Carbamoyloximes as novel non-competitive mGlu5 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.06.075

Hit-to-lead optimization of a HTS hit led to new carbamoyloxime derivatives. After identification of an advanced hit (8d) the CYP enzyme inhibitory activity of this class of compounds was successfully eliminated. Systematic exploration of different parts of the advanced hit led us to some promising lead compounds with mGluR5 affinities comparable to that of MPEP.

Full text of DOI:10.1016/j.bmcl.2010.06.075

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4371–4375
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbamoyloximes as novel non-competitive mGlu5 receptor antagonists
János Galambos, Gábor Wágner, Katalin Nógrádi, Attila Bielik, László Molnár, Amrita Bobok, Attila Horváth,
Béla Kiss, Sándor Kolok, József Nagy, Dalma Kurkó, Mónika L. Bakk, Mónika Vastag, Katalin Sághy,
*
}
István Gyertyán, Krisztina Gál, István Greiner, Zsolt Szombathelyi, György M. Keseru, György Domány
Gedeon Richter Plc, Budapest 10, PO Box 27, H-1475, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Hit-to-lead optimization of a HTS hit led to new carbamoyloxime derivatives. After identification of an
advanced hit (8d) the CYP enzyme inhibitory activity of this class of compounds was successfully elim-
inated. Systematic exploration of different parts of the advanced hit led us to some promising lead com-
pounds with mGluR5 affinities comparable to that of MPEP.
Received 20 April 2010
Revised 11 June 2010
Accepted 11 June 2010
Available online 17 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keyword:
mGluR5 non-competitive antagonist
carbamoyloxime
In the early 1990s, a new family of receptors mediating the
intracellular metabolic effects of glutamate via coupling to second-
ary messenger systems was cloned. There are eight metabotropic
glutamate (mGlu1–mGlu8) receptor subtypes known to date
which have been clustered into three groups (I–III)1 according to
their amino acid sequence, pharmacology, and second-messenger
coupling. In contrast to the glutamate-gated ion channels (NMDA,
AMPA, and kainate receptors), which are responsible for fast excit-
atory transmission, mGlu receptors have been shown to play a
modulatory role in the glutamatergic synaptic transmission either
by modulating the ion channel activity or by influencing neuro-
transmitter release.2 Since mGlu receptors are G-protein coupled,
they obviously constitute a new attractive group of ‘druggable’ tar-
gets for the treatment of various CNS disorders.3 The recent discov-
ery of small molecules that selectively bind to group I (mGlu1 and
mGlu5) and group II (mGlu2 and mGlu3) receptors4 has signifi-
cantly facilitated the understanding of their roles in brain physiol-
ogy and pathophysiology.
Identification of the first selective, non-competitive mGluR5
antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP, 1)5
(Fig. 1) initiated the pharmacological evaluation of this class of
compounds. Subsequent behavioral studies revealed that mGlu5
receptor antagonists exhibit profound anxiolytic effects in animal
models and are remarkably free of the adverse effects characteris-
tic of either benzodiazepines or iGlu receptor antagonists.6 These
preclinical data added support for the mGlu5 receptor as a poten-
tially important therapeutic target for anxiety.7
Before metabotropic glutamate receptor subtypes were cloned,
the non-GABAergic agent fenobam (2) (Fig. 1) was investigated in a
double-blind, placebo-controlled clinical trial in which it showed
efficacy and onset of action comparable with that of diazepam.8
In 2005 Roche reported that fenobam, like MPEP, is a negative allo-
steric modulator of mGluR59 that provided clinical proof of princi-
ple for the mGluR5 approach in anxiety.
There is a large unmet medical need for new anti-anxiety agents
that relieve symptoms quickly yet lack (benzodiazepine-like) side-
effects. Recent findings have suggested an important role for the
mGlu5 receptor in anxiolysis. Consequently, several pharmaceuti-
cal companies have initiated mGluR5 discovery programmes. The
most important chemotypes of negative allosteric modulator of
mGluR5 were reviewed recently.10
High throughput screening (HTS) of our corporate compound
collection resulted in several hit clusters, which were further opti-
mized as reported in our previous communication11 and several
patent applications.12 This Letter describes the optimization of
one structural family, the carbamoyloximes, represented by com-
* Corresponding author.
E-mail address: gy.domany@richter.hu (G. Domány).
Figure 1. Proof of concept mGluR5 antagonists.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.075

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J. Galambos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4371–4375
Figure 2. The HTS hit and its isomer served as starting point of hit-to-lead optimization. Binding and functional data (value SD (N)) and CYP inhibitory activity (inhibition%)
were obtained as described in Refs. 13–15.
pound 3 (Fig. 2), which showed moderate affinity to mGluR5.13
From functional assays these compounds were shown to be non-
competitive antagonists.14 The original HTS hit (3) was resynthe-
sized yielding a 4:1 mixture of E and Z isomers having a Ki value
of 156 nM. Isomers were separated and the E isomer (4) shown
to be the more active. The hit-to-lead process was started with
compound 4.
Multiple objectives were set for our hit-to-lead optimization,
for this purpose, the hit compound 4 was divided into four regions
(Fig. 2). These included identification of the optimal substitution
pattern of the aromatic ring (A), optimal replacements for the
imidazole ring (I) and the cyclohexane moiety (C), and finding
the best spacer unit (S) and the active enantiomer. In order to an-
swer these questions we systematically investigated the respective
regions of our hit.
The synthesis of carbamoyloximes 8 was realized by preparing
oximes 6 from suitable ketones 5 stirred under mild conditions in
aqueous methanol with hydroxylamine (Scheme 1). The Z and E
isomers of oximes 6 were then separated by crystallization or by
column chromatography. In the second step, carbamoyloximes
(8) were prepared from oximes 6 and commercially available
appropriate phenylisocyanates 7 in dichloromethane. The final
products were purified by crystallization or by column chromatog-
raphy to >95% purity (HPLC, 1H, and 13C NMR). Depending on the Y
substituents, the cyclic ketones 5 were synthesized by different
pathways. If Y was a nitrogen heterocycle connected via its nitro-
Scheme 1. Reagents and conditions: (a) NH2OHÁHCl, NaOAc, MeOH, H2O, 45 °C, 2–8 h, then separation of Z and E isomers, 50–80%; (b) dichloromethane, rt, 6–24 h, 40–70%;
(c) CH2O, AcOH, 75 °C, 10 h, 50–70%; (d) NaOH, H2O, rt, 12 h, 50–70%; (e) H2 (1 bar), Pd/C, MeOH, rt, 3 h, 80–90%.

J. Galambos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4371–4375
4373
Table 1
Table 3
Early optimization of 4
Replacement of imidazole to different heteroaryl groups
Compd
X
mGluR5
CYP inhib.15
Compd
X
Y
rmGluR5
CYP inhib.15
14
14
Ki13 (nM)
IC50 (nM)
Ki13 (nM)
IC50 (nM)
8a
8b
8c
8d
(+)-8d
(–)-8d
CF3
CN
F
Cl
Cl
78 26 (2)
38 10.8 (2)
30.6 4.78 (2)
10.8 2.28 (2)
8.78 (1)
775 175 (3)
561 115 (3)
92.3 37 (7)
116.5 60 (9)
76 0 (3)
61/57/58
50/32/53
62/52/69
61/53/61
50/51/45
—
8o
8p
8q
8r
8s
8t
Cl
Cl
Cl
F
2-Furanyl
438 (1)
130 26 (9) 7/0/0
3-Thiophenyl 13.8 4.78 (3) 477 89 (3) 14/0/0
2-Thiophenyl 9.1 1.18 (2) 109 15 (4) 0/0/0
2-Thiophenyl 12.3 3.38 (2)
46 16 (5) 17/0/1
48 14 (4) 13/0/1
181 43 (4) 18/0/4
Me
2-Thiophenyl
8.3 1.88 (2)
88 7.8 (2)
Cl
>1000
—
MeO 2-Thiophenyl
Y = 1-imidazolyl; R1, R2 = cyclohexane, formed with the intermediate two carbon
atoms; value SD (N).
R1, R2 = cyclohexane, formed with the intermediate two carbon atoms; value SD
(N).
gen atom (5a), cyclic ketones 9 and N-containing heterocycles 10
were reacted with formaldehyde in acetic acid. If Y was an aryl
or heteroaryl group (5b), the reaction of cyclic ketones 9 and suit-
able arylaldehydes 11 was performed with NaOH in water to afford
(8k–m) in which the activity depended on the position of the pyr-
idine nitrogen. The 2-pyridyl analog 8m was the only compound
lacking CYP inhibitory property that showed moderate in vitro
activity. Compound 8l was the most active derivative, but showed
moderate CYP inhibition.
a,b-unsaturated ketones 12, that were reduced with palladium un-
der hydrogen in methanol. The structures of all intermediates and
end products were confirmed by IR, NMR, and MS spectroscopy.16
Early optimization of 4 yielded compound 8d as an advanced
hit. In accord with the results of others,10,17 we found that substit-
uents at position 3 of the phenylcarbamoyl moiety increased the
affinity to mGlu5 receptors (Table 1). Compound 8d showed im-
proved affinity relative to MPEP and was active in vivo as well
(Fig. 3).18 Its enantiomers were separated by preparative HPLC
revealing the (+) enantiomer as the eutomer.
Although both the in vitro and in vivo activity of 8d was prom-
ising, its CYP inhibitory activity15 prompted us to either substitute
the imidazole moiety or to replace it by other N-heterocycles.
Unfortunately, most modifications of the imidazole ring re-
sulted in either inactive compounds (8e–i) or active compounds
with retained CYP inhibitory activity (8k, 8l and 8n) (Table 2). Sub-
stitution of the imidazole by pyridine resulted in active compounds
Another option was to substitute the imidazole for different
heteroaryl groups that—in most cases—eliminated the CYP inhibi-
tion yet maintained, or in some cases increased, the affinity to
mGluR5 receptors (Table 3). The good activity of furan (8o) and
the excellent activities of the thiophene (8p–s) derivatives were
somewhat surprising. These results suggested that the presence
of at least one well positioned H-bond acceptor could replace the
basic N (H-bond donor when protonated) in active compounds.
Elimination of the CYP inhibitory activity was successful in the case
of the highly active compounds 8q–s.
It was interesting that mGluR5 affinity was maintained by
replacing the imidazole with (substituted)phenyl groups. In these
compounds essentially hydrophobic forces are expected to play a
major role in binding. Non-specific hydrophobic contacts resulted
in moderately active compounds (8u, 8w). Substituents on the
phenyl ring decreased activity (8v, 8x, 8y) (Table 4).
MPEP (1)
8d
100
100
++
++
+++
80
60
40
20
0
80
++
++
60
40
20
0
+
5
control 2.5
10
20
40
control
1
3
10
dose, mg/kg ip.
dose, mg/kg ip.
Figure 3. Effect of MPEP (1) and 8d in the Vogel punished drinking test in rats. +, ++, and +++: p <0.05, p <0.01, and p <0.001 versus control, respectively.
Table 2
Replacement of imidazole to N-heterocycles
Compd
X
Y
rmGluR5
CYP inhib.15
14
Ki13 (nM)
IC50 (nM)
8e
8f
8g
8h
8i
8j
8k
8l
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
1-Piperidinyl
Inactive
>1000
>1000
>1000
>1000
43.7 19.5 (4)
117 (1)
3.35 0.5 (2)
40.4 16.2 (7)
16.1 4.6 (2)
—
—
—
—
—
—
—
—
4-Phenyl-piperazin-1-yl
Tetrahydro-isoquinoline-2-yl
4-Morpholinyl
2-Methyl-imidazol-1-yl
4-Methyl-imidazol-1-yl
4-Pyridyl
3-Pyridyl
2-Pyridyl
4-Pyrrolyl
—
6/0/44
42/0/58
56/60/46
22/12/50
0/0/27
44/0/14
311 56 (4)
—
15 7 (9)
65 14 (3)
194 (1)
8m
8n
R1, R2 = cyclohexane, formed with the intermediate two carbon atoms; value SD (N).

4374
J. Galambos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4371–4375
Next we aimed to investigate the role of the cyclohexane moi-
enantiomeric constraints this part of the carbamoyloximes is
rather sensitive to conformational changes.
ety in mGluR5 binding. The analog of 8d with an unsubstituted
cyclohexane ring (13) lost activity. Changing the benzylcyclohex-
ane moiety to tricyclic systems (14–16) also eliminated mGluR5
affinity. Other modifications of the cyclohexane ring (e.g., tetrahy-
dronaphthyl 17, cyclopentyl 18, and propyl 19, 20) generally re-
sulted in much weaker compounds (Fig. 4). These observations
support our hypothesis that in addition to diastereomeric and
In the final part of our hit-to-lead program we modified the car-
bamoyloxime spacer. Thus, reduction of the oxime to hydroxyl-
amine (21) decreased the affinity. Modification of the
carbamoyloxime to semicarbazone (22) also resulted in an inactive
compound (Fig. 5), indicating the carbamoyloxime moiety as a
privileged moiety for mGluR5 binding.
Figure 4. Modification of the cyclohexane ring; value SD (n = 2).
Figure 5. Modifications of the spacer (n = 2).
Table 4
Replacement of imidazole to (substituted) phenyl groups
Table 5
Functional activity at mGluR1 (group I) and mGluR2 (group II)19
Compd
X
Y
rmGluR5 Ki (nM)
CYP
inhib.15
Ki13 (nM)
IC50 (nM)
14
Compd
mGluR1 inhib.% (10
lM)
mGluR2 inhib.% (10 lM)
MPEP
8d
(+)-8d
8o
8p
8q
15.7 8.1 (2)
30.6 10.1 (2)
26.9 14.2 (2)
20.9 11.9 (2)
15.2 4.3 (2)
11.0 4.2 (2)
10.0 3.5 (4)
À6.2 3.0 (4)
À6.7 2.9 (4)
33.3 7.6 (4)
22.8 9.3 (4)
32.0 4.9 (4)
8u
8v
8w
8x
8y
Me Phenyl
Me 3-F-phenyl
29.7 10.8 (3) 152 39 (4)
0/0/0
56.4 4 (2)
16 1.4 (2)
39.5 8.8 (2)
>1000
224 67 (15) 9/0/0
Cl
Cl
Cl
Phenyl
151 37 (3)
127 27 (5)
—
9/0/0
0/0/0
—
3-F-phenyl
4-Me2N-
phenyl
Value SD (N).
R1, R2 = cyclohexane, formedwith the intermediatetwo carbon atoms;value SD (N).

J. Galambos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4371–4375
4375
Kuhn, R. Bioorg. Med. Chem. Lett. 2002, 2, 407.) with modifications. Rat cerebro-
cortical membrane preparation was used to determine the binding
characteristics of reference compounds and novel compounds to the rat
mGluR5. As radioligand [3H]-M-MPEP (2 nM) was used. The non-specific
Functional activity data show high selectivity against other
mGluR subtypes both in the same group I and also in group II as
exemplified by mGluR1 and mGluR2, respectively (Table 5).
In summary, hit-to-lead optimization of a HTS hit identified car-
bamoyloxime derivatives as a novel class of non-competitive
mGlu5 receptor antagonists. Early optimization of the validated
hit (4) by identification of the optimal substitution pattern of the
aromatic ring, resulted in an advanced hit (8d). Further optimiza-
tion of 8d successfully eliminated its CYP enzyme inhibitory activ-
ity. Modification of the cyclohexane ring, or the spacer diminished
the affinity. Replacement of the imidazole ring by aryl or hetero-
aryl groups yielded active mGluR5 antagonists one of which (8q)
was identified as a promising lead with affinity, and selectivity
comparable to that of MPEP.
binding was determined in the presence of 10 lM M-MPEP. The ligand
displacement by the compounds was determined in duplicates or triplicates.
For IC50 (Ki) determinations concentration–displacement curves were
generated consisting of minimum of six concentrations. Ki values (i.e.,
inhibition constants) were calculated using the Cheng–Prusoff equation:
Ki = IC50/[1 + (L/Kd)], where [L] is the radioligand concentration and Kd the
affinity of the labeled ligand for receptor. Kd was determined from the
Scatchard plot.
14. Functional activity at mGluR5 was measured in primary rat neuronal cultures
of neocortical origin, taking advantage of the high expression level of mGluR5
in this brain area (Romano, C.; Sesma, M. A.; McDonald, C. T.; O’Malley, K.; Van
den Pol, A. N.; Olney, J. W. J. Comp. Neurol. 1995, 355, 455.). The neocortical cell
cultures were prepared as described in Nagy et al. (Nagy, J.; Horváth, C.; Farkas,
S.; Kolok, S.; Szombathelyi, Z. Neurochem. Int. 2004, 44, 17.) Functional activity
at mGluR5 was measured by Ca2+-fluorometry according to Nagy et al. with
modifications. Briefly, cells were isolated from E17 rat embryos, seeded in 96-
well plates and cultured at least for 5 days before being subjected to Ca2+
-
-
References and notes
measurements. For the Ca2+-measurements cells were loaded with the Ca2+
sensitive dye, fluo-4/AM. Baseline and agonist evoked signals were recorded
with a plate reader fluorometer. Agonist was (S)-3,5-dihydroxyphenylglycine.
For IC50 determination sigmoidal (4-parameter) concentration–inhibition
curves were fitted to the percent inhibition data derived from at least three
independent experiments using GraphPad Prism software.
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R. D.; Gentsch, C. J. Pharmacol. Exp. Ther. 2000, 295, 1267.
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15. The CYP enzyme inhibition was determined according to Schoene et al.
(Schoene, B.; Fleischmann, R. A.; Remmer, H. Eur. J. Clin. Pharmacol. 1972, 4, 65.)
and Chhabra et al. (Chhabra, R. S.; Gram, T. E.; Fouts, J. R. Toxicol. Appl.
Pharmacol. 1972, 22, 50.). CYP (cytochrome P450) enzyme inhibition is
calculated as
% of inhibition of CYP mediated enzyme reactions such as
aminopyrine-N-demethylation/p-nitroanisole-O-demethylation/aniline-
hydroxylation, respectively, in rat liver microsomes at nominal 10
compound concentration.
16. Galambos, J.; Vastag, M.; Bobok, A. Á.; Keseru, G. M.; Gál, K.; Benko, B.; Rill, A.;
Demeter, Á. WO 2007072089, 2007.
lM test
}
}
17. Gasparini, F.; Bilbe, G.; Gomez-Mancilla, B.; Spooren, W. Curr. Opin. Drug Discov.
Devel. 2008, 11, 655.
18. The Vogel punished drinking test was according to Vogel et al. (Vogel, J. R.;
Beer, B.; Clody, D. E. Psychopharmacology 1971, 100, 138.) with modifications.
Rats are deprived of drinking water for 48 h prior to test. 24 h prior to test they
are placed into the test chambers equipped with a metal water spout mounted
on the wall of the chamber and a metal grid floor for delivering electric shocks.
During a 5-min adaptation period, they have free access to the drinking spout.
On the day of the measurement, the animals are treated with the test
compounds then placed into the test chambers where they have free access to
drinking water for a 30-s unpunished period. After that, electric shocks (1 mA,
1 s) are applied through the drinking spout following every 10 licks during a 5-
min punished period. Number of licks and shocks delivered are recorded and
stored in a computer. Anxiolytic or anxiogenic activity is reflected by increased
or decreased number of accepted shocks, respectively. Test compounds are
studied minimally at three dose levels because of the occurrence of bell-shaped
dose–response curves.
11. Wágner, G.; Wéber, C.; Nyéki, O.; Nógrádi, K.; Bielik, A.; Molnár, L.; Bobok, A.;
Horváth, A.; Kiss, B.; Kolok, S.; Nagy, J.; Kurkó, D.; Gál, K.; Greiner, I.;
}
Szombathelyi, Z.; Keseru, G. M.; Domány, G. Bioorg. Med. Chem. Lett. 2010, 20,
3737.
}
12. (a) Gál, K.; Wéber, C.; Wágner, G. A.; Horváth, A.; Nyéki, G.; Vastag, M.; Keseru,
G. M. WO 2007039781, 2007.; (b) Gál, K.; Wéber, C.; Wágner, G. A.; Bobok, A.
}
Á.; Nyéki, G.; Vastag, M.; Keseru, G. M.; Háda, V.; Kóti, J. WO 2007039782,
}
19. Functional activity at mGluR1 and mGluR2 was measured by Ca2+-fluorometry
according to Kurkó et al. (Kurkó, D.; Bekes, Z.; Gere, A.; Baki, A.; Boros, A.;
Kolok, S.; Bugovics, G.; Nagy, J.; Szombathelyi, Z.; Ignácz-Szendrei, G.
Neurochem. Int. 2009, 55, 467.) with slight modifications. Briefly, cells were
seeded in 96-well plates 24 h before being subjected to Ca2+-measurements.
For the Ca2+-measurements cells were loaded with the Ca2+-sensitive dyes,
fluo-4/AM (mGluR1) or FLIPR Calcium 5 kit (mGluR2). All buffers contained
2007.; (c) Nógrádi, K.; Wágner, G. A.; Keseru, G. M.; Bielik, A.; Gáti, T.; Háda, V.;
Kóti, J.; Gál, K.; Vastag, M.; Bobok, A. Á. WO 2007072090, 2007.; (d) Nógrádi, K.;
}
Keseru, G. M.; Bielik, A.; Gáti, T.; Gál, K.; Vastag, M.; Bobok, A. Á. WO
}
2007072091, 2007.; (e) Keseru, G. M.; Wéber, C.; Bielik, A.; Bobok, A. Á.; Gál, K.;
Meszlényiné, S. M.; Molnár, L.; Vastag, M. WO 2007072093, 2007.; (f) Nógrádi,
}
K.; Wágner, G. A.; Keseru, G. M.; Bielik, A.; Gáti, T.; Háda, V.; Kóti, J.; Gál, K.;
Vastag, M.; Bobok, A. Á. WO 2007072094, 2007.; (g) Nógrádi, K.; Wágner, G. A.;
}
glutamate–pyruvate transaminase (3
lg/ml) and sodium pyruvate (2 mM).
Keseru, G. M.; Bielik, A.; Gáti, T.; Háda, V.; Kóti, J.; Gál, K.; Vastag, M.; Bobok, A.
Baseline and agonist evoked signals were recorded with
a
plate reader
Á. WO 2007072095, 2007.
fluorometer. Agonist was (S)-3,5-dihydroxyphenylglycine (mGluR1) or
glutamate (mGluR2), administered at their respective EC80 concentrations.
13. The mGluR5 receptor binding was determined according to Gasparini et al.
(Gasparini, F.; Andres, H.; Flor, P. J.; Heinrich, M.; Inderbitzin, W.; Lingenhöhl,
K.; Müller, H.; Munk, V. C.; Omilusik, K.; Stierlin, C.; Stoehr, N.; Vranesic, I.;