Tetrahydrobiisoquinoline Derivatives by Reaction of Dopamine with

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Chem. Res. Toxicol. 2004, 17, 1190-1198

Tetrahydrobiisoquinoline Derivatives by Reaction of Dopamine with Glyoxal: A Novel Potential Degenerative Pathway of Catecholamines under Oxidative Stress Conditions Paola Manini,† Lucia Panzella,† Idolo Tedesco,‡ Fabio Petitto,‡ Gian Luigi Russo,‡ Alessandra Napolitano,† Anna Palumbo,§ and Marco d’Ischia*,† Department of Organic Chemistry and Biochemistry, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, Via Cinthia 4, I-80126 Naples, Italy, Institute of Food Sciences, National Research Council, via Roma 52 A/C, 83100 Avellino, Italy, and Laboratory of Biochemistry and Molecular Biology, Zoological Station “Anton Dohrn”, Villa Comunale, I-80121 Naples, Italy Received December 23, 2003

In 0.1 M phosphate buffer, pH 7.4, dopamine reacts with glyoxal, a cytotoxic and genotoxic R-oxoaldehyde produced by oxidative degradation of carbohydrates, to give three main products, two of which could be isolated and identified as the isomeric tetrahydrobiisoquinolines 1 and 2 by extensive two-dimensional NMR and mass spectrometric analysis. Time course studies indicated that 1 is the first intermediate in the process and changes slowly to 2 via an unstable species that escaped all efforts at isolation and structural identification. Products 1 and 2 were detected also among the species formed by the interaction of dopamine with oxidized carbohydrates, such as glucose, ribose, and fructose. Mechanistic evidence suggests that the formation of 1 proceeds by an unusual reaction pathway involving intramolecular cyclization of a double Schiff base intermediate followed by glyoxal-induced oxidation of the resulting octahydrobiisoquinoline intermediate (4). Subsequent conversion of 1 to 2 would involve a complex redox mechanism depending on an initial oxidation step. Product 2 was only poorly toxic to PC12 cells, whereas its methylated derivative 3 was as toxic as salsolinol, an established neurotoxin. Overall, these results throw light on a novel pathway of dopamine modification of potential relevance to the mechanisms underlying neurodegenerative changes in Parkinson’s disease and other disorders characterized by a prooxidant state.

Introduction Dopamine is a major catecholamine neurotransmitter in brain areas concerned with arousal, cognitive, reward, and emotional behaviors and in neural circuits that carry information about movement (1). The selective and progressive degeneration of neurons in the substantia nigra is the underlying cause of Parkinson’s disease, a disabling movement disorder characterized by tremor, rigidity, and a stooped posture. Although the etiology of Parkinson’s disease remains largely unclear, evidence has accrued for a pathogenetic role of oxidative stress-dependent dopamine toxicity leading to necrotic (2, 3) and apoptotic neuronal loss (4). A catecholamine toxicity mechanism associated with oxidative stress has also been implicated as a possible contributory factor in the pathogenesis of vitiligo (5-7). Potential routes of dopamine toxicity include monoamine oxidase-mediated oxidation and autoxidation, leading to potentially cytotoxic quinones and reactive oxygen species (8-11), the formation of cysteinyldopamine conjugates as mediators of DNA modifications * To whom correspondence should be addressed. Tel: +39-081674132. Fax: +39-081674393. E-mail: [email protected]. † University of Naples “Federico II”. ‡ National Research Council. § Zoological Station “Anton Dohrn”.

and caspase-3 activity (12-15), and exposure to nitric oxide-derived species (16-23). Another oxidative stress-dependent mechanism that may interfere with dopamine activity would involve interaction with reactive aldehyde species, particularly glyoxal. This is a mutagenic and cytotoxic metabolite that arises from the degradation of glucose and glycated proteins (24). Other sources of glyoxal may be oxidatively degraded nucleosides, nucleotides, and nucleic acids and lipid peroxidation (25, 26). The excess formation of glyoxal can elicit apoptosis and cell growth arrest via irreversible reactions with nucleic acids and proteins (27) and has been implicated in the development of diabetic complications, e.g., retinopathy, neuropathy, and nephropathy, as well as in atherosclerosis and aging. Against the adverse effects of glyoxal, cells have developed the GSH-dependent glyoxalase system (28). Under oxidative stress conditions, a depletion of GSH with consequent failure of the glyoxalase system and/or an elevation of cytoplasmic levels of dopamine (29), e.g., following L-DOPA therapy (30), may set favorable circumstances for a covalent interaction of the catecholamine with glyoxal. Consistent with this view is an early observation on the reaction of biogenic amines with glyoxal (31) as well as the extensive literature dealing with Pictet-Spengler condensations of dopamine

10.1021/tx034268q CCC: $27.50 © 2004 American Chemical Society Published on Web 08/20/2004

Tetrahydrobiisoquinolines from Dopamine and Glyoxal

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Table 1. NMR Data for Compounds 1-3 1a carbon

δC

1 3 4 4a 5 6 7 8 8a CH3O CH3O

154.7 41.9 25.4 134.0 115.8 176.6 144.8 116.4 115.2

a

δH (mult, J Hz) 3.83 (t, 8.0) 2.94 (t, 8.0)

2a 1H-13C

HMBC

7.02 2.94 3.83, 6.74 7.02

6.74 (s) 7.02 6.74, 7.02 7.02 (s) 6.74

δC

δH (mult, J Hz)

170.0 42.0 25.4 136.1 117.1 156.2 145.0 120.0 115.0

3.76 (t, 7.8) 2.93 (t, 7.8)

3b 1H-13C

HMBC

7.29 2.93 3.76, 6.75 7.29

6.75 (s) 7.29 6.75, 7.29 7.29 (s) 6.75

δC 164.5 47.2 24.7 131.3 109.9 151.3 147.1 110.0 118.5 55.6 55.7

δH (mult, J Hz) 3.71 (t, 8.0) 2.56 (t, 8.0)

1H-13C

HMBC

7.25 2.56 3.71 7.25

6.58 (s) 7.25 6.58, 7.25 7.25 (s) 6.58 3.76 3.78

Spectra registered in D2O. b Spectra registered in CDCl3.

with aldehydes leading to neurotoxic tetrahydroisoquinolines (32-34). Our past studies, moreover, demonstrated that dopamine, like its biogenetic precursor L-DOPA, reacts with D-glyceraldehyde, as well as with D-glucose, under biomimetic conditions to give tetrahydroisoquinoline products (35-37). In the present study, we report the complete structural characterization of two main products formed by the reaction of dopamine with glyoxal under biologically relevant conditions and disclose the unusual mechanistic features of their formation pathway. The feasibility of this chemistry in the frame of dopamine-carbohydrate interactions in an oxidizing environment was briefly addressed, and preliminary data on the generation and toxicity of the new compounds in PC12 cells were reported.

Experimental Procedures Materials. Dopamine hydrochloride, 2-(3,4-dimethoxyphenyl)ethylamine, glyoxal (hydrate in trimeric form), 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline hydrobromide, diethyloxalate, POCl3, HBr (48%, water solution), sodium borohydride, D-glucose, D-fructose, D-ribose, L-cysteine, N-acetyl-Lcysteine, reduced GSH, ascorbic acid, mannitol, potassium ferricyanide, Fe(NH4)(SO4)2, CuSO4‚7H2O, superoxide dismutase (SOD) from bovine liver (superoxide:superoxide oxidoreductase, EC 1.15.1.1), and catalase from bovine liver (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) were used. 6,7-Dihydroxy-1,2,3,4tetrahydroisoquinoline was synthesized as reported (11). The PC12 cell line was obtained from a rat pheochromocytoma (38) and was maintained in culture as reported (39). Briefly, cells were grown at 37 °C under 5% CO2 in RPMI 1640 supplemented with 10% (v/v) heat-inactivated horse serum, 5% (v/v) heat-inactivated fetal bovine serum, antibiotics (50 units/ mL of penicillin and 50 µg/mL of streptomycin), and glutamine (2 mM). Methods. UV and IR spectra were performed using a diode array and an FT-IR spectrophotometer, respectively. Electrospray (ESI+) mass spectra were recorded with a quadrupole mass spectrometer. For electron impact (EI) mass spectra, samples were ionized with a 70 eV beam, and the source was taken at 230 °C. 1H NMR spectra were obtained at 400 MHz with tert-butyl alcohol (δ 1.23) as the internal standard; 13C NMR spectra were run at 100 MHz; 1H-1H COSY, 1H-13C HMQC, and 1H-13C HMBC NMR experiments were recorded at 400 MHz. Analytical and preparative TLC were performed on silica gel plates F254 (0.25 and 0.5 mm, respectively) using chloroform/methanol 99:1 (v/v) as the eluant (eluant I). Column chromatography was performed on silica gel (60-230 mesh) using chloroform as the eluant (eluant II). Analytical and preparative HPLC were performed with an instrument equipped with a UV detector set at two different wavelengths: 280 and 380 nm. Octadecylsilane-coated columns (4.6 mm × 250 mm or

10 mm × 250 mm, 5 µm particle size) were used for analytical or preparative runs, respectively. Flow rates of 1.0 or 15 mL/min were used. Elution conditions were as follows: 0.1 M formic acid (solvent A), acetonitrile (solvent B), 0% solvent B, 5 min, from 0 to 30% solvent B gradient, 15 min (eluant III) and 0.1 M formic acid/acetonitrile 98:2 (v/v) (eluant IV). Molecular mechanics (MM+) and AM1/PM3 calculations were carried out with Hyperchem 5.0 package produced by Hypercube Inc. (Waterloo, Ontario, Canada). Syntheses of 2 and 3. Compound 2 was synthesized according to a previously reported procedure with slight modifications (40). In brief, a solution of 2-(3,4-dimethoxyphenyl)ethylamine (120 mg, 0.66 mmol) in toluene (5 mL) was treated with diethyloxalate (438 mg, 3.0 mmol) predissolved in toluene (1 mL). The mixture was kept under reflux for 5 h and then evaporated under reduced pressure. The oily residue was fractionated by a silica gel column (eluant II) to afford pure N,N′bis[2-(3,4-dimethoxyphenyl)ethyl]ethanediamide (Rf 0.78, eluant I, 130 mg, 95% yield). 1H NMR (40) (400 MHz, acetone-d6) δ (ppm): 2.83 (4H, t, J ) 7.0 Hz), 3.49 (4H, t, J ) 7.0 Hz), 3.77 (6H, s), 3.79 (6H, s), 6.74 (2H, dd, J ) 8.0, 1.6 Hz), 6.83 (2H, d, J ) 8.0 Hz), 6.86 (2H, d, J ) 1.6 Hz). 13C NMR (400 MHz, acetone-d6) δ (ppm): 35.3 (CH2), 41.6 (CH2), 56.0 (CH3), 62.9 (CH3), 112.8 (CH), 113.4 (CH), 121.4 (CH), 132.3 (C), 148.9 (C), 150.2 (C), 161.4 (C). N,N′-Bis[2-(3,4-dimethoxyphenyl)ethyl]ethanediamide (109 mg, 0.26 mmol) was dissolved in a solution of ethanol/dichloromethane 3:11 (v/v) (700 µL). The mixture was taken to 60 °C, treated with POCl3 (226 µL, 2.42 mmol), and then kept at 130 °C. After 5 h, light petroleum (550 µL) was added and the mixture was kept under vigorous stirring overnight. Then, the mixture was filtered under reduced pressure and the solid was dissolved in water/methanol (1.7 mL/50 µL) and treated with K2CO3 (350 mg) to adjust the pH to 10. The mixture was extracted with chloroform (2 mL × 3), and the combined organic layers were dried over sodium sulfate, treated with decolorizing carbon, and finally evaporated under reduced pressure to afford pure 3 (60 mg, 61% yield). Compound 3: UV (40) λmax (H2O or phosphate buffer, pH 3): 234, 288, 318 nm. 1H NMR (40) (400 MHz, CDCl3) δ (ppm): see Table 1. 13C NMR (400 MHz, CDCl3) δ (ppm): see Table 1. Compound 3 (200 mg, 0.53 mmol) was dissolved in HBr (1.8 mL), and the mixture was kept under reflux. After 6 h, the reaction was stopped and the precipitate was separated by filtration and washed first with water and then with acetone to afford 2 hydrobromide (180 mg, 62% yield). Compound 2: UV λmax (0.5 M HCl): 244, 311, 359 nm. log 3.09, 3.12, 3.15. FT-IR (CHCl3) νmax: 1620 cm-1. 1H NMR (40) (400 MHz, D2O) δ (ppm): see Table 1. 13C NMR (400 MHz, D2O) δ (ppm): see Table 1. ESI+/MS (m/z): 325 [M + H]+. EI/MS (m/z): 162 [M/2]+. 6,6′,7,7′-Tetrahydroxy-1,1′,2,2′,3,3′,4,4′-octahydro-1,1′-biisoquinoline (4). Compound 2 (100 mg, 0.31 mmol) was dissolved in methanol (10 mL) and treated with sodium borohydride (20 mg) to afford 4 (92 mg, 90% yield). 1H NMR data were in accord with those reported in the literature (40).

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Reaction of Dopamine with Glyoxal: General Procedure. A solution of dopamine (20-500 µM) in 0.1 M phosphate buffer (pH 7.4) was treated with glyoxal (20-500 µM) and was kept in a water bath thermostated at 37 °C. Aliquots of the reaction mixture were periodically withdrawn, acidified to pH 2 with 3 M HCl, and analyzed by HPLC using eluant III. Similar experiments were performed under an argon atmosphere and with the addition of 1 M equivalent of K3Fe(CN)6. In other experiments, additives, i.e., L-cysteine (500 µM), N-acetyl-Lcysteine (500 µM), reduced GSH (500 µM), ascorbic acid (500 µM), DMSO (500 mM), mannitol (100 mM), SOD (1000 U/mL), and catalase (1000 U/mL), were added to the reaction mixtures containing dopamine and glyoxal both at concentrations of 500 µM. Identification and quantitation of reaction products 1 and 2 were carried out by comparing retention times and integrated peak areas with external calibration curves for authentic samples. Isolation of 7,7′-Dihydroxy-3,3′,4,4′-tetrahydro-1,1′-biisoquinoline-6,6′(2H,2′H)dione (1). A solution of dopamine (500 mg, 2.6 mmol) in 400 mL of 0.1 M phosphate buffer (pH 7.4) was kept in a water bath thermostated at 37 °C and treated with glyoxal (552 mg, 2.6 mmol). After 5 min, the reaction mixture was acidified to pH 2 with 3 M HCl and evaporated under reduced pressure. The oily residue was subjected to preparative HPLC (eluant IV), which afforded pure 1 (tR 15.6 min, eluant III, 200 mg, 47% yield). UV λmax (0.5 M HCl): 243, 301, 349 nm. log 3.48, 3.32, 3.32. FT-IR (CHCl3) νmax: 1652, 1589 cm-1. 1H NMR (400 MHz, D2O) δ (ppm): see Table 1. 13C NMR (400 MHz, D2O) δ (ppm): see Table 1. ESI+/MS (m/z): 325 [M + H]+. EI/MS (m/z): 162 [M/2]+. Isolation of 6,6′,7,7′-Tetrahydroxy-3,3′,4,4′-tetrahydro1,1′-biisoquinoline (2). A solution of dopamine (500 mg, 2.6 mmol) in 400 mL of 0.1 M phosphate buffer (pH 7.4) was treated with glyoxal (552 mg, 2.6 mmol) and was kept in a water bath thermostated at 37 °C. After 2 h, the reaction mixture was acidified to pH 2 with 3 M HCl and evaporated under reduced pressure. The oily residue was subjected to preparative HPLC (eluant IV), which afforded pure 2 (tR 12 min, eluant III, 350 mg, 83% yield). Reaction of Dopamine with Autooxidized Carbohydrates: General Procedure. A solution of the carbohydrate (250 mM) in 0.1 M phosphate buffer (pH 7.4) was kept in a water bath thermostated at 37 °C for a week, with or without Fe3+ or Cu2+ salts (2.5 mM). Aliquots were periodically withdrawn and added to a solution of dopamine (50 µM to 2.5 mM) in 0.1 M phosphate buffer (pH 7.4). At 1 h reaction time, the mixture was acidified to pH 2 with 3 M HCl and analyzed by HPLC using eluant III. PC12 Cell Viability Assay. PC12 cell viability was estimated by the crystal violet method as previously described (39). Briefly, 0.15 × 106 cells/mL were cultured for 48 h in the presence of 2, 3, norsalsolinol, and salsolinol at different concentrations (50-500 µM). All molecules were dissolved in methanol at a 10 mM concentration. At the end of the incubation, PC12 cells were fixed in 10% (v/v) formalin buffered at pH 7, stained with 0.1% (v/v) crystal violet for 30 min, rinsed extensively, and air-dried. The uptaken dye was extracted with 10% acetic acid, and the optical density at 590 nm was determined. Within each experiment, the time points were determined in triplicate; each growth curve was performed at least twice. Dopamine-Glyoxal Interaction in PC12 Cells. An amount of 1 × 106 PC12 cells/mL (3 mL final volume) was loaded with dopamine and glyoxal to a final concentration of 500 µM each and cultured for 4 h. At the end of the incubation, the medium was saved for further analysis while the cells were washed twice in PBS, resuspended in 0.2 mL of PBS, and lysed by sonication. After centrifugation, the cell lysates were subjected to HPLC analysis (eluant III) for product determination.

Manini et al.

Figure 1. HPLC elution profile of the products formed by reaction of dopamine (500 µM) with glyoxal (500 µM) in 0.1 M phosphate buffer, pH 7.4, at 37 °C and 90 min reaction time. Elution conditions: eluant III, flow rate 1 mL/min; UV detection set at λ ) 280 and 380 nm.

Results Reaction of Dopamine with Glyoxal: Product Isolation and Structural Characterization. In a preliminary series of experiments, the reaction of dopamine with glyoxal was investigated in 0.1 M phosphate buffer, pH 7.4, at 37 °C. At 500 µΜ substrate concentration, HPLC analysis showed the generation of a welldefined pattern of products (Figure 1) comprising, besides dopamine, three main species designated A, B, and C. The reaction was then carried out on a preparative scale, and the resulting mixture was separated by preparative HPLC on reverse phase. By this approach, products A and C could be readily isolated in pure form and were subjected to extensive spectral analysis. The 1H NMR spectra of A and C in D2O displayed a relatively simple pattern of resonances, comprising two triplets (2H each) in the aliphatic region, ascribable to the protons on the ethylamine chains, and only two singlets (1H each) in the aromatic region, suggesting a cyclized structure. The 13C NMR spectra of A and C were similar and displayed nine signals, namely, two high field resonances relative to the CH2 carbons of the ethylamine chains, two aromatic CH resonances, and five signals due to quaternary carbons in the low field region of the spectrum. The latter group included in both cases two signals at around δ 145 and 155 and one rather deshielded signal at δ 170/176. The mass spectra registered in the ESI+ mode showed for both compounds a pseudomolecular ion peak at m/z 325, compatible with 2:1 dopamine-glyoxal adducts, whereas the EI/MS spectra revealed a major fragmentation peak at m/z 162. Of particularly diagnostic value were the pH-dependent chromophores of A and C. In 0.1 M HCl, compound A displayed three absorption maxima at λmax ) 243, 301, and 349 that shifted bathochromically to 261, 316, and 384 nm, whereas compound C exhibited similar absorption maxima at λmax ) 244, 311, and 359 nm shifting to 270, 326, and 400 at pH 7.4. This behavior was strongly reminiscent of that of 1-methyl-6,7-dihydroxy-3,4-dihydroisoquinoline (41). The FT-IR spectra of A and C showed significant differences in the double bond stretching region: whereas A displayed two bands at 1652 and 1589 cm-1, characteristic of enaminone groups (42), C displayed in the same region only one band at 1620 cm-1, suggestive of conju-


Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1193

gated aromatic imines (43). Overall, these data allowed the formulation of A and C as 7,7′-dihydroxy-3,3′,4,4′tetrahydro-1,1′-biisoquinoline-6,6′(2H,2′H)dione (1) and 6,6′,7,7′-tetrahydroxy-3,3′,4,4′-tetrahydro-1,1′-biisoquinoline (2), respectively. The identity of compound C with 2 was secured by comparing its chromatographic and spectral properties with those of an authentic sample, prepared by a Bischler-Napieralski synthesis (40), as well as of the methylated derivative 3. Figure 2. Time course of product formation by reaction of dopamine (500 µM) with glyoxal (500 µM) in 0.1 M phosphate buffer, pH 7.4, at 37 °C. Compound 1 ([); 2 (9). Product concentrations were determined by the integration of peaks in HPLC chromatograms. Peak areas were corrected using measured 380 for each species. Shown are the mean ( SD values for three separate experiments.

A summary of two-dimensional NMR correlation data with signal assignments for compounds 1-3 is given in Table 1. Consistent with the proposed tetrahydrobiisoquinoline nature, both compounds 1 and 2 could be reduced on treatment with sodium borohydride to give a product displaying an o-diphenolic chromophore (maximum at around 290 nm) and eluting earlier than 1 and 2 on a reverse phase column. This species was identified as the expected octahydrobiisoquinoline 4 by spectral analysis in comparison with literature data (40). The pH-dependent chromophores of 1 and 2 suggested that at physiological pH, e.g., 7.4, both structures exist at least in part in the ionized form. Consistent with this conclusion, the chromophore of 3, to which ionization was precluded, was identical to that of 2 at acidic pH. Unfortunately, all attempts at isolating the species eluted under peak B met with failure. This species was highly unstable and could not be obtained in pure form despite a careful search for appropriate chromatographic conditions, being apparently converted to 2. The UV spectrum displayed absorption maxima at 247, 308, and 359 nm in acidic medium and at 253, 338, 380, and 557 nm at pH 7.4, suggesting a quinonoid structure. However, attempts to obtain a derivative by the addition of ophenylenediamine, an o-quinone trapping reagent, to the reaction mixture at a time when the concentration of B was peaking met with failure. Reaction of Dopamine with Glyoxal: Mechanistic Issues. Kinetic analysis showed that the reaction of dopamine with glyoxal is first order with respect to dopamine, with an apparent rate constant of 5.33 × 10-3 s-1 as determined under pseudo-first-order conditions with a 10-fold excess of glyoxal. Figure 2 shows the temporal profile of the reaction with the catecholamine and glyoxal at 500 µM concentration. Product 1 was rapidly generated in the early stages, peaked at 120 min, and decreased below detection limits after 300 min. Product 2 was formed not earlier than 60 min, and its concentration increased throughout the reaction course (4 h), while compound B became detectable after 20 min of reaction time and attained a maximum concentration at 180 min, decreasing below detection limits after 4 h (not shown). When dopamine and glyoxal were reacted at 50 µM concentration, the reaction proceeded at a slower rate leading to products 1 and 2 as the main detectable species after 4 h. At 20 µM concentration, 1 was the main species

after 4 h, while B and 2 were still below detection limits and became visible only after more prolonged periods of time. HPLC traces recorded periodically during the reaction failed to show even the transient generation of other species: 1, 2, and B were the only detectable products at 280 or 380 nm as analytical wavelengths. Table 2 reports product formation as a function of substrate concentration, temperature, and reaction conditions. The formation ratio of 1:2 was found to vary significantly with the temperature of the reaction mixture. After 2 h, with dopamine and glyoxal at 500 µM concentration, the 1:2 ratio varied from 1.4 at 37 °C to 0.2 at 60 °C, whereas no 2 was detected at 0 °C. Time course studies suggested that B is produced en route from 1 to 2. Consistent with this interpretation, the incubation of pure 1 in 0.1 M phosphate buffer, pH 7.4, at 37 °C gave after 1 h both B and 2 as the major products. Notably, when the reaction of dopamine with glyoxal was carried out under an argon atmosphere, 1 was the main product, with no detectable B or 2 (Table 2). However, the addition of potassium ferricyanide after 2 h caused complete consumption of 1 with formation of B and then 2. Overall, these data indicated that the formation of 1 proceeds very rapidly and does not require molecular oxygen, although the compound is at a four electron higher oxidation state than 4, the expected PictetSpengler condensation product of dopamine with glyoxal. Most likely, the critical oxidation step leading to 1 is brought about by glyoxal itself. It should be noted that the biisoquinoline system contains one molecule of the dialdehyde and two molecules of dopamine, implying that when equimolar amounts of the substrates are used some glyoxal would be available to effect oxidation of the Pictet-Spengler adduct. This conclusion was corroborated by a separate experiment in which the octahydrobiisoquinoline 4, prepared by borohydride reduction of synthetic 2, was exposed to glyoxal (1 M equivalent) and was found to be rapidly converted to 2 as the main product by HPLC analysis. When the reaction of 500 µM dopamine with glyoxal was carried out with half molar equivalents of glyoxal, HPLC analysis (Table 2) showed slower reaction kinetics with small amounts of 1 and B but little or no 2. Under these latter conditions, appreciable pigment formation was observed. These data suggested that whatever the dopamine/glyoxal molar ratio, part of the glyoxal was invariably engaged with

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Table 2. Compounds 1, 2, and Residual Dopamine Concentrations by Reaction of Dopamine with Glyoxala dopamine (µM)

glyoxal (µM)

conditions/ additives

20 50 500 500 500 500 500 500

20 50 500 250 500 500 500 500

37 °C 37 °C 37 °C 37 °C 60 °C 0 °C Ar atmosphere Ar atmosphere/ K3Fe(CN)6 (500 µM)

1 (µM)b

2 (µM)b

0.43 ( 0.02c 7.10 ( 0.30c 97.00 ( 4.00 45.00 ( 2.00 63.00 ( 3.00 32.00 ( 2.00 324.00 ( 15.0

c 3.4 ( 0.1c 71.0 ( 3.0 315.0 ( 15.0 101.0 ( 5.0

residual dopamine (µM)b 12.5 ( 0.6c 27.3 ( 1.3c 140.0 ( 7.0 200.0 ( 9.0 141.0 ( 7.0 229.0 ( 10.0 138.0 ( 6.0 142.0 ( 7.0

a All reactions were carried out in 0.1 M phosphate buffer, pH 7.4. Product concentrations were determined at 2 h reaction time unless otherwise stated. b Average of three determinations ( SD. c Determined at 4 h reaction time.

Table 3. Effect of Additives on the Reaction of Dopamine with Glyoxala

additives

1 (µM)b,c

2 (µM)b,c

residual dopamine (µM)b,c

DMSO (500 mM) mannitol (100 mM) SOD (1000 U/mL) catalase (1000 U/mL) cysteine (500 µM) N-acetylcysteine (500 µM) GSH (500 µM) ascorbate (500 µM)

75.0 ( 3.0 66.0 ( 3.0 74.0 ( 4.0 242.0 ( 10.0 72.0 ( 3.0 14.0 ( 0.7 184.0 ( 9.0 158.0 ( 8.0 139.0 ( 7.0

24.0 ( 1.0 19.0 ( 1.0 22.0 ( 1.0 ) 23.0 ( 1.0 ) ) 7.0 ( 0.3 22.0 ( 1.0

179 ( 9 151 ( 7 153 ( 7 152 ( 7 153 ( 7 239 ( 10 174 ( 7 185 ( 9 170 ( 7

noned

a All reactions were carried out in 0.1 M phosphate buffer, pH 7.4. b Product concentrations were determined at 90 min reaction time. c Average of three determinations ( SD. d [Dopamine] ) [glyoxal] ) 500 µM.

adduct oxidation and that unreacted dopamine undergoes a slow oxidation to melanin. The ability of glyoxal to oxidize tetrahydroisoquinolines was confirmed in a similar experiment in which salsolinol (500 µM) was reacted with an equimolar amount of glyoxal in 0.1 M phosphate buffer, pH 7.4. Spectrophotometric monitoring of the reaction showed smooth oxidation of the compound to the corresponding dihydroisoquinoline. In the absence of glyoxal, no chromophoric change was detected during a comparable period of time. The data in Table 2 indicated that an oxidative activation step was critical for the subsequent conversion of 1 to its tautomer 2. To address this intriguing point, the effect of some common scavengers of reactive oxygen species and antioxidants on product formation was investigated. As shown in Table 3, SOD completely suppressed the formation of 2 and compound B, inducing the accumulation of 1, whereas hydroxyl radical trappers, such as DMSO, mannitol, and catalase did not affect the reaction course. A similar effect was observed on addition of biologically relevant thiol compounds such as GSH and N-acetylcysteine, which inhibited or suppressed the formation of 2. Cysteine, on the other hand, inhibited the formation of 1 due to its ability to form stable cyclic adducts with glyoxal (44). Ascorbate favored the accumulation of 1 with only modest conversion to B and 2. Reaction of Dopamine with Autoxidized Carbohydrates. In another set of experiments, the formation of tetrahydrobiisoquinolines from the interaction of dopamine with oxidized carbohydrates was briefly investigated. The reaction was typically carried out by exposing the catecholamine to mixtures obtained by autoxi-

Figure 3. Effect of metal ions on the formation of compounds 1 (a) and 2 (b) by reaction of dopamine (2.5 mM) with an autoxidized mixture of glucose (250 mM), fructose (250 mM), and ribose (250 mM). Autoxidation reactions were stopped at 3 days; incubation mixtures were analyzed 1 h after dopamine addition. No additives (black bars), Fe3+ (2.5 mM) added (white bars), and Cu2+ (2.5 mM) added (gray bars). Product concentrations were determined by integration of peaks in HPLC chromatograms. Peak areas were corrected using measured 380 for each species. Shown are the mean ( SD values for three separate experiments.

dation of three representative sugars, viz. glucose, fructose, and ribose. Autoxidation was carried out by leaving the carbohydrate (250 mM) in 0.1 M phosphate buffer, pH 7.4, at 37 °C for 7 days, under reported conditions (45). In some cases, catalytic amounts of transition metal ions of biological relevance, e.g., Fe3+ and Cu2+, were added (46, 47) to enhance carbohydrate degradation. Aliquots of the reaction mixtures were periodically withdrawn and allowed to react with dopamine at varying concentrations. HPLC analysis revealed in all cases after 1 h of incubation the formation of the typical pattern of glyoxalderived products 1, 2, and B. These latter were clearly detectable at dopamine concentrations higher than 1 mM. As shown in Figure 3, the yields of formation of 1 (panel a) and 2 (panel b) varied with the nature of the carbohydrate, under comparable conditions. In the absence of metal ions, glucose gave lesser amounts of 1 and 2, indicating a lower susceptibility to oxidative breakdown to yield glyoxal, whereas ribose proved the most effective precursor, leading up to 20 times higher yields


Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1195 Scheme 1. Proposed Mechanism of Formation of Compound 1 by Reaction of Dopamine with Glyoxal

Figure 4. Viability of PC12 cells (0.15 × 106/mL) incubated for 48 h with 2, 3, norsalsolinol, and salsolinol. Substrate concentrations: 50 (black bars), 100 (white bars), and 500 (grey bars) µM. Each time point was determined in triplicate ( SD.

of 1 and 2 as compared to glucose. The addition of metal ions during autoxidations markedly increased dopamine-glyoxal adduct yields from all carbohydrates examined. Moreover, after 1 h of reaction time, Cu2+ promoted the conversion of 1 to 2, whereas Fe3+ was much less efficient, 1 still being the major product. Preliminary Biological Assays. The effect of 2 on PC12 viability was briefly tested in comparison with 3, using norsalsolinol and salsolinol as positive controls (48-52). The data in Figure 4 indicate a significant toxicity of 2 only at relatively high concentrations. Interestingly enough, however, the tetramethyl derivative 3 was found to be as toxic as salsolinol after 48 h of incubation. The higher activity of 3 as compared to 2 can be reasonably ascribed to its more lipophilic character and the absence of appreciable ionization at neutral pH. The in vitro generation of dopamine-glyoxal adducts within cells was then assessed in separate experiments in which PC12 cells were loaded with dopamine and glyoxal at 500 µM concentration each. After 4 h of incubation time, HPLC analysis of the cell lysate showed the formation of 1 (1.8 nmol/106 cells) and 2 (2.0 nmol/106 cells). Under such conditions, cell viability was not affected to any appreciable extent.

Discussion The generation of elevated levels of glyoxal is an established biochemical correlate in a variety of pathological states associated with oxidative stress (53). In this study, we show that glyoxal can react with dopamine to give the previously unknown tetrahydrobiisoquinoline compounds 1 and 2. Two features of the reported chemistry are worthy of comment, namely, the generation of isoquinoline derivatives at a higher oxidation state than expected on the basis of a simple Pictet-Spengler type condensation and the apparent requirement of oxidative activation for isomerization of 1 to 2. The Pictet-Spengler condensation of dopamine with aldehyde compounds involves formation of a transient Schiff base followed by intramolecular attack of the electron rich o-diphenolic ring to the imine functionality leading to 6,7-dihydroxytetrahydroisoquinoline systems (54). As a rule, these adducts are relatively stable under the aerobic conditions of the reaction and are only slowly converted to the corresponding dihydroisoquinolines. The observed formation of a tetrahydrobiisoquinoline derivative, 1, instead of the expected octahydrobiisoquinoline product even under an argon atmosphere, and mecha-

nistic experiments, would call into play a dual role of glyoxal as both carbonyl counterpart for dopamine and oxidizing agent for the first formed Pictet-Spengler product. The ability of R-oxoaldehydes to act as oxidants is documented in the literature and is illustrated by the reported generation of free radicals during glycation of amino acids with methylglyoxal (55). Most likely, the octahydrobiisoquinoline 4 initially formed by PictetSpengler cyclization of the double Schiff base is oxidized by the unreacted glyoxal in the medium to yield the tetrahydrobiisoquinoline 1. Viability of this route would be supported by the observed ability of glyoxal to oxidize 4 and salsolinol. The production of reactive oxygen species by aerobic oxidation of glyoxal radical anion could then provide an important contribution to these oxidation steps. The proposed mechanistic scheme for the reaction of dopamine with glyoxal leading to 1 is illustrated in Scheme 1. The mechanism of conversion of 1 to 2 is also intriguing. Because 1 and 2 are at the same oxidation level, their conversion would involve a mere tautomerization step. Yet, the bulk of available evidence, including the effects of additives, would argue in favor of a sequence of redox processes. A plausible explanation would be that 1 is reluctant to isomerize at neutral pH but is susceptible to oxidation to give a quinonoid derivative, which would be prone to isomerize to a labile intermediate, designated X (Scheme 2). This latter would then be reduced to 2, e.g., by a disproportionation reaction. A predominance of the quinone methide tautomers of 6,7-dihydroxy-3,4dihydroisoquinolines vs the imine forms at neutral pH has been reported (56). Preliminary data for minimized structures of 1 and 2 obtained by molecular mechanics (MM+ force field) and optimized using the semiempirical AM1 and PM3 algorithms showed that both compounds tend to adopt similar conformations in which the isoquinoline rings form dihedral angles N-C1-C1′-N′ approximating 90° for 1 and 2, respectively, with C1-C1′ bond lengths of about 1.50 Å (Figure 5). This

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Figure 5. Energy-minimized (PM3) structures of compounds 1 and 2.

Scheme 2. Proposed Mechanism of Formation of Compound 2 from 1

implies that the C1-C1′ bond in both 1 and 2 has largely single bond character and that interaction between the π-electron systems of the two isoquinoline rings is not of primary importance. In the proposed conversion path (Scheme 2), the oxidized counterpart of X, denoted Y, would be engaged with a redox cycling process whose outcome would be the oxidation of 1 with concomitant regeneration of X. Oxidants such as superoxide and ferricyanide, but not hydrogen peroxide, can promote the conversion of 1 to X, whereas SOD and thiol compounds would prevent it. Cysteine, on the other hand, would act as a trap for glyoxal to give thiazolidine intermediates, as reported (44). It would be tempting to suggest the identity of B with X, but the elusive nature of the product would make this conclusion more a matter of surmise rather than of direct proof. The generation of 1 and 2 by interaction of dopamine with oxidized carbohydrates clearly reflects a similar condensation with glyoxal or chemically equivalent degradation fragments. The oxidative breakdown of sugars to give R-dicarbonyl intermediates is central to glycation reactions, including AGE formation, and is amply documented in the literature (24, 57). The underlying chemistry encompasses a combination of oxidative and nonoxidative processes, e.g., reverse aldol condensations, that are markedly susceptible to catalysis by transition metal cations acting both as redox agents, favoring oxygen radical formation, and Lewis acids. Apparent differences in the kinetics of biisoquinoline formation and product patterns would therefore reflect the intrinsic structural characteristics of the sugars examined and their relative

Manini et al.

susceptibility to the influence of metal ions. Although free transition metal ions are not likely to exist in vivo, their effects on glycations reactions are usually considered to hasten the processes and to probe certain mechanistic facets connected, e.g., with oxygen radical formation (46, 47). In this regard, the superior ability of Cu(II) ions to promote glycation reactions was already noted (47) and is consistent with the results reported in the present paper. On the basis of previous studies on PictetSpengler condensations of catecholamine compounds (35-37, 58), we envisage a dual role of metal ions both as enhancers of glyoxal formation from oxidized carbohydrates and as promoters of Schiff base cyclization in the construction of the biisoquinoline skeleton. In conclusion, the interaction of glyoxal with dopamine described in the present paper hints at a novel possible contributory mechanism underlying oxidative stressmediated impairment of dopamine neurotransmission in neuronal degeneration and other disease states associated with altered dopamine metabolism and function. Although the proposed interaction appears to produce toxic responses only under select conditions in which the concentrations of dopamine and glyoxal rise significantly (59-61), the isolation of compounds 1 and 2 from cells loaded with dopamine and glyoxal would support the possible biological relevance of the reported chemistry.

Acknowledgment. This work was carried out in the frame of the projects “Vitiligine: Studio Sui Meccanismi Patogenetici e Sulle Modalita` di Approccio Terapeutico” (IFO convenzione 121, Italian Ministry of Health) and “Constitutional Bone Marrow Failures: Molecular Characterization, Pathogenesis and Novel Therapeutic Approaches” (MURST, PRIN 2003). We thank the Centro Interdipartimentale di Metodologie Chimico-Fisiche (CIMCF, University of Naples Federico II) for NMR and mass spectra. We thank Silvana Corsani for technical assistance.

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Tetrahydrobiisoquinoline Derivatives by Reaction of Dopamine with

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