Synthesis and biological evaluation of 2-(3′-(1H-tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl)glycine (S-TBPG), a novel mGlu1 receptor antagonist

Article abstract of DOI:10.1016/S0968-0896(00)00270-4

The design and synthesis of 2-(3′-(1H-tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl)glycine (S-TBPG), a novel mGluR1 antagonist is reported. S-TBPG is characterized by the bioisosteric replacement of the distal carboxy group of 2-(3′-carboxybicyclo[1.1.1]pent-1-yl)glycine (S-CBPG) by a tetrazolyl moiety. Despite a moderate reduction in potency, S-TBPG is a selective mGluR1 antagonist (69 μM), with no activity at other mGluR subtypes. The interesting biological profile of S-TBPG, coupled with its peculiar chemical structure, is discussed in terms of the structure-activity relationship (SAR) of mGluR1 antagonists.

Full text of DOI:10.1016/S0968-0896(00)00270-4

Bioorganic & Medicinal Chemistry 9 (2001) 221±227
Synthesis and Biological Evaluation of 2-(30-(1H-Tetrazol-5-yl)
bicyclo[1.1.1]pent-1-yl)glycine (S-TBPG), a Novel mGlu1
Receptor Antagonist
Gabriele Costantino,a Katiuscia Maltoni,a Maura Marinozzi,a Emidio Camaioni,a
Laurent Prezeau,b Jean-Philippe Pinb and Roberto Pellicciaria,*
a
Á
Dipartimento di Chimica e Tecnologia del Farmaco. Universita di Perugia, Via del Liceo 1, 06123 Perugia, Italy
bCentre National de la Recherche Scienti®que, UPR 9023 Ð CCIPE, 141 Rue de la Cardonille, 34094 Montpellier, France
Received 12 July 2000; accepted 22 August 2000
AbstractÐThe design and synthesis of 2-(30-(1H-tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl)glycine (S-TBPG), a novel mGluR1 antagonist
is reported. S-TBPG is characterized by the bioisosteric replacement of the distal carboxy group of 2-(30-carboxybicyclo [1.1.1]pent-
1-yl)glycine (S-CBPG) by a tetrazolyl moiety. Despite a moderate reduction in potency, S-TBPG is a selective mGluR1 antagonist
(69 mM), with no activity at other mGluR subtypes. The interesting biological pro®le of S-TBPG, coupled with its peculiar chemical
structure, is discussed in terms of the structure±activity relationship (SAR) of mGluR1 antagonists. # 2001 Elsevier Science Ltd.
All rights reserved.
Introduction
Recent reports, in particular, point out an inherent role
for mGlu1 subtype in the induction and in the progres-
sion of the post-ischemic neuronal damage,2 as well as
the notion that mGlu1 antagonists might be clinically
employed as neuroprotective agents following ischemia
and oxygen/glucose deprivation.3
Metabotropic glutamate (mGlu) receptors constitute a
heterogeneous family of G-protein coupled receptors
activated by synaptically released l-glutamic acid (1).1
At least eight genes encode for molecularly diverse
receptor subtypes that, when functionally expressed in
heterologous systems, couple to a variety of e€ector sys-
tems. On the basis of the deduced sequence homology
and the transduction mechanisms, the eight mGlu
receptor subtypes so far cloned have been classi®ed into
three groups. Group I includes mGlu1 and mGlu5
which are positively coupled to phospholipase C (PLC);
group II (mGlu2and mGlu3) and group III (mGlu4,
mGlu6, mGlu7, and mGlu8) are both negatively coupled
to adenylyl cyclase (AC) but are endowed with di€erent
pharmacology, di€erent neuronal and/or glial localiza-
tion and share little sequence homology with each other.
The search for selective mGlu1 antagonists, as well as
for other subtype selective antagonists, has been for
many years a knotty problem. The class of carboxy-
phenylglycines (CPGs, 2±5, Chart 1), ®rst reported by
Watkins in 1993,4 has provided the source for mGlu
receptor antagonists. CPGs have played a fundamental
role in the study of mGlu receptors since they were the
®rst antagonists with selectivity over glutamate iono-
tropic receptors; nonetheless, CPGs of the ®rst genera-
tion were generally endowed with low potency and poor
selectivity. Several research laboratories have since then
addressed the problem of improving the potency and
the selectivity of the CPG class of compounds. As a
result, CPG derivatives such as 4, endowed with low to
sub-micromolar range potency and group or subtype
selectivity, are now available.5
The molecular and functional diversity of the mGlu
family has fostered the search for potent and subtype
selective agonists and antagonists which may be of help
in proving the physiopathological role of individual
subtypes en route towards clinically useful drugs.
In this frame, we focused our attention on the task of
de®ning the structure±activity relationship (SAR) for
the class of carboxyphenylglycines with the aim of dis-
closing the structural features responsible for selective
*Corresponding author. Tel.: +39-075-585-5128; fax: +39-075-585-
5124; e-mail: rp@unipg.it
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00270-4

222
G. Costantino et al. / Bioorg. Med. Chem. 9 (2001) 221±227
(2) and the glycine moiety. The rigidi®cation of such
bonds into a ®ve-membered ring yielded AIDA (6), a
conformationally constrained analogue endowed with a
lower potency than the parent compound 4-CPG (2) but
with an increased selectivity, with no activity at group
II, group III and mGlu5 receptor subtypes.6
The second structural feature to be investigated was the
role played by the aromatic ring of CPGs. Indeed, we
thought that the aromatic ring may either serve as a
spacer between pharmacophoric groups or be involved
in more speci®c interactions. Thus, we designed and
synthesized S-CBPG (7),7 where the propellane moiety
adequately substitutes for the aromatic ring in keeping
the pharmacophore groups in a coplanar, linear dis-
position but is endowed with a very di€erent physico-
chemical pro®le. The good activity of 7 as mGlu1
receptor antagonist was the con®rmation that the aro-
matic ring is not strictly necessary for the activity. S-
CBPG (7) is, however, signi®cantly shorter than 4-CPG
(2) and we wondered whether this reduction in length
could have a€ected the potency of S-CBPG (7). Thus,
we reported the synthesis of ACUDA (8),8 where the
unusual cubyl moiety is able to keep the pharmaco-
phoric groups still in a coplanar disposition as the aro-
matic ring or the propellane moiety. Interestingly,
ACUDA (8) has exactly the same length as 4-CPG (2).
When tested as mGlu ligand, however, 8 was shown to
be only a weak mGlu1 antagonist. The lost of anity of
8 with respect to 4-CPG (2) or S-CBPG (7) must there-
fore be ascribed to the increase in the volume of the
spacer. It should be noted, however, that S-CBPG (7) and
ACUDA (8) are characterized by di€erent distances
between pharmacophoric groups. Indeed, since S-CBPG
(7) is signi®cantly shorter than ACUDA (8) and 4-CPG
(2) itself, the SAR study requires the e€ect of the distance
between pharmacophoric groups to be investigated in the
case of propellane derivatives. Thus, we have envisaged
the synthesis of a novel propellane derivative, 2-(30-(1H-
tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl)glycine (S-TBPG,
9), characterized by the substitution of the distal
carboxylate by a tetrazole moiety. Indeed, the tetrazole
Chart 1.
antagonism towards individual mGlu receptor subtypes
(Chart 2). Thus, our approach was to dissect the structure
of 4-carboxyphenylglycine (4-CPG, 2) into two main
features and to investigate their respective role in a€ecting
potency and selectivity as group I mGlu receptor antago-
nists. The ®rst structural feature we took into account was
the rotatable bond between the aromatic ring of 4-CPG
Chart 2.

G. Costantino et al. / Bioorg. Med. Chem. 9 (2001) 221±227
223
ring is a suitable bioisoster in terms of acidity and
hydrogen bond capability but it is signi®cantly longer
than the carboxylate group. The synthesis and the pre-
liminary biological evaluation of 9 are reported herein.
20 can be attributed with an S-con®guration at the
a-aminonitrilic center. The two aminonitriles 20 and 21
were submitted to oxidative cleavage with lead tetra-
acetate, acidic (HCl) hydrolysis and ion exchange chro-
matography to a€ord (S)-2-(30-(1H-tetrazol-5-yl)bicyclo
[1.1.1]pent-1-yl)glycine (9, S-TBPG) and (R)-2-(30-(1H-
tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl)glycine (10, R-TBPG)
in 75 and 70% yield, starting from the respective amino-
nitrile derivatives.
Chemistry
The synthesis of S- and R-TBPG (9 and 10, respectively),
reported in Scheme 1, involves the diacid 11 as a key
intermediate.
Pharmacological Evaluation
Brie¯y, 11 was prepared basically as described by Michl9
and previously reported by us.7 Esteri®cation of 11 via
the corresponding acyl chloride a€orded the diester 12,
which was converted into the half ester 13 under mild
alkaline conditions (86%).10 Methyl-3-carbamoylbi-
cyclo[1.1.1]pentane-1-carboxylate (14) was accessed
from the monoester 13 through the corresponding acyl
chloride and dehydration of 14 by SOCl2 gave the nitrile
15 (94%).10 This compound was converted to the 1-tri-
butyltin-substituted tetrazole 16 using tri-n-butyltin
azide (n-BuSnN3) in xylene (98%).11 The Sn±N bond
was readily cleaved by treatment with gaseous hydrogen
chloride in MeOH to give the corresponding free tetra-
zole 17, which was then protected as trityl derivative12
using trityl bromide and Et3N (64%). DIBAH reduc-
tion of 18 yielded the aldehyde 19,13 which was then
reacted with (R)-a-phenylglycinol in MeOH followed by
treatment of the resulting Shi€ base with TMSCN to
give a 2.5:1 (1H NMR) mixture of the two expected
aminonitriles 20 and 21, which were separated by ¯ash
chromatography in 28 and 10% yields, respectively.
According to the known inductive e€ect of chiral
a-phenylglycinol when employed in diastereoselective
Strecker reaction, the more abundant aminonitrile has
the newly formed chiral center opposite in sign to that
of the a-phenylglycinol employed.14 Thus, aminonitrile
The e€ect of S-TBPG (9) was examined on HEK 293 cells
transiently expressing mGluR1a, mGluR5a, mGluR2or
mGluR4a. The same read out of receptor activation was
used for any mGluR subtypes. The assay consisted of
measuring the IP accumulated in the cells upon agonist
treatment. Though group I mGluRs (mGluR1a, R5a)
naturally coupled to phospholipase C, the group II and
III (mGluR2and R4a, respectively) do not. The coupl-
ing of these latter mGluRs to PLC was made possible
by co-expressing these receptors with the chimeric G-
protein a subunit in which the carboxyl terminal 9 resi-
dues of Gq were replaced by those of Gi2.15,16 We have
previously reported that the pharmacological pro®les of
mGluR2and mGluR4 determined using this assay were
identical to those determined by measuring the inhibi-
tion of cAMP formation.16
Discussion
As shown in Figure 1, l-glutamic acid at 1 mM stimu-
lated IP formation in cells expressing mGluR1a or R5a
alone, or mGluR2or R4a with Gqi9. When applied at a
concentration of 1000 mM S-TBPG (9) did not sig-
ni®cantly stimulate IP formation in cells expressing
Scheme 1. (a) (i) SOCl2 re¯ux; (ii) MeOH, re¯ux; (b) THF, NaOH/CH3OH; (c) (i) C2O2Cl2, Et2O; (ii) NH3, CH2Cl2; (d) SOCl2; (e) (n-Bu)3SnN3,
xylene, 110ꢀ; (f) HCl (g)/CH3OH; (g) TrBr, CHCl3/DMF; (h) DIBAH, toluene, À78 ꢀC; (i) (i) (R)-2-phenylglycinol, MeOH, rt; (ii) TMSCN, 0 ꢀC,
then rt; (iii) ¯ash chromatography; (m) (i) Pb(OAc)4, CH2Cl2:MeOH (1:1, v/v), 0 ꢀC; (ii) 6 N HCl, re¯ux; (iii) Dowex 50X2-200, 10% Py.

224
G. Costantino et al. / Bioorg. Med. Chem. 9 (2001) 221±227
mGluR1a, R5a, R2, or R4a. To test a possible antago-
nistic e€ect of S-TBPG (9) on rat mGluRs, cells expres-
sing these receptors were stimulated with a Glu
concentration around its EC50 value (1mM for mGluR1a,
5 mM for mGluR5a, 20mM for mGluR2, and 30mM for
mGluR4a) (Fig. 2).
mGluR5a, R2, or R4a. However, a strong inhibition of
the Glu e€ect (98.6Æ5.8% inhibition; n=3) was observed
with 1000mM S-TBPG (9), with an IC50 of 68.9Æ10.7 mM,
as determined by inhibiting the Glu (1mM)-induced
stimulation of mGluR1a by increasing concentrations of
S-TBPG (9).
S-TBPG (9) was applied at a concentration of 1000mM
5 min before and during the stimulation with Glu. No
inhibition of the Glu-e€ect was observed in cells expressing
These data indicate S-TBPG (9) as a novel, moderately
potent but subtype selective mGluR1 antagonist.
Although more potent and selective mGlu1 antagonists
Figure 1. (a) S-TBPG inhibits l-Glu-induced stimulation of mGluR1a. Cells expressing the di€erent receptors were incubated with S-TBPG and
then stimulated with l-Glu (concentration around the EC50 of l-Glu on each receptor). Only the cells expressing mGluR1a did not show any
increase in the production of IP upon Glu stimulation, thus indicating that S-TBPG acted as an antagonist at this receptor. (b) S-TBPG has no
agonist activity on mGluR1a, R5a, R2and R4a. Cells expressing the mGluR1a or mGluR5a, or mGluR2or R4a with Gqi9 were stimulated with l-
Glu (1000 mM) or S-TBPG (1000 mM). No signi®cant e€ect on any of these receptors was observed for S-TBPG, while Glu stimulated each receptor
3.5- to 7.5-fold in three experiments.
Figure 2. The IC50 of S-TBPG on mGluR1a was estimated by inhibiting Glu (1 mM)-induced stimulation of mGluR1a expressing cells by increasing
concentrations of S-TBPG.

G. Costantino et al. / Bioorg. Med. Chem. 9 (2001) 221±227
225
have recently appeared in the literature, the activity of
Experimental
S-TBPG (9) deserves some consideration in relation to
the SAR for mGlu1 antagonists. In particular, S-TBPG
(9) was designed with the aim of evaluating the e€ect of
lengthening the distance between pharmacophoric
groups in S-CBPG (7) analogues. The introduction of
the tetrazole ring in place of the carboxylate moiety
increases this pharmacophore distance of about 1 A
(Fig. 3).
Chemistry
General methods. Methanol and toluene were distilled
over magnesium and sodium, respectively. All reactions
were carried out under an argon atmosphere. Melting
points were determined by the capillary method on a Buchi
535 electrothermal apparatus and are uncorrected. 1H and
13C NMR spectra were taken on a Bruker AC 200 spec-
trometer. Proton chemical shifts are reported in ppm
down®eld from tetramethylsilane, except with D2O which
was also used as an internal standard. Carbon chemical
shifts are reported in ppm using acetone as internal stan-
dard. The abbreviations used are as follows: s, singlet; dd,
double doublet; m, multiplet. Flash chromatography was
performed on Merck silica gel (0.040±0.063 mm). Spe-
ci®c rotations were recorded on a Jasco Dip-360 digital
polarimeter.
When compared with the activity of S-CBPG (7), S-
TBPG (9) is about 2.5-fold less potent as mGlu1
antagonist and does not show any partial agonism at
mGlu5. These data indicate that the substitution of
the carboxylate moiety by the tetrazole ring is detri-
mental for the activity. However, since S-TBPG (9)
has the same length as 4-CPG (2), the decrease in the
activity should be mainly ascribed to the lower acidity
of the tetrazole ring with respect to the carboxylate
moiety. Hence, it can be concluded that the e€ect of
increasing the length between pharmacophore groups
up to the `standard' distance of CPGs is somehow
compensated by the lower pKa. This observation allows
us to complete the SARs for this class of mGlu1
antagonists. The propellane nucleus is well accepted by
the active site and can be considered as a bioisoster of
the phenyl ring. The increase in the size of the spacer, as
in the case of ACUDA (8), is detrimental for the activ-
ity, thus suggesting the existence of a region of limited
space in the active site. The distance between pharma-
cophore groups seems not to be an essential issue.
Indeed, the optimal distance between the distal acidic
group and the a-aminoacidic moiety, owned by CPGs,
can be lowered as in the case of S-CBPG (7) providing
that the acidity of the distal moiety is kept.
Methyl-3-(1-tri-n-butylstannyltetrazol-5-yl)bicyclo[1.1.1]-
pentane-1-carboxylate (16). Tri-n-butyltin azide (19.9 g,
0.06 mol) was added to a solution of the nitrile 15 (4.5 g,
0.03 mol) in xylene (30 mL). The resulting mixture was
heated at 110 ꢀC under argon. After 60 h the solvent was
evaporated o€, acetonitrile was added (50 mL) and the
solution was washed with hexane (8Â20 mL). The ace-
tonitrile phase was dried (Na2SO4) and concentrated to
1
yield the desired tetrazole 16 (13.9 g, 95%): H NMR
(CDCl3) d 1.00±1.60 (27H, m, 3Ân-Bu); 2.50 (6H, s,
3ÂCH2); 3.65 (3H, s, CO2CH3); 13C NMR (CDCl3) d
13.2, 17.1, 26.6, 27.7, 33.5, 38.8, 51.3, 53.9, 160.1, 169.5.
Methyl 3-(1H-tetrazol-5-yl)bicyclo[1.1.1]pentane-1-car-
boxylate (17). A solution of 16 (10.7 g, 0.02mol) in dry
MeOH (300 mL) was treated with anhydrous hydrogen
chloride. After 3 h the solvent was evaporated o€ and the
residue was triturated with hexane to give the free tetra-
In conclusion, we have reported S-TBPG (9) as a novel,
subtype selective, mGlu1 antagonist.
1
zole 17 (3.74 g, 88%): H NMR (CDCl3) d 2.65 (6H, s,
3ÂCH2), 3.70 (3H, s, CO2CH3), 9.15 (1H, s, NH); 13C
NMR (CDCl3) d 31.4, 39.5, 52.1, 54.2, 154.6, 169.4.
S-TBPG (9) has allowed us to extend the structure±
activity relationship for mGlu1 antagonists and repre-
sents a new example of an mGlu1 antagonist with no
e€ect at mGlu5 receptors to be utilized as a pharmaco-
logical tool to study the role of mGlu1 subtypes in
physiopathological conditions.
Methyl 3-(1-Trityltetrazol-5-yl)bicyclo[1.1.1]pentane-1-
carboxylate (18). A solution of EtN3 (4.6 g, 45 mmol) in
CHCl3:DMF (24 mL, 2:1) was dropped over a period of
45 min on to a vigorously stirred solution of 17 (4.4 g,
Figure 3. Comparison between 4-CPG (2), S-CBPG (7), S-ACUDA (8) and S-TBPG (9) in terms of distances between pharmacophoric groups.

226
G. Costantino et al. / Bioorg. Med. Chem. 9 (2001) 221±227
23 mmol) and trityl bromide (8.2 g, 25 mmol) in CHCl3:
DMF (115 mL, 2:1). After 1.5 h the reaction mixture was
neutralized with 2N HCl and the solvent was evaporated
o€. The residue was taken up in Et2O (300 mL) and the
organic phase was washed with H2O (4Â40 mL), dried
(Na2SO4) and evaporated to give a semisolid product
which was puri®ed by ¯ash chromatography. Elution
with petroleum ether:EtOAc (90:10) gave 18 (6.3 g, 64%):
mp 184±187 ꢀC; 1H NMR (CDCl3) d 2.50 (6H, s, 3ÂCH2),
3.70 (3H, s, CO2CH3), 7.0±7.50 (15H, s, aromatics).
phase was evaporated to dryness and the residue was
puri®ed by ion exchange chromatography (Dowex
50WX2-200). Elution with 10% pyridine a€orded 9
(189 mg, 75%): mp 282±284 ꢀC; H NMR (D2O) d 2.21
1
(6H, s, 3ÂCH2), 3.65 (1H, s, CH); 13C NMR (D2O) d
38.8, 51.2, 54.3, 154.4, 170.9; [a]D20 +11.4 (c 1, H2O).
(R)-2-[3-(1H-Tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl]glycine
(10). Lead (IV) acetate (370 mg, 0.70 mmol) was added
to a 0 ꢀC solution of the nitrile 21 (386 mg, 0.70 mmol) in
anhydrous MeOH±CH2Cl2 (8 mL, 1:1). After 10 min
phosphate bu€er pH 7 was added (12mL) and the
resulting mixture was ®ltered with the aid of Celite. After
evaporation of the solvent the residue was re¯uxed in 6 N
HCl (20 mL) for 12 h. The reaction mixture was allowed
to cool to room temperature and washed with diethyl
ether (2Â10 mL). The aqueous phase was evaporated to
dryness and the residue was puri®ed by ion exchange
chromatography (Dowex 50WX2-200). Elution with
10% pyridine a€orded 10 (103 mg, 70%): mp 280±
3-(1-Trityltetrazol-5-yl)bicyclo[1.1.1]pentane-1-carbox-
aldehyde (19). A À78 ꢀC solution of DIBAL-H (1.5 M
in toluene, 8.70 mL) was dropped over 30 min to a
À78 ꢀC solution of 18 (5.8 g, 13.0 mmol) in dry toluene
(150 mL). After 30 min the reaction was quenched with
methanol (20 mL) and saturated NH4Cl (100 mL) and
then allowed to warm up to room temperature. The
organic phase was separated and the aqueous phase was
extracted with ethyl acetate (3Â40 mL). The combined
organic phases were washed with water (2Â50 mL), dried
(Na2SO4), evaporated and puri®ed by ¯ash chromato-
graphy. Elution with petroleum ether:EtOAc (80:20)
gave 19 (3.5 g, 64%): mp 159±163 ꢀC; 1H NMR (CDCl3)
d 2.45 (6H, s, 3ÂCH2), 6.90±7.40 (15H, s, aromatics),
9.55 (1H, s, CHO); 13C NMR (CDCl3) d 34.6, 45.1,
52.8, 83.1, 127.7, 128.3, 130.2, 141.3, 162.9, 197.9.
282 ꢀC; H NMR (D2O) d 2.21 (6H, s, 3ÂCH2), 3.70
1
(1H, s, CH); 13C NMR (D2O) d 38.6, 51.0, 54.1, 154.0,
170.7; [a]D20 À15.3 (c 1, H2O).
Biology
Culture and transfection of HEK 293 cells
(2S)- and (2R)-N-[(R)-ꢀ-Phenylglycinyl]-2-[30-(1-trityl-
1H-tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl]glycinonitriles (20
and 21). (R)-a-Phenylglycinol (1.0 g, 7.4 mmol) was
added to a solution of the aldehyde 1 (3.0g, 7.4 mmol) in
dry MeOH (150mL) and the resulting mixture was stirred
at room temperature for 3 h. After cooling to 0 ꢀC,
TMSCN (1.5 g, 14.8mmol) was added. After 12h, the
solvent was evaporated o€ and the residue was puri®ed by
¯ash chromatography. Elution with petroleum ether:
HEK 293 cells were cultured in Dulbecco's modi®ed
Eagle's medium (DMEM, Gibco BRL, Paris, France)
supplemented with 10% fetal calf serum and transfected
by electroporation as previously described.16 Electro-
poration was carried out in a total volume of 300 mL with
10 mg carrier DNA, plasmid DNA containing mGluR1
(0.3 mg), mGluR2(2 mg), or mGluR4a (5 mg) and 10 mil-
lion cells.17 To allow mGluR2or mGluR4a to activate
PLC, these receptors were co-expressed with the chimeric
G-protein Gqi9 as previously described.16,18 Because
glutamate concentration in the cultured medium was
found to profoundly a€ect its functioning, mGluR5a was
co-expressed with the high anity glutamate transporter
EAAC1.19
EtOAc (80:20) gave 20 (0.38 g, 28%): mp 72±75 ꢀC; H
1
NMR (CDCl3) d 2.20 (6H, s, 3ÂCH2), 3.40 (1H, s,
CHCN), 3.40±3.55 (1H, m, CHPh), 3.70 (1H, dd, J=3.8
and 9.3 Hz, CHaOH), 4.05 (1H, dd, J=3.8 and 9.3 Hz,
CHbOH), 7.00±7.50 (20H, s, aromatics); 13C NMR
(CDCl3) d 33.6, 40.2, 48.9, 51.2, 63.0, 67.2, 83.2, 118.1,
127.8, 128.3, 128.8, 130.3, 138.4, 141.3, 163.0; [a]D20 À36.0
(c 1, MeOH). Subsequent elutionꢀwith the same solvent
Determination of inositol phosphate (IP) accumulation
1
gave 21 (0.14 g, 10%): mp 75±78 C; H NMR (CDCl3)
Determination of inositol phosphate accumulation in
transfected cells was performed after labeling the cells
overnight with [3H]-myo-inositol (23.4 Ci/mol, NEN,
France). The stimulation was conducted for 30 min in a
medium containing 10mM LiCl and indicated concentra-
tion of antagonists or agonists. The basal IP formation
was determined after 30 min incubation in the presence of
10 mM LiCl and the glutamate degrading enzyme glu-
tamate pyruvate transaminase (1 U/mL) and 2mM pyr-
uvate to avoid the possible action of glutamate released
from the cells. Results are expressed as the amount of IP
produced over the radioactivity present in the membranes.
d 2.25 (6H, s, 3ÂCH2), 3.50±3.80 (2H, m, CHPh and
CHaOH), 3.80 (1H, s, CHCN), 3.95 (1H, dd, J=4.3 and
7.5 Hz, CHbOH), 6.90±7.40 (20H, s, aromatics); 13C
NMR (CDCl3) d 33.6, 40.6, 49.5, 51.4, 63.2, 66.5, 83.1,
118.1, 127.4, 127.7, 128.2, 128.8, 130.2, 139.4, 141.3,
162.8; [a]D20 À16.0 (c 1, MeOH).
(S)-2-[3-(1H-Tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl]glycine
(9). Lead (IV) acetate (635 mg, 1.44 mmol) was added
to a 0 ꢀC solution of the nitrile 20 (670 mg, 1.20 mmol)
in anhydrous MeOH:CH2Cl2 (12mL, 1:1). After 10 min
phosphate bu€er pH 7 was added (12mL) and the
resulting mixture was ®ltered with the aid of Celite.
After evaporation of the solvent the residue was
re¯uxed in 6 N HCl (20 mL) for 12 h. The reaction mix-
ture was allowed to cool to room temperature and
washed with diethyl ether (2Â10 mL). The aqueous
References
1. Pellicciari, R.; Costantino, G. Curr. Opin. Chem. Biol. 1999,
3, 433.

G. Costantino et al. / Bioorg. Med. Chem. 9 (2001) 221±227
227
2. Bordi, F.; Ugolini, A. Prog. Neurobiol. 1999, 59, 55.
3. Pellegrini-Giampietro, D. E.; Peruginelli, F.; Meli, E.;
Cozzi, A.; Albani-Torregrossa, S.; Pellicciari, R.; Moroni, F.
Neuropharmacology 1999, 38, 1607.
4. Eaton, S. A.; Jane, D. E.; Jones, P. L.; Porter, R. H.; Pook,
P. C.; Sunter, D. C.; Udvarhelyi, P. M.; Roberts, P. J.; Salt, T.
E.; Watkins, J. C. Eur. J. Pharmacol. 1993, 244, 195.
5. Clark, B. P.; Baker, S. R.; Goldswothy, J.; Harris, J. R.;
Kingston, A. E. Bioorg. Med. Chem. Lett. 1997, 7, 2777.
6. Pellicciari, R.; Luneia, R.; Costantino, G.; Marinozzi, M.;
Natalini, B.; Jakobsen, P.; Kanstrup, A.; Lombardi, G.; Mor-
oni, F.; Thomsen, C. J. Med. Chem. 1995, 38, 3717.
7. Pellicciari, R.; Raimondo, M.; Marinozzi, M.; Natalini, B.;
Costantino, G.; Thomsen, C. J. Med. Chem. 1996, 39, 2874.
8. Pellicciari, R.; Costantino, G.; Giovagnoni, E.; Mattoli, L.;
Brabet, I.; Pin, J. P. Bioorg. Med. Chem. Lett. 1998, 8, 1569.
9. Kaszynski, P.; Michl, J. J. Org. Chem. 1988, 53, 4593.
10. Della, E. W.; Taylor, D. K. J. Org. Chem. 1994, 59, 2986.
11. Sisido, K.; Nabika, K.; Isida, T.; Kozima, S. J. Organo-
metal. Chem. 1971, 33, 337.
12. Mutter, M.; Hersperg, R. Synthesis 1989, 198.
13. Zakharkin, L. I.; Khorlina, I. M. Tetrahedron Lett. 1962,
14, 619.
14. Chakraborty, T. K.; Hussain, K. A. Tetrahedron 1995, 51,
9179.
15. Conklin, B. R.; Farfel, Z.; Lustig, K. D.; Julius, D.;
Bourne, H. R. Nature 1993, 363, 274.
16. Gomeza, J.; Mary, S.; Brabet, I.; Parmentier, M.-L.; Resti-
tuito, S.; Bockaert, J.; Pin, J.-P. Mol. Pharmacol. 1996, 50, 92 3.
17. Brabet, I.; Parmentier, M.-L.; De Colle, C.; Bockaert, J.;
Acher, F.; Pin, J.-P. Neuropharmacology 1998, 37, 1043.
18. Parmentier, M.-L.; Joly, C.; Restituito, S.; Bockaert, J.;
Grau, Y.; Pin, J.-P. Mol. Pharmacol. 1998, 53, 778.
19. Kanay, Y.; Hediger, M. A. Nature 1992, 360, 467.