Novel N-substituted indol-3-ylglyoxylamides probing the LDi and L1/L2 lipophilic regions of the benzodiazepine receptor site in search for subtype-selective ligands

Article abstract of DOI:10.1021/jm0607707

Novel N-substituted indol-3-ylglyoxylamides (10-37) were synthesized and evaluated as ligands of the benzodiazepine receptor (BzR). In an effort to achieve affinity-based selectivity among BzR subtypes, these compounds were designed to probe the L_Di<

Full text of DOI:10.1021/jm0607707

J. Med. Chem. 2007, 50, 1627-1634
1627
Novel N-Substituted Indol-3-ylglyoxylamides Probing the LDi and L1/L2 Lipophilic Regions of
the Benzodiazepine Receptor Site in Search for Subtype-Selective Ligands†
Giampaolo Primofiore,*,∆ Sabrina Taliani,∆ Federico Da Settimo,∆ Anna Maria Marini,∆ Concettina La Motta,∆
Francesca Simorini,∆ Maria Paola Patrizi,∆ Valentina Sergianni,∆ Ettore Novellino,‡ Giovanni Greco,‡ Barbara Cosimelli,‡
Vincenzo Calderone, Marina Montali, Franc¸ois Besnard,§ 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, Synthe´labo, 10 rue des Carrie`res, 92500 Rueil-Malmaison, France, and Dipartimento di Psichiatria, Neurobiologia,
Farmacologia e Biotecnologie, UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
ReceiVed June 29, 2006
Novel N-substituted indol-3-ylglyoxylamides (10-37) were synthesized and evaluated as ligands of the
benzodiazepine receptor (BzR). In an effort to achieve affinity-based selectivity among BzR subtypes, these
compounds were designed to probe the LDi and L2 lipophilic regions. Taking the R1-selective benzylin-
dolylglyoxylamides Ia and Ib as leads, we varied the substituent on the benzylamide phenyl ring (compounds
10-23) or replaced the benzyl moiety with alkyl groups (compounds 24-37). The above structural changes
gave no shift of selectivity from the R1 toward the R2 or R5 subtypes, thus confirming that a ligand which
occupies the LDi region probably exhibits R1 selectivity, despite its interactions with other lipophilic areas
in the receptor binding cleft. Compound 11 (N-(p-methylbenzyl)-5-nitroindol-3-ylglyoxylamide), which
selectively binds with a full agonist efficacy at the R1 receptor subtype and displays sedative action, can be
regarded as an interesting potential zolpidem-like sedative-hypnotic agent.
Introduction
GABA-induced chloride influx, respectively. Spanning these
efficacy extremes are partial agonists, antagonists (which bind
to the BzR but have little or no effect on the chloride flux, being
devoid of any appreciable intrinsic efficacy), and partial inverse
agonists.5,6
GABA (γ-aminobutyric acid), the major inhibitory neu-
rotransmitter in the central nervous system (CNS), operates
through three different receptor types: the ionotropic GABAA
and GABAC receptors and the metabotropic GABAB receptor.1,2
The GABAA receptor plays a fundamental role in several
neurological functions including modulation of anxiety, sleep,
and muscle tone, as well as in processes like memory and
learning.3 This membrane-bound heteropentameric protein
receptor may be assembled from at least 21 subunits belonging
to 8 different classes (6R, 4â, 4γ, 1ꢀ, 1δ, 3F, 1θ, and 1π), which
form a chloride channel.1,3 Three types of subunits (R, â, and
γ in a predominant 2:2:1 stoichiometry) are required for the
construction of recombinant receptors which most closely mimic
the biochemical, electrophysiological, and pharmacological
functions of native GABAA receptors obtained from mammalian
brain cells.4
The benzodiazepine receptor site (BzR) is located at the
interface of the R and γ subunits of the GABAA complex and
binds with a high affinity benzodiazepine derivatives, like
diazepam, and other structurally different classes of compounds.
BzR ligands can allosterically modulate the affinity of GABA
for the GABAA receptor.4 On the basis of their modulatory
effects, they are classified as agonists (positive allosteric
modulators, with anxiolytic, anticonvulsant, sedative-hypnotic,
and myorelaxant activities) or inverse agonists (negative allos-
teric modulators, with anxiogenic, somnolytic, proconvulsant,
or even convulsant activities), potentiating or decreasing the
The majority of benzodiazepine-sensitive GABAA receptor
subtypes in the brain are R1âxγ2, R2âxγ2, R3âxγ2, and R5âxγ2,
while the R4âxγ2 and R6âxγ2 subtypes do not respond to
benzodiazepines and are therefore called benzodiazepine-
insensitive receptors.7,8 Since the γ2 subunit does not vary, and
the â subunit type does not seem to influence the pharmacology
of benzodiazepines, the R subunit is the main determinant of
affinity and efficacy of BzR ligands7-9 (therefore BzR subtypes
take their names from the R subunit).
The regional heterogeneity of the BzR subtypes (Rl, R2, R3,
and R5) has been suggested as the basis for the multiplicity of
pharmacological properties of most agonists belonging to the
class of benzodiazepines. In particular, R1, which is the dominant
subtype, is present in both cerebellum and cortex and thus
mediates the sedative/muscle relaxant effects; R2 is moderately
abundant in the cortex and hippocampus and is related to the
anxiolytic and anticonvulsant activities; the R5 type is scarce
and widely expressed only in the hippocampus and is associated
with impairment of cognition and memory; finally, the role of
R3 remains unclear or, at least, it seems to be partly involved
in mediating anxiety behavior.7-10 Consequently, selective R1
agonists represent ideal sedative and hypnotic agents devoid of
side effects on cognition and memory performances; an agonist
that selectively modulates R2 and R3 receptors, but does not
affect the R1 subtype, would represent a nonsedative anxiolytic;
an inverse agonist selectively acting at the R5 receptor may have
therapeutic utility as a cognition-enhancing agent, lacking the
unwanted side effects associated with inverse agonist activity
* Author to whom correspondence should be addressed. Phone:
+390502219546. Fax: +390502219605. E-mail: primofiore@farm.unipi.it.
† Dedicated to Professor Fulvio Gualtieri, University of Florence, on the
occasion of his 70th birthday.
∆ Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡ Universita` “Federico II” di Napoli.
§ Synthe´labo.
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
nologie, Universita` di Pisa.
10.1021/jm0607707 CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/03/2007

1628 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Primofiore et al.
Chart 1. Structures of N-(Benzyl)indol-3-ylglyoxylamides I,
Imidazobenzodiazepines II, and Pyrazoloquinolines III
Figure 1. Hypothetical binding modes A and B of indole BzR ligands
(ref 15) within the framework of Cook’s pharmacophore/topological
model (ref 13).
Table 1. Inhibition of [3H]Flumazenil Specific Binding to Bovine Brain
Membranes of Indol-3-ylglyoxylylamide Derivatives Ia,b and 10-37
at the other subtypes, including anxiogenesis or convulsant or
proconvulsant activity.8,11,12
Structure-activity relationships (SARs) of widely different
classes of BzR ligands were rationalized by Cook and co-
workers through a pharmacophore/topological model made up
of the following interaction sites within the receptor binding
cleft: H1 and H2 as hydrogen bond donors, A2 as a hydrogen
bond acceptor, L1, L2, L3, and LDi as lipophilic regions, and S1,
S2, and S3 as sterically forbidden sites.13
Ki (nM) or
% inhibition
no.
R5
R
(10 µM)a,b
Iac
Ibc
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
H
CH2-C6H5
NO2 CH2-C6H5
CH2-C6H4-4′-CH3
NO2 CH2-C6H4-4′-CH3
CH2-C6H4-4′-CH2CH3
NO2 CH2-C6H4-4′-CH2CH3
CH2-C6H4-4′-Br
120 ( 11
117 ( 12
606 ( 57
88 ( 6
2160 ( 180
42% ( 3
93 ( 8
H
H
Some years ago, we presented the N-(benzyl)indol-3-ylgly-
oxylamides I as a new class of BzR ligands (Chart 1).14,15 These
compounds display an enhanced affinity for the BzR R1 subtype,
compared with that of the R2 and R5 isoforms. SARs of indole
derivatives soon revealed that the effects of the R5 and X
substituents on R1 affinity are interdependent. Specifically, the
affinity is favored by X ) hydroxy/methoxy or halogens,
depending on whether the 5-position of the indole nucleus is
substituted (R5 ) Cl/NO2) or not (R5 ) H), respectively. Thus,
while affinity in the 5-Cl/NO2 series is optimized by X ) 3′,4′-
(OH)2 or 3′,4′-(OMe)2, in the 5-H series the highest affinity is
achieved with electron-attracting X ) 4′-Cl. These puzzling data
were interpreted by assuming that in their interaction with the
receptor site, our ligands might adopt two alternative binding
modes called A and B (Figure 1).15 The 5-Cl/NO2 indoles bind
according to mode A, by interacting with the A2 site (through
the indole NH), with the H1 and H2 sites (through the C ) O2
and C ) O1), and with the L1, L2, and LDi lipophilic regions
(filled by the CH2, the side chain phenyl ring, and the benzene
moiety of the indole nucleus, respectively). In this orientation,
the affinity is increased by hydroxy and methoxy X substituents,
due to a hydrogen bond formed between the 3′-OH or 3′-OMe
and a bifunctional hydrogen bond acceptor/donor (HBA/D)
group located on the surface of the S1 site, as recently proposed
by our research group.16
H
NO2 CH2-C6H4-4′-Br
H
NO2 CH2-C6H4-4′-CtCSi(CH3)3
H
31% ( 3
29% ( 2
21% ( 2
3% ( 1
CH2-C6H4-4′-CtCSi(CH3)3
CH2-C6H4-4′-CtC-CH2Si(CH3)3
NO2 CH2-C6H4-4′-CtC-CH2Si(CH3)3
CH2-C6H4-4′-CtCH
NO2 CH2-C6H4-4′-CtCH
CH2-C6H4-4′-CtC-CH3
NO2 CH2-C6H4-4′-CtC-CH3
(CH2)3CH3
NO2 (CH2)3CH3
(CH2)4CH3
NO2 (CH2)4CH3
(CH2)5CH3
NO2 (CH2)5CH3
CH(CH3)2
NO2 CH(CH3)2
0% ( 2
H
164 ( 10
28% ( 1
3275 ( 299
0% ( 3
H
H
3720 ( 30
110 ( 9
H
1480 ( 11
4100 ( 40
1650 ( 13
53% ( 4
2690 ( 15
57.8 ( 4
34% ( 4
412 ( 9
H
H
H
CH(CH3)CH2CH3
NO2 CH(CH3)CH2CH3
H
NO2 C(CH3)3
H
C(CH3)3
7% ( 2
2800 ( 27
43% ( 3
5% ( 2
CH2CH(CH3)2
NO2 CH2CH(CH3)2
diazepam
flumazenil
clonazepam
10 ( 1
0.90 ( 0.05
0.85 ( 0.02
a Ki values are means ( SEM of three determinations carried out in
triplicate. b Percentage inhibition values of specific [3H]flumazenil binding
at 10 µM concentration are means ( three determinations carried out in
triplicate. c Data taken from ref 14.
Binding mode B requires R5 ) H (substituents bulkier than
a hydrogen would be repelled by the sterically forbidden region
S2) and is favored by electron-attracting X groups. This binding
mode relies on the following interactions: C ) O2 and C )
O1 are hydrogen-bound to the H1 and H2 sites; the L1, L2, and
by research focusing on differences in the recognition properties
of the BzR subtypes. Specifically, it has been suggested that
the basic topology of these subtypes is highly conserved, with
the exception of the L2 and LDi lipophilic pockets, whose
different dimensions might play a role in determining selectivity
profiles.17 In particular, it has been proposed that the LDi and
L2 regions are wider in the R1 and R5 binding sites, respectively,
with respect to the other subtypes. Consequently, full occupation
of LDi or L2 may account for R1 and R5 selectivity, respec-
tively.17,18 An excellent example of R5-selective ligands is given
by imidazobenzodiazepines II (Chart 1) featuring bulky sub-
L
Di lipophilic regions are occupied by the pyrrole and benzene
moieties of the indole nucleus, and the side chain phenyl ring,
respectively; a hydrogen bond is donated by the indole NH to
the HBA/D group belonging to the S1 site.16
In an effort to identify novel ligands possessing selectivity
at the BzR subtypes, we have now turned our attention to
N-substituted indol-3-ylglyoxylamides filling the LDi and L2
lipophilic regions to different extents. Our approach was guided

N-Substituted Indol-3-ylglyoxylamides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1629
Scheme 1. Synthesis of 4-Substituted Benzylamine Derivatives 4-7
Scheme 2. Synthesis of Indole Derivatives 16-23
halides described for the first time by Sonogashira et al. in
1975,21 which is one of the most straightforward methods for
the preparation of arylalkynes and conjugated enynes.22
On the basis of this evaluation, a possible route for the
synthesis of derivatives 16-23 could consist of a coupling
reaction between the appropriate, commercially available,
terminal trimethylsilylalkynes and the 4′-bromo derivatives 14
and 15, to give compounds 16-19, which by subsequent
removal of the protecting group could generate products 20-
23.
However, despite varying many parameters, such as solvent,
catalyst, and base, no satisfactory results were obtained, as only
traces of the final products 16-19 were isolated, probably due
to the low solubility of the 4′-bromo derivatives 14 and 15 in
the reaction solvent.
The key to the successful synthesis of derivatives 16-23 was
the preparation of the amine intermediates 4-7 (Scheme 1),
which possess good solubility properties, making the coupling
reaction more efficient.
stituents in the 8-position, such as ethynyl and (trimethylsylyl)-
ethynyl, which fit into the L2 region.17,18 Yu et al. have proposed
that simultaneous occupation of L2 and LDi seems to promote
R2 selectivity, in accordance with the binding data of some
pyrazoloquinolines III (Chart 1) substituted in both 4′- and
8-positions with lipophilic bulky groups.19 However, the factors
determining R2 selectivity are not so clearly defined, as the same
research group subsequently proposed that a BzR ligand which
potently interacts with the LDi region displays R1 selectivity,
despite occupation of other receptor lipophilic areas.17,18
Taking the benzylindolylglyoxylamides Ia and Ib as reference
compounds (Table 1),14 we decided to probe the steric recogni-
tion properties of the LDi and L2 pockets in two different ways:
by varying the substituent on the benzylamide phenyl ring
(compounds 10-23 in Table 1) or by replacing the benzyl
moiety with alkyl groups (compounds 24-37 in Table 1). This
paper describes the synthesis, biological evaluation, and SARs
of the new indole derivatives 10-37.
Chemistry. The synthetic procedure used for the preparation
of compounds 10-15 and 24-37 involved the reaction in mild
conditions of the indolylglyoxylyl chlorides 8 and 920 with the
appropriate amine in the presence of triethylamine in toluene
solution (Supporting Information).
For the synthesis of indole derivatives 16-23, we focused
our attention on the palladium-catalyzed C-C bond forming
process. Initially we took into consideration the palladium-
catalyzed coupling of terminal alkynes with aryl or alkenyl
Synthesis of alkynes 6 and 7 was achieved in three steps
starting from the commercially available 4-bromobenzylamine,
which was first protected as its carbamate 1,23 and then coupled
with the appropriate trimethylsilylalkyne, using an improved
Sonogashira coupling procedure, reported by Thorand and
Krause (Scheme 1).24 It should be noted that a careful choice
of the composition of the reaction mixture and the mode of
addition of the reactants are crucial to obtain the intermediate
products 2 and 3 with good to excellent yields. Following this
method, the coupling reaction was carried out in THF instead
of in an amine as the solvent; the side homocoupling reaction
of the alkyne was prevented almost completely by slow addition
of the alkyne; a slight excess of trimethylsilylalkyne (1.5 equiv)
was used; the palladium catalyst (5 mol %) was stabilized by
the addition of triphenylphosphine; finally, it turned out to be
useful to employ a very small amount (ca. 1 mol %) of copper
iodide and to add this as the last component to the reaction
mixture.24 The coupling reaction was carried out for 72-96 h
at room temperature in a nitrogen atmosphere, following the
reaction by GC analysis. The suspension obtained was worked
up to yield the crude products 2 and 3, which were purified by
flash-chromatography (Scheme 1, Experimental Section). Simple
deprotection of the Boc group, performed using 3 M HCl in
ethyl acetate at room temperature for 96 h (GC analysis),25
furnished good yields of the hydrochloride salts of trimethyl-
silylalkyne derivatives 4 and 5 (Experimental Section), which

1630 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Primofiore et al.
Table 2. Inhibition of [3H]Flumazenil Specific Binding of Selected
Compounds on Rat R1â2γ2, R2â2γ2, and R5â3γ2 GABAA/Bz Receptor
Subtypesa
10-15 and 24-37, giving the desired compounds 16-23 with
high yields (Scheme 2).
All target products 10-37 were purified by recrystallization
from the appropriate solvent after filtration, when necessary,
through a silica gel column, and their structures were confirmed
by IR, 1H NMR, MS, and elemental analyses (Supporting
Information).
Biological Studies. The binding affinity of the newly
synthesized indolylglyoxylamide derivatives 10-37 at the BzR
in bovine brain membranes was determined by competition
experiments against the radiolabeled antagonist [3H]flumazenil27
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 1).
Ki (nM) or % inhibition (10 µM)b,c
no.
R1â2γ2
R2â2γ2
R5â3γ2
Ia
346 ( 29
65 ( 5
31.3 ( 2
559 ( 44
17 ( 1
39% ( 3
32% ( 3
0%
1600 ( 145
81 ( 7
414 ( 31
16% ( 2
7% ( 1
553 ( 45
175 ( 12
765 ( 63
46% ( 5
44% ( 4
0%
Ib
11
12
14
20
22
24
25
31
40% ( 3
1445 ( 120
1154 ( 99
13% ( 3
28% ( 2
943 ( 80
498 ( 37
35% ( 3
250 ( 19
881 ( 78
1175 ( 95
125 ( 10
15 ( 1
zolpidem
50 ( 3
Subtype selectivities of selected compounds (11, 12, 14, 20,
22, 24, 25, and 31) and benzyl derivatives Ia and Ib were
evaluated by assaying their ability to displace [3H]flumazenil
in membranes from HEK293 cells expressing rat R1, R2, and
R5 BzR subtypes (Table 2).15
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 Experimental Section. b Ki values are means
( SEM of three determinations carried out in triplicate. c Percentage
inhibition values of specific [3H]flumazenil binding at 10 µM concentration
are means ( SEM of three determinations carried out in triplicate.
The functional efficacy of compound 11 was determined by
measuring its modulatory effect on 36Cl- influx through the ion
channel pore at a GABA concentration evoking 20% maximum
influx (EC20) in cloned HEK293 cells expressing the R1 BzR
subtype, as described by Harris et al.28 A concentration 100
times greater than the compound Ki value provided the maximal
effect on GABA-evoked 36Cl- influx.29 Efficacy results are
shown in Figure 2, in which the nonselective full agonist
diazepam was included as a standard.
In order to evaluate a potential sedative activity, the phar-
macodynamic profile of the selected compound 11 was exam-
ined by means of a behavioral test on mice. In particular, the
influence of compound 11 (10 mg/kg ip) on the spontaneous
motor activity of the animals was analyzed and compared with
that induced by the same dose of the R1-selective reference drug
zolpidem, through the well-known test of “intermittent observa-
tions” (Figure 3).30
Figure 2. Maximal efficacy of compound 11 on rat recombinant R1â2γ2
GABAA receptor subtype. Results are expressed as percentages of
increase in response to GABA at EC20 (mean ( SEM) from at least
five independent experiments.
Results and Discussion
Table 1 lists the affinity values of the new indole derivatives
10-37 and those of the reference indoles Ia and Ib. These
binding data show that the same amide side chain produces
different effects on affinity, depending on whether the 5-position
of the indole nucleus is substituted (R5 ) NO2) or not (R5 )
H). This trend of the affinity confirms that 5-NO2 indolylgly-
oxylamides bind to the BzR differently from their 5-H coun-
terparts (according to modes A and B, respectively) (Figure 1).
The much higher affinity of the 5-H derivative 14 bearing a
bromine in the 4′-position of the phenyl ring with respect to its
5-NO2 analogue 15 is in agreement with previous findings that
an electron-attracting group in the para position of the phenyl
ring strongly disfavors binding mode A (compare 15 with Ib),
while it slightly favors binding mode B (compare 14 with Ia).15
A methyl group in the 4′-position of the phenyl ring slightly
increases affinity if R5 ) NO2 (compare 11 with Ib), whereas
it lowers affinity 5-fold if R5 ) H (compare 10 with Ia). When
the 4′-methyl is replaced by the bulkier 4′-ethyl, 4′-ethynyl, and
4′-(1-propynyl) groups, affinity is lost by the 5-NO2 derivatives
13, 21, and 23 but is retained at a submicromolar or micromolar
value by the 5-H derivatives 12, 20, and 22. The excessive
dimensions of the 4′-trimethylsilylalkynyl substituents are
probably responsible for the inactivity of both 5-NO2 and 5-H
derivatives 16-19.
Figure 3. Histograms indicate the values of counts/h recorded in the
groups of mice treated with vehicle, zolpidem, or compound 11. The
vertical bars indicate the standard errors. The asterisks represent the
levels of statistical significance.
were utilized in the next reaction without further purification.
Silyl cleavage was performed using tetrabutylammonium fluo-
ride (TBAF) 0.1 M in THF at room temperature.26 After 1 h
(GC analysis) the reaction mixture was worked up to afford
analytically pure amines 6 and 7, which were characterized as
hydrochloride salts (Experimental Section).
The condensation of amines 4-7 with the appropriate
indolylglyoxylyl chloride 8 and 9 was achieved using the same
experimental procedure utilized for the preparation of derivatives
According to the SARs so far outlined, the LDi region appears
to be longer than the L2 one because the former can accom-

N-Substituted Indol-3-ylglyoxylamides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1631
modate the lengthy 4′-substituents of 5-H indoles 12, 20, and
22 (oriented in binding mode B), whereas the latter cannot host
the same 4′-substituents of the 5-NO2 counterparts 13, 21, and
23 (oriented according to binding mode A). When the size of
the 4′-substituent increases above a certain threshold, as in
compounds 16-19, both the LDi and the L2 pockets reveal their
steric limits and affinity is lost in both 5-NO2 and 5-H series.
represented by compound 11, which displays a Ki of 31.3 nM
at the R1 subtype and no appreciable binding at the R2 or R5
isoforms.
These data indicate that our indolylglyoxylamides, due to their
strong interaction with the LDi lipophilic region (in either of
the binding modes A or B), maintain R1 selectivity indepen-
dently of the extent of the interaction with the L1/L2 lipophilic
pockets. This is in agreement with literature reports, where the
same effect was observed in several series of ligands, such as
â-carbolines31 and pyrazolo[4,3-c]quinolin-3-ones.17,18,32
Compound 11 was selected to be screened for functional
efficacy, and as shown in Figure 2, its efficacy on coapplication
with GABA at the BzR subtype was similar to that of standard
diazepam (72% ( 2% and 78% ( 3% for compound 11 and
diazepam, respectively), showing that the main feature of
compound 11 is a full agonist efficacy profile at this receptor
subtype.
Due to its efficacy profile, compound 11 should exert sedative
effects in vivo. This hypothesis was preliminarily assessed by
a behavioral model based on the observation of the spontaneous
motor activity of mice,30 employing the R1-selective zolpidem33
as the reference drug (Figure 3). The control groups of mice
(vehicle) showed a spontaneous motor activity which was
measured as 74 ( 5 counts/h. The spontaneous motor activity
was found to decrease significantly and markedly in the groups
of mice treated with zolpidem or compound 11 (9 ( 2 and 28
( 4 counts/h, respectively). These results demonstrate that
compound 11, although less active than zolpidem, is a sedative-
hypnotic agent.
In the same way as observed for N-benzyl compounds 11-
23, steric hindrance is a crucial determinant of affinity also for
the N-alkyl derivatives 24-37. However, in the latter subset,
affinity seems to depend not only on the overall length and width
but also on the shape of the side chain. Particularly, in the 5-NO2
series (binding according to mode A) the n-butyl group perfectly
replaces the benzyl one, as compound 25 is equipotent to Ib,
but insertion of one or two additional methylene units in the
alkyl chain lowers or completely disrupts affinity (see com-
pounds 27 and 29). The same N-substituents are still tolerated
when included in the 5-H counterparts 24, 26, and 28 (binding
with micromolar affinity according to mode B), confirming that
the LDi region is longer than the L2 one, which should have a
precise length, with the result that affinity is abolished when
the amide side chain increases above a certain threshold.
R-Branching is tolerated within the L1/L2 pocket when
included in the 5-NO2 derivatives 31, 33, and 35 (binding with
high or moderate affinity according to mode A), while it is
sterically repelled by the LDi region when included in the 5-H
counterparts 30, 32, and 34 (binding with low or very poor
affinity according to mode B), in agreement with earlier work
on N-(R-methylbenzyl)indol-3-ylglyoxylamide derivatives.15
Finally, the shape of the â-branched N-i-butyl groups of
compounds 36 and 37 is unsuited for both LDi and L1/L2
lipophilic cavities.
In conclusion, none of the structural changes so far made in
the indolylglyoxylamides have yielded any shift of selectivity
from the R1 subtype toward the R2 or R5 subtypes, thus
confirming that a ligand which favorably occupies the LDi region
exhibits R1 selectivity, despite its interactions with other
lipophilic areas in the receptor binding cleft. Compound 11 has
been identified as an affinity-based R1-selective ligand display-
ing a full agonist efficacy profile in vitro and sedative-hypnotic
activity in vivo.
It is worth pointing out that the N-benzyl side chain can be
successfully replaced by the N-alkyl moieties in the series of
5-NO2 indoles (compare compounds 25, 31, and 33 with Ib)
but not in the series of 5-H indoles, which are all less potent
than the reference compound Ia. These data might be related
to the different shapes and volumes of the L1/L2 and LDi regions.
As far as our indoles are concerned, the L1/L2 pocket might
host either the aromatic or the aliphatic N-side chains of 5-NO2
derivatives (oriented in binding mode A) as well as the fused
benzene ring of the 5-H derivatives (oriented in binding mode
B). By contrast, the LDi cavity is able to accommodate the fused
benzene ring of the 5-NO2 indoles as well as the N-benzyl side
chains of 5-H ligands but is not sterically suited for several
N-alkyl chains of the 5-H indoles. In other words, besides being
long (compared with the L2 one), the LDi region appears to be
mainly flat and can be roughly described as a “benzyl-like”
cavity. In fact, any deviation from this “best” substituent in the
5-H indoles so far investigated has unfavorable effects on
affinity. Accordingly, several molecular modeling studies17,18
have shown that most of the BzR ligands fill the LDi region
with aromatic moieties (e.g., a fused benzene ring of â-carbo-
lines, CGS compounds, dihydropyridodiindoles) or small alkyl
groups (e.g., a methyl of zolpidem and of the triazolopyrimidine
CL 218872).
Experimental Section
Chemistry. Melting points were determined using a Reichert
Ko¨fler hot-stage apparatus and are uncorrected. Infrared spectra
were recorded with an FTIR spectrometer Nicolet/Avatar in Nujol
mulls. Routine nuclear magnetic resonance spectra were recorded
in DMSO-d6 solution on a Varian Gemini 200 spectrometer
operating at 200 MHz. Evaporation was performed in vacuo (rotary
evaporator). 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. GC analysis
was performed with a Shimadzu 17A gas chromatograph equipped
with a flame ionization detector, SPB-5 Supelco column (15 m ×
0.53 mm); operating conditions: low isotherm 170 °C (5 min), high
isotherm 280 °C (15 min), temperature scaled up 9 °C/min, injection
temperature 250 °C, detector temperature 260 °C, carrier nitrogen
12 cm/s. Elemental analyses were performed by our analytical
laboratory and agreed with theoretical values to within (0.4%.
All 4-substituted benzylamines are commercially available
products with the exception of products 4-7. N-Boc-4′-bromoben-
zylamine 1 was prepared in accordance with ref 23.
General Procedure for the Synthesis of N-Substituted 5-Sub-
stituted Indol-3-ylglyoxylamide Derivatives 10-37. Triethylamine
(3.0 mmol) was added dropwise to a stirred suspension, cooled at
0 °C, of indolylglyoxylyl chloride 8 and 920 (2.5 mmol) and the
appropriate amine (2.75 mmol) in 50 mL of dry toluene. The
reaction mixture was left to warm to room temperature, stirred for
24-36 h (TLC analysis), and then filtered. The precipitate collected
was triturated with a saturated NaHCO3 aqueous solution, washed
Selected representative indole derivatives (11, 12, 14, 20, 22,
24, 25, and 31), together with the reference compounds Ia and
Ib, were tested for their affinity at R1, R2, and R5 BzR subtypes.
All these ligands showed fair to high selectivity affinities for
R1 over R2 and R5 subtypes (Table 2). A good correlation exists
between the affinities at wild-type and R1-subtype receptors.
The best result in terms of both R1 affinity and selectivity is

1632 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Primofiore et al.
with water, and collected again to give a first portion of crude
product. The toluene solution was evaporated to dryness, and the
residue was treated with saturated NaHCO3 aqueous solution,
washed with water, and collected to yield an additional amount of
crude product. The quantities of amide derivatives obtained from
the initial insoluble precipitate or from the toluene solution were
variable, depending upon the solubility of the various compounds.
All products 10-37 were finally purified by recrystallization from
the appropriate solvent. Yields, recrystallization solvents, melting
points, and analytical and spectral data are reported in the
Supporting Information.
General Procedure for the Synthesis of (4-Trimethylsilyla-
lkyn-1-ylbenzylamine)carbamic Acid tert-Butyl Ester Deriva-
tives 2 and 3. Bis(triphenylphosphine)palladium(II) dichloride
(0.182 g, 0.26 mmol), triphenylphosphine (0.034 g, 0.13 mmol),
and triethylamine (1.10 mL, 7.9 mmol) were added to a stirred
mixture of 1.50 g (5.25 mmol) of N-Boc-4-bromobenzylamine 123
in 18 mL of dry THF under an atmosphere of nitrogen. A solution
of 7.9 mmol of the appropriate alkyne (trimethylsilylacetylene,
3-trimethylsilyl-1-propyne) in 6 mL of dry THF was then added
over 1 h. The mixture was stirred for 20 min at room temperature,
and 0.012 g (0.063 mmol) of CuI was then added. After being stirred
under an atmosphere of nitrogen and at room temperature for 72-
96 h (GC analysis), the suspension obtained was filtered at reduced
pressure, and the solvent was evaporated. The semisolid residue
was then purified by flash-chromatography (eluting system: pe-
troleum ether 60-80 °C/ethyl acetate:8/2), yielding analytically pure
products 2 and 3.
4-Ethynylbenzylamine Hydrochloride 6‚HCl (yield 81%, mp
) 222-224 °C). IR, ν cm-1: 3300, 2040, 1100, 840. 1H NMR, δ
ppm: 4.06 (s, 2H, CH2), 4.26 (s, 1H, CH), 7.45 (d, 2H, 3-H, 5-H),
7.53 (d, 2H, 2-H, 6-H), 8.35 (bs, 3H, NH3+, exch with D2O). Anal.
(C9H10ClN) C, H, N.
4-(Prop-1-ynyl)benzylamine Hydrochloride 7‚HCl (yield 86%,
mp ) 253-255 °C). IR, ν cm-1: 3400, 2050, 1110, 830. 1H NMR,
δ ppm: 2.04 (s, 3H, CH3), 4.00 (s, 2H, CH2), 7.43-7.48 (m, 4H,
Ar-H), 8.41 (bs, 3H, NH3+, exch with D2O). Anal. (C10H12ClN)
C, H, N.
Biological Procedures. 1. Radioligand Binding Studies. [3H]-
Flumazenil (specific activity 70.8 Ci/mmol) was obtained 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 accordance
with ref 34. 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.35
[3H]Flumazenil binding studies were performed as previously
reported.15
HEK293 cells stably expressing rat GABAA receptor subtypes
(R1â2γ2, R3â2γ2, R5â3γ2) were maintained, as previously described36
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 streptomycin (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
homogenization 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.36
(4-Trimethylsilylethyn-1-ylbenzylamine)carbamic Acid tert-
Butyl Ester 2 (yield 91%, mp ) 91-92 °C). IR, ν cm-1: 3320,
1
2160, 1680, 1520, 840. H NMR, δ ppm: 0.26 (s, 9H, Si(CH3)3),
1.47 (s, 9H, C(CH3)3), 4.32 (d, 2H, CH2), 4.84 (bs, 1H, CONH,
exch with D2O), 7.25 (d, 2H, 3-H, 5-H), 7.44 (d, 2H, 2-H, 6-H).
Anal. (C17H25NO2Si) C, H, N.
[3H]Flumazenil binding assays to transfected cell membranes
were carried out as previously described.36 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 of 10-9 to 10-5 M. Nonspecific binding was defined
by 10-5 M diazepam. Assays were incubated to equilibrium for 1
h at 4 °C.
(4-Trimethylsilylprop-1-ynylbenzylamine)carbamic Acid tert-
Butyl Ester 3 (yield 89%, mp ) 30 °C). IR, ν cm-1: 3340, 2210,
1
1710, 1500, 850. H NMR, δ ppm: 0.16 (s, 9H, Si(CH3)3), 1.46
(s, 9H, C(CH3)3), 1.70 (s, 2H, CH2Si), 4.13 (d, 2H, CH2NH), 4.85
(bs, 1H, CONH, exch with D2O), 7.27 (d, 2H, 3-H, 5-H), 7.32 (d,
2H, 2-H, 6-H). Anal. (C18H27NO2Si) C, H, N.
2. Functional Efficacy Studies. 36Cl- uptake was measured in
transfected HEK293 cells as previously described with minor
modifications.37 In brief, cells were plated into each well at a density
of 10 × 105 cells/well and were grown for 2 days at 37 °C with
5% CO2 in DMEM/nut mix F-12 with Glut-I (GIBCO), supple-
mented with 10% fetal bovine serum, penicillin (100 units/mL),
and streptomycin (100 µg/mL). Cells were initially washed at room
temperature with 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-piperazin ethane sulfonic acid, adjusted to a pH
of 7.4 with Tris base. Cells were then incubated in 0.2 mL of a
36Cl- solution (0.4 µCi/mL) containing the compound, with or
without 100 nM GABA. Influx was terminated after 8 s by
aspiration of incubation solution and immediate washing with 0.2
mL of ice-cold picrotoxin stop buffer. Then, 1.6 mL of 0.2 N NaOH
was rapidly placed and 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 spectrom-
etry. Values for 36Cl- influx were expressed as nanomol/mg protein.
3. Behavioral Test. The spontaneous motor activity was evalu-
ated through a slight modification of the test of “intermittent
observations”.30 Briefly, 27 adult male BalbC mice (about 40 g of
body weight) were used for the pharmacological test, which was
performed in three different sessions. In each session, nine mice
(about 40 g) were divided into groups of three mice. Mice of the
first group received the reference drug zolpidem (10 mg/kg ip),
those of the second group received compound 11 (10 mg/kg ip),
and those of the third group received the equivalent amount of
vehicle (10 mL/kg of 10% DMSO, 60% PEG 400, 30% physi-
ological saline solution). In order to avoid any possible influence
General Procedure for the Synthesis of 4-(Trimethylsilyla-
lkyn-1-yl)benzylamine Derivatives 4 and 5. Compounds 2 and 3
(0.48 mmol) were dissolved in 25 mL of ethyl acetate and treated
with 10 mL of 3 M HCl. After being stirred at room temperature
for 96 h (GC analysis), the mixture was treated with water, basified
with solid K2CO3, and extracted with CHCl3. The organic phase
was dried over Na2SO4 and evaporated at reduced pressure, yielding
practically pure amino derivatives 4 and 5 as hydrochloride salts
(Supporting Information), which were utilized in the next reaction
without further purification.
4-(Trimethylsilylethynyl)benzylamine Hydrochloride 4‚HCl
(yield 80%, mp ) 238-240 °C). IR, ν cm-1: 3320, 2160, 1245,
1
840. H NMR, δ ppm: 0.25 (s, 9H, 3CH3), 3.94 (s, 2H, CH2),
+
7.40 (d, 2H, 3-H, 5-H), 7.46 (d, 2H, 2-H, 6-H), 8.50 (bs, 3H, NH3
exch with D2O). Anal. (C12H18ClNSi) C, H, N.
,
4-(Trimethylsilylprop-1-ynyl)benzylamine Hydrochloride 5‚
HCl (yield 83%, mp ) >300 °C). IR, ν cm-1: 3390, 2060, 1250,
1
840. H NMR, δ ppm: 0.17 (s, 9H, 3CH3), 1.76 (s, 2H, CH2Si),
4.00 (s, 2H, CH2NH3+), 7.29-7.43 (m, 4H, Ar-H), 8.44 (bs, 3H,
NH3+, exch with D2O). Anal. (C13H20ClNSi) C, H, N.
General Procedure for the Synthesis of 4-Alkyn-1-ylbenzy-
lamine Derivatives 6 and 7. The trimethylsilyl compounds 4 and
5 (10.0 mmol) in 1.5 mL of dry THF were treated with 0.33 mL of
0.1 M Bu4NF in THF (3.3 mmol). The suspension was stirred at
room temperature for 1 h (GC analysis), after which the mixture
was added to H2O (10 mL) and extracted with CHCl3. The organic
phase was dried over Na2SO4 and evaporated at reduced pressure,
furnishing the analytically pure derivatives 6 and 7, which were
utilized in the next reaction without further purification. Samples
of 6 and 7 were characterized as hydrochloride salts.

N-Substituted Indol-3-ylglyoxylamides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1633
(16) Primofiore, G.; Da Settimo, F.; Marini, A. M.; Taliani, S.; La Motta,
C.; Simorini, F.; Novellino, E.; Greco, G.; Cosimelli, B.; Ehlardo,
M.; Sala, A.; Besnard, F.; Montali, M.; Martini, C. Refinement of
the Benzodiazepine Receptor Site Topology by Structure-Activity
Relationships of New N-(Heteroarylmethyl)indol-3-ylglyoxylamides.
J. Med. Chem. 2006, 49, 2489-2495.
of the circadian rhythm, the experiments were performed only
between 9:00 a.m. and 1:00 p.m. Each group was placed in a glass
cage (20 cm × 40 cm). Twenty minutes after the ip injection, a
video movie was recorded for 1 h by means of a video camera
placed above the cages.
(17) Huang, Q.; He, X.; Ma, C.; Liu, R.; Yu, S.; Dayer, C. A.; Wenger,
G. R.; McKernan, R.; Cook, J. M. Pharmacophore/Receptor Models
for GABAA/BzR Subtypes (R1â3γ2, R5â3γ2, and R6â3γ2) via a
Comprehensive Ligand-Mapping Approach. J. Med. Chem. 2000, 43,
71-95.
(18) He, X.; Huang, Q.; Ma, C.; Yu, S.; McKernan, R.; Cook, J. M.
Pharmacophore/Receptor Models for GABAA/BzR R2â3γ2, R3â3γ2
and R4â3γ2 Recombinant Subtypes. Included Volume Analysis and
Comparison to R1â3γ2, R5â3γ2 and R6â3γ2. Drug Des. DiscoVery
2000, 17, 131-171.
(19) Yu, S.; He, X.; Ma, C.; McKernan, R.; Cook, J. M. Studies in Search
of R2 Selective Ligands for GABAA/BzR Receptor Subtypes. Part
I. Evidence for the Conservation of Pharmacophoric Descriptors for
DS Subtypes. Med. Chem. Res. 1999, 9, 186-202.
(20) Da Settimo, A.; Primofiore, G.; Marini, A. M.; Ferrarini, P. L.;
Franzone, J. S.; Cirillo, L.; Reboani, M. C. N-(indol-3ylglyoxylyl)-
methionine Derivatives: Preparation and Gastric Antisecretory
Activity. Eur. J. Med. Chem. 1988, 23, 21-24.
Data collection was performed with a blind procedure by three
different observers. By means of the video movie, the observers
looked at each cage for 1 s with 1 min intervals and counted if
none, one, two, or all three mice showed any characteristic
spontaneous motor activity (such as locomotion, rearing, grooming,
or sniffing). Each observer collected the experimental results in
the absence of the other observers and without knowing their
findings. The data were obtained as counts/h and were expressed
as means ( standard error, deriving from the results collected by
the three observers in all the experimental sessions. The data were
statistically analyzed by the two-way Student’s t-test for unpaired
data. A value of P < 0.05 was considered as representative of
statistically significant differences.
Acknowledgment. This work was financially supported by
(21) Sonogashira, K.; Tohda, Y.; Hagihara, N. A. Convenient Synthesis
of Acetylenes: Catalytic Substitutions of Acetylenic Hydrogen with
Bromoalkenes, Iodoarenes, and Bromopyridines. Tetrahedron Lett.
1975, 50, 4467-4470.
(22) Sonogashira, K. Development of Pd-Cu Catalyzed Cross-Coupling
of Terminal Acetylenes with sp2-Carbon Halides. J. Organomet.
Chem. 2002, 653, 46-49.
(23) Howell, S. J.; Spencer, N.; Philp, D. Recognition-Mediated Regio-
control of a Dipolar Cycloaddition Reaction. Tetrahedron 2001, 57,
4945-4954.
MIUR (COFIN 2005, ex 40%).
Supporting Information Available: Physical (Table 1),
spectral (Table 2), and analytical data (Appendix) of 10-37.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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