Refinement of the benzodiazepine receptor site topology by structure-activity relationships of new N-(heteroarylmethyl)indol-3- ylglyoxylamides

Article abstract of DOI:10.1021/jm0511841

N-(Heteroarylmethyl)indol-3-ylglyoxylamides (1-26) were synthesized and evaluated as ligands of the benzodiazepine receptor (BzR) to probe the hydrogen bonding properties of the so-called S₁ site of the BzR by means of suitable heterocyclic side chains. SARs were developed in light of our hypothesis of binding modes A and B. Pyrrole and furan derivatives adopting mode A (2, 8, 10, 20, 22) turned out to be more potent (K_i values < 35 nM) than their analogues lacking hydrogen bonding heterocyclic side chains. These data suggest that the most potent indoles interact with a hydrogen bond acceptor/donor (HBA/D) group located within the S₁ site of the BzR. Compounds 1, 2, 8, 19, 20, and 22, tested at recombinant rat α₁β₂γ₂. α ₂β₂γ₂, and α₅β ₃γ₂ BzRs, elicited selectivity for the α₁β₂γ₂ isoform. On the basis of published mutagenesis studies and the present SARs, we speculate that the S₁ HBA/D group might be identified as the hydroxyl of α₁-Tyr209 or of other neighboring amino acids.

Full text of DOI:10.1021/jm0511841

J. Med. Chem. 2006, 49, 2489-2495
2489
Refinement of the Benzodiazepine Receptor Site Topology by Structure-Activity Relationships
of New N-(Heteroarylmethyl)indol-3-ylglyoxylamides
Giampaolo Primofiore,§,* Federico Da Settimo,§ Anna Maria Marini,§ Sabrina Taliani,§ Concettina La Motta,§
Francesca Simorini,§ Ettore Novellino,† Giovanni Greco,† Barbara Cosimelli,† Marina Ehlardo,† Annalisa Sala,†
Franc¸ois Besnard,‡ Marina Montali,| and Claudia Martini|
Dipartimento di Scienze Farmaceutiche, UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Chimica Farmaceutica e
Tossicologica, UniVersita` di Napoli “Federico II”, Via Domenico Montesano, 49, 80131 Napoli, Italy, Department of Molecular and
Functional Genomics, 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 NoVember 23, 2005
N-(Heteroarylmethyl)indol-3-ylglyoxylamides (1-26) were synthesized and evaluated as ligands of the
benzodiazepine receptor (BzR) to probe the hydrogen bonding properties of the so-called S1 site of the BzR
by means of suitable heterocyclic side chains. SARs were developed in light of our hypothesis of binding
modes A and B. Pyrrole and furan derivatives adopting mode A (2, 8, 10, 20, 22) turned out to be more
potent (Ki values < 35 nM) than their analogues lacking hydrogen bonding heterocyclic side chains. These
data suggest that the most potent indoles interact with a hydrogen bond acceptor/donor (HBA/D) group
located within the S1 site of the BzR. Compounds 1, 2, 8, 19, 20, and 22, tested at recombinant rat R1â2γ2,
R2â2γ2, and R5â3γ2 BzRs, elicited selectivity for the R1â2γ2 isoform. On the basis of published mutagenesis
studies and the present SARs, we speculate that the S1 HBA/D group might be identified as the hydroxyl
of R1-Tyr209 or of other neighboring amino acids.
Introduction
The R1âxγ2 subtypes are the dominant ones, seeing that they
are present in both cerebellum and cortex, the R2âxγ2 and R3âxγ2
are moderately abundant and are found mainly in the cortex
and hippocampus, whereas the R5âxγ2 receptors are scarce, as
they are widely expressed only in the hippocampus. This
regional heterogeneity of the GABAA receptor subtypes in the
brain has suggested that the different subtypes might be
associated with different physiological effects. Actually, the R1
containing BzR should be responsible for the sedative action,
the R2 subtype should mediate the anxiolytic activity and the
myorelaxation effects, the role of the R3 subtype remains
unclear, or at least, it seems to be partly involved in mediating
anxiety behavior, and finally, the R5 subtype has been associated
with cognition processes such as learning and memory.5-8
GABA (γ-aminobutyric acid) is the major inhibitory neu-
rotransmitter in the central nervous system (CNS) and operates
through three different receptor types, the ionotropic GABAA
and GABAC receptors, and the metabotropic GABAB recep-
tors.1,2 Besides playing an important role in central transmission
processes, these receptors, especially the GABAA receptors, have
increasingly attracted interest as therapeutic targets, due to their
involvement with several neurological and psychiatric disorders
such as anxiety, sleeplessness, epilepsy, and amnesia.3 The
GABAA receptor is a ligand-gated chloride ion channel with a
heteropentameric structure made up of a total of 21 subunits
(R1-6, â1-4, γ1-4, θ, π, ꢀ, F1-3, and δ) of which 16 have been
found in the mammalian CNS.1,3 The major isoform of GABAA
receptors contains R, â, and γ subunits, with R and â subunits
being necessary for the GABA-activated ion-channel function,
and γ subunits giving sensitivity to benzodiazepines (Bz). It
has been proposed that the stoichiometry of the three subunits
in the pentamer is 2R2â1γ, with the Bz binding site (BzR)
occurring at the interface of the R and γ subunits.4 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.5,6 The
â subunit type does not seem to influence the pharmacology of
Bz, whereas it has been demonstrated5-7 that the R subunit is
the main determinant of BzR ligand action selectivity (therefore,
BzR subtypes take their names from the R subunit). In situ
mRNA hybridization, subunit specific immunoprecipitation, and
immunoaffinity chromatography have made it possible to
determine the distribution of the GABAA subtypes in the brain.
BzR ligands modulate GABAA receptors increasing or
decreasing the action of the inhibitory GABA neurotransmitter,
which, by binding to its receptor, determines the opening of
the chloride channel. Depending on the type of efficacy, BzR
ligands have been classified as agonists (anxiolytic, anticon-
vulsant, sedative-hypnotic, and myorelaxant agents), antagonists
(nil efficacy), and inverse agonists (anxiogenic, somnolytic,
proconvulsant, or even convulsant agents).9,10
Thus, a better knowledge of the structure of the site of action
of the GABAA/BzR complex should aid in the design and
synthesis of high affinity subtype-selective ligands, which may
lead to the selective treatment of anxiety, sleep disorders,
convulsions, and memory deficits with fewer side effects.
Unfortunately, our understanding of the GABAA/BzR complex
functioning is limited by the lack of high-resolution three-
dimensional structural information. An acetylcholine-binding
protein (AChBP) homologous to the N-terminal extracellular
portion of the nicotinic acetylcholine receptor was crystallized
by Breijc et al. in 2001.11 This nicotinic acetylcholine receptor
belongs, with GABAA, glycine, and the 5-HT3 receptors, to the
same gene family, the ligand-gated ion channels (LGICs).12,13
Thus, GABAA receptors possess a structural and, to a small
extent, sequential homology with the crystallized protein.
* To whom all correspondence should be addressed. Tel.: +390502219546.
Fax: +390502219605. E-mail: primofiore@farm.unipi.it.
§ 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/jm0511841 CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/30/2006

2490 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Primofiore et al.
Scheme 1
Several models of the extracellular, ligand-binding domain of
the GABAA/BzR complex have recently been proposed on the
basis of the AChBP structure.14-17 Despite these advances, no
comprehensive models have so far been obtained. Before serious
modeling can be performed, more information about the
orientation of the residues in the BzR binding site has to be
available.18
Some help to clarify the manner in which ligands bind to the
GABAA/BzR may be offered by the use of the techniques of
chemical synthesis, radioligand binding, and receptor mapping.
The pharmacophore/topological model proposed by Cook et al.19
greatly improved our understanding of the structure-affinity
relationships of BzR ligands in terms of ligand-receptor
interactions. This model consists of several BzR (sub)sites: (i)
a hydrogen bond acceptor (A2), (ii) a hydrogen bond donor (H1),
(iii) a hydrogen bond donor/acceptor (H2/A3), (iv) four lipophilic
pockets (L1, L2, L3, and LDi), and (v) sterically forbidden sites
(S1, S2, and S3) as boundaries of the receptor binding cleft.
Some years ago, we presented a new class of BzR ligands
designed as open chain analogues of â-carbolines I, the
N-(benzyl)indol-3-ylglyoxylamides II (Chart 1).20 In these indole
S2) and is favored by electron-attracting X groups. This binding
mode relies on the following interactions: CdO2 and CdO1
are hydrogen-bound to the H1 and H2 sites; the L1, L2, and LDi
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
a heteroatom belonging to the S1 site.
To verify our hypothesis of a hydrogen bond function located
on the surface of the S1 site, and with the aim of exploiting this
interaction to improve the affinity of indolylglyoxylamides, we
prepared and carried out biological assays on a series of
N-(heteroarylmethyl)indol-3-ylglyoxylamides 1-26. The N-(pyr-
rol-2-ylmethyl)indol-3-ylglyoxylamides 1 and 2 formally derive
from benzylamides II by replacement of the side phenyl ring
with a pyrrole moiety capable of donating a hydrogen bond.
The N-methylated analogues 3 and 4 were designed as negative
controls to verify the effect of the hydrogen bonding properties
of 1 and 2 on the affinity. Moreover, to assess whether the
hydrogen bond acceptor group on S1 could also act as a
hydrogen bond donor, the N-(fur-2-ylmethyl)indol-3-ylglyoxyl-
amides 7-10, featuring an acceptor oxygen atom, were prepared
and tested. To gain information about the location of the
hypothesized hydrogen bond acceptor/donor group (HBA/D) at
the S1 site, the 3-pyrrolylmethyl 19, 20, the 3-furylmethyl 21,
22, the 2-pyridylmethyl 13, 14, the 3-pyridylmethyl 15, 16, and
the 4-pyridylmethyl 17, 18 derivatives were also investigated.
Finally, to probe the dimensions and the physicochemical
properties of the S1 site environment, the 2-indolylmethyl 5, 6,
the 2-thienylmethyl 11, 12, the 3-thienylmethyl 23, 24, and the
2-imidazolylmethyl 25, 26 derivatives were also synthesized and
biologically evaluated.
Chemistry. The general synthetic procedure used in the
preparation of compounds 1-26 involved the acylation of indole
and 5-nitroindole with oxalyl chloride, in accordance with a
published procedure.22 The indolylglyoxylyl chlorides obtained
were allowed to react in mild conditions with the appropriate
amine in the presence of triethylamine in toluene solution
(Scheme 1). The amines employed for the synthesis of
compounds 1-26 were commercially available or obtained in
accordance with published procedures (see Experimental Sec-
tion), except for the unknown 3-aminomethylpyrrole, which was
prepared by slightly modifying the literature procedure for the
obtainment of 2-aminomethylpyrrole,23 involving the reduction
of pyrrole-3-carbaldehyde oxime with sodium in ethanol solu-
tion.
Chart 1
derivatives, the effects of the R1 and X substituents on potency
were found to be interdependent. Specifically, the affinity was
favored by hydroxy/methoxy or halogens as X substituents,
depending on whether the 5-position of the indole nucleus was
substituted (R1 ) Cl/NO2) or not (R1 ) H), respectively. Thus,
while affinity in the 5-Cl/NO2 series was optimized by X )
3′,4′-(OMe)2 (Ki ) 11 nM), in the 5-H series, the highest
potency was reached with X ) 4′-Cl (Ki ) 67 nM). The
structure-affinity relationships of compounds II were subse-
quently rationalized by assuming that in their interaction with
the receptor site, they adopt two alternative binding modes called
A and B (Figure 1).21 The 5-Cl/NO2 ligands bind to the BzR in
Figure 1. The hypothetical binding modes A and B of indole BzR
ligands within the framework of Cook’s pharmacophore/topological
model.19
All target products were purified by recrystallization from
the appropriate solvent, and their structures were confirmed by
IR, 1H NMR, and elemental analysis. The physical and spectral
data of all the newly synthesized compounds 1-26 are reported
in the Supporting Information.
Binding Studies. The binding affinity of each newly syn-
thesized indole derivative at the BzR in bovine brain membranes
was determined by competition experiments against the radio-
labeled antagonist [3H]flumazenil24 and was expressed as the
Ki value only for those compounds inhibiting radioligand binding
mode A, by interacting with the A2 site (through the indole NH),
with the H1 and H2 sites (through the CdO2 and CdO1), 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 ligand affinity is
enhanced by hydroxy and/or methoxy X substituents.
Binding mode B requires R1 ) H (substituents bulkier than
a hydrogen would be repelled by the sterically forbidden region

Benzodiazepine Receptor Site Topology
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2491
Table 1. Inhibition of [3H]Flumazenil Specific Binding to Bovine Cortical Membranes by Indolylglyoxylamides 1-26
a Ki values are means ( SEM of three determinations. b Data taken from ref 21.
by more than 80% at a fixed concentration of 10 µM (Table 1).
For comparison purposes, the binding data of benzylindol-3-
ylglyoxylamides IIa-d are also included in Table 1.21
The subtype selectivity of the most active compounds (1, 2,
8, 19, 20, and 22) was tested by assaying their ability to displace
[3H]flumazenil in membranes from HEK293 cells expressing
rat R1â2γ2, R2â2γ2, and R5â3γ2 GABAA/Bz receptor subtypes
(Table 2).21
of the pyrrole nitrogen, as compound 4 is 4-fold less potent
than 2 and therefore equipotent to the benzylamide derivative
IIb.
Figure 2 shows a low resolution model of interaction between
the pyrrole derivative 2 and the BzR, where the putative
hydrogen bonding function of the S1 site is represented as the
side chain of a hydroxyl-bearing amino acid.
The 5-H derivative 1 (hypothesized to adopt binding mode
B at the BzR) could in principle interact with the A2 acceptor
site through the pyrrole NH. However, the affinity value of 1
(Ki ) 531 nM) similar to, even if slightly lower than, that of
the benzylamide IIa (Ki ) 120 nM), which bears a side phenyl
ring with no hydrogen bond ability, leads to suppose that the
pyrrole moiety of 1 is not likely engaged in any hydrogen bond
with the BzR. Furthermore, the pyrrole moiety, which substitutes
the more lipophilic side chain phenyl ring of IIa, establishes a
Results and Discussion
As shown by the data reported in Table 1, the 5-NO2 pyrrole
derivative 2, which is supposed to interact with the BzR in mode
A, is 3.5-fold more potent than benzylamide IIb, thus suggesting
that the pyrrole NH could donate a hydrogen bond to an acceptor
group located at the S1 site of the BzR. This hypothesis is
supported by the detrimental effect resulting from methylation

2492 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Primofiore et al.
Figure 2. Hypothetical binding mode A at the BzR of compounds 2 and 20, 8 and 22, assumed to form a hydrogen bond with the HBA/D function
located at the S1 site.
Table 2. Inhibition of [3H]Flumazenil Specific Binding on Rat R1â2γ2,
pyrrole and furan derivatives 2 and 20, 8 and 22 might be
R2â2γ2, and R5â3γ2 GABAA/Bz Receptor Subtypes by Selected
oriented within the BzR binding cleft.
The similar affinity displayed by thiophene derivatives 11,
12, 23, and 24 with respect to that of benzylamides IIa and IIb
can be explained by considering that thiophene and benzene
are similar in several physicochemical properties, such as size,
lipophilicity (logP values are 1.89 and 2.13, respectively), and
incapacity to form hydrogen bonds.
Compoundsa
Ki (nM)b or % inhibition (10 µM)c
no.
R1â2γ2
R2â2γ2
R5â3γ2
1
2
8
19
20
22
271 ( 15
73.5 ( 6
20 ( 3
272 ( 18
9.2 ( 0.8
56.6 ( 4
50 ( 3
47% ( 5
585 ( 43
155 ( 12
46% ( 4
154 ( 13
236 ( 19
765 ( 63
1500 ( 143
5% ( 1
28% ( 3
681 ( 53
1600 ( 152
9% ( 2
The introduction of 2-pyridyl, 3-pyridyl, and 4-pyridyl rings
into the glyoxylamide side chain gave compounds 13/14, 15/
16, and 17/18. The 5-H derivatives 13, 15, and 17 showed a
potency no better than that of the benzylamide IIa, thus
confirming the hypothesis of binding mode B in which the
ligands do not form any hydrogen bond with the A2 acceptor
site. Analogously to our hypothesis for the 5-NO2 furan
derivatives 8, 10, and 22, the interaction at the BzR of the 5-NO2
pyridine derivatives 14, 16, and 18 should in theory be favored
by a hydrogen bond formed between the ligand pyridine nitrogen
and the receptor HBA/D function. However, the pyridine
derivatives 14, 16, and 18 turned out to be significantly less
potent than the furan analogues 8, 10, and 22. These findings
might be attributed to the different hydrogen bonding geometric
properties of nitrogen and oxygen as acceptors in aromatic
heterocycles. Nobeli and co-workers investigated the direction-
ality and relative strengths of hydrogen bonds between C-OH
and aromatic rings containing nitrogen and/or oxygen heteroa-
toms, using crystal structure data and theoretical calculations.26
These authors showed that the hydrogen bonds in the C-OH‚
‚‚O(furan) system are more widely scattered around the aromatic
ring plane compared with the C-OH‚‚‚N(pyridine) system.
More specifically, the interaction energy is destabilized up to
5.0 kJ/mol upon deviations of the C-OH hydrogen from the
pyridine plane by 25-35° or from the furan plane by 50°. In
the quoted article, Nobeli’s group concluded that “substituting
oxygen for nitrogen heterocycles will generally have a marked
effect on the binding of a protein ligand”.26 In light of the above-
mentioned studies, given that the HBA/D group at the S1 site
might correspond to an OH placed out of the plane of the ligand
side chain heterocycle, only the furan oxygen, but not the
pyridine nitrogen, in addition to the pyrrole NH, would form a
significantly attractive hydrogen bond with the receptor protein.
These concepts are schematized in Figure 3 by means of three-
dimensional models of ligand moieties illustrating the putative
HBA/D function. The model discussed so far might also explain
zolpidem
35% ( 3
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. c Percentage inhibition values of specific
[3H]flumazenil binding at 10 µM concentration are means ( SEM of three
determinations.
less effective lipophilic interaction with the receptor binding
site. The much lower potency of 3 (Ki ) 4590 nM) with respect
to 1 should therefore be due to a poor steric complementarity
of this compound with the LDi region.
Compounds 5 and 6, which contain an indole moiety in the
side chain, were designed to maintain the hydrogen bond donor
ability, while increasing lipophilicity. The poor affinity displayed
by these compounds appears to stem from their excessive overall
bulk compared with the corresponding pyrrole derivatives 1 and
2. Taken together, these data suggest that the BzR binding cleft
possesses precise dimensions and cannot accommodate bulky
groups into the L2 and LDi lipophilic regions simultaneously,
as earlier proposed by Cook et al.25
As mentioned, the 2-furylmethyl derivatives 7-10 were
designed to probe the hydrogen bond donor ability of the S1
site. The Ki values of the 5-NO2 furans 8 and 10 were similar
to that exhibited by the corresponding 5-NO2 pyrrole 2, thus
suggesting that a bifunctional hydrogen bond acceptor/donor
(HBA/D) group may exist at the S1 site.
The shift of the methylene spacer from position 2 to 3 of the
pyrrole or furan ring gave compounds 19, 20, and 21, 22, which
possess a similar affinity to that of their 2-substituted analogues.
We speculate that the side chains of the 5-NO2 compounds 2,
8, 20, and 22 possess the right geometry, in the binding
orientation A, to interact properly with the HBA/D group at
the S1 site. Figure 2 illustrates our hypothesis of how 5-NO2

Benzodiazepine Receptor Site Topology
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2493
Figure 3. Three-dimensional models illustrating our hypothesis of a hydrogen bond between ligands featuring a pyrrole or a furan moiety (but not
pyridine) in the side chain and a hydroxyl-bearing amino acid located in the vicinity of the S1 site of the BzR binding cleft.
the high potency of the 3′,4′-dimethoxyphenyl-bearing 5-NO2
derivative IId, as the result of a hydrogen bond formed between
the 3′-OMe and the HBA/D function.
to the L1 lipophilic area, which is the only common region of
interaction for all BzR ligands in Cook’s pharmacophore/
topological model.31 As S1 is part of the boundaries of the L1
region, we tentatively hypothesize that the hydroxyl moiety of
R1-Tyr209 might represent the HBA/D group. The role of other
amino acid-bearing side chains with hydrogen bond donor/
acceptor capabilities, located in the proximity of R1-Tyr209,
cannot be ruled out until accurate three-dimensional information
is obtained about the BzR binding site. In this connection, it
has been demonstrated that Ser20432 and Thr20629 influence
affinities for benzodiazepine binding site ligands.
The differences in affinity between the benzylamide IIb and
the ligands which are thought to form a hydrogen bond with
the putative receptor hydroxy group (compounds 2, 8, 10, 20,
and 22) vary from 3.5-fold to 7.7-fold. Such differences seem
to be consistent with the energies associated with “neutral-
neutral” hydrogen bonds which stabilize the ligand-receptor
affinity up to a maximum of 15-fold, compared with the much
stronger contributions to the affinity (up to 3000-fold) that come
from “charge-reinforced” hydrogen bonds.33
An additional point supporting the existence of an HBA/D
group at the S1 site is the markedly higher potency of the 5-NO2
ligands compared with the 5-H counterparts, whenever the
glyoxylamide side chain forms a hydrogen bond with the BzR
in binding mode A (compare the Ki values of the couples IId/
IIc, 8/7, 10/9, 20/19, and 22/21). In contrast, the couples of
5-NO2/5-H derivatives which are not engaged in such a
hydrogen bond do not display the affinity gap favoring the
5-NO2 ligand (compare the Ki values of the couples IIb/IIa,
12/11, and 24/23).
The inactivity of imidazole derivatives 25 and 26, which
feature both hydrogen bond accepting and donating functions
in the heteroaryl ring, might be ascribed to their high intrinsic
hydrophilicity (the logP of imidazole is -0.08) as well as their
propensity to be partly protonated at physiological pH (the pKb
of imidazole is 7.0).
A number of compounds (1, 2, 8, 19, 20, and 22) were tested
for their ability to displace [3H]flumazenil from recombinant
rat R1â2γ2, R2â2γ2, and R5â3γ2 GABAA/Bz receptor subtypes
(Table 2). All of the ligands had enhanced affinities for the
R1â2γ2 isoform compared with the R2â2γ2 and R5â3γ2 subtypes.
It should be noted that the 5-H derivatives 1 and 19, hypoth-
esized to bind according to mode B, showed a high R1â2γ2
selectivity over the R2â2γ2 subtype, but a lower selectivity over
the R5â3γ2 subtype; on the other hand, the 5-NO2 derivatives
2, 8, 20, and 22, hypothesized to adopt binding mode A,
displayed a very good R1â2γ2 selectivity over the R5â3γ2
subtype, but only a moderate selectivity over the R2â2γ2 subtype.
It has been suggested that the basic topology of the BzR
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.27 In particular, it is
hypothesized that the LDi and L2 regions are wider in the R1â2γ2
and R5â2γ2 binding sites, respectively, with respect to the other
subtypes. Consequently, full occupation of LDi or L2 may
account for R1â2γ2 and R5â2γ2 selectivity, respectively.27 Finally,
simultaneous occupation of LDi and L2 may promote R2
selectivity.28 This is consistent with the general R1â2γ2 selectiv-
ity profile displayed by our N-(heteroarylmethyl)indol-3-yl-
glyoxylamides filling the LDi region in either of the two possible
binding modes A and B. Additionally, the moderate affinity at
the R2â2γ2 subtype shown by the 5-NO2 derivatives 2, 8, 20,
and 22 might be related to simultaneous occupation of the LDi
and L2 regions in binding mode A, whereas the 5-H derivatives
1 and 19, which adopt binding mode B, display a significant
affinity at the R5â3γ2 subtype thanks to a strong interaction of
the bulky indole ring with the L2 pocket.
In conclusion, the binding affinity data of the novel N-(het-
eroarylmethyl)indol-3-ylglyoxylamides 1-26 supported the
existence of a hydrogen bond acceptor/donor (HBA/D) group
located within the S1 site of the BzR. The affinity results at the
GABAA/BzR receptor subtypes, performed on the more sig-
nificant compounds of the series (1, 2, 8, 19, 20, and 22, Table
2), indicate for these ligands a general R1â2γ2 selectivity over
the other subtypes. Interestingly, those compounds adopting
binding mode A, such as 2, 8, 20, and 22, also display a
significant affinity at the R2â2γ2 subtype, probably because they
simultaneously occupy both LDi and L2 regions, whereas ligands
binding according to mode B (1 and 19), show some affinity at
the R5â3γ2 subtype, thanks to the full occupation of the L2 region
by the bulky indole moiety.
Experimental Section
Chemistry. Melting points were determined using a Reichert
Ko¨fler hot-stage apparatus and are uncorrected. Boiling points were
determined using a Bu¨chi Melting Point B-540. IR spectra were
recorded with a Pye Unicam Infracord Model PU956 in Nujol mulls.
1
Routine H NMR spectra were determined on a Varian Gemini
200 spectrometer using DMSO-d6 as the solvent. Evaporation was
performed in vacuo (rotary evaporator). Anhydrous sodium sulfate
was always used as the drying agent. Analytical TLC was carried
out on Merck 0.2 mm precoated silica gel (60 F-254) aluminum
sheets, with visualization by irradiation with a UV lamp. Elemental
analyses were performed by our Analytical Laboratory and agreed
with theoretical values to within (0.4%.
2-Aminomethylfurane, 2-aminomethyl-5-methylfurane, 2-ami-
nomethylthiophene, 2-aminomethylpyridine, 3-aminomethylpyri-
dine, 4-aminomethylpyridine are commercially available products.
Indol-3-ylglyoxylyl chloride,22 5-nitroindol-3-ylglyoxylyl chloride,22
2-aminomethyl-1H-pyrrole,23 2-aminomethyl-1-methyl-1H-pyr-
role,34 2-aminomethylindole,35 3-aminomethylfuran,34 3-aminom-
ethylthiophene,34 2-aminomethylimidazole,36 and pyrrole-3-carbox-
aldehyde37 were prepared in accordance with reported procedures.
3-Aminomethyl-1H-pyrrole. A solution of 1H-pyrrole-3-car-
boxaldehyde37 (0.810 g, 8.5 mmol), hydroxylamine hydrochloride
On the basis of mutagenesis studies, Buhr and Amin,29,30
independently postulated that the phenyl moiety of the side chain
of R1-Tyr209 is important for the binding of agonist, antagonist,
and inverse-agonist BzR ligands. Thus, this R1-Tyr209 phenyl
ring should interact favorably with a common feature of these
ligands, and, consequently, should be located in a region close

2494 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Primofiore et al.
(0.945 g, 13.6 mmol), and K2CO3 (0.764 g, 5.5 mmol) in 25.0 mL
of water was refluxed for 30 min. After cooling, the white crystals
that precipitated were collected by filtration and recrystallized from
water to give 0.751 g of pure 1H-pyrrole-3-carboxaldehyde
oxime: yield 94%; mp 150-152 °C (lit. ref 38 mp 149 °C). The
oxime obtained was dissolved in 13.0 mL of absolute ethanol, and
the solution was maintained in a nitrogen atmosphere. Metallic
sodium (2.17 g, 94.0 mmol) was then added, and the mixture was
refluxed with stirring until all the sodium had reacted (about 1 h).
After cooling, the suspension was treated with 10.0 mL of water
and concentrated under reduced pressure, and the residue was
extracted with diethyl ether. After drying over Na2SO4, evaporation
of the solvent gave analytically pure 3-aminomethyl-1H-pyrrole as
a colorless oil (yield 64%; bp 154 °C). IR ν cm-1: 3350, 3280,
1600, 1580, 1440, 780. 1H NMR (DMSO-d6): δ 3.20 (bs, 2H, NH2);
3.53 (s, 2H, CH2); 5.96-5.98 (m, 1H, 4-H); 6.45-6.63 (m, 2H,
2-H, 5-H); 10.45 (bs, 1H, NH). Anal. (C5H8N2), C, H, N.
General Procedure for the Synthesis of N-[(5-Substituted
indol-3-yl)glyoxyl]amide Derivatives 1-26. Triethylamine (3.0
mmol) was added dropwise to a stirred suspension, cooled at 0 °C,
of 5-substituted indolylglyoxylyl chloride22 (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
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 1-26 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.
Supporting Information Available: Physical (Table 1), spectral
(Table 2), and analytical data of 1-26. This material is available
free of charge via the Internet at http://pubs.acs.org.
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MIUR (COFIN 2003, ex-40%).

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