Novel irreversible fluorescent probes targeting the 18 kDa translocator protein: Synthesis and biological characterization

Article abstract of DOI:10.1021/jm100100q

The 18 kDa translocator protein (TSPO) is a mitochondrial protein whose basal density is altered in several diseases, with the result that the evaluation of its expression levels by means of molecular imaging techniques represents a promising diagnostic approach. Experimental procedures using a labeled ligand often cause loss of the bound probe, and consequently high affinity ligands covalently binding the receptor protein are needed to overcome this problem. We have previously described a series of N,N-dialkyl-(2- phenylindol-3-yl)glyoxylamides as potent and selective TSPO ligands. Starting from these derivatives, we designed novel TSPO irreversible ligands bearing an electrophilic isothiocyanato group (7, 8), together with an irreversible NBD-fluorescent probe (18). The TSPO affinity of the new irreversible ligands was measured on rat tissue homogenates by [³H]Ro 5-4864 radiobinding kinetic assays, all compounds showing high affinities for the target protein. Further biological characterization of the fluorescent irreversible TSPO probe 18 was carried out by using fluorescent spectroscopy in human glioma cells.

Full text of DOI:10.1021/jm100100q

J. Med. Chem. 2010, 53, 4085–4093 4085
DOI: 10.1021/jm100100q
Novel Irreversible Fluorescent Probes Targeting the 18 kDa Translocator Protein: Synthesis and
Biological Characterization
Sabrina Taliani,*,†, Eleonora Da Pozzo,‡, Marusca Bellandi,† Sara Bendinelli,‡ Isabella Pugliesi,† Francesca Simorini,†
Concettina La Motta,† Silvia Salerno,† Anna Maria Marini,† Federico Da Settimo,† Barbara Cosimelli,§ Giovanni Greco,§
Ettore Novellino,§ and Claudia Martini‡
†
‡
Dipartimento di Scienze Farmaceutiche, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Psichiatria, Neurobiologia,
ꢀ
Farmacologia e Biotecnologie, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy, and Dipartimento di Chimica Farmaceutica e Tossicologica,
§
ꢀ
Universita di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy. These authors have equally contributed to the research.
ꢀ
Received January 25, 2010
The 18 kDa translocator protein (TSPO) is a mitochondrial protein whose basal density is altered in several
diseases, with the result that the evaluation of its expression levels by means of molecular imaging techniques
represents a promising diagnostic approach. Experimental procedures using a labeled ligand often cause loss
of the bound probe, and consequently high affinity ligands covalently binding the receptor protein are
needed to overcome this problem. We have previously described a series of N,N-dialkyl-(2-phenylindol-
3-yl)glyoxylamides as potent and selective TSPO ligands. Starting from these derivatives, we designed novel
TSPO irreversible ligands bearing an electrophilic isothiocyanato group (7, 8), together with an irreversible
NBD-fluorescent probe (18). The TSPO affinity of the new irreversible ligands was measured on rat tissue
homogenates by [3H]Ro 5-4864 radiobinding kinetic assays, all compounds showing high affinities
for the target protein. Further biological characterization of the fluorescent irreversible TSPO
probe 18 was carried out by using fluorescent spectroscopy in human glioma cells.
Introduction
changes in TSPO receptor levels have been found in anxiety
and mood disorders.12,17-19
The 18 kDa translocator protein (TSPOa) is an evolutio-
narily well-conserved protein consisting of 169 amino acids,1,2
which is mainly located at the contact sites between the outer
and inner mitochondrial membranes.3,4 TSPO is also ex-
pressed at low levels in other subcellular compartments such
as plasma membranes and the nuclear fraction of cells.5
Photolabeling studies indicated that this receptor is strictly
associated in a trimeric complex with the 32 kDa voltage-
dependent anion channel (VDAC) and the 30 kDa adenine
nucleotide translocase (ANT),6 thus forming the mitochon-
drial permeability transition pore (MPTP).
TSPO is involved in a variety of biological processes including
cholesterol transport, steroidogenesis, calcium homeostasis, lipid
metabolism, mitochondrial oxidation, cell growth and differen-
tiation, apoptosis induction, and regulation of immune func-
tions.7,8 Recent evidence suggests that on glial cells, TSPO may
regulate the biosynthesis of neurosteroids, leading to the hypo-
thesis of its potential role as a key determinant for the treatment
of neuropathological conditions.9-12
The evaluation of TSPO expression and distribution, by
means of specific molecular probes may thus represent a
promising diagnostic marker of the above-listed diseases.20,21
Typically, a probe is a derivative of a known ligand, designed
to allow visualization of the target protein while maintaining
the affinity and selectivity of the parent compound. A number
of radiolabeled ligands targeting TSPO have been developed
as powerful tools to image and measure the expression levels
of this protein in both humans and animals.22-28 On the other
hand, fluorescent ligands represent a safer, faster, and less
expensive alternative to radioligands for usage in biomedical
research.29-31 In this field, we have recently developed novel
highly potent and selective fluorescent probes targeting TSPO
with the general formula II, designed as derivatives of the
highly potent N,N-dialkyl-2-phenylindol-3-ylglyoxylamide
ligands I,32,33 featuring the fluorescent moiety linked to
the N-alkyl chain (see Chart 1).31 For these compounds, we
selected the well-known 7-nitrobenz-2-oxa-1,3-diazol-4-yl
(NBD) group as the fluorophore because its small size does
not generally affect affinity of the parent ligand.31
TSPO basal expression is up-regulated in a number of
human pathologies, including a variety of tumors and neuro-
pathologies, such as gliomas and neurodegenerative disorders
(Huntington’s and Alzheimer’s diseases), as well as various
forms of brain injury and inflammation.13-16 Furthermore,
Tissue experimental procedures using a labeled ligand often
cause the alteration of the chemical equilibrium and the
subsequent loss of the bound fluorescent ligand. To overcome
this problem, some high affinity ligands which bind covalently
to the receptor protein have been developed.34
*To whom all correspondence should be addressed. Phone: 39 050
2219547. Fax: 39 050 2219605. E-mail: taliani@farm.unipi.it.
aAbbreviations: TSPO, 18 kDa translocator protein; NBD, 7-nitro-
benz-2-oxa-1,3-diazol-4-yl; PBS, phosphate-buffered saline; SEM, stan-
dard error of mean.
The aim of this work was to design, synthesize, and bio-
logically characterize a series of 2-phenylindol-3-ylglyoxyl-
amides and to test their validity as new TSPO probes. All of
these compounds are characterized by the presence of a
r
2010 American Chemical Society
Published on Web 05/03/2010
pubs.acs.org/jmc

4086 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Taliani et al.
Chart 1. Structures of Known (I and II) and Novel (7, 8, and 18) TSPO Ligands
Scheme 1. Synthesis of Irreversible Ligands 7 and 8
chemoreactive isothiocyanate group, able to bind the recep-
tor protein irreversibly and covalently (compounds 7-8,
Chart 1). Moreover, compound 18 (Chart 1), featuring the
NBD-fluorescent moiety, was synthesized to develop an
irreversible fluorescent probe. The presence of these two
devices (a chemoreactive group and a fluorescent chromo-
phore) on a single molecule may offer a multiplicity of
advantages, both in protein purification/characterization
and in protein cell visualization/density determination. To
the best of our knowledge, no TSPO molecular probes with
these characteristics have been described to date.
For the rational design of the irreversible derivatives 7, 8,
and 18, we take into consideration our pharmacophore/
topological model (Chart 1)31-33 made up of three lipophilic
pockets L1, L3, and L4, occupied by the 2-phenyl group, and
the arylalkyl substituents on the amide nitrogen, respectively,
and an H-bond donor group interacting with the amide
carbonyl group. In this view, the position 5 of the indole
nucleus seemed suitable to support the chemoreactive group
without affecting the affinity or the selectivity for TSPO.
Among the possible chemoreactive moieties, the isothiocya-
nate function has proven to be extremely versatile as an
electrophilic moiety for irreversible ligands. It can easily be
synthesized from a primary amino group; furthermore, its
high reactivity toward amino and sulfhydryl groups, along
with its low reactivity toward water and other hydroxyl
functions, accounts for its successful applications in receptor
studies.35-40
palladium to yield the corresponding amines 5 and 6, which
were then reacted with thiophosgene in a biphasic CH2Cl2/
aqueous NaHCO3 system to give the desired isothiocyanato
derivatives 7 and 8.
In this paper, we describe the synthesis, the spectro-
scopic, and the biological characterization of novel irre-
versible 2-phenylindol-3-ylglyoxylamide TSPO ligands 7,
8, and 18.
The synthesis of the irreversible fluorescent probe 18 was
achieved by the convergent procedure outlined in Schemes 2
and 3. The first key intermediate 5-(N-BOC-amino)-2-phenyl-
indol-3-ylgyloxyl chloride 12 was obtained in three steps
starting from the 5-nitro-2-phenylindole 9,33 (Scheme 2) which
was first catalytically hydrogenated toyield the corresponding
amine 10, subsequently protected with the BOC group to give
11, and then reacted with oxalyl chloride. The N-BOC-N0-(7-
nitrobenz-2-oxa-1,3-diazol-4-yl)hexamethylendiamine hy-
drochloride 15, prepared as described in our previous work,31
was condensed with compound 12 in anhydrous dimethylfor-
mamide in the presence of triethylamine at room temperature,
Chemistry
The target N,N-dialkyl-5-isothiocyanato-2-phenylindol-3-
ylglyoxylamides 7 and 8 were synthesized through the 5-nitro-
2-phenylindol-3-ylglyoxylamides 3 and 4, which in turn were
obtained following the experimental procedures described in
our previous work (Scheme 1).33 The 5-nitro-2-phenylindol-3-
ylglyoxylamides 3 and 4 were catalytically hydrogenated over

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4087
Scheme 2. Synthesis of Intermediate Glyoxylyl Chloride 12
Figure 1. Absorption spectra of compound 18 at 10 μM in solu-
tions varying from aqueous to 60% dioxane-water (v/v) PBS.
Scheme 3. Synthesis of Irreversible and Fluorescent Ligand 18
Figure 2. The influence of the polarity of the medium on the
fluorescence of compound 18 was investigated by the addition of
dioxane (indicated in % v/v) in PBS measured at a λmax of 470 nm.
environment. Product 18, dissolved in dimethyl sulfoxide
(DMSO), was diluted at a final concentration of 10 μM in
different assay solutions from aqueous to 60% v/v dioxane-
water phosphate-buffered saline (PBS).31 The percentage of
DMSO did not exceed 1% of the final assay volume solution.
Figure 1 shows the absorption spectra of compound 18. In
an aqueous solution, the spectra were characterized by two
maximal absorptions at 331 and 482 nm, showing extinction
coefficients ε = 7963 M-1 cm-1 and ε = 4078 M-1 cm-1
,
respectively. When compound 18 was dissolved in PBS solu-
tion containing 20% of dioxane, the spectrum proved to be
practically unchanged, with the exception of a slight decrease
in the extinction coefficient at the absorption maximum at the
lower wavelength from ε = 7963 M-1 cm-1 to ε = 6523 M-1
cm-1. The further increase in hydrophobicity of the medium
caused a small hypsochromic shift of both maximal absorp-
tions, accompanied by a significant hyperchromic effect (40%
dioxane-water (v/v) PBS: λmax = 328 nm ε = 11036 M-1
cm-1, λmax = 478 nm ε = 6415 M-1 cm-1; 60% dioxane-
water (v/v) PBS: λmax = 327nm ε = 11832 M-1 cm-1, λmax
474 nm ε = 6181 M-1 cm-1).
=
Figure 2 shows the emission spectra of compound 18. As
expected, the decrease in polarity of the environment (from
aqueous buffer to 60% dioxane in PBS) induced an approxi-
mately 30-fold increase in quantum yield. This phenomenon
was accompanied by a slight blue-shift of the emission maxi-
mum from 542 to 538 nm.
yielding the fluorescent compound 16 (Scheme 3). Simple
deprotection of the BOC group with trifluoroacetic acid in
anhydrous CH2Cl2 gave the 5-amino derivative 17, which was
then reacted with thiophosgene, yielding the desired irrever-
sible fluorescent compound 18 (Scheme 3).
Spectroscopic Properties of Irreversible Fluorescent Ligand 18
Biological Studies
The ultraviolet absorption and the emission spectra of
compound 18 were measured to investigate the spectroscopic
properties of the ligand and how these were affected by the
The biological characterization of the newly developed
irreversible ligands 7, 8, and 18 started with the evaluation
of their equilibrium binding parameters, followed by the

4088 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Taliani et al.
Table 1. Values of IC50 and Ki for Compounds 7, 8, and 18 Obtained
from Three Independent Equilibrium Binding Assaysa
compd
IC50 (nM)
Ki (nM)
7
8
45.6 ( 0.4
59.7 ( 0.6
42.2 ( 0.4
37.9 ( 0.4
49.5 ( 0.5
37.6 ( 0.4
23 ( 3.1
18
Ro 5-4864
a The IC50 values were converted to an absolute inhibition constant
Ki, using the Cheng-Prusoff equation.45 Data are shown as values (
SEM.
Figure 3. Displacement of [3H]Ro 5-4864 by compound 18 in rat
kidney mitochondria membranes. Bound radioactivity is expressed
as a percentage of specific binding in the presence of competitor
molecule. Points represent the mean values ( SEM of triplicate
determinations pooled from three independent experiments.
investigation of the potential irreversibility of their binding to
the protein.
The affinity of the newly synthesized indolylglyoxylamides
for TSPO was determined in rat kidney membranes by
competition experiments against [3H]Ro 5-4864.41 It has been
reported that the interaction between TSPO and irreversible
ligands bearing an isothiocyanato group probably takes place
in accordance with the following equation (eq 1):42,43
Figure 4. Time course of compound 18 binding to receptor. The
semilogarithmic representation of the decrease in [3H]Ro 5-4864
binding to TSPO by pretreatment (0, 10, 30, 60, 90, and 180 min)
with various concentrations of compound 18 is shown. Points
represent the mean values ( SEM of duplicate determinations
pooled from three independent experiments.
Kd
/
K2
f
D þ R
DR
s
DR
ð1Þ
irrev
in literature.44,47 Results showed that mitochondrial mem-
brane pretreatment with newly synthesized TSPO ligands
inhibited the [3H]Ro 5-4864 binding to TSPO. This inhibition
was time- and ligand concentration-dependent, as exemplified
in Figure 4, which refers to compound 18 (see Supporting
Information for derivatives 7 and 8), where B is binding after
pretreatment of membranes with different concentrations of
TSPO ligands for several times, and B0 is control binding.
When the B and B0 ratio was plotted on a logarithmic scale
versus incubation times, the decrease in the ln B/B0 values
appeared to be linearly related to the preincubation time.
Therefore, inactivation proceeded according to apparent first-
The binding process begins with a first reversible step,
followed by the formation of an irreversible molecular com-
plex. Kinetic and mathematical analysis of this two-step
reaction were developed in order to evaluate the order rate
and kinetic constants (Kd and K2) of our TSPO ligands.44
Moreover, the irreversible fluorescent probe18 was assayed
in experiments aimed at labeling TSPO in U87MG human
glioma cells by means of fluorescence spectroscopy.
Results
As a first step, the equilibrium binding parameters (IC50
and Ki values) of the test compounds were derived by com-
petition experiments against [3H]Ro 5-4864 in rat kidney
membranes. Ro 5-4864 was used as the reference standard,
showing a Ki value of 23 nM, in accordance with literature
data.45 The curve of [3H]Ro 5-4864 displacement by the
irreversible fluorescent ligand 18 is shown in Figure 3,
while the analogous graphs for compounds 7 and 8 are
included in the Supporting Information. Binding data for
the three ligands 7, 8, and 18 are listed in Table 1; all
compounds showed affinity values in the nanomolar range,
comparable to that of the reference standard Ro 5-4864
(Ki from 37.6 to 49.5 nM).
To investigate the potential covalent binding of compounds
7, 8, and 18 to TSPO, the samples derived from competition
radiobinding assays were diluted more then 10 times with
fresh buffer at the end of the incubation time; data did not
markedly change with respect to those obtained in the un-
diluted samples (data not shown). This observation indicates
that the TSPO-ligand interaction is irreversible, and it im-
mediately disqualifies the application of the Cheng-Prusoff
equation46 in binding parameter analysis.
order kinetics. The apparent first-order rate constant (Kobs
was defined by eq 2:
)
B
In
¼ - Kobs ꢀ t
ð2Þ
B0
Thus, a mathematical analysis was conducted to calculate
the dissociation constant (Kd) of the reversible receptor-
ligand complex and the pseudo first-order rate constant (K2)
for the irreversible interaction between the receptor and the
irreversible ligand, as previously reported by Liu-Chen and
colleagues.44 The mathematical equation describing the pro-
cess is shown below (eq 3):
1
Kd
1
1
¼
þ
ð3Þ
3
Kobs
K2 ½Dꢁ K2
Kobs is the observed first-order rate constant for irreversible
binding, and it is derived from the slope of the different straight
lines shown in Figure 4. The plotting of 1/Kobs versus 1/[D]
(where D is the ligand concentration) gave a straight line that
intercepted the ordinate and abscissa axes at 1/K2 and -1/Kd,
respectively. The slope of the line was the ratio Kd/K2 (Figure 5).
With the aim of evaluating the rate order of the binding
reaction, a kinetic analysis method was developed, as reported

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4089
Analogous graphs for compounds 7 and 8 are supplied in the
Supporting Information. K2 and Kd values of each compound
are listed in Table 2. Interestingly, all the Kd values are in the
nanomolar range (Kd from 3.17 to 8.31 nM), confirming an
effective interaction with the receptor.
To estimate the ability of compound 18 to permeate cell
membranes, a number of its physicochemical properties were
calculated and reported in Table 3.48 Values obtained were
consistent with an adequate distribution of 18 into the cell.
The ability of the probe to specifically and irreversibly label
TSPO was evaluated in live cells of human glioma, using
fluorescence spectroscopy. For this purpose, U87MG glioma
cells were incubated with different concentrations of 18 (100,
250, and 500 nM) after pretreatment with buffer (control) or
with 10 μM Ro 5-4864. Incubation was performed at 90 min,
a time in which the irreversible bound is formed (see eqs 2
and 3), and at a shorter time (20 min). As expected, at the
shortest time, the detected values of fluorescence intensity were
smaller than the mean values obtained after 90 min (panels A
and B of Figure 6, respectively). Interestingly, at 20 min time
incubation, Ro 5-4864 was able to displace compound 18 in a
similar extent for all the concentrations assayed, demonstrat-
ing the specificity of ligand 18-TSPO binding. Conversely,
in the experiments performed at the longest time, Ro 5-4864
showed to be unable to displace 18, demonstrating the irrever-
sibility of the bond.
Figure 5. Calculation of K2 and Kd for ligand 18. On the basis of
data obtained by three different experiments carried out in dupli-
cate, the values were calculated in accordance with eq 2. Data are
shown as mean values ( SEM.
Table 2. Values of K2 and Kd for the Irreversible Binding of Compounds
7, 8, and 18 to TSPOa
compd
K2 (nM)
Kd (nM)
7
0.003 ( 0.001
0.015 ( 0.002
0.012 ( 0.002
3.17 ( 0.31
5.07 ( 0.61
8.31 ( 0.89
8
18
a Data, obtained from three independent kinetic assays, are shown as
Conclusions
values ( SEM.
We designed novel irreversible TSPO ligands featuring
the 2-phenylindol-3-ylglyoxylamide scaffold. The binding of
these compounds to the receptor was investigated in detail,
characterizing both order rate and kinetic constants. Experi-
mental data showed that all the new derivatives effectively
bind the receptor by means of an irreversible covalent inter-
action. Fluorescence spectroscopy experiments performed
on derivative 18, featuring an NBD fluorophore group,
revealed its ability to specifically and irreversibly label TSPO
Table 3. Physicochemical Predicted Properties of Compound 1848
bond
compd ClogPa ASA (A) PSA (A) Nd count rbe MWf(Da)
b
c
ꢁ
˚
ꢁ
˚
18
4.485
847.530
170.945 67
46
13
583.620
a Calculated n-octanol/water partition coefficient. b Total solvent
accessible surface area. c Polar surface area. d Number of atoms. e Num-
ber of rotatable bonds. f Molecular weight.
Figure 6. Human glioma cell staining with the irreversibile fluorescent TSPO ligand 18. The cells were incubated with compound 18 for 20 min
(A) and 90 min (B), in the absence or presence of 10 μM Ro 5-4864 (gray bars). Data were obtained from three independent assays and are
shown as mean values of fluorescence intensity ( SEM.

4090 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Taliani et al.
in live U87MG glioma cells. All these novel irreversible TSPO
ligands represent useful tools in the investigation of the
physiological role of TSPO and in the detection of its expres-
sion levels.
In particular, the fluorescent probe 18 may be employed as
diagnostic marker to evaluate the TSPO expression in peri-
pheral cells, post-mortem, and biopsy tissues from patients
affected by diseases in which the TSPO density is altered.
Actually, irreversible probes offer the advantage of a lasting
detection in visualization techniques that generally require
multiple washes to remove the nonspecific signal, steps in
which reversible probes may be lost.
NH). Anal. Calcd for C28H37N3O2 (%): C, 75.13; H, 8.33; N,
9.39. Found: C, 74.98; H, 8.27; N, 9.26.
General Procedure for the Synthesis of N,N-Dialkyl-[5-iso-
thiocyanato-(2-phenylindol-3-yl)glyoxylamide Derivatives 7 and 8.
A stirred solution of 5 and 6 (0.52 mmol) in 10 mL of NaHCO3
6% was added to 10 mL of CH2Cl2. After 20 min of vigorous
stirring at 0 °C, thiophosgene (0.03 mL, 0.52 mmol) was added
dropwise. The reaction mixture was left under stirring for 24 h at
room temperature (TLC analysis), and the organic solvent was
removed under reduced pressure and the crude residue was
purified by washing with cold ether to afford derivatives 7 and 8.
N,N-Dipropyl-[5-isothiocyanato-2-phenylindol-3-yl)glyoxyl-
amide 7. Yield 71%; mp 77-79 °C. IR (nujol, cm-1): 3187,
2095, 2047, 1617, 1211, 1088. 1H NMR (DMSO-d6, ppm):
0.68-0.79 (m, 6H, 2CH3), 1.18-1.29, (m, 2H, CH2CH2-
CH3), 1.42-1.52 (m, 2H, CH2CH2CH3), 2.91 (t, 2H, J = 7.6
Hz, NCH2), 3.04 (t, 2H, J = 7.6 Hz, NCH2), 7.34 (d, 1H, J =
8.4 Hz, Ar-H), 7.53-7.57 (m, 6H, Ar-H), 8.33 (s, 1H, Ar-H),
12.79 (s exch, 1H, NH). MS m/e %: 405 (Mþ, 5), 277 (10), 129
(15), 83 (100). Anal. Calcd for C23H23N3O2S (%): C, 68.12; H,
5.72; N, 10.36. Found: C, 68.29; H, 5.65; N, 10.47.
Complementary investigations using irreversible and rever-
sible fluorescent TSPO ligands, in in vitro and in vivo studies,
respectively, may offer a useful platform for the development
of new diagnostic approaches.
Experimental Section
Chemistry. Melting points were determined using a Reichert
N,N-Dihexyl-[5-isothiocyanato-2-phenylindol-3-yl)glyoxylamide 8.
Yield: 80% oil. IR (nujol, cm-1): 3180, 2116, 2061, 1617, 1258,
1091, 746. 1H NMR (DMSO-d6, ppm): 0.73-0.90 (m; 6H,
2CH3), 1.09-1.43 (m, 16H, 8CH2), 2.97-3.08 (m, 4H, 2NCH2),
7.35 (d, 1H, J = 8.4 Hz, Ar-H), 7.53-7.60 (m, 6H, Ar-H), 8.00
(s, 1H, Ar-H), 12.78 (s exch, 1H, NH). MS m/e %: 489, (Mþ, 5),
277 (100), 85 (15). Anal. Calcd for C29H35N3O2S (%): C, 71.13; H,
7.20; N, 8.58. Found: C, 70.75; H, 7.03; N, 8.63.
5-Amino-2-phenylindole 10. A mixture of 5-nitro-2-phenyl-
indole 9 (0.543 g, 4.0 mmol) and 10% Pd/C (0.28 g) in 150 mL of
absolute ethanol was hydrogenated at room temperature and
pressure. Once hydrogen absorption ceased (3-4 h), the catalyst
was filtered off and the solvent was evaporated to afford a violet-
colored amorphous solid. The residue was purified by crystal-
lization from toluene to afford 0.666 g (80%) of 10, mp 223-
225 °C. IR (nujol, cm-1): 3347, 3413, 1631, 1537, 1071, 757. 1H
NMR (DMSO-d6, ppm): 4.87 (s exch, 2H, NH2), 6.52 (d, 1H,
J = 8.2 Hz, Ar-H), 6.63 (s, 1H, Ar-H), 6.70 (s, 1H, Ar-H), 7.10
(d, 1H, J = 8.4 Hz, Ar-H), 7.22-7.29 (m, 1H, Ar-H), 7.38-7.45
(m, 2H, Ar-H), 7.78 (d, 2H, J = 7.4 Hz, Ar-H), 11.06 (bs exch,
1H, NH). Anal. Calcd for C14H12N2 (%): C, 80.74; H, 5.81; N,
13.45. Found: C, 80.60; H, 5.83; N, 13.26.
5-N-Boc-amino-2-phenylindole 11. Di-tert-butyldicarbonate
(0.348 g, 1.6 mmol) was added to a stirred solution, cooled at
0 °C, of 5-amino-2-phenylindole 10 (0.330 g, 1.6 mmol) in a
mixture of acetone (6 mL) and H2O (3 mL). The reaction
mixture was stirred at room temperature for 24 h (TLC ana-
lysis). The solvent was reduced to half volume under reduced
pressure, and the precipitate that formed was collected and
purified by crystallization from toluene (67%); mp 165-
167 °C. IR (nujol, cm-1): 3437, 3326, 3289, 1686, 1531, 1050,
754. 1H NMR (DMSO-d6, ppm): 1.48 (s, 9H, C(CH3)3), 6.81 (s,
1H, Ar-H), 7.10-7.15 (m, 1H, Ar-H), 7.23-7.32 (m, 2H, Ar-H),
7.40-7.47 (m, 2H, Ar-H), 7.65 (s, 1H, Ar-H), 7.80-7.85 (m, 2H,
Ar-H), 9.05 (s exch, 1H, CONH), 11.36 (s exch, 1H, NH). Anal.
Calcd for C19H20N2O2 (%): C, 74.00; H, 6.54; N, 9.08. Found:
C, 74.20; H, 6.63; N, 9.26.
5-N-Boc-amino-2-phenylindol-3-ylglyoxyl chloride 12. Oxalyl
chloride (0.07 mL, 0.8 mmol), in 2 mL of anhydrous THF, was
added dropwise over 10-15 min to a cooled (0 °C) stirred
solution of 11 (0.184 g, 0.6 mmol) in 10 mL of freshly distilled
THF. The mixture was maintained at room temperature for 2 h.
The organic solvent was removed under reduced pressure, and
the residue was washed with three portions of dry distilled THF.
The glyoxyl chloride 12 obtained was particularly unstable, and
thus it was utilized in the following reaction without any further
purification.
€
Kofler hot-stage apparatus and are uncorrected. Infrared
spectra were recorded with a Nicolet/Avatar FT-IR spectro-
meter in Nujol mulls. Routine nuclear magnetic resonance
spectra were recorded in DMSO-d6 solution on a Varian
Gemini 200 spectrometer operating at 200 MHz. Mass spectra
were obtained on a ThermoQuest Finnigam GCQplus spec-
trometer using a direct injection probe and an electron
beam energy of 70 eV. 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. Combustion analyses on target compounds
were performed by our Analytical Laboratory in Pisa. All
compounds showed g95% purity. Ultraviolet absorption
spectra were measured with a Perkin-Elmer UV/Vis spectro-
photometer Lambda 5. Fluorescence spectra were recorded
with a Gemini XS Molecular Device Spectramax fluorescence
spectrophotometer.
5-Nitro-(2-phenylindol-3-yl)glyoxyl chloride 2,33 N,N-dialkyl-
[5-nitro-(2-phenylindol-3-yl)glyoxyl]amide derivatives 3 and 4,33
5-nitro-2-phenylindole 9,33 and N0-(7-nitrobenz-2-oxa-1,3-diazol-
4-yl)-1,6-diaminohexane hydrochloride 1531 were prepared in acc-
ordance with our previous works.
General Procedure for the Synthesis of N,N-Dialkyl-[5-amino-
(2-phenylindol-3-yl)glyoxyl]amide Derivatives 5 and 6. A mixture
of the appropriate N,N-dialkyl-[5-nitro-(2-phenylindol-3-yl)-
glyoxyl]amide derivative 3 and 4 (0.73 mmol) and 10% Pd/C
(0.05 g) in 150 mL of absolute ethanol was hydrogenated at room
temperature and pressure. Once hydrogen absorption ceased
(4-6 h), the catalyst was filtered off and the solution was
evaporated to dryness at reduced pressure. The semisolid pro-
ducts 5 and 6 obtained were utilized in the following reaction
without any further purification.
N,N-Dipropyl-[5-amino-(2-phenylindol-3-yl)glyoxyl]amide 5.
Yield 91%. IR (nujol, cm-1): 3365, 3221, 1627, 1214, 1088, 1043.
1H NMR (DMSO-d6, ppm): 0.64-0.74 (m, 6H, 2CH3), 1.08-1.20
(m, 4H, CH2CH2CH3), 2.82 (t, 2H, J = 8.0 Hz, NCH2), 2.98 (t,
2H, J = 8.0 Hz, NCH2), 5.31 (bs exch, 2H, NH2), 6.63 (d, 1H, J =
9.0 Hz, Ar-H), 7.15 (d, 1H, J = 7.6 Hz, Ar-H), 7.34 (s, 1H, Ar-H);
7.44-7.48 (m, 5H, Ar-H), 12.04 (s exch, 1H, NH). Anal. Calcd for
C22H25N3O2 (%): C, 72.70; H, 6.93; N, 11.56. Found: C, 72.87; H,
6.83; N, 11.65.
N,N-Dihexyl-[5-amino-(2-phenylindol-3-yl)glyoxyl]amide 6.
Yield 89%. IR (nujol, cm-1): 3365, 3290, 1627, 1600, 1211, 1094.
1H NMR (DMSO-d6, ppm): 0.74-0.90 (m; 6H, 2CH3),
1.10-1.43 (m, 16H, 8CH2), 2.87 (t, 2H, J = 7.2 Hz, NCH2),
3.02 (t, 2H, J = 7.2 Hz, NCH2), 4.92 (bs exch, 2H, NH2), 6.61
(d, 1H, J = 6.6 Hz, Ar-H), 7.14 (d, 1H, J = 8.4 Hz, Ar-H), 7.32
(s, 1H, Ar-H), 7.44-7.48 (m, 5H, Ar-H), 12.00 (bs exch, 1H,
N-[5-(N-Boc-amino)-2-phenylindol-3-ylglyoxyl]-N’-(7-nitrobenz-
2-oxa-1,3-diazol-4-yl)-1,6-diaminohexane 16. A solution of amine

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4091
15 (0.084 g, 0.3 mmol) in 2-3 mL of dry DMF was added dropwise
to a stirred solution, cooled at 0 °C, of glyoxyl chloride 12 (0.199 g,
0.5 mmol) in 3 mL of the same solvent, followed by addition of a
solution of triethylamine (0.16 mL, 1.2 mmol). The reaction
mixture was allowed to warm to room temperature, stirred for
12 h (TLC analysis), and then filtered to eliminate the triethylamine
hydrochloride formed. The organic solvent was evaporated to
dryness, and the residue was washed with water. The suspension
was filtered, and the brown-colored amorphous solid obtained was
washed with cold diethyl ether to afford 0.253 g (80%) of 16; mp
153-155 °C. IR (nujol, cm-1): 3324, 1696, 1580,1290, 1153, 897,
726. 1H NMR (DMSO-d6, ppm): 1.06-1.80 (m, 19H, CH2(CH2)4-
CH2, C(CH3)3, CONHCH2), 2.68-2.74 (m, 2H, CH2NH), 6.42
(d, 1H, J = 9.2 Hz, 50-H), 7.34-7.53 (m, 7H, Ar-H), 8.28 (s, 1H,
Ar-H), 8.42 (bs exch, 1H, COCONH), 8.51 (d, 1H, J = 9.0 Hz,
60-H), 9.28(s, 1H, ArNHCO), 9.57 (bs exch, 1H, CH2NH), 12.21(s
exch, 1H, NH). Anal. Calcd for C28H37N3O2 (%): C, 61.77; H,
5.50; N, 15.28. Found: C, 61.90; H, 5.63; N, 15.16.
Time Course of Binding of the New Ligands to the TSPO
Receptor: Irreversible Binding. The binding studies were carried
out essentially as previously described.47 Briefly, rat kidney mem-
branes were incubated with each compound (concentration range:
1 nM to 1 μM) for different times (0, 10, 30, 60, 90, and 180 min) in
binding assay buffer. Incubations were stopped by the addition of
1 mL of ice-cold 50 mM Tris/HCl buffer (pH 7.4), and then the
samples were centrifuged for 15 min at 13000g at 4 °C. Pellets
were washed twice with cold binding buffer. Then, samples were
processed for determination of protein content. [3H]Ro 5-4864
binding assays were performed in a final volume of 500 μL of
binding assay buffer containing membranes (65 μg of protein/
tube), as mentioned above.
Fluorescent Labeling of Human Glioma Cells. U87MG cells
were cultured in 96-well plates (4 ꢀ 104 cells/well) in RPMI 1640
complete medium. After 24 h, the cells were incubated with
different concentrations of 18 (100, 250, and 500 nM) in cell
culture medium, for 20 and 90 min, under 5% CO2 at 37 °C. In
parallel, some samples were incubated with Ro 5-4864 (10 μM)
prior to add compound 18. To remove the excess of unbound
compound, the cells were washed four times with cell medium
with cysteine, a molecule able to react with the isothiocyanate
group. After washing, PBS was added to the cells and the
fluorescence intensity (Ex: 485 nm; Em: 535 nm) from each
sample was measured by Victor Wallac 2 (Perkin-Elmer, Boston,
MA). All data are presented as means ( SEM, derived from at
least three independent experiments done in duplicate.
N-(5-Amino-2-phenylindol-3-ylglyoxyl)-N’-(7-nitrobenz-2-oxa-
1,3-diazol-4-yl)-1,6-diaminohexane 17. Trifluoroacetic acid (2 mL,
20 mmol) was added dropwise to a stirred solution of derivative 16
(0.192 g, 0.3 mmol) in 10 mL of anydrous CH2Cl2, cooled at 0 °C.
The mixture was stirred at room temperature for 12 h (TLC
analysis). Then the reaction mixture was concentrated under
reduced pressure. The resulting solid residue was taken up with
water and cooled in an ice bath. Upon the addition of a 5%
solution of NaHCO3, a precipitate formed which was filtered to
provide 0.148 g (91%) of 17; mp 195-197 °C. IR (nujol, cm-1):
Data Analyses. Data were analyzed by use of the GraphPad
Prism software (GraphPad Software, version 4.0; San Diego,
CA). Statistical analyses were performed by one-way ANOVA
(with post hoc Bonferroni test).
1
3338, 3242, 1621, 1587, 1135, 801, 723. H NMR (DMSO-d6,
ppm): 1.04-1.36 (m, 8H, CH2(CH2)4CH2), 1.60-1.69 (m, 2H,
CONHCH2), 2.68-2.74 (m, 2H, CH2NH), 6.42 (d, 1H, J = 9.6
Hz, 50-H), 6.76-6.80 (m, 1H, Ar-H), 7.25-7.55 (m, 9H, Ar-H,
NH2), 8.36 (bs exch, 1H, COCONH), 8.52 (d, 1H, J = 9.8 Hz,
60-H), 9.58 (bs exch, 1H, CH2NH), 12.11 (s exch, 1H, NH). Anal.
Calcd for C28H27N7O5 (%): C, 62.10; H, 5.03; N, 18.10. Found: C,
62.27; H, 4.83; N, 18.19.
Acknowledgment. This work was supported by the Univer-
sity of Pisa (grant “Progetto d’Ateneo 2007”) and by the Tuscan
Cancer Institute (Istituto Toscano Tumori, ITT, grant 2007).
N-(5-Isothiocyanato-2-phenylindol-3-ylglyoxyl)-N’-(7-nitrobenz-
2-oxa-1,3-diazol-4-yl)-1,6-diaminohexane 18. A stirred solution of
17 (0.162 g, 0.3 mmol) in 20 mL of 6% NaHCO3 was added to
20 mL of CH2Cl2. After 20 min of vigorous stirring at 0 °C,
thiophosgene (0.1 mL, 1 mmol) was added dropwise. The reaction
mixture was stirred for 4 h. A precipitate formed which was
collected by vacuum filtration and washedwithcoldether to afford
0.117 g (67%) of 18; mp 152-154 °C (dec). IR (nujol, cm-1): 3345,
Supporting Information Available: Displacement of [3H]Ro
5-4864 by compounds 7 and 8 in rat kidney mitochondria
membranes, Time course of compounds’ binding to the recep-
tor, and the calculation of K2 and Kd for ligands 7 and 8. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1
References
2123, 2041, 1648, 1614, 1586, 1177, 1105, 811, 719. H NMR
(DMSO-d6, ppm): 1.15-1.36 (m, 8H, CH2(CH2)4CH3), 1.60-1.69
(m, 2H, CONHCH2), 2.72-2.80 (m, 2H, CH2NH), 6.41 (d, 1H,
J = 7.8 Hz, 50-H), 7.31-7.56 (m, 8H, Ar-H,CONH), 8.01 (s, 1H,
Ar-H), 8.50 (d, 1H, J=7.2 Hz, 60-H), 9.57 (bs exch, 1H, CH2NH),
12.68 (s exch, 1H, NH). MS m/e %: 589, (Mþ, 5), 277 (5), 83 (100).
Anal. Calcd for C29H25N7O5S (%): C, 59.68; H, 4.32; N, 16.80.
Found: C, 59.81; H, 4.13; N, 17.06.
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