New insights into the oxidation pathways of apomorphine

Article abstract of DOI:10.1039/b204605a

A detailed study of the oxidative behaviour of apomorphine in aqueous media is reported. Resorting to the synthesis of apomorphine derivatives it was possible to identify all the anodic oxidation peaks of apomorphine, which are related to the oxidation of

Full text of DOI:10.1039/b204605a

View Article Online / Journal Homepage / Table of Contents for this issue
New insights into the oxidation pathways of apomorphine
a
a
b
c
Jorge M. P. J. Garrido, Cristina Delerue-Matos, Fernanda Borges,* Tice R. A. Macedo and
Ana M. Oliveira-Brett
d
a
2
CEQUP/Departamento de Engenharia Química, Instituto Superior de Engenharia do Porto,
Rua S. Tomé, 4200-485 Porto, Portugal
CEQOFFUP/Departamento de Química Orgânica, Faculdade de Farmácia,
Universidade do Porto, 4050-047 Porto, Portugal. E-mail: fborges@ff.up.pt;
Fax: ϩ351222003977; Tel: ϩ351222078952
b
c
Faculdade de Medicina, Departamento de Farmacologia, Universidade de Coimbra,
3
004-535 Coimbra, Portugal
d
Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra,
004-535 Coimbra, Portugal
3
Received (in Cambridge, UK) 14th May 2002, Accepted 30th July 2002
First published as an Advance Article on the web 23rd August 2002
A detailed study of the oxidative behaviour of apomorphine in aqueous media is reported. Resorting to the synthesis
of apomorphine derivatives it was possible to identify all the anodic oxidation peaks of apomorphine, which are
related to the oxidation of the catechol and tertiary amine groups. These findings were revealed to be important since
they could lead to a better understanding of the biological interactions of apomorphine and gain insight into its
metabolic pathways. During the voltammetric studies, it was also found that apomorphine forms a complex with
borate through the catechol group leading to an increase of its oxidation potential. This property could be very useful
with regard to the stabilization of apomorphine solutions since it could drastically reduce its autoxidation.
5
products, besides oxoapomorphine, could be formed by oxid-
Introduction
ation has led us to undertake a detailed study of the electro-
Apomorphine has been known as an emetic agent for over a
chemical oxidation behaviour of apomorphine. In order to
century. Nevertheless, due to its dopaminergic activity it has
identify all the oxidation processes and the products formed the
received renewed attention because it was found to have bene-
syntheses of two apomorphine derivatives, oxoapomorphine
ficial effects in the treatment of idiopathic Parkinson’s disease,
and diacetylapomorphine (Fig. 1), were carried out as well as
which is related to the progressive degeneration of the central
studies of their electrochemical behaviour.
nervous system. In addition to Parkinson’s disease, recent
studies have indicated potential new uses for apomorphine,
namely in the treatment of erectile dysfunction. However, its
1
inherent instability complicates its application in clinical prac-
tice. In fact, aqueous solutions of apomorphine undergo spon-
taneous oxidative decomposition turning green in the presence
of light and air. These findings, together with reports of large
inter-patient variability in apomorphine pharmacokinetics,
have led to an increase of studies regarding interactions of
the drug and its metabolites with plasma and tissues. The
pharmacokinetic profile of apomorphine could be influenced
by covalent interactions through its catechol moiety, which
after oxidation gives an electrophilic quinone that can also
2
interact with proteins and other tissue components. Moreover,
it is known that quinones represent a class of toxicological
intermediates, which can lead to a variety of hazardous effects
in vivo, including acute cytotoxicity, immunotoxicity and
3
carcinogenesis. The mechanism by which quinones cause these
effects is quite complex and not well established. It is worth
noting that the presence of an N-methyl group in apomorphine
that could be oxidized may also influence its biological activity,
since it is known that a change in the N-methyl group could
result in a significant decrease in the affinities to 5-HT1A
Fig. 1 Structural formulae of apomorphine and its derivatives.
Experimental
Apparatus
4
receptors.
A better understanding of the biological interactions and the
metabolic pathways of apomorphine could be achieved through
detailed knowledge of its electrochemical oxidation behaviour.
The knowledge that apomorphine metabolites could be
involved in coupling reactions with nucleophilic groups of
All experiments were performed using a 663 VA Metrohm cell
containing a glassy carbon working electrode (Metrohm, d =
2.0 mm), a glassy carbon rod counter electrode (Metrohm) and
a Ag/AgCl (3 M KCl) reference electrode (Metrohm) attached
to a Autolab PSTAT 10 potentiostat/galvanostat running with
model GPES (EcoChimie, Netherlands). Experiments were
2
proteins, together with the possibility that other oxidation
DOI: 10.1039/b204605a
J. Chem. Soc., Perkin Trans. 2, 2002, 1713–1717
This journal is © The Royal Society of Chemistry 2002
1713

View Article Online
made without deaeration of the solutions. A Metrohm E520
pH-meter and a glass electrode of the same make were used for
the pH measurements.
(N–CH ), 33.7 (CH ), 28.6 (CH ), 20.5 (CH ), 20.4 (CH );
3
2
2
3
3
ϩ
ؒ
ϩ
ؒ
EI-MS: m/z 351 ([M] , 62), 308 ([M Ϫ COCH ] , 67), 266
3
ϩ
ؒ
([M Ϫ COCH ] , 100), 250 (9), 224 (26), 206 (13), 165 (12),
3
1
Melting points were measured using a Köfler microscope. H
152 (9).
13
1
and C NMR ( H decoupled) spectra were acquired, at room
temperature, on a Brüker AMX 300 spectrometer operating
at 300.13 and 75.47 MHz, respectively. Chemical shifts are
expressed as δ (ppm) values relative to tetramethylsilane (TMS)
as internal reference and coupling constants (J) are given in Hz;
Results and discussion
Although in aqueous solutions the degradation of apo-
morphine is a very rapid process accelerated with increasing
pH, little is known about its mechanism. The study of the
oxidative degradation mechanism of apomorphine and identifi-
cation of all the electrochemical oxidation data obtained was
only possible after the synthesis of the apomorphine deriv-
atives oxoapomorphine and diacetylapomorphine (Fig. 1). The
oxoapomorphine contains the ortho-quinone group formed
during the autoxidation of apomorphine and enables the
clarification of the oxidative behaviour of the catechol of apo-
morphine. In the compound diacetylapomorphine the catechol
group is blocked by acetyl groups, consequently preventing
oxidation and even the formation of oxoapomorphine. The
study of the electrochemical oxidation of diacetylapomorphine
enables the identification of the peak related to the oxidation of
the tertiary amine present in apomorphine.
DMSO-d was used as the sample solvent. Assignments were
6
also made from DEPT (distortionless enhancement by polar-
ization transfer) (see underlined values). Electron impact mass
spectra (EI-MS) were recorded on a VG AutoSpec instrument
and data are reported as m/z (% of relative intensity of the most
important fragments).
Thin layer chromatography (TLC) was carried out on alu-
minium sheets precoated with silica gel 60 F254 with layer
thickness 0.2 mm (Merck). The following chromatographic
systems were used chloroform–methanol–diethylamine (8 : 1.5 :
0
.5), ethyl acetate–hexane (9 : 1). The spots were visualised by
UV detection (254 nm) and iodine vapour. Solvents were
evaporated in a Büchi Rotavapor. Petroleum ether used was in
the boiling range 40–60 ЊC.
Reagents and solutions
Synthesis of the apomorphine derivatives oxoapomorphine and
diacetylapomorphine
Apomorphine was obtained from Sigma as the hydro-
chloride and was used without further purification. All chemi-
cals and solvents were reagent grade and were used as received.
The synthesis of oxoapomorphine was based on the procedure
7
of Linde and Ragab which uses mercuric chloride as the oxid-
Ϫ1
Deionised water with conductivity less than 0.1 µS cm was
ant reagent. Since the use of mercury is nowadays problematic
for environmental reasons, a new synthetic procedure based
used throughout. Buffer solutions employed were 0.2 M in the
6
8
pH range 1.2–12.2.
on the use of silver oxide and ultrasound was developed in
this work, and is described in the Experimental section.
Diacetylapomorphine was synthesised by the classic method
of acetylation (see Experimental section).
The C NMR spectrum of oxoapomorphine confirmed the
presence of aromatic, methylene, methine and methyl carbons
and supported the occurrence of carbonyl functional groups at
δ 183.3 and 176.0 ppm. The absence of the signals at δ 61.2 and
Synthesis of oxoapomorphine
7
Using the method of Linde and Ragab. An aqueous solution
of apomorphine hydrochloride (250 mg) was added to 25 mL
13
of 5% HgCl and 50 mL of 0.2 M citric acid–phosphate buffer
2
(
pH 6). The solution was warmed at 70 ЊC for 30 min. After
cooling, green crystals were obtained and filtered. It was found
by TLC that the reaction was not complete since some starting
material was present.
5
1.1 ppm in DEPT data confirmed the presence of a double
13
bond between C-6a and C-7 [ C NMR data of apomorphine:
45.0 (C–OH), 143.2 (C–OH), 132.3, 129.9, 128.7, 127.4 (CH),
126.9 (CH), 126.7 (CH), 124.5, 119.7, 118.5 (CH), 114.4 (CH),
61.2 (CH), 51.1 (CH ), 40.9 (N–CH ), 30.5 (CH ), 25.5 (CH )].
1
Using the ultrasound method. A mixture of apomorphine
hydrochloride (100 mg) and silver oxide (370 mg) in ethanol
was subjected to ultrasonication for 4.5 h, at 25 ЊC. The mixture
was filtered and the solvent evaporated. The crude product was
purified using flash chromatography (FC): silica gel 60 (0.040–
2
3
2
2
The mass spectrum of the quinone is in accordance with the
proposed structure showing two characteristic features of
this class of compounds: the loss of carbon monoxide and
the formation of a peak with two mass units higher than the
0
.063 mm, Merck) and ethyl acetate–hexane (9 : 1). Fractions
9
containing the main product were combined and recrystallized
from ethanol to give oxoapomorphine as blue-green crystals
molecular weight. Based on the spectral data the structure of
oxoapomorphine was proposed as shown in Fig. 1, which is in
13
10
(
1
(
90%; mp > 300 ЊC). C NMR: 183.3 (C᎐O), 176.0 (C᎐O),
52.7, 147.2, 139.7, 134.7, 133.3, 131.5 (CH), 129.5, 128.7
CH), 124.3 (CH), 123.7 (CH), 119.8, 108.0 (CH), 49.9 (CH ),
accordance with that anticipated by Erhardt et al. The proton
NMR spectrum was identical to that reported by Linde and
7
Rabad. It is noteworthy that the oxidant agent employed in this
2
ϩ
ؒ
4
0.1 (N–CH ), 28.2 (CH ); EI-MS: m/z 265 ([M ϩ 2] , 76), 264
work was not the reagent described in the literature for the
3
2
ϩ
ϩ
ؒ
ؒ
ϩ
ؒ
7
(
[M ϩ 1] , 19), 263 ([M] , 25), 235 ([M Ϫ CO] , 100), 206 (5).
synthesis of the compound.
The chemical identity of diacetylapomorphine (Fig. 1) was
established by NMR and MS. The presence of signals in the
Synthesis of diacetylapomorphine
13
C NMR spectrum at δ 168.4 (CH C᎐O), 168.0 (CH C᎐O),
3
᎐
3
᎐
Apomorphine hydrochloride (250 mg) was dissolved in a mix-
ture of 1.5 mL of dry pyridine and 15 mL of acetic anhydride
and maintained with stirring at room temperature, during 24 h.
After reaction the solution was poured into a beaker contain-
ing ice. The crude product was extracted with chloroform
1
41.9 (COCOCH ), 139.0 (COCOCH ) and 20.5 (CH ), 20.4
3 3 3
1
(
CH ) and the absence of signals in the H NMR spectrum at
3
δ 9.8 and 8.8 (C–OH) and the presence of two acetyl groups at
δ 2.5 and 2.3 led to the structural elucidation of this compound.
The mass spectrum of the compound is in agreement with the
proposed structure.
(
3 × 20 mL). The combined organic phases were washed
with water (20 mL) and dried with anhydrous sodium sulfate.
After evaporation of the solvent, a residue was obtained and
recrystallized with diethyl ether and petroleum ether. Diacetyl-
apomorphine was obtained as white crystals (88%, mp 127–
Effect of pH
The anodic oxidation of apomorphine is a complex mechanism
which is pH dependent and three oxidation processes can be
observed. As will be demonstrated below these processes could
be identified after studying the electrochemical oxidation of the
13
1
28 ЊC). C NMR: 168.4 (CH C᎐O), 168.0 (CH C᎐O), 141.9
3
3
(
COCOCH ), 139.0 (COCOCH ), 135.1, 135.0, 133.6, 129.6,
3
3
1
28.9, 127.9, 126.6, 126.1, 124.1, 122.1, 65.1, 52.2 (CH ), 43.6
2
1
714
J. Chem. Soc., Perkin Trans. 2, 2002, 1713–1717

View Article Online
synthesised apomorphine derivatives oxoapomorphine and
diacetylapomorphine (Fig. 1).
The electrochemical behaviour of apomorphine was studied
over a wide pH range, between 1.2 and 12.2, at a glassy
carbon working electrode using differential pulse voltammetry
(
Figs. 2 and 3). The first peak (I) in Fig. 2, in very acid media,
Fig. 2 Differential pulse voltammograms in pH 6 of 0.1 mM
solutions: (—) apomorphine, ( ؒ ؒ ؒ ) oxoapomorphine and (---)
diacetylapomorphine in 0.2 M ionic strength phosphate buffer. Scan
Ϫ1
rate 5 mV s
.
Scheme 1 Oxidation mechanism of apomorphine.
Fig. 3 Plot of Ep vs. pH from differential pulse voltammograms of
0
9
.1 mM apomorphine in 0.2 M ionic strength buffer electrolyte. For pH
and 10 results are presented using borate buffers (᭿) and ammonia
Fig. 4 Plot of Ep vs. pH from differential pulse voltammograms of
.1 mM diacetylapomorphine in 0.2 M ionic strength buffer electrolyte.
Scan rate 5 mV s . Slope 59.2 mV per pH unit.
Ϫ1
buffers (ᮀ). Scan rate 5 mVs . Slope 59.2 mV per pH unit.
0
Ϫ1
corresponds to the oxidation of catechol, the only peak starting
at pH 1, E = ϩ0.5 V. The second peak (II), starting at pH 3,
p
E = ϩ1.1 V, corresponds to the oxidation of the tertiary amine.
p
The third peak (III), starting at pH 6, E = Ϫ0.1 V, corresponds
p
to the oxidation of the species in equilibrium with oxoapo-
morphine, occurring as a result of spontaneous homogenous
oxidative decomposition of apomorphine to oxoapomorphine
(
Scheme 1).
The anodic wave observed starting at pH 1, E = ϩ0.5 V,
p
the peak potential decreasing as the pH increases, was easily
attributed to the oxidation of the catechol group after the
voltammetric behaviour of the apomorphine derivatives, oxo-
apomorphine and diacetylapomorphine was studied. In both
compounds the absence of the apomorphine oxidation peak at
ϩ0.5 V in acid media (Figs. 4 and 5) was observed. These results
led to the conclusion that this peak is related to the oxidation of
the catechol group present in apomorphine giving oxoapo-
morphine. Moreover, this result is also in agreement with the
data found in the literature, which gives oxoapomorphine as
5
the only oxidation product of apomorphine.
Fig. 5 Plot of Ep vs. pH from differential pulse voltammograms of
By studying the oxidation behaviour of diacetylapomorphine
Fig. 4) it was possible to identify the apomorphine oxidation
0
.1 mM oxoapomorphine in 0.2 M ionic strength buffer electrolyte.
Ϫ1
(
Scan rate 5 mV s . Slope 59.2 mV per pH unit.
J. Chem. Soc., Perkin Trans. 2, 2002, 1713–1717
1715

View Article Online
peak starting at pH 3, E = ϩ1.1 V. This peak is due to the
studied so far was observed which has important implications
on solution stability. As mentioned earlier aqueous solutions of
apomorphine undergo spontaneous oxidative decomposition
turning green in the presence of light and air and its instability
deters application in clinical practice.
Comparing the oxidation patterns of apomorphine for pH 8,
9, 10 and 11 in Fig. 3, it is surprising to observe a drastic change
for pH 9 and 10. In fact, for these pH values not only did the
peak around 0.0 V, corresponding to oxidation of the catechol
group disappear, but a new peak also appeared at ϩ0.5 V
(Fig. 7). This clearly indicates the occurrence of an interaction
between the catechol group in apomorphine and the supporting
electrolyte.
p
oxidation of the tertiary amine group present in the apo-
morphine molecule. This was rapidly confirmed because in
diacetylapomorphine the catechol position is blocked by acetyl
groups (Fig. 1) and presents only one anodic wave, initially
appearing at ϩ1.1 V and decreasing in potential as the pH
increased (Fig. 4). The oxidation of the tertiary amine led to the
11
formation of a secondary amine and an aldehyde. These
results undoubtedly show that the second peak of apo-
morphine, starting at pH 3, E = ϩ1.1 V, is due to the oxidation
p
of the tertiary amine group present in the apomorphine
molecule (Fig. 3) and not to further oxidation of oxoapo-
morphine. In aqueous solution apomorphine rapidly undergoes
spontaneous oxidative decomposition to oxoapomorphine,
5
this process being accelerated by oxygen and high pH. The
apomorphine oxidation wave starting at pH 6, E = Ϫ0.1 V,
p
could be related to an oxidation process involving the catechol
group of the intermediate (Scheme 1) generated in situ in
oxoapomorphine solutions (Fig. 3).
Another important observation is that even at pH lower than
6
this apomorphine oxidation peak appeared if several consecu-
tive scans were done. In fact, consecutive scans showed the
appearance in the second scan of the oxidation peak at Ϫ0.3 V
and growth of the peak occurred during the following scans
while the apomorphine oxidation peak at ϩ0.5 V does not
increase with time. This could be easily explained by the ability
of the new redox couple, formed by oxidation of the catechol
group of apomorphine (see Scheme 1), to adsorb very strongly
5
on the solid electrodes and to the reversibility of the oxoapo-
morphine peak occurring at Ϫ0.3 V, clearly shown in the square
wave voltammogram of Fig. 6. The square wave voltammogram
Fig. 7 Differential pulse voltammograms of 0.1 mM apomorphine at
pH 9 with 0.2 M ionic strength buffer: (—) borate and ( ؒ ؒ ؒ ) ammonia.
Ϫ1
Scan rate 5 mV s
.
At pH 9 and 10 the supporting electrolyte used was borate
buffer. In the literature the formation of complexes between
boric acid or borate and catechols or substituted catechols is
12,13
described,
which easily explains the shift in the oxidation
potential of the complexed catechol to more positive values.
Nevertheless, to prove that this interaction was due to the
composition of the buffer used, the voltammetric oxidation
of apomorphine was studied again but now using ammonia
buffer at pH 9 and 10. The results obtained (Fig. 3) fit
perfectly to the line of E vs. pH. This clearly shows that the
p
oxidation peak for the apomorphine catechol group is at the
potential of ϩ0.1 V in ammonia buffer, whereas the oxidation
of the catechol group in the apomorphine–borate complex is
at a much higher potential of ϩ0.5 V, in borate buffer. This
finding suggests that the use of borate buffer for storage of
apomorphine would prevent autoxidation of apomorphine
in an aqueous stock solution, due to the increase of the
apomorphine–complex oxidation potential. The implication
Fig. 6 Square-wave voltammogram of 0.1 mM oxoapomorphine at
pH 7 with 0.2 M ionic strength buffer electrolyte. (I = I Ϫ I , I = total
t
f
b
t
current, I = forward current and I = backward current). Frequency
f
b
1
50 Hz; pulse amplitude 50 mV.
that stabilisers are not needed in apomorphine–borate buffer
shows similar values for the forward and backward current and
peak potentials, characteristic of reversible electron transfer
reactions. The most relevant data regarding this oxoapo-
morphine oxidation peak comes from the electrochemical study
of oxoapomorphine with pH (Fig. 5). The oxidation behaviour
of oxoapomorphine studied by differential pulse voltammetry
showed an anodic peak, over the whole pH range, at the same
oxidation potential as the peak appearing in a solution of
apomorphine. This is very good evidence for the fact that
14,15
solutions
means a widening of possible applications in
clinical practice.
Conclusions
The oxidation mechanism of apomorphine in aqueous solution
was found to be complex and strongly pH dependent; a detailed
study of the oxidation behaviour of apomorphine and its
derivatives with pH was performed. The synthesis of the
apomorphine derivatives, oxoapomorphine and diacetylapo-
morphine, was undertaken and enabled the identification of all
the anodic peaks found for the apomorphine oxidation and
verified that they are related to the oxidation of catechol
and tertiary amine groups. This could lead to important
advances in understanding the metabolic pathways and bio-
logical interactions of apomorphine. Moreover it was found
the apomorphine oxidation wave starting at pH 6, E =Ϫ0.1 V,
p
is related with the above-mentioned oxidative process (see
Scheme 1).
Effect of supporting electrolyte
During the apomorphine voltammetric study over a wide pH
range a very interesting aspect of its oxidation mechanism not
1
716
J. Chem. Soc., Perkin Trans. 2, 2002, 1713–1717

View Article Online
that apomorphine forms a complex with borate through
the catechol group which has a higher oxidation potential
than in ammonia buffer. This means improved apomorphine
stabilization in borate buffer solutions, due to the increase of
the apomorphine–complex oxidation potential, and suggests
the use of these conditions for storage without the need for
stabilisers.
4 M. H. Hedberg, J. M. Jansen, G. Nordvall, S. Hjorth, L. Unelius and
A. M. Johansson, J. Med. Chem., 1996, 39, 3491.
5
6
H.-Y. Cheng, E. Strope and R. N. Adams, Anal. Chem., 1979, 51,
2
243.
A. M. Oliveira-Brett, M. M. M. Grazina, T. R. A. Macedo,
C. Oliveira and D. Raimundo, J. Pharm. Biomed. Anal., 1993, 11,
203.
7 H. H. A. Linde and M. S. Ragab, Helv. Chim. Acta, 1968, 51, 683.
C. A. Hall III, S. L. Cuppett and P. Dussault, J. Agric. Food Chem.,
998, 46, 1303.
8
1
Acknowledgements
9 K.-P. Zeller, in The Chemistry of the Hydroxyl Group: Part 1,
ed. S. Patai, Interscience, London, 1971, pp. 231–255.
10 P. W. Erhardt, R. V. Smith, T. T. Sayther and J. E. Keiser,
J. Chromatogr., 1976, 116, 218.
J. M. P. J. G. would like to thank the PRODEP Program for a
PhD grant.
1
1 J. R. L. Smith and D. Masheder, J. Chem. Soc., Perkin Trans. 2, 1977,
732.
1
1
2 K. Yoshino, M. Kotaka, M. Okamoto and H. Kakihana, Bull.
Chem. Soc. Jpn., 1979, 52, 3005.
References
1
2
3
H. Padma-Nathan and F. Giuliano, Urol. Clin. North Am., 2001, 28,
21.
R. V. Smith, R. B. Velagapudi, A. M. McLean and R. E. Wilcox,
J. Med. Chem., 1985, 28, 613.
J. L. Bolton, M. A. Trush, T. M. Penning, G. Dryhurst and
T. J. Monks, Chem. Res. Toxicol., 2000, 13, 135.
13 R. Pizer and L. Babcock, Inorg. Chem., 1977, 16, 1677.
14 E. Sam, P. Augustijns and N. Verbeke, J. Chromatogr., B: Biomed.
Appl., 1994, 658, 311.
15 R. Van der Geest, P. Kruger, J. M. Gubbens-Stibbe, T. Van Laar,
H. E. Bodde and M. Danhof, J. Chromatogr., B: Biomed. Appl.,
1997, 702, 131.
3
J. Chem. Soc., Perkin Trans. 2, 2002, 1713–1717
1717