New fluorescent 2-phenylindolglyoxylamide derivatives as probes targeting the peripheral-type benzodiazepine receptor: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1021/jm061137o

Fluorescent ligands for the peripheral-type benzodiazepine receptor (PBR) featuring the 7-nitrobenz-2-oxa-1,3-diazol-4-yl moiety were synthesized, based on N,N-dialkyl-2-phenylindol-3-ylglyoxylamides, a potent, selective class of PBR ligands previously de

Full text of DOI:10.1021/jm061137o

404
J. Med. Chem. 2007, 50, 404-407
New Fluorescent 2-Phenylindolglyoxylamide Derivatives as Probes Targeting the
Peripheral-Type Benzodiazepine Receptor: Design, Synthesis, and Biological Evaluation
Sabrina Taliani,*,† Francesca Simorini,† Valentina Sergianni,† Concettina La Motta,† Federico Da Settimo,† Barbara Cosimelli,‡
Enrico Abignente,‡ Giovanni Greco,‡ Ettore Novellino,‡ Leonardo Rossi,§ Vittorio Gremigni,§ Francesca Spinetti,|
Beatrice Chelli,| and Claudia Martini|
Dipartimento di Scienze Farmaceutiche, UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Chimica Farmaceutica e
Tossicologica, UniVersita` di Napoli “Federico II”, Via D. Montesano, 49, 80131 Napoli, Italy, Dipartimento di Morfologia Umana e Biologia
Applicata, UniVersita` di Pisa, Via Roma 55, 56126 Pisa, Italy, and Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie,
UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
ReceiVed September 29, 2006
Fluorescent ligands for the peripheral-type benzodiazepine receptor (PBR) featuring the 7-nitrobenz-2-oxa-
1,3-diazol-4-yl moiety were synthesized, based on N,N-dialkyl-2-phenylindol-3-ylglyoxylamides, a potent,
selective class of PBR ligands previously described by us. All the new ligands are moderately to highly
potent at the PBR, with a complete selectivity over the central benzodiazepine receptor. Results from
fluorescence microscopy showed that these probes specifically labeled the PBR at the mitochondrial level
in C6 glioma cells.
Introduction
recognized by the ligands. Fluorescent agents with high
specificity and attractive spectroscopic properties are therefore
needed in the field of biomedical research.9
The peripheral-type benzodiazepine receptor (PBR) is an 18
KDa protein located on the outer mitochondrial membrane,1
which is directly or indirectly implicated in numerous cellular
functions, including calcium homeostasis, cell proliferation,
regulation of mitochondrial permeability transition, apoptosis,
steroid biosynthesis, and immunomodulation.2,3 Although the
acronym PBR is widely accepted in the scientific community,
In this paper, we report the synthesis, the physical charac-
terization, and the biological application of new PBR fluorescent
probes with the general formula II, designed on the basis of
N,N-dialkyl-2-phenylindol-3-ylglyoxylamides I,10 bearing the
fluorescent moiety linked to the N-alkyl chain (Figure 1).
According to our pharmacophore/topological model (Figure 1),10
the fluorescent group of derivatives II should interact with one
of the lipophilic pockets termed L3 and L4 in the receptor
binding cleft, thus maintaining the high affinity of the parent
ligand I. The length of the spacer alkyl chain was modified to
introduce some flexibility that would favor self-adaptation of
the ligands into the receptor binding site.
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. Moreover,
NBD-containing compounds typically exhibit a low quantum
yield in an aqueous solution, but they become highly fluorescent
in nonpolar solvents or when bound to membranes or to
hydrophobic clefts in proteins.
4
Papadopoulos et al. have proposed the revised nomenclature
“translocator protein (18 KDa)” or “TSPO” for this protein,
which represents more accurately its structure, subcellular role,
and putative molecular functions.
Clinical investigations have revealed that PBR basal expres-
sion is up-regulated in a number of human pathologies, including
a variety of tumors, and PBR expression appears to be related
to the tumor malignancy grade. Furthermore, significantly
enhanced PBR expression has been observed in neurodegen-
erative diseases (Huntington’s and Alzheimer’s diseases and
multiple sclerosis), as well as in various forms of brain injury
and inflammation.3 In vivo studies have shown that, following
neuronal injury, increased PBR expression is primarily localized
on activated microglial cells, whereas undetectable expression
has been reported in resting microglia, suggesting that the up-
regulation is a consequence of cell activation.5 All these findings
have stimulated the development of new radiolabeled ligands
targeting PBR as powerful tools to image and measure the
expression level of this protein in both humans and animals.5-8
Fluorescently labeled ligands represent a safer, faster, and
less-expensive alternative to radioligands in probing the ligand-
receptor complex. As a further advantage, fluorescent probes
can be displaced from their binding sites of a target protein by
nonfluorescent ligands, allowing the identification of the sites
Chemistry
The procedure for the obtainment of derivatives 1-6 repre-
sents an improved modification of those used by Kozikowski
et al.11 and Chen et al.12 for the preparation of analogous PBR
fluorescent probes (Scheme 1). The appropriate mono N-Boc-
protected diamines were condensed with NBD-Cl to obtain
compounds 7-9. Deprotection of the Boc group furnished the
hydrochloride salts of amino derivatives 10-12, which were
directly condensed with 2-phenylindolylglyoxylyl chlorides 13
and 14,10 yielding the desired fluorescent compounds 1-6
(Supporting Information).
* To whom all correspondence should be addressed. Tel: 39 050
2219547. Fax: 39 050 2219605. E-mail: taliani@farm.unipi.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡ Dipartimento di Chimica Farmaceutica e Tossicologica, Universita`
“Federico II” di Napoli.
Biological Results
The affinity of fluorescent probes 1-6 at the PBR was
determined by competition experiments against [3H]PK 11195
performed on rat kidney mitochondrial membranes (Table 1).
All the newly synthesized ligands are moderately to highly
§ Dipartimento di Morfologia Umana e Biologia Applicata, Universita`
di Pisa.
| Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
nologie, Universita` di Pisa.
10.1021/jm061137o CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/22/2006

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 405
Table 1. Receptor Binding Affinity of Compounds 1-6 for PBR
Figure 1. 2-Phenylindolglyoxylamide derivatives I10 in the pharma-
cophore/topological model and general formula of fluorescent probes
no.
n
X
Kia (nM)
1
2
3
4
2
2
4
4
6
6
H
F
H
F
H
F
994 ( 95
835 ( 80
42 ( 4.00
23 ( 2.00
17 ( 1.50
12 ( 1.00
0.37 ( 0.13
9.3 ( 0.50
23 ( 3.10
0.5-7
II.
Scheme 1. Synthesis of Fluorescent Probes 1-6
5
6
Ipb
PK 11195
Ro 5-4864
Alpidem
a The concentration of tested compounds that inhibited [3H]PK 11195
binding at rat kidney mitochondrial membranes (IC50) by 50% was
determined with six concentrations of the displacers, each performed in
triplicate. Ki values are the means ( SEM of three determinations. b Data
taken from ref 10.
Finally, the PBR/central benzodiazepine receptor (BzR)
selectivity of our ligands was evaluated by binding studies
carried out on rat brain membranes using [3H]flumazenil as the
radioligand. All compounds showed a high selectivity for the
PBR over the BzR, as they inhibited the binding of [3H]-
flumazenil by less than 10% at the fixed 10 µM concentration
(data not reported).
The most potent fluorescent probe, compound 6, was selected
in experiments aimed at labeling PBR in C6 glioma cells. For
this purpose, cells were incubated with different concentrations
of 6 in cell culture medium and were subsequently processed
for microscopy analysis.11 An intracellular fluorescent labeling
was detectable using compound 6 at 1 µM, and a consistent,
reproducible staining was obtained by applying the fluorescent
ligand at 10 µM. In these conditions, cells appeared to be
uniformly stained in the cytoplasm (Figure 2A,B), and no
significant differences were observed with higher ligand con-
centrations. These data are in agreement with our previous
observations that in C6 glioma cells the PBR is mainly expressed
on mitochondria.13 To test the specificity of the fluorescent
pattern, cells were preincubated with different concentrations
of the nonfluorescent lead Ip, or PK 11195, and were then
stained with 10 µM 6. The fluorescent staining was significantly
displaced by both Ip and PK 11195 at 10 µM concentration
(Figure 2C,D). The specificity of the staining was further
demonstrated by the complete displacement of 6 staining
observed when the cells were preincubated with 50 µM PK
11195 (Figure 2E).
potent at the PBR, with Ki values varying from the submicro-
molar to the nanomolar range. Compounds 1 and 2, featuring a
short ethylene chain, showed a moderate affinity, whereas probes
3-6 with chains of 4 or 6 methylene units displayed nanomolar
affinities, probably resulting from a successful accommodation
of their side chains into the L3 or L4 lipophilic pocket of the
receptor binding site. All compounds 1-6 are N-monosubsti-
tuted glyoxylamides unable to occupy either L3 or L4 subsites.
Such a partial occupation could account for the lower affinity
of compound 6 with respect to its lead Ip.10 In agreement with
the structure-activity relationship data of N,N-dialkyl-2-phe-
nylindol-3-ylglyoxylamides I,10 the presence of an electron-
withdrawing fluorine in the 4′-position determines a slight gain
in affinity, confirming a possible involvement of the 2-phenyl
ring in a π-stacking interaction with an electron-rich aromatic
ring within the L1 pocket.
Spectroscopic Properties of Fluorescent Ligand 6. The UV
(see Supporting Information) and the emission spectra of 6 were
measured to investigate the spectroscopic properties of the ligand
and how these were affected by the environment. Compound
6, 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). The percentage of DMSO did not exceed 1% of the final
assay volume solution.
Radioligand binding assays were also performed on C6
glioma cell mitochondrial membranes. In these tests, all the
derivatives 1-6 demonstrated affinity values similar to those
observed in experiments on rat kidney membranes (data not
shown).
Figure 3 shows the emission spectra of compound 6. As
expected, the decrease in polarity of the environment (from
aqueous buffer to 60% dioxane in PBS) induced a 9-fold

406 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Brief Articles
Figure 2. Rat C6 glioma cells stained with 10 µM 6 (A,B); cells stained with 10 µM 6 in the presence of 10 µM PK 11195 (C); cells stained with
10 µM 6 in the presence of 10 µM Ip (D); and cells stained with 10 µM 6 in the presence of 50 µM PK 11195 (E).
for 36-48 h (TLC analysis) and then filtered. The precipitate was
triturated with a saturated NaHCO3 aqueous solution, washed with
water, and collected to give a first portion of crude product. The
organic solution was evaporated to dryness, and the residue was
treated with saturated NaHCO3 aqueous solution, washed with
water, and collected to yield an additional amount of crude product.
All products 1-6 were purified by washing with cold diethyl ether,
as the recrystallization process led to partial decomposition of the
products (Supporting Information).
Fluorescent Labeling of C6 Glioma Cells. For fluorescence
microscopy observation, rat C6 glioma cells were cultured on 96-
well plates in Dulbecco’s Modified Eagle’s medium, as previously
described.13 Cells were grown to subconfluence and incubated with
different concentrations (0.5, 1, 5, 10, 20 µM) of the fluorescent
PBR ligand 6 in cell culture medium for 30 min under 5% CO2 at
37 °C. In parallel, some samples were preincubated for 30 min
with increasing concentrations (ranging from 10 µM to 100 µM)
of PK 11195 or Ip and were then stained by adding compound 6
Figure 3. The influence of the polarity of the medium on the
fluorescence of compound 6 was investigated by the addition of dioxane
(indicated in % v/v) in PBS measured at a λmax of 470 nm.
at a final concentration of 10 µM. The cells were then quickly
washed with PBS, fixed in 4% paraformaldehyde, and subsequently
observed under the fluorescence microscope Axiovert-25 (Zeiss).
Excitation was at 458 nm, and fluorescence detection was with a
band-pass filter of 516-565 nm. Images of cells were acquired
with ACT-2U 1.2 (Nikon).
increase in quantum yield. This phenomenon was accompanied
by a blue-shift of the emission maximum from 534 to 540 nm.
Acknowledgment. This work was supported by MIUR
Conclusions
(cofin. 2004).
Compound 6 can be considered to be a new fluorescent probe
eligible for investigating the PBR. In view of its chemical and
optical properties, 6 has a great potential as a fluorophore to
replace PBR radioligands in studies on the localization, expres-
sion level, structure and physiological/pathological roles of this
receptor.
Supporting Information Available: Ultraviolet absorption
spectra of compound 6; general chemistry directions; synthesis of
compounds 7-12; Tables 1 and 2, including yields, physical, and
spectral data of compounds 1-12; analytical data of compounds
1-12; and general biological directions, including [3H]PK 11195
binding to rat kidney and C6 glioma cell mitochondria membranes
and [3H]flumazenil binding to rat cerebral cortex membranes. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Experimental Section
Chemistry. General directions are in the Supporting Information.
General Procedure for the Synthesis of N-{[(7-Nitrobenz-2-
oxa-1,3-diazol-4-yl)amino]alkyl}-2-(4-substituted)phenylindol-
3-ylglyoxylamide Derivatives 1-6. A solution of NEt3 (0.58 mL,
4.2 mmol) in 3.0 mL of anhydrous THF was added dropwise to a
stirred suspension, at 0 °C under a nitrogen atmosphere, of the
indolylglyoxyl chloride 13 and 1410 (2.1 mmol) and the amine
hydrochloride 10-12 (1.9 mmol) in 25.0 mL of the same solvent.
The reaction mixture was left to warm to room temperature, stirred
References
(1) James, M. L.; Selleri, S.; Kassiou, M. Development of ligands for
the peripheral benzodiazepine receptor. Curr. Med. Chem. 2006, 13,
1991-2001.
(2) Casellas, P.; Galiegue, S.; Basile, A. S. Peripheral benzodiazepine
receptors and mitochondrial function. Neurochem. Int. 2002, 40, 475-
486.

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 407
(3) Papadopoulos, V.; Lecanu, L.; Brown, R. C.; Han, Z.; Yao, Z. X.
Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis,
neuropathology and neurological disorders. Neuroscience 2006, 138,
749-756.
(4) Papadopoulos, V.; Baraldi, M.; Guilarte, T. R.; Knudsen, T. B.;
Lacape`re, J.-J.; Lindemann, P.; Norenberg, M. D.; Nutt, D.; Weizman,
A.; Zhang, M.-R.; Gavish, M. Translocator protein (18 kDa): New
nomenclature for the peripheral-type benzodiazepine receptor based
on its structure and molecular function. Trends Pharmacol. Sci. 2006,
27, 402-409.
(5) Kassiou, M.; Meikle, S. R.; Banati, R. B. Ligands for peripheral
benzodiazepine binding sites in glial cells. Brain Res. ReV. 2005,
48, 207-210.
(6) Thominiaux, C.; Dolle`, F.; James, M. L.; Bramoulle`, Y.; Boutin, H.;
Besret, L.; Gre´goire, M.-C.; Valette, H.; Bottlaender, M.; Tavitian,
B.; Hantraye, Ph.; Selleri, S.; Kassiou, M. Improved synthesis of
the peripheral benzodiazepine receptor ligand [11C]DPA-713 using
[11C]methyl triflate. Appl. Radiat. Isot. 2006, 64, 570-573.
(7) Cappelli, A.; Matarrese, M.; Moresco, R. M.; Valenti, S.; Anzini,
M.; Vomero, S.; Turolla, E. A.; Belloli, S.; Simonelli, P.; Filannino,
M. A.; Lecchi, M.; Fazio, F. Synthesis, labelling, and biological
evaluation of halogenated 2-quinolinecarboxamides as potential
radioligands for the visualization of peripheral benzodiazepine
receptors. Bioorg. Med. Chem. 2006, 14, 4055-4066.
tential radioligands for visualization of peripheral benzodiazepine
receptors. J. Med. Chem. 2001, 44, 579-585.
(9) McGrath, J. C.; Arribas, S.; Daly, C. J. Fluorescent ligands for the
study of receptors. Trends Pharmacol. Sci. 1996, 17, 393-399.
(10) Primofiore, G.; Da Settimo, F.; Taliani, S.; Simorini, F.; Patrizi, M.
P.; Novellino, E.; Greco, G.; Abignente, E.; Costa, B.; Chelli, B.;
Martini, C. N,N-Dialkyl-2-phenylindol-3-ylglyoxylamides. A new
class of potent and selective ligands at the peripheral benzodiazepine
receptor. J. Med. Chem. 2004, 47, 1852-1855.
(11) Kozikowski, A. P.; Kotoula, M.; Ma, D.; Boujrad, N.; Tu¨ckmantel,
W.; Papadopoulos, V. Synthesis and biology of a 7-nitro-2,1,3-
benzoxadiazol-4-yl derivative of 2-phenylindole-3-acetamide:
A
fluorescent probe for the peripheral-type benzodiazepine receptor.
J. Med. Chem. 1997, 40, 2435-2439.
(12) Chen, Y.; Zheng, X.; Dobhal, M. P.; Gryshuk, A.; Morgan, J.;
Dougherty, T. J.; Oseroff, A.; Pandey, R. K. Methyl pyropheophor-
bide-R analogues: Potential fluorescent probes for the peripheral-
type benzodiazepine receptor. Effect of central metal in photosen-
sitizing efficacy. J. Med. Chem. 2005, 48, 3692-3695.
(13) Chelli, B.; Rossi, L.; Da Pozzo, E.; Costa, B.; Spinetti, F.; Rechichi,
M.; Salvetti, A.; Lena, A.; Simorini, F.; Vanacore, R.; Scatena, F.;
Da Settimo, F.; Gremigni, V.; Martini, C. PIGA (N,N-Di-n-butyl-5-
chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide), a new mitochon-
drial benzodiazepine-receptor ligand, induces apoptosis in C6 glioma
cells. ChemBioChem 2005, 6, 1082-1088.
(8) Matarrese, M.; Moresco, R. M.; Cappelli, A.; Anzini, M.; Vomero,
S.; Simonelli, P.; Verza, E.; Magni, F.; Sudati, F.; Soloviev, D.;
Todde, S.; Carpinelli, A.; Kienle, M. G.; Fazio, F. Labelling and
evaluation of N-[11C]methylated quinoline-2-carboxamides as po
JM061137O