ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
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Electrochemical Studies of the Oxidation Pathways of Apomorphine H.-Y. Cheng, E. Strope, and R. N. Adams" Department of Chemistry, University of Kansas, Lawrence, Kansas 66045
The oxidative reaction mechanisms of apomorphine in aqueous buffer are reported, based on electrochemical data and product analysis. I n the absence of strong nucleophiles, an irreversible chemical reaction follows the initlai 2e-/2H+ oxidation of apomorphine and eventually produces a new redox couple. Thls new redox couple adsorbs irreversibly on the Nujdcarbon paste electrode surface and exhibits properties of electrocatalysis on the oxidation of ascorbic acid. I n the presence of excess glutathione, a strong nucleophile, other reactions predominate.
with an underlying intention of better understanding its biological interactions in brain. Major efforts are on the elucidation of the reaction mechanism, the identification of products, and the probing of interactions between a reactive intermediate and glutathione. Preliminary results on the electrochemistry of an irreversibly adsorbed product on the Nujol-carbon paste electrode are also included.
11, OXO-APM
EXPERIMENTAL Instrumentation. Cyclic voltammograms of moderate scan rate were obtained using the Princeton Applied Research (PAR) model 174 polarographic analyzer and a Houston Omnigraphic X-Y recorder. Coulometry and bulk electrolysis were performed utilizing the PAR model 173polarographic analyzer equipped with a model 179 digital coulometer. A home-built potentiostat interfaced to the Hewlett-Packard 2100A minicomputer was employed for fast electrochemical experiments and thin-layer coulometry. A Cary 14 spectrophotometer was used to obtain UV-visible spectra. Electrodes and Cells. Cyclic voltammetry was run on the conventional Nujol-graphite (30/70 w/w) paste electrode with a surface area of -2 mm2. Unless otherwise specified, the electrode was freshly packed for each cyclic voltammetric run to minimize the undesirable electrochemicalsignah which arise from surface-adsorbed species. Bulk electrolysis utilized either the platinum gauze or carbon cloth electrode. The hanging mercury drop electrode (HMDE) was used in the fast cyclic voltammetric experiment to estimate the rate of the follow-up reaction. The reference electrode was a SCE and the auxiliary electrode was a piece of platinum wire. Thin-layer coulometric experiments were done in a cell fashioned from a 10-mL beaker and two 1 X 1 X l/lo inch quartz plates. A 11/2X 3/16 inch length of Au grid (100 lines/inch), positioned between the quartz slides, serves as the working electrode. This cell is basically the same design as Heineman's thin-layer electrode; detailed procedures for cell construction are available (9). The volume of solution around the Au grid was calibrated with standard solutions of 4-methylcatechol in pH 6.0 buffer and with solutions of hydroquinone in 0.1 N H2S04. Chemicals. Apomorphine was obtained from Mallinckrodt as the hydrochloride (USP) and was used without further purification. Glutathione, in reduced form, was purchased from Sigma. All other chemicals were reagent grade and were used as received. Oxidation Product. Several bulk electrolysis experiments of apomorphine in pH 6.0 or 7.4 MacIlvaine buffer were done to obtain enough electrochemicaloxidation product. The potential was set at 0.6 V vs. SCE and the electrolysis was interrupted several times to allow the blue-green product covering the Pt gauze electrode to be washed off with CHCl,. The crude extract was washed with 0.5 M HC1 and filtered to remove traces of HzO,then evaporated down on a rotary evaporator. The yields of crude oxidation product from these electrolyses varied from 2470to 55%, with pH 6.0 solutions giving higher yields. The crude electrolysis product was recrystallized from hot benzene. Thin-layer chromatography was used to check for purity. Elemental analysis WBB performed by Micro-Tech Laboratories.
No work has been reported on the electrochemistry of apomorphine. This study investigates the redox behavior of apomorphine in aqueous solutions of near physiological p H
RESULTS AND DISCUSSION Reaction Mechanism. Figure 1 is a cyclic voltammogram of apomorphine in phosphate buffer a t p H 7.4. Two anodic
Apomorphine (I) was synthesized from naturally occurring morphine over a century ago and was soon recognized for its powerful emetic effect in animals and humans (1, 2).
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Recently a number of research developments, mainly the work on dopamine which led to the introduction of treatment for Parkinson's disease, have caused a great deal of interest in this compound (3). The dopamine backbone may be traced in the structure (heavier lines, structure I), and it is thought to derive its pharmacological activity from this sub-structure. Aqueous solutions of apomorphine rapidly undergo spontaneous oxidative decomposition and turn green. This process is accelerated by oxygen and high pH, but little is known of its mechanism. There have been conflicting reports on the qualitative and quantitative changes of pharmacological activity by the development of color in old solutions ( 4 , 5 ) . The chemical identity of the oxidation products has been studied by several workers (6-8). The oxidation of aqueous solutions of apomorphine with O2 or HgC12 a t about neutral p H all resulted in a similar product. Structure I1 was postulated, based on spectroscopic data of UV-vis, IR, NMR, and mass spectrometry. These workers also found that a t higher p H (10-14), various other products were formed.
I CH3
0003-2700/79/0351-2243$01.00/0 0 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL.
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51, NO. 13,NOVEMBER 1979
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Figure 1. Cyclic voltammetry of apomorphine in pH 7.4 phosphate buffer. Scan rate, 3 V/min; [APM] = M. Applied potential vs. SCE
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Flgure 3. Fate of oxidized apomorphine at pH 4.5. Cyclic voltammograms were taken: (a) Before electrolysis, 4.37t rig apomrphinsHC1 in 10 mL 1.2M HCI. (b) After 2 e- electrolysis, oxidized apomorphine in 1.2M HCI. (c) Immediately after adequate amount of buffer was added to allow the pH to reach 4.5. 1:l ratio of apomorphine:oxoapomorphine
0 2
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Flgure 2. Fast scan cyclic voltammetry of
-
M apomorphine in pH 6.0 buffer. Scan rates, from top to bottom: 5 V/min, 100 V/min, 1000 V/min
waves (02,03) appear a t +0.15 and +0.82 V vs. SCE, respectively, on the first scan in the positive direction. Upon scan reversal, only one corresponding reduction peak (R2)is visible, while a new redox couple (Ol,Rl) appears a t -0.2 V on subsequent scans. The ratios of R2/02 and R1/O1 depend greatly on scan rates, as shown in Figure 2, indicating a rate-limiting chemical reaction following the initial oxidation. The formation of the follow-up product, R1, which appears at less positive potential than its parent compound, can be eliminated by scanning at lo00 V/min in a pH 6 solution. The rate of this follow-up reaction is also pH-dependent, as evidenced by the gradual disappearance of R1/O1 in more acidic solutions. In acid solution (see, for example, Figure 3a, run in 1.2 M HCl), there is no sign of follow-up reactions. The voltammetric peak O3 (see Figure 1) starts to merge into background as the scan rate increases to greater than 6 V/min or at pH below 6, making electrochemical investigation difficult. However, within the limited window of available experimental manipulation, one can correlate the magnitude
of O3 with the production of the peaks O1/R1, particularly in the presence of external nucleophiles. This will be discussed in detail later. The redox potential of apomorphine shifts toward more positive direction as the solution becomes more acidic. Both anodic and cathodic peak potentials show E, vs. pH variations with linear slopes of -75 mV/pH and -61 mV/pH, respectively, in the pH range 2-6. In spite of the serious distortion of the voltammetric peak due to absorption occurring a t the carbon paste electrode, one can safely assume a %electron2-proton process for the initial oxidation of apomorphine. The peak separation between O1 and O2 is approximately 300 mV and remains constant a t this pH region. Coulometric experiments utilizing a precalibrated thin-layer cell were run on solutions of apomorphine in 0.1 N HzS04, and 2.07 f 3% electrons/molecule was obtained for the initial oxidation process. The resulting solution was yellowish brown. The electrolysis of apomorphine in 1.2 M HC1 was stopped after 2 electrons per molecule had passed into the solution. An adequate amount of buffer was then added to the remaining yellowish brown solution to allow the pH to reach about 4.5. The solution very quickly turned bluish green, resembling the color developed in old solutions of apomorphine. Figure 3 shows the cyclic voltammograms taken before the electrolysis, after the electrolysis, and after the buffer was added. Notice that the initial potentials and scan directions are different in Figure 3a, b, and c. It is concluded that at higher pH 1mol of the oxidized apomorphine (Figure 3b) will rapidly product mol of the reduced APM and '/z mol of the final product. (In Figure 3c, O2 corresponds to the oxidation of the reduced APM and R1corresponds to the reduction of the final product.) The drawn-out voltammetric peaks and slightly larger than 1:l ratio of 0 1 : 0 2 are the consequence of strong adsorption of the final product on the electrode. Identification of this electrochemical oxidation product in neutral pH solution was carried out as follows: apomorphine (11-16 mM) was electrolyzed in pH 6 or 7.4 buffer using a Pt gauze electrode. The blue-green oxidation product, adsorbed
lALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
on the electrode surface, was washed off with CHC13 and recrystallized from hot benzene. This product exhibited the same voltammetric behavior and adsorption pattern as the redox couple O1/R1. The UV-visible spectrum in isoamyl acetate was identical with that of the chemical oxidation product (11),oxo-APM (6). Elemental analysis of this dark purple electrochemical oxidation product yielded the following data: Calculated: C, 77.53; H, 4.98; N, 5.32; 0, 12.16. Found: C, 77.66; H, 4.77; N, 5.23; 0, 12.33. Calculation based on molecular formula (CI7Hl3NO2). A plausible mechanism can thus be concluded based on the positive identification on oxo-APM as the final product. An
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