The synthesis of isotopically labeled (+)-2-amino-bicyclo[3.1.0]hexane-2,6- carboxylic acid and its 2-oxa-and 2-thia-analogs

Article abstract of DOI:10.1002/jlcr.956

As part of a program aimed at the design of conformationally constrained analogs of glutamic acid, (+)-2-aminobicyclo[3.1.0]hexane-2,6-carboxylic acid (1), identified as a highly potent, selective, group II metabotropic glutamate receptor agonist has been synthesized and studied clinically. Heterocyclic analogs of 1 were subsequently synthesized in which the C-2 methylene has been replaced by an oxygen atom (2) or a sulfur atom (3). C-14 labeled isotopomers of 1, 2 and 3 have been synthesized to facilitate pre-clinical ADME studies. A tritium labeled isotopomer of 1 was also synthesized for use in in vitro experiments. A stable labeled isotopomer of rac-1 was prepared for use as an internal standard for bioanalytical assays. The key step in each of these syntheses was the reaction of chiral ketone 4, 5 or 6 with K¹⁴CN/ (NH₄)₂CO₃ using the Bucherer-Berg protocol. In the preparation of the stable labeled isotopomer, rac-4-[¹³C ₂] was prepared in two steps from ethyl bromoacetate-[UL- ¹³C₂]; subsequent reaction of rac-4-[¹³C ₂] with K¹³CN/¹⁵NH₄Cl/Na ₂CO₃, followed by hydrolysis of the hydantoin yielded rac-1-[¹³C₃, ¹⁵N]. Copyright

Full text of DOI:10.1002/jlcr.956

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 605–620.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.956
Research Article
The synthesis of isotopically labeled (+)-2-amino-
bicyclo[3.1.0]hexane-2,6-carboxylic acid and its 2-oxa-
and 2-thia-analogs
William J. Wheeler*, Douglas D. O’Bannon, Joseph H. Kennedy, James A.
Monn, Roger W. Tharp-Taylor, Matthew J. Valli and Fengjiun Kuo
Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis,
IN 46285, USA
Dedicated to the memory of my friend and mentor, Winston S. Marshall (1938–2005)
Summary
As part of a program aimed at the design of conformationally constrained analogs of
glutamic acid, (+)-2-aminobicyclo[3.1.0]hexane-2,6-carboxylic acid (1), identified as a
highly potent, selective, group II metabotropic glutamate receptor agonist has been
synthesized and studied clinically. Heterocyclic analogs of 1 were subsequently
synthesized in which the C-2 methylene has been replaced by an oxygen atom (2) or a
sulfur atom (3). C-14 labeled isotopomers of 1, 2 and 3 have been synthesized to
facilitate pre-clinical ADME studies. A tritium labeled isotopomer of 1 was also
synthesized for use in in vitro experiments. A stable labeled isotopomer of rac-1 was
prepared for use as an internal standard for bioanalytical assays. The key step in each
of these syntheses was the reaction of chiral ketone 4, 5 or 6 with K14CN/(NH4)2CO3
using the Bucherer–Berg protocol. In the preparation of the stable labeled isotopo-
mer, rac-4-[13C2] was prepared in two steps from ethyl bromoacetate-[UL-13C2];
subsequent reaction of rac-4-[13C2] with K13CN/15NH4Cl/Na2CO3, followed by
hydrolysis of the hydantoin yielded rac-1-[13C3,15N]. Copyright # 2005 John Wiley &
Sons, Ltd.
Key Words: mGlu agonist; carbon 14; tritium; Bucherer–Berg reaction
Introduction
Metabotropic glutamate receptors (mGlu) are G-protein-coupled receptors,
which comprise eight different subtypes.1 These are also divided into three
*Correspondence to: William J. Wheeler, Eli Lilly and Company, Lilly Corporate Center, DC1543,
Indianapolis, IN 46285, USA. E-mail: wheeler william joe dr@lilly.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 14 March 2005
Revised 30 March 2005
Accepted 31 March 2005

606
W. J. WHEELER ET AL.
groups (Group 1, 2 and 3) on the basis of their amino acid sequence homo-
logy, pharmacology and the signal transduction pathways to which they
are coupled.2 Monn et al. have reported on the design, synthesis and
biological evaluation of 1, a novel, conformationally constrained analog of
glutamic acid with potent mGlu2,3 agonist activity.3 Evaluation of 1 in
humans4 for use in the treatment of anxiety followed pre-clinical ADME
studies5 for which 1-[14C] as well as rac-1-[13C3,15N] was required.
Subsequently, Monn et al. described the synthesis and biological evalua-
tion of the heterocyclic analogs 2 and 3 which were very potent mGlu2,3
agonists as well.6,7 In preparation for pre-clinical ADME studies, carbon-14
isotopomers of 2 and 3 were also required. The syntheses of 1-[14C], 2-[14C],
3-[14C] and 1-[3H] as well as rac-1-[13C3,15N] have been outlined in pre-
liminary reports.8,9 In this manuscript, their syntheses will be discussed in
further detail.
H
H
H
O
S
HO2C
HO2C
HO2C
CO2H
CO2H
CO2H
H
NH2
H
H
NH2
NH2
1
2
3
An earlier report of the synthesis of 1-[3H] by another method was published
by Malherbe et al., although no experimental details were given.10
Discussion
In the preparation of 1-[14C], rac-4, prepared as detailed by Monn et al.,3 was
hydrolysed in NaOH to yield ketoacid rac-7, and subsequently resolved as its
S-1-phenethylamine salt 8 (Scheme 1). Treatment of 8 with HCl yielded (+)-7.
Reaction of (+)-7 with K14CN/(NH4)2CO3/EtOH/H2O yielded the hydantoin
as a mixture of diastereomers 9 and 10. Re-crystallization of the mixture from
i-PrOH/H2O yielded the desired chromatographically pure hydantoin 9 in
58% yield. A suspension of 9 in 48% HBr was refluxed for 20 h; the pH was
adjusted to 3 by the addition of NaOH to afford crystalline 1-[14C] (64%,
HPLC showed slight contamination with 9). The impure material was
redissolved in 1 N HCl and treated dropwise with 1 N NaOH to pH 3
whereupon a white crystalline solid formed to yield 1-[14C] (43% yield). This
material was diluted with 1 as described above to yield 1-[14C] (77% yield);
specific activity 37.87 mCi/mg. The radiochemical purity (RCP) was 99.81%
and the material co-eluted with authentic 1 on HPLC; the chiral purity was
98.98%.
Ethyl(-)-4-oxobicyclo[3.2.1]hex-2-ene-6-carboxylate(11, Scheme 2) was pre-
pared as described by Massey et al. from (+)-4 by conversion, first to the TMS
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF ANALOGS OF GLUTAMIC ACID
607
H
H
H
H
H
X
1. NaOH
S-1-phenethyl-
amine, EtOH
EtO2C
HO2C
HO2C
2. HCl
X = CH2
H
O
O
O
rac-4; X = CH2
rac-5; X = O
rac-6; X = S
rac-7
8 (as S-
phenethyl-
amine salt)
HCl
H
H
H
(NH4)2CO3
HO2C
HO2C
HO2C
O
O
+
K14CN, EtOH
H2O
H
H
H
O
*
HN
*
HN
NH
NH
(+)-7
O
O
9
10
1. 48% HBr
2. NaOH to
pH 3
3. recry.
H
H
1. 1/HCl
HO2C
HO2C
*
*
H
CO2H
2. NaOH to pH 3
H
CO2H
H2N
H2N
1-[14C]
14C
1-[14C]
*
=
Scheme 1.
3H
O
3H
H
H
H
H
H
H
T2, 10% Pd-C
EtOAc
(NH4)2CO3
EtO2C
3H
EtO2C
EtO2C
3H
O
KCN, EtOH
H2O
O
HN
NH
11
12
O
13
NaOH, 105oC
3H
H
H
HO2C
3H
CO2H
H2N
1-[3H2]
Scheme 2.
enol ether by reaction with TMSI/Et3N, followed by treatment with
Pd(OAc)2.11 Ketoester 11 is also an intermediate in the asymmetric synthesis
of 1, reported by Dominguez et al.12 Treatment of 11 with tritium gas/5%
Pd-C in EtOAc and HPLC purification of the crude product yielded
Copyright # 2005 John Wiley & Sons, Ltd.
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608
W. J. WHEELER ET AL.
acetone
-
(CH3)2+S13CH213CO2Et Br
Br13CH213CO2Et + (CH3)2S
14
cyclopent-2-enone
DBU/CH3CN
H
H
H
15NH4Cl/Na2CO3
*
*
*
*
EtO2C
EtO2C
O
H
H
K13CN, EtOH
H2O
15N
*
O
NH
rac-4-[13C2]
O
15
1. 48% HBr
2. NaOH to
pH 3
H
*
*
HO2C
*
H
CO2H
H215N
rac-1-[13C3,15N]
13C
*
=
Scheme 3.
ethyl (+)-2-oxobicyclo[3.2.1]hexane-6-carboxylate-[3,4-3H2] (12).y Further re-
action of 12 with KCN/(NH4)2CO3/EtOH/H2O as described above and HPLC
purification of hydantoin 13, followed by NaOH hydrolysis and HPLC
purification yielded 1-[3H2] (specific activity 38 Ci/mmol).
A mixture of ethyl bromoacetate-[1,2-13C] and dimethylsulfide was stirred in
acetone for 44 h to yield ethyl (dimethylsulfuranylidene)acetate-[1,2-13C2]
bromide (14, Scheme 3). Subsequent reaction of 14 with 2-cyclopentenone/
CH3CN/DBU yielded rac-4-[13C2] in 64% after chromatography. Further
reaction of rac-4-[13C2] with a mixture of NH4Cl-[15N], Na2CO3, and K13CN
in water using the Bucherer–Berg protocol yielded a 97:3 mixture of 15 and the
undesired diastereomers in 26% yield. Due to the unavailability of
(NH4)2CO3-[15N], we envisioned its in situ preparation by the reaction of
NH4Cl-[15N] and Na2CO3. We believe this to be the first example of using
NH4Cl-[15N] in the Bucherer–Berg reaction. Hydrolysis of the hydantoin 15 in
48% HBr and subsequent adjustment of the pH of the reaction mixture to 4
with NaOH yielded rac-1-[13C3, 15N] in 28% yield.
y The tritiation and subsequent chemistry was conducted at Amersham Biosciences, Cardiff, Wales, UK.
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF ANALOGS OF GLUTAMIC ACID
609
The 1-oxa-(2-[14C]), and 1-thia (3-[14C]), (Scheme 4) analogs were
synthesized in a manner similar to that described above for 1-[14C]. In order
to stem the losses accompanied by a resolution conducted in the last
step, chiral precursors (+)-5 and (+)-6 were required. Chiral (+)-5 was
prepared in a six step synthesis from (S)-(-)-1,2,4-butanetriol as described
by Massey et al.;13 (+)-6 was obtained by preparative chromatography
of rac-6 (prepared as described by Monn et al.3) on a 5 cm Chiralpak
AD chiral column eluting with 10% ethanol in acetonitrile. As described
above, reaction of (+)-5/(+)-6 with K14CN/(NH4)2CO3/EtOH/H2O yielded
the two hydantoins 16/17 in 52% (100% de) and 26% (99% de) yield,
respectively. Hydrolysis of the hydantoins 16/17 in refluxing 2 N
NaOH, followed by acidification to pH 3.0–3.2 yielded the crystalline
2-[14C] and 3-[14C] in 38 and 63% yields. The RCPs were 99.92%
and 99.0%, respectively. The specific activity of 3-[14C] was 97.9 mCi/mg.
There was a problem obtaining a reproducible specific activity for
2-[14C], which may have arisen from residual contamination by in-
organics. The material was recrystallized after dilution with authentic
2 (56% yield); the specific activity was 36.89 mCi/mg and the RCP was
99.79%.
OH
HO
OH
S-(-)-1,2,4-butanetriol
ref. 12, X = O
H
H
H
H
(NH4)2CO3
X
X
separation by
X
EtO2C
EtO2C
EtO2C
O
K14CN, EtOH
H2O
chiral HPLC
X = S
H
*
H
HN
O
O
NH
X = O, (+)-5
X = S, (+)-6
X = S, rac-6
O
X = O, 16
X = S, 17
1. NaOH to
pH 3
2. recry.
H
X
HO2C
*
H
CO2H
H2N
X = O, 2-[14C]
X = S, 3-[14C]
*
=
14C
Scheme 4.
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610
W. J. WHEELER ET AL.
Results
We have described the syntheses of two isotopomers of 1 (1-[14C] and 1-[3H])
as well as the isotopomer of rac-1 (rac-1-[13C3,15N]). In addition, we have
described the synthesis of the isotopomers of two heterocyclic analogs of 1 (2-
[14C] and 3-[14C]). In contrast to the published syntheses of 1, 2 and 3, which
required a resolution at the hydantoin stage, these preparations used the chiral
precursors (+)-7, 11, (+)-5 and (+)-6, which obviated the late stage resolutions.
The synthesis of rac-1-[13C3,15N] employed the use of in situ generated
(15NH4)2CO3, which is not commercially available, for the Bucherer–Berg
reaction.
Experimental
NMR spectra were obtained on a General Electric QE-300 or a Varian
500 MHz nuclear magnetic resonance spectrometer. Chemical shifts are
reported in parts per million (ppm) downfield from tetramethylsilane. Mass
spectra were recorded on a Nermag R30-10 triple stage quadrupole
mass spectrometer (DCI), a VG Analytical VG-ZAB3F mass spectrometer
(FAB), a Varian Associates MAT 731 mass spectrometer (FD) or a Waters
Micromass ZQ single quadrupole mass spectrometer (ES-MS). Flash
chromatography was performed on silica gel. RCP was assessed by
HPLC with radiochemical detection on a Packard Flow Scintillation
Analyzer. RCP was further assessed by TLC-autoradiography. The radio-
activity was detected on Kodak X-ray film BB-5; the lanes of the TLC were
cut, mixed with methanol and scintillation fluid and counted. Microanalyses
were conducted in the Physical Chemistry Department of the Lilly Research
Laboratories.
Synthesis of (ꢀ)-2-oxobicyclo[3.1.0]-hexane-6-carboxylic acid ðrac-7Þ
Ethyl 2-oxobicyclo[3.1.0]-hexane-6-carboxylate (rac-4) was saponified by base
hydrolysis using NaOH and subsequently acidified with HCl to yield rac-7 in
yields of 92–95%.14 Several individual lots of rac-7 were mixed (1150.8 g,
8.212 mol) and dissolved in 4.1 l of 1 N NaOH at 15–308C. After stirring for
10–20 min to complete dissolution, the mixture was cooled to 15–208C and
slowly acidified to pH 1, by treatment with HCl (12 N, 800 ml). A thick slurry
formed and the solid was collected by filtration and washed with water to yield
(ꢀ)-2-oxobicyclo-[3.1.0]-hexane-6-carboxylic acid (rac-7). The solid and NaCl
(0.5 kg) was suspended in 2 l of water, stirred for 15 min and then extracted
with EtOAc (4.0 l). The EtOAc solution was dried (anhydrous MgSO4) and
concentrated in vacuo. The residue was crystallized from acetone (2.5 l) to yield
(ꢀ)-2-oxobicyclo[3.1.0]-hexane-6-carboxylic acid (rac-7, 837 g, 72.7% yield,
97.4% by HPLC, vida infra).
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF ANALOGS OF GLUTAMIC ACID
611
Synthesis of (1S, 5R, 6S)-2-oxobicyclo[3.1.0]-hexane-6-carboxylic acid,
(S)-a-methyl-benzylamine salt ð8Þ
An EtOH/H2O solution (4.2 l, 8:1) of ( ꢀ )-2-oxobicyclo[3.1.0]-hexane-
6-carboxylic acid (rac-7, 837 g, 5.97 mol) at 15–208C was treated in a
portionwise manner with (S)-a-methylbenzylamine (724 g, 5.97 mol); the
temperature was kept below 308C by an ice bath. The resulting mixture was
stirred overnight. The solid was collected by filtration and washed with
acetone (4 ꢁ 0.4 l) to yield (+)-2-oxobicyclo[3.1.0]-hexane-6-carboxylic acid,
(S)-a-methylbenzylamine salt (8, 323.8 g, 20.7% yield, theoretical ¼ 50%), mp
142.5–147.58C.
Synthesis of (1S, 5R, 6S)-2-oxobicyclo[3.1.0]-hexane-6-carboxylic acid ððþÞ-7Þ
(1S, 5R, 6S)-2-Oxobicyclo[3.1.0]-hexane-6-carboxylic acid, (S)-a-methylben-
zylamine salt (8, 277.85 g, 0.863 mol) was added to a stirred solution of 1200 ml
of 1 N HCl (1.2 mol, 1.13 eq.). After stirring for 15 min, the solution was
extracted with EtOAc (4 ꢁ 3 l). The combined extracts were dried (anhydrous
MgSO4) and concentrated in vacuo to yield (1S, 5R, 6S)-2-oxobicyclo[3.1.0]-
hexane-6-carboxylic acid ((+)-7, 157.9 g). The crude material was crystallized
from water to yield (+)-7 (108.2 g, 72.6%), mp 128–134.58C; HPLC purity
1
99.7%; H-NMR (CDCl3) d 1.98 (t, J ¼ 3 Hz, 1H, 6-H), 2.03 (m, 1H, 3b-H),
2.11 (m, 1H, 3a-H), 2.15 (m, 1H, 4b-H), 2.24 (m, 1H, 4a-H), 2.33 (dd, J ¼ 5:5
and 2.3 Hz, 1-H) and 2.55 (dt, J ¼ 3:4 and 5.5 Hz, 5-H); 13C-NMR (CDCl3) d
22.5 (C-4), 26.0 (C-6), 29.9 (C-5), 31.7 (C-3), 36.2 (C-1), 176.05 (carboxyl-
C ¼ O) and 211.3 (C ¼ O); IR (CHCl3) showed carbonyl resonances at
1733.91 and 1703.51 cmꢂ1; UV (95% EtOH), lM 277 ðeM ¼ 58:54Þ; Titration
(66% DMF) pKa 4.72, 6.23; ½aꢃD ¼ 74:78 (c ¼ 10:06, CH3OH); FD-MS,
M+ 140. Karl Fischer titration indicated 0.31% water. The chemical purity
was 99.7% by HPLC (0.3% S-MBA) on a Zorbax SB Phenyl column
(4.6 ꢁ 250 mm) with gradient elution at 2.0 ml/min (column temperature
408C) and UV detection at 210 nm. RT ððþÞ-7Þ ¼ 3:05 min; RT ðS-MBAÞ ¼
2:35 min.
Time (min)
Solvent A
Solvent B
0
20
25
30
35
10
10
80
80
10
90
90
20
20
90
Solvent A: acetonitrile.
Solvent B: 0.1 M NaH2PO4, adjusted to pH 2.5 with H3PO4.
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W. J. WHEELER ET AL.
The chiral purity was 98.7% as determined by HPLC on a ChiralPak AD
column (5 mm, 4.6 ꢁ 250 mm), eluting with 40% n-propanol in hexane(column
temperature at 408C) at 1.0 ml/min with UV detection at 210 nm.
RT ((+)-7Þ ¼ 6:5 min; RT ðð-Þ-7Þ ¼ 4:5 min.
Analysis calculated for C7H8O3: C, 60.00; H, 5.75. Found: C, 60.09; H, 5.76.
Synthesis of (-)-20,50-dioxospiro[bicyclo[3.1.0]hexane 2,40-imidazolidine]-6-
carboxylic-[2-carbonyl-14C] acid, 9
A mixture of (1S, 5R, 6S)-2-oxobicyclo-[3.1.0]-hexane-6-carboxylic acid ((+)-
7, 0.560 g, 4 mmol), potassium cyanide-[14C] (200 mCi, specific activity 50 mCi/
mmol, 4 mmol), and ammonium carbonate (0.920 g, 10 mmol) in 10 ml of
ethanol/water (1:1) was stirred at room temperature for 68 h. The mixture was
diluted with 25 ml of water and concentrated in vacuo to an amorphous foam.
This material was re-dissolved in water and acidified to pH 2 with 1 N HCl.
After stirring for 1 h at room temperature, the white crystalline material was
collected by filtration, washed with water, and dried in vacuo to yield (-)-20,50-
dioxospiro[bicyclo-[3.1.0]hexane-2,40-imidazolidine]-6-carboxylic-[2-carbonyl-14C]
acid (9, 0.598 g, 71%); ES-MS: [M+Na]+, m=z ¼ 233=235 (major). The
white solid was suspended in i-propyl alcohol/water (6 ml/0.4 ml) and
stirred at reflux. Water was added until all the material was immersed in the
solution (ca. 2 ml); the mixture was allowed to cool to room temperature
and stirred for 1 h. The white solid was collected by filtration, washed
with i-propyl alcohol, and dried in vacuo to yield (-)-20,50-dioxo-spiro-[bi-
cyclo-[3.1.0]-hexane-2,40-imidazolidine]-6-carboxylic-[2-carbonyl-14C] acid (9,
0.352 g, 42%). This material co-elutes with authentic unlabeled isotopomer
of 9 on HPLC (vide infra). The chiral purity was determined on the final
product.
Synthesis of (1S, 2S, 5R, 6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic-
[2-carboxyl-14C] acid, monohydrate (1-[14C] monohydrate)
A suspension of (-)-20,50-dioxospiro-[bicyclo[3.1.0]hexane-2,40-imidazolidine]-
6-carboxylic-[2-carbonyl-14C] acid (9, 0.352 g, 1.68 mmol) in 48% HBr (5.5 ml)
was stirred at gentle reflux for 16 h. HPLC (vida infra) showed >99%
conversion to the desired product. Stirring at reflux was continued for an
additional 4 h and the mixture was allowed to cool to room temperature. An
additional 10 ml of water was added to redissolve the HBr salt; the pH was
adjusted to 3 by the dropwise addition of 5 N NaOH. The solution was
concentrated to ca. 8 ml in vacuo, seeded with authentic 1, and stirred as a solid
slowly crystallized. After stirring at room temperature for 3 h, the mixture was
chilled at 48C overnight. The white solid was collected by filtration, washed
with water, and dried in vacuo (0.200 g, 64%). HPLC showed some
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF ANALOGS OF GLUTAMIC ACID
613
contamination with unreacted 9. This material was dissolved in 2 ml of 1 N
HCl, filtered, and treated dropwise with 5 N NaOH to pH 3. The resulting
mixture was stirred until crystals were formed and 4 h thereafter. The white
solid was collected by filtration, washed with water, and dried in vacuo to yield
(1S, 2S, 5R, 6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic-[2-carboxyl-
14C] acid, monohydrate (1-[14C] monohydrate, 0.134 g, 43%).
To adjust the specific activity, a mixture of 1 (0.690 g) and 1-[14C] (0.134 g)
was dissolved in 1 N HCl (8.24 ml) at room temperature with stirring. The pH
was carefully adjusted to 3.0 by the dropwise addition of 5 N NaOH (at ca. pH
2.6, material began to crystallize). After the pH adjustment was complete, the
mixture was stirred at room temperature for 4 h. The white solid was collected
by filtration, washed with water, and dried in vacuo to yield (1S, 2S, 5R, 6S)-2-
aminobicyclo[3.1.0]hexane-2,6-dicarboxylic-[2-carboxyl-14C] acid, monohy-
drate (1-[14C] monohydrate, 0.631 g, 77%); ES-MS: [M+H]+, m=z ¼ 186
1
(major)/188; H-NMR (D2O) d 1.49 (m, 1H, 3b-H), 1.85 (m, 1H, 2b-H) and
1.95–2.32 (m, 5H). This spectrum was superimposable with that of 1 in
D2O;3specific activity 37.87 mCi/mg. This material co-eluted with authentic 1
monohydrate by radio-HPLC on a Zorbax SB-phenyl column (5 mm,
4.6 ꢁ 250 mm), eluting with 0.1 N NaH2PO4 (pH 2.1)/acetonitrile (93:7) at
1 ml/min (column temperature 408C), UV detection at 220 nm. The sample was
dissolved at 1 mg/ml (in buffer); 20 ml was injected. The RCP using the same
conditions with radiochemical detection was 99.82%. The RCP by TLC/
Autoradiography (silica gel, EtOAc/CH3CN/HOAc/H2O, 21:7:7:9) was
99.6%. The chiral purity was 98.98% as determined by HPLC after
derivatization with Marfey’s reagent15 on a Zorbax 300 SB C-18 column
(4.6 ꢁ 250 mm), eluting with H2O (pH 3.0 with H3PO4)/acetonitrile in the
following gradient: 90:10 at 0 min, increasing to 83:17 over the next 5 min. The
concentration was held at 83:17 for 40 min and then returned to the original
concentration in 1 min. The flow rate was 1 ml/min at ambient temperature;
UV detection at 340 nm. A 2 mg/ml sample of 1-[14C] was derivatized; a 20 ml
sample was injected.
Synthesis of ethyl (1R, 5S, 6S)-4-oxobicyclo[3.1.0]hexane-6-carboxylate-
[2,3-3H2] ð12Þ
An ethyl acetate (2 ml) solution of ethyl (1R, 5S, 6S)-4-oxobicyclo[3.1.0]hex-2-
ene-6-carboxylate (11, 0.0497 g) and 10% Pd-C (0.0281 g) was stirred for
5 h under tritium gas (30 Ci). The catalyst was removed by filtration and the
labile tritium was removed by several evaporations from ethanol (under
vacuum) to yield ethyl (1R, 5S, 6S)-4-oxobicyclo[3.1.0]hexane-6-carboxylate-
[2,3-3H2] (12, 9.6 Ci). This material was purified by reversed phase HPLC on a
Partisil ODS column, eluting with a water/CH3OH gradient to yield purified
12 (6.1 Ci).
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W. J. WHEELER ET AL.
Synthesis of ethyl (-)-20,50-dioxospiro-[bicyclo-[3.1.0]hexane-2,40-imidazoli-
dine]-6-carboxylate-[2,3-3H3] ð13Þ
Ethyl (1R, 5S, 6S)-4-oxobicyclo[3.1.0]hexane-6-carboxylate-[2,3-3H2] (12,
9.6 Ci, ca. 0.15 mmol) was mixed with KCN (0.011 g, 0.169 mmol), (NH4)2CO3
(0.0296 g, 0.238 mmol), water (0.4 ml) and EtOH (1 ml) and heated at 358C for
18 h. The reaction was quenched by the addition of TFA (0.1 ml) and
concentrated in vacuo. The residue was re-dissolved in EtOH (3 ꢁ 1 ml) and
concentrated. This material was purified by reversed phase HPLC on a Partisil
ODS column, eluting with a water/CH3CN/TFA gradient to yield ethyl (-)-
20,50-dioxospiro-[bicyclo[3.1.0]hexane-2,40-imidazolidine]-6-carboxylate-[2,3-
3H3] (13, 2.78 Ci).
Synthesis of (1S, 2S, 5R, 6S)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylic-
[2,3-3H2] acid (1-[3H2])
A NaOH (1 N, 1 ml) solution of ethyl (-)-20,50-dioxospiro-[bicyclo-[3.1.0]hex-
ane-2,40-imidazolidine]-6-carboxylate-[2,3-3H3] (13, 2.78 Ci) was stirred at
1058C for 1 h. TLC indicated that no starting material remained, so the
reaction was quenched by the addition of HCl (12 N, 0.1 ml) and concentrated
in vacuo. The residue was re-dissolved in 1:1 EtOH/H2O (3 ꢁ 1 ml) and
concentrated to yield (1S, 2S, 5R, 6S)-2-aminobicyclo[3.1.0]hexane-2,6-
dicarboxylic-[2,3-3H2] acid (1-[3H2], 2.1 Ci). After an initial purification of a
portion of the crude product by preparative TLC on KC18 plates (n-BuOH/
H2O/HOAc), the product was further purified by reversed phase HPLC on a
Partisil ODS column, eluting with a water/CH3CN/TFA gradient. Final
purification on a Primesphere silica column, eluting with EtOAc/CH3CN/
HOAc yielded (1S, 2S, 5R, 6S)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylic-
[2,3-3H2] acid (1-[3H2], 19 mCi); specific activity ¼ 38 Ci=mmol; FAB-MS:
[M+H]+, m=z ¼ 190. The product co-eluted on HPLC with authentic 1;
Waters Novapak C-18 column (3.9 ꢁ 150 mm) with gradient elution at 1 ml/
min (0–100% B over 30 min) with UV (230 nm) and Radiochemical Detection
(Solvent A ¼ 10 mM TFA/50 mM heptane sulfonic acid (HSA), Solvent B ¼
10 mM TFA plus 50 mM HSA/CH3CN, 1:3). The RCP in this same system
was 95.5%. The chiral purity was 96.0% as determined by HPLC on a
Chirobotic T column (5 mm, 4.6 ꢁ 250 mm), eluting with 0.1% aqueous TFA/
THF (80:20) at 1 ml/min with UV detection at 230 nm.
Synthesis of ethyl (dimethylsulfuranylidene)acetate-[1,2-13C2] bromide ð14Þ
Ethyl bromoacetate-[13C2] (2.0 g, 11.83 mmol) was dissolved in 5 ml of freshly
opened acetone and a slight excess of dimethylsulfide (0.821 g, 0.97 ml,
13.21 mmol). The solution became cloudy after about 1 h and the reaction
mixture was then stirred at ambient temperature for 44 h. The white
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SYNTHESIS OF ANALOGS OF GLUTAMIC ACID
615
precipitate was collected by filtration under a stream of nitrogen, washed with
cold acetone and dried in vacuo to yield ethyl (dimethylsulfuranylidene)ace-
tate-[1,2-13C2] bromide (14, 2.04 g, 74.6% yield); 1H-NMR (DMSO/d6) d 1.31
(t, 3H, CH3), 3.50 (s, 6H, S+(CH3)2), 4.3 (q, 2H, OCH2Me), 5.31 (d,
¼ 100:8 Hz, 13CH2).
13
CꢂH
J
Synthesis of ethyl rac-2-oxobicyclo[3.1.0]hexane-6-carboxylate-[carboxyl,
6-13C2] (rac-4-[13C2])
A suspension of ethyl (dimethylsulfuranylidene)acetate-[1,2-13C2] bromide (14,
2.04 g, 8.9 mmol) in CH3CN (8 ml) was stirred and treated with DBU (1.63 g,
1.60 ml, 10.7 mmol). After 15 min, 2-cyclopenten-1-one (0.735 g, 0.75 ml,
8.95 mmol) was added and stirring was continued for 44 h. Hydrochloric acid
(1 N, 10 ml) and Et2O (25 ml) were added. The aqueous layer was re-extracted
with Et2O (3 ꢁ 25 ml) and the combined Et2O extracts were washed with brine,
dried (anhydrous MgSO4) and concentrated in vacuo. The dark oil was
purified by chromatography on silica gel (30 ꢁ 70 mm) by eluting with 500 ml
of hexanes/EtOAc (60:40). The solvent was removed and the remaining oil was
re-dissolved in hexanes (10 ml) and chilled to ꢂ208C, whereupon a yellow
crystalline solid formed. The crystals were collected by filtration and washed
with hexanes to yield of ethyl rac-2-oxobicyclo[3.1.0] hexane-6-carboxylate-
1
[carboxyl, 6-13C2] acid (rac-4-[13C2], 0.967 g, 64% yield); H-NMR (CDCl3) d
1.30 (t, J ¼ 7 Hz, 3H, CH3), 2.0–2.4 (m, 6H), 2.48–2.55 (m, 1 H), 4.15 (q,
J ¼ 7 Hz, CH2Me); 13C-NMR (CDCl3) d 26.39 (13CH), 169.80 (13C ¼ O); ES-
MS, [M+H]+, m=z ¼ 171, [M+NH4]+, m=z ¼ 188.
Synthesis of ethyl (-)-20,50-dioxospiro-[bicyclo[3.1.0]hexane-2,40-imidazoli-
dine]-6-carboxylate-[ carboxyl, 50-carbonyl, 6-13C3, 20-15N] ð15Þ
An aqueous (1.8 ml) suspension of Na2CO3 (1.44 g, 9.52 mmol) and 15NH4Cl
(1.02 g, 19.04 mmol) was sonicated for 15 min. Ethanol (5.5 ml) was added
followed by ethyl rac-2-oxobicyclo[3.1.0]-hexane-6-carboxylate-[carboxyl,
6-13C2] (rac-4, 0.81 g, 4.76 mmol) and the reaction mixture was heated to
408C. After 15 min, K13CN (0.347 g, 5.24 mmol) was added and heating was
continued for ca. 15 h. The solution was allowed to cool to room temperature,
diluted with H2O (3.5 ml) and then chilled to 58C. A white precipitate formed
which was collected by filtration (0.472 g, inorganic salts). Three additional
crops of white solid formed and collected by filtration and analysed by HPLC
(see below):
A : 0:427 g ð88:3 : 11:5 desired : undesired diastereomersÞ
B : 0:285 g ð54:6 : 45:2 desired : undesired diastereomersÞ
C : 0:156 g ð36:6 : 63:1 desired : undesired diastereomersÞ
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W. J. WHEELER ET AL.
Crop A (0.427 g) was suspended in EtOH (5 ml) and H2O (0.9 ml) and heated
until dissolved. Upon cooling to room temperature, some crystals formed and
the mixture was diluted with 5 ml of H2O and chilled in an ice bath. After, 2 h,
the solid was collected by filtration, washed with H2O and dried to yield ethyl
(-)-20,50-dioxospiro-[bicyclo[3.1.0]hexane-2,40-imidazolidine]-6-carboxylate-
1
[carboxyl, 50-carbonyl, 6-13C3, 20-15N] (15, 0.304 g, 26% yield, de ¼ 94%); H-
NMR (DMSO/d6) d 1.14 (t, J ¼ 7 Hz, 3H, CH3), 1.30 (m, 1H, 2 eq.-H), 1.4-
2.2 (m, 6H), 4.02 (q, J ¼ 7 Hz, 2H, CH2Me), 7.95 (dd, J ¼ 14:4, 86.4 Hz,
20-15NH), 10.5 (bs, 1H, 40-NH); 13C-NMR (DMSO/d6) d 20.8 (6-13C), 172.0
(50-13C ¼ O), 177.29 (6-13C ¼ O); HPLC on a Zorbax SB phenyl column
(4.6 ꢁ 250 mm), eluting with 0.1 M NaH2PO4 at pH 2.1/CH3CN (80:20) at
2 ml/min with UV detection at 220 nm.
Treatment of crop B as described above only increased the de to 40%.
Synthesis of 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic-[6, dicarbox-
yl-13C3,15N] acid (rac-1-[13C3,15N])
A mixture of ethyl (-)-20,50-dioxospiro-[bicyclo[3.1.0]hexane-2,40-imidazoli-
dine]-6-carboxylate-[carboxyl, 50-carbonyl, 6-13C3, 20-15N] (15, 0.286 g,
1.18 mmol) and 48% HBr (15 ml) was heated at reflux for 24 h (HPLC
showed 90% conversion to the desired product). The solvent was removed
under vacuum and the residue was re-dissolved in H2O (2 ml). The pH of the
solution was adjusted to 3.0 by the addition of 0.1 N NaOH and then cooled in
an ice bath. After 1 h, a white solid began to crystallize. The suspension was
stirred in the cold for an additional 2 h. The solid was collected by filtration
and washed with cold H2O to yield 2-aminobicyclo[3.1.0]hexane-2,6-dicar-
boxylic-[6,dicarboxyl-13C3,15N] acid (rac-1-[13C3,15N], 0.062 g, 28% yield);
13C-NMR (D2O) d 24.68 (6-13C), 176.74 (2-13C¼¼O), 180.52 (6-13C¼¼O); ES-
MS: [M+H]+, m=z ¼ 190; [M+Na]+, m=z ¼ 212. HR-ES-MS: Analysis
calculated for C153C3H NO4: [M + H]+ 190.0832. Found:190.0833.
15
11
Synthesis of ethyl (-)-20,50-dioxospiro-[2-oxabicyclo[3.1.0]hexane-4,40-imida-
zolidine]-6-carboxylate-[50-carbonyl-14C] ð16Þ
A mixture of potassium cyanide-[14C] (50 mCi, specific activity 55.5 mCi/
mmol, 0.9 mmol), potassium cyanide (0.171 g, 2.63 mmol), and ammonium
carbonate (0.850 g, 8.50 mmol) in 4 ml of methanol was stirred at room
temperature for 0.5 h. A methanol (16 ml) solution of ethyl (1R, 5R, 6R)-4-
oxo-2-oxabicyclo[3.1.0]hexane-6-carboxylate ((+)-5, 0.600 g, 3.53 mmol) was
added and stirring was continued at room temperature. After 24 h, the mixture
was concentrated to ca. 10 ml, chilled to 0–58C, and diluted with water (15 ml).
The pH was adjusted to 7 with 5 N HCl, seeded with unlabeled product, and
then stirred until crystalline and 1 h thereafter. The off-white crystalline
material was collected by filtration, washed with water (2 ꢁ 10 ml), and dried in
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SYNTHESIS OF ANALOGS OF GLUTAMIC ACID
617
vacuo to yield ethyl (-)-20,50-dioxospiro-[2-oxabicyclo[3.1.0]hexane-4,
40-imidazolidine]-6-carboxylate-[50-carbonyl-14C] (16, 0.439 g, 52%). This
material co-elutes with unlabeled product by HPLC on a Zorbax
SB-phenyl column (4.6 ꢁ 250 mm) eluting with 0.1 M NaH2PO4/acetonitrile
(97:3) at 1 ml/min; UV at 220 nm. HPLC showed that this material was
100% de.
Synthesis of (1R, 4R, 5S, 6R)-(-)-4-amino-2-oxabicyclo[3.1.0]-hexane-4,6-
dicarboxylic-[4-carboxyl-14C] acid, (2-[14C])
A solution of ethyl (-)-20,50-dioxospiro-[2-oxabicyclo[3.1.0]hexane-4,40-imida-
zolidine]-6-carboxylate-[50-carbonyl-14C] (16, 0.439 g, 1.84 mmol) in NaOH
(9.2 ml, 2 N, 18.3 mmol) was stirred at just below reflux for 24 h. HPLC
(see below) showed a significant amount of the corresponding hydantoin acid
as well as the desired product. Stirring at reflux was continued for an
additional 16 h and the mixture was allowed to cool to room temperature.
HPLC showed that the slightly cloudy solution was completely converted to
the desired product. The pH was adjusted to 9.2–9.3 with 5 N HCl and the
precipitate was collected by filtration. The solid was washed with water
(2 ꢁ 5 ml) and the filtrate was concentrated to ca. 5 ml in vacuo. The pH was
adjusted to 3.2 with 5 N HCl (from pH 3.5, 1 N HCl was used) and stirred for
2 h (additional HCl was added as necessary to maintain the pH at 3.2).
The mixture was chilled in an ice bath and stirred for an additional 2 h.
The white solid was collected by filtration, washed with water, and dried in
vacuo to yield (1R, 4R, 5S, 6R)-(-)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-
dicarboxylic-[4-carboxyl-14C] acid, (2-[14C], 0.1298 g, 38%); ES-MS:
[M+H]+, m=z ¼ 186 (major)/188. This material co-eluted with authentic 2
by radio-HPLC on a Zorbax SB-CN column (5 mm, 4.6 ꢁ 250 mm), eluting
with 0.1% trifluoroacetic acid/acetonitrile (97:3) at 1 ml/min (column
temperature ambient), UV detection at 220 nm. The sample was dissolved at
1 mg/ml (in buffer); 20 ml was injected. The RCP using the same conditions
with radiochemical detection was 99.92%. There was a problem getting a
reproducible specific activity that may have arisen from residual inorganics, so
the material was recrystallized again. The white solid (0.1298 g) was mixed
with 2 (0.1298 g) and dissolved in 2 N NaOH (1.5 ml, 3 mmol). The solution
was diluted with water (1.1 ml) and the pH of the resulting stirred solution was
adjusted to 3.5 with 5 N HCl. At pH ¼ 4, material began crystallizing. The
final adjustment of the pH to 3.2 was made with 1 N HCl. The mixture was
stirred for 2 h, whereupon the white solid was collected by filtration, washed
with water (2 ꢁ 2 ml), and dried in vacuo to yield (1R, 4R, 5S, 6R)-(-)-4-amino-
2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic-[4-carboxyl-14C] acid, (2-[14C],
0.1459 g, 56%); specific activity ¼ 36:89 mCi=mg; RCP ¼ 99:79% (see above
for HPLC details).
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618
W. J. WHEELER ET AL.
Preparation of ethyl (1R, 5S, 6S)-4-oxo-2-thiabicyclo[3.1.0]hexane-6-carbox-
ylate ((+)-6)
An EtOH/CH3CN (10:90, 80 ml) solution of 4-oxo-2-thiabicyclo[3.1.0]hexane-
6-carboxylate (rac-6, 4.0 g, 21.5 mmol) was purified by chiral HPLC on a
Chiralpak AD column (5 ꢁ 24 cm) eluting with 10% EtOH/CH3CN at 100 ml/
min with UV detection at 240 nm. Eight samples of 10 ml were injected and the
pure fractions of each compound was collected and concentrated in vacuo. The
samples from the individual injections containing (+)-6 were combined and
concentrated in vacuo to yield ethyl (1R, 5S, 6S)-4-oxo-2-thiabicyclo[3.1.0]-
hexane-6-carboxylate ((+)-6, 1.7 g, 43%): 1H-NMR (CDCl3) d 1.26 (t,
J ¼ 7 Hz, 3H, CH3), 2.32 (t, J ¼ 3:5 Hz, 1H, 6-H), 2.58 (dd, J ¼ 6:5 and
3.5 Hz, 1H, 5-H), 3.20 (dd, J ¼ 6:5 and 3.5 Hz, 1H, 1-H), 3.31 (bd, J ¼ 18 Hz,
1H, 3b-H), 3.26 (d, J ¼ 18 Hz, 1H, 3a-H and 4.15 (q, J ¼ 7 Hz, 2H, CH2Me);
13C-NMR (CDCl3) d 14.1 (CH3), 29.3 (C-1), 30.8 (C-6), 34.9 (C-3), 61.7 (CH2),
168.8 (ester C ¼ O) and 206.4 (ketone C ¼ O). Upon analysis by analytical
chiral HPLC on a Chiralpak AD column (4.6 ꢁ 250 mm) eluting with 10%
EtOH/CH3CN at 1.0 ml/min with UV detection at 210 nm, (+)-6 eluted with a
retention time of 5.51 min ðee ¼ 99:9%Þ.
In addition to the pure (+)-6 enantiomer, (-)-6 (1.9 g, 47%) was recovered;
its retention time was 7.30 min on the analytical column ðee ¼ 90%Þ.
Synthesis of ethyl (-)-20,50-dioxospiro-2-thiabicyclo[3.1.0]hexane-4,40-imidazo-
lidine]-6-carboxylate-[50-carbonyl-14C] ð17Þ
A mixture of ammonium carbonate (0.375 g, 2 mmol), 50 mCi of potassium
cyanide-[14C], and 0.083 g (1.28 mmol) of potassium cyanide in 5 ml of
anhydrous methanol was stirred at room temperature for 10 min and then (1R,
5S, 6S)-4-oxo-2-thiabicyclo[3.1.0]hexane-6-carboxylate ((+)-6, 0.375 g,
2 mmol) was added as a yellow solid in one portion. The solution was stirred
heterogeneously at room temperature overnight (about 23 h). The solution was
diluted by adding 3 ml of water, and adjusted to pH 3.0 with 5 N HCl. A
yellowish precipitate formed and was filtered, washed with 2.5 ml of water and
3 ml of i-PrOH, and dried in the air to give the ethyl (-)-20,50-dioxospiro-2-
thiabicyclo[3.1.0]-hexane-4,40-imidazolidine]-6-carboxylate-[50-carbonyl-14C]
(17, 0.168 g, 26%) as an orange solid. HPLC analysis indicated it to be 99.2%
chemical purity and 99.0% de.
Synthesis of (1R, 4S, 5S, 6S)-(-)-4-amino-2-thiabicyclo[3.1.0]-hexane-4,6-
dicarboxylic-[4-carboxyl-14C] acid, (3-[14C])
Ethyl (-)-20,50-dioxospiro-2-thiabicyclo[3.1.0]hexane-4,40-imidazolidine]-6-car-
boxylate-[50-carbonyl-14C] (17, 0.168 g) was dissolved in 4 ml of 2 N NaOH at
room temperature, then heated to reflux (1208C, oil bath) for about 20 h. The
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SYNTHESIS OF ANALOGS OF GLUTAMIC ACID
619
solution was cooled to room temperature, diluted with 10 ml of water, and
adjusted to pH 9.3 with 5 N HCl. The white precipitate was filtered and the
solid was washed with 5 ml of water. The combined filtrates were concentrated
and the volume was adjusted to about 5 ml. The solution was then adjusted to
pH 3.0 with 5 N HCl and stirred at room temperature until some white
precipitate appeared. The solution was stirred in an ice bath for another 2 h,
and the salt was then filtered, washed with 5 ml of water and 1 ml of i-PrOH
and air dried. Further drying in the vacuum oven at 408C for 2 h gave the
desired product (1R, 4S, 5S, 6S)-(-)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-
dicarboxylic-[4-carboxyl-14C] acid (3-[14C], 0.084 g, 63%) as a white solid;
specific activity ¼ 97:9 mCi=mg and RCP ¼ 99:0% by HPLC and TLC-
autoradiography (CH3CN/EtOAc/HOAc/H2O 21:7:7:9, Rf ¼ 0:32). This
material co-eluted with authentic 3 by radio HPLC on a Zorbax SB-phenyl
column (4.6 ꢁ 250 mm), eluting with 0.1 M NaH2PO4 (pH 2.14)/CH3CN
(90:10, 408C) at 1 ml/min with radiochemical and UV detection (220 nm). The
retention time was 2.99 min. ES-MS: ½M þ Hꢃþ ¼ 204=206.
Acknowledgements
The authors would like to especially thank Dr Palaniappan Kulanthaivel for
NMR data acquisition and interpretation, Mr Dean Clodfelter for mass
spectral data acquisition, and Mr Douglas Schmidt for high resolution mass
spectral data acquisition.
References
1. Hollmanm M, Heinemann S. Annu Rev Neurosci 1994; 17: 31–108.
2. Conn PJ, Pin PJ. Annu Rev Pharmacol Toxicol 1997; 37: 205–237.
3. Monn JA, Valli MJ, Massey SM, Wright RA, Salhoff CR, Johnson BG, Howe T,
Alt CA, Rhodes GA, Robey RL, Griffey KR, Tizanno JP, Kallman MJ, Helton
DR, Schoepp DD. J Med Chem 1997; 40: 528–537.
4. Grillon C, Cordova J, Levine LR, Morgan CA. Psychopharmacology (Berlin)
2003; 168: 446–454.
5. Johnson JT, Mattiuz EL, Chay SH, Herman JL, Wheeler WJ, Kassahun K,
Swanson SP, Phillips DL. Drug Disp Metab 2002; 30: 27–33.
6. Monn JA, Valli MJ, Massey SM, Hansen MM, Kress TJ, Wepsiec JP, Harkness
AR, Grutsch JL, Wright RA, Johnson BG, Andis SL, Kingston A, Tomlinson R,
Lewis R, Griffey KR, Tizanno JP, Schoepp DD. J Med Chem 1999; 42:
1027–1040.
7. Kingston AE, O’Neill MJ, Lam A, Bates KR, Monn JA, Schoepp DD. Eur
J Pharmacol 1999; 377: 155–165.
8. Wheeler WJ, Monn JA, Valli MJ, O’Bannon DD. Abstracts of the 5th
International Conference for Isotopes, Brussels, Belgium, April 2005.
9. Wheeler WJ, Spence MC, Kuo F. Abstracts of the 5th International Conference for
Isotopes, Brussels, Belgium, April 2005.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 605–620

620
W. J. WHEELER ET AL.
10. Malherbe P, Knoflach F, Broger C, Ohresser S, Kratzeisen C, Adam G, Stadler
H, Kemp JA, Mutel V. Mol Pharmacol 2001; 60: 944–954.
11. Massey SM, Monn JA, Valli MJ. US Patent 5958960; 28 September, 1999.
12. Dominguez C, Ezquerra J, Prieto L, Espada M, Petregal C. Tetrahedron
Asymmetry 1997; 8: 511–514.
13. Massey SM, Monn JA, Valli MJ. US Patent 5688826; 18 November, 1997.
14. Monn JA, Schoepp DD. US Patent 5750566, 12 May, 1998.
15. B’Hymer C, Montes-Bayon M, Caruso JA. J Sep Sci 2003; 26: 7–19.
Copyright # 2005 John Wiley & Sons, Ltd.
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