Translocator protein (TSPO) ligand-Ara-C (cytarabine) conjugates as a strategy to deliver antineoplastic drugs and to enhance drug clinical potential

Article abstract of DOI:10.1021/mp100235w

The aim of this work was to evaluate TSPO ligand-Ara-C conjugation as an approach for the selective delivery of the antineoplastic agent to brain tumors as well as for overcome P-gp resistance induction observed for the majority of cytotoxic agents, enhancing the drug clinical potential. To this end, the novel N-imidazopyridinacetyl-Ara-C conjugates 3a-c, 10 and 15 have been prepared and evaluated for their cytotoxicity against glioma cell lines. In contrast to that observed for 3a-c and 10, the conjugate 15 resulted stable in both phosphate buffer and physiological medium. In all cases, the release of free Ara-C from hydrolyzed conjugates was checked by HPLC and ESI-MS analysis. Conjugates 10 and 15 displayed very high in vitro TSPO affinity and selectivity, and, hence, they may possess potential for targeted brain delivery. Due to the favorable features displayed by the conjugate 15, it was further evaluated on glioma cell lines, expressing high levels of TSPO, in the presence and in the absence of specific nucleoside transport (NT) inhibitors. In contrast to that observed for the free Ara-C, the presence of NT inhibitors did not reduce the cytotoxic activity of 15. Moreover, conjugate 15, as N⁴-acyl derivative of Ara-C, should be resistant to inactivation by cytidine deaminase, and it may possess enhanced propensity to target brain tumor cells characterized by a reduced expression of NTs. In addition, this conjugate behaves as a clear P-gp modulator and thereby may be useful to reverse MDR. Transport studies across the MDCKII-MDR1 monolayer indicated that conjugate 15 should overcome the BBB by transcellular pathway. All these features may be useful for enhancing the clinical potential of the nucleoside drug Ara-C.

Full text of DOI:10.1021/mp100235w

articles
Translocator Protein (TSPO) Ligand-Ara-C (Cytarabine)
Conjugates as a Strategy To Deliver Antineoplastic
Drugs and To Enhance Drug Clinical Potential
Nunzio Denora,† Valentino Laquintana,† Adriana Trapani,† Angela Lopedota,†
Andrea Latrofa,† James M. Gallo,‡ and Giuseppe Trapani*,†
Dipartimento Farmaco-Chimico, Facolta` di Farmacia, UniVersita` degli Studi di Bari, Via
Orabona 4, 70125 Bari, Italy, and Department of Pharmacology and Systems Therapeutics,
Mount Sinai School of Medicine, One GustaVe L. LeVy Place, New York,
New York 10029, United States
Received July 14, 2010; Revised Manuscript Received September 29, 2010; Accepted
October 19, 2010
Abstract: The aim of this work was to evaluate TSPO ligand-Ara-C conjugation as an approach
for the selective delivery of the antineoplastic agent to brain tumors as well as for overcome
P-gp resistance induction observed for the majority of cytotoxic agents, enhancing the drug
clinical potential. To this end, the novel N-imidazopyridinacetyl-Ara-C conjugates 3a-c, 10
and 15 have been prepared and evaluated for their cytotoxicity against glioma cell lines. In
contrast to that observed for 3a-c and 10, the conjugate 15 resulted stable in both phosphate
buffer and physiological medium. In all cases, the release of free Ara-C from hydrolyzed
conjugates was checked by HPLC and ESI-MS analysis. Conjugates 10 and 15 displayed
very high in vitro TSPO affinity and selectivity, and, hence, they may possess potential for
targeted brain delivery. Due to the favorable features displayed by the conjugate 15, it was
further evaluated on glioma cell lines, expressing high levels of TSPO, in the presence and
in the absence of specific nucleoside transport (NT) inhibitors. In contrast to that observed
for the free Ara-C, the presence of NT inhibitors did not reduce the cytotoxic activity of 15.
Moreover, conjugate 15, as N4-acyl derivative of Ara-C, should be resistant to inactivation
by cytidine deaminase, and it may possess enhanced propensity to target brain tumor cells
characterized by a reduced expression of NTs. In addition, this conjugate behaves as a
clear P-gp modulator and thereby may be useful to reverse MDR. Transport studies across
the MDCKII-MDR1 monolayer indicated that conjugate 15 should overcome the BBB by
transcellular pathway. All these features may be useful for enhancing the clinical potential
of the nucleoside drug Ara-C.
Keywords: Ara-C (cytarabine); TSPO conjugates; stability; cytotoxicity; BBB; transport studies;
brain tumors
proposed1 are abundant in peripheral organs like kidney,
liver, lung as well as in glial cells of the central nervous
Introduction
Peripheral-type benzodiazepine receptors for which the
name “translocator protein (TSPO)” has recently been
* To whom correspondence should be addressed. Mailing address:
Dipartimento Farmaco-Chimico, Universita` degli Studi di Bari,
Via Orabona 4, 70125 Bari, Italy. Phone: (039) 080-5442764.
Fax: (039) 0805442724. E-mail: trapani@farmchim.uniba.it.
† Universita` degli Studi di Bari.
(1) 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.
‡ Mount Sinai School of Medicine.
10.1021/mp100235w  2010 American Chemical Society
Published on Web 10/19/2010
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Denora et al.
system (CNS).2 The pharmacological action of TSPOs is
completely different from the central-type benzodiazepine
receptors (CBRs), which are located in neurons in the CNS.
TSPOs are located primarily on mitochondrial membranes2
within cells and are composed of at least three subunits: the
isoquinoline-binding protein, a voltage-dependent anion
channel, and an adenine nucleotide carrier.3 The trimeric
complex is a component of the mitochondrial permeability
transitional (MPT) pore, which plays an important role in
the passage of various biogenic compounds between the
cytoplasm and mitochondrial intermembrane.4 There is
growing experimental evidence suggesting that TSPOs are
involved in the regulation of cholesterol transport into
mitochondria and in steroidogenesis5 as well as in various
neurological diseases such as Alzheimer’s disease, Hunting-
ton’s disease, epilepsy and stroke-induced brain injury.6,7 It
has also been shown that TSPO expression selectively
increases in many tumor types, such as brain, colon, breast,
and ovary, which suggests a possible role of these receptors
in the tumorigenesis.3,8-11 Moreover, there is evidence
indicating TSPO-specific ligands induce apoptosis and cell
cycle arrest in cancer cells,3,12-15 which is consistent with
the association of TSPOs with other mitochondrial proteins
(i.e., the anti- and proapoptotic members of the Bcl-2 family)
of the MPT pore known to be involved in apoptosis. The
increased expression of TSPOs in neoplastic cells opens up
the possibility to evaluate TSPO ligands as diagnostic
imaging agents in oncology and to use them as receptor-
mediated drug carriers to selectively target anticancer drugs
to tumors. To date, the known TSPO-selective ligands belong
to different chemical classes such as benzodiazepines (Ro-
54864),16 isoquinolines (PK-11195),17 2-aryl-3-indoleaceta-
mides (FGIN-1-27),18 and N-phenoxyphenyl-N-isopropoxy-
benzyl-acetamides (DAA1097)19 (Figure 1). However, it
should be noted that none of these TSPO ligands contain
hydrophilic groups or organic functions such as amino,
hydroxy and carboxylic groups useful for further conjugation.
Development of conjugable forms of TSPO ligands would
allow their use as starting material for the formulation of
anticancer drug delivery systems and as diagnostic imaging
agents as well. In this context, we have recently synthesized
the first examples of conjugable imidazopyridinacetamide
TSPO ligands endowed with high affinity and selectivity for
TSPO (Figure 1).20-22
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It is known that malignant brain tumors are among the
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interest to devise TSPO-based drug delivery systems to
selectively target brain tumors and overcome the BBB.
The proof-of-concept of the potential of TSPO ligand-
anticancer drug conjugate for targeted brain delivery was
demonstrated by Guo et al. using a TSPO ligand-gemcitabine
(GEM) conjugate in a preclinical brain tumor model.24 The
approximate 2-fold enhancement in brain tumor penetration
compared with GEM alone was likely due to both the
increased lipophilicity of the conjugate leading to enhanced
transport across BBB27 and, possibly, due to TSPO-mediated
delivery to tumor cells. Based on these results, several efforts
have been devoted to the identification and evaluation of new
TSPO ligand-anticancer drug conjugates.28,29
Ara-C [cytarabine, cytosine arabinose, 1-(ꢀ-D-arabino-
furanosyl)cytosine, Figure 1] is a pyrimidine nucleoside
analogue employed for the treatment of various cancers
including acute and chronic myeloblastic leukemia, colon,
breast and ovary carcinoma.30 AraC has also been part of
combination chemotherapeutic regimens for various brain
tumors including primary CNS lymphoma, and recurrent or
refractory malignant gliomas.31 In particular, promising
results for the treatment of brain tumors were obtained using
sustained delivery systems of cytarabine represented by a
liposomal formulation, (i.e., Depocyte).32
Ara-C is rapidly converted to an inactive and more soluble
metabolite 1-(ꢀ-D-arabinofuranosyl)uracil (Ara-U) by cy-
Figure 1. The chemical structures of the known
TSPO-selective ligands and Ara-C (1).
(24) Guo, P.; Ma, J.; Li, S.; Guo, Z.; Adams, A. L.; Gallo, J. M.
Targeted delivery of a peripheral benzodiazepine receptor ligand-
gemcitabine conjugate to brain tumors in a xenograft model.
Cancer Chemother. Pharmacol. 2001, 48, 169–176.
(25) Black, K. L.; Ikezaki, K.; Toga, A. W. Imaging of brain tumors
using peripheral benzodiazepine receptor ligands. J. Neurosurg.
1989, 71, 113–118.
penetration of antineoplastic agents across the blood-brain
barrier (BBB). The structure of the normally tight junctions
of the BBB in brain tumors may be compromised, leading
to a disrupted and “leaky” barrier. However, other areas,
particularly at the outer rim of the tumor, may have a more
normal BBB where it is still a challenge to achieve effective
drug delivery.23 Moreover, cytotoxic anticancer drugs possess
limited specificity and cause lethal damage to healthy cells.
Based on these considerations and the observation that
TSPOs can be overexpressed in brain tumors,24-26 it is of
(26) Black, K. L.; Ikezaki, K.; Santori, E.; Becker, D. P.; Vinters, H. V.
Specific high-affinity binding of peripheral benzodiazepine receptor
ligands to brain tumors in rat and man. Cancer 1990, 65, 93–97.
(27) Harivardhan Reddy, L.; Couvreur, P. Novel approaches to deliver
gemcitabine to cancers. Curr. Pharm. Des. 2008, 14, 1124–1137.
(28) Kupczyk-Subotkowaka, L.; Siahaan, T. J.; Basile, A.; Friedman,
H. S.; Higgins, P. E.; Song, D.; Gallo, J. M. Modulation of
melphalan resistance in glioma cells with a peripheral benzodi-
azepine receptor ligand-melphalan conjugate. J. Med. Chem. 1997,
40, 1726–1730.
(29) Trapani, G.; Laquintana, V.; Latrofa, A.; Ma, J.; Reed, K.; Serra,
M.; Biggio, G.; Liso, G.; Gallo, J. M. Peripheral benzodiazepine
receptor ligand-melphalan conjugates for potential selective drug
delivery to brain tumors. Bioconjugate Chem. 2003, 14, 830–839.
(30) Sun, Y.; Sun, J.; Shi, S.; Jing, Y.; Yin, S.; Chen, Y.; Li, G.; Xu,
Y.; He, Z. Synthesis, transport and pharmacokinetics of 5′-amino
acid ester prodrugs of 1-ꢀ-D-arabinofuranosylcytosine. Mol.
Pharmaceutics 2009, 6, 315–325.
(20) Laquintana, V.; Denora, N.; Musacchio, T.; Lasorsa, M.; Latrofa,
A.; Trapani, G. Peripheral benzodiazepine receptor ligand-PLGA
polymer conjugates potentially useful as delivery systems of
apoptotic agents. J. Controlled Release 2009, 137, 185–195.
(21) Trapani, G.; Laquintana, V.; Denora, N.; Trapani, A.; Lopedota,
A.; Latrofa, A.; Franco, M.; Serra, M.; Pisu, M. G.; Floris, I.;
Sanna, E.; Biggio, G.; Liso, G. Structure-activity relationships
and effects on neuroactive steroids in a series of 2-phenylimidazo-
[1,2-a]pyridineacetamide peripheral benzodiazepine receptors
ligands. J. Med. Chem. 2005, 48, 292–305.
(22) Denora, N.; Laquintana, V.; Pisu, M. G.; Dore, R.; Murru, L.;
Latrofa, A.; Trapani, G.; Sanna, E. 2-Phenyl-imidazo[1,2-a]py-
ridine compounds containing hydrophilic groups as potent and
selective ligands for peripheral benzodiazepine receptors: syn-
thesis, binding affinity and electrophysiological studies. J. Med.
Chem. 2008, 51, 6876–6888.
(23) Laquintana, V.; Trapani, A.; Denora, N.; Wang, F.; Gallo, J. M.;
Trapani, G. New strategies to deliver anticancer drugs to brain
tumors. Expert Opin. Drug DeliVery 2009, 6, 1017–1032.
(31) Kripp, M.; Hofheinz, R. D. Treatment of lymphomatous and
leukemic meningitis with liposomal encapsulated cytarabine. Int.
J. Nanomed. 2008, 3, 397–401.
(32) Benesch, M.; Siegler, N.; Hoff, K. V.; Lassay, L.; Kropshofer,
G.; Mu¨ller, H.; Sommer, C.; Rutkowski, S.; Fleischhack, G.;
Urban, C. Safety and toxicity of intrathecal liposomal cytarabine
(Depocyte) in children and adolescents with recurrent or refractory
brain tumors: a multi-institutional retrospective study. Anti-Cancer
Drugs 2009, 20, 794–799.
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tosine nucleoside deaminase that is widely distributed in
tissues including hepatic and intestinal cells. Ara-C requires
intracellular conversion to a triphosphate anabolite for
biological activity that is initiated by the formation of a 5′-
monophosphate moiety by deoxycytidine kinase. Many
nucleoside analogues, including Ara-C, enter cells Via
specific nucleoside transporters (NTs). In this regard, it is
also well-known that the clinical potential of Ara-C can be
impaired by a deficient cellular drug uptake due to a reduced
expression of NTs in cancer cells.33 The complexities of Ara-
C’s membrane transport attributes, rapid clearance, and intra-
cellular activation have promoted many efforts to modify its
transport and metabolic properties to circumvent such deleteri-
ous attributes. Besides the use of high-dose protocols to ensure
biological activity, many Ara-C prodrugs, such as polymeric-
Ara-C conjugates, have been extensively examined.34-37 In
particular, N4-amide- as well as 3′ and 5′ ester-Ara-C
prodrugs have been synthesized with the aim to increase the
Ara-C biological activity and/or to modify its pharmacoki-
netic properties.35,36
IR spectra were obtained on a Perkin-Elmer Spectrum
one system spectrophotometer (KBr pellets for solid). H
1
NMR spectra were determined on a Varian VX Mercury
instrument operating at 300 MHz. Chemical shifts are
given in δ values. The mass spectra of all new compounds
were obtained using an Agilent 1100 LC-MSD trap system
VL instrument using methanol/ammonium formate 7 mM
9:1 (v/v). All compounds showed appropriate IR, 1H NMR
and mass spectra. Elemental analyses were performed on
a Hewlett-Packard 185 C, H, N analyzer and agreed with
theoretical values within (0.40%. Thin layer chromatog-
raphy (TLC) analyses were performed on silica gel plate
60 F254 (Merck). Silica gel 60 (Merck 70- 230 mesh)
was used for column chromatography. All the follow-
ing reactions were performed under a nitrogen atmosphere.
High-Performance Liquid Chromatography (HPLC)
Analyses. HPLC analyses were performed with a Waters
Associates model 600 pump equipped with a Waters 2996
photodiode array detector and Empower software or with
a 990 variable wavelength UV detector. For kinetic studies
on conjugates 3a-c, 10, and 15, a reversed phase
Symmetry C18 (25 cm ×3.9 mm; 5 µm particles) column
in conjunction with a SecurityGuard Phenomenex precol-
umn was eluted with mixtures of methanol and deionized
water 80/20 (v/v). The volume injected was 20 µL. The
flow rate of 0.8 mL/min was maintained, and the column
effluent was monitored continuously at 254 nm. Quanti-
fication of the compounds was carried out by measuring
the peak areas in relation to those of standards chromato-
graphed under the same conditions. Stability studies were
carried out at controlled temperature at 37 °C ((0.2 °C)
in a water bath.
There have been no prior efforts to prepare TSPO
ligand-Ara-C conjugates, which, in addition to an enhanced
transport across BBB and a tumor targeting effect, might
increase drug stability and prolong its efficacy. Therefore,
the aim of this study was to synthesize and evaluate the
cytotoxicity of imidazopyridinacetamide TSPO ligand-Ara-C
(TSPO-Ara-C) conjugates in glioma cells as an approach that
may serve to selectively deliver antineoplastic agent to brain
tumors and improve the therapeutic potential.
Materials and Methods
Materials. The starting cytarabine hydrochloride 1, di-
tert-butyl dicarbonate (DBDC), 1-hydroxybenzotriazole (HO-
BZT), dicyclohexylcarbodiimide (DCC), triethylamine (TEA),
1,1′-carbonyldiimidazole (CDI), N-(3-(dimethylamino)pro-
pyl)-N′-ethylcarbodiimide (EDC) and 4-(dimethylamino)py-
ridine (DMAP) were purchased from Sigma-Aldrich (Milan,
Italy). The preparation of the imidazopyridine compounds
2a-c, 8 and 11 has previously been reported.20-22
For transport studies on compound 15 and diazepam, a
reversed phase Phenomenex Synergi Hydro (250 × 4.6
mm; 4 µm particles) column in conjunction with a
SecurityGuard Phenomenex precolumn was used with
mixtures of methanol/25 mM ammonium acetate buffer
pH 4.8 70/30 (v/v). A volume of 20 µL of the sample
was injected, and the flow rate of 0.8 mL/min was
maintained. The column effluent was monitored continu-
ously at 254 nm. Quantification of the compounds was
carried out as mentioned above. Analyses of Ara-C
samples were performed similarly with a change in mobile
phase containing 25 mM ammonium acetate buffer pH
4.8/methanol 95/5 (v/v), and the column effluent was
monitored continuously at 275 nm.
Apparatus. Melting points were determined in open
capillary tubes with a Bu¨chi apparatus and are uncorrected.
(33) Galmarini, C. M.; Myhren, F.; Sandvold, M. L. CP-4055 and CP-
4126 are active in ara-C and gemcitabine-resistant lymphoma cell
lines. Br. J. Haematol. 2008, 144, 263–275.
(34) Schiavon, O.; Pasut, G.; Moro, S.; Orsoline, P.; Guiotto, A.;
Veronese, F. M. PEG-Ara-C conjugates for controlled release.
Eur. J. Med. Chem. 2004, 39, 123–133.
(35) Wipf, P.; Li, W. Prodrugs of Ara-C. Drug Future 1994, 19, 49–
54.
Synthetic Procedures for TSPO ligand-Ara
C
Conjugates 3a-c, 10, 15. General Procedure for the
Preparation of Conjugates 3a-c. A solution of the ap-
propriate imidazo[1,2-a]pyridine-3-acetic acid 2 (0.85
mmol) and CDI (1.0 mmol) in DMF (10 mL) was stirred
at room temperature (rt) for 15 min. Then, cytarabine
hydrochloride 1 (0.82 mmol) was added and stirring was
prolonged overnight. Subsequently, the solvent was evapo-
(36) Choe, Y. H.; Conover, C. D.; Wu, D.; Royzen, M.; Greenwald,
R. B. Anticancer drug delivery systems: N4-acyl poly(ethyleneg-
lycol) prodrug of Ara-C I. Efficacy in solid tumors. J. Controlled
Release 2002, 79, 41–53.
(37) Sun., Y.; Sun, J.; Shi, S.; Jing, Y.; Yin, S.; Chen, Y.; Li, G.; Xu,
Y.; He, Z. Synthesis, transport and pharmacokinetics of 5′-amino
acid ester prodrugs of 1-b-D-arabinofuranosylcytosine. Mol.
Pharmaceutics 2009, 6, 315–325.
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rated under reduced pressure and the residue was purified
by silica gel column chromatography [chloroform/methanol 85/
15 (v/v) as eluent] to give the corresponding conjugate 3.
2-(6,8-Dichloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridin-3-
yl)-N-(1-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tet-
rahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)aceta-
mide (3a). Yield: 25%. Mp: 176 °C dec. IR (KBr): 3233,
1722, 1654 cm-1. 1H NMR (DMSO-d6) δ: 3.5-3.7 (m, 2H,
5′-CH2O Ara-C), 3.7-4.1 (m, 3H, 2′, 3′,4′-CHO Ara-C), 4.40
(s, 2H, CH2CO), 5.0-5.1 (m, 1H, 5′-OH Ara-C), 5.4-5.5
(m, 2H, 2′-OH + 3′-OH Ara-C), 6.05 (d, J ) 3.8 Hz, 1H,
1′-CHO(N) Ara-C), 7.10 (d, J ) 7.4 Hz, 1H, 5-CH Ara-C),
7.56 (d, J ) 7.8 Hz, 2H, Ar), 7.7-7.8 (m, 3H, Ar), 8.07 (d,
J ) 7.4 Hz, 1H, 6-CHN Ara-C), 8.86 (d, J ) 1.8 Hz, 1H,
Ar), 11.2 (bs, 1H, NHCO). MS (ESI) m/z: 580 [M + H]+.
Anal. (C24H20Cl3N5O6) C, H, N.
aqueous KOH. The reaction progress was monitored by TLC.
After 1 h the reaction was nearly complete to give compound
4 as the main product together with lower amounts of
compound 5. Then, the solvent was removed under reduced
pressure and the residue purified by silica gel column
chromatography (chloroform as eluent) to give compounds
4 and 5.
4. Yield: 48%. Oil. IR (KBr): 3243, 1749, 1648 cm-1. 1H
NMR (CDCl3) δ: 1.33 (s, 9H, CH3), 1.44 (s, 18H, CH3),
4.1-4.2 (m, 1H, 4′-CHO), 4.3-4.5 (m, 2H, 5′-CH2O),
4.9-5.0 (m, 1H, 3′-CHO), 5.2-5.3 (m, 1H, 2′-CHO), 5.78
(d, J ) 7.4 Hz, 1H, 5-CH), 6.2-6.4 (m, 1H, 1′-CHO(N)),
7.46 (d, J ) 7.4 Hz, 1H, 6-CHN). MS (ESI) m/z: 542 [M -
H]-.
5. Yield: 15%. Oil. IR (KBr): 1747, 1681 cm-1. 1H NMR
(CDCl3) δ: 1.33 (s, 9H, CH3), 1.44 (s, 18H, CH3), 4.1-4.2
(m, 1H, 4′-CHO), 4.3-4.5 (m, 2H, 5′-CH2O), 4.9-5.0 (m,
1H, 3′-CHO), 5.2-5.3 (m, 1H, 2′-CHO), 5.78 (d, J ) 7.4
Hz, 1H, 5-CH), 6.2-6.4 (m, 1H, 1′-CHO(N)), 7.46 (d, J )
7.4 Hz, 1H, 6-CHN).
2-(6,8-Dichloro-2-(phenyl)imidazo[1,2-a]pyridin-3-yl)-N-(1-
((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-
2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide (3b). Yield:
23%. Mp:140 °C dec. IR (KBr): 3388, 1721, 1648 cm-1. 1H
NMR (DMSO-d6) δ: 3.5-3.7 (m, 2H, 5′-CH2O Ara-C),
3.7-4.1 (m, 3H, 2′, 3′,4′-CHO Ara-C), 4.41 (s, 2H, CH2CO),
5.0-5.1 (m, 1H, 5′-OH Ara-C), 5.4-5.5 (m, 2H, 2′-OH +
3′-OH Ara-C), 6.05 (d, J ) 3.8 Hz, 1H, 1′-CHO(N) Ara-C),
7.10 (d, J ) 7.4 Hz, 1H, 5-CH Ara-C), 7.3-7.5 (m, 3H,
Ar), 7.6-7.8 (m, 3H, Ar), 8.07 (d, J ) 7.4 Hz, 1H, 6-CHN
Ara-C), 8.86 (m, 1H, Ar), 11.3 (bs, 1H, NHCO). MS (ESI)
m/z: 546 [M + H]+. Anal. (C24H21Cl2N5O6) C, H, N.
2-(6-Chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridin-3-yl)-
N-(1-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahy-
drofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide (3c).
Yield: 65%. Mp: 182-184 °C. IR (KBr): 3218, 1714, 1663
Preparation of ((2R,3R,4S,5R)-5-(4-(tert-Butoxycarbonylami-
no)-2-oxopyrimidin-1(2H)-yl)-3,4-bis(tert-butoxycarbonyloxy)-
tetrahydrofuran-2-yl)methyl 2-(6,8-Dichloro-2-(4-chlorophe-
nyl)imidazo[1,2-a]pyridin-3-yl)acetate (6) and (2R,3S,4R,5R)-
2-(4-(2-(6,8-Dichloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridin-
3-yl)acetamido)-2-oxopyrimidin-1(2H)-yl)-4-(piValoyloxy)-5-
(piValoyloxymethyl)tetrahydrofuran-3-yl 3,3-Dimethylbutanoate
(7). To a stirred suspension of the 2-(4-chlorophenyl)-6,8-
dichloroimidazo [1,2-a]pyridine-3-acetic acid 2a (212 mg,
0.6 mmol) in anhydrous CH2Cl2 (20 mL) were added HO-
BZT (90 mg, 0.66 mmol) and DCC (136 mg, 0.66 mmol) at
rt. After 30 min, compound 4 (296 mg, 0.55 mmol) was
added, and stirring was prolonged for 72 h. Then, ethyl ether
was added and the resulting dicyclohexylurea precipitate
removed by filtration. The filtrate was concentrated, and the
residue was purified by silica gel column chromatography
[chloroform/methanol 85/15 (v/v) as eluent)] to give the
protected conjugates 6 and 7.
1
cm-1. H NMR (DMSO-d6) δ: 3.5-3.7 (m, 2H, 5′-CH2O
Ara-C), 3.7-4.1 (m, 3H, 2′, 3′,4′-CHO Ara-C), 4.41 (s, 2H,
CH2CO), 5.0-5.1 (m, 1H, 5′-OH Ara-C), 5.4-5.5 (m, 2H,
2′-OH + 3′-OH Ara-C), 6.05 (d, J ) 3.8 Hz, 1H, 1′-CHO(N)
Ara-C), 6.95 (t, J ) 7.4 Hz, 1H, Ar),7.10 (d, J ) 7.4 Hz,
1H, 5-CH Ara-C), 7.4-7.6 (m, 3H, Ar), 7.75 (d, J ) 8.5
Hz, 2H, Ar), 8.07 (d, J ) 7.4 Hz, 1H, 6-CHN Ara-C), 8.45
(d, J ) 6.9 Hz, 1H, Ar), 11.3 (bs, 1H, NHCO). MS (ESI)
m/z: 546 [M + H]+. Anal. (C24H21Cl2N5O6) C, H, N.
Preparation of (2R,3S,4R,5R)-2-(4-Amino-2-oxopyrimidin-
1(2H)-yl)-4-(piValoyloxy)-5-(piValoyloxymethyl)tetrahydrofu-
ran-3-yl3,3-Dimethylbutanoate(4)andtert-Butyl1-((2R,3S,4R,5R)-
3,4-Bis(tert-butoxycarbonyloxy)-5-((tert-butoxycarbonyloxy)-
methyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-
ylcarbamate (5). To a stirred solution of cytarabine hydro-
chlolride 1 (0.5 g, 1.8 mmol) in 20 mL of 1 N aqueous KOH,
DBDC (3.9 g, 1.8 mmol) in 40 mL of dioxane was added
dropwise. The reaction mixture was stirred at rt for 16 h
and monitored by TLC (CHCl3/CH3OH 85:15). Then, the
solvent was removed under reduced pressure and the residue
was extracted with EtOAc (3 × 50 mL). The combined
organic extracts were washed with water, dried over Na2SO4
and concentrated to dryness. The resulting residue and DBDC
(3.9 g, 1.8 mmol) were dissolved in 40 mL of dioxane. To
this solution with stirring and at rt was added 40 mL of 1 N
6. Yield: 42%. Mp: 152 °C dec. IR (KBr): 1747, 1665
1
cm-1. H NMR (CDCl3) δ: 1.31 (s, 9H, CH3), 1.50 (s, 9H,
CH3), 1.51 (s, 9H, CH3), 4.11 (s, 2H, CH2CO), 4.2-4.7 (m,
4H, 5′-CH2O and 3′,4′-CHO Ara-C), 5.0-5.1 (m, 1H, 2′-
CHO Ara-C), 6.35 (d, J ) 3.0 Hz, 1H, 1′-CHO(N) Ara-C),
7.15 (d, J ) 7.7 Hz, 1H, 5-CH Ara-C), 7.31 (d, J ) 1.6 Hz,
1H, Ar), 7.44 (d, J ) 8.5 Hz, 2H, Ar), 7.3-7.5 (m, 3H,
6-CH Ara-C and Ar), 8.13 (d, J ) 1.8 Hz, 1H, Ar). MS
(ESI) m/z: 880 [M - H]-.
7. Yield: 49%. Mp: 148 °C dec. IR (KBr): 3442, 1736,
1652 cm-1. 1H NMR (CDCl3) δ: 1.42 (s, 9H, CH3), 1.44 (s,
9H, CH3), 1.48 (s, 9H, CH3), 3.8-4.0 (m, 2H, CH2CO),
4.2-4.6 (m, 4H, 5′-CH2O and 3′,4′-CHO Ara-C), 5.0-5.1
(m, 1H, 2′-CHO Ara-C), 6.13 (d, J ) 4.9 Hz, 1H, 1′-CHO(N)
Ara-C), 6.97 (d, J ) 7.7 Hz, 1H, 5-CH Ara-C), 7.29 (d, J )
1.6 Hz, 1H, Ar), 7.39 (d, J ) 8.5 Hz, 2H, Ar), 7.58 (d, J )
8.5 Hz, 2H, Ar), 7.75 (d, J ) 7.7 Hz, 1H, 6-CHN Ara-C),
8.18 (d, J ) 1.6 Hz, 1H, Ar). MS (ESI) m/z: 880 [M - H]-.
VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 2259

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Denora et al.
Deprotection Reaction of Compound 7. To a stirred
solution of 7 (100 mg) in CH2Cl2 (10 mL) at rt was added
TFA (5 mL), and the reaction progress was monitored by
TLC (CHCl3/CH3OH 85:15 v/v as eluent) for 2 h. Then, the
stirring was prolonged overnight and, subsequently, the
solvent was removed under reduced pressure to give the
crude conjugate 3a in almost quantitative yield.
Procedure for the Preparation of Conjugate 10. A solution
of the 2-(6,8-dichloro-2-(4-hydroxyphenyl)imidazo[1,2-a]py-
ridin-3-yl)-N,N-dipropylacetamide 8 (0.24 mmol) and 4-ni-
trophenyl chloroformate (0.48 mmol) in anhydrous THF (10
mL) was stirred at rt for 6 h in the presence of TEA. Then,
to the obtained intermediate 9, a solution of cytarabine
hydrochloride 1 (0.24 mmol) in 2 mL of anhydrous DMF
was added and stirring was prolonged overnight. Afterward,
solvent was evaporated under reduced pressure and the
residue was purified by silica gel column chromatography
[chloroform/methanol 95/5 (v/v) as eluent] to give the
corresponding conjugate 10.
solvent was evaporated under reduced pressure. The residue
was taken up with water, and the cooled aqueous solution
was acidified with dilute HCl until pH 4 was reached. The
resulting precipitate was the pure compound 13, which was
isolated by filtration.
Methyl 6-(2-(4-Chlorophenyl)-3-(2-(dipropylamino)-2-oxo-
ethyl)imidazo[1,2-a]pyridin-8-ylamino)-6-oxohexanoate (12).
Yield: 67%. Mp: 136 °C dec. IR (KBr): 3393, 1736, 1694,
1
1639 cm-1. H NMR (CDCl3) δ: 0.75 (t, J ) 7.4 Hz, 3H,
CH3), 0.84 (t, J ) 7.4 Hz, 3H, CH3), 1.4-1.6 (m, 4H, CH2),
1.6-2.0 (m, 4H, CH2), 2.37 (t, J ) 7.15 Hz, 2H, CH2CON),
2.53 (t, J ) 7.15 Hz, 2H, CH2COO), 3.11 (t, J ) 7.4 Hz,
2H, CH2NCO), 3.28 (t, J ) 7.4 Hz, 2H, CH2NCO), 3.67 (s,
3H, CH3O), 4.05 (s, 2H, CH2CON), 6.83 (t, J ) 7.1 Hz,
1H, Ar), 7.45 (d, J ) 8.5 Hz, 2H, Ar), 7.60 (d, J ) 8.5 Hz,
2H, Ar), 7.90 (d, J ) 7.4 Hz, 1H, Ar), 8.19 (d, J ) 7.4
Hz, 1H, Ar), 8.71 (b, 1H, NH). MS (ESI) m/z: 565 [M +
K]+. Anal. (C28H35ClN4O4) C, H, N.
6-(2-(4-Chlorophenyl)-3-(2-(dipropylamino)-2-oxoethyl)imi-
dazo[1,2-a]pyridin-8-ylamino)-6-oxohexanoic Acid (13). Yield:
88%. Mp: 125 °C dec. IR (KBr): 1659, 1638, 1551 cm-1.
1H NMR (CDCl3) δ: 0.75 (t, J ) 7.4 Hz, 3H, CH3), 0.84 (t,
J ) 7.4 Hz, 3H, CH3), 1.4-1.6 (m, 4H, CH2), 1.6-1.8 (m,
4H, CH2), 2.35 (t, J ) 6.8 Hz, 2H, CH2CON), 2.56 (t, J )
6.8 Hz, 2H, CH2COO), 3.08 (t, J ) 7.7 Hz, 2H, CH2NCO),
3.27 (t, J ) 7.7 Hz, 2H, CH2NCO), 3.98 (s, 2H, CH2CON),
6.89 (t, J ) 7.1 Hz, 1H, Ar), 7.43 (d, J ) 8.5 Hz, 2H, Ar),
7.46 (d, J ) 8.5 Hz, 2H, Ar), 7.91 (d, J ) 6.9 Hz, 1H, Ar),
8.30 (d, J ) 6.9 Hz, 1H, Ar), 9.66 (b, 1H, NH). MS (ESI) m/z:
511 [M - H]-. Anal. (C27H33ClN4O4) C, H, N.
4-(6,8-Dichloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenyl 1-((2R,3S,4S,5R)-3,4-Dihydroxy-5-(hy-
droxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-
4-ylcarbamate (10). Yield: 23%. Mp: 156 °C dec. IR (KBr):
1
3327, 1704, 1627 cm-1. H NMR (DMSO-d6) δ: 0.7-0.9
(m, 6H, CH3), 1.4-1.7 (m, 4H, CH2), 3.1-3.4 (m, 4H,
-CH2NCO), 3.1-3.4 (m, 2H, 5′-CH2O Ara-C), 3.8-4.1 (m,
3H, 2′, 3′,4′-CHO Ara-C), 4.28 (s, 2H, -CH2CON), 6.12
(d, J ) 3.57 Hz, 1H, 1′-CHO(N) Ara-C), 6.82 (d, J ) 7.4
Hz, 1H, 5-CH Ara-C), 7.52 (d, J ) 7.4 Hz, 2H, Ar), 7.6-7.7
(m, 3H, Ar), 8.05 (d, J ) 7.4 Hz, 1H, 6-CHN Ara-C), 8.60
(d, J ) 1.6 Hz, 1H, Ar). MS (ESI) m/z: 711 [M + Na]+.
Anal. (C31H34Cl2N6O8) C, H, N.
N-(2-(4-Chlorophenyl)-3-(2-(dipropylamino)-2-oxoethyl)imi-
dazo[1,2-a]pyridin-8-yl)-6-oxo-6-(2-thioxothiazolidin-3-yl)hex-
anamide (14). Yield: 72%. Mp: 165 °C dec. IR (KBr): 1698,
Procedure for the Preparation of Conjugate 15. A mixture
of compound 13 (0.36 mmol), 2-mercaptothiazoline (0.43
mmol), EDC · HCl (0.39 mmol), and DMAP (0.43 mmol) in
anhydrous CH2Cl2 (20 mL) was stirred for 4 h at rt. The
mixture was concentrated in Vacuo, and then the residue was
purified by silica gel column chromatography [chloroform/
acetone 8/2 (v/v) as eluent] to give the pure compound 14.
Then, a mixture of 14 (0.07 mmol) and 1 (0.14 mmol) in
anhydrous pyridine was stirred at 50 °C overnight. After that,
the reaction mixture was concentrated in Vacuo and the
residue purified by silica gel column chromatography
[chloroform/methanol 85/15 (v/v) as eluent] to give the
corresponding conjugate 15. Compound 13, in turn, was
synthesized following a two step procedure. First, the 2-(8-
amino-2-(4-chlorophenyl)imidazo[1,2-a]pyridin-3-yl)-N,N-
dipropylacetamide 11 (0.13 mmol) was dissolved in anhy-
drous THF at rt in the presence of K2CO3 (0.65 mmol) and
methyl adipoyl chloride (0.26 mmol). The resulting solution
was stirred at rt for 2 h. After that period, the solvent was
removed under reduced pressure and the residue dissolved
in ethyl acetate and extracted with brine. The organic phase
was dried on anhydrous Na2SO4 and evaporated to dryness,
giving compound 12. Second, to a solution of the methyl
ester 12 in dioxane (10 mL) was added NaOH 0.1 N (10
mL). The mixture was stirred at 50 °C for 1 h, and then, the
1
1636 cm-1. H NMR (CDCl3) δ: 0.74 (t, J ) 7.4 Hz, 3H,
CH3), 0.84 (t, J ) 7.4 Hz, 3H, CH3), 1.4-1.6 (m, 4H, CH2),
1.7-2.0 (m, 4H, CH2), 2.5-2.6 (m, 2H, CH2S), 3.11 (t, J )
7.7 Hz, 2H, CH2NCO), 3.2-3.4 (m, 2H, CH2NCO), 3.2-3.4
(m, 4H, CH2CON), 4.06 (s, 2H, CH2CON), 4.57 (t, J ) 7.4
Hz, 2H, CH2N), 6.82 (t, J ) 7.1 Hz, 1H, Ar), 7.27 (d, J ) 8.5
Hz, 2H, Ar), 7.45 (d, J ) 8.5 Hz, 2H, Ar), 7.90 (d, J ) 7.4 Hz,
1H, Ar), 8.17 (d, J ) 7.4 Hz, 1H, Ar), 8.56 (b, 1H, NH). MS
(ESI) m/z: 636 [M + Na]+. Anal. (C30H36ClN5O3S2) C, H, N.
N1-(2-(4-Chlorophenyl)-3-(2-(dipropylamino)-2-oxoethyl)imi-
dazo[1,2-a]pyridin-8-yl)-N6-(1-((2R,3S,4S,5R)-3,4-dihydroxy-
5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropy-
rimidin-4-yl)adipamide (15). Yield: 66%. Mp: 125 °C dec.
IR (KBr): 1643, 1553 cm-1. 1H NMR (DMSO-d6) δ:0.7-0.9
(m, 6H, CH3), 1.4-1.6 (m, 4H, CH2), 2.4-2.6 (m, 4H, CH2),
3.2-3.4 (m, 4H, CH2NCO), 3.2-3.4 (m, 4H, CH2CON),
3.5-3.7 (m, 2H, CH2O Ara-C), 3.7-4.2 (m, 3H, CHO Ara-
C), 4.19 (s, 2H, CH2CON), 6.02 (d, J ) 3.85 Hz, 1H, 1′-
CHO(N) Ara-C), 6.87 (t, J ) 6.9 Hz, 1H, Ar), 7.19 (d, J )
7.4 Hz, 1H, 5-CH Ara-C), 7.51 (d, J ) 8.5 Hz, 2H, Ar),
7.67 (d, J ) 8.5 Hz, 2H, Ar), 7.8-8.0 (m, 2H, Ar), 8.03 (d,
J ) 7.4 Hz, 1H, 6-CH Ara-C), 9.77 (s, 1H, NH), 10.8 (s,
1H, NH). 13C NMR (DMSO-d6) δ: 11.6, 11.9, 21.1, 22.4,
24.9, 25.3, 29.7, 36.5, 36.9, 40.1, 47.7, 49.3, 49.6, 61.7, 75.3,
2260 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6

TSPO Ligand-Ara-C Conjugates
articles
76.8, 86.4, 87.7, 94.9, 111.9, 112.8, 118.3, 120.2, 127.7,
129.2, 130.3, 132.9, 133.9, 138.9, 141.0, 147.4, 155.2, 162.8,
168.1, 173.3, 174.4. MS (ESI) m/z: 760 [M + Na]+. Anal.
(C36H44ClN7O8) C, H, N.
[3H]Flunitrazepam Binding. The tissues were thawed and
homogenized with a Polytron PT 10 in 50 volumes of ice-
cold 50 mM Tris-HCl buffer (pH 7.4) and centrifuged twice
at 20000g for 10 min. The pellet was reconstituted in 50
volumes of Tris-HCl buffer and was used for the binding
assay. Aliquots of 400 µL of tissue homogenate (0.4-0.5
mg of protein) were incubated in presence of [3H]fluni-
trazepam at a final concentration of 0.5 nM, in a total
incubation volume of 1000 µL. The drugs were added in
100 µL aliquots. After a 60 min incubation at 0 °C, the assay
was determined by rapid filtration through glass-fiber filter
strips (Whatman GF/B). The filters were rinsed with 2 to 4
mL portions of ice-cold Tris-HCl buffer as described above.
Radioactivity bound to the filters was quantitated by liquid
scintillation spectrometry. Nonspecific binding was deter-
mined as binding in the presence of 5 µM diazepam and
represented about 10% of total binding.
Stability Studies. Chemical Hydrolysis. The hydrolysis
of the Ara-C conjugates 3, 10 and 15 was studied at pH 7.4
in 0.05 M phosphate buffer at 37 ( 0.2 °C in a water bath.
The reaction was carried out by adding 100 µL of a stock
solution of the conjugates (5 mg/mL in methanol) to 5 mL
of the buffer solution preheated at 37 °C. The final
concentration of the compounds was about 1 × 10-5 M. The
resulting solutions were vortexed and maintained in a water
bath at constant temperature of 37 ( 0.2 °C. Aliquots of
400 µL were removed at appropriate intervals and either
immediately analyzed or frozen at -20 °C until analyzed
by HPLC. Each sample was filtered through a 0.2 µm
membrane filter (cellulose acetate) and then analyzed by
HPLC. Pseudo-first-order rate constants for the hydrolysis
of 3, 10 and 15 were determined from the slopes of linear
plots of the logarithms of residual starting material against
time.
Stability in Physiological Medium. The stability in physi-
ological medium of the Ara-C conjugates 3, 10 and 15 was
studied at 37 °C in 0.05 M phosphate buffer and 0.14 M
NaCl at pH 7.4, containing 50% v/v of human serum. The
reaction was carried out by adding 100 µL of the stock
solution of compound in methanol (5 mg/5 mL) to 1.6 mL
of preheated serum solution, and the mixture was maintained
in water bath at 37 ( 0.2 °C. Aliquots of 100 µL were
withdrawn at appropriate intervals and added to 500 µL of
cold acetonitrile in order to deproteinize the serum. After
mixing and centrifugation for 10 min at 2000 rpm, 20 µL of
the clear supernatant was analyzed by HPLC. Pseudo-first-
order rate constants for the degradation of compounds 3, 10
and 15 were determined from the slopes of linear plots of
the logarithms of remaining Ara-C conjugate against time.
Degradation products of Ara-C conjugates 3, 10 and 15 in
serum, were characterized by direct infusion of an aliquot
of deproteinized supernatant diluted 1:10 with blank by ESI
(both positive and negative mode).
BiologicalMethods.AdultmaleorfemaleSprague-Dawley
CD rats (Charles River, Como, Italy) with body masses of
200-250 g at the beginning the experiments were maintained
under an artificial 12 h light/dark cycle (light on 08.00 to
20.00 h) at a constant temperature of 23 ( 2 °C and 65%
humidity. Food and water were freely available, and the
animals were acclimatized for >7 days before use. Experi-
ments were performed between 08.00 and 14.00 h. Animal
care and handling througouth the experimental procedure
were performed in accordance with the European Communi-
ties Council Directive of 24 November 1986 (86/609/EEC).
The experimental protocol was approved by the Animal
Ethical Committee of the University of Cagliari (Italy).
In Vitro Receptor Binding Assays. After sacrifice the brain
was rapidly removed, the cerebral cortex was dissected and
tissues were stored at -80 °C until assay.
[3H]PK 11195 Binding. The tissues were thawed and
homogenized in 50 volumes of Dulbecco’s phosphate
buffered saline (PBS) pH 7.4 at 4 °C with a Polytron PT 10
(setting 5, for 20s). The homogenate was centrifuged at
40000g for 30 min, and the pellet was resuspended in 50
volumes of PBS and recentrifugated. The new pellet was
resuspended in 20 volumes of PBS and used for the assay.
[3H]PK 11195 binding was determined in a final volume of
1000 µL of tissue homogenate (0.15-0.20 mg protein), 100
µL of [3H]PK 11195 (sp act. 85.5 Ci/mmol, New England
Nuclear) at final assay concentration of 1 nM, 5 µL of drug
solution or solvent and 795 µL of PBS buffer (pH 7.4 at 25
°C). Incubations (25 °C) were initiated by addition of
membranes and were terminated 90 min later by rapid
filtration through glass-fiber filter strips (Wathaman GF/B),
which were rinsed with five 4 mL portions of ice-cold PBS
buffer using a cell harvester filtration manifold (Brandel).
Filter bound radioactivity was quantified by liquid scintil-
lation spectrometry. Nonspecific binding was defined as
binding in the presence of 10 µM unlabeled PK 11195
(Sigma).
Cytotoxicity Assays of Conjugates 3a-c, 10 and 15
against Human Glioma Cells Expressing High Levels of
TSPO. SF126, SF188, RG2, and C6 glioma cells expressing
high levels of TSPO were cultured in 96-well plates (1000
to 3000 cells/well) for 24 h prior to drug treatment. The wells
containing culture medium were utilized as control. After
the growth period, the cells were treated with either Ara-C
or one of the conjugates 3a-c, 10 and 15 at different
concentrations for 72 h. Cell lines were also treated with
vehicle (DMSO) only as controls. After the drug incubation
period cytotoxicity assays were performed with CCK-8
(Alexis Biochemicals) according to the instructions from the
manufacturer, followed by colorimetric measurements ob-
tained with a microplate reader.
Cytotoxicity Assays of Conjugate 15 and Ara-C against
C6 Glioma Cells in the Presence of Nucleoside Transport
Inhibitors. C6 glioma cells were cultured in Ham’s/F12
nutrient supplemented with 10% heat inactivated FBS, 2 mM
L-glutamine, 100 U/mL penicillin and 100 µg/mL strepto-
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Denora et al.
[drug]acceptor
mycin. Cells were seeded in 96-well plates at a density of
∼10000 cells/well, and, after 1 day of incubation at 37 °C
in a humidified atmosphere with 5% CO2, the culture medium
was replaced with the same volume of fresh complete
medium or with medium containing different concentrations
of the tested compounds in the presence or not of the
nucleoside transport inhibitor. The nucleoside transport
inhibitors S-(4-nitrobenzyl)-6-thioinosine (NBTI, Sigma-
Aldrich) and dipyridamole (Sigma-Aldrich) were used at
subtoxic levels (100 µM and 30 µM respectively), while
Ara-C and compound 15 were added at serial dilutions in
the appropriate range concentrations. Untreated cells were
used as positive control, and cells incubated with a 2% (w/
v) SDS solution were used as negative control. In each well,
the final volume was 200 µL. The cells were then allowed
to proliferate for 72 h at 37 °C in a humidified CO2-controlled
atmosphere. Cytotoxicity values (IC50) for 15 were deter-
mined using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay.38 At the end of the
incubation period, 10 µL of a 0.5% (w/v) MTT/PBS solution
was added to each well and the incubation was prolonged
for a further 4 h. Then, medium was removed and replaced
with 150 µL of a DMSO/ethanol (1:1) solution per well. The
absorbance of the individual well was measured by micro-
plate reader (Wallac Victor3, 1420 Multilabel Counter,
Perkin-Elmer). Each drug concentration was tested in
triplicate, and the experiments were repeated three times.
In Vitro Assays To Predict Compound 15 P-Glyco-
VA
Papp
)
×
(1)
(
)
(
)
area × time
[drug]initial
where VA is the volume in the acceptor well, area is the
surface area of the membrane, time is the total transport
time, [drug]acceptor is the concentration of the drug measured
by UV spectroscopy and [drug]initial is the initial drug
concentration in the AP or BL chamber.
Calcein AM Inhibition Assay. This experiment was carried
out as described by Feng et al.39 with minor modifications.
Madin-Darby canine kidney (i.e., MDCK) cells, retrovirally
transfected with the human MDR1 cDNA (MDCKII-MDR1)
(kindly provided by Prof. P. Borst, NKI-AVL Institute,
Amsterdam, The Netherlands), were cultured in DMEM high
glucose supplemented with 10% heat inactivated FBS, 2 mM
glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin
at 37 °C in a humidified 5% CO2 atmosphere. Cells were
seeded at a density of ∼50,000 cells per well into black 96-
well plates and allowed to become confluent overnight.
Compound 15 was added to monolayers in 100 µL of culture
medium and incubated at 37 °C for 30 min. Calcein AM
was added in 100 µL of PBS to yield a final concentration
of 2.5 µM, and the plate was incubated for a further 30 min.
Cells were then washed three times with ice-cold PBS. PBS
was added to each well, and the plate was read with a Victor3
fluorometer (Wallac Victor3, 1420 Multilabel Counter, Per-
kin-Elmer) at excitation and emission wavelengths of 485
and 535 nm, respectively. In these experimental conditions,
calcein cell accumulation, in the absence and in the presence
of the tested compound 15, was evaluated and fluorescence
basal level was estimated by untreated cells. In treated wells
the increase of fluorescence with respect to basal level was
measured. IC50 values were determined by fitting the
fluorescence increase percentage versus log[dose]. Elacridar,
a known inhibitor of P-gp, was used as control.40
P-gp ATPase ActiVity Assay. The experiment was per-
formed as reported in the technical sheet of ATPlite 1step
kit for luminescence ATP detection. MDCKII-MDR1 cells
were seeded into 96-well microplates in 100 µL of complete
medium at a density of 2 × 104 cells/well. The plate was
incubated overnight in a humidified atmosphere of 5% CO2
at 37 °C. Then, the medium was removed and 100 µL of
the complete medium in the presence or absence of different
concentrations of conjugate 15 ranging from 1 to 100 µM
was added. The plate was incubated for 2 h, and then 50 µL
of mammalian cell lysis solution was added to all wells and
the plate stirred for 5 min. In all wells, 50 µL of substrate
solution was added, the plate stirred for 5 min was dark
adapted for 10 min and in the end the luminescence was
measured by using a Victor3 luminometer (Wallac Victor3,
1420 Multilabel Counter, Perkin-Elmer).
39
protein Interaction. Monolayer Efflux Studies. Apical to
basolateral (Papp, AP) and basolateral to apical (Papp, BL)
permeability of conjugate 15 were measured using human
colonic carcinoma cells (Caco-2) monolayer grown on a
MultiScreen assay system (Millipore). After 21 days of cell
growth, the medium was removed from filter wells and from
the receiver plate. The formation of confluent Caco-2
monolayer with tight junctions was confirmed by TEER
values. The filter wells were filled with 75 µL of fresh Hanks’
balanced salt solution (HBSS) buffer and the receiver plate
with 250 µL per well of the same buffer. This procedure
was repeated twice, and the plates were incubated at 37
°C for 30 min. Compound 15 was dissolved in HBSS pH
7.4 and sterile filtered. For AP-to-BL or BL-to-AP flux
studies, the drug solution was added in the AP chamber
(75 µL) or in the BL chamber (250 µL), respectively. The
plates were incubated at 37 °C for 120 min. After
incubation time, samples were removed from the apical
and basolateral side of the monolayer and then analyzed
by UV spectroscopy. The apparent permeability, in units
of nm/s, was calculated using eq 1,
(38) Hansen, M.; Nielsen, S.; Berg, K. Re-examination and further
development of a precise and rapid dye method for measuring
cell growth/cell kill. Immunol. Methods 1989, 119, 203–210.
(39) Feng, B.; Mills, J. B.; Davidson, R. E.; Mireles, R. J.; Janiszewski,
J. S.; Troutman, M. D.; de Morais, S. M. In vitro P-glycoprotein
assays to predict the in vivo interactions of P-glycoprotein with
drugs in the central nervous system. Drug Metab. Dispos. 2008,
36, 268–275.
Transport Studies on 15 through MDCKII-MDR1
Monolayer. Transport studies on 15 were carried out using
MDCKII-MDR1 cells monolayer grown on a 12-well Tran-
(40) Hyafil, F.; Vergely, C.; Du Vignaud, P.; Grand-Perret, T. In vitro
and in vivo reversal of multidrug resistance by GF120918, an
acridonecarboxamide derivative. Cancer Res. 1993, 53, 4595–4602.
2262 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6

TSPO Ligand-Ara-C Conjugates
articles
Scheme 1a
swell insert (diameter 12 mm) as previously described.41 All
experiments were done at 37 °C. Medium was aspirated from
both the apical (AP) and basolateral (BL) chambers of each
insert, and cell monolayers were washed three times (10 min
per wash) with DPBS pH 7.4. The formation of confluent
MDCKII-MDR1 monolayer with tight junctions was con-
firmed by microscopy and TEER values. Diazepam (75 µM)
and fluorescein isothiocyanate dextran (FD4, Sigma) (200
µg/mL) were used as markers for the transcellular and
paracellular pathways, respectively. The assay medium was
as follows: 0.4 mM K2HPO4, 25 mM NaHCO3, 3 mM KCl,
122 mM NaCl, 10 mM glucose. The pH was 7.4, and the
osmolarity was 300 mOsm as determined by a freeze point
based osmometer. Except for FD4, which was solubilized
directly in the assay medium, the other compounds were first
dissolved in DMSO and then diluted with the assay medium
to a final concentration of 75 µM. Next, the tested solutions
were added to the donor side (0.5 mL for the AP chamber
and 1.5 mL for the BL chamber) and fresh assay medium
was placed in the receiver compartment. The percentage of
DMSO never exceeded 1% (v/v) in the samples. During a
period of 180 min, aliquots of 300 µL were withdrawn from
the receiver side at scheduled intervals, and replaced with
fresh assay medium to maintain sink conditions. Samples
were stored at -20 °C until further analysis. The apparent
permeability coefficient (Papp) was calculated according to
eq 2,
a (a) CDI, DMF.
VA × dC
area × [drug]initial × dt
Results
Papp
)
(2)
Synthetic Procedures. As shown in Scheme 1, the new
TSPO ligand-Ara-C conjugates 3a-c were prepared by
condensation of the corresponding imidazopyridine-acetic
acids 2a-c with Ara-C · HCl 1. The condensation was
successfully achieved by using CDI as dehydrating agent in
anhydrous THF (Scheme 1). The Ara-C conjugates 3a-c
were obtained in moderate to good yields and were fully
where VA × dc/dt is the linear appearance rate of mass in
the receiver solution, area is the filter/cell surface area and
[drug]initial is the initial tested compound concentration in the
AP or BL chamber.
Compound 15, diazepam and Ara-C samples were ana-
lyzed by HPLC as described above. The FD4 samples were
analyzed with a Victor3 fluorometer (Wallac Victor3, 1420
Multilabel Counter, Perkin-Elmer) at excitation and emission
wavelengths of 485 and 535 nm, respectively. Each com-
pound was tested in triplicate, and the experiments were
repeated three times. Following the described protocol,
transport studies on 15 were also carried out in the presence
of known P-gp inhibitors such as verapamil (50 and 100 µM)
and elacridar (2 µM).
Statistical Analysis. The statistical analysis was ac-
complished using one-way analysis of variance (ANOVA)
followed by the Tukey post hoc tests (GraphPad Prism
version 4 for Windows, GraphPad Software, San Diego, CA).
Differences were considered statistically significant at p <
0.05.
1
characterized by IR, H NMR, mass spectra, and elemental
1
analyses. An interesting feature of the H NMR spectra of
the Ara-C-imidazoacetamides 3a-c concerns the signals due
to the C(5)- and C(6)-protons in conjugates 3a-c. These
protons resonate at lower fields than the corresponding
protons of Ara-C (∆δ 1.04 ppm and 0.15 ppm, respectively).
This may be attributed to the deshielding effect of the N4-
carbonyl group in 3a-c.
To confirm the structural assignments done, we decided
to prepare 3a by an alternative and unambiguous synthesis
which involves the use of Boc protected Ara-C derivatives
as shown in Scheme 2. Treatment of 1 with DTBDC in
dioxane and successive alkalinization with KOH 1 N gave
a mixture of tri- and tetra-Boc protected Ara-C derivatives
(i.e., compounds 4 and 5). Actually, when DBDC was added
to a solution of 1 in dioxane-aqueous KOH, the product
profile changed over the time.42 To avoid the complete
(41) Denora, N.; Laquintana, V.; Lopedota, A.; Serra, M.; Dazzi, L.;
Biggio, G.; Pal, D.; Mitra, A. K.; Latrofa, A.; Trapani, G.; Liso,
G. Novel L-dopa and dopamine prodrugs containing a 2-phenyl-
imidazopyridine moiety. Pharm. Res. 2007, 24, 1309–1324.
(42) Guo, Z.; Gallo, J. M. Selective protection of 2′,2′-difluorodeoxy-
cytidine (Gemcitabine). J. Org. Chem. 1999, 64, 8319–8322.
VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 2263

articles
Denora et al.
Scheme 2a
transacylation reaction to give the intermediate primary
alcohol 4a, which, in turn, can be esterified with 2a, leading
to compound 6. Cleavage of the Boc groups in compound 7
was accomplished with TFA to give in almost quantitative
yield 3a identical in all the physicochemical features to that
prepared according to Scheme 1.
Preparation of conjugate 10 was accomplished according
to the synthetic sequences showed in Scheme 3. Treatment
of the TSPO ligand 822 with 4-nitrophenyl chloroformate in
anhydrous THF and in the presence of TEA gave the
corresponding carbonate 9, which in turn was condensed with
1, yielding the carbamate conjugate 10 in moderate yield.
In Scheme 4 is shown the preparation of conjugate 15
involving the reaction of the TSPO ligand 1120 with methyl
adipoyl chloride in the presence of K2CO3 to give the ester
12, which was hydrolyzed to the corresponding acid 13. To
obtain the selective amidification of the N4-amino group of
Ara-C 1, compound 13 was first activated with 2-mercap-
tothiazoline to yield the active thiazolidine thione 1435 and
then condensed with 1 in anhydrous pyridine at 50 °C to
produce the desired conjugate 15.
Hydrolysis in Buffer and Physiological Media. The
hydrolysis of the derivatives 3a-c, 10 and 15 was determined
in 0.05 M phosphate buffer at pH 7.4 as well as in 50%
(v/v) dilute human serum solution at 37 °C. All the
experiments were done in duplicate and half-lives were
measured by the disappearance of the Ara-C conjugate. All
the amide derivatives 3a-c were relatively stable in 0.05 M
phosphate buffer at pH 7.4 and their half-lives exceeding
13 h (Table 1). Conversely, compounds 3a-c were found
to be susceptible to enzyme-catalyzed hydrolysis in serum
with half-lives in the range of 2-4.6 h (Table 1).
As for conjugates 10 and 15 a marked difference in their
stability behavior was noted. In fact, compound 15 resulted
to be very stable in both buffer and physiological medium
with half-lives of 144 and 18 h, respectively, while conjugate
10 was found unstable in both conditions. It should be noted
that with the aim to prepare compounds hopefully character-
ized by a moderate stability, so as to have a balance between
systemic stability and rapid drug release, we attempted to
prepare compounds structurally similar to 15 but possessing
a shorter spacer. Thus, the known emisuccinate 1622 and
emiglutarate 1722 (Scheme 4), after activation reaction and
condensation with 1 under the same conditions used for 13,
gave large amounts of the corresponding imide compounds
18 and 19, respectively. To gain insights on the degradation
pathway of the conjugates 3a-c, 10 and 15, the main
degradation products in buffer and physiological medium
were examined by LC-mass spectrometry (LC-MS). Thus,
the ESI LC-MS analysis in negative mode of the mixture
obtained from the stability studies in physiological medium
of compound 3a after 24 h showed the presence of the Ara-C
(m/z 268.7) and the imidazopyridinemethyl ion (m/z 309)
but no trace of the starting material 3a (m/z 578). In contrast,
the ESI LC-MS analysis in negative mode for 10 and 15
after 24 h showed the presence of the Ara-C (m/z 268.7)
a (a) Dioxane-1 N aqueous KOH, DBDC; (b) DCC, HO-BZT; (c) TFA.
formation of the unreactive tetra-Boc protected compound
5, it is necessary to add the DBDC stepwise. Compounds 4
and 5 were obtained prolonging the reaction time for 16 h
at room temperature, and the mixture was separated by
column chromatography on silica gel. The condensation of
2a with 4 to give the desired compound 7 in 49% yield was
successfully achieved by using DCC and HO-BZT as a
dehydrating agent in CH2Cl2 at room temperature. Further-
more, formation of the isomer compound 6 was also found
to occur in 42% yield. Likewise compound 4 undergoes a
2264 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6

TSPO Ligand-Ara-C Conjugates
articles
Scheme 3a
a (a) 4-Nitrophenyl chloroformate, anhydrous THF, TEA; (b) DMF, TEA.
though less than Ara-C (Table 2). The high TSPO affinity
and selectivity observed for conjugate 15 coupled with
its relevant stability in both phosphate buffer and serum
supported an evaluation of its cytotoxicity profile against
glioma cell lines expressing high levels of TSPO. In
particular, conjugate 15 was tested against SF188, RG2
and C6 glioma cells, and the results observed are also
reported in Table 2. As can be seen, conjugate 15 resulted
in Vitro once again less cytotoxic than Ara-C against these
glioma cell lines. However, the appreciable lipophilicity
of 15 could enhance the BBB penetration by passive
diffusion independently of NT proteins and, hence, its
administration may overcome drug resistance in cancer
cells with a deficiency in NT.33 In addition, since the stable
conjugate 15 is characterized by high affinity and selectiv-
ity for TSPO, it may possess enhanced propensity to target
tumor cells in ViVo. Both these features of the Ara-C
derivative 15 could be advantageous to improve the
clinical potential of this nucleoside drug. Therefore, we
considered it of interest to evaluate the cytotoxicity of 15
and Ara-C against C6 glioma cells in the presence of
potent NT inhibitors as well as to assess the transport
properties across an in Vitro BBB model of the conjugate.
and the starting imidazopyridines 8 (m/z 419) and 11 (m/z
384), respectively.
Affinities of Imidazopyridine Derivatives for
Peripheral and Central Benzodiazepine Receptors. The
affinities of the conjugates 3a-c, 10 and 15 for CBR and
TSPO were evaluated by measuring their ability to compete
with [3H]flunitrazepam and [3H]PK 11195 binding, respec-
tively, to membrane preparations from rat cerebral cortex.
Their affinities were compared with those of unlabeled PK
11195. The measured binding affinities for CBR and TSPO
are shown in Table 2.
The analysis of the binding affinities of the entire set of
compounds 3a-c indicated that conjugates possessed low
selectivity and affinity, being in the micromolar range for
TSPO. Unlike conjugates 3, compounds 10 and 15 showed
high affinity and selectivity for TSPO (2.22 nM and 2.13
nM, respectively, Table 2).
Cytotoxicity Studies. The results of the cytotoxicity assays
of the Ara-C conjugates 3a-c, 10 and 15 conducted against
glioma cancer cells are also shown in Table 2. In this
regard, it should be noted that TSPO expression has been
well characterized in glioma cancer cell lines.43 The SF126
glioma cell line was the most resistant to the conjugates
3a-c, while compounds 10 and 15 resulted active even
(43) Yamasaki, T.; Kumata, K.; Yanamotoa, K.; Hatori, A.; Takei,
M.; Nakamura, Y.; Koike, S.; Ando, K.; Suzuki, K.; Zhang, M. R.
Imaging of peripheral-type benzodiazepine receptor in tumor: in
vitro binding and in vivo biodistribution of N-benzyl-N-[11C]m-
ethyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)ac-
etamide. Nucl. Med. Biol. 2009, 36, 801–809.
Cytotoxicity Assays of Conjugate 15 and Ara-C against
C6 Glioma Cells in the Presence of Nucleoside Transport
Inhibitors. The effects of compound 15 and Ara-C on C6
glioma cells were evaluated in the presence or absence of
the well-known NT inhibitors S-(4-nitrobenzyl)-6-thioinosine
VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 2265

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Scheme 4a
a (a) Methyl adipoyl chloride, K2CO3, anhydrous THF; (b) 100 nM NaOH/1,4-dioxane, 50° C; (c) EDC, DMAP, 2-mercaptothiazoline, CH2Cl2; (d) anhydrous
pyridine, 50° C.
(NBTI) and dipyridamole at subtoxic concentrations of 100
µM and 30 µM, respectively.44 The IC50 values of compound
15 and Ara-C, presented in Table 3, were 1.28 ( 0.06 and
0.005 ( 0.001 µM, respectively. Thus, the presence of the
transport inhibitors did not reverse the cytotoxic activity of
compound 15, while the cytotoxicity of Ara-C was reduced
of 1300-1400-fold by dipyridamole, whereas the inhibitory
effect of Ara-C on cell proliferation was reduced 200-300-
fold by NBTI. These results clearly demonstrated that the
transport of compound 15 is not affected by the presence of
NT inhibitors and, moreover, it may overcome the drug
resistance to Ara-C due to deficient NT systems.
In Vitro P-Glycoprotein Assays To Predict the
Interaction of P-gp with 15. It is important to establish if
a compound interacts with P-gp as a substrate, a modulator,
or an inhibitor. A wide range of methodologies have been
used to characterize drug interaction with P-gp. These
(44) Breistøl, K.; Balzarini, J.; Sandvold, M. L.; Myhren, F.; Martinsen,
M.; De Clercq, E.; Fodstad, Ø. Antitumor activity of P-4055
(elaidic acid-cytarabine) compared to cytarabine in metastatic and
s.c. human tumor xenograft models. Cancer Res. 1999, 59, 2944–
2949.
2266 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6

TSPO Ligand-Ara-C Conjugates
articles
Table 1. Lipophilicity and Chemical and Enzymatic
Stability of Conjugates 3a-c, 10, 15 and Ara-C
monolayer with functional tight junctions was confirmed by
microscopy, TEER values and flux of FD4 and diazepam.
The transport was followed for 3 h and the average MDCK
TEER value was approximately 800 Ω/cm2. The results
reported in Table 4 indicate that compound 15 is character-
ized by Papp value greater than the paracellular marker FD4
and Ara-C as well but lower than that of the transcellular
marker diazepam. In particular, compound 15 Papp value
resulted 7.8 times greater than free Ara-C and, in agreement
with its lipophilic character (Table 1, CLOGP 2.84), it should
overcome the BBB by the transcellular pathway. In addition,
it should be noted that when the transport experiments on
15 were repeated in the presence of well-known P-gp
inhibitors, such as verapamil and elacridar, no significant
difference in Papp values was observed.
b
t1/2 (h)
phosphate buffer
0.05 M, pH 7.4
diluted
serum
compd
CLOGPa
3a
3b
3c
2.72
2.00
13.5
16.4
30
4.5
2
2.00
4.6
c
10
3.34
0.5
15
2.84
144
18
Ara-C
-2.19
a Estimated according to ChemDraw Ultra 10.0 software.
b Based on the loss of starting material. c Unstable.
methods can employ intact cells or purified protein, and a
combination of different approaches are often required to
identify the mechanism of interaction. A number of P-gp
expressing cell lines can be used to assess the interaction
and transport of new chemical entities with the protein
including Caco-2 and MDCK. The Caco-2 cell line is well-
known to display high levels of P-gp, which plays an
important role in the efflux of drugs. Therefore, at first we
evaluated whether conjugate 15 is substrate, modulator or
inhibitor of P-gp using the calcein AM fluorescent, the
ATPase assays and the bidirectional transport study on
Caco-2 cell lines.39 Calcein AM is a lipophilic MDR1 and
MRP1 substrate able to cross the cell membrane. In the cell
compartment it is hydrolyzed by endogenous cytoplasmic
esterases yielding highly fluorescent calcein, which, in turn,
is not a MDR1 and MRP1 substrate, and it cannot cross the
cell membrane Via passive diffusion. Thus, a rapid increase
in the fluorescence of cytoplasmic calcein can be monitored.
As reported in Table 4, the observed IC50 value for 15 in
the calcein AM assay was 67 µM. In the P-gp ATPase
activity assay, drug-stimulated P-gp ATPase activity was
estimated by measuring the level of inorganic phosphate
released from ATP. In this assay it was found that conjugate
15 was unable to deplete ATP. Caco-2 monolayer grown on
permeable filters are commonly employed to characterize
P-gp substrates. The Papp values in both apical to basolateral
(Papp, AP) and basolateral to apical (Papp, BL) directions on
Caco-2 monolayer were used to evaluate the BL/AP ratio.
In these experiments carried out on conjugate 15, the BL/
AP ratio was found to be 9.6.
Discussion
The main aim of the present work was to synthesize TSPO
ligand-AraC conjugates and to evaluate their in Vitro
receptor binding, chemical stability, cytotoxic and transport
properties. For this purpose, the TSPO ligand-Ara-C
conjugates 3a-c, 10 and 15 were designed and prepared. In
particular, in conjugates 3a-c the hydrophilic Ara-C moiety
was introduced on the amide nitrogen at the 3-position of
the imidazopyridine nucleus, while the same moiety was
linked through appropriate spacers at 8- and at the para-
position of the 2-phenylimidazopyridine skeleton for 15 and
10, respectively. On the other hand, it must be noted that
3a-c, 10 and 15, as N4-acyl derivatives of Ara-C, should
display a metabolic stability against the cytosine nucleoside
deaminase greater than the parent drug. In fact, although
acylation of the N-amino group of Ara-C is known to be a
difficult task due to the low nucleophilicity of the aromatic
NH2, N4 derivation of Ara-C has been shown to prevent
inactivation by cytidine deaminase.36 In the case of 15 the
obstacle of the low nucleophilicity of the aromatic NH2 was
efficiently overcome by employing the intermediate acylthi-
azolidinethione 14, which is remarkably reactive and selec-
tive toward the N4-amino group.
The differences in binding affinity and selectivity observed
for the conjugates herein studied are consistent with the
structure-affinity relationship analysis22 of the 2-phenylimi-
dazo[1,2-a]pyridine derivatives that suggested the substitution
with three chlorine atoms on the imidazopyridine nucleus
would lead to a favorable interaction with the corresponding
complementary site of the receptor. However, it has been
pointed out that introduction of hydrophilic substituents (such
as Ara-C moiety) at the 8- and at the para-position of the
2-phenylimidazopyridine skeleton leads to compounds en-
dowed with high affinity and selectivity, and it can explain
the observed high receptor binding affinity and selectivity
of conjugates 10 and 15, whereas the presence of hydrophilic
substituents on the amide nitrogen at the 3-position of the
Transport Studies on 15 through MDCKII-MDR1
Monolayer. To obtain information on the ability of compound
15 to cross the BBB, transport studies involving MDCKII-
MDR1 monolayer were carried out. This approach constitutes
a well established in Vitro method to estimate drug perme-
ability across the BBB.45,46 On the other hand, MDCKII-
MDR1, as Caco-2 cell lines, are well-known to overexpress
P-gp. In these studies, the formation of confluent MDCK
(45) Garberg, P.; Ball, M.; Borg, N.; Cecchelli, R.; Fenart, L.; Hurst,
R. D.; Lindmark, T.; Mabondzo, A.; Nilsson, J. E.; Raub, T. J.;
¨
¨
Stanimirovic, D.; Terasaki, T.; Oberg, J. O.; Osterberg, T. In vitro
models for the blood-brain barrier. Toxicol. in Vitro 2005, 19,
299–334.
(46) Mensch, J.; Oyarzabal, J.; Mackie, C.; Augustijns, P. In vivo, in
vitro and in silico methods for small molecule transfer across BBB.
J. Pharm. Sci. 2009, 98, 4429–4468.
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Table 2. Affinity for Rat Cerebrocortical CBR and TSPO and Cytotoxicity against Glioma Cells of Compounds 3a-c, 10, 15
and Ara-C
receptor binding assays: IC50 (nM)a
cytotoxicity assays: IC50 (µM)a
compd
CBR
TSPO
SF126
SF188
RG2
C6
3a
3b
3c
10
15
(24%)b
(45%)b
(20%)b
(27%)b
2.22d
2.13d
e
ISc
2480
IS
6140
IS
>10000
>10000
e
6.90 ( 3.79
10.00 ( 2.77
1.25 ( 0.07
1.73 ( 0.85
3.00 ( 0.90
1.37 ( 0.64
Ara-C
0.003 ( 0.002
0.003 ( 0.003
0.005 ( 0.001
a Data are means ( SD of three separate experiments performed in duplicate (CV < 10%) and are reported in nM, unless otherwise
expressed. b Values in parentheses are the percentages of inhibition of specific [3H]flunitrazepam binding determined at 40 µM
concentration of the tested compound and of specific [3H]PK11195 at 1 × 10-5 M for CBR and TSPO affinity, respectively. c IS, insensitive.
d For comparison purpose the IC50 of the reference TSPO ligand is reported and namely 2.1 nM. e No receptor binding affinity, predicted
IC50 > 10000 nM.
Table 3. Effects of Nucleoside Transport Inhibition on the
in Vitro Cytotoxic Effect of Cytarabine Hydrochloride and
Compound 15 on C6 Glioma Cell Line
examined by LC-MS, does not satisfactorily explain the
cytotoxicity of 10 being much lower than that of Ara-C. On
the other hand, the stability of 15 being greater than that of
10 may also account for the lower cytotoxicity of the former
against the same glioma cell line. In our opinion, the
cytotoxicity data can be rationalized assuming that the trend
observed should be mainly due to a different intracellular
uptake and activation by cellular enzymes in combination
with the hydrolysis pathway to give the free drug, the latter
occurring to a lower extent. Definitive conclusions in this
regard can be reached after a detailed study on the conju-
gates’ degradation kinetics.
IC50 (µM)
nucleoside transport
inhibitor
Ara-C
compd 15
without inhibitor
+NBTIa
+dipyridamoleb
0.005 ( 0.001
1.07 ( 0.46
7.08 ( 0.64
1.28 ( 0.06
0.85 ( 0.05
1.34 ( 0.02
a NBTI was used at
a subtoxic concentration of 100 µM.
b Dipyridamole was used at a subtoxic concentration of 30 µM.
The studies aimed to predict the interaction of P-gp with
15 were motivated by the fact that P-gp serves as a drug
efflux pump on the BBB and, possibly, on tumor cells,
whereby it may act on lipophilic substrates. A variety of in
Vitro assays have been used to classify compounds as P-gp
substrates, modulators or inhibitors. Substrates are molecules
actively transported by the protein and therefore have a
higher concentration outside the cell with respect to the
cytosol. Modulators interact at the binding sites, therefore
reducing substrate binding through a negative allosteric
interaction. Inhibitors interfere with the substrate thereby
blocking P-gp translocation.47 Modulators and inhibitors
exert the same final biological effect, restoring cell sensitivity
to chemotherapeutic agents, and have been the subject of
numerous investigations.47 The calcein AM fluorescent, the
ATPase assays and the bidirectional transport study on
Caco-2 cell line are well established for classification
purposes.39,47 Results from these assays conducted on 15,
taken together, indicate that the conjugate 15 behaves as a
clear P-gp modulator.47 In fact, it has been recently evidenced
that a BL/AP ratio from 18 to 20 and a ratio <2 identifies
substrates and inhibitors, respectively, while modulators show
intermediate ratios ranging from 2 to 18.47 The BL/AP ratio
of 9.6 allow us to classify 15 as a P-gp modulator. On the
other hand, the fact that the Papp values observed for 15 are
imidazopyridine nucleus was shown detrimental for affinity
and selectivity as observed for compounds 3.20-22
As for the results of stability studies, particularly those
aimed to gain information on the main degradation products
in buffer and physiological medium by LC-MS, it should
be pointed out that the analytical approach followed provides
only qualitative information. So, the actual degradation
pathway(s) is (are) unknown and the extent of the 3a-c, 10
and 15 cleavage at the amide bond level leading to free Ara-C
is unknown as well. The marked decrease in cytotoxicity of
conjugates should be mainly due to an intracellular uptake
of conjugates and 5′-monophosphate activation by deoxy-
cytidine kinase lower than what occurs for the free drug. In
fact, Ara-C is able to enter cells by NT, but we do not know
if such a process could account for transport of an intact
conjugate. The alternative explanation that 3a-c, 10 and 15
toxicity is connected with their hydrolysis to give the free
Ara-C is not in agreement with the stability data because it
is to be expected the cytotoxicity of the essentially unstable
conjugate 10 should be comparable to or even better than
that of free drug. In fact, the hydrolysis of compound 10
produces also compound 8. This TSPO ligand is a proapo-
ptotic agent which, in combination with Ara-C, may cause
a synergistic growth inhibition of cancer cells. Such an effect
may also occur for compound 15 but not for compounds
3a-c, which led to the formation of compounds 2a-c
endowed with weak TSPO affinity and proapoptotic activity.
Therefore, the pathway that 3a-c, 10 and 15 toxicity is
connected with their hydrolysis to give the free Ara-C, as
suggested by the presence of the free drug in the mixture
(47) Colabufo, N. A.; Berardi, F.; Cantore, M.; Contino, M.; Inglese,
C.; Niso, M.; Perrone, R. Perspectives of P-glycoprotein modulat-
ing agents in oncology and neurodegenerative diseases: pharma-
ceutical, biological, and diagnostic potentials. J. Med. Chem. 2010,
53, 1883–1897.
2268 MOLECULAR PHARMACEUTICS VOL. 7, NO. 6

TSPO Ligand-Ara-C Conjugates
articles
Table 4. Transport across MDCKII-MDR1 Cells and on Compound 15 and Evaluation of Its Interaction with P-Glycoprotein
by Three Specific Biological Assays
transport study on MDCKII-MDR1a
P-gp interaction specific biological assaysa
compd
15
Ara-C
Papp (AP) (cm/s)
Papp (BL) (cm/s)
2.21 ( 0.48 × 10-5
Papp (BL/AP)
9.6c
calcein IC50 (µM)
ATPase activation
no
6.01 ( 1.23 × 10-6 (3.7)b
7.72 ( 1.78 × 10-7
5.09 ( 0.52 × 10-5
2.96 ( 0.45 × 10-10
67d
diazepam
FD4
a Data are means ( SD of three determination. b The value in parentheses is the permeability BL/AP ratio. c The Papp in both AP and BL
directions was determined on Caco-2 monolayer. d Elacridar, a known inhibitor of P-gp, was used as the positive control (IC50 0.01 µM).
not significantly influenced by the presence of well-known
P-gp inhibitors, such as verapamil and elacridar, confirms
that this compound is not a P-gp substrate.
across the MDCKII-MDR1 monolayer indicated that
conjugate 15 should overcome the BBB by transcellular
pathway. Therefore, based on the in Vitro properties of
conjugate 15, which consisted of its high affinity and
selectivity for TPSO combined and chemical and meta-
bolic stability, its further evaluation in ViVo as a means
to target brain tumors is warranted.
In conclusion, synthetic routes toward imidazopyridine-
TSPO ligand-Ara-C conjugates 3, 10 and 15 have been
developed. These new compounds displayed variable in
Vitro TSPO binding ranging from low/moderate (3a-c)
to high (10 and 15) affinity and selectivity. Receptor
binding affinity data combined with the stability results
induced us to focus our attention on compound 15. First
of all, as N4-acyl derivative of Ara-C should be resistant
to inactivation by cytidine deaminase. The evaluation of
its cytotoxicity profile against further glioma cell lines
expressing high levels of TSPO showed an activity less
than that of Ara-C. However, in contrast to that observed
for free parent drug, the presence of NT inhibitors did
not affect the cytotoxic activity of 15. In Vitro studies
suggest that 15 behaves as a clear P-gp modulator and
thereby may be useful to reverse MDR. Transport studies
Acknowledgment. This work was supported by a grant
from Ministero dell’Universita` e della Ricerca (PRIN 2004
and 2006 MIUR to G.T.). Thanks are due to Prof. Giovanni
Biggio (University of Cagliari, Italy) and Dr. Steven Johnson
(University of Pennsylvania, Philadelphia, PA) for their help
in receptor binding and cytotoxicity studies, respectively. We
thank Mr. Giovanni Dipinto and Mr. Antonio Palermo for
their skillful technical assistance in recording mass spectra
and NMR spectra, respectively.
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