Synthesis and characterization of 8-ethynyl-1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: New potent non-competitive metabotropic glutamate receptor 2/3 antagonists. Part 1

Article abstract of DOI:10.1016/j.bmcl.2007.10.026

A series of 1,3-dihydrobenzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Attachment of an 8-(2-aryl)-ethynyl-moiety produced compounds inhibiting the binding of [³H]-LY354740 to rat mGluR2 with low n

Full text of DOI:10.1016/j.bmcl.2007.10.026

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6811–6815
Synthesis and characterization of 8-ethynyl-1,3-dihydro-
benzo[b][1,4]diazepin-2-one derivatives: New potent
non-competitive metabotropic glutamate
receptor 2/3 antagonists. Part 1
Thomas J. Woltering,a,* Geo Adam,a Alexander Alanine,a Jurgen Wichmann,a
¨
b
b,ꢀ
Frederic Knoflach, Vincent Mutel and Silvia Gattib
´ ´
aF. Hoffmann-La Roche Ltd, Pharma Discovery Chemistry, CNS CH-4070 Basel, Switzerland
bF. Hoffmann-La Roche Ltd, Pharma Research, CNS CH-4070 Basel, Switzerland
Received 30 August 2007; revised 10 October 2007; accepted 10 October 2007
Available online 17 October 2007
Abstract—A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists.
Attachment of an 8-(2-aryl)-ethynyl-moiety produced compounds inhibiting the binding of [3H]-LY354740 to rat mGluR2 with low
nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3.
Ó 2007 Elsevier Ltd. All rights reserved.
L-Glutamic acid (L-Glu) is an excitatory neurotransmit-
ter in the mammalian central nervous system (CNS) and
activates several classes of glutamate receptors. These
are divided into two major groups termed ionotropic
(iGluR) and metabotropic (mGluR) glutamate recep-
tors.1 The family of mGluRs consists of at least eight
subtypes, grouped according to their sequence homol-
ogy, pharmacology, and intracellular coupling. Group
I receptors (mGluR1 and 5) are positively coupled to
phospholipase C, whereas group II (mGluR2 and 3)
and group III (mGluR4, 6, 7, and 8) receptors are nega-
tively coupled to adenyl cyclase. In recent years the
mGluRs were recognized as valuable therapeutic tar-
gets.2 In particular mGluR2/3 agonists show antipsy-
chotic properties and mGluR2/3 antagonists may be
useful as antidepressants and cognitive enhancers as
demonstrated in different animal models.3,4
Our program focused initially on non-amino acid
mGluR2/3 ligands, including non-competitive ones,
with the aim to increase receptor selectivity and improve
pharmacokinetic properties. Random screening was per-
formed using a forskolin-stimulated cAMP production
in mGluR2-transfected CHO cells. Compounds with
antagonistic properties were further characterized using
the inhibition of (1S,3R)-ACPD (10 lM)-stimulated
[35S]GTPcS binding using membranes from rat
mGluR2-transfected CHO cells.5 This study revealed
two promising structural classes (1 and 2, Fig. 1), which
have been evaluated until moderately potent and selec-
tive compounds were identified in both classes.6,7 Inter-
estingly when compounds of type 2 were tested in
Me
N
O
Me
N
H
N
O
N
O
Me
Me
N
O
S
N
N
Cl
N
O
Me
Cl
Cl
Cl
Keywords: Metabotrophic glutamate receptors; mGluR2; LY354740;
1,3-Dihydro-benzo[b][1,4]diazepin-2-one; mGluR2/3 antagonists;
Non-competitive antagonists.
1
2
3
*
Figure 1. Random screening hits. 1, 1-[2-(2,6-Dichloro-phenyl)-7-
methyl-5H-thiazolo[3,2-a]pyrimidin-6-yl]-ethanone IC50 30 lM; 2, E-
1-[2-(2,4-Dichlorophenyl)-2-2isobutoxyvinyl]-1H-[1,2,4]-triazole IC50
6 lM [35S]GTPcS binding assay.
Corresponding author. Tel.: +41 616880407; fax: +41 616888714;
e-mail: thomas.woltering@roche.com
ꢀ
Present address: Addex Pharmaceuticals SA, 12 Chemin des Aulx,
CH-1228 Plan-les-Ouates, Switzerland.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.026

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T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6811–6815
displacement studies they were able to inhibit concentra-
tion dependently both [3H]-DCG IV and [3H]-LY354740
binding to rat mGluR25,7,8 and appeared to be selective
versus AMPA and NMDA ionotropic receptors. There-
fore, a second random screening was carried out using
the [3H]-LY354740 binding assay which discovered
among other hits the compound 3 (Fig. 1). Herein we re-
port on the characterization of compounds obtained
from the screening hit 3—a 1,3-dihydro-benzo[b][1,4]
diazepin-2-one—with an inhibition of [3H]-LY354740
binding to rat mGlu2 receptors with IC50 values ranging
from 6.4 lM to low nanomolar affinity. Selectivity and
specificity data are also provided.
unsymmetrically 7,8-substituted 1,3-dihydro-benzo[b]
[1,4]diazepin-2-ones 14.10
The concentration-dependent displacement of the [3H]-
LY354740 binding to rat mGluR2 in the experimental
conditions described by Schweizer et al.8 was then used
to determine the structure activity relationship (SAR)
of compounds obtained after the screening hit 3
(>95% purity). Compound 14m and derivatives par-
tially inhibit [3H]-LY354740 binding to mGluR2 with
a residual of 25% specific bound (Fig. 2b). Considering
the non-competitive nature of this inhibition (Fig. 2a)
IC50 values are reported (Table 1) as relative measure
of affinity.8
We were attracted to compounds of type 3 due to the
high levels of chemical tractability through their assem-
bly via condensation reactions of 1,2-phenylenedi-
amines and b-ketoesters (Scheme 1). The central
building block for the variation of the phenylenedi-
amine part was prepared by iodination9 of 5-chloro-
2-nitroaniline 4 furnishing 5-chloro-4-iodo-2-nitroani-
line 5. Mono Boc protection was most conveniently
achieved by a two-step procedure: first bis-protection
with Boc2O/cat. DMAP and second selective removal
of one Boc group by treatment with TFA. The chlorine
was replaced by O- (in compound 5) and N-nucleo-
philes (in compound 6) in an SNAr fashion, whereas
the iodine in compounds of type 8 was amenable for
palladium-catalyzed cross couplings, for example,
Sonogashira-type reactions with terminal alkynes to
give compounds of type 9. Finally very mild reduction
of the nitro group was performed with SnCl2Æ2H2O in
pyridine/DMF at ambient temperature to produce the
mono Boc protected 1,2-phenylenediamine building
blocks 10. The regioselective synthesis was achieved
by simple condensation of mono Boc protected 1,2-
phenylenediamines 10 and b-ketoesters 11 or [1,3]-
dioxinones 12 in refluxing toluene to the corresponding
b-ketoamides 13, which were deprotected and concom-
itantly cyclized by treatment with TFA yielding the
a
b
110
120
100
90
80
70
60
50
40
30
20
10
100
80
60
40
20
0
-20
-7
-6
-5
-4
-9
-8
-7
-6
-5
10
10
10
10
10
10
10
10
10
[compound 14m] (M)
[1S,3R-ACPD] (M)
Figure 2. (a) Effect of the non-competitive antagonist 14m on the
functional responses induced by increasing concentrations of the
mGluR2 agonist (1S,3R)-ACPD in the [35S]GTPc S assay. Increasing
concentrations of 14m reduce the maximal response to the agonist with
no changes in EC50 values. EC50 values: (1S,3R)-ACPD
12.1 lM, + 14m (10 nM) 11.8 lM, + 14m (30 nM) 12.6 lM. [j,
(1S,3R)-ACPD (n = 6); d, 14m 3 nM (n = 4); m, 14m 10 nM (n = 3);
., 14m 30 nM (n = 4); Ç, 14m 100 nM (n = 3)]. (also cf. Ref. 12). (b)
Indirect measurement of the affinity of 14m for the recombinant rat
mGluR2 expressed in CHO cells. Inhibition curve is normalized as %
of the maximum specific bound. Compound 14m partially inhibits the
radioligand binding to mGluR2 with a residual 25% of specific bound
([3H]-LY354740, 10 nM). The binding of this radioligand to mGluR2
is GTPcS sensitive and it is blocked by pertussin toxin.
NO2
NH2
I
NO2
NH2
I
NO2
a
b,c
b,c
Cl
Cl
Cl
NHBoc
4
5
6
d
e
R8
R7
NO2
I
NO2
NH2
I
NO2
f
R7
NHBoc
RO
NHBoc
7 R = 1º alkyl
8 R7 = NR'R", OR
9
R',R" = 1º & 2º alkyl
O
O
H
N
O
R8
RO
Ar
H
N
R8
R7
NH2
NHBoc
R8
R7
Ar
i
11 R = t-Bu, Et
g
R7
O
O
or
N
NHBoc
R4
O
O
h
10
O
Ar
13
14
12
Scheme 1. General synthesis of unsymmetrically 7,8-substituted 1,3-dihydro-benzo[b][1,4]diazepin-2-ones 14. Reagents and conditions: (a) ICl,
NaOAc, AcOH, 23–80 °C, 95%; (b) Boc2O, DMAP, THF, rt, 99%; (c) TFA, DCM, 0 °C, 99%; (d) ROH, KOH, DMSO, rt, 75–95%; (e) R0R00NH,
DMSO, rt, 80–98%; (f) alkyne, PdCl2(PPh3)2, PPh3, CuI, Et3N, THF, 50–65 °C, 50–95%; (g) SnCl2Æ2H2O, pyridine, DMF, 50–90%; (h) toluene,
reflux, 40–95%; (i) TFA [optional anisole], DCM, rt.

T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6811–6815
6813
Table 1. Activities of the 1,3-dihydro-benzo[b][1,4]diazepin-2-ones 14 (R4, R7, and R8 refer to the positions of Rs in Scheme 1)
Compound R4
R8
R7
Rat mGluR2 [3H]-LY354740 bindinga IC50 (lM)
14a
14b
14c
14d
14e
14f
14g
14h
14i
MeO
Cl
Me
Me
H
H
H
H
H
H
H
H
H
H
6.58
2.82
CF3
CN
H
Me
Me
1.53
2.90
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
0.026
0.962
0.080
0.268
0.154
0.358
MeO
Cl
CF3
I
14j
CONH2
O
X = O
X = NMe
14k
Ph-C„C–
H
0.360
N
X
14l
1.04
14m
14n
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
Ph-C„C–
H
0.034
0.076
0.064
0.068
0.034
0.030
0.480
0.032
0.300
0.870
2-Cl–C6H4-C„C–
4-Me-C6H4-C„C–
4-MeO-C6H4-C„C–
4-F-C6H4-C„C–
2-F-C6H4-C„C–
2,4-Di-F-C6H3-C„C–
2-Thiophenyl-C„C–
2-Thiazolyl-C„C–
2-Pyridyl-C„C–
HO(Me)2C-C„C–
H2C@C(Me)-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
H
14o
14p
H
H
14q
14r
H
H
14s
14t
H
H
14u
14v
H
H
14w
14x
14y
H
26
H
NMe2
0.400
0.060
0.378
0.278
0.040
0.446
0.160
0.082
0.092
0.028
0.600
2.4
14z
X = O
X = NMe
X = S
14aa
14ab
14ac
14ad
14ae
14af
14ag
14ah
14ai
14aj
14ak
N
X = SO
X = SO2
X = CHOMe
OMe
X
OCH2CH2OMe
OCH2CO2H
OCH2CONH2
OCH2CONHBut
OCH2CN
Ph-C„C–
Ph-C„C–
Ph-C„C–
Ph-C„C–
0.026
0.018
a Values are means of three experiments.
In the R4 position a clear preference for meta-substitu-
tion at the phenyl ring was quickly established (14a–d).
With the introduction of a phenylacetylene moiety in
the 8-position we found an enhanced affinity compared
to 3 (14e) and more thoroughly investigated the struc-
ture activity relationship (SAR) at the phenyl in 4-posi-
tion. The functional group tolerance proved to be wide,
ranging from simple halides (14g and 14i), methoxy
(14f), trifluoromethyl (14h) to carboxamides (14j–l),
but among the substituted compounds the cyano group
showed the highest affinity (14m).
introduction of a nitrogen containing heterocycle, for
example, 2-thiazolyl (14u) or 2-pyridyl (14v), resulted
in an approximately 10- and 30-fold drop in affinity.
Using a smaller non-aromatic substituent with a tertiary
alcohol as in 14w produced an almost inactive com-
pound, but elimination of water from 14w generated
the olefin 14x and a regain in potency was observed.
As a conclusion, the acetylenic groups in 8-position were
preferably chosen from lipophilic aromatic residues like
phenyl-, 2- or 4-F-phenylacetylene (14m, 14q and 14r).
To complete the overall SAR, we decorated 14m with
a MeO group in 6- or with a Cl in 9-position, which
led to completely inactive compounds (data not shown).
Keeping this nitrile constant we explored the variation
of the alkyne in the 8-position. Substituted phenyl resi-
dues produced very active molecules, for example, a 2-
chloro- (14n), a 4-methyl- (14o) or a 4-methoxy-phenyl
(14p) were almost equal in affinity. Fluoro substitution
in 2- and 4-position of the phenyl group led to com-
pounds with equal properties (14q and 14r) like 14m,
as well as the 2-thiophenyl-group (14t). 2,4-Di-fluoro
substitution however led to the less favored 14s. The
The addition of more polar substituents in 7-position
was made to reduce the high lipophilicity of the com-
pounds (14m clogP 4.44; calculated log(coctanol/cwater)).
As described in Scheme 1, N-nucleophiles were intro-
duced in the 7-position by SNAr reaction. The simple
lipophilic dimethylamino group (14y clogP 4.62) was
well tolerated, but the larger and more polar morpho-

6814
T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6811–6815
lino group (14z clogP 4.23) and the more basic N-meth-
ylpiperazinyl group (14aa clogP 4.20) resulted in loss of
affinity. For the substituents in the 7-position not the
size but more likely the polarity seemed to be the domi-
nant factor for affinity since the even larger but most
lipophilic 14ae (clogP 5.14) proved to be a very potent
compound.
a
b
100
80
60
40
20
0
100
80
60
40
20
0
-8
-7
-6
-8
-7
-6
10
10
10
10
10
10
The restricted scope in the 7-position was also nicely
demonstrated by the examination of the thiomorpholino
compound and its oxide derivatives (14ab–ad). The most
lipophilic 14ab (clogP 5.08) was most active, the most
polar sulfoxide 14ac (clogP 2.94) the least active,
whereas the sulfone 14ad with intermediate polarity
(clogP 3.06) again showed intermediate properties.
[compound 14m] (M)
[compound 14m] (M)
c
100
80
60
40
20
As for the oxygen-linked moieties the small simple meth-
oxy group (14af, clogP 4.52) and the larger 2-
(methoxy)ethoxy group (14ag, clogP 4.25) are both well
tolerated. The attachment of a carboxylic acid group via
an oxygen linker as in 14ah (clogP 3.78) produced a
moderately active compound.
0
-6
-5
-4
10
10
10
[glutamate] (M)
Figure 3. Concentration–response curves for the inhibition of GIRK
currents by 14m in CHO cells stably expressing GIRKs and transiently
transfected with: (a) rat mGluR2 (Glutamate 10 lM) and (b) rat
mGluR3 (glutamate 1 lM). (c) Glutamate concentration–response
curves in CHO cells expressing GIRKs and rat mGluR2 generated in
the absence (d, control) and presence of 14m (j, 100 nM) showing the
non-competitive nature of block of the compound. This effect was
reversible.
A correlation between polarity of groups in the 7-posi-
tion with the in vitro activity was obtained in the corre-
sponding amide series. The primary amide (14ai clogP
3.01) is about 100-fold less active than the less polar
N-tert-butylamide (14aj, clogP 4.84) and the corre-
sponding nitrile (14ak, clogP 3.54), therefore exhibiting
the same trend of activities as observed for the thiomor-
pholino compound and its oxides (14ab–ad).
Acknowledgments
We cordially thank J. Beck, N. Benedetti, D. Buchy, M.
Burn, S. Chaboz, M. Dellenbach, A. Maier, A. Nilly, H.
Scha¨r, and W. Vifian for their skillful technical
assistance.
We further assessed the selectivity of 14m (among other
compounds) and this compound neither activated nor
inhibited glutamate-stimulated rat mGlu1a and mGlu5a
receptors (using a Ca2+ mobilization functional assay,
when tested at 30 lM final concentration) nor the bind-
ing of [3H]-L-AP4 to rat mGlu8a receptor. Compound
14m is also devoid of any affinity at the NMDA and
GABAA receptors (data not shown).
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The non-competitive nature of 14m was exemplified by
measuring the effect on the binding of [35S]GTPcS in-
duced by (1S,3R)-ACPD (Fig. 2a),11,12 which partially
inhibits [3H]-LY354740 binding to mGluR2 with a resid-
ual of 25% specific bound (Fig. 2b). The antagonist prop-
erties of 14m were also evaluated electrophysiologically in
CHO cells stably expressing the human Kir3.1 and
Kir3.2c GIRK subunits and transiently transfected with
either rat mGlu2 or rat mGlu3 receptors (Fig. 3). In these
cells, glutamate induced an inward K+ current that was
reversible and dependent on the glutamate concentration,
which was concentration-dependently inhibited by 14m.
In summary, we have explored the SAR of a novel series
of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives
which are selective, non-competitive antagonists at
mGluR2/3. Attachment of an 8-(2-aryl)-ethynyl moiety
produced compounds inhibiting the binding of [3H]-
LY354740 to rat mGluR2 with low nanomolar affinity.
These compounds represent a very promising structural
class of non-amino acid antagonists with very high
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