Synthesis and biological studies of glycosyl dopamine derivatives as potential antiparkinsonian agents

Article abstract of DOI:10.1016/S0008-6215(00)00073-2

A new approach to deliver dopamine into the central nervous system, based on the use of D-glucose as transportable agent, has been studied. Glycosyl dopamine derivatives bearing the sugar moiety linked to either the amino group or the catechol ring of dopamine through amide, ester or glycosidic bonds were synthesised as potential antiparkinsonian agents. Studies on the binding to dopamine D₂ receptor, in vitro stability, and locomotive effect in mice of the synthetic glycoconjugates are reported. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00073-2

www.elsevier.nl/locate/carres
Carbohydrate Research 327 (2000) 353–365
Synthesis and biological studies of glycosyl dopamine
derivatives as potential antiparkinsonian agents
Caridad Ferna´ndez a, Ofelia Nieto a, Emilia Rivas b, Gisela Montenegro b,
Jose´ A. Fontenla b, Alfonso Ferna´ndez-Mayoralas a,*
a Instituto de Qu´ımica Orga´nica General, CSIC, Juan de la Cier6a 3, E-28006 Madrid, Spain
b Departamento de Farmacolog´ıa, Facultad de Farmacia, Uni6ersidad de Santiago de Compostela,
E-15706 Santiago de Compostela, Spain
Received 26 January 2000; accepted 28 February 2000
Abstract
A new approach to deliver dopamine into the central nervous system, based on the use of
D
-glucose as
transportable agent, has been studied. Glycosyl dopamine derivatives bearing the sugar moiety linked to either the
amino group or the catechol ring of dopamine through amide, ester or glycosidic bonds were synthesised as potential
antiparkinsonian agents. Studies on the binding to dopamine D2 receptor, in vitro stability, and locomotive effect in
mice of the synthetic glycoconjugates are reported. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Glycosides; Neurologically active compounds; Neurotransmitter; Glucose carrier
1. Introduction
with levodopa, a variety of problems may
appear. These limitations have stimulated the
Parkinson’s disease is a progressive disorder
search for new strategies [3,4]. One of these
of the central nervous system (CNS) [1,2],
may rely on dopamine-derivatives able to pen-
whose major symptoms are stiffness of the
etrate the BBB, by making use of specific
muscles, difficulty in moving, and tremors. In
transport systems. It is known that glucose —
biochemical terms, the disease is characterised
the brain’s source of energy — and other
by a reduced concentration of the neurotrans-
hexoses are transported across the BBB by the
mitter, dopamine in the brain. A possible ther-
glucose carrier GLUT-1 [5,6]. This proteic
apy based on the use of dopamine itself is
transporter is located in the membrane of
hampered by the fact that this substance is
brain capillary endothelial cells composing the
unable to cross the blood brain barrier (BBB).
BBB. Thus, one could envisage a new ap-
The usual treatment of this disorder is the use
proach to deliver dopamine into the CNS by
linking the neurotransmitter to a sugar
molecule so that the resulting glycoconjugate
may cross the BBB using this carrier-mediated
transport [7]. Upon reaching the CNS, the
prodrug should be enzymatically cleaved to
release the active compound (Fig. 1). Some
authors have studied this approach in peptide
drugs [8] and cytotoxic agents [9,10] delivery
into the CNS.
of levodopa, a dopamine precursor, which
reverses symptoms. Levodopa enters the CNS
trough active transport and is enzymatically
cleaved in the brain to release dopamine. In
fact, levodopa is actually a prodrug for do-
pamine. However, during chronic treatment
* Corresponding author.
E-mail address: iqofm68@fresno.csic.es (A. Ferna´ndez-
Mayoralas).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00073-2

354
C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
2. Results and discussion
Chemical synthesis.—The glycosyl dopa-
mine derivatives were obtained in short and
effective syntheses involving 4–7 steps from
dopamine. The synthesis of 1–3 (Scheme 2)
was carried out from a common succinamic
acid derivative 8, which was coupled with the
corresponding substituted monosaccharide.
Previously, 8 was obtained from the N-Boc
protected dopamine 6 [11] by benzylation of
the phenolic groups, followed by removal of
the Boc group, to give amine 7, and subse-
quent treatment with succinic anhydride. The
glucose and galactose derivatives with a free
hydroxyl group at C-6 (intermediates 10 and
12, respectively) were easily obtained by
Fig. 1. Schematic representation of the new strategy to deliver
dopamine into the CNS.
Following this strategy, we have synthesised
several glycosyl derivatives of dopamine as
potential antiparkinsonian agents. Sugar and
neurotransmitter are linked through ester,
amide and glycosidic bonds. These linkages
can be cleaved enzymatically in the brain tis-
sue to release dopamine. A series of com-
pounds presents the amino group of dopamine
linked to the C-6 (1 and 2, Scheme 1) or
C-1(b) (3) position of the sugar through a
succinyl linker. In another series, the sugar is
attached to the phenolic groups of dopamine
through a glycosidic bond (4 and 5). We re-
port herein the synthesis, in vitro stability
studies, binding to dopamine D2 receptor,
and behavioural activity, in an experimental
model of Parkinson’s disease, of compounds 1
to 5.
trimethylsilylation of benzyl b-D-glucopyrano-
side (9) and -galactose, and selective
D
methanolysis of the primary silyl group. The
methanolysis was first tried under basic condi-
tions (K2CO3–MeOH) [12] giving a mixture of
partially silylated products. A cleaner reac-
tion, however, was obtained when the persily-
lated
compounds
were
treated
with
AcOH–MeOH [9]. Esterification of 10 with
acid 8 in the presence of DCC and subsequent
removal of the silyl groups gave 11, which
after hydrogenolysis led to target 1. Similar
transformations on 12 furnished the galactose-
containing product 2. The glycosyl dopamine
derivative 3 was obtained by reaction of 8
Scheme 1.

C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
355
Scheme 2. Reagents and conditions. (a) BnBr, K2CO3, DMF, 60 °C, 24 h; (b) 5% CF3COOH, CH2Cl2, rt, 5 h, 81% two steps; (c)
succinic anhydride, Py, rt, 3 h, 94%; (d) (Me)3SiCl, HMDS, rt, overnight, 74%; (e) 5% AcOH, MeOH–acetone, 0 °C, 1 h, 75%;
(f) (1) 8, DCC, DMAP, CH2Cl2, rt, 4 h; (2) 2% CF3COOH, CH3NO2, 0 °C, 1 h, 69% for 11, 78% for 13; (g) H2, MeOH, PdꢀC,
rt, 1 h, 99%; (h) 8, CH2Cl2, 5% DMF, Ar, rt, 5 days, 40%; (i) H2, MeOH–AcOEt, PdꢀC, rt, 3 h, 99%.
with a-trichloroacetimidate 14 [13] giving the
b-glucopyranosyl ester 15 in a stereospecific
manner, followed by debenzylation.
Glycosides 4 and 5 were obtained from the
N-protected dopamine 16 (Scheme 3). The
reaction of 16 with glucose peracetate 17 pro-
moted by BF3·OEt2, gave glycosylation at
HO-3 (18) and HO-4 (19) of the catechol ring
in a 3:1 ratio. Deprotection steps on 18 and
19, separately, afforded 4 and 5, respectively
as their trifluoroacetate salts.
Binding to dopamine D2 receptor.—To test
for the possibility that the intact glycoconju-
gates might be pharmacologically active, the
affinity of compounds 1–5 for dopamine D2
receptor was evaluated. None of the glycosyl
dopamine derivatives inhibited the binding of
3H-spiperone to D2 receptor, showing pKi val-

356
C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
the TM3 aspartic acid and TM5 serine, re-
spectively, of the receptor [14].
Stability studies.—In order to determine the
stability and ability to deliver dopamine after
brain uptake, compounds 1, 4, and 5 were
incubated in the presence of specific enzymes,
rat plasma, and rat brain extracts. The pre-
sumed activation pathways for 1, 4, and 5 are
outlined in Scheme 4. The degradation route
for 1 involves the formation of the acid inter-
mediate 20 through an esterase-catalysed hy-
drolysis, and subsequent cleavage of the amide
bond, by amidase or protease activation, to
give dopamine. On the other hand, glycosides
4 and 5 should give rise to the neurotransmit-
ter through an activation mechanism catalysed
by glucosidase.
The progress of the incubations were moni-
tored by high-performance liquid chromatog-
raphy (HPLC; UV detector, u=280 nm)
using a reverse-phase column. Table 1 reports
the half-life (t1/2) of these compounds in the
different incubation media, together with that
of dopamine. Compound 1 was highly stable
in water, where the half-life exceeded 15 days.
In the presence of esterase (60 units/mL) and
protease (30 units/mL) 1 underwent hydrolysis
(t1/2=7 and 43 h, respectively). In plasma, 1
showed a moderate stability (t1/2=3 h); how-
ever, it slowly decomposed in brain extracts.
In all the incubation media, the degradation
of glycosylꢀsuccinyl dopamine 1 led to the
formation of the expected acid derivative 20.
Scheme 3. Reagents and conditions. (a) BF3·OEt2, CH2Cl2,
Ar, rt, 4 h, 18% for 18 and 54% for 19. (b) 0.1 M NaMeO–
MeOH, rt, 1.5 h, 88% for 18, 97% for 19; (c) H2, CF3COOH
(1.6 equiv), MeOH, PdꢀC, rt, 1 h, 99%.
ues below 5. This result is in agreement with
the generally accepted assumption that the
amino and m-hydroxyl groups of the do-
pamine are essential for the interaction with
Scheme 4.

C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
357
Table 1
tively). In this medium, the amount of re-
leased dopamine during the incubation of 4
and 5 was low and constant, probably due to
the slow activation of the prodrugs as com-
pared to the degradation rate of dopamine
(t1/2 of dopamine in brain extract, 3 h).
Half-life of compounds 1, 4, 5, and dopamine in different
incubation media
Incubation
mean
Half-lives (h)
1
4
5
Dopamine
In 6i6o acti6ity.—Compounds 1–5 were
tested for their ability to induce a recovery of
motor activity in reserpinized mice (1 h). They
were administered intraperitoneally at a dose
of 10 and 100 mg/kg, and amphetamine (2.5
mg/kg, i.p.) was used as the reference drug.
The results for locomotive activity (cm/h) for
the 100 mg/kg dose are shown in Fig. 2. While
amphetamine increased the locomotive activ-
ity in mice (5842.149949.77, PB0.01), some
glycosyl derivatives caused a slight modifica-
tion during the hour following administration;
however, this was not significant enough as to
exhibit antiparkinsonian properties. Several
factors can be responsible for the lack of in
vivo activity of the glycosyl dopamine deriva-
tives. One of them may be the inability of the
compounds to cross the BBB. Also, their slow
bioconversion into dopamine, as it has been
observed during in vitro experiments, indi-
cates poor properties of the compounds as
prodrugs. Studies about the affinity of the
glycosyl derivatives for the glucose carrier
GLUT-1, and the synthesis of new prodrugs
with modified linkages that may undergo
faster enzyme catalysed hydrolysis, are cur-
rently in progress.
Water
\144
\144 \144
27
–
–
Esterease a
Protease b
b-Glucosidase c
Plasma
7
43
–
–
–
2
–
–
1.5
–
3
\144 \144
2
Brain extract
21 d
132 e 67 e
3 e
a From bovine liver (60 units/mL).
b a-Chymotrypsin from bovine pancreas (30 units/mL).
c From almonds (20 units/mL).
d Rat brain extract soluble fractions.
e Rat brain homogenate, prepared following Ref. [18].
Fig. 2. Locomotor activity of compounds 1–5.
This intermediate accumulated during the in-
cubation but no appreciable release of do-
pamine was observed for the time that all 1
was consumed. The amide bond in 20 and in 1
was found to be highly resistant under the
assayed conditions. Other prodrugs described
in the literature met with highly stable amide
bonds under in vitro conditions [15,16].
Glycosides 4 and 5 released dopamine in the
presence of b-glucosidase from almonds (20
units/mL). However, they were very stable in
plasma, as was expected, due to the low levels
of b-glucosidase found in the blood [17]. In
brain extract with glucosidase activity, as de-
termined following a described procedure [18],
glycosides 4 and 5 were slowly hydrolysed,
showing differences in their rates of hydrolysis
(t1/2 for 4 and 5, 5.6 and 2.8 days, respec-
3. Experimental
General methods.—THF was distilled from
Na/benzophenone and CH2Cl2 from CaH2,
immediately prior to use. N,N-Dimethylfor-
mamide was distilled over CaH2 and was kept
,
over molecular sieves (4 A) under argon. Un-
less stated otherwise, materials were obtained
from commercial sources (Aldrich, Sigma,
Fluka) and used without further purification.
All reactions were monitored by thin-layer
chromatography (TLC), carried out on 0.20
mm E. Merck pre-coated Silica Gel 60 F-254
plates by using UV light (254/365 nm) and
10% H2SO4 in EtOH or a soln of ammonium
molybdate and ceric ammonium sulfate in wa

358
C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
3,4-Dibenzyloxyphenylethylamoniun trifluoro-
acetate (7).—To a mixture of 6 (6.15 g, 24.2
mmol) in anhyd DMF (40 mL) containing
K2CO3 (21.36 g, 217.8 mmol) was added benzyl
bromide dropwise (7.20 mL, 60.53 mmol). The
reaction was stirred at room temperature (rt),
under argon for 24 h. After this time, the mixture
was filtered through Celite and the solid washed
with Et2O (30 mL). The combined filtrate and
washings were washed with ice-water (4×25
mL), an aq soln of 1 N HCl (3×15 mL) and
brine. The organic layer was dried (Na2SO4) and
concentrated to give a residue which was
purified by flash chromatography. Elution with
7:1“3:1 hexane–EtOAc afforded N-tert-
butoxycarbonyl - 3,4 - dibenzyloxyphenylethyl-
amine (8.92 g, 85%) as a white solid; mp
106–108 °C; Rf 0.52 (3:1:0.05 hexane–EtOAc–
ter–H2SO4 5% as developing agent. E. Merck
Silica Gel 60 (230–400 mesh) was used for flash
columnchromatography. AnalyticalHPLCwas
carried out with a Water 600E multisolvent
delivery system equipped with a reverse-phase
(C18) column (Lichrospher 100RP-18, 14.5 cm,
5 mm, E. Merck), using as eluent, different
mixtures of MeCN, MeOH and trifluoroacetic
acid 5% in water, with a UV detector (Waters
484) at 280 nm. Melting points were measured
with a Reicher Jung Thermovar microscoper
and are uncorrected. Optical rotations were
determined with a Perkin–Elmer 241-MC po-
1
larimeter. H and 13C NMR spectra were
recorded in CDCl3 or CD3OD on a Varian
Gemini 200, a Bruker AMX-200, a Varian
INOVA 300 or a Varian 400 spectrometer.
Chemical shifts are given in ppm (l). Micro-
analyseswereperformedwithanHeraeusCHN-
O analyzer.
1
AcOH); H NMR (200 MHz, CDCl3): l 7.39
(m, 10 H, aromatics), 6.89 (d, 1 H, J 1.7 Hz,
H-2 Ar), 6.71 (dd, J 8.0 and 1.7 Hz, H-6 Ar),
5.15 (s, 4 H, 2 CH2ꢀPh), 4.45 (s, 1 H, NH), 3.32
(m, 2 H, CH2ꢀNH), 2.70 (t, 2 H, J 6.9 Hz,
CH2ꢀAr), 1.45 (s, 9 H, C(CH3)3); 13C NMR (50
MHz, CDCl3): l 155.8 (ꢀCOꢀ), 149.1 (C-3 Ar),
147.8 (C-4 Ar), 137.5 and 137.3 (C ipso Bn),
132.5 (C-1 Ar), 128.5–127.3 (10 C aromatics),
121.7 (C-6 Ar), 116.0 and 115.6 (C-5 and C-2
Ar), 79.2 (C(CH3)3), 71.6 and 71.5 (CH2ꢀPh),
41.8 (CH2ꢀNH), 35.7 (CH2ꢀAr), 28.4
(C(CH3)3); Anal. Calcd for C27H31NO4: C,
74.80; H, 7.21; N, 3.14. Found C, 74.58; H, 7.25;
N, 3.23.
N-tert-Butoxycarbonyl-3,4-dihydroxiphenyl-
ethylamine (6).—To a solution of dopamine
hydrochloride (5 g, 26.4 mmol) in Et3N and
MeOH (1:9, 143 mL) was added di-tert-butyldi-
carbonate (6.331 g, 29 mmol) and the mixture
was stirred at 40–50 °C for 30 min. After
tert-butoxycarbonylation was complete (as ev-
idenced by TLC using 3:1:0.5 hexane–EtOAc–
MeOH), stirring was continued for 30 min more,
the solvent was then evaporated under reduced
pressure, and the residue treated with ice-cold
dilute HCl (pH 2.15, 7×10−3 M) for 10 min
and extracted immediately with EtOAc. The
organic layer was dried (MgSO4), then filtered,
and concentrated. The residue was purified by
flash chromatography (4:1:0.5“2:1:0.5 hex-
ane–EtOAc–MeOH) to yield 6 as a solid (6.15
g, 92%); mp 136–138 °C; Rf 0.36 (2:1:0.15
hexane–EtOAc–AcOH); 1H NMR (200 MHz,
CD3OD): l 6.86 (d, J 8.0 Hz, 1 H, H-5 Ar), 6.82
(d, J 2.0 Hz, 1 H, H-2 Ar), 6.70 (dd, 1 H, J 1.9
and 8.0 Hz, H-6 Ar), 3.37 (t, 2 H, J 7.5 Hz,
CH2ꢀNH), 2.77 (t, 2 H, J 7.5 Hz, CH2ꢀAr), 1.61
(s, 9 H, C(CH3)3); 13C NMR (50 MHz, CD3OD):
l 158.4 (ꢀCOꢀ), 146.2 (C-3 Ar), 144.65 (C-4 Ar),
132.3 (C-1 Ar), 121.1 (C-6 Ar), 116.9 and 116.4
(C-2, C-5 Ar), 80.0 (C(CH3)3), 43.3 (CH2ꢀNH),
36.6 (CH2ꢀAr), 28.8 (C(CH3)3); Anal. Calcd for
C13H19NO4: C, 61.63; H, 7.56; N, 5.53. Found:
C, 61.20; H, 7.55; N, 5.39.
This compound (7.34 g, 16.95 mmol) was
treated with 5% TFA in CH2Cl2 (190 mL) at
rt for 5 h. After this time, the solvent was
removed under vacuum (co-evaporation with
toluene) to obtain 7 as a white solid (8.48 g,
95%); mp 159–162 °C; Rf 0.32 (5:1 CH2Cl2–
1
MeOH); H NMR (300 MHz, CD3OD): l
7.65–7.47 (m, 10 H, aromatics), 7.20 (d, 1 H,
J 8.2 Hz, H-5 Ar), 7.14 (d, 1 H, J 2.1 Hz, H-2
Ar), 7.00 (dd, 1 H, J 8.2 and 2.1 Hz, H-6 Ar),
5.32 and 5.30 (2s, 4 H, CH2ꢀPh), 3.30 (t, 2 H,
J 7.6 Hz, CH2ꢀNH), 3.04 (t, 2 H, J 7.6
Hz, CH2ꢀAr); 13C NMR (50 MHz, CD3OD):
l 150.6 (C-3 Ar), 149.6 (C-4 Ar), 138.7 and
138.6 (C ipso Bn), 131.3 (C-1 Ar), 129.4–
128.6 (10 C, aromatics), 122.9 (C-6 Ar), 117.1
and 117.0 (C-2 and C-5 Ar), 72.52 (CH2ꢀPh),

C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
359
11.8 Hz, CH2ꢀPh), 4.36 (d, 1 H, J1,2 7.32 Hz,
H-1), 3.76 (m, 1 H, H-6%), 3.58 (m, 1 H,
H-6), 3.44–3.32 (m, 3 H, H-2,-3,-4), 3.24 (m,
1 H, H-5), 0.18–0.10 (27 H, SiꢀCH3); 13C
NMR (50 MHz, CDCl3): l 128.3 (2 C aro-
matics), 128.1 (2 C aromatics), 127.8 (C ipso
Bn), 103.0 (C-1), 78.1–71.7 (C-2,-3,-4,-5),
71.7 (CH2ꢀPh), 62.3 (C-6), 1.3, 1.1 and 0.9 (9
C, SiꢀCH3).
41.95 (CH2ꢀNH), 34.04 (CH2ꢀAr); Anal.
Calcd for C24H24F3NO4: C, 64.42; H, 5.41; N,
3.13. Found: C, 64.21; H, 5.18; N, 3.24.
N-(3,4-Dibenzyloxyphenylethyl)succinamic
acid (8).—A solution of 7 (1 g, 2.706 mmol)
in pyridine (1.08 mL) was treated with suc-
cinic anhydride (542 mg, 5.412 mmol). The
reaction mixture was stirred at rt for 3 h,
then the solvent was co-evaporated with
toluene, and the residue was purified by flash
chromatography (15:1“10:1“5:1 CH2Cl2–
MeOH) to give 8 as a white solid (1.103 g,
94%); mp 160–163 °C; Rf 0.40 (10:1 CH2Cl2–
Benzyl 6-O-[N-(3,4-dibenzyloxyphenylethyl)-
succinamyl]-i-
D-glucopyranoside
(11).—A
stirred solution of acid 8 (53 mg, 0.122
mmol) in dry CH2Cl2 (1.52 mL) was treated
with compound 10 (54 mg, 0.111 mmol),
DCC (26.3 mg, 0.128 mmol) and DMAP (1
mg, 0.008 mmol). The mixture was kept at rt
for 3 h and then filtered through Celite. The
filtrate was diluted with CH2Cl2 and washed
with an aq soln of 5% AcOH and water,
dried (Na2SO4) and concentrated. The residue
was treated with a 2% soln of TFA in ni-
tromethane (5.24 mL) for 1 h at 0 °C and
then NaHCO3 (178 mg) was added. The reac-
tion mixture was filtered and concentrated.
The residue was purified by flash chromatog-
raphy (50:1“30:1 CH2Cl2–MeOH) to give
11 as a solid (76 mg, 69%); mp 46–49 °C;
[h]2D0 −24.4° (c 1, CHCl3); Rf 0.52 (9:1
CH2Cl2–MeOH); 1H NMR (300 MHz,
CDCl3): l 7.25–7.45 (m, 15 H, aromatics),
6.87 (d, 1 H, J 8.2 Hz, H-5 Ar), 6.77 (d, 1 H,
J 1.9 Hz, H-2 Ar), 6.67 (dd, 1 H, J 8.2 and
1.9 Hz, H-6 Ar), 5.67 (m, 1 H, NH), 4.89 (d,
1 H, J 11.7 Hz, CH2ꢀPh), 4.59 (d, 1 H, J 5.6
Hz, CH2ꢀPh), 4.36 (d, 1 H, J 7.3 Hz, H-1),
4.53 (dd, 1 H, J6,6% 12.0 Hz, J5,6 4.1 Hz, H-6),
4.24 (dd, 1 H, J6,6% 12.0 Hz, J5,6% 1.7 Hz,
H-6%), 3.36–3.54 (m, 6 H, H-2,-3,-4,-5,
CH2ꢀNH), 2.58–2.67 (m, 4 H, CH2 succ.),
2.35 (m, 2 H, CH2ꢀAr); 13C NMR (50 MHz,
CDCl3): l 173.2 (ꢀCONHꢀ), 171.7 (ꢀCOOꢀ),
137.3, 137.2 and 137.0 (3 C ipso Bn), 149.0
and 147.7 (C-3,-4 Ar), 132.1 (C-2 Ar), 127.3–
128.5 (15 C aromatics), 121.63 (C-6 Ar),
115.8 and 115.5 (C-2,-5 Ar), 101.8 (C-1),
76.1, 73.8, 73.7, 69.6 (C-2,-3,-4,-5), 71.5, 71.3
and 71.1 (CH2ꢀPh), 63.4 (C-6), 40.7
(CH2ꢀNH), 34.9 (CH2ꢀAr), 30.8 and 29.4
(CH2 succ.). Anal. Calcd for C39H43NO10: C,
68.31; H, 6.33; N, 2.04. Found: C, 68.05; H,
6.20; N, 1.92.
1
MeOH); H NMR (200 MHz, CDCl3): l 7.39
(m, 10 H, aromatics), 6.89 (d, 1 H, J 8.1 Hz,
H-5 Ar), 6.80 (d, 1 H, J 2.0 Hz, H-2 Ar),
6.71 (dd, 1 H, J 8.1 and 2.0 Hz, H-6 Ar),
5.55 (s, 1 H, NH), 5.17 and 5.15 (2s, 4 H, 2
CH2ꢀPh), 3.45 (m, 2 H, CH2ꢀNH), 2.70 (t, 2
H, J 6.8 Hz, CH2ꢀAr), 2.65 (t, 2 H, J 6.8 Hz,
CH2 succ.), 2.38 (t, 2 H, J 6.8 Hz, CH2
succ.); Anal. Calcd for C26H27NO5: C, 72.04;
H, 6.26; N, 3.23. Found: C, 71.75; H, 6.19;
N, 3.06.
Benzyl 2,3,4-tri-O-trimethylsilyl-i- -gluco-
D
pyranoside (10).—A mixture of chloro-
trimethylsilane (1.410 mL, 11.1 mmol) and
hexamethyl disilazane (0.78 mL, 3.70 mmol)
was carefully added to a vigorously stirred
solution of benzyl b- -glucopyranoside (9,
D
500 mg, 1.85 mmol) in pyridine (8 mL) at
0 °C. The temperature was slowly raised to
20 °C and the solution was stirred overnight.
Solvent and excess reagents were evaporated
under reduced pressure. The resulting white
solid was dissolved in pentane, washed with
water, dried (MgSO4) and concentrated to
afford benzyl 2,3,4,6-tetra-O-trimethylsilyl-b-
D-glucopyranoside (764 mg, 74%) as a syrup.
To a solution of this compound (737 mg) in
acetone (3 mL) were added MeOH (4.10 mL)
and AcOH (0.15 mL), and the mixture was
stirred for 2 h at 0 °C. After the addition of
solid NaHCO3 (242 mg), the mixture was
concentrated and the residue was purified by
flash chromatography (9:1 hexane–EtOAc) to
yield 10 as a solid (481.5 mg, 75%); mp 53–
55 °C; [h]2D0 −20° (c 1, CHCl3); Rf 0.64 (3:1
hexane–EtOAc); 1H NMR (300 MHz,
CDCl3): 7.27–7.37 (m, 5 H, aromatics), 4.81
(d, 1 H, J 11.8 Hz, CH2ꢀPh), 4.64 (d, 1 H, J

360
C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
(d, 1 H, J 1.9 Hz, H-2 Ar), 6.64 (dd, 1 H, J 1.8
6-O-[N-(3,4-Dihydroxyphenylethyl)-succina-
myl]-h,i- -glucopyranose (1).—A mixture of
and 8.2 Hz, H-6 Ar), 5.24 and 4.35 (2d, 1 H,
J 3.1 and 7.0 Hz H-1), 5.06 and 5.04 (2s, 4 H,
CH2ꢀPh), 2.61 (2 H, t, J 7.3 Hz, CH2CON);
13C NMR (50 MHz, 1:1 CDCl3–CD3OD): l
172.6 and 172.0 (2 C, CO), 148.5 and 147.0 (2
C, C-3 and C-4 Ar), 136.8 and 136.7 (2 C, C
ipso Bn), 132.3 (C-1 Ar), 121.3 (C-6 Ar), 115.5
and 115.2 (C-2 and C-5 Ar), 71.2 and 71.0 (2
C, CH2ꢀPh), 72.9–67.2 (C-2,-3,-4,-5), 63.5 and
63.1 (C-6), 40.4 (CH2ꢀAr), 34.9 (CH2ꢀNH),
29.9 and 28.8 (2 CH2 succ.); Anal. Calcd for
C32H37NO10: C, 64.53; H, 6.26; N, 2.35.
Found: C, 64.20; H, 6.12; N, 2.20.
D
11 (24 mg, 0.035 mmol) Pd/C (40 mg) and
MeOH (3 mL) was stirred under hydrogen at
rt for 1 h. The catalyst was removed by filtra-
tion and the solvent evaporated to give a
syrup, which was dissolved in 95:5 water–
Me3CN and freeze-dried to afford 1 as a white
powder (15 mg, 99%); mp 80–85 °C, Rf 0.34
(4:1:1 EtOAc–AcOH–water); [h]2D0 +28.9° (c
1
0.7, MeOH); H NMR (300 MHz, CD3OD): l
6.87 (d, 1 H, J 8.0 Hz, H-5 Ar), 6.83 (d, 1 H,
J 2.0 Hz, H-2 Ar), 6.72 (dd, 1 H, J 8.0 and 2.0
Hz, H-2 Ar), 5.27 and 4.67 (2d, 1 H, J 3.6 and
7.8 Hz, H-1), 4.41 and 4.39 (2dd, 1 H, H-6),
4.53 and 4.58 (2dd, 1 H, H-6%), 2.65 (t, 2 H, J
6.7 Hz, CH2CON); 13C NMR (50 MHz,
CD3OD): l 174.3 and 174.2 (2 C, CO), 146.2
and 144.7 (C-3 and C-4 Ar), 132.1 (C-1 Ar),
121.0 (C-6 Ar), 116.8 and 116.3 (C-2 and C-5
Ar), 98.3 (C-1b), 94.0 (C-1a), 77.9–70.6 (4 C,
C-2,-3,-4,-5), 65.1 and 65.0 (C-6), 42.3
(CH2NH), 35.8 (CH2Ar), 31.4 and 30.4 (2 C,
CH2 succ.); Anal. Calcd for C18H25NO10: C,
52.05; H, 6.07; N, 3.37. Found: C, 52.25; H,
5.95; N, 3.22.
6-O-[N-(3,4-Dihydroxyphenylethyl)-succina-
myl]-h,i- -galactopyranose (2).—Compound
D
2 was obtained by catalytic hydrogenation of
13 (94 mg, 0.16 mmol), as described for 1,
using 10% Pd/C (183 mg) and MeOH (10
mL). Work-up and final lyophilization
analogously to 1, afforded 2 as a white solid
(65.7 mg, 99%), mp 97–100 °C; Rf 0.30 (4:1:1
EtOAc–AcOH–water); [h]2D0 +25.8° (c 0.4,
1
MeOH); H NMR (400 MHz, CD3OD): l
6.90 (d, 1 H, J 8.0 Hz, H-5 Ar), 6.83 (d, 1 H,
J 2.0 Hz, H-2 Ar), 6.72 (dd, 1 H, J 2.0 and 8.0
Hz, H-6 Ar), 5.33 and 4.62 (2d, 1 H, J 3.3 Hz
and 7.6 Hz, H-1), 3.54 (m, 2 H, CH2ꢀNH),
2.65–2.84 (m, 6 H, CH2ꢀAr); 13C NMR (50
MHz, CD3OD): l 174.7 and 174.5 (2 C, CO),
145.8 and 144.3 (C-3 and C-4 Ar), 132.3 (C-1
Ar), 121.3 (C-6 Ar), 116.6 and 117.0 (C-2 and
C-5), 76.0–69.1 (4 C, C-2,-3,-4,-5), 65.2 and
64.9 (C-6), 42.2 (CH2ꢀNH), 35.6 (CH2ꢀAr),
31.4 and 30.4 (2 C, CH2 succ.); Anal. Calcd
for C18H25NO10: C, 52.05; H, 6.07; N, 3.37.
Found: C, 52.31; H, 5.93; N, 3.19.
1,2,3,4-Tetra-O-trimethylsilyl-h- -galacto-
D
pyranose (12).—Compound 12 was prepared
according to the procedure described for the
synthesis of 10. After column chromatography
(10:1“8:1 hexane–EtOAc), compound 12
was obtained as a solid, in 75% yield; mp
54–55 °C; Rf 0.60 (3:1 hexane–EtOAc); [h]D20
1
+92° (c 1, CHCl3); H NMR (200 MHz,
CDCl3): l 5.11 (d, 1 H, J 1.8 Hz, H-1),
3.87–3.85 (m, 3 H, H-2,-3,-4), 3.96 (m, 1 H,
H-5), 3.80 (m, 1 H, H-6), 3.61 (m, 1 H, H-6%),
0.16–0.12 (36 H, m, (CH3)3Si; 13C NMR (50
MHz, CDCl3): l 94.5 (C-1), 73.9–69.7 (C-2,-
3,-4,-5), 63.2 (C-6), 0.1–0.7 (12 C, (CH3)3Si).
6-O-[N-(3,4-Dibenzyloxyphenylethyl)succ-
1-O-[N-(3,4-Dibenzyloxyphenylethyl)-succ-
inamyl] - 2,3,4,6 - tetra - O - benzyl - i -
D
- gluco-
pyranose (15).—A solution of 14 (Ref. [13],
394 mg, 0.577 mmol) in CH2Cl2 and DMF
(95:5, 13.1 mL) was treated with acid 8 (227
mg, 0.524 mmol) at rt and under argon for 5
days. Then, CH2Cl2 was added (10 mL) and
the mixture was washed with ice-water (4×5
mL), dried (Na2SO4) and concentrated. The
residue was purified by flash chromatography
(5:1“4:1“3:2 hexane–EtOAc) to give 15 as
a white solid (220 mg, 40%); mp 75–78 °C; Rf
0.395 (1:1 hexane–EtOAc); [h]2D0 +10.4° (c 1,
CHCl3); 1H NMR (300 MHz, CDCl3): l
inamyl]-i-D-galactopyranose
(13).—Com-
pound 13 was prepared following a similar
procedure as described for 11. After purifica-
tion by flash chromatography (15:1 CH2Cl2–
MeOH), 13 was obtained as a solid (99 mg,
78%); mp 160–163 °C; Rf 0.35 (7:1 CH2Cl2–
MeOH); [h]2D0 +26.6° (c 0.8, 1:1 CHCl3–
MeOH); 1H NMR (400 MHz, 1:1
CDCl3–CD3OD): l 7.38–7.23 (m, 10 H, aro-
matics), 6.81 (d, 1 H, J 8.2 Hz, H-5 Ar), 6.76

C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
361
filtered, and concentrated to give a residue,
which was purified by flash chromatography
(2:1“1:1 hexane–EtOAc), affording com-
pound 16 as a solid (2.11 g, 70%); mp 125–
7.04–7.38 (m, 30 H, aromatics), 6.81 (d, 1 H,
J 8.2 Hz, H-5 Ar), 6.76 (d, 1 H, J 2.1 Hz,
H-2 Ar), 6.61 (dd, 1 H, J 2.1 Hz and 8.2 Hz,
H-6 Ar), 5.53 (d, 1 H, J 7.9 Hz, H-1), 5.38
(m, 1 H, NH), 5.07 and 5.06 (2s, 4 H, 2
CH2ꢀPh), 4.83–4.36 (m, 8 H, 4 CH2ꢀPh),
3.69–3.45 (m, 6 H, H-2,-3,-4,-6,-6%), 3.34 (m,
2 H, CH2NH), 2.62–2.55 (m, 4 H, CH2 succ.
and CH2ꢀAr), 2.24 (m, 2 H, CH2 succ.); 13C
NMR (50 MHz, CDCl3): l 171.4 (COO),
170.7 (CONH), 147.8 and 149.1 (C-3 and
C-4 Ar), 137.3–138.4 (C ipso Bn), 132.3 (C-1
Ar), 121.6 (C-6 Ar), 115.6, 115.9 (C-5, C-2
Ar), 94.3 (C-1), 84.8, 80.9, 77.2, 75.6 (C-2,-
3,-4,-5), 75.6–71.3 (CH2Ph); 68.1 (C-6), 40.7
(CH2NH), 35.1 (CH2Ar), 30.8 and 29.7 (2
CH2 succ.); Anal. Calcd for C60H58NO10: C,
75.61; H, 6.13; N, 1.47. Found: C, 75.31; H,
6.09; N, 1.52.
127 °C;
Rf
0.35
(2:1:0.15
hexane–
EtOAc–AcOH); 1H NMR (200 MHz,
CHCl3): l 6.67 (d, 1 H, J 8.1 Hz, H-5 Ar),
6.65 (d, 1 H, J 3.0 Hz, H-2 Ar), 6.53 (m, 1
H, H-6 Ar), 5.06 (s, 2 H, CH2Ph), 3.27 (t, 2
H, J 7.5 Hz, CH2NH), 2.62 (t, 2 H, J 7.7
Hz, CH2Ar); 13C NMR (50 MHz, CD3OD):
159.1 (CO), 146.5 (C-3 Ar), 145.0 (C-4 Ar),
138.7 (C ipso), 132.3 (C-1 Ar), 129.7–129.0
(4 C, aromatics), 121.4 (C-6 Ar), 117.2 and
116.6 (C-2 and C-5 Ar), 67.5 (CH2ꢀPh), 44.0
(CH2ꢀNH), 36.8 (CH2ꢀAr); Anal. Calcd for
C16H17NO3: C, 70.83; H, 6.32; N, 5.16.
Found: C, 70.71; H, 6.39; N, 5.02.
N-Benzyloxycarbonyl-3,4-dihydroxy-3-O-(2,
3,4,6 - tetra - O - acetyl - i -
D
- glucopyranosyl)-
1 - O - [N - (3,4 - Dihydroxyphenylethyl) - succ-
phenylethylamine (18) and N-benzyloxycar-
inamyl]-i- -glucopyranose (3).—A solution
D
bonyl - 3,4 - dihydroxy - 4 - O - (2,3,4,6 - tetra - O-
of 15 (146 mg, 0.153 mmol) was hy-
drogenolyzed in 3:2 MeOH–EtOAc, in the
presence of Pd/C (175 mg). After 3 h, work-
up was carried out as described for 1, giving
3 as a solid (63 mg, 99%); Rf 0.32 (4:1:1
EtOAc–AcOH–water); [h]2D0 −4° (c 1,
acetyl - i -
(19).—To a solution of 16 (1.5 g, 5.22
mmol) and b- -glucose pentaacetate (17, 3.06
D
- glucopyranosyl)phenylethylamine
D
g, 7.83 mmol) in dry CH2Cl2 (53 mL) was
added BF3·OEt2 dropwise (3.2 mL, 26.10
mmol), stirring under argon for 4 h. Then,
the mixture was diluted with CH2Cl2 and
washed with satd NaHCO3, water and dried
(NaSO4). Evaporation of the solvent, fol-
lowed by purification by silica gel chro-
1
MeOH); H NMR (300 MHz, CD3OD): l
6.87 (d, 1 H, J 8.1 Hz, H-5 Ar), 6.83 (d, 1
H, J 2.0 Hz, H-2 Ar), 6.72 (dd, J 8.1 and 2.0
Hz, H-6 Ar), 5.67 (d, 1 H, J 8.1 Hz, H-1),
2.68 (t, 2 H, J 6.7 Hz, CH2CON); 13C NMR
(50 MHz, CD3OD): l 174.0 and 173.1 (2
CO), 146.2 and 144.7 (C-3 and C-4 Ar),
132.0 (C-1 Ar), 121.3 (C-6 Ar), 116.8 and
116.3 (C-2 and C-5 Ar), 95.8 (C-1), 78.8–
71.0 (4 C, C-2,-3,-4,-5), 62.3 (C-6), 42.4
(CH2NH), 35.9 (CH2ꢀAr), 31.2 and 30.4 (2
CH2 succ.); Anal. Calcd for C18H25NO10: C,
52.04; H, 6.07; N, 3.37. Found: C, 52.27; H,
5.93; N, 3.15.
N-Benzyloxycarbonyl-3,4-dihydroxyphenyl-
ethylamine (16).—To a solution of dopamine
hydrochloride (2 g, 10.5 mmol) in MeOH (33
mL) and Et3N (4.4 mL, 31.6 mmol), was
added benzylchloroformate dropwise (1.9
mL) at 0 °C. Then, the stirred solution was
allowed to attain rt and the stirring was con-
tinued for 2 h. The reaction mixture was
neutralised with amberlite IR 150 (H+)
matography
(2:1“1:1
hexane–EtOAc)
afforded compounds 18 (0.58 g, 18%) and 19
(1.74 g, 54%) as solids; 18: mp 42–45 °C; Rf
0.40, (1:1 hexane–EtOAc); [h]2D0 −13.2° (c 1,
1
CHCl3), H NMR (400 MHz, CDCl3): l 7.32
(m, 5 H, aromatics), 6.85 (m, 2 H, H-5 and
H-6 Ar), 6.80 (s, 1 H, H-2 Ar), 5.94 (s, 1 H,
OH), 4.93 (d, 1 H, J 7.3 Hz, H-1), 4.81 (s, 1
H, NH), 4.26 (dd, 1 H, J5,6 5.1 Hz, J6,6% 12.4
Hz, H-6), 4.18 (dd, 1 H, J5,6% 2.4 Hz, J6,6%
12.4 Hz, H-6%), 3.82 (m, 1 H, H-5), 3.41 (m,
2 H, CH2ꢀNH), 2.72 (t, 2 H, J 6.59 Hz,
CH2ꢀAr), 2.05 (m, 12 H, 4 CH3, Ac); 13C
NMR (50 MHz, CDCl3). l 177.1 (CO),
170.6–169.3 (4 C, CO, Ac), 156.2 (C-3 Ar),
145.9 (C-4 Ar), 144.0 (C ipso Bn), 136.4 (C-1
Ar), 130.7–128.5 (5 C, aromatics), 125.4 (C-6
Ar), 118.0 (C-2 Ar), 116.3 (C-5 Ar), 101.4

362
C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
125.3 (C-6 Ar), 119.5 (C-2 Ar), 117.8 (C-5 Ar),
104.4 (C-1), 78.7, 77.9, 75.1, 71.8 (C-2,-3,-4,-
5), 62.9 (C-6), 42.3 (CH2ꢀNH), 34.2
(CH2ꢀAr); Anal. Calcd for C16H22F3NO9: C,
44.76; H, 5.16; N, 3.26. Found: C, 44.62; H,
5.20; N, 3.14. ESMS: [M−CF3COO−] 316.3.
(C-1), 72.2–68.0 (C-2,-3,-4,-5), 66.6 (CH2Ph),
61.6 (C-6), 42.0 (CH2ꢀNH), 35.3 (CH2ꢀAr);
Anal. Calcd for C30H35NO13: C, 58.34; H,
5.71; N, 2.27. Found: C, 58.20; H, 5.90; N,
2.10. 19: mp 87–89 °C, Rf 0.30 (1:1 hexane–
1
EtOAc), [h]2D0 −65.7° (c 1, CHCl3); H NMR
3,4-Dihydroxy-4-O-(i- -glucopyranosyl)-
D
(200 MHz, CDCl3): l 7.36 (m, 5 H, aromat-
ics), 6.88 (d, 1 H, J 8.1 Hz, H-5 Ar), 6.79 (d,
1 H, J 2.09 Hz, H-2 Ar), 6.63 (dd, 1 H, J 8.2
and 2.1 Hz, H-6 Ar), 6.02 (s, 1 H, OH), 5.11
(s, 2 H, CH2Ph), 4.91 (m, 1 H, H-1), 4.31 (dd,
1 H, J6,6% 12.4 Hz, J6,5 5.3 Hz, H-6), 4.19 (dd,
1 H, J6,6% 12.4 Hz, J5,6% 2.63 Hz, H-6%), 3.83 (m,
1 H, H-5), 3.43 (m, 2 H, CH2ꢀNH), 2.74 (m, 2
H, CH2Ar), 2.11 (m, 12 H, 4 CH3, Ac); 13C
NMR (50 MHz, CDCl3): 177.1 (NHꢀCOꢀ),
170.5–169.3 (ꢀCOꢀ, Ac), 156.2 (C-3 Ar),
147.4 (C-4 Ar), 142.8 (C ipso Bn), 136.4 (C-1
Ar), 128.0–128.4 (5 C, aromatics), 120.41 (C-6
Ar), 118.0 (C-2 Ar), 101.6 (C-1), 72.1, 71.2
and 68.0 (C-2,-3,-4,-5), 66.5 (CH2Ph), 61.6
(C-6), 42.0 (CH2NH), 35.4 (CH2Ar), 20.6–
20.5 (4 C, CH3, Ac); Anal. Calcd for
C30H35NO13: C, 58.34; H, 5.71; N, 2.27.
Found: C, 58.12; H, 6.00; N, 2.33.
phenylethylamonium trifluoroacetate (5).—
Compound 5 was prepared from 19 as de-
scribed for 4. Thus, 5 was obtained as a syrup
(1.2 g, 99%); Rf 0.25 (4:2:1 EtOAc–AcOH–
1
water); [h]2D0 −39.7° (c 1, MeOH); H NMR
(300 MHz, CD3OD): l 7.36 (d, 1 H, J 8.3 Hz,
H-5 Ar), 6.97 (d, 1 H, J 2.2 Hz, H-2 Ar), 6.88
(dd, 1 H, J 2.2 and 8.3 Hz, H-6 Ar), 4.91 (d,
1 H, J 7.6 Hz, H-1), 3.32 (t, 2 H, J 7.6 Hz,
13
CH2NH), 3.03 (t, 2 H, J 7.6 Hz, CH2Ar); C
NMR (50 MHz, CD3OD): l 148.7 (C-3 Ar),
145.9 (C-4 Ar), 133.4 (C-1 Ar), 121.1 (C-6 Ar),
119.3 (C-2 Ar), 117.5 (C-5 Ar), 104.3 (C-1),
78.2, 77.5, 74.8 and 71.2 (C-2,-3,-4,-5), 62.3
(C-1), 41.9 (CH2ꢀNH), 33.9 (CH2ꢀAr); Anal.
Calcd for C16H22F3NO9: C, 44.76; H, 5.16; N,
3.26. Found: C, 44.62; H, 5.22; N, 3.31.
ESMS: [M−CF3COO−] 316.3.
3,4-Dihydroxy-3-O-(i-
D
-glucopyranosyl)-
D2 receptor binding assays.—[3H]spiperone
(104 Ci/mmol) were obtained from Amersham
International (England), unlabelled R(+)-
SCH23390·HCl from Research Biochemicals
Inc. (RBI, USA), and unlabelled spiperone,
sulpiride·HCl and haloperidol from Sigma
(USA). The reference drugs were stored in 1
mM soln at −20 °C. The new and reference
drugs were diluted to the required concentra-
tion on ice, immediately before binding as-
says. Male Sprague–Dawley rats were killed
by decapitation and their brains were rapidly
removed and dissected on an ice-cold plate.
Striatal membrane preparations were obtained
by homogenization (Polytron homogenizer,
setting 6.10 s) in 50 mM TrisꢀHCl (pH 7.7 at
25 °C; about 100 mL per mg of tissue) contain-
ing 5 mM EDTA; the homogenates were cen-
trifuged (49,000g for 15 min at 4 °C, Sorvall
RC-26 plus), resuspended in 50 mM TrisꢀHCl
buffer (pH 7.4 at 25 °C), and centrifuged
again (same conditions), and the final pellets
were stored at −80 °C pending use. Just be-
fore binding assays, the pellets were resus-
pended (1.25 mg original wet weight per 750
mL) in 50 mM TrisꢀHCl buffer (pH 7.4 at
phenylethylamonium trifluoroacetate (4).—
Compound 18 (490 mg, 0.793 mmol) was dis-
solved in NaMeO (0.1 M, 15 mL) and the
mixture was stirred for 1.5 h at rt. Then the
reaction mixture was neutralized with amber-
lite IR-120 (H+), filtered and concentrated.
The residue was purified by silica gel chro-
matography (7:1 EtOAc–MeOH) to yield the
deacetylated glycoside (314 mg, 88%); Rf 0.67
(4:1 EtOAc–MeOH) which was dissolved (180
mg, 0.40 mmol) in MeOH (6 mL), and treated
with TFA (26 mL, 1.6 equiv) and Pd/C (90
mg) and stirred under hydrogen at rt for 1 h.
The catalyst was removed by filtration and the
solvent and excess of TFA were evaporated
giving 4 as a syrup (170 mg, 99%); Rf 0.28
(4:2:1 EtOAc–AcOH–water); [h]2D0 −35.7° (c
1
1, MeOH); H NMR (300 MHz, CD3OD): l
7.30 (m, 1 H, H-2 Ar), 7.02 (m, 2 H, H-5 and
H-6 Ar), 4.96 (d, 1 H, J 7.4 Hz, H-1), 4.13
(dd, 1 H, J6,6% 12.0 Hz, J6,5 2.1 Hz, H-6), 3.88
(dd, 1 H, J6,6% 12.0 Hz, J6%,5 6.2 Hz, H-6%), 3.33
(2 H, t, J 8.0 Hz, CH2NH), 3.04 (2 H, t, J 8
13
Hz, CH2Ar); C NMR (50 MHz, CD3OD): l
147.9 (C-3 Ar), 147.2 (C-4 Ar), 129.6 (C-1 Ar),

C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
363
Protease. To 500 mL of a solution of 1 (1.0
mM) in phosphate buffer (0.1 M, pH 8.0),
were added 190 mL of buffer, 60 mL of an
enzyme solution (0.5 units/mL) in phosphate
buffer and 250 mL of a solution of CaCl2 (40
mM) in water. Aliquots (40 mL) were removed
and analysed by HPLC.
Glucosidase. Compound 4 or 5 was dis-
solved in phosphate buffer (0.1 M, pH 5.0) at
a concentration of 10 mM. To 50 mL of this
solution, 750 mL of buffer preincubated at
37 °C and 200 mL of a glucosidase solution
(0.1 units/mL) in buffer (pH 5), were added.
The samples were incubated and analysed by
HPLC.
25 °C) containing 120 mM NaCl, 5 mM KCl,
2 mM CaCl2 and 1 mM MgCl2.
Striatal membrane preparation (750 mL
aliquots) were added to ice-cold tubes contain-
ing: (a) 100 mL of [3H]spiperone, (b) 50 mL of
ketanserin (final concentration 50 nM) to
block 5-HT2A receptors; and either (c) 100 mL
of buffer (for total binding assay) or (d) 100
mL of sulpiride (final concentration 10 mM) to
allow quantification of unspecific binding by
[3H]spiperone, or (e) 100 mL of the compounds
to be tested (final assay vol was 1 mL). All
assays were performed in duplicate. Incuba-
tions (15 min at 37 °C) were stopped by rapid
vacuum filtration through GF-52 glass fibre
filters (Schleicher and Schuell) in a Brandel
M-30 cell harvester. The filters were rinsed
three times with 3 mL of ice-cold 50 mM
TrisꢀHCl buffer (pH 7.4), and radioactivity
was determined by liquid scintillation count-
ing in a Beckman LS-6000LL apparatus.
Statistical analysis.—Competition analyses
in binding assays were carried out with the aid
of the Graphpad Prism software (v 2.1)
(GraphPad, San Diego, CA), and Ki values
were calculated as Ki =IC50/(1+L/Kd), where
L is the concentration and Kd the apparent
dissociation constant of the ligand.
Stability studies.—All incubations were car-
ried at 37 °C in a shaker (Adolf Ku¨hner AG
CH-4127) at 200 rpm. At various time inter-
vals, aliquots were removed and immediately
analysed by HPLC. The mobile phase was
95:5 water 0.1% TFA–MeCN for compound
1 and dopamine and 97.5:2.5 water 0.1%
TFA–MeOH for compounds 4 and 5. The
flow rate was fixed at 0.5 mL/min for all the
stability experiments. Bovine liver esterase
(EC 3.1.1.1), a-chymotrypsin from bovine
pancreas (EC 3.4.21.1), and b-glucosidase
from almonds (EC 3.2.1.21) were purchased
from Sigma.
Stability in plasma.—To 10 mL of rat
blood was added 90 mL of EDTA and the
suspension was centrifuged at 10,000g for 15
min. Plasma was kept in eppendorf tubes at
4 °C and it was used immediately for the
stability studies. Bioactivation experiments
were initiated by adding a solution (50 mL, 10
mM in phosphate buffer 0.1 M, pH 7.01) of
compounds 1, 4, 5 or dopamine, to 950 mL of
plasma preincubated at 37 °C (final com-
pounds concentration 0.5 mM). Aliquots (50
mL) were removed and 0.8 N HClO4 (50 mL)
was added. The resulting mixtures were cen-
trifuged (15 000 g, 15 min, 4 °C) to remove
precipitated proteins. A 50 mL aliquot of the
supernatant layer was subjected to HPLC
analyses (mobile phase for compound 1:
95:5“93:7“70:30“95:5 water 0.1% TFA–
Me3CN; for compounds 4 and 5: 97.5:2.5
water 0.1% TFA–MeOH; for dopamine: 95:5
water 0.1% TFA–Me3CN.
Stability in rat brain extract
Compound 1 and dopamine. The entire rat
brain was removed after incision of the skull,
rinsed with phosphate buffer, dried, weighed
(6.78 g) and then homogenized in phosphate
buffer (0.1 M, pH 7.3) 1:4 w/v brain–phos-
phate buffer, using a mechanical tissue ho-
mogeniser for 5 min, at 4 °C. The homogenate
was centrifuged at 60 000 g for 35 min at
4 °C, and the supernatant was aliquoted and
stored at −80 °C until it was to be used for
the experimental studies. Compound 1 or do-
pamine (50 mL of stock solution 10.0 mM)
was incubated at 37 °C with brain preparation
(950 mL) (final concentration 0.5 mM). At
Enzyme stability studies
Esterase. To 500 mL of a stock solution of 1
(1.0 mM) in phosphate buffer (0.1 M, pH 7.2)
preincubated at 37 °C, 380 mL of buffer and
120 mL of an esterase solution (0.5 units/mL)
in buffer, were added. Aliquots (40 mL) were
removed and immediately analysed by HPLC
as described above.

364
C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
Drugs and chemicals.—All compounds were
administered in 0.01 mL/g injections intraperi-
toneally. Reserpine (Sigma, St Louis, MO)
was dissolved in 1% AcOH in water and new
drugs were prepared in saline, immediately
prior to the experiments. Methanol, citric acid
monohydrate, perchloric acid, and NaCl were
all of analytical grade (E. Merck, Darmstadt,
Germany). Dopamine hydrochloride, 3,4-di-
hydroxyphenylacetic acid (DOPAC), 3-
methoxytyramine HCl (3-MT), homovanillic
acid (HVA) and 1-octanesulphonic acid
(sodium salt) were purchased from Sigma.
Statistical analysis and graphics.—Pharma-
cological calculations were obtained by using
the PCS program (Pharmacologic Calculation
System; Tallarida and Murray, 1987) or
PRISM program. The statistical significance of
differences between means were determined by
the Newman–Keuls test, and the differences
with probabilities lower than 0.05 were con-
sidered statistically significant.
various time points, 50 mL were removed and
treated as described for plasma experiments.
Compounds 4 and 5. For these compounds a
brain homogenate with b-glycosidase activity
was obtained as described by Withers and
co-workers [18]. A rat brain was rinsed in
distilled water, blotted and weighed (1.8 g);
then 4.5 mL of 25 mM citrate–50 mM phos-
phate buffer, pH 5.0, containing EDTA (1
mM), dithiothreitol (4 mM) and Triton X-100
(0.1% v/v) was added and the tissues were
treated for 1 min with a mechanical tissue
homogeneizer. b-Glucosidase activity of the
homogenate was measured and expressed as
nmol of p-nitrophenyl b- -glucopyranoside
D
hydrolysed per h and per mg of tissue; the
resulting value, 4.45 nmol/h mg, is in good
agreement with published data. Tissue ho-
mogenates were frozen and stored at −80 °C.
For the stability experiments 300 mL of a
stock solution of 4 or 5 (1.0 mM) in buffer
(pH 5) was added to 300 mL of the brain
homogenate and incubated at 37 °C. At vari-
ous time intervals, aliquots (50 mL) were re-
moved and treated as described for plasma
experiments.
Acknowledgements
We thank Dr M.L. de Ceballos for her
interest and helpful discussions. Financial sup-
port by the DGICYT (grant PB96-0833), the
Xunta de Galicia (grant XUGA-20308B96,
and fellowship to E.R.), Agencia Espan˜ola de
Cooperacio´n Iberoamericana (fellowship to
G.M.), and the Ministerio de Educacion y
Cultura (fellowship to C.F.) is gratefully
acknowledged.
Beha6iour experiments.—Mice (weighing
2595 g) were housed in groups of 12 (mice)
under regulated conditions (light/dark cycle
between 8.00 and 20.00 h at 2191 °C) in
standard Makrolon cages (215×465×145
mm). The animals received standard labora-
tory chow and tap water ad lib until the
beginning of the experiments. Experimental
animals were pretreated with 5 mg/kg of reser-
pine intraperitoneally, 18 h prior to experi-
ments. Locomotive activity, rearing and
velocity, were measured always at the same
time of day (9–17 h), to avoid variation due
to circadian rhythms, with a video computer-
ised animal observation system (EthoVision
V. 1.90, Noldus Information Technology, Wa-
geningen, The Netherlands). Activity (total
distance travelled in cm), rearing (changes up-
per 15% in corporal surface) and velocity,
were recorded in foursquare test arenas (50×
50×30 cm) via a video camera fixed on the
ceiling above the arenas. Animals were tested
for 1 h and the recorded information was
relayed to a monitor and a video tracking
motion analysis.
References
[1] D.Y. Melvin, Ad6. Neurobiol., 60 (1993) 11–17.
[2] F. Hucho, Neurochemistry: Fundamentals and Concepts,
VCH, Weinheim, 1986.
[3] Y. Madrid, L.F. Langer, H. Brem, R. Langer, Ad6.
Pharmacol., 22 (1991) 299–324.
[4] K.L. Audus, P.J. Chikhale, W.D. Miller, S.E. Thomp-
son, R.T. Borchardt, Ad6. Drug Res., 23 (1992) 1–64.
[5] W.H. Oldendorf, Am. J. Physiol., 221 (1971) 1629–1639.
[6] M. Mueckler, Eur. J. Biochem., 219 (1994) 713–725.
[7] L. Prokai, Prog. Drug Res., 51 (1998) 95–131.
[8] R. Polt, F. Porreca, L.Z. Szabo`, E.J. Bilsky, P. Davis,
T.J. Abbruscato, T.P. Davis, R. Horvath, H.I. Yama-
mura, V.J. Hruby, Proc. Natl. Acad. Sci. USA, 91 (1994)
7114–7118.
[9] T. Halmos, M. Santarromana, K. Antonakis, D. Scher-
man, Eur. J. Pharmacol., 318 (1996) 477–484.

C. Ferna´ndez et al. / Carbohydrate Research 327 (2000) 353–365
365
[14] M. Baginski, F. Claudi, G. Giorgini, J.A. Fontenla, E.
Rosa, M. Caedellini, Bioorg. Chem., 24 (1996) 358.
[15] A.S. Horn, C.J. Grol, D.J. Dijkstra, J. Med. Chem., 21
(1978) 825–828.
[16] H. Frey, W. Loscher, Neuropharmacology, 19 (1980)
217–220.
[17] D.B.A. de Bont, R.G.G. Leenders, H.J. Haisma, I. van
der Meulen-Muileman, H.W. Scheeren, Bioorg. Med.
Chem., 5 (1997) 405–414.
[18] J.D. McCarter, M.J. Adam, N.G. Hartman, S.G. With-
ers, Biochem. J., 301 (1994) 343–348.
[10] C. Uriel, M.-J. Egron, M. Santarromana, D. Scherman,
K. Antonakis, J. Herscovici, Bioorg. Med. Chem., 4
(1996) 2081–2090.
[11] R.B. Walker, J.W. Ayres, J.H. Block, A. Lock, J.
Pharm. Sci., 67 (1978) 558–559.
[12] T.E.C.L. Rønnow, M. Meldal, K. Bock, Carbohydr.
Res., 260 (1994) 323–328.
[13] R.R. Schmidt, W. Kinzy, Ad6. Carbohydr. Chem.
Biochem., 50 (1994) 21–123.
.