Chem. Res. Toxicol. 2003, 16, 493-501
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Stability of the Putative Neurotoxin Tryptamine-4,5-dione Monika Z. Wrona,† Xiang-Rong Jiang,† Yashige Kotake,‡ and Glenn Dryhurst*,† Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, and Free Radicals in Biology Group, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 Received August 22, 2002
Tryptamine-4,5-dione (1) is formed by oxidation of the neurotransmitter 5-hydroxytryptamine by reactive oxygen and reactive nitrogen species, and on the basis of in vitro and in vivo studies, it has been proposed to be a neurotoxin that may contribute to the selective neurodegeneration in Alzheimer’s disease and the serotonergic neurotoxicity of methamphetamine. Several investigators have noted that under the conditions employed in the past to synthesize 1 and explore its in vitro and in vivo biological properties, the dione is somewhat unstable. In the present study, the stability of 1 has been investigated in a number of media employed in previous investigations to synthesize the dione and evaluate its biological properties. At low concentrations (e200 µM), 1 is most stable in artificial cerebrospinal fluid (aCSF, pH 6-6.5) in which it decomposes e10% over 24 h forming primarily 3-(2-aminoethyl)-6-[3′-(2-aminoethyl)indol-4′,5′-dione-7′-yl]-5-hydroxyindole-4,7-dione (10). In phosphate buffer or 0.5 M NH4Cl solutions at pH 7.4 and in acidic solution (e.g., 0.01 M HCl), such low concentrations of 1 also decompose to 10 although somewhat more rapidly than in aCSF. As the concentration of 1 is increased in all of these media, its decomposition becomes more rapid and shifts toward formation of 7,7′-bi-(5-hydroxytryptamine-4-one) (9) and its autoxidation product 7,7′-bitryptamine-4,5-dione (11). At 20 mM concentrations in aCSF or at pH 7.4, 1 rapidly decomposes to a dark, uncharacterized, presumably polymeric precipitate. However, in 0.01 M HCl solution g20 mM, 1 rapidly and almost quantitatively dimerizes to 9. The initial reaction of 1, which leads to the ultimate formation of 9 or 11 and 10, is the nucleophilic addition of water to the C(7) position of the dione to form 4,5,7-trihydroxytryptamine (2). Oxidation of 2 by 1 and/or molecular oxygen forms radical species, the predominant form of which has been detected by electron spin resonance spectroscopy using a spin stabilization method. Subsequent reactions of radical intermediates lead to the formation of 9 or 11 and 10. The results of this investigation are discussed in terms of previous in vitro and in vivo biological properties of 1 and its possible role in the serotonergic neurotoxicity of methamphetamine and neurodegenerative diseases.
Introduction Compound 11 is a major product of the in vitro oxidation of the neurotransmitter 5-HT (serotonin) (1, 2). Quinones are often cytotoxic and have found uses as anticancer and antibacterial drugs (3, 4). Their toxicity is attributed to the alkylation of critical protein thiol and amino groups and/or to redox cycling reactions, which generate cytotoxic ROS (5). Indeed, in vitro, dione 1 reacts rapidly with Cys, GSH, and Cys residues of proteins (1, 6-8). Interest in 1 derives from the possibility that it may be an endogenously formed neurotoxin produced as a result of the oxidation of 5-HT by ROS and/ or RNS, which may contribute to the degeneration of * To whom correspondence should be addressed. Tel: (405)325-4811. Fax: (405)325-6111. E-mail:
[email protected]. † University of Oklahoma. ‡ Oklahoma Medical Research Foundation. 1 Abbreviations: aCSF, artificial cerebrospinal fluid; AD, Alzheimer’s disease; CSF, cerebrospinal fluid; HFSC, hyperfine coupling constant; 5-HT, 5-hydroxytryptamine; MA, methamphetamine; MeCN, acetonitrile; MeOH, methanol; NHE, normal hydrogen electrode; NO•, nitric oxide; ONOO-, peroxynitrite; O2-•, superoxide radical anion; RNS, reactive nitrogen species; ROS, reactive oxygen species; SCE, saturated calomel electrode; SOD, superoxide dismutase; 1, tryptamine4,5-dione; TFA, trifluoracetic acid; TPH, tryptophan hydroxylase.
serotonergic neurons in disorders such as AD or evoked by drugs such as MA and 3,4-methylenedioxymethamphetamine (9, 10). Indeed, an abnormal oxidized form of 5-HT has been detected in the CSF of AD patients but not in age-matched controls (11, 12). While such oxidized forms of 5-HT have not been unambiguously identified, one such aberrant metabolite exhibits chromatographic and redox (electrochemical) properties that suggest it might be 1 or a structurally similar compound (11-13). Furthermore, microinjections of relatively concentrated solutions (up to 21 mM) of a compound believed to be 1 into the brains of rats evoke neurotoxic effects particularly in medial limbic systems that degenerate in AD (12-14). In vitro, 1 irreversibly inhibits several mitochondrial respiratory enzyme complexes (9, 15), Gua nucleotide-binding regulatory proteins (16), and TPH (17) probably by covalent modification of active site Cys residues of these proteins. Dione 1 was initially identified as a product of the controlled potential electrochemical oxidation of e30 µM 5-HT in aqueous 0.01 M HCl solution (18). At such low concentrations of 5-HT, the electrochemical oxidation reaction forms 1 as the sole product whereas at higher
10.1021/tx020080f CCC: $25.00 © 2003 American Chemical Society Published on Web 03/14/2003
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concentrations many additional products are formed (18). Most studies aimed at exploring the biological properties of 1 have used electrochemical methods to prepare e30 µM solutions of the dione in 0.01 M HCl that are then adjusted to the desired pH. However, even low concentrations of 1 (e30 µM) in 0.01 M HCl solution are only moderately stable (13, 18, 19) although the resulting decomposition products have not been identified. In recent studies, we have also noted that low concentrations of 1 decompose in the media employed by us and other investigators to study the in vitro and in vivo biological properties of the dione. Such media include phosphate buffer (11, 15) and aqueous 0.5 M NH4Cl at pH 7.4 (8, 12). Furthermore, the instability of 1 is particularly apparent at a millimolar concentration of the dione. This is of concern because the proposition that 1 may be neurotoxic has been based on experiments in which up to 21 mM concentrations of the dione were administered into rat brain (12-14). In this paper, information is provided concerning the stability of 1 in various aqueous media that have been or might be employed in studies aimed at investigating its neurotoxic and other neurobiological properties. The products of decomposition have been isolated and identified, and information bearing on reaction pathways that might lead to these products is presented.
Materials and Methods Chemicals. 5-HT, SOD, diethylenetriaminepentaacetic acid (DTPA), nitroblue tetrazolium (NBT), Gly, and TFA were obtained from Sigma; HPLC grade MeCN and MeOH were obtained from Fisher Scientific (Springfield, NJ); and potassium nitrosodisulfonate was obtained from Aldrich (Milwaukee, WI). All other chemicals were of the highest purity commercially available and were used without further purification. Spectroscopy. 1H and 13C NMR spectra were recorded on either a Varian XL-300 or XL-400 spectrometer. FAB-MS were obtained with a VG Instruments (Manchester, UK) ZAB-E spectrometer. UV-visible spectra were recorded on a HewlettPackard model 8455A diode array spectrophotometer. ESR spectra were obtained with a Bruker ER 300 spectrometer with the following settings: modulation amplitude, 0.1 G; time constant, 40 ms; field sweep rate, 40 G/83 s; and microwave power attenuation of 18 db (200 mw at 0 db). Semiquinone anion radical was best produced by rapidly dissolving 1 (HCl salt, 1.0 mg) in 200 µL of 70% ethanol in water containing 100 mM sodium hydroxide and 200 mM magnesium sulfate. The resulting solution was immediately transferred to a quartz flat cell, and the ESR spectrum was recorded. The entire procedure involving dissolution of 1, transfer of the solution to a quartz flat cell, and instrument tuning was achieved in less than 1 min. HFSCs for hydrogen and nitrogen atoms in the radical were determined using computer spectral simulation. Preparative HPLC. Preparative HPLC employed a Gilson (Middleton, WI) 712 binary gradient system equipped with a HM Holochrome UV detector (set at 254 nm) and a reversed phase column (Bakerbond C18, 25 cm × 2.1 cm, 10 µm particle size, J. T. Baker, Phillipsburg, NJ) protected with a guard column (5 cm × 0.9 cm) packed with the same stationary phase. Preparative HPLC, used to purify the decomposition products of 1, employed a binary gradient mobile phase system. Solvent A was 5% MeCN/95% deionized H2O (v/v) adjusted to pH 2.1 with TFA. Solvent B was 40% MeCN/60% H2O adjusted to pH 2.1 with TFA. The gradient was 0-40 min, linear gradient from 100% solvent A to 32% solvent B; 40-42 min, linear gradient to 100% solvent B. The flow rate was 10 mL min-1. Analytical HPLC. Analytical HPLC with UV detection (HPLC-UV, detector set at 254 nm), employed to monitor the decomposition of 1 and the resultant products formed in various
Wrona et al. media, utilized a reversed phase column (Phenomenex RP-18, 5 µm, 100 mm × 3.2 mm, Torrence, CA) and a Rheodyne (Cotati, CA) 7125 injector equipped with a 50 µL sample loop. A binary gradient mobile phase system was used consisting of solvent A and solvent C (50% MeCN/50% H2O adjusted to pH 2.1 with TFA). The gradient was 0-20 min, linear gradient from 100% solvent A to 25% solvent C; 20-22 min, linear gradient to 100% solvent C. The flow rate was 0.6 mL min-1. Synthetic Procedures. In the section that follows, the procedures employed to synthesize decomposition products of 1 are described together with spectroscopic evidence in support of their proposed structures. Typically, the purification step using preparative HPLC was carried out at least twice. The purity of each compound was based on the results of highresolution FAB-MS, NMR spectra, and analytical HPLC-UV analysis. Synthesis of 1. The procedures employed for synthesis of 1, based on the oxidation of 5-HT by potassium nitrosodisulfonate in aqueous solution and purification by preparative HPLC methods, have been described in detail elsewhere (15). Compound 1 was isolated as a hygroscopic red-brown solid with an elemental analysis of C (48.40%), H (5.18%), N (10.85%), Cl (13.98%) corresponding to 1. HCl‚1.2H2O (calcd: 48.37% C, 5.40% H, 11.29% N, and 14.30% Cl). FAB-MS (glycerol matrix): m/z 191.0825 (MH+, 7%, C10H11N2O2); calcd m/z 191.0821. 1H NMR (D O, 300 MHz): δ 7.24 (d, J ) 9.9 Hz, 1H, C(7)-H), 2 6.78 (s, 1H, C(2)-H), 5.89 (d, J ) 9.9 Hz, 1H, C(6)-H), 3.20 (t, J ) 6.9 Hz, 2H, C(β)-H2), 2.89 (t, J ) 6.9 Hz, 2H, C(R)-H2). 1H NMR (Me2SO-d6, 300 MHz): δ 12.14 (bs, 1H, N(1)-H), 7.98 (bs, 3H, NH3+), 7.35 (d, J ) 9.9 Hz, 1H, C(7)-H), 6.91 (s, 1H, C(2)H), 5.92 (d, J ) 9.9 Hz, 1H, C(6)-H), 3.02 (t, J ) 7.2 Hz, 2H, C(β)-H2), 2.89 (t, J ) 7.2 Hz, 2H, C(R)-H2). Synthesis of 3-(2-Aminoethyl-6-[3′-(2-aminoethyl)-indol4′,5′-dione-7′-yl]-5-hydroxyindole-4,7-dione (10). A solution of 1 (200 µM) in 200 mL of 0.01 M HCl or 0.1 M phosphate buffer (pH 7.4) exposed to the atmosphere was stirred at room temperature overnight. During this period, the initially bright purple color of 1 changed to red. The product solution was then pumped into the preparative HPLC system. The solution eluted under the chromatographic peak corresponding to 10, retention time (tR) ) 32.6 min, was collected, immediately frozen, and then lyophilized to give a red-brown solid. A freshly chromatographed solution of 10 dissolved in the HPLC mobile phase (apparent pH 2.1) had a red-brown color and UV-visible spectrum with λmax ) 480, 354, 292, and 232 nm. FAB-MS (3-nitrobenzyl alcohol matrix): m/z 395.1338 (MH+, 4%, C20H19N4O5); calcd m/z 395.1335. 1H NMR (D2O, 400 MHz): δ 6.70 (s, 1H, C(2)-H), 6.47 (s, 1H, C(2′)-H), 5.68 (s, 1H, C(6′)-H), 3.04 (m, 4H, C(β)H2, C(β′)-H2), 2.79 (t, J ) 6.8 Hz, 2H, C(R)-H2), 2.66 (t, J ) 6.8 Hz, 2H, C(R′)-H2). 1H NMR (Me2SO-d6, 300 MHz): δ 12.53 (bs, 1H, N(1)-H), 11.67 (bs, 1H, N(1′)-H), 8.37 (bs, 3H, NH3+), 8.25 (bs, 3H, NH3+), 7.09 (s, 1H, C(2)-H), 6.87 (s, 1H, C(2′)H), 5.86 (s, 1H, C(6′)-H), 3.08 (m, 4H, C(β)-H2, C(β′)-H2), 2.97 (m, 4H, C(R)-H2, C(R′)-H2). Synthesis of 7,7′-Bi-(5-hydroxytryptamine-4-one) (9). Concentrated TFA (0.2 mL) was added to a 21 mM solution of 1 in 1.0 mL of deionized water. After it was stirred for 3 min, the solution was diluted with water until the pH was 2.1. The resulting solution was then pumped into the preparative HPLC system. The solution eluted under the peak corresponding to 9 (tR ) 42.9 min) was collected in a flask suspended in a bath of dry ice and acetone, which was continuously purged with nitrogen. After it was lyophilized, 9 was obtained as a deep red fluffy solid. A freshly chromatographed solution of 9 dissolved in the HPLC mobile phase (pH 2.1) was a deep purple color with λmax ) 544, 366, 304, and 222 nm. FAB-MS (glycerol matrix): m/z 383.1693 (MH2H+, 4%, C20H23N4O4); calcd m/z 383.1719. 1H NMR (D2O, 300 MHz): δ 6.87 (s, 2H, C(2)-H, C(2′)-H), 6.48 (s, 2H, C(6)-H, C(6′)-H), 3.28 (t, J ) 6.9 Hz, 4H, C(β)-H2, C(β′)-H2), 2.99 (t, J ) 6.9 Hz, 4H, C(R)-H2, C(R′)-H2).
Stability of Tryptamine-4,5-dione Synthesis of 7,7′-Bitryptamine-4,5-dione (11). Dione 1 (21 mM) was dissolved in 1.0 mL of water, and then, 0.2 mL of concentrated TFA was added. After it was stirred for 3 min, the solution was diluted with water until the pH was 2.1. The solution, which contained 9, was carefully adjusted to pH 4.0 with 1 M ammonium hydroxide. Within 30 min, the deep purple color of 9 changed to blue-purple, characteristic of 11. This solution was pumped into the preparative HPLC system and chromatographed. The solution eluted under the peak corresponding to 9 (tR ) 37.4 min) and was collected, frozen, and freeze-dried to give a red-brown solid. A freshly chromatographed solution of 11 dissolved in the HPLC mobile phase (pH 2.1) was blue-purple and exhibited a UV-visible spectrum with λmax ) 556, 366, 302, and 236 nm. FAB-MS (3-nitrobenzyl alcohol matrix): m/z 381.1600 (MH2H+, 11%, C20H21N4O4); calcd m/z 381.1582. 1H NMR (Me2SO-d6, 300 MHz): δ 12.06 (bs, 2H, N(1)-H, N(1′)-H), 8.05 (bs, 6H, 2 NH3+), 6.97 (s, 2H, C(2)-H, C(2′)-H), 6.05 (s, 2H, C(6)-H, C(6′)-H), 3.04 (m, 4H, C(β)-H2, C(β′)-H2), 2.95 (m, 4H, C(R)-H2, C(R′)-H2). 13C NMR (D2O, 400 MHz): δ 183.5, 174.2, 138.8, 134.6, 124.0, 123.8, 122.2, 119.6, 38.1, 24.0. Synthesis of 5-Methoxy-5′-hydroxy-7,7′-bitryptamine-4one (14) and 7,7′-Bi-(5-methoxytryptamine-4-one) (16). Dione 1 (21 mM) was dissolved in 1.0 mL of MeOH followed by addition of 0.2 mL of concentrated TFA, and the solution was stirred with a Teflon-coated magnetic stirring bar. After 10 min, the deep purple solution was diluted with deionized water until the pH was 2.1 and then pumped into the preparative HPLC system and chromatographed. The solutions eluted under the peaks corresponding to 14 (tR ) 48.8 min) and 16 (tR ) 50.7 min) were collected separately and lyophilized to give deep redpurple fluffy solids. A freshly chromatographed solution of 14 dissolved in the HPLC mobile phase (pH 2.1) was bright purple and exhibited a UV-visible spectrum with λmax ) 548, 368, and 300 nm. FAB-MS (glycerol matrix) of 14: m/z 397.1882 (MH2H+, 8%, C21H25N4O4); calcd m/z 397.1876. 1H NMR (D2O, 400 MHz) of 14: δ 6.83 (s, 1H, C(2)-H), 6.75 (s, 1H, C(2′)-H), 6.46 (s, 1H, C(6)-H), 6.22 (s, 1H, C(6′)-H), 3.51 (s, 3H, OCH3), 3.18 (t, 2H, J ) 6.8 Hz, C(β)-H2), 3.14 (t, 2H, J ) 6.8 Hz, C(β′)-H2), 2.92 (t, 2H, J ) 6.8 Hz, C(R)-H2), 2.85 (t, 2H, J ) 6.8 Hz, C(R′)H2). A freshly chromatographed solution of 16 dissolved in the HPLC mobile phase (pH 2.1) was bright purple and exhibited a UV-visible spectrum with λmax ) 552, 372, and 298 nm. FABMS (glycerol matrix) of 16: m/z 409.1890 (MH+, 3%, C22H25N4O4); calcd m/z 409.1876 and 411.2035 (MH2H+, 11%, C22H27N4O4); calcd m/z 411.2032. 1H NMR (D2O, 400 MHz) of 16: δ 6.94 (s, 2H, C(2)-H, C(2′)-H), 6.46 (s, 2H, C(6)-H, C(6′)-H), 3.51 (s, 6H, 2 × OCH3), 3.20 (t, J ) 6.8 Hz, 4H, C(β)-H2, C(β′)-H2), 2.92 (t, J ) 6.8 Hz, 4H, C(R)-H2, C(R′)-H2). Superoxide (O2-•) Assay. The method employed to detect O2-• generation during the decomposition of 1 was based on the SOD inhibitable reduction of NBT to nitroblue formozan (NBF), monitored at 560 nm, in 2 M potassium glycinate at pH 10 (20, 21). To 700 µL of NBT (0.25 mM) in glycinate buffer was added 100 µL of freshly prepared 1 so that the final concentration of the dione was 7, 13, or 26 µM. The changes in absorbance at 560 nm were periodically measured for 30 min at room temperature. Parallel experiments were carried out in the presence of 1200 units of SOD (from bovine erythrocytes) to determine the SOD inhibitible portion of NBT reduction to NBF.
Results Synthesis of 9 or 11 and 10. Compound 10 was synthesized by incubating a stirred solution of 1 (0.2 mM) for 24 h in either 0.01 M H Cl or pH 7.4 phosphate buffer. Preparative HPLC was then employed to obtain a solution of 10, which, following lyophilization, was isolated as a red-brown solid. At higher concentrations (g1 mM) of 1 in aqueous 0.01 M HCl, 0.5 M HCl, or 20% TFA, dimer 9 replaced 10 as
Chem. Res. Toxicol., Vol. 16, No. 4, 2003 495 Table 1. Time-Dependent Decomposition of 30 µM 1 in Various Media Expressed as Its HPLC-UV Peak Height Relative to that Measured at Time Zero time (h) time zero 0 0.5 4 24 0 1 4 24 0 1 4 24 0 1 4 24 a
medium as percentage of peak height at 0.01 M HCl
phosphate buffer, pH 7.4
0.5 M NH4 Cl solution, pH 7.4
aCSF, pH 6.5
HPLC-UV peak height for 1 100 ( 6a 91 ( 6 83 ( 6 51 ( 2 100 ( 10 97 ( 13 83 ( 12 55 ( 2 100 ( 5 81 ( 6 68 ( 2 31 ( 4 100 ( 6 98 ( 4 96 ( 4 90 ( 6
Mean ( SD of g3 replicate experiments.
the major reaction product. At relatively high concentration (g20 mM) in these media, 1 was converted almost quantitatively into 9 within
