Synthesis, SARs, and pharmacological characterization of 2-Amino-3 or 6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent, selective, and orally active group II metabotropic glutamate receptor agonists

J . Med. Chem. 2000, 43, 4893-4909

4893

Syn th esis, SARs, a n d P h a r m a cologica l Ch a r a cter iza tion of 2-Am in o-3 or

6-flu or obicyclo[3.1.0]h exa n e-2,6-d ica r boxylic Acid Der iva tives a s P oten t,

Selective, a n d Or a lly Active Gr ou p II Meta botr op ic Glu ta m a te Recep tor

Agon ists

Atsuro Nakazato,*^,†Toshihito Kumagai,^†Kazunari Sakagami,^†Ryoko Yoshikawa,^†Yoshiko Suzuki,^†

Shigeyuki Chaki,^†Hisanaka Ito,^‡Takeo Taguchi,^‡Shigetada Nakanishi,^§and Shigeru Okuyama^†

1st Laboratory, Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Ohmiya, Saitama

330-8530, J apan, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, J apan, and

Department of Biological Sciences, Kyoto University Faculty of Medicine, Sakyo, Kyoto 606-8501, J apan

Received August 10, 2000

(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (4, LY354740), a highly selective and

orally active group II metabotropic glutamate receptor (mGluR) agonist, has increased interest

in the study of group II mGluRs. Our interest focused on a conformationally constrained form

of compound 4, because it appeared that the rigid form resulted in not only selectivity for group

II mGluR but was orally active. Therefore, we introduced a fluorine atom to compound 4, based

on the molecular size (close resemblance to hydrogen atom) and electronegativity (effects on

the electron distribution in the molecule) of this atom and carbon-fluorine bond energy.

Compound (+)-7 (MGS0008), the best compound among 3-fluoro derivatives 7-10, retained

the agonist activity of compound 4 for mGluR2 and mGluR3 ((+)-7: EC₅₀) 29.4 ( 3.3 nM and

45.4 ( 8.4 nM for mGluR2 and mGluR3, respectively; 4: EC₅₀) 18.3 ( 1.6 nM and 62.8 ( 12

nM for mGluR2 and mGluR3, respectively) and increased the oral activity of compound 4 ((+)-

7: ED₅₀) 5.1 mg/kg and 0.26 mg/kg for phencyclidine (PCP)-induced hyperactivy and PCP-

induced head-weaving behavior, respectively; 4: ED₅₀) >100 mg/kg and 3.0 mg/kg for PCP-

induced hyperactivity and PCP-induced head-weaving behavior, respectively). In addition, a

compound [³H]-(+)-7 binding study using mGluR2 or 3 expressed in CHO cells was successful

((+)-7: K_i) 47.7 ( 17 nM and 65.9 ( 7.1 nM for mGluR2 and mGluR3, respectively; 4: K_i)

23.4 ( 7.1 nM and 53.5 ( 13 nM for mGluR2 and mGluR3, respectively). On the basis of a

successful result of compound 7, we focused on the introduction of a fluorine atom on the C6

position of compound 4. (1R,2S,5R,6R)-2-Amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic

acid ((-)-11) exhibited a high degree of agonist activity for group II mGluRs equal to that of

compound 4 or 7 ((-)-11: K_i) 16.6 ( 5.6 and 80.9 ( 31 nM for mGluR2 and mGluR3,

respectively). Our interest shifted to modification on CH₂at C4 position of compound 11, since

replacement of the CH₂group with either an oxygen atom or sulfur atom yielded compound 5

or 6, resulting in increased agonist activity. We selected a carbonyl group instead of CH₂at

the C4 position of compound 11. The carbonyl group might slightly change the relative

conformation of three functional groups, the amino group and two carboxylic acids, which have

important roles in mediating the interaction between group II mGluRs and their ligand,

compared with the CH₂group of 4, oxygen atom of 5, and sulfur atom of 6. (1R,2S,5S,6S)-2-

Amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid monohydrate ((+)-14, MGS0028)

exhibited a remarkably high degree of agonist activity for mGluR2 (K_i) 0.570 ( 0.10 nM) and

mGluR3 (K_i) 2.07 ( 0.40 nM) expressed in CHO cells but not mGluR4, 6, 7, 1a, or 5 expressed

in CHO cells (K_i) >100 000 nM). Furthermore, compound (+)-14 strongly inhibited phencyc-

lidine (PCP)-induced head-weaving behavior (ED₅₀) 0.090 µg/kg) and hyperactivity (ED₅₀

)

0.30 mg/kg) in rats. Thus, (+)-7 and (+)-14 are potent, selective, and orally active group II

mGluR agonists and might be useful not only for exploring the functions of mGluRs but in the

treatment of schizophrenia.

In tr od u ction

Normal functioning of glutamatergic synapses is re-

quired for all major functions of the brain.^1,2At present,

glutamate receptors are broadly classified into two

types: the ionotropic glutamate receptors (iGluRs), in

which the receptors have an ion channel structure, and

the metabotropic glutamate receptors (mGluRs), which

are coupled to G-proteins. iGluRs are classified phar-

macologically into three subtypes: N-methyl-D-aspartic

acid (NMDA), R-amino-3-hydroxy-5-methyl isoxazole-4-

L-Glutamate (1, Chart 1) is a neurotransmitter at the

vast majority of excitatory synapses in the brain.

^†Taisho Pharmaceutical Co., Ltd.

^‡Tokyo University of Pharmacy and Life Science.

^§Kyoto University Faculty of Medicine.

* Correspondence to Atsuro Nakazato, Ph.D., 1st Laboratory,

Medicinal Research Laboratories,Taisho Pharmaceutical Co., Ltd.,

1-403 Yoshino-cho, Ohmiya, Saitama 330-8530, J apan. Tel: +81-48-

663-1111. Fax: +81-48-652-7254. E-mail: S10968@ccm.taisho.co.jp.

Published on Web 11/22/2000

4894 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25

Nakazato et al.

Ch a r t 1

(2S,3S,4S)-2-(carboxycyclopropyl)glycine (2, L-CCG-1) is

a potent agonist for group II mGluRs but possesses

agonist effects at group I mGluRs at somewhat higher

concentrations.^16,17Recently, a highly selective group II

mGluR agonist, (+)-2-aminocyclo[3.1.0]hexane-2,6-di-

carboxylic acid (4), has been found to have oral activities

in mice in both the elevated plus maze model of anxiety

and in the compound 3 (ACPD)-induced limbic seizure

model.²⁰Furthermore, compound 4 had potent antip-

sychotic effects in an animal model designed specifically

to mimic the glutamatergic dysfunction observed in

schizophrenia¹¹and drug addiction.¹³More recently,

selective group II mGluR agonists, 2-oxa-4-aminobicyclo-

[3.1.0]hexane-4,6-dicarboxylic acid (5, LY379268) and

2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid

(6, LY389795), have been presented.²¹

It was our goal to develop novel, potent, selective, and

orally active agonists for group II mGluRs. We were

interested in introduction of fluorine atom(s) to com-

pound 4, based on the molecular size and electronega-

tivity of fluorine and carbon-fluorine bond energy.

Fluorine is not a sterically demanding substituent, since

sterically, with its small van der Waals radius, it closely

resembles hydrogen. In molecules where conformational

recognition is important, minimal steric disturbance by

a substituent is especially significant. The electronega-

tivity of fluorine can have pronounced effects on the

electron distribution in molecules, affecting the basicity

or acidity of neighboring groups, dipole moments within

molecules, and the overall reactivity and stability of

neighboring functional groups. Once introduced, the

high carbon-fluorine bond energy renders the substitu-

ent relatively resistant to metabolic transformation.²²

Given the nature of fluorine, the C3 or C6 position of

compound 4, an orally active, potent and selective group

II mGluR agonist, was selected for introduction to

fluorine atom(s), since it was expected that the intro-

duced fluorine atom(s) would directly influence func-

tional groups, carboxylic acid, and amino group at C2

or carboxylic acid at C6. Among compounds 7-11

containing fluorine atom(s) at C3 or C6, (+)-(1S,2S,3S,5R,

6S)-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicarboxy-

lic acid (+)-7 and (-)-(1R,2S,5R,6R)-2-amino-6-fluorobi-

cyclo[3.1.0]hexane-2,6-dicarboxylic acid (-)-11 had the

best inhibitory effect of cAMP formation, with the same

EC₅₀value as compound 4, and compound (+)-7 showed

higher oral activity in laboratory animals than com-

pound 4.

Furthermore, the successful modifications of CH₂at

the C4 position of compound 4 with either an oxygen

atom (5) or sulfur atom (6) aroused our interest in

replacement of the CH₂with a carbonyl group. The

chemical modification based on replacement of the C4-

CH₂of compound 11 with a carbonyl group produced

(+)-(1R,2S,5S,6S)-2-amino-6-fluoro-4-oxobicyclo[3.1.0]-

hexane-2,6-dicarboxylic acid monohydrate ((+)-14) as

the best group II mGluR agonist.

In this paper, we report the synthesis, structure-

activity relationships (SARs), and biological activities

of compounds 7-14, which have fluorine atom(s) incor-

porated on the C3 or C6 position of compound 4.

propionate (AMPA), and kainate. mGluRs are classified

into eight subtypes, identified as subtype 1 through

subtype 8, which are classified into three groups (I-

III) on the basis of their sequence homology, signal

transduction mechanisms, and pharmacology.^3-9

Group I mGluRs (mGluR1 and mGluR5) are positively

coupled to phospholipase C, and their activation pro-

duces phosphoinositide turnover and diacylglycerol

within the target neuron. In contrast, both group II

(mGluR2 and mGluR3) and group III mGluRs (mGluR4,

and mGluR6-mGluR8) are located in glutamatergic

terminals and negatively coupled to the activity of

adenyl cyclase.^9,10,18Accordingly, by inhibiting the

glutamatergic system, agonists for group II and/or group

III might be useful in the treatment of many diseases

and conditions such as schizophrenia,¹¹anxiety,¹²and

drug addiction,¹³and for protection from excitotoxic-

ity.^14,15

(1S, 3R)-1-Aminocyclopentane-1,3-dicarboxylic acid

(3, ACPD) is a selective agonist for group I and group

II mGluRs and activates group I and group II mGluRs

at similar concentrations.¹⁰In vitro electrophysiological

data have demonstrated that compound 3 produces

membrane depolarization and increases the firing rate

of midbrain dopamine neurons.⁹Unilateral microinjec-

tion of compound 3 into the dorsal striatum produced

circling behavior contralateral to the site of microinjec-

tion that appeared to be mediated by dopamine. Simi-

larly, microinjection of compound 3 into the nucleus

accumbens induced dopamine-dependent locomotor ac-

tivity, and haloperidol blocked this compound 3 (ACPD)-

induced hyperactivity. In contrast, in an in vivo mi-

crodialysis study, application of compound 3 to the

nucleus accumbens blocked prefrontal cortex stimula-

tion-induced increases in dopamine release in the

nucleus accumbens. Compound 3 also blocked ventral

tegmental area stimulation-induced increase in dopam-

ine release in the nucleus accumbens. It has been

postulated that agonists of group II mGluRs might

indirectly enhance net glutamatergic transmission in

brain areas involved in schizophrenia by reducing

synaptic excitation of inhibitory GABAergic neurons.¹⁹

Ch em istr y

Syn th esis of 2-Am in o-3-flu or obicyclo[3.1.0]h ex-

a n e-2,6-d ica r boxylic Acid s. The syntheses of com-

Synthesis of Group II mGluR Agonists

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4895

Sch em e 1^a

a

Reagents and conditions: (a) EtO₂CCH₂SMe₂‚Br, DBU, PhCH₃; (b) (i) LHMDS, TMSCl, THF, (ii) (PhSO₂)₂NF, CH₂Cl₂; (c) (NH₄)₂CO₃,

KCN, EtOH-H₂O; (d) 60% aq H₂SO₄; (e) (i) 2 M aq NaOH, (ii) (R)-PhCH(NH₂)Me, acetone-H₂O and then 1 M aq HCl; (f) 2.5 or 3.0 M

aq NaOH. Method A: a, b. Method B: c, d. Method C: c, f. Method D: e, d.

Sch em e 2^a

a

Reagents and conditions: (g) (i) LHMDS, TMSCl, THF, (ii) Pd(OAc)₂, MeCN; (h) TBHP, Triton B, PhMe; (i) KF‚HF, ethylene glycol;

(j) H₂, Pd/C, EtOH; (c) (NH₄)₂CO₃, KCN, EtOH-H₂O; (d) 60% aq H₂SO₄. Method E: g-j. Method D: c, d.

pounds (()-7, (+)-7, (-)-7, (()-8, (()-9, and (()-10 are

shown in Scheme 1, and the stereospecific syntheses of

compounds (+)-7 and (-)-7 are depicted in Scheme 2.

Racemic ethyl 2-oxobicyclo[3.1.0]hexane-6-carboxylate

(()-16 obtained by a known procedure²⁰was treated

with trimethylsilyl chloride in the absence of lithium

bisilanamide, and the resulting silyl enolate was flu-

orinated with N-fluoro-benzenesulfonamide (NFSI)^23,24

to yield a mixture of monofluoro compounds (()-17 and

(()-18, and difluoro compound (()-19 after chromatog-

raphy (method A). Treatment of lithium enolate²³of

compound (()-16 with NFSI resulted in poor yield. The

mixture of (()-17 and (()-18 was reacted under

Bucherer-Bergs conditions to yield three hydantoins,

(()-20, (()-21, and (()-22, after chromatography and

recrystallization. Similarly, hydantoin (()-24 was pre-

pared from compound (()-19 by treatment with am-

monium carbamate and potassium cyanide. Hydantoins

(()-20, (()-21, (()-22, and (()-24 were hydrolyzed under

acidic or basic conditions to yield compounds (()-7, (()-

8, (()-9, and (()-10, respectively (method B or C)

(Scheme 1).

The relative stereochemistries of the fluorine atom

and hydantoin of compound (()-20, (()-21, or (()-22

were determined by NOE analysis (Figure 1). In com-

pound (()-21, NOEs were observed between the H_3âand

H

_6â, H_3âand amide H, and H_6âand amide H. On the

other hand, no NOEs were observed between H_3Rand

_6â, or between H_3Rand amide H of compound (()-20.

H

NOEs were observed between H_1Rand amide H, and

H_3âand H_6âof the racemic diastereoisomer (()-22 of

(()-21.

Optically pure compounds (+)-7 and (-)-7 were

produced by acidic hydrolysis of carboxylic acids (+)-23

and (-)-23 afforded by selective saponification of ester

of compound (()-20 under basic conditions followed by

optical resolution by treatment with (R)- or (S)-1-

phenylethylamine (method D) (Scheme 1).

In addition to the above resolution, optical compound

(+)-7 was stereospecifically afforded from optical com-

pound (+)-16 via key intermediate (-)-27 as shown in

Scheme 2. Racemic ethyl 2-oxobicyclo[3.1.0]hexane-6-

carboxylate (()-16 was separated by chiral HPLC²⁵to

yield the known optical compound (+)-16 and its enan-

4896 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25

Nakazato et al.

F igu r e 1. NOEs of compounds (()-20-(()-22, (()-34, (()-38, and (()-42.

Sch em e 3^a

a

Reagents and conditions: (k) THPO-(CH₂)₄-Br (30), NaH, DMF; (l) J one’s reagent; (m) (i) (COCl)₂, hexane, reflux, (ii) CH₂N₂, Et₂O,

(iii) Cu(TBS)₂, PhH, reflux; (n) (i) 1 N aq NaOH, (ii) KCN, (NH₄)₂CO₃, EtOH-H₂O; (d) 60% H₂SO₄, 140-150 °C. Method F: k, l. Method

G: m. Method H: n, d.

tiomer (-)-16.²⁶Treatment of optically active compound

(+)-12 with trimethylsilyl chloride (TMSCl) under basic

conditions using lithium bis(trimethylsilyl)amide (LH-

MDS) followed by palladium acetate²⁷afforded enone

compound (-)-25, which was stereoselectively epoxi-

dated by tert-butyl hydroperoxide (TBHP)^28,29in the

presence of Triton B to yield epoxide (+)-26. Fluorina-

tion of epoxide (+)-26 with potassium hydrogen difluo-

ride^28,30in ethylene glycol yielded key intermediate (-)-

27 and its ester exchanger (-)-28. Hydrogenation of

vinyl fluoride (-)-27 with palladium on carbon induced

stereoselective progress, yielding (-)-17 (method E),

which was derived to compound (+)-7 by two steps,

formation of hydantoin under Bucherer-Bergs condi-

tions followed by hydrolysis of both the hydantoin and

ester under acidic conditions (method D).

Syn th esis of 2-Am in o-6-flu or obicyclo[3.1.0]h ex-

a n e-2,6-d ica r boxylic Acid s. The syntheses of com-

pounds (()-11, (+)-11, (-)-11, (()-12, (()-13, (()-14, (+)-

14, and (-)-14 are shown in Schemes 3-7.

Racemic ethyl 6-fluoro-2-oxobicyclo[3.1.0]hexane-6-

carboxylate (()-33 was obtained by intermolecular

reaction using bis(N-tert-butylsalicylaldimine)copper-

(II)^32,33between the double bond and diazomethyl group

produced by the treatment of compound 32 with oxalyl

chloride followed by diazomethane (method G). Ethyl

(Z)-2-fluoro-5-carboxy-2-pentenate 32 was stereoselec-

tively prepared by coupling ethyl phenylsulfinylfluoro-

acetate 29³⁴with 1-bromo-4-tetrahydropyranyloxybu-

tane 30³⁵in the presence of sodium hydride followed

by oxidation with J one’s reagent (method F). After

hydrolysis of the ethyl ester of compound (()-33, the

resulting carboxylic acid was reacted under Bucherer-

Bergs conditions to yield hydantoin (()-34 as a single

product. The direct treatment of compound (()-33 under

Bucherer-Bergs conditions resulted mainly in amida-

Compound [³H]-(+)-7 was prepared in the same

manner as for preparation of (+)-7 from (-)-17 after

compound (-)-27 was hydrogenated by tritium gas in

the absence of 10% Pd/CaCO₃.³¹

Synthesis of Group II mGluR Agonists

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4897

Sch em e 4^a

a

Reagents and conditions: (l) J one’s reagent; (m) (i) (COCl)₂, hexane, reflux, (ii) CH₂N₂, Et₂O, (iii) Cu(TBS)₂, PhH, reflux; (n) (i) 1 N

aq NaOH, (ii) KCN, (NH₄)₂CO₃, EtOH-H₂O; (d) 60% H₂SO₄, 140-150 °C. Method G: m. Method H: n, d. Method I: l.

Sch em e 5^a

a

Reagents and conditions: (g) (i) LHMDS, TMSCl, THF. (ii) Pd(OAc)₂, MeCN; (h) TBHP, Triton B, PhMe; (o) (PhSe)₂, NaBH₄, AcOH,

EtOH; (p) (i) 1 N aq NaOH, (ii) KCN, (NH₄)₂CO₃, EtOH-H₂O, (iii) EtOH, EDC‚HCl, DMAP, DMF; (d) 60% H₂SO₄, 140-150 °C; (q) (i)

TBSCl, imidazole, DMF, (ii) HS(CH₂)₂SH, BF₃‚Et₂O, CHCl₃; (r) DMSO, DCC, Py-TFA; (d) (i) 1 N aq NaOH, (ii) KCN, (NH₄)₂CO₃, EtOH-

H₂O. Method J : g, h, o. Method K: p, d. Method L: q, r. Method H: n, d.

Sch em e 6^a

compound (()-11 (methods G and H) after oxidizing

compound 35 with J one’s reagent (method I) (Scheme

4). The relative stereochemistry of compound (()-38 was

determined by NOEs which were observed between H_3â

and H_Me, H_amideand H_Me, and H_3âand H_amide(Figure

1).

The key intermediate (()-41 for both (()-13 and (()-

14 was prepared by four steps, treatment with trimeth-

ylsilyl chloride (TMSCl) under basic conditions using

LHMDS followed by palladium acetate,³⁷stereoselective

epoxidation by TBHP^28,29in the presence of Triton B,

and regiospecific reduction of R,â-epoxy carbonyl com-

pound (()-40 using benzeneselenol generated in situ by

reduction of diphenyl diselenide (PhSe)₂with sodium

borohydride in the presence of acetic acid³⁷(method J ).

Compound (()-13 was prepared by acidic hydrolysis of

compound (()-42, which was derived by hydrolysis of

the ester, treatment under Bucherer-Bergs conditions,

and then esterification of carboxlic acid for purification

by silica gel chromatography (method K) (Scheme 5).

The relative stereochemistry of compound (()-13 was

determined by NOE analysis of precursor (()-42. NOEs

between H_3âand H_4âand H_3âand H_amidewere observed,

but not between H_3Rand H_amide(Figure 1). For ketone

(()-44, compound (()-43, which was produced from

compound (()-41 by protection of hydroxyl group with

tert-butyldimethylsilyl (TBS) group followed by thioket-

alyzation (TBS-O bond was cleaved by workup), was

oxidized by dimethylsufoxide (DMSO) and dicyclohexyl-

carbodiimide (DCC) in the presence of pyridine and

a

Reagents and conditions: (s) (i) (R)-(+)-1-phenylethylamine,

EDC‚HCl, HOBt, DMF, (ii) silica gel chromatography; (d) 60%

H₂SO₄, 140-150 °C. Method M: s, d.

tion of the ethyl ester of compound (()-33. Compound

(()-34 was hydrolyzed under acidic conditions to yield

compound (()-11 (method H) (Scheme 3). The relative

stereochemistry of compound (()-34 was supported by

NOE analysis (Figure 1). Moderate and weak NOEs

were observed between H_4Rand H_5Rand H_4âand H_5R

respectively. Furthermore, the observation of NOEs

between H_3âand H_amideand H_3âand H_4âbut not between

_3Rand H_amidesupported the streochemistry of (1RS,2SR,

5RS,6RS).

6-Methyl compound (()-12 was synthesized from

ethyl (E)-2-methyl-6-(tetrahydropyran-2-yl)oxy-2-hex-

enate 35³⁶in the same manner as for synthesis of

,

H

4898 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25

Nakazato et al.

Sch em e 7^a

a

Reagents and conditions: (t) chiral HPLC; (n) (i) 1 N aq NaOH, (ii) KCN, (NH₄)₂CO₃, EtOH-H₂O; (d) 60% H₂SO₄, 140-150 °C; (g)

(i) LHMDS, TMSCl, THF, (ii) Pd(OAc)₂, MeCN; (h) TBHP, Triton B, PhMe; (o) (PhSe)₂, NaBH₄, AcOH, EtOH; (q) (i) TBSCl, imidazole,

DMF, (ii) HS(CH₂)₂SH, BF₃‚Et₂O, CHCl₃; (r) DMSO, DCC, Py-TFA; (u) (i) (R)-(+)-1-phenylethylamine, EDC‚HCl, HOBt, DMF, (ii) silica

gel chromatography. Method N: t. Method H: n, d. Method J : g, h, o. Method L: q, r. Method O: u, d.

F igu r e 2. Stereoview of slightly polar isomer 46‚MeOH from the X-ray crystallograph. Solvent molecule (MeOH) is omitted for

clarity.

trifluoroacetic acid^38,39(method L). Ketone (()-44 was

derived to compound (()-14 using method H (Scheme

5).

45 was unsuccessful, and this compound was derived

to amide (1R,2S,5R,6S)-46 () slightly polar isomer 46)

followed by purification (Scheme 7).

Optical isomer (+)-14 or (-)-14 was prepared by

hydrolysis of highly polar isomer 46 or slightly polar

isomer 46 produced by coupling (()-45 with (R)-(+)-1-

phenylethylamine using 3-ethyl-1-[3-(dimethylamino)-

propyl]carbodiimide hydrochloride (EDC‚HCl) and 1-hy-

droxybenzotriazole hydrate (HOBt‚H₂O) followed by

separation of the two diastereomers using silica gel

chromatography (method M) (Scheme 6). The X-ray

finding for the slightly polar isomer 46 suggested that

the stereochemistry of slightly polar isomer 46 was

(1S,2R,5R,6R) (Figure 2).

Furthermore, optical isomer (+)-11, (-)-11, or (+)-14

was prepared from optical isomer (-)-33 or (+)-33,

which was resolved optically by chiral HPLC (method

N).⁴⁰Treatment of (-)-33 and (+)-33 under conditions

of method H yielded optical isomers (-)-11 and (+)-11,

respectively. On the other hand, compound (+)-14 was

obtained from optical isomer (+)-33 by methods J , L, H

(n), and O. The purification of compound (1R,2S,5R,6S)-

Resu lts a n d Discu ssion

Agon ist an d An tagon ist Activities of Com pou n ds

7-14. Agonist activities were evaluated by measuring

agonist-dependent inhibition of forskolin-induced cyclic

AMP (cAMP) formation in mGluR2-, mGluR3-, mGluR4-,

mGluR6-, and mGluR7-expressing cells,⁴¹by measuring

D-myo Ins (1,4,5) P₃formation in mGluR1a-expressing

cells^42,43and by determining intracellular concentration

of Ca²⁺in mGluR5-expressing cells.^44,45Antagonist

activities were measured with 30 µM glutamic acid

present.

The agonist and antagonist activities of derivatives

7-14 and standard agonist 4 or standard antagonist

48 for mGluRs2 and mGluRs3 expressed in CHO cells

are shown in Table 1.

Racemic (()-7, a compound with fluorine atom(s)

incorporated on 4, demonstrated high activity as a

Synthesis of Group II mGluR Agonists

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4899

Ta ble 1. Agonist and Antagonist Activities of Compounds

7-14 and Standards 4 and 48

position. The C2 diastereomer (()-9 of (()-8 exhibited

low agonist activity for mGluR2 (EC₅₀) 4490 ( 400

nM), but higher than that of (()-8. Concerning the

structure-activity relationships of compounds 4, 5, and

6, the importance of C2 stereochemisty for the activity

for group II mGluRs has already been reported.^20,21

Notably, however, the C3 stereochemistry of the fluorine

atom more strongly agonist influences activity than C2

stereochemisty ((()-7 vs (()-8, and (()-8 vs (()-9).

Similarly, the difluoro compound (()-10 exhibited no

agonist or antagonist activity for mGluR2 expressed in

CHO cells (EC₅₀) >100 000 nM). This lack of activity

might be due to R-fluorine atom of (()-10, since com-

pound (()-8 with a fluorine atom incorporated on the

R-side of C3 exhibited neither agonist nor antagonist

activities for mGluR2 expressed in CHO cells.

EC₅₀( SEM (nM)^a

agonist activity^b

antagonist activity^c

compd

mGluR2 mGluR3

mGluR2

mGluR3

(()-7

(-)-7

(()-8

(()-9

(()-10

(()-11

(-)-11

(()-12

(()-13

(()-14

(+)-14

(-)-14

4

64.7 ( 9.3

29.4 ( 3.3

2640 ( 290

>100 000

4490 ( 400

>100 000

34.2 ( 6.3

1120 ( 200

16.6 ( 5.6

>100 000

21.0 ( 3.3

1.26 ( 0.2

0.570 ( 0.10

94.7 ( 10

18.3 ( 1.6

NT^d

45.4 ( 8.4

>100 000

NT^d

17 100

>100 000

36 200

NT^d

80.9 ( 31

>100 000

NT^d

>100 000

NT^d

2.07 ( 0.40

>100 000

NT^d

Racemic (()-11, a compound with a fluorine atom

incorporated on the C6 of (()-4, demonstrated high

activity as a mGluR2 agonist for suppression of forsko-

lin-stmulated cAMP accumulation in CHO cells with an

EC₅₀value of 34.2 ( 6.3 nM. This agonist activity was

determined to be highly stereoselective. Enantiomer (-)-

11 (EC₅₀) 16.6 ( 5.6 nM) exhibited approximately 67-

fold higher agonist activity than the corresponding

inactive enantiomer (+)-11 (EC₅₀) 1120 ( 200 nM) for

mGluR2 expressed in CHO cells. Furthermore, enanti-

omer (-)-11 exhibited a high degree of agonist activity

for mGluR3-expressing CHO cells (EC₅₀) 80.9 ( 31

nM) but not for mGluR4, mGluR6, mGluR7, mGluR1a,

or mGluR5 expressed in CHO cells (EC₅₀) >100 000

nM), and it exhibited no clear antagonist activity for

mGluR1a and mGluRs2-7 (EC₅₀) >100 000 nM). In

contrast, compound (()-12, with a methyl group incor-

porated on the C6 of (()-4, exhibited neither agonist nor

antagonist activities for mGluR2 expressed in CHO cells

(EC₅₀) >100 000 nM). The dramatic difference in

activity between compounds (()-11 and (()-12 might

depend on differences in steric hindrance and/or electron

density between the fluorine atom and methyl group.

62.8 ( 12

NT^d

48

50.3 ( 22

100 ( 16

a

EC₅₀values represent the means of 3-7 separate experiments

obtained from 5-7 concentrations of each compound, run in

b

duplicate. Compounds (+)-7, (-)-11, and (+)-14 exhibited no

agonist activities for mGluR1a, mGluR4-mGluR7 expressed in

CHO cells (ED₅₀) >100 000 nM). ^cCompounds (+)-7, (-)-11, and

(+)-14 exhibited no antagonist activities for mGluR1a, mGluR4,

mGluR6, and mGluR7 expressed in CHO cells (ED₅₀) >100 000

d

nM). Not tested.

Ch a r t 2

mGluR2 agonist for suppression of forskolin-stmulated

cAMP accumulation in CHO cells, with an EC₅₀value

of 64.7 ( 9.3 nM. This agonist activity was determined

to be highly stereoselective. Enantiomer (+)-7 (EC₅₀

)

29.4 ( 3.3 nM) exhibited approximately 90-fold higher

agonist activity than the corresponding inactive enan-

tiomer (-)-7 (EC₅₀) 2640 ( 290 nM) in mGluR2

expressed in CHO cells. Furthermore, enantiomer (+)-7

exhibited a degree of high agonist activity for mGluR3-

expressing CHO cells (EC₅₀) 45.4 ( 8.4 nM) but not

for mGluRs4, 6, 7, 1a, and 5 expressed in CHO cells

(EC₅₀) >100 000 nM), and it exhibited no clear

As a next step, the C4 methylene of compound (()-

11 was modified. The incorporation of a hydroxyl group

on R-site of the C4 methylene did not significantly

decrease forskolin-stimulated cAMP formation via

mGluR2 expressed in CHO cells (EC₅₀value of 21.0 (

3.3 nM (compound (()-13)). Notably, however, the

replacement of C4 methylene with a carbonyl group

greatly enhanced agonist suppression of forskolin-

stimulated cAMP accumulation via mGluR2 expressed

in CHO cells ((()-14: EC₅₀) 1.26 ( 0.2 nM). Compound

(+)-14 demonstrated great and stereospecific activity as

an agonist for mGluR2 and mGluR3 (EC₅₀) 0.570 (

0.10 nM and 2.07 ( 0.40 nM for mGluR2 and mGluR3,

respectively), but had no agonist effect on mGluR1a or

mGluR4-mGluR7 (EC₅₀) >100 000 nM) and no an-

tagonist effect on mGluR1a or mGluR2-mGluR7 (EC₅₀

) >100 000 nM). This agonist activity was approxi-

mately 165-fold that of the corresponding inactive

enantiomer (-)-14 (EC₅₀) 94.7 ( 10 nM) for mGluR2

expressed in CHO cells. Compared with compounds (-)-

8, the greatly enhanced agonist activity of (+)-14 might

depend on the slight variation caused by the conversion

of C4 methylene group into carbonyl group: the relative

conformation of three functional groups (one amino

group and two carboxylic acids) and/or the electron

antagonist activity for mGluRs1a and 2-7 (EC₅₀

)

>100 000 nM). These findings suggested that enanti-

omer (+)-7 was a potent and selective agonist for group

II mGluRs. In contrast, it is quite interesting that the

C3-diastereomer (()-8 of compound (()-7 exhibited no

agonist or antagonist activities for mGluR2 expressed

in CHO cells (EC₅₀) >100 000 nM). The conspicuous

difference in activity between (()-7 and (()-8 might not

be due to steric hindrance by the incorporated fluorine

atom alone, since derivative 47 (Ro 65-3479) (Chart 2),

a compound with a hydroxyl group incorporated on the

C3 position of 4, exhibited high affinities for both

mGluRs2 and mGluR3.⁵¹These results suggest that the

conspicuous decrease in agonist activity of (()-8 for

mGluR2 might be due to the electrophilicity of the

incorporated fluorine atom, and that the stereochemis-

try of the fluorine atom might influence the electron

density of amino and/or carboxylic groups at the C2

4900 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25

Nakazato et al.

Ta ble 2. Competition by Compounds (+)-7, (-)-8, and (+)-11

and Standards 4 and 48 for [³H]-(+)-7 Binding to mGluR2 or

mGluR3 Expressed in CHO Cells

fluorine substituent effect, high electronegativity, large

carbon-fluorine bond energy, and/or increase in lipo-

philicity.

K_i( SEM (nM)^a

PCP-induced head-weaving behavior in rats was very

strongly inhibited by oral administration of (+)-14 (ED₅₀

) 0.090 µg/kg). The oral activity of this compound was

much stronger than those of compounds 4 and 7.

Furthermore, PCP-induced hyperactivity in rats was

antagonized by oral administration of compound (+)-

14 (ED₅₀) 0.30 mg/kg) more strongly than by com-

pounds 4 and 7. Its oral efficacy suggested that com-

pound (+)-14 might be the best agonist for oral activity

ever presented and useful in the treatment of the

schizophrenia.

compd

mGluR2

mGluR3

(+)-7

(-)-11

(+)-14

4

47.7 ( 17

65.9 ( 7.1

41.7 ( 7.1

3.62 ( 1.6

53.5 ( 13

4.11 ( 1.6

22.5 ( 7.3

3.30 ( 0.31

23.4 ( 7.1

3.72 ( 0.43

48

a

K_ivalues represent the means of 3 or 4 separate experiments

obtained from 5-7 concentrations of each compound, run in

duplicate.

density of molecular. The degree of structural similarity

among compounds 5, 6, and (+)-14 might be higher than

that between compounds (-)-11 and (+)-14, since

compounds 5, 6, and (+)-14 might have more planarity

in the five-member ring than compounds 4 and (-)-11.

At any rate, compound (+)-14 might be the best agonist

for group II mGluRs ever presented.

[³H]-(+)-7 Bin d in g to m Glu R2 Exp r essed in CHO

Cells. The binding of [³H]-(+)-7 (34 Ci/mmol) was

performed according to the method previously described

with minor modifications using CHO cells stably ex-

pressing mGluR2 or mGluR3.⁴⁶Binding data of group

II mGluR antagonist (2S)-2-amino-2-((1S,2S)-2-carboxy-

cycloprop-1-yl)-3-(9-xanthyl)propanoic acid 48 (LY-

341495)^52,53and group II mGluR agonists 4,²⁰(+)-7, (-)-

11, and (+)-14 are shown in Table 2.

Compound 4 and 48 inhibited [³H]-7 binding with K_i

values of 23.4 ( 7.1 and 3.72 ( 0.43 nM for mGluR2

expressed in CHO cells, and 53.5 ( 13.5 and 4.11 ( 1.6

nM for mGluR3 expressed in CHO cells, respectively.

The binding of [³H]-(+)-7 was inhibited by compounds

(+)-7 and (-)-11 with K_ivalues of 47.7 ( 17 and 22.5 (

7.3 nM for mGluR2, and 65.9 ( 7.1 and 41.7 ( 7.1 nM

for mGluR3, respectively.

Con clu sion s

Compounds (+)-7 (MGS0008), a compound with a

fluorine atom incorporated on the C3 position of 4, is

an orally active and highly selective group II mGluR

agonist. Compound (+)-7 successfully overcame the low

oral activity of 4 due to incorporation of a fluorine atom.

Notably, however, the stereochemistry of the incorpo-

rated fluorine atom dramatically affected the in vitro

activity for mGluR2 expressed in CHO cells ((()-7 vs

(()-8). Compound (+)-14 (MGS0028), which was pro-

duced by both incorporation of a fluorine atom on the

C6 position and replacement of the C4-methylene with

a carbonyl group from compound 4, is a potent and

selective group II mGluR agonist and has great oral

activity. PCP-induced hyperactivity and head-weaving

behavior in rats were potently antagonized by the oral

administration of compound (+)-14.

These findings suggest that potent, selective, and

orally active mGluR agonists (+)-7 and (+)-14 might be

useful not only for exploring the functions of group II

mGluRs but in the treatment of schizophrenia. Fur-

thermore, tritium labeled [³H]-(+)-7 might be useful for

examining the physiological significance of the mGluRs.

Compound (+)-14 inhibited potently the binding of

[³H]-7 with K_ivalues of 3.30 ( 0.31 and 3.62 ( 1.6 nM

for mGluR2 and mGluR3 expressed in CHO cells,

respectively. The binding affinity of compound (+)-14

might be highest among group II mGluR agonists ever

presented. Furthermore, [³H]-(+)-7 may be an excellent

ligand for examination of the physiological roles played

by mGluRs.

Exp er im en ta l Section

Ch em istr y. Melting points were determined on a Yanaco

MP-500D melting point apparatus and are uncorrected. Proton

nuclear magnetic resonance (NMR) spectra were obtained

using a Varian Gemini 2000 (200 MHz) or Varian Unity Inova

300 (300 MHz) spectrometer. Chemical shifts are reported in

parts per million relative to tetramethylsilane as an internal

standard. Mass spectra (MS) were obtained on a Shimadzu

Profile (EI and CI), J EOL J MS-SX102 (FAB) or Micromass

Platform LC (IonSpray and ES). Optical rotations were

determined with a J AS.CO DIP-360 polarimeter and are

reported at the sodium D-line (589 nm). Elemental analyses

were performed by a Perkin-Elmer 2400 (carbon, hydrogen,

and nitrogen) or Yokogawa IC7000P (halogen and sulfur).

Chiral HPLC (resolution and analysis) was performed with a

Gilson Model 305 and/or 306 Piston Pump, 806 Manometric

Module, 115 Variable Wavelength Detector and Erma ERC-

3322 Degasser. Analytical thin-layer chromatography was

conducted on precoated silica gel 60 F₂₅₄plates (Merck). Silica

gel (C-200, 100-200 mesh (Wako Pure Chemical)) was used

for column chromatography, using the solvent systems (volume

ratios) indicated below.

Beh a vior a l P h a r m a cologica l Stu d ies. The effects

of group II mGluR agonists 4, (+)-7, and (+)-14 on PCP-

nduced hyperactivity and head-weaving behavior in rats

are shown in Table 3. It was recently found that PCP-

induced head-weaving behavior in rats was inhibited

by intraperitoneal administration of 4.¹¹Oral adminis-

tration of 4 (ED₅₀) 3.0 mg/kg) inhibited PCP-induced

head-weaving behavior in rats as well. Compound (+)-7

(ED₅₀) 0.26 mg/kg) more strongly inhibited PCP-

induced head-weaving behavior in rats than 4. Further-

more, PCP-induced hyperactivity in rats was antago-

nized by oral administration of (+)-7 (ED₅₀) 5.1 mg/

kg), but not by oral administration of 4 (ED₅₀> 100 mg/

kg). These results suggested that PCP-induced head-

weaving behavior is a sensitive method for screening

of group II mGluR agonists and that the introduction

of a fluorine atom clearly increased the oral activity.

The enhanced oral activity of (+)-7 might be due to a

Eth yl (1SR,3SR,5RS,6SR)-3-F lu or o-2-oxobicyclo[3.1.0]-

h exa n e-6-ca r boxyla te ((()-17), Eth yl (1SR,3RS,5RS,

6SR)-3-F lu or o-2-oxob icyclo[3.1.0]h exa n e-6-ca r b oxyla t e

((()-18), a n d Eth yl (1SR,5RS,6SR)-3,3-Diflu or o-2-oxobi-

cyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-19). A solution of

ethyl (1SR,5RS,6SR)-2-oxobicyclo[3.1.0]hexane-6-carboxylate

((()-16) (6.60 g, 39.3 mmol) in tetrahydrofuran (THF) (150 mL)

Synthesis of Group II mGluR Agonists

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4901

Ta ble 3. Effects of Standard 4 and Compounds (+)-7 and (+)-14 on PCP-Induced Hyperactivity and Head-Weaving Behavior in Rats

PCP-induced hyperactivity

%

PCP-induced head-weaving behavior

ED₅₀

number of

%

ED₅₀

compd

mg/kg

N

total counts

inhibition

(mg/kg)

µg/kg

N

head-weavings

inhibition

(µg/kg)

4 (LY354740)

>100

3000

vehicle

10

30

6

64700 ( 13000

40600 ( 11600

43800 ( 13300

36000 ( 18400

vehicle

1000

3000

6

38.5 ( 2.69

33.0 ( 3.98

19.4 ( 4.63

6.50 ( 1.58

37

32

44

14

50

83

100

10000

(+)-7

5.1

260

vehicle

3

10

30

6

54700 ( 10200

40600 ( 12100

11300 ( 3770

1850 ( 621

vehicle

100

300

6

39.3 ( 5.71

25.5 ( 8.27

21.0 ( 7.19

7.50 ( 1.70

26

79

97

35

47

81

1000

(+)-14

0.30

0.090

vehicle

6

59800 ( 9670

38500 ( 10400

27700 ( 8040

26100 ( 5920

7760 ( 3330

vehicle

0.03

0.1

6

34.1 ( 2.80

27.1 ( 4.75

16.2 ( 5.15

4.88 ( 2.10

0.1

0.3

1

36

54

56

87

21

52

86

0.3

3

was added dropwise to a solution of lithium bis(trimethylsilyl)-

amide (LHMDS) prepared by treatment of 1,1,1,3,3,3-hexa-

methyl disilazane (HMDS) (7.70 g, 47.6 mmol) with 1.54 M

solution of n-butyllithium in hexane (30.9 mL, 47.6 mmol) in

150 mL of THF, at -75 °C under a nitrogen atmosphere. After

the mixture was stirred for 1 h at this temperature, chloro-

trimethylsilane (TMSCl) (7.50 mL, 59.4 mmol) was added, and

the reaction mixture was stirred for 1 h at room temperature.

After concentration of the reaction mixture under reduced

pressure, anhydrous hexane was added to the residue, the

resulting inorganic salts were filtered off, and the filtrate was

concentrated under reduced pressure.

To a solution of the above residue in CH₂Cl₂(66 mL) was

added N-fluorobenzenesulfonimide (NFSI) (15.00 g, 47.6 mmol),

and the mixture was stirred at room temperature for 16.5 h.

After the reaction mixture was washed twice with water, the

organic phase was dried (MgSO₄), filtered, concentrated under

reduced pressure, and chromatographed on silica gel (hexane/

CH₂Cl₂/AcOEt 60:4:1) to yield a mixture of (()-17 and (()-18

(4.32 g) ((()-17/(()-18 1:3) as colorless oil and (()-19 (2.04 g,

25% yield) as a colorless oil.

Eth yl (1SR,2SR,3SR,5RS,6SR)-2-Sp ir o-5′-h yd a n toin -3-

flu or obicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-20), Eth yl

(1SR,2SR,3RS,5RS,6SR)-2-Sp ir o-5′-h yd a n t oin -3-flu or o-

bicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-21), a n d Eth yl

(1SR,2RS,3RS,5RS,6SR)-2-Sp ir o-5′-h yd a n t oin -3-flu or o-

bicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-22). A mixture of

the mixture of (()-17 and (()-18 (4.84 g, 26.0 mmol), am-

monium carbonate (6.25 g, 65.0 mmol), and potassium cyanide

(1.86 g, 28.6 mmol) in a mixed solvent of water (26 mL) and

EtOH (38 mL) was stirred at 35 °C for 37 h. After the reaction

mixture was cooled to room temperature, water (31 mL) was

added, stirring was continued for 2.5 h with ice-cooling, and

the resulting crystal was collected by filtration to yield 2.10 g

of the first crystal as a mixture of diastereomers (()-20, (()-

21, and (()-22 (5:6:1).⁵⁴With ice-cooling, concentrated HCl was

added to the filtrate to adjust its pH to 1.0, and the resulting

crystal was collected by filtration to yield 2.00 g of the second

crystal as a mixture of diastereomers (()-20, (()-21, and (()-

22 (4:6:11).⁵⁴The first crystal was chromatographed on silica

gel (CHCl₃/MeOH 100:1) to yield nonpolar diastereomer (()-

20 (0.61 g) and a mixture of two polar diastereomers (()-21

and (()-22 (0.55 g) (including about 25% diastereomer (()-

22,⁵⁷with the same R_fvalue for diastereomers (()-21 and (()-

22). The nonpolar diastereomer (()-20 (0.61 g) was purified

by recrystallization from a mixture of water and EtOH (1:1)

to yield (()-20 (0.52 g, 8% isolated yield) as a colorless

crystal: mp 265-269 °C; ¹H NMR (200 MHz, DMSO-d₆) δ 1.19

(3 H, t, J ) 7.0 Hz), 1.95-2.46 (5 H, m), 4.06 (2 H, q, J ) 7.0

Hz), 4.81 (1 H, dd, J ) 52 Hz, 5.1 Hz), 8.44 (1 H, s), 10.91 (1

H, s); MS (EI) m/z 256 (M⁺); Anal. (C₁₁H₁₃FN₂O₄) C, H, F, N.

Mixture of (()-17 and (()-18: ¹H NMR (200 MHz, CDCl₃)

δ 1.28 (3 H × 3/4, t, J ) 7.2 Hz), 1.29 (3 H × 1/4, t, J ) 7.2

Hz), 2.11-2.79 (5 H, m), 4.18 (2 H, q, J ) 7.2 Hz), 4.51 (1 H

× 1/4, dd, J ) 51 Hz, 8.1 Hz), 4.58 (1 H × 3/4, dt, J ) 51 Hz,

8.1 Hz); MS (FAB) (Pos) m/z 187 (M⁺+ 1).

(()-19: ¹H NMR (200 MHz, CDCl₃) δ 1.30 (3 H, t, J ) 7.1

Hz), 2.42-2.80 (5 H, m), 4.20 (2 H, q, J ) 7.1 Hz); MS (ion

spray) (Nega) m/z 203 (M⁺- 1).

Eth yl (1SR,5RS,6SR)-3,3-Diflu or o-2-oxobicyclo[3.1.0]-

h exa n e-6-ca r boxyla te ((()-19). A solution of a mixture of

(()-17 and (()-18 (1.30 g, 6.98 mmol) in THF (6.5 mL) was

added dropwise to a solution of LHMDS prepared by treatment

of HMDS (1.40 g, 8.3 mmol) with 1.54 M solution of n-

butyllithium in hexane (5.0 mL, 7.7 mmol) in THF (26 mL),

at -75 °C under a nitrogen atmosphere. After the mixture was

stirred for 1 h at this temperature, TMSCl (1.30 mL, 10.3

mmol) was added, and the reaction mixture was stirred for 1

h at room temperature. After concentration of the reaction

mixture under reduced pressure, anhydrous hexane was added

to the residue, the resulting inorganic salts were filtered off,

and the filtrate was concentrated under reduced pressure.

The residue was dissolved in CH₂Cl₂(13 mL); to this was

added NFSI (3.30 g, 10.5 mmol), and the mixture was stirred

at room temperature for 5 h. After the reaction solution was

washed twice with water, the organic phase was dried (Na₂-

SO₄), filtered off the desiccant, concentrated under reduced

pressure, and then chromatographed on silica gel (hexane/

AcOEt 15:1) to yield (()-19 (0.39 g, 27% yield) as a colorless

oil: ¹H NMR (200 MHz, CDCl₃) δ 1.30 (3 H, t, J ) 7.1 Hz),

2.42-2.80 (5 H, m), 4.20 (2 H, q, J ) 7.1 Hz); MS (ion spray)

(Nega) m/z 203 (M⁺- 1).

Also, the mixture of higher-polar diastereomers (()-21 and

(()-22 (0.55 g) was recrystallized from a mixture of water and

EtOH (1:1) to yield (()-21 (0.37 g, 6% isolated yield) as a

colorless crystal: mp 195-199 °C; ¹H NMR (200 MHz, DMSO-

d₆) δ 1.18 (3 H, t, J ) 7.1 Hz), 1.85-2.43 (5 H, m), 4.05 (2 H,

q, J ) 7.1 Hz), 4.70 (1 H, dt, J ) 52 Hz, 8.0 Hz), 8.21 (1 H, s),

10.83 (1 H, s); MS (EI) m/z 256 (M⁺); Anal. (C₁₁H₁₃FN₂O₄) C,

H, F, N.

The second crystal was washed with AcOEt to remove

inorganic material, and the filtrate was concentrated under

reduced pressure. The residue was recrystallized twice from

a mixture of water and EtOH (1:1). The combined filtrate was

concentrated under reduced pressure, and the residue was

chromatographed on silica gel (CHCl₃/MeOH 100:1), com-

pletely removing the aforesaid nonpolar diastereomer (()-20.

The resulting crystal (0.25 g) of polar diastereomer (()-22

(including about 10% polar diastereomer (()-21) was recrys-

tallized from a mixture of water and EtOH (1:1) to yield (()-

22 (0.18 g, 3% isolated yield) as a colorless crystal: mp 165-

1

170 °C; H NMR (300 MHz, DMSO-d₆) δ 1.18 (3 H, t, J ) 7.1

Hz), 1.81-2.17 (4 H, m), 2.36 (1 H, dd, J ) 13 Hz, 7.2 Hz),

3.95-4.11 (2 H, m), 4.90 (1 H, ddd, J ) 51 Hz, 8.9 Hz, 7.2

4902 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25

Nakazato et al.

Hz), 8.54 (1 H, s), 10.87 (1 H, s); MS (EI) m/z 256 (M⁺); Anal.

(C₁₁H₁₃FN₂O₄) C, H, F, N.

Hz), 8.44 (1 H, m), 10.88 (1 H, s), 12.30 (1 H, brs); MS (FAB)

(Nega) m/z 227 (M⁺- 1); Anal. (C₉H₉FN₂O₄) C, H, F, N.

Eth yl (1SR,2SR,5RS,6SR)-2-Sp ir o-5′-h yd a n toin -3,3-d i-

flu or obicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-24). A mix-

ture of (()-19 (1.65 g, 8.1 mmol), ammonium carbonate (1.78

g, 18.5 mmol), and potassium cyanide (0.60 g, 9.2 mmol) in a

mixture (12 mL) of water and EtOH (1:2.5) was stirred at 35

°C for 9 days. To the mixture was added water (1 mL), and

the resulting mixture was stirred for 2 h with ice-cooling. The

resulting precipitate was collected by filtration to yield (()-

24 (1.05 g, 47% yield) as a colorless crystal: mp 209-213 °C;

¹H NMR (200 MHz, DMSO-d₆) δ 1.19 (3 H, t, J ) 7.0 Hz),

1.85-1.89 (1 H, m), 2.00-2.08 (1 H, m), 2.15-2.27 (1 H, m),

2.33-2.50 (1 H, m), 2.55-2.86 (1 H, m), 4.07 (2H, q, J ) 7.0

Hz), 8.49 (1 H, s); MS (EI) m/z 274 (M⁺); Anal. (C₁₁H₁₂F₂N₂O₄)

C, H, F, N.

(1S,2S,3S,5R,6S)-2-Sp ir o-5′-h yd a n toin -3-flu or obicyclo-

[3.1.0]h exan e-6-car boxylic Acid ((+)-23) an d (1R,2R,3R,5S,

6R)-2-Sp ir o-5′-h yd a n toin -3-flu or obicyclo[3.1.0]h exa n e-6-

ca r boxylic Acid ((-)-23). Compound (()-23 (1.80 g, 7.89

mmol) was stirred at 55 °C in a mixture (26 mL) of acetone

and water (8:5), and after adding (R)-(+)-1-phenylethylamine

(0.96 g, 7.89 mmol), it was stirred for 15 h at room tempera-

ture. The resulting crystals were filtered to yield (R)-(+)-1-

phenylethylamine salt of (+)-23 (1.30 g, 47% yield) as a

colorless crystal. To a suspension of 1.20 g of the salt in water

(15 mL), 1 M HCl was added to adjust to pH 1.0, and the

mixture was stirred at room temperature for 14 h. The

resulting crystal was isolated by filtration to yield (+)-23 (0.65

g, 82% yield) as a colorless crystal. The filtrate was further

chromatographed on AG50W-X8 anion-exchange resin (Bio-

Rad) (1 M acetic acid) to yield (+)-23 (0.06 g, 8% yield) as a

colorless crystal: mp 330 °C (decomposed); ¹H NMR (200 MHz,

DMSO-d₆) δ 1.85-2.44 (5 H, m), 4.80 (1 H, dd, J ) 52 Hz, 5.3

Hz), 8.44 (1 H, m), 10.88 (1 H, s), 12.30 (1 H, brs); MS (FAB)

(Nega) m/z 227 (M⁺); [R]_D²²) + 36.8° (c ) 0.20, MeOH); Anal.

(C₉H₉FN₂O₄) C, H, F, N.

The filtrate of resolution ((R)-(+)-1-phenylethylamine salt)

was concentrated under reduced pressure. A mixture of the

resulting crystal (1.3 g) in water (17 mL) was adjusted to pH

1.0 by addition of 1 N HCl and was stirred at room temper-

ature. After 4 h, the resulting crystal was collected by

filtration, yielding 0.81 g of crystals. The filtrate was chro-

matographed on AG50W-X8 anion-exchange resin (Bio-Rad),

(1 M AcOH), yielding 0.08 g of crystals. The combined crystals

(0.89 g, 3.88 mmol) were dissolved in a mixture of acetone and

water (8:5) (13 mL) at 55 °C. (S)-(-)-1-Phenylethylamine (0.47

g, 3.88 mmol) was added to the solution, and the mixture was

stirred at room temperature for 15 h. The precipitated crystals

were filtered to yield (S)-(-)-1-phenylethylamine salt of (-)-

23 as a colorless crystal (1.10 g, 85% yield).

(1SR,2SR,3RS,5RS,6SR)-2-Am in o-3-flu or obicyclo[3.1.0]-

h exa n e-2,6-d ica r boxylic Acid ((()-8). Compound (()-21

(300 mg, 1.2 mmol) was dissolved in 3 M aqueous NaOH

solution (2.50 mL), and the mixture was heated at reflux for

16 h. The reaction mixture was cooled to room temperature

and filtered through a glass filter. After the filtrate was

brought to pH 3 with concentrated HCl, it was chromato-

graphed on AG1-X8 cation-exchange resin (Bio-Rad) (0.1 M

AcOH to 3 M AcOH) to yield (()-8 (51 mg, 21% yield) as a

colorless crystal: mp 230 °C (decomposed); ¹H NMR (300 MHz,

trifluoroacetic acid-d) δ 2.23-2.24 (1 H, m), 2.56-2.96 (4 H,

m), 5.15 (1 H, dt, J ) 52 Hz, 7.5 Hz); MS (CI) m/z 204 (M⁺

1); Anal. (C₈H₁₀FNO₄) C, H, F, N.

+

(1SR,2SR,5RS,6SR)-2-Am in o-3,3-d iflu or obicyclo[3.1.0]-

h exa n e-2,6-d ica r boxylic Acid ((()-10). Compound (()-24

(97 mg, 0.35 mmol) was dissolved in aqueous 2.5 M NaOH

solution (1.0 mL), and the mixture was heated at reflux for

24 h. The reaction mixture was cooled to room temperature

and filtered through a glass filter. After the filtrate was

brought to pH 3 with concentrated HCl, it was chromato-

graphed on AG1-X8 cation-exchange resin (Bio-Rad) (0.5 M

AcOH to 2 M AcOH) to yield (()-10 (44 mg, 56% yield) as a

colorless crystal: mp 250 °C (decomposed); ¹H NMR (300 MHz,

trifluoroacetic acid-d) δ 2.46 (1 H, brs), 2.63-2.90 (3H, m),

3.01-3.12 (1H,m); MS (CI) m/z 222 (M⁺+ 1); Anal. (C₈H₉F₂-

NO₄‚1/2H₂O) C, H, F, N.

(1SR,2SR,3SR,5RS,6SR)-2-Am in o-3-flu or obicyclo[3.1.0]-

h exa n e-2,6-d ica r boxylic Acid ((()-7). A solution of (()-20

(100 mg, 0.39 mmol) in aqueous 60% H₂SO₄(1.50 mL) was

stirred at 140 °C for 12 h. The reaction solution was then

cooled to room temperature and brought to pH 8 with 5 M

aqueous NaOH. The resulting mixture was chromatographed

on AG1-X8 cation-exchange resin (Bio-Rad) (0.1 M AcOH to 2

M AcOH) to yield (()-7 (20 mg, 25% yield) as a colorless

crystal: mp 160 °C (decomposed); ¹H NMR (300 MHz, tri-

fluoroacetic acid-d) δ 2.49 (1H, brs), 2.59-3.06 (4 H, m), 5.40

(1 H, dd, J ) 52 Hz, 5.3 Hz); MS (CI) m/z 204 (M⁺+ 1); Anal.

(C₈H₁₀FNO₄) C, H, F, N.

This salt was transformed to a free form by treatment with

1 M HCl in the same fashion as for preparation of (+)-23 to

yield (-)-23 (0.58 g, 86% yield) as a colorless crystal. The

filtrate was chromatographed on AG50W-X8 anion-exchange

resin (Bio-Rad), (1 M AcOH) to yield (-)-23 (0.07 g, 10% yield)

as a colorless crystal: mp 328 °C (decomposed); ¹H NMR (200

MHz, DMSO-d₆) δ 1.85-2.44 (5 H, m), 4.80 (1 H, dd, J ) 52

Hz, 5.3 Hz), 8.44 (1 H, m), 10.88 (1 H, s), 12.30 (1 H, brs); MS

(FAB) (Nega) m/z 227 (M⁺); [R]_D²²) - 37.5° (c ) 0.20, MeOH);

Anal. (C₉H₉FN₂O₄) C, H, F, N.

(1S,2S,3S,5R,6S)-2-Am in o-3-flu or obicyclo[3.1.0]h exan e-

2,6-d ica r boxylic Acid ((+)-7). Compound (+)-23 (0.60 g, 2.63

mmol) was dissolved in 10 mL of aqueous 60% H₂SO₄, and it

was stirred at 140 °C for 2 days. The reaction solution was

cooled to room temperature and then brought to pH 8 by

addition of aqueous 5 M NaOH. The mixture was chromato-

graphed on AG1-X8 cation-exchange resin (Bio-Rad) (0.1 M

AcOH to 2 M AcOH) to yield (+)-7 (0.34 g, 64% yield)⁵⁵as a

colorless crystal: mp 200 °C (decomposed); ¹H NMR (300 MHz,

trifluoroacetic acid-d) δ 2.49 (1H, brs), 2.59-3.06 (4 H, m), 5.40

(1SR,2RS,3RS,5RS,6SR)-2-Am in o-3-flu or obicyclo[3.1.0]-

h exa n e-2,6-d ica r boxylic Acid ((()-9). A solution of (()-22

(150 mg, 0.59 mmol) in aqueous 60% H₂SO₄(2.3 mL) was

stirred at 140 °C for 13 h. The reaction solution was then

cooled to room temperature and brought to pH 8 with an

aqueous 5 M NaOH. The resulting mixture was chromato-

graphed on AG1-X8 cation-exchange resin (Bio-Rad) (0.1 M

AcOH to 2 M AcOH) to yield (()-9 (59 mg, 49% yield) as a

colorless crystal: mp 220 °C (decomposed); ¹H NMR (300 MHz,

trifluoroacetic acid-d) δ 2.33 (1 H, brs), 2.54-2.89 (4 H, m),

(1 H, dd, J ) 52 Hz, 5.3 Hz); MS (CI) m/z 204 (M⁺+ 1); [R]_D

22

) +58.6° (c ) 0.20, 1 M HCl); Anal. (C₈H₁₀FNO₄) C, H, F, N.

(1R,2R,3R,5S,6R)-2-Am in o-3-flu or obicyclo[3.1.0]h exan e-

2,6-d ica r boxylic Acid ((-)-7). Compound (-)-23 (0.58 g, 2.54

mmol) was reacted as for preparation of (+)-7 to yield (-)-7

(0.37 g, 73% yield)⁵⁵as a colorless crystal: mp 200 °C

(decomposed); ¹H NMR (300 MHz, trifluoroacetic acid-d) δ 2.49

(1H, brs), 2.59-3.06 (4 H, m), 5.40 (1 H, dd, J ) 52 Hz, 5.3

5.42-5.59 (1 H, m); MS (CI) m/z 204 (M⁺+ 1); Anal. (C₈H₁₀

FNO₄) C, H, F, N.

-

Hz); MS (CI) m/z 204 (M⁺+ 1); [R]_D) -59.4° (c ) 0.20, 1 M

22

HCl); Anal. (C₈H₁₀FNO₄) C, H, F, N.

(1SR,2SR,3SR,5RS,6SR)-2-Sp ir o-5′-h yd a n toin -3-flu or o-

bicyclo[3.1.0]h exa n e-6-ca r boxylic Acid ((()-23). A mixture

of (()-20 (2.20 g, 8.59 mmol) in an aqueous 2 M NaOH was

stirred at room temperature. After 2 h, its pH was adjusted

to 1.0 by adding concentrated HCl. The resulting crystal was

isolated by filtration to yield (()-23 (1.81 g, 93% yield) as a

colorless crystal: mp 319 °C (decomposed); ¹H NMR (200 MHz,

DMSO-d₆) δ 1.85-2.44 (5 H, m), 4.80 (1 H, dd, J ) 52 Hz, 5.3

E t h yl (1S,5R,6S)-2-Oxob icyclo[3.1.0]h ex-3-en e-6-ca r -

boxyla te ((-)-25). A solution of (+)-16²⁵(8.58 g, 51.0 mmol)

in THF (126 mL) was added dropwise to a solution of LHMDS

prepared by treatment of HMDS (9.88 g, 61.2 mmol) with 1.61

M solution of n-butyllithium in hexane (38.0 mL, 61.2 mmol)

in THF (126 mL), at -75 °C under a nitrogen atmosphere.

After the mixture was stirred for 1 h at this temperature,

Synthesis of Group II mGluR Agonists

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4903

TMSCl (9.70 mL, 76.8 mmol) was added, and the reaction

mixture was stirred for 1 h at room temperature. After

concentration of the reaction solution under reduced pressure,

anhydrous hexane was added to the residue, the resulting

inorganic salts were filtered off, and the filtrate was concen-

trated under reduced pressure.

The separated organic phase was dried (Na₂SO₄), concentrated

under reduced pressure, and chromatographed on silica gel

(CHCl₃/MeOH 80:1-60:1) to yield (+)-20 (232 mg, 61% yield)

as a colorless crystal: mp 279-282 °C; ¹H NMR (200 MHz,

DMSO-d₆) δ 1.19 (3 H, t, J ) 7.0 Hz), 1.95-2.46 (5 H, m), 4.06

(2 H, q, J ) 7.0 Hz), 4.81 (1 H, dd, J ) 52 Hz, 5.1 Hz), 8.44 (1

H, s), 10.91 (1 H, s); MS (EI) m/z 256 (M⁺); [R]_D) +30.1° (c

25

A mixture of the above residue and Pd(OAc)₂(12.6 g, 56.1

mmol) in MeCN (126 mL) was stirred at room temperature

for 16 h. To the mixture was added Et₂O, and the resulting

mixture was filtered through Celite. The filtrate was concen-

trated under reduced pressure, chromatographed on silica gel

(hexane/AcOEt 8:1), and then recrystallized from a mixture

of hexane and iso-PrOH (9:1) to yield (-)-25 (7.26 g, 86% yield)

as colorless crystal: mp 97-99 °C; ¹H NMR (200 MHz, CDCl₃)

δ 1.26 (3 H, t, J ) 7.0 Hz), 2.27 (1 H, t, J ) 2.6 Hz), 2.63 (1 H,

m), 2.96 (1 H, dt, J ) 4.8 Hz, 2.6 Hz), 4.15 (2 H, q, J ) 7.0

Hz), 5.74 (1 H, d, J ) 5.7 Hz), 7.61 (1 H, dd, J ) 5.7 Hz, 2.6

) 0.12, MeOH); Anal. (C₁₁H₁₃FN₂O₄) C, H, F, N.

(1S,2S,3S,5R,6S)-2-Am in o-3-flu or obicyclo[3.1.0]h exan e-

2,6-d ica r boxylic Acid ((+)-7). A mixture of (+)-20 (120 mg,

0.47 mmol) in aqueous 60% H₂SO₄(1.8 mL) was stirred at 140

°C for 2 days. The reaction solution was cooled to room

temperature and then brought to pH 8 by addition of aqueous

5 M NaOH. The mixture was chromatographed on AG1-X8

cation-exchange resin (Bio-Rad) (0.1 M AcOH to 2 M AcOH)

to yield (+)-7 (75 mg, 79% yield)⁵⁵as a colorless crystal: mp

265-269 °C; mp 200 °C (decomposed); ¹H NMR (300 MHz,

trifluoroacetic acid-d) δ 2.49 (1H, brs), 2.59-3.06 (4 H, m), 5.40

Hz); MS (CI) m/z 167 (M⁺+ 1); [R]_D) -278° (c ) 0.42, CH₂-

25

(1 H, dd, J ) 52 Hz, 5.3 Hz); MS (CI) m/z 204 (M⁺+ 1); [R]_D

22

Cl₂).

) +58.6° (c ) 0.20, 1 M HCl); Anal. (C₈H₁₀FNO₄) C, H, F, N.

Eth yl (1S,3R,4R,5R,6S)-3,4-Ep oxy-2-oxobicyclo[3.1.0]-

h ex-3-en e-6-ca r b oxyla t e ((+)-26). To a solution of (-)-25

(7.00 g, 42.1 mmol) in toluene (47 mL) was added aqueous 70%

tert-butyl hydroperoxide (9.3 mL, 72.2 mmol) and 10% ben-

zyltrimethylammonium hydroxide in MeOH (3.5 mL, 1.9

mmol), and then the mixture was stirred at room temperature

for 30 min. The reaction mixture was partitioned between

AcOEt and water. The separated water phase was extracted

with AcOEt. The combined organic phase was dried (MgSO₄)

and concentrated under reduced pressure. The residue was

recrystallized from AcOEt to yield (+)-26 (5.50 g, 73% yield)

as a colorless crystal: mp 131-136 °C; ¹H NMR (200 MHz,

CDCl₃) δ 1.28 (3 H, t, J ) 7.0 Hz), 2.09 (1 H, t, J ) 3.1 Hz),

2.21 (1 H, ddt, J ) 5.3 Hz, 2.4 Hz, 1.1 Hz), 2.96 (1 H, m), 3.25

(1 H, dt, J ) 2.4 Hz, 1.1 Hz), 4.00 (1 H, t, J ) 2.4 Hz), 4.17 (2

Eth yl (Z)-2-F lu or o-6-(tetr a h yd r op yr a n -2-yl)oxy-2-h ex-

en a te (31). A solution of ethyl phenylsulfinylfluoroacetate 29³⁴

(130 g, 056 mol) and 1-bromo-4-tetrahydropyranyloxybutane

30³⁵(147 g, 0.62 mol) in N,N-dimethylformamide (DMF) (140

mL) was added dropwise to a suspension of 60% NaH/oil (23.7

g, 0.59 mol) in DMF (700 mL) over 45 min with ice-cooling,

and the resulting mixture was stirred at room temperature

for 2 h and at 110 °C for 2 h. The reaction mixture was cooled

to room temperature, poured into saturated aqueous NH₄Cl,

and extracted with a mixture of AcOEt and hexane (2:1). The

extract was washed with water and saturated brine, dried

(Na₂SO₄), concentrated under reduced pressure, and then

chromatographed (hexane/AcOEt 25:1-25:2) to yield 31 (77.9

g, 53% yield) as a light yellow oil: ¹H NMR (200 MHz, CDCl₃)

δ 1.33 (3 H, t, J ) 7.1 Hz), 1.46-1.90 (8 H, m), 2.30-2.41 (2

H, m), 3.33-3.57 (2 H, m), 3.72-3.90 (2 H, m), 4.28 (2 H, q, J

) 7.1 Hz), 4.57-4.60 (1 H, m), 6.17 (1 H, dt, J ) 33.3, 7.8 Hz);

MS (CI) (Pos) m/z 261 (M⁺+ 1).

Eth yl (Z)-2-F lu or o-5-ca r boxy-2-p en ten a te (32). To a

cooled solution of 31 (7.30 g, 28.0 mmol) in acetone (73 mL) in

an ice-bath, 8 N J ones’ reagent (30 mL) was added dropwise

over 20 min. After the mixture was stirred for 2.5 h at room

temperature, an excess of J ones’ reagent was quenched by the

addition of 2-propanol (280 mL) with ice-cooling. The reaction

mixture was partitioned between AcOEt and water. The

separated water phase was extracted with AcOEt twice. The

combined organic phase was washed with water and saturated

brine, dried (Na₂SO₄), concentrated under reduced pressure,

and then chromatographed (hexane/AcOEt 2:1-1:1) to yield

32 (4.69 g, 88% yield) as a colorless oil: ¹H NMR (200 MHz,

CDCl₃) δ 1.34 (3 H, t, J ) 7.1 Hz), 2.46-2.60 (8 H, m), 4.29 (2

H, q, J ) 7.1 Hz), 6.03-6.27 (1 H, m); MS (CI) (Pos) m/z 191

(M⁺+ 1).

Eth yl (1RS,5RS,6RS)-6-F lu or o-2-oxobicyclo[3.1.0]h ex-

a n e-6-ca r boxyla t e ((()-33). A solution of 32 (4.69 g, 24.7

mmol) and (COCl)₂(6.5 mL, 74.5 mmol) in hexane (50 mL)

was heated at reflux for 3 h and then concentrated under

reduced pressure. A solution of the residue in Et₂O (50 mL)

was added to a solution of CH₂N₂in Et₂O, which was prepared

by treatment of a solution of N-methylnitrosourea (12.70 g,

124 mmol) in Et₂O with a solution of KOH (40.8 g, 618 mmol)

in water (150 mL). The resulting mixture was stirred for 10

min with ice cooling, stirred at room temperature for 1 h, and

then concentrated under reduced pressure. The residue was

dissolved in benzene (55 mL), and the solution was added

dropwise to a solution of bis(N-tert-butylsalicylaldimine)-

copper(II)^32,33(Cu(TBS)₂) (0.41 g, 0.99 mmol) in benzene (600

mL) over 30 min under heating reflux. The mixture was cooled

to room temperature, concentrated under reduced pressure,

and then chromatographed (hexane/acetone 9:1) to yield (()-

33 (0.39 g, 27.3% yield) as a colorless oil: ¹H NMR (200 MHz,

CDCl₃) δ 1.33 (3 H, t, J ) 7.1 Hz), 2.05-2.55 (4 H, m), 2.59 (1

H, d, J ) 6.6 Hz), 2.70-2.77 (1 H, m), 4.30 (2 H, q, J ) 7.1

Hz); MS (ion spray) (Pos) m/z 187 (M⁺+ 1).

H, q, J ) 7.0 Hz); MS (CI) m/z 183 (M⁺+ 1); [R]_D) +12.2°

25

(c ) 0.41, CH₂Cl₂).

Eth yl (1S,5R,6S)-3-F lu or o-2-oxobicyclo[3.1.0]h ex-3-

en e-6-ca r boxyla te ((-)-27) a n d 2-Hyd r oxyeth yl (1S,5R,

6 S )-3 -F l u o r o -2 -o x o b i c y c l o [3 .1 .0 ]h e x -3 -e n e -6 -c a r -

boxyla te ((-)-28). A suspension of (+)-26 (5.45 g, 30.0 mmol)

and HF‚KF (23.3 g, 300 mmol) in ethylene glycol (82 mL) was

stirred at 130 °C for 2 h under a nitrogen atmosphere. The

mixture was partitioned between ice-water and CHCl₃. The

separated organic phase was dried (MgSO₄), concentrated

under reduced pressure, and chromatographed on silica gel

(hexane/AcOEt 6:1-1:1-3:2) to yield (-)-27 (1.01 g, 18% yield)

as a colorless oil and (-)-28 (1.67 g, 28% yield) as a colorless

oil.

(-)-27: ¹H NMR (200 MHz, CDCl₃) δ 1.28 (3 H, t, J ) 7.0

Hz), 2.48 (1 H, dt, J ) 3.1 Hz, 0.7 Hz), 2.58 (1 H, m), 2.81 (1

H, m), 4.17 (2 H, q, J ) 7.0 Hz), 6.91 (1 H, m); MS (CI) m/z

25

185 (M⁺+ 1); [R]_D) -96.8° (c ) 0.43, CH₂Cl₂).

(-)-28: ¹H NMR (200 MHz, CDCl₃) δ 1.72-1.92 (1 H, br s),

2.54 (1 H, t, J ) 3.0 Hz), 2.61 (1 H, m), 2.84 (1 H, m), 3.80-

3.92 (2 H, m), 4.23-4.30 (2 H, m), 6.92 (1 H, m); MS (CI) m/z

201 (M⁺+ 1); [R]_D) -181° (c ) 0.41, CH₂Cl₂).

25

Eth yl (1S,3S,5R,6S)-3-Flu or o-2-oxobicyclo[3.1.0]h exan e-

6-ca r boxyla te ((-)-17). A suspension of (-)-27 (0.80 g, 4.3

mmol) and 5% Pd on carbon (80 mg) in EtOH (8 mL) was

stirred under a hydrogen atmosphere. Filtration through

Celite, concentration under reduced pressure, and then chro-

matography on silica gel (hexane/AcOEt 6:1) yielded (-)-17

(0.61 g, 75% yield) as a colorless oil: ¹H NMR (200 MHz,

CDCl₃) δ 1.29 (3 H, t, J ) 7.2 Hz), 2.10-2.70 (5 H, m), 4.18 (2

H, q, J ) 7.2 Hz), 4.51 (1 H, dd, J ) 51.0 Hz, 8.1 Hz); MS (IC)

m/z 187 (M⁺+ 1); [R]_D) -11.7° (c ) 0.45, CH₂Cl₂).

22

Eth yl (1S,2S,3S,5R,6S)-2-Sp ir o-5′-h yd a n toin -3-flu or o-

bicyclo[3.1.0]h exa n e-6-ca r boxyla te ((+)-20). A mixture of

((-)-17) (280 mg, 1.50 mmol), ammonium carbonate (360 mg,

3.75 mmol), and potassium cyanide (110 mg, 1.69 mmol) in a

mixture (3.7 mL) of water and EtOH (2:3) was stirred at 35

°C for 1.5 days. The mixture was concentrated under reduced

pressure and partitioned between AcOEt and saturated brine.

4904 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25

Nakazato et al.

(1R S ,2S R ,5R S ,6R S )-2-Sp ir o-5′-h yd a n t oin -6-flu or o-

bicyclo[3.1.0]h exa n e-6-ca r boxylic Acid ((()-34). A mixture

of (()-33 (256 mg, 1.38 mmol) and 1 N NaOH (1.40 mL, 1.40

mmol) in EtOH (2.60 mL) was stirred for 10 min with ice

cooling. To the mixture, 1 N HCl was added dropwise to pH 1,

and the resulting mixture was partitioned between AcOEt and

saturated brine. The water phase was extracted with AcOEt

twice, and the combined organic phase was dried (Na₂SO₄) and

then concentrated under reduced pressure. A mixture of the

residue, (NH₄)₂CO₃(796 mg, 8.28 mmol), and KCN (277 mg,

4.25 mmol) in a mixture of EtOH and water (1:1) (2.0 mL)

was stirred at 55 °C for 8.5 h. After cooling in an ice bath, the

reaction mixture was acidified by treatment with concentrated

HCl. The resulting mixture was chromatographed on AG50W-

X8 cation-exchange resin (Bio-Rad) (H₂O) to yield (()-34 (310

mg, 99% yield) as a colorless crystal: mp 256 °C (decomposed);

¹H NMR (200 MHz, DMSO-d₆) δ 1.49-1.70 (1 H, m), 1.93-

2.40 (5 H, m), 8.08 (1 H, s), 10.71 (1 H, s); MS (CI) (Pos) m/z

229 (M⁺+ 1); Anal. (C₉H₉FN₂O₄) C, H, F, N.

(1R S ,2S R ,5R S ,6R S )-2-Am in o-6-flu or ob icyclo[3.1.0]-

h exa n e-2,6-d ica r boxylic Acid ((()-11). A solution of (()-

34 (200 mg, 0.88 mmol) in aqueous 60% H₂SO₄(3.0 mL) was

stirred at 140 °C for 6 days. The reaction solution was then

cooled to room temperature and brought to pH 8 with 5 M

aqueous NaOH. The resulting mixture was chromatographed

on AG50W-X8 cation-exchange resin (Bio-Rad) (H₂O-50%

aqueous THF-10% aqueous pyridine) to yield (()-11 (61 mg,

34% yield) as a colorless crystal: mp 225 °C (decomposed); ¹H

NMR (300 MHz, trifluoroacetic acid-d) δ 2.15-2.28 (1 H, m),

2.57 (1 H, dd, J ) 13.5, 8.6 Hz), 2.67-2.94 (4 H, m); MS (ion

spray) (Nega) m/z 202 (M⁺- 1); Anal. (C₈H₁₀FNO₄‚1/2H₂O)

C, H, F, N.

105 mmol) in THF (230 mL) was added dropwise to a solution

of LHMDS prepared by treatment of HMDS (20.3 g, 125 mmol)

with 1.61 M solution of n-butyllithium in hexane (78 mL, 125

mmol) in THF (230 mL), at -75 °C under a nitrogen atmo-

sphere. After the mixture was stirred for 1 h at this temper-

ature, TMSCl (19.8 mL, 157 mmol) was added, and the

reaction mixture was stirred for 1.5 h at room temperature.

After concentration of the reaction solution under reduced

pressure, anhydrous hexane was added to the residue, the

resulting inorganic salts were filtered off, and the filtrate was

concentrated under reduced pressure.

A mixture of the above residue and Pd(OAc)₂(25.9 g, 115

mmol) in MeCN (240 mL) was stirred at room temperature

for 16 h. To the mixture was added Et₂O, and resulting

mixture was filtered through Celite. The filtrate was concen-

trated under reduced pressure and then chromatographed

(hexane/AcOEt 9:1-5:1) to yield (()-39 (17.1 g, 89% yield) as

a colorless oil: ¹H NMR (200 MHz, CDCl₃) δ 1.34 (3 H, t, J )

7.3 Hz), 2.78 (1 H, dt, J ) 0.6, 5.8 Hz), 3.22 (1 H, dd, J ) 2.9,

5.8 Hz), 4.31 (2 H, q, J ) 7.3 Hz), 6.07 (1 H, dd, J ) 0.6, 5.6

Hz), 7.42 (1 H, ddd, J ) 0.6, 2.9, 5.6 Hz); MS (CI) (Pos) m/z

185 (M⁺+ 1).

E t h yl (1RS,3RS,4RS,5SR,6RS)-3,4-E p oxy-6-flu or o-2-

oxobicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-40). To a solu-

tion of (()-39 (13.8 g, 74.9 mmol) in toluene (70 mL) was added

aqueous 70% tert-butyl hydroperoxide (32.9 mL, 256 mmol)

and a solution of 40% aqueous benzyltrimethylammonium

hydroxide (3.2 mL, 7.56 mmol) in EtOH (10 mL), and the

mixture was then stirred at room temperature for 4 h. The

reaction mixture was poured into 20% aqueous Na₂S₂O₃cooled

in an ice bath and extracted with AcOEt twice. The combined

organic phase was washed with saturated brine, dried (Na₂-

SO₄), concentrated under reduced pressure, and then chro-

matographed (hexane/AcOEt 8:1-6:1) to yield (+)-40 (14.7 g,

98% yield) as a colorless crystal: mp 47-48 °C; ¹H NMR (200

MHz, CDCl₃) δ 1.34 (3 H, t, J ) 7.3 Hz), 2.50 (1 H, ddt, J )

0.8, 2.4, 6.0 Hz), 3.19 (1 H, dt, J ) 0.8, 6.0 Hz), 3.53 (1 H, dt,

J ) 0.8, 2.4 Hz), 4.02 (1 H, tt, J ) 0.8, 2.4 Hz), 4.32 (2 H, q, J

) 7.3 Hz); MS (EI) (Pos) m/z 200 (M⁺); Anal. (C₉H₉FO₄) C, H,

F.

Eth yl (E)-2-Meth yl-5-ca r boxy-2-p en ten a te (36). In a

manner similar to the preparation of 32 from 31, 36 (3.47 g,

76% yield) was obtained from ethyl (E)-2-methyl-6-(tetra-

hydropyran-2-yl)oxy-2-hexenate 35³⁶(10.0 g, 24.4 mmol) and

8 N J ones’ reagent (50 mL) in acetone (100 mL), as a colorless

oil: ¹H NMR (200 MHz, CDCl₃) δ 1.30 (3 H, t, J ) 7.0 Hz),

1.87 (3 H, d, J ) 1.5 Hz), 2.52 (4 H, d, J ) 3.3 Hz), 4.23 (2 H,

q, J ) 7.0 Hz), 6.70 (1 H, m); MS (CI) (Pos) m/z 187 (M⁺+ 1).

Eth yl (1RS,5RS,6SR)-6-Meth yl-2-oxobicyclo[3.1.0]h ex-

a n e-6-ca r boxyla te ((()-37). In a manner similar to the

preparation of (()-33 from 32, (()-37 (2.82 g, 84% yield) was

obtained from 36 (3.40 g, 18.3 mmol) by treatment with

(COCl)₂(4.8 mL, 55.0 mmol) and then a solution of CH₂N₂in

Et₂O followed by Cu(TBS)₂(301 mg, 0.73 mmol), as a colorless

oil: ¹H NMR (200 MHz, CDCl₃) δ 1.25 (3 H, t, J ) 7.0 Hz),

1.34 (3 H, s), 1.96-2.55 (6 H, m), 4.13 (2 H, q, J ) 7.0 Hz);

MS (EI) m/z 182 (M⁺).

(1R S ,2S R ,5R S ,6S R )-2-Sp ir o-5′-h yd a n t oin -6-m e t h yl-

bicyclo[3.1.0]h exan e-6-car boxylic Acid ((()-38). After treat-

ment of (()-37 (200 mg, 1.06 mmol) with 1 N NaOH (1.10 mL,

1.10 mmol), 1 N HCl, and then (NH₄)₂CO₃(611 mg, 6.36 mmol)

and KCN (207 mg, 3.18 mmol) followed by concentrated HCl

in a manner similar to the preparation of (()-34 from (()-33,

the resulting mixture was chromatographed on AG50W-X8

cation-exchange resin (Bio-Rad) (H₂O) to yield (()-38 (111 mg,

46% yield) as a colorless crystal: mp 280 °C (decomposed); ¹H

NMR (200 MHz, DMSO-d₆) δ 1.34 (3 H, s), 1.42-1.57 (1 H,

m), 1.75-2.16 (5 H, m), 7.96 (1 H, s), 10.66 (1 H, s); MS (ES)

(Nega) m/z 223 (M⁺- 1); Anal. (C₁₀H₁₂N₂O₄) C, H, N.

E t h yl (1RS,4SR,5SR,6RS)-6-F lu or o-4-h yd r oxy-2-oxo-

bicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-41). Under a ni-

trogen atmosphere, to a solution of (()-40 (5.63 g, 28.1 mmol)

in EtOH (80 mL) was added a solution prepared by the

treatment of diphenyl diselenide (PhSe)₂(13.7 g 43.9 mmol)

with sodium borohydride (3.19 g, 84.3 mmol) in EtOH (110

mL) followed by acetic acid (0.70 mL, 12.1 mmol),³²and the

resulting mixture was stirred at 35 °C for 2.5 h. The reaction

mixture was diluted with AcOEt (380 mL) into which oxygen

was passed at room temperature for 5 min. The mixture was

washed with half-saturated brine, and the separated water

phase was extracted with AcOEt. The combined organic phase

was dried (Na₂SO₄), concentrated under reduced pressure, and

chromatographed (hexane/AcOEt 4:1-3:1-1:1) to yield (()-

41 (4.30 g, 76% yield) as a light yellow oil: ¹H NMR (200 MHz,

CDCl₃) δ 1.34 (3 H, t, J ) 7.1 Hz), 2.05 (1 H, d, J ) 5.1 Hz),

2.30 (1 H, dd, J ) 3.5, 19.2 Hz), 2.63 (1 H, dt, J ) 5.9, 19.2

Hz), 2.72 (1 H, d, J ) 5.9 Hz), 2.85 (1 H, dd, J ) 2.1, 5.9 Hz),

4.31 (2 H, q, J ) 7.1 Hz), 4.76 (1 H, t, J ) 5.1 Hz); MS (EI)

(Pos) m/z 202 (M⁺).

Eth yl (1RS,2SR,4SR,5SR,6SR)-2-Sp ir o-5′-h yd a n toin -6-

flu or o-4-h yd r oxybicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-

42). After treatment of (()-41 (720 mg, 3.56 mmol) with 1 N

NaOH (3.70 mL, 3.70 mmol) in EtOH (3.70 mL), to this

mixture was added (NH₄)₂CO₃(860 mg, 8.95 mmol) and KCN

(260 mg, 3.99 mmol), and the resulting mixture was then

stirred at 35 °C for 3 days. The cooled mixture in an ice bath

was adjusted to pH 1 by treatment with concentrated HCl and

chromatographed on AG50W-X8 cation-exchange resin (Bio-

Rad) (H₂O) to yield crude (()-21 (440 mg) as a light yellow

solid. The solid was treated with EtOH (90 mg, 1.95 mmol),

4-(dimethylamino)pyridine (20 mg, 0.16 mmol), and 1-ethyl-

3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC‚

HCl) (380 mg, 1.98 mmol) in DMF (3.9 mL) at room temper-

(1RS,2SR,5RS,6SR)-2-Am in o-6-m et h ylb icyclo[3.1.0]-

h exa n e-2,6-d ica r boxylic Acid ((()-12). A solution of (()-

38 (120 mg, 0.54 mmol) in 2 N NaOH (1.4 mL) was stirred at

reflux for 4 days. The reaction solution was cooled to room

temperature and chromatographed on AG50W-X8 cation-

exchange resin (Bio-Rad) (H₂O-H₂O/THF (1:1)-10% aqueous

pyridine) to yield (()-12 (84 mg, 79% yield) as a colorless

crystal: mp 245 °C (decomposed); ¹H NMR (300 MHz, D₂O) δ

1.36 (3H, s), 1.64-1.75 (1 H, m), 1.92-1.99 (1 H, m), 2.14-

2.33 (4 H, m); MS (ES) (Nega) m/z 198 (M⁺- 1); Anal. (C₉H₁₃

NO₄‚1/3H₂O) C, H, N.

-

Eth yl (1RS,5RS,6RS)-6-F lu or o-2-oxobicyclo[3.1.0]h ex-

3-en e-6-ca r boxyla te ((()-39). A solution of (()-33 (19.5 g,

Synthesis of Group II mGluR Agonists

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4905

ature 16 h, poured into 1 N HCl, and extracted with CHCl₃.

The extract was dried (Na₂SO₄), concentrated under reduced

pressure, and chromatographed (CHCl₃/MeOH 50:1-40:1)

followed by recrystallization from EtOH to yield (()-42 (153

mg, 16% yield) as a colorless crystal: mp 196 °C (decomposed);

¹H NMR (300 MHz, DMSO-d₆) δ 1.21 (3 H, t, J ) 7.2 Hz),

1.90-2.08 (2 H, m), 2.26 (1 H, dd, J ) 1.8, 7.2 Hz), 2.45 (1 H,

dd, J ) 1.8, 7.2 Hz), 4.17 (2 H, q, J ) 7.2 Hz), 4.33 (1 H, dd,

J ) 5.6, 8.8 Hz), 4.75 (1 H, d, J ) 8.8 Hz), 8.13 (1 H, s), 11.00

(1 H, s); MS (ES) (Nega) m/z 271 (M⁺- 1); Anal. (C₁₁H₁₃FN₂O₅‚

H₂O) C, H, F, N.

concentrated HCl, and the resulting precipitate was collected

by filtration to yield (()-45 (1.18 g, 79% yield) as a colorless

crystal: mp 287 °C (decomposed); ¹H NMR (200 MHz, DMSO-

d₆) δ 2.37-2.50 (2 H, m), 2.68 (1 H, dd, J ) 1.9, 6.9 Hz), 2.76

(1 H, dd, J ) 4.2, 15.4 Hz), 3.28-3.50 (4 H, m), 8.10 (1 H, s),

10.78 (1 H, s); MS (ES) (Nega) m/z 317 (M⁺- 1); Anal. (C₁₁H₁₁

-

FN₂O₄S₂) C, H, F, N, S.

(1RS,2SR,5SR,6SR)-2-Am in o-6-flu or o-4-oxobicyclo[3.1.0]-

h exa n e-2,6-d ica r boxylic Acid ((()-14). In a manner similar

to the preparation of (()-11 from (()-34, (()-14 (41 mg, 12%

yield) was obtained from (()-45 (500 mg, 24.4 mmol) in 60%

H₂SO₄(12 mL) as a light yellow crystal: mp 170 °C (decom-

posed); ¹H NMR (300 MHz, trifluoroacetic acid-d) δ 3.16 (1 H,

dd, J ) 4.6, 19.5 Hz), 3.45 (1 H, dd, J ) 4.6, 19.5 Hz), 3.46 (1

H, d, J ) 6.6 Hz), 3.67 (1 H, d, J ) 6.6 Hz); MS (ES) (Nega)

m/z 216 (M⁺- 1); Anal. (C₈H₈FNO₅?H₂O) C, H, F, N.

N-(R)-1-P h en yleth yl-(1R,2S,5R,6S)-2-spir o-5′-h ydan toin -

4,4-et h ylen ed it h io-6-flu or ob icyclo[3.1.0]h exa n e-6-ca r -

boxyla m id e (sligtly p ola r isom er 46) a n d N-(R)-1-

P henylethyl-(1S,2R,5S,6R)-2-spiro-5′-hydantoin-4,4-ethylene-

d it h io-6-flu or ob icyclo[3.1.0]h e xa n e -6-ca r b oxyla m id e

(h igh ly p ola r isom er 46). To a solution of (()-45 (5.70 g,

17.9 mmol), (R)-(+)-1-phenylethylamine (2.60 g, 21.5 mmol),

and 1-hydroxybenzotriazole monohydrate (3.40 g, 22.2 mmol)

was added EDC‚HCl (4.10 g, 21.4 mmol) in DMF (240 mL)

with ice cooling. After being stirred for 2 h, the mixture was

stirred at room temperature for 16 h and partitioned between

AcOEt and 1 N HCl. The separated water phase was extracted

with AcOEt three times, and the combined organic phase was

washed with saturated brine, dried (Na₂SO₄), and concentrated

under reduced pressure. The residue was chromatographed

(CHCl₃/MeOH 50:1) to yield slightly polar isomer 46 (3.50 g,

46% yield) and highly polar isomer 46 (3.50 g, 46% yield) as

colorless crystals.

(1RS,2SR,4SR,5SR,6SR)-2-Am in o-6-flu or o-4-h yd r oxy-

bicyclo[3.1.0]h exa n e-2,6-d ica r boxylic Acid ((()-13). In a

manner similar to the preparation of (()-11 from (()-34, (()-

13 (17 mg, 15% yield) was obtained from (()-42 (140 mg, 0.51

mmol) by treatment with 60% H₂SO₄(4 mL), as a colorless

crystal: mp 220 °C (decomposed): ¹H NMR (300 MHz, pyri-

dine-d₆-D₂O 1:1) δ 2.56-2.75 (3 H, m), 2.92 (1 H, dd, J ) 1.2,

6.9 Hz), 4.56 (2 H, d, J ) 5.4 Hz); MS (ES) (Nega) m/z 218

(M⁺- 1); Anal. (C₈H₁₀FNO₅‚H₂O) C, H, F, N.

Eth yl (1RS,4RS,5RS,6SR)-2,2-Eth ylen d ith io-6-flu or o-

4-h yd r oxybicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-43). To

a solution of (()-41 (2.28 g, 13.9 mmol) and tert-butyldimeth-

ylsilyl chloride (TBSCl) (2.50 g, 16.6 mmol) in DMF (14 mL)

was added imidazole (1.04 g, 15.3 mmol), and the resulting

mixture was stirred for 16 h with ice cooling. The mixture was

poured into water and extracted with a mixture of AcOEt and

hexane (1:1). The extract was washed with water and satu-

rated brine, dried (Na₂SO₄), concentrated under reduced

pressure, and chromatographed (hexane/AcOEt 15:1) to yield

ethyl (1RS,4SR,5SR,6RS)-4-tert-butyldimethylsilyloxy-6-fluoro-

2-oxobicyclo[3.1.0]hexane-6-carboxylate (3.81 g, 86% yield) as

a colorless oil: ¹H NMR (200 MHz, CDCl₃) δ 0.11 (3 H, s), 0.13

(3 H, s), 0.90 (9 H, s), 1.33 (3 H, t, J ) 7.1 Hz), 2.21 (1 H, dd,

J ) 4.0, 19.1 Hz), 2.57 (1 H, dt, J ) 5.6, 19.1 Hz), 2.60-2.72

(4 H, m), 4.31 (2 H, q, J ) 7.1 Hz), 4.66 (1 H, d, J ) 5.6 Hz);

MS (CI) (Pos) m/z 317 (M⁺+ 1).

To a solution of (1RS,4SR,5SR,6RS)-4-tert-butyldimethyl-

silyloxy-6-fluoro-2-oxobicyclo[3.1.0]hexane-6-carboxylate (3.70

g, 11.7 mmol) and 1,2-ethylenedithiol (1.20 mL, 14.0 mmol)

in CHCl₃(37 mL), BF₃‚OEt₂(0.44 mL, 3.5 mmol) was added

dropwise at room temperature, and the resulting mixture was

stirred at room temperature for 16 h. The reaction mixture

was washed with saturated NaHCO₃and saturated brine,

dried (Na₂SO₄), concentrated under reduced pressure, and

chromatographed (hexane/AcOEt 4:1-2:1) to yield (()-43 (3.21

g, 99% yield) as a colorless oil: ¹H NMR (200 MHz, CDCl₃) δ

1.32 (3 H, t, J ) 7.1 Hz), 2.07 (1 H, d, J ) 7.1 Hz), 2.38-2.69

(4 H, m), 3.33-3.45 (4 H, m), 4.27 (2 H, q, J ) 7.1 Hz), 4.50 (1

H, dd, J ) 5.5, 7.1 Hz); MS (EI) (Pos) m/z 278 (M⁺).

E t h yl (1RS,5RS,6SR)-4,4-E t h ylen ed it h io-6-flu or o-2-

oxobicyclo[3.1.0]h exa n e-6-ca r boxyla te ((()-44). To solu-

tion of (()-43 (3.10 g, 11.1 mmol) and dicyclohexylcarbodiimide

(DCC) (9.00, 43.4 mmol) in dimethylsufoxide (DMSO) (116 mL)

was added pyridine (1.2 mL) and trifluoroacetic acid (0.6 mL),

and the resulting mixture was stirred at room temperature

for 16 h. The precipitate in the mixture was filtered, and the

filtrate was partitioned between AcOEt and water. The

separated organic phase was washed with water and saturated

brine, dried (Na₂SO₄), concentrated under reduced pressure,

and chromatographed (hexane/AcOEt 7:1-5:1) to yield (()-

44 (2.60 g, 85% yield) as a colorless crystal: mp 72-74 °C; ¹H

NMR (CDCl₃) δ 1.35 (3 H, t, J ) 7.1 Hz), 2.79 (1 H, d, J ) 6.3

Hz), 2.86-3.08 (2 H, m), 3.18 (1 H, dd, J ) 1.9, 6.3 Hz), 3.38-

3.53 (4 H, m), 4.31 (2 H, q, J ) 7.1 Hz); MS (EI) (Pos) m/z 276

(M⁺); Anal. (C₁₁H₁₃FO₃S₂) C, H, F, S.

Slightly polar isomer 46: R_f0.74 (CHCl₃/MeOH 9:1); mp

1

288-289 °C; H NMR (200 MHz, DMSO-d₆) δ 1.45 (3 H, d, J

) 7.0 Hz), 2.34 (1 H, dd, J ) 2.6, 6.8 Hz), 2.46 (1 H, m), 2.57

(1 H, dd, J ) 2.3, 6.8 Hz), 2.78 (1 H, dd, J ) 4.0, 15.2 Hz),

3.34 (4 H, s), 5.04 (1 H, m), 7.23-7.41 (5 H, m), 8.10 (1 H, s),

8.81 (1 H, d, J ) 8.4 Hz), 10.77 (1 H, s); MS (ES) (Nega) m/z

420 (M⁺- 1); [R]_D²⁶) +62.6° (c ) 0.21, MeOH); Anal. (C₁₉H₂₀

FN₃O₃S₂) C, H, F, N, S.

-

Highly polar isomer 46: R_f0.69 (CHCl₃/MeOH 9:1); mp 315-

316 °C; ¹H NMR (200 MHz, DMSO-d₆) δ 1.44 (3 H, d, J ) 7.0

Hz), 2.36-2.52 (3 H, m), 2.78 (1 H, dd, J ) 4.0, 15.2 Hz), 3.29

(4 H, s), 5.00 (1 H, m), 7.20-7.42 (5 H, m), 8.07 (1 H, s), 8.79

(1 H, d, J ) 8.4 Hz), 10.80 (1 H, s); MS (ES) (Nega) m/z 420

(M⁺- 1); [R]_D) +52.6° (c ) 0.24, MeOH); Anal. (C₁₉H₂₀

-

26

FN₃O₃S₂) C, H, F, N, S.

(1S,2R,5R,6R)-2-Am in o-6-flu or o-4-oxobicyclo[3.1.0]h ex-

a n e-2,6-d ica r boxylic Acid ((-)-14). In a manner similar to

the preparation of (()-11 from (()-34, (-)-14 (40 mg, 28%

yield)⁵⁶was obtained from slightly polar isomer 46 (281 mg,

0.67 mmol) in 60% H₂SO₄(5.2 mL) as a light yellow crystal:

1

mp 175 °C (decomposed); H NMR (300 MHz, trifluoroacetic

acid-d) δ 3.16 (1 H, dd, J ) 4.6, 19.5 Hz), 3.45 (1 H, dd, J )

4.6, 19.5 Hz), 3.46 (1 H, d, J ) 6.6 Hz), 3.67 (1 H, d, J ) 6.6

Hz); MS (ES) (Nega) m/z 216 (M⁺- 1); [R]_D) -75.1° (c )

24

0.16, 1 M HCl); Anal. (C₈H₈FNO₅‚H₂O) C, H, F, N.

(1R,2S,5S,6S)-2-Am in o-6-flu or o-4-oxobicyclo[3.1.0]h ex-

a n e-2,6-d ica r boxylic Acid ((+)-14). In a manner similar to

the preparation of (()-11 from (()-34, (+)-14 (1.22 g, 45%

yield)⁵⁶was obtained from highly polar isomer 46 (5.30 g, 12.6

mmol) in 60% H₂SO₄(96 mL) as a light yellow crystal: mp

175 °C (decomposed); ¹H NMR (300 MHz, trifluoroacetic acid-

d) δ 3.16 (1 H, dd, J ) 4.6, 19.5 Hz), 3.45 (1 H, dd, J ) 4.6,

19.5 Hz), 3.46 (1 H, d, J ) 6.6 Hz), 3.67 (1 H, d, J ) 6.6 Hz);

MS (ES) (Nega) m/z 216 (M⁺- 1); [R]_D) +73.2° (c ) 0.13, 1

24

(1RS,2SR,5RS,6SR)-2-Sp ir o-5′-h yd a n toin -6-flu or o-4,4-

eth ylen edith iobicyclo[3.1.0]h exan e-6-car boxylic Acid ((()-

45). In a manner similar to the preparation of (()-42 from (()-

41, (()-44 (1.30 g, 4.70 mmol) was treated with 1 N NaOH

(5.00 mL, 5.00 mmol) in EtOH (5.0 mL), and then (NH₄)₂CO₃

(1.13 mg, 11.8 mmol) and KCN (0.34 mg, 5.22 mmol). The

reaction mixture was adjusted to pH 1 by the addition of

M HCl); Anal. (C₈H₈FNO₅‚H₂O) C, H, F.

(1R,2S,5R,6R)-2-Sp ir o-5′-h yd a n t oin -6-flu or ob icyclo-

[3.1.0]h exa n e-6-ca r boxyla te ((-)-34). In a manner similar

to the preparation of (()-34 from (()-33, (-)-34 (427 mg, 90%

yield) was obtained from (-)-33⁴⁰(389 g, 2.1 mmol) as a light

1

yellow crystal: mp 311-314 °C (decomposed); H NMR (200

4906 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25

Nakazato et al.

MHz, DMSO-d₆) δ 1.49-1.70 (1 H, m), 1.93-2.40 (5 H, m),

8.08 (1 H, s), 10.71 (1 H, s); MS (CI) (Pos) m/z 229 (M⁺+ 1);

dithiol in the same manner as for preparation of (()-43 from

ethyl (()-(1RS,4SR,5SR,6RS)-4-tert-butyldimethylsilyloxy-6-

fluoro-2-oxobicyclo[3.1.0]hexane-6-carboxylate, to yield (+)-43

(60.48 g, 94% yield) as a colorless oil: ¹H NMR (200 MHz,

CDCl₃) δ 1.32 (3 H, t, J ) 7.1 Hz), 2.07 (1 H, d, J ) 7.1 Hz),

2.38-2.69 (4 H, m), 3.33-3.45 (4 H, m), 4.27 (2 H, q, J ) 7.1

Hz), 4.50 (1 H, dd, J ) 5.5, 7.1 Hz); MS (EI) (Pos) m/z 278

26

[R]_D) -77.3° (c ) 0.41, 1 M NaOH); Anal. (C₉H₉FN₂O₄) C,

H, F, N.

(1S,2R,5S,6S)-2-Spir o-5′-h ydan toin -6-flu or obicyclo[3.1.0]-

h exa n e-6-ca r boxyla te ((+)-34). In a manner similar to the

preparation of (()-34 from (()-33, (+)-34 (444 mg, 88% yield)

was obtained from (+)-33⁴⁰(414 g, 2.2 mmol) as a light yellow

crystal: mp 311-313 °C (decomposed); ¹H NMR (200 MHz,

26

(M⁺); [R]_D) +44.9° (c ) 0.25, CHCl₃).

E t h yl (1R,5R,6S)-4,4-E t h ylen ed it h io-6-flu or o-2-oxo-

bicyclo[3.1.0]h exa n e-6-ca r boxyla te ((-)-44). In a manner

similar to the preparation of (()-44 from (()-43, (-)-44 (45.58

g, 76% yield) was obtained from (+)-43 (60.00 g, 0.116 mol) as

a colorless crystal: mp 87-89 °C; ¹H NMR (200 MHz, CDCl₃)

δ 1.35 (3 H, t, J ) 7.1 Hz), 2.79 (1 H, d, J ) 6.3 Hz), 2.86-

3.08 (2 H, m), 3.18 (1 H, dd, J ) 1.9, 6.3 Hz), 3.38-3.53 (4 H,

DMSO-d₆) δ 1.49-1.70 (1 H, m), 1.93-2.40 (5 H, m), 8.08 (1

26

H, s), 10.71 (1 H, s); MS (CI) (Pos) m/z 229 (M⁺+ 1); [R]_D

)

+77.9° (c ) 0.43, 1 M NaOH); Anal. (C₉H₉FN₂O₄) C, H, F, N.

(1R,2S,5R,6R)-2-Am in o-6-flu or ob icyclo[3.1.0]h exa n e-

2,6-d ica r boxylic Acid ((-)-11). In a manner similar to the

preparation of (()-11 from (()-34, (-)-11 (239 mg, 73% yield)⁵⁶

was obtained from (-)-34 (350 mg, 1.5 mmol) as a light yellow

crystal: mp 233 °C (decomposed); ¹H NMR (300 MHz, trifluo-

roacetic acid-d) δ 2.15-2.28 (1 H, m), 2.57 (1 H, dd, J ) 8.6,

13.5 Hz), 2.67-2.94 (4 H, m); MS (IonSpray) (Nega) m/z 202

26

m), 4.31 (2 H, q, J ) 7.1 Hz); MS (EI) (Pos) m/z 276 (M⁺); [R]_D

) -42.8° (c ) 0.14, CHCl₃); Anal. (C₁₁H₁₃FO₃S₂) C, H, F, S.

N-(R)-1-P h en yleth yl-(1R,2S,5R,6S)-2-spir o-5′-h ydan toin -

4,4-et h ylen ed it h io-6-flu or ob icyclo[3.1.0]h exa n e-6-ca r -

boxyla m id e ((1R,2S,5R,6S)-46). After treatment of (-)-44

(47.39 g, 0.171 mol) with 1 N NaOH (180 mL, 0.180 mol) in

EtOH (180 mL), to the mixture were added (NH₄)₂CO₃(41.2

g, 0.429 mol) and KCN (12.3 g, 0.189 mmol), and the resulting

mixture was then stirred at 35 °C for 5 days. The cooled

mixture in an ice bath was adjusted to pH 1 by the treatment

of concentrated HCl and chromatographed on AG50W-X8

cation-exchange resin (Bio-Rad) (H₂O-50% aqueous H₂O) to

yielded crude (1R,2S,5R,6S)-45 (51.2 g) as a light yellow solid.

The solid was treated with (R)-(+)-1-phenylethylamine (23.4

g, 0.193 mol), 1-hydroxybenzotriazole monohydrate (30.8 g,

0.201 mol), and EDC‚HCl (34.8 g, 0.182 mol) in DMF (1.08 L)

at room temperature for 13 h and the mixture was partitioned

between AcOEt and 1 N HCl. The separated water phase was

extracted with AcOEt three times, and the combined organic

phase was washed with saturated brine, dried (Na₂SO₄), and

concentrated under reduced pressure. The residue was chro-

matographed (CHCl₃/MeOH 50:1-20:1) to yield (1R,2S,5R,6S)-

(M⁺- 1); [R]_D) -58.8° (c ) 0.14, H₂O); Anal. (C₈H₁₀FNO₄‚

26

1/2H₂O) C, H, F, N.

(1S,2R,5S,6S)-2-Am in o-6-flu or ob icyclo[3.1.0]h exa n e-

2,6-d ica r boxylic Acid ((+)-11). In a manner similar to the

preparation of (()-11 from (()-34, (+)-11 (233 mg, 72% yield)⁵⁶

was obtained from (+)-34 (350 mg, 1.5 mmol) as a light yellow

crystal: mp 228 °C (decomposed); ¹H NMR (300 MHz, trifluo-

roacetic acid-d) δ 2.15-2.28 (1 H, m), 2.57 (1 H, dd, J ) 8.6,

13.5 Hz), 2.67-2.94 (4 H, m); MS (IonSpray) (Nega) m/z 202

(M⁺- 1); [R]_D) +57.5° (c ) 0.16, H₂O); Anal. (C₈H₁₀FNO₄‚

26

1/2H₂O) C, H, F, N.

E t h yl (1R,5R,6R)-6-F lu or o-2-oxob icyclo[3.1.0]h ex-3-

en e-6-ca r boxyla te ((+)-39). In a manner similar to the

preparation of (()-39 from (()-33, (+)-39 (76.10 g, 89% yield)

was obtained from (+)-33 (86.40 g, 0.46 mol) as a colorless oil:

¹H NMR (200 MHz, CDCl₃) δ 1.34 (3 H, t, J ) 7.3 Hz), 2.78 (1

H, dt, J ) 0.6, 5.8 Hz), 3.22 (1 H, dd, J ) 2.9, 5.8 Hz), 4.31 (2

H, q, J ) 7.3 Hz), 6.07 (1 H, dd, J ) 0.6, 5.6 Hz), 7.42 (1 H,

ddd, J ) 0.6, 2.9, 5.6 Hz); MS (CI) (Pos) m/z 185 (M⁺+ 1);

1

46 (42.6 g, 59% yield): mp 287-289 °C; H NMR (200 MHz,

26

[R]_D) +430° (c ) 0.34, CHCl₃).

DMSO-d₆) δ 1.45 (3 H, d, J ) 7.0 Hz), 2.34 (1 H, dd, J ) 2.6,

6.8 Hz), 2.46 (1 H, m), 2.57 (1 H, dd, J ) 2.3, 6.8 Hz), 2.78 (1

H, dd, J ) 4.0, 15.2 Hz), 3.34 (4 H, s), 5.04 (1 H, m), 7.23-

7.41 (5 H, m), 8.10 (1 H, s), 8.81 (1 H, d, J ) 8.4 Hz), 10.77 (1

Eth yl (1R,3R,4R,5S,6R)-3,4-Epoxy-6-flu or o-2-oxobicyclo-

[3.1.0]h exa n e-6-ca r boxyla te ((+)-40). In a manner similar

to the preparation of (()-40 from (()-39, (+)-40 (72.21 g, 88%

yield) was obtained from (+)-39 (75.60 g, 0.41 mol) as a

colorless oil: ¹H NMR (200 MHz, CDCl₃) δ 1.34 (3 H, t, J )

7.3 Hz), 2.50 (1 H, ddt, J ) 0.8, 2.4, 6.0 Hz), 3.19 (1 H, dt, J

) 0.8, 6.0 Hz), 3.53 (1 H, dt, J ) 0.8, 2.4 Hz), 4.02 (1 H, tt, J

) 0.8, 2.4 Hz), 4.32 (2 H, q, J ) 7.3 Hz); MS (EI) (Pos) m/z 200

H, s); MS (ES) (Nega) m/z 420 (M⁺- 1); [R]_D) +63.2° (c )

24

0.23, MeOH); Anal. (C₁₉H₂₀FN₃O₃S₂) C, H, F, N,S.

(1R,2S,5S,6S)-2-Am in o-6-flu or o-4-oxobicyclo[3.1.0]h ex-

a n e-2,6-d ica r boxylic Acid ((+)-14). In a manner similar to

the preparation of (()-11 from (()-34, (+)-14 (10.04 g, 43%

yield)⁵⁶was obtained from (1R,2S,5R,6S)-46 (45.0 g, 0.107 mol)

followed by recrystallization from water as a colorless crys-

tal: mp 175 °C (decomposed); ¹H NMR (trifluoroacetic acid-d)

δ 3.16 (1 H, dd, J ) 4.6, 19.5 Hz), 3.45 (1 H, dd, J ) 4.6, 19.5

Hz), 3.46 (1 H, d, J ) 6.6 Hz), 3.67 (1 H, d, J ) 6.6 Hz); MS

(M⁺); [R]_D) +84.2° (c ) 0.28, CHCl₃).

26

Eth yl (1R,4S,5S,6R)-6-F lu or o-4-h yd r oxy-2-oxobicyclo-

[3.1.0]h exa n e-6-ca r boxyla te ((+)-41). In a manner similar

to the preparation of (()-41 from (()-40, (+)-41 (17.09 g, 71%

yield) was obtained from (+)-40 (24.00 g, 0.12 mol) as a

colorless oil: ¹H NMR (200 MHz, CDCl₃) δ 1.34 (3 H, t, J )

7.1 Hz), 2.05 (1 H, d, J ) 5.1 Hz), 2.30 (1 H, dd, J ) 3.5, 19.2

Hz), 2.63 (1 H, dt, J ) 5.9, 19.2 Hz), 2.72 (1 H, d, J ) 5.9 Hz),

2.85 (1 H, dd, J ) 2.1, 5.9 Hz), 4.31 (2 H, q, J ) 7.1 Hz), 4.76

(1 H, t, J ) 5.1 Hz); MS (EI) (Pos) m/z 202 (M⁺); [R]_D²⁶) +79.4°

(c ) 0.26, CHCl₃).

(ES) (Nega) m/z 216 (M⁺- 1); [R]_D) +78.7° (c ) 0.65, 1 M

23

HCl); Anal. (C₈H₈FNO₅‚H₂O) C, H, F, N.

P h a r m a cology. Cell Cu ltu r e. CHO cell lines stably

expressing mGluR1a, mGluR2, mGluR3, mGluR4, mGluR6,

and mGluR7 were cultured in DMEM supplemented with 10%

dialyzed fetal bovine serum, 2 mM glutamine, 1% proline, 1

mM sodium pyruvate, 1 mM succinic acid, 50 U/mL penicillin,

and 50 µg/mL streptomicin. Cells were maintained at 37 °C

in a humidified atmosphere of 5% CO₂in air.

Mea su r em en ts of Cyclic AMP F or m a tion .⁴¹Agonist

activities for mGluR2, mGluR3, mGluR4, mGluR6, and mGluR7

were evaluated by measuring agonist-dependent inhibition of

forskolin-induced cyclic AMP (cAMP) formation in mGluR2-,

mGluR3-, mGluR4-, mGluR6-, and mGluR7-expressing cells.

Briefly, CHO cells expressing either mGluR2, mGluR3, mGluR4,

mGluR6, or mGluR7 were seeded in 96-well plates at a density

of 1.26 × 10⁴cells per well and grown for 2 days. The medium

was changed to fresh medium without 2 mM glutamine for 4

to 5 h. The cells were preincubated with PBS containing 1 mM

3-isobutyl-1-methylxanthine (IBMX) (PBS-IBMX) for 20 min

at 37 °C. The reaction was started by replacing the medium

Eth yl (1R,4R,5R,6S)-2,2-Eth ylen d ith io-6-flu or o-4-h y-

d r oxybicyclo[3.1.0]h exa n e-6-ca r boxyla te ((+)-43). In a

manner similar to the preparation of ethyl (1RS,4SR,5SR,6RS)-

4-tert-butyldimethylsilyloxy-6-fluoro-2-oxobicyclo[3.1.0]hexane-

6-carboxylate from (()-41, ethyl (+)-(1R,4S,5S,6R)-4-tert-

butyldimethylsilyloxy-6-fluoro-2-oxobicyclo[3.1.0]hexane-6-

carboxylate (73.10 g, 100% yield) was obtained from (+)-41

(46.70 g, 0.23 mol) as a colorless oil: ¹H NMR (200 MHz,

CDCl₃) δ 0.11 (3 H, s), 0.13 (3 H, s), 0.90 (9 H, s), 1.33 (3 H, t,

J ) 7.1 Hz), 2.21 (1 H, dd, J ) 4.0, 19.1 Hz), 2.57 (1 H, dt, J

) 5.6, 19.1 Hz), 2.60-2.72 (4 H, m), 4.31 (2 H, q, J ) 7.1 Hz),

4.66 (1 H, d, J ) 5.6 Hz); MS (CI) (Pos) m/z 317 (M⁺+ 1);

26

[R]_D) +48.2° (c ) 0.28, CHCl₃).

The carbonyl group of ethyl (+)-(1R,4S,5S,6R)-4-tert-bu-

tyldimethylsilyloxy-6-fluoro-2-oxobicyclo[3.1.0]hexane-6-car-

boxylate (73.00 g, 0.23 mol) was protected with 1,2-ethylene-

Synthesis of Group II mGluR Agonists

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4907

with fresh PBS-IBMX containing 10 µM forskolin and various

concentrations of test agents. After incubation for 15 min

(mGluR2, mGluR3, and mGluR4) or 30 min (mGluR6 and

mGluR7) at 37 °C, the reaction was terminated with ice-cold

100% ethanol and allowed to settle on ice for 40 min. The

supernatants were evaporated, and cAMP levels were deter-

mined by a cAMP enzymeimmunoassay (EIA) system (RPN

225, Amersham). Antagonist activities were measured with

30 µM glutamic acid present.

Gaithersburg, MD). Aquasol-2 scintillator (Du Pont/New En-

gland Neclear) (10 mL) was added, and filter-bound radioac-

tivity was counted in

a liquid scintillation spectrometer

(LS6000TA, Beckman Instruments Inc., Fullerton, CA). Non-

specific binding was determined in the presence of 10 µM of

compound 4.

For determination of the equilibrium dissociation constant

(K_d), the crude membrane preparation was incubated with

0.5-100 nM of [³H]-7. Saturation binding data were analyzed

by Scatchard plot analysis, and the K_dand the maximal

number of binding sites (B_max) were calculated.

In the competitive binding assay, the reaction was carried

out using 3.0 nM of [³H]-7. The concentration of the test

compound that caused 50% inhibition of specific binding of

[³H]-7 (IC₅₀value) was determined from each concentration-

response curve. K_ivalues for each test compound were

calculated using the K_dvalue obtained from Scatchard analy-

sis.

P h en cyclidin e (P CP )-In du ced Hyper activity in Rats.^47,48

Animals were housed individually in transparent acrylic cages

(47 × 28.5 × 29.5 cm) and acclimatized for 60 min with a

SCANET apparatus (Neuroscience Inc. J apan) placed in a

sound-proof box. Test compounds were administered orally 180

min before the i.p. administration of PCP (5 mg/kg). Im-

mediately after PCP administration, locomotor activity was

recorded every 10 min for 120 min. Values are expressed as

means ( S.E.M. Data were analyzed by ANOVA, and the

significance of differences between groups was determined

using Dunnett’s test. The total count for the vehicle-treated

control group was defined as 100%, the percent inhibition in

each group was calculated, and ED₅₀values were determined.

P CP -In d u ced Hea d -Wea vin g Beh a vior in R a t s.^11,49,50

Animals were placed individually in a clear acrylic cage (40.5

× 24.5 × 19.5 cm) and allowed a minimum of 60 min to

acclimatize to the new environment. Test compounds were

administered orally 60 min before the i.p. administration of

PCP (7.5 mg/kg). Immediately after PCP administration, the

number of head-weavings (slow, side-to-side or lateral head

movement) was counted every 10 min for 30 min. Values are

expressed as means ( S. E. M. Data were analyzed by ANOVA,

and the significance of differences between groups was deter-

mined using Dunnett’s test. The total count for the vehicle-

treated control group was defined as 100%, the percent

inhibition in each group was calculated, and ED₅₀values were

determined.

Mea su r em en ts of D-m yo In s (1,4,5) P ₃F or m a tion .^42,43

Agonist activities for mGluR1a were evaluated by measuring

D-myo Ins (1,4,5) P₃formation in mGluR1a-expressing cells.

Briefly, CHO cells expressing mGluR1a were seeded in 6-well

plates at a density of 3.72 × 10⁵cells per well and grown for

2 days. The medium was changed to fresh medium without 2

mM glutamine for 4 to 5 h. The cells were preincubated with

incubation buffer (20 mM HEPES, 150 mM NaCl, 1.5 mM KCl,

1.8 mM CaCl₂, 0.8mM MgSO₄, 25 mM glucose, and 10 mM

LiCl) (pH 7.4) for 20 min at 37 °C. The reaction was started

by replacing the medium with fresh incubation buffer contain-

ing various concentrations of test agents. After incubation for

15 s at room temperature, the reaction was terminated with

ice-cold 20% perchloric acid and incubated on ice for 20 min.

The cells were scraped, and cell suspensions were centrifuged

at 15 000 rpm for 10 min. The supernatants were neutralized

to pH 7.5 by titrating with ice-cold 1.5 M KOH containing 60

mM HEPES and recentrifuged to remove KClO₄precipitate.

The resulting supernatants were evaporated, and the content

of Ins (1,4,5) P₃was quantitatively determined using a D-myo-

Inositol 1,4,5-triphosphate (IP₃) [³H] assay system (TRK 1000,

Amersham). Antagonist activities were measured with 30 µM

glutamic acid present.

Mea su r em en t s of In t r a cellu la r Con cen t r a t ion of

Ca ^{2+ 44,45}Intracellular concentrations of Ca²⁺were determined

.

with the Ca²⁺-sensitive dye fura 2-AM. mGluR5-expressing

CHO cells were plated on dishes (Sumilon, MS-11900) and

cultured for 2 days, and the medium was changed to fresh

medium without 2 mM glutamine for 4 to 5 h. The cells were

loaded with 4 µM fura 2-AM in D-MEM cell culture medium

without ^L-glutamine for 30 min at 37 °C. After loading fura

2-AM, the cells were washed once with 20 mM HEPES buffer

(pH 7.4) containing 130 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂,

1 mM MgCl₂, 10 mM glucose, and 0.1% BSA and then scraped

from the well. The cells were washed with the buffer twice

and suspended in the buffer to obtain a concentration of 8 ×

10⁵cells/mL. The intensity of cell-associated fura 2-AM fluo-

resence was imaged in a CAF-100 (Nihonbunko). At the end

of each incubation, 0.02% Triton X-100 and 0.01 M EGTA were

added to measure maximal and minimal fluoresence values,

respectively. The intracellular Ca²⁺concentration was calcu-

lated from the ratio of 340/380 nm intensities. MGS compounds

were added to the cells with/without 10 µM glutamate.

Ack n ow led gm en t. The authors thank the fourth

Laboratory of Medicinal Laboratories in Taisho Phar-

maceutical Co., Ltd., for spectral data and elemental

analyses.

Su p p or tin g In for m a tion Ava ila ble: Elemental analysis

data. This material is available free of charge via the Internet

at http://pubs.acs.org.

[³H]-(+)-7 Bin d in g.⁴⁶The binding of [³H]-(+)-7 (34 Ci/

mmol) was performed according to the method previously

described with minor modifications. CHO cells stably express-

ing mGluR2 or 3 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 48000g 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 (pH 7.4) by resuspension

and recentrifugation. The pellet obtained was then suspended

in 50 mM Tris-HCl buffer (pH 7.4), containing 2 or 4 mM

MgCl₂, and served as a crude membrane preparation.

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nm). The separated enantiomers, (+)-33 and (-)-33, were

evaluated on CHIRALPAK AD (0.46 × 25 cm), using n-hexane/

2-propanol 3:1 as eluent, UV detection at 210 nm, flow rate of

27

1.0 mL/min, and column at room temperature. (+)-33: [R]_D

)

+27.98° (c ) 0.13, CHCl₃), t_R) 5.7 min. (-)-33: [R]_D²⁷) -30.33°

(c ) 0.16, CHCl₃), t_R) 9.1 min.

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min, column temperature: room temperature, detection: UV210

nm). The separated enantiomers, (()-16 and (-)-16, were

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2-propanol 3:1 as eluent, UV detection at 210 nm, flow rate of

1.0 mL/min, and column temperature of room temperature, and

23

each exhibited a single peak on the HPLC chart. (+)-16: [R]_D

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(54) The ratio was determined by comparing the integration volumes

of H_amide(see Figure 1) of (()-20, (()-21, or (()-22.

(55) The optical purities of (+)-7 and (-)-7 were evaluated on

CROWNPAK CR(+) (Daisel, two columns (0.4 × 15 cm) con-

nected in series, using eluent of pH 1 aqueous HClO₄, UV

detection at 208 nm, flow rate of 0.3 mL/min, and column

temperature of 2 °C. Compounds (+)-7 (t_R) 19.4 min) and (-)-7

(t_R) 23.0 min) each exhibited a single peak on the HPLC chart.

(56) The optical purities of (+)-14, (-)-14, (+)-11, and (-)-11 were

evaluated on CROWNPAK CR(+) (Daisel, two columns (0.4 ×

15 cm) connected in series, using eluent of 0.1 M aqueous HClO₄,

UV detection at 208 nm, flow rate of 0.25 mL/min (for (+)-14

and (-)-14) or 0.30 mL/min (for (+)-11 and (-)-11), and column

temperature of 6 °C. (+)-14 (t_R) 18.6 min), (-)-14 (t_R) 27.8

min), (+)-11 (t_R) 36.9 min), and (-)-11 (t_R) 25.9 min) each

exhibited a single peak on the HPLC chart.

J M000346K

Article Doi

Synthesis, SARs, and pharmacological characterization of 2-Amino-3 or 6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent, selective, and orally active group II metabotropic glutamate receptor agonists

DOI: 10.1021/jm000346k

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