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

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

proposed¹are 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).²The 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.³The 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.⁴There is

growing experimental evidence suggesting that TSPOs are

involved in the regulation of cholesterol transport into

mitochondria and in steroidogenesis⁵as well as in various

neurological diseases such as Alzheimer’s disease, Hunting-

ton’s disease, epilepsy and stroke-induced brain injury.^6,7It

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.^3,8-11Moreover, there is evidence

indicating TSPO-speciﬁc ligands induce apoptosis and cell

cycle arrest in cancer cells,^3,12-15which 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),¹⁶isoquinolines (PK-11195),¹⁷2-aryl-3-indoleaceta-

mides (FGIN-1-27),¹⁸and 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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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.²⁴The

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 BBB²⁷and, 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.³⁰AraC has also been part of

combination chemotherapeutic regimens for various brain

tumors including primary CNS lymphoma, and recurrent or

refractory malignant gliomas.³¹In 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).

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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.²³Moreover, 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,^24-26it is of

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H. S.; Higgins, P. E.; Song, D.; Gallo, J. M. Modulation of

melphalan resistance in glioma cells with a peripheral benzodi-

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receptor ligand-melphalan conjugates for potential selective drug

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acid ester prodrugs of 1-ꢀ-^D-arabinofuranosylcytosine. Mol.

Pharmaceutics 2009, 6, 315–325.

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A.; Latrofa, A.; Franco, M.; Serra, M.; Pisu, M. G.; Floris, I.;

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and effects on neuroactive steroids in a series of 2-phenylimidazo-

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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.³³The 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.^34-37In

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.

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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 Na₂SO₄and 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^-5M. 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-

VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 2261

articles

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.³⁸At 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-

V_A

P_app

)

×

(1)

(

)

(

)

area × time

[drug]_initial

where V_Ais the volume in the acceptor well, area is the

surface area of the membrane, time is the total transport

time, [drug]_acceptoris the concentration of the drug measured

by UV spectroscopy and [drug]_initialis 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.³⁹with 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% CO₂atmosphere. 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. IC₅₀values 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 × 10⁴cells/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 Victor³luminometer (Wallac Victor³,

1420 Multilabel Counter, Perkin-Elmer).

39

protein Interaction. Monolayer Efﬂux Studies. Apical to

basolateral (P_app, AP) and basolateral to apical (P_app, 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 1^a

swell insert (diameter 12 mm) as previously described.⁴¹All

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 (P_app) was calculated according to

eq 2,

^a(a) CDI, DMF.

V_A× dC

area × [drug]_initial× dt

Results

P_app

)

(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 V_A× dc/dt is the linear appearance rate of mass in

the receiver solution, area is the ﬁlter/cell surface area and

[drug]_initialis 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 Victor³ﬂuorometer (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.⁴²To 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 2^a

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 8²²with 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 11²⁰with methyl

adipoyl chloride in the presence of K₂CO₃to 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 14³⁵and

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 16²²and

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 CH₂Cl₂at 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 3^a

^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.³³In 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.⁴³The 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

articles

Denora et al.

Scheme 4^a

^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.⁴⁴The IC₅₀values 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 P_appvalue greater than the paracellular marker FD4

and Ara-C as well but lower than that of the transcellular

marker diazepam. In particular, compound 15 P_appvalue

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 P_appvalues was observed.

b

t_1/2(h)

phosphate buffer

0.05 M, pH 7.4

diluted

serum

compd

CLOGP^a

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

^aEstimated according to ChemDraw Ultra 10.0 software.

^bBased on the loss of starting material. ^cUnstable.

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.³⁹Calcein 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 IC₅₀value 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 P_appvalues in both apical to basolateral

(P_app, AP) and basolateral to apical (P_app, 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, N⁴derivation of Ara-C has been shown to prevent

inactivation by cytidine deaminase.³⁶In the case of 15 the

obstacle of the low nucleophilicity of the aromatic NH₂was

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 analysis²²of 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.^45,46On 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.

VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 2267

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Denora et al.

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

2.22^d

2.13^d

e

IS^c

2480

IS

6140

IS

>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

^aData are means ( SD of three separate experiments performed in duplicate (CV < 10%) and are reported in nM, unless otherwise

expressed. ^bValues 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^-5M for CBR and TSPO afﬁnity, respectively. ^cIS, insensitive.

^dFor comparison purpose the IC₅₀of the reference TSPO ligand is reported and namely 2.1 nM. ^eNo 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

+NBTI^a

+dipyridamole^b

0.005 ( 0.001

1.07 ( 0.46

7.08 ( 0.64

1.28 ( 0.06

0.85 ( 0.05

1.34 ( 0.02

^aNBTI was used at

a subtoxic concentration of 100 µM.

^bDipyridamole 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.⁴⁷Modulators and inhibitors

exert the same ﬁnal biological effect, restoring cell sensitivity

to chemotherapeutic agents, and have been the subject of

numerous investigations.⁴⁷The calcein AM ﬂuorescent, the

ATPase assays and the bidirectional transport study on

Caco-2 cell line are well established for classiﬁcation

purposes.^39,47Results from these assays conducted on 15,

taken together, indicate that the conjugate 15 behaves as a

clear P-gp modulator.⁴⁷In 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 18.⁴⁷The BL/AP ratio

of 9.6 allow us to classify 15 as a P-gp modulator. On the

other hand, the fact that the P_appvalues 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-MDR1^a

P-gp interaction speciﬁc biological assays^a

compd

15

Ara-C

P_app(AP) (cm/s)

P_app(BL) (cm/s)

2.21 ( 0.48 × 10^-5

P_app(BL/AP)

9.6^c

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

67^d

diazepam

FD4

^aData are means ( SD of three determination. ^bThe value in parentheses is the permeability BL/AP ratio. ^cThe P_appin both AP and BL

directions was determined on Caco-2 monolayer. ^dElacridar, a known inhibitor of P-gp, was used as the positive control (IC₅₀0.01 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.

MP100235W

VOL. 7, NO. 6 MOLECULAR PHARMACEUTICS 2269

Article Doi

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

DOI: 10.1021/mp100235w

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