Synthesis, structure and affinity of novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones for CNS central and peripheral benzodiazepine receptors

Article abstract of DOI:10.1016/j.ejmech.2009.12.027

A series of novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones (7-15) was synthesized and their in vitro affinity for both the central benzodiazepine receptor (CBR) and the peripheral benzodiazepine receptor (PBR) of rat brain was studied. Racemic mixture of 7-bromo-3-(2-methoxy)ethoxy-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one (13) was separated into enantiomers 14, 15 by chiral HPLC. Absolute configuration of R-enantiomer 15 was determined by the method of X-ray diffraction analysis. The affinity of S-enantiomer 14 for CBR (I{cyrillic, ukrainian}C₅₀ = 245 nM) is 20-fold higher than the affinity of R-enantiomer 15 (I{cyrillic, ukrainian}C₅₀ = 4930 nM). A high selectivity for CBR versus PBR (I{cyrillic, ukrainian}C₅₀ (PBR) > 10000 nM) was shown by all reported compounds. Compound 12 was revealed as a potent (IC₅₀ = 9 nM) and selective CBR ligand among the synthesized 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones.

Full text of DOI:10.1016/j.ejmech.2009.12.027

European Journal of Medicinal Chemistry 45 (2010) 1346–1351
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structure and affinity of novel
3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones for CNS central and
peripheral benzodiazepine receptors
,
,
,
1
,
b
Sergey Andronati a 1, Ekaterina Semenishyna a 1, Victor Pavlovsky a , Yuriy Simonov ,
*
Svetlana Makan a 1, Irina Boyko a 1, Natalya Burenkova a 1, Maria Gdaniec c, Pascal Cardinael d,
,
,
,
Jean-Philippe Bouillon d, Alexander Mazepa a
,
1
a A.V. Bogatsky Physico-Chemical, Institute of the National Academy of Sciences of Ukraine, 86 Lustdorfskaya doroga, 65080 Odessa, Ukraine
b Institute of Applied Physics of Academy of Sciences of Moldova, MD-2028, 5 Academicheskaya str., Chisinau, Republic of Moldova
c Chemical Department of A. Mitzkevitch University, 60-780 Poznan, Poland
d Sciences et Me´thodes Se´paratives EA 3233, Universite´ de Rouen, IRCOF, F-76821 Mont-Saint-Aignan Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones (7-15) was synthesized and their in
vitro affinity for both the central benzodiazepine receptor (CBR) and the peripheral benzodiazepine
receptor (PBR) of rat brain was studied. Racemic mixture of 7-bromo-3-(2-methoxy)ethoxy-5-phenyl-
1,2-dihydro-3H-1,4-benzodiazepin-2-one (13) was separated into enantiomers 14, 15 by chiral HPLC.
Absolute configuration of R-enantiomer 15 was determined by the method of X-ray diffraction analysis.
The affinity of S-enantiomer 14 for CBR (_C50 ¼ 245 nM) is 20-fold higher than the affinity of R-enan-
tiomer 15 (_C50 ¼ 4930 nM). A high selectivity for CBR versus PBR (_C50 (PBR) > 10000 nM) was shown by
all reported compounds. Compound 12 was revealed as a potent (IC50 ¼ 9 nM) and selective CBR ligand
among the synthesized 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones.
Received 24 December 2008
Received in revised form
17 August 2009
Accepted 11 December 2009
Available online 22 December 2009
Keywords:
3-alkoxy-1,2-dihydro-3H-1,4-bezodiazepin-
2-ones
Ó 2009 Elsevier Masson SAS. All rights reserved.
Enantiomer
Central benzodiazepine receptor
Peripheral benzodiazepine receptor
Affinity
X-ray analysis
1. Introduction
and neuronal nicotinic acetylcholine receptors [4], glycine recep-
tors [5], the zinc-activated ion channel [6], as well as the
Central benzodiazepine receptors (CBR) [1] and peripheral
benzodiazepine receptors (PBR) [2] which differ by their distribu-
tion, molecular organization and functions are the main biological
targets of benzodiazepines (BD) in the brain.
5-hydroxytryptamine type 3 receptor [7]. These cl-LGIC are
pentamers, either composed of five identical subunits (homomers)
or different subunits (heteromers) [8,9].
GABAA receptor subunits represent eight classes with multiple
CBR are presented in the central nervous system (CNS). CBR are
part of the heteropentameric GABAA-receptor-associated chloride
ion channel, located on postsynaptic membranes of neurons [1].
Ionotropic GABAA receptors are the members of the Cys-loop family
of ligand-gated ion channel (cl-LGIC) [3], which includes muscle
isoforms: a1–6
receptors include two
Receptors with different subtype composition are associated with
different physiological effects [11,12]. BD requires abg2-type
receptors (a1b2g2
azepine-binding site located between
Noteworthy, a4bg2- and a6bg2-type receptors are insensitive to
most BD, as well as any receptors containing the subunit [14].
,
b1–4
,
g1–4
d
,
3
,
p
,
q
,
r1–3 [10]. The most of GABAA
subunits and one subunit.
a
subunits, two
b
g
,
a2b2g2
,
a3b3g2, and a5b2g2) with the benzodi-
a
and g2 subunits [13].
d
Abbreviations: CBR, central benzodiazepine receptor; PBR, peripheral benzodi-
azepine receptor; BD, benzodiazepines; GABA,
loop family of ligand-gated ion channel.
g-aminobutiryc acid; cl-LGIC, Cys-
PBR are an individual receptor class due to their unique struc-
ture, cell localization and physiological functions mediated by them
[2,15]. PBR were initially discovered in kidneys [16], later – in
different organs and tissues, including the CNS [2]. PBR are
5-transmembrane 169-amino acid polypeptides. These receptors
* Corresponding author. Tel.: þ380 48 765 92 30; fax: þ380 765 96 02.
E-mail addresses: victor_pavlovsky@mail.ru (V. Pavlovsky), jean-philippe.
bouillon@univ-rouen.fr (J.-P. Bouillon).
1
E-mail: medchem_department@ukr.net.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.027

S. Andronati et al. / European Journal of Medicinal Chemistry 45 (2010) 1346–1351
1347
are localized mainly on the mitochondrial outer membrane of
peripheral tissues and CNS glial cells [2,15]. PBR are associated with
numerous biological functions including regulation of cellular
proliferation, immunomodulation, anion transport, porphyrin
transport, heme biosynthesis, apoptosis, regulation of cholesterol
transport, steroidogenesis [2,15,17]. In addition, PBR ligands might
exert antianxiety effects via activation of neurosteroid synthesis
[18]. The ligands binding to PBR induces cholesterol transport into
mitochondria and steroid production by glial cells, resulting to the
synthesis of pregnane neurosteroids such as allopregnanolone and
pregnanolone, which positively modulate the GABAA receptor
functions and provoke anxiolytic effects [18].
The search for novel selective CBR ligands as selective anxio-
lytics is still interesting for practical medicine and theoretical
investigation of molecular mode of action of 1,2-dihydro-3H-1,4-
benzodiazepin-2-ones.
3-Hydroxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones as CBR
ligands (7-chloro-3-hydroxy-5-phenyl-1,2-dihydro-3H-1,4-benzo-
of rat brain was evaluated. Racemic mixture of 7-bromo-3-(2-
methoxy)ethoxy-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one
(13) was separated into enantiomers 14, 15 by chiral HPLC to study
influence of stereocentre configuration on affinity for CBR. Absolute
configuration of R-enantiomer 15 was determined by the method of
X-ray diffraction analysis. S-Enantiomer 14 is 20-fold higher affinity
ligand for CBR than R-enantiomer 15.
2. Chemistry
3-Alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones 7-13 were
synthesized by the substitution reaction of 3-chloro-1,2-dihydro-
3H-1,4-benzodiazepin-2-ones 4-6 with corresponding alcohols to
study the structure - affinity for benzodiazepine receptors rela-
tionship. 3-Chloro derivatives 4-6 were obtained through the
treatment of 3-hydroxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones
1-3 with thionylchloride (Scheme 1).
The structures of 3-alkoxy derivatives 7-13 were identified by
the methods of IR spectroscopy, mass spectrometry and NMR
spectroscopy.
diazepin-2-one
hydroxy-1,2-dihydro-3H-1,4-benzodiazepin-2-one
(oxazepam),
7-chloro-5-(2’-chloro)phenyl-3-
(lorazepam),
7-chloro-5-(2’-chloro)phenyl-3-hydroxy-1-methyl-1,2-dihydro-3H-l,4-
benzodiazepin-2-one (lormetazepam), 7-chloro-3-hydroxy-l-
methyl-5-phenyl-1,2-dihydro-3H-l,4-benzodiazepin-2-one (tema-
zepam)) are applied for treatment of neurological disorders such as
convulsions, anxiety, sleep disturbance [19–24]. Novel selective
CBR ligand 7-bromo-5-(2’-chloro)phenyl-3-hemisuccinylhydroxy-
1,2-dihydro-3H-1,4-benzodiazepin-2-one (cinazepam), (Ki 72 nM)
[25] is hypnotic and anxiolytic agent with decreased side effects
[26,27].
The modulation of the GABAA-receptor-associated channel by 3-
subsituted benzodiazepines depends on both the substituent nature
and stereocentre configuration. As it has been demonstrated S-
enantiomers of 3-hydroxy-1,2-dihydro-3H-1,4-benzodiazepine deri-
vatives has higher affinity than R-enantiomers [28]. S-Enantiomer
of 7-chloro-3-O-(N,N-dimethylcarbamyl)-1-methyl-5-phenyl-1,2-
dihydro-3H-1,4-benzodiazepin-2-one (camazepam) exhibits 14-fold
higheraffinityforbenzodiazepinereceptorsthantheR-enantiomerin
rat brain [29]. The ratio of IC50(R-enantiomer)/IC50(S-enantiomer) for
Compounds 7-13 were obtained as racemic mixtures. Never-
theless, enantiomers of 7-bromo-3-(2-methoxy)ethoxy-5-phenyl-
1,2-dihydro-3H-1,4-benzodiazepin-2-one (13) were separated by
HPLC on chiral column. Enantiomeric separation of benzodiaze-
pines, such as oxazepam and temazepam, have been already ach-
ieved with chirobiotic stationary phases [31,32]. Therefore,
enantioseparation of compounds 14 and 15 was performed with
chirobiotic T column using ethanol as mobile phase. The resolution
value obtained (Rs > 4) was sufficient to realize the enantiomeric
semi-preparative separation of 13. Each enantiomer (14 or 15) was
obtained with 99% enantiomeric excess (Scheme 2).
Enantiomers rotate plate of the polarized light and have
respective rotation angles: S-enantiomer 14 ½a D25
þ 147:2 (c 0.83,
ꢀ
CH3OH); R-enantiomer 15 ½a D25
ꢁ 148:1 (c 1, CH3OH).
ꢀ
3. Results and discussion
3.1. Crystal and molecular structure
acetate, hemisuccinate, and a-methyl-b-phenyl-propionate esters of
oxazepam is 1.7, 3.8 and 81.2, respectively [30].
Absolute configuration of R-enantiomer 15 was determined by
the method of X-ray diffraction analysis (Fig. 1).
The main distances between atoms, valence angles and crystal
data in 7-member heterocycle 15 are given in the Tables 1 and 2.
Nine novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones
were synthesized. Structure-activity relationship of the 3-alkoxy
derivatives was studied and their in vitro affinity for CBR and PBR
H
H
N
H
O
O
O
HO R3
N
N
SOCl2
OR3
Cl
OH
R1
R1
R1
N
R2
N
R2
N
R2
1-3
4-6
7-13
1, 4: R1 = Br, R2 = H
7: R1 = Br, R2 = H, R3 = C H
;
;
2
5
2, 5: R1 = Br, R2 = Cl
8: R1 = Br, R2 = Cl, R3 = C H
;
;
5
2
3, 6: R1 = CH3, R2 = Cl
9: R1 = CH3, R2 = , R3 = C H
;
;
5
2
10: R1 = Br, R2 = , R3 = (CH ) OH
;
2 2
11: R1 = Br, R2 = Cl, R3 = (CH ) OH
;
2 2
12: R1 = Br, R2 = Cl, R3 = (CH ) OCH
;
3
2 2
13: R1 = Br, R2 = , R3 = (CH ) OCH
;
3
2 2
Scheme 1.

1348
S. Andronati et al. / European Journal of Medicinal Chemistry 45 (2010) 1346–1351
H
H
N
O
O
H
N
O
N
O
O
+
O
N
Br
N
Br
N
Br
O CH3
15 (-) R
O CH3
C6H5
O CH3
C6H5
C6H5
13
14 (+) S
Scheme 2.
The skeleton of molecule 15 is similar to the 7-bromo-5-(2’-chloro)-
phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one (phenazepam) one
[33], its hydroxy derivative [34], lorazepam [35], 7-nitro-5-(2’-chlor-
o)phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one (clonazepam)
[36], 7-chloro-5-(2’-chloro)phenyl-1,2-dihydro-3H-1,4-benzodia-
zepin-2-one [37] and 7-bromo-1,2-dihydro-5-(2-pyridyl)-3H-1,4-
benzodiazepin-2-one (bromozepam) [38].
Comparison of the compounds pairs containing 5-phenyl and 5-
(2’-chloro)phenyl substituents allows to note the increase of
affinity for the CBR with of the chlorine atom introduction into
ortho-position of 5-phenyl substituent. Affinity increased in 56-fold
in the case of compounds 12 and 13, and in 11-fold in the case
compounds of 10 and 11, that prove previous conclusion [39].
Derivative 12 is the most potent CBR ligand (_C50 ¼ 9 ꢃ 1 nM)
Double bond between atoms N(4)–C(5) is of 1.293 Å, and it is
similar to above listed compounds. In 7-membered heterocycle
among the investigated compounds and it is
compound for pharmacological research.
a promising
delocalization of
p
-electronic density covers fragment O(2) ¼ C(2)–
Racemic compound 13 has a low affinity for the CBR
N(1)–C(10)–C(11)–C(5)–N(4). Bonds C(2)–C(3) ¼ 1.530 Å and C(3)–
(_C50 ¼ 510 nM). S-Enantiomer 14 has 20-fold higher affinity for
CBR (_C50 245 nM) as compared to R-enantiomer 15 (_C50 4930 nM)
(Fig. 3).
N(4) ¼ 1.446 Å are not involved in delocalization. 7-Membered cycle
has
a boat conformation with two-sided angles between
base N(1)C(2)N(4)C(5) and fragments C(2)C(3)N(4) and
N(1)C(10)C(11)C(5), that are of 117.2ꢂ and 33.7ꢂ, respectively. Distance
C(3)–O(3) is equal to 1.403 Å, and this bond has equatorial location
relatively to the cycle. Angle between aromatic fragment and phenyl
substituent in the 5th position is of 58.6ꢂ, that is close to the found
value for phenazepam (75.4ꢂ) and for other similar structures [33].
Molecules of R-enantiomer 15 are packed into a chain due to the
intermolecular hydrogen bonds between groups N(1)–
H$$$O(2) ¼ 2.958(2) Å (N–H ¼ 0.82(2) Å, O$$$H ¼ 2.21(2) Å, the
angel near H is of 153(2)ꢂ). Noteworthy, there is no dimer associates
formation, that is not typical for 1,4-benzodiazepin-2-ones deriv-
atives unsubstituted at the first position (Fig. 2).
4. Conclusion
Binding to CBR and PBR of synthesized 3-alkoxy derivatives was
studied and it was found that all of these compounds are selective
CBR ligands. R- and S-enantiomers of 3-(2-methoxy)ethoxy-5-
phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one (14, 15) were
separated by chiral HPLC. Absolute configuration of R-enantiomer
15 was determined by the method of X-ray diffraction analysis. S-
Enantiomer 14 is a most potent ligand of the CBR (_C50 ¼ 245 nM) as
compared to R-enantiomer 15 (_C50 ¼ 4930 nM). This fact proves
that binding to the CBR is stereospecific process. It was found, that
3-alkoxy-5-(2’-chloro)phenyl derivatives demonstrate higher
affinity for CBR than 3-alkoxy-5-phenyl-1,4-benzodiazepin-2-ones.
The compound 12 demonstrates the highest affinity for CBR.
3.2. Affinity for the central and peripheral benzodiazepine receptors
All synthesized compounds were studied for their in vitro
binding affinity for CBR and PBR by means of a binding assay using
[3H]flumazenil (Ro15-1788) and [3H]PK11195 as radioligands,
respectively. Binding data are expressed as IC50 (nM) (Table 3).
All studied compounds demonstrate affinity for the CBR of the
rat brain within wide range of values (_C50 from 9 nM up to
5000 nM) (Table 3).
5. Experimental protocols
5.1. Chemistry
1H-NMR spectra were recorded on a Bruker spectrometer at
300 MHz frequency, in CDCl3 and DMSO-d6, internal standard TMS,
at 25 ꢂC. The IR spectra (in KBr pellets) were recorded on a Specord
75-IR. Mass spectra were obtained using the electron ionization
method on mass-spectrometer VG 7070EQ (ionizing voltage is
70 eV, temperature of the ionizing chamber is 200 ꢂC). Values of
Studied compounds 7-15 didn’t demonstrate affinity for the PBR
of the rat brain. All compounds did not display significant inhibitory
effects (IC50 > 1 m
M) on the [3H]PK11195 binding (Table 3).
Table 1
Bond lengths (Å) between atoms in 7-member heterocycle of compound 15.
Bond
Length (Å)
O(33)–C(34)
O(33)–C(32)
C(2)–O(2)
C(2)–N(1)
C(2)–C(3)
1.406(3)
1.425(3)
1.224(2)
1.362(3)
1.530(3)
1.293(3)
1.478(3)
1.413(2)
0.82(2)
1.403(21)
1.434(2)
1.395(3)
1.446(3)
1.496(3)
C(5)–N(4)
C(5)–C(11)
N(1)–C(10)
N(1)–H(1N1)
O(3)–C(3)
O(3)–C(31)
C(34)–H(12A)
C(31)–C(32)
C(31)–H(10A)
Fig. 1. Molecular structure of R-7-bromo-3-(2-methoxy)ethoxy-5-phenyl-1,2-dihydro-
3H-1,4-benzodiazepin-2-one (15).

S. Andronati et al. / European Journal of Medicinal Chemistry 45 (2010) 1346–1351
1349
Table 2
Valence angles (
Table 3
u
ꢂ) in 7-member heterocycle of compound 15.
In vitro binding affinity (_C50, nM) for CBR and PBR of compounds 7-15.
Valence angles
uꢂ
H
N
O
C(34)–O(33)–C(32)
O(2)–C(2)–N(1)
O(2)–C(2)–C(3)
111.36(16)
122.17(19)
123.6(2)
114.25(16)
124.42(17)
117.3(2)
118.32(19)
126.09(16)
114.4(16)
119.0(16)
112.31(14)
122.19(18)
116.95(18)
108.54(17)
122.31(18)
106.74(15)
111.00(16)
107.37(14)
109.79(17)
OR3
R1
N
R2
N(1)–C(2)–C(3)
N(4)–C(5)–C(11)
N(4)–C(5)–C(51)
C(11)–C(5)–C(51)
C(2)–N(1)–C(10)
C(2)–N(1)–H(1N1)
C(10)–N(1)–H(1N1)
C(3)–O(3)–C(31)
C(11)–C(10)–N(1)
C(5)–N(4)–C(3)
O(3)–C(31)–C(32)
C(10)–C(11)–C(5)
O(3)–C(3)–N(4)
O(3)–C(3)–C(2)
No
R1
R2
R3
_C50, nM
CBRa
PBRb
7 (R,S)
8 (R,S)
9 (R,S)
10 (R,S)
11 (R,S)
12 (R,S)
13 (R,S)
14 (S)
Br
Br
CH3
Br
Br
Br
Br
Br
Br
H
Cl
H
H
Cl
Cl
H
H
H
C2H5
C2H5
C2H5
(CH2)2OH
(CH2)2OH
(CH2)2OCH3
(CH2)2OCH3
(CH2)2OCH3
(CH2)2OCH3
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
52 ꢃ 3
>1000
540 ꢃ 15
48 ꢃ 2
9 ꢃ 1
N(4)–C(3)–C(2)
O(33)–C(32)–C(31)
510 ꢃ 40
245 ꢃ 21
4 930 ꢃ 58
15 (R)
exact molecular ions masses were measured on mass-spectrometer
VG 7070EQ with resolution 10 000 using the peak matching
method. Thin layer chromatography was performed on plates
Silufol UV-254, in acetonitrile–chloroform–hexane (1:1:3) system
and in benzene–acetonitrile–hexane–methanol (25:15:5:1) system,
a
The concentration of tested compounds that inhibited [3H]flumazenil specific
binding to rat brain synaptosomal membranes (IC50) by 50% was determined with
eight concentrations of the displacers, each performed in triplicate. IC50 values are
the mean ꢃ SEM of three determinations.
b
[3H]PK11195 was incubated in the presence 1
mM of the compounds examined.
using UV-light at
l
¼ 254 nm. Semi-preparative purification and
analytical separation were performed on a Varian 9010 HPLC pump
coupled to a Varian 9050 UV detector. Analytical Chiral HPLC
separation: stationary phase: CHIROBIOTIC T (150 mm ꢄ 4.6 mm;
5.3. General procedure for the synthesis of the 3-alkoxy-1,2-
dihydro-3H-1,4-benzodiazepin-2-ones (7-13)
5 mm) purchased from Interchim (Monluçon, France), mobile phase:
To the suspension of 3-chloro derivatives 4-6 (1.4 mmol) in
anhydrous chloroform (10 ml) appropriate alcohol (7.5 mmol) was
added. The result mixture was refluxed and left for 24 h. Reaction
mixture was washed with water until pH 7. Solvent was evaporated
under reduced pressure. The resulting oil was crystallized from
acetonitrile or ethanol.
100% EtOH at 1 mL/min, UV detector:
l
¼ 254 nm, compound 14
tr ¼ 2.56 min, compound 15 tr ¼ 5.14 min. Semi-preparative puri-
fication: stationary phase: CHIROBIOTIC T (250 mm ꢄ 10 mm;
5 mm) purchased from Interchim (Monluçon, France), mobile phase:
100% EtOH at 4 mL/min, UV detector:
fractions were evaporated to dryness under vacuum.
l
¼ 254 nm. The collected
5.3.1. Compound 7
5.2. General procedure for the synthesis of the 3-chloro derivative
(4-6)
Yield: 52 %; m.p. ¼ 218–222 ꢂC; EI–MS m/z 358 [Mþ]; HRMS
calcd for C17H15BrN2O2 [M]þ m/z, 358,031; found, 358,033; 1H NMR
(CDCl3, 300 MHz) d: 10.04 (s, 1H), 7.60–7.18 (m, 8H), 4.77 (s, 1H),
A well pounded 3-hydroxy-1,4-benzodiazepine 1-3 (1.5 mmol)
and thionylchloride (10 ml) were added in round-bottomed flask.
Reaction mixture was reflux for 15 min after effervescence finished
and left for 12 h at room temperature. Thionylchloride was evapo-
rated under reduced pressure. Anhydrous chloroform (10 ml) was
added toresidue and evaporated. This operationwas twice repeated.
Obtained residue was used in next stage without purification.
4.08–3.98 (m, 1H), 3.76–3.66 (m, 1H), 1.39 (t, 3H); IR (KBr):
n
(cmꢁ1): 3173, 3106, 2929, 1702, 1604.
5.3.2. Compound 8
Yield: 46 %; m.p. ¼ 205–208 ꢂC; EI–MS m/z 391 [Mþ]; HRMS
calcd for C17H14BrClN2O2 [M]þ m/z, 391,992; found, 391,994;1H
Fig. 2. Crystal structure of the R-7-bromo-3-(2-methoxy)ethoxy-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepine-2-one (15).

1350
S. Andronati et al. / European Journal of Medicinal Chemistry 45 (2010) 1346–1351
The structure was solved by direct methods (3007 reflections
with F(hkl) ꢅ 2 (F), R(int) ¼ 0.0169, final R1 ¼0.0202,
1/IC50, nM
s
0.004
0.0035
0.003
0.0025
0.002
0.0015
0.001
0.0005
0
wR2 ¼ 0.0447) and was refined by full-matrix least-squares tech-
niques based on F2. The non-H atoms were refined with anisotropic
displacement parameters. In all cases, positions of H-atoms on
carbon atoms were calculated and were allowed to ride. Other
hydrogen atoms were found from difference Fourier maps and
refined isotropically. Calculation was performed using SHELX-97
[40] crystallographic software package. Molecules of the 15 have R-
configuration with Flack parameter – 0.0033 (56). CCDC reference
number is 705472.
5.5. Biological methods
5.5.1. Materials
Adult male Wistar rats with a body weight of 180–220 g were
maintained under an artificial 12-h-light/dark cycle (light on
08.00–20.00 h). Food and water were freely available until the time
of the experiment. Animal care and handling throughout the
experimental procedures were in accordance with the European
Communities Council Directive of 24 November 1986 (86/609/EEC).
The experimental protocol were approved by the Animal Ethical
Committee of the University of Cagliari.
13
15
14
Fig. 3. The affinity for CBR of CNS for racemate 13, R-enantiomer 15 and
S-enantiomer 14.
NMR (CDCl3, 300 MHz) d: 9.78 (s, 1H), 7.59–7.15 (m, 7H), 4.82 (s,
1H), 4.11–4.00 (m, 1H), 3.75–3.65 (m, 1H),1.38 (t, 3H); IR (KBr):
n
(cmꢁ1): 3216, 3155, 2924, 2862, 1718, 1691, 1605.
5.5.2. In vitro receptor binding assays
5.5.2.1. [3H]Flumazenil binding. Affinity of compounds 7-15 for CBR
of rat brain was determined by modified method [41] and values of
_C50 were evaluated.
5.3.3. Compound 9
Yield: 57 %; m.p. ¼ 223–225 ꢂC; EI–MS m/z 294 [Mþ]; HRMS
calcd for C18H18N2O2 [M]þ m/z, 294,136; found, 294,139; 1H NMR
(CDCl3, 300 MHz) d: 9.65 (s, 1H), 7.49–7.05 (m, 8H), 4.76 (s, 1H),
4.11–3.65 (m, 2H), 2.55 (s, 3H), 1.38 (t, 3H); IR (KBr):
3150, 1670, 1605.
Animals were anesthetized and decapitated, the cerebral cortex
was quickly extracted and homogenized in 30 ml of 0.05 M ice-cold
citrate buffer (pH 7.1 at 4 ꢂC) with a Dounce homogenizer. The
homogenate was centrifuged at 20 000 g for 15 min at 4 ꢂC. The
pellet was resuspended in initial volume of the same buffer and
centrifuged again under the same conditions. The process of
homogenization and centrifugation was repeated for 4 times.
Supernatant was decanted, residue was resuspended in 0.05 M of
ice-cold citrate buffer (pH 7.1 at 4 ꢂC) to obtain the suspension with
wet membranes concentration of 50 mg/ml.
n
(cmꢁ1): 3266,
5.3.4. Compound 10
Yield: 81 %; m.p. ¼ 224–229 ꢂC; EI–MS m/z 374 [Mþ]; HRMS
calcd for C17H15BrN2O3 [M]þ m/z, 374,026; found, 374,028;1H NMR
(DMSO-d6, 300 MHz)
1H), 4.65 (br. s, 1H), 3.83–3.77 (m, 1H), 3.7–3.67 (m, 1H), 3.64–3.61
(m, 2H); IR (KBr):
(cmꢁ1): 3411, 3191, 3030, 2911, 2851, 1701, 1605.
d: 10.82 (s, 1H), 7.80–7.21 (m, 8H), 4.81 (s,
n
Binding of [3H]flumazenil (Ro15-1788) was carried out in the
final incubation volume of 0.5 ml, containing 0.1 ml of radioligand
([3H]flumazenil) at concentration of 1 nM, 0.1 ml of tested
5.3.5. Compound 11
Yield: 44 %; m.p. ¼120–124 ꢂC; EI–MS m/z 408 [Mþ]; HRMS
calcd for C17H14BrClN2O3 [M]þ m/z, 407,987; found, 407,989;1H
compound at concentration of 1 mM, 0.1 ml of 0.05 M citrate buffer
(pH 7.1 at 4 ꢂC), and 0.2 ml of membrane suspension.
NMR (DMSO-d6, 300 MHz)
d: 10.93 (s, 1H), 8.31 (s, 1H), 7.75–7.07
All assay tubes were incubated for 60 min at 4 ꢂC. The incuba-
tion was quenched by the addition of 6 ml of 0.05 M ice-cold citrate
buffer (pH 7.1 at 4 ꢂC) to samples and rapid filtration on a 12-
position harvester using Whatman GF/C filters. The filters were
washed with 6 ml of the same buffer. The dried filters were placed
into the scintillation vials. The vials were filled with 10 ml of
standard Optifase scintillator (LKB, Sweden), kept for 24 h at 20 ꢂC,
and then the radioactivity was counted with a scintillation counter
Rackbeta 1219 Spectral.
(m, 7H), 4.86 (s, 1H), 3.84–3.78 (m, 1H), 3.69–3.65 (m, 1H), 3.62–
3.58 (m, 2H); IR (KBr):
n
(cmꢁ1): 3340, 3175, 3102, 2933, 1683, 1610.
5.3.6. Compound 12
Yield: 27%; m.p. ¼179–188 ꢂC; EI–MS m/z 422 [Mþ]; HRMS calcd
for C18H16BrClN2O3 [M]þ m/z, 422,003; found, 422,006;1H NMR
(CDCl3, 300 MHz)
4.18–4.12 (m, 1H), 3.97–3.90 (m, 1H), 3.97–3.64 (m, 2H), 3.36 (s,
3H); IR (KBr):
(cmꢁ1): 3418, 3209, 2883, 1710, 1672, 1628.
d: 9.62 (s, 1H), 7.59–7.13 (m, 7H), 4.94 (s, 1H),
n
To determine the non-specific binding of radioligand, the
5.3.7. Compound 13
incubation was carried out in the presence of 10
flumazenil. Specific binding was determined as
between general and non-specific bindings. The value of non-
specific radioligand binding (SB0) was less than 10% from total
value. IC50 values of the different compounds were determined by
m
M of unlabeled
Yield: 42 %; m.p. ¼ 204–208 ꢂC; EI–MS m/z 388 [Mþ]; HRMS
a
difference
calcd for C18H17BrN2O3 [M]þ m/z, 388,042; found, 388,044;1H NMR
(CDCl3, 300 MHz)
4.12–4.10 (m, 1H), 3.98–3.94 (m, 1H), 3.77-3.70(m, 2H), 3.37 (s, 3H);
IR (KBr):
(cmꢁ1): 3210, 2916, 2874, 1706, 1671, 1607.
d: 9.81 (s, 1H), 7.61–7.15 (m, 8H), 4.89 (s, 1H),
n
testing a series of eight increasing concentrations (0.1 nM–10 mM).
5.4. Crystal structure determination of compound 15
5.5.3. In vitro receptor binding assays
5.5.3.1. [3H]PK11195 binding. Binding assays were determined by
modified method [42].
Crystal 15 monoclinic, space group P21, a ¼ 9.5342(3),
b ¼ 8.2524(3), c ¼ 11.1086(5) Å,
Z ¼ 2, rcalc ¼ 1.491gcm-3. (130 K).
b
¼ 97.23(3)ꢂ, V ¼ 867.08(6) Å3,
After sacrifice the brain was rapidly removed, the cerebral cortex
was dissected, and tissues were used for the assay. The tissues were

S. Andronati et al. / European Journal of Medicinal Chemistry 45 (2010) 1346–1351
1351
homogenized in 30 ml of ice-cold Tris-HCl buffer (pH 7.4 at 4 ꢂC)
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20 000 g for 30 min, and the pellet was resuspended in 30 ml of ice-
cold Tris-HCl buffer (pH 7.4 at 4 ꢂC) and recentrifuged. The new
pellet was resuspended in 30 ml of ice-cold Tris-HCl buffer (pH 7.4
at 4 ꢂC) and used for the assay. [3H]PK11195 binding was deter-
mined in a final volume of 0.5 ml, containing 0.1 ml of radioligand
([3H]PK11195) at concentration of 1 nM, 0.1 ml of tested compound
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