Synthesis and biological evaluation of fenobam analogs as mGlu5 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.12.033

Optimization of affinity and microsomal stability led to identification of the potent, metabolically stable fenobam analog 4l. Robust in vivo efficacy of 4l was demonstrated in four different models of anxiety. Additionally, a ligand based pharmacophore a

Full text of DOI:10.1016/j.bmcl.2006.12.033

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1307–1311
Synthesis and biological evaluation of fenobam analogs as
mGlu5 receptor antagonists
Georg Jaeschke,* Richard Porter, Bernd Buttelmann, Simona M. Ceccarelli,
¨
Wolfgang Guba, Bernd Kuhn, Sabine Kolczewski, Jo¨rg Huwyler,
Vincent Mutel, Jens-Uwe Peters, Theresa Ballard, Eric Prinssen,
Eric Vieira, Jurgen Wichmann and Will Spooren
¨
Pharmaceutical Division, Discovery Research, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland
Received 17 October 2006; revised 1 December 2006; accepted 3 December 2006
Available online 15 December 2006
Abstract—Optimization of affinity and microsomal stability led to identification of the potent, metabolically stable fenobam analog
4l. Robust in vivo efficacy of 4l was demonstrated in four different models of anxiety. Additionally, a ligand based pharmacophore
alignment of fenobam and MPEP is proposed.
Ó 2007 Elsevier Ltd. All rights reserved.
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 (iGlu) and metabo-
tropic glutamate receptors (mGlu).1 The mGlu receptors
are classified based on their homology, pharmacology,
and second messengers in three groups.2 The mGlu5
receptor belongs to group I mGlu receptors, which are
coupled to phospholipase C leading to the activation
of phosphoinositide (PI) hydrolysis and elevation of
Ca2+ levels. The high expression of mGlu5 receptor in
the limbic areas of the brain suggests a potential role
of this receptor in psychiatric disorders, such as anxiety.
brane domain.3 MPEP is active in a wide range of pre-
clinical anxiety models such as the stress-induced
hyperthermia, Vogel conflict and plus maze test.4
We have serendipitously discovered in a screening
campaign that fenobam [N-(3-chlorophenyl)-N0-(4,5-
dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] 2 is a
potent, subtype-selective, and non-competitive mGlu5
receptor antagonist.5 Fenobam entered clinical trials
for anxiety in the late seventies and proved to be similar-
ly active as benzodiazepines in a double blind placebo-
controlled clinical trial, but did not show the same
liabilities such as ethanol interaction and sedation.6
With studies using point mutated mGlu5 receptors and
3-D receptor pharmacophore-based modeling, we have
demonstrated recently that the two structurally diverse
allosteric antagonists MPEP and fenobam have similar
contact sites on the mGlu5 receptor binding crevice.7
O
H
N
N
O
N
N
N
H
Cl
1
2
In this paper, we would like to report a SAR of fenobam
analogs8 and to propose a ligand based pharmacophore
alignment of fenobam and MPEP. In addition, we de-
scribe the biological evaluation of fenobam analog 4l
with improved metabolic stability.
MPEP (2-methyl-6-(phenylethynyl)-pyridine) 1 has been
reported to be an mGlu5 receptor antagonist and acts
via an allosteric binding site located in the transmem-
To establish a SAR, we decided to vary the 3-chlorophe-
nyl substituent of fenobam. Synthesis was accomplished
by either direct condensation of creatinin with the corre-
sponding aniline in the presence of CDI or via a stepwise
Keywords: mGluR5; Fenobam; Anxiety; MPEP; Pharmacophore.
*
Corresponding author. Tel.: +41 61 68 82034; fax: +41 61 68
88714; e-mail: georg.jaeschke@roche.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.033

1308
G. Jaeschke et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1307–1311
Table 1. Binding affinity and functional activity of fenobam analogs 2,
4a–f
O
O
H
N
O
N
Compound
R
3H-MPEP FLIPR CL (r/h)
(nM)
a
N
N
N
O
(nM)
(ll/min/mg prot)
NH2
3
4a
4b
2
Ph
2-Cl–Ph
3-Cl–Ph
1360
2520
61
3300
6800
—
—
38
>5000
1100
44/100
—
b
4c
4d
4e
4f
4-Cl–Ph >5000
c
2-Py
3-Py
4-Py
587
>5000
1120
22/47
5/20
9/11
O
>5000
>5000
H
N
O
R
N
N
N
Table 2. Binding affinity and functional activity of MPEP analogs 1,
6a–f
2, 4a-l
Compound
R
3H-MPEP (nM)
FLIPR (nM)
Scheme 1. Synthesis of fenobam analogs 2, 4a–l. Reagents and
conditions: (a) phenylchloroformate, THF, reflux, 17%; (b) aniline,
DMF, 50 °C, 30–70%; (c) aniline, CDI, DMF, 90 °C or reflux, 20–
65%, 1% for 4b.
1
Ph
4
5
29
15
6a
6b
6c
6d
6e
6f
2-Cl–Ph
3-Cl–Ph
4-Cl–Ph
2-Py
3
7
286
39
10
26
1020
400
66
procedure using the phenoxycarbamate intermediate 3
(Scheme 1).
3-Py
4-Py
213
To allow a direct comparison with the SAR of MPEP,
we also synthesized a representative subset of MPEP
analogs varying the phenyl substituent via Sonogashira
coupling reaction using intermediate 59 (Scheme 2).
are least potent in both series. Substitution of the phenyl
ring by a pyridine in the MPEP series leads only to a
moderate drop in affinity, which is less pronounced than
substitution of the 3-Cl-phenyl ring by a pyridine in the
fenobam series. We believe that the similarities in the
SAR of fenobam and MPEP support the hypothesis that
the phenyl ring of MPEP and the 3-chloro phenyl
substituent of fenobam occupy a similar position in
the allosteric mGlu5 binding site. A ligand based
pharmacophore model supporting this hypothesis is
proposed further below (see modeling section).
The fenobam analogs show a steep structure–activity
relationship. The 3-chloro substituent is of crucial
importance and the unsubstituted phenyl analog is sig-
nificantly less active. Shifting the chloro substituent to
the ortho- and para-position also leads to a drop in
affinity. The 2- and 4-pyridine derivatives maintain some
activity, whereas the 3-pyridine derivative is inactive
(Table 1). Hepatic stability (determined in vitro using
rat and human liver microsomes) is low for fenobam,
an observation that can be rationalized by assuming
hydroxylation of the para-position on the phenyl.10
The more polar pyridine derivatives 4d–f show a higher
microsomal stability (Table 1).
As mentioned above, a substituent in the meta-position
clearly increases affinity in the fenobam series. We there-
fore investigated the influence of a chloro- or methyl-
substituent in combination with ortho- or para-pyridine
moieties (Table 3). Interestingly, the influence of the
meta substituents is less pronounced than for the phenyl
substituents, but in case of the ortho pyridine derivatives
4g and 4i compounds with improved potency were ob-
tained. Microsomal clearance is medium to high for
these compounds in both rat and human, and is higher
than for the respective para pyridine derivatives 4h
and 4j. This observation is again in line with a potential
hydroxylation of the para-position for the ortho pyri-
dine derivatives. To prove the theory of oxidative
In the MPEP series modifications of the phenyl ring are
much better tolerated at the mGlu5 receptor compared
to fenobam analogs. In particular, the influence of the
3-chloro substituent is of lower importance than for
fenobam and a chloro substituent in the 2-position is
also tolerated (Table 2). Nevertheless, the SAR of the
MPEP derivatives shows some qualitative overlap with
fenobam analogs as, for example, the 3-chloro deriva-
tives are most potent whereas the 4-chloro derivatives
Table 3. Binding affinity, functional activity, and microsomal stability
of fenobam analogs 4g–l
Si
R
Compound
R
3H-MPEP FLIPR CL (r/h)
(nM)
a
N
N
(nM)
(ll/min/mg
prot)
4g
4h
4i
3-Cl-2-Py
3-Cl-4-Py
166
663
320
2670
700
78
—
—
53/42
31/15
37/29
14/2
5
1, 6a-f
3-Me-2-Py
3-Me-4-Py
3-Thienyl
5-Cl-3-thienyl11
681
1600
4016
434
4j
Scheme 2. Synthesis of MPEP derivatives 1, 6a–f. Reagents and
conditions: (a) ArI, CuI, Bu4NF; Pd(PPh3)Cl2, PPh3, triethylamine,
THF, 50 °C, 36–80%.
4k
4l
ꢀ/ꢀ
27/55

G. Jaeschke et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1307–1311
1309
metabolism, we replaced the 2-chloro phenyl substituent
***
140
120
100
80
60
40
of fenobam with the isosteric 3-thienyl substituent
leading to the fenobam derivative 4k. We hypothesized
that the moderate potency of 4k could be improved by
introduction of an additional chloro substituent in the
5-position leading to 4l. Indeed 4l not only retains rea-
sonable potency but also shows improved metabolic sta-
bility compared with fenobam.11
20
0
Veh
3
10
30
Therefore, we profiled 4l in additional models of
anxiety.
4l, mg/kg, p.o.
Figure 2. Drinking times (sec) in the Vogel conflict test (punished
drinking) as a measure of anxiolytic-like response. The drinking times
after oral doses of 3, 10, and 30 mg/kg of 4l (pretreatment time = 60 min)
are compared with vehicle. Data are means and SEM; statistics:
***p < 0.001 versus vehicle (Mann–Whitney U test, one-tailed).
We evaluated 4l in four models for assessing anxiolytic-
like activity. In stress-induced hyperthermia, exaggerat-
ed responses of the autonomic nervous system to stress
are measured using body temperature measurements in
mice.4b Compound 4l (3, 10, and 30 mg/kg, po) signifi-
cantly reduced stress-induced hyperthermia at 10 and
30 mg/kg, while having no effect on the baseline temper-
ature (Fig. 1).
0.5
A
B
*
2400
1800
1200
600
0
**
Compound 4l was evaluated in the Vogel conflict
test,13,4d in which drinking is suppressed in water-re-
stricted rats by a brief electrical shock every second of
cumulative drinking time. Compound 4l, after an oral
dose of 30 mg/kg po, but not 3 or 10 mg/kg po, signifi-
cantly increased drinking time, consistent with an anxio-
lytic-like profile (Fig. 2).
0.4
0.3
0.2
0.1
0.0
*
In the conditioned emotional response (CER) test4d in
rats, 4l significantly and dose-dependently reversed the
suppression of lever pressing by stimuli previously asso-
ciated with foot shock, at 3, 10, and 30 mg/kg po
(Fig. 3A). There was no significant effect of treatment
on the total number of lever presses made during the
1 h session (Fig. 3B) suggesting that behavioral disrup-
tion did not occur at this dose–range (although there
was a tendency for a reduction at 30 mg/kg po).
V
3
10 30
4l, mg/kg, p.o.
V
3
10 30
4l, mg/kg, p.o.
Figure 3. (A) Suppression ratio in the CER test as a measure of
anxiety; (B) number of lever presses during the 60-min session as a
measure of non-specific effects. Data are expressed as means SEM.
Statistics: *p < 0.05, **p < 0.01 versus vehicle (A, Wilcoxon rank sum
test; B, paired t test with Bonferroni correction).
In the conflict test4d in rats, 4l significantly and dose-de-
pendently reversed the suppression of lever pressing
associated with foot shock at 3, 10, and 30 mg/kg po
(Fig. 4A). There was no significant effect of treatment
on the lever pressing during the unpunished periods
(Fig. 4B) suggesting that behavioral disruption did not
occur at this dose–range.
1.4
A
B 50
1.2
1.0
0.8
0.6
0.4
0.2
0.0
*
40
**
30
20
10
0
1.0
0.8
0.6
*
0.4
0.2
0.0
***
***
V
3
10
30
V
3
10
30
4l, mg/kg, p.o.
4l, mg/kg, p.o.
0
3
10
30
Figure 4. (A) Punished lever presses per minute in the conflict test as a
measure of anxiety; (B) unpunished lever presses per minute as a
measure of non-specific effects. Data are expressed as means SEM.
Statistics: *p < 0.05, **p < 0.01 versus vehicle (paired t test with
Bonferroni correction).
4l, mg/kg, p.o.
Figure 1. Stress-induced hyperthermia (SIH). Data are means and
SEM; statistics: ***p < 0.001 versus vehicle (Dunnet, two-tailed).

1310
G. Jaeschke et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1307–1311
In conclusion, robust efficacy of 4l in four different mod-
els of anxiety was seen.
Cl is very likely to disrupt this H-bond. Similarly,
replacements of the 3-Cl-phenyl ring with pyridine sub-
stitutions are not tolerated within the fenobam as op-
posed to the MPEP series, since fenobam has less
translational and rotational degrees of freedom than
MPEP due to this additional directed hydrogen bond.
Therefore, the introduction of an additional H-bond
acceptor via pyridine substitutions cannot be properly
accommodated by reorientations within the binding
pocket.
Fenobam can exist in two tautomeric forms, depicted in
Figure 5, maintaining the energetically favorable C@O
bond. To better understand the binding mode to the
mGlu5 receptor, we calculated the relative stabilities of
the two tautomers using quantum chemical methods.
Geometry optimization at the AM1 level of low-energy
conformers of the two tautomers, followed by single-
point energy calculation with the B3LYP/cc-pVDZ
method, revealed that fenobam with the imino-imidazo-
lidinone motif is substantially more stable than the cor-
In conclusion, we could establish in the present report a
ligand based pharmacophore alignment between MPEP
and fenobam, and characterize in vitro as well as in vivo
several fenobam analogs including analog 4l with im-
proved metabolic stability. In view of their anxiolytic
properties in different animal models, the presented
fenobam analog 4l could be of potential interest for
the treatment of psychiatric disorders.
responding
amino-dihydroimidazolone
tautomer
(DE = 19.1 kcal/mol in the gas phase, DE = 10.3 kcal/
mol in a PCM continuum solvent description of
water).13 The large preference for the right tautomer in
Figure 5 can be understood by the energetically favor-
able formation of a 6-membered ring through an intra-
molecular hydrogen bond.
Acknowledgments
The pharmacophore-guided superposition of MPEP and
fenobam is illustrated in Figure 6. Both the central ethy-
nyl and amide linkers of MPEP and fenobam, respec-
tively, are spacers for correctly positioning the benzene
rings and the corresponding pyridine or imidazolidinone
moieties. As noted in a homology modeling study by
Malherbe et al.7 the common determinants of the MPEP
and fenobam binding pockets are hydrophobic interac-
tions with the (chloro)benzene ring and hydrogen bonds
with the polar pyridine and imidazolidinone substitu-
ents. The pyridine nitrogen of MPEP overlaps with the
doubly bonded nitrogen of the most stable fenobam tau-
tomer, which leaves the carbonyl oxygen of the imidazo-
lidinone ring free for an additional H-bond with S3.39.
The excellent technical assistance of Daniel Rueher,
Antonio Ricci, Francoise Kahn, Severine Weil-Bandi-
nelli, and Sean Durkin is gratefully acknowledged.
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O
O
H
N
O
N
O
N
N
N
N
Cl
N
N
Cl
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3-(1-methyl-4-oxo-2-imidazolin-2-yl)urea), see: Hunkeler,