A novel conjugated agent between dopamine and an A2a adenosine receptor antagonist as a potential anti-Parkinson multitarget approach

Article abstract of DOI:10.1021/mp200489d

We propose a potential antiparkinsonian prodrug DP-L-A_2AANT (2) obtained by amidic conjugation of dopamine (1) via a succinic spacer to a new triazolo-triazine A_2A adenosine receptor (AR) antagonist A _2AANT (3). The affinity of 2 and its hydrolysis products-1, 3, dopamine-linker DP-L (4) and A_2AANT-linker L-A_2AANT (5)-was evaluated for hA₁, hA_2A, hA_2B and hA₃ ARs and rat striatum A_2AARs or D₂ receptors. The hydrolysis patterns of 2, 4 and 5 and the stabilities of 1 and 3 were evaluated by HPLC analysis in human whole blood and rat brain homogenates. High hA_2A affinity was shown by compounds 2 (K_i = 7.32 ± 0.65 nM), 3 (K_i = 35 ± 3 nM) and 5 (K_i = 72 ± 5 nM), whose affinity values were similar in rat striatum. These compounds were not able to change dopamine affinity for D₂ receptors but counteracted the CGS 21680-induced reduction of dopamine affinity. DP-L (4) was inactive on adenosine and dopaminergic receptors. As for stability studies, compounds 4 and 5 were not degraded in incubation media. In human blood, the prodrug 2 was hydrolyzed (half-life = 2.73 ± 0.23 h) mainly on the amidic bound coupling the A_2AANT (3), whereas in rat brain homogenates the prodrug 2 was hydrolyzed (half-life > eight hours) exclusively on the amidic bound coupling dopamine, allowing its controlled release and increasing its poor stability as characterized by half-life = 22.5 ± 1.5 min.

Full text of DOI:10.1021/mp200489d

Article
pubs.acs.org/molecularpharmaceutics
A Novel Conjugated Agent between Dopamine and an A2A
Adenosine Receptor Antagonist as a Potential Anti-Parkinson
Multitarget Approach
Alessandro Dalpiaz,† Barbara Cacciari,*,† Chiara Beatrice Vicentini,† Fabrizio Bortolotti,†
Giampiero Spalluto,‡ Stephanie Federico,‡ Barbara Pavan,§ Fabrizio Vincenzi,∥ Pier Andrea Borea,∥
and Katia Varani∥
†Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy
‡Department of Pharmaceutical Sciences, University of Trieste, Trieste, Italy
§Department of Biology, General Physiology Section, University of Ferrara, Ferrara, Italy
∥Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Ferrara, Italy
ABSTRACT: We propose a potential antiparkinsonian prodrug
DP-L-A2AANT (2) obtained by amidic conjugation of dopamine
(1) via a succinic spacer to a new triazolo-triazine A2A adenosine
receptor (AR) antagonist A2AANT (3). The affinity of 2 and its
hydrolysis products1, 3, dopamine-linker DP-L (4) and
A2AANT-linker L-A2AANT (5)was evaluated for hA1, hA2A,
hA2B and hA3 ARs and rat striatum A2AARs or D2 receptors. The
hydrolysis patterns of 2, 4 and 5 and the stabilities of 1 and 3
were evaluated by HPLC analysis in human whole blood and rat
brain homogenates. High hA2A affinity was shown by compounds
2 (Ki = 7.32 0.65 nM), 3 (Ki = 35 3 nM) and 5 (Ki = 72
5
nM), whose affinity values were similar in rat striatum. These
compounds were not able to change dopamine affinity for D2
receptors but counteracted the CGS 21680-induced reduction of dopamine affinity. DP-L (4) was inactive on adenosine and
dopaminergic receptors. As for stability studies, compounds 4 and 5 were not degraded in incubation media. In human blood, the
prodrug 2 was hydrolyzed (half-life = 2.73 0.23 h) mainly on the amidic bound coupling the A2AANT (3), whereas in rat brain
homogenates the prodrug 2 was hydrolyzed (half-life > eight hours) exclusively on the amidic bound coupling dopamine,
allowing its controlled release and increasing its poor stability as characterized by half-life = 22.5 1.5 min.
KEYWORDS: A2A/D2 receptor heteromers, A2A antagonist, controlled release, dopamine, HPLC, human blood, hydrolysis, prodrug,
rat brain homogenates, stability
peripheral phenomena, such as nausea, vomiting and
hypotension, due to the activation of dopaminergic receptors,4
and a series of central complications that arise during long-term
use of L-DOPA, which dramatically increase in severity as the
disease evolves. These complications depend on the progressive
loss of the drug’s ability to relieve motor impairments (wearing
off), on the induction of excessive and abnormal purposeless
movements, which interfere with the normal motor activity
(dyskinesias), and on fluctuations in the intensity of the motor
stimulation effects of L-DOPA (“on/off phenomena”).5 The
mechanisms triggering these phenomena are not clearly
understood, even if they are attributed to abnormal permanent
plastic changes in striatal synapses induced by L-DOPA
INTRODUCTION
■
Parkinson’s disease (PD) is a chronic neurological disorder
characterized by tremors, muscular rigidity, bradykinesia, poor
balance and difficulty in walking. This disease results from the
progressive and irreversible degeneration of dopaminergic
neurons in the substantia nigra of the brain, with a consequent
depletion of dopamine production. PD is clearly revealed with a
70−80% reduction in nigrostriatal dopamine concentration.1
Dopamine replacement currently represents the major
therapeutic strategy to alleviate the neurological symptoms
associated with PD. At present, from a pharmacologiacal point
of view, dopamine is administered as L-DOPA, its metabolic
precursor, which is able to cross the blood−brain barrier and is
converted into the active drug by enzymatic decarboxylation.2
Even if L-DOPA is effective in ameliorating the motor
symptoms of patients in the early stage of PD, important side
effects seriously limit the therapeutic potential of this drug.3
Administration of L-DOPA is associated with both acute
Received: September 29, 2011
Revised: January 27, 2012
Accepted: January 31, 2012
Published: January 31, 2012
© 2012 American Chemical Society
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Figure 1. Design of the prodrug DP-L-A2AANT (2) obtained by the conjugation of dopamine (1) with a new A2A antagonist (A2AANT, 3) via a
succinic linker (L).
treatment.6 Other unwanted central effects related to L-DOPA
therapy include hallucinations and mental confusion, due to
stimulation of central dopaminergic receptors.7 Finally, L-
DOPA and dopamine would appear to be able to enhance
neuronal degeneration by way of oxidative metabolism.8 L-
DOPA is, therefore, usually administered in association with
other drugs, such as direct dopamine agonists or agents able to
delay dopamine catabolism, with a view to reducing the L-
DOPA dose required to act against the symptoms of PD. On
the other hand, the drugs coadministered with L-DOPA are
often not adequately effective in counteracting the parkinsonian
motor disabilities and can induce unwanted effects which limit
their therapeutic usage.9 As a consequence, novel pharmaco-
logical approaches to the management of PD are required.
In the past decade increasing interest has been focused on
A2A adenosine receptors (ARs) as an important pharmaco-
logical target in PD. Indeed, preclinical and clinical studies have
evidenced the ability of A2A antagonists, such as istradefylline,
to amplify the therapeutic effects of L-DOPA and reduce motor
complications (wearing off, dyskinesias and on/off phenom-
ena) deriving from its pulsatile long-term treatment.10,11 It is
known that A2AARs are coexpressed with dopaminergic D2
receptors in striatopallidal GABA neurons, where they form
heterodimeric complexes able to decrease the D2 affinity for
dopamine when the A2AARs are stimulated.10,12,13 The A2A
antagonists can therefore enhance the therapeutic index of L-
DOPA and D2 agonists by blocking the A2AARs in these A2A−
D2 heteromers.13,14 Moreover, A2A antagonists are able to
reduce the L-DOPA induced dyskinesias by restoring the
appropriate balance between A2AARs and D2 receptors.15−17
Finally, the neuroprotective effects of A2A antagonists are
considered as potentially useful in preventing the onset and
development of PD.18−20 The A2A antagonists emerge,
therefore, as a class of efficacious antiparkinsonian drugs for
the future, whose coadministration with L-DOPA appears
incisive for both the early stage and long-term treatment of PD.
Formulations containing both L-DOPA or dopamine and A2A
antagonists should be developed with a view to enhancing the
therapeutic action via the A2A−D2 heteromers.
1). The conjugation was obtained by amidic bonds. We chose,
as A2A antagonist, the 7-amino-5-(aminomethyl)-
cyclohexylmethyl-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a]-1,3,5-
triazine trifluoroacetate A2AANT (3), a new triazolo-triazine
derivative which does not have the limitation of aqueous
solubility and is characterized by the presence of an amino
group available for the coupling.21 DP-L-A2AANT (2) was
considered as a potential prodrug of both dopamine (1) and
the A2AANT (3). The affinity of DP-L-A2AANT (2) and its
potential hydrolysis products dopamine (1), dopamine-linker
DP-L (4), A2AANT (3) and antagonist-linker L-A2AANT (5)
were tested on human A1, A2A, A2B and A3ARs expressed in
CHO cells. Binding experiments were also performed on
A2AARs and D2 receptors expressed in rat striatum. In this case
the influence of the compounds on the dopamine affinity
toward D2 receptors in the presence and in the absence of the
A2A agonist CGS 21680 was also tested. Finally, we evaluated
the hydrolysis pattern of the prodrug DP-L-A2AANT (2) in
water, phosphate buffer, human whole blood and rat brain
homogenates by monitoring the degradation and appearance
over time of its hydrolysis products dopamine (1), DP-L (4),
A2AANT (3) and L-A2AANT (5), whose stabilities were
accurately determined according to the above incubation
media.
MATERIALS AND METHODS
■
Materials. [3H]-1,3-Dipropyl-8-cyclopentylxanthine ([3H]-
DPCPX, specific activity 120 Ci/mmol), [125I]-4-aminobenzyl-
5′-N-methylcarboxamidoadenosine ([125I]-AB-MECA, specific
activity 2000 Ci/mmol) and [3H]-spiperone (specific activity,
16 Ci/mmol) were purchased from Perkin-Elmer Life and
Analytical Sciences (Boston, MA, USA). [3H]-4-(2-((7-amino-
2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl)amino)-
ethyl)phenol ([3H]-ZM 241385, specific activity 27 Ci/mmol)
was obtained from American Radiolabeled Chemicals (St.
Louis, MO, USA). [3H]-cAMP, specific activity 21 Ci/mmol,
was purchased from GE Healthcare (Chalfont St Giles, U.K.).
Dopamine, forskolin, Ro 20-1724, DPCPX, R-phenylisopropy-
ladenosine (R-PIA), ZM 241385 and heptafluorobutyric acid
(HFBA) were obtained from Sigma (St. Louis, MO, USA). The
dopamine-glutaric derivative (DP-glu, 6) and the 5-amino-4-
cyano-1-[3-(4-methoxyphenyl)propyl]pyrazole (AN-Pyr, 7)
In this paper we propose a new, potential antiparkinsonian
compound DP-L-A2AANT (2) made up of dopamine (1)
coupled via a succinic spacer to a new A2A antagonist (Figure
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were synthesized as previously described.22,23 Methanol,
acetonitrile and water were high-performance liquid chroma-
tography (HPLC)-grade from Merck (Darmstadt, Germany).
All other reagents were of analytical grade and obtained from
commercial sources. Male Wistar rats were purchased from
Harlan SRC (Milan, Italy).
cyclohexyl)methyl)amino)-4-oxobutanoic Acid (5). A
solution of 7-amino-5-(aminomethyl)cyclohexylmethyl-amino-
2-(2-furyl)-1,2,4-triazolo[1,5-a]-1,3,5-triazine trifluoroacetate
(3, 0.033 mmol) and succinic anhydride (0.039 mmol) in dry
pyridine (0.5 mL) was stirred at room temperature for 24 h.
Then, the solvent was removed under vacuum, and the residue
was acidified to pH 5 with a 10% aqueous citric acid solution
and extracted with ethyl acetate several times. The organic layer
was dried, filtered and evaporated under reduced pressure to
give the compound as a colorless foam (80% yield). [M + H]+
Synthesis. Reactions were routinely monitored by thin-
layer chromatography (TLC) on silica gel (precoated F254
Merck plates) and products visualized with iodine or potassium
permanganate solution. Infrared spectra (IR) were measured on
a Perkin-Elmer 257 instrument. 1H NMR spectra were
determined in CDCl3 or DMSO-d6 solutions with a Bruker
AC 200 spectrometer, peak positions are given in parts per
million (δ) downfield from tetramethylsilane as internal
standard, and J values are given in hertz. Light petroleum
ether refers to the fractions boiling at 40−60 °C. Melting points
were determined on a Buchi-Tottoli instrument and are
uncorrected. Chromatographies were performed using Merck
200−400 and 60−200 mesh silica gel. All reported products
1
= 443. H NMR (CD3OD): 1.45−1.91 (m, 10H), 2.45−2.61
(m, 4H), 2.99−3.31 (m, 6H), 6.59 (dd, 1H, J = 2, J = 4), 7.1
(bs, 1H), 7.51−7.55 (m, 1H), 7.68 (d, 1H, J = 2), 7.95−7.97
(m, 1H), 7.57 (d, 1H, J = 4). 13C NMR (CD3OD): 27.37,
29.82, 30.13, 30.38, 31.48, 36.41, 36.90, 39.06, 39.34, 44.27,
45.66, 112.78, 126.04, 145.76, 147.13, 149.02, 174.51, 176.23,
210.46. IR (neat) cm−1: 3510−3050, 1685, 1470. Anal.
(C20H26N8O4), C, H, N.
Membrane Preparation of CHO Cells Transfected with
A1, A2A, A2B or A3ARs. The expression of the human A1, A2A,
A2B and A3ARs in CHO cells has been previously described.24
Briefly, for membrane preparation the cells were grown
adherently and maintained in Dulbecco’s modified Eagle's
medium with nutrient mixture F12 (DMEM/F12) without
nucleosides, containing 10% fetal calf serum, penicillin (100 U/
mL), streptomycin (100 μg/mL), L-glutamine (2 mM) and
Geneticin (G418, 0.2 mg/mL) at 37 °C in 5% CO2/95% air.
For membrane preparation the culture medium was removed
and the cells were washed with phosphate buffer solution
(PBS) and scraped off T75 flasks in ice-cold hypotonic buffer
(5 mM Tris HCl, 2 mM EDTA, pH 7.4). The cell suspension
was homogenized with Polytron (Kinematica Inc., Bohemia,
NY, USA), the homogenate was spun for 10 min at 1000g and
the supernatant was centrifuged for 30 min at 100000g. The
membrane pellet was resuspended in 50 mM Tris HCl buffer,
pH 7.4, containing 10 mM MgCl2, incubated with 2 UI/mL of
adenosine deaminase for 30 min at 37 °C and centrifuged
again.
Preparation of Rat Striatum Membranes. Rat striatum
was homogenized in 50 mM Tris HCl buffer, pH 7.4, with a
Polytron and centrifuged for 20 min at 48000g.25 To study
A2AARs, the membrane pellet was resuspended in 50 mM Tris
HCl buffer, pH 7.4, containing 10 mM MgCl2 and incubated
with 2 IU/mL adenosine deaminase for 30 min at 37 °C.
Similar aliquots of membranes were suspended in 50 mM Tris
HCl buffer, pH 7.4, with the aim to investigate D2DRs.
Competition Binding Experiments in CHO Cells. The
tested compounds were used in the range 1 nM to 1 μM in
competition binding experiments in CHO cells transfected with
human ARs. Briefly, hA1CHO membranes (60 μg of protein/
assay) and [3H]-DPCPX (1 nM) as radioligand were incubated
for 90 min at 25 °C.24 Nonspecific binding was determined in
the presence of DPCPX 1 μM. Competition binding experi-
ments in hA2ACHO membranes (60 μg of protein/assay) were
carried out by using [3H]-ZM 241385 (2 nM) as radioligand
and were incubated for 60 min at 4 °C.24 Nonspecific binding
was determined in the presence of ZM 241385 1 μM.
Competition binding experiments to hA3ARs were conducted
in membranes (80 μg of protein/assay) and [125I]-AB-MECA
(0.5 nM) at 37 °C for 60 min, and R-PIA 50 μM was used to
evaluate the nonspecific binding.24
1
showed IR, 13C NMR and H NMR spectra in agreement with
the assigned structures. Organic solutions were dried over
anhydrous sodium sulfate. Elemental analyses were performed
by the microanalytical laboratory of the Department of
Chemistry, University of Trieste, and they were within 0.4%
of the theoretical values for C, H, and N.
Synthesis of 4-[[2-(3,4-Dihydroxyphenyl)ethyl]-
amino]-4-oxobutanoic Acid (4). To a solution of dopamine
hydrochloride (2.64 mmol) in dry pyridine (7.5 mL) was added
succinic anhydride (3.2 mmol), and the mixture was stirred for
12 h at room temperature. After evaporation under reduced
pressure, the crude reaction product was partitioned between
ethyl acetate and water. The organic phase was dried, filtered
and then evaporated under vacuum. The residue was purified
by chromatography (CH2Cl2/MeOH 15:4) to afford the right
1
compound as a colorless oil (70% yield). [M + H]+ = 254. H
NMR (DMSO-d6): 2.29 (m, 2H), 2.35−2.43 (m, 4H), 3.07−
3.17 (m, 2H), 6.40 (dd, 1H), 6.54 (d, 1H, J = 2), 6.60 (d, 1H, J
= 7.8), 7.86 (m, 1H). 13C NMR (DMSO-d6): 29.07, 29.32,
34.59, 40.55, 115.35, 115.81, 119.07, 130.12, 143.38, 144.91,
170.63, 173.77. IR (neat) cm−1: 3220, 1685. Anal.
(C12H15NO5), C, H, N.
Synthesis of N1-((4-(((7-Amino-2-(furan-2-yl)-[1,2,4]-
triazolo[1,5-a][1,3,5]triazin-5-yl)amino)methyl)-
cyclohexyl)methyl)-N4-(3,4-dihydroxyphenethyl)-
succinamide (2). A solution of 7-amino-5-(aminomethyl)-
cyclohexylmethyl-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a]-1,3,5-
triazine trifluoroacetate (0.109 mmol) in DMF (2 mL) and
triethylamine (0.109 mmol, 15.4 mL) was added dropwise to a
solution of 4-[[2-(3,4-dihydroxyphenyl)ethyl]amino]-4-oxobu-
tanoic acid (4, 0.132 mmol), HOBt (0.132 mmol), and WSC
(0.132 mmol) in DMF (2 mL); the mixture was stirred at room
temperature for 24 h. After the evaporation of the solvent
under reduced pressure, the residue was purified by flash
cromatography on NH3 saturated silica gel (ethyl acetate/
methanol 8:2) to furnish the compound as a colorless oil (80%
yield). [M + H]+ = 578.3. 1H NMR (DMSO-d6): 1.2−1.85 (m,
10H), 2.27−2.38 (m, 4H), 2.81−3.19 (m, 10H), 6.4 (dd, 1H, J
= 2, J = 4), 6.52−6.75 (m, 3H), 7.1 (d, 1H, J = 4), 7.23 (bs,
1H), 7.56−7.88 (m, 3H), 8.07 (bs, 1H), 8.61 (s, 1H), 8.73 (s,
1H). IR (neat) cm−1: 3480−3050, 1697, 1456. Anal.
(C28H35N9O5), C, H, N.
Synthesis of 4-(((4-(((7-Amino-2-(furan-2-yl)-[1,2,4]-
triazolo[1,5-a][1,3,5]triazin-5-yl)amino)methyl)-
Competition Binding Experiments in Rat Striatum.
The tested compounds, dopamine, ZM 241385 and butaclamol,
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were used in the range 1 nM to 1 μM in competition binding
experiments in rat striatum. Competition binding experiments
to A2AARs (60 μg of protein/assay) were carried out by using
[3H]-ZM 241385 (2 nM) as radioligand and were incubated for
60 min at 4 °C.24 Nonspecific binding was determined in the
presence of ZM 241385 1 μM.
Competition binding experiments to D2DRs (40 μg of
protein/assay) were performed by incubating [3H]-spiperone
(1 nM) and rat striatum membranes for 15 min at 37 °C. The
affinity of dopamine (1 nM to 1 μM) in the absence and in the
presence of CGS 21680 (100 nM) was evaluated. Moreover,
the effect of A2AANT 3, L-A2A ANT 5 and the prodrug DP-L-
A2A ANT 2 at 1 μM concentration on the affinity of dopamine
was also investigated in the absence and in the presence of CGS
21680 (100 nM). Nonspecific binding was determined in the
presence of butaclamol 1 μM.25
Saturation Binding Experiments in Rat Striatum.
Saturation binding experiments to A2AARs in rat striatum (60
μg of protein/assay) were carried out by using [3H]-ZM
241385 (0.01−20 nM) as radioligand and were incubated for
60 min at 4 °C.25 Nonspecific binding was determined in the
presence of ZM 241385 1 μM. Additional saturation binding
experiments were performed in the absence and in the presence
of dopamine (10 μM).
Saturation binding experiments to D2 receptors in rat
striatum aliquots (60 μg of protein/assay) were performed by
using [3H]-spiperone as radioligand.25 The tissue membranes
were incubated for 15 min at 37 °C with 8 to 10 concentrations
of the radioligand [3H]-spiperone (0.05−5 nM). Nonspecific
binding was determined in the presence of butaclamol 1 μM.
Additional saturation binding experiments were performed in
the absence and in the presence of CGS 21680 (100 nM) or
the tested compounds at the 1 μM concentration.
counted in a 2810 TR liquid scintillation counter Packard
(Perkin-Elmer Life and Analytical Sciences, Boston, MA, USA).
HPLC Analysis. The quantification of the prodrug DP-L-
A2AANT (2) and its potential hydrolysis products dopamine
(1), DP-L (4), A2AANT (3) and L-A2AANT (5) was performed
by HPLC. The chromatographic apparatus consisted of a
modular system (model LC-10 AD VD pump and model SPD-
10A VP variable wavelength UV−vis detector; Shimadzu,
Kyoto, Japan) and an injection valve with a 20 μL sample loop
(model 7725; Rheodyne, IDEX, Torrance, CA). Separation was
performed at room temperature on a reverse phase column
(Phenomenex Synergi Polar RP 80 Å, 150 × 4.6 mm, 4 μm,
ChemTek Analytica, Anfola Emilia, Bologna, Italy). Data
acquisition and processing were carried out with a personal
computer using Class-VP software (Shimadzu).
For dopamine (1) and DP-L (4) the detector was set at 280
nm. The mobile phase consisted of a mixture of 0.1% HFBA
and methanol with a ratio of 85:15 (v/v). The flow rate was 0.8
mL/min. DP-glu (6) was employed as internal standard for
plasma and brain homogenate samples (see below). The
retention times of dopamine (1), DP-L (4) and DP-glu (6)
were 6.3, 12.4, and 17.5 min, respectively. For the A2AANT (3)
the detector was set at 260 nm. The mobile phase consisted of a
mixture of 20 mM phosphate buffer (pH 2.5) and acetonitrile
with a ratio of 85:15 (v/v). The flow rate was 0.8 mL/min. The
DP-glu (6) was employed as internal standard for plasma and
brain homogenate samples (see below). The retention times of
A2AANT (3) and DP-glu (6) were 4.15 and 6.5 min,
respectively.
For the prodrug DP-L-A2AANT (2) and L-A2AANT (5) the
detector was set at 248 nm. The mobile phase consisted of a
mixture of water, methanol and acetonitrile with a ratio of
55:22:23 (v/v/v). The flow rate was 1 mL/min. AN-Pyr (7)
was employed as internal standard for plasma and brain
homogenate samples (see below). The retention times of L-
A2AANT (5), DP-L-A2AANT (2), and AN-Pyr (7) were 4.15,
5.45, and 10.3 min, respectively.
At the end of the incubation time in competition or
saturation binding experiments, bound and free radioactivity
were separated by filtering the assay mixture through Whatman
GF/B glass fiber filters in Brandel cell harvester (Brandel,
Unterfohring, Germany). Filter bound radioactivity was
The chromatographic precision for each compound was
evaluated by repeated analysis (n = 6) of the same sample (25
μM). For all compounds dissolved in the aqueous phase, the
calibration curves of peak areas versus concentration were
generated in the range 0.5 to 100 μM.
Kinetic Analysis in Water and Phosphate Buffer. The
prodrug DP-L-A2AANT (2) and its potential hydrolysis
products dopamine (1), DP-L (4), A2AANT (3) and L-
A2AANT (5) were incubated at 37 °C in water (HPLC grade,
pH 6.0) or in 50 mM phosphate buffer (pH 7.4). Six milliliters
of water or buffer were spiked with compound solutions
resulting in a final concentration of 100 μM. At regular time
intervals 200 μL samples were withdrawn and 10 μL aliquots
were immediately injected into the HPLC apparatus. All the
values were obtained as the mean of three independent
experiments.
Kinetic Analysis in Whole Blood. The prodrug DP-L-
A2AANT (2) and its potential hydrolysis products dopamine
(1), DP-L (4), A2AANT (3) and L-A2AANT (5) were incubated
at 37 °C in heparinized human whole blood obtained from
healthy volunteers. Six milliliters of whole blood was spiked
with compound solutions resulting in a final concentration of
100 μM. At regular time intervals, 400 μL samples were
withdrawn and immediately centrifuged at 1500g at 4 °C for 15
min. 100 μL of the plasma obtained by centrifugation was
quenched in 200 μL of ethanol (4 °C); 100 μL of internal
̈
counted in a 2810 TR liquid scintillation counter Packard
(Perkin-Elmer Life and Analytical Sciences, Boston, MA, USA).
Measurement of cAMP Levels. CHO cells transfected
with the human A2BARs were suspended in a 0.5 mL incubation
mixture containing NaCl 150 mM, KCl 2.7 mM, NaH2PO4
0.37 mM, MgSO4 1 mM, CaCl2 1 mM, glucose 5 mM, Hepes
100 mM, MgCl2 100 mM, pH 7.4 at 37 °C. Then 2.0 IU of
adenosine deaminase/mL and 0.5 mM Ro 20-1724 as
phosphodiesterase inhibitor were added and preincubated for
10 min in a shaking bath at 37 °C. The effect of the novel
compounds at different concentrations (1 nM to 1 μM) was
evaluated. The reaction was terminated by the addition of cold
6% trichloroacetic acid (TCA). The TCA suspension was
centrifuged at 2000g for 10 min at 4 °C, and the supernatant
was extracted four times with water saturated diethyl ether. The
final aqueous solution was tested for cyclic AMP levels by a
competition protein binding assay where samples of cyclic
AMP standards (0−10 pmol) were added to each test tube
containing trizma base 0.1 M, aminophylline 8.0 mM,
mercaptoethanol 6.0 mM, pH 7.4 and [3H]-cyclic AMP.26
The binding protein, previously prepared from beef adrenals,
was added to the samples and incubated at 4 °C for 150 min. At
the end of the incubation time and after the addition of
charchoal, the samples were centrifuged at 2000g for 10 min.
The clear supernatant was mixed in Ultima Gold solution and
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Scheme 1. Synthesis of Compounds DP-L (4) and L-A2AANT (5)
standard [100 μM DP-glu (6) for the analysis of dopamine (1)
and DP-L (4); 500 μM DP-glu (6) for the analysis of A2AANT
(3); 100 μM AN-Pyr (7) for the analysis of prodrug DP-L-
A2AANT (2) and L-A2AANT (5)] was then added. After
centrifugation at 13000g for 10 min, 300 μL aliquots were
reduced to dryness under a nitrogen stream. Two hundred
microliters of mobile phase was added and, after centrifugation,
10 μL was injected into the HPLC system. All the values were
obtained as the mean of three independent incubation
experiments.
The accuracy of the analytical method was determined by
recovery experiments, comparing the peak areas extracted from
blood test samples at 4 °C (n = 6) with those obtained by
injection of an equivalent concentration of the analytes
dissolved in their mobile phase. For all compounds analyzed,
the calibration curves were constructed by employing eight
different concentrations in whole blood at 4 °C ranging from 2
to 100 μM and expressed as peak area ratios of the compounds
and their internal standard versus concentration.
affinity or KD values, as well as the maximum densities of
specific binding sites, Bmax, were calculated for a system of one
or two binding site populations by nonlinear curve fitting using
the program Ligand (Kell Biosoft, Ferguson, MO) (Munson
and Rodbard, 1980).28 Functional experiments were calculated
by nonlinear regression analysis using the equation for a
sigmoid concentration−response curve (GraphPAD Prism, San
Diego, CA).
Kinetic Calculations. The half-life values of the com-
pounds showing a first order kinetic degradation were
calculated from an exponential decay plot of its concentrations
versus incubation time, using the computer program GraphPad
Prism. The same software was employed for the linear
regression of the corresponding semilogarithmic plots.
RESULTS
■
Synthesis. The desired compounds were synthesized as
summarized in Schemes 1 and 2. The compound N1-((4-(((7-
Kinetic Analysis in Rat Brain Homogenates. The brains
of male Wistar rats were immediately isolated after their
decapitation, and homogenized in 5 volumes (w/v) of Tris HCl
(50 mM, pH 7.4, 4 °C) with an ultra-Turrax (IKA Werke
GmbH & Co. KG, Staufen, Germany) using 3 × 15 s bursts.
The supernatant obtained after centrifugation (3000g for 15
min at 4 °C) was decanted off and stored at −80 °C before its
employment for kinetic studies. The total protein concentration
in the tissue homogenate was determined by using a Bio-Rad
method,27with bovine albumin as reference standard, and
resulted as 7.2 0.4 μg of protein/μL.
Scheme 2. Synthesis of Compound DP-L-A2AANT (2)
The prodrug DP-L-A2AANT (2) and its potential hydrolysis
products dopamine (1), DP-L (4), A2AANT (3) and L-
A2AANT (5) were incubated at 37 °C in 3 mL rat brain
homogenates, resulting in a final concentration of 100 μM. At
regular time intervals, 100 μL samples were withdrawn and
immediately quenched in 200 μL of ethanol (4 °C); 100 μL of
internal standard (the same as described for blood samples)
was then added. After centrifugation at 13000g for 10 min, 300
μL aliquots were reduced to dryness under a nitrogen stream.
Two hundred microliters of mobile phase was added, and, after
centrifugation, 10 μL was injected into the HPLC system. All
the values were obtained as the mean of three independent
incubation experiments.
The accuracy of the method and the calibration curves
referred to rat brain homogenates were obtained as described
for the blood samples.
Data and Statistical Analysis of Binding Experiments.
The protein concentration of membrane employed for binding
experiments was determined by the Bio-Rad method.27
Dissociation equilibrium constants for saturation binding,
amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)-
a m i n o ) m e t h y l ) c y c l o h e x y l ) m e t h y l ) - N 4 - ( 3 , 4 -
dihydroxyphenethyl)succinamide (2) was prepared following
standard coupling conditions with hydroxybenzotriazole
(HOBt) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
(WSC) in dimethylformamide (DMF), starting from com-
pound 7-amino-5-(aminomethyl)cyclohexylmethyl-amino-2-(2-
furyl)1,2,4-triazolo[1,5-a]-1,3,5-triazine trifluoroacetate (3) and
4-[[2-(3,4-dihydroxyphenyl)ethyl]amino]-4-oxobutanoic acid
(4). Compound 3 was prepared as described in our previous
work.21 The derivatives DP-L (4) and 4-(((4-(((7-amino-2-
(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)-
methyl)cyclohexyl)methyl)amino)-4-oxobutanoic acid (L-
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Table 1. Affinity (Ki, nM) or Potency (IC50, nM) Values of the Examined Compounds and Dopamine in CHO Membranes
a
Transfected with hA1, hA2A, hA2B and hA3ARs and in Rat Striatum Membranes Expressing A2AARs and D2 Receptors
in vitro experiments
A2AANT (3)
L-A2AANT (5)
DP-L-A2AANT (2)
dopamine (1)
DP-L (4)
3
hA1ARs H-DPCPX binding (Ki, nM)
>5000
>5000
>5000
>10000
>5000
>5000
>5000
>5000
>5000
>5000
3
hA2AARs H-ZM241385 binding (Ki, nM)
72
5
35
3
7.32 0.65
>5000
>10000
hA2BARs cAMP assay (IC50, nM)
hA3ARs 125I-AB-MECA binding (Ki, nM)
>5000
>5000
>5000
>5000
>10000
>5000
>10000
3
rat striatum A2A ARs H-ZM241385 binding (Ki, nM)
50
2
24
2
2.07 0.23
>5000
>10000
3
rat striatum D2 receptors H-spiperone binding (Ki, nM)
>5000
>5000
2667 120
a
Data are expressed as mean (n = 4 experiments) SEM.
A2AANT (5)) were synthesized in high yield through the
reaction of dopamine hydrochloride or compound 3 with
succinic anhydride in dry pyridine. The DP-L was prepared as
already described in the literature and further purified. Our
spectroscopic data confirmed the structure, but they do not
correspond to the data reported by Bonina et al.29
striatum or hA2ACHO cells, respectively. As expected,
butaclamol was not able to interact with A2AARs.
Saturation binding experiments in hA2ACHO cell membranes
were performed to evaluate affinity (KD) and receptor density
(Bmax) of the A2AARs in the absence and in the presence of DP
10 μM. Figure 3A illustrates saturation binding curves relative
Affinity of Examined Compounds versus ARs. Com-
petition binding experiments to human A1, A2A and A3ARs, by
using the examined compounds and dopamine in CHO cell
membranes, were performed (Table 1). The novel compounds
showed affinity values only for A2AARs revealing Ki values in
the nanomolar range (Figure 2A). Additional competition
Figure 3. Saturation curves (A) and Scatchard plot (B) of [3H]-ZM
241385 binding on A2AARs in rat striatum membranes. Each value
represents the mean SEM of four separate experiments performed in
duplicate.
Figure 2. Affinity values of the examined compounds obtained by
using competition binding experiments versus hA2AARs in CHO
membranes (A) and in rat striatum membranes (B). Each value
represents the mean SEM of four separate experiments performed in
duplicate.
to A2AARs showing affinity of 0.65 0.03 nM and a very high
receptor density of 1515 120 fmol/mg protein. Similarly, in
the presence of DP 10 μM the affinity of A2AARs was in the
nanomolar range (Ki = 0.68
0.02 nM) and the receptor
density was 1507 115 fmol/mg protein, suggesting that DP
was not able to modify A2A affinity or density. Scatchard plot
analysis revealed the presence of a high affinity binding site as
suggested by the linearity of the lines (Figure 3B). Computer
analysis of the data failed to show a significantly better fit to a
two site than to a one site binding model, indicating that in our
experimental conditions one class of high affinity binding site is
primarily present.
binding experiments were also performed in rat striatum where
the compounds examined showed affinity values closely
associated with those found in hA2ACHO cells (Table 1,
Figure 2B). Interestingly, the prodrug DP-L-A2AANT (2)
showed a very high affinity versus A2AARs with a Ki of 2.07
0.23 or 7.32
0.65 nM in rat striatum or hA2ACHO cells,
respectively (Table 1, Figure 2). The affinity of a well-known
A2A antagonist ZM 241385 versus A2AARs was also investigated
and showed a Ki of 0.67 0.08 nM or of 0.85 0.09 nM in rat
Affinity of Examined Compounds versus D2 Recep-
tors. Table 2 reports the results obtained by saturation and
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affinity and density in rat striatum in the absence and in the
presence of CGS 21680 (100 nM). The well-known A2A agonist
was not able to modify KD or Bmax values as reported in Figure
4A and 4B. The compounds examined at the 1 μM
concentration did not modify binding parameters of [3H]-
spiperone in rat striatum (Table 2A). Competition binding
experiments to D2 receptors were also performed in rat
striatum with the aim to evaluate affinity values of DP (Ki =
2667 120 nM) and butaclamol (Ki = 3.93 0.12 nM). In
contrast ZM 241385, a well-known A2A antagonist, did not
interact with D2 receptors expressing in rat striatum (Ki > 1000
nM). In addition, no affinity values were found for the novel
compounds versus D2 receptors (Table 1). The affinity value of
dopamine (1) was evaluated in the presence of CGS 21680
showing a significant increase in the Ki value to 14433 nM,
corresponding to reduced affinity (Table 2B, Figure 5).
Table 2. (A) Affinity (KD, nM) and Receptor Density (Bmax
fmol/mg protein) of D2 Receptors in Rat Striatum
Membranes and (B) Affinity Values (Ki, nM) of the
,
Examined Compounds on D2 Receptors in the Absence and
in the Presence of CGS 21680 (100 nM) in Rat Striatum
a
Membranes
(A) Affinity (KD, nM) and Receptor Density (Bmax, fmol/mg protein)
[3H]-spiperone saturation binding
experiments
KD (nM)
Bmax (fmol/mg protein)
control
0.25 0.02
0.23 0.02
0.24 0.02
0.25 0.01
0.23 0.02
102
107
101
103
100
8
9
9
8
7
+ CGS 21680 (100 nM)
+ A2AANT (3) (1 μM)
+ L-A2AANT (5) (1 μM)
+ DP-L-A2AANT (2) (1 μM)
(B) Affinity Values (Ki, nM)
[3H]-spiperone competition binding
experiments
Ki (nM) + CGS 21680 (100
Ki (nM)
nM)
dopamine
2667 120
2650 104
2700 153
2633 174
14433 233
2767 117
2623 101
2730 114
+ A2AANT (3) (1 μM)
+ L-A2AANT (5) (1 μM)
+ DP-L-A2AANT (2)
(1 μM)
a
Data are expressed as mean (n = 4 experiments) SEM.
competition binding experiments versus D2 receptors in rat
striatum. Figure 4 shows D2 receptor binding characteristics as
Figure 5. Affinity value of dopamine (1) in the absence and in the
presence of DP-L-A2AANT2 by using competition binding experi-
ments versus D2 receptors in rat striatum membranes. The affinity
values were calculated both in the absence (A) or in the presence (B)
of CGS 21680 (100 nM). Each value represents the mean SEM of
four separate experiments performed in duplicate.
Interestingly, the compounds examined at the 1 μM
concentration blocked the effect of CGS 21680 restoring the
affinity of dopamine (1) to control values (Table 2B, Figure 5).
HPLC and Stability Studies. The aim of our work was to
evaluate the potential hydrolysis pattern of the prodrug DP-L-
A2AANT (2) in different media such as water, phosphate buffer,
human whole blood and rat brain homogenates. For this
purpose it was necessary to detect and quantify, in all
incubation media, not only the prodrug but also its potential
hydrolysis products dopamine (1), DP-L (4), A2AANT (3) and
L-A2AANT (5). In order to do so, an efficacious analytical
method was developed based on the employment of a reverse
phase HPLC column, characterized by a polar ether-linked
phenyl phase, able to provide a satisfactory polar and aromatic
reversed phase selectivity of the compounds analyzed by us. In
particular, the analytical HPLC method for dopamine (1) and
Figure 4. Saturation curves (A) and Scatchard plot (B) of [3H]-
spiperone binding on D2 receptors in rat striatum membranes. Each
value represents the mean
performed in duplicate.
SEM of four separate experiments
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Figure 6. Degradation profile of the prodrug DP-L-A2AANT (2) and its potential hydrolysis products in human whole blood. The inset shows the
semilogaritmic plot of the prodrug profile; its linearity (n = 7, r = 0.985, P < 0.0001) provides evidence of a degradation following apparent first order
kinetics (half-life = 2.73 0.23 h). Data are reported as the mean SD of three independent experiments.
Figure 7. Appearance in whole blood of the compounds derived by the hydrolysis of the prodrug DP-L-A2AANT (2) during its incubation at 37 °C.
The values are reported as the percentage of the overall amount of incubated prodrug. Data are reported as the mean SD of three independent
experiments.
its derivative DP-L (4) was optimized with a mobile phase
containing heptafluorobutyric acid, allowing their satisfactory
retention at 15% (v/v) methanol concentration in the mobile
phase.
The chromatographic precision for all the compounds
dissolved in aqueous phase and their internal standards were
represented by the relative standard deviation (RSD) values
ranging, among the different molecules, between 0.87% and
1.36%. The calibration curves of the compounds incubated in
aqueous phase were linear over the range of 0.5−100 μM (n =
8, r > 0.998, P < 0.0001). The average recoveries SD of the
compounds from human whole blood and rat brain
dopamine (1), which decomposed following apparent first
order kinetics (data not shown) with a half-life of 6.3 0.4 h.
Dopamine (1) incubated in human whole blood was degraded
following a biphasic pattern, which was relatively rapid in the
first stage (more than 60% of the drug degraded in 30 min) and
slower in the second phase, where about 11% of incubated drug
was degraded within eight hours (Figure 6). The A2AANT (3)
incubated in whole blood showed a degradation pattern similar
to that of dopamine (1), even if the rates were different: in
particular, about 40% of incubated antagonist was degraded in
30 min, and then a further decrease of about 25% was
registered within eight hours (Figure 6). High stability was,
instead, registered in human whole blood for the derivatives
DP-L (4) and L-A2AANT (5), whose amounts had not
decreased during eight hours of incubation at 37 °C (Figure
6). Finally, the prodrug DP-L-A2AANT (2) appeared degraded
in human whole blood following pseudo first order kinetics
(Figure 6, half-life = 2.73 0.23 h), as confirmed by the linear
pattern of the semilogarithmic plot (n = 7, r = 0.985, P <
0.0001) reported in the inset of Figure 6, and suggesting,
therefore, a prodrug degradation governed by hydrolysis
processes. In order to verify this hypothesis, the blood samples
related to DP-L-A2AANT (2) incubation were also analyzed for
the quantification of its potential hydrolysis products dopamine
(1), DP-L (4), A2AANT (3) and L-A2AANT (5). The results of
this investigation are shown in Figure 7, where the appearance
over time of the hydrolysis products is reported as a percentage
of the overall amount of prodrug incubated in whole blood. In
homogenates ranged between 61.2
2.9% and 97
4.8%.
The concentrations of the prodrug DP-L-A2AANT (2) and its
potential hydrolysis products were therefore referred to as peak
area ratio with respect to their internal standards. The precision
of the method based on peak area ratio was represented by
RSD values ranging between 1.2% and 1.6% among the
analyzed molecules. The calibration curves referred to the
compounds incubated in human whole blood and rat brain
homogenates were linear over the range 2−100 uM (n = 8, r >
0.996, P < 0.0001). No interferences were observed from
plasma or brain homogenate extract components.
The prodrug DP-L-A2AANT 2 and its potential hydrolysis
products dopamine (1), DP-L (4), A2AANT (3) and L-
A2AANT (5) were not degraded in water during incubation for
eight hours at 37 °C. Similarly, the compounds were not
degraded in phosphate buffer (pH 7.4) with the exception of
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Figure 8. Degradation profile of the prodrug DP-L-A2AANT (2) and its potential hydrolysis products in rat brain homogenates. The inset shows the
semilogaritmic plot of the compounds; their linearity (n ≥ 6, r > 0.970, P ≤ 0.001) evidences a degradation of dopamine (1), A2AANT (3) and
prodrug DP-L-A2AANT (2), following apparent first order kinetics (half-life = 22.5 1.5 min for dopamine (1), higher than eight hours for A2AANT
(3) and prodrug DP-L-A2AANT (2)). Data are reported as the mean SD of three independent experiments.
particular, it can be observed that the amounts of A2AANT (3)
and DP-L (4), delivered over time, are relatively high, since
their values are near to those derived from the degradation
pattern of the prodrug. Indeed, the amounts of A2AANT (3)
and DP-L (4) delivered within eight hours corresponded to
about 50% and 65%, respectively, of the incubated prodrug DP-
L-A2AANT (2), with respect to its 75% degradation. Figure 7
also provides evidence that, after 4 h of incubation, dopamine
(1) and L-A2AANT (5) became detectable in whole blood, and
that after eight hours their amounts corresponded to 4 and 6%,
respectively, of the incubated prodrug. It is important to
underline that DP-L (4) and L-A2AANT (5) were characterized
by their inability to be degraded in blood (Figure 6) and that
the sum of their amounts delivered during the prodrug DP-L-
A2AANT (2) incubation in whole blood appeared compatible
with its degradation pattern. Our results indicate, therefore, that
the prodrug degradation in human blood is mainly governed by
the hydrolysis of the N1 amide group obtained by amidation of
the A2AANT (3) with the linker and weakly governed by the
hydrolysis of N4 amide group obtained by amidation of
dopamine (1) with the linker.
were also analyzed for the quantification of its hydrolysis
product. The results of this investigation are reported in Figure
9, where the appearance over time of the hydrolysis products is
Figure 9. Appearance of rat brain homogenates of the compounds
derived by the hydrolysis of the prodrug DP-L-A2AANT (2) during its
incubation at 37 °C. The values are reported as the percentage of the
overall amount of incubated prodrug. Data are reported as the mean
SD of three independent experiments.
The potential ability of the prodrug DP-L-A2AANT (2) to be
hydrolyzed in central nervous system (CNS) environments was
investigated by incubating it in rat brain homogenates. Also in
this case, not only the prodrug but also its potential hydrolysis
products were detected. As reported in Figure 8, dopamine (1)
showed a relatively fast degradation in rat brain homogenates,
following pseudo first order kinetics with a half-life of 22.5
1.5 min. The A2AANT (3) and the prodrug DP-L-A2AANT (2)
also appeared degraded following pseudo first order kinetics,
even if slower than dopamine (1) (Figure 8), and showed half-
life values higher than eight hours. In particular, after eight
hours of incubation, the degradations of the prodrug DP-L-
A2AANT (2) and A2AANT (3) were 41% and 37%, respectively.
The pseudo first order kinetics related to the degradation of the
three compounds was confirmed by the linearity of the
semilogarithmic plots (n ≥ 6, r > 0.970, P ≤ 0.001) reported in
the inset of Figure 8. As found in human whole blood, the
derivatives DP-L (4) and L-A2AANT (5) showed high stability
in rat brain homogenates, since their amounts did not decrease
during eight hours of incubation at 37 °C (Figure 8).
reported as a percentage of the overall amount of prodrug
incubated in rat brain homogenates. Neither the A2AANT (3)
nor the derivative DP-L (4) was detected within eight hours
incubation of the prodrug DP-L-A2AANT (2). On the other
hand, L-A2AANT (5) and dopamine (1) appeared detectable
after one and two hours of incubation, respectively, and after
eight hours their amounts delivered appeared to be 13% and
39% of the incubated prodrug. It is interesting to observe that
the amounts of the delivered L-A2AANT (5), that was not
degraded in rat brain homogenates, appeared compatible with
the prodrug DP-L-A2AANT (2) degradation pattern. On the
other hand, the amounts of dopamine (1) were lower,
according to its relatively high instability in rat brain
homogenates. Our results indicate, therefore, that the prodrug
degradation in rat brain homogenates is governed exclusively by
the hydrolysis of the amide group obtained from the amidation
of the dopamine (1) with the linker. This process allowed us to
obtain a controlled and prolonged release of dopamine (1): a
comparison of Figure 8 with Figure 9 evidences that relatively
high amounts of free dopamine were totally degraded within 3
In order to verify whether the degradation of the prodrug
DP-L-A2AANT (2) in rat brain homogenates was related to
hydrolysis processes, the samples obtained from its incubation
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h, whereas the prodrug appeared able to perform a prolonged
delivery of small DP amounts for at least eight hours.
At present, the relevance of the A2A−D2 heteromeric
complexes in PD pathophysiology and treatment is widely
investigated, suggesting that these heteromers remain in the
dorsal striatopallidal GABA pathway after degeneration of the
nigrostriatal pathway.38 Thus, the supersensitive D2 receptors
remain under strong antagonistic A2AARs control.51 As a
consequence, the DP/adenosine bivalent action could represent
a novel concept in PD pharmacotherapy.
DISCUSSION
■
Adenosine antagonist and dopamine interactions have been
widely reported in CNS in both behavioral and biochemical
studies.30 In vitro and in vivo developments of selective A2A
antagonists, like istradefylline or preladenant, have yielded the
greatest improvement in the mobility of PD patients by
simultaneous activation of D2 receptors and inhibition of
A2AARs.31−34 On the other hand, it has been observed that the
administration of A2AAR antagonists in monotherapy does not
improve motor symptoms in PD, suggesting a need for
dopamine receptor activation.35,36 It has also been postulated
that the interaction between A2AARs and D2 receptors may
involve the formation of heteromeric complexes.37 Various
studies are present in the literature showing that A2A
antagonists exert marked efficacy on locomotor activity in
various models of PD.38 In a well-known in vitro model, widely
used for PD studies represented by PC 12 cells, treatment with
adenosine agonists mediated a reduction in D2 receptors and a
decrease in the dopamine affinity.14 In addition, in PC 12 cells
the A2A antagonists, inhibiting dopamine uptake, increased the
concentrations of dopamine available to interact with D2
receptors and a consequent marked activation of dopaminergic
pathway providing a significant improvement as far as
symptoms are concerned.25 Moreover, it has been demon-
strated that A2AARs are upregulated in PD patients in
comparison with healthy subjects.25 Thus, A2A antagonists
might block the A2AARs by reducing the activity in the indirect
pathway known to produce motor inhibition.39 On the other
hand, it has been reported that overstimulation of A2AARs
could be due to the uncontrolled discharge of corticostriatal
terminals leading to the increase in adenosine levels through
the ATP catabolic degradation.40 It is well-known that the
activation of D2 leads to endocannabinoid production that is
able to activate presynaptic CB1 receptors at corticostriatal
terminals. Since presynaptic A2AAR activation inhibits CB1
receptors in the striatum, it prevents the CB1-mediated
inhibition of motor activity and of depolarization-induced
glutamate release.41 Postsynaptically, A2AAR stimulation
inhibits the endocannabinoid production mediated by D2
activation.42 Thus, A2AAR and D2 receptors might act in
concert to regulate endocannabinoid function in the striatum.
As a consequence, the coadministration of A2A antagonists with
L-DOPA or D2 agonists appears efficacious in preventing the
motor complications, such as dyskinesias, induced by pulsatile
long-term treatment of dopaminergic drugs. In this case the A2A
antagonists most likely contribute to restoring the appropriate
balance between A2A ARs and D2 receptors.11,15−17 It could be
of interest to note that patients with advanced PD treated with
levodopa/carbidopa, formulated as intestinal gel (DUODOPA)
to avoid pulsatile L-DOPA administration, showed evident
improvements in motor fluctuations and dyskinesias.43−45
Ongoing studies are looking into potential future therapies,
including medications that provide both monoaminooxidase B
and glutamate inhibition, a sustained-release levodopa prodrug,
a carbidopa subcutaneous patch, an oral neurotrophic factor
inducer and different antidyskiniesia medications.36,46−50
Nowadays, there is an urgent need for other therapies that
provide anti-Parkinsonian benefits but which, at the same time,
avoid dyskinesia, and reduce the progression of the pathology.
The idea of building a molecular conjugate able to interact in
a dual synergic way on different biological targets is quite
common in medical chemistry. Novel compounds characterized
by the presence of A2A antagonist and dopaminergic
pharmacophores that, either directly or after their release, can
perform the bivalent action of A2AAR blockade and D2 receptor
activation could constitute highly interesting drug candidates.
To this end, we decided to conjugate an A2A antagonist and the
dopamine, the natural ligand of dopaminergic receptor, through
the linkage with an opportune spacer. We chose to link the A2A
antagonist and the dopamine to the spacer through amidic
bonds since these moieties can be cleaved by endogenous
enzymes, with consequent release of the two drugs, even if they
are slightly more resistant than ester functions. This approach
can potentially induce a controlled release of dopamine, and
may, therefore, considerably limit its typical pulsatile pattern in
the brain that generally follows L-DOPA administration.
Moderate and nonpulsating tones of dopamine in the CNS
can therefore limit its neuronal toxicity and avoid the long-term
side effects derived from pulsatile treatments. Succinic acid
seemed the best candidate as a spacer given the high reactivity
of its anhydride and its good toxicological tolerability, if
released into the bloodstream. Our research team has been
working toward the development of various structures such as
A2A antagonists.51 Recently, we synthesized several triazolo-
triazine derivatives endowed with a good affinity and selectivity
for A2AARs.21 Among these new structures, the 7-amino-5-
(aminomethyl)cyclohexylmethyl-amino-2-(2-furyl)-1,2,4-
triazolo[1,5-a]-1,3,5-triazine trifluoroacetate (3) was selected as
a better analogue to be conjugated through its amine group on
the side chain to the succinic acid. Compound 3 has a good
pharmacological profile with a high affinity for the receptor
(hA2A 72 nM) and very high selectivity versus all the other
human A1, A2B, and A3ARs. The presence of the amine group
on the side chain allowed us to link the succinic acid without
influencing the other parts of the molecule that are necessary to
the receptor binding, such as the amino group at position 7.21
Moreover, in accordance with our previous experience, we
hypothezised that a steric hindrance at the side chain could
induce an affinity improvement.52
The prodrug DP-L-A2AANT (2) was not able to interact
toward D2 receptors, but it did show high affinity toward
A2AARs, with Ki values of 2.07 or 7.32 nM in rat striatum or
hA2ACHO cells, respectively, 1 order of magnitude lower than
those of the A2AANT (3). Moreover, the L-A2AANT (5),
potentially derived from hydrolysis of DP-L-A2AANT (2),
showed affinity values toward A2AARs similar to those of the
A2AANT (3). The latter compound and its derivatives 2 and 5
appeared, moreover, highly selective for A2AARs and unable to
interact with the D2 receptors. These results were predictable
since it is known, from docking studies, that the side chain
protrudes from the binding cavity and hydrophilic substitutions
can interact with the hydrophilic portion of the extracellular
loops.21 The compounds examined were not able to modify the
affinity and potency studied by means of 3H-spiperone
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saturation binding experiments versus D2 receptors, suggesting
that their presence did not change the kinetic parameters of
these receptors. Interestingly, the compounds tested at the 1
μM concentration blocked the reduction in the dopamine
affinity by CGS 21680, restoring the affinity of dopamine to
control values.
Recently, a novel designed synthesized family of hetero-
bivalent ligands, containing a D2 agonist and an A2A antagonist
acting as a probe for A2A−D2 heteromers, has been studied.
These data suggest the cooperative effect derived from the
simultaneous interaction of heterobivalent ligands with
receptors in brain striatum, but not in cotransfected cells with
the single homomers, thus evidencing the presence of the A2A−
D2 heteromers in striatum tissue.53
high stability also in rat brain homogenates, unlike the other
compounds analyzed, dopamine (1), the A2AANT (3) and the
prodrug DP-L-A2AANT (2) that were all degraded following
pseudo first order kinetics. The degradation rate of dopamine
appeared relatively fast, considering that the total amounts
incubated disappeared within 3 h. These data reflect the
metabolic processes that involve dopamine in CNS,59 where its
degradation can be a source of free radicals.60 Concerning these
metabolic processes, it has been hypothesized that the high
dopamine concentrations induced in the brain by L-DOPA
therapy may potentially contribute to the progression of
oxidative damage of dopaminergic neurons.59,61−63 We have
demonstrated that in rat brain homogenates the prodrug 2 is
hydrolyzed exclusively on the amidic group coupling dopamine
(1), allowing its controlled release. These data suggest that, in
the CNS, the prodrug 2 may allow us to obtain a moderate
dopamine tone for higher interval times than those derived
from L-DOPA therapy. This behavior may be useful in limiting
not only the neuronal toxicity caused by the oxidative
metabolism of dopamine but also the side effects derived
from pulsatile L-DOPA long-term treatments, indicating the
prodrug 2 as a potential alternative to the DUODOPA systems.
These beneficial effects may be corroborated by the A2A
antagonistic activity of the prodrug 2 itself and its main
hydrolysis product, the L-A2AANT (5), whose stability in rat
brain homogenates has been shown to be higher than that of its
parent antagonist (3). Indeed, the neuroprotective effects of
A2A antagonists on CNS and their ability to potentiate the
therapeutic effects of dopamine and limit the motor
complications derived from long-term L-DOPA treatment are
well-known.10,15,16,18−20
Several prodrugs able to release dopamine by enzymatic
hydrolysis have been found to induce antiparkinsonian effects
on rats,29,64−66 suggesting the existence, in the brain, of
enzymes able to obtain the release of dopamine at extracellular
level. These phenomena allow us to hypothesize that the
release of dopamine observed in brain homogenates may
involve extracellular enzymatic systems.
Taking into account the aspects described above, the prodrug
DP-L-A2AANT (2) appears to be a good candidate for nasal
administration, a promising way for the cerebral uptake of
potent neuroactive agents.67,68 We have demonstrated that
microparticulate formulations based on chitosan can be useful
in promoting the CNS entry of neuroactive drugs via the nasal
pathway.69,70 These new results may help guide and improve
future pharmacological treatment in PD, even if further studies
are necessary to better investigate the in vivo administration and
effects of the prodrug 2.
With the aim to evaluate the hydrolysis pattern of the
prodrug DP-L-A2AANT (2) in physiologic environments (and
thus its potential bivalent action on A2A−D2 heteromers
obtained by the release of dopamine (1)), we first compared
the stabilities of the prodrug 2 and its potential hydrolysis
products (dopamine (1), DP-L (4), A2AANT (3) and L-
A2AANT (5)) in water, phosphate buffer, human whole blood
and rat brain homogenates; then, over time, we detected the
appearance of the hydrolysis products in the fluids where the
prodrug 2 appeared degraded. All the compounds analyzed
were not found to be degraded in water, whereas in phosphate
buffer only dopamine (1) showed degradation over time. This
result confirms previous studies on dopamine (1) stability54
and suggests that its chemical degradation, probably oxidative,55
is influenced by the pH and/or ionic strength of the incubation
medium. Dopamine was also degraded in human whole blood,
in conformity with data in the literature reporting a severe
degradation of dopamine (1) the day after its incubation at 37
°C in human plasma.55 The A2AANT (3) showed a biphasic
degradation pattern over time in whole blood, suggesting its
poor stability at peripheral level in the body. The prodrug DP-
L-A2AANT (2) was degraded in whole blood following pseudo
first order kinetics. We have demonstrated that this degradation
is related to a hydrolysis process which is mainly related to the
amide group coupling the A2AANT (3) with the linker, and
very poorly to the amide group coupling dopamine (1). These
data suggest that, at the peripheral level, the prodrug could
release limited amounts of dopamine, thus avoiding induction
of its typical peripheral side effects. The release process from
the prodrug DP-L-A2AANT (2) of the A2AANT (3) could,
moreover, contribute to increasing its half-life. The other main
hydrolysis product of prodrug (2), the DP-L (4), was totally
inactive toward the dopaminergic receptors. Surprisingly, this
compound and its homologous L-A2AANT (5) were not
degraded in human whole blood. This behavior, apparently
paradoxical if we take into account the hydrolysis pattern of the
prodrug (2), may be attributed to the presence of the carboxylic
groups belonging to these compounds, that, at physiologic pH,
are negatively charged. The presence of this charge can inhibit
the enzymatic hydrolysis activity on the amide groups. A similar
phenomenon has been demonstrated in the case of aspirin,
which, in plasma, is normally not hydrolyzed to salicylic acid.
On the other hand, the esterification of the carboxylic acid
group of aspirin renders its O-acetyl ester highly susceptible to
plasma-mediated hydrolysis.56,57 This phenomenon is due to
butyrylcholinesterase, a dominant esterase in human plasma
that is not able to induce the hydrolysis of negatively charged
substrates, but is extremely efficient in processing neutral
molecules.58 The linker-coupled compounds 4 and 5 showed
CONCLUSIONS
■
The prodrug DP-L-A2AANT (2) was designed with a view to
conjugating the beneficial effects against PD obtained by a
combined action of dopamine and A2A antagonists in CNS.
This action is focused on striatal A2A−D2 heteromers. The
prodrug 2, able to act as a potent and selective A2A antagonist,
does not release dopamine in human whole blood by
hydrolysis, most likely reducing the probability of inducing
the typical dopaminergic side effects at the peripheral level. In
rat brain homogenates the prodrug 2 is able to control the
release of dopamine and, therefore, to increase its poor stability.
Both the prodrug and hydrolysis product L-A2AANT (5), the
latter characterized by high stability in physiologic media, are
able to increase the dopamine affinity toward striatal D2
601
dx.doi.org/10.1021/mp200489d | Mol. Pharmaceutics 2012, 9, 591−604

Molecular Pharmaceutics
Article
Disease. Relevance for L-Dopa Induced Dyskinesias. J. Neurol. Sci.
2006, 248, 16−22.
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combined effects suggest the prodrug approach as promising
for both early and long-term pharmacological treatment of PD.
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Gago, B.; Roberts, D. C.; Lange, U.; Genedani, S.; Ferraro, L.; de la
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Receptor− Receptor Interactions within Receptor Mosaics. Impact on
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AUTHOR INFORMATION
■
Corresponding Author
*Department of Pharmaceutical Sciences, via Fossato di
Mortara 19, I-44121, Ferrara, Italy. Tel: +39 0532 455273.
Fax: +39 0532 455256. E-mail: barbara.cacciari@unife.it.
Notes
The authors declare no competing financial interest.
(19) Xu, K.; Bastia, E.; Schwarzschild, M. Therapeutic Potential of
Adenosine A(2A) Receptor Antagonists in Parkinson’s Disease.
Pharmacol. Ther. 2005, 105, 267−310.
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M. Targeting Adenosine A2A Receptors in Parkinson’s Disease. Trends
Neurosci. 2006, 29, 647−654.
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