9H-Xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and (2H-tetrazol-5-yl)-amides as potent, orally available mGlu1 receptor enhancers

Article abstract of DOI:10.1016/j.bmcl.2005.05.135

Small molecule mGluR1 enhancers based on the lead compound (9H-xanthene-9-carbonyl)-carbamic acid butyl ester derived from random-screening hit diphenylacetyl-carbamic acid ethyl ester were designed and synthesized as useful pharmacological tools for the study of the physiological roles mediated by mGlu1 receptors. The synthesis and the structure-activity relationship of this new class of positive allosteric modulators of mGlu1 receptors will be discussed in detail.

Full text of DOI:10.1016/j.bmcl.2005.05.135

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4628–4631
9H-Xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and
(2H-tetrazol-5-yl)-amides as potent, orally available
mGlu1 receptor enhancersq
a
a
a
Eric Vieira,a, Jorg Huwyler, Synese Jolidon, Frederic Knoflach,
*
`
´ ´
¨
Vincent Mutelb and Jurgen Wichmanna
¨
aPharma Division, Discovery Research, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland
bAddex Pharmaceuticals, 12 Chemin Des Aulx, CH-1228 Plan Les Ouates, Geneva, Switzerland
Received 26 January 2005; revised 20 May 2005; accepted 20 May 2005
Available online 15 August 2005
Abstract—Small molecule mGluR1 enhancers based on the lead compound (9H-xanthene-9-carbonyl)-carbamic acid butyl ester
derived from random-screening hit diphenylacetyl-carbamic acid ethyl ester were designed and synthesized as useful pharmacolog-
ical tools for the study of the physiological roles mediated by mGlu1 receptors. The synthesis and the structure–activity relationship
of this new class of positive allosteric modulators of mGlu1 receptors will be discussed in detail.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
(enhancers) of mGlu1 receptors (Fig. 1).5,6 Recent re-
ports of allosteric modulators of mGlu27,8 and
mGlu59,10 receptors have raised considerable interest.4
Here, we report on the development of heterocyclic
derivatives—based on the xanthyl-carbamate 2—which
are suitable for in vivo use. The random-screening hit
1 was identified initially by using recombinant mGlu1
receptors expressed at very high levels. The constitu-
tive activity of the receptor is such that the compound
elicits a response in the absence of glutamate site
ligands. However, in physiologically more relevant
recombinant systems with a lower level of receptor
expression, the compound potentiated the agonist-
stimulated response without any detectable intrinsic
activity. Using this screening hit as a starting point,
we have discovered 2 as a potent and selective positive
allosteric modulator of mGlu1 receptors.5,6 To eluci-
L-Glutamate, the major excitatory amino acid neuro-
transmitter in the central nervous system, binds to
and activates several classes of receptors which are
divided into two groups termed ionotropic (iGluR)
and metabotropic glutamate receptors (mGluR)1. The
latter family comprises eight subtypes of G-protein
coupled receptors, grouped according to pharmacolo-
gy and coupling to second messengers.2 The primary
transduction mechanism of group I mGlu receptors
(mGluR1 and mGluR5) is the stimulation of phospho-
inositide (PI) hydrolysis, whereas group II (mGluR2
and mGluR3) and group III (mGluR4, mGluR6,
mGluR7, and mGluR8) receptors inhibit forskolin-
stimulated cyclic AMP accumulation.3 A role for
group I mGlu receptor activation has been implicated
in physiological processes including pain perception,
learning, and memory, as well as in certain psychiatric
and neurological disorders.4 Recently, we described
a series of carbamates like the screening hit 1, which
behave as selective positive allosteric modulators
q
This work was presented at the 4th international meeting on
metabotropic glutamate receptors, Taormina (Sicily, Italy) 15–20
September 2002.
Corresponding author. Tel.: +41 61 688 2464; fax: +41 61 688
*
Figure 1. Representative mGluR1 enhancers: screening hit 1 and lead
structure 2.
8714; e-mail: eric.vieira@roche.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.135

E. Vieira et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4628–4631
4629
Figure 2. Structures oxadiazoles 3 and tetrazoles 4.
date the putative binding site of these compounds,
chimeras and point mutants of rmGlu1a and rmGlu5a
were prepared. The compounds enhanced the gluta-
mate-induced current in all dimeric receptors contain-
ing the TM region of rmGlu1a which is required for
the enhancing effect.5 Hydrolysis and decarboxylation
of the carbamate moiety of 2 by esterases in blood
plasma leads to formation of inactive amides. To
overcome this problem, the corresponding ester bio-
isosteres 1,2,4-oxadiazoles 3 and tetrazoles 4 have
been prepared (Fig. 2).
Scheme 2. Synthesis of oxadiazoles 3a–f and tetrazoles 4a–e. Reagents
and conditions: (a) Py, 0 °C to rt, 50–90%; (b) CDI, 0 °C to rt, 25–80%.
ed. The best overall properties in terms of potency,
efficacy, and metabolic stability were observed for the
methyl- and ethyl-substituted derivatives (Table 1).
The methyl-substituted 1,2,4-oxadiazole 3a and the
ethyl-substituted tetrazole 4b showing the best overall
profiles were selected for further evaluation regarding
pharmacology and in vivo PK analysis.
2. Chemistry
4. Pharmacology
Syntheses of the amino-oxadiazoles 5a–f and amino-
tetrazoles 6a–e were realized using methods described
in the literature11–13 (Scheme 1).
The activities of the compounds at rat mGlu1 receptors
were assessed using intracellular Ca2+ measurements on
rat mGlu1a transiently transfected HEK-293 cells
expressing recombinant mGlu1 receptors at very high
levels. The constitutive activity of the receptor is such
that the compounds elicit an agonist response in the ab-
sence of glutamate site ligands. [Ca2+]i measurements
were performed after incubation of the cells with Fluo-
3 AM (Molecular Probes, Eugene, OR, USA) for 1 h
and four washes with assay buffer (DMEM supplement-
ed with Hankꢀs salt and 20 mM HEPES). [Ca2+]i mea-
surements were done using a fluorometric imaging
plate reader (FLIPR, Molecular Devices Corporation,
La Jolla, CA, USA). Fluorescence ratio values were cal-
culated as described.16 EC50 values for the enhancers are
the mean of separate values from at least three individual
experiments. The agonist effect is normalized to the max-
imum response induced by 10 lM of glutamate (Table 1).
Reaction of the aminooxadiazoles 5a–f with xanthene-
9-carboxylic acid chloride 8 yielded, after conventional
workup and purification, the desired compounds 3a–f
in good yields.14 The aminotetrazoles 6a–e and 7a–e
were obtained by alkylation of 2-aminotetrazole and
separation of the regioisomers by column chromatog-
raphy. Reaction of 6a–e with xanthene-9-carboxylic
acid 9 using carbonyl diimidazole (CDI) as coupling
reagent formed the adducts 4a–e in acceptable yields15
(Scheme 2).
3. Structure–activity relationship
In both the series, small alkyl and cycloalkyl substitu-
ents on the heterocyclic moiety led to compounds with
higher activities, whereas larger groups were less tolerat-
5. Selectivity
Using various functional models, it was found that 3a
was devoid of any enhancing effect at rat mGlu2,
mGlu4, mGlu5, and mGlu8, and human GABA-B
receptors. In addition, 3a had no activity in radioligand
binding assays at major adenosine; adrenergic; GABA-
A; glycine; histamine; muscarinic; nicotinic; opiate;
purinergic and 5-HT receptors, and adenosine; norepi-
nephrine; GABA and 5-HT uptake sites.
6. Electrophysiology
Scheme 1. Synthesis of oxadiazoles 5a–f and tetrazoles 6a–e. Reagents:
(a) NaOH, EtOH, 5–20%; (b) NaOH, H2O, acetone, quant.; (c)
NH2OHÆHCl, Py, EtOH, 8–55%; (d) R–X, K2CO3, EtOH, 17–25%.
Figure 3 shows the effect of 3a on glutamate-activated
inward current in a CHO cell stably expressing

4630
E. Vieira et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4628–4631
Table 1. Structure–activity relationship of oxadiazoles 3a–3f and tetrazoles 4a–4e
a
R
mGluR1 EC50
(nM)
Agonist effect
(%)a
In vitro metabolism
(rat microsomes) (ll/min/mg protein)
In vitro metabolism
category
Solubility (pH 6.5)
(lg/ml)
3a
3b
3c
3d
3e
3f
Me
Et
52
6
100
57
90
80
80
54
65
80
80
70
28
42
78
Intermediate
Intermediate
High
12
0.056
<1
4
n-Pr
i-Pr
Cyprop
i-Bu
Me
25
22
23
10
180
65
29
45
34
97
130
227
17
High
High
<1
7
Low
Low
4a
4b
4c
4d
4e
16
41
35
21
4
Et
Intermediate
High
n-Pr
i-Pr
i-Bu
92
63
Intermediate
High
4
n.m.
229
a See details in pharmacology. n.m. indicates not measured.
Table 2. Pharmacokinetics of selected compounds 3a and 4b (data are
mean values, n = 2)
Dose route of
administration
DMPK parameters
3a
4b
10 (mg/kg)
iv bolus
Half-life (h)
0.32
0.49
Cl (ml/min/kg)
Vss (L/kg)
60.0
1.82
49.2
2.09
10 (mg/kg)
po bolus
Cmax (ng/ml)
1174
1130
1.5
Figure 3.
Tmax(h)
Brain/plasma
CSF/plasma
1.5
1.5
GIRKs and transiently transfected with the rat
mGlu1a receptor cDNA. Glutamate (3 lM) induced
a current that was potentiated 10-fold by 3a. The ef-
fect of 3a was reversible and greater than a saturating
concentration of glutamate (100 lM). Compound 3a
had no effect when applied alone. This effect was sim-
ilar to that observed for screening hit 1.5 Figure 4
shows the concentration–response curve for gluta-
mate-induced current potentiation by 3a. The current
amplitudes were normalized to the control responses
(3 lM glutamate) and fitted individually for each cell
(n = 3). The EC50 was 220 nM.
0.08
15 mg/kg/h
iv infusion
Brain/plasma
CSF/plasma
0.16
0.06
Oral bioavailability at low doses was up to 100% for
both compounds. In the case of compound 3a, a mean
maximum plasma concentration (Cmax) of 1174 ng/ml,
a mean brain level of 1761 ng/ml, and a CSF concentra-
tion of 94 ng/ml were achieved after 1.5 h (Tmax) at an
oral dose of 10 mg/kg (Table 2).
7. Pharmacokinetics
Although similar maximal plasma concentrations were
observed upon oral dosing of the less lipophilic com-
pound 4b, an infusion experiment of this compound at
a dose of 15 mg/kg/h showed very low steady-state
brain tissue concentrations indicative of poor brain
penetration. Several compounds of both the series
were evaluated in a functional P-glycoprotein assay
and did not stimulate P-gp ATPase activity.
Both the methyl-substituted 1,2,4-oxadiazole 3a and the
ethyl-substituted tetrazole 4b have an intermediate to
high plasma clearance (Cl) and an intermediate volume
of distribution (Vss) (Table 2).
8. Discussion
On the basis of the potent and selective positive alloste-
ric modulator 2, two series of heterocyclic derivatives
have been discovered: 1,2,4-oxadiazoles 3 and tetrazoles
4. In both the series, several derivatives with high activ-
ities at rat mGlu1 receptors have been prepared. In each
series, one compound with the most promising in vitro
profile was selected for further evaluation. With respect
to the PK profile, the methyl-substituted 1,2,4-oxadiaz-
Figure 4.

E. Vieira et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4628–4631
4631
8. Johnson, M. P.; Baez, M.; Jagdmann, G. E., Jr.; Britton, T.
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ole 3a could serve as a suitable tool to further study
the role of positive allosteric modulation of mGlu1
receptors in vivo.
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