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

Article abstract of DOI:10.1016/j.bmcl.2007.12.005

A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Replacement of a cyano group by a five-membered heterocycle produced compounds inhibiting the binding of [³H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. Further modification to improve the physicochemical properties led eventually to compounds with the ability to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus.

Full text of DOI:10.1016/j.bmcl.2007.12.005

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1091–1095
Synthesis and characterization of 8-ethynyl-1,3-dihydro-
benzo[b][1,4]diazepin-2-one derivatives: Part 2. New potent
non-competitive metabotropic glutamate receptor 2/3 antagonists
Thomas J. Woltering,a,* Geo Adam,a Jurgen Wichmann,a Erwin Goetschi,a
¨
b
b,ꢁ
James N. C. Kew,b,ꢀ Frederic Knoflach, Vincent Mutel and Silvia Gattib
´ ´
aF. Hoffmann-La Roche Ltd, Pharma Discovery Chemistry, CH-4070 Basel, Switzerland
bF. Hoffmann-La Roche Ltd, Pharma Research CNS Psychiatry, CH-4070 Basel, Switzerland
Received 9 November 2007; revised 3 December 2007; accepted 4 December 2007
Available online 8 December 2007
Abstract—A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists.
Replacement of a cyano group by a five-membered heterocycle produced compounds inhibiting the binding of [3H]-LY354740 to rat
mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. Further modification to
improve the physicochemical properties led eventually to compounds with the ability to reverse LY354740-mediated inhibition
of field excitatory postsynaptic potentials in the rat dentate gyrus.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The glutamate receptors are divided into two major
groups: ionotropic (iGluR: ligand-gated ion channels)
and metabotropic (mGluR: G-protein coupled recep-
tors) glutamate receptors.1 From mGluRs eight sub-
types were identified and grouped into three groups
according to their sequence homology, agonist pharma-
cology, and second messenger coupling. Group I recep-
tors (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 neg-
atively coupled to the activity of adenyl cyclase. Target-
ing mGluRs has been recognized as a valuable approach
for the treatment of CNS disorders, such as depression,
anxiety, and schizophrenia.2 It was shown that mGluR2/
3 agonists exhibit antipsychotic properties and mGluR2/
3 antagonists may be useful as antidepressants and cog-
nitive enhancers as demonstrated in different animal
models.3,4
Recently we reported about the development of the ran-
dom screening hit 1—a compound from the chemical
class of 1,3-dihydro-benzo[b][1,4]diazepin-2-ones—into
compounds with low nanomolar affinity (Fig. 1) assayed
by partial inhibition of [3H]-LY354740 binding to rat
mGluR25 (Fig. 2a).6 The in vitro binding characteristics
of this selective mGluR2/3 radioligand with agonist
properties were described by Schweitzer et al.5a The
specific binding of [3H]-LY354740 was dependent on
the addition of divalent cations, it showed the presence
of a single binding site, and was partially inhibited
by GTPcS. Saturation isothermal curves on [3H]-
LY354740 binding to rat mGluR2 and functional stud-
ies carried out with 1 and derivatives on (1S,3R)-ACPD
Ph
H
N
1
O
3
H
N
O
8
Me
Keywords: Metabotropic glutamate receptors; mGluR2; LY354740;
Non-competitive antagonists; 1,3-Dihydro-benzo[b][1,4]diazepin-2-
ones; Excitatory postsynaptic potentials.
2
7
4
N
N
3'
3'
CN
2
*
1
Corresponding author. Tel.: +41 616880407; fax: +41 616888714;
e-mail: thomas.woltering@roche.com
IC50 = 6.4 μM
IC50 = 34 nM
ꢀ
Present address: GlaxoSmithKline, New Frontiers Science Park
North Harlow, Essex CM19 5AW, UK.
Figure 1. Development of the random screening hit 1 into compounds,
like, for example, 2, with low nanomolar affinity in inhibition of [3H]-
LY354740 binding to rat mGluR2.6
ꢁ
Present address: Addex Pharmaceuticals SA, 12 Chemin des Aulx,
CH-1228 Plan-les-Ouates, Switzerland.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.005

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T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1091–1095
a
b
100
90
80
70
60
50
40
30
20
10
100
80
60
40
20
0
-20
-5
-9
-8
-7
-6
-5
-7
-6
-4
-3
10
10
10
10
10
10
-ACPD] (M)
10
10
[1
10
10
[compound] (M)
S,3
R
Figure 2. (a) 2 (j), 15o (d), and 15q (m) inhibition of specific [3H]-LY354740 binding to the recombinant rat mGluR2 permanently expressed in
CHO cells. Inhibition curves are normalized as % of the maximum specific bound. All compounds partially inhibit the radioligand binding to
mGluR2 with a residual of about 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. (b) (1S,3R)-ACPD-induced inhibition of cAMP production: the non-competitive mGluR2 antagonist 15b reduces
maximal response and causes rightward shift of dose–response curve of (1S,3R)-ACPD [+ (1S,3R)-ACPD (n = 5); (d) 15b 1 nM (n = 2); (m) 15b
3 nM (n = 2); (.) 15b 10 nM (n = 6); (ꢀ) 15b 30 nM (n = 3)] (also cf. Ref. 7).
inhibition of forskolin stimulated cAMP production
(Fig. 2b), on the 35S-GTPcS binding induced by
(1S,3R)-ACPD, and on the glutamate induced GIRK
currents, respectively, showed the presence of a non-
competitive antagonist activity. The conclusion that
these mGluR2 antagonists do not interact with the
orthosteric site of the receptor is also in agreement with
the observation that they do not displace [3H]-
LY341495, a competitive antagonist to mGluR2.7 The
capacity of 1 and derivatives to partially displace [3H]-
LY354740 is most likely due to an effect on coupling effi-
ciency of mGluR2. This property, in the described
experimental conditions, was used in association to
functional studies to optimize the structure–activity rela-
tionship for this class of compounds.
here exemplified by the preparation of compound 15o
(Scheme 1). The o-phenylenediamine part utilized 5-
chloro-4-iodo-2-nitroaniline 38 and the b-ketoester part
used 3-imidazol-1-yl-benzoic acid 109 as starting materi-
als. Nucleophilic displacement of the Cl in 3 with allyl
alcohol gave 4 wherein—after introduction of the Boc-
group—the allyl group was switched to tert-butyl by
Pd-catalyzed deprotection with morpholine and intro-
duction of the t-Bu group by the method of Widmer10
to give intermediate 7. Sonogashira-type coupling with
4-fluorophenylacetylene was followed by reduction of
the nitro group and yielded the mono-Boc protected o-
phenylenediamine building block 9. For the b-ketoester
part the known 3-imidazol-1-yl-benzoic acid 109 was
converted via its methyl ester 11 into the t-Bu b-ketoes-
ter 12 following a protocol of Ohta.11 To increase the
reactivity of the t-Bu b-ketoester 12 for the condensation
with the o-phenylenediamine building block 9, it was
transformed into the 2,2-dimethyl-[1,3]dioxin-4-one 13
by treatment with TFAA/TFA in acetone as described
by Winkler.12 Both pieces 9 and 13 were then condensed
in refluxing toluene to the corresponding b-ketoamide
14o, which was doubly deprotected and concomitantly
cyclized by treatment with TFA yielding the unsymmet-
rically 7,8-substituted 1,3-dihydro-benzo[b][1,4]diaze-
pin-2-one 15o.
The extension of the methyl group in 8-position to a
phenylacetylene residue as well as the attachment of a
cyano group in meta-position on the phenyl in 4-posi-
tion resulted in the very potent compound 2. Neverthe-
less these modifications led to very lipophilic molecules
(2: clogP 4.44; calculated log(coctanol/cwater)) and there-
fore we sought for further alteration of the substituents
enabling the evaluation of compounds from this class in
different models. Our main focus was the search for a
suitable nitrile replacement as well as the exploration
of the 7-position.
We found that five-membered heterocycles were suitable
replacements for the cyano group, especially the
employment of imidazoles (15a–c) and 1,2,3-triazoles
The regioselective synthesis of 1,3-dihydro-benzo[b]-
[1,4]diazepin-2-ones has already been described6 and is
F
I
NO2
NH2
I
NO2
NH2
I
NO2
a
b,c
f
NX2
O
O
R
NHBoc
Cl
F
t-BuO
g
NHBoc
F
3
4
O
O
O
8
9
X = O
X = H
H
N
5
6
7
R = allyl
d
N
N
R = H
k
N
H
e
l
R = But
HO
N
t-BuO
NHBoc
14o
N
O
O
O
N
N
N
N
RO2C
N
tBuO2C
N
N
j
15o
i
O
10 R = H
h
13
12
11 R = Me
Scheme 1. Synthesis of compound 15o. Reagents and conditions: (a) allyl alcohol, KOH, DMSO, rt, 95%; (b) Boc2O, DMAP, THF, rt, 99%; (c)
TFA, DCM, 0 ꢁC, 75%; (d) morpholine, Pd(PPh3)4, THF, rt, 98%; (e) Me2NCH(OBut)2, toluene, 80 ꢁC, 73%; (f) 4-F–C6H4C„CH, PdCl2(PPh3)2,
PPh3, CuI, Et3N, THF, 60 ꢁC, 38%; (g) SnCl2Æ2H2O, pyridine, DMF, rt, 68%; (h) concd H2SO4, MeOH, reflux, 98%; (i) t-BuOAc, LDA, LiOBut,
THF, ꢀ78 ꢁC, 95%; (j) TFAA, TFA, acetone, ꢀ78 to 23 ꢁC, 65%; (k) toluene, reflux, 68%; (l) TFA, DCM, rt, 65%.

T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1091–1095
Table 1. Activities of the 8-ethynyl-1,3-dihydro-benzo[b][1,4]diazepin-2-ones 15
1093
0
Compound
R3
R8
R7
Rat mGluR2 [3H]-LY354740
bindinga IC50 (lM)
Rat mGluR2 (1S,3R)-ACPD
inhibition of forskolin stimulated
cAMPb IC50 (lM)
2
CN
Ph–C„C–
Ph–C„C–
H
H
0.034
0.013
0.017
N
15a
0.010
N
N
N
15b
15c
4-F–C6H4–C„C–
2-F–C6H4–C„C–
H
H
0.009
0.020
0.009
0.016
N
N
N
Me
15d
15e
4-F–C6H4–C„C–
Ph–C„C–
H
H
0.098
0.170
nt
nt
N
Me
N
Me
N
Me
N
15f
15g
15h
15i
Ph–C„C–
H
0.730
0.098
0.006
0.072
0.023
0.064
0.046
0.018
0.140
0.026
0.036
0.020
0.378
nt
N
N
N
N
4-F–C6H4–C„C–
4-F–C6H4–C„C–
4-F–C6H4–C„C–
Ph–C„C–
H
nt
N
N
N
H
0.012
nt
N
N
N
N
H
N
N
N
N
N
N
15j
OCH2CH2OMe
OCH2CH2OH
OCH2CH2OH
OCH2CN
N(Me)CH2CH2OH
OH
0.015
nt
N
N
N
N
N
N
15k
15l
Ph–C„C–
4-F–C6H4–C„C–
Ph–C„C–
nt
15m
15n
15o
15p
15q
0.013
nt
4-F–C6H4–C„C–
4-F–C6H4–C„C–
Ph–C„C–
0.011
nt
N
N
N
NMe2
N
N
N
4-F–C6H4–C„C–
4-F–C6H4–C„C–
OH
0.017
nt
N
N
N
15r
OH
OH
0
R3 , R7, and R8 refer to the positions of Rs in Figure 1.
nt, not tested.
a Values are means of at least two independent experiments.
b Values are means of three independent experiments.

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T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1091–1095
(15h,p,q) led to very potent mGluR2/3 antagonists
(Table 1). Interestingly the N-linked 1-imidazoles (e.g.,
15a or 15e) were far more active than its C-linked 2-iso-
mer (15f). With regard to the methyl substitution on the
1-imidazoles it appeared only to some extent to be toler-
ated resulting a ca. 10-fold drop in affinity (15a vs 15e;
15b vs 15d). The order of activity depending on the
heterocycles incorporated was determined as follows:
1-imidazole ꢁ 1,2,3-triazole > 1-tetrazole ꢁ 1,2,4-triazole.
Overall, the unsubstituted 1-imidazole and 1,2,3-triazole
proved to be most favorable (e.g., 15a, 15b, 15c, and 15h
all exhibit IC50 6 20 nM).
a
100
80
60
40
20
0
-8
-7
-6
10
10
10
[compound] (M)
b
100
80
60
40
20
0
The structural class of the 1,3-dihydro-benzo[b][1,4]dia-
zepin-2-ones offered in addition to the (2-aryl)-ethynyl-
moiety in 8-position and the meta-attached heterocycle
on the phenyl group in 4-position a third exit vector—
the 7-position—where the introduction of further sub-
stituents was allowed as previously described.6 The most
successful ones in altering physicochemical properties to
reduce the overall lipophilicity without loss of mGluR2
affinity were small oxygen- or nitrogen-linked C2 units
bearing polar head groups like OH (15k,l,n), OMe
(15j) or CN (15m). It was found that a 2-hydroxyethyl
residue is more active when O-linked (15l) rather than
N-linked (15n) to the core structure, but in general even
the relatively small 2-hydroxyethyl group with the free
OH (15k) resulted in loss of activity (ca. 3-fold) in com-
parison with the less polar OMe (15j) and CN (15m).
Replacing the 2-hydroxyethyl group in 15n by a simple
methyl group produced the rather non-polar 15p (bear-
ing the NMe2 group) but was accompanied with a regain
in affinity. In analogy cutting off the 2-hydroxyethyl
group in 15l yielded the free phenol 15o—a very potent
imidazole compound. The corresponding 1,2,3-triazole
compound 15q proved to be equipotent.
-8
-7
-6
10
10
[compound] (M)
10
Figure 3. Concentration–response curves for the inhibition of GIRK
currents by 15o (n) and 15q (m) in CHO cells stably expressing GIRKs
and transiently transfected with: (a) rat mGluR2 (glutamate 10 lM)
and (b) rat mGluR3 (glutamate 1 lM).
120
a
100
80
60
5 ms
40
control
LY354740
20
Further increase in polarity by introduction of hydroxy-
methyl group in the 5-position on the 1,2,3-triazole in
15q (clogP 3.80) resulted in 15r (clogP 2.88) and ca.
20-fold loss of affinity.
LY354740 + 15o
0
0
60
120
time (min)
180
240
b
100
80
60
40
20
In conclusion, only limited allowance for the introduc-
tion of more polar groups was found—the 1-imidazole
or 1,2,3-triazole in combination with free phenol in 7-
position appeared to be the maximum in order to
achieve highly potent compounds.
As described earlier 15o and 15q partially inhibit [3H]-
LY354740 binding to mGluR2 (Fig. 2a).13 To demon-
strate non-competitive nature of the antagonism at
mGluR2 by the newly generated compounds, we show
hereby the effect of increasing concentrations of 15b
on the (1S,3R)-ACPD-induced inhibition of forskolin
stimulated cAMP production (Fig. 2b).7,13
0
0.01
0.1
1
10
[LY354740] (μM)
Figure 4. Antagonist activity of compound 15o on the LY354740-
mediated inhibition of synaptic transmission in the rat hippocampus:
(a) Compound 15o reverses the LY354740-mediated inhibition of field
excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of
the medial perforant path and recorded in the dentate gyrus (—
compound 15o (1 lM), ---- LY354740 (1 lM); mean SE, n = 4). The
inset shows representative fEPSPs under control conditions, in the
presence of 1 lM LY354740, and in the presence of 1 lM LY354740
and 1 lM 15o at 180 min. (b) The concentration-dependent inhibition
of fEPSPs by LY354740 (IC50 = 0.23 lM, slope = 1.2) was completely
blocked following 3-h pre-incubation of hippocampal slices with 15o [•
control (n = 7); ꢂ +30 nM 15o (n = 4)].
The antagonist properties of 15o and 15q were also eval-
uated electrophysiologically in CHO cells stably express-
ing the human Kir3.1 and Kir3.2c GIRK subunits and
transiently transfected with either rat mGluR2 or rat
mGluR3 (Fig. 3).13 In these cells, glutamate concentra-
tion-dependently induced a reversible inward K+ cur-
rent, which was inhibited by both 15o and 15q.

T. J. Woltering et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1091–1095
1095
Swanson, C. J.; Bures, M.; Johnson, M. P.; Linden, A.-
M.; Monn, J. A.; Schoepp, D. D. Nat. Rev. Drug Discov.
2005, 4, 131; (c) Higgins, G. A.; Miczek, K. A. Psycho-
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Pharmacol. Exp. Ther. 2001, 299, 12.
We assessed the selectivity of 15o and 15q and these
compounds neither activated nor inhibited glutamate-
stimulated rat mGluR1a and mGluR5a (using a Ca2+
mobilization functional assay, when tested at 30 lM fi-
nal concentration). Compound 15o was also inactive in
the binding of [3H]-L-AP4 to rat mGluR8a, while 15q
showed an IC50 of 5 lM. In addition both compounds
were devoid of any affinity at NMDA and GABAA
receptors (data not shown).
3. Spinelli, S.; Ballard, T.; Gatti-McArthur, S.; Richards, G.
J.; Kapps, M.; Woltering, T.; Wichmann, J.; Stadler, H.;
Feldon, J.; Pryce, C. R. Psychopharmacology 2005, 179, 292.
4. Higgins, G. A.; Ballard, T. M.; Kew, J. N. C.;
Richards, J. G.; Kemp, J. A.; Adam, G.; Woltering,
T.; Nakanishi, S.; Mutel, V. Neuropharmacology 2004,
46, 907.
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K.; Malherbe, P.; Ohresser, S.; Stadler, H.; Wichmann, J.;
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Chaboz, S.; Kemp, J. A.; Klingelschmidt, A.; Messer, J.;
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Using compound 15o we could also demonstrate antag-
onism at native group II metabotropic glutamate recep-
tors by reversal of LY354740-mediated inhibition of
fEPSPs in the dentate gyrus (Fig. 4).14
In summary, by replacement of a nitrile with a five-mem-
bered heterocycle we were able to develop a series of 1,3-
dihydro-benzo[b][1,4]diazepin-2-one derivatives which
are selective and potent non-competitive antagonists at
recombinant group II metabotropic glutamate receptors.
Further optimization of physicochemical properties is re-
quired before approaching in vivo studies.
8. (a) Adam, G.; Alanine, A.; Goetschi, E.; Mutel, V.;
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Chem. Abstr. 2001, 134, 311234; (b) Adam, G.; Goet-
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World Patent WO02/083652, 2002; Chem. Abstr. 2002,
137, 325447.
9. Hagedorn, A. A., III; Erhardt, P. W.; Lumma, W. C., Jr.;
Wohl, R. A.; Cantor, E.; Chou, Y. L.; Ingebretsen, W. R.;
Lampe, J. W.; Pang, D.; Pease, C. A.; Wiggins, J. J. Med.
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Acknowledgments
We cordially thank N. Benedetti, D. Buchy, M. Burn, S.
Chaboz, M. Dellenbach, A. Maier, A. Nilly, H. Scha¨r,
M.-C. Pflimlin, R. Haab, and M. Weber for their skilful
technical assistance.
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E.; Wichmann, J.; Woltering, T.; Kemp, J.; Mutel, V.
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