High affinity central benzodiazepine receptor ligands: Synthesis and biological evaluation of a series of phenyltriazolobenzotriazindione derivatives

Article abstract of DOI:10.1021/jm0408722

A series of 2-phenyl[1,2,3]triazolo[1,2-α][1,2,4]benzotriazin-1,5(6H) -diones (PTBTs), VII, were prepared and tested at the central benzodiazepine receptor (BzR). The skeleton of these B compounds was designed by formally combining the N-C=O moieties of t

Full text of DOI:10.1021/jm0408722

2936
J. Med. Chem. 2005, 48, 2936-2943
High Affinity Central Benzodiazepine Receptor Ligands: Synthesis and
Biological Evaluation of a Series of Phenyltriazolobenzotriazindione
Derivatives
Giampaolo Primofiore,§,* Federico Da Settimo,§ Sabrina Taliani,§ Silvia Salerno,§ Ettore Novellino,†
Giovanni Greco,† Barbara Cosimelli,† Franc¸ois Besnard,‡ Barbara Costa,| Marina Montali,| and Claudia Martini|
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Chimica
Farmaceutica e Tossicologica, Universita` di Napoli “Federico II”, Via Domenico Montesano, 49, 80131 Napoli, Italy,
Department of Molecular and Functional Genomics, Sanofi-Synthe´labo, 10 rue des Carrie`res, 92500 Rueil-Malmaison, France,
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, Via Bonanno 6,
56126 Pisa, Italy
Received August 10, 2004
A series of 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones (PTBTs), VII, were
prepared and tested at the central benzodiazepine receptor (BzR). The skeleton of these
compounds was designed by formally combining the N-CdO moieties of the known BzR ligands,
triazoloquinoxalines (IV) and triazinobenzimidazoles (ATBIs) (VI). Most of the PTBTs displayed
submicromolar/nanomolar potency at the BzR. The 9-chloro derivatives (45-49) were generally
found to be more potent than their 9-unsubstituted counterparts (37-44). Compound 45 turned
out to be the most potent of the PTBTs (Ki 2.8 nM). A subset of compounds (37, 42, 45, 49),
when tested for their affinity on recombinant rat R1â2γ2, R2â2γ2, and R5â3γ2 GABAA/Bz
receptor subtypes, showed enhanced affinities for the R1â2γ2 isoform, with compounds 45 and
49 exhibiting the highest selectivity. Moreover, compounds 45 and 49 were found to display a
full agonist efficacy profile at R1 and R2 receptor subtypes, and an antagonist efficacy at
R5-containing receptors.
Introduction
Recent studies clearly suggest that a particular
pharmacological response might be associated with an
action at a specific GABAA receptor subtype. It has been
demonstrated that the alpha subunit is the one which
may influence the ligands’ efficacy and their selective
pharmacological actions. In particular, R1 subtypes
containing GABAA receptors mediate the sedative ac-
tion; R2 subtypes may be involved in anxiolytic-like
activity and in myorelaxation effects; R5 ones might be
associated with cognition and memory, while the role
of R3-containing receptors remains unclear or, at least,
it seems they are partly involved in mediating anxiety
behavior.6-8
Benzodiazepine receptor (BzR) ligands are structur-
ally different compounds that bind to the γ-amino-
butyric acidA (GABAA)/BzR complex. They exhibit a wide
variety of pharmacological actions, ranging in a con-
tinuum from full agonist (agents potentiating the af-
finity of GABA for the GABAA receptor, with anxiolytic,
anticonvulsant, sedative-hypnotic, and myorelaxant
activities), through antagonist (nil efficacy) to inverse
agonist (agents reducing the affinity of GABA for the
GABAA receptor, with anxiogenic, somnolytic, pro-
convulsant, or even convulsant activities).1-3
The GABAA/BzR complex is a membrane-bound het-
eropentameric protein that may be assembled from at
least 21 subunits belonging to eight different classes
(6R, 4â, 4γ, 1ꢀ, 1δ, 3F, 1θ, and 1π), which form a chloride
channel.1,4 It has been found that a fully functional
GABAA/BzR receptor must contain an R subunit, a
â subunit, and a γ subunit, and it is currently ac-
cepted that the predominant stoichiometry in vivo is
(R)2(â)2(γ).5 The major Bz-sensitive GABAA receptor
subtypes in the brain are R1âXγ2, R2âXγ2, R3âXγ2, and
R5âXγ2, the benzodiazepine binding site being located
between the alpha and gamma subunits.5,6
Among the wide variety of nonbenzodiazepine ligands
binding to the BzR the following classes are the most
potent: 2-arylpyrazoloquinolines (I),9,10 â-carbolines
(II),11 pyridodiindoles (III)12 and triazoloquinoxalines
(IV)13 (Chart 1). In the past 10 years, our research group
has reported several N-benzylindolylglyoxylamide de-
rivatives (V) (Chart 1) as BzR ligands designed by
taking â-carbolines II as the reference structure.14,15
Compounds V were generally found to be less potent
than ligands of classes I-IV, probably owing to their
greater conformational flexibility. For this reason, we
started research aimed at devising new BzR ligands
characterized by more rigid molecular scaffolds. As a
result of these efforts, we presented 3-aryl[1,2,4]triazino-
[4,3-a]benzimidazol-4(10H)-ones VI (ATBIs) (Chart 1)
as BzR ligands possessing nanomolar affinity values and
a higher potency compared with indolylglyoxylamides
V.16 We now report the synthesis and the biological
evaluation of a series of derivatives of the 2-phenyl-
[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-dione
* To whom all correspondence should be addressed. Tel.:
+390502219561. Fax: +390502219605. E-mail: primofiore@
farm.unipi.it.
§ Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
† Dipartimento di Chimica Farmaceutica e Tossicologica, Universita`
“Federico II” di Napoli.
‡ Department of Molecular and Functional Genomics, Sanofi-Syn-
the´labo.
| Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Bio-
tecnologie, Universita` di Pisa.
10.1021/jm0408722 CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/11/2005

Central Benzodiazepine Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2937
Chart 1
materials or led to complex mixtures that required
tedious purification procedures and provided the cor-
responding amino derivatives 32-36 with poor chemical
yields.
To solve this problem, we modified the reduction
conditions and found that the desired products 32-36
were formed with good yields and a higher purity when
the catalyst (10% Pd/C) was first activated overnight
on hydrogen.
Due to their low stability on heating, the nitro
(11-23) and the amino (24-31) compounds were not
recrystallized, but were purified by washing with cold
petroleum ether 60-80 °C and/or by flash chromatog-
raphy. The chloro-substituted amino derivatives
32-36 were particularly instable, so they were not
isolated and characterized, but directly used in the next
reaction.
The cyclocondensation of all the amines 24-36 to the
final tricyclic compounds 37-49 was performed using
triphosgene22 in anhydrous tetrahydrofuran solution at
room temperature.
The tricyclic derivatives 37-49 are stable products,
and they were purified by recrystallization from the
appropriate solvent. With the exception of the labile
products 32-36, the structures of all the newly synthe-
sized compounds were confirmed by analytical and
spectral data (Tables1 and 2).
Binding Studies. The binding affinity of each newly
synthesized PTBT derivative at the BzR in bovine brain
membranes was determined by competition experiments
against the radiolabeled antagonist [3H]flumazenil23
and was expressed as the Ki value only for those
compounds inhibiting radioligand binding by more than
80% at a fixed concentration of 10 µM (Table 3).
(PTBT) family with the general formula VII (Chart 1).
These derivatives maintain the 6,6,5-tricyclic heteroaro-
matic system analogous to that of pyrazoloquinolines
(I).9,10 Moreover, the triazolobenzotriazindione skeleton
of VII was designed by formally combining the
N-CdO moieties of triazoloquinoxalines (IV)13 and of
ATBIs (VI),16 as shown in Figure 1.
PTBTs seem to fulfill all the requirements necessary
for a high affinity at the BzR, according to the phar-
macophore/topological model proposed by Cook et al.17
In Figure 2 the PTBT derivative 37 (VII, X ) R ) H) is
superimposed on the 2-phenylpyrazoloquinoline
(I, R ) H). In particular, the receptor H-bond sites A2
(donor), H1 and H2 (acceptors) are supposed to interact
with NH(6), the 5-CdO oxygen (possibly together with
N3 in a three-centered H-bond), and the 1-CdO oxygen,
respectively. Additionally, the fused benzene and the
pendant phenyl rings might fill the lipophilic pockets
Subtype selectivity of the most active compounds
(37, 42, 45, 49) was tested by assaying their ability to
displace [3H]flumazenil in membranes from HEK293
cells expressing rat R1â2γ2, R2â2γ2, and R5â3γ2 GABAA/
Bz receptor subtypes (Table 4).15
The functional efficacy of the most potent compounds
45 and 49 was determined on cloned rat R1â2γ2,
R2â2γ2, and R5â3γ2 GABAA/Bz receptor subtypes ex-
pressed in HEK293 cells by measurement of the modu-
latory effect on the GABA EC20 ion current using
36Cl- influx through the ion channel pore, as described
by Harris et al.24 (Figure 3). For each recombinant
receptor subtype, we first examined the concentration
dependence of the modulatory effects elicited by com-
pounds 45 and 49 on GABA 20% maximum influx (data
not shown). In each case concentrations 100 times
greater than the compounds’ Ki values provided the
maximal effect on GABA-evoked 36Cl- influx.25 These
concentrations were used in each experiment. The
nonselective full agonist diazepam was included as the
standard in the determination of efficacy.
L
Di and L1, respectively. Although no structural analogy
with benzylindolylglyoxylamides can be invoked, a
chlorine was introduced in the 9-position of the PTBT
system to verify whether a higher polarization of the
NH(6) bond due to electron-withdrawing effects might
strengthen the putative H-bond with the receptor ac-
ceptor site A2, as had already been observed for the
5-chloro-substituted benzylindolylglyoxylamides V.14,15
Finally, a small number of substituents were inserted
on the 2-phenyl ring (H, F, Cl, Br, MeO) to probe the
L1/L2 lipophilic pocket and its surroundings.
Chemistry. The general procedure used for the
synthesis of [1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,
5(6H)-dione derivatives 37-49 is shown in Scheme 1.
Ionic 1,3-dipolar cycloaddition of the appropriate azide
1 and 2,18,19 to ethyl phenylacetates 3-10,20,21 furnished
the 1-(2-nitrophenyl)-4-aryl-5-oxo[1,2,3]triazoles 11-23.
Catalytic reduction of compounds 11-18 in ethanolic
solution afforded the corresponding amines 24-31.
Reduction of the nitro group of the chloro derivatives
19-23 under the same conditions gave only the starting
Results and Discussion
The binding results shown in Table 3 indicate that
the majority of the PTBTs display from submicromolar
to nanomolar potency at the BzR, the 2′-fluorophenyl
derivative 43 and the 3′,4′-dimethoxyphenyl derivative
44 being the only compounds devoid of any appreciable
affinity.

2938 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Primofiore et al.
Figure 1. Design of compounds VII based on the formal assembly of substructures highlighted in compounds IV13 and VI.16
higher affinity of the 9-chloro derivatives might be
attributable to both a stronger H-bond between the
NH(6) and the receptor acceptor site A2, due to a higher
polarization of the NH bond, and a better interaction
of the 9-chloro with the lipophilic receptor area LDi,
which is modulated by the overall fit of the molecule in
the Bz-receptor recognition site.
Substituents on the 2-phenyl ring modulate affinity.
The highest affinity ligands are 4′-H and 4′-MeO
derivatives in the 9-Cl and 9-H subsets: 45 (Ki 2.8 nM)
and 37 (Ki 22 nM), and, respectively, 49 (Ki 6 nM) and
42 (Ki 11 nM). In both subsets, insertion in position
4′ of a halogen (38-40, 46, 47) or a methyl (41, 48)
lowers affinity. It is worth noticing that Cl, Br and Me
are all more lipophilic and bulkier than H, but F has
approximately the same lipophilicity and atomic radius
as H. Thus, it seems difficult to establish a clear
correlation between the affinity and the lipophilic or
steric properties of the 4′-substituent. Likewise, elec-
tronic effects cannot explain the structure-affinity re-
lationships, since both MeO and Me are classified as
electron-donating groups.
Figure 2. Overlay of the PTBT derivative 37 (blue) on the
2-phenylpyrazoloquinoline CGS 82167 of class I (red). The two
molecular models are embedded within the pharmacophore/
topological model proposed by Cook and co-workers17 for BzR
ligands. A2 is an H-bond donor site, H1 and H2 are H-bond
acceptor sites, L1, L2 and LDi are lipophilic pockets.
Scheme 1
In an attempt to improve the affinity of the 4′-MeO
derivative 42, we prepared compound 44, featuring
an additional MeO group in the 3′-position. This
3′,4′-(MeO)2 derivative turned out to be inactive in the
binding assays. No appreciable affinity was displayed
by the 2′-F derivative 43, either. The lack of affinity
found for compounds 43 and 44 seems to indicate that
the L1 pocket possesses precise dimensions that can well
accommodate only a phenyl group with no ortho- or
meta-substituents, as already pointed out by Savini et
al. for a series of pyrazolo[4,3-c]quinolin-3-one deriva-
tives substituted in the ortho position of the 2-phenyl
ring.26
The PTBTs were applied to Cook’s model,17 hypoth-
esizing that the 2-phenyl ring and the 4′-substituent
might fill the L1 and L2 lipophilic pockets, respectively,
of the BzR (see Figure 2). In the 2-arylpyrazoloquinoline
series I,9,10 it is well-known that H, Cl, and MeO in the
4′-position of the phenyl ring are associated with sub-
nanomolar affinity, while the effects of a 4′-Me have not
been disclosed in the literature, although this structure
was included in two patents.27,28 Thus, the low affinity
shown by compounds 38-41 was unexpected on the
basis of the alignment depicted in Figure 2. Actually,
the unfavorable effects of the 4′-Me and the 3′,4′-(MeO)2
groups on the pendant phenyl ring had already been
observed in the recently reported series of ATBIs VI.16
The 9-chloro derivatives are generally more potent
than their 9-unsubstituted counterparts to an extent
comprised between 2- and 8-fold. Exceptions to this
trend are the 4′-fluorophenyl derivatives 38 and 46,
which exhibit almost identical Ki values. The favorable
effect of the 9-chloro reaches a maximum when the
2-phenyl ring is not substituted (compare 45 vs 37). The

Central Benzodiazepine Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2939

2940 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Primofiore et al.
Table 2. Physical Properties and Spectral Data of Compounds 37-49
yield
(%)
mpa
(°C)
IR
1H NMR
(δ, ppm)
MS
m/e (%)
no.
X
R
formulab
(cm-1
)
37
H
H
41 240-241
C
C
C
C
C
15H10N4O2
3080, 1750, 1650, 7.16-7.41 (m, 3H, H-7, H-8, H-9);
1600, 1260, 1110, 7.49-7.60 (m, 3H, H-3′, H-4′, H-5′);
278 (M+, 18);
(EtOH)
250 (35);
960
8.29 (dd, 2H, H-2′, H-6′);
222 (52);
8.67 (dd, 1H, H-10)
194 (40); 90 (100)
296 (M+, 78);
268 (46);
38
39
40
41
H
H
H
H
4′-F
4′-Cl
4′-Br
35 228-230
15H9FN4O2
3100, 1750, 1660, 7.16-7.45 (m, 5H, Ar-H);
(EtOH)
1600, 1300, 1160, 8.34 (dd, 2H, H-2′, H-6′);
770
8.66 (dd, 1H, H-10);
11.69 (bs, 1H, NH)
240 (68); 212 (100)
67 225-227
15H9ClN4O2 3350, 1710, 1660, 7.17-7.38 (m, 3H, H-7, H-8, H-9);
312 (M+, 18);
284 (42);
(DMF/H2O)
1600, 1340, 1280, 7.63 (d, 2H, H-3′, H-5′); 8.30 (d,
750
2H,H-2′, H-6′); 8.67 (dd, 1H, H-10);
11.72 (bs, 1H, NH)
256 (67); 228 (54);
89 (100)
33 237-238
15H9BrN4O2 3100, 1740, 1640, 7.17-7.38 (m, 3H, H-7, H-8, H-9);
356 (M+,3); 328 (19);
300 (32);
(EtOH)
1600, 1320, 1260, 7.77 (d, 2H, H-3′, H-5′); 8.23 (d,
740
2H, H-2′, H-6′); 8.66 (dd, 1H, H-10);
272 (10); 89 (100)
11.75 (bs, 1H, NH)
4′-CH3
45 243-245
16H12N4O2
3150, 1740, 1650, 2.38 (s, 3H, CH3); 7.16-7.39
292 (M+, 84)
264 (64);
236 (90); 208 (100)
(EtOH)
1590, 1340, 1260, (m, 5H, Ar-H); 8.19 (d, 2H, H-2′,
720
H-6′); 8.66 (dd, 1H, H-10); 11.61
(bs, 1H, NH)
42
43
44
H
H
H
4′-OCH3
2′-F
38 253-254
C16H12N4O3
C15H9FN4O2
C17H14N4O4
3050, 1740, 1630, 3.83 (s, 3H, OCH3); 7.09-7.39 (m,
1590, 1290, 1160, 5H, Ar-H); 8.24 (d, 2H, H-2′, H-6′);
308 (M+, 100);
280 (49);
(EtOH)
750
8.65 (dd, 1H, H-10); 11.60 (bs, 1H, NH) 252 (89); 224 (37)
72 220-222
3100, 1750, 1620, 7.17-7.59 (m, 6H, Ar-H); 8.24-8.32 (m, 296 (M+, 11); 268 (58);
(EtOH)
1600, 1320, 1260, 1H, Ar-H); 8.65 (dd, 1H, H-10); 11.77
770 (bs, 1H, NH)
240 (92); 212 (89);
107 (100)
3′,4′-(OCH3)2 70 235-238
3075, 1720, 1670, 3.83 (s, 3H, 3′-OCH3); 3.85 (s, 3H,
338 (M+, 34); 310 (43);
282 (97); 251 (7); 178 (100)
(DMF/H2O)
1600, 1380, 1260, 4′-OCH3); 7.10-7.32 (m, 4H, Ar-H);
760
7.75 (s, 1H, H-2′); 7.97 (dd, 1H,
H-6′); 8.65 (dd, 1H, H-10); 11.58
bs,1H, NH)
45 Cl
H
33 228-230
C15H9ClN4O2 1730, 1680, 1610, 7.18 (d, 1H, H-7); 7.44 (dd, 1H, H-8);
312 (M+, 100); 284 (35);
278 (47); 256 (29);
228 (51)
(EtOH)
1310, 730
7.52-7.61 (m, 3H, H-3′, H-4′, H-5′);
8.27 (dd, 2H, H-2′, H-6′); 8.65 (d, 1H,
H-10); 11.78 (bs, 1H, NH)
46 Cl 4′-F
59 208-210
C
C
C
C
15H8ClFN4O2 1735, 1670, 1600, 7.17-7.45 (m, 4H, Ar-H); 8.32 (dd,
330 (M+, 9); 296 (63);
268 (26); 240 (44); 212
(79); 103 (100)
(EtOH)
1320, 1220, 730
2H, H-2′, H-6′); 8.66 (d, 1H, H-10);
11.70 (bs, 1H, NH)
47 Cl 4′-Cl
48 Cl 4′-CH3
49 Cl 4′-OCH3
49 168-170
15H8Cl2N4O2 1730, 1665, 1610, 7.16-7.64 (m, 4H, Ar-H); 8.28
348 (M+ + 1,7); 312 (60);
284 (39); 278 (100);
256 (25); 228 (45)
(EtOH)
1340, 1260, 760
(d, 2H, H-2′, H-6′); 8.65 (d, 1H, H-10);
11.67 (bs, 1H, NH)
54 245-247
16H12N4O3
1720, 1675, 1600, 2.38 (s, 3H, CH3); 7.17-7.38
1290, 1180, 760
326 (M+, 3); 292 (100);
(EtOH)
(m, 5H, Ar-H); 8.19 (d, 2H, H-2′, H-6′); 264 (27); 236 (17);
8.66 (d, 1H, H-10); 11.44 (bs, 1H, NH)
208 (63)
49 239-241
16H11ClN4O3 1650, 1600, 1330, 3.83 (s, 3H, OCH3); 7.12 (d, 2H, H-3′,
342 (M+, 8); 308 (100);
280 (21); 252 (21);
224 (29)
(DMF/H2O)
1030, 750
H-5′); 7.19-7.39 (m, 2H, H-7, H-8);
8.24 (d, 2H, H-2′, H-6′); 8.65 (d, 1H,
H-10); 12.60 (bs, 1H, N-H)
a Recrystallization solvents are indicated in parentheses. b Elemental analyses for C, H, N were within (0.4% of the calculated values.
However, the puzzling structure-affinity relationships
in this class of ATBIs were ascribed to tautomeric
equilibria involving the NH(10) proton responsible for
the bond with the receptor A2 site. It is worth noting
that in the case of the PTBT series, among compounds
with different affinity, we did not observe the establish-
ment of any tautomerism either by NMR or IR spec-
troscopy.
The most potent compounds (37, 42, 45, 49), when
tested for their affinity at R1â2γ2, R2â2γ2, and R5â3γ2
GABAA/Bz receptor subtypes,15 showed enhanced af-
finities for the R1â2γ2 isoform and exhibited a certain
selectivity over both R2â2γ2, and R5â3γ2 subtypes
(Table 4).
region of the receptor binding site.29 The moderate R1
selectivity shown by PTBTs is consistent with the
binding mode proposed in Figure 2 whereby the LDi site
is occupied by the fused benzene ring.
Compounds 45 and 49 were selected to be screened
for functional efficacy and, as shown in Figure 3, their
efficacy on coapplication with GABA at the R1â2γ2 and
R2â2γ2 receptor subtypes was comparable to that of
standard diazepam (110 ( 9%, 118 ( 10%, and
113 ( 9%, for compounds 45, 49, and diazepam at
R1â2γ2, respectively; 105 ( 10%, 108 ( 9% and
110 ( 6%, for compounds 45, 49 and diazepam at
R2â2γ2, respectively), showing that the main feature
of compounds 45 and 49 is their full agonist efficacy
profile at both these receptor subtypes. By contrast, in
comparison to the modulation induced by standard
diazepam, compounds 45 and 49 did not substantially
It has been proposed that one of the features of a BzR
ligand necessary to display R1 selectivity is that of
allowing a favorable occupation of the lipophilic LDi

Central Benzodiazepine Receptor Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2941
Table 3. Inhibition of [3H]Flumazenil Specific Binding to
Bovine Brain Membranes of PTBT Derivatives 37-49
These products should consequently exert their effects
only through modulation of the R1 and R2 subtypes,
bearing a functional agonist efficacy at these subtypes
and an antagonist behavior at the R5 subtype. Thus,
these results might indicate hypnotic with anxiolytic
properties for compounds 45 and 49, possibly without
any effect on learning or memory processes.
In conclusion, we have described 2-phenyl[1,2,3]-
triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones (PT-
BTs) as a novel class of BzR ligands. Compounds 45
(X ) Cl and R ) 4′-H) and 49 (X ) Cl and R )
4′-OCH3) displayed the highest potency at the BzR
(Ki 2.8 nM and 6 nM, respectively). When tested for their
affinity on recombinant rat R1â2γ2, R2â2γ2, and R5â3γ2
GABAA/Bz receptor subtypes, these ligands proved to
be about 4-10-fold selective for the R1â2γ2 over the
R2â2γ2, and R5â3γ2 subtypes. Moreover, they showed
a full agonist efficacy on the R1 and R2 receptor
subtypes, and an antagonist efficacy on R5-containing
receptors. This affinity and efficacy selectivity profile
makes compounds 45 and 49 a valuable starting point
for further research aimed at obtaining novel zolpidem-
like30 sedative agents.
no.
X
R
Kia (nM)
37
38
39
40
41
42
43
44
45
46
47
48
49
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
22 ( 2
54 ( 3
169 ( 10
224 ( 15
616 ( 59
11 ( 1
4′-F
4′-Cl
4′-Br
4′-CH3
4′-OCH3
2′-F
3′,4′-(OCH3)2
H
>10000
>10000
2.8 ( 0.15
56 ( 3
4′-F
4′-Cl
38 ( 2
4′-CH3
4′-OCH3
185 ( 15
6 ( 0.5
diazepam
flumazenil
clonazepam
10 ( 1
0.90 ( 0.05
0.85 ( 0.02
a Ki values are means ( SEM of three determinations.
Experimental Section
Chemistry. Melting points were determined using a Re-
ichert Ko¨fler hot-stage apparatus and are uncorrected. Infra-
red spectra were recorded with a Nicolet/Avatar FT-IR spec-
trometer in Nujol mulls. Routine nuclear magnetic resonance
spectra were recorded in DMSO-d6 solution on a Varian
Gemini 200 spectrometer operating at 200 MHz. Mass spectra
were obtained on a Hewlett-Packard 5988 A spectrometer
using a direct injection probe and an electron beam energy of
70 eV. Evaporation was performed in vacuo (rotary evapora-
tor). Analytical TLC was carried out on Merck 0.2 mm
precoated silica gel aluminum sheets (60 F-254). Silica gel 60
(230-400 mesh) was used for column chromatography. El-
emental analyses were performed by our Analytical Laboratory
and agreed with theoretical values to within (0.4%.
Ethyl phenylacetate 3, ethyl 4-methylphenylacetate 7, ethyl
4-methoxyphenylacetate 8 and ethyl 3,4-dimethoxyphenyl-
acetate 10 are commercially available products. The following
compounds were prepared in accordance with reported proce-
dures: 2-azidonitrobenzene 1,18 2-azido-4-chloronitrobenzene
2,19 ethyl 4-fluorophenylacetate 4,20 ethyl 4-chlorophenyl-
acetate 5,20 ethyl 4-bromophenylacetate 6,20 ethyl 2-fluoro-
phenylacetate 9.21
Table 4. Inhibition of [3H]Flumazenil Specific Binding of
Selected Compounds on Rat R1â2γ2, R2â2γ2 and R5â3γ2
GABAA/Bz Receptor Subtypesa
Ki (nM)b or % inhibition (10 µM)c
no.
R1â2γ2
R2â2γ2
R5â3γ2
37
42
45
49
9.14 ( 1
3.76 ( 0.3
1.74 ( 0.1
1.82 ( 0.1
50 ( 3
27.6 ( 2
14.3 ( 1
13.9 ( 1
18.0 ( 2
765 ( 63
24.1 ( 2
17.2 ( 2
10.3 ( 1
7.57 ( 0.5
35% ( 3
zolpidem
a The ability of the compounds to displace [3H]flumazenil was
measured in membranes from HEK293 cells expressing the
R1â2γ2, R2â2γ2 and R5â3γ2 subtypes, as described in the Experi-
mental Section. b Ki values are means ( SEM of three determina-
tions. c Percentage inhibition values of specific [3H]flumazenil
binding at 10 µM concentration are means ( SEM of three
determinations.
General Procedure for the Synthesis of 1-(2-Nitro-5-
substituted-phenyl)-4-(substituted-phenyl)-5-oxo-1,2,3-
triazole Derivatives 11-23. The appropriate ethyl phenyl-
acetate 3-10 (8.5 mmol) was added dropwise, at -10 °C, under
stirring and in a nitrogen atmosphere, to an ethanolic solution
of sodium ethoxide (0.196 g, 8.5 mmol of sodium in 4.5 mL of
absolute ethanol). Then, the azido derivatives 1 and 2
(8.2 mmol) was added in a single portion to the resulting
mixture. Stirring was continued at -10 °C overnight, and then
at room temperature for 1-6 h (TLC analysis). The suspension
obtained was evaporated to dryness at reduced pressure and
at a temperature not higher than 30-40 °C; the resulting
semisolid residue was treated with ice and water. The aqueous
mixture was washed three times with dichloromethane, cooled
at 0 °C and acidified to pH 5 with dilute hydrochloric acid.
The precipitate which formed, made up of the crude products
11-23, was collected and purified by washing with cold
petroleum ether 60-80 °C. Yields, melting points and analyti-
cal and spectral data are reported in Table 1.
Figure 3. Maximal efficacy of compounds 45 and 49 on rat
recombinant R1â2γ2, R2â2γ2 and R5â3γ2 GABAA receptor
subtypes. Results are expressed as percentages of increase in
response to GABA at EC20 (mean ( SEM) from at least five
independent experiments.
affect GABA-evoked Cl- influx at the R5â3γ2 GABAA/
Bz receptor subtype, suggesting an intrinsic antagonist
activity (6 ( 2%, 12 ( 2%, and 111 ( 12%, for com-
pounds 45, 49 and diazepam at R5â3γ2, respectively).
General Procedure for the Synthesis of 1-(2-Amino-
phenyl)-4-(substituted-phenyl)-5-oxo-1,2,3-triazole De-
rivatives 24-31. A mixture of the appropriate 1-(2-nitro-
phenyl)-4-(substituted-phenyl)-5-oxo-1,2,3-triazole derivative

2942 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Primofiore et al.
11-18 (0.285 mmol) and 10% Pd/C (0.02 g) in 150 mL of
absolute ethanol was hydrogenated at room temperature and
pressure. Once hydrogen absorption ceased (2-4 h), the
catalyst was filtered off and the solution was evaporated to
dryness at reduced pressure and at a temperature not higher
than 30-40 °C. The resulting residue made up of the crude
amines 24-31 was purified by flash-chromatography (eluting
system: toluene/acetonitrile/glacial acetic acid in various
ratios). Yields, melting points and analytical and spectral data
are reported in Table 1.
General Procedure for the Synthesis of 1-(2-Amino-
5-chlorophenyl)-4-(substituted-phenyl)-5-oxo-1,2,3-tria-
zole Derivatives 32-36. The amino derivatives 32-36 were
prepared following the same procedure described for the
unsubstituted analogues 24-31, but the catalyst (10% Pd/C)
was first activated overnight on hydrogen. The usual workup
of the reaction mixture gave compounds 32-36, which were
particularly unstable; they were not isolated and characterized,
and they were utilized in the following reaction without any
further purification.
binding was defined by 10-5 M diazepam. Assays were
incubated to equilibrium for 1 h at 4 °C.
2. Functional Efficacy Studies. 36Cl- uptake was mea-
sured in transfected HEK293 cells by minor modification of a
previously described method.24 In brief, coverlips were placed
on 8-10/cell culture plates (100 mm) and were incubated
overnight under germicidal light in a solution containing
0.01 mg/mL poly-lysine in 0.1 M boric acid (pH 8.4). The
following day, coverlips were washed twice with phosphate-
buffered saline and were placed on individual six-well cell
culture plates. DMEM/Nut Mix F-12 with Glut-I (GIBCO),
supplemented with 10% fetal bovine serum, penicillin
(100 units/mL) and streptomycin (100 µg/mL), were added to
each well. Cells were then plated into each well at a density
of 50 × 105 cells/well and were grown on the coverlips for 3
days at 37 °C with 5% CO2. Cells were washed twice
(10 s/wash) in wash buffer 136 mM NaCl, 5.4 mM KCl,
1.4 mM MgCl2, 1.2 mM CaCl2, 1 mM NaH2PO4, and 20 mM
4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid, adjusted
to a pH of 7.4 with Tris base. All steps were conducted at room
temperature. Cells were then dipped into
2 mL of a
General Procedure for the Synthesis of 9-Substituted-
2-(substituted-phenyl)-[1,2,3]triazolo[1,2-a][1,2,4]-
benzotriazin-1,5(6H)-dione Derivatives 37-49. Triphos-
gene (0.060 g, 0.2 mmol) and triethylamine (0.17 mL,
1.22 mmol) were successively added to a stirred and ice-cooled
solution of the appropriate 1-(2-amino-5-substituted-phenyl)-
4-(substituted-phenyl)-5-oxo-1,2,3-triazole derivative 24-36
(0.5 mmol) in 9.0 mL of anhydrous tetrahydrofuran. The
mixture was stirred under a nitrogen atmosphere at 0 °C for
1 h and then at room temperature for 12-36 h (TLC analysis).
The suspension obtained was filtered, and the solid was
washed with water to give a first portion of crude tricyclic
product. The reaction mother liquors were reduced to half
volume to precipitate an additional amount of crude product.
The quantities of tricyclic derivatives obtained from the initial
insoluble precipitate and from the concentrated tetrahydro-
furan solution were variable, depending upon the solubility
of the particular compound. All the newly synthesized tricyclic
products 37-49 were purified by recrystallization from the
appropriate solvent. Yields, recrystallization solvents, melting
points and analytical and spectral data are reported in Table
2.
36Cl- solution (2 µCi/mL) containing the drug(s) to be studied,
with or without GABA. Influx was terminated after 8 s by
transfer of coverlips to 500 mL ice-cold assay buffer (with
0.1 mM picrotoxin). Coverlips were drained rapidly and placed
in 1.6 mL of 0.2 N NaOH in 20 mL scintillation vials and were
left overnight. A 0.1 mL aliquot was removed and assayed for
protein determination. The remaining 1.5 mL was neutralized
with 0.3 mL of 1 N acetic acid and 20 mL of BioSafe II was
added for counting by liquid scintillation spectrometry. Values
for 36Cl- influx were expressed as nanomol/mg protein.
Molecular Modeling. Molecular modeling was performed
using the software package SYBYL version 6.5 (Tripos Inc.)
running on a Silicon Graphics R8000 Indigo 2 workstation.
The model of the ligand CGS 8216 (Figure 2) corresponds to
the crystal structure retrieved from the Cambridge Structural
Database34 (CSD) with the entry code COVLII using ConQuest
version 1.6. The model of PTBI derivative 37 (Figure 2) was
constructed using the SYBYL fragment library and optimizing
the trial geometry with the semiempirical quantum-mechanics
AM1 method35 available within MOPAC version 6.00 (QCPE).
MOPAC was run under default settings using the keyword
“MMOK”. To evaluate the reliability of the geometry-optimiza-
tion, we searched the CSD looking for compounds similar to
37 through structure-based queries. The results of the searches
were consistent with the overall coplanar geometry of 37
yielded by the AM1 method. Examples of hits similar to 37
are two compounds filed in the CSD with the entry codes
DMOCDO1036 and GOZLUC,37 both featuring entirely copla-
nar geometries. Overlay of the molecular models was per-
formed as described in a previous study.38
Biological Procedures. 1. Radioligand Binding Stud-
ies. [3H]Flumazenil (specific activity 70.8 Ci/mmol) was ob-
tained from NEN Life Sciences Products. All other chemicals
were of reagent grade and were obtained from commercial
suppliers.
Bovine cerebral cortex membranes were prepared in ac-
cordance with ref 31. The membrane preparations were
subjected to a freeze-thaw cycle, washed by suspension and
centrifugation in 50 mM Tris-citrate buffer pH 7.4 (T1) and
then used in the binding assay. Protein concentration was
assayed by the method of Lowry et al.32
Acknowledgment. This work was financially sup-
ported by MIUR (Cofin 2003, ex 40%).
[3H]Flumazenil binding studies were performed as previ-
Supporting Information Available: Microanalytical data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
ously reported.15
HEK293 cells stably expressing rat GABAA receptor sub-
types (R1â2γ2, R3â2γ2, R5â3γ2) were maintained, as previ-
ously described,33 in DMEM/Nut Mix F-12 with Glut-I
(GIBCO), supplemented with 10% fetal bovine serum,
L-glutamine (2 mM), penicillin (100 units/mL) and streptomy-
cin (100 µg/mL) in a humidified atmosphere of 5% CO2/95%
air at 37 °C. Cells were harvested and then centrifuged at
500g. The crude membranes were prepared after homogeniza-
tion in 10 mM potassium phosphate, pH 7.4, and differential
centrifugation at 48 000g for 30 min at 4 °C. The pellets were
washed twice in this manner before final resuspension in
10 mM potassium phosphate, pH 7.4, containing 100 mM
potassium chloride.33
[3H]Flumazenil binding assays to transfected cell mem-
branes were carried out as previously described.33 In brief, the
cell line membranes were incubated in a volume of 500 µL
which contained [3H]flumazenil at a concentration of 1-2 nM
and the test compound in the range 10-9-10-5 M. Nonspecific
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