Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides as potent, orally available mGlu1 receptor enhancers

Article abstract of DOI:10.1016/j.bmcl.2009.01.108

Small molecule mGluR1 enhancers, which are 9H-xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and (2H-tetrazol-5-yl)-amides, have been previously reported. Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides with improved pharmacokinetic proper

Full text of DOI:10.1016/j.bmcl.2009.01.108

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1666–1669
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides as potent,
orally available mGlu1 receptor enhancers
à
*
Eric Vieira , Jörg Huwyler , Synèse Jolidon, Frédéric Knoflach, Vincent Mutel , Jürgen Wichmann
Pharma Division, Discovery Research, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Small molecule mGluR1 enhancers, which are 9H-xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and
(2H-tetrazol-5-yl)-amides, have been previously reported. Fluorinated 9H-xanthene-9-carboxylic acid
oxazol-2-yl-amides with improved pharmacokinetic properties have been 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 class of positive allosteric modulators of mGlu1
receptors will be discussed in detail.
Received 18 December 2008
Revised 30 January 2009
Available online 6 February 2009
Keywords:
Metabotropic glutamate receptor 1
mGluR1
Ó 2009 Elsevier Ltd. All rights reserved.
GPCR
Positive allosteric modulator
PAM
Enhancer
L
-Glutamate, the major excitatory amino acid neurotransmitter
could be of interest in indications where reduced mGluR1 function
in this brain area leads to motor impairment (cerebellar ataxia).9–12
Another interesting finding is the in vitro reversal of synaptic plas-
ticity in mouse dopaminergic (DA) neurons induced by cocaine
with the mGluR1 enhancer Ro 67-7476,13,14 which could find
potential use in the field of drug addiction.
Recently, we described a series of carbamates that behave as
selective positive allosteric modulators (enhancers) of mGlu1
receptors.15 Potent orally available mGluR1 enhancers 1 and 2
(Fig. 1) have also been reported in a preceding paper.16 The com-
pounds were initially screened using cells having recombinant
mGlu1 receptors transiently expressed at very high levels. In this
system, the constitutive activity of the receptor is such that the
compound elicits an agonist response in the absence of glutamate
site ligands. However, in recombinant systems with a lower level
of receptor expression, the compound potentiated the agonist-
stimulated response without any detectable intrinsic activity.
in the central nervous system, binds to and activates several clas-
ses of receptors which are divided into two groups termed ionotro-
pic (iGluR) and metabotropic glutamate receptors (mGluR).1 The
latter family comprises eight subtypes of G-protein coupled recep-
tors (GPCRs), grouped according to pharmacology and coupling to
second messengers.2 The primary transduction mechanism of
group I mGlu receptors (mGluR1 and mGluR5) is the stimulation
of phosphoinositide (PI) hydrolysis, leading to mobilization of
intracellular calcium stores and increase of the intracellular cal-
cium concentration. Group II (mGluR2 and mGluR3) and group III
(mGluR4, mGluR6, mGluR7 and mGluR8) receptors inhibit for-
skolin-stimulated cyclic AMP accumulation.3 A role for group I
mGlu receptor activation has been proposed in physiological pro-
cesses including pain perception, learning and memory, as well
as in certain psychiatric and neurological disorders.4 Positive allo-
steric modulators (PAMs) of the mGluR5 receptor have gained
recent interest due to their possible use for the treatment of
schizophrenia.5–7 Although both mGluR1 and mGluR5 belong to
group 1 mGluR’s, the expression patterns of these receptors in
the brain are quite different, mGluR1 expression being for example
much higher in cerebellar Purkinje cells.8 Thus mGluR1 PAMs
* Corresponding author. Tel.: +41 61 688 2464; fax: +41 61 688 8714.
E-mail address: eric.vieira@roche.com (E. Vieira).
Institute of Pharma Technology, University of Applied Sciences, Northwestern
Switzerland, Gründenstrasse 40, CH-4132 Muttenz, Switzerland.
à
Addex Pharmaceuticals, 12 Chemin Des Aulx, CH-1228 Plan-Les-Ouates,
Switzerland.
Figure 1. Structures of oxadiazole- and tetrazole derivatives 1 and 2.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.108

E. Vieira et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1666–1669
1667
Although these lead compounds were orally available, improve-
ment of pharmacokinetic (PK) properties, for example, clearance,
half-life, and bioavailability, was desirable. Replacement of the
oxadiazole or tetrazole moiety with the oxazole ring system led
to compounds with similar activities. Metabolism of compound 1
was found to take place on the methyl substituent of the oxadiaz-
ole ring as well as on positions 2 and 7 of the xanthene moiety. To
reduce metabolism at these positions, the synthesis of fluorinated
analogues was envisaged.
Syntheses of the trifluoromethylated 2-amino-oxazoles 3 and 4
were realized by reacting commercially available trifluorobromoace-
tone with 5 equiv of urea17 or cyanamide18 in refluxing tert-butanol
to afford the corresponding 4- and 5-trifluoromethyl-2-amino-oxaz-
oles in 70% and 14% yields, respectively, (Scheme 1). The synthesis of
selectively fluorinated xanthene-9-carboxylic acid derivatives is de-
scribed in Schemes 2 and 3.18 Friedel–Crafts acylation of appropri-
ately substituted para-substituted anisoles (5) with 2-fluorobenzoic
acid chlorides (6) leads selectively to ortho-acylation products with
concomitant deprotection of the methoxy group, to form the corre-
sponding 2-hydroxy-benzophenones 7b–e and 7g in good yields. In
the case of compound 7f this reaction did not proceed as desired. It
was necessary to proceed by esterification of phenol 8 to yield ester
9 which was transformed into ketone 7f via Fries rearrangement.
Cyclisation of ketones 7b–g proceeded smoothly to yield the corre-
sponding xanthones 10b–g.19 Borane-dimethylsulfide reduction in
tetrahydrofuran (THF) of the xanthones to the corresponding xanth-
enes 11b–g followed by deprotonation using lithium diisopropyl-
amide (LDA) and carboxylation with carbon dioxide afforded the
corresponding carboxylic acids 12b–g in acceptable yields. For com-
pounds 12d–g, the bromine atom which was used to block the para
position of anisole 5 during the Friedel–Crafts acylation or the Fries
rearrangement procedure was removed via hydrogenation. One ob-
tains the desired acids 13d–g in nearly quantitative yields. Reaction
of the aminooxadiazoles 3 and 4 with xanthene-9-carboxylic acid
chloride afforded amides 14a and 15a in 69% and 75% yields, respec-
tively. Reaction of the aminooxadiazoles 3 and 4 with the xanthene-
9-carboxylic acid derivatives 12b–c and 13d–g in presence of the
coupling reagent CBMIT (1,10-carbonylbis (3-methylimidazolium)
triflate) in nitromethane20 yielded the carboxamides 14b–g and
15b–g in acceptable yields. The asymmetrical compounds (entries
b, d, and e) have been tested as racemic mixtures.
Scheme 2. Synthesis of benzophenones 7b–g. Reagents and conditions: (a) AlCl3,
1,2-dichloromethane, 50 °C,50–95%; (b) Et3N, CH2Cl2, rt, 92%; (c) AlCl3, 150 °C, 30%.
Since the activation of mGluR1 leads to an increase of intracel-
lular calcium concentration, the functional activities of the com-
pounds at rat mGlu1 receptors were assessed using a fluorimetric
intracellular Ca2+ ([Ca2+]I, FLIPR) assay.21,22 The compounds show-
ing agonistic activity in the standard screening assay were selected
for further evaluation of their enhancing activity in systems with
receptor expression corresponding to physiological levels in neu-
rons (electrophysiology). The values show the effect of the tested
compounds on glutamate-activated inward K+ currents in a CHO
Scheme 3. Synthesis of carboxamides 14a–g and 15a–g. Reagents and conditions:
(a) K2CO3, 2-butanone, reflux 99%; (b) BH3.Me2S, THF, 2 h, 45 °C, quant.; (c) 1.
(iPr)2NLi, THF, ꢀ70 °C; 2. CO2, 45–87%; (d) H2, 5% Pd/C, Et3N, THF, 93–100%; (e) 1.
carbonyl diimidazole, MeOTf, CH3NO2, 10 °C, 10 min; 2. 12b–g, 10 °C- rt, 30 min; 3.
3 or 4, rt, 15 h, 30–80%.
cell stably expressing GIRKs24 and transiently transfected with
the rat mGlu1a receptor cDNA. In this setup, glutamate (3 lM) in-
duced a current that was potentiated by an mGluR1 enhancer.13
The effect of the compounds was reversible and greater than that
obtained with a saturating concentration of glutamate (100
No effect was observed in the absence of glutamate. The current
amplitudes were normalized to the control responses (3 M gluta-
lM).
l
mate) and fitted individually for each cell (n = 3). The EC50 values
[nM] as well as the efficacies (effect expressed in percentage of
control response) are shown in Table 1.
Using various functional models it was found that, in analogy to
compounds 1 and 2,16 14a was devoid of any enhancing effect at
rat mGlu2, mGlu4, mGlu5 and mGlu8 and human GABA-B recep-
tors. In addition, 14a had no activity in radioligand binding assays
at major adenosine, adrenergic, GABA-A, glycine, histamine, mus-
carinic, nicotinic, opiate, purinergic and 5-HT receptors or at aden-
osine, norepinephrine, GABA and 5-HT uptake sites. Similarly to
the reference compound 1, most of the synthesized compounds
Scheme 1. Synthesis of trifluoromethylated oxazoles
conditions: (a) tBuOH, 8 h, reflux, 70%; (b) tBuOH, 5 h, rt, 14%.
3 and 4. Reagents and

1668
E. Vieira et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1666–1669
Table 1
Activities (rat) of oxazoles 14a–g, 15a–g (n.m. = not measured)
Compd
R1, R2
FLIPR assay
mGluR1 EC50 (nM)
Electrophysiology
mGluR1 EC50 (nM)
In vitro clear. (rat microsomes)
Effect (%)
Effect (%)
Cl int. (ll/min/mg prot.)
1
14a
Ref.1
H, H
52
56
40
44
40
19
47
Inactive
38
100
86
63
48
50
220
202
n.m.23
90
260
102
161
n.m.
394
67
140
n.m.
131
162
n.m.
n.m.
1128
n.m.23
300
570
1191
707
n.m.
3055
1219
629
n.m.
910
42
108
61
9
rac-14b
14c
rac-14d
rac-14e
14f
14g
15°
rac-15b
15c
rac-15d
rac-15e
15f
H, 2-F
2,7-F
H, 3-F
H, 4-F
4,5-F
3,6-F
H, H
H, 2-F
2,7-F
H, 3-F
H, 4-F
4,5-F
3,6-F
34
33
18
38
50
40
26
28
32
12
n.m.
84
51
n.m.
104
57
205
n.m.
50
20
124
Inactive
21
25
Inactive
36
n.m.
568
n.m.
15g
Table 2
Pharmacokinetics of selected compounds in Wistar rats
Compd
Dose (iv/po) (mg/kg)
Cl (ml/min/kg)
Vss (L/kg)
t1/2 (h)
Brain/plasma ratio
Cmax (ng/ml)
1
10/10
10/10
3/6
5/10
1/10
60
1.7
0.67
0.9
0.99
0.43
0.32
0.91
2.46
3.7
1.5
0.8
0.5
n.m.
0.2
1330
1330
1810
1440
2070
14a
14c
rac-14e
14f
19.6
7.4
3.3
3.79
3.9
show affinities in the nanomolar range and good efficacies against
rat recombinant mGluR1 in the standard screening assay. The low-
er potencies obtained in the electrophysiological experiments may
be due to the lower receptor expression levels in this model. With
the exception of 14d, substitution at position 3 of the xanthene
moiety is generally not well tolerated. The compounds with the
best overall activity profile were further characterized with respect
to their metabolic stability in vitro as well as their PK properties in
Wistar rats. Brain to plasma ratios were determined by iv-infusion
experiments.25
Although the in vitro clearance in rat microsomes of the tri-
fluoromethyloxazole 14a is higher than for the methyl substi-
tuted oxadiazole 1, the rat in vivo clearance of 14a is much
lower, leading to a three fold increase in half-life compared to
1 (Table 2). Bioavailabilities of these compounds are in the
acceptable range (15–100%), with plasma levels ranging between
1300 and 2100 ng/ml after po administration. Intermediate vol-
umes of distribution as well as variable brain penetration are ob-
served. In comparison to the unsubstituted xanthene derivative
14a, additional fluorination of the xanthene moiety leads in most
cases to further reduction of the in vitro as well as the in vivo
clearance values. The half-life of these compounds is also mark-
edly increased. The volume of distribution is in the intermediate
range, while brain penetration seems to decrease for the fluorox-
anthene derivatives.
pounds could serve as suitable tools to further study the role of po-
sitive allosteric modulation of mGlu1 receptors in vivo.
Acknowledgments
The skillful synthetic work of Nadia Bitter,§ Martin Kuratli,§ and
Patrick Scheifele– is gratefully acknowledged.
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Based on the potent and selective positive allosteric modulator
1, two series of trifluoromethyloxazole derivatives with improved
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been prepared and the compounds with the most promising
in vitro profiles were selected for further PK evaluation.
13. Knoflach, F.; Mutel, V.; Jolidon, S.; Kew, J. N. C.; Malherbe, P.; Vieira, E.;
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Recently, acute oral administration of compound 14a consis-
tently improved motor performance in a rat model of experimental
autoimmune encephalomyelitis (EAE).26 With respect to their PK
profile and brain penetrating properties, several of these com-
§
Present address: Pharma Division, Discovery Research, F. Hoffmann-La Roche Ltd,
CH-4070 Basel, Switzerland.
–
Present address: Novartis AG, Basel, Switzerland.

E. Vieira et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1666–1669
1669
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21. [Ca2+]I measurements were performed on rat mGlu1a-transiently transfected
HEK-293 cells after incubation of the cells with the calcium sensitive fluorescent
indicator Fluo-3 AM (Molecular Probes, Eugene, OR, USA) for 1 h and 4 washes
with assay buffer (DMEM supplemented with Hank’s salt and 20 mM HEPES).
[Ca2+]I measurements were done using a fluorometric imaging plate reader
(FLIPR, Molecular Devices Corporation, La Jolla, CA, USA). Fluorescence ratio
valueswere calculatedas described.22 EC50 valuesforthe enhancersarethemean
of separate values from at least three individual experiments. DMEM, Dulbecco’s
Modified Eagle Medium, is a very common basic cell culture medium usually
supplemented with various ingredients such us growth factors, animal serum,
vitamins etc. Hanks’ Balanced Salt Solution is a preservative-free medium for cell
22. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 3440.
23. Compound 14b was not evaluated in electrophysiology due to its high in vitro
clearance compared to the other fluoro derivatives.
24. GIRK is an abbreviation for G-protein coupled inwardly rectifying K+ channel.
In a cell, activation of G-protein coupled receptors leads to opening of K+
channels by the coupling G-protein. Electrophysiological measurements of the
induced potassium currents allow direct quantification of GPCR activation. For
more details, see Ref. 13.
25. The iv and po and infusion experiments are performed using two animals per
group. For the iv and po experiments, blood sampling and iv bolus of compounds
were realized via implanted jugular vein catheters. Blood samples were taken at
0.3, 0.5, 1, 2, 4, 8, and 24 h after administration. For the infusion experiments
(1 mg/kg/h, via catheter), blood samples were taken sublingual at 1, 3 and 5 h
post administration. Brain tissue samples were obtained by decapitation of the
animals following isofluran anesthesia. Sample analysis of blood samples or
brain tissue homogenate was performed using standard LC–MS methodology.
26. Fazio, F.; Notartomaso, S.; Aronica, E., et al J. Neuropharmacol. 2008, 5(4),
491.