Deepening the Topology of the Translocator Protein Binding Site by Novel N,N-Dialkyl-2-arylindol-3-ylglyoxylamides

Article abstract of DOI:10.1021/acs.jmedchem.5b00689

As a continuation of our studies on 2-phenylindol-3-ylglyoxylamides as potent and selective translocator protein (TSPO) ligands, two subsets of novel derivatives, featuring hydrophilic group (OH, NH₂, COOH) at the para-position of the pendent 2

Full text of DOI:10.1021/acs.jmedchem.5b00689

Article
pubs.acs.org/jmc
Deepening the Topology of the Translocator Protein Binding Site by
Novel N,N‑Dialkyl-2-arylindol-3-ylglyoxylamides
Elisabetta Barresi,†,∥ Agostino Bruno,‡,∥ Sabrina Taliani,*,† Sandro Cosconati,*,§ Eleonora Da Pozzo,†
Silvia Salerno,† Francesca Simorini,† Simona Daniele,† Chiara Giacomelli,† Anna Maria Marini,†
Concettina La Motta,† Luciana Marinelli,‡ Barbara Cosimelli,‡ Ettore Novellino,‡ Giovanni Greco,‡
Federico Da Settimo,† and Claudia Martini†
†Dipartimento di Farmacia, Universita
‡Dipartimento di Farmacia, Universita
̀
̀
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
§DiSTABiF, Seconda Universita
̀
di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
S
* Supporting Information
ABSTRACT: As a continuation of our studies on 2-phenylindol-3-ylglyoxylamides
as potent and selective translocator protein (TSPO) ligands, two subsets of novel
derivatives, featuring hydrophilic group (OH, NH2, COOH) at the para-position of
the pendent 2-phenyl ring (8−16) or different 2-aryl moieties, namely, 3-thienyl, p-
biphenyl, 2-naphthyl (23−35), were synthesized and biologically evaluated, some
of them showing Ki values in the subnanomolar range and the 2-naphthyl group
performance being the best. The resulting SARs confirmed the key role played by
interactions taking place between ligands and the lipophilic L1 pocket of the TSPO
binding site. Docking simulations were performed on the most potent compound
of the present series (29) exploiting the recently available 3D structures of TSPO
bound to its standard ligand (PK11195). Our theoretical model was fully
consistent with SARs of the newly investigated as well of the previously reported 2-
phenylindol-3-ylglyoxylamide derivatives.
modulation, and inflammation.1,4,6 This protein is ubiquitously
INTRODUCTION
■
expressed, with higher levels in tissues that produce steroids
and that are mitochondrially enriched such as myocardium,
skeletal muscle, and renal tissue;1 other peripheral tissues,
including liver and lung, express TSPO to less extent.1 In the
central nervous system (CNS), TSPO is mainly located in glial
cells, but it is also present in neurons.7 Such a distribution in
the CNS may be related to the crucial role that TSPO plays in
the regulation of cholesterol translocation from the outer to the
inner mitochondrial membrane, where it is converted into
pregnenolone, the precursor of all neurosteroids.8 A number of
these endogenous molecules, such as pregnenolone and
allopregnanolone, are able to rapidly inhibit neuron excitability
as a result of a positive allosteric modulation of type A receptors
for GABA (GABAA), the major inhibitory neurotransmitter in
the brain.9
Altered expression of TSPO has been linked to multiple
diseases, including anxiety, cancer, ischemia−reperfusion injury,
brain injury, and neurodegenerative conditions such as
Alzheimer’s and Parkinson’s diseases.10 In this view, TSPO
ligands have potential as diagnostic tools for the state and
progression of these related-diseases,10,11 as well as therapeutic
antiproliferative and neuroprotective agents.12−14
The translocator protein (TSPO), first described as peripheral
benzodiazepine receptor (PBR),1,2 is an 18 kDa protein mainly
expressed in the outer mitochondrial membranes, where it
interacts with a number of associated proteins including the
voltage-dependent anion channel (VDAC, 32 kDa), the
adenine nucleotide transporter (ANT, 30 kDa), the PBR
associated protein 1 (PRAX-1, 220−250 kDa), the steroido-
genesis regulatory protein (StAR, 37 kDa), and the TSPO and
protein kinase A regulatory subunit RIα-associated protein
(PAP7, 52 kDa).1 Additional subcellular locations of TSPO
have been identified such as nucleus, lysosome, Golgi
apparatus, peroxisomes, and plasma membrane.1,3 TSPO is an
evolutionarily well-conserved and tryptophan-rich 169 amino
acids protein organized into five transmembrane helices linked
by hydrophobic loops, with a carboxy terminal and short
amino-terminal tails located outside and inside the mitochon-
dria, respectively.1 Site-directed mutagenesis experiments,4
together with a recent NMR study,5 demonstrated that the
portion of the receptor that recognizes the ligands is located at
the bottom of the transmembrane bundle, also contacting the
first cytoplasmatic loop.
TSPO is involved in numerous cellular processes related to
the regulation of mitochondrial cholesterol translocation,
porphyrin transport and heme synthesis, cellular proliferation
and apoptosis, regulation of mitochondrial functions, immuno-
Received: May 5, 2015
© XXXX American Chemical Society
A
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a
Chart 1. Structures of Known (1−7) and Newly Synthesized (8−35) TSPO Ligands
a
Structures 7−35 have been drawn embedded within a pharmacophore/topological model of ligand−TSPO interaction.25,26,28
Since identification of TSPO by means of the benzodiaze-
pines diazepam and Ro5-4864 1 (Chart 1),2 structurally
different classes of highly potent and selective TSPO ligands
have been reported (Chart 1), including the isoquinoline-
carboxamides, of which the 1-(2-chlorophenyl)-N-methyl-N-(1-
methylpropyl)-1-isoquinolinecarboxamide (PK11195, 2) is
widely considered as a prototypical TSPO ligand,15 imidazo-
pyridines (alpidem, 3), indoleacetamides (FGIN-1-27, 4),16
tetrahydrocarbazolecarboxamides (GE-180, 5),17−19 and puri-
neacetamides (AC-5216, 6).20−24
In this context, we disclosed the N,N-dialkyl-2-phenylindol-3-
ylglyoxylamides 7 as a class of potent and selective TSPO
ligands (Chart 1), the majority of which showed Ki values in the
nanomolar/subnanomolar range and were able to stimulate
steroid biosynthesis in rat C6 glioma cells to an extent similar
to or higher than that of classic TSPO ligands Ro5-4864 and
PK11195.25−27 A large number of derivatives 7 was synthesized
and biologically evaluated, featuring different combinations of
R1−R5 substituents. The structure−affinity relationships
(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 H1
(Chart 1).25,26,28 According to our model, the amide carbonyl
oxygen of the oxalyl bridge engages an H-bond with the donor
site H1; the two lipophilic substituents on the amide nitrogen,
R1, and R2 interact through hydrophobic contacts with the L3
and/or L4 lipophilic pockets; the 2-phenyl moiety establishes a
putative π-stacking interaction within the L1 pocket. The
optimum binding affinity to TSPO in series 7 requires two
lipophilic substituents on the amide nitrogen (R1, R2 = methyl,
ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, benzyl, etc.) to fit
both in the L3 and L4 lipophilic pockets. Symmetrically (R1 =
R2) and asymmetrically (R1 ≠ R2) N,N-disubstituted indoles
were investigated revealing that (i) an aromatic moiety (R1/R2)
is equivalent to an aliphatic one of similar size in taking
hydrophobic contacts with the L3 or L4 lipophilic pocket; (ii)
the L3 and L4 pockets are probably different in their
dimensions as substitution pattern on the amide nitrogen
with R1 and R2 of different sizes yielded high affinity derivatives.
Moreover, the R3 substituent has to feature electron-with-
drawing properties to reinforce the putative π-stacking
interaction within the L1 pocket, and R4 has to be both
electron-withdrawing and very small for optimal binding, a
combination of properties only featured by the fluorine atom.
Conversely, substitutions at position 7 of the indole nucleus
(R5) do not produce any affinity gain.25,26
As stated above, TSPO may also represent a marker for
related disease progression so that the 2-phenylindolyl-
glyoxylamide scaffold has been studied as a novel chemotype
for the development of specific TSPO molecular probes. Novel
reversible and irreversible fluorescent probes targeting TSPO
were developed which featured the 7-nitrobenz-2-oxa-1,3-
diazol-4-yl (NBD) group as a fluorophore.29,30 Furthermore,
a new 11C-radiolabeled probe from class 7, the N,N-di-n-propyl-
B
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(N1-methyl-2-(4′-nitrophenyl)indol-3-yl)glyoxylamide, was
synthesized and evaluated with positron emission tomography
in monkey, showing it to enter the brain and give a high
proportion of TSPO-specific binding, auguring well for its
future application in humans as biomarker of neuroinflamma-
tion.31
Scheme 1. Synthesis of Indolylglyoxylamide Derivatives 8−
22
As a continuation of our studies on 2-phenylindol-3-
ylglyoxylamides as potent and selective TSPO ligands, our
attention has been now focused on the effects on binding
affinity of different aryl moieties at the 2-position of the indole
nucleus. The aim of our project was to probe the L1 lipophilic
pocket and its surroundings to better define the whole topology
of the TSPO binding site. For this purpose, a first subset of
novel indole derivatives (8−16) was designed, featuring
hydrophilic group (OH, NH2, COOH) at the para-position
of the pendent 2-phenyl ring. Actually, Trapani and colleagues
reported that the introduction of polar and ionizable
substituents at the para- and meta-positions of the 2-phenyl
ring of 2-phenylimidazopyridine TSPO ligands yielded
compounds showing very high affinity and selectivity for
TSPO, with Ki values in the low nanomolar/subnanomolar
range.32 To expand the SARs, derivatives 17−22 (Ar = p-
CH3O-C6H4 and p-CH3OOC-C6H4, respectively), which are
the intermediates in the synthesis of compounds 8−10 and
14−16 (see Scheme 1), were biologically evaluated. The
second subset is represented by indoles 23−35, which are
functionalized at 2-position with aryl moieties different from
the variously substituted phenyl ring, that is 3-thienyl (23−25),
and the highly steric-demanding p-biphenyl (26−28) or 2-
naphthyl (29−35) group.
All the novel indole derivatives 8−35 were biologically
evaluated for their binding affinity to TSPO.
Finally, a 3D model of the rTSPO was developed employing
the newly published NMR model of mTSPO (PDB code
2MGY),5 and docking studies were conducted using compound
29 as reference ligand in order to rationalize the SARs within
the 2-arylindol-3-ylglyoxylamide TSPO ligands described so far.
RESULTS AND DISCUSSION
■
Chemistry. The target N,N-dialkyl-2-(4-substituted-
phenyl)indol-3-ylglyoxylamides 8−16, N,N-dialkyl-2-(thien-3-
yl)indol-3-ylglyoxylamides 23−25, N,N-dialkyl-2-(p-biphenyl)-
indol-3-ylglyoxylamides 26−28, and N,N-dialkyl-2-(naphth-2-
yl)indol-3-ylglyoxylamides 29−35 were synthesized through
the 2-arylindoles 36−38 and 42−44, which were in turn simply
obtained, with the exception of the commercially 2-(biphenyl-
4-yl)indole 43 and 2-(naphth-2-yl)indole 44, with a one-step
Fischer indole synthesis by reacting phenylhydrazine hydro-
chloride and the appropriate acetyl derivative, Schemes 1 and 2.
Under acid catalysis [excess of polyphosphoric acid (PPA)], the
phenylhydrazone initially formed undergoes a number of
isomerizations/rearrangements and finally eliminates NH3 to
give the indole products.
in a nitrogen atmosphere (Scheme 1). At the end of the
reaction, methanol was added to hydrolyze the excess of BBr3,
and crude compounds were recovered as a solid precipitated
after evaporation under reduced pressure.
The N,N-dialkyl-[2-(4-nitrophenyl)indol-3-yl]glyoxylamide
derivatives 7d−f were catalytically hydrogenated over palladium
to yield the corresponding 2-phenylindolylglyoxylamide ana-
logues 11−13 bearing a NH2 at para-position of the 2-phenyl
ring (Scheme 1).
Finally, hydrolysis of the methyl ester group of compounds
20−22 with lithium hydroxide monohydrate in a solution of
methanol/water (3:1) gave the target compounds 14−16
(Scheme 1).
Scheme 2 outlined the general procedure for the synthesis of
N,N-dialkyl-2-(aryl)indol-3-ylglyoxylamides 23−35. The 2-
arylindoles 42−44 were acylated with oxalyl chloride, in
anhydrous diethyl ether, at room temperature, to obtain the
corresponding indolylglyoxylyl chlorides 45−47. The subse-
quent condensation with the appropriate amines, in the
presence of triethylamine, in dry toluene solution, yielded the
target derivatives 23−35.
Acylation of the appropriate 2-(4-substituted-phenyl)indole
36−38 with oxalyl chloride, in anhydrous diethyl ether, at room
temperature, yielded the corresponding 2-(4-substituted-
phenyl)indolylglyoxylyl chlorides 39−41, which were allowed
to react with the appropriate dialkylamine, in the presence of
triethylamine, in dry toluene solution, at room temperature, to
give compounds 7d−f, 17−22 (Scheme 1).
The indolylglyoxylamide derivatives featuring a OH group in
R3 (8−10) were achieved through a demethylation of the
methoxy group of compounds 17−19 by treatment with BBr3
The reaction yields, the chemical physical constants, and
spectroscopic data of compounds 8−35 are listed in the
Experimental Section.
C
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Scheme 2. Synthesis of Indolylglyoxylamide Derivatives 23−
35
Table 1. TSPO Binding Affinity of N,N-Dialkyl-2-(4′-
substituted-phenyl)indol-3-ylglyoxylylamide Derivatives 8−
22 and 7a−f
a
compd
R1 = R2
R3
Ki (nM)
8
(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
OH
OH
OH
16.1 1.0
25.7 2.6
6.3 0.5
44.4 5.0
133 13
4.2 0.4
343 10
9
10
11
12
13
14
15
16
17
18
19
20
21
22
NH2
NH2
NH2
COOH
COOH
COOH
OCH3
OCH3
OCH3
COOCH3
COOCH3
COOCH3
H
406
184
4
1
5.8 0.5
20.3 2.2
4.0 0.4
3.1 0.3
2.7 0.3
3.3 0.3
12.2 1.0
7.5 0.7
1.4 0.2
0.95 0.10
0.27 0.10
0.23 0.10
23.0 3.1
9.3 0.5
b
7a
7b
b
H
b
b
7c
H
7d
NO2
b
7e
NO2
The (2-arylindol-3-yl)glyoxylyl chlorides 39, 41, 45−47 have
never been described before in the literature and were
characterized as their corresponding ethyl ester derivatives
(see Experimental Section).
b
7f
NO2
Ro5-4864 (1)
PK11195 (2)
a
The concentration of tested compounds that inhibited [3H]PK11195
Biological Studies. The binding affinity of all the newly
synthesized 2-arylindol-3-ylglyoxylamide derivatives 8−35 for
the TSPO was determined by binding competition experiments
against [3H]PK11195,25,26 carried out in rat kidney membrane
homogenates (Tables 1 and 2). Because of the well-established
TSPO versus central benzodiazepine receptor (BzR) selectivity
of N,N-dialkyl-2-phenylindol-3-ylglyoxylamides,25,26 a few
randomly selected derivatives were evaluated for their BzR
affinity by experiments against [3H]flumazenil in rat cerebral
cortex membrane homogenates.25,26 The tested compounds
showed no significant binding properties in this assay
(inhibition percentages at 10 μM concentration ranging from
0% to 23%, data not shown).
The TSPO binding affinities of compounds 8−22 and 23−
35, expressed as Ki values, are listed in Tables 1 and 2,
respectively, together with the Ki values of the standard TSPO
ligands Ro5-4864 and PK11195 (Chart 1). The binding data of
some of the previously investigated indole derivatives25,26 are
included for comparison at the bottom of Table 1 (7a−f) and
Table 2 (7a−c,g−j).
In the 8−16 series, the amide nitrogen is symmetrically
disubstituted (R1 = R2, Table 1) with n-propyl, n-butyl, and n-
hexyl chains and the 2-phenyl moiety is decorated by small
hydrophilic groups inserted at the para-position (R3 = OH,
NH2, COOH, Table 1); derivatives 17−22 (R3 = OCH3,
COOCH3, Table 1), which are the intermediates in the
synthesis of compounds 8−10 and 14−16, respectively (see
Scheme 1), were also biologically evaluated to expand the
SARs.
binding to rat kidney mitochondrial membranes by 50% (IC50) was
determined with eight concentrations of the displacers, each
performed in triplicate. Ki values are the mean
determinations. Data taken from ref 26.
SEM of three
b
In general, the introduction of a hydrophilic group at the
para-position of the 2-phenyl ring (OH, NH2, COOH) did not
determine any significant improvement of the affinity,
suggesting that the interaction at the level of the L1 pocket is
mostly lipophilic (Table 1). In particular, the insertion of an
OH group at the para-position of the 2-phenyl ring is tolerated
for affinity (8 Ki = 16.1 nM, 9 Ki = 25.7 nM, 10 Ki = 6.3 nM, vs
7a Ki = 12.2 nM, 7b Ki = 7.5 nM, 7c Ki = 1.4 nM, respectively).
Conversely, a decrease of the binding affinity is observed
introducing a NH2 (11 Ki = 44.4 nM, 12 Ki = 133 nM, 13 Ki =
4.2 nM, vs 7a Ki = 12.2 nM, 7b Ki = 7.5 nM, 7c Ki = 1.4 nM,
respectively) or, to a major extent, a COOH group (14 Ki =
343 nM, 15 Ki = 406 nM, 16 Ki = 184 nM vs 7a Ki = 12.2 nM,
7b Ki = 7.5 nM, 7c Ki = 1.4 nM, respectively, Table 1). In
addition, in all the subsets, compounds bearing n-hexyl chains
on the symmetrically disubstituted amide nitrogen are more
potent than the others. A different trend is observed for
derivatives 17−19 and 20−22, featuring a p-methoxy and p-
methoxycarbonyl substituent, respectively, that show, with the
exception of compound 18 (Ki = 20.3 nM), appreciable and
comparable affinity (Ki values in the low nanomolar range),
whatever is the length of the side alkyl chains, Table 1.
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Table 2. TSPO Binding Affinity of N,N-Dialkyl-2-arylindol-3-ylglyoxylylamide Derivatives 23−35, 7a−c, and g−j
a
compd
R1
R2
Ar
Ki (nM)
23
24
25
26
27
28
29
30
31
32
33
34
35
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)3CH3
(CH2)4CH3
(CH2)3CH3
CH2C6H5
thien-3-yl
thien-3-yl
thien-3-yl
p-biphenyl
p-biphenyl
p-biphenyl
naphth-2-yl
naphth-2-yl
naphth-2-yl
naphth-2-yl
naphth-2-yl
naphth-2-yl
naphth-2-yl
C6H5
1.2 0.1
2.8 0.3
0.89 0.10
0.53 0.05
5.5 1.0
1.8 0.2
0.31 0.04
0.54 0.06
0.52 0.06
0.56 0.06
0.37 0.04
0.51 0.05
0.51 0.05
12.2 1.0
7.5 0.7
CH3
CH2CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
CH3
b
7a
7b
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)3CH3
(CH2)4CH3
(CH2)3CH3
CH2C6H5
b
C6H5
b
7c
C6H5
1.4 0.2
b
7g
7h
C6H5
53.3 4.0
12.1 1.0
12.6 1.0
11.0 1.0
23.0 3.1
9.3 0.5
b
CH3
C6H5
b
7i
7j
CH2CH3
CH2CH3
C6H5
b
C6H5
Ro5-4864 (1)
PK11195 (2)
a
The concentration of tested compound that inhibited [3H]PK11195 binding to rat kidney mitochondrial membranes by 50% (IC50) was
b
determined with eight concentrations of the displacers, each performed in triplicate. Ki values are the mean SEM of three determinations. Data
taken from ref 26.
The data listed in Table 2 indicate that the replacement of
the 2-phenyl group with a thien-3-yl (23−25), a p-biphenyl
(26−28), or a naphth-2-yl (29−35) moiety produces, with few
exceptions, superior affinity TSPO ligands, with Ki values in the
subnanomolar range. As the presence of a naphthyl group
seemed to be particularly favorable for affinity, this subset was
further investigated by the synthesis of a number of
asymmetrically N,N-disubstituted indoles (32−35, Table 2),
to more deeply probe the L3 and L4 lipophilic pockets of the
TSPO binding site (R1 = methyl, ethyl; R2 = n-butyl, n-pentyl,
benzyl). Interestingly, within the subset of the 2-(naphth-2-yl)-
substituted derivatives 29−35, all compounds showed almost
identical Ki values in the subnanomolar range, ranging from
0.30 to 0.56 nM (Table 2). Thus, the nature of the groups
bound to the amide nitrogen does not significantly influence
the binding of ligands 29−35 at TSPO. These data suggest a
key role played by the lipophilic interaction at the level of the
L1 pocket that, reaching its optimum with a naphthyl group,
makes less significant the contribution to the binding of the
remaining lipophilic interactions established by the ligand
within the receptor binding site.
Computational Studies: rTSPO Model Construction
and Docking Calculations. A survey in the available
literature demonstrates that, to date, several X-ray33,34 and
solution5 (NMR) structures were deposited in the Protein Data
Bank (PDB).35 Among them, we decided to choose the
solution one (mTSPO) as a template for the construction of
our rTSPO model, given the higher sequence identity shared by
the two proteins (95%). Therefore, several 3D models of the
rTSPO were generated using the newly published NMR model
of mTSPO (PDB code 2MGY),5 by applying the following
protocol: (i) 20 NMR snapshots are available in the structure of
the mTSPO, and we used each of them as a template for the
construction of the rTSPO; (ii) for each template, 20 models
were generated making a total of 400 rTSPO models, grouped
in 20 groups (one for each NMR snapshot); (iii) then the best
rTSPO models in each group were selected (i.e., the models
giving the highest score according to the modeler native scoring
function and featuring the best Ramachandran plots) for a total
of 20 rTSPO structures. The selected 20 rTSPO models were
refined through the protein preparation protocol available in
Maestro, version 9.8.36
Because of the well-established steroidogenic effect of
derivatives from the 2-phenylindol-3-ylglyoxylamide class,25,26
compound 29 was selected and routinely evaluated for its
ability to stimulate pregnenolone formation in rat C6 glioma
cells,25,26 showing an increase percentage in pregnenolone
production vs control similar to that of the reference standard
PK1119525,26 (see Supporting Information for method details).
Comparison between the rTSPO model (see Docking
Studies) and its corresponding mTSPO NMR snapshot
template is depicted in Supporting Information Figure S1,
with the corresponding Ramachandran plots.
Docking was performed using Glide tool of Maestro 9.836 on
the 20 selected mTSPO structures. To set up the docking
studies the following considerations were made: (i) the
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stoichiometry of the protein−ligand interaction is 1:1;5 (ii) the
indolylglyoxylamide derivatives are competing for the same
PK11195 binding site.25−27,29 On the basis of these
observations, the docking studies were conducted on the
monomeric rTSPO and using compound 29 as reference ligand
(i.e., the one endowed with the best binding properties).
Among the 20 29/rTSPO generated complexes, we considered
only those in which (i) the ligand was occupying the same
PK11195 binding regions (17 complexes), (ii) there was
agreement with the SARs reported by us in literature25−27,29,37
and in this work (two complexes). Of these latter complexes,
we considered the one having the highest glide score (one
complex) (Supporting Information Figure S2).
16) (see Supporting Information Figure S4 for a qualitative
correlation). This observation highlights that a cooperative
effect between the nature of the substituents on the N,N-alkyl
branches and the electronic nature of the substituents in R3
exists, and in turn a proper balance is required. Indeed, in the
case of the N,N-dihexyl derivatives, the hydrophobic nature of
the N,N-alkyl branches predominates over the electronic effects
of the R3 substituents.
The extension and the chemical nature of the cleft lodging in
the aforementioned group (Figure 1a−d) would also explain
why substitution with a biphenyl (26) results in comparably
high affinities, while the presence of the smaller phenyl/thienyl
groups results in lower binding (29 = 26 > 23 > 7a).
Binding Mode and the SARs of the Indolylglyox-
ylamide Derivatives. Glide software36 predicts 29 to fairly
recapitulate the experimental TSPO/PK11195 interactions (see
Supporting Information Figure S3) so that the ligand is nicely
adapted in the mainly hydrophobic binding pocket of rTSPO
(see Figure 1a). In the proposed binding mode of 29 to TSPO,
According to the present binding mode (Figure 1a) the N-
propyl branch in the cisoid configuration protrudes toward the
membrane environment, while the second N-propyl branch
establishes hydrophobic interactions with V26, W107, and
L150.
The proposed theoretical model (Figure 1a) is also in
agreement with our previous works where fluorescent probes
were developed.29 In particular, we derivatized our 2-phenyl-
indol-3-ylglyoxylamides with a 7-nitrobenz-2-oxa-1,3-diazol-4-yl
(NBD) group that is well-known for displaying low quantum
yields in water while becoming highly fluorescent in more
lipophilic environments, such as membranes or hydrophobic
receptor pockets.30 Indeed, according to our model, the
attached NBD linker would be hosted outside the receptor in
the membrane environment, thus explaining why our
previously published TSPO NBD-labeled fluorescent probes29
are so efficient in staining rat and human glioma cells.
The position of the N,N-disubstituted branches would also
explain why bulkier substituents on the ligand amide moiety do
not significantly affect the activity for the naphthyl derivatives
(29 vs 30−35). Interestingly, the amide branches accommodate
in a similar region with respect to the amide branches of the
PK11195 (Supporting Information Figure S3), and we already
demonstrated in our previous works that bulkier substituents
are well tolerated in this region.5,21,22,24,26,27,35 Different from
the naphthyl series (29−35), the biphenyl one seems to be
affected by the presence of bulkier substituents on the amide
branch of the glyoxylamide moiety (26 vs 27, 28). This effect
can be explained by the different bulkiness and shape of the
biphenyl moiety with respect to the naphthyl one (Figure 1b−d
and Supporting Information Figure S5), which in turn does not
tolerate bulkier substituents on the amide group.
Furthermore, on the basis of the proposed binding mode
(Figure 1a), the hydrophobic residues A23, W53, L49, and
W143 surround the indole ring of 29. This agrees with our
previous works showing that substitutions at positions 5 and 7
of the indole ring are tolerated.25−27 Finally, in our previous
works, based on pharmacophoric models, it was suggested that
the distal carbonyl oxygen of the oxalyl bridge engages an H-
bond with the donor site H1 of the pharmacophore/topological
model (Chart 1), in the TSPO binding site.25−27 On the
contrary, the present binding mode lacks any polar interaction
between the glyoxylamide branch and the receptor binding site
(Figure 1a). In this regard, it can be argued that the presence of
the amide/glyoxylamide groups in TSPO binders, rather than
engaging specific interactions with the receptor, has a role in
constraining the flexibility of the ligand branch so as to adopt
the bioactive conformation. We cannot rule out the presence of
water molecules inside the TSPO binding pocket that mediate
polar interactions between the amide/glyoxylamide and the
Figure 1. (a) Binding mode of 29 (orange sticks) into the rTSPO
binding site (white cartoon and sticks). (b) Bottom view of 29 binding
mode (the TSPO structure is highlighted in blue and gray surfaces in
this as well as in pictures c and d). (c) Bottom view of 7a binding
mode. (d) Bottom view of 29 (orange sticks), 20 (purple sticks), and
26 (cyan sticks) binding modes.
the naphthyl moiety is placed inside a roomy aromatic cage
(herein referred to as L1 pocket), establishing hydrophobic
interaction with V110, W95, W107, L114, W143, F146, and
L150 residues. This is in agreement with our previous
works21,22,24 showing that, among the indolylglyoxylamides,
p-substitution of the phenyl ring at position 2 of the indole ring
results in higher affinity with respect to the unsubstituted
phenyl moiety (i.e., 7a vs 7d). Interestingly, given the aromatic/
hydrophobic nature of the L1 region, it is not surprising that R3
polar and/or electron-donating groups are generally less
tolerated than hydrophobic and/or electron-withdrawing
ones. In particular, in the proposed binding mode (Figure
1a−c), the aromatic ring at position 2 of the indole ring
establishes a parallel displaced π−π interaction with W107, thus
explaining the general correlation between the R3 electron-
withdrawing effect and the affinity for rTSPO observed for the
N,N-dipropyl (7d > 20 > 17 > 7a > 8 > 11 > 14) and for the
N,N-dibutyl (7e > 21 > 7b > 18 > 9 > 12 > 15) series but less
evident for the N,N-dihexyl one (7f > 7c > 22 > 13 > 19 > 10 >
F
DOI: 10.1021/acs.jmedchem.5b00689
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
General Procedure for the Synthesis of [2-(4-Substituted-
phenyl)indol-3-yl]glyoxylyl Chloride Derivatives 39 and 41
and (2-Arylindol-3-yl)glyoxylyl Chloride Derivatives 45−47.
Oxalyl chloride (8.0 mmol) was added dropwise at 0 °C to a well-
stirred mixture of the appropriate indole 36, 38, 42−44 (4.0 mmol) in
freshly distilled diethyl ether (10 mL). The mixture was maintained at
room temperature for 2−24 h (TLC analysis). The precipitate formed
was collected and washed with portions of diethyl ether (20 mL) to
give the desired glyoxylyl chlorides, which were dried over P2O5 in
vacuo and directly used in the subsequent reaction. All glyoxylyl
chloride derivatives were characterized by conversion into their
corresponding ethyl esters.
protein binding site. The same interaction (H-bond with the
H1 site) was also postulated in a previous paper for the
PK11195/TSPO pharmacophore/topological model38 and was
not lately verified by the NMR experimental structure.5
CONCLUSIONS
■
Novel 2-arylindol-3-ylglyoxylamides were synthesized and
biologically evaluated to clarify the effect of different aryl
moieties at the 2-position of the indole nucleus on modulation
of affinity to TSPO and thus to probe the L1 lipophilic pocket
and its surroundings.
[2-(4-Methoxyphenyl)indol-3-yl]glyoxylyl Chloride (39).
Yield, 72%.
Within the first subset of novel indole derivatives (8−16),
the introduction of a hydrophilic group at the para-position of
the 2-phenyl ring (OH, NH2, COOH) does not determine any
significant improvement of the affinity; conversely, the
replacement of the 2-phenyl group with thien-3-yl (23−25),
p-biphenyl (26−28), or naphth-2-yl (29−35) moiety produces,
with few exceptions, superior affinity TSPO ligands, with Ki
values in the subnanomolar range. Of note, within the subseries
of the 2-(naphth-2-yl)-substituted derivatives 29−35, all
compounds showed almost identical Ki subnanomolar values,
independent from the nature of the groups bound to the amide
nitrogen. Taken together, the SARs suggest a key role of the
lipophilic interaction at the level of the L1 pocket that, when it
reaches its optimum with a naphthyl group, makes less
significant all the other lipophilic interactions of the molecule
within the receptor protein.
A 3D model of the rTSPO was developed employing the
newly published NMR model of mTSPO (PDB code 2MGY).5
Docking studies were conducted using compound 29 as
reference ligand, and the proposed binding mode highlighted
the cooperative effects between the nature of the substituents
on the N,N-alkyl branches and the electronic features of the
moiety at indole 2-position, rationalizing the SARs within the 2-
arylindol-3-ylglyoxylamide TSPO ligands described so far.
Ethyl [2-(4-Methoxyphenyl)-1H-indol-3-yl](oxo)acetate 39a.
Yield, 70%; mp 194−196 °C. 1H NMR (DMSO-d6, ppm): 0.99 (t, J =
7.2 Hz, 3H, CH2CH3); 3.68 (q, J = 7.2 Hz, 2H, CH2CH3); 3.85 (s, 3H,
OCH3); 7.12 (d, J = 6.8 Hz, 2H, 3′-H, 5′-H); 7.26−7.33 (m, 2H, Ar-
H); 7.49−7.52 (m, 3H, Ar-H); 8.12−8.15 (m, 1H, 4-H); 12.52 (bs,
1H, NH, exch with D2O). Anal. Calcd for C19H17NO4: C, 70.58; H,
5.30; N, 4.33. Found: C, 70.45; H, 5.17; N, 4.27.
Methyl 4-{3-[Chloro(oxo)acetyl]-1H-indol-2-yl}benzoate
(41). Yield, 80%
Methyl 4-{3-[Ethoxy(oxo)acetyl]-1H-indol-2-yl}benzoate
41a. Yield, 70%; mp 152−154 °C. 1H NMR (DMSO-d6, ppm):
0.97 (t, J = 7.2 Hz, 3H, CH2CH3); 3.63 (q, J = 7.2 Hz, 2H, CH2CH3);
3.92 (s, 3H, OCH3); 7.32−7.36 (m, 2H, Ar-H); 7.54−7.57 (m, 1H,
Ar-H); 7.74 (d, J = 8.4 Hz, 2H, 2′-H, 6′-H); 8.11−8.16 (m, 3H, Ar-
H); 12.80 (bs, 1H, NH, exch with D2O). Anal. Calcd for C20H17NO5:
C, 68.37; H, 4.88; N, 3.99. Found: C, 68.51; H, 4.71; N, 4.05.
Oxo[2-(3-thienyl)-1H-indol-3-yl]acetyl Chloride (45). Yield,
65%.
Ethyl Oxo[2-(3-thienyl)-1H-indol-3-yl]acetate 45a. Yield, 72%;
mp 168−170 °C. 1H NMR (DMSO-d6, ppm): 1.06 (t, J = 7.2 Hz, 3H,
CH2CH3); 3.82 (q, J = 7.2 Hz, 2H, CH2CH3); 7.28−7.33 (m, 2H, Ar-
H); 7.37−7.40 (m, 1H, Ar-H); 7.48−7.51 (m, 1H, Ar-H); 7.77−7.79
(m, 1H, Ar-H); 7.93−7.94 (m, 1H, Ar-H); 8.07−8.09 (m, 1H, 4-H);
12.59 (bs, 1H, NH, exch with D2O). Anal. Calcd for C16H13NO3S: C,
64.20; H, 4.38; N, 4.68. Found: C, 64.08; H, 4.47; N, 4.75.
(2-Biphenyl-4-yl-1H-indol-3-yl)(oxo)acetyl Chloride (46).
Yield, 80%.
Ethyl (2-Biphenyl-4-yl-1H-indol-3-yl)(oxo)acetate 46a. Yield,
90%; mp 145−147 °C. H NMR (DMSO-d6, ppm): 0.98 (t, J = 7.2
EXPERIMENTAL SECTION
1
■
Chemistry. 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. Melting points were
Hz, 3H, CH2CH3); 3.67 (q, J = 7.2 Hz, 2H, CH2CH3); 7.31−7.36 (m,
2H, Ar-H); 7.42−7.46 (m, 1H, Ar-H); 7.52−7.55 (m, 3H, Ar-H); 7.67
(d, J = 8.2 Hz, 2H, 3′-H, 5′-H); 7.76−7.78 (m, 2H, Ar-H); 7.88 (d, J =
8.2 Hz, 2H, 2′-H, 6′-H); 8.16−8.18 (m, 1H, 4-H); 12.70 (bs, 1H, NH,
exch with D2O). Anal. Calcd for C24H19NO3: C, 78.03; H, 5.18; N,
3.79. Found: C, 78.19; H, 5.33; N, 3.61.
determined using a Reichert Kofler hot-stage apparatus and are
̈
uncorrected. Routine nuclear magnetic resonance spectra were
recorded in DMSO-d6 solution on a Varian Gemini 200 spectrometer
operating at 200 MHz. The NMR spectra of tertiary amides show the
presence of two different conformational isomers (ratio about 1:1) in
equilibrium (see refs 26 and 37 for details). Elemental analyses were
performed by our analytical laboratory and agreed with theoretical
values to within (0.4%).
[2-(2-Naphthyl)-1H-indol-3-yl](oxo)acetyl Chloride (47).
Yield, 82%.
Ethyl [2-(2-Naphthyl)-1H-indol-3-yl](oxo)acetate 47a. Yield,
1
79%; mp 143−145 °C. H NMR (DMSO-d6, ppm): 0.79 (t, J = 7.2
Hz, 3H, CH2CH3); 3.34 (q, J = 7.2 Hz, 2H, CH2CH3); 7.32−7.38 (m,
2H, Ar-H); 7.56−7.58 (m, 1H, Ar-H); 7.64−7.66 (m, 2H, Ar-H);
7.70−7.72 (m, 1H, Ar-H); 8.04−8.06 (m, 2H, Ar-H); 8.09−8.14 (m,
2H, Ar-H); 8.19−8.21 (m, 1H, 4-H); 12.76 (bs, 1H, NH, exch with
D2O). Anal. Calcd for C22H17NO3: C, 76.95; H, 4.99; N, 4.08. Found:
C, 77.10; H, 4.83; N, 4.17.
2-(p-Biphenyl-4-yl)indole 43 and 2-(naphth-2-yl)indole 44 are
commercially available. 2-(4-Methoxyphenyl)indole 36,39 2-(4-
nitrophenyl)indole 37,26 2-(3-thienyl)-1H-indole 42,40 [2-(4-
nitrophenyl)indol-3-yl]glyoxylyl chloride 40,26 N,N-di-n-propyl-[2-(4-
nitrophenyl)indol-3-yl]glyoxylamide 7d,26 N,N-di-n-butyl-[2-(4-
nitrophenyl)indol-3-yl]glyoxylamide 7e,26 N,N-di-n-hexyl-[2-(4-
nitrophenyl)indol-3-yl]glyoxylamide 7f26 were prepared in accordance
with reported procedures.
Methyl-4-(1H-indol-2-yl)benzoate 38. An amount of 1 g of
polyphosphoric acid (PPA) was added to a mixture of phenylhydrazine
hydrochloride (0.541 g, 5.0 mmol) and methyl 4-acetylbenzoate
(0.891 g, 5.0 mmol). The reaction was maintained at 60 °C for 4 h and
then poured into ice (∼100 g). The precipitated solid was collected by
filtration and purified by recrystallization from toluene (30 mL). Yield,
78%; mp 204−207 °C, lit. ref 41 mp 206.3−207.5 °C.
General Procedure for the Synthesis of N,N-Dialkyl-[2-(4-
substituted-phenyl)indol-3-yl]glyoxylamide Derivatives 17−
22 and N,N-Dialkyl-[(2-arylindol)-3-yl]glyoxylamide Deriva-
tives 23−35. A solution of the appropriate amine (2.0 mmol) in 5
mL of dry toluene was added dropwise to a stirred suspension, cooled
at 0 °C, of the glyoxylyl chlorides 39−41, 45−47 (2.0 mmol) in 50
mL of the same solvent, followed by the addition of triethylamine (2.0
mmol). The reaction mixture was left under stirring for 2−24 h at
room temperature (TLC analysis) and then filtered. The collected
precipitate was triturated with a 5% NaHCO3 aqueous solution (10
mL), washed with water (10 mL), and collected again to give a first
G
DOI: 10.1021/acs.jmedchem.5b00689
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
portion of crude products. The toluene was removed under reduced
pressure, and the oily residue dissolved with CHCl3 (20−30 mL). The
organic solution was washed with diluted HCl (10 mL), a 5%
NaHCO3 aqueous solution (10 mL), and water (10 mL), dried
(MgSO4), and evaporated to dryness to yield an additional amount of
crude products, which were finally purified by flash chromatography
(CHCl3 as eluent).
2-[2-(4-Methoxyphenyl)-1H-indol-3-yl]-2-oxo-N,N-dipropy-
lacetamide (17). Yield, 72%; mp 121−123 °C. 1H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.68, 0.76 (2t, J = 7.2 Hz,
6H, 2(CH2)2CH3); 1.21−1.26, 1.39−1.47 (2m, 4H, 2CH2CH2CH3);
2.93−3.06 (m, 4H, 2NCH2); 3.83 (s, 3H, OCH3); 7.06 (d, J = 8.8 Hz,
2H, 3′-H, 5′-H); 7.18−7.30 (m, 2H, Ar-H); 7.44−7.56 (m, 3H, Ar-H);
7.99−8.04 (m, 1H, 4-H); 12.34 (bs, 1H, NH, exch with D2O). 13C
NMR (DMSO-d6, ppm, mixture of conformational isomers): 11.38;
11.76; 20.55; 21.71; 46.03; 49.76; 55.82; 109.84; 112.39; 113.94;
121.20; 122.71; 123.44; 123.66; 127.35; 131.81; 136.14; 147.75;
160.90; 168.18; 187.62. Anal. Calcd for C23H26N2O3: C, 72.99; H,
6.92; N, 7.40. Found: C, 73.12; H, 6.80; N, 7.33.
Methyl 4-{3-[(Dihexylamino)(oxo)acetyl]-1H-indol-2-yl}-
1
benzoate (22). Yield, 65%; oil. H NMR (DMSO-d6, ppm, mixture
of conformational isomers): 0.66−1.46 (m, 22H, Aliph-H); 2.85−2.97
(m, 4H, 2NCH2); 3.81 (s, 3H, OCH3); 7.18−7.21 (m, 2H, Ar-H);
7.39−7.42 (m, 1H); 7.64 (d, J = 7.8 Hz, 2H, 2′-H, 6′-H); 7.96−8.00
(m, 3H, Ar-H); 12.53 (bs, 1H, NH, exch with D2O). 13C NMR
(DMSO-d6, ppm, mixture of conformational isomers): 14.19; 14.33;
22.28; 22.47; 26.03; 26.60; 26.87; 28.14; 31.09; 31.45; 44.18; 47.76;
52.76; 110.61; 112.67; 121.43; 123.04; 124.17; 127.14; 129.16; 130.79;
130.91; 135.96; 136.39; 145.98; 166.29; 167.67; 187.61. Anal. Calcd
for C30H38N2O4: C, 73.44; H, 7.81; N, 5.71. Found: C, 73.56; H, 7.72;
N, 5.65.
2-Oxo-N,N-dipropyl-2-[2-(3-thienyl)-1H-indol-3-yl]-
1
acetamide (23). Yield, 78%; mp 69−71 °C. H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.65, 0.71 (2t, J = 7.2 Hz,
6H, 2(CH2)2CH3); 1.30−1.50 (m, 4H, 2CH2CH2CH3); 3.00−3.14
(m, 4H, 2NCH2); 7.21−7.27 (m, 2H, Ar-H); 7.46−7.50 (m, 2H, Ar-
H); 7.69−7.73 (m, 1H, Ar-H); 7.89−7.93 (m, 1H, 4-H); 8.13 (s, 1H,
2′-H); 12.45 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6,
ppm, mixture of conformational isomers): 11.36; 11.81; 20.64; 21.73;
46.09; 49.72; 109.87; 112.48; 120.92; 122.75; 123.85; 126.53; 127.05;
128.74; 129.23; 131.42; 136.05; 142.02; 168.49; 187.32. Anal. Calcd
for C20H22N2O2S: C, 67.77; H, 6.26; N, 7.90. Found: C, 67.91; H,
6.19; N, 7.95.
N,N-Dibutyl-2-[2-(4-methoxyphenyl)-1H-indol-3-yl]-2-oxoa-
1
cetamide (18). Yield, 75%; mp 119−121 °C. H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.72, 0.84 (2t, J = 7.0 Hz,
6H, 2(CH2)3CH3); 1.02−1.13 (m, 6H, Aliph-H); 1.35−1.43 (m, 2H,
Aliph-H); 2.98−3.07 (m, 4H, 2NCH2); 3.83 (s, 3H, OCH3); 7.06 (d, J
= 8.8 Hz, 2H, 3′-H, 5′-H); 7.20−7.28 (m, 2H, Ar-H); 7.44−7.55 (m,
3H, Ar-H); 8.03−8.06 (m, 1H, 4-H); 12.53 (bs, 1H, NH, exch with
D2O). 13C NMR (DMSO-d6, ppm, mixture of conformational
isomers): 13.89; 14.11; 19.70; 20.23; 29.28; 30.40; 43.89; 47.55;
55.77; 109.87; 112.37; 113.91; 121.24; 122.70; 123.38; 123.67; 127.38;
131.86; 136.17; 147.79; 160.88; 168.00; 187.70. Anal. Calcd for
C25H30N2O3: C, 73.86; H, 7.44; N, 6.89. Found: C, 73.72 ; H, 7.51; N,
6.99.
N,N-Dibutyl-2-oxo-2-[2-(3-thienyl)-1H-indol-3-yl]acetamide
(24). Yield, 79%; mp 119−121 °C. 1H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.66−1.09 (m, 10H, Aliph-H);
1.22−1.35 (m, 4H, 2CH2CH2CH2CH3); 3.00−3.32 (m, 4H, 2NCH2);
7.16−7.30 (m, 2H, Ar-H); 7.45−7.49 (m, 2H, Ar-H); 7.68−7.72 (m,
1H, Ar-H); 7.88−7.92 (m, 1H, 4-H); 8.11 (s, 1H, 2′-H); 12.43 (bs,
1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm, mixture of
conformational isomers): 13.82; 14.21; 19.66; 20.24; 29.41; 30.44;
43.98; 47.59; 109.85; 112.46; 120.93; 122.73; 123.86; 126.55; 127.04;
128.79; 129.23; 131.38; 136.04; 141.98; 168.37; 187.37. Anal. Calcd
for C22H26N2O2S: C, 69.08; H, 6.85; N, 7.32. Found: C, 69.19; H,
6.76; N, 7.39.
N,N-Dihexyl-2-[2-(4-methoxyphenyl)-1H-indol-3-yl]-2-oxoa-
cetamide (19). Yield, 65%; oil. 1H NMR (DMSO-d6, ppm, mixture of
conformational isomers): 0.73−1.36 (m, 22H, Aliph-H); 2.98−3.05
(m, 4H, 2NCH2); 3.81 (s, 3H, OCH3); 7.05 (d, J = 6.8 Hz, 2H, 3′-H,
5′-H); 7.20−7.25 (m, 2H, Ar-H); 7.43−7.54 (m, 3H, Ar-H); 8.01−
8.04 (m, 1H, 4-H); 12.33 (bs, 1H, NH, exch with D2O). 13C NMR
(DMSO-d6, ppm, mixture of conformational isomers): 14.20; 14.36;
22.26; 22.47; 26.02; 26.64; 27.09; 28.19; 31.08; 31.46; 43.57; 47.79;
55.77; 109.87; 112.38; 113.90; 121.26; 121.65; 122.32; 123.35; 127.35;
131.87; 136.19; 148.12; 159.34; 168.00; 187.91. Anal. Calcd for
C29H38N2O3: C, 75.29; H, 8.28; N, 6.06. Found: C, 75.13; H, 8.21; N,
6.00.
N,N-Dihexyl-2-oxo-2-[2-(3-thienyl)-1H-indol-3-yl]acetamide
(25). Yield, 82%; oil. 1H NMR (DMSO-d6, ppm, mixture of
conformational isomers, mixture of conformational isomers): 0.66−
1.12 (m, 14H, Aliph-H); 1.26−1.39 (m, 8H, Aliph-H); 3.00−3.20 (m,
4H, 2NCH2); 7.18−7.29 (m, 2H, Ar-H); 7.45−7.51 (m, 2H, Ar-H);
7.68−7.72 (m, 1H, Ar-H); 7.87−7.93 (m, 1H, 4-H); 8.14 (s, 1H, 2′-
H); 12.44 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm,
mixture of conformational isomers): 14.17; 14.37; 22.21; 22.50; 25.98;
26.63; 27.22; 28.14; 31.00; 31.48; 44.23; 47.48; 109.76; 112.49;
120.88; 122.67; 123.82; 126.50; 127.02; 128.81; 129.21; 131.40;
136.07; 141.90; 168.43; 187.32. Anal. Calcd for C26H34N2O2S: C,
71.19; H, 7.81; N, 6.39. Found: C, 71.05; H, 7.89; N, 6.32.
2-(2-Biphenyl-4-yl-1H-indol-3-yl)-2-oxo-N,N-dipropylaceta-
Methyl 4-{3-[(Dipropylamino)(oxo)acetyl]-1H-indol-2-yl}-
1
benzoate (20). Yield, 65%; mp 139−141 °C; H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.66−0.77 (m, 6H,
2(CH2)2CH3); 1.20−1.27, 1.45−1.49 (2m, 4H, 2CH2CH2CH3);
2.93−3.10 (m, 4H, 2NCH2); 3.92 (s, 3H, OCH3); 7.27−7.31 (m,
2H, Ar-H); 7.50−7.54 (m, 1H, Ar-H); 7.74 (d, J = 8.0 Hz, 2H, 2′-H,
6′-H); 8.02−8.09 (m, 3H, 4-H); 12.64 (bs, 1H, NH, exch with D2O).
13C NMR (DMSO-d6, ppm, mixture of conformational isomers):
11.38; 11.71; 20.46; 21.77; 46.03; 49.77; 52.83; 110.49; 112.79;
121.27; 123.02; 124.10; 127.13; 129.10; 130.74; 130.82; 136.12;
136.51; 146.10; 166.37; 167.95; 187.37. Anal. Calcd for C24H26N2O4:
C, 70.92; H, 6.45; N, 6.89. Found: C, 7.79; H, 6.53; N, 6.95.
1
mide (26). Yield, 70%; mp 155−157 °C. H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.67−0.73 (m, 6H, 2-
(CH2)2CH3); 1.15−1.20, 1.45−1.51 (2m, 4H, 2CH2CH2CH3);
2.92−3.10 (m, 4H, 2NCH2); 7.29−7.32 (m, 2H, Ar-H); 7.51−7.55
(m, 4H, Ar-H); 7.68−7.81 (m, 6H, Ar-H); 8.06−8.08 (m, 1H, 4-H),
12.52 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm,
mixture of conformational isomers): 11.40; 11.77; 20.93; 21.71; 46.01;
49.75; 110.29; 112.55; 121.31; 122.90; 123.91; 126.68; 127.24; 127.28;
128.39; 129.53; 130.33; 130.99; 136.26; 139.94; 141.80; 147.31;
168.02; 187.68. Anal. Calcd for C28H28N2O2: C, 79.22; H, 6.65; N,
6.60. Found: C, 79.56; H, 6.81; N, 6.44.
Methyl 4-{3-[(Dibutylamino)(oxo)acetyl]-1H-indol-2-yl}-
1
benzoate (21). Yield, 71%; mp 119−121 °C; H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.69−0.80 (m, 6H,
2(CH2)3CH3); 1.00−1.15 (m, 4H, 2(CH2)2CH2CH3); 1.25−1.41
(m, 4H, 2CH2CH2CH2CH3); 2.95−3.09 (m, 4H, 2NCH2); 3.89 (s,
3H, OCH3); 7.25−7.30 (m, 2H, Ar-H); 7.48−7.52 (m, 1H, Ar-H);
7.72 (d, J = 8.0 Hz, 2H, 2′-H, 6′-H); 8.04−8.09 (m, 3H, 4-H); 12.59
(bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm, mixture of
conformational isomers): 13.90; 14.01; 19.70; 20.16; 29.12; 30.41;
43.93; 47.55; 52.78; 110.63; 112.70; 121.42; 123.06; 124.17; 127.17;
129.17; 130.81; 130.90; 136.01; 136.42; 146.08; 166.32; 167.69;
187.56. Anal. Calcd for C26H30N2O4: C, 71.87; H, 6.96; N, 6.45.
Found: C, 71.72; H, 6.89; N, 6.54.
2-(2-Biphenyl-4-yl-1H-indol-3-yl)-N,N-dibutyl-2-oxoaceta-
1
mide (27). Yield, 67%; mp 153−155 °C. H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.71−0.78 (m, 6H, 2-
(CH2)3CH3); 1.07−1.15 (m, 6H, Aliph-H); 1.41−1.44 (m, 2H,
Aliph-H); 2.98−3.11 (m, 4H, 2NCH2); 7.27−7.34 (m, 2H, Ar-H);
7.42−7.57 (m, 4H, Ar-H); 7.66−7.86 (m, 6H, Ar-H); 8.07−8.11 (m,
1H, 4-H); 12.51 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6,
ppm, mixture of conformational isomers): 13.93; 13.99; 19.72; 20.18;
29.08; 30.45; 43.95; 47.65; 110.27; 112.55; 121.33; 122.87; 123.90;
126.58; 127.18; 127.34; 128.40; 129.50; 130.33; 131.02; 136.33;
H
DOI: 10.1021/acs.jmedchem.5b00689
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
139.77; 141.64; 147.31; 167.93; 187.72. Anal. Calcd for C30H32N2O2:
C, 79.61; H, 7.13; N, 6.19. Found: C, 79.91; H, 7.27; N, 5.92.
2-(2-Biphenyl-4-yl-1H-indol-3-yl)-N,N-dihexyl-2-oxoaceta-
Calcd for C25H24N2O2: C, 78.10; H, 6.29; N, 7.29. Found: C, 78.23;
H, 6.21; N, 7.32.
N-Methyl-2-[2-(2-naphthyl)-1H-indol-3-yl]-2-oxo-N-pentyla-
1
1
cetamide (33). Yield, 65%; mp 153−155 °C. H NMR (DMSO-d6,
mide (28). Yield, 65%; mp 50−52 °C. H NMR (DMSO-d6, ppm,
ppm, mixture of conformational isomers): 0.70−1.00 (m, 7H, Aliph-
H); 1.08−1.42 (m, 2H, CH2CH2(CH2)2CH3); 2.25, 2.82 (2s, 3H,
NCH3); 2.66, 3.12 (2t, J = 7.2 Hz, 2H, NCH2); 7.28−7.36 (m, 2H, Ar-
H); 7.51−7.55 (m, 1H, Ar-H); 7.64−7.69 (m, 3H, Ar-H); 8.01−8.05
(m, 3H, Ar-H); 8.15 (s, 1H, 1′-H); 8.16−8.18 (m, 1H, 4-H); 12.60
(bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm, mixture of
conformational isomers): 14.23; 14.28; 14.23; 22.06; 22.14; 25.60;
27.42; 28.47; 28.72; 31.03; 35.04; 45.75; 49.65; 110.56; 110.59;
112.56; 121.46; 121.50; 123.04; 124.02; 124.05; 127.18; 127.26;
127.39; 127.44; 127.50; 127.61; 127.65; 127.86; 128.13; 128.67;
128.69; 128.74; 129.91; 130.12; 132.53; 132.62; 133.56; 133.67;
136.33; 136.39; 147.80; 148.01; 167.47; 167.76; 187.76; 188.01. Anal.
Calcd for C26H26N2O2: C, 78.36; H, 6.58; N, 7.03. Found: C, 78.49;
H, 6.62; N, 6.97.
mixture of conformational isomers): 0.72−0.75 (m, 6H, 2-
(CH2)5CH3); 1.07−1.14 (m, 14H, Aliph-H); 1.41−1.44 (m, 2H,
Aliph-H); 2.92−3.08 (m, 4H, 2NCH2); 7.25−7.34 (m, 2H, Ar-H);
7.42−7.52 (m, 4H, Ar-H); 7.66−7.85 (m, 6H, Ar-H); 8.07−8.10 (m,
1H, 4-H), 12.51 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6,
ppm, mixture of conformational isomers): 14.21; 14.24; 22.31; 22.48;
26.07; 26.69; 28.18; 31.13; 31.38; 39.40; 44.20; 47.84; 110.30; 112.51;
121.40; 122.86; 123.91; 126.55; 127.15; 127.33; 128.38; 129.47;
130.29; 131.04; 136.30; 139.70; 141.61; 147.26; 167.88; 187.77. Anal.
Calcd for C34H40N2O2: C, 80.28; H, 7.93; N, 5.51. Found: C, 80.29;
H, 8.15; N, 5.40.
2-[2-(2-Naphthyl)-1H-indol-3-yl]-2-oxo-N,N-dipropylaceta-
1
mide (29). Yield, 60%; mp 171−172 °C. H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.51, 0.71 (2t, J = 7.2 Hz, 6H,
2(CH2)2CH3); 0.86−0.98, 1.41−1.52 (2m, 4H, 2CH2CH2CH3); 2.79,
3.08 (2t, J = 7.0 Hz, 4H, 2NCH2); 7.28−7.32 (m, 2H, Ar-H); 7.51−
7.56 (m, 1H, Ar-H); 7.64−7.72 (m, 3H, Ar-H); 8.00−8.09 (m, 4H, Ar-
H); 8.17 (s, 1H, 1′-H); 12.60 (bs, 1H, NH, exch with D2O). 13C NMR
(DMSO-d6, ppm, mixture of conformational isomers): 11.39; 11.56;
20.18; 21.75; 45.94; 49.81; 110.46; 112.57; 121.33; 122.92; 123.94;
127.15; 127.29; 127.57; 127.60; 127.82; 128.07; 128.76; 128.85;
130.06; 132.65; 133.65; 136.35; 147.62; 168.00; 187.68. Anal. Calcd
for C26H26N2O2: C, 78.36; H, 6.58; N, 7.03. Found: C, 78.49; H, 6.63;
N, 6.95.
N-Butyl-N-ethyl-2-[2-(2-naphthyl)-1H-indol-3-yl]-2-oxoace-
1
tamide (34). Yield, 63%; mp 179−181 °C. H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.53−1.04 (m, 10H, Aliph-
H); 2.81−2.87 (m, 2H, Aliph-H); 3.20−3.33 (m, 2H, Aliph-H); 7.27−
7.32 (m, 2H, Ar-H); 7.51−7.53 (m, 1H, Ar-H); 7.61−7.70 (m, 3H, Ar-
H); 8.00−8.04 (m, 3H, Ar-H); 8.12−8.14 (m, 1H, 4-H); 8.15 (s 1H,
1′-H); 12.58 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6,
ppm, mixture of conformational isomers): 12.28; 13.91; 13.96; 14.02;
19.74; 19.95; 28.81; 30.54; 38.84; 42.58; 43.26; 47.47; 121.34; 121.44;
122.90; 123.92; 127.14; 127.16; 127.39; 127.45; 127.58; 127.80;
127.86; 128.10; 128.14; 128.75; 128.77; 128.89; 128.93; 130.10;
130.20; 132.64; 132.67; 133.61; 133.67; 136.52; 147.80; 147.98;
167.70; 187.66; 187.83. Anal. Calcd for C26H26N2O2: C, 78.36; H,
6.58; N, 7.03. Found: C, 78.49; H, 6.49; N, 7.07.
N,N-Dibutyl-2-[2-(2-naphthyl)-1H-indol-3-yl]-2-oxoaceta-
1
mide (30). Yield, 79%; mp 98−100 °C. H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.54, 075 (2t, J = 7.2 Hz, 6H,
2(CH2)3CH3); 0.63−0.70, 0.83−0.90 (2m, 4H, 2(CH2)2CH2CH3);
1.09−1.14, 1.37−1.42 (2m, 4H, 2CH2CH2CH2CH3); 2.79, 3.09 (2t, J
= 7.6 Hz, 4H, 2NCH2); 7.27−7.35 (m, 2H, Ar-H); 7.50−7.53 (m, 1H,
Ar-H); 7.60−7.70 (m, 3H, Ar-H); 8.00−8.05 (m, 3H, Ar-H); 8.11−
8.14 (m, 1H, 4-H); 8.16 (s, 1H, 1′-H); 12.56 (bs, 1H, NH, exch with
D2O). 13C NMR (DMSO-d6, ppm, mixture of conformational
isomers): 13.89; 13.94; 19.72; 19.96; 28.78; 30.43; 43.81; 47.59;
110.55; 112.53; 121.43; 122.93; 123.96; 127.16; 127.34; 127.57;
127.59; 127.87; 128.14; 128.77; 130.20; 132.67; 133.68; 136.36;
147.66; 167.80; 187.83. Anal. Calcd for C28H30N2O2: C, 78.84; H,
7.09; N, 6.57. Found: C, 78.71; H, 7.05; N, 6.49.
N-Benzyl-N-ethyl-2-[2-(2-naphthyl)-1H-indol-3-yl]-2-oxoace-
1
tamide (35). Yield, 70%; mp 83−85 °C. H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.26, 0.91 (2t, J = 7.0 Hz, 3H,
CH3); 2.73, 3.22 (2q, J = 7.0 Hz, 2H, NCH2CH3); 4.17, 4.41 (2s, 2H,
NCH2C6H5); 7.00−7.04 (m, 2H, Ar-H); 7.18−7.35 (m, 5H, Ar-H);
7.53−7.77 (m, 4H, Ar-H); 7.97−8.06 (m, 4H, Ar-H); 8.23−8.25 (m,
1H, 4-H); 12.65 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6,
ppm, mixture of conformational isomers): 11.47; 13.96; 38.40; 42.85;
46.73; 50.96; 110.02; 110.80; 112.62; 112.67; 121.22; 121.46; 122.96;
123.08; 123.98; 124.08; 127.25; 127.38; 127.56; 127.59; 127.71;
127.83; 127.96; 128.14; 128.21; 128.27; 128.54; 128.74; 128.80;
128.83; 128.85; 130.07; 130.20; 132.66; 132.69; 133.67; 133.69;
136.42; 136.76; 137.36; 147.72; 148.17; 167.80; 168.39; 187.29;
187.68. Anal. Calcd for C29H24N2O2: C, 80.53; H, 5.59; N, 6.48.
Found: C, 80.41; H, 5.49; N, 6.57.
N,N-Dihexyl-2-[2-(2-naphthyl)-1H-indol-3-yl]-2-oxoaceta-
1
mide (31). Yield, 88%; oil. H NMR (DMSO-d6, ppm, mixture of
conformational isomers): 0.70−0.88 (m, 10H, Aliph-H); 1.08−1.41
(m, 12H, Aliph-H); 2.82, 3.10 (2t, J = 7.6 Hz, 4H, 2NCH2); 7.27−7.35
(m, 2H, Ar-H); 7.51−7.59 (m, 1H, Ar-H); 7.61−7.71 (m, 3H, Ar-H);
8.00−8.04 (m, 3H, Ar-H); 8.10−8.12 (m, 1H, 4-H); 8.16 (s, 1H, 1′-
H); 12.57 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm,
mixture of conformational isomers): 14.22; 14.31; 22.31; 22.36; 26.07;
26.41; 26.58; 28.18; 31.14; 31.19; 44.08; 47.82; 110.50; 112.54;
121.42; 122.89; 123.94; 127.14; 127.33; 127.57; 127.85; 128.12;
128.76; 130.20; 132.66; 133.66; 136.38; 147.61; 167.80; 187.82. Anal.
Calcd for C32H38N2O2: C, 79.63; H, 7.94; N, 5.80. Found: C, 79.75;
H, 7.97; N, 5.86.
General Procedure for the Synthesis of N,N-Dialkyl-[2-(4-
hydroxyphenyl)indol-3-yl]glyoxylamide Derivatives 8−10. A
stirred suspension of the appropriate derivative 17−19 (0.5 mmol) in
10 mL of dry dichloromethane was cooled to −10 °C, and an excess
(0.2 mL) of BBr3 was added dropwise. The mixture was left under
stirring for 30 min at −10 °C and subsequently at room temperature
for 1 h under nitrogen atmosphere. Finally, the solution was cooled
again, and methanol (5 mL) was added to hydrolyze the excess of
BBr3. The solvent was evaporated at reduced pressure, and the solid
precipitate was washed several times with methanol (3 × 5 mL). The
residues obtained were purified by crystallization from toluene
(compound 8, 20 mL) or by flash chromatography (compounds 9
and 10, ethyl acetate−petroleum ether 60−80 °C in varying v/v ratios
as eluting system).
N-Butyl-N-methyl-2-[2-(2-naphthyl)-1H-indol-3-yl]-2-oxoa-
1
cetamide (32). Yield, 70%; mp 74−76 °C. H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.56−0.92 (m, 5H,
(CH2)2CH2CH3); 1.12−1.56 (m, 2H, CH2CH2CH2CH3); 2.24, 2.82
(2s, 3H, NCH3); 2.67, 3.13 (2t, J = 7.2 Hz, 2H, NCH2); 7.29−7.36
(m, 2H, Ar-H); 7.51−7.55 (m, 1H, Ar-H); 7.61−7.69 (m, 3H, Ar-H);
8.01−8.05 (m, 3H, Ar-H); 8.15 (s, 1H, 1′-H); 8.17−8.19 (m, 1H, 4-
H); 12.62 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm,
mixture of conformational isomers): 13.89; 13.99; 19.60; 19.74; 28.08;
29.93; 31.04; 35.02; 45.49; 49.44; 110.57; 110.59; 112.56; 121.47;
121.51; 123.04; 124.02; 124.05; 127.18; 127.20; 127.27; 127.39;
127.46; 127.50; 127.63; 127.65; 127.86; 128.14; 128.66; 128.68;
128.74; 128.75; 129.92; 130.12; 132.53; 132.62; 133.56; 133.69;
136.33; 136.39; 147.83; 148.01; 167.48; 167.74; 187.78; 188.03. Anal.
2-[2-(4-Hydroxyphenyl)-1H-indol-3-yl]-2-oxo-N,N-dipropyla-
1
cetamide (8). Yield, 59%; mp 210−212 °C. H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.64−0.82 (m, 6H,
2(CH2)2CH3); 1.27−1.46 (m, 4H, 2CH2CH2CH3); 2.95−3.02 (m,
4H, 2NCH2); 6.86 (d, J = 8.4 Hz, 2H, 3′-H, 5′-H); 7.17−7.25 (m, 2H,
Ar-H); 7.40−7.44 (m, 3H, Ar-H); 8.00−8.03 (m, 1H, 4-H); 9.90 (bs,
1H, OH, exch with D2O); 12.27 (bs, 1H, NH, exch with D2O). 13C
NMR (DMSO-d6, ppm, mixture of conformational isomers): 11.39;
11.84; 20.58; 21.72; 46.10; 49.82; 109.60; 112.32; 115.22; 121.15;
I
DOI: 10.1021/acs.jmedchem.5b00689
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
121.74; 122.63; 123.55; 127.40; 131.80; 136.08; 148.31; 159.32;
168.22; 187.64. Anal. Calcd for C22H24N2O3: C, 72.50; H, 6.64; N,
7.69. Found: C, 72.39; H, 6.59; N, 7.77.
113.33; 117.94; 120.90; 122.33; 123.23; 127.61; 131.35; 136.13;
149.22; 150.64; 168.42; 187.43. Anal. Calcd for C28H37N3O2: C, 75.13;
H, 8.33; N, 9.39. Found: C, 75.02; H, 8.39; N, 9.28.
N,N-Dibutyl-2-[2-(4-hydroxyphenyl)-1H-indol-3-yl]-2-oxoa-
General Procedure for the Synthesis of N,N-Dialkyl-[2-(4-
carboxyphenyl)indol-3-yl]glyoxylamide Derivatives 14−16.
Lithium hydroxide monohydrate (0.3 mmol) was added to a
suspension of the appropriate ester derivative 20−23 (0.5 mmol) in
20 mL of a MeOH/H2O (3:1) solution. The mixture was stirred under
reflux at 80 °C overnight. The solid precipitate was eliminated through
vacuum filtration, and the solution was acidified with 10% HCl to pH
5. The acid precipitated was collected by filtration and did not need
any further purification.
1
cetamide (9). Yield, 68%; mp 250−252 °C. H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.68−0.84 (m, 6H,
2(CH2)3CH3); 1.02−1.35 (m, 8H, 2CH2(CH2)2CH3); 1.21−1.36,
2.96−2.99 (2m, 4H, 2NCH2); 6.83 (d, J = 6.2 Hz, 2H, 3′-H, 5′-H);
7.19−7.21 (m, 2H, Ar-H); 7.37−7.40 (m, 3H, Ar-H); 7.98−8.00 (m,
1H, 4-H); 9.84 (bs, 1H, OH, exch with D2O); 12.22 (bs, 1H, NH,
exch with D2O). 13C NMR (DMSO-d6, ppm, mixture of conforma-
tional isomers): 13.99; 14.14; 19.75; 20.27; 29.50; 30.57; 44.07; 47.93;
111.04; 111.38; 115.25; 119.50; 121.55; 123.29; 123.83; 126.45;
132.47; 137.17; 149.73; 159.25; 167.54; 187.95. Anal. Calcd for
C24H28N2O3: C, 73.44; H, 7.19; N, 7.14. Found: C, 73.59; H, 7.28; N,
7.26.
N,N-Dihexyl-2-[2-(4-hydroxyphenyl)-1H-indol-3-yl]-2-oxoa-
cetamide (10). Yield, 55%; oil. 1H NMR (DMSO-d6, ppm, mixture of
conformational isomers): 0.68−0.88 (m, 6H, 2(CH2)5CH3); 1.04−
1.23 (m, 16H, Aliph-H); 2.95−3.05 (m, 4H, 2NCH2); 6.85 (d, J = 8.4
Hz, 2H, 3′-H, 5′-H); 7.18−7.22 (m, 2H, Ar-H); 7.38−7.45 (m, 3H,
Ar-H); 7.97−8.00 (m, 1H, 4-H); 9.88 (bs, 1H, OH, exch with D2O);
12.27 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm,
mixture of conformational isomers): 14.13; 14.23; 22.30; 22.56; 26.07;
26.71; 27.19; 28.21; 31.12; 31.47; 44.28; 47.81; 109.50; 112.24;
115.16; 121.20; 121.65; 122.54; 123.48; 127.37; 131.87; 136.06;
148.23; 159.34; 168.01; 187.90. Anal. Calcd for C28H36N2O3: C, 74.97;
H, 8.09; N, 6.24. Found: C, 74.85; H, 7.98; N, 6.30.
4-{3-[(Dipropylamino)(oxo)acetyl]-1H-indol-2-yl}benzoic
1
Acid (14). Yield, 85%; mp 292−294 °C. H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.64−0.75 (m, 6H, 2-
(CH2)2CH3); 1.15−1.26, 1.43−1.47 (2m, 4H, 2CH2CH2CH3);
2.91−3.11 (m, 4H, 2NCH2); 7.27−7.31 (m, 2H, Ar-H); 7.49−7.51
(m, 1H, Ar-H); 8.10 (d, J = 7.8 Hz, 2H, 3′-H, 5′-H); 8.03−8.07 (m,
3H, Ar-H); 12.63 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-
d6, ppm, mixture of conformational isomers): 11.38; 11.73; 20.43;
21.76; 46.03; 49.78; 110.48; 112.69; 121.32; 123.05; 124.12; 127.09;
129.24; 130.64; 132.10; 135.51; 136.31; 146.27; 167.40; 167.89;
187.50. Anal. Calcd for C23H24N2O4: C, 70.39; H, 6.16; N, 7.14.
Found: C, 70.22; H, 6.25; N, 7.18.
4-{3-[(Dibutylamino)(oxo)acetyl]-1H-indol-2-yl}benzoic Acid
(15). Yield, 87%; mp 148−150 °C. 1H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.68−0.81 (m, 6H, 2-
(CH2)3CH3); 1.03−1.40 (m, 8H, 2CH2(CH2)2CH3); 2.93−3.04 (m,
4H, 2NCH2); 7.25−7.28 (m, 2H, Ar-H); 7.46−7.50 (m, 1H, Ar-H);
7.68 (d, J = 6.8 Hz, 2H, 3′-H, 5′-H); 8.00−8.08 (m, 3H, Ar-H); 12.56
(bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm, mixture of
conformational isomers): 13.91; 14.07; 19.70; 20.17; 29.09; 30.42;
43.93; 47.57; 110.57; 112.65; 121.44; 123.05; 124.14; 127.17; 129.31;
130.71; 132.11; 135.48; 136.33; 146.32; 167.37; 167.68; 187.64. Anal.
Calcd for C25H28N2O4: C, 71.41; H, 6.71; N, 6.66. Found: C, 71.27;
H, 6.79; N, 6.57.
4-{3-[(Dihexylamino)(oxo)acetyl]-1H-indol-2-yl}benzoic Acid
(16). Yield, 79%; mp 242−244 °C. 1H NMR (DMSO-d6, ppm,
mixture of conformational isomers): 0.73−0.84 (m, 6H, 2-
(CH2)5CH3); 1.00−1.38 (m, 16H, Aliph-H); 2.93−3.03 (m, 4H,
2NCH2); 7.24−7.27 (m, 2H, Ar-H); 7.46−7.50 (m, 1H, Ar-H); 7.68
(d, J = 7.8 Hz, 2H, 3′-H, 5′-H); 8.00−8.05 (m, 3H, Ar-H); 12.56 (bs,
1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm, mixture of
conformational isomers): 14.20; 14.32; 22.29; 22.48; 26.04; 26.59;
26.88; 28.17; 31.10; 31.38; 44.19; 47.80; 110.53; 112.64; 121.42;
123.01; 124.12; 127.15; 129.28; 130.70; 132.09; 135.47; 136.35;
146.25; 167.33; 167.70; 187.63. Anal. Calcd for C29H36N2O4: C, 73.08;
H, 7.61; N, 5.88. Found: C, 72.95; H, 7.71; N, 5.96.
General Procedure for the Synthesis of N,N-Dialkyl-[2-(4-
aminophenyl)indol-3-yl]glyoxylamide Derivatives 11−13. Pd/
C 10% (0.05 g) was added to a suspension of the appropriate
derivative 7d−f26 (0.65 mmol) in 150 mL of absolute ethanol. The
mixture was hydrogenated under stirring at room temperature. Once
hydrogen absorption ceased, the catalyst was filtered off and the
solution was evaporated to dryness at reduced pressure. The crude
products obtained were finally purified by recrystallization from
toluene (20−30 mL).
2-[2-(4-Aminophenyl)-1H-indol-3-yl]-2-oxo-N,N-dipropyla-
1
cetamide (11). Yield, 62%; mp 197−199 °C. H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.67, 0.81 (2t, J = 7.2 Hz,
6H, 2(CH2)2CH3); 1.29−1.49 (m, 4H, 2CH2CH2CH3); 2.97−3.08
(m, 4H, 2NCH2); 5.57 (bs, 2H, NH2, exch with D2O); 6.62 (d, J = 8.4
Hz, 2H, 3′-H, 5′-H); 7.17−7.43 (m, 5H, Ar-H); 7.92−7.97 (m, 1H, 4-
H); 12.09 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm,
mixture of conformational isomers): 11.41; 11.89; 20.60; 21.67; 46.08;
49.77; 108.90; 112.14; 113.23; 117.84; 120.93; 122.39; 123.25; 127.64;
131.29; 136.10; 149.30; 150.87; 168.50; 187.45. Anal. Calcd for
C22H25N3O2: C, 72.70; H, 6.93; N, 11.56. Found: C, 72.82; H, 6.87;
N, 11.62.
Biological Methods. Materials. [3H]PK11195 (S.A. 85.5 Ci/
mmol) and [3H]Ro15-1788 (S.A. 83.4 Ci/mmol) were purchased
from PerkinElmer Life Sciences. Culture medium, fetal bovine serum
(FBS), L-glutamine, and antibiotics were purchased from Euroclone
SpA (Milano, Italy). PK11195 and Ro5-4864 were obtained from
Sigma-Aldrich. All other reagents were obtained from commercial
suppliers.
2-[2-(4-Aminophenyl)-1H-indol-3-yl]-N,N-dibutyl-2-oxoace-
1
tamide (12). Yield, 61%; mp 225−227 °C. H NMR (DMSO-d6,
ppm, mixture of conformational isomers): 0.64, 0.87 (2t, J = 7.2 Hz,
6H, 2(CH2)3CH3); 0.98−1.36 (m, 8H, 2CH2(CH2)2CH3); 3.00−3.06
(m, 4H, 2NCH2); 5.52 (bs, 2H, NH2, exch with D2O); 6.61 (d, J = 8.4
Hz, 2H, 3′-H, 5′-H); 7.15−7.41 (m, 5H, Ar-H); 7.93−7.96 (m, 1H, 4-
H); 12.08 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm,
mixture of conformational isomers): 13.88; 14.23; 19.72; 20.28; 29.34;
30.38; 43.90; 47.56; 108.92; 112.11; 113.22; 117.74; 120.97; 122.38;
123.27; 127.66; 131.35; 136.11; 149.33; 150.89; 168.34; 187.54. Anal.
Calcd for C24H29N3O2: C, 73.63; H, 7.47; N, 10.73. Found: C, 73.52;
H, 7.56; N, 10.79.
[3H]PK11195 Binding to Rat Kidney Mitochondrial Membranes.
For binding studies, crude mitochondrial membranes were incubated
with 0.5 nM [3H]PK11195 in the presence of a compound
concentration range (1 pM to 10 μM) in 50 mM Tris-HCl, pH 7.4,
as previously described.25,26 For the active compounds, the IC50 values
were determined and Ki values were derived in accordance with the
equation of Cheng and Prusoff.42
2-[2-(4-Aminophenyl)-1H-indol-3-yl]-N,N-dihexyl-2-oxoace-
1
tamide (13). Yield, 68%; mp 147−149 °C. H NMR (DMSO-d6,
[3H]Ro15-1788 Binding to Rat Cerebral Cortex Membranes. Rat
cerebral cortex membranes were prepared as previously described.2
After differential centrifugation, the crude membrane fraction obtained
was subjected to washing procedures to remove endogenous GABA.43
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.44
ppm, mixture of conformational isomers): 0.68−0.89 (m, 6H,
2(CH2)3CH3); 0.93−1.24 (m, 16H, Aliph-H); 2.97−3.12 (m, 4H,
2NCH2); 5.58 (bs, 2H, NH2, exch with D2O); 6.60 (d, J = 8.6 Hz, 2H,
3′-H, 5′-H); 7.16−7.41 (m, 5H, Ar-H); 7.90−7.93 (m, 1H, 4-H);
12.08 (bs, 1H, NH, exch with D2O). 13C NMR (DMSO-d6, ppm,
mixture of conformational isomers): 14.20; 14.37; 22.26; 22.54; 26.05;
26.71; 27.19; 28.13; 31.08; 31.45; 44.21; 47.83; 108.82; 112.12;
J
DOI: 10.1021/acs.jmedchem.5b00689
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Computational Studies. Homology Modeling. Recently the
group of Zweckstetter released the NMR structure of the mouse
TSPO (mTSPO) in complex with the PK11195 modulator,5 thus
paving the way for a better definition of the interaction mode between
TSPO and its synthetic modulators. Our group in the past decades
embarked in the discovery of new promising TSPO modulators,
describing different ligand classes able to interact with rat TSPO
(rTSPO) at the subnanomolar range, with the indolylglyoxylamide
class being the most promising one.25−27,29,30 mTSPO and rTSPO
share a 95% sequence identity, making mTSPO a suitable template for
homology modeling approaches. Therefore, we decided to build a
three-dimensional (3D) rTSPO model by using the Modeller 9.13
software and mTSPO as template (see Supporting Information Figure
S6 for the sequence alignment). The final rTSPO model was
subsequently used to perform docking studies and to rationalize the
SARs of the new series of indolylglyoxylamide derivatives described
herein.
Docking Studies. The selected 20 rTSPO models were used to
dock the new series of indolylglyoxylamide derivatives reported in this
work. All the docking studies were performed employing the Glide
tool implemented in Maestro 9.8.36 The ligand used for the validation
step of the rTSPO structures was represented by our lead in the new
series of the indolylglyoxylamide (Chart 1, compound 29, Table 2).
The 3D structures of 29 was generated with the Maestro fragment
Build tool and then geometrically optimized with Macromodel.36 The
rTSPO structures were prepared through the Protein Preparation
Wizard of the Maestro 9.836 graphical user interface which assigns
bond orders, adds hydrogen atoms, and generates appropriate
protonation states.
ABBREVIATIONS USED
■
BzR, central benzodiazepine receptor; CNS, central nervous
system; SAR, structure−affinity relationship; TSPO, trans-
locator protein
REFERENCES
■
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The docking grid box was centered on the residues lining the
PK11195 binding pocket, with a grid box dimension equal to 24 Å ×
24 Å × 24 Å (see Supporting Information Figure S7). In detail the
residues considered to center the docking grid are A23, V26, L49, A50,
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between the mTSPO and rTSPO, a part A110V). Finally, docking runs
were carried out using the standard precision (SP) method. Finally, all
the compounds reported in Tables 1 and 2 were docked using the
refined rTSPO−29 complex applying the same docking protocol.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00689.
Biological methods for evaluation of steroidogenic
activity of compound 29 and Figures S1−S7 (PDF)
Molecular formula strings (CSV)
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C.; Papadopoulos, V.; Rupprecht, R.; Schumacher, M.; Groyer, G.
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protein 18 kDa. J. Neuroendocrinol. 2012, 24, 71−81.
AUTHOR INFORMATION
■
Corresponding Authors
*S.T.: phone, +39-0502219547; fax, +39-0502219605; e-mail,
sabrina.taliani@farm.unipi.it.
*S.C.: phone, +39-0823274789; fax, +39-0823274605; e-mail,
sandro.cosconati@unina2.it.
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Author Contributions
∥E.B. and A.B. contributed equally
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This study was financially supported by MIUR (Futuro in
Ricerca 2010, PRIN 2010-2011), University of Pisa.
K
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Journal of Medicinal Chemistry
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