Design, synthesis and biological evaluation of 7-substituted 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f] [1,4]diazepines as safe anxiolytic agents

Article abstract of DOI:10.1016/j.ejmech.2020.112405

A series of 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate esters were synthesized and tested as central benzodiazepine receptor (CBR) ligands by the ability to displace [³H]flumazenil from rat cortical membranes. All the compounds showed high affinity with IC₅₀ values ranging from 5.19 to 16.22 nM. In particular, compounds 12b (IC₅₀ = 8.66 nM) and 12d (IC₅₀ = 5.19 nM) appeared as the most effective ligands being their affinity values significantly lower than that of diazepam (IC₅₀ = 18.52 nM). Compounds 12a-f were examined in vivo for their pharmacological effects in mice and five potential benzodiazepine (BDZ) actions were thus taken into consideration: anxiolytic, anticonvulsant, anti-amnesic, hypnotic, and locomotor activities. All the new synthesized compounds were able to induce a significant antianxiety effect and, among them, compound 12f protected pentylenetetrazole (PTZ)-induced convulsions in a dose-dependent manner reaching a 40% effect at 30 mg/kg. In addition, all the compounds were able to significantly prevent the memory impairment evoked by scopolamine, while none of them was able to interfere with pentobarbital-evoked sleep and influence motor coordination. Moreover, title compounds did not affect locomotor and exploratory activity at the same time and doses at which the anti-anxiety effect was observed. Finally, molecular docking simulations were carried out in order to assess the binding mode for compounds 12a-f. The obtained results demonstrated that these compounds bind the BDZ binding site in a similar fashion to flumazenil.

Full text of DOI:10.1016/j.ejmech.2020.112405

Journal Pre-proof
Design, synthesis and biological evaluation of 7-substituted 4-phenyl-6H-
imidazo[1,5-a]thieno[3,2-f] [1,4]diazepines as safe anxiolytic agents
Angela Di Capua, Annalisa Reale, Marco Paolino, Giulia Chemi, Simone Brogi,
Andrea Cappelli, Gianluca Giorgi, Fedora Grande, Lorenzo Di Cesare Mannelli, Carla
Ghelardini, Rosanna Matucci, Antonio Garofalo, Maurizio Anzini
PII:
S0223-5234(20)30376-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112405
EJMECH 112405
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 February 2020
Revised Date: 24 April 2020
Accepted Date: 27 April 2020
Please cite this article as: A. Di Capua, A. Reale, M. Paolino, G. Chemi, S. Brogi, A. Cappelli, G. Giorgi,
F. Grande, L. Di Cesare Mannelli, C. Ghelardini, R. Matucci, A. Garofalo, M. Anzini, Design, synthesis
and biological evaluation of 7-substituted 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f] [1,4]diazepines as
safe anxiolytic agents, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112405.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Masson SAS.

Graphical Abstract

Design, Synthesis and Biological Evaluation of 7-Substituted 4-Phenyl-
6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepines as Safe Anxiolytic
Agents
Angela Di Capua,a,¥ Annalisa Reale,a Marco Paolino,a Giulia Chemi,a,§ Simone Brogi,b Andrea
Cappelli,a Gianluca Giorgi,a Fedora Grande,c Lorenzo Di Cesare Mannelli,d Carla Ghelardini,d
Rosanna Matucci,d Antonio Garofalo,c and Maurizio Anzinia,*
a
Dipartimento di Biotecnologie, Chimica e Farmacia, (Dipartimento d’Eccellenza 2018-2022),
Università di Siena, Via Aldo Moro 2, 53100 Siena, Italy;
b Dipartimento di Farmacia, Università di Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy;
c
Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria,
Edificio Polifunzionale, 87036 Arcavacata di Rende (CS), Italy;
d Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino – Sezione di
Farmacologia e Tossicologia, Università di Firenze, Viale G. Pieraccini 6, I-50139 Firenze, Italy.
¥Present address: Department of Chemistry and Biochemistry, The Ohio State University,
Columbus, Ohio 43210, United States.
§Present address: Wellcome Centre for Anti-Infectives Research, Drug Discovery Unit, Division of
Biological Chemistry and Drug Discovery, University of Dundee, DD1 5EH Dundee, United
Kingdom.
Corresponding Author:
*Maurizio Anzini Tel. +390577234173, email: maurizio.anzini@unisi.it
Declarations of interest: none
1

Abstract
A series of 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate esters were
synthesized and tested as central benzodiazepine receptor (CBR) ligands by the ability to displace
[3H]flumazenil from rat cortical membranes. All the compounds showed high affinity with IC50
values ranging from 5.19 to 16.22 nM. In particular, compounds 12b (IC50 = 8.66 nM) and 12d
(IC50 = 5.19 nM) appeared as the most effective ligands being their affinity values significantly
lower than that of diazepam (IC50 = 18.52 nM). Compounds 12a-f were examined in vivo for their
pharmacological effects in mice and five potential benzodiazepine (BDZ) actions were thus taken
into consideration: anxiolytic, anticonvulsant, anti-amnesic, hypnotic, and locomotor activities. All
the new synthesized compounds were able to induce a significant antianxiety effect and, among
them, compound 12f protected pentylenetetrazole (PTZ)-induced convulsions in a dose-dependent
manner reaching a 40% effect at 30 mg/kg. In addition, all the compounds were able to significantly
prevent the memory impairment evoked by scopolamine, while none of them was able to interfere
with pentobarbital-evoked sleep and influence motor coordination. Moreover, title compounds did
not affect locomotor and exploratory activity at the same time and doses at which the anti-anxiety
effect was observed. Finally, molecular docking simulations were carried out in order to assess the
binding mode for compounds 12a-f. The obtained results demonstrated that these compounds bind
the BDZ binding site in a similar fashion to flumazenil.
2

1. Introduction
Anxiety may be a mood, a sensation or an emotional response. Anyhow, when it induces a reaction
to a particular intrapsychic feeling or feelings, accompanied by physiological changes, that may or
may not be outwardly manifested, it must be considered a disease.
Anxiety is per se a functional state until it allows to organise responses aimed at situations that
raise difficulties, while it becomes pathological when it lasts longer than the situations that have
evoked it or when it results in an exaggerated reaction. In pathological conditions, the growth of
anxiety is accompanied by a decrease of physical and mental performances [1].
Anxiety disorders are the most common psychiatric disorders worldwide and represent a
considerable burden in primary and secondary medical care [2].
According to the Diagnostic and Statistical Manual of Mental Disorders DSM IV-Tr (APA 2000)
the main debilitating anxiety disorders are: generalized anxiety disorder (GAD), panic disorder
(PD), obsessive-compulsive disorder (OCD), social anxiety disorder (SAD), and post-traumatic
stress disorder (PTSD) [3].
There are many psychotropic drugs and psychotherapies available for the management of anxiety
disorders above cited, but overall clinical outcomes and the standard of care for most patients are
far from optimal. In most cases current treatments, suffer from modest efficacy or low tolerability
[4].
Among the commonly used anxiolytics, only 1,4-benzodiazepines (BDZs) and β-adrenergic
antagonists are effective in the acute phase of such disorders [5]. Chronic treatment by
noradrenaline and serotonin re-uptake inhibitors (SNRI), selective serotonin re-uptake inhibitors
(SSRI) or 5-HT1A antagonists is required to produce and sustain anxiolytic effects over time [6].
Since the 1960s, when BDZs were discovered, they became the primary choice for the treatment of
many psychological disorders.
Although biochemistry alterations involved in anxiety have not yet been fully defined, a
considerable impulse certainly came from the knowledge of the mechanism of action of BDZs. In
fact, these drugs were shown to be able to specifically antagonize the classic symptomatology of
anxiety also in its reflections of somatization.
Serendipitously discovered in the late 1950s by Dr Leo H. Sternbach at Hoffmann-La Roche, BDZs
were found to be endowed with an array of therapeutically useful activities, including anxiolysis,
sedation, seizure suppression, sedative-hypnotic (sleep-inducing) and muscle relaxation
accompanied by an advantageous toxicological profile [7].
Between 1969 and 1982 diazepam (Valium®
) (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-
benzodiazepin-2-one) (1, Figure 1) was the most prescribed drug in the United States, with over 2.3
3

billion doses sold in 1978 [8]. Although these drugs are generally well tolerated, some risk of
dependence during long-term use and unwanted sedative effects are possible [9].
Only in 1974 different research groups independently demonstrated that there was a highly specific
potentiation of γ-aminobutyric acid (GABA) by means of BDZs, the mechanism of action of which
was unknown until 1977. Later, these compounds were proven to be able to interact with a specific
binding site in the central nervous system (CNS), named central benzodiazepine receptor (CBR),
which turned out to be an integral part of the type-A GABA receptor complex (GABAA) [10].
GABAA are receptors essential for the regulation of CNS functions. They belong to the Cys-loop
receptors, an important superfamily of ligand-gated ion channels (LGICs), including nicotinic
acetylcholine (nACh), 5-hydroxytryptamine subtype 3 (5-HT3) and strychnine-sensitive glycine
receptors [11].
The structure of GABAA consists of five subunits arranged pseudo-symmetrically around the
chloride ion channel. In particular, GABAA are generally hetero-oligomers and at least 19 related
subtypes are present in mammals comprising a variety of subunits that include α (subtypes 1–6), β
(1–4), γ (1–3), δ, ε, θ, π and ρ (1-3) [12].
GABAA heterogeneity is expected to provide the basis for flexibility in signal transduction and
drug-induced allosteric modulation.
However, the most common form of the GABAA contains α, β, and γ subunits and recombinant
receptors including these subunits mimic the biological, electrophysiological and pharmacological
properties of the native receptor. The BDZ binding site occurs at the interface of the α and γ
subunits and, although the γ2-subunit is essential to convey classical BDZ sites to recombinant
receptors, the α-subunit variants largely determine the pharmacological profiles [13]. Thus, type I
and type II, also referred to as ω1 and ω2 benzodiazepine receptors, are formed upon co-expression
of the subunits α1βxγ2 and α2- or α3βxγ2, respectively [13], while receptors containing the α4- or α6-
subunits have very low affinities for classical benzodiazepines, such as diazepam (1) [14].
Indeed, circuits operating with GABAA receptors, containing the α2-subunit, were found to mediate
anxiolysis while those containing the α1-subunit mediate sedation [15].
Although BDZ are not able to selectively target GABAA containing the α1, α2, α3, or α5 subunits
these compounds have been used in clinical practice for decades and are still among the most
widely prescribed drugs for the treatment of anxiety disorders even if their use is limited by side
effects and the risk of drug-dependence [8].
Alprazolam (Xanax®) (8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine)
(2), originally manufactured by Pfizer as a short acting anxiolytic, widely used for the treatment of
4

panic disorder with or without agoraphobia [16], surpassed diazepam in popularity in the late '80s
and to date is among the first fifty prescribed drugs [17].
This drug structurally belongs to the 1,2-annelated 1,4-benzodiazepines [18]. This class of
compounds, also referred to as tricyclic BDZ [19], deriving from the condensation of an additional
ring on the 1,2-bond of the classical 1,4-benzodiazepine scaffold, resulted in very interesting CBR
ligands [18]. An extensive chemical work aimed at the fusion of various heterocycles to this
position. In particular, the fusion of a s-triazole nucleus gave rise to new drugs of interest as
hypnotics such as triazolam (8-chloro-6-(2-chlorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-
a][1,4]benzodiazepine) (3) and estazolam (8-chloro-6-(2-chlorophenyl)-4H-[1,2,4]triazolo[4,3-
a][1,4]benzodiazepine) (4). The fusion of an imidazole to the same position generated imidazo-
benzodiazepines (iBDZ) such as
midazolam (8-chloro-6-(2-fluorophenyl)-1-methyl-4H-
imidazo[1,5-a][1,4]benzodiazepine) (5), used in anesthesia, and flumazenil (Anexate®) (ethyl 8-
fluoro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate, Ro 15-1788) (6) [20],
endowed with highly specific and competitive antagonistic activity at the CBR. This latter resulted
as an efficient antidote in the emergency departments to relieve from coma and respiratory
depression during the BDZ overdose.
iBDZ also include imidazenil (6-(2-bromophenyl)-8-fluoro-4H-imidazo[1,5-a][1,4]benzodiazepine-
3-carboxamide) (7) and the tetracyclic bretazenil (t-butyl-8-bromo-11,12,13,13a-tetrahydro-9-oxo-
9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate) (8). Imidazenil, a CBR partial
agonist with unusual profile of effects, although proposed as an effective anxiolytic and a potent
non-sedating anticonvulsant lacking many of the side effects associated with classical BDZ, was
never marketed for therapeutic use in humans [21].
Etizolam
(Pasaden®)
(4-(2-chlorophenyl)-2-ethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-
a][1,4]diazepine) [22] (9) and brotizolam (Lendormin®) (2-bromo-4-(2-chlorophenyl)-9-methyl-
6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4] diazepine (10) [23], bioisosteric analogues of
triazoleBDZ, showed interesting hypnotic, antianxiety and muscle relaxant properties. These
compounds enlarged the family of 1,2-annelated 1,4-BDZ giving rise to the new class of
thienotriazolodiazepines. In particular, etizolam is prescribed as a short-acting drug, with less side-
effects typical of BDZ full agonists, while brotizolam is widely used in anesthesia practice (Figure
1).
5

Figure 1. Structure of Diazepam and comparison of imidazo[1,5-a][1,4]benzodiazepine esters
11a,b and the most relevant tri- and tetracyclic compounds endowed with CBR activity.
Recently, the synthesis and evaluation of two imidazo[1,5-a][1,4]benzodiazepine esters (11a,b)
(Figure 1) were reported [24,25]. As a part of our continuing interest in the synthesis of novel
analogues [26–29], with the aim to in-depth investigate the binding mode to CBR, the synthesis of a
series of novel 7-substituted 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepines (12a-f) (Table
1) is herein reported. The biological activity of the title compounds was assayed in vitro to prove
that this new tricyclic BDZ belong to a privileged scaffold able to efficaciously interact with CBR
[30,31]. The anxiolytic effect was further investigated in vivo in order to assess an improved safety
for these compounds. Moreover, a computational investigation of the synthesized compounds was
performed to establish the binding mode to GABAA.
6

2. Results and Discussion
2.1 Chemistry
Table 1. Structures of 7-substituted 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepines 12a-f.
Cpd
12a
X
H
Y
H
R
Et
F
H
H
F
H
F
Et
12b
12c
12d
12e
12f
Et
H
H
F
t-Bu
t-Bu
t-Bu
H
A thiophene framework is present in several biologically active compounds of both natural and
synthetic origins [32]. Accordingly, the interest towards this heterocycle has spread in different
fields of chemistry, spanning from biodiagnostic to modern drug design [33,34].
In particular, the chemistry of 2-aminothiophenes has received much attention upon their
convenient availability through the versatile synthetic method developed by Gewald and co-workers
[35].
The core structure is formed in the multicomponent reaction (MCR) between a ketone or aldehyde,
an activated nitrile and sulfur in the presence of a suitable base.
Generally, there are four basic variations of this method and up to fifteen modifications reported to
accomplish the synthesis of highly functionalized 2-aminothiophenes [35]. The last version from
the basic Gewald’s method represents the latest improvement of the reaction. This synthesis is
based on the condensation and subsequent cyclization of an α-activated acetonitrile with the stable
dimeric forms of an α-sulfanylcarbonyl compound–(un)substituted 1,4-dithiane-2,5-diols, requiring
a stoichiometric amount of amine [36]. A series of (2-aminothiophen-3-yl)(phenyl)methanones 15a-
c was accordingly obtained by this procedure and used as starting materials for the synthesis of title
compounds (12a-f), as sketched in Scheme 1.
Briefly, the reaction of the appropriate benzoylacetonitrile (13a-c) with 2,5-dihydroxy-1,4-dithiane
(mercaptoacetaldehyde dimer, 14) in the presence of diethylamine, gave 2-amino-3-
benzoylthiophenes (15a-c) [36], which by means of the sequential treatment with bromoacetyl
bromide in dichloromethane and then with liquid ammonia in ethanol at reflux afforded the
expected 5-phenyl-1H-thieno[3,2-f][1,4]diazepin-2(3H)-ones (18a-c).
7

The imidazo-annulation of thienodiazepinones (18a-c) was accomplished via the intermediate enol
phosphate (19) by means of ethyl- or tert-butyl isocyanoacetate, in the presence of potassium tert-
butoxide, and provided the expected 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-
carboxylates (thienoimidazoesters) (12a-f) [24,37].
Scheme 1. Reagents and conditions: i) Et2NH, EtOH, 60 °C, 1 h; ii) BrCOCH2Br, CH2Cl2, NaHCO3
20%, rt 1 h; iii) liq. NH3 reflux, 4 h; iv) EtOH, reflux, 4 h; v) ClPO(OEt)2, t-BuOK, dry THF, 0 °C,
30 min; vi) NCCH2COOR, t-BuOK, 0 °C, 1 h/rt, 24 h.
2.2 In vitro assay
The binding affinity of 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylates
(12a-f) at the benzodiazepine receptor in rat cortical membranes was determined by means of
competition experiments against the radiolabeled antagonist [3H]flumazenil and expressed as the
IC50; all these compounds inhibited radioligand binding by more than 80% at a fixed concentration
of 10 μM. The results are reported in Table 2.
8

Table 2. Inhibition of [3H]flumazenil specific binding to rat cortical membranes of the title and
reference compounds.
Compound
12a
IC50 (nM)
11.65 ± 0.05
8.66 ± 0.08
16.22 ± 0.16
5.19 ± 0.09
9.61 ± 0.31
10.15 ± 0.29
4.5b
12b
12c
12d
12e
12f
Etizolam
Brotizolam
Flumazenil
Diazepam
2.6 ± 0.6 c
2.3 ± 0.6d
18.52 ± 0.5
aBrain cortex membranes were incubated with 0.2 nM [3H]flumazenil ([3H]Ro 15-1788]) in absence and
presence of increasing concentrations of the indicated compounds. Non-specific binding was determined in
the presence of 10 µM of diazepam. Concentration-response curves were analyzed using the curve-fitting
program GraphPad Prism. Ten concentrations of displacers were examined each in duplicate. Each value was
the average ± S.E.M. of 4-6 independent experiments. bSee ref.[22]. cSee ref. [38]. dSee ref. [39].
Table 2 summarizes the biological data and it appears that all the newly synthesized compounds are
more effective than diazepam (IC50 = 18.52 nM) with IC50 ranging from 5.19 to 16.22 nM. In
particular, 12b and 12d seem to be the most active compounds showing IC50 values of 8.66 and
5.19 nM, respectively, lower than that of diazepam, while the same compounds are both equipotent
with etizolam (4.50 nM) [22], but, to some extent less effective than brotizolam (2.6 nM) [38]. On
the basis of the structure-affinity relationships it appears that the condensation of thiophen[3,2-
f][1,4]diazepine scaffold with the imidazole ring as the carboisoster of triazole nucleus present in
etizolam and brotizolam, still leads to active ligands able to bind CBR in nanomolar concentration
range.
Beside the hydrophobic interaction of the whole molecule, the tert-butyl- or ethyl ester moiety at
position 3 seems to be essential for a fruitful interaction at the CBR binding site for all the tested
compounds. This behavior seems to be due to polar contacts between the carboxyalkyl group and
T207 and T142 side chains by means of two hydrogen bonds, in analogy with what previously
reported for compounds 11a,b [24].
9

The effect elicited by the fluorine atom at 3- or 4-position of the pendant 4-phenyl ring seems to be
less important in both groups of thienoimidazo esters, where halogenated compounds 12c and 12e,f
result less active with respect to unsubstituted derivatives 12a and 12d.
Furthermore, this behavior appears to be contradictory when the single class of thienoimidazo ester
is considered. In fact, in the series of imidazoethyl esters 12a-c, 3-fluorine derivative 12b shows to
be more active than both the unsubstituted compound 12a and its 4-fluorine analog 12c.
On the basis of these results, it is possible to conclude that the presence of a fluorine atom in
compounds 12e-f, regardless the specific position, is able to somehow modulate the interaction at
CBR binding site. The presence of the fluorine atom in the para-position of the pendant phenyl
ring (compounds 12c and 12f) may constitute a metabolic drawback to a probable hydroxylation by
means of cytochrome P-450, leading to better pharmacokinetic properties.
2.3 Molecular Modelling studies
The binding mode of the developed derivatives was investigated by means of a molecular docking
protocol (see Experimental section for further details). We used a recently solved structure of a
synaptic GABAA receptor composed by α1β2γ2 subunits appropriately prepared, along with the
synthesized ligands (Table 1), employing Glide software. The output was depicted in Figure 2.
Figure 2. In panel A-F are reported the representative poses of compounds 12a-f (represented as
colored sticks), respectively into the BDZ binding site at GABAA receptor (PDB ID: 6D6U, in light
green cartoon). The interacting residues within the binding site are represented by lines (the
residues F100, H102, Y160, S205, S206, T207 and Y210 belonging to the α1 subunit, while the
10

residues Y58, F77, T142 from γ2 subunit). H-bonds are represented as black dotted line. The picture
was generated by PyMOL (The PyMOL Molecular Graphics System, version 1.8.4.0, Schrödinger,
LLC, New York, 2018).
Concisely, our developed compounds bind the BDZ binding site in a similar fashion compared to
that experimentally found for flumazenil. This latter establishes polar contacts with the sidechain of
T142 by its ethyl ester group. The tricyclic system is involved in π-π stacking with F77 by its
imidazole moiety and with Y210 by the fluorobenzene portion. Regarding our compounds, we
observed that they are able to target the mentioned residues. In fact, the series 12a-c (Figure 2A-C)
targets T142 by means of a H-bond with the sidechain of T142 established by the nitrogen of
imidazole. In addition, 12a-c interact by π-π stacking with F77 and Y210 through the imidazole and
thiophene rings, respectively. Interestingly, compounds 12a-c could be able to target S205 with the
nitrogen of the hepta-ring and the sidechain of S206 with the ethyl ester group. This latter was
replaced by a tert-butyl ester group in compounds 12d-f. This group is responsible for the
differences observed in the binding modes with respect to those found for 12a-c. The tert-butyl
ester group forms a H-bond with T142. In compounds 12d-f, we observed a different pattern of
interaction compared to that found for the series 12a-c. In particular, the imidazole moiety from the
tricyclic scaffold establishes a π-π stacking with F77, while the thiophene targets Y210, and the
tricyclic moiety interacts with S205. All three compounds lack the bond with S206 as observed for
12a-c. The docking scores reported in Table S1 revealed that the compounds have a similar SP
score, supporting the similar experimentally determined binding potencies. On the contrary,
flumazenil showed an SP score -1 kcal/mol lower than that of our derivative. This could be an
indication of its improved activity against GABAA (Table 2). Moreover, also the ligand efficiency
values (Table S1) confirm the observed trend regarding the activity of the mentioned compounds.
2.4 In vivo assay
Compounds 12a-f were examined in mice in vivo to evaluate their pharmacological effects. Five
potential benzodiazepine actions were considered: potential anxiolytic effects screened using
light/dark choice test, the anticonvulsant action evaluated by means of the new compounds against
pentylenetetrazole (PTZ)-induced convulsions, possible anti-amnesic properties in the passive
avoidance test, pentobarbital-induced sleep test, and finally motor abilities verified by the rotarod as
well as the hole-board tests.
Using the light/dark box test, the time spent by the animal in the lighted compart was evaluated as
an index of anxiolytic effect. Diazepam (10 mg/kg subcutaneously - s.c.) increased the time up to
11

165.6 ± 9.1 s in comparison to 122.3 ± 8.5 s of controls (saline-treated) (Table 3). All the new
synthesized compounds were able to induce a significant antianxiety effect when dosed at 30 mg/kg
s.c., in particular 12a, 12b and 12f were as effective as diazepam. 12f maintained its efficacy also at
the lower dose of 10 mg/kg whereas the other compounds did not.
Table 3. Anti-anxiety effect, light-dark box test.
Treatment
Dose (mg/kg)
Persistent time in light box (min)
VEHICLE
122.3 ± 8.5
10
30
10
30
10
30
10
30
131.3 ± 7.9
166.2 ± 8.8*
111.3 ± 7.9
163.9 ± 6.4*
109.8 ± 6.7
151.2 ± 8.3*
113.6 ± 9.1
151.4 ± 8.5*
12a
12a
12b
12b
12c
12c
12d
12d
10
30
10
118.3 ± 8.8
143.8 ± 8.7*
145.6 ± 9.3*
12e
12e
12f
30
10
169.4 ± 8.2*
165.6± 9.1*
12f
DIAZEPAM
All compounds were administered p.o. 30 min before the test. Each value represents the mean ± S.E.M. of 10
mice; P<0.05 vs saline.
As shown in Table 4, PTZ (90 mg/kg intraperitoneally - i.p.) induced convulsions that respond to
diazepam, as a reference compound its effect was considered as 100%. 12f dose-dependently
protected PTZ-induced convulsion reaching the 40% when dosed at 30 mg/kg. The same dose of
12b and 12d protected by 20%.
Table 4. Effect on PTZ-induced convulsions.
Treatment
Dose (mg/kg)
% Protection on PTZ induced convulsions
30
10
12a
12

30
30
30
30
10
30
10
20
0
12b
12c
20
0
12d
12e
20
40
100
12f
12f
D
IAZEPAM
All compounds were administered p.o. 30 min before the test, PTZ (90 mg/kg) was injected immediately
before the test and animals were observed for 30 min. Each value represents the mean of 10 mice.
The passive avoidance test allows to evaluate the improvement of memory ability in the presence of
a scopolamine (1.5 mg/kg i.p.)-induced deficit. All compounds (30 mg/kg) were able to
significantly prevent the memory impairment evoked by scopolamine, while 12a was active also at
10 mg/kg (Table 5). None of the new synthesized compounds was able to interfere with
pentobarbital-evoked sleep, a kind of inhibition sensitive to amphetamine (Table 6). Interestingly,
the tested compounds did not influence motor coordination as measured by the rota-rod test (Table
7), moreover neither locomotor activity nor exploratory activity were altered by compounds at the
same time and doses at which the anti-anxiety effect was observed (Table 8).
Table 5. Anti-amnesic effect, passive avoidance test
Dose (mg/kg)
Training session
Retention session
Treatment
V
EHICLE
+
VEHICLE
-
16.6 ± 3.1
14.2 ± 2.2
13.6 ± 2.5
18.1 ± 3.0
102.9 ± 8.8
46.2 ± 8.9
V
EHICLE + SCOPOLAMINE
12a + SCOPOLAMINE
12a + SCOPOLAMINE
-
10
30
54.1 ± 8.2
88.6 ± 9.5**
12b + SCOPOLAMINE
12b + SCOPOLAMINE
12c + SCOPOLAMINE
10
30
10
14.9 ± 2.7
17.4 ± 2.5
13.9 ± 3.1
39.2 ± 6.8
75.3 ± 8.1**
55.8 ± 7.4
12c + SCOPOLAMINE
30
14.7 ± 3.0
89.2 ± 9.3**
12d + SCOPOLAMINE
12d + SCOPOLAMINE
10
30
15.4 ± 3.1
17.2 ± 2.2
46.9 ± 8.5
70.3 ± 8.1**
12e + SCOPOLAMINE
10
18.2 ± 2.8
51.4 ± 7.7
12e + SCOPOLAMINE
12f + SCOPOLAMINE
12f + SCOPOLAMINE
30
10
30
15.6 ± 2.0
12.8 ± 2.9
16.5 ± 2.3
72.5 ± 8.1**
67.1 ± 8.2*
85.9 ± 8.0**
13

All compounds were administered p.o. 30 min before the test. Scopolamine (1.5 mg/kg i.p.) was
administered immediately after punishment. Each value represents the mean the mean ± S.E.M of 10 mice.
*P< 0.05; **P< 0.01 in comparison with vehicle + scopolamine.
Table 6. Effect against pentobarbital-induced sleep
Dose (mg/kg)
Induction time (min)
Endurance time (min)
Treatment
V
EHICLE
-
4.4 ± 1.1
3.9 ± 1.0
27.4 ± 2.3
23.8 ± 5.1
30
12a
30
30
30
30
30
2.0
5.2 ± 1.3
5.5 ± 2.1
4.4 ± 1.3
3.5 ± 1.3
4.7 ± 1.5
24.9 ± 4.3
29.2 ± 4.4
28.8 ± 3.1
25.9 ± 3.0
27.2 ± 3.6
22.2± 2.2*
12b
12c
12d
12e
12f
A
MPHETAMINE
The new compounds were administered p.o., amphetamine s.c., 30 min before the test. Pentobarbital was i.p.
injected at 40 mg/kg. Each value represents the mean the mean ± S.E.M of 10 mice. *P< 0.05 in comparison
with vehicle.
Table 7. Effect on motor coordination, rotarod test
F
ALLS IN 30
S
30 min
Treatment
Dose (mg/kg)
Pre-test
V
EHICLE
12a
5.5 ± 0.5
4.7± 0.4
5.3 ± 0.5
5.2 ± 0.5
5.1± 0.4
4.9± 0.5
5.5 ± 0.5
2.2 ± 0.2
1.9 ± 0.2
2.5 ± 0.4
2.2 ± 0.3
2.4 ± 0.4
1.9 ± 0.4
2.1 ± 0.3
30
30
30
30
30
30
12b
12c
12d
12e
12f
Compounds were administered p.o. 30 min before the test. Each value represents the mean ± S.E.M of 10
mice.
Table 8. Effect on locomotor and explorative activities, hole-board test
Treatment
CMC
12a
Dose (mg/kg)
n° of movements
31.2 ± 8.2
n° of movements
18.3 ± 6.2
30
30
28.2 ± 6.9
19.1 ± 6.5
12b
27.3 ± 6.2
15.3 ± 4.4
14

12c
12d
12e
12f
30
30
30
30
34.5 ± 7.6
33.4 ± 6.7
29.5 ± 7.7
26.7 ± 5.5
15.8 ± 7.4
15.9 ± 6.2
17.5 ± 7.1
17.5 ± 7.1
Compounds were administered p.o. 30 min before the test. Each value represents the mean ± S.E.M of 10
mice.
3. Conclusion
A
series of hitherto unknown 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepines-7-
carboxylates was designed and synthesized as central benzodiazepine receptor ligands. All the title
compounds showed high affinity for CBR with IC50 values ranging from 5.19 to 16.22 nM and they
resulted almost equal or even more potent than diazepam (IC50 18.52 nM). Various animal models
used to assess the pharmacological effects of novel compounds demonstrated that all of the novel
compounds were able to induce a significant antianxiety effect, and among them compound 12f
showed dose-dependently protected PTZ-induced convulsion reaching a 40% effect at a 30 mg/kg
dose. In addition, all the compounds were able to significantly prevent the memory impairment
evoked by scopolamine, while none of them was able to interfere with pentobarbital-evoked sleep
and influence motor coordination. Moreover, neither locomotor activity nor exploratory activity
were altered by title compounds at the same time and doses at which the anti-anxiety effect was
observed. Finally, molecular docking simulations performed to assess the binding modes of
compounds 12a-f, demonstrated that these compounds interact with the BDZ binding site in a
similar fashion compared to what experimentally found for flumazenil. The results obtained in in
vitro and in vivo test demonstrated that, when opportunely decorated, the tricyclic structure of 4H-
imidazo[1,5-a]thieno[3,2-f][1,4]diazepines (12a-f) represents a molecular framework able to
provide new 1,2-annelated 1,4-BDZ as useful ligands for CBR that may serve as the basis for the
development of new drug candidates.
4. Materials and Methods
4.1 Chemistry
All chemicals used were of reagent grade. Yields refer to purified products and are not optimized.
Melting points were determined in open capillaries on a Gallenkamp apparatus and are uncorrected.
Merck silica gel 60 (230–400 mesh) was used for column chromatography. Merck TLC plates and
1
13
silica gel 60 F254 were used for TLC. H- and C-NMR spectra were recorded with a Bruker AC
15

400 spectrometer in the indicated solvent (TMS as internal standard). The values of the chemical
shifts are expressed in ppm, and the coupling constants (J) are expressed in Hz. Mass spectra were
recorded using an Agilent 1100 LC/MSD operating with an electrospray source. Purity of
compounds 12a-f was assessed by RP-HPLC and was found to be higher than 95%. A Zorbax
Eclipse XDB-C8 column (4.6x150 mm, 5 μm) was used in the HPLC analysis with acetonitrile–
water-methanol (20:30:50) as the mobile phase at a flow rate of 0.5 mL/min. UV detection was
achieved at 254 nm.
4.1.1 General Procedure for the Synthesis of 4-Phenyl-6H-imidazo[1,5-a]thieno[3,2-
f][1,4]diazepine-7-carboxylates (12a-f).
A mixture of the suitable thienodiazepin-2-one 18a-c (1.2 mmol) and potassium tert-butoxide (2.7
mmol) in dry THF (30 mL) was stirred at 0 °C for 10 min under argon atmosphere. Then, diethyl
chlorophosphate (4.6 mmol) was added and the reaction mixture was stirred for additional 30 min.
Subsequently, a suitable isocyanoacetate (6.1 mmol) was added and the reaction was conducted 1 h
at 0 °C and then, overnight at room temperature. Acetic acid (1.8 mL) was added, and the mixture
was stirred for additional 20 min and then poured onto crushed ice to give a brownish solid. After
filtration, the solid was dissolved in dichloromethane and the organic layer washed with brine, dried
over Na2SO4, filtered, and concentrated at reduced pressure. The residue was purified by flash
chromatography with the suitable eluent to afford the expected imidazo ester, which was
recrystallized from the proper solvent [25].
4.1.2 Ethyl 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate (12a).
The title compound was prepared starting from 18a following the above-cited general procedure
(0.20 g, 49% yield) and using a mixture CH2Cl2 /EtOAc 1:1 (v/v) as eluent. An analytical sample
1
was prepared by recrystallization from cyclohexane/EtOAc (m.p. 190.0-191.5 °C). H NMR (400
MHz, CDCl3) δ (ppm): 1.41 (t, 3H, J = 7.0), 4.40 (q, 2H, J = 7.0), 5.13 (bs, 2H), 6.98 (d, 1H, J =
5.6), 7.16 (d, 1H, J = 5.6), 7.36-7.43 (m, 3H), 7.56 (d, 2H, J = 7.2), 7.81 (s, 1H). MS(ESI): m/z
337.8 (M + H+).
16

Figure 3. Crystallographic structure of compound 12a. Ellipsoids enclose 50% probability.
4.1.3 Ethyl 4-(3-fluorophenyl)-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate
(12b).
The title compound was prepared starting from 18b following the above-cited general procedure
(0.19 g, yield 45%) and using a mixture CH2Cl2 /EtOAc 1:1 (v/v) as eluent. An analytical sample
was obtained as yellow needles by recrystallization from cyclohexane/EtOAc (m.p. 230.0-231.0
°C). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.41 (t, 3H, J = 7.0), 4.41 (q, 2H, J = 7.0), 5.13 (bs, 2H),
6.98 (d, 1H, J = 5.6), 7.12 (m, 1H), 7.18 (d, 1H, J = 5.6), 7.32 (m, 3H), 7.81 (s, 1H). MS(ESI): m/z
377.8 (M + Na+).
4.1.4 Ethyl 4-(4-fluorophenyl)-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate
(12c).
The title compound was prepared starting from 18c following the above-cited general procedure
(0.21 g, yield 49%) and using a mixture CH2Cl2 /EtOAc 1:1 (v/v) as eluent. An analytical sample
was obtained as yellow needles by recrystallization from cyclohexane/EtOAc (m.p. 225.0-226.0
°C). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.38 (t, 3H, J = 7.0), 4.38 (q, 2H, J = 7.0), 5.08 (bs, 2H),
6.95 (d, 1H, J = 5.6), 7.03 (t, 2H, J = 8.6), 7.16 (d, 1H, J = 5.6), 7.55 (t, 2H, J = 4.0), 7.79 (s, 1H).
MS(ESI): m/z 393.9 (M + K+).
4.1.5 tert-Butyl 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate (12d).
The title compound was prepared starting from 18d following the above-cited general procedure
(0.25 g, yield 57%) and using a mixture CH2Cl2 /EtOAc 1:1 (v/v) as eluent. An analytical sample
17

was obtained as yellow needles by recrystallization from cyclohexane/EtOAc (m.p. 144.0-145.0
°C). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.60 (s, 9H), 5.09 (bs, 2H), 6.95 (d, 1H, J = 5.6), 7.13 (d,
1H, J = 5.6), 7.29-7.40 (m, 3H), 7.55 (d, 2H, J = 7.2), 7.77 (s, 1H). MS(ESI): m/z 365.8 (M + H+).
4.1.6 tert-Butyl 4-(3-fluorophenyl)-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate
(12e).
The title compound was prepared starting from 18e following the above-cited general procedure
(0.27 g, yield 59%) and using a mixture CH2Cl2 /EtOAc 1:1 (v/v) as eluent. An analytical sample
was obtained as yellow needles by recrystallization from cyclohexane/EtOAc (m.p. 175.0-176.0
1
°C). H NMR (400 MHz, CDCl3) δ (ppm): 1.60 (s, 9H), 5.09 (bs, 2H), 6.96 (d, 1H, J = 5.6), 7.12
(m, 1H), 7.16 (d, 1H, J = 5.6), 7.32 (m, 3H), 7.78 (s, 1H). MS(ESI): m/z 405.8 (M + Na+).
4.1.7 tert-Butyl 4-(4-fluorophenyl)-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate
(12f).
The title compound was prepared starting from 18f following the above-cited general procedure
(0.27 g, yield 59%) and using a mixture CH2Cl2 /EtOAc 1:1 (v/v) as eluent. An analytical sample
was obtained as yellow needles by recrystallization from cyclohexane/EtOAc (m.p. 190.0-191.0
1
°C). H NMR (400 MHz, CDCl3) δ (ppm): 1.61 (s, 9H), 5.08 (bs, 2H), 6.95 (d, 1H, J = 5.6), 7.05
(m, 2H), 7.16 (d, 1H, J = 5.6), 7.58 (m, 2H), 7.78 (s, 1H). MS(ESI): m/z 405.8 (M+ Na+).
4.2 X-Ray Crystallography
A single crystal of 12a and 18c was submitted to X-ray data collection by means of a Siemens P4
four circled diffractometer at 293 K equipped with a graphite monochromated Mo KR radiation (λ)
0.71069 Å. The structure was solved by direct methods implemented in the SHELXS-97 program
[40]. The refinements were carried out by full-matrix anisotropic least-squares on F2 for all
reflections for non-H atoms by means of the SHELXL-97 program. Crystallographic data
(excluding structure factors) of these crystal structures have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication numbers CCDC 1967523 (12a) and
CCDC 1917567 (18c). Copies of the data can be obtained, free of charge, on application to CCDC,
12 Union Road, Cambridge CB2 1EZ, UK [fax: 144-(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk].
4.3 Radioligand Binding Studies
18

[3H]flumazenil (specific activity 70.8 Ci/mmol) was obtained from Perkin-Elmer Life and
Analytical Science (Monza, Italy). All other chemicals were reagent grade and were obtained from
commercial suppliers.
Membrane preparation
Membranes were prepared essentially as previously described [41]. Briefly, cerebral cortex from
male CD-1 albino mice (22-24 g) were homogenized using a glass/teflon homogenizer in 10
volumes of ice cold 0.32 M sucrose containing protease inhibitors. The homogenate was
centrifuged at 1,000g for 10 min at 4 °C. The resulting pellet was discarded and the supernatant was
centrifuged at 30,000g for 15 min at 4 °C. Then the pellet was resuspended and dispersed with an
Ultra-Turrax homogenizer (IKA Labortechnik, Staufen, Germany, setting 3 for 30 sec) in 10
volumes of 50 mM Tris-citrate buffer at pH 7.4 containing protease inhibitors and centrifuged again
at 30,000g for 15 min at 4 °C. This washing step was repeated twice. The resulting crude
membranes were frozen (about 0.2 g of tissue each tube) at -80 °C. After freezing, the membranes
were defrozen and washed by means of a procedure previously described [41]: the pellet was
resuspended with 0.32 volumes of 50 mM Tris-citrate buffer at pH 7.4 containing 0.01% of tritonX-
100 using an Ultra Turrax for 30 sec at the half of the maximum speed. The suspension was
incubated under gentle agitation at 4 °C for 60 min and then centrifuged 30,000g for 30 min at 4 °C.
At the end of the process the pellet was resuspended in the same buffer by an Ultra Turrax and
centrifuged again: this washing step was repeated three times and finally the suspension was used in
the binding assay. The protein content was defined according to the method of Bradford [41] using
the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Munchen, West Germany) and bovine
serum albumin was used as the standard.
[3H]flumazenil binding
Binding tests were carried out in duplicate in a final volume of 0.5 mL in Tris-citrate buffer;
aliquots of the membrane suspension containing about 0.5 mg/mL of proteins were added to the
incubation mixture with approximately 0.2 nM [3H]flumazenil and with or without different
concentrations of the tested compounds (0.01 nM-100 µM) [25]. Nonspecific binding was defined
in the presence of 10 μM cold diazepam. After incubation in ice for 90 min, the binding reaction
was interrupted by adding 5 mL of ice-cold water, the bound radioactivity was separated by rapid
filtration through glass-fiber paper MGB (Sartorius Italy S.r.l., Bagno a Ripoli, Firenze, Italy) in a
Brandel harvester and washed with 3 × 5 mL of ice-cold water.
The filters were dried and placed in vials containing 4 mL of Filter Count (Perkin Elmer Life and
Analytical Sciences, Monza, Italy) scintillation fluid and counted with a liquid scintillation counter
19

(TriCarb 1900TR, Perkin Elmer Life and Analytical Sciences, Monza, Italy). Specific binding was
obtained by subtracting non-specific binding from total binding and was approximately 85–90% of
the total binding. Data from competition curves were analyzed by a least-squares curve-fitting
computer program (GraphPad Prism, version 5.02) and the IC50 values were derived. Data are
presented as mean ± S.E.M.
4.4 Pharmacological Methods
The ethical policy of the University of Florence complies with the Guide for the Care and Use of
Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85-23, revised
1996). Formal approval to conduct the described experiments was obtained from the Animal
Subjects Review Board of the University of Florence. All experiments were carried out according to
the Directive 2010/63/EU of the European parliament and the European Union council (22
September 2010) on the protection of animals used for scientific purposes. All efforts were made to
minimize animal suffering and to reduce the number of animals involved. Male CD-1 albino mice
(22-24 g) and male Swiss Webster (20-26 g) (Envigo, Italy) were used. Twelve mice were housed
per cage and fed a standard laboratory diet, with tap water ad libitum for 12 h/12 h light/dark cycles
(lights on at 7:00). The cages were brought into the experimental room the day before the
experiment, for acclimatization purposes. All experiments were performed between 10:00 and
15:00.
Light/Dark Box Test. The apparatus (50 cm long, 20 cm wide, and 20 cm high) consisted of two
equal acrylic compartments, one dark and one light, illuminated by a 60 W bulb lamp and separated
by a divider with a 10 cm × 3 cm opening at floor level. Each mouse was tested by placing it in the
center of the lighted area, away from the dark one, and allowing it to explore the novel environment
for 5 min. The number of transfers from one compartment to the other and the time spent in the
illuminated side were measured. This test exploited the conflict between the animal’s tendency to
explore a new environment and its fear of bright light [42].
Pentylenetetrazole (PTZ)-Induced Seizure. PTZ (90 mg/kg s.c.) was injected 30 min after the
administration of drugs. The frequency of the occurrence of clonic generalized convulsions was
noted over a period of 30 min [43].
Passive-Avoidance Test. The test was performed according to the step-through method described
by Jarvik et al. [44]. The apparatus consisted of a two-compartment acrylic box with a lighted
compartment connected to a darkened one by a guillotine door. As soon as the mouse entered the
dark compartment, it received a thermal shock punishment. The latency times for entering the dark
20

compartment were measured in the training test and after 24 h in the retention test. The maximum
entry latency allowed in the training and retention sessions was, respectively, 60 and 180 s.
Pentobarbital-Induced Sleep Test. Pentobarbital (40 mg/kg i.p.) was injected 30 min after drug
administration. The duration of a loss of the righting reflex was measured as the sleep time. The
induction and endurance times were recorded.
Rota-Rod Test. The integrity of the animals’ motor coordination was assessed using a rota-rod
apparatus (Ugo Basile, Varese, Italy) at a rotating speed of 16 rpm. The treatment was performed
before the test. The numbers of falls from the rod were counted for 30 s, 30 min after drug
administration, and the test was performed according to the method described by Vaught et al. [45].
Hole-Board test. The locomotor activity was evaluated by using the hole-board test [46]. Mice
were placed on the center of the board one by one and allowed to move about freely for a period of
5 min each. Two photobeams, crossing the plane from mid-point to mid-point of opposite sides,
thus dividing the plane into 4 equal quadrants, automatically signaled the movement of the animal
(counts in 5 min) on the surface of the plane (locomotor activity). Miniature photoelectric cells, in
each of the 16 holes, recorded (counts in 5 min) the exploration of the holes (exploratory activity)
by the mice.
Drugs. Diazepam (Valium 10) (Roche, Italy), pentylenetetrazole (PTZ), amphetamine and
pentobarbital (Sigma-Aldrich, Italy), were used. All drugs were dissolved into isotonic (NaCl 0.9%)
saline solution and injected s.c./i.p. The new compounds were administered by the p.o. route and
were suspended in 1% carboxymethylcellulose sodium salt and sonicated immediately before use.
Drug concentrations were prepared in such a way that the necessary dose could be administered in a
10 mL/kg volume of carboxymethylcellulose (CMC) 1% by the p.o., i.p., or s.c. routes.
Statistical Analysis. All experimental results are given as the mean (SEM). An analysis of
variance, ANOVA, followed by Fisher’s protected least significant difference procedure for post
hoc comparison, were used to verify significance between two means of behavioral results. The data
were analyzed with the StataView software for Macintosh (1992). P values of less than 0.05 were
considered significant.
4.5 Computational details
Protein and ligands preparation. A recently solved three-dimensional structures of GABAA
receptor (PDB ID: 6D6U [47], in complex with flumazenil) was used for the computational study.
The protein was downloaded from PDB, compounds used for the crystallization were removed from
the available experimental structures. The resulting protein submitted to protein preparation
protocol in Maestro drug discovery suite release 2018 (Maestro release 2018, Schrödinger LLC,
21

New York, NY, 2018) as already described by us [48–50]. Ligands were built in Maestro using
available drawing tools, while flumazenil was extracted from 6D6U. Compounds were energy
minimized by means of MacroModel (Schrödinger release 2018, Schrödinger LLC, New York, NY,
2018) and subsequently treated by LigPrep (Schrödinger release 2018, Schrödinger LLC, New
York, NY, 2018) in order to obtain the most probable ionization state at cellular pH (7.4 ± 0.2)
[51,52].
Molecular Docking. Glide software (Schrödinger release 2018, Schrödinger LLC, New York, NY,
2018) was used for performing the computational investigation. We used the protein and the
ligands, prepared as explained in the previous paragraph, for molecular docking calculation using
Glide standard precision (SP) scoring function [53,54]. Energy grid was prepared using default
protein atom scaling factor value (1.0 Å) within a cubic box centered on the co-crystallized ligand
(flumazenil). After grid generation, molecular docking was performed in SP mode without
constraints. For post-docking minimization, the number of poses considered was set to 100. After
that, Glide SP score was evaluated. In order to assess the validity of docking protocol, flumazenil
was used as reference compound for a re-docking procedure as depicted in Supporting Information.
The docking results revealed a similar accommodation for the above-mentioned reference
compound with respect to the crystal structure with low RMSD (data not shown) [55].
Supplementary Materials
Experimental details for the synthesis and characterization of intermediates 15a-c, 16a-c, 17a-c, and
18a-c; additional figures from computational study. Supplementary material related to this article
can be found, in the online version, at doi:
Author Contribution
A.D.C., A.R., M.P., A.C., F.G., A.G. performed the synthesis and characterization of the
compounds; G.C, S.B. performed the computational studies; G.G. performed the X-ray studies;
L.D.C.M., C.G., R.M. performed the in vitro and in vivo pharmacological studies; M.A. designed
and coordinated the work.
Conflict of Interest
Declarations of interest: none
22

Acknowledgements
The authors wish to thank Dr. Francesco Berrettini (University of Siena) for X-ray acquisition data.
ABBREVIATIONS USED
CBR, central benzodiazepine receptor; BDZ, benzodiazepine; GABA, γ-aminobutyric acid; CNS,
central nervous system; CMC, carboxymethylcellulose; RMSD, root-mean-square deviation; iBDZ,
imidazobenzodiazepine; PDB, protein data bank; PTZ, pentylenetetrazole.
23

REFERENCES
[1] M. Garner, H. Möhler, D.J. Stein, T. Mueggler, D.S. Baldwin, Research in anxiety disorders:
From the bench to the bedside, Eur. Neuropsychopharmacol. 19 (2009) 381–390.
doi:https://doi.org/10.1016/j.euroneuro.2009.01.011.
[2] B. Bandelow, S. Michaelis, Epidemiology of anxiety disorders in the 21st century, Dialogues
Clin. Neurosci. 17 (2015) 327–335. https://www.ncbi.nlm.nih.gov/pubmed/26487813.
[3] S.B. GUZE, Diagnostic and Statistical Manual of Mental Disorders, 4th ed. (DSM-IV), Am.
J. Psychiatry. 152 (1995) 1228. doi:10.1176/ajp.152.8.1228.
[4] M.J. Millan, The neurobiology and control of anxious states, Prog. Neurobiol. 70 (2003) 83–
244. doi:https://doi.org/10.1016/S0301-0082(03)00087-X.
[5] T. P. Dooley, Treating Anxiety with either Beta Blockers or Antiemetic Antimuscarinic
Drugs: A Review, Ment. Health Fam. Med. 11 (2015) 89–99. doi:10.25149/1756-
8358.1102013.
[6] D.S. Baldwin, M. Garner, Chapter 5.2 How effective are current drug treatments for anxiety
disorders, and how could they be improved?, in: R.J. Blanchard, D.C. Blanchard, G. Griebel,
D.B.T.-H. of B.N. Nutt (Eds.), Handb. Anxiety Fear, Elsevier, 2008: pp. 395–411.
doi:https://doi.org/10.1016/S1569-7339(07)00018-5.
[7] L.H. Sternbach, The benzodiazepine story, J. Med. Chem. 22 (1979) 1–7.
doi:10.1021/jm00187a001.
[8] B. Dell’osso, M. Lader, Do benzodiazepines still deserve a major role in the treatment of
psychiatric disorders? A critical reappraisal, Eur. Psychiatry. 28 (2013) 7–20.
doi:https://doi.org/10.1016/j.eurpsy.2011.11.003.
[9] M. Lader, Benzodiazepines revisited—will we ever learn?, Addiction. 106 (2011) 2086–
2109. doi:10.1111/j.1360-0443.2011.03563.x.
[10] R. Squires, GABA and Benzodiazepine Receptors, CRC Press, Boca Raton, FL, 1988.
[11] C.N. Connolly, K.A. Wafford, The Cys-loop superfamily of ligand-gated ion channels: the
impact of receptor structure on function, Biochem. Soc. Trans. 32 (2004) 529–534.
doi:10.1042/bst0320529.
[12] R.W. Olsen, W. Sieghart, GABAA receptors: Subtypes provide diversity of function and
pharmacology, Neuropharmacology. 56 (2009) 141–148.
doi:https://doi.org/10.1016/j.neuropharm.2008.07.045.
[13] E.A. Barnard, P. Skolnick, R.W. Olsen, H. Mohler, W. Sieghart, G. Biggio, C. Braestrup,
A.N. Bateson, S.Z. Langer, International Union of Pharmacology. XV. Subtypes of γ-
24

Aminobutyric Acid&lt;sub&gt;A&lt;/sub&gt; Receptors: Classification on the Basis of
Subunit Structure and Receptor Function, Pharmacol. Rev. 50 (1998) 291 LP – 314.
http://pharmrev.aspetjournals.org/content/50/2/291.abstract.
[14] H. Lüddens, D.B. Pritchett, M. Köhler, I. Killisch, K. Keinänen, H. Monyer, R. Sprengel,
P.H. Seeburg, Cerebellar GABAA receptor selective for a behavioural alcohol antagonist,
Nature. 346 (1990) 648–651. doi:10.1038/346648a0.
[15] K. Löw, F. Crestani, R. Keist, D. Benke, I. Brünig, J.A. Benson, J.-M. Fritschy, T. Rülicke,
H. Bluethmann, H. Möhler, U. Rudolph, Molecular and Neuronal Substrate for the Selective
Attenuation of Anxiety, Science (80-. ). 290 (2000) 131 LP – 134.
doi:10.1126/science.290.5489.131.
[16] R. Noyes, G.D. Burrows, J.H. Reich, F.K. Judd, M.J. Garvey, T.R. Norman, B.L. Cook, P.
Marriott, Diazepam versus alprazolam for the treatment of panic disorder, J. Clin. Psychiatry.
57 (1996) 349—355. http://europepmc.org/abstract/MED/8752017.
[17] V.A. Fuentes, D.M. Pineda, C.N.K. Venkata, Comprehension of Top 200 Prescribed Drugs
in the US as a Resource for Pharmacy Teaching, Training and Practice, Pharm. . 6 (2018).
doi:10.3390/pharmacy6020043.
[18] W. Haefely, E. Kybuz, M. Gerecke, H. Mohler, Recent advances in the molecular
pharmacology of Benzodiazepine receptors and in the structure-activity relationships of their
agonists and antagonists, Adv. Drug Res. 14 (1985) 165–322.
[19] L. Luan, Z. Song, Z. Li, Q. Wang, Synthesis of new tricyclic 5,6-dihydro-4H-
benzo[b][1,2,4]triazolo[1,5-d][1,4]diazepine derivatives by [3+ + 2]-
cycloaddition/rearrangement reactions, Beilstein J. Org. Chem. 14 (2018) 1826–1833.
[20] W. Hunkeler, ChemInform Abstract: Benzodiazepines, the Story of the Antagonist
Flumazenil and of the Partial Agonist Bretazenil, ChemInform. 24 (1993).
doi:10.1002/chin.199332313.
[21] J. Höjer, S. Baehrendtz, G. Matell, L.L. Gustafsson, Diagnostic utility of flumazenil in coma
with suspected poisoning: a double blind, randomised controlled study, BMJ. 301 (1990)
1308–1311. doi:10.1136/bmj.301.6764.1308.
[22] E. Sanna, D. Pau, F. Tuveri, F. Massa, E. Maciocco, C. Acquas, C. Floris, S.N. Fontana, G.
Maira, G. Biggio, Molecular and Neurochemical Evaluation of the Effects of Etizolam on
GABAA Receptors under Normal and Stress Conditions, Arzneimittelforschung. 49 (1999)
88–95. doi:10.1055/s-0031-1300366.
[23] M.S. Langley, S.P. Clissold, Brotizolam, Drugs. 35 (1988) 104–122. doi:10.2165/00003495-
198835020-00002.
25

[24] M. Anzini, C. Braile, S. Valenti, A. Cappelli, S. Vomero, L. Marinelli, V. Limongelli, E.
Novellino, L. Betti, G. Giannaccini, A. Lucacchini, C. Ghelardini, M. Norcini, F. Makovec,
G. Giorgi, R. Ian Fryer, Ethyl 8-Fluoro-6-(3-nitrophenyl)-4H-imidazo[1,5-
a][1,4]benzodiazepine-3-carboxylate as Novel, Highly Potent, and Safe Antianxiety Agent, J.
Med. Chem. 51 (2008) 4730–4743. doi:10.1021/jm8002944.
[25] M. Anzini, S. Valenti, C. Braile, A. Cappelli, S. Vomero, S. Alcaro, F. Ortuso, L. Marinelli,
V. Limongelli, E. Novellino, L. Betti, G. Giannaccini, A. Lucacchini, S. Daniele, C. Martini,
C. Ghelardini, L. Di Cesare Mannelli, G. Giorgi, M.P. Mascia, G. Biggio, New Insight into
the Central Benzodiazepine Receptor–Ligand Interactions: Design, Synthesis, Biological
Evaluation, and Molecular Modeling of 3-Substituted 6-Phenyl-4H-imidazo[1,5-
a][1,4]benzodiazepines and Related Compounds, J. Med. Chem. 54 (2011) 5694–5711.
doi:10.1021/jm2001597.
[26] M. Anzini, A. Cappelli, S. Vomero, G. Giorgi, T. Langer, G. Bruni, M.R. Romeo, A.S.
Basile, Molecular Basis of Peripheral vs Central Benzodiazepine Receptor Selectivity in a
New Class of Peripheral Benzodiazepine Receptor Ligands Related to Alpidem, J. Med.
Chem. 39 (1996) 4275–4284. doi:10.1021/jm960325j.
[27] M. Matarrese, R.M. Moresco, A. Cappelli, M. Anzini, S. Vomero, P. Simonelli, E. Verza, F.
Magni, F. Sudati, D. Soloviev, S. Todde, A. Carpinelli, M.G. Kienle, F. Fazio, Labeling and
Evaluation of N-[11C]Methylated Quinoline-2-carboxamides as Potential Radioligands for
Visualization of Peripheral Benzodiazepine Receptors, J. Med. Chem. 44 (2001) 579–585.
doi:10.1021/jm001004h.
[28] A. Cappelli, G. Bini, S. Valenti, G. Giuliani, M. Paolino, M. Anzini, S. Vomero, G. Giorgi,
A. Giordani, L.P. Stasi, F. Makovec, C. Ghelardini, L. Di Cesare Mannelli, A. Concas, P.
Porcu, G. Biggio, Synthesis and structure-activity relationship studies in translocator protein
ligands based on a pyrazolo[3,4-b]quinoline scaffold, J. Med. Chem. 54 (2011) 7165–7175.
[29] A. Cappelli, M. Anzini, F. Castriconi, G. Grisci, M. Paolino, C. Braile, S. Valenti, G.
Giuliani, S. Vomero, A. Di Capua, L. Betti, G. Giannaccini, A. Lucacchini, C. Ghelardini, L.
Di Cesare Mannelli, M. Frosini, L. Ricci, G. Giorgi, M.P. Mascia, G. Biggio, Design,
Synthesis, and Biological Evaluation of Imidazo[1,5-a]quinoline as Highly Potent Ligands of
Central Benzodiazepine Receptors, J. Med. Chem. 59 (2016) 3353–3372.
doi:10.1021/acs.jmedchem.6b00034.
[30] K. Navjeet, An insight intomedicinal and biological significance of privileged scaffold: 1,4-
Benzodiazepine, Int. J. Pharma Bio Sci. 4 (2013) 318–337.
[31] L. Yet, Privileged Structures in Drug Discovery: Medicinal Chemistry and Synthesis, first
26

edit, John Wiley & Sons, Inc., Ithaca, NY, 2018.
[32] T.S. Jagodziński, Thioamides as Useful Synthons in the Synthesis of Heterocycles, Chem.
Rev. 103 (2003) 197–228. doi:10.1021/cr0200015.
[33] K. Doré, S. Dubus, H.-A. Ho, I. Lévesque, M. Brunette, G. Corbeil, M. Boissinot, G. Boivin,
M.G. Bergeron, D. Boudreau, M. Leclerc, Fluorescent Polymeric Transducer for the Rapid,
Simple, and Specific Detection of Nucleic Acids at the Zeptomole Level, J. Am. Chem. Soc.
126 (2004) 4240–4244. doi:10.1021/ja038900d.
[34] Z. Puterová, A. Krutošíková, D. Véghc, Gewald reaction: Synthesis, properties and
applications of substituted 2-aminothiophenes, Arkivoc. 2010 (2010) 209–246.
doi:10.3998/ark.5550190.0011.105.
[35] K. Gewald, Zur Reaktion von α-Oxo-mercaptanen mit Nitrilen, Angew. Chemie. 73 (1961)
114. doi:10.1002/ange.19610730307.
[36] C.E. Tranberg, A. Zickgraf, B.N. Giunta, H. Luetjens, H. Figler, L.J. Murphree, R. Falke, H.
Fleischer, J. Linden, P.J. Scammells, R.A. Olsson, 2-Amino-3-aroyl-4,5-alkylthiophenes:ꢀ
Agonist Allosteric Enhancers at Human A1 Adenosine Receptors, J. Med. Chem. 45 (2002)
382–389. doi:10.1021/jm010081p.
[37] R.I. Fryer, Z.-Q. Gu, C.-G. Wang, Synthesis of novel, substituted 4H-imidazo[1,5-
a][1,4]benzodiazepines, J. Heterocycl. Chem. 28 (1991) 1661–1669.
doi:10.1002/jhet.5570280704.
[38] Y. Watanabe, T. Shibuya, S. Khatami, B. Sakafsky, Comparison of Typical and Atypical
Benzodiazepines on the Central and Peripheral Benzodiazepine Receptors, Jpn. J. Pharmacol.
42 (1986) 189–197. doi:10.1254/jjp.42.189.
[39] H. Möhler, W.P. Burkard, H.H. Keller, J.G. Richards, W. Haefely, Benzodiazepine
Antagonist Ro 15–1788: Binding Characteristics and Interaction with Drug-Induced Changes
in Dopamine Turnover and Cerebellar cGMP Levels, J. Neurochem. 37 (1982) 714–722.
doi:10.1111/j.1471-4159.1982.tb12546.x.
[40] G.M. Sheldrick, A short history of {\it SHELX}, Acta Crystallogr. Sect. A. 64 (2008) 112–
122. doi:10.1107/S0108767307043930.
[41] C. Martini, A. Lucacchini, G. Ronca, S. Hrelia, C.A. Rossi, Isolation of Putative
Benzodiazepine Receptors from Rat Brain Membranes by Affinity Chromatography, J.
Neurochem. 38 (1982) 15–19. doi:10.1111/j.1471-4159.1982.tb10848.x.
[42] D.M. Walsh, S.C. Stratton, F.J. Harvey, I.J.M. Beresford, R.M. Hagan, The anxiolytic-like
activity of GR159897, a non-peptide NK2 receptor antagonist, in rodent and primate models
of anxiety, Psychopharmacology (Berl). 121 (1995) 186–191. doi:10.1007/BF02245629.
27

[43] M. Malcangio, A. Bartolini, C. Ghelardini, F. Bennardini, P. Malmberg-Aiello, F. Franconi,
A. Giotti, Effect of ICV taurine on the impairment of learning, convulsions and death caused
by hypoxia, Psychopharmacology (Berl). 98 (1989) 316–320. doi:10.1007/BF00451681.
[44] M.E. Jarvik, R. Kopp, An Improved One-Trial Passive Avoidance Learning Situation,
Psychol. Rep. 21 (1967) 221–224. doi:10.2466/pr0.1967.21.1.221.
[45] J.L. Vaught, K. Pelley, L.G. Costa, P. Setler, S.J. Enna, A comparison of the antinociceptive
responses to the GABA-receptor agonists THIP and baclofen, Neuropharmacology. 24
(1985) 211–216. doi:https://doi.org/10.1016/0028-3908(85)90076-0.
[46] H. Takeda, M. Tsuji, T. Matsumiya, Changes in head-dipping behavior in the hole-board test
reflect the anxiogenic and/or anxiolytic state in mice, Eur. J. Pharmacol. 350 (1998) 21–29.
doi:https://doi.org/10.1016/S0014-2999(98)00223-4.
[47] S. Zhu, C.M. Noviello, J. Teng, R.M. Walsh, J.J. Kim, R.E. Hibbs, Structure of a human
synaptic GABAA receptor, Nature. 559 (2018) 67–72. doi:10.1038/s41586-018-0255-3.
[48] L. Zaccagnini, S. Brogi, M. Brindisi, S. Gemma, G. Chemi, G. Legname, G. Campiani, S.
Butini, Identification of novel fluorescent probes preventing PrPSc replication in prion
diseases, Eur. J. Med. Chem. 127 (2017) 859–873.
doi:https://doi.org/10.1016/j.ejmech.2016.10.064.
[49] A. Di Capua, C. Sticozzi, S. Brogi, M. Brindisi, A. Cappelli, L. Sautebin, A. Rossi, S. Pace,
C. Ghelardini, L. Di Cesare Mannelli, G. Valacchi, G. Giorgi, A. Giordani, G. Poce, M.
Biava, M. Anzini, Synthesis and biological evaluation of fluorinated 1,5-diarylpyrrole-3-
alkoxyethyl ether derivatives as selective COX-2 inhibitors endowed with anti-inflammatory
activity, Eur. J. Med. Chem. 109 (2016) 99–106.
doi:https://doi.org/10.1016/j.ejmech.2015.12.044.
[50] C.C. Maquiaveli, J.F. Lucon-Júnior, S. Brogi, G. Campiani, S. Gemma, P.C. Vieira, E.R.
Silva, Verbascoside Inhibits Promastigote Growth and Arginase Activity of Leishmania
amazonensis, J. Nat. Prod. 79 (2016) 1459–1463. doi:10.1021/acs.jnatprod.5b00875.
[51] M. Brindisi, J. Senger, C. Cavella, A. Grillo, G. Chemi, S. Gemma, D.M. Cucinella, S.
Lamponi, F. Sarno, C. Iside, A. Nebbioso, E. Novellino, T.B. Shaik, C. Romier, D. Herp, M.
Jung, S. Butini, G. Campiani, L. Altucci, S. Brogi, Novel spiroindoline HDAC inhibitors:
Synthesis, molecular modelling and biological studies, Eur. J. Med. Chem. 157 (2018) 127–
138. doi:https://doi.org/10.1016/j.ejmech.2018.07.069.
[52] A. Vallone, S. D’Alessandro, S. Brogi, M. Brindisi, G. Chemi, G. Alfano, S. Lamponi, S.G.
Lee, J.M. Jez, K.J.M. Koolen, K.J. Dechering, S. Saponara, F. Fusi, B. Gorelli, D. Taramelli,
S. Parapini, R. Caldelari, G. Campiani, S. Gemma, S. Butini, Antimalarial agents against
28