Novel L-Dopa and dopamine prodrugs containing a 2-phenyl-imidazopyridine moiety

Article abstract of DOI:10.1007/s11095-007-9255-y

Purpose. The aim of this study was to gain insight into the feasibility of enhancing the delivery of L-Dopa and dopamine to the brain by linking these neurotransmitters and L-Dopa ethyl ester to 2-phenyl-3-carboxymethyl- imidazopyridine compounds giving r

Full text of DOI:10.1007/s11095-007-9255-y

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Pharmaceutical Research, Vol. 24, No. 7, July 2007 ( 2007)
DOI: 10.1007/s11095-007-9255-y
Research Paper
Novel L-Dopa and Dopamine Prodrugs Containing
a 2-Phenyl-imidazopyridine Moiety
Nunzio Denora,1 Valentino Laquintana,1 Angela Lopedota,1 Mariangela Serra,2 Laura Dazzi,2 Giovanni Biggio,2
Dhananjay Pal,3 Ashim K. Mitra,3 Andrea Latrofa,1 Giuseppe Trapani,1,4 and Gaetano Liso1
Received November 28, 2006; accepted January 26, 2007; published online April 3, 2007
Purpose. The aim of this study was to gain insight into the feasibility of enhancing the delivery of L-
Dopa and dopamine to the brain by linking these neurotransmitters and L-Dopa ethyl ester to 2-phenyl-
3-carboxymethyl-imidazopyridine compounds giving rise to the so-called Dopimid compounds.
Materials and Methods. A number of Dopimid compounds were synthesized and both stability and
binding studies to dopaminergic and benzodiazepine receptors were performed. To evaluate whether
Dopimid compounds are P-gp substrates, [3H]ritonavir uptake experiments and bi-directional transport
studies on confluent MDCKII-MDR1 monolayers were carried out. The brain penetration properties of
Dopimid compounds were estimated by the Clark_s computational model and evaluated by investigation
of their transport across BBMECs monolayers. The dopamine levels following the intraperitoneal
administration of the selected Dopimid compounds were measured in vivo by using brain microdialysis
in rat.
Results. Tested compounds were adequately stable in solution buffered at pH 7.4 but undergo faster
cleavage in dilute rat serum at 37-C. Receptor binding studies showed that Dopimid compounds are
essentially devoid of affinity for dopaminergic and benzodiazepine receptors. [3H]ritonavir uptake
experiments indicated that selected Dopimid compounds, like L-Dopa and dopamine hydrochloride, are
not substrates of P-gp and it was also confirmed by bi-directional transport experiments across MDCKII-
MDR1 monolayers. By Clark_s model a significant brain penetration was deduced for L-Dopa ethyl
ester and dopamine derivatives. Transport studies involving BBMECs monolayers indicated that some
of these compounds should be able to cross the BBB. Interestingly, the rank order of apparent
permeability (Papp) values observed in these assays parallels that calculated by the computational
approach. Brain microdialysis experiments in rat showed that intraperitoneal acute administration of
some Dopimid compounds induced a dose-dependent increase in cortical dopamine output.
Conclusion. Based on these results, it may be concluded that some Dopimid compounds can be
proposed as novel L-Dopa and dopamine prodrugs.
KEY WORDS: blood brain barrier; imidazopyridine-compounds; L-Dopa and dopamine; microdialysis;
prodrugs.
INTRODUCTION
administration of DA cannot be useful for the treatment of
PD since DA is unable to cross the blood brain barrier
L-Dopa (3,4-dihydroxyphenyl-L-alanine) is a precursor (BBB) due to its hydrophilic nature and the absence of active
of dopamine (DA), which is deficient in the brains of patients transport mechanism (1). Instead, L-Dopa enters into the
suffering from the progressive disorder of the central nervous CNS through active transport and it is enzymatically decar-
system (CNS) known as Parkinson_s disease (PD). Peripheral boxylated in the brain giving rise to DA. Actually, L-Dopa
can be considered a prodrug of DA. However, L-Dopa is
characterized by a low water solubility, sensitive to chemical
and enzymatic oxidations and suffers from extensive periph-
1 Dipartimento Farmaco-Chimico, Facolta` di Farmacia, Universita`
degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy.
eral decarboxylation (2,3). After a good initial response,
complications are associated with long-term L-Dopa therapy
2 Dipartimento di Biologia Sperimentale, Sezione di Neuroscienze,
including motor fluctuations, dyskinesias, mental changes
Universita` di Cagliari, Cittadella Universitaria Monserrato, SS 554
and loss of efficacy (4). In this regard, it has been suggested
Km 4.5 Monserrato, Cagliari, Italy.
that drugs enhancing the g-aminobutyric acid (GABA)
transmission could be helpful in PD (5). In fact, it has been
suggested that GABAA receptors contribute to modulation of
the activity of mesocortical and mesolimbic dopaminergic
neurons (6).
3 Division of Pharmaceutical Sciences, School of Pharmacy, Univer-
sity of Missouri-Kansas City, 5005 Rockhill Road,
Kansas City, Missouri 64110 USA.
4 To whom correspondence should be addressed. (e-mail: trapani@
farmchim.uniba.it)
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1309
0724-8741/07/0700-1309/0
2007 Springer Science + Business Media, LLC

1310
Denora et al.
Among the strategies aimed at enhancing the delivery of of the high blood brain barrier (BBB) crossing ability shown
biologically active substances to the brain, the prodrug by most phenyl-imidazopyridine derivatives (e.g., log Cbrain/
approach is commonly used to improve physicochemical, Cblood of alpidem is 0.4, (8)). Moreover, it has been reported
biopharmaceutical, and drug delivery properties. Thus, a that selective gabaergic agonists such as zolpidem could be
promoiety is covalently attached to the bioactive molecule, useful in patients who have complications associated with
and the resulting prodrug is converted to the parent drug in long-term L-Dopa treatment (9). Therefore, we considered
the body exhibiting the pharmacological effect. Therefore, it to combine by covalent-bonding of L-Dopa and DA to 2-
may be useful to manipulate the molecules L-Dopa and DA phenyl-imidazopyridine-3-acetic acids. The rationale was to
to obtain compounds with increased lipophilicity, and there- have pharmacologically inert prodrugs, which upon delivery
by allow them to gain access to the CNS. In this paper, we into the brain, are cleaved to the active L-Dopa and DA and
describe the synthesis and properties of compounds 1–8 and phenyl-imidazopyridine moieties. These last could interact at
9–12 (Dopimid compounds, Table I) which are characterized GABA-BZR complex and it can influence the dopaminergic
by an L-Dopa or DA moiety, respectively, and linked to neurons. A number of advantages were expected as good
appropriately substituted 2-phenyl-imidazopyridine-3-acetic lipophilicity, BBB penetration, delivery enhancement at the
acids. These potential prodrugs have been prepared in order target site and reduced complications associated with L-Dopa
to take advantage of two features concerning imidazopyr- treatment. Synthesis, in vitro studies for assessing the BBB
idine compounds. The first one is of a pharmacodynamic penetration, binding data and results of a biodistribution
nature and is based on the high affinity and selectivity for the study are herein reported and discussed.
GABA-benzodiazepine receptor (GABA-BZR) complex
shown by most phenyl-imidazopyridine compounds, as ex-
emplified by alpidem and zolpidem (7,8). The second feature MATERIALS AND METHODS
is that the lipophilic phenyl-imidazopyridine moiety could
serve as a carrier for L-Dopa and DA leading to increased
Melting points were determined in open capillary tubes
brain levels of these last two compounds taking into account with a Bu¨chi apparatus and are uncorrected. IR spectra were
Table I. Chemical Structures of Compounds 1–12, 16 a–d and 19 a–d
Dopimid Compound
R1
R2
R3
R4
COOH
COOH
R5
R6
Mp (-C)
Yield (%)
1
2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
Cl
H
H
Cl
Cl
H
H
Cl
Cl
H
H
Cl
Cl
H
H
Cl
Cl
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
168–173
212 dec.
230 dec.
165–170
107–110
210 dec.
87–90
95
96
98
96
63a
85a
73a
77a
32
45
35
40
45
30
21
44
27
40
34
35
3
COOH
COOH
4
5
COOCH2CH3
COOCH2CH3
6
7
COOCH2CH3
COOCH2CH3
8
168–170
147–150
127–131
125–130
85–90
9
H
10
11
12
16a
16b
16c
16d
19a
19b
19c
19d
H
H
H
COOCH2CH3
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
OTBDMS
87–90
90–93
COOCH2CH3
COOCH2CH3
COOCH2CH3
110–115
105–107
183–185
165–167
147–150
156–161
H
H
H
H
a Yields obtained following Method B.

Novel L-Dopa and Dopamine Prodrugs
1311
obtained on a Perkin–Elmer 283 spectrophotometer (KBr was monitored by TLC [petroleum ether/ethyl acetate 7/3
1
pellets for solids or nujol for liquids). H NMR spectra were (v/v) as eluent]. The solvent was removed under reduced
determined on a Varian VX Mercury operating at 300 MHz pressure and the resulting residue was purified by silica gel
instrument. Chemical shifts are given in d values downfield column chromatography using petroleum ether/ethyl acetate
from Me4Si as internal standard. Mass spectra were recorded 7/3 (v/v) as eluent. In this way, the corresponding 3,4-bis(tert-
on a Hewlett-Packard 5995c GC-MS low-resolution spec- butyldimethylsilyloxy)-L-Dopa ethyl ester compounds 16 a–d
trometer or were obtained using an Agilent 1100 LC-MSD trap were obtained. Subsequently, complete deprotection of
system VL instrument using methanol/7 mM ammonium TBDMS groups was accomplished by using TFA. In short,
formiate 9/1 (v/v). Elemental analyses were carried out with a the appropriate compound 16 a–d (0.46 mmol) was dissolved
Hewlett Packard 185 C, H, N analyzer and results were within in 5 ml of 95% aqueous TFA. The reaction was carried out
T0.40% of the theoretical values. Silica gel 60 (Merck 70–230 under nitrogen atmosphere for 12–24 h at RT and the
mesh) was used for column chromatography. All the following progress of the reaction was monitored by TLC [petroleum
reactions were performed under a nitrogen atmosphere and the ether/ethyl acetate 7/3 (v/v) as eluent]. Then, the mixture was
progress of the reaction was monitored by thin-layer chroma- evaporated at RT under a stream of nitrogen and the crude
tography (TLC) by using Kieselgel 60 F254 (Merck) plates. product was taken up with diethyl ether. The organic phase
The preparation of the starting 2-phenylimidazo[1,2-a]pyr- was evaporated under reduced pressure to give the
idine-3-acetic acids 15 a–d was accomplished following a corresponding L-Dopa ethyl ester compounds 5–8.
reported procedure (10). Dopamine hydrochloride 17, L-Dopa,
tert-butyldimethylsilyl chloride (TBDMSCl), 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU), ethyl-1,2-dihydro-2-ethoxy-1-
quinolinecarboxylate (EEDQ), 1,10-carbonyldiimidazole
(CDI), triethylamine (TEA) and trifluoroacetic acid (TFA)
were purchased from Sigma-Aldrich (Italy).
Reagents used for the preparation of the buffers were of
analytical grade. Fresh deionized water from all glass
apparatus was used in the preparation of all the solutions.
Method B (Fig. 1). A solution of the suitably substituted
imidazo[1,2-a]pyridine-3-acetic acid 15 a–d (1.4 mmol) and
CDI (0.34 g, 2.1 mmol) in anhydrous DMF (20 ml) was stirred
at RT. After 15 min, L-Dopa ethyl ester hydrochloride 13
(0.55 g, 2.1 mmol) was added, and the stirring was prolonged
for 8 h. Solvent was evaporated under reduced pressure and
the residue purified on silica gel column chromatography
[ethyl acetate/petroleum ether 8/2 (v/v) as eluent] to give the
appropriate L-Dopa ethyl ester conjugate 5–8.
Ethyl-2-{2-[6,8-dichloro-2-(4-chlorophenyl)imidazo[1,2-
a]pyridine-3-yl]acetylamino}-3-[3,4-bis(tert-butyldimethylsily-
Synthesis of Ethyl 2-amino-3-[3,4-bis
(tert-butyldimethylsilyloxy)phenyl]propanoate 14
loxy)phenyl]propanoate (16a). IR (KBr): 1,738, 1,678 cmj1
;
1H NMR (CDCl3) d: 0.14 (s, 6 H, CH3Si), 0.16 (s, 6 H,
CH3Si), 0.95 (s, 9 H, C(CH3)3), 0.97 (s, 9 H, C(CH3)3), 1.29
(m, 3 H, CH3CH2), 2.9–3.1 (m, 2 H, CH2CH), 3.98 (s, 2 H,
CH2CONH), 4.24 (m, 2 H, CH2CH3), 4.78 (m, 1 H, CHCH2),
6.02 (br d, 1 H, NHCO), 6.4–6.7 (m, 3 H, Ar, L-Dopa
moiety), 7.3–7.6 (m, 5 H, Ar), 8.08 (d, 1 H, Ar).
The preparation of the compound 14 (Fig. 1) was
accomplished according to a previously reported method
(11). Briefly, a stirred mixture of L-Dopa (1 g, 5.07 mmol) in
ethanol (40 ml) was saturated with HCl gas and heated at
50-C for 3 h. Excess of HCl gas was removed under a stream
of nitrogen and then the solvent was evaporated under
reduced pressure. The resulting residue was dried under
vacuum to provide compound 13 in quantitative yield (12).
To a stirred suspension of L-Dopa ethyl ester hydrochlo-
ride 13 (2.3 g, 8.8 mmol) in acetonitrile (20 ml), TEA (1.2 ml,
8.8 mmol) was added. After cooling to 0-C, TBDMSCl (4 g,
26 mmol) and DBU (3.9 ml, 26 mmol) were added. Stirring
was continued for additional 18 h at room temperature (RT)
and the progress of the reaction was monitored by TLC
[chloroform/ethyl acetate 7/1 (v/v) as eluent]. The solvent
was removed under reduced pressure and then chloroform
was added to the residue. The organic phase was washed with
water, dried (Na2SO4) and evaporated to dryness. The crude
ester 14 was purified on silica gel column chromatography
[chloroform/ethyl acetate 7/1 (v/v) as eluent].
Ethyl-2-{2-[6-chloro-2-(4-chlorophenyl)imidazo[1,2-
a]pyridine-3-yl]acetylamino}-3-[3,4-bis(tert-butyldimethylsily-
loxy)phenyl]propanoate (16b). IR (KBr): 3,289, 1,737,
1
1,650 cmj1; H NMR (CDCl3) d: 0.12 (s, 6 H, CH3Si), 0.13
(s, 6 H, CH3Si), 0.94 (s, 9 H, C(CH3)3), 0.96 (s, 9 H,
C(CH3)3), 1.22 (t, 3 H, CH3CH2), 2.9–3.1 (m, 2 H, CH2CH),
3.85 (s, 2 H, CH2CONH), 4.1–4.2 (m, 2 H, CH2CH3), 4.78 (m,
1 H, CHCH2), 6.09 (br d, 1 H, NHCO), 6.4–6.7 (m, 3 H, Ar,
L-Dopa moiety), 7.2–7.5 (m, 6 H, Ar), 8.06 (d, 1 H, Ar).
Ethyl-2-{2-[6,8-dichloro-2-phenylimidazo[1,2-a]pyridine-
3-yl]acetylamino}-3-[3,4-bis(tert-butyldimethylsilyloxy)phe-
1
nyl]propanoate (16c). IR (KBr): 3,325, 1,738, 1,674 cmj1; H
NMR (CDCl3) d: 0.13 (s, 6 H, CH3Si), 0.14 (s, 6 H, CH3Si), 0.95
(s, 9 H, C(CH3)3), 0.97 (s, 9 H, C(CH3)3), 1.22 (t, 3 H,
CH3CH2), 2.9–3.0 (m, 2 H, CH2CH), 3.88 (s, 2 H, CH2CONH),
4.1–4.2 (m, 2 H, CH2CH3), 4.7–4.8 (m, 1 H, CHCH2), 6.01 (br
d, 1 H, NHCO), 6.4–6.7 (m, 3 H, Ar, L-Dopa moiety), 7.3–7.6
(m, 6 H, Ar), 8.09 (d, 1 H, Ar).
General Procedure for Preparation of Dopimid
Compounds 5–8
Ethyl-2-{2-[6-chloro-2-phenylimidazo[1,2-a]pyridine-3-
Method A (Fig. 1). To a stirred solution of the required yl]acetylamino}-3-[3,4-bis(tert-butyldimethylsilyloxy)phenyl]-
imidazo[1,2]pyridine-3-acetic acid 15 a–d (1.4 mmol) in propanoate (16d). IR (KBr): 3,314, 1,740, 1,682 cmj1; 1H NMR
anhydrous THF (20 ml), EEDQ (0.41 g, 1.68 mmol) and (CDCl3) d: 0.13 (s, 6 H, CH3Si), 0.15 (s, 6 H, CH3Si), 0.94 (s, 9
coumpound 14 (0.63 g, 1.4 mmol) were added. After 10 min, H, C(CH3)3), 0.96 (s, 9 H, C(CH3)3), 1.20 (t, 3 H, CH3CH2),
TEA (0.3 ml, 2.1 mmol) was added drop by drop, and stirred 2.8–3.0 (m, 2 H, CH2CH), 3.90 (s, 2 H, CH2CONH), 4.16 (q,
for prolonged time at RT for 15 h. The progress of reaction 2 H, CH2CH3), 4.7–4.9 (m, 1 H, CHCH2), 6.05 (br d, 1 H,

1312
Denora et al.
NHCO), 6.4–6.7 (m, 3 H, Ar, L-Dopa moiety), 7.2–7.7 (m, 7 H, CH2CH), 3.9–4.2 (m, 4 H, CH2), 4.65 (m, 1 H, CHCH2), 5.97 (br
Ar), 8.09 (d, 1 H, Ar).
d, 1 H, NHCO), 6.0–6.6 (m, 3 H, Ar, L-Dopa moiety), 7.3–7.6
(m, 5 H, Ar), 8.08 (d, 1 H, Ar); MS (ESI) m/z 562 [M + H]+.
Ethyl-2-{2-[6-chloro-2-(4-chlorophenyl)imidazo[1,2-
Ethyl-2-{2-[6,8-dichloro-2-(4-chlorophenyl)imidazo[1,2-
a]pyridine-3-yl]acetylamino}-3-(3,4-dihydroxyphenyl)propa-
noate (5). IR (KBr): 3,324, 1,732, 1,660 cmj1 1H NMR a]pyridine-3-yl]acetylamino}-3-(3,4-dihydroxyphenyl)propa-
;
(DMSO d6) d: 1.2–1.3 (m, 3 H, CH3CH2), 2.7–2.9 (m, 2 H, noate (6). IR (KBr): 3,318, 1,732, 1,660 cmj1
;
1H NMR
Method A
R2
OH
OH
OTBDMS
OTBDMS
OH
O
OH
O
N
R3
N
+
R1
O
O
OH
OCH2CH3
NH2 HCl
OCH2CH3
.
OH
NH2
NH2
14
13
15 a-d
R2
R2
N
N
R3
R3
N
N
R1
R1
OH
OTBDMS
OTBDMS
O
O
HN
HN
OH
O
O
H3CH2CO
5-8
H3CH2CO
16 a-d
R2
N
R3
OH
OH
N
R1
a) R1 = R2 = R3 = Cl
b) R1 = R3 = Cl; R2 = H
c) R1 = R2 = Cl; R3 = H
d) R1 = Cl; R2 = R3 = H
O
HN
O
HO
1-4
Method B
13 + 15 a-d
5-8
Fig. 1. Synthetic scheme for preparation of Dopimid compounds 5–8.

Novel L-Dopa and Dopamine Prodrugs
1313
(DMSO d6) d: 1.04 (t, 3 H, CH3CH2), 2.7–3.0 (m, 2 H, Synthesis of 2-(3,4-di-tert-butyldimethylsilyloxyphenyl)
CH2CH), 4.0–4.1 (m, 4 H, CH2), 4.4–4.5 (m, 1 H, CHCH2), ethanamine (18)
6.4–6.7 (m, 3 H, Ar, L-Dopa moiety), 7.2–7.8 (m, 6 H, Ar),
8.5–8.6 (m, 1 H, Ar), 8.81 (br d, 1 H, NHCO); MS (ESI) m/z
526 [M + H]+.
Dopamine hydrochloride 17 (2 g, 0.0105 mol) was added
to a stirred solution of TBDMSCl (4.8 g, 0.0316 mol) in
anhydrous acetonitrile (20 ml). The resulting suspension
under stirring was cooled with an ice bath and then DBU
(4.7 ml, 0.0316 mol) was added drop-wise during 10 min
reaction. The ice bath was removed after 1 h and stirred for
further 18 h at RT. After this period, solvent was removed
under reduced pressure to give compound 18 (Fig. 2) as an oil
(3.6 g, 90% yield).
Ethyl-2-{2-[6,8-dichloro-2-phenylimidazo[1,2-a]pyridine-
3-yl]acetylamino}-3-(3,4-dihydroxyphenyl)propanoate (7). IR
(KBr): 3,401, 1,732, 1,656 cmj1; 1H NMR (DMSO d6) d: 1.22
(t, 3 H, CH3CH2), 2.9–3.0 (m, 2 H, CH2CH), 3.8–4.0 (m, 2 H,
CH2CONH), 4.1–4.2 (m,
2 H, CH2CH3), 4.7–4.8
(m, 1 H, CHCH2), 6.2–6.6 (m, 3 H, Ar, L-Dopa moiety), 7.3–
7.6 (m, 6 H, Ar), 8.0–8.1 (m, 1 H, Ar); MS (ESI) m/z 526
[M+ H]+.
1
IR (Nujol): 3,404 cmj1; H NMR (CDCl3) d: 0.15 (s, 12
H, SiCH3), 0.95 (s, 18 H, C(CH3)3), 2.91 (m, 2 H, CH2Ar),
3.11 (m, 2 H, CH2NH2), 6.6–6.7 (m, 3 H, Ar); MS m/z : 381
(M+, 12), 193 (base).
Ethyl-2-{2-[6-chloro-2-phenylimidazo[1,2-a]pyridine-3-
yl]acetylamino}-3-(3,4-dihydroxyphenyl)propanoate (8). IR
;
(KBr): 3,335, 1,729, 1,651 cmj1 1H NMR (DMSO d6) d:
1.0–1.2 (m, 3 H, CH3CH2), 2.8–3.0 (m, 2 H, CH2CH), 3.9–4.1
(m, 4 H, CH2), 4.3–4.4 (m, 1 H, CHCH2), 6.4–6.7 (m, 3 H, Ar,
L-Dopa moiety), 7.2–7.7 (m, 7 H, Ar), 8.4–8.5 (m, 1 H, Ar);
MS (ESI) m/z 494 [M + H]+.
General Procedure for Preparation of 2-(2-phenylimidazo
[1,2-a]pyridine-3-yl)-N-[2-(3,4-di-tert-butyldimethylsilyloxyphenyl)
ethyl]acetamide (19 a–d)
General Procedure for Preparation of Dopimid
Compounds 1–4
To a stirred solution of the suitably substituted 2-
phenylimidazo[1,2-a]pyridine-3-acetic acid 15 a–d (2.1 mmol)
in anhydrous THF (20 ml), EEDQ (0.62 g, 2.52 mmol) and
To a solution of the appropriate ethyl ester 5–8 (0.57 mmol) 3,4-Bis(tert-butyldimethylsilyloxy)-phenetylamine 18 (0.8 g,
in dioxane (20 ml), HCl 1N (5 ml) was added. The mixture 2.1 mmol) were added. The reaction mixture was stirred at
was stirred at 50-C for 24 h and then, the solvent was RT for 10 min and then TEA (0.44 ml, 3.15 mmol) was added
evaporated under reduced pressure. The resulting residue drop-wise. Stirring was continued for additional 24 h at RT
was taken up with aqueous NaHCO3 and extracted with ethyl and then the solvent was evaporated under reduced pressure.
acetate (3 ꢀ 20 ml). The cooled aqueous solution was The resulting crude residue was purified by column chroma-
acidified with dilute HCl and the resulting precipitate was tography on silica gel [petroleum ether/ethyl acetate 9/1 (v/v)
pure L-Dopa conjugate 1–4, which was isolated by filtration. as eluent] to give the appropriate compound 19 a–d (Fig. 2).
2-{2-[6,8-dichloro-2-(4-chlorophenyl)imidazo[1,2-a]pyri-
din-3-yl]acetylamino}-3-(3,4-dihydroxyphenyl)propanoic acid yl]-N-[2-(3,4-di-tert-butyldimethylsilyloxyphenyl)ethyl]acet-
(1). IR (KBr): 3,342, 1,731, 1,660 cmj1 1H NMR (DMSO amide (19a). IR (KBr): 3,306, 1,649 cmj1; 1H NMR (CDCl3)
2-[6,8-dichloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-3-
;
d6) d: 2.7–2.9 (m, 2 H, CH2CH), 4.03 (s, 2 H, CH2CONH), 4.37 d: 0.14 (s, 6 H, CH3Si), 0.17 (s, 6 H, CH3Si), 0.95 (s, 9 H, C(CH3)3),
(m, 1 H, CHCH2), 6.4–6.7 (m, 3 H, Ar, L-Dopa moiety), 7.4– 0.97 (s, 9 H, C(CH3)3), 2.65 (t, J=6.7 Hz, 2 H, CH2Ar), 3.45
7.8 (m, 5 H, Ar), 8.6–8.7 (m, 1 H, Ar), 8.75 (br d, 1 H, NHCO); (q, J= 6.7 Hz, 2 H, CH2NHCO), 3.83 (s, 2 H, CH2CONH), 5.85
MS (ESI) m/z 534 [M + H]+.
(br s, 1 H, NHCO), 6.4–6.7 (m, 3 H, Ar, dopamine moiety), 7.3–7.6
(m, 5 H, Ar), 8.08 (d, 1 H, Ar); Anal. (C35H46O3N3Cl3Si2) C, H, N.
2-[6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-3-
2-{2-[6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridin-
3-yl]acetylamino}-3-(3,4-dihydroxyphenyl)propanoic acid
(2). IR (KBr): 3,238, 1,730, 1,666 cmj1 1H NMR (DMSO yl]-N-[2-(3,4-di-tert-butyldimethylsilyloxyphenyl)ethyl]acet-
;
d6) d: 2.7–3.0 (m, 2 H, CH2CH), 4.09 (m, 2 H, CH2CONH), amide (19b). IR (KBr): 3,293, 1,651 cmj1; 1H NMR (CDCl3)
4.3–4.4 (m, 1 H, CHCH2), 6.4–6.7 (m, 3 H, Ar, L-Dopa d: 0.12 (s, 6 H, CH3Si), 0.16 (s, 6 H, CH3Si), 0.93 (s, 9 H,
moiety), 7.6–7.9 (m, 6 H, Ar), 8.81 (br d, 1 H, NHCO), 8.9– C(CH3)3), 0.96 (s, 9 H, C(CH3)3), 2.69 (t, 2 H, CH2Ar), 3.49 (q,
9.0 (m, 1 H, Ar); MS (ESI) m/z 500 [M + H]+.
2 H, CH2NHCO), 3.84 (s, 2 H, CH2CONH), 6.02 (br s, 1 H,
NHCO), 6.4–6.7 (m, 3 H, Ar, dopamine moiety), 7.2–7.5 (m, 6
H, Ar), 8.06 (m, 1 H, Ar); Anal. (C35H47O3N3Cl2Si2) C, H, N.
2-[6,8-dichloro-2-phenylimidazo[1,2-a]pyridine-3-yl]-N-
2-{2-[6,8-dichloro-2-phenylimidazo[1,2-a]pyridin-3-yl]a-
cetylamino}-3-(3,4-dihydroxyphenyl)propanoic acid (3). IR
;
(KBr): 3,238, 1,730, 1,666 cmj1 1H NMR (DMSO d6) d:
2.7–3.0 (m, 2 H, CH2CH), 3.9–4.0 (m, 2 H, CH2CONH), 4.1– [2-(3,4-di-tert-butyldimethylsilyloxyphenyl)ethyl]acetamide
4.2 (m, 1 H, CHCH2), 6.4–6.7 (m, 3 H, Ar, L-Dopa moiety), (19c). IR (KBr): 3,304, 1,650 cmj1; 1H NMR (CDCl3) d: 0.14
7.3–7.8 (m, 6 H, Ar), 8.0–8.2 (br m, 1 H, NHCO), 8.4–8.5 (m, (s, 6 H, CH3Si), 0.16 (s, 6 H, CH3Si), 0.95 (s, 9 H, C(CH3)3), 0.98
1 H, Ar); MS (ESI) m/z 500 [M + H]+.
(s, 9 H, C(CH3)3), 2.64 ( t, 2 H, CH2Ar), 3.46 (q, 2 H,
CH2NHCO), 3.86 (s, 2 H, CH2CONH), 5.85 (br s, 1 H, NHCO),
6.4–6.7 (m, 3 H, Ar, dopamine moiety), 7.3–7.6 (m, 6 H, Ar), 8.09
(m, 1 H, Ar); Anal. (C35H47O3N3Cl2Si2) C, H, N.
2-{2-[6-chloro-2-phenylimidazo[1,2-a]pyridin-3-yl]acety-
lamino}-3-(3,4-dihydroxyphenyl)propanoic acid (4). IR
;
(KBr): 3,391, 1,725, 1,661 cmj1 1H NMR (DMSO d6) d:
2.7–3.0 (m, 2 H, CH2CH), 4.13 (s, 2 H, CH2CONH), 4.3–4.5
2-[6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl]-N-[2-
(m, 1 H, CHCH2), 6.4–6.7 (m, 3 H, Ar, L-Dopa moiety), 7.5– (3,4-di-tert-butyldimethylsilyloxyphenyl)ethyl]acetamide
8.0 (m, 7 H, Ar), 8.80 (br d, 1 H, NHCO), 9.0–9.1 (m, 1 H, (19d). IR (KBr): 3,295, 1,655 cmj1; 1H NMR (CDCl3) d: 0.13
(s, 6 H, CH3Si), 0.16 (s, 6 H, CH3Si), 0.94 (s, 9 H, C(CH3)3), 0.97
Ar); MS (ESI) m/z 466 [M + H]+.

1314
Denora et al.
OH
OTBDMS
OTBDMS
R2
OH
N
R3
N
+
R1
O
.
OH
NH2 HCl
NH2
15 a-d
17
18
R2
N
R3
OTBDMS
OTBDMS
N
a) R1 = R2 = R3 = Cl
R1
b) R1 = R3 = Cl; R2 = H
c) R1 = R2 = Cl; R3 = H
d) R1 = Cl ; R2 = R3 = H
O
HN
19 a-d
R2
N
R3
OH
OH
N
R1
O
HN
9-12
Fig. 2. Synthetic scheme for preparation of Dopimid compounds 9–12.
(s, 9 H, C(CH3)3), 2.63 ( t, 2 H, CH2Ar), 3.46 (q, 2 H, under nitrogen atmosphere for 24 h at RT. Then, the solvent
CH2NHCO), 3.90 (s, 2 H, CH2CONH), 5.85 (br s, 1 H, NHCO), was evaporated at RT under a stream of nitrogen and the
6.4–6.7 (m, 3 H, Ar, dopamine moiety), 7.2–7.7 (m, 7 H, Ar), resulting residue was taken up with diethyl ether. The organic
8.09 (m, 1 H, Ar); Anal. (C35H48O3N3ClSi2) C, H, N.
phase was dried over Na2SO4 and the solvent was evaporated
under reduced pressure to give the corresponding dopamine
conjugate 9–12 (Fig. 2). Yields of these Dopimid compounds
were approximately quantitative.
General Procedure for Preparation of 2-(2-phenylimidazo
[1,2-a]pyridine-3-yl)-N-[2-(3,4-dihydroxyphenyl)
ethyl]acetamide (9–12)
2-[6,8-dichloro-2-(4-chlorophenyl)imidazo[1,2-a]pyri-
dine-3-y]-N-[2-(3,4-dihydroxyphenyl)ethyl]acetamide (9). IR
1
Complete deprotection of the 2-(2-phenylimidazo[1,2- (KBr) 3,295, 1,667 cmj1; H NMR (DMSOd6) d: 2.53 (t, 2 H,
a]pyridine-3-yl)-N-[2-(3,4-di-tert-butyldimethylsilyloxyphe- CH2Ar), 3.24 (q, 2 H, CH2NHCO), 3.99 (s, 2 H, CH2CONH),
nyl)ethyl] acetamide 19 a–d was accomplished by dissolving 6.4–6.6 (m, 3 H, Ar, dopamine moiety), 7.5–7.9 (m, 4 H, Ar)
the suitably substituted trimethylsilyl derivative (200 mg) in 7.69 (d, 1 H, Ar), 8.68 (d, 1 H, Ar); MS (ESI): m/z 490 [M + H]+;
95% aqueous TFA (1.5 ml). The reaction was carried out Anal. (C23H18O3N3Cl3) C, H, N.

Novel L-Dopa and Dopamine Prodrugs
1315
0.05 M phosphate buffer saline (0.14 M NaCl) at pH 7.4,
containing 50% v/v of rat serum. The stability of L-Dopa
in physiological medium was carried out by adding 10 ml of
a stock solution in phosphate buffer pH 8.0 (4.2 mg/ml) to
1.6 ml of the preheated serum solution. The final
concentration was about 100 mM. The resulting test solu-
tions were maintained at 37 T 0.2-C in a shaker water bath.
Aliquots of 100 ml were withdrawn at appropriate intervals
and treated with 500 ml of cold acetonitrile in order to
precipitate the serum proteins. After mixing and centrifu-
gation for 5 min at 13,200 rpm, the clear supernatant (20
ml) was analyzed by HPLC. Pseudo-first-order rate con-
stants for degradation of the derivatives were determined
from the slopes of linear plots of the logarithms of residual
dopamine or L-Dopa conjugate against time.
For assessment of L-Dopa stability a reversed phase
Phenomenex Synergi Hydro C18 (25 cm ꢀ 3.9 mm; 5 mm
particles) column in conjunction with a precolumn (Sentry
Guard Symmetry C18, 20 ꢀ 3.9 mm) was used with 20 mM
K2HPO4 buffer pH 2.5. The injected volume was 20 ml. The
flow rate of 0.8 ml/min was maintained and the column
effluent was monitored continuously at 280 nm.
2-[6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-
3-yl]-N-[2-(3,4-dihydroxyphenyl)ethyl]acetamide (10). IR
1
(KBr) 3,305, 1,667 cmj1; H NMR (DMSOd6) d: 2.60 (t, 2 H,
CH2Ar), 3.33 (q, 2 H, CH2NHCO), 4.10 (s, 2 H, CH2CONH),
6.5–6.7 (m, 3 H, Ar, dopamine moiety), 7.6–8.0 (m, 6 H, Ar),
9.03 (m, 1 H, Ar); MS (ESI): m/z 456 [M + H]+; Anal.
(C23H19O3N3Cl2) C, H, N.
2-[6,8-dichloro-2-phenylimidazo[1,2-a]pyridine-3-yl]-N-
[2-(3,4-dihydroxyphenyl)ethyl]acetamide (11). IR (KBr)
;
3,323, 1,661 cmj1 1H NMR (DMSOd6) d: 2.50 (t, 2 H,
CH2Ar), 3.33 (q, 2 H, CH2NHCO), 4.10 (s, 2 H, CH2CONH),
6.4–6.6 (m, 3 H, Ar, dopamine moiety), 7.4–7.7 (m, 6 H, Ar),
8.68 (m, 1 H, Ar); MS (ESI): m/z 456 [M + H]+; Anal.
(C23H19O3N3Cl2) C, H, N.
2-[6-chloro-2-phenylimidazo[1,2-a]pyridine-3-y]-N-[2-
(3,4-dihydroxyphenyl)ethyl]acetamide (12). IR (KBr) 3,293,
1,669 cmj1; 1H NMR (DMSOd6) d: 2.5–2.6 (m, 2 H, CH2Ar),
3.2–3.3 (m, 2 H, CH2NHCO), 4.03 (s, 2 H, CH2CONH), 6.4–
6.6 (m, 3 H, Ar, dopamine moiety), 7.5–7.9 (m, 7 H, Ar), 8.9–
9.0 (m, 1 H, Ar); MS (ESI): m/z 422 [M + H]+; Anal.
(C23H20O3N3Cl) C, H, N.
Stability Studies
Receptor Binding Assays
Chemical Hydrolysis
Adult male or female Sprague–Dawley CD rats (Charles
Stability studies were carried out at controlled temper- River, Como, Italy) with body masses of 200–250 g at the
ature (37 T 0.2-C) in a water bath shaken at 150 rpm. The beginning the experiments, were maintained under an
hydrolysis of Dopimid compounds 1, 2, 5, 6, 9, and 10 was artificial 12-h-light/dark cycle (light on 08.00–20.00 h) at a
studied at pH 7.4 in 0.05 M phosphate buffer at 37-C. The constant temperature of 23 T 2-C and 65% humidity. Food
experiments were carried out by adding 50 ml of a stock and water were freely available, and the animals were
solution in DMSO (1.5, 2.6, 3.6, 2.8, 3.7, and 2.7 mg/ml for 1, acclimatized for >7 days before use. Experiments were
2, 5, 6, 9, and 10, respectively), to 5 ml of the buffer solution performed between 08.00 and 14.00 h. Animal care and
preheated at 37-C. The final concentration of the resulting handling throughout the experimental procedure were per-
solutions was about 50 mM. The test solutions of the formed in accordance with the European Communities
compounds were vortexed and maintained in a shaker water Council Directive of 24 November 1986 (86/609/EEC). The
bath at constant temperature of 37 T 0.2-C. At appropriate experimental protocol was approved by the Animal Ethical
intervals, aliquots of 100 ml were removed and immediately Committee of the University of Cagliari.
analyzed by high-performance liquid chromatography
(HPLC). Pseudo-first-order rate constants for the hydrolysis [3H]Flunitrazepam Binding
of the derivatives were determined from the slopes of linear
plots of the logarithms of residual compound against time.
Cerebral cortex was homogenized with a Polytron PT 10
HPLC analyses were performed with a Waters Associates in 50 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4)
Model 600 pump equipped with a Waters 990 variable and centrifuged twice at 20,000 ꢀ g for 10 min. The pellet was
wavelength UV detector and a 20 ml loop injection valve. reconstituted in 50 volumes of Tris-HCl buffer and was used
A reversed phase Symmetry C18 (25 cm ꢀ 3.9 mm; 5 mm for the binding assay. Aliquots of 400 ml of tissue homog-
particles) column in conjunction with a precolumn (Sentry enate (0.4–0.5 mg of protein) were incubated in presence of
Guard Symmetry C18, 20 ꢀ 3.9 mm) was eluted with [3H]flunitrazepam (spec. act. 7.4 Ci/mmol, New England
mixtures of methanol and 50 mM acetate buffer pH 4.7 Nuclear) at a final concentration of 0.5 nM, in a total
70/30 (v/v). The injected volume was 20 ml. The flow rate incubation volume of 1,000 ml. Drugs were dissolved in
of 0.8 ml/min was maintained and the column effluent was DMSO and serial dilutions were made up in DMSO and
monitored continuously at 254 nm. The compounds were added in 100 ml aliquots. After a 60 min incubation at 0-C,
estimated by measuring the peak areas or peak heights in the assay was determined by rapid filtration through glass-
relation to those of standards chromatographed under the fiber filter strips (Whatman GF/B). The filters were rinsed
same conditions.
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 (Ultima Gold,
Camberra Packard). Nonspecific binding was determined as
Stability in Physiological Medium
The stability of Dopimid compounds 1, 2, 5, 6, 9, 10 and binding in the presence of 5 mm diazepam, and represented
L-Dopa in physiological medium was studied at 37-C in about 10% of total binding.

1316
Denora et al.
[3H]PK 11195 Binding
MDR1) was a gift from Professor Piet Borst (The Netherlands
Cancer Institute, Amsterdam, The Netherlands). The growth
After sacrifice brain and ovary were rapidly removed, medium Dulbecco_s Modified Eagle Medium (DMEM),
cerebral cortex was dissected and both tissues were stored at trypsin/EDTA solution, calf serum, minimum essential medi-
j80-C until assay. The tissues were thawed and homoge- um (MEM) and non-essential amino acids were obtained from
nized in 50 volumes of Dulbecco_s phosphate buffered Gibco (Invitrogen, Grand Island, NY, USA). Penicillin
saline (PBS) pH 7.4 at 4-C with a Polytron PT 10 (setting (10,000 U/ml), streptomycin (10,000 mg/ml), sodium bicarbon-
5, for 20s). The homogenate was centrifuged at 40,000 ꢀ g ate, HEPES and quinidine (P-gp inhibitor) were purchased
for 30 min, and the pellet was resuspended in 50 volumes of from Sigma Chemical Co. (St. Louis, MO, USA). [3H]Ritonavir
PBS and recentrifugated. The new pellet was resuspended in (specific activity 1.0 Ci/mmol) was obtained from Moravek
20 volumes of PBS and used for the assay. [3H]PK 11195 Biochemicals (Brea, CA, USA). [3H]Diazepam (specific
binding was determined in a final volume of 1,000 ml tissue activity 76 Ci/mmol), [14C]mannitol (specific activity 55
homogenate (0.15–0.20 mg protein), 100 ml of [3H]PK 11195 mCi/mmol) and [3H]L-Dopa (specific activity 50 Ci/mol)
(spec. act. 85.5 Ci/mmol, New England Nuclear) at final assay were purchased from Perkin Elmer Life Science Products
concentration of 1 nM, 5 ml of drug solution or solvent and (Boston, MA, USA) and from American Radiolabeled
795 ml of PBS buffer (pH 7.4 at 25-C). Drugs were dissolved Chemicals Inc. (St. Louis, MO, USA), respectively. Culture
in dimethylsulfoxide and serial dilutions were made up in flasks (75 cm2 growth area), polyester Transwells\ (pore size
dimethylsulfoxide and added in 5 ml aliquots. Incubations 0.4 mm and 6.5 mm diameter) and 12 well plates were
(0-C) were initiated by addition of membranes and were obtained from Corning Costar (Cambridge, MA, USA).
terminated 90 min later by rapid filtration through glass- Buffer components and other solvents were obtained from
fiber filter strips (Whatman GF/B), which were rinsed with Fisher Scientific Co. (Fair Lawn, NJ, USA).
two 4 ml of ice-cold PBS buffer using Cell Harvaster filtration
manifold (Brandel). Filter bound radioactivity was quantified Uptake Studies
by liquid scintillation spectrometry (Ultima Gold, Camberra
Packard). Non specific binding was defined as binding in the
presence of 10 mM unlabelled PK 11195 (Sigma).
Uptake studies were performed using confluent cells
monolayer 6–8 days post seeding. Medium was aspirated and
cells were washed thrice with Dulbecco_s phosphate buffered
saline (DPBS), pH 7.4. Uptake was initiated by adding 1 ml
of drug solution at increasing concentration (10, 25, 50 and
75 mM, respectively), in DPBS 0.01% (v/v) Cremophor EL
5% (v/v) DMSO, in the presence or absence of competing
substrates, to the wells. Incubation was carried out for a
period of 10 min at 37-C in a humidified atmosphere with
5% CO2. At the end of the incubation period drug solution
was removed and the cells monolayer was washed thrice with
ice-cold stop solution. Cells were lysed overnight by adding 1
ml 0.1% w/v Triton X-100 and 0.3 N sodium hydroxide at
RT. Aliquots of 500 ml were withdrawn from each well and
transferred to scintillation vials containing 5 ml scintillation
cocktail. Samples were then analyzed by liquid scintillation
spectrophotometry using a Beckman scintillation counter
(Model LS-6500, Beckman Instruments, Inc.). Rate of uptake
was normalized to the protein content of each well. Amount
of protein in the cell lysate was quantified by the method of
Bradford utilizing Bio-Rad protein estimation kit (Bio-Rad,
Hercules, CA, USA).
DA D2-like Receptor Binding Assay
Rats were killed by decapitation, and the striatum was
immediately dissected on ice. The tissue was rapidly homog-
enized in 200 vol (200 ml per gram of wet tissue) of ice-cold
50 mM Tris-HCl (pH 7.7 at 25-C), designated buffer A, with
the use of a Teflon pestle and glass homogenizer (two series
of 15 strokes with a 1-min interval between series). The
homogenate was centrifuged for 10 min at 43,000 ꢀ g, and
the resulting pellet was resuspended and homogenized in 200
vol of buffer B (buffer A containing 120 mM NaCl, 5 mM
KCl, 2 mM CaCl2, 1 mM EDTA, and 5.7 mM ascorbic acid)
before use in the binding assay. The binding of [3H]YM-
09151 was determined in a final volume of 1 ml consisting of
400 ml of membrane suspension (1.5–2.0 mg of protein), 100 ml
of 0.4 nM [3H]YM-09151 (84 Ci/mmol; New England Nuclear),
5 ml of drug (10j11–10j4 M, dissolved and diluted in DMSO) or
solvent, and buffer B to volume. Nonspecific binding was
determined in the presence of 50 mM L-sulpiride (Knoll S.p.A.,
Milan, Italy). After incubation for 60 min at 25-C in the
dark, the binding reaction mixtures were passed through
glass-fiber (Whatman GF/B) filters with the use of a Cell-
Harvester filtration manifold (M24; Brandel, Gaithersburg,
MD). Filters were then rinsed twice with 4 ml portions of
ice-cold buffer A and transferred to vials containing 3.5 ml
of scintillation cocktail (Ultima Gold, Camberra Packard).
Radioactivity associated with the filters was quantified with a
scintillation spectrometer.
All uptake experiments were performed at least in
quadruplicate (n = 4) and results are expressed as meanT SD.
The permeability coefficient experiments were carried out in
triplicate (n = 3) and results are expressed as mean T SD.
Statistical analyses were performed using Student_s t test
between two mean values. A probability of less than 0.05
(p < 0.05) was considered to be statistically significant.
Bi-directional Transport Studies on MDCKII-MDR1
Monolayers
Cell Culture
Bi-directional transport studies were carried out
using MDCKII-MDR1 cells monolayers grown on 12 well
Madin–Darby Canine Kidney (i.e., MDCK) cells, retro- Transwell\ insert (diameter 6.5 mm). All experiments were
virally transfected with the human MDR1 cDNA (MDCKII- done at 37-C in air. Medium was aspirated from both the

Novel L-Dopa and Dopamine Prodrugs
1317
apical (AP) and basolateral (BL) chambers of each insert and (20 mM phosphate buffer, pH 2.5), flow rate (1 ml/min) and
cells monolayers were washed three times (10 min per wash) wavelength (l = 280 nm).
with DPBS pH 7.4. The formation of confluent MDCKII-
MDR1 monolayers with tight junctions was confirmed Computational Calculations of Physicochemical
by microscopy and TEER values. [3H]Diazepam and and Permeability Characteristics of Dopimid
[13C]mannitol were used as markers for the transcellular Compounds 1–12
and paracellular pathway, respectively, and as an internal
control to verify tight junction integrity during the assay. Test
drug solution in DPBS 0.01% (v/v) Cremophor EL 5% (v/v) by using C Log P (v. 4, BioByte Corp) software. To estimate the
DMSO at a concentration of 100 mM was added to the donor BBB penetration of Dopimid compounds 1–12, their log Cbrain
Lipophilicity of the examined compounds was estimated
/
side (0.5 ml for the AP chamber and 1.5 ml for the BL Cblood (log BB) values were calculated according to the
chamber) and fresh DPBS was placed in the receiver equation: log BB ¼ ꢂ0:0148ðꢃ 0:001Þ PSA þ 0:152ðꢃ 0:036Þ
compartment. For AP-to-BL or BL-to-AP flux studies, the C log P þ 0:139ðꢃ 0:073Þ (14) in which PSA is the polar sur-
2
drug solution was added in the AP chamber or in the BL face area (defined as the surface area in A occupied by
˚
chamber, respectively. Aliquots (200 ml) were withdrawn nitrogen and oxygen atoms and polar hydrogens connected
from the receiver side at various time intervals of 180 min, to these heteroatoms) and Clog P is the calculated log Po/w
and replaced with fresh DPBS pH 7.4 to maintain sink (octanol/water partition coefficient). Calculation of the polar
conditions. Samples were stored at j80-C until further surface area was made by using a simple protocol proposed by
analysis. Statistical analyses were performed using analysis Ertl et al., (15) allowing the so-called topological PSA
of variance (ANOVA) followed by the Tukey post hoc tests (TPSA). The lipophilicity indexes of Dopimid compounds 1–
(GraphPad Prism v. 4 for Windows, GraphPad Software, San 12, expressed as log k0 = log (tR j t0)/t0, were obtained eluting
Diego, CS). Differences were considered statistically significant these compounds on a reversed phase Symmetry C18 (25 cm ꢀ
at p < 0.05.
3.9 mm; 5 mm particles) column in conjunction with a
The apparent permeability coefficient (Papp) was calcu- precolumn (Sentry Guard Symmetry C18, 20 ꢀ 3.9 mm) with
lated according to the following equation:
mixtures of methanol and acetate buffer pH 4.7 70/30 (v/v).
The injected volume was 20 ml. The flow rate was maintained
at of 0.8 ml/min and the column effluent was monitored
continuously at 254 nm. High-performance liquid chroma-
tography (HPLC) analyses were performed with a Waters
V ꢁ dC
Papp
¼
ð1Þ
A ꢁ C0 ꢁ dt
where V ꢁ ðdC=dtÞ is the linear appearance rate of mass in Associates Model 600 pump equipped with a Waters 990
the receiver solution, A is the filter/cell surface area and C0 is variable wavelength UV detector. The column effluent was
the initial concentration of the test compounds. The net monitored continuously at 254 nm. HPLC mobile phase was
efflux of a test compound was assessed by calculating the prepared using HPLC-grade methanol.
ratio of Papp in the BL-to-AP direction vs. Papp in the AP-to-
BL direction (Papp BL-to-AP/Papp AP-to-BL). A ratio of substan-
tially greater than 1.0 indicates net efflux (13).
[3H]Diazepam, [14C]mannitol and [3H]L-Dopa samples
were analyzed by liquid scintillation spectrophotometry using
Isolation and Culture of BBMEC
The bovine brain microvessel endothelial cells (i.e.,
a Beckman scintillation counter (Model LS-6500, Beckman BBMECs) were isolated from the gray matter of bovine
Instruments, Inc.). Dopimid compounds 1, 2, 5, 6, 9, 10, and brains following an isolation procedure described previously
dopamine hydrochloride samples were assayed by HPLC. (16,17). Briefly two or more freshly excised bovine brains
HPLC analyses were performed with a HP 1050 pump were purchased from Steve_s Meat Market (DeSoto, KS,
equipped with a Waters 990 variable wavelength UV USA) and transported to the laboratory in ice-cold MEM.
detector and an Alcott autosampler (model 718AL HPLC). Subsequently, all the procedures for isolation were per-
For transport studies on Dopimid compounds 1, 2, 5 and 6, a formed in aseptic condition using sterile materials and lam-
reversed phase Phenomenex Synergi Hydro C18 (25 cm ꢀ 4.6 mm; inar flow hood. The large surface capillaries and meninges
4 mm particles) column in conjunction with a precolumn were removed from the brains and the gray matter was
(Sentry Guard Symmetry C18, 20 ꢀ 3.9 mm) was used with scraped off and minced into small pieces (1–2 mm). The gray
mixtures of methanol and deionized water 70/30 (v/v). A matter suspension was treated with dispase for 30 min in the
50 ml of the sample was injected and the flow rate of 0.8 ml/ shaker bath set at 37-C and 100 oscillations per min. The pH
min was maintained. The column effluent was monitored of the suspension was adjusted to 7.4, by adding MEM pH 9–
continuously at 254 nm. Quantification of the compounds 10. The suspension was returned to the same setting for an
was carried out by measuring the peak areas in relation to another hour. The suspension was then centrifuged at
those of chromatographic standards under the same 3,200 rpm (Beckman centrifuge, rotor-JA 14) for 10 min at
conditions. Analyses of Dopimid compounds 9 and 10 were 4-C. Three distinct layers: a red pellet at the bottom, a large
performed similarly with a change in mobile phase containing semisolid pinkish layer in the middle and brown liquid
methanol and deionized water 30/70 (v/v). The flow rate of supernatant at the top were thus obtained. The brown liquid
0.6 ml/min was maintained and the column effluent was supernatant was discarded and the rest of the suspension was
monitored continuously at 254 nm. Flux of dopamine mixed with 13% complete dextran solution and centrifuged
hydrochloride was evaluated by using the reported HPLC at 7,730 rpm (Beckman centrifuge, rotor-JA14) for 10 min.
conditions, except for the composition of elution mixture On centrifugation, a thick red pellet was obtained containing

1318
Denora et al.
erythrocytes and micro vessels. These micro vessels were (diameter 6.5 mm). All experiments were done at 37-C in air.
further treated with collagenase/dispase for approx 2.5 h at Medium was aspirated from both the apical AP and BL
shaker bath to separate the endothelial cells from the fat, chambers of each insert and cells monolayers were washed
myelin, and other cells. Micro vessel endothelial cells were three times (10 min per wash) with DPBS pH 7.4. These
collected from second layer of the percoll gradient, rinsed transport experiments were studied by using Dopimid
with MEM and stored in freezing medium containing 70% of compounds 1, 2, 5, 6, 9, and 10 at 100 mM and confluent
MEM-F12 medium, 20% horse serum and 10% DMSO at BBMEC monolayers grown on 12 well Transwell\ on one
j80-C and then in liquid nitrogen.
side of collagen-coated membrane inserts and astrocytes on
The isolated brain micro vessel endothelial cells were the bottom of six well plastic dishes. Again [3H]diazepam and
thawed at 37-C, rinsed and resuspended in culture medium [13C]mannitol were used as markers for the transcellular and
containing 45% v/v MEM, 45% v/v Ham_s F-12 nutrient paracellular pathway, respectively, and as an internal control
mixture, 10% v/v horse serum, 100 mg/ml penicillin-G, 100 mg/ml to verify tight junction integrity during the assay. [3H]L-Dopa
streptomycin, 100 mg/ml heparin, 2.5 mg/ml amphoter icin-B, and dopamine hydrochloride were also included in transport
50 mg/ml polymyxin-B and seeded on collagen-fibronectin experiments. The drug solution was added in the AP chamber
coated 12 well plates at density of 50,000 cells cm2. The cells at a concentration of 100 mM and fresh DPBS was placed in
were incubated at 37-C, 5% CO2 and 95% humidity for 3–4 the BL chamber. Drug solutions were prepared in DPBS
days undisturbed. After that the cells were fed with the 0.01% (v/v) Cremophor EL 5% (v/v) DMSO. Volumes of the
aforementioned medium without amphotericin-B and AP and the BL chambers were 0.5 and 1.5 ml, respectively.
polymyxin-B every other day until formation of a confluent Aliquots (200 ml) were withdrawn from the receiver side at
monolayer. Cells were used for uptake studies usually after various time intervals to 180 min, and replaced with fresh
12–14 days.
DPBS pH 7.4 to maintain sink conditions. Samples were
stored at j80-C until further analysis. The permeability
coefficient (Papp) was determined according to Eq. 1 as
described above. Samples collected were analyzed by HPLC
or counted on a liquid scintillation counter under the same
conditions as illustrated previously for transport studies on
MDCKII-MDR1 cells monolayers.
Co-culture of BBMECs and Astrocytes
In order to establish some of the cellular environment
complexities that exist in vivo, we used an in vitro model of
BBB by growing endothelial cells on one side of collagen-
coated membrane inserts and astrocytes on the bottom of six
well plastic dishes, as previously described (18,19). Human
astrocytes were purchased from ATCC. These cells were
tested negative for HIV-1 and hepatitis-B DNA by PCR.
Cells were grown to confluence in 7–10 days in RPMI-1640
medium supplemented with 10 mg/ml human recombinant
epidermal growth factor (hEGF), 10 mg/ml insulin, 50 mg/ml
gentamicin, 50 mg/ml amphothericin-B and 5% v/v fetal
bovine serum (FBS), in 75 cm2 culture flasks at 37-C and a
water saturated atmosphere containing 5% CO2.
Astrocytes were plated, with a density of 50,000 cells/cm2,
on the under side of a collagen-coated polyester Transwell\
membrane in the specific medium supplemented with all the
factors described above. After 48 h, the astrocyte medium in
the basal compartment was replaced. The medium in the
apical chamber was removed and was replaced with the
BBMECs specific medium, described above, supplemented
with 0.5% v/v of endothelial growth factor (ECGF).
BBMECs were seeded in the apical chamber at 50,000 cells/
cm2. The Transwell\ plates were then incubated at 37-C in an
atmosphere containing 5% CO2. Under these experimental
conditions, BBMECs formed a confluent monolayer within
15 days. Cell confluence was assessed by light microscopy
and transepithelial electric resistance (TEER) and flux of
[14C]mannitol were measured as markers of cell monolayers
integrity. TEER values of the cell monolayers were
approximately 200 ohmIcm2 and [14C]mannitol transport
was <0.5%/h in representative cell monolayers.
Brain Microdialysis
Dopimid compounds 5, 6, 9 were dissolved in distilled
water and injected intraperitoneally in a volume of 3 ml/kg
of body mass. Rats were anesthetized with chloral hydrate
(0.4 g/kg, i.p.) and a concentric dialysis probe was inserted at
the level of the medial prefrontal cortex (A + 3.2, ML + 0.8,
V j5.3 relative to the bregma) according to the Paxinos atlas
(20). The active length of the dialysis membrane (Hospal
Dasco, Bologna, Italy) was restricted to 2 mm.
Experiments were performed õ24 h after probe implan-
tation, as previously described (21). Samples were collected
every 20 min for all the experiments. The detection limit for
dopamine was 2 fmol per injection. The average neurotrans-
mitter concentration in the last three samples before
treatment was taken as 100%, and all posttreatment values
were expressed as means T SEM relative to the basal value.
The mean in vitro recovery of the probes was 24 T 3%. All
probes were tested before implantation, and those with a
recovery value outside of this range were not used. The
absolute concentration of dopamine was not corrected for
this value. At the end of each experiment, the placement of
the probe was verified histologically. All rats in which the
probe was located outside of the prefrontal cortex were
excluded from the analysis. Data of microdialysis studies are
presented as means T SEM. Comparisons among groups were
performed by two-way analysis of variance (ANOVA) for
repeated measures, with factors being treatment and time
points. For statistical analysis, the raw baseline values of
dopamine concentration were used. Absolute basal dopa-
mine concentrations are reported in the Results section. Post
Transport Studies on BBMECs Monolayers
Permeability experiments were carried out using confluent hoc comparisons were performed by Neuman–Keuls test. A
BBMECs monolayers grown on 12 well Transwell\ insert p value of <0.05 was considered statistically significant.

Novel L-Dopa and Dopamine Prodrugs
1319
RESULTS AND DISCUSSION
both at 37-C. Pseudo first-order kinetics were always
observed and the half-lives of starting materials computed
(Table II). They were found in the range 11–30 h when
phosphate buffer was used while, in dilute rat serum, the half-
Synthesis of Dopimid Compounds 1–12
As reported in Fig. 1, two methods were employed to lives were in the ranges 18–20 min and 2.42–2.96 h for 5, 6
prepare the compounds 16 a–d. The Method A involving at and 1, 2, 9, 10, respectively. It should be noted that L-Dopa
first the condensation of the appropriate imidazopyridinacetic decomposition similarly occurred, with a half-life of 1 h
acid 15 a–d (10) with 3,4-bis(tert-butyldimethylsilyloxy)-L- (Table II). To gain information on the degradation pathway,
Dopa ethyl ester 14 in anhydrous THF and in the presence of the main degradation products of the representative Dopimid
EEDQ as dehydrating agent leading to the corresponding compounds 1, 5, and 9 in physiological medium were
compounds 16 a–d. Removal of the silyl groups of compounds characterized by LC-mass spectrometry (LC-MS). Thus, the
16 a–d to obtain the corresponding Dopimid compounds 5– ESI LC-MS analysis in negative mode of the mixture from
8 was accomplished by using TFA. Compounds 5–8 were also degradation of 1 in physiological medium after 2 h showed
prepared in a straightforward fashion by condensation of the the presence of a peak at m/z 532 attributable to [M-H]j and
appropriate imidazopyridinacetic acid 15 a–d with the ethyl another peak at m/z 309 attributable to imidazopyridinmethyl
ester hydrochloride 13 in anhydrous DMF and in the presence ion. By ESI LC-MS analysis in negative mode of the mixture
of CDI (Method B). By these methods, Dopimid compounds obtained from the stability studies in physiological medium
5–8 were obtained in moderate to good yields. However, in of compound 5 it was detected after 30 min the presence of
terms of step_s number and yields, Method B proved to be the compound 1 [M-H]j (m/z 532) and traces of the
overall more advantageous. The esters 5–8 were subsequently imidazopyridinmethyl ion (m/z 309). In the case of 9, under
hydrolysed with HCl 1N in dioxane to give the corresponding analogous conditions and only after 2 h, ESI LC-MS analysis
carboxylic acids 1–4. As for the synthesis of dopamine revealed the peak attributable to the imidazopyridinmethyl
derivatives 9–12 (Fig. 2), it was accomplished by condensation ion (m/z 309) together with the one of the intact compound 9
of the required imidazopyridinacetic acid 15 a–d with the O- [M-H]j (m/z 488). Therefore, all of these results suggest that
silyl protected dopamine 18 to give the silylated compounds in physiological medium, the ester compound 5 undergoes a
19 a–d. Removal of their O-silyl groups by TFA readily rapid cleavage to give the corresponding acid 1 which, as
afforded the dopamine derivatives 9–12. All the compounds the dopamine derivative 9, is hydrolyzed at the amide bond
1
were characterized by IR, H NMR and mass spectroscopy. level leading to the corresponding imidazopyridinacetic
Table I reports yields and melting points of the compounds 1– acid 15a.
12, 16 a–d and 19 a–d.
Radioligand Binding Assays
Stability Studies
GABAA receptors are allosterically modulated by sev-
The hydrolysis rate of representative Dopimid com- eral types of compounds among which the benzodiazepines
pounds such as 1, 2, 5, 6, 9, and 10 was examined in 0.05 M (22). Moreover, there is overwhelming evidence that binding
phosphate buffer at pH 7.4 and in diluted rat serum solution sites of benzodiazepines can be located both at central
Table II. Stability Data in Phosphate Buffer and Diluted Rat Serum, Physicochemical Parameters and Estimation of BBB Penetration
for Dopimid Compounds 1–12
t1/2 (h) in 0.05M
Phosphate Buffer
(pH 7.4)
t1/2 in 50%
Lyophilized
Rat Serum
RP-HPLC
b
˚
Comp.
CLOG Pa
log k0
TPSAb (A2)
Log BBc
e
d
1
4.46
3.75
3.75
3.03
5.13
4.42
4.42
3.70
4.53
3.81
3.81
3.54
j0.135
j0.271
j0.223
j0.522
j0.122
j0.271
j0.260
j0.393
j0.155
j0.292
j0.264
j0.485
124
124
124
124
113
443
113
113
87
j1.02
j1.13
j1.12
j1.24
j0.76
j0.86
j0.86
j0.97
j0.46
j0.57
j0.57
j0.61
19.5 T 0.3
31.0 T 1.4
2.9 T 0.2 (h)
3.0 T 0.3 (h)
e
d
2
3
4
d
d
d
5
18.9 T 4.3
23.6 T 5.7
18.2 T 1.8 (min)
20.4 T 1.8 (min)
e
6
7
8
d
d
9
10
11.1 T 2.6
24.3 T 6.3
2.6 T 0.4 (h)
2.4 T 0.4 (h)
e
d
87
87
11
12
87
e
L-Dopa
60.0 T 1.2 (min)
a
Calculated according to CLOG P software.
Calculated according to Ertl_s method (15).
b
c
Calculated according to Clark_s model and by using TPSA (14).
Data are means T SD of three separate experiments.
Data are means T SD of two separate experiments.
d
e

1320
Denora et al.
(CBR) and peripheral (PBR) level (23) and the pharmaco- [3H] Ritonavir Uptake and Bi-directional Transport Studies
logical effects mediated by these two receptor subtypes are on MDCKII-MDR1 Cells
very different. Alpidem interacts with both CBRs and PBRs.
Previous works from these laboratories (24,25) pointed out
MDCK cell lines, as Caco-2 cells, are known to express
that substitution of the alpidem at position 8 with lipophilic P-glycoprotein (P-gp) which plays an important role in the
substituents and the presence of one chlorine atom at the efflux transport of drugs from the brain-to-blood (28).
para position of the phenyl ring at C(2) are key structural Therefore, the design of drugs that are not P-gp substrates
features for high binding affinity and selectivity toward is considered a valuable approach for obtaining higher BBB
PBRs.
permeability (29). Thus, we were particularly interested to
The binding of Dopimid compounds 1–12 to the CBR see if Dopimid compounds 1, 2, 5, 6, 9, and 10 are substrates
and PBR receptors was evaluated by measuring their ability of P-gp. To this purpose, at first a study aimed at evaluating
to inhibit [3H]flunitrazepam and [3H]PK 11195 (26) binding the effect of the L-Dopa and dopamine derivatives on the
to membrane preparations arising from the cerebral cortex. uptake characteristics of a P-gp model substrate such as the
Their effects were compared with those of unlabelled [3H]ritonavir was carried out by using MDCKII-MDR1 cells
flunitrazepam and PK 11195, a selective ligand for PBRs which are characterized by a high expression of P-gp (13).
(26). The affinities of compounds 1–12 to D2-like receptors was The formation of confluent MDCK monolayers with
measured by binding assays in vitro by using [3H]YM-09151 as functional tight junctions was confirmed by microscopy,
radioligand and membrane preparations from cerebral TEER values and flux of [14C]mannitol. The average
(striatum) homogenate. The inhibitory concentration (IC50) MDCK TEER value during this study was approximately
values determined are listed in Table III together with that of 250 ohmIcm2 and [14C]mannitol transport was <0.5%/h in
the atypical antipsychotic clozapine for comparison.
representative cell monolayers. The uptake experiments were
As can be seen, Dopimid compounds 1–12 displayed no performed by adding 1 ml of a [3H] ritonavir solution
or low affinity for both CBR and PBR. Only compounds 5, 9 (0.5 mCi/ml) to drug solution at increasing concentration
and 10, 12 possess a submicromolar binding affinity towards (10, 25, 50 and 75 mM, respectively). Incubation was carried
CBR and PBR subtypes, respectively. None of examined out for a period of 10 min. In these tests the known P-gp
compounds showed affinity for dopaminergic receptors. substrate quinidine, L-Dopa and dopamine hydrochloride
Moreover, these binding results suggest that compounds 1– were also included and the results obtained are shown in Fig.
12 could be considered as inactive (or poorly active) 3. As can be seen, Dopimid compounds 1, 2, 5, 6, 9, and 10 or
derivatives of the active compound (i.e., L-Dopa or dopa- L-Dopa or dopamine hydrochloride, exhibited no inhibitory
mine) and this is the requisite of a true prodrug as proposed effect on the [3H]ritonavir uptake, while [3H]ritonavir uptake
by Albert (27).
increased (about 50%), in the presence of quinidine. These
data indicate that compounds 1, 2, 5, 6, 9, and 10 are not
substrates of P-gp.
Next, to further support that compounds 1, 2, 5, 6, 9, and
10 are not P-gp substrates, transport studies involving these
compounds were performed on MDCK cells. Transport
studies were conducted both in AP-to-BL and BL-to-AP
direction and the results are reported in Table IV. Given that
Dopimid compounds 1, 2, 5, 6, 9, and 10 showed not
significant differences in Papp values between AP-to-BL and
BL-to-AP direction, it constitutes further evidence that these
compounds are not substrates of P-gp.
Table III. Binding Data for CBR, PBR and DA Receptors
of Dopimid Compounds 1–12
a,b
IC50 (nM)
Compound
CBR
PBR
[3H] YM 09151
1
(24%)
(74%)
(13%)
(45%)
(44%)
>10,000
(55%)
(49%)
>10,000
381.5
>10,000
225.4
5
>10,000
>10,000
>10,000
>10,000
327
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
–
2
3
Computational Calculations of Physicochemical
and Permeability Characteristics of Dopimid
Compounds 1–12
4
5
6
>10,000
4,760
>10,000
165
7
To be effective as therapeutic agents, compounds acting at
CNS level should possess optimal BBB penetration properties.
These are related to molecular lipophilicity indexes such as the
partition coefficient (log P) and capacity factor log k0 (15,30).
Therefore, the log P values of Dopimid compounds 1–12 when
distributed between n-octanol and water were calculated by
using the C log P computer program and the log k0 values of
these compounds estimated by measuring their retention times
in RP-HPLC (Table II). The obtained partition coefficients and
capacity factors are correlated by a linear relationship with
acceptable statistics (n=12, r2 = 0.71, s=0.07). Log P value of
2.1 is generally considered optimal for penetration of the BBB
(31). As can be seen from Table II, compounds 1–12 possess log
8
9
10
>10,000
9,776
5,435
–
11
12
flunitrazepam
PK 11195
clozapine
–
4.27
–
4,119
–
–
a Data are means T SD of three separate experiments performed in
duplicate.
b Values in parentheses are the percentages of inhibition of specific
[3 H]flunitrazepam binding determined at 40 mM concentration of
the tested compound for CBR affinity.

Novel L-Dopa and Dopamine Prodrugs
1321
180
∗
160
140
120
100
80
60
40
20
0
1
2
5
6
9
10
Fig. 3. Effect of Dopimid compounds 1, 2, 5, 6, 9, 10, quinidine, L-Dopa and dopamine hydrochloride on the [3H]ritonavir uptake. The bars
represent the uptake of [3H]ritonavir as percent of control in the presence of the tested compounds.
P values in the range 3.03–5.13 and hence are lipophilic enough readily, while compounds with log BB < j1.0 are poorly
to cross the BBB. However, it has been pointed out that the log distributed to the brain. As can be seen from the data
P is an important factor, although it correlates poorly with the reported in Table II, Dopimid compounds 5, 6, 9 and 10 but
log Cbrain/Cblood (log BB) which is a more appropriate measure not 1–4 should be able to cross the BBB.
of the degree of BBB penetration. To estimate BBB
penetration by compounds 1–12, we used Clark_s model (14) Transport Across Co-cultures of BBMECs
which relates log BB to polar surface area (PSA). This last was and Astrocytes Monolayers
computed with a fragment-based approach, known as
topological PSA (TPSA) approach (15). It is generally To obtain more accurate information on the ability of
accepted that compounds with log BB > 0.3 cross the BBB Dopimid compounds 1, 2, 5, 6, 9, and 10 to cross the BBB,
Table IV. Bi-directional Transport Across MDCKII-MDR1 Cells on Dopimid Compounds 1,2,5,6,9, and 10
Papp ꢀ 10j6 (cm/s)a
Compound
Efflux Ratio Papp (BA)/(AB)
Apical-Basolateral
Basolateral-Apical
1
0.36 T 0.034
0.38 T 0.022
1.91 T 0.350
2.26 T 0.290
1.47 T 0.320
1.24 T 0.950
0.39 T 0.240
1.49 T 0.360
0.19 T 0.180
2.30 T 0.210
0.38 T 0.026
0.24 T 0.045
1.05
0.63
0.78
0.93
1.21
1.58
–
2
5
1.49 T 0.190
2.10 T 0.220
1.78 T 0.520
1.96 T 0.260
6
9
10
Dopamine
[3H]L-Dopa
Mannitol
Diazepam
–
–
–
–
–
–
–
a Data are means T SD of three determination.

1322
Denora et al.
Table V. Transport Across BBMEC on Dopimid Compounds
1,2,5,6,9, and 10
ab
ab
200
150
100
50
Compound
Papp ꢀ 10j6 (cm/s)a
ab
a
1
0.33 T 0.440
0.21 T 0.100
2.77 T 0.210
2.10 T 0.310
3.86 T 0.38*
2.98 T 0.19*
0.21 T 0.280
1.92 T 0.140
0.19 T 0.140
1.72 T 0.090
a
a
a
a
2
a
5
6
9
10
Dopamine
[3H]L-Dopa
Mannitol
Diazepam
0
60
Time (min)
120
a Data are means T SD of at least three determinations.
*p < 0.001 vs dopamine.
Fig. 5. Effect of acute administration of compound 6 on cortical
extracellular concentration of dopamine in the rat medial prefrontal
cortex. The drug has been administered intraperitoneally at the dose
of 10 (open bars) or 20 (hatched bars) mg/kg. Data are means T SEM
of at least five rats per dose, are expressed as a percentage of basal
transport studies involving co-culture of BBMECs and
astrocytes were carried out. This approach constitute a well
established in vitro method to estimate compound perme-
ability across the BBB (32). The results indicate that
dopamine derivatives 9 and 10 are characterized by Papp
values greater than dopamine hydrochloride (up to tenfold,
Table V). As expected, the esters 5 and 6 showed BBMEC
monolayers permeability greater than L-Dopa which, in turn,
exhibited a transport greater than dopamine hydrochloride.
The rank order of Papp values observed in these assays was
9, 10 > 5 Ê 6 Ê L-Dopa >>1 and 2 = dopamine (Table V). This
trend parallels the rank order of BBB penetration calculated
by the computational approach (Table II).
values and represent the samples collected every 20 min after drug
administration. ap < 0.01 vs basal values; bp < 0.05, b p < 0.01 vs
previous dose.
0
istration of compounds 5, 6, and 9 to gain complete evidence
for a prodrug-type mechanism. As shown in Fig. 4, the acute
administration of compound 5 (10–20 mg/kg, i.p.) induced a
dose-dependent increase in cortical dopamine output. The
maximal increase (+197%), obtained with the dose of 20 mg/
kg, was observed 120 min after drug injection and persisted for
more than 240 min. ANOVA revealed a significant main effect
of treatment [F(3,111) = 4.369; p< 0.001]; a significant main
effect of repeated measures [F(6,111) = 1.369; p< 0.001]; and a
significant interaction between factors [F(12,111) = 0.259;
p< 0.001].
Microdialysis Studies
Microdialysis is a well-established sampling method for
determining neurotransmitter concentrations in the brains of
freely-moving animals (33). In this work, we decided to use
such a very sensitive analytical methodology for determining
the dopamine levels following the intraperitoneal (i.p.) admin-
The acute administration of compound 6 (10–20 mg/kg,
i.p.) induced a smaller and less persistent effect on cortical
dopamine output. The maximal increase (+90% over basal
values) was observed 40 min after the injection of the drug
350
ab
ab
200
150
100
50
ab'
300
a
a
250
ab'
a
a
200
150
100
50
0
60
120
Time (min)
180
240
0
60
120
Time (min)
180
240
Fig. 4. Effect of acute administration of compound 5 on cortical Fig. 6. Effect of acute administration of compound 9 on cortical
concentration of dopamine in the rat medial prefrontal cortex. The concentration of dopamine in the rat medial prefrontal cortex. The
drug has been administered intraperitoneally at the dose of 10 (open drug has been administered intraperitoneally at the dose of 10 (open
bars) or 20 (hatched bars) mg/kg. Data are means T SEM of at least bars) or 20 (hatched bars) mg/kg. Data are means T SEM of at least
five rats per dose, are expressed as a percentage of basal values and five rats per dose, are expressed as a percentage of basal values and
represent the samples collected every hour after drug administration. represent the samples collected every hour after drug administration.
0
0
ap < 0.01 vs basal values; bp < 0.05, b p < 0.01 vs previous dose.
ap < 0.01 vs basal values; bp < 0.05, b p < 0.01 vs previous dose.

Novel L-Dopa and Dopamine Prodrugs
1323
and returned to basal values in 80 min. ANOVA revealed a compounds 5, 6, and 9 induce a dose- and time-dependent
significant main effect of treatment [F(3,111) = 12.345; increase in the dopamine levels in the rat medial prefrontal
p < 0.001]; a significant main effect of repeated measures cortex. These results, taken together, lead to the conclusion
[F(6,111) = 0.957; p < 0.001]; and a significant interaction that Dopimid compounds 5, 6, and 9 may be considered as
between factors [F(12,111) = 1.520; p < 0.001] (Fig. 5).
Acute administration of compound 9 (10–20 mg/kg, i.p.)
elicited a dose- and time-dependent increase in the concen-
tration of dopamine in the rat medial prefrontal cortex. This
novel potential L-Dopa and dopamine prodrugs.
increase was maximal (õ100% over basal values) 120 min ACKNOWLEDGMENTS
after drug administration and returned to basal values in 240
min (Fig. 6). ANOVA revealed a significant main effect of
This work was supported by a grant from Ministero
treatment [F(3,111) = 58.596; p < 0.001]; a significant main dell_Universita` e della Ricerca Scientifica e Tecnologica
effect of repeated measures [F(6,111) = 2.532; p < 0.001]; and (MIUR) (COFIN 2003 of G.L.). We thank Mr. Giovanni
a significant interaction between factors [F(12,111) = 2.486; Dipinto for skilful technical assistance in recording mass
p < 0.001]. For Figs. 4 and 6 as the effect of the compounds spectra. The authors would like to express their thanks to
tested was persistent, only the data collected at 60, 120, 180 Dr. Soumyajit Majumdar from the Department of Pharma-
and 240 min were reported.
ceutics, School of Pharmacy, the University of Mississippi,
Our microdialysis studies show that the compounds for his helpful discussions.
tested induce a dose- and time-dependent increase in the
dopamine levels in the rat medial prefrontal cortex. Our
results are very similar to those observed by Eltayb et al.,
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