A method for the synthesis of 2-amino-4-fluorobicyclo[3.1.0]hexane-2,6- dicarboxylic acid-[3H2]

Article abstract of DOI:10.1002/jlcr.843

A process for double deuterium labeling of an alcohol was developed. The process was utilized in the subsequent tritium labeling of a secondary alcohol with high specific activity (24 Ci/mmol) by reduction of the corresponding ketone using sodium borotrit

Full text of DOI:10.1002/jlcr.843

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 571–581.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.843
Research Article
A method for the synthesis of 2-amino-4-fluoro-
bicyclo[3.1.0]hexane-2,6-dicarboxylic acid-[3H2]
Fengjiun Kuo*, Palaniappan Kulanthaivel, Gregory A. Rener, Ping Yi
and William J. Wheeler
Lilly Research Laboratories, A Division of Eli Lilly and Company Lilly Corporate
Center, Indianapolis, IN 46285, USA
Summary
A process for double deuterium labeling of an alcohol was developed. The process was
utilized in the subsequent tritium labeling of a secondary alcohol with high specific
activity (24 Ci/mmol) by reduction of the corresponding ketone using sodium
borotritide. The starting ketone was first brominated with pyridinium tribromide;
the resulting alpha bromoketone was then reduced in THF/alcohol in the presence of
Ni(OAc)2. The alcohol was then converted to 2-amino-4-fluorobicyclo[3.1.0]hexane-
2,6-dicarboxylic acid-[3H2], an mGluR agonist. Copyright # 2004 John Wiley &
Sons, Ltd.
Key Words: tritium labeling of secondary alcohol; sodium borotritide reduction;
mGluR agonist
Introduction
Metabatropic glutamate receptors (mGluR) are a family of G-protein coupled
excitatory amino acid receptors. The family has eight distinctive receptor sub-
types, divided into three families based on sequence similarity, primary signal
transduction pathways and pharmacology. (1R, 2S, 4R, 5R, 6R)-2a-amino-4a-
fluorobicyclo[3.1.0]-hexane-2,6-dicarboxylic acid, 20, is a potent mGluR2,3
agonist (Group II).1 To support in vitro receptor binding studies, a tritium
isotopomer with high specific activity (over 20 Ci/mmol) was required. In
this report, the synthesis of this material is described. The key to the tritia-
tion of 20 was to find a method for the incorporation of tritium into the
protected alcohol 3. Once 3 was available, its conversion to 20 was
easily accomplished. Alcohol 3 was easily obtained by reduction of the
*Correspondence to: F. Kuo, Eli Lilly and Company, Lilly Corporate Center, DC1543, Indianapolis,
IN 46285, USA. E-mail: kuo fengjiun@lilly.com
Copyright # 2004 John Wiley & Sons, Ltd.
Received 15 March 2004
Revised 21 April 2004
Accepted 26 April 2004

572
F. KUO ET AL.
corresponding ketone 2, which was prepared from an intermediate (1) used in
the synthesis of 20.
Discussion
Direct reduction of 2 with sodium borotritide (NaBT4) should give a mono-
tritium labeled product 3, but the specific activity would not meet the
requirements, since commercially available NaBT4 has a specific activity of
40–60 Ci/mmol, which would correspond to about 10–15 Ci/mmol in the
product. Tritium hydrogen exchange at the position alpha to the ketone in 2,
followed by reduction of 2-½3Hꢀ with NaBT4 would also yield 3-½3Hꢀ; however,
the specific activity would still not be high enough due to the limitations of
commercially available tritiated water (5–15 Ci/ml). Clearly, a new method to
introduce two tritium atoms was required.
It is known that enol esters or silyl enol ethers of ketones can be reduced by
hydrogenation to yield the corresponding acetates or silyl ethers,2 which upon
cleavage would give secondary alcohols containing two new hydrogen atoms.
Reaction of ketone 2 with TBSOTf yielded silyl enol ether 5 (Scheme 1). No
Scheme 1.
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A METHOD FOR THE SYNTHESIS OF 2-AMINO-
573
reaction occurred when 5 was treated with H2/5% Pd-C (or 5% Rh/Al2O3) in
EtOAc at room temperature.
Alternatively, ketone 2 was converted to enol acetate 6 by reaction with iso-
propenyl acetate. Unfortunately, no reaction occurred upon reaction of 6 with
H2/5% Pd-C at room temperature. Higher temperature or pressure may be
required due to the steric hindrance of the molecule.
In view of the results described above, we envisioned that if a leaving group
at the position alpha to the ketone in 2 could be substituted with tritium, then
subsequent reduction of the ketone with NaBT4 would provide 3-½3Hꢀ with the
desired specific activity. Although the reduction of alpha-bromoketones
usually yields bromohydrins, in some cases the debromination product has
been reported.3 In a detailed study, on the reduction of 7, Goto and Kishi,4
reported that a trace of nickel ion alters the course of the borohydride
reduction of an alpha-bromoketone and yields a de-brominated product 9
instead of the expected bromohydrin 8. Similar de-bromination of an alpha-
bromoketone was observed by Sarma et al.5 during their studies on the
in situ nickel boride generated from nickel chloride and sodium borohydride
(Scheme 2). It was proposed that further reduction of ketone 9 to the alcohol
10 should occur if the nickel salt could be removed from the reaction media
after forming the ketone intermediate.
Scheme 2.
Bromoketone 11 was prepared by the bromination of ketone 2 with
pyridinium tribromide.6 The bromoketone 11 was then treated with NaBH4 in
a mixed solvent of 1:1 methanol/THF in the presence of nickel acetate
tetrahydrate at room temperature (Scheme 3). The reaction was monitored by
LC-MS. The de-bromination product 2 was detected as the major product
along with minor amounts of alcohol 3 as well as a trace amount of
bromohydrin 12 (and minor amounts of the methyl ester of 2). The crude
reaction mixture was passed through a short column of silica gel and eluted
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F. KUO ET AL.
Scheme 3.
with EtOAc to remove the nickel salt. The crude product 2 was treated with
NaBH4 in EtOH to yield 3 as a mixture of diastereomer isomers in a 1:1 ratio
as analyzed by LC-MS. In order to eliminate the possibility of trans-
esterification, the reaction was carried out in THF alone; bromohydrin 12
became the major product, along with minor amounts of alcohol 3,
presumably because of the poor solubility of Ni(OAc)2 ꢁ 4H2O in THF.
When the reduction of 11 was carried out in 1:1 THF/EtOH using NaBD4/
Ni(OAc)2 ꢁ 4H2O (Scheme 4), the deuterated alcohol 13 became the major
product as a mixture of two diastereomers in about a 1:1 ratio (as seen by LC-
MS). Only a trace amount of the corresponding bromohydrin 14 was detected.
One possible explanation might be that while nickel acetate had similar
solubility in either ethanol or methanol, sodium borohydride has a 4 fold
better solubility in methanol than in ethanol,7 and it reacts with methanol at
an appreciable rate and only slowly with ethanol.8 Thus, most of the excess
sodium borohydride was consumed by methanol before it could reduce the
ketone, but it survived longer in ethanol and was able to reduce the ketone.
That might explain why Goto and Kishi4 did not detect the formation of
alcohol 10 in their experiments when methanol was used as part of the solvent.
Further analysis of the deuterium incorporation in 13 by LC-MS indicated
that both diastereomers had about 80% of di-deuterium and 20% of mono-
deuterium labeled, which corresponds to about 22.5 Ci/mmol of specific
activity in tritium if a sodium borotritide (NaBT4) of 50 Ci/mmol was used. In
a similar experiment, when less NaBD4 was used, the reaction stopped at the
ketone stage (as shown previously). Although the ketone intermediate was
further reduced to alcohol 13 with additional NaBD4, the ratio of the labeling
changed to 70% D1 and 30% D2. This suggests that H–D exchange occurred
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A METHOD FOR THE SYNTHESIS OF 2-AMINO-
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Scheme 4.
in ketone intermediate. Very reproducible results, 70 to 80% D2 and 20 to
30% D1, were obtained when an excess of NaBD4 was used at the onset of
the reduction. Apparently, to minimize the H–D exchange, and thus maximize
the deuterium incorporation, an excess of reducing agent was required in the
beginning of the reaction.
Reaction of a-bromoketone 11 with NaBT4/Ni(OAc)2/EtOH/THF at room
temperature (Scheme 5),y yielded a mixture of the alcohols (15; 16) and a trace
of the bromohydrin (17; 18). Purification of the reaction mixture and
subsequent reaction of 15 with Deoxo-Fluor1 ([Bis-(2-methoxyethyl)amino]-
sulfur trifluoride)/CH2Cl2 yielded 19.1 Following HPLC purification,z the
radiochemical purity of 20 was 98.0%; the specific activity was 24 Ci/mmol.
3
ES-MS showed a [M+H]+ at m=z 208. The H-NMR spectrum in CDCl3
(Figure 1) confirms the introduction of tritium in the 3-a and 4 positions.
Conclusion
A general and simple method has been developed for the synthesis of a double-
deuterium-(or tritium) labeled alcohol from the corresponding ketone 2.
Ketone 2 was brominated with pyridinium tribromide in acetic acid to give
3-bromoketone 11, and this ketone 11 was reduced directly to the
corresponding alcohol 15 or 16 by an excess of sodium borodeuteride or
sodium borotritide in ethanol/THF in the presence of nickel acetate. The
ketone 2 from debromination was the major product if the reaction was
carried out in methanol, and bromohydrin 12 was the major product if the
reaction was carried out in THF alone.
Experimental
To monitor the deuterium labeling, LC-MS analysis was run on LCQ Classic
Mass Spectrometer with inline PDA UV detector, using a Metachem Polaris
C18-A column (150 ꢂ 2 mm, 5 m). NMR spectra were obtained on a General
Electric QE-400 nuclear magnetic resonance spectrometer at 400 MHz.
y The tritium labeling was performed by Amersham Bioscience.
z HPLC conditions: Column: Hypercarb 7 m, (100 ꢂ 4.6 mm). Solvent A: 0.1% trifluoroacetic acid in water.
Solvent B: 0.1% trifluoroacetic acid in acetonitrile. Gradient: 0% B for 15 min then to 100% B over 20 min.
Flow rate: 1 ml/min UV detector at 200 nm. RT ¼ 7:43 min.
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F. KUO ET AL.
Scheme 5.
PPM
2
8
6
10
4
3
Figure 1. H-NMR Spectrum for compound 20. The chemical shift for the 3b
proton occurs at d2.76 ppm
Chemical shifts are reported in parts per million (ppm) downfield from
tetramethylsilane. Direct chemical ionization mass spectra (DCI-MS) and
electron impact mass spectra (EI-MS) were recorded on a Nermag R30-10
triple stage quadrapole mass spectrometer. Flash chromatography was
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A METHOD FOR THE SYNTHESIS OF 2-AMINO-
577
performed on Biotage system, or as described by Still et al., using E.M. Science
silica gel 60 (230–400 mesh).
Synthesis of (1S, 2S, 5R, 6R)-4-oxo-2-(R-1-phenylethylamino)bicyclo[3.1.0]-
hexane-2,6-dicarboxylic acid, diethyl ester (4a)
(
A mixture of 1 (0.750 g, 2.07 mmol) and crushed 3 A molecular sieves (0.975 g)
in CH2Cl2 (45 ml) was treated with pyridinium chlorochromate (PCC, 1.375 g,
6.30 mmol). The mixture was stirred for 25 h and then treated with Hi-Flo
supercel. After stirring for 5 min, the mixture was filtered. The filter cake was
washed with an additional 25 ml of CH2Cl2 and the combined filtrate was
concentrated in vacuo. The crude product was crystallized from EtOH to yield
4 (0.566 g, 71%). ES-MS: [M+Na]+, m=z ¼ 382; [M+H]+, m=z ¼ 360.
HR ES-MS: Anal. Calculated for C20H26NO5 ([M+H]+) 360.1811. Found:
360.1835.
Synthesis of (1S, 2S, 5R, 6R)-2-amino-4-oxobicyclo[3.1.0]hexane-2,6-dicar-
boxylic acid, diethyl ester (4b)
An ethanol (80 ml) solution of 4 (0.425 g, 1.23 mmol), HCl (1 N, 1.2 ml) was
treated with 10% Pd-C (0.028 g) and hydrogenated at 60 psi for 24 h. The
mixture was filtered and concentrated to dryness to yield 4b as a white solid
(0.291 g, 81%). ES-MS: [M+H]+, m=z ¼ 256. The NMR spectrum in CD3OD
was consistent with the product with no aromatic resonances.
1H-NMR spectrum (CD3OD) d 1.32 (m, 7 H, ester-CH3’s and H-3a), 2.52
(d, 1 H, J=3 Hz, H-5), 2.70 (d, 1 H, J=3 Hz, H-1), 2.75 (m, 1 H, H-6), 2.92 (m,
1 H, H-3b), 4.24 and 4.40 (t, 2 H, ester CH2’s).
HR ES-MS: Anal. Calculated for C12H18NO5 ([M+H]+) 256.1185. Found:
256.1185.
Synthesis of (1S, 2S, 5R, 6R)-2-(9H-fluoren-9-ylmethoxycarbonyl)-4-oxo-
bicyclo[3.1.0]-hexane-2,6-dicarboxylic acid, diethyl ester (2)
A mixture of 4b (0.291 g, 1 mmol), Fmoc-OSu (0.337 g, 1 mmol), and NaOH
(1 N, 1 ml) was dissolved in dioxane (10 ml) and stirred at room temperature
for 2 days. The reaction mixture was diluted with EtOAc and washed with
water. The organic layer was dried (anhydrous MgSO4) and concentrated in
vacuo. The crude product (0.570 g) was purified by Biotage chromatography
on silica gel, eluting with Et2O/heptane (8:2). Fractions 1–7 were combined
and concentrated in vacuo to yield 2 as a white solid (0.496 g), which was
crystallized from EtOAc/hexanes to yield crystalline 2 (0.425 g, 89%). ES-MS:
[M+H]+, m=z ¼ 478; ES-MS: [M+Na]+, m=z ¼ 500.
HR ES-MS: Anal. Calculated for C27H27NO7Na ([M+Na]+) 500.1685.
Found: 500.1669.
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Synthesis of (1S, 2S, 4RS, 5R, 6R)-2-(9H-fluoren-9-ylmethoxycarbonyl)-4-
hydroxy-bicyclo[3.1.0]-hexane-2,6-dicarboxylic acid, diethyl ester (3a, 3b)
A DME (2 ml) solution of 2 (0.048 g, 0.10 mmol) and NaBH4 (0.004 g,
0.10 mmol) was stirred at room temperature for 2 days. The reaction mixture
was quenched with acetone (1 ml) and diluted with EtOAc. HCl (1 N, 1 ml)
was added and the two phase mixture was stirred for 1 h. The layers were
separated and the organic layer was washed with H2O, dried (anhydrous
MgSO4), and concentrated in vacuo. The crude product showed 2 spots on
TLC (silica gel, Et2O saturated with H2O) and no unreacted starting material.
ES-MS showed a [M+H]+, m=z ¼ 480; ES-MS: [M+Na]+, m=z ¼ 502. The
two diastereomers of 3 were separated by HPLC (column: Discovery C18,
10 ꢂ 250 mm, 5 mm. Flow rate=4 ml/min. Mobile phase A: 10 mM ammonium
acetate, mobile phase B: methanol, isocratic at A/B=30/70 for 18 min, then
flush the column at A/B=10/90) (RT ¼ 12:4 min for C4a-OH ð3aÞ isomer and
RT ¼ 13:7 min for C4b-OH (3b) isomer).
1
3a: H-NMR spectrum (CD3OD) d 1.22 (t, 3 H, ester-CH3), 1.24 (t, 3 H,
ester-CH3), 1.25 (m, 1 H, 3a-H), 2.04 (m, 1 H, 6-H), 2.14 (m, 1 H, 5-H), 2.36
(m, 1 H, 1-H), 2.53 (m, 1 H, 3b-H) 4.05 (m, 2 H, Fmoc-CH2), 4.10 (q, 2 H,
ester-CH2), 4.11 (q, 2 H, ester-CH2), 4.32 (m, 1 H, Fmoc-CH), 4.61 (m, 1 H, 4-
H), 7.30 (m, 2 H, 20-H, 90-H), 7.38 (t, 2 H, 40-H, 70-H), 7.65 (m, 2 H, 30-H, 80-H)
and 7.79 ppm (d, 2 H, 50-H, 60-H); ES-MS: [M+H]+, m=z ¼ 480.
1
3b: H-NMR spectrum (CD3OD) d 1.22 (t, 3 H, ester-CH3), 1.25 (t, 3 H,
ester-CH3), 1.58 (m, 1 H, 6-H), 1.73 (m, 1 H, 3b-H), 2.09 (m, 1 H, 5-H), 2.27
(m, 1 H, 3a-H), 4.11 (m, 2 H, Fmoc-CH2), 4.16 (m, 2 H, ester CH2), 4.17 (2 H,
ester CH2), 4.26 (m, 1 H, 4-H), 4.32 (m, 1 H, Fmoc-CH), 7.30 (m, 2 H, 20-H, 90-
H), 7.38 (t, 2 H, 40-H, 70-H), 7.65 (m, 2 H, 30-H, 80-H) and 7.79 ppm (d, 2 H, 50-
H, 60-H); ES-MS: [M+H]+, m=z ¼ 480.
HR ES-MS: Anal. Calculated for C27H29NO7Na ([M+Na]+) 502.1842.
Found: 502.1825.
Synthesis of (1S, 2S, 4RS, 5R, 6R)-2-(9H-fluoren-9-ylmethoxycarbonyl)-4-
hydroxy-bicyclo[3.1.0]-hexane-2,6-dicarboxylic-[3,4-2H2] acid, diethyl ester
(13a, 13b)
A solution of ketone 2 (4.8 mg) and pyridinium tribromide (4.1 mg) in 2 ml of
glacial acetic acid was stirred at 708C (oil bath) for 3 h. TLC (silica gel, 3:1
hexanes/EtOAc) showed only trace amount of the ketone 2 remaining. The
solution was diluted with ethyl acetate, washed with saturated solution of
NaHCO3 and water. The organic layer was concentrated, and the residue was
dissolved in ethanol and concentrated (this was repeated three times). The final
residue was dried in vacuo overnight to give a crude bromoketone 11. This
crude product was used in the next step without purification. ES-MS:
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[M+H]+=556/558, two diastereomers. The crude bromoketone, 11, was
dissolved in anhydrous ethanol to a total volume of 4 ml. To a 0.5 ml aliquot
of this ethanol solution was added a catalytic amount of Ni(OAc)2 ꢁ 4H2O,
followed by 0.5 ml of anhydrous THF. To the above solution was then added
an excess of NaBD4 in one portion at room temperature. After stirring for
10 min, TLC (silica gel, 3:1 hexanes/EtOAc) indicated no bromoketone 2
remained. Two new spots with lower Rf were detected. The reaction was
quenched by adding 10% acetic acid dropwise. The solution was passed
through
a
short column of silica gel and concentrated. ES-MS:
[M+H]+=481/482 for D1 and D2 isomers in a ratio of 20 to 30% D1 and
70 to 80% D2 isomers. Two diasteriomeric alcohols 13 were in a ratio of about
1:1 by ES-MS. In another experiment, when less NaBD4 was used, the reaction
stopped at the ketone stage. Although the ketone intermediate was further
reduced to alcohol with additional NaBD4, the ratio of the labeling changed to
70% D1 and 30% D2.
Synthesis of (1S, 2S, 4R, 5R, 6R)-2-(9H-fluoren-9-ylmethoxycarbonyl)-4-
hydroxy-bicyclo[3.1.0]-hexane-2,6-dicarboxylic acid, diethyl ester (non-labeled
19)
To a solution of 3b (0.050 g) in 1 ml of anhydrous CH2Cl2 at ꢃ208C was added
12 ml (3 drops via a micro-syringe) of Deoxo-Fluor1 ([Bis-(2-methoxyethyl)a-
mino]sulfur trifluoride) in 5 min. The reaction was monitored by TLC (silica
gel, 3:1 hexanes/EtOAc). After stored in a –208C cooling bath for 90 min, TLC
indicated some alcohol remained. Additional drops of Deoxo-Fluor1 were
added. No further progress was observed even after another 3 h. The solution
was stored in refrigerator overnight. A solution of 15% of Na2CO3 was added
dropwise to the flask to quench the reaction in an ice bath. The resulting
solution was further diluted with water, and the aqueous layer was extracted
twice with CH2Cl2. The combined extracts were dried over Na2SO4, filtered,
and concentrated. The residue was chromatographed (silica gel, 4:1 hexanes/
EtOAc with 2% of CH2Cl2) to afford a desired non-labeled product 19 as a
white form (0.035 g, 72.8%). ES-MS: [M+H]+=482.
HR ES-MS: Anal. Calculated for C27H28FNO6Na ([M+Na]+) 504.1798.
Found: 504.1786.
Synthesis of 2a-amino-4a-fluoro-bicyclo-[3.1.0]hexanes-2,6-dicarboxylic acid
A solution of non-labeled 19 (0.003 g in 0.5 ml of dioxane) was stirred with
0.5 ml of diethylamine at room temperature overnight. TLC (silica gel, 3:1
hexanes/EtOAc) indicated no starting material remained, ES-MS:
[M+H]+=260. The solution was concentrated in vacuo to remove diethyla-
mine. The residue was taken into 0.5 ml of dioxane (or methanol) and 0.1 ml of
1 N NaOH, and the resulting solution was stirred at room temperature
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F. KUO ET AL.
overnight. The reaction was quenched by adding 0.1 ml of 1 N HCl. ES-MS
indicated no starting material remained. The crude product, [MꢃH]ꢃ=202,
co-eluted with an authentic sample1 of LY459477 on HPLC (TosoHaas Amide
80, 4.6 ꢂ 250 mm, mobile phase: 0.1% TFA in 80:20 CH3CN/H2O, isocratic at
0.5 ml/min for 15 min, then flush the column at 90/10 CH3CN/H2O containing
0.1% of TFA, UV at 205 nm but very weak at 1 mg/ml, RT ¼ 9:5 min).
1H-NMR spectrum (D2O/DCl): d 1.79 (m, 1 H, 3a-H), 2.44 (m, 1 H, 6-H), 2.52
(m, 1 H, 1-H), 2.65 (m, 1 H, 5-H), 2.76 (m, 1 H, 3b-H), 5.68 (m, 1 H, 4-H).
Synthesis of (1S, 2S, 4S, 5R, 6R)-2-(9H-fluoren-9-ylmethoxycarbonyl)-4-
hydroxy-bicyclo[3.1.0]-hexane-2,6-dicarboxylic-[3,4-3H2] acid, diethyl ester
(15-[3,4-3H2])
A solution of 11 (prepared as described above from 0.033 g, 0.069 mmol of
ketone 2) in EtOH (2 ml) was treated with NaBT4 (5 Ci, 64 Ci/mmol,
0.078 mmol) and stirred for 10 min.y Another 5 Ci of NaBT4 was added and
stirring was continued for an additional 10 min. The reaction mixture was
quenched with 50% aqueous HOAc (200 ml) and the labile tritium was
removed by rotary evaporations with CH3OH. The product was eluted
through a silica sep-pac with EtOAc/hexane (3:2) and the eluate was
evaporated to dryness and re-dissolved in EtOH. This product was further
purified by chromatography on silica gel, eluting with EtOAc/hexane to yield
pure 15-½3; 4-3H2ꢀ (198 mCi).
Synthesis of (1R, 2S, 4R, 5R, 6R)-2-(9H-fluoren-9-ylmethoxycarbonyl)-
4-fluoro-bicyclo[3.1.0]-hexane-2,6-dicarboxylic-[3,4-3H2] acid, diethyl ester
(19-[3,4-3H2])
Deoxoflour (2 ml) was added to a stirred solution of 15-½3; 4-3H2ꢀ (198 mCi) in
CH2Cl2 (1.3 ml) at ꢃ208C.y No reaction was observed after several hours and
the reaction mixture was allowed to warm to room temperature and treated
with additional deoxoflour (2 ꢂ 10 ml) over 2 h. After standing overnight, an
additional 10 ml of deoxoflour was added. About 80% of the starting material
had been consumed, so the reaction was quenched by the addition of 15%
aqueous Na2CO3 (100 ml), diluted with H2O and extracted with CH2Cl2. The
organic extract was washed with H2O and dried (anhydrous Na2SO4). The
crude product was purified by chromatography on silica gel (18 ꢂ 250 mm),
eluting with EtOAc/hexane (1:1) to yield 19-½3; 4-3H2ꢀ (55 mCi).
(1R, 2S, 4R, 5R, 6R)-2a-amino-4a-fluorobicyclo[3.1.0]-hexane-2,6-dicar-
boxylic-[3,4-3H2] acid, 20
Sodium hydroxide (1 N) was added to a methanol solution of 19-½3; 4-3H2ꢀ
(55 mCi).y The reaction mixture was stirred at room temperature overnight
and subsequently quenched by the addition of 2 N HCl (100 ml). The resulting
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A METHOD FOR THE SYNTHESIS OF 2-AMINO-
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solution was diluted with H2O and washed with Et2O. The aqueous solution
was concentrated and re-dissolved in H2O and purified by HPLC on a
Hypercarb column (7 m, 10 ꢂ 150 mm) eluting with a gradient from H2O/TFA
to CH3CN/TFA. The desired fractions were evaporated and the residue re-
dissolved in H2O/EtOH (1:1) to yield 20-½3; 4-3H2ꢀ (25 mCi). The radio-
chemical purity was 98.0%z and the specific activity (determined by mass
3
spectrometry) was 24 Ci/mmol. The H-NMR spectrum(D2O) showed two
resonances at d 1.79 (3a-3H) and 5.68 (4-3H) ppm.
Acknowledgements
The authors would like to thank Dr Eric Moher for providing the starting
material for the synthesis of ketone 2.
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Copyright # 2004 John Wiley & Sons, Ltd.
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