Novel codrugs with GABAergic activity for dopamine delivery in the brain

Article abstract of DOI:10.1016/j.ijpharm.2012.08.023

This study investigates the use of codrugs of the GABAergic agent 2-phenyl-imidazo[1,2-a]pyridinacetamide and dopamine (DA) or ethyl ester L-Dopa (LD) as a strategy to deliver DA and simultaneously activate GABA-receptors in the brain. For this purpose, both DA and LD ethyl ester were linked by carbamate bond to imidazo[1,2-a]pyridine acetamide moieties to yield two DA- and two LD-imidazopyridine derivatives. These compounds were evaluated in vitro to assess their stability, binding affinities and cell membrane transport, and in vivo to assess their bio-availability via microdialysis studies. The two DA derivatives were adequately stable in buffered solution, but underwent cleavage in diluted human serum. By contrast, the LD derivatives were unstable in buffered solution. Receptor binding studies showed that the DA-imidazopyridine carbamates had binding affinity for benzodiazepine receptors in the nanomolar range. Brain microdialysis experiments indicated that intraperitoneal administration of the DA derivatives sustained DA levels in rat striatum over a 4-h period. These results suggest that DA-imidazopyridine carbamates are new DA codrugs with potential application for DA replacement therapy.

Full text of DOI:10.1016/j.ijpharm.2012.08.023

International Journal of Pharmaceutics 437 (2012) 221–231
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Novel codrugs with GABAergic activity for dopamine delivery in the brain
Nunzio Denoraa,1 , Tommaso Cassanob,1 , Valentino Laquintanaa , Antonio Lopalcoa , Adriana Trapania ,
Concetta Stefania Cimminob, Leonardo Laconcab, Andrea Giuffridac, Giuseppe Trapania,∗
a Department of Pharmacy, University of Bari, Bari 70125, Italy
b Department of Clinical and Experimental Medicine, University of Foggia, Foggia 71100, Italy
c Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 April 2012
Received in revised form 13 August 2012
Accepted 16 August 2012
Available online 23 August 2012
This study investigates the use of codrugs of the GABAergic agent 2-phenyl-imidazo[1,2-
a]pyridinacetamide and dopamine (DA) or ethyl ester L-Dopa (LD) as a strategy to deliver DA and
simultaneously activate GABA-receptors in the brain. For this purpose, both DA and LD ethyl ester were
linked by carbamate bond to imidazo[1,2-a]pyridine acetamide moieties to yield two DA- and two LD-
imidazopyridine derivatives. These compounds were evaluated in vitro to assess their stability, binding
affinities and cell membrane transport, and in vivo to assess their bio-availability via microdialysis studies.
The two DA derivatives were adequately stable in buffered solution, but underwent cleavage in diluted
human serum. By contrast, the LD derivatives were unstable in buffered solution. Receptor binding stud-
ies showed that the DA-imidazopyridine carbamates had binding affinity for benzodiazepine receptors
in the nanomolar range. Brain microdialysis experiments indicated that intraperitoneal administration
of the DA derivatives sustained DA levels in rat striatum over a 4-h period.
Keywords:
Blood–brain barrier
Codrugs
Drug targeting
MDCKII-MDR1 cell
Microdialysis
P-glycoprotein
These results suggest that DA-imidazopyridine carbamates are new DA codrugs with potential appli-
cation for DA replacement therapy.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
2009; Stella et al., 2007; Rautio et al., 2008a) to enhance the brain
concentration of CNS active drugs is the development of their corre-
Many substances acting in the central nervous system (CNS),
e.g., dopamine (DA), do not cross the blood–brain barrier (BBB)
due to their unfavourable physicochemical properties and/or the
absence of specific transporters within the brain capillaries to carry
them across the BBB. Only active molecules with low molecular
weight and high lipophilicity can cross the BBB by passive diffu-
sion (Pardridge, 2001). A well-established strategy (Denora et al.,
sponding prodrugs. To be effective, a CNS-targeted prodrug should
have optimal physicochemical characteristics enabling passive dif-
fusion, or serve as a substrate for one of the influx transport proteins
expressed at the BBB (Rautio et al., 2008b; Trapani et al., 2012). For
example L-Dopa (LD) is a prodrug of DA taken up to the brain by the
neutral amino acid transporter 1 (LAT1), which is highly expressed
at the BBB. Once in the brain, LD is rapidly converted into DA by
aromatic amino-acid decarboxylase (Nutt and Woodward, 1986).
Since its introduction in the late 1960s, LD remains the gold
standard of Parkinson’s disease (PD) pharmacotherapy (Warren
Olanow, 2008). However, chronic administration of LD leads to the
development of motor complications, e.g., motor fluctuations and
dyskinesias, in the majority of PD patients (Ahlskog and Muenter,
2001). The molecular mechanisms underlying the development of
these motor complications remain poorly understood, and several
factors including pulsatile stimulation of DA receptors (Cenci, 2007)
and reduced GABAergic output to the thalamus (Daniele et al.,
1997) have been shown to play a role. In this context, drugs affecting
␥-aminobutyric acid (GABA) transmission could play a favourable
role given their ability to modulate the activity of mesocortical
and mesolimbic dopaminergic neurons (Jankovic and Marsden,
1988). As a result, selective GABAergic agonists, such as the 2-
phenyl-imidazopyridinacetamide compound Zolpidem, has shown
Abbreviations: ANOVA, analysis of variance; BBB, blood–brain barrier; BBMECs,
bovine brain microvessel endothelial cells; CBR, central benzodiazepine Recep-
tor; CDI, 1,1ꢀ-carbonyldiimidazole; CNS, central nervous system; DA, dopamine;
DMSO, dimethylsulfoxide; ESI, electrospray ionization mass spectrometry; GABA␥,
-aminobutyric acid; HPLCh, igh-performance liquid chromatography; LAT1, neu-
tral amino-acid transporter 1; LC–MS, LC–mass spectrometry; LD, L-Dopa; MDCK,
Madin–Darby canine kidney cells; MDCKII-MDR1, Madin–Darby canine kidney cells
retrovirally transfected with the human MDR1 cDNA; MFB, medial forebrain bundle;
PSA, polar surface area; TEA, triethylamine; TFA, trifluoroacetic acid; THF, anhy-
drous tetrahydrofurane; TPSA, topological PSA; TSPO, translocator protein 18 kDa;
6-OHDA, 6-hydroxydopamine.
∗
Corresponding author at: Dipartimento di Farmacia, Università degli Studi di
Bari, Via Orabona 4, 70125 Bari, Italy. Tel.: +39 080 5442764; fax: +39 080 5442724.
E-mail addresses: giuseppe.trapani@uniba.it, trapani@farmchim.uniba.it
(G. Trapani).
1
These are co-first authors.
0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2012.08.023

222
N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
beneficial effects in patients undergoing long-term LD treatment
(Daniele et al., 1997; Jankovic and Marsden, 1988).
results were within 0.40% of the theoretical values. Silica gel
60 (Merck 70–230 or 230–400 mesh) was used for column chro-
matography. All the following reactions were performed under a
nitrogen atmosphere and the progress of the reactions was mon-
itored by thin-layer chromatography (TLC) by using Kieselgel 60
F254 (Merck) plates.
We have recently reported that linking DA and LD ethyl ester
to 2-phenylimidazopyridine moieties yields the compounds 1–3
(Table 1), which increase cortical DA output and act as novel DA
and LD prodrugs (Denora et al., 2007). These prodrugs were engi-
neered to take advantage of both the GABAergic agonism, shown by
most phenyl-imidazopyridinacetamide compounds, such as Alpi-
dem and Zolpidem (Table 1), and the lipophilic nature of the
imidazopyridine moiety. It is noteworthy that the toxicity of these
compounds mainly depends on the substitution on the imidazopy-
ridinacetamide nucleus. Therefore, despite the similar chemical
structures, Alpidem, but not Zolpidem, has shown hepatotoxic
effects and consequently it has been withdrawn from the market
(Berson et al., 2001).
To investigate the possibility to deliver DA in the brain while
simultaneously increasing GABAergic transmission, we studied
compounds 4–7 (Table 1), in which DA and LD are linked through a
carbamate bond at the para position of the phenyl group on the imi-
dazopyridinacetamide nucleus. These conjugates are structurally
very different from compounds 1–3, in which DA and LD are linked
by the amide nitrogen of the imidazopyridinacetamide structure.
Based on previous structure-affinity relationship studies (Trapani
et al., 2005), compounds 4–7 are expected to have affinity for GABA-
receptors, unlike compounds 1–3 previously studied.
In this work, we combined, by reversible covalent-bonding, DA
12 and LD ethyl ester 13 with appropriate 2-phenyl-imidazo[1,2-
a]pyridine acetamides 8 and 9 to give compounds 4–7. Compounds
8 or 9 were treated in anhydrous THF with bis-(4-nitrophenyl) car-
bonate and TEA to give the intermediates 10 or 11, respectively
(Fig. 1). Then, addition of a DA hydrochloride solution (12) or LD
ethyl ester hydrochloride (13) in anhydrous DMF to a solution of
the suitable intermediate (10 or 11) in anhydrous THF yielded the
desired DA derivatives 4 and 5 and the LD ethyl ester compounds
6 and 7, respectively, in moderate amount (Table 1). The starting
imidazopyridinacetamide 8 was prepared according to synthetic
procedures reported elsewhere (Denora et al., 2008), while the
imidazopyridinacetamide 9 was synthesized according to a previ-
ously reported procedure with slight modifications (Fig. 2) (Denora
et al., 2008). In particular, condensation in n-butanol of 5-cholro-2-
amino-pyridine 14 with the butyl-bromoketo ester 15 yielded the
butyl imidazopyridine acetates 16. Compound 16 was hydrolyzed
with NaOH 1 N in n-butanol to give the corresponding acid 17. The
demethylation of the 2-p-methoxyphenylimidazopyridine acetic
acid 17 was accomplished with HBr (48%, w/w) at reflux, giving rise
to the corresponding 2-p-hydroxyphenyl acid 18. The subsequent
condensation of this last compound with the suitable dialkylamine
hydrochloride in anhydrous THF in the presence of CDI gave com-
pound 9. All synthesized compounds were fully characterized by
FT-IR, 1H NMR and mass spectroscopy.
In this study, we report the synthesis, stability, binding affin-
ity and transport of compounds 4–7 and discuss their in vivo
effects on striatal dopaminergic neurotransmission. In addition, we
investigated the pharmacological actions of compounds 4 and 5
in 6-hydroxydopamine (6-OHDA)-lesioned rats, a well-established
animal model of PD.
2.3. General procedure for preparation of the dopamine
derivatives 4 and 5
2. Materials and methods
2.1. Materials
Bis-(4-nitrophenyl) carbonate (2.0 mmol) and TEA (1.2 mmol)
were added to a stirred solution of compound 8 (0.42 g, 1.0 mmol)
or 9 (0.40 g, 1.0 mmol) in anhydrous THF (30 ml), and stirring was
prolonged for 4 h at 50 ◦C under nitrogen atmosphere. The reac-
tion progress was monitored by TLC [light petroleum ether/ethyl
acetate 1/1 (v/v) as eluent] and, at its completion, solvent was
evaporated under reduced pressure, the residue was taken up with
20 ml of HCl 0.1 N, extracted with ethyl acetate (3× 20 ml) and
dried (Na2SO4) to give the intermediates 10 or 11, respectively,
which were characterized without further purification by FT-IR,
1H NMR and LC–MS analysis. Next, a solution of DA hydrochlo-
ride (12) (0.20 g, 1.0 mmol) in anhydrous DMF (2.0 ml) was added
drop wise into a stirred solution of the appropriate crude inter-
mediate (0.9 mmol) dissolved in 10 ml of anhydrous THF. The
mixture was stirred at room temperature (rt) for 12 h. Solvent
was evaporated under reduced pressure, and the residue was dis-
solved in CHCl3 (20 ml), washed with water (3× 30 ml) and dried
(Na2SO4). Evaporation of the solvent gave a residue, which was
purified by silica gel chromatography [light petroleum ether/ethyl
acetate 3/7 (v/v) as eluent] to give the desired compounds
4 or 5.
Compounds
N,N-di-n-propyl-[2-(6,8-dichloro-2-(4-
and
hydroxyphenyl)imidazo[1,2-a]pyridin-3-yl)]acetamide
8
the LD ethyl ester derivative 13 (Fig. 1) were prepared accord-
ing to synthetic procedures reported elsewhere (Denora et al.,
2007, 2008). Compound N,N-di-n-propyl-[2-(6-chloro-2-(4-
hydroxyphenyl)imidazo[1,2-a]pyridin-3-yl)]acetamide 9 (Fig. 1)
was synthesized according to a previously reported procedure
with slightly modifications (Denora et al., 2008). The starting DA
hydrochloride 12, bis-(4-nitrophenyl) carbonate, triethylamine
(TEA), anhydrous tetrahydrofurane (THF), dimethylsulfoxide
(DMSO), 1,1ꢀ-carbonyldiimidazole (CDI), 5-chloro-2-amino-
pyridine, trifluoroacetic acid (TFA), human serum, Rhodamine
123, Calcein AM, and other chemicals were purchased from
Sigma–Aldrich (Milan, Italy). Chemicals and solvents were used
as received. Equithesin was prepared as previously described
(Morgese et al., 2009). Desipramine hydrochloride, LD methyl
ester, 6-OHDA hydrochloride, benserazide and amphetamine were
purchased from Sigma–Aldrich (Milan, Italy).
2.2. Synthetic procedures
4-(6,8-Dichloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenyl-3,4-dihydroxyphenethylcarbamate (4): IR
Melting points were determined in open capillary tubes with a
Büchi apparatus and were uncorrected. FT-IR analyses were carried
out on dried powder samples by using KBr discs and a Perkin-Elmer
1600 FT-IR spectrophotometer (Spectrum One). 1H NMR spectra
were determined on a Varian VX Mercury operating at 300 MHz
instrument. Mass spectra were recorded on a Hewlett–Packard
5995c GC–MS low-resolution spectrometer. Elemental analyses
were carried out with a Hewlett–Packard 185 C, H, N analyzer and
(KBr): 3400, 1730, 1630 cm−1 1H NMR (DMSO-d6) ı: 0.7–0.9
;
(m, 6H, CH3), 1.4–1.7 (m, 4H, CH2), 2.59 (t, J = 7.7 Hz, 2H, CH2Ar),
3.2–3.4 (m, 6H, CH2N and CH2NHCO), 4.27 (s, 2H, CH2CO), 6.4–6.6
(m, 3H Ar), 7.17 (d, J = 8.5 Hz, 2H, Ar), 7.59 (d, J = 8.5 Hz, 2H, Ar),
7.65 (d, J = 1.6 Hz, 1H, Ar), 8.59 (d, J = 1.6 Hz, 1H, Ar); MS (ESI) m/z
597.0 [M−H]−; Anal. (C30H32Cl2N4O5) C, H, N.
4-(6-Chloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenyl-3,4-dihydroxyphenethylcarbamate (5): IR

N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
223
Table 1
Chemical structures of Alpidem and Zolpidem as well as melting points and yields of compounds 1–7.
N
N
Cl
CH3
N
N
Cl
H3C
O
O
N
N
Alpidem
Zolpidem
N
Y
Y
N
O
O
Cl
N
N
NH
R
X
X
O
O
R
N
NH
OH
OH
OH
OH
1-3
4-7
Compound
X
Y
R
MW (Da)
Mp (◦C)
Yield (%)
1a
2a
3a
4
5
6
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
H
COOCH2CH3
COOCH2CH3
H
H
561
527
489
598
564
670
636
107–110
210 dec.
147–150
89–91
83–86
74–76
63
85
32
28
35
29
26
H
Cl
H
COOCH2CH3
COOCH2CH3
7
73–76
a
Ref. Denora et al. (2007).
(KBr): 3400, 1735, 1630 cm−1 1H NMR (DMSO-d6) ı: 0.7–0.9
;
and the residue was dissolved in CHCl3 (20 ml), washed with
water (3× 30 ml) and dried (Na2SO4). Evaporation of the solvent
gave a residue, which was purified by silica gel chromatography
[light petroleum ether/ethyl acetate 3/7 (v/v) as eluent] to give the
desired compounds 6 and 7.
(m, 6H, CH3), 1.4–1.7 (m, 4H, CH2), 2.59 (t, J = 7.7 Hz, 2H, CH2Ar),
3.2–3.4 (m, 6H, CH2N and CH2NHCO), 4.24 (s, 2H, CH2CO), 6.4–6.6
(m, 3H Ar), 7.15 (d, J = 8.5 Hz, 2H, Ar), 7.2–7.4 (m, 1H, Ar), 7.5–8.0
(m, 3H, Ar), 8.4–8.6 (m, 1H, Ar); MS (ESI) m/z 563.0 [M−H]−; Anal.
(C30H33ClN4O5) C, H, N.
Ethyl-2-((4-(6,8-dichloro-3-(2-(dipropylamino)-2-
oxoethyl)imidazo[1,2-a]pyridine-2-yl)phenoxy)carbonylamino)-
3-(3,4-dihydroxyphenyl)propanoate (6): IR (KBr): 3400, 1730,
4-(6,8-Dichloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenyl 4-nitrophenyl carbonate (10): IR (KBr): 1780,
1650 cm−1 1H NMR (DMSO-d6) ı: 0.7–0.9 (m, 6H, CH3), 1.2–1.4
;
1740, 1620 cm−1 1H NMR (CDCl3) ı: 0.8–1.0 (m, 6H, CH3), 1.4–1.6
;
(m, 3H, CH3), 1.4–1.7 (m, 4H, CH2), 2.6–2.8 (m, 2H, CH2Ar), 2.8–3.4
(m, 4H, CH2N), 3.77 (s, 2H, CH2CO), 3.9–4.4 (m, 3H, CHN and
CH2COO), 6.4–6.6 (m, 3H Ar), 6.82 (d, J = 8.5 Hz, 2H, Ar), 7.0–7.7
(m, 3H, Ar), 8.56 (m, 1H, Ar); MS (ESI) m/z 668.8 [M−H]−; Anal.
(C33H36Cl2N4O7) C, H, N.
(m, 4H, CH2), 3.2–3.4 (m, 4H, CH2N), 4.20 (s, 2H, CH2CO), 6.8–7.0
(m, 4H, Ar), 7.5–7.6 (m, 3H, Ar), 8.1–8.2 (m, 2H, Ar), 8.3–8.4 (m, 1H,
Ar); MS (ESI) m/z 584.9 [M+H]+; Anal. (C28H26Cl2N4O6) C, H, N.
4-(6-Chloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenyl 4-nitrophenyl carbonate (11): IR (KBr): 1780,
1,740 1,610 cm−1 1H NMR (CDCl3) ı: 0.8–1.0 (m, 6H, CH3), 1.5–1.7
;
Ethyl-2-((4-(6-chloro-3-(2-(dipropylamino)-2-
oxoethyl)imidazo[1,2-a]pyridine-2-yl)phenoxy)carbonylamino)-
3-(3,4-dihydroxyphenyl)propanoate (7): IR (KBr): 3435, 1739,
(m, 4H, CH2), 3.2–3.4 (m, 4H, CH2N), 4.18 (s, 2H, CH2CO), 6.8 7.0
(m, 4H, Ar), 7.2–7.3 (m, 1H, Ar), 7.4–7.6 (m, 3H, Ar), 8.1–8.2 (m,
2H, Ar), 8.3–8.4 (m, 1H, Ar); MS (ESI) m/z 550.9 [M+H]+; Anal.
(C28H27ClN4O6) C, H, N.
1648 cm−1 1H NMR (DMSO-d6) ı: 0.7–0.9 (m, 6H, CH3), 1.2–1.4
;
(m, 3H, CH3), 1.4–1.7 (m, 4H, CH2), 2.6–2.8 (m, 2H, CH2Ar), 2.8–3.4
(m, 4H, CH2N), 3.95 (s, 2H, CH2CO), 3.9–4.4 (m, 3H, CHN and
CH2COO), 6.4–6.6 (m, 3H, Ar), 7.20 (d, J = 8.5 Hz, 2H, Ar), 7.2–7.6
(m, 4H, Ar), 8.50 (m, 1H, Ar); MS (ESI) m/z 658.9 [M+Na]+; Anal.
(C33H37ClN4O7) C, H, N.
2.4. General procedure for preparation of the LD derivatives 6
and 7
Compound 9: Yield: 50%; mp 75–77 ◦C; IR (KBr): 1610 cm−1
;
Bis-(4-nitrophenyl) carbonate (2.0 mmol) and TEA (1.2 mmol)
were added to a stirred solution of compound 8 (0.42 g, 1.0 mmol) or
9 (0.40 g, 1.0 mmol) in anhydrous THF (30 ml) to yield the interme-
diate compounds 10 and 11, respectively, as above described. Next,
a solution of LD ethyl ester hydrochloride 13 (0.26 g, 1.0 mmol) in
anhydrous DMF (2.0 ml) was added drop wise to a stirred solu-
tion of the appropriate crude intermediate (0.9 mmol) dissolved
in 10 ml of anhydrous THF. The mixture was stirred at room tem-
perature for 12 h. Solvent was evaporated under reduced pressure,
1H NMR (DMSO-d6) ı: 0.8–1.0 (m, 6H, CH3), 1.5–1.7 (m, 4H, CH2),
3.2–3.4 (m, 4H, CH2N), 4.18 (s, 2H, CH2CO), 6.81 (d, J = 8.2 Hz, 2H,
Ar), 7.2–7.3 (m, 1H, Ar), 7.40 (d, J = 8.2 Hz, 2H, Ar), 7.57 (d, J = 8.5 Hz,
1H, Ar), 8.47 (m, 1H, Ar); MS m/z 385 (M+, 17), 257 (base); Anal.
(C21H24ClN3O2) C, H, N.
Compound 17: Yield: 90%; mp 220 ◦C dec; IR (KBr): 3445,
1656 cm−1 1H NMR (DMSO-d6) ı: 3.33 (s, 3H, CH3O), 4.10 (s, 2H,
;
CH2CO), 7.03 (d, J = 8.2 Hz, 2H, Ar), 7.2–7.4 (m, 1H, Ar), 7.6–7.7

224
N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
Y
Y
N
N
a
N
N
OH
O
O
N
N
X
O
X
O
O
NO2
10 X= Y= Cl
8 X= Y= Cl
11 X= Cl, Y= H
9 X= Cl, Y= H
.
HO
HO
NH2 HCl
R
b
12 R= H
13 R= COOC2H5
Y
N
O
O
N
NH
R
X
O
N
OH
OH
4 X= Y= Cl, R=H
5 X= Cl, Y= H, R= H
6
X= Y= Cl, R=C2H5
7 X= Cl, Y= H, R= C2H5
Fig. 1. Synthetic scheme for preparation of DA and LD compounds 4–7. (a) THF, bis-(4-nitrophenyl) carbonate and TEA, 50 ◦C and (b) THF/DMF 5/1 (v/v), rt.
O
Br
N
O
a
Cl
OCH3
N
O
+
Cl
OCH3
N
NH2
O
O
15
14
16
b
N
N
N
N
OH
N
OH
OCH3
Cl
N
N
d
Cl
Cl
c
O
O
O
OH
OH
9
18
17
Fig. 2. Synthetic scheme for preparation of compound 9: (a) n-butanol, reflux; (b) n-butanol, NaOH 1 N, 50 ◦C; (c) HBr 48%, reflux; (d) anhydrous DMF, CDI, TEA and N,Nꢀ
dipropyl amine, rt.

N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
225
(m, 3H, Ar), 8.67 (s, 1H, Ar); MS (ESI) m/z 314.8 [M−H]−; Anal.
(C16H13ClN2O3) C, H, N.
procedures are reported elsewhere (de Candia et al., 2005). To
determine pKa values, an appropriate amount of compound 4 or
5 was dissolved in 20 ml of ionic-strength adjusted and argon-
degassed water (0.15 M KCl), to achieve final sample concentration
in the range of 0.5–1 mM. All titrations were performed in the pH
range between 1.8 and 12.2 at 25 0.5 ◦C, using standardized 0.5 M
HCl and 0.5 M carbonate-free KOH as titrating agents. All experi-
ments were carried out in triplicate and under a slow argon flow
to avoid CO2 absorption at high pHs. Electrode calibrations (Four-
PlusTM parameters) were performed everyday by a blank titration,
and Bjerrum plots were used to calculate precise pKa values. Due to
the low aqueous solubility of the tested compounds, potentiomet-
ric titrations were carried out with methanol as co-solvent, ranging
from 12 to 45%, and final aqueous pKas were extrapolated by using
the Yasuda–Shedlowsky plot (observed pKa + log [H2O] against the
inverse of the dielectric constant ε of the mixture).
Compound 18: Yield: 90%; mp 230 ◦C dec; IR (KBr): 3398,
1608 cm−1 1H NMR (DMSO-d6) ı: 4.11 (s, 2H, CH2CO), 7.00 (d,
;
J = 8.2 Hz, 2H, Ar), 7.2–7.4 (m, 1H, Ar), 7.6–7.7 (m, 3H, Ar), 8.65 (s,
1H, Ar); MS (ESI) m/z 301.0 [M−H]−; Anal. (C15H11ClN2O3) C, H, N.
2.5. Stability studies
2.5.1. Chemical hydrolysis
Stability studies were carried out at 37 0.2 ◦C in a shaking
(150 rpm) water bath. The hydrolysis rates of compounds 4–7 were
studied at pH 7.4 in isotonic (0.14 M NaCl) 0.05 M phosphate buffer
solution at 37 ◦C. The experiments were carried out by adding 50 ␮l
of a stock solution of the tested compounds 4–7 in methanol, to 5 ml
of the buffer solution preheated at 37 ◦C. The final concentration of
the resulting solutions was about 50 ␮M. The stock solutions were
vortexed and maintained in a shaking water bath at constant tem-
perature of 37 0.2 ◦C. At appropriate intervals, aliquots of 100 ␮l
were removed and immediately analyzed by high-performance liq-
uid chromatography (HPLC). Pseudo-first-order rate constants for
the hydrolysis of the derivatives were determined from the slopes
of linear plots of the logarithms of residual compound against
time. HPLC analyses were performed with a Waters Associates
Model 600 pump equipped with a Waters 990 variable wavelength
UV detector and a 20 ␮l loop injection valve. A reversed phase
Phenomenex Synergi Hydro C18 (25 cm × 3.9 mm; 5 ␮m particles)
column in conjunction with a precolumn (Sentry Guard Symme-
try C18, 20 × 3.9 mm) was eluted with mixtures of methanol and
0.01% TFA in water 65/35 (v/v). All analyses were performed in iso-
cratic conditions. The injected volume was 20 ␮l. The flow rate was
0.8 ml/min and the column effluent was monitored continuously
at 254 nm. The compounds were estimated by measuring the peak
areas or heights in relation to those obtained from the standards
chromatographed under the same conditions. Hydrolysis products
of compounds 4–7 were characterized by electrospray ionization
(ESI) mass spectrometry, both in positive and negative mode, via
direct injection of an aliquot of the stock solution in an Agilent
1100 LC–MSD trap system VL, using methanol/7 mM ammonium
formiate 9/1 (v/v) as vehicle.
To determine the potentiometric experimental octanol/water
partition coefficient, the apparent pKa values were determined
in the biphasic system constituted by 20 ml of 0.15 M KCl and
0.25 ml of water-saturated octanol. All data were processed using
the RefinementPro software (Sirius Analytical Instruments Ltd.,
Forest Row, East Sussex, UK).
2.7. Receptor binding assays
The in vitro receptor binding assays for Central Benzodiazepine
Receptor (CBR) and Translocator protein 18 kDa (TSPO) receptors
were carried out as previously reported (Denora et al., 2010).
2.8. BBMEC permeability assays
2.8.1. Isolation and cell culture of brain endothelial cells
Bovine brain microvessel endothelial cells (BBMECs) were iso-
lated as previously described (Audus and Borchardt, 1987) and
cultured at a density of approximately 50,000 cells/cm2 onto 24-
well plates or 100 mm culture dishes as previously reported (Rose
et al., 1998).
2.8.2. Rhodamine 123 uptake assay
In this assay, Rhodamine 123, a P-gp and breast cancer resistance
protein (BCRP) substrate, was used (Fontaine et al.,1996; Mao and
Unadkat, 2005). The effect of the test compound on Rhodamine
123 uptake was determined by monitoring intracellular fluores-
cence. BBMECs were as reported above and used to assay for P-gp
interactions (for details, see Rose et al., 1998).
2.5.2. Stability in physiological medium
The stability of compounds 4 and 5 was studied by adding 10 ␮l
of the stock solution of 4 and 5 in DMSO to 1.6 ml of preheated
serum solution [0.05 M phosphate buffer saline (0.14 M NaCl) at
pH 7.4, containing 50% (v/v) of human serum], and the mixture
was maintained in water bath at 37 0.2 ◦C. Aliquots of 100 ␮l
were collected at appropriate intervals and added to 500 ␮l of cold
acetonitrile in order to deproteinize the serum. After mixing and
centrifuging at 2000 × g for 10 min, 20 ␮l of the clear supernatant
was analyzed by HPLC as described above. Pseudo-first-order rate
constants for the degradation of compounds 4 and 5 were deter-
mined from the slopes of linear plots of the logarithms of remaining
compound against time. Degradation products of the tested com-
pounds in serum were characterized by ESI (both positive and
negative mode) via direct injection of an aliquot of deproteinized
supernatant (diluted 1:10 with blank) into an Agilent 1100 LC–MSD
trap system VL mass spectrometer, using methanol/7 mM ammo-
nium formiate 9/1 (v/v) as vehicle.
2.8.3. Transport studies on BBMEC monolayers
These studies were carried out as previously described by Audus
et al. (1996, 1998).
2.9. In vitro assays to assess compounds 4 and 5 P-glycoprotein
interaction
To assess whether compounds 4 and 5 interacted with P-
glycoprotein, we carried out bi-directional transport studies on
Madin–Darby Canine kidney (MDCK) cells, retrovirally transfected
with the human MDR1 cDNA (MDCKII-MDR1), the Calcein AM inhi-
bition assay and the P-gp ATPase activity assay following previously
reported procedures (Denora et al., 2010). It should be noted that
the transport experiments were performed for a 120 min period
and that compounds 4 and 5, respectively were analyzed by HPLC.
The HPLC tracers showed high-intensity peaks corresponding to
compounds 4 and 5 together with peaks of low intensity due to the
respective parent compounds (i.e., 8 and 9, respectively).
2.6. Experimental pKa and log P determinations of compounds 4
and 5 by potentiometric titrations
Experimental pKas and log Ps were determined by potentio-
metric titrations on Sirius GLpKa (Sirius Analytical Instruments
Ltd., Forest Row, East Sussex, UK). Details on instrument and

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N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
2.10. Brain microdialysis
box to observe drug-induced rotational behaviour over a 3-h period
by a trained researcher blind to the treatment schedule.
At the end of each observation, rats were euthanatized with
Equithesin and their brains rapidly dissected to isolate the striatum
from both hemispheres. Striatal samples were sonicated in ice-cold
perchloric acid 0.1 M and then centrifuged at 10,000 × g for 8 min
at 4 ◦C. Supernatants were collected and used for DA assay by HPLC,
as previously described (Cassano et al., 2009).
Microdialysis experiments were carried out as previously
described (Morgese et al., 2009). Briefly, male Wistar rats
(225–250 g; Harlan, San Pietro al Natisone, Udine, Italy) were anes-
thetized with equithesin (3 ml/kg, i.p.) [Napentobarbital (0.972 g),
chloral hydrate (4.251 g), magnesium sulphate (2.125 g), ethanol
(12.5 ml), and propylene glycol (42.6 ml) in distilled water (total
volume, 100 ml)] and positioned on a stereotaxic frame (Kopf
Instruments, Tujunga, CA). A microdialysis probe of concentric
design (AN69 Hospal S.p.A; 20 kDa cut-off; membrane length,
3 mm) was surgically implanted into the striatum according to the
following stereotaxic coordinates: AP +1.0, ML 2.8 from bregma
and DV −6.8 from dura (Paxinos and Watson, 1998). The mem-
branes were tested for in vitro recovery of DA one day before surgery
to verify that recoveries were within the desired range (∼30% for
DA).
Twenty-four hours after surgery, the microdialysis probe
was perfused with Krebs Ringer solution (NaCl 145 mmol/l, KCl
2.7 mmol/l, CaCl2·2H2O 1.2 mmol/l MgCl2 6H2O 1 mmol/l, Na2HPO4
2 mmol/l, pH 7.4) at a constant flow rate of 2 ␮l/min and per-
fusates were collected every 20 min. After a wash-out period of
2 h, 4 samples were collected to determine the baseline levels
of the neurotransmitters studied (no more than 10% difference
among 4 consecutive samples). Rats were acutely treated with com-
pound 4 or 5 (dissolved in a volume of 3 ml/kg and administered
at 10–20 mg/kg, i.p.), or vehicle (saline/Tween 80/PEG, 90/5/5, i.p.)
and consecutive microdialysates were collected every 20 min over a
4 h period. The correct placement of the dialysis probes was verified
histologically at the end of each experiment.
2.13. Statistical analysis
Microdialysis data were expressed as mean S.E.M. and ana-
lyzed by two-way analysis of variance (ANOVA) for repeated
measures with “treatment” and “time” as the between and within
variables, respectively. DA levels in tissue samples collected from
6-OHDA rats were analysed by two-way ANOVA with “hemi-
sphere” and “treatment” as variables. Violations of the sphericity
assumption were corrected using the Greenhouse–Geisser epsilon
correction to adjust the degrees of freedom. The effect of treatments
on rotational behaviour was analysed by one-way ANOVA. Bonfer-
roni’s multiple comparison test was used for post hoc comparisons.
The threshold for statistical significance was set at p < 0.05.
3. Results
3.1. Stability studies
The half-live values of DA derivatives 4 and 5 were calculated
by taking into account that the loss of the starting material fol-
lows the first order kinetics. Half-lives (Table 2) were about 3–5 h
in phosphate buffer and 10–40 min in dilute human serum. LD ethyl
ester derivatives 6 and 7, were very unstable in phosphate buffered
solution, yielding the starting imidazopyridine compounds 8 and
9, respectively, as shown by HPLC and LC–mass spectrometry
(LC–MS). Because of their chemical instability, we excluded these
derivatives from further evaluation. The ESI LC–MS analysis in neg-
ative mode of the mixture obtained from degradation of 4 and 5 in
physiological medium after 30 min showed the presence of peaks
attributable to imidazopyridinacetamides (8 and 9, respectively), as
well as to DA. Therefore, these results suggest that, in physiological
medium, the carbamate linkage of compounds 4 and 5 undergoes a
rapid cleavage restoring DA and the suitable 2-phenyl-imidazo[1,2-
a]pyridine acetamide compound 8 or 9.
Analysis of the extracellular levels of DA was performed by an
HPLC apparatus (BAS, Bioanalytical Systems, Kenilworth Warwick-
shire, United Kingdom) connected to an electrochemical detector
(INTRO, Antec Leyden, The Netherlands), as previously described
(Cassano et al., 2009).
Animal care and all experiments were conducted in accordance
with the guidelines of the European Communities Council Directive
of 24 November 1986 (86/609/EEC) and the Italian Department of
Health (D.L. 116/92).
2.11. 6-OHDA lesions
DA-denervating lesions were performed by unilateral injection
of 6-OHDA into the left medial forebrain bundle (MFB) as previ-
ously reported (Morgese et al., 2009). Briefly, on the day of the
surgery (day-21), male Wistar rats (225–250 g; Harlan, San Pietro al
Natisone, Udine, Italy) were anesthetized with equithesin (3 ml/kg,
i.p.) and infused unilaterally into the left MFB (AP −4.3, ML +1.6,
DV −8.3, tooth bar −2.4 relative to bregma and midline, in mm
according to Paxinos and Watson, 1998), with 2 ␮l of 6-OHDA
(4 ␮g/␮l dissolved in 0.1% ascorbate saline) through a stainless
steel needle, at a flow rate of 0.5 ␮l/min. All rats were pre-treated
with desipramine (25 mg/kg, i.p.) 30 min before 6-OHDA to prevent
damage to noradrenergic neurons. Two weeks after surgery (day-7)
6-OHDA-treated rats received an acute injection of amphetamine
(2.5 mg/kg, i.p.) to assess the efficacy of the lesion (Morgese et al.,
2009).
3.2. Prediction of BBB penetration of compounds 4–7
The partition coefficient (log P) values of compounds 4–7 were
calculated (Clog P) by using the Software ChemDraw Ultra 10.0,
while the Exp log P and the Exp pKa of compounds 4 and 5 were
obtained by potentiometric titrations using the Sirius GLpKa instru-
ment (Table 2). The Exp log P values of 4 and 5 were slightly
lower than the calculated ones. The chemical instability of com-
pounds 6 and 7, prevented the assessment of their Exp log P values.
The experimental Log D7.4 values of compounds 4 and 5 are also
reported in Table 2. Both the calculated and the experimental log P
values suggest that compounds 4–7 should cross the BBB (Norinder
and Haeberlein, 2002; Mehdipour and Hamidi, 2009). However,
the degree of BBB penetration for CNS active substances is more
appropriately measured by the log Cbrain/Cblood (log BB). The log BB
for compounds 4–7 was calculated according to the Clark’s equa-
tion: log BB = −0.0148 ( 0.001) PSA + 0.152 ( 0.036) C log P + 0.139
2.12. Treatment, assessment of rotational behaviour and striatal
DA assay
Three weeks after surgery (day 0), 6-OHDA-treated rats
received a single i.p. injection of either LD + the inhibitor of
aromatic aminoacid decarboxylase, benserazide (25 mg/kg and
12.5 mg/kg, respectively), or compound 4 (20 mg/kg) or compound
5 (20 mg/kg). Then animals were individually placed in a Plexiglas
( 0.073) (Clark, 1999a), where PSA is the polar surface area, a
molecular descriptor that can be also useful for the prediction of the
intestinal absorption (Clark, 1999b; Trapani et al., 2001). PSA was
computed by the Ertl’s approach known as topological PSA (TPSA)
(Ertl et al., 2000). It should be considered that log BB values > 0.3 are

N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
227
Table 2
Physicochemical parameters and estimation of BBB penetration, stability data in phosphate buffer and dilute human serum for compounds 4–7.
a
a
Compound
t1/2 (h) Phosphate buffer 0.05 M (pH 7.4)
t1/2 (min) diluted serum
C Log Pc
Exp log P d
TPSAf
Log BB g
4
5
6
7
3.87 0.52
33.65 0.46
5.58
4.86
5.62
4.91
0.17
−2.82
5.38
2.83
4.81 (4.79)
114.7
114.7
141
−0.71
−0.82
−1.09
−1.20
−0.88
−2.02
0.38
4.94 0.45
9.78 0.71
4.33 (4.30)
b
b
e
b
b
e
141
DA
LD
Alpidem
Zolpidem
70.5
116.6
39
39
−0.01
a
Based on the loss of starting material. Data are means SD of three separate experiments
Unstable.
Estimated according to ChemDraw Ultra 10.0 software.
b
c
d
Experimental Log P determined by Sirius GLpKa instrument. In parentheses Exp log D7.4 values. The pKa values measured by Sirius GLpKa instrument were the following:
compound 4, pKa1 (m-OH) 10.11, pKa2 (p-OH) 8.71, pKa3 (imidazole nitrogen) 2.65; compound 5, pKa1 (m-OH) 9.71, pKa2 (p-OH) 8.45, pKa3 (imidazole nitrogen) 3.33.
e
The chemical instability of this compound prevented the assessment of the Exp log P value.
Calculated according to Ertl’s method (Ertl et al., 2000).
Calculated according to Clark’s model and by using TPSA (Clark, 1999a,b).
f
g
shown by compounds that cross the BBB efficiently, while values
< −1.0 are characteristic of compounds that do not enter the brain.
As shown in Table 2, compounds 4 and 5, but not 6 and 7, should
be able to cross the BBB.
As shown in Table 3, compound 4 showed nanomolar bind-
ing affinity for TSPO (IC50 1.32 nM), while the DA derivative 5 had
high affinity for both CBR (IC50 78 nM) and TSPO (IC50 1.14 nM).
Interestingly, compounds 4 and 5 retained the binding affinity of
the parent imidazopyridine compounds 8 and 9, respectively. In
particular, compound 8 showed high affinity and selectivity for
TSPO (Denora et al., 2008), while compound 9 bound to TSPO (IC50
1.0 nM) and CBR (IC50 22.2 nM) with high affinity (Table 3). Com-
pound 8 was tested at the concentration of 1 and 10 ␮M using
the voltage–clamp technique to measure its modulatory action on
GABA-evoked Cl− currents in Xenopus oocytes expressing human
␣1ˇ2␥2 GABAA receptors. Using this approach, we found that 8
modulated GABAA receptor activity and produced a 30% change
over the control response (Denora et al., 2008).
3.3. Radioligand binding assays
GABA transmission is modulated by several types of compounds,
among which the benzodiazepines are one of the best characterized
examples (Casellas et al., 2002). There are two pharmacologically
distinct benzodiazepine receptors in the CNS, central (CBR) and
peripheral (PBR) benzodiazepine receptors; the latter have been
recently renamed Translocator protein 18 kDa (TSPO). The CBR is
part of the ionotropic GABA receptor located on the plasma mem-
brane of GABAergic neurons, while TSPO is found on the outer
membrane of mitochondria and mainly in glial cells (Casellas et al.,
2002; Majewska et al., 1986). On the other hand, TSPO is involved
in the synthesis of neurosteroids which have been suggested to be
endogenous modulators of GABAA neurotransmission (Rupprecht
et al., 2010). The binding of DA derivatives 4 and 7 and their pre-
cursors 8 and 9 to CBR and TSPO was evaluated by measuring their
ability to inhibit [3H]-flunitrazepam and [3H]-PK 11195 binding to
membrane preparations from the rat cerebral cortex. The calcu-
lated inhibitory concentration (IC50) values are listed in Table 3
together with that of Diazepam and PK 11195 for comparison.
3.4. Transport across BBMECs monolayers
To assess the ability of compounds 4 and 5 to cross the BBB, we
carried out transport studies involving BBMECs monolayers. The
results presented in Table 3 indicate that DA derivatives 4 and 5
possess Papp values greater than that presented by [14C]-sucrose, a
marker that should not readily cross the cell monolayer. Further-
more, using a Rhodamine 123 uptake assay (Fontaine et al., 1996),
we found that the tested compounds did not affect the intracellular
concentration of Rhodamine 123, suggesting that they should not
be a substrate for P-gp expressed on BBMECs (data not shown).
Table 3
Binding data for CBR and TSPO 18 kDa receptors of compounds 4–7, and BBMEC
permeability values of compounds 4 and 5.
3.5. In vitro interaction of P-gp with compounds 4 and 5
After transfecting MDCKII cells with the human MDR1 cDNA
to yield high levels of P-gp expression, we assessed whether
compounds 4 and 5 interacted with P-gp using the Calcein AM fluo-
rescent assay, the P-gp ATPase activity assay and the bi-directional
transport test. As reported in Table 4, the observed IC50 values for
compounds 4 and 5 in the Calcein AM assay were below 100 ␮M.
In the P-gp ATPase assay, both compounds were unable to deplete
ATP. Finally, we calculated the Papp values in both apical to baso-
lateral (Papp, AP) and basolateral to apical (Papp, BL) directions and
found that the BL/AP ratios for compounds 4 and 5 were 1.48 and
1.07, respectively.
Compound
IC50 (nM) a
CBR b
Papp (cm/s) d
TSPO 18 kDa c
4
5
6
7
8
9
DA
LD
>105
78
1.32
1.14
1.31
1.0
1.58 0.55 × 10−6
1.27 0.08 × 10−6
>105
22.2
e
>105
1.31e
1.0
22.2
0.21 0.28 × 10−6 f
1.92 0.14 × 10−6 f
PK 11195
Diazepam
–
6.2
1.74
–
a
Data are means SD of three separate experiments performed in duplicate.
Diazepam a selective ligand for CBR was used for comparison.
b
c
3.6. Microdialysis studies
PK 11195 a selective ligand for TSPO 18 kDa was used for comparison.
d
Data are means SD of three separate experiments. [14C]-sucrose was used as
To assess whether the administration of 4 or 5 influenced brain
DA transmission, we monitored the striatal DA output in freely-
moving rats for 4 h after single systemic injections (10–20 mg/kg,
i.p.) of 4 or 5. For compound 4 (Fig. 3A), two-way ANOVA revealed a
a low permeability paracellular marker and its Papp (0.22 0.03 × 10−6) was always
less than the tested compounds.
e
From Denora et al. (2008).
From Denora et al. (2007).
f

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N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
Table 4
Bi-directional transport studies across MDCKII-MDR1 cells on compounds 4 and 5, and evaluation of their interaction with P-glycoprotein (P-gp) by specific biological assays.
Transport study on MDCKII-MDR1 a P-gp interaction specific biological assays a
Compound
Papp (AP) (cm/s)
Papp (BL) (cm/s)
Papp (BL/AP)
Calcein IC50 (␮M)
ATPase activation
4
5
2.87 0.75 × 10−7*
2.87 0.15 × 10−7*
1.43 0.03 × 10−5
2.72 0.22 × 10−10
4.25 0.30 × 10−7
1.48
1.07
>100
>100
No
No
3.08 0.15 × 10−7
Diazepam
–
–
FD4b
a
Data are means SD of three determinations and were analyzed by one-way analysis of variance (ANOVA) and post hoc test was made by Bonferroni’s multiple comparison
test. The threshold for statistical significance was set at p < 0.05.
b
Diazepam and fluorescein isothiocyanate dextran 4000 (FD4, Sigma) were used as markers for the transcellular and paracellular pathways, respectively.
p < 0.001 vs FD4.
*
Table 5
significant effect of treatment [F2255 = 4.108, p < 0.05], a significant
Effects of acute administration of LD, compounds 4 and 5 on contralateral turning
in 6-OHDA-lesioned rats.
main effect of repeated measures [F15,255 = 7.851, p < 0.001], and a
significant interaction between factors [F30,255 = 3.202, p < 0.001].
Levodopa
(25 mg/kg)
Compound 4
(20 mg/kg)
Compound 5
(20 mg/kg)
The post hoc test showed that 4 (10–20 mg/kg, i.p.) increased
extracellular DA levels dose dependently. Indeed, we observed a
significant DA elevation 100 min after administration of the highest
dose, which peaked at 120 min. The DA increase was still significant
4 h after treatment (Fig. 3A).
Statistical analysis for compound 5 showed a significant effect
of treatment [F2240 = 10.23, p < 0.01], a significant main effect of
repeated measures [F15,240 = 8.112, p < 0.001], and a significant
interaction between factors [F30,240 = 3.236, p < 0.001]. Sixty min
after administration of the highest dose, we observed a significant
Total turns/3 h
1120 100.2***
1.8 0.5
6.3 0.8
Data are expressed as mean S.E.M. of n = 6 animals per group. One way-ANOVA
followed by Bonferroni’s multiple comparison test.
***
p < 0.001 vs compounds 4 and 5.
increase of DA levels, which peaked at 80 min. As in the case of
compound 4, DA levels were still significantly elevated 4 h after
drug administration (Fig. 3B).
3.7. Effects of LD, and compounds 4 and 5 in 6-OHDA-treated rats
Table 5 shows the effect of treatments on rotational behaviour
in 6-OHDA-treated rats during the 3-h observation period. Statisti-
cal analysis showed that 6-OHDA rats receiving LD (25 mg/kg) had
a significantly higher number of rotations contralateral to the den-
ervated hemisphere compared to 6-OHDA animals injected with
4 (20 mg/kg) or 5 (20 mg/kg) [F2,17 = 124.1, p < 0.001]. The Bon-
ferroni’s multiple comparison test showed no difference between
groups receiving compounds 4 and 5.
At the end of the behavioural test, striatal DA levels were
analysed in both hemispheres (ipsi- and contra-lateral to the 6-
OHDA-lesion) (Fig. 4). Two-way ANOVA revealed a significant
main effect of “hemisphere” [F1,47 = 8.683, p < 0.01] and “treatment”
Fig. 4. Effect of acute vehicle, LD (25 mg/kg, i.p.), 4 or 5 (20 mg/kg, i.p.) injections
on striatal DA levels in the ipsilateral (open bar) and contralateral (filled bar) hemi-
spheres of 6-OHDA-treated rats. Data are expressed as mean S.E.M. of n = 6 animals
per group. Two-way ANOVA followed by Bonferroni’s multiple comparison test.
***p < 0.001 ipsi vs contra.
Fig. 3. Effect of single (10–20 mg/kg, i.p.) 4 (A) and 5 (B) injections on DA outflow
over 4 h in striatum of awake rats. Data are expressed as mean S.E.M. of n = 6–8
animals per group. Two-way ANOVA followed by Bonferroni’s multiple compari-
son test. *p < 0.05, **p < 0.01, ***p < 0.001 vs respective vehicle; p < 0.05 vs 10 mg/kg.
Arrow indicates the time of vehicle, 4 and 5 administration.

N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
229
[F3,47 = 5.748, p < 0.01], and no significant interaction between the
two variables [F3,47 = 0.810, n.s.]. Post hoc multiple comparison
test indicated that DA levels were significantly lower (p < 0.001)
in the denervated vs intact striatum, in animals receiving either
acute LD (25 mg/kg) or vehicle. Administration of 4 (20 mg/kg) or 5
(20 mg/kg) increased DA levels in both hemispheres and attenuated
the disparity in DA concentrations between them.
a Calcein AM IC50 higher than 100 ␮M) indicate that 4 and 5 are
unambiguous non-substrates (Feng et al., 2008) and lack active
transport, ruling out the possibility of active brain uptake.
The microdialysis experiments were performed to determine
the extracellular concentrations of striatal DA in freely-moving rats
following peripheral (i.p.) administration of compounds 4 and 5.
The striatum is the main target region for symptomatic treatment of
PD and it is known to be rich in dopaminergic nerve terminals. Fol-
lowing the administration of compound 5, DA levels were greater
than those recorded after injection of compound 4 (Fig. 3).
Interestingly, we have previously demonstrated that neither an
acute nor a chronic (for 11 days) treatment with an equimolar dose
(6 mg/kg) of LD, co-administered with the inhibitor of aromatic
aminoacid decarboxylase benserazide (12.5 mg/kg), increased DA
extracellular levels in the striata of both 6-OHDA- and intact rats
(Morgese et al., 2009).
An important aspect is that compounds 4 and 5 are essentially
unstable in serum and nevertheless, they are able to increase stri-
atal DA concentration. At this stage the mechanism through which
4 and 5 exert their effect is unclear, but we hypothesize that both
compounds can be rapidly transported across the BBB. Once the
BBB has been crossed, the diverse pharmacokinetic profiles of each
compound may have accounted for the differences in DA release
observed at both doses. In fact, even though 4 showed better BBB
penetration properties than 5, the latter was less stable than 4 in
physiological medium and consequently it might have released a
much higher amount of DA. This hypothesis is consistent with the
earlier DA elevation observed after administration of compound 5
(60 min vs 100 min). On the other hand, the sustained DA release
observed with compound 5 suggests that the hydrolytic degrada-
tion of 4 and 5 in the brain parenchyma might be slower than in
human serum, even though the rank order was similar (i.e., 5 less
stable than 4).
The microdialysis results were significantly different from those
observed with compounds 1–3 in a previous study (Denora et al.,
2007). Unlike the imidazopyridine derivatives 4 and 5, which
yielded sustained DA levels (4 h), DA elevation after systemic
administration of 1–3 (12 mg/kg, i.p.) peaked at 80 min and wore
off within 120 min after drug injection (Denora et al., 2007).
To investigate the potential for therapeutic actions, we stud-
ied the effects of 4 and 5 administrations in 6-OHDA-treated rats,
a well-established animal model of PD (Deumens et al., 2002).
The 6-OHDA-induced unilateral lesion of the MFB causes motor
asymmetry (rotation) in rats due to the DA imbalance across
the two striata. Subcutaneous administration of the DA-releasing
agent D-amphetamine creates a DA imbalance that favours the
intact nigrostriatal projections, thus producing ipsilateral rota-
tions (Deumens et al., 2002). Conversely, dopaminergic agonists
like apomorphine induce contralateral rotations by stimulating
upregulated dopamine D2 receptors in the denervated striatum
(Ungerstedt, 1971). Similarly to apomorphine, administration of LD
to 6-OHDA rats produced contralateral rotational behaviour. On the
contrary, when 6-OHDA rats were challenged with 4 or 5 (20 mg/kg,
i.p.), the rotational asymmetry was almost absent (Table 5).
As expected, basal DA levels were significantly decreased in the
denervated striatum of 6-OHDA-treated rats (Fig. 4). In these ani-
mals, we found no increase in DA content in both hemispheres three
hours after acute LD (25 mg/kg, i.p.) injection. This result is in line
with another study showing that even higher doses of LD (33 mg/kg
and 66 mg/kg, i.p.) do not increase DA levels in this animal model
(Rodriguez et al., 2007). However, compound 4 or 5 (20 mg/kg, i.p.)
increased DA content in ipsi- and contralateral striata and, more
interestingly, attenuated the imbalance in DA levels across the two
hemispheres. In our opinion, the latter condition might account for
the significant attenuation of motor asymmetry observed after 4 or
5 administration.
4. Discussion
Our study shows that the GABAergic agent–DA–LD ethyl
ester-conjugation represents
a new strategy to deliver the
neurotransmitter DA to the brain while increasing GABAergic
transmission. The GABAergic agents used in the study were imi-
dazopyridine compounds structurally analogues to Alpidem and
Zolpidem, a drug showing beneficial effects in patients undergo-
ing long-term LD treatment (Daniele et al., 1997; Jankovic and
Marsden, 1988). The choice of the imidazopyridine moiety was
also based on its lipophilic nature, which served as carrier for the
DA and LD ethyl ester derivatives through the brain. In particular,
compounds 4–7 were designed to be structurally different from
compounds 1–3 (Denora et al., 2007).
The DA- and LD-derivatives 4, 5 and 6, 7 showed different sta-
bility profiles, the latter being very unstable in both buffered and
physiological media in contrast to that we observed for the former.
This marked instability was not found in the previously examined
LD-derivatives 1 and 2 (Denora et al., 2007), indicating that the car-
bamate in ␣ to the ester group is much more labile than the amide
moiety in the same position.
Interestingly, compounds 4 and 5 were slightly larger in size
than 1–3. In fact, as shown in Table 1, the molecular weights of these
compounds were slightly greater than 500 Da, i.e., the accepted
maximum value for BBB drug penetration by passive diffusion. Like-
wise, the C log P values of compounds 4 and 5 were slightly greater
than those of 1–3. Nevertheless, although the log BB values (−0.71
and −0.82 for 4 and 5, respectively) were predictive of higher BBB
permeability, it should be taken into account that increasing the
lipophilicity may result in extensive serum protein binding, which
can influence the action of these compounds, as well as their dis-
tribution and elimination (Smith and Lockman, 2011).
Another difference between the imidazopyridine compounds
4, 5 and 1–3 is their activity at the benzodiazepine receptors. In
particular, the imidazopyridine 4 showed affinity for TSPO 18 kDA
only, while 5 exhibited affinity for both receptor subtypes (i.e., CBR
and TSPO 18 kDA). These results are consistent with the structure-
activity relationships developed for this series of GABAergic agents
(Denora et al., 2008). Substitution of the imidazopyridine nucleus
with a lipophilic group, such as a chlorine atom, at the 8-position
improves affinity and selectivity toward TSPO. On the other hand,
the lack of a lipophilic substituent at this position produces com-
pounds that bind with high affinity to both TSPO and CBR. Therefore,
to gain high affinity to both receptor subtypes, we included in the
study compound 4 and 6 which have two chlorine atoms at 6-
and 8-position of the imidazopyridine nucleus. In addition, mono-
substitution of the amide nitrogen significantly decreases affinity
(Denora et al., 2008) and may explain the very low binding observed
for imidazopyridine 1–3. To estimate the predictive ability of our
compounds to cross the BBB, we used in vitro and in silico mod-
els. Interestingly, the rank order of BBB penetration for 4 and 5
measured in BBMECs monolayers parallelled that calculated by
computational approach. By these methods, compound 4 showed
better log BB and higher Papp values than compound 5. We also
studied the interaction of P-gp with compounds 4 and 5 as P-gp
serves as a drug efflux pump on the BBB. Altogether, the results
from these assays (a BL/AP ratio less than 2, no ATP depletion and

230
N. Denora et al. / International Journal of Pharmaceutics 437 (2012) 221–231
Finally, a question arises on how compounds 4 and 5 should
Berson, A., Descatoire, V., Sutton, A., Fau, D., Maulny, B., Vadrot, N., Feldmann, G.,
Berthon, B., Tordjmann, T., Pessayre, D., 2001. Toxicity of alpidem, a peripheral
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be classified. Unlike compounds 1–3, these compounds cannot be
considered typical “DA prodrugs” given their activity at benzo-
diazepine receptors. Indeed, the term “prodrug”, as proposed by
Albert (1958), refers to an “inactive” derivative, obtained by linking
a drug to a promoiety that requires an in vivo chemical or enzymatic
reaction yielding the parent drug. Instead, compounds 4 and 5 con-
sist of two pharmacologically active substances that are coupled
together in a single molecule. Such derivatives are called “mutual
prodrugs” or “codrugs” and offer the advantage of co-delivery when
it is desirable to have two drugs at the same site simultaneously
(Das et al., 2010). A codrug has a distinct advantage over the use
of either parent drugs alone because it incorporates their chemical
properties while allowing a better pharmacokinetic profile and/or a
sustained delivery over an extended period of time (Einmahl et al.,
1999). Our findings suggest that 4 and 5 are able to reach the CNS
and that the lipophilic and pharmacologically active imidazopy-
ridine moieties (8 and 9 respectively) could serve as DA carrier. In
addition, the phenyl-imidazopyridine portion is important because
it plays both a pharmacodynamic and a pharmacokinetic role (i.e.,
BBB crossing ability). Finally, the sustained DA release observed in
the microdialysis experiments is consistent with the concept of a
“mutual prodrug” or “codrug”.
The benefit of producing a codrug with a GABAergic moiety is
in line with recent studies suggesting that enhancing GABA trans-
mission can be helpful in subpopulations of patients with severe
PD (Daniele et al., 1997; Jankovic and Marsden, 1988).
The codrug approach has been already applied to the treatment
of PD to deliver DA or LD together with antioxidant molecules
[e.g. (R)-␣-lipoic acid, glutathione] (Di Stefano et al., 2009), or in
combination with a potent catechol-O-methyl transferase inhibitor
(e.g. Entacapone) (Leppanen et al., 2002; Sozio et al., 2012). In this
regard, it is noteworthy that the latter codrug is characterized by
a half life of 0.4–0.7 h (see http://www.drugbank.ca/) that is com-
parable with the values observed for the codrugs examined in this
study.
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This work was supported by
a grant from Ministero
dell’Università e della Ricerca Scientifica e Tecnologica (MIUR) and
from NS050401 (to AG). We thank Dr. M. De Candia (Department of
Pharmacy, University of Bari) for his help with the Exp log P mea-
surements and Mr Giovanni Dipinto and Mr Antonio Palermo for
their skilful technical assistance in recording mass spectra and NMR
spectra, respectively.
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