1.94 g, 10.59 mmol) in tetrahydrofuran (8 ml) was
added through cannula. The reaction mixture was stirred at ꢂ78 8C for 1 h,
then diluted with saturated aqueous sodium bisulfate (10–13 ml), and
extracted with ethyl acetate (ca. 50 ml). The extract was washed with brine
(2 ꢀ 5 ml), dried over sodium sulfate, and evaporated under vacuum. Flash
chromatography of the residue (column 5 ꢀ 27 cm, eluting with ethyl acetate/
hexanes, 2:3, ca. 2000 ml) gave 3 (1.233 g, 34%) as a yellow solid, Rf ¼ 0:39
(ethyl acetate/hexanes, 2:3). 1H-NMR (d, ppm): 2.77 (dd, J ¼ 13:2 and 9.3 Hz,
1 H), 3.28 (dd, J ¼ 13:2 and 3.4 Hz, 1 H), 4.20–4.27 (m, 2 H), 4.40 (AB dd,
J ¼ 35:7 and 15.6 Hz, 2 H), 4.69 (m, 1 H), 7.15 (d, J ¼ 7:8 Hz, 2 H), 7.26–7.33
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 85–100

SYNTHESIS OF 14C-
93
(m, 3 H), 7.50 (d, J ¼ 8:3 Hz, 2 H), 8.22 (d, J ¼ 8:3 Hz, 2 H). IR (KBr, n,
cmꢂ1): 744, 1196, 1347, 1384, 1520, 1695, 1788. UV (EtOH, l, nm): 268 (e
9784). MS (ESI, %): 340 (M+, 20), 339 (1 0 0), 194 (13), 162 (42). Analytically
Calculated for C18H16N2O5: C, 63.53; H, 4.74; N, 8.23; Found: C, 63.71; H,
4.72; N, 8.01.
4R-Benzyl-3-(2R-(4-nitrophenyl)propionyl)-2-oxazolidinone, 4
To a solution of 3 (681 mg, 2.0 mmol) in tetrahydrofuran (12 ml) was added a
solution of methyl iodide (283 mg, 1.99 mmol) in toluene (2 ml). The mixture
was cooled to ꢂ78 8C and a solution of sodium bis(trimethylsilyl)amide (1 M
in tetrahydrofuran, 2.0 ml, 2.0 mmol) was added dropwise. The resultant dark
red mixture was stirred for 15 min at ca. ꢂ70 8C and allowed to reach room
temperature. After 3.5 h the reaction mixture was diluted with saturated
aqueous sodium bisulfate (ca. 10 ml), and extracted with ethyl acetate (ca.
50 ml). The extract was washed with brine (2 ꢀ 10 ml), dried over sodium
sulfate, and evaporated under vacuum. Flash chromatography of the residue
(column 3 ꢀ 30 cm, eluting with ethyl acetate/hexanes, 3:7, ca. 500 ml) gave 4
(230 mg, 33%) as a light yellow solid, Rf ¼ 0:49 (ethyl acetate/hexanes, 3:7).
1H-NMR (d, ppm): 1.57 (d, J ¼ 6:8 Hz, 3H), 2.81 (dd, J ¼ 13:2 and 9.3 Hz,
1H), 3.34 (dd, J ¼ 13:2 and 3.4 Hz, 1H), 4.09–4.17 (m, 2H), 4.62 (m, 1H), 5.22
(q, J ¼ 6:8 Hz, 1H), 7.21 (d, J ¼ 7:8 Hz, 2H), 7.29–7.37 (m, 3H), 7.54 (d,
J ¼ 8:3 Hz, 2H), 8.18 (d, J ¼ 8:3 Hz, 2H).
4R-Benzyl-3-(2R-(4-nitrophenyl)propionyl-3-[14C])-2-oxazolidinone, 4a
In the same manner as described above, starting from 2 (590 mg, 1.734 mmol)
in tetrahydrofuran (10 ml), methyl iodide-[14C] (100 mCi, 58 mCi/mmol,
1.724 mmol) in toluene (2 ml), and sodium bis(trimethylsilyl)amide (1 M in
tetrahydrofuran, 1.73 ml, 1.73 mmol), 4a (195 mg, 32%) was obtained.
The repetition of the same experimental procedure with the same amounts
of the reagents gave the second lot of 4a (209 mg, 34%). The two lots were
combined and repurified by flash chromatography (column 3 ꢀ 30 cm, eluting
with ethyl acetate/hexanes, 25:75, ca. 500 ml) to obtain 4a (400 mg), which was
used in the next step.
4R-Benzyl-3-(2R-(4-nitrophenyl)propionyl)-2-oxazolidinone,4, and 4R-benzyl-
1-(2S-(4-nitrophenyl)propionyl)-2-oxazolidinone, 5
To a solution of 2-(4-nitrophenyl)propionic acid (390 mg, 2.0 mmol) and
triethylamine (335 ml, 2.4 mmol) in tetrahydrofuran (30 ml) at ꢂ78 8C was
added trimethylacetyl chloride (280 ml, 2.27 mmol) dropwise. After 10 mins the
reaction mixture was allowed to reach 0–5 8C and cooled back to ꢂ78 8C. In a
separate flask, to a solution of (R)-(+)-4-benzyl-2-oxazolidinone (354 mg,
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94
B. A. CZESKIS ET AL.
2.0 mmol) and triphenylmethane (ꢁ10 mg) in tetrahydrofuran (8 ml) at
ꢂ78 8C was added n-butyllithium (1.6 M in hexane) until yellowish color
persisted (ca. 1.3 ml). After 10 mins the resulting solution was transferred
through a cannula into the suspension of mixed anhydride prepared as
described above. The reaction mixture was allowed to reach ca. ꢂ20 8C over
the period of 30 minutes, then diluted with saturated aqueous sodium bisulfate
(ca. 10 ml), and extracted with ethyl acetate (ca. 50 ml). The extract
was washed with brine (2 ꢀ 10 ml), dried over sodium sulfate, and evaporated
under vacuum. Flash chromatography of the residue (column 3 ꢀ 30 cm,
eluting with ethyl acetate/hexanes, 3:7, ca. 500 ml followed by ethyl acetate/
hexanes, 2:3, ca. 500 ml) gave 4 (169 mg, 24%), Rf ¼ 0:49 (ethyl acetate/
hexanes, 3:7), and 5 (224 mg, 32%), Rf ¼ 0:34 (ethyl acetate/hexanes, 3:7).
1
1
For 4: H-NMR is the same as described above. For 5: H-NMR (d, ppm):
1.55 (d, J ¼ 6:8 Hz, 3H), 2.59 (dd, J ¼ 13:2 and 9.3 Hz, 1H), 3.14 (dd, J ¼ 13:2
and 3.4 Hz, 1H), 4.14 (m, 1H), 4.23 (m, 1H), 4.74 (m, 1H), 5.19 (q, J ¼ 6:8 Hz,
1H), 7.02 (m, 2H), 7.21–7.26 (m, 3H), 7.59 (d, J ¼ 8:8 Hz, 2H), 8.21
(d, J ¼ 8:8 Hz, 2H).
Benzyl 4-(dibenzylamino)phenylacetate, 7
To a mixture of 4-aminophenylacetic acid (6) (1.51 g, 10.0 mmol) and
potassium carbonate (14.0 g, 101 mmol) in dimethylformamide (30 ml) was
added benzyl bromide (4.0 ml, 33.6 mmol). The reaction mixture was stirred
for 16 h at room temperature, then diluted with water (20 ml), and extracted
with ethyl acetate (ca. 50 ml). The extract was washed with brine (2 ꢀ 10 ml),
dried over sodium sulfate, and evaporated under vacuum. Flash chromato-
graphy of the residue (column 3 ꢀ 30 cm, eluting with ethyl acetate/hexanes,
1:9, ca. 500 ml) gave 7 (3.772 g, 88%) as a yellow solid, Rf ¼ 0:42 (ethyl
1
acetate/hexanes, 1:9). H-NMR (d, ppm): 3.54 (s, 2H), 4.64 (s, 4H), 5.11 (s,
2H), 6.69 (d, J ¼ 8:6 Hz, 2H), 7.08 (d, J ¼ 8:6 Hz, 2H), 7.24–7.34 (m, 15H). IR
(KBr, n, cmꢂ1): 1152, 1453, 1495, 1522, 1617, 1730. UV (EtOH, l, nm): 261 (e
25422). MS (ESI, %): 422 (M+, 100). Analytically Calculated for C29H27NO2:
C, 82.63; H, 6.46; N, 3.32; Found: C, 82.76; H, 6.50; N, 3.38.
4-(Dibenzylamino)phenylacetic acid, 8
A mixture of 7 (1.0 g, 2.37 mmol) and aqueous sodium hydroxide (1 N, 5 ml,
5.0 mmol) in tetrahydrofuran (7 ml) and methanol (3 ml) was stirred at room
temperature for 3 h, then acidified with aqueous hydrochloric acid (5 N,
5.2 ml), and extracted with ethyl acetate (ca. 40 ml). The extract was washed
with brine (2 ꢀ 5 ml), dried over magnesium sulfate, and evaporated under
vacuum to give a mixture of 8 with benzyl alcohol, which was removed by the
azeotropic distillation with water (2 ꢀ 5 ml). The residual water, in turn, was
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF 14C-
95
removed by the azeotropic distillation with toluene (2 ꢀ 3 ml) to obtain 8
1
(527 mg, 67%) as a colorless solid, Rf ¼ 0:45 (ethyl acetate/hexanes, 1:1). H-
NMR (d, ppm): 3.52 (s, 2H), 4.64 (s, 4H), 6.69 (d, J ¼ 8:6 Hz, 2H), 7.07 (d,
J ¼ 8:6 Hz, 2H), 7.23-7.34 (m, 10H).
4R-Benzyl-3-(4-(dibenzylamino)phenylacetyl)-2-oxazolidinone, 9
To a solution of 8 (225 mg, 0.68 mmol) and dimethylformamide (1 drop) in
dichloromethane (5 ml) at 0–5 8C was added oxalyl chloride (180 ml,
2.06 mmol) dropwise. The mixture was allowed to reach room temperature,
stirred for 20 min, evaporated under vacuum, and re-evaporated with toluene
(1 ml) to obtain the acid chloride. In a separate flask, to a solution of (R)-(+)-
4-benzyl-2-oxazolidinone (120 mg, 0.68 mmol) and triphenylmethane (ꢁ5 mg)
in tetrahydrofuran (2 ml) at ꢂ78 8C was added n-butyllithium (1.6 M in
hexane) until yellowish color persisted (ca. 0.6 ml). After 10 mins a solution of
the acid chloride in tetrahydrofuran (1.5 ml) was added through a cannula.
The reaction mixture was allowed to reach room temperature over the period
of 1 h, then diluted with saturated aqueous sodium bisulfate (3 ml), and
extracted with ethyl acetate (ca. 20 ml). The extract was washed with brine
(2 ꢀ 3 ml), dried over sodium sulfate, and evaporated under vacuum. Flash
chromatography of the residue (column 3 ꢀ 25 cm, eluting with ethyl acetate/
hexanes, 3:7, ca. 500 ml) gave 9 (190 mg, 57%) as a light yellow solid, Rf ¼
1
0:45 (ethyl acetate/hexanes, 3:7). H-NMR (d, ppm): 2.68 (dd, J ¼ 13:3 and
9.3 Hz, 1H), 3.17 (dd, J ¼ 13:3 and 3.2 Hz), 4.04–4.15 (m, 4H), 4.58 (m, 5H),
6.64 (d, J ¼ 8:5 Hz, 2H), 7.06 (d, J ¼ 8:5 Hz, 4H), 7.15–7.27 (m, 13H). IR
(KBr, n, cmꢂ1): 744, 1196, 1347, 1384, 1520, 1695, 1788. UV (EtOH, l, nm):
268 (e 9784). MS (ESI, %): 340 (M+, 21), 339 (1 0 0), 194 (13), 162 (42).
Analytically Calculated for: C18H16N2O5: C, 63.53; H, 4.74; N, 8.23; Found:
C, 63.71; H, 4.72; N, 8.01.
R-2-(4-Nitrophenyl)-1-propanol, 10
To a solution of 4 (229 mg, 0.646 mmol) in tetrahydrofuran (2 ml) was added a
solution of sodium borohydride (100 mg, 2.64 mmol) in water (0.7 ml). The
reaction mixture was stirred for 3 h at room temperature. The excess of
hydride was destroyed by slow addition of aqueous hydrochloric acid (1 N, ca.
2.9 ml). The mixture was further diluted with water (1 ml) and extracted with
ethyl acetate (ca. 30 ml). The extract was washed with brine (2 ꢀ 5 ml), dried
over sodium sulfate, and evaporated under vacuum. Flash chromatography of
the residue (column 3 ꢀ 20 cm, eluting with ethyl acetate/hexanes, 1:1, ca.
600 ml) gave 10 (107 mg, 91%) as a light yellow oil, Rf ¼ 0:43 (ethyl acetate/
hexanes, 1:1). 1H-NMR (d, ppm): 1.32 (d, J ¼ 7:3 Hz, 3H), 3.09 (m, 1H), 3.78
(m, 2H), 7.42 (d, J ¼ 8:8 Hz, 2H), 8.19 (d, J ¼ 8:8 Hz, 2H).
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R-2-(4-Nitrophenyl)-1-propanol-3-[14C], 10a
In the same manner as described above, starting from 4a (400 mg, 1.12 mmol)
in tetrahydrofuran (3.5 ml), and sodium borohydride (173 mg, 4.57 mmol) in
water (1.2 ml), 10a (179 mg, 87%) was obtained.
R-2-(4-Nitrophenyl)-1-propyl azide, 11
Using DPPA. To a solution of 10 (200 mg, 1.1 mmol) and triphenylphosphine
(304 mg, 1.16 mmol) in tetrahydrofuran (3 ml) at 0–5 8C was added diethyla-
zodicarboxylate (DEAD) (183 ml, 1.16 mmol) dropwise. After 5 mins diphe-
nylphosphoryl azide (DPPA) (250 ml, 1.16 mmol) was added to the reaction
mixture, which was allowed to reach room temperature, stirred for 16 h, and
evaporated under vacuum. Flash chromatography of the residue (column
3 ꢀ 30 cm, eluting with ethyl acetate/hexanes, 1:9, ca. 700 ml) gave 11 as an
inseparable mixture (218 mg) with DPPA in a ratio of ca. 2:1, Rf ¼ 0:34 (ethyl
1
acetate/hexanes, 1:9). For 11: H-NMR (d, ppm): 1.36 (d, J ¼ 7:3 Hz, 3H),
3.13 (m, 1H), 3.48 (d, J ¼ 7:3 Hz, 2H), 7.40 (d, J ¼ 8:8 Hz, 2H), 8.20 (d,
J ¼ 8:8 Hz, 2H).
Using hydrazoic acid. To a solution of 10 (200 mg, 1.1 mmol) and
triphenylphosphine (579 mg, 2.2 mmol) in tetrahydrofuran (3.5 ml) at 0–5 8C
was added a solution of hydrazoic acid14 (ca. 1.2 M in toluene, 1.8 ml, ca.
2.2 mmol). After 1 min DEAD (346 ml, 2.2 mmol) was added to the reaction
mixture, which was allowed to reach room temperature, stirred for 16 h, and
evaporated under vacuum. Flash chromatography of the residue (column
3 ꢀ 30 cm, eluting with ethyl acetate/hexanes, 1:9, ca. 700 ml) gave 11 (211 mg,
1
93%) as a colorless oil, Rf and H-NMR of which were identical to described
above.
N-(R-2-(4-Nitrophenyl)-1-propyl)phthalimide, 12
To a solution of 10 (105 mg, 0.58 mmol), phthalimide (90 mg, 0.61 mmol), and
triphenylphosphine (228 mg, 0.87 mmol) in tetrahydrofuran (2 ml) was added
DEAD (137 ml, 0.87 mmol) dropwise. The reaction mixture was stirred at room
temperature for 18 h and evaporated under vacuum. Flash chromatography of
the residue (column 3 ꢀ 23 cm, eluting with ethyl acetate/hexanes, 3:7, ca.
500 ml) gave 12 (159 mg, 88%) as a white solid, Rf ¼ 0:43 (ethyl acetate/
hexanes, 3:7). 1H-NMR (d, ppm): 1.36 (d, J ¼ 6:8 Hz, 3H), 3.49 (m, 1H), 3.78–
3.94 (m, 2H), 7.42 (d, J ¼ 8:8 Hz, 2H), 7.70 (m, 2H), 7.79 (m, 2H), 8.12 (d,
J ¼ 8:8 Hz, 2H).
N-(R-2-(4-Nitrophenyl)-1-propyl-3-[14C])phthalimide, 12a
In the same manner as described above, starting from 10a (179 mg,
0.977 mmol) in tetrahydrofuran (3 ml), phthalimide (151 mg, 1.026 mmol),
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SYNTHESIS OF 14C-
97
triphenylphosphine (384 mg, 1.464 mmol), and DEAD (230 ml, 1.461 mmol),
12a (275 mg, 90%) was obtained.
R-2-(4-Nitrophenyl)-1-propylamine, 13
From azide 11 with triphenylphosphine. To a solution of 11 (58 mg, 0.28 mmol)
and triphenylphosphine (90 mg, 0.34 mmol) in tetrahydrofuran (1 ml) was
added water (50 ml). The mixture was heated at 50–60 8C for 3 h, and
evaporated under vacuum. Flash chromatography of the residue (column
2 ꢀ 20 cm, eluting with chloroform/methanol/ammonium hydroxide, 100:10:1,
ca. 200 ml) gave 13 (45 mg, 89%) as an oil, Rf ¼ 0:38 (chloroform/methanol/
ammonium hydroxide, 100:10:1). 1H-NMR (d, ppm): 1.14 (broad s, 2H), 1.29
(d, J ¼ 6:5 Hz, 3H), 2.91 (m, 3H), 7.38 (d, J ¼ 8:8 Hz, 2H), 8.18 (d,
J ¼ 8:8 Hz, 2H).
From azide 11 with sodium borohydride. To a solution of 11 (100 mg,
0.48 mmol) and copper(II) sulfate pentahydrate (12 mg, 0.048 mmol) in
methanol (2 ml) was added sodium borohydride (18 mg, 0.48 mmol) in
portions. After 3 h the reaction mixture was treated with saturated aqueous
sodium bicarbonate (ca. 0.l ml), and evaporated under vacuum. The residue
was diluted with water (ca. 1 ml), and extracted with extracted with ethyl
acetate (ca. 10 ml). The extract was washed with brine (2 ꢀ 2 ml), dried over
sodium sulfate, and evaporated under vacuum. Flash chromatography of the
residue (column 2 ꢀ 20 cm, eluting with chloroform/methanol/ammonium
1
hydroxide, 100:10:1, ca. 200 ml) gave 13 (63 mg, 73%), Rf and H-NMR of
which were identical to described above.
From imide 12. To a solution of 12 (156 mg, 0.5 mmol) in toluene (2.2 ml)
was added hydrazine (220 ml, 7.0 mmol) dropwise. The reaction mixture was
heated at 80–90 8C for 1 h and cooled to room temperature. The supernatant
solution was decanted, and the residual solid was washed with toluene
(2 ꢀ 1 ml). The combined solution was evaporated under vacuum to give 13
1
(86 mg, 95%), Rf and H-NMR of which were identical to described above.
R-2-(4-Nitrophenyl)-1-propylamine-3-[14C], 13a
In the same manner as described above, starting from 12a (275 mg, 0.88 mmol)
in toluene (3.9 ml), and hydrazine (390 ml, 12.42 mmol), 13a (151 mg, 94%) was
obtained.
R-2-(4-Nitrophenyl)-1-(2-propanesulfonamido)-propane, 14
To a solution of 13 (84 mg, 0.466 mmol), triethylamine (290 ml, 2.08 mmol),
and 4-dimethylaminopyridine (6 mg, 0.049 mmol) in dichloromethane (2 ml) at
0–5 8C was added isopropylsulfonyl chloride (63 ml, 0.56 mmol) dropwise. The
reaction mixture was stirred at room temperature for 18 h, and subjected
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directly to flash chromatography (column 2 ꢀ 20 cm, eluting with ethyl
acetate/hexanes, 1:1, ca. 200 ml) to give 14 (117 mg, 88%) as a light yellow
1
solid, Rf ¼ 0:36 (ethyl acetate/hexanes, 1:1). H-NMR (d, ppm): 1.29 (d,
J ¼ 6:8 Hz, 3H), 1.31 (d, J ¼ 6:8 Hz, 3H), 1.34 (d, J ¼ 6:8 Hz, 3H), 3.05–3.15
(m, 2H), 3.24–3.41 (m, 2H), 3.92 (broad t, J ¼ 6:8 Hz, 1H), 7.40 (d,
J ¼ 8:8 Hz, 2H), 8.20 (d, J ¼ 8:8 Hz, 2H).
R-2-(4-Nitrophenyl)-1-(2-propanesulfonamido)-propane-3-[14C], 14a. In the
same manner as described above, starting from 13a (151 mg, 0.829 mmol) in
dichlomethane (3.6 ml), triethylamine (515 ml, 3.695 mmol), 4-dimethylamino-
pyridine (10 mg, 0.082 mmol), and isopropylsulfonyl chloride (112 ml,
0.997 mmol), 14a (203 mg, 85%) was obtained.
R-2-(4-Aminophenyl)-1-(2-propanesulfonamido)-propane, 15. A mixture of 14
(114 mg, 0.398 mmol) and 10% palladium on carbon (25 mg) in ethanol (4 ml)
was stirred under the balloon pressure of hydrogen for 16 h, then diluted with
ethyl acetate (2 ml), filtered through hyflo supercel, and evaporated under
vacuum. Flash chromatography of the residue (column 2 ꢀ 20 cm, eluting with
ethyl acetate/hexanes, 55:45, ca. 200 ml) gave 15 (90 mg, 88%) as an oil, Rf ¼
0:32 (ethyl acetate/hexanes, 3:2). 1H-NMR (d, ppm): 1.24 (d, J ¼ 6:8 Hz, 3H),
1.26 (d, J ¼ 6:8 Hz, 3H), 1.30 (d, J ¼ 6:8 Hz, 3H), 2.85 (m, 1H), 3.03–3.16 (m,
2H), 3.31 (m, 1H), 3.63 (broad s, 2H), 3.82 (broad s, 1H), 6.65 (d, J ¼ 8:3 Hz,
2H), 6.99 (d, J ¼ 8:3 Hz, 2H).
R-2-(4-Aminophenyl)-1-(2-propanesulfonamido)-propane-3-[14C], 15a
In the same manner as described above, starting from 14a (203 mg,
0.704 mmol) in ethanol (7 ml), and 10% palladium on carbon (45 mg), 15a
(153 mg, 84%) was obtained.
R-2-(4-(3,5-Difluorobenzoylamino)phenyl)-1-(2-propanesulfonamido)-propane
LY450108
To a solution of 15 (88 mg, 0.343 mmol) and triethylamine (55 ml, 0.395 mmol)
in dichloromethane (3.6 ml) was added 3,5-difluorobenzoyl chloride (49 ml,
0.389 mmol) dropwise. The reaction mixture was stirred at room temperature
for 16 h, then diluted with saturated aqueous sodium bisulfate (ca. 2 ml), and
extracted with ethyl acetate (ca. 20 ml). The extract was washed with brine
(2 ꢀ 3 ml), dried over sodium sulfate, and evaporated under vacuum. Flash
chromatography of the residue (column 2 ꢀ 20 cm, eluting with ethyl acetate/
hexanes, 45:55, ca. 200 ml) gave LY450108 (123 mg, 90%) as a white solid,
1
Rf ¼ 0:52 (ethyl acetate/hexanes, 1:1), ½aꢃD ¼ þ28:88 (c 10.4, MeOH). H-
NMR (d, ppm): 1.28 (d, J ¼ 6:8 Hz, 3H), 1.29 (d, J ¼ 6:8 Hz, 3H), 1.31 (d,
J ¼ 6:8 Hz, 3H), 2.97 (m, 1H), 3.09 (sept, J ¼ 6:8 Hz, 1H), 3.17–3.24 (m, 1H),
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SYNTHESIS OF 14C-
99
3.32–3.39 (m, 1H), 3.88 (broad s, 1H), 7.00 (t, J ¼ 8:3 Hz, 1H), 7.23 (d,
J ¼ 8:8 Hz, 2H), 7.40 (d, J ¼ 7:8 Hz, 2H), 7.58 (d, J ¼ 8:8 Hz, 2H), 7.82
(broad s, 1H).
R-2-(4-(3,5-Difluorobenzoylamino)phenyl)-1-(2-propanesulfonamido)-propane-
3-[14C], LY450108-[14C]
In the same manner as described above, starting from 15a (153 mg,
0.592 mmol) in dichloromethane (6.2 ml), triethylamine (95 ml, 0.682 mmol),
and 3,5-difluorobenzoyl chloride (85 ml, 0.674 mmol), LY450108-[14C] (221 mg,
94%) was obtained. A sample after dilution (1:1) with non-labeled LY450108,
and recrystallization from ethyl acetate/hexane, 2:3, had specific activity
72.9 mCi/mmol, radiochemical purity 99.4%, and optical purity 99.8%.
R-2-(4-(3,5-Difluorobenzoylamino)phenyl)-1-(2-propanesulfonamido)-propane-
3-[3H], LY450108-[3H]
LY450108 (0.005 g) and Crabtree’s catalyst (0.007 g) were dissolved in CH2Cl2
(3 ml). The mixture was stirred under 5 Ci of tritium gas for 5 h. Labile tritium
was removed by repeated rotary evaporation with EtOH. The catalyst was
removed by filtration of and EtOAc solution of the residue through a silica
Sep-pak. The filtrate was purified by reversed phase HPLC on an Untrasphere
ODS column eluting with a H2O/CH3CN/TFA gradient system. Analysis of
the purified LY450108ꢂ[3H] by HPLC chromatography showed a radio-
chemical purity of 98.8%. The specific activity as determined by mass
3
spectrometry was 96 Ci/mmol (3.55 TBq/mmol). H-NMR (CDCl3) d 7.5,
7.75; FAB-MS: [M + H]+, m=z ¼ 405.
Acknowledgements
Authors are grateful to Andy Fray and Jim Aikins of Chemical Process
Research and Development for the supplying of some of the intermediates and
experimental procedures.
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