Synthesis and in vitro studies on a potential dopamine prodrug

Article abstract of DOI:10.1691/ph.2008.8585

Dopamine delivery to the central nervous system (CNS) undergoes the permeability limitations of blood-brain barrier (BBB) which is a selective interface that excludes most water-soluble molecules from entering the brain. Neutral amino acids permeate the BBB by specific transport systems. Condensation of dopamine with neutral amino acids could afford potential prodrugs able to interact with the BBB endogenous transporters and easily enter the brain. The synthesis and characterization of the dopamine derivative 2-amino-N-[2-(3,4- dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (7) is described. The chemical and enzymatic stability of 7 was evaluated. The molecular weight (300 Da) and Log P_app (0.76) indicated that the physico-chemical characteristics of compound 7 are adequate to cross biological membranes. Compound 7 was enzymatically cleaved to free dopamine in rat brain homogenate (t_1/2 = 460 min). In human plasma, the t_1/2 of 7 was estimated comparable to that reported for L-DOPA. In view of a possible oral administration of 7, studies of its chemical behavior under conditions simulating those of the gastrointestinal tract showed that no dopamine production occurred; furthermore, 7 is able to permeate through a simulated intestinal mucosal membrane. The collected data suggest that compound 7 could be considered a very valuable candidate for subsequent in vivo evaluation.

Full text of DOI:10.1691/ph.2008.8585

ORIGINAL ARTICLES
Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita` degli Studi di Palermo, Palermo, Italy
Synthesis and in vitro studies on a potential dopamine prodrug
L. I. Giannola, V. De Caro, G. Giandalia, M. G. Siragusa, L. Lamartina
Received April 15, 2008, accepted May 23, 2008
Libero Italo Giannola, Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita` di Palermo, Via
Archirafi, 32, 90123 Palermo, Italy
litagian@unipa.it
Pharmazie 63: 704–710 (2008)
doi: 10.1691/ph.2008.8585
Dopamine delivery to the central nervous system (CNS) undergoes the permeability limitations of
blood-brain barrier (BBB) which is a selective interface that excludes most water-soluble molecules
from entering the brain. Neutral amino acids permeate the BBB by specific transport systems. Conden-
sation of dopamine with neutral amino acids could afford potential prodrugs able to interact with the
BBB endogenous transporters and easily enter the brain. The synthesis and characterization of the
dopamine derivative 2-amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (7) is de-
scribed. The chemical and enzymatic stability of 7 was evaluated. The molecular weight (300 Da) and
Log Papp (0.76) indicated that the physico-chemical characteristics of compound 7 are adequate to
cross biological membranes. Compound 7 was enzymatically cleaved to free dopamine in rat brain
homogenate (t1/2 ¼ 460 min). In human plasma, the t1/2 of 7 was estimated comparable to that re-
ported for l-DOPA. In view of a possible oral administration of 7, studies of its chemical behavior
under conditions simulating those of the gastrointestinal tract showed that no dopamine production
occurred; furthermore, 7 is able to permeate through a simulated intestinal mucosal membrane. The
collected data suggest that compound 7 could be considered a very valuable candidate for subse-
quent in vivo evaluation.
1. Introduction
tive disease (Tolosa et al. 1998). The drug uptake into the
brain is followed by aromatic l-amino acid decarboxylase
enzymatic conversion to dopamine (Pardridge 1977, 1995;
Oldendorf et al. 1983). Unfortunately, upon continuous
treatment, the clinical response to oral l-DOPA is variable
and unreliable since its irregular absorption and first-pass
metabolism. Following oral administration, the bioavailabil-
ity of l-DOPA is estimated about 5 to 15% and less than
1% of the dose reaches the brain unchanged (Standaert and
Young 1996). Even if the l-DOPA dose is reduced, and the
bioavailability is enhanced by co-administration of decar-
boxylase inhibitors, plasma half-life is only modestly pro-
longed, and a reduction in the frequency of drug adminis-
tration is not possible (Reynolds 1993).
An attractive, non invasive way to enhance brain bioavail-
ability of dopamine could be the prodrug approach, which
consists in covalent linking of actives with specific mole-
cules able to shuttle the drug into CNS (Anderson 1996).
Various dopamine prodrugs have been described; among
them acyl- (Casagrande and Ferrari 1973; Borgman et al.
1973) glycosyl- (Ferna´ndez et al. 2000, 2003), amido-
(Bodor et al. 1981) and other derivatives have been re-
ported (Di Stefano et al. 2008). In vivo studies have identi-
fied specific carrier systems for the transfer of essential
neutral amino acids throughout the ECs (Terasaki and
Tsuji 1994). In particular, it has been described that phe-
nylalanine is transported using the leucine system (L)
Drug delivery to the brain is one of the most challenging
fields of research and development for pharmaceutical pro-
ducts. The drug transfer from blood to brain is severely
restricted by the blood brain barrier (BBB) as the tight
junctions, sandwiched between the cerebral endothelial
cells (ECs), form a selective diffusion barrier which ex-
cludes most blood-borne substances from entering
(Scherrmann 2002; Ballabh et al. 2004; Lo et al. 2001;
Ghersi-Egea et al. 1994; Wolburg and Lippoldt 2002; Hu-
ber et al. 2001). The rate at which molecules cross the
BBB is related to their physico-chemical characteristics;
lipid solubility, hydrogen bonding, charge, molecular size,
ionization profile, flexibility or other parameters play an
important role (Crivori et al. 2000; Abraham et al. 1994).
Dopamine is one of the most important neurotransmitters in
the central nervous system (CNS) and its striatal depletion
is responsible of clinical signs of Parkinson’s disease (PD)
(Antolin et al. 2002). Owing to the high hydrophilicity and
the absence of a specific transport system within the mem-
brane of ECs, dopamine is unable to cross the BBB, thus
its use is precluded in treatment of PD. On the other hand,
the dopamine precursor l-DOPA is actively transported into
the brain through the LNAA carrier (Pahwa and Koller
1998). Currently, PD therapy is basically symptomatic and
l-DOPA is the treatment of choice for this neurodegenera-
704
Pharmazie 63 (2008) 10

ORIGINAL ARTICLES
large neutral amino acid (LNAA) carrier (Egleton and Da-
mine itself. DCC was chosen as coupling agent (Slebioda
et al. 1990). The main product of the fist step was identi-
fied as 2-(boc)amino-N-[2-(3,4-dimethoxy-phenyl)-ethyl]-
3-phenyl-propionamide (4). An important role in the reac-
tion pathway is played by the volume of solvent used:
high amount of CH2Cl2 promote solubilization of 3
which, in turn, reacts with activated 2 and originates the by-
product 5 (Scheme). To compound 5 was attributed the
structure of [1-benzyl-2-(1,3-dicyclohexyl-ureido)-2-oxo-
ethyl]-carbamic acid tert-butyl ester which was confirmed
by reacting compound 3 with compound 1 in the same ex-
perimental conditions. By removing of the Boc-protection
group from 4 (second step), compound 6 was obtained. De-
methylation of 6 (McOmie et al. 1968; Chen et al. 1998;
Ragot et al. 1999) afforded the desired 2-amino-N-[2-(3,4-
dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (7). Spec-
tral data of synthesized compounds were in agreement with
the proposed structures (Tables 1 and 2).
vis 1997). In the same way, amino acidic derivatives of
dopamine could interact with amino acid transporters pre-
sent in the BBB and, once reached the CNS, could be
enzymatically cleaved releasing dopamine into the brain
(Anderson 1996; Bodor and Buchwald 1999).
In this preliminary study we developed the synthesis and
characterization of an amino acidic dopamine derivative
obtained by covalently binding the drug to the essential
neutral amino acid phenylalanine. Since the ECs are selec-
tive for amino acids of the L series (Bickel et al. 2001;
Egleton and Davis 1997; Pardridge 1995), in this study
was used l-phenylalanine. In order to determine the prop-
erties of the synthesized compound as prodrug, in vitro
stability studies have been performed in plasma and rat
brain extracts. Finally, in view of p.o. administration of
the dopamine amino acidic derivative, the behavior in gas-
trointestinal environment and the aptitude to permeate
through the intestinal membrane barrier were evaluated.
The drug lipophilicity is an important factor conditioning
brain uptake and the apparent partition coefficient (Papp
)
could be used as simple descriptor of ability to cross the
BBB: values of log Papp within ꢀ0.2 to 1.3 have been
described as optimal for cerebral transport; on the other
hand higher values than these could reduce the rate of
transport inside the membrane (Levin 1980; Gimenez
et al. 2004). In view of a potential application of com-
2. Investigations, results and discussion
The amino acidic derivative of dopamine was successfully
obtained by synthesis involving three main steps
(Scheme). Compound 1 was selected as a stable dopamine
derivative to avoid oxygen and light instability of dopa-
Scheme
Boc
HN
H3CO
+
OH
H3CO
NH2
O
(1)
(2)
DCC
CH2Cl2
O
H
H3CO
H3CO
N Boc
O
HN
+
N
N
H
H
(4)
(3)
Compound 2
+
CH2Cl2
TFA
CH2Cl2
DCC
O
Boc
O
HN
H3CO
H3CO
NH2
HN
N
H
N
O
(6)
(5)
BBr3
CH2Cl2
CH3OH
O
HO
HO
NH2
HN
(7)
Pharmazie 63 (2008) 10
705

ORIGINAL ARTICLES
Table 1: Yield, m.p., UV, IR, MS of compounds 4, 5, 6 and 7
Compd. Yield
(%)
m.p.
(ꢁC)
UV (methanol)
(lmax, nm)
IR (cmꢀ1
)
MS
(Mþ)
––OH ––NH2 ––NH––CO ––NH––CO NH––CO BOC––CO C––O––C
N––CO––N
4
5
6
7
50
10
68
89
150–152 279.4
85–87 258.5
104–105 279.4
218–220 283.0
––
––
––
––
––
3346
3306
1520
1522
1510
1510
1657 1682
1654 1682
1239, 1167
1226, 1170 1716
428
471
328
3289 3349
1637
1670
––
––
––
––
––
––
3521 3230–3100
301 (M þ 1)þ
pound 7 as prodrug, the octanol-water Papp was deter-
mined as preliminary parameter. Log Papp of compound 7
was determined as 0.76 thus suggesting that the synthe-
sized product possesses adequate characteristics to perme-
ate membranes.
The aptitude of compound 7 to undergo cleavage by cere-
bral enzymes and produce dopamine in situ was evaluated
using rat brain homogenate. We experienced that com-
pound 7 slowly decreased in concentrations while raising
amounts of dopamine were formed (Fig. 1). Experimental
data were curve-fitted to the most common kinetic equations
and the best fit was obtained using the Michaelis-Menten
relationship thus suggesting a typical enzymatic saturation
kinetic of cleavage. By extrapolation the t1/2 was estimated
about 460 min which is considerably higher than that re-
ported for dopamine degradation (t1/2 ¼ 180 min) in the
1
Table 2: 13C and H (in parenthesis) NMR data of compounds 4, 5, 6 and 7
O
c3
c2
O
O
s
c1
RO
RO
NHR'
r
c4
NHBoc
i
t
a
NH
c2'
a
HN
l
N
b
c
b
c
i
c5
c6
c1'
m
o
h
q
h
d
e
c3'
n
c6'
d
e
p
g
g
c5'
c4'
f
f
Compd.
4
6
7
Compd.
5
C-a
C-b
170.97
56.15
(4.24)
38.76
173.84
56.17
(3.57)
169.30
55.72
(4.08)
C-a
C-b
170.72
54.51
(4.61)
39.06
C-c
40.77
38.63
C-c
(3.01, 3.05)
136.77
129.29
(7.18)
(2.69, 3.22)
137.62
129.07
(7.20)
(3.03, 3.13)
135.50
130.51
(7.30)
(2.83, 3.05)
136.16
129.14
C-d
C-e, i
C-d
C-e, i
(7.17)
C-f, h
C-g
128.62
(7.27)
126.91
(7.26)
128.41
(7.30)
126.54
(7.22)
129.99
(7.24)
128.74
(7.31)
C-f, h
C-g
128.57
(7.32)
127.96
(7.31)
C-l
152.91
(C-a)NH
C-m
(5.76)
40.68
(7.37)
40.17
(8.27)
42.25
(C-l)NH
C-1
(5.08)
50.02
(3.40)
35.11
(2.61)
131.06
(3.48)
35.13
(2.73)
131.35
(3.34)
35.52
(2.56)
131.58
(3.65)
32.38
(1.26, 1.93)
25.25
(1.31, 1.68)
25.40
(1.17, 1.60)
54.51
(4.11)
31.28
(1.35, 1.91)
25.95
C-n
C-o
C-p
C-q
C-r
C-s
C-t
C-2,6
C-3,5
C-4
111.80
(6.63)
149.10
111.84
(6.70)
148.80
116.78
(6.65)
146.19
C-10
147.76
147.46
144.77
C-20,60
C-30,50
C-40
111.40
(6.76)
120.55
(6.57)
55.85
(3.85)
55.90
(3.84)
111.26
(6.78)
120.45
(6.68)
55.64
(3.83)
55.69
(3.83)
116.39
(6.68)
121.03
(6.49)
(1.29, 1.70)
25.86
(1.13, 1.60)
R ¼(C-q)OCH3
R ¼(C-r)OCH3
*
R ¼ OH
( )
R0¼ H; ––NH2
(4.79)
(7.26)
R0¼ Boc; ––NH
(5.01)
(Boc)––NH
(5.08)
R0¼ Boc: 155.25; 80.15; 28.23 (1.39).
Boc: 155.83; 80.20; 28.14 (1.39).
*
( ) Rapidly exchangeable protons
706
Pharmazie 63 (2008) 10

ORIGINAL ARTICLES
0,14
0,12
0,10
0,08
0,06
0,04
0,02
0
0,14
0,12
0,1
Compound 7
Dopamine
Compound 7
Dopamine
0,08
0,06
0,04
0,02
0
40
60
time (min)
0
20
80
100
120
0
50
100
time (min)
150
200
Fig. 2: Profiles of disappearance of compound 7 and simultaneous forma-
tion of dopamine in human plasma at 37 ꢂ 0.5 ꢁC. Values are pre-
sented as means ꢂ SD (n ¼ 3)
Fig. 1: Time courses of compound 7 cleavage and simultaneous formation
of dopamine in rat brain homogenate at 37 ꢂ 0.5 ꢁC. Values are
presented as means ꢂ SD (n ¼ 3)
mine was observed. UV measurements experienced that
absorption peak of aqueous solution of 7 occurred at
l ¼ 281.0 nm, no shifts in the pH range 1.2 to 6.5 were
observed. This behavior remained unchanged up to 6 h,
thus indicating high stability at pH conditions of gastro-
jejunum tract (Fig. 3a and b). For solutions at pH > 7,
after about 15 min, the appearance of a broad peak centered
at l ¼ 458.5 nm was observed which increased with time
suggesting a degradation process (Fig. 3c and d). The degra-
dation trend was followed graphically reporting the absor-
bance values at l ¼ 458.5 nm vs time (Fig. 4). The rate of
formation of the species that absorbs at l ¼ 458.5 nm in-
creased with pH, indicating a base-catalyzed process.
Likewise the most dihydroquinone derivatives, dopamine
in alkaline media is easily auto-oxidized in presence of
molecular oxygen. During this chain process, dopamine is
transformed into a semiquinone species by hydroxyl ions.
same experimental conditions (Fernandez et al. 2000). This
result suggests that compound 7 could be considered as a
reservoir of dopamine into the CNS.
Stability was evaluated in plasmatic environment follow-
ing the in vitro disappearance of 7 from human plasma
and simultaneous appearance of dopamine (Fig. 2). As
above, the hydrolysis occurred following typical enzy-
matic saturation kinetics. By extrapolation, the t1/2 in hu-
man plasma was estimated about 28 min. It is noteworthy
that this value is comparable to that reported for l-DOPA
(Gennaro 2000).
In view of a possible oral administration of compound 7,
studies of chemical stability of 7 in conditions simulating
those of gastrointestinal tract (37 ꢁC, pH 1.2 to 8.0) were
performed. Experimental data showed that the peptide
bond of 7 remains uncleaved and no production of dopa-
Fig. 3:
UV measurements of aqueous solution of 7,
scanned every 15 min up to 360 min, at a) pH
1.2, b) pH 6.5, c) pH 7.4, d) pH 8.0. An in-
crease in absorbance was detected for solu-
tions at pH > 7 at l ¼ 280.5 and l ¼ 458.5: it
was 0.0020 ꢂ 0.0003 and 0.0015 ꢂ 0.0005
respectively for solutions at pH 7.4 whereas
the increase was 0.0080 ꢂ 0.0001 and 0.0050
ꢂ 0.0002 respectively for solutions at pH 8
Pharmazie 63 (2008) 10
707

ORIGINAL ARTICLES
These preliminary data suggest that 7 could be able to
0,20
0,15
0,10
0,05
0
cross membranes of the duodenum/jejunum/ileum tract.
The collected data suggest that compound 7 fulfills the
prodrug criteria and could be considered a very valuable
candidate for subsequent evaluation in vivo.
pH7.4
pH8
3. Experimental
3.1. Materials and apparatus
Trifluoroacetic acid (TFA), 2-(3,4-dimethoxyphenyl)-ethylamine (1), egg
lecithin (phosphatidylcoline), n-dodecane and all components of buffer so-
lutions were purchased from Sigma-Aldrich-Chemie (Steinheim, Ger-
many). N-Boc-l-phenylalanine (2), N,N0-dicyclohexylcarbodiimide (DCC)
and 1M CH2Cl2 solution of boron tribromide (BBr3) were from Acros
(Milan, Italy). HPLC grade solvents were obtained from Baker (Milan,
Italy). All other chemicals and solvents were of analytical grade and were
used without further purification. The process of reactions was monitored
by TLC using Alugram1 SIL G/UV254 (Macherey-Nagel) pre-coated alu-
minium sheets (0.20 mm layer thickness) and visualised using an UV
lamp. Melting points (m.p.) were determined with a Bu¨chi 530 capillary
apparatus and are uncorrected. IR spectra were recorded with a Perkin-
Elmer 1720 FT spectrophotometer as nujol mulls and UV spectra with a
Shimadzu UV-Vis 1700 PharmaSpec spectrophotometer. In chemical and
enzymatic stability tests, HPLC analyses were performed with a Shimadzu
LC-10ADVP instrument equipped with a binary pump LC-10ADVP, a UV
SPD-M20A Diode Array detector, a 20 ml injector and a computer integrat-
ing apparatus (EZ Start 7.3 software). Chromatographic separation was
achieved on a reversed-phase column Discovery HS-F5 (Supelco, 5 mm,
25 cm ꢃ 4.6 mm), mobile phase consisted of a mixture of 0.01% TFA in
distilled water and acetonitrile (gradient profile: 95 : 5 ! 93 : 7 ! 70 : 30
! 95 : 5). The flow rate was set at 1 mL/min and the UV wavelength at
280.5 nm. In these conditions, the retention times for dopamine and com-
pound 7 were 11 min and 34 min, respectively. Standard curves were used
for quantification of integrated areas under the peaks. The calibration
curves were performed at the concentration range of 2 to 20 mg/100 mL.
In these conditions the detection limits (LOD) were < 0.002 mg/mL. 1H
and 13C NMR spectra were recorded in CDCl3 solutions on a Bruker AC
250 spectrometer operating in FT mode at 250.13 and 62.89 MHz, respec-
tively. Only for compound 7 spectra were recorded in CD3OD solution as
it was very scarcely soluble in CDCl3. 1H and 13C NMR chemical shift
values were measured relative to CDCl3 centered respectively at 7.25 and
77.00 ppm downfield from TMS or relative to CD3OD centered respec-
tively at 3.31 and 49.00 ppm downfield from TMS. 13C NMR chemical
shift values were determined from proton fully decoupled spectra with
the assignment supported by 1D and 2D experiments performed using
the standard Bruker pulse sequences DEPT.AUR, XHDEPT.AUR and
COLOC.AUR. Electrospray ionization-mass (ESI-MS) spectrometry was
recorder on a Autospec Tof Ultima, MicroMass Magnetic Sector Orthogo-
nal Tof Spectrometer (spray voltage 4000 V; cone voltage 20 V, capillary
temperature 40 ꢁC, collision gas: argon 1ꢃ10ꢀ6 atm). Experimental data
were elaborated using Kaleidagraph and Curve Expert 1.34 for Windows
software.
0
50
100
150
200
time (min)
250
300
350
400
Fig. 4: Time courses of absorbance of peak at l ¼ 458.5 nm of aqueous
solutions of 7 at pH 7.4 and 8.0. Values are presented as means
ꢂ SD (n ¼ 5)
The semiquinone derivative is oxidized to an open-chain
quinone which, in turn, undergoes intramolecular-pH de-
pendent cyclization to dopaminochrome and others bypro-
ducts. This very fast degradation pathway has been de-
scribed on the basis of UV spectrophotometric studies
(Klegeris et al. 1995; Terland et al. 1997). In analogy, in
our experiments, the appearance of the peak at
l ¼ 458.5 nm at pH > 7 is indicative of the same base-
catalysed autoxidation mechanism for compound 7.
In p.o. drug administration, a further limiting step to bioa-
vailability could be permeation across the gastro-enteric
mucosal tissue. The Papp value of compound 7 suggested
that it has suitable characteristics to cross the intestinal
membrane (Scherrmann 2002; Balimane et al. 2000). In
this study, the aptitude of compound 7 to permeate
through a membrane simulating the intestinal barrier was
evaluated (Zhu et al. 2002). Diffusion of 7 through the
membrane was reported as cumulative amount of perme-
ated 7 per unit area vs time (Fig. 5). At the steady state
the flux (Js) and the permeability coefficient (Kp) do not
change significantly (t-test) with the pH and resulted
20.08 ꢂ 1.71 mg/cm2 h and 0.10 ꢂ 0.01 cm/h respectively.
3.2. Synthetic pathway
0.10
0.09
0.08
0.07
0.06
0.05
3.2.1. Synthesis of 2-(boc)amino-N-[2-(3,4-dimethoxy-phenyl)-ethyl]-
3-phenyl-propionamide (4)
To a dry CH2Cl2 solution (10 mL) of 1 (0.92 g, 0.005 mol) and 2 (1.33 g,
0.005 mol), DCC (1.25 g, 0.005 mol) was added as coupling agent. The
mixture was maintained at 0 ꢁC in an ice bath with constant stirring for
3 h. The reaction was monitored by TLC using petroleum ether/chloro-
form/ethyl acetate ¼ 10/65/25 as eluent. The mixture was then filtered to
separate the solid N,N0-dicyclohexylurea (3) formed from DCC. The fil-
tered solution was evaporated under reduced pressure. The resulting oily
mass was treated with anh. ice-cooled acetone (20 mL) to separate addi-
tional 3. The acetone solution was evaporated under reduced pressure and
the crude resulting mass was purified by crystallization from cyclohexane
followed by recrystallization from ethanol/water (1/5). Two recrystalliza-
tions were sufficient to obtain a pure sample of a product identified as
2-(boc)amino-N-[2-(3,4-dimethoxy-phenyl)-ethyl]-3-phenyl-propionamide
(4). Yield, m.p. and spectral data are given in Tables 1 and 2. MS: m/z (relative
abundance %) 428 (16, Mþ), 164 (100, (CH3O)2C6H3-CH2-CH2þ), 151 (20,
(CH3O)2C6H3-CH2þ), 120 (24, C6H5-CH2-CH-NH2þ), 104 (4, C6H5-CH2-
CHþ), 91 (23, C6H5-CH2þ), 77 (5, C6H5þ), 57 (13, ter-butylþ).
y = 0.028246 + 0.020879x R = 0.99853
0.04
0.03
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
time (h)
From cyclohexane mother liquors, after evaporation, a by-product was se-
parated which was recrystallized twice from ethyl acetate/petroleum ether
(1/3) and identified as [1-benzyl-2-(1,3-dicyclohexyl-ureido)-2-oxo-ethyl]-
carbamic acid tert-butyl ester (5).
Fig. 5: Cumulative amount (mg) of compound 7 permeated across simu-
lated duodenum/jejunum/ileum barrier per unit area versus time.
Values are presented as means ꢂ SD (n ¼ 5)
Yield, m.p. and spectral data are given in Table 1 and 2.
708
Pharmazie 63 (2008) 10

ORIGINAL ARTICLES
3.2.2. Hydrolysis of compound 4 to 2-amino-N-[2-(3,4-dimethoxy-phenyl)-
ethyl]-3-phenyl-propionamide (6)
37 ꢂ 0.5 ꢁC. At appropriate time intervals samples (25 ml) were taken,
quenched by adding the same volume of acetonitrile, and centrifuged
(15,000ꢃg, 10 min). The supernatant was analysed by HPLC as above
described. The experiments were carried out for 2 h and repeated three
times.
To a solution of TFA (5 mL) in CH2Cl2 (10 mL) were added 400 mg
(0.0009 mol) of compound 4. The mixture was allowed to react in an ice
bath for 4 h with constant stirring. The reaction was monitored by TLC
using ethyl acetate/methanol/chloroform ¼ 50/20/30 as eluent. At the end
of the reaction, to the mixture was added dropwise a saturated water solu-
tion of NaHCO3 until pH 8 was reached. The organic phase was separated
and treated with solid anh. Na2SO4 to eliminate water residues, filtered and
evaporated under reduced pressure. The residual solid mass was purified
by crystallization from cyclohexane. Two recrystallizations were sufficient
to obtain a pure sample of a product identified as 2-amino-N-[2-(3,4-di-
methoxy-phenyl)-ethyl]-3-phenyl-propionamide (6). Yield, m.p. and spec-
tral data are given in Tables 1 and 2.
3.4.3. Stability in buffer solutions simulating biological fluids
Chemical stability of compound 7 was studied at 37 ꢂ 0.5 ꢁC in 50 mM
sodium citrate/HCl (pH 1.2), phosphate (pH 6.5, 7.4, 8.0) buffers adjusted
to an ionic strength of m ¼ 0.5 by addition of a calculated amount of so-
dium chloride. Experiments were initiated by adding 15 mg of compound
7 in 100 mL of the appropriate buffer solution. In all experiments, to eval-
uate the peptide bond stability, an aliquot of solution was kept in a water
bath and every 15 min samples (50 ml) were withdrawn and immediately
analysed by HPLC, as above described. Moreover, to assess potential mo-
lecular rearrangements, a further aliquot of the solution (3 mL) was placed
in a quartz UV cell, maintained at constant temperature, and scanned every
15 min (l ranging from 200 to 800 nm). The experiments were carried out
for 6 h and repeated five times.
3.2.3. Demethylation of compound 6 to 2-amino-N-[2-(3,4-dihydroxy-
phenyl)-ethyl]-3-phenyl-propionamide (7)
To a dry CH2Cl2 (20 mL) solution of 6 (100 mg, 0.00030 mol) BBr3
(1.8 mL, 0.00180 mol) was added and the mixture was stirred for 2 h at
room temperature. Methanol (20 mL) was then added and the reaction set
aside at room temperature for further 2 h under constant stirring. The reaction
was monitored by TLC using a mixture of chloroform/methanol ¼ 80/20 as
eluent. At the end of the reaction, the mixture was evaporated under re-
duced pressure. To the crude mass was added excess methanol and the
solution was evaporated off under reduced pressure, to eliminate trimethyl-
borate formed. The residue was then heated to eliminate methyl bromide
and hydrobromic acid. The oily mass obtained was washed with ethyl
acetate (10 mL ꢃ 5) to obtain a solid product. The TLC showed only one
chromatographic spot. To the product was assigned the structure of 2-ami-
no-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (7) which
was obtained as hydrobromide salt. Yield, m.p. and spectral data are given in
Tables 1 and 2. MS: m/z (relative abundance %) 301 (100, M þ 1þ), 137 (22,
(HO)2C6H3-CH2-CH2þ), 123 (4, (HO)2C6H3-CH2þ), 120 (76, C6H5-CH2-CH-
NH2þ), 104 (11, C6H5-CH2-CHþ), 91 (16, C6H5-CH2þ), 77 (6, C6H5þ).
3.5. In vitro permeation of compound 7 through artificial intestinal bar-
rier
A solution of compound 7 (0.2 mg/mL in phosphate buffer pH 5.0, 6.0
and 7.0 solutions simulating duodenum/jejunum/ileum juices respectively)
was placed in the donor compartment of a horizontal Franz-type diffusion
cell. In the acceptor compartment was placed phosphate buffer pH 7.4 so-
lution simulating plasma. As artificial intestinal barrier, a filtration nitrate
cellulose membrane (Whatman, International Ltd. Maidstone, England),
wetted with egg lecithin dispersed in n-dodecane (1% w/v), was used (Zhu
et al. 2002). At regular time intervals (10 min), samples were withdrawn
from the acceptor compartment. To avoid saturation phenomena and main-
tain the “sink” conditions, the sample volume taken out was replaced by
fresh fluid. Transfer of 7 from the donor to the acceptor compartment was
monitored by HPLC measurements of the amount of 7 that reached the
acceptor fluid. The experiments were carried out for 4 h and repeated five
times. The flux value (Js) and the permeability coefficient (Kp) were calcu-
lated at the steady state per unit area by linear regression analysis of per-
meation data (Giannola et al. 2007).
3.3. Determination of apparent partition coefficient (Papp) of compound 7
Apparent partition coefficient (Papp) of compound 7 (2.5 mg) was determined,
at 20 ꢁC and at pressure of 1.013 ꢃ 105 Pa, in n-octanol (20 mL)/0.02 M
phosphate buffer pH 7.4 solution (20 mL) and expressed as Log Papp. Follow-
ing 5 min shaking at time intervals of 10 min for 3 h in a separator funnel,
the mixture reached equilibrium. After partition, the organic phase was
separated from the aqueous one, dried with anh. solid Na2SO4 and filtered.
The concentration of 7 in the octanol and aqueous layers was determined
by UV spectrophotometric analysis using the appropriate calibration curve
and blank. The calibration curves were performed at the concentration
range 2 to 12 mg/100 mL in both media. Papp was calculated according to
equation:
3.6. Statistical analysis
The statistical significance was determined by Student’s t-test. A probabil-
ity level of 0.05 or smaller was used to indicate statistical significance.
Acknowledgement: The authors wish to thank the MIUR, Rome, for finan-
cial support.
ꢀ
ꢁ
Ci ꢀ Cw Vw
Papp
¼
References
Cw
Vo
Abraham MH, Chada S, Mitchell R (1994) Hydrogen bonding. 33 Factors
that influence the distribution of solutes between blood and brain. J
Pharm Sci 83: 1257–1268.
Anderson BD (1996) Prodrugs for improved CNS delivery. Adv Drug De-
liv Rev 19: 171–202.
where Ci and Cw represent the drug concentration before and after parti-
tion, respectively. Vw and Vo are the volumes of the aqueous and the oil
phase. The experiments were repeated five times.
Antolin I, Mayo JC, Sainz RM, de los Angeles del Brio M, Herrera F,
Martin V, Rodriguez C (2002) Protective effect of melatonin in a
chronic experimental model of Parkinson’s disease. Brain Res 943:
163–173.
Balimane PV, Chong S, Morrison RA (2000) Current methodologies used
for evaluation of intestinal permeability and absorption. J Pharmacol
Toxicol Meth 44: 301–312.
Ballabh P, Braun A, Nedergaard M (2004) The blood-brain barrier: an
overview. Structure, regulation, and clinical implications. Neurobiol Dis-
ease 16: 1–13.
Bickel U, Yoshikawa T, Pardridge WM (2001) Delivery of peptides and
proteins through the blood-brain barrier. Adv Drug Deliv Rev 46: 247–
279.
Bodor N, Farag HH, Brewster ME (1981) Site-specific, sustained release
of drugs to the brain. Science 214: 1370–1372.
Bodor N, Buchwald P (1999) Recent advances in the brain targeting of
neuropharmaceuticals by chemical delivery systems. Adv Drug Deliv
Rev 3: 229–254.
Borgman RJ, McPhillips JJ, Stitzel RE, Goodman IJ (1973) Synthesis and
pharmacology of centrally acting dopamine derivatives and analogs in
relation to Parkinson’s disease. J Med Chem 16: 630–633.
Casagrande C, Ferrari G (1973) 3,4–O-Diacyl derivatives of dopamine.
Farmaco 28: 143–148.
3.4. Stability of compound 7
3.4.1. Stability in rat brain homogenate
Sprague-Dawley rats (Stefano Morini, San Polo d’Enza, Italy) weighing
300–350 g were housed in our institutional animal care facility under di-
rection of a licensed veterinary. Procedures were conducted according to
local and international guidelines on the ethical use of animals. Rats were
sacrificed by decapitation and the brains were immediately removed,
weighed and homogenized in buffer solution (nuclei buffer containing:
50 mM Na2HPO4, 50 mM NaH2PO4, 50 mM KCl, 0.15 mM spermine,
0.5 mM spermidine, 2 mM EDTA, 0.5 mM EGTA, 0.32 M sucrose). The
homogenate was centrifuged at 60,000 ꢃ g for 40 min at 0 ꢁC and the sur-
natant was subdivided in aliquots of 400 ml which were stored at ꢀ20 ꢁC
until used. Experiments started by adding 50 ml of stock solution of com-
pound 7 (2.075 mg/mL in phosphate buffer pH 6.5 solution) to a 400 ml
aliquot of brain homogenate previously equilibrated at 37 ꢂ 0.5 ꢁC. At
appropriate time intervals, samples (25 ml) were withdrawn and, after
quenching the reaction by adding 20 ml of acetonitrile, were analyzed by
HPLC as above described. The experiments were carried out for 3 h and
repeated three times.
3.4.2. Stability in human plasma
The stability of compound 7 was investigated in 80% human plasma di-
luted in PBS pH 7.4. The reaction was initiated by adding 100 ml of stock
solution of 7 (2.05 mg/mL in isotonic buffer pH 7.4) to 800 ml of pre-
heated (37 ꢂ 0.5 ꢁC) plasma. The mixture was kept in a water bath at
Chen HJC, Applewhite T, Jayachandran B, Kirk KL (1998) Synthesis of
4,6-difluoro-5-hydroxy-(a-methyl)tryptamine and 4,6-difluoro-5-hydro-
xy-(b-methyl)tryptamine as potential selective monoamine oxidase B in-
hibitor. J Fluorine Chem 92: 41–44.
Pharmazie 63 (2008) 10
709

ORIGINAL ARTICLES
Crivori P, Cruciani G, Carrupt PA, Testa B (2000) Predicting blood-brain
barrier permeation from three-dimensional molecular structure. J Med
Chem 43: 2204–2216.
Di Stefano A, Sozio P, Cerasa LS (2008) Antipakinson prodrugs. Mole-
cules 13: 46–68.
Pahwa R, Koller WC (1998) Advances in the treatment of Parkinson’s
disease. Drugs Today 34: 95–105.
Pardridge WM (1977) Kinetics of competitive inhibition of a neutral ami-
no acid transport across the blood-brain barrier. J Neurochem 28: 103–
108.
Egleton RD, Davis TP (1997) Bioavailability and transport of peptides and
peptide drugs into the brain. Peptides 18: 1431–1439.
Ferna`ndez C, Nieto O, Rivas E, Montenegro G, Fontenla JA, Ferna`ndez-
Mayoralas A (2000) Synthesis and biological studies of glycosyl dopa-
mine derivatives as potential antiparkinsonian agents. Carbohydr Res
327: 353–365.
Ferna´ndez C, Nieto O, Fontenla JA, Rivas E, de Ceballos ML, Ferna´ndez-
Mayoralas A (2003) Synthesis of glycosyl derivatives as dopamine pro-
drugs: interaction with glucose carrier GLUT-1. Org Biomol Chem 1:
767–771.
Pardridge WM (1995) Transport of small molecules through the blood-
brain barrier: biology and methodology. Adv Drug Deliv Rev 15: 5–36.
Ragot JP, Steeneck C, Alcaraz ML, Taylor RJK (1999) The synthesis of
1,8-dihydroxtnaphtalene-derived natural products: palmarumycin CP1,
palmarumycin CP2, palmarumycin C11, CJ-12,371, deoxypreussomerin
A and novel analogues. J Chem Soc, Perkin Trans I, 1073–1082.
Reynolds JEF (1993) Martindale, The Extra Pharmacopoeia, 30th Ed, Phar-
maceutical Press, London, UK, p. 844.
Scherrmann JM (2002) Drug delivery to brain via the blood-brain barrier.
Vasc Pharmacol 38: 349–354.
Ghersi-Egea JF, Leninger-Muller B, Suleman G, Siest G, Minn A (1994)
Localization of drug-metabolizing enzyme activities to blood-brain inter-
faces and circumventricular organs. J Neurochem 62: 1089–1096.
Giannola LI, De Caro V, Giandalia G, Siragusa MG, Campisi G, Florena
AM, Ciach T (2007) Diffusion of naltrexone across reconstituted human
oral epithelium and histomorphological features. Eur J Pharm Biopharm
65: 238–246.
Gimenez F, Fernandez C, Mabondzo A (2004) Transport of HIV protease
inhibitors through the blood-brain barrier and interactions with the ef-
flux proteins, P-glycoprotein and multidrug resistance proteins. J Acquir
Immune Defic Syndr 36: 649–658.
Slebioda M, Wodecki Z, Kolodziejczyk AM (1990) Formation of optically
pure N-acyl-N,N0-dicyclohexylurea in N,N0-dicyclohexyl-carbodiimide-
mediated peptide synthesis. Int J Pept Protein Res 35: 539–541.
Standaert DG, Young AB (1996) Treatment of central nervous system de-
generative disorders. In: Hardman JG, Limbird LE, Molinoff PB, Rud-
don RW, Gilman AG (eds) Goodman & Gilman’s The Pharmacological
Basis of Therapeutics, 9th ed., McGraw-Hill Companies, New York,
p. 503–519
Terasaki T, Tsuji A (1994) Drug delivery to the brain utilizing blood-brain
barrier transport systems. J Control Release 29: 163–169.
Terland O, Flatmark T, Tangera˚s A, Gronberg M (1997) Dopamine oxida-
tion generates an oxidative stress mediated by dopamine semiquinone
and unrelated to reactive oxygen species. J Mol Cell Cardiol 29: 1731–
1738.
Huber JD, Egleton RD, Davis TP (2001) Molecular physiology and patho-
physiology of tight junctions in the blood-brain barrier. Trends Neurosci
24: 719–725.
Klegeris A, Korkina LG, Greenfield SA (1995) Autoxidation of dopamine:
a comparison of luminescent and spectrophotometric detection in basic
solution. Free Radic Biol Medic 18: 215–222.
Tolosa E, Marti MJ, Valldeoriola F, Molinuevo JL (1998) History of levo-
dopa and dopamine agonists in Parkinson’s disease treatment. Neurology
50: S2–S10.
Levin VA (1980) Relationship of octanol/water partition coefficient and
molecular weight to rat brain capillary permeability. J Med Chem 23:
682–684.
White HS (2000) Skeletal muscle relaxants. In: Gennaro AR (ed.) Reming-
ton: The Science and Practice of Pharmacy, 20th Ed., Lippincott Wil-
liams and Wilkins, Philadelphia, p. 1341–1342.
Lo EH, Singhal AB, Torchilin VP, Abbott NJ (2001) Drug delivery to
damage brain. Brain Res Rev 38: 140–148.
Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier:
development, composition and regulation. Vasc Pharmacol 38: 323–337.
Zhu C, Jiang L, Chen TM, Hwang KK (2002) A comparative study of
artificial membrane permeability assay for high throughput profiling of
drug absorption potential. Eur J Med Chem 37: 399–407.
McOmie JFW, Watts ML, West DE (1968) Demethylation of aryl methyl
ethers by boron tribromide. Tetrahedron Lett 24: 2287–2292.
Oldendorf WH, Crane PD, Braun LD, Wade LA, Diamond JM (1983)
Blood-Brain Barrier transport of basic amino acids is selectively inhib-
ited at low pH. J Neurochem 40: 797–800.
710
Pharmazie 63 (2008) 10