Anxiolytic-like effects of N,N-dialkyl-2-phenylindol-3-ylglyoxylamides by modulation of translocator protein promoting neurosteroid biosynthesis

Article abstract of DOI:10.1021/jm8003224

Novel N,N-disubstituted indol-3-ylglyoxylamides (1-56), bearing different combinations of substituents R₁-R₅, were synthesized and evaluated as ligands of the translocator protein (TSPO), the 18 kDa protein representing the minimal f

Full text of DOI:10.1021/jm8003224

5798
J. Med. Chem. 2008, 51, 5798–5806
Anxiolytic-like Effects of N,N-Dialkyl-2-phenylindol-3-ylglyoxylamides by Modulation of
Translocator Protein Promoting Neurosteroid Biosynthesis
Federico Da Settimo,§ Francesca Simorini,*,§ Sabrina Taliani,§ Concettina La Motta,§ Anna Maria Marini,§ Silvia Salerno,§
Marusca Bellandi,§ Ettore Novellino,† Giovanni Greco,† Barbara Cosimelli,† Eleonora Da Pozzo,‡ Barbara Costa,‡
Nicola Simola,# Micaela Morelli,#,∆ and Claudia Martini‡
Dipartimento di Scienze Farmaceutiche and Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, UniVersita` di Pisa,
Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersita` di Napoli “Federico II”,
Via D. Montesano, 49, 80131 Napoli, Italy, and Dipartimento di Tossicologia, UniVersita` di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
ReceiVed March 21, 2008
Novel N,N-disubstituted indol-3-ylglyoxylamides (1-56), bearing different combinations of substituents
R1-R5, were synthesized and evaluated as ligands of the translocator protein (TSPO), the 18 kDa protein
representing the minimal functional unit of the “peripheral-type benzodiazepine receptor” (PBR). Most of
the new compounds showed a nanomolar/subnanomolar affinity for TSPO and stimulated steroid biosynthesis
in rat C6 glioma cells with a potency similar to or higher than that of classic TSPO ligands such as PK
11195. Moreover, when evaluated in vivo by means of the elevated-plus-maze (EPM) paradigm in the rat,
compound 32, the best-performing derivative in terms of TSPO affinity and pregnenolone production, showed
clear anxiolytic effects. The results of this study suggested that the novel N,N-disubstituted indol-3-
ylglyoxylamides may represent a promising class of compounds potentially suited for the treatment of anxiety
disorders.
Introduction
The PBR is involved in a variety of biological processes,
including calcium homeostasis, lipid metabolism, mitochondrial
oxidation, cell growth and differentiation, apoptosis induction,
and regulation of immune functions.5,8 The basal expression of
this receptor is up-regulated in a number of neuropathologies,
including gliomas and neurodegenerative disorders, such as
Alzheimer’s disease, as well as in various forms of brain injury
and inflammation.9 Changes in the PBR receptor levels have
also been found in patients affected by generalized anxiety,
panic, post-traumatic stress, obsessive-compulsive disorders,
and separation anxiety.5,10,11
Anxiety and sleep disorders can be controlled by the
administration of benzodiazepines, such as diazepam and
chlordiazepoxide, which produce their therapeutic effects by
enhancing the affinity of γ-aminobutyric acid (GABAa) toward
the central benzodiazepine receptor (BzR). The clinical useful-
ness of these relatively safe drugs is limited by unwanted side
effects, including ataxia, ethanol potentiation, dizziness, amnesia,
and the risk of dependence with long-term use.1,2 Therefore,
there is still a need for new and safer anxiolytic agents.
Several benzodiazepines bind to a receptor distinct from the
BzR, originally identified outside the central nervous system
(CNS) and therefore called “peripheral-type benzodiazepine
receptor” (PBR).3 The PBR was subsequently described as a
multimeric complex made up of three subunits: a protein of 18
kDa hosting the diazepam binding site (PBR), a 32 kDa voltage-
dependent anion channel (VDAC) and a 30 kDa adenine
nucleotide carrier (ANC).4,5 Although the PBR has a wide
distribution in several peripheral tissues, especially those
producing steroids,4,5 it is also present in the CNS, where it is
mainly expressed in glial cells and, at lower levels, in neurons.6
Inside the cell, the PBR is primarily localized in the outer/inner
mitochondrial membrane as a component of the megachannel
responsible for mitochondrial permeability transition (MPT).7
The PBR is involved in the regulation of cholesterol trans-
location from the outer to the inner mitochondrial membrane,
a process known to be the rate-determining step in steroid
biosynthesis.4 For this reason, Papadopoulos et al. proposed
“translocator protein” (TSPO) as a new name for the 18 kDa
protein representing the minimal functional unit of the PBR.12
This term is used throughout the present paper.
High-affinity TSPO ligands, such as PK 11195,13 alpidem,14
and FGIN-1-2715,16 (Chart 1), increase the levels of the so-called
neurosteroids, such as pregnenolone and allopregnanolone,
acting as positive allosteric modulators of GABA neurotrans-
mission. Thus, neurosteroidogenic TSPO ligands may represent
a significant advance in the development of novel pharmaco-
logical agents displaying an anxiolytic-like profile, without the
unwanted side effects typical of classic benzodiazepines.9
We have recently described the N,N-dialkyl-2-phenylindol-
3-ylglyoxylamides I (Chart 1)17 as a new class of potent and
selective TSPO ligands. The SARs of these compounds were
rationalized in light of a pharmacophore/topological model made
up of three lipophilic pockets (L1, L3, and L4) and an H-bond
donor group (Figure 1).18 The optimum binding affinity to TSPO
requires two lipophilic substituents on the amide nitrogen (R1
) R2 ) n-propyl, n-butyl, n-hexyl, etc.). Moreover, substituents
in the 4′ and 5 positions of the 2-phenylindole scaffold (R3 and
R4, respectively) modulate potency in a manner dependent on
the nature of the R1 and R2 substituents.17 Several of our indole
* To whom correspondence should be addressed. Phone: +39 50
2219555. Fax: +39 50 2219605. E-mail: simorini@farm.unipi.it.
§
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
Universita` “Federico II” di Napoli.
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
†
‡
nologie, Universita` di Pisa.
#
Universita` di Cagliari.
Present Address: Istituto di Neuroscienze, CNR Sezione di Cagliari,
∆
S.p. 8 Monserrato-Sestu, 09042 Monserrato (CA), Italy.
a Abbreviations: TSPO, translocator protein; PBR, peripheral-type ben-
zodiazepine receptor; EPM, elevated plus-maze; GABA, γ-aminobutyric
acid; MPT, mitochondrial permeability transition; VDAC, voltage-dependent
anion channel; ANC, adenine nucleotide carrier.
10.1021/jm8003224 CCC: $40.75
2008 American Chemical Society
Published on Web 08/26/2008

N,N-Dialkyl-2-phenylindol-3-ylglyoxylamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5799
Table 1. Receptor Binding Affinity of Compounds 1-27 for TSPO and
Their Stimulatory Effects on Pregnenolone Biosynthesis
Chart 1. Structures of TSPO Ligands
increase in
pregnenolone
production vs
compd
R1 ) R2
R3
R4
R5
Ki (nM)a
control (%)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
(CH2)2CH3 NO2
(CH2)3CH3 NO2
(CH2)5CH3 NO2
(CH2)2CH3 CF3
(CH2)3CH3 CF3
(CH2)5CH3 CF3
H
H
H
H
H
H
NO2
NO2
NO2
OCH3
OCH3
OCH3
F
F
F
F
F
F
Cl
Cl
Cl
H
0.95 ( 0.1
0.27 ( 0.07
0.23 ( 0.1
1.69 ( 0.2
1.16 ( 0.1
1.0 ( 0.1
20.2 ( 2.02
21.6 ( 2.15
30.3 ( 9.15
328 ( 45
65.2 ( 3.4
35.5 ( 8.7
2.67 ( 0.48
4.00 ( 0.15
0.37 ( 0.12
6.73 ( 1.39
4.36 ( 0.05
0.95 ( 0.1
2.83 ( 0.08
3.05 ( 0.45
7.75 ( 1.55
14.0 ( 1.5
3.40 ( 0.3
2.4 ( 0.3
70 ( 7
64 ( 2
88 ( 7
66 ( 7
60 ( 6
85 ( 5
91 ( 2
102 ( 10
115 ( 10
45 ( 2
36 ( 3
87 ( 7
66 ( 7
88 ( 9
36 ( 4
17 ( 1
91 ( 8
40 ( 2
42 ( 4
37 ( 4
135 ( 4
0
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
H
H
H
H
H
H
H
H
H
F
F
F
F
F
F
22
23
24
25
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0
0
CH3 25.0 ( 3.0
CH3 6.0 ( 0.6
CH3 1.90 ( 0.1
H
H
H
129 ( 13
71 ( 8
63 ( 5
8 ( 1
23 ( 3
30 ( 3
48 ( 5
41 ( 4
26
derivatives turned out to be more effective than standard TSPO
ligands, such as PK 11195 and FGIN-1-27, in stimulating
pregnenolone biosynthesis. Furthermore, the introduction of a
fluorophore group linked to the 2-phenylindolylglyoxylamide
scaffold gave fluorescent probes useful for investigating the
localization and the expression level of TSPO.19
27
Iac
12.2 ( 1.0
7.5 ( 0.7
1.40 ( 0.2
9.3 ( 0.5
23 ( 3.1
0.5-7
Ibc
Icc
H
PK 11195
Ro5-4864
alpidem
The present paper describes the refinement of our TSPO
pharmacophore/topological model through the synthesis and the
biological evaluation of novel indole derivatives with the general
formula I, bearing different combinations of substituents R1-R5.
Some of these compounds (1-27, Table 1) bear linear alkyl
chains on the amide nitrogen (R1 ) R2 ) n-propyl, n-butyl,
n-hexyl) and modifications to the basic 2-phenylindol-3-ylgly-
oxylamide scaffold, consisting of the introduction of small
groups in the para position of the 2-phenyl ring (R3 ) F, NO2,
CF3) and the 5-position (R4 ) F, Cl, NO2, OCH3) and the
7-position (R5 ) Cl, CH3) of the indole ring. A series of N-ethyl-
N-benzyl substituted indoles (28-42, Table 2) was designed
taking as a reference AC-5216 (Chart 1), a TSPO ligand that
displays antianxiety activity without the undesirable effects
caused by typical benzodiazepines.20 We also investigated a
subset of asymmetrical N,N-disubstituted indoles (43-56, Table
3) to probe the L3 and L4 lipophilic pockets of the TSPO
binding site (R1 ) methyl, ethyl; R2 ) ethyl, n-butyl, n-pentyl;
R3 ) H, Cl; R4 ) H, Cl).
a The concentration of test compounds that inhibited [3H]PK11195
binding to rat kidney mitochondrial membranes (IC50) by 50% was
determined with six concentrations of the displacers, each performed in
triplicate. Ki values are the mean ( SEM of three determinations. b C6
glioma cells were incubated for 2 h at 37 °C in the presence of each
compound. Pregnenolone was quantified by enzymatic immunoassay, as
described in the text. The values represent the mean ( SEM of at least
three determinations. For comparison, the effects of PK 11195 and Ro5-
4864 on pregnenolone production are also included. c Data taken from
ref 17.
whether their ability to increase neurosteroid synthesis in vitro
was associated with the production of anxiolytic effects in vivo.
Chemistry
The target N,N-dialkyl-2-phenylindol-3-ylglyoxylamides 1-
56 were synthesized through the 2-phenylindoles 67-81, which
in turn were obtained, with the exception of the commercially
available 2-phenylindole 67 and 2-(4-fluorophenyl)indole 68,
following experimental procedures reported in literature. The
preparation of indoles 69-72 (see Experimental Section) was
performed essentially as described in our previous work.17
2-(4-Nitrophenyl)indole 73 and 2-(4-trifluoromethylphenyl)in-
dole 74 were prepared with a one-step Fischer indole synthesis
by warming phenylhydrazine hydrochloride and 4′-nitro- or 4′-
(trifluoromethyl)acetophenone with an excess of polyphosphoric
Most of the compounds were evaluated for their ability to
induce pregnenolone biosynthesis in rat C6 glioma cells. The
best-performing ligands in terms of TSPO affinity and preg-
nenolone production were tested in a rat anxiety model to verify

5800 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Da Settimo et al.
Figure 1. TSPO pharmacophore/topological model.18
Table 2. Receptor Binding Affinity of Compounds 28-42 for TSPO
The general synthetic procedure used in the preparation of
derivatives 1-56 is shown in Scheme 2 and involved the
acylation of the appropriate 2-phenylindole 67-81 with oxalyl
chloride, in anhydrous ethyl ether, at room temperature to obtain
the corresponding 2-phenylindolylglyoxylyl chlorides 82-96.22
The acyl chlorides 88-92, 94-96 have never been described
before in literature and were characterized as their corresponding
ethyl ester derivatives (Supporting Information). The indolylg-
lyoxylyl chlorides 82-96 were allowed to react at room
temperature with the appropriate dialkylamine or with N-ethyl-
N-benzylamine in the presence of triethylamine in dry toluene
solution to give products 1-56, which were purified by
recrystallization from the appropriate solvent or by flash
and Their Stimulatory Effects on Pregnenolone Biosynthesis
increase in pregnenolone
production vs
compd
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
R3
R4
R5
Ki (nM)a
control (%)b
H
F
Cl
H
Cl
CH3
NO2
CF3
H
H
H
F
F
H
H
H
H
H
Cl
Cl
H
H
H
NO2
OCH3
F
H
11 ( 1.0
20 ( 2
96 ( 8
136 ( 11
85 ( 8
171 ( 14
86 ( 7
103 ( 8
nd
0
nd
nd
nd
H
H
H
H
H
H
H
H
H
H
H
H
Cl
1.68 ( 0.12
1.30 ( 0.05
4.6 ( 0.5
1
chromatography. Their structures were confirmed by IR, H
NMR, and elemental analysis (Supporting Information).
3.33 ( 0.3
2.64 ( 0.1
0.55 ( 0.02
1.0 ( 0.1
18.3 ( 0.15
69.5 ( 3.6
1.33 ( 0.2
1.67 ( 0.37
4.01 ( 0.26
5.0 ( 0.4
Biological and Pharmacological Studies
The binding affinity of all the newly synthesized indolylg-
lyoxylamides 1-56 at the TSPO was determined in rat kidney
membranes by competition experiments against [3H]PK 11195.17
Because of the well-established TSPO versus BzR selectivity
of N,N-dialkyl-2-phenylindol-3-ylglyoxylamides,17 a few ran-
domly selected indole derivatives were evaluated for their BzR
affinity, using membranes from rat brain tissues and [3H]Ro15-
1788 as the radioligand. The compounds tested showed no
significant binding properties in this assay (data not shown).
A subset of TSPO high-affinity ligands was examined for
the ligands’ ability to stimulate pregnenolone formation from
rat C6 glioma cells at 40 µM concentration. This high
concentration of compounds with respect to their nanomolar
affinity is necessary to induce pregnenolone production, as
demonstrated by previously reported analogous studies.17,18,23
The best-performing compounds, 21 and 32, were evaluated for
their potential in vivo anxiolytic activity by means of the
elevated-plus-maze (EPM) paradigm in the rat. Both compounds
were tested at a dose of 30 mg/kg (ip), prompted by similar in
vivo experiments on 2-phenylimidazo[1,2-a]pyridine derivatives
possessing TSPO affinity.24 Additionally, the effects of com-
pound 21 were also evaluated at a dose of 50 mg/kg (ip).
F
Cl
H
H
0
0
0
CH3 2.30 ( 0.2
PK 11195
Ro5-4864
alpidem
9.3 ( 0.5
48 ( 5
41 ( 4
23 ( 3.1
0.5-7
a The concentration of test compounds that inhibited [3H]PK11195
binding to rat kidney mitochondrial membranes (IC50) by 50% was
determined with six concentrations of the displacers, each performed in
triplicate. Ki values are the mean ( SEM of three determinations. b C6
glioma cells were incubated for 2 h at 37 °C in the presence of each
compound. Pregnenolone was quantified by enzymatic immunoassay, as
described in the text. The values represent the mean ( SEM of at least
three determinations. For comparison, the effects of PK 11195 and Ro5-
4864 on pregnenolone production are also included.
acid (PPA) (Scheme 1). Nitration of 2-phenylindole 67 furnished
the 5-nitro-2-phenylindole 75 (Scheme 1). The indole derivatives
76-81 (see Experimental Section) were obtained through a
Houlihan21 modification of the Madelung indole synthesis from
the appropriate amides 61-66, obtained by acylation of the
corresponding 2-methylanilines (Scheme 1; all experimental data
of amides 61-66 and indoles 73-75 are reported in the
Supporting Information). In brief, amides 61-66 were trans-
formed into the corresponding indole derivatives by reaction
with 3 molar equiv of n-butyllithium in tetrahydrofuran at -20
°C and were then stirred at room temperature for 24-48 h. The
crude indole derivatives recovered after workup were purified
by flash chromatography and/or by recrystallization from the
appropriate solvent (Supporting Information).
Results and Discussion
The binding affinities of compounds 1-27, 28-42, and 43-
56, expressed as Ki values, are given in Tables 1, 2, and 3,
respectively, together with the Ki values of the standard TSPO
ligands PK 11195, Ro5-4864, and alpidem (Chart 1). The
binding data of some of the previously investigated indole
derivatives17 are included for comparison at the bottom of
Table 1 (Ia-c) and Table 3 (Ia,d-f). Tables 1-3 also include

N,N-Dialkyl-2-phenylindol-3-ylglyoxylamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5801
Scheme 1. Synthesis of 2-Phenylindoles 73-81
the neurosteroidogenic activity of most of the newly synthesized
compounds, expressed as percent value vs control of the increase
in pregnenolone production.
in the 7-position of the indole nucleus (R5) did not produce
any gain in affinity (compare 22-27 with their unsubstituted
counterparts Ia-c).17
In series 1-27, the amide nitrogen is symmetrically disub-
stituted (R1 ) R2) with n-propyl, n-butyl, and n-hexyl chains.
These compounds generally showed a high affinity for TSPO
in the nanomolar/subnanomolar range. With the exception of
derivatives 9 and 21, the highest potency was achieved with R1
) R2 ) n-hexyl. Introduction of a 4′-NO2 group on the 2-phenyl
ring gave the most potent indolylglyoxylamides within this
subset: 2 and 3 with a Ki of 0.27 and 0.23 nM, respectively. A
4′-CF3 group was likewise beneficial for potency (compare 4-6
with Ia-c), although to a lesser extent compared with the 4′-
NO2 group. These data confirmed the favorable influence on
affinity exerted by an electron-withdrawing R3 substituent. This
effect was ascribed by us to a putative π-stacking interaction
between the 2-phenyl and an electron-rich aromatic ring within
the L1 pocket.17 The affinity was lowered by an NO2 or an
OCH3 group in the 5-position of the indole nucleus (R4)
(compare 7-12 with Ia-c) or, in contrast, enhanced by an F
in the same position (compare 13-15 with Ia-c). These data
suggest that R4 has to be electron-withdrawing and also very
small for optimal binding to TSPO, a combination of properties
featured only by fluorine. Substitutions in the 4′- and 5-positions
were merged to yield 16-18 (R3 ) R4 ) F) and 19-21 (R3 )
F, R4 ) Cl). Two halogens in both the 4′- and 5-positions did
not increase affinity in an additive manner, all the 4′,5-dihalo
derivatives being almost as potent or slightly less potent than
the 5-fluoro derivatives 13-15. Insertion of an electron-
withdrawing (Cl) or an electron-donating (CH3) lipophilic group
Compounds 1-21 displayed fair to high steroidogenic
activity, with 21 performing best (135% increase in preg-
nenolone production under conditions in which the activities
of PK 11195 and Ro 5-4864 were 48% and 41%, respectively).
Consequently, 21 was selected for in vivo studies (vide infra).
It is noted that TSPO affinity did not generally correlate with
the steroidogenic activity (this holds true for all the indoles
studied in the present paper, as well for those previously
described17). On the other hand, the nature of the 7-substituent
was decisive in the pregnenolone biosynthesis assay, as the
7-chloro derivatives 22-24 were devoid of any activity, whereas
the 7-methyl derivatives 25-27 were good stimulators of
pregnenolone production. The precise molecular determinants
of the above differences in activity are unknown. At the moment,
we can only speculate about an electronic-based triggering effect
exerted by R5 on the translocation function of the TSPO.
Further attempts to optimize potency focused on asymmetri-
cally N,N-substituted indoles (R1 * R2).
A first subset of N-benzyl-N-ethyl derivatives (28-42 in
Table 2) revealed that this combination of R1 and R2 led to
affinities roughly similar to those exhibited by N,N-di-n-butyl
derivatives. Such a conclusion can be drawn by comparing the
Ki values of 28-42 with our previously reported symmetrically
N,N-substituted indoles.17 Thus, it is likely that the aromatic
moiety of the N-benzyl chain is accommodated within the
lipophilic pocket L3, which also hosts the phenyl ring of PK
11195, in agreement with our pharmacophore/topological model

5802 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Da Settimo et al.
Scheme 2. Synthesis of N,N-Dialkyl-2-phenylindol-3-ylglyoxylamides 1-56
depicted in Figure 1.18 Such a benzyl gives rise to the same
type of hydrophobic interactions established by an aliphatic
moiety of similar size. In this subset, the effects of R3-R5 on
the affinity were similar to those observed in the series of
symmetrical N,N-dialkyl derivatives 1-27 listed in Table 1.
Thus, 34 (R3 ) NO2, R4 ) R5 ) H) was the most potent (Ki )
0.55 nM), whereas 37 (R3 ) H, R4 ) 5-OCH3, R5 ) H) was
the least potent (Ki ) 69.5 nM).
The highest levels of steroidogenic activity were displayed
by 30, 32, and 34. Among these three compounds, 32 was tested
for its in vivo anxiolytic properties (vide infra).
Table 3 lists the binding data of asymmetrical N,N-dialkyl
derivatives (43-56) designed to evaluate whether differences
in the lengths of R1 and R2 could optimize their fit into the
lipophilic pockets L3 and L4. For this purpose, the unsubstituted
and the 4′,5-dichloro, the 4′-chloro, and the 5-chloro substitution
patterns were chosen as representative within the previously
investigated N,N-dialkylindoles (see for comparison Ia,d-f in
Table 3).17 Four combinations of R1 and R2 were selected:
methy/ethyl (43 and 44), methyl/n-butyl (45, 48, 51, 54), methyl/
n-pentyl (46, 49, 52, 55), and ethyl/n-butyl (47, 50, 53, 56).
The 4′,5-dichloro derivatives 48 (R1 ) CH3, R2 ) (CH2)3CH3,
Ki ) 0.15 nM) and 49 (R1 ) CH3, R2 ) (CH2)4CH3, Ki ) 0.18
nM) turned out to be the most potent indolylglyoxylamides so
far described by us.17 These results indicate that the L3 and L4
pockets differ in their dimensions, and that R1 and R2 should

N,N-Dialkyl-2-phenylindol-3-ylglyoxylamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5803
Table 3. Receptor Binding Affinity of Compounds 43-56 for TSPO
and Their Stimulatory Effects on Pregnenolone Biosynthesis
increase in
pregnenolone
production vs
compd
43
44
45
46
47
48
49
50
51
52
53
54
R1
R2
R3 R4 Ki (nM)a
940 ( 120
Cl Cl 9.54 ( 1.29
control (%)b
CH3
CH3
CH3
CH3
CH2CH3
CH3
CH3
CH2CH3
CH3
CH3
CH2CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
H
H
nd
0
(CH2)3CH3
(CH2)4CH3
(CH2)3CH3
(CH2)3CH3 Cl Cl 0.15 ( 0.02
(CH2)4CH3 Cl Cl 0.18 ( 0.02
(CH2)3CH3 Cl Cl 0.36 ( 0.04
(CH2)3CH3 Cl
(CH2)4CH3 Cl
(CH2)3CH3 Cl
(CH2)3CH3
(CH2)4CH3
(CH2)3CH3
H
H
H
H
H
H
53.3 ( 4.0
12.1 ( 1.0
12.6 ( 1.0
32 ( 3
36 ( 4
43 ( 5
67 ( 2
31 ( 2
11 ( 2
nd
H
H
H
11 ( 1.0
3.4 ( 0.4
3.6 ( 0.4
65 ( 7
32 ( 3
70 ( 7
76 ( 8
72 ( 6
8 ( 1
49 ( 6
40 ( 5
10 ( 2
48 ( 5
41 ( 4
Figure 2. Effects of the administration of compounds 21 and 32 on
EPM performance in rats. Administration of 32 (30 mg/kg ip)
significantly modified rats’ EPM performance by increasing both the
entries in open arms (A) and the time spent by rats in the open arms
(B). On the other hand, administration of 21 (30 mg/kg ip) elicited
only a slight, but not significant, enhancement in both entries (A) and
time spent by the rats (B) in the EPM open arms: (/) P < 0.05 compared
with control rats; N ) 5-12.
H
H
H
H
Cl 3.9 ( 0.5
Cl 3.6 ( 0.5
Cl 1.8 ( 0.2
55
56
Iac
(CH2)2CH3 (CH2)2CH3
H 12.2 ( 1.0
Idc
(CH2)2CH3 (CH2)2CH3 Cl Cl 0.62 ( 0.06
(CH2)2CH3 (CH2)2CH3 Cl 4.65 ( 0.52
(CH2)2CH3 (CH2)2CH3
Iec
H
Ifc
H
Cl 2.80 ( 0.3
9.3 ( 0.5
23 ( 3.1
PK 11195
Ro5-4864
alpidem
0.5-7
to an in vivo anxiolytic effect of the test compound. An increase
in EPM open arm exploration is, in fact, a distinctive effect
elicited by anxyolitic agents.25 The effects observed here
probably depend on the ability of 32 to increase the in vivo
neurosteroid levels, as is suggested by its effect on pregnenolone
production in C6 glioma cells and its lack of affinity for the
BzR.
a The concentration of test compounds that inhibited [3H]PK11195
binding to rat kidney mitochondrial membranes (IC50) by 50% was
determined with six concentrations of the displacers, each performed in
triplicate. Ki values are the mean ( SEM of three determinations. b C6
glioma cells were incubated for 2 h at 37 °C in the presence of each
compound. Pregnenolone was quantified by enzymatic immunoassay, as
described in the text. The values represent the mean ( SEM of at least
three determinations. For comparison, the effects of PK 11195 and Ro5-
4864 on pregnenolone production are also included. c Data taken from
ref 17.
The administration of 21 to rats produced different results
with respect to those observed following treatment with 32.
Indeed, at a dose of 30 mg/kg, ip, 21 modified neither the
number of entries nor the time spent by rats in open arms of
the EPM, and in addition, it did not influence rats’ spontaneous
exploratory activity, as revealed by the lack of difference in
arm entries between 21-treated and control rats (9.8 ( 1 for 21
and 10.3 ( 0.7 for control rats). On the other hand, when
administered at the higher dose of 50 mg/kg ip, 21 suppressed
the spontaneous exploratory behavior of rats, as shown by a
significant reduction in the number of arm entries (2 ( 0.4 for
21 and 10.3 ( 0.7 for control rats, P < 0.05) performed by the
animals over the 5 min test session. The results obtained
following administration of 21 appear to be unexpected, since
in vitro studies pointed out the effectiveness of this ligand in
eliciting pregnenolone synthesis from C6 glioma cells, although
at a lower level than that displayed by 32 (135% vs 171%
increase in pregnenolone production vs control; see Tables 1
and 2). In this connection, however, it is emphasized that the
in vivo effects of pharmacologically active substances are
influenced by several factors and may diverge from those
expected on the basis of in vitro experiments. For example, it
might be possible that 21 and 32 are metabolized to different
extents. Actually, AC-5216 (Chart 1), a TSPO ligand that
displays antianxiety activity,20 shares with 32 the same ethyl/
benzyl substitution pattern on the amide nitrogen.
be likewise different in their size. The 4′-chloro and 5-chloro
derivatives (51-56) displayed Ki values between those shown
by their unsubstituted counterparts (45-47) and by the 4′,5-
dichloro derivatives (48-50). Taken together, these data suggest
that each chlorine favorably contributes to the affinity to a
similar extent.
Most of the compounds in Table 3 showed a low/medium
percentage increase in pregnenolone production vs control,
despite their high affinities, thus confirming the lack of
correlation between potency and steroidogenic activity.
The best-performing compounds in terms of pregnenolone
production, 21 and 32, were evaluated for their potential in vivo
anxiolytic effects by means of the EPM paradigm in the rat,
which employs an elevated four-arm maze with two open and
two enclosed arms and measures the animal’s preference for
dark, enclosed places (closed arms) over bright, exposed places
(open arms).25 An increase in both number of entries and time
spent by the rats in open arms was observed following
administration of 32 (30 mg/kg, ip) with respect to rats treated
with the vehicle (Figure 2). Notably, 32 was not found to
influence the spontaneous exploration of the apparatus by the
rats, as the total number of arm entries (in both open and closed
arms) performed by rats treated with 32 did not significantly
differ from that performed by control rats (9.29 ( 1 for 32 and
10.3 ( 0.7 for control rats, ns). Therefore, the modification in
EPM performance observed seemed to be clearly attributable
In conclusion, the SAR data deriving from the biological
results of the newly synthesized N,N-dialkyl-2-phenylindol-3-
ylglyoxylamide TSPO ligands 1-56 led to a refinement of our

5804 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Da Settimo et al.
HCl solution. The organic layer was separated, and the aqueous
phase was extracted with CH2Cl2. The combined organic layers
were dried over MgSO4, filtered, and evaporated to dryness,
furnishing the crude indoles 76-81, which were purified by flash
chromatography (eluting system, cyclohexane-ethyl acetate or
petroleum ether-dichloromethane in varying ratios) and/or by
recrystallization from the appropriate solvent. Yields and melting
points of indoles 76-81 are reported in the Supporting Information.
General Procedure for the Synthesis of Substituted (2-Phe-
nylindol-3-yl)glyoxylyl Chloride Derivatives 88-96. Oxalyl
chloride (0.65 mL, 7.5 mmol) was added dropwise, at 0 °C to a
well-stirred mixture of the appropriate indole 73-81 (3.0 mmol)
in 10 mL of freshly distilled ethyl ether. The mixture was
maintained at room temperature for 2-24 h (TLC analysis). The
precipitate formed was collected and washed with portions of
petroleum ether 30-50 °C to give the acyl chlorides 88-96, which
were dried over P2O5 in vacuo and directly used in the subsequent
reaction. All the acyl chloride derivatives, except for compound
93, were characterized by conversion to their corresponding ethyl
esters. Yields and melting points of derivatives 88-96 and
analytical and spectral data of esters of compounds 88-92, 94-96
are reported in the Supporting Information.
TSPO pharmacophore/topological model: (1) the R3 substituent
has to be electron-withdrawing to reinforce a putative π-stacking
interaction between the 2-phenyl and an electron-rich aromatic
ring within the L1 pocket; (2) R4 has to be both electron-
withdrawing and very small for optimal binding, a combination
of properties featured only by fluorine; (3) substitutions in the
7-position of the indole nucleus (R5) do not produce any gain
in affinity; (4) an aromatic moiety (R1/R2) is equivalent to an
aliphatic moiety of similar size in interacting hydrophobically
with the L3 or L4 lipophilic pocket; (5) the L3 and L4 pockets
are probably different in their dimensions, as the best-performing
substitution pattern on the amide nitrogen is obtained with R1
and R2 of different sizes.
Most of the new compounds exhibited a high affinity for
TSPO, with Ki values in the nanomolar/subnanomolar range,
and stimulated steroid biosynthesis in rat C6 glioma cells to an
extent similar to or higher than that of classic TSPO ligands
such as PK 11195, Ro5-4864, and alpidem. Furthermore, in the
EPM test, the indole derivative 32 elicited an anxiolytic activity.
Taken together, the results of the present study suggest that the
novel N,N-dialkyl-2-phenylindol-3-ylglyoxylamides may rep-
resent promising pharmacological tools suited for the treatment
of anxiety disorders.
General Procedure for the Synthesis of N,N-Dialkyl-2-(phe-
nylindol-3-yl)glyoxylamide Derivatives 1-56. A solution of the
appropriate amine (2.5 mmol) in 5 mL of dry toluene was added
dropwise to a stirred suspension, cooled at 0 °C, of indolylglyoxylyl
chloride 82-96 (2.5 mmol) in 50 mL of the same solvent, followed
by the addition of a solution of triethylamine (0.3 mL, 2.5 mmol)
in 5 mL of dry toluene. The reaction mixture, allowed to warm to
room temperature, was stirred for 2-24 h (TLC analysis) and then
filtered to eliminate the triethylamine hydrochloride formed. The
toluene solution was washed with water, dried with MgSO4, and
evaporated to dryness to yield the crude compounds 1-56. In the
case of the less soluble products, a first portion of the crude
compound precipitated together with triethylamine hydrochloride
and was collected after washing the solid with water. All products
1-56 were purified by recrystallization from the appropriate solvent
or by flash chromatography (eluting system, petroleum ether-ethyl
acetate or petroleum ether-dichloromethane in varying ratios).
Yields, recrystallization solvents, melting points, and spectral data
of compounds 1-56 are listed in the Supporting Information.
Biological Methods. Materials. [3H]PK 11195 (S.A. 85.5 Ci/
mmol) and [3H]Ro15-1788 (S.A. 83.4 Ci/mmol) were purchased
from Perkin-Elmer Life Sciences. Culture medium, fetal bovine
serum (FBS), L-glutamine, and antibiotics were purchased from
Cambrex Bio Science. PK 11195 and Ro5-4864 were obtained from
Sigma-Aldrich. 1,2,3,4-Tetrahydro-4-oxo-7-chloro-2-naphthylpy-
ridine (SU10603) and 2R,4R,5R,17ꢀ-4,5-epoxy-17-hydroxy-3-
oxoandrostane-2-carbonitrile (Trilostane) were kindly given by
Novartis Pharma Spa and Dr. Zister, University of Dublin. All
reagents were obtained from commercial suppliers.
[3H]PK 11195 Binding to Rat Kidney Mitochondrial Mem-
branes. For binding studies, crude mitochondrial membranes were
incubated with 0,6 nM [3H]PK 11195 in the presence of a
compound concentration range (0.1 nM to 10 µM) in 50 mM Tris-
HCl, pH 7.4, as previously described.17 For the active compounds,
the IC50 values were determined and Ki values were derived in
accordance with the equation of Cheng and Prusoff.26
[3H]Ro15-1788 Binding to Rat Cerebral Cortex Membranes.
Rat cerebral cortex membranes were prepared as previously
described.3 After differential centrifugation, the crude membrane
fraction obtained was subjected to washing procedures to remove
endogenous GABA.27 The washed membranes were incubated with
0.4 nM [3H]Ro15-1788 for 90 min at 0 °C in 500 µL of 50 mM
Tris-citrate buffer, pH 7.4, as previously described.28
Experimental Section
Chemistry. Melting points were determined using a Reichert
Ko¨fler hot-stage apparatus and are uncorrected. Infrared spectra
were recorded with a Nicolet-Avatar 360 FT-IR spectrophotometer
in Nujol mulls. Routine nuclear magnetic resonance spectra were
recorded in DMSO-d6 solution on a Varian Gemini 200 spectrom-
eter 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. Elemental
analyses were performed by our analytical laboratory and agreed
with theoretical values to within (0.4%.
2-Phenylindole 67 was from Sigma-Aldrich; 2-(4-fluorophe-
nyl)indole 68 was from Lancaster.
The literature data and synthetic procedures regarding the
following compounds are described in our previous work:17 4-
chloro-N-(2-methylphenyl)benzamide 57; N-(4-chloro-2-methylphe-
nyl)benzamide 58; 4-chloro-N-(4-chloro-2-methylphenyl)benzamide
59; 4-methyl-N-(2-methylphenyl)benzamide 60; 2-(4-chlorophenyl)in-
dole 69; 5-chloro-2-phenylindole 70; 5-chloro-2-(4-chlorophenyl)indole
71; 2-(4-methylphenyl)indole 72; (2-phenylindol-3-yl)glyoxylyl chlo-
ride 82; [2-(4-fluorophenyl)indol-3-yl]glyoxylyl chloride 83; [2-(4-
chlorophenyl)indol-3-yl]glyoxylyl chloride 84; (5-chloro-2-phe-
nylindol-3-yl)glyoxylyl chloride 85; [5-chloro-2-(4-chlorophenyl)indol-
3-yl]glyoxylyl chloride 86; [2-(4-methylphenyl)indol-3-yl]glyoxylyl
chloride 87.
The following compounds were prepared in accordance with
reported procedures: N-(4-methoxy-2-methylphenyl)benzamide 61;
N-(4-fluoro-2-methylphenyl)benzamide 62; 4-fluoro-N-(4-fluoro-
2-methylphenyl)benzamide 63; 4-fluoro-N-(4-chloro-2-methylphe-
nyl)benzamide 64; N-(2-chloro-6-methylphenyl)benzamide 65; N-(2,6-
dimethylphenyl)benzamide 66; 2-(4-nitrophenyl)indole 73; 2-(4-
trifluoromethylphenyl)indole 74; 5-nitro-2-phenylindole75 (all
experimental data of amides 61-66 and indoles 73-75 are reported
in the Supporting Information).
General Procedure for the Synthesis of Substituted 2-Phe-
nylindole Derivatives 76-81. n-Butyllithium 1.6 M solution in
hexane (9.3 mL, 15 mmol) was added dropwise over 10-15 min
to a stirred solution of the appropriate benzamide 61-66 (5.0 mmol)
in 25 mL of dry THF and maintained under nitrogen at a
temperature from -20 to 0 °C; then the stirred mixture was allowed
to warm to room temperature over 1-2 h. The resulting dark-red
solution was stirred at room temperature for 24-48 h (TLC
analysis), cooled in an ice bath, and acidified to pH 5-6 with 2 N
Cell Culture. Rat glioma C6 cells were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10% FBS, 2 mM
L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin.
Cultures were maintained in a humidified atmosphere of 5% CO2/
95% air at 37 °C.

N,N-Dialkyl-2-phenylindol-3-ylglyoxylamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5805
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Steroid Biosynthesis. The pregnenolone production measure-
ment in C6 cells, exposed to the novel compounds or PK 11195
(40 µM), was performed by competitive enzyme-linked immunoas-
say (ELISA). Briefly, C6 cells were seeded in 96-well plates at a
density of about 10 000 cells/well in a final volume of 100 µL and
the pregnenolone secretion into the medium was performed as
previously described.29 The final concentration of DMSO and
ethanol was constant for all the wells within each experiment and
did not exceed 0.5% (v/v), a concentration that on its own had no
effect on steroid production. At the end of the incubation period
(2 h), the cell medium was used in an enzyme immunoassay for
the direct quantitative determination of pregnenolone, under the
conditions recommended by the supplier (Pregnenolone ELISA,
the EiAsy Way, IBL Hamburg, Germany).
Pharmacology. Subjects. Male Sprague-Dawley rats (Charles
River, Calco, Italy) weighing 180-200 g were used in this study.
Animals were housed in groups of 6 per cage in a room with the
temperature of 24 ( 1 °C and maintained under a 12 h light/dark
cycle (lights on at 8:00 a.m.). Rats had free access to water and
food except during the measurement of elevated-plus-maze
performance.
All experiments were conducted in accordance with the
guidelines for care and use of experimental animals of the
European Communities Directive (86/609/EEC; D.L., 27.01.1992,
number 116).
Drugs. 21 and 32 were suspended in 0.5% methylcellulose +
1% Tween-80 in distilled water and were administered ip (1 mL/
100 g of body weight).
Elevated-Plus-Maze Performance. The elevated plus-maze
represents a reliable experimental tool to evaluate anxiety in
rodents.30,31 Briefly, the apparatus was made of white PVC and
consisted of two opposite open arms (length 50 cm, width 10 cm)
and two opposite closed arms (length 50 cm, width 10 cm), the
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arms converged to a central square (10 cm × 10 cm), thus
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50 cm from the floor. Rats, having no prior experience of the
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total amount of time spent in the arms, respectively. A rat was
considered inside a specific arm when having all the four paws
inside the arm. A reduced preference for the open arms is considered
as an index of anxiety, and drugs bearing anxiolytic activity increase
the preference of the rats for the open arms.25
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Statistical Analysis. Values are reported as the mean ( SEM.
Differences between experimental groups were evaluated by
Student’s t test. Significance was set at p < 0.05.
(18) Campiani, G.; Nacci, V.; Fiorini, I.; De Filippis, M. P.; Garofalo, A.;
Ciani, S. M.; Greco, G.; Novellino, E.; Williams, D. C.; Zisterer, D. M.;
Woods, M. J.; Mihai, C.; Manzoni, C.; Mennini, T. Synthesis,
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Acknowledgment. This work was supported by MIUR
(cofin. 2004).
(19) Taliani, S.; Simorini, F.; Sergianni, V.; La Motta, C.; Da Settimo, F.;
Cosimelli, B.; Abignente, E.; Greco, G.; Novellino, E.; Rossi, L.;
Gremigni, V.; Spinetti, F.; Chelli, B.; Martini, C. New fluorescent
2-phenylindolglyoxylamide derivatives as probes targeting the peripheral-
type benzodiazepine receptor: design, synthesis, and biological evalu-
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Supporting Information Available: General procedure for the
synthesis of amides 61-66; physical properties and spectral data
of benzamides 61-66, 2-phenylindole derivatives 73-81, (2-
phenylindol-3-yl)glyoxylyl chlorides 88-96, and their correspond-
ing ethyl esters 97-104; Tables S1-S6 including yields, physical
properties, and spectral data of indolylglyoxylamides 1-56; analyti-
cal data of target compounds 1-56. This material is available free
of charge via the Internet at http://pubs.acs.org.
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alkylphenyl)alkanamides and related compounds. A modified Made-
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Franzone, J. S.; Cirillo, R.; Reboani, M. C. N-(Indol-3-ylglyoxylyl) -
methionine derivatives: preparation and gastric anti-secretory activity. Eur.
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