Synthesis, in vitro pharmacology, and pharmacokinetic profiles of 2-[1-amino-1-carboxy-2-(9H-xanthen-9-yl)-ethyl]-1-fluorocyclopropanecarb oxylic acid and its 6-heptyl ester, a potent mGluR2 antagonist

Article abstract of DOI:10.1016/j.bmc.2008.02.066

In this paper, we describe the synthesis of (+)-(1R^*,2R^*)-2-[(1S^* )-1-amino-1-carboxy-2-(9H-xanthen-9-yl)-ethyl]-1-fluorocyclopropanecarbo xylic acid (+)-16a, a compound, that is, fluorinated at the alpha position of the carboxylic acid in the cyclopropane ring of a group II mGluRs antagonist, 1 (LY341495), using a previously reported stereoselective cyclopropanation reaction. The fluorinated compound (+)-16a exhibited almost the same affinity (IC₅₀ = 3.49 nM) for mGluR2 as 1 but had a superior pharmacokinetic profile. Furthermore, a marked elevation of the plasma levels of (+)-16a was observed following the administration of a prodrug, (+)-17.

Full text of DOI:10.1016/j.bmc.2008.02.066

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4359–4366
Synthesis, in vitro pharmacology, and pharmacokinetic profiles
of 2-[1-amino-1-carboxy-2-(9H-xanthen-9-yl)-ethyl]-1-fluoro-
cyclopropanecarboxylic acid and its 6-heptyl ester,
a potent mGluR2 antagonist
Kazunari Sakagami,a,* Akito Yasuhara,a Shigeyuki Chaki,b Ryoko Yoshikawa,b
Yasunori Kawakita,c Akio Saito,d,ꢀ Takeo Taguchid and Atsuro Nakazatoe
aMedicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi,
Saitama 331-9530, Japan
bMolecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho,
Kita-ku, Saitama-shi, Saitama 331-9530, Japan
cDrug Safety and Pharmacokinetics Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi,
Saitama 331-9530, Japan
dSchool of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
eStrategic Planning of Drug Discovery, R&D Headquarters of Pharmaceutical Operation, Taisho Pharmaceutical Co., Ltd,
1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
Received 10 January 2008; revised 20 February 2008; accepted 21 February 2008
Available online 26 February 2008
Abstract—In this paper, we describe the synthesis of (+)-(1R*,2R*)-2-[(1S*)-1-amino-1-carboxy-2-(9H-xanthen-9-yl)-ethyl]-1-fluoro-
cyclopropanecarboxylic acid (+)-16a, a compound, that is, fluorinated at the alpha position of the carboxylic acid in the cyclopro-
pane ring of a group II mGluRs antagonist, 1 (LY341495), using a previously reported stereoselective cyclopropanation reaction.
The fluorinated compound (+)-16a exhibited almost the same affinity (IC50 = 3.49 nM) for mGluR2 as 1 but had a superior phar-
macokinetic profile. Furthermore, a marked elevation of the plasma levels of (+)-16a was observed following the administration of a
prodrug, (+)-17.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
dissimilarities in their coupling mechanisms, molecular
structures, sequences homology, and the pharmacology
of the receptors.3–9 Group I mGluRs (mGluR1 and 5)
activate phospholipase C, while both group II mGluRs
(mGluR2 and 3) and group III mGluRs (mGluR4, 6,
7 and 8) inhibit adenylate cyclase.9–11 mGluRs have
received significant attention as therapeutic targets for
L-Glutamate is a major excitatory neurotransmitter in
the mammalian central nervous system (CNS).1,2 Gluta-
mate receptors are classified into two categories, iono-
tropic glutamate receptors (iGluRs) and metabotropic
glutamate receptors (mGluRs). iGluRs have an ion
channel structure, whereas mGluRs belong to the family
of G-protein coupled receptors. mGluRs have been
further categorized into three groups (group I–III) com-
prising eight subtypes (mGluR1–8) based on similarities/
the treatment of CNS disorders and conditions.12–19
A
number of antagonists of group II mGluRs have been
identified, as shown in Figure 1.20–23 Compounds 1–3
show very high affinities for mGluR2 and are potent
functional antagonists of human mGluRs.
Keywords: Group II mGluRs; Antagonist; Fluorinated compound;
Prodrug.
We previously reported the synthesis, in vitro pharma-
cological profiles, and structure–activity relationships
of 2–3, as well as the pharmacokinetic profiles of several
typical compounds.22,23 In particular, 2 (MGS0039)
exhibited a high binding affinity for group II mGluRs,
*
Corresponding author. Tel.: +81 48 663 1111; fax: +81 48 652
7254; e-mail: kazunari.sakagami@po.rd.taisho.co.jp
ꢀ
Present address: Showa Pharmaceutical University, 3-3165 Higashi-
tamagawagakuen, Machida, Tokyo 194-8543, Japan.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.066

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K. Sakagami et al. / Bioorg. Med. Chem. 16 (2008) 4359–4366
substitution reaction. According to our procedure for
the preparation of the cyclopropane ring system, the
radical addition of fluoroiodoacetate to 7 in the presence
of triethyl borane produced 8 as a mixture of the two
diastereomers. The cyclization of 8 using lithium hexam-
ethyldisilazane (LHMDS) as a base produced 9, in a
highly cis-selective manner in 98% yield.26 The deprotec-
tion of the tert-butyldiphenylsilyl group of 9 using
tetrabutylammonium fluoride (TBAF) gave 10 in 92%
yield. We adopted a convergent method for the coupling
of the cyclopropane and xanthene moiety using a
palladium-catalyzed reaction of the corresponding zinc
reagent with acid chloride to obtain 14. A carboxylic acid
obtained from 10 by Jones oxidation was allowed to react
with SOCl2 to yield acid chloride 11. According to the
reported method for the preparation of zinc reagents,27
zinc reagent 13 was synthesized from iodide 1220 using
zinc powder in the presence of cuprous chloride. Then,
the zinc reagent 13 was reacted with 11 in the presence
of Pd(PPh3)4 togive 14in 68% yield (threesteps from com-
pound 10). Compound 14 was allowed to react under
Bucherer–Bergs conditions28 to yield two hydantoin dia-
stereomers. To separate the diastereomeric mixtures, we
converted the two hydantoin diastereomers to p-meth-
oxybenzylhydantoins 15. Then the two diastereomers
15a (a highly polar isomer) and 15b were separated by
silica gel column chromatography.
Figure 1. Group II mGluRs antagonists and their prodrugs.
but its oral bioavailability was 10.9%. However, we
recently reported the pharmacokinetics of prodrugs of
2 and noted that the oral administration of ester-type
prodrugs improved the bioavailability of 2.24 Com-
pound 4 exhibited an approximately 10-fold enhance-
ment in the plasma and brain concentrations of 2 as a
result of the high reactivity of the ethyl ester during
hydrolysis, arising from the electronegativity of the fluo-
rine atom. In contrast, compound 5, a non-fluorinated
analogue, exhibited a poor pharmacokinetic profile.
We also reported the results of both in vitro and
The hydrolysis of ( )-15a and ( )-15b with 1 M NaOH
at 200 ꢁC in a sealed apparatus for between 44 h and 5.5
days gave ( )-16a and ( )-16b. The harsh conditions
required to complete these reactions gave a complicated
mixture, resulting in a low yield. Compound ( )-15a was
resolved into two enantiomers, (+)-15a and (ꢀ)-15a,
using HPLC. Finally, the hydrolysis of (+)-15a and
(ꢀ)-15a under the above-mentioned conditions yielded
(+)-16a and (ꢀ)-16a, respectively. The selective esterifi-
cation of (+)-16a at the C-1 carboxy group in the cyclo-
propane ring with n-heptanol was performed in the
presence of SOCl2 at 70 ꢁC to obtain (+)-17 in 78%
yield.
in vivo studies indicating that heptyl ester
6
(MGS0210), a prodrug of 2, exhibited an optimal phar-
macokinetic profile. In these previous papers, we con-
cluded that a fluorine atom at the C-6 position of the
bicyclo[3.1.0] hexane ring played an important role in
the binding affinity to mGluR2, strongly influencing
the compounds’ pharmacokinetic profiles.
With this background in mind, we focused on a com-
pound fluorinated at the alpha position of carboxylic
acid in the cyclopropane ring of the group II mGluRs
antagonist 1 (LY341495). Although general methods
for the mild and selective introduction of a fluorine
atom into organic molecules are limited,25 we previously
reported a convenient stereoselective preparation meth-
od for 1-fluorocyclopropane-1-carboxylate.26 Thus, we
examined the synthesis of the fluorinated compound
using our reported synthetic method for 1-fluorocyclo-
propane-1-carboxylate. In this paper, we report the syn-
thesis, in vitro pharmacology, and pharmacokinetic
profiles of the fluorinated compound and its prodrug,
heptyl ester.
The relative configurations of the two diastereomers
were determined as follows. X-ray diffraction analysis
of carboxylic acid 18, which was obtained from 15b by
hydrolysis with LiOHÆH2O (Scheme 2), showed that
18 is (1RS,2RS)-1-fluoro-2-[(4RS)-1-(4-methoxyben-
zyl)-2,5-dioxo-4-(9H-xanthen-9-ylmethyl)-imidazolidin-
4-yl]cyclopropanecarboxylic acid (Fig. 2). Both 15a and
15b have the same relative configuration for their cyclo-
propane ring system; therefore, the X-ray diffraction
analysis of 18 suggested that the stereochemistry of
15a, a diasteromer of 15b, was 1RS, 2RS, 4SR.
2. Chemistry
3. Results and discussion
Scheme 1 illustrates the synthesis of fluorinated com-
pounds ( )-16a, ( )-16b, (+)-16a, (ꢀ)-16a, and its pro-
drug (+)-17. We previously reported a stereoselective
preparation for 1-fluorocyclopropane-1-carboxylate
through the iodo-atom transfer radical addition of fluor-
oiodoacetate to alkenes, followed by an intramolecular
3.1. Pharmacological profile studies
The mGluR2 binding affinities of the fluorinated com-
pounds ( )-16a, ( )-16b, (+)-16a and (ꢀ)-16a were eval-
uated by examining [3H]-(1S,2S,3S,5R,6S)-2-amino-3-
fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid binding

K. Sakagami et al. / Bioorg. Med. Chem. 16 (2008) 4359–4366
4361
Scheme 1. Reagents and conditions: (a) ICFHCO2Et, Et3B, CH2Cl2, rt, 6.5 days, 98%; (b) LHMDS, THF, ꢀ66 ꢁC; 4.5 h, 98%; (c) TBAF, THF, rt,
1 h, 92%; (d) i—Jones reagent, acetone, 4 ꢁC, 40 min, ii—SOCl2, rt, 18 h; (e) Zn, CuCl, DMF, benzene, 60 ꢁC, 3 h; (f) 11, PdCl2(PPh3)2, benzene, rt,
3 h, 68% (three steps, from 10); (g) i—1 M NaOH, EtOH, rt; 30 min, ii—KCN, (NH4)2CO3, 60 ꢁC, 5 days, 77%, iii—p-methoxybenzyl chloride,
K2CO3, DMF, 125 ꢁC, 12 h, 15a (29%), 15b (47%); (h) 1 M NaOH, 200 ꢁC (in a stainless sealed apparatus), ( )-16a (44 h, 18%), ( )-16b (5.5 days,
20%), (+)-16a (3 days, 51%), (ꢀ)-16a (3 days, 14%); (i) chiral HPLC; (j) n-heptanol, SOCl2, 70 ꢁC, 4 h, 78%.
fully synthesized (+)-16a, a compound, that is, fluori-
O
nated at the alpha position of the carboxylic acid in
the cyclopropane ring of the group II mGluRs antago-
nist 1, and found that (+)-16a exhibited a high affinity
for mGluR2.
H
HO2C
F
a
HN
15b
( )-
O
N
O
MPM
18
3.2. Pharmacokinetic evaluation
Scheme 2. Reagents and conditions: (a) LiOHÆH2O, THF, H2O, rt,
30 min, 89%.
Table 2 shows the pharmacokinetic parameters of (+)-
16a and its prodrug (+)-17 following the oral adminis-
tration to male Crl:CD(SD) rats at a dose of 10 mg/
kg. Compound (+)-16a exhibited higher plasma concen-
in CHO cells that stably expressed mGluR2. The IC50
values obtained for mGluR2 are shown in Table 1.
Compound ( )-16a exhibited an approximately 47-fold
higher binding affinity than its diastereomer ( )-16b.
Furthermore, enantiomer (+)-16a also exhibited a high
binding affinity for mGluR2 (IC50 = 3.49 nM). The
enantiomer (+)-16a showed almost the same affinity to
mGluR2 as 1, and the IC50 was 2.90 nM. We success-
trations (AUC0–1: 3310 ng h/mL) than 1 (AUC0–1
;
284 ng h/mL).21 Furthermore, the plasma levels of the
parent compound (+)-16a following the oral administra-
tion of the prodrug (+)-17 to rats were much higher
(AUC0–1; 78,000 ng h/mL) than those observed follow-
ing the oral administration of (+)-16a. The introduction
of a fluorine atom to 1 (LY341495) allowed the high

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K. Sakagami et al. / Bioorg. Med. Chem. 16 (2008) 4359–4366
Figure 2. Stereoview of 18 from the X-ray crystallograph.
Table 1. Binding affinity for mGluR2
S9 fractions. It would, therefore, be necessary to exam-
ine some prodrugs of (+)-16a for further progression.
Compound
Affinitya IC50 (nM)
( )-16a
( )-16 b
(+)-16a
4.45
211
4. Conclusions
3.49
>10,000
2.90
(ꢀ)-16a
LY34149521
In summary, we successfully synthesized (+)-16a, which
contains a fluorine atom at the C-1 position of 1, using a
previously reported stereoselective cyclopropanation
reaction.26 Compound (+)-16a exhibited almost the
same affinity for mGluR2 as 1.
a Affinity for mGluR2 was determined by the binding study utilizing
[3H]-(1S,2S,3S,5R,6S)-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicar-
boxylic acid. IC50 values were calculated from the concentration–
response curves obtained in duplicate.
The introduction of the fluorine atom at the C-1 posi-
tion of the cyclopropane ring in 1 improved the oral bio-
availability. Furthermore, the plasma levels of (+)-16a
were elevated following the administration of prodrug
(+)-17. However, further structural conversions of pro-
drug (+)-17 are required to obtain a favorable pharma-
cokinetic profile in humans because of the low
transformation ratio of (+)-17 to the parent compound
(+)-16a, as observed in the in vitro study using liver
S9 fractions.
Table 2. Pharmacokinetic parameters for (+)-16a and (+)-17 following
the oral administration to rats at the dose of 10 mg/kg
Compound
(+)-16a
(+)-17a
LY34149521
Tmax (h)
Cmax (ng/mL)
t1/2 (h)
2.67
308
2.00
0.50
64.1
2.64
284
8640
3.87
5.05
3310
AUC0–1 (ng h/mL)
78,000
a Pharmacokinetic parameters of the parent compound (+)-16a after
oral administration (+)-17 to rats.
affinity for mGluR2 to be retained and improved the
oral bioavailability. Furthermore, the plasma levels of
(+)-16a were significantly elevated following the admin-
istration of the prodrug.
5. Experimental
Melting points were determined on a Yanaco MP-500D
melting point apparatus, and are uncorrected. H and
1
19F NMR spectra were obtained using a Varian Gemini
2000, Varian Unity Inova 300, JEOL Alpha500, Lamb-
da500, or ECA500. Chemical shifts are reported in parts
per million relative to tetramethylsilane (TMS), sodium
3-trimethylsilylpropionate-2,2,3,3-d4 (TMSP) or trichlo-
rofluoromethane as the internal standard. Mass spectra
(MS) were obtained on a Micromass Platform LC (ESI).
High-resolution spectra were recorded on a Micromass
GCT instrument or Micromass Q-TOF2 instrument.
Optical rotations were determined with a JASCO DIP-
360 polarimeter and are reported at the sodium D-line
(589 nm). Elemental analyses were performed on a Per-
kin-Elmer 2400. Silica gel [C-200, 100–200 mesh (Wako
Pure Chemical)] was used for the column chromatogra-
phy using the solvent systems (volume ratios) indicated
below.
We next investigated the in vitro metabolic stabilities of
prodrug (+)-17 using rat, monkey, and human liver S9
fractions. Table 3 shows the transformation ratio of
(+)-17 to (+)-16a. Prodrug (+)-17 yielded the carboxylic
acid form (+)-16a when it was incubated with rat, mon-
key, or human liver S9 fractions. However, the conver-
sion ratios of (+)-17 in the liver S9 fractions were
relatively low, with the exception of that in the rat liver
Table 3. Transformation ratio (%) from prodrug (+)-17 to carboxylic
acid (+)-16a by liver S9 fractions
Rat S9
68.1
Monkey S9
7.30
Human S9
12.1

K. Sakagami et al. / Bioorg. Med. Chem. 16 (2008) 4359–4366
4363
5.1. 5-(tert-Butyldiphenylsilanyloxy)-2-fluoro-4-iodopen-
tanoic acid ethyl ester (8)
5.4. (1RS,2RS)-1-Fluoro-2-(2-9H-xanthen-9-yl-acetyl)-
cyclopropanecarboxylic acid ethyl ester (14)
Triethylborane (1.01 M in hexanes, 88.8 mL, 89.7 mmol)
was added to a mixture of alloxy-tert-butyldiphenylsi-
lane 7 (26.6 g, 89.7 mmol) and ethyl fluoroiodoacetate
(52.0 g, 224 mmol) in CH2Cl2 (380 mL), and the reaction
mixture was stirred for 6 h. To the reaction mixture was
added triethylborane (1.01 M hexane soln, 44.4 mL,
44.8 mmol), and stirred for 3 h. To the reaction mixture
was added triethylborane (1.01 M hexane soln, 44.4 mL,
44.8 mmol), and stirred for 3 days. To the reaction mix-
ture was added triethylborane (1.01 M hexane soln,
44.4 mL, 44.8 mmol), and stirred for 3 days. The reaction
mixture was washed with H2O and brine, dried over
MgSO4, concentrated under reduced pressure, and chro-
matographed on silica gel (hexane/EtOAc = 15:1) to
yield 8 (46.3 g, 98% as a diastereomeric mixture) as a col-
orless oil. 1H NMR (300 MHz, CDCl3) d: 1.06–1.10 (9H,
m), 1.32 (3H, t, J = 7.2 Hz), 2.14–2.79 (2H, m), 3.80–3.94
(2H, m), 4.19–4.38 (3H, m), 4.93–5.24 (1H, m), 7.33–7.51
(6H, m), 7.56–7.74 (4H, m); HRMS (ESI) m/z 551.0895
(M+Na)+, calcd for C23H30FINaO3Si: 551.0891.
8 M Jones reagent (130 mL) was added to a mixture of
10 (21.2 g, 131 mmol) and acetone (260 mL) with ice-
cooling, and the mixture was stirred for 40 min.
Saturated aqueous NaHSO3 was added to the reaction
mixture and extracted with EtOAc three times. The
combined organic layer was washed with brine, dried
over MgSO4, and concentrated under reduced pressure.
SOCl2 (180 mL) was added to the residue and the
mixture was stirred at room temperature for 18 h. The
mixture was concentrated under reduced pressure to
give crude 11.
A
mixture of 9-iodomethyl-9H-xanthene20 (66.4 g,
206 mmol), zinc powder (67.4 g, 1.03 mmol), CuCl
(5.10 g, 51.5 mmol), DMF (70 mL), and benzene
(640 mL) was stirred at 60 ꢁC for 3 h under nitrogen
atmosphere. After cooling, a mixture of crude 11,
Pd(PPh3)4 (14.5 g, 20.6 mmol), and benzene (160 mL)
was added to the reaction mixture and stirred at room
temperature for 3 h. The reaction mixture was diluted
with EtOAc, and filtered through Celiteꢂ pad. The fil-
trate was washed with 10% aqueous NaHSO4, saturated
aqueous NaHCO3, and brine. The organic layer was
dried over MgSO4, concentrated under reduced pressure,
and chromatographed on silica gel (hexane/
EtOAc = 10:1–8:1) to yield 14 [31.7 g, 68% (three steps,
from 10)] as a colorless oil. 1H NMR (300 MHz, CDCl3)
d: 1.29 (3H, t, J = 7.2 Hz), 1.59 (1H, dt, J = 9.5, 6.3 Hz),
2.16 (1H, ddd, J = 18.4, 8.1, 6.3 Hz), 2.49 (1H, ddd,
J = 9.5, 8.1, 2.8 Hz), 2.96 (1H, dd, J = 17.1, 6.1 Hz),
3.04 (1H, dd,J = 17.1, 7.0 Hz), 4.23 (2H, q, J = 7.2 Hz),
4.67 (1H, dd, J = 7.0, 6.1 Hz), 7.02–7.32 (8H, m); HRMS
(ESI) m/z 377.1184 (M+Na)+, calcd for C21H19FNaO4:
377.1165.
5.2. (1RS,2RS)-2-(tert-Butyldiphenylsilanyloxymethyl)-
1-fluorocyclopropancarboxylic acid ethyl ester (9)
LHMDS (29% in THF, 171 mL, 297 mmol) was
added to a solution of 8 (52.3 g, 99.0 mmol) in THF
(600 mL) at ꢀ66 to ꢀ61 ꢁC, and the mixture was stir-
red at ꢀ66 ꢁC for 4.5 h. 1 M HCl was added to the
reaction mixture, and extracted with Et2O. The organ-
ic layer was washed with saturated aqueous NaHCO3
and brine, dried over Na2SO4, concentrated under re-
duced pressure, and chromatographed on silica gel
(hexane/EtOAc = 20:1) to yield 9 (38.7 g, 98%) as a
colorless oil. 1H NMR (300 MHz, CDCl3) d: 1.05
(9H, s), 1.07–1.19 (1H, m), 1.31 (3H, t, J = 7.1 Hz),
1.47 (1H, ddd, J = 10.5, 8.7, 6.5 Hz), 1.86–1.98 (1H,
m), 3.75 (1H, ddd, J = 11.2, 7.9, 1.2 Hz), 3.90 (1H,
ddd, J = 11.2, 5.8, 1.3 Hz), 4.27 (2H, q, J = 7.1 Hz),
7.33–7.47 (6H, m), 7.63–7.71 (4H, m); HRMS (ESI)
m/z 401.1967 (M+H)+, calcd for C23H30FO3Si:
401.1948.
5.5. (1RS,2RS)-1-Fluoro-2-[(4SR)-1-(4-methoxybenzyl)-
2,5-dioxo-4-(9H-xanthen-9-ylmethyl)-imidazolidin-4-yl]-
cyclopropanecarboxylic acid 4-methoxybenzyl ester (15a)
and (1RS,2RS)-1-fluoro-2-[(4RS)-1-(4-methoxybenzyl)-
2,5-dioxo-4-(9H-xanthen-9-ylmethyl)-imidazolidin-4-yl]-
cyclopropanecarboxylic acid 4-methoxybenzyl ester (15b)
5.3. (1RS,2RS)-1-Fluoro-2-hydroxymethyl-cyclopropane-
carboxylic acid ethyl ester (10)
A mixture of 14 (31.6 g, 89.2 mol), 1 M NaOH
(98.1 mL, 98.1 mmol), and EtOH (200 mL) was stirred
at room temperature for 30 min. Ammonium carbonate
(77.1 g, 803 mmol), KCN (29.0 g, 446 mmol), and H2O
(102 mL) were added to the reaction mixture, and the
mixture was stirred at 60 ꢁC for 5 days. The reaction
mixture was washed with Et2O, and adjusted with 6 M
HCl to pH 1. After stirring the mixture with ice-cooling
for 1 h, the precipitation was filtered and washed with
H2O and IPE to yield a mixture of two hydantoin diaste-
reomers (27.3 g, 77%). A mixture of the precipitation
(2.37 g, 5.98 mmol), KHCO3 (1.67 g, 16.7 mmol), 4-
methoxybenzyl chloride (1.87 mL, 13.8 mol), DMF
(18 mL) was stirred at 125 ꢁC for 12 h. The reaction mix-
ture was diluted with Et2O, and washed with H2O. The
aqueous layer was extracted with Et2O twice. The com-
bined organic layer was dried over MgSO4, concentrated
under reduced pressure, and chromatographed on silica
TBAF (1 M in THF, 472 mL, 472 mmol) was added to a
mixture of 9 (126 g, 315 mmol) and THF (780 mL), and
the mixture was stirred at room temperature for 1 h. The
mixture was concentrated under reduced pressure, and
the residue was dissolved with EtOAc. The organic layer
was wash with saturated aqueous NH4Cl. The aqueous
layer was extracted with EtOAc. The combined organic
layer was washed with brine, dried over MgSO4, concen-
trated under reduced pressure, and chromatographed on
silica gel (hexane/EtOAc = 2:1–1:2) to yield 10 (46.7 g,
1
92%) as a colorless oil. H NMR (300 MHz, CDCl3) d:
1.19–1.29 (1H, m), 1.32 (3H, t, J = 7.2 Hz), 1.51–1.64
(2H, m), 1.90–2.07 (1H, m), 3.67 (1H, dd, J = 11.1,
8.9 Hz), 3.93–4.07 (1H, m), 4.27 (2H, q, J = 7.15 Hz);
MS (ESI) m/z 185 (M+H)+.

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K. Sakagami et al. / Bioorg. Med. Chem. 16 (2008) 4359–4366
gel (hexane/AcOEt = 3:1–1:1) to yield 15a (1.12 g, 29%)
and 15 b (1.79 g, 47%) as a colorless solid. ( )-15a; H
din-4-yl]-cyclopropanecarboxylic acid 4-methoxybenzyl
ester ((ꢀ)-15a)
1
NMR (300 MHz, CDCl3) d: 1.34–1.62 (2H, m), 1.74
(1H, ddd, J = 10.5, 8.9, 4.4 Hz), 2.33 (1H, dd, J = 14.9,
7.2 Hz), 2.44 (1H, dd, J = 14.9, 4.7 Hz), 3.71 (3H, s),
3.81 (3H, s), 4.25 (1H, dd, J = 7.2, 4.7 Hz), 4.32 (1H,
d, J = 14.5 Hz), 4.40 (1H, d, J = 14.5 Hz), 4.49 (1H, s),
5.06 (1H, d, J = 12.4 Hz), 5.11 (1H, d, J = 12.4 Hz),
6.70–6.92 (4H, m), 7.03–7.29 (12H, m); HRMS (ESI)
m/z: 637.2343 (M+H)+, calcd for C37H34FN2O7:
637.2350. ( )-15b; 1H NMR (300 MHz, CDCl3) d:
1.17–1.30 (2H, m), 1.93–2.06 (1H, m), 2.24 (1H, dd,
J = 14.8, 5.4 Hz), 2.67 (1H, dd, J = 14.8, 6.0 Hz), 3.74
(3H, s), 3.81 (3H, s), 4.10 (1H, dd, J = 6.0, 5.4 Hz),
4.19 (1H, d, J = 14.5 Hz), 4.38 (1H, d, J = 14.5 Hz),
4.54 (1H, s), 5.07 (1H, d, J = 11.8 Hz), 5.12 (1H, d,
J = 11.8 Hz), 6.76–6.90 (4H, m), 6.97–7.15 (6H, m),
7.17–7.29 (6H, m); HRMS (ESI) m/z 637.2338
(M+H)+, calcd for C37H34FN2O7: 637.2350.
Compound ( )-15a (720 mg, 1.13 mmol) was resolved
using CHIRALPAK AD (DAICEL CHEMICAL
IND., LTD, 2.0 · 25 cm, eluent; hexane/2-propa-
nol = 1:2, flow rate; 4.0 mL/min., temperature; room
temperature, detect; UV 220 nm) to (+)-15a (294 mg)
and (ꢀ)-15a (299 mg).
1
(+)-15a; H NMR (300 MHz, CDCl3) d: 1.34–1.62 (2H,
m), 1.74 (1H, ddd, J = 10.5, 8.9, 4.4 Hz), 2.33 (1H, dd,
J = 14.9, 7.2 Hz), 2.44 (1H, dd, J = 14.9, 4.7 Hz), 3.71
(3H, s), 3.81 (3H, s), 4.25 (1H, dd, J = 7.2, 4.7 Hz),
4.32 (1H, d, J = 14.5 Hz), 4.40 (1H, d, J = 14.5 Hz),
4.49 (1H, s), 5.06 (1H, d, J = 12.4 Hz), 5.11 (1H, d,
J = 12.4 Hz), 6.70–6.92 (4H, m), 7.03–7.29 (12H, m);
28
MS (ESI) m/z 635 (MꢀH)ꢀ; ½aꢂ +66.0ꢁ (c = 0.577,
D
(CHIRALPAK
CHCl3);
tR = 17.5 min
AD,
0.46 · 25 cm, eluent; hexane/2-propanol = 1:2, flow rate;
1.0 mL/min, temperature; room temperature, detect; UV
220 nm).
5.6. (1RS,2RS)-2-[(1SR)-1-amino-1-carboxy-2-(9H-xan-
then-9-yl)ethyl]-1-fluorocyclopropancarboxylic acid (( )-
16a)
1
(ꢀ)-15a; H NMR (300 MHz, CDCl3) d: 1.34–1.62 (2H,
A mixture of ( )-15a (278 mg, 0.437 mol) and 1 M
NaOH (8.4 mL) was stirred at 200 ꢁC for 44 h in a stain-
less sealed apparatus. After cooling, the mixture was ad-
justed to pH 1 with 1 M HCl. The mixture was
chromatographed on AG50W-X8 cation-exchange resin
(Bio-Rad) (H2O ꢁ 50% aqueous THF ꢁ 10% aqueous
pyridine) yield to crude product (61 mg). THF (1 mL)
was added to the crude product and stirred at room tem-
perature for 6 h. The solid was collected and washed
with THF to give ( )-16a (29 mg, 18%) as a white solid:
m), 1.74 (1H, ddd, J = 10.5, 8.9, 4.4 Hz), 2.33 (1H, dd,
J = 14.9, 7.2 Hz), 2.44 (1H, dd, J = 14.9, 4.7 Hz), 3.71
(3H, s), 3.81 (3H, s), 4.25 (1H, dd, J = 7.2, 4.7 Hz), 4.32
(1H, d, J = 14.5 Hz), 4.40 (1H, d, J = 14.5 Hz), 4.49 (1H,
s), 5.06 (1H, d, J = 12.4 Hz), 5.11 (1H, d, J = 12.4 Hz),
6.70–6.92 (4H, m), 7.03–7.29 (12H, m); MS (ESI) m/z
27
D
635 (MꢀH)ꢀ; ½aꢂ
ꢀ65.8ꢁ (c = 0.975, CHCl3);
tR = 12.3 min (CHIRALPAK AD, 0.46 · 25 cm, eluent;
hexane: 2-propanol = 1: 2, flow rate; 1.0 mL/min., tem-
perature; room temperature, detect; UV 220 nm).
1
mp > 270 ꢁC (decomposed). H NMR (300 MHz, D2O)
d: 1.34–1.55 (2H, m), 1.84–2.00 (1H, m), 2.10 (1H, dd,
J = 15.0, 7.7 Hz), 2.29 (1H, dd, J = 15.0, 5.8 Hz), 4.23
(1H, dd, J = 7.7, 5.8 Hz), 7.15–7.50 (8H, m); 19F NMR
(470 MHz, D2O) d: ꢀ197.1; MS (ESI) m/z 370 (MꢀH)ꢀ.
Anal. Calcd for C20H18FNO5Æ0.3H2O: C, 63.76; H, 4.98;
N, 3.72; F, 5.04. Found: C, 63.88; H, 4.89; N, 3.77; F,
5.02.
5.9. (+)-(1R*,2R*)-2-[(1S*)-1-Amino-1-carboxy-2-(9H-
xanthen-9-yl)ethyl]-1-fluorocyclopropancarboxylic acid
((+)-16a)
In a manner similar to the preparation of ( )-16a from
( )-15a, (+)-16a (679 mg, 51%, as a white solid) was ob-
tained from (+)-15a (1.21 mg, 1.90 mmol). Mp > 270 ꢁC
1
(decomposed); H NMR (300 MHz, D2O) d: 1.34–1.55
5.7. (1RS,2RS)-2-[(1RS)-1-amino-1-carboxy-2-(9H-xan-
then-9-yl)ethyl]-1-fluorocyclopropancarboxylic acid (( )-
16b)
(2H, m), 1.84–2.00 (1H, m), 2.10 (1H, dd, J = 15.0,
7.7 Hz), 2.29 (1H, dd, J = 15.0, 5.8 Hz), 4.23 (1H, dd,
J = 7.7, 5.8 Hz), 7.15–7.50 (8H, m); 19F NMR
(470 MHz, D2O) d: ꢀ197.1. MS (ESI) m/z 370 (MꢀH)ꢀ;
Anal. Calcd for C20H18FNO5Æ1.7 H2O: C, 59.76; H,
In a manner similar to the preparation of ( )-16a from
( )-15a, ( )-16b (40 mg, 20%, as a white solid) was ob-
tained from ( )-15b (343 mg, 0.539 mmol). Mp > 295 ꢁC
5.37; N, 3.48; F, 4.73. Found: C, 59.87; H, 5.36; N,
26
D
3.54; F, 4.77; ½aꢂ +27.1ꢁ (c = 0.335, 1 M NaOH).
1
(decomposed); H NMR (300 MHz, D2O) d: 1.39–1.53
(1H, m), 1.69–1.88 (2H, m), 2.23 (1H, dd, J = 15.1,
8.0 Hz), 2.48 (1H, dd, J = 15.1, 5.8 Hz), 4.26 (1H, dd,
J = 8.0, 5.8 Hz), 7.18–7.47 (8H, m); 19F NMR
(470 MHz, D2O) d: ꢀ197.4; MS (ESI) m/z 370 (MꢀH)ꢀ;
Anal. Calcd for C20H18FNO5ÆH2O: C, 61.69; H, 5.18; N,
3.60; F, 4.88. Found: C, 61.41; H, 4.98; N, 3.54; F, 4.78.
5.10. (ꢀ)-(1S*,2S*)-2-[(1R*)-1-Amino-1-carboxy-2-(9H-
xanthen-9-yl)ethyl]-1-fluorocyclopropancarboxylic acid
((ꢀ)-16a)
In a manner similar to the preparation of ( )-16a from
( )-15a, (ꢀ)-16a (22 mg, 14%, as a white solid) was ob-
tained from (ꢀ)-15a (266 mg, 0.418 mmol). Mp >
270 ꢁC (decomposed); 1H NMR (300 MHz, D2O) d:
1.34–1.55 (2H, m), 1.84–2.00 (1H, m), 2.10 (1H, dd,
J = 15.0, 7.7 Hz), 2.29 (1H, dd, J = 15.0, 5.8 Hz), 4.23
(1H, dd, J = 7.7, 5.8 Hz), 7.15–7.50 (8H, m); 19F NMR
(470 MHz, D2O) d: ꢀ197.1. MS (ESI) m/z 370 (MꢀH)ꢀ;
5.8. (+)-(1R*,2R*)-1-Fluoro-2-[(4S*)-1-(4-methoxyben-
zyl)-2,5-dioxo-4-(9H-xanthen-9-ylmethyl)-imidazolidin-4-
yl]-cyclopropanecarboxylic acid 4-methoxybenzyl ester
((+)-15a) and (ꢀ)-(1S*,2S*)-1-fluoro-2-[(4R*)-1-(4-methoxy-
benzyl)-2,5-dioxo-4-(9H-xanthen-9-ylmethyl)-imidazoli-

K. Sakagami et al. / Bioorg. Med. Chem. 16 (2008) 4359–4366
4365
Anal. Calcd for C20H18FNO5Æ0.1 H2O: C, 64.37; H, 4.92;
N, 3.75; F, 5.09. Found: C, 64.26; H, 4.92; N, 3.58; F, 5.06;
½aꢂ ꢀ25.8ꢁ (c = 0.329, 1 M NaOH).
5.11. (+)-(1R*,2R*)-2-[(1S*)-1-Amino-1-carboxy-2-(9H-
xanthen-9-yl)ethyl]-1-fluorocyclopropanecarboxylic acid
heptyl ester ((+)-17)
For a typical binding experiment, the reaction was initi-
ated by incubating 0.5 mL of the crude membrane prepa-
ration with [3H]MGS0008. The reaction mixture was
incubated for 1 h at 25 ꢁC. The reaction was terminated
by rapid filtration through Whatman GF/C glass fiber fil-
ters presoaked with 0.3% polyethyleneimine, after which
the filters were washed three times with 3 mL of ice-cold
50 mM Tris–HCl buffer (pH 7.4), using a multicell har-
vester M-48 (Perkin-Elmer Life and Analytical Science
Inc., Wellesly, MA, USA). Aquazol-2 scintillator (Per-
kin-Elmer Life and Analytical Science Inc., Wellesly,
MA, USA) (10 mL) was added, and filter-bound radioac-
tivity was counted in a liquid scintillation spectrometer
(LS6000TA, Perkin-Elmer Life and Analytical Science
Inc., Wellesly, MA, USA). Nonspecific binding was deter-
mined in the presence of 10 lM LY354740.
24
D
A mixture of (+)-16a (281 mg, 0.757 mmol), SOCl2
(220 lL, 3.03 mmol), and 1-heptanol (2.8 mL) was stir-
red at 70 ꢁC for 4 h. SOCl2 was removed under reduced
pressure at 80 ꢁC. EtOH (2.2 mL) and propylene oxide
(2.2 mL) was added to the residue, and the mixture
was refluxed for 1 h. After removing the solvent, Et2O
was added and stirred at room temperature for 12 h.
The precipitation was collected and washed with Et2O
and IPE to yield (+)-17 (277 mg, 78%) as a white solid.
1H NMR (300 MHz, CD3OD) d: 0.90 (3H, t,
J = 6.7 Hz), 1.26–1.53 (10H, m), 1.57–1.69 (2H, m),
2.05 (1H, dd, J = 14.6, 6.5 Hz), 2.10–2.17 (1H, m),
2.22 (1H, dd, J = 14.6, 6.5 Hz), 4.07–4.21 (2H, m),
4.30 (1H, t, J = 6.45 Hz), 7.06–7.19 (4H, m), 7.21–7.33
(2H, m), 7.44–7.53 (2H, m); 19F NMR (470 MHz,
In the competition binding assay, the reaction was car-
ried out using 3.0 nM [3H]MGS0008. The concentration
of the test compound that caused 50% inhibition of spe-
cific binding of [3H]MGS0008 (IC50 value) was deter-
mined from each concentration–response curve.
CD3OD) d: ꢀ206.4; HRMS (ESI) m/z 468.2201
5.14. Pharmacokinetic evaluation
27
D
(MꢀH)ꢀ, calcd for C27H31FNO5: 468.2186; ½aꢂ
+10.6ꢁ (c = 0.537, pyridine).
Each compound was administered to male Crl:CD(SD)
rats (Charles River, Japan) orally at a dose of 10 mg/kg.
Blood samples were taken from the tail vein at 0.5, 1, 2,
4, 6, 8, and 24 h post-dose into EDTA-containing tubes.
Immediately plasma was separated by centrifugation
(11,200g, 4 ꢁC, 2 min), and then plasma was mixed with
5 M HCl (plasma/5 M HCl = 50:1). The plasma samples
were frozen with liquid N2 immediately and stored at
ꢀ80 ꢁC until bioanalysis.
5.12. (1RS,2RS)-1-Fluoro-2-[(4RS)-1-(4-methoxybenzyl)-
2,5-dioxo-4-(9H-xanthen-9-ylmethyl)-imidazolidin-4-yl]-
cyclopropanecarboxylic acid (18)
A mixture of ( )-15b (940 mg, 1.48 mol), LiOHÆH2O
(93 mg, 2.21 mmol), THF (11 mL), and H2O (5.5 mL)
was stirred at room temperature for 30 min. The reac-
tion mixture was washed with EtOAc, and adjusted with
1 M HCl to pH 2. The aqueous layer was extracted with
CHCl3. The organic layer was dried over MgSO4 and
concentrated under reduced pressure to yield 18
To 50-lL aliquot of plasma sample, 400 lL of aceto-
nitrile/methanol (9:1) solution containing internal stan-
dard was mixed, and centrifuged at 11,200g, 4 ꢁC,
15 min. The resulting supernatant was analyzed by li-
quid chromatography tandem mass spectrometry (LC–
MS/MS) on an Agilent ZORBAX SB-C18 column
(5 lm, 50 · 2.1 mm). The analytes were eluted with
mobile phase in linear gradient by increasing acetoni-
trile concentration in 0.1% acetic acid from 5% to
95% over 4 min at flow rate of 250 lL/min. Tandem
mass spectrometric detection was carried out using
TurboIonSpray (AB/MDS Sciex API3000) in positive
ion mode.
1
(682 mg, 89%). H NMR (300 MHz, CDCl3) d: 1.27–
1.40 (m, 2H), 1.96–2.09 (m, 1H), 2.27 (dd, J = 14.8,
5.4 Hz, 1H), 2.71 (dd, J = 14.7, 6.0 Hz, 1H), 3.75 (s,
3H), 4.12 (t, J = 5.8 Hz, 1H), 4.20 (d, J = 14.6 Hz,
1H), 4.38 (d, J = 14.6 Hz, 1H), 5.39 (s, 1H), 6.76–6.84
(m, 2H), 6.97–7.30 (m, 10H); HRMS (ESI) m/z:
517.1786 (M+H)+, calcd for C29H26FN2O6: 517.1775.
5.13. [3H]-(1S,2S,3S,5R,6S)-2-Amino-3-fluorobicyclo-
[3.1.0]hexane-2,6-dicarboxylic acid ([3H]MGS0008)
binding
5.15. Metabolic stability study
The binding of [3H]MGS0008 (34 Ci/ mmol) was per-
formed according to the method previously described.12
CHO cells stably expressing mGluR2 were collected by
centrifugation at 1000 rpm for 5 min. The cells were
homogenized with 50 mM Tris–HCl buffer (pH 7.4)
and centrifuged at 48,000g for 20 min at 4 ꢁC. The pellet
was suspended in 50 mM Tris–HCl buffer (pH 7.4), and
incubated at 37 ꢁC for 15 min, after which the pellet was
washed twice with 50 mM Tris–HCl buffer (pH 7.4) by
resuspension and recentrifugation. The pellet obtained
was then suspended in 50 mM Tris–HCl buffer (pH
7.4) containing 2 mM MgCl2 and served as a crude
membrane preparation.
Liver S9 fractions (1 mg protein/mL) from rat (BD Bio-
science), monkey (Daiichi), and human (Xenotech) were
incubated with 3 lM prodrug in the presence of an
NADPH generating system (0.16 mM NADP+,
2.4 mM MgCl2, and 1.5 mM glucose-6-phosphate) in
0.25 M phosphate buffer containing 69 mM KCl (pH
7.4) for 60 min at 37 ꢁC. All experiments were per-
formed in triplicate. After incubation, a twofold volume
of DMSO was added to incubation medium, and the
tube was vortexed and centrifuged at 2150g, 4 ꢁC,
10 min. The resulting supernatant was subjected to
LC–MS/MS analysis.

4366
K. Sakagami et al. / Bioorg. Med. Chem. 16 (2008) 4359–4366
14. Moghaddam, B.; Adams, B. W. Science 1998, 281, 1349.
Acknowledgments
15. Grillon, C.; Cordova, J.; Levine, L. R.; Morgan, C. A.
Psychopharmacology 2003, 168, 446.
16. Tizzano, J. P.; Griffey, K.; Schoepp, D. D. Pharmacol.
Biochem. Behav. 2002, 73, 367.
17. Helton, D. R.; Tizzano, J. P.; Monn, J. A.; Schoep,
D. D.; Kallman, M. J. J. Pharmacol. Exp. Ther. 1998,
284, 651.
The authors thank Professor Sigetada Nakanishi of the
Department of Biological Sciences, Faculty of Medicine,
Graduate School of Biostudies, Kyoto University, for
providing us with the CHO cell line that stably ex-
pressed rat mGluR2.
18. Monn, J. A.; Valli, M. J.; Massey, S. M.; Wright, R. A.;
Salhoff, C. R.; Johnson, B. G.; Howe, T.; Alt, C. A.;
Rhodes, G. A.; Robey, R. L.; Griffey, K. R.; Tizzano, J.
P.; Kallman, M. J.; Helton, D. A.; Schoepp, D. D. J. Med.
Chem. 1997, 40, 528.
19. Shekhar, A.; Keim, S. R. Neuropharmacology 2000, 39,
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20. Ornstein, P. L.; Bleisch, T. J.; Arnold, M. B.; Wright, R.
A.; Johnson, B. G.; Schoepp, D. D. J. Med. Chem. 1998,
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