Reactions of the Putative Neurotoxin Tryptamine-4, 5-dione with l

Xiang-Rong Jiang, Monika Z. Wrona, Susan S. Alguindigue, and Glenn Dryhurst*. Department of ... Soumya Mukherjee , Manas Seal , Somdatta Ghosh Dey...
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Chem. Res. Toxicol. 2004, 17, 357-369

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Reactions of the Putative Neurotoxin Tryptamine-4,5-dione with L-Cysteine and Other Thiols Xiang-Rong Jiang, Monika Z. Wrona, Susan S. Alguindigue, and Glenn Dryhurst* Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 Received September 16, 2002

Tryptamine-4,5-dione (1) is formed by oxidation of 5-hydroxytryptamine by reactive oxygen and reactive nitrogen species. Dione 1 is a powerful electrophile that can covalently modify cysteinyl residues of proteins and deactivate key enzymes. Thus, 1 has been suggested to play a role in the degeneration of serotonergic neurons in brain disorders such as Alzheimer’s disease or evoked by amphetamine drugs. However, if formed in the brain, it is also likely that 1 would react with low molecular weight thiols such as cysteine (CySH) and glutathione (GSH). The resulting metabolites might not only contribute to the degeneration of serotonergic neurons but also, perhaps, serve as biomarkers of such neurodegeneration. In this investigation, it is shown that in oxygenated buffer at pH 7.4 dione 1 reacts with CySH and other low molecular weight sulfhydryls such as GSH, N-acetylcysteine, and cysteamine to form, first, the corresponding 7-S-thioethers of the dione. However, unlike the glutathionyl and N-acetylcysteinyl conjugates of 1, the 7-S-cysteinyl conjugate is very unstable at pH 7.4 forming a number of novel products, the nature of which are dependent on the relative concentrations of 1 and CySH. These products have been isolated, and spectroscopic and other evidence is provided in support of their proposed chemical structures.

Introduction MA1 can evoke neurotoxic effects on serotonergic and dopaminergic terminals in the brains of rodents and nonhuman primates (1, 2). A related amphetamine, MDMA, is a selective serotonergic neurotoxin in most species (2). The mechanism(s) underlying the neurotoxicity of MA and MDMA is not understood. Nevertheless, several processes triggered by MA and MDMA have been identified as important steps in the mechanism. The first appears to be a transient MA- or MDMA-induced impairment of serotonergic and dopaminergic ATP production (2-4). The resultant neuronal depolarization, interference by MA and MDMA with the vesicular storage of 5-HT and DA (5, 6) and reversal of the 5-HT transporter (5-HTT) and DA transporter (DAT) (7-9), together mediate a massive release of 5-HT and DA but a decrease of extracellular levels of their usual metabolites (8, 10). Extracellular concentrations of 5-HT and DA subsequently return to basal levels although without any increases of their normal metabolites. These observations suggest that as the MA- or MDMA-induced neuronal energy impairments subside increasing ATP production returns the reversed 5-HTT and DAT to their normal function with consequent reuptake of released 5-HT and * To whom correspondence should be addressed. Tel: 405-325-4811. Fax: 405-325-6111. E-mail: [email protected]. 1 Abbreviations: AMT, R-methyl-p-tyrosine; CME, L-cysteine methyl ester; CySH, L-cysteine; DA, dopamine; DAT, dopamine plasma membrane transporter; GSH, glutathione; γ-GT, γ-glutamyl transpeptidase; 5-HT, 5-hydroxytryptamine; 5-HTT, 5-hydroxytryptamine plasma membrane transporter; MA, methamphetamine; MDMA, 3,4-methylenedioxymethamphetamine; MeCN, acetonitrile; MeOH, methanol; MPP+, 1-methyl-4-phenylpyridinium ion; NAC, N-acetyl-L-cysteine; NO•, nitric oxide; ONOO-, peroxynitrite; O2•-, superoxide; SCE, saturated calomel reference electrode; TFA, trifluoroacetic acid; TPH, tryptophan hydroxylase.

DA, respectively (11). Elevated production of intraneuronal O2•- (12, 13), NO• (14, 15), and thence ONOO- (1618) has also been implicated as a key process in the neurotoxic mechanism induced by MA and MDMA. Multiple small doses or a large single dose of MA or MDMA cause a delayed but irreversible inhibition of TPH (19, 20), which, together with serotonergic terminal damage, have been attributed to direct effects of elevated intraneuronal NO• (21) or ONOO- (22). However, other factors implicated with MA- and MDMA-induced irreversible TPH inactivation and serotonergic neurotoxicity are not easily reconciled with this explanation. For example, 5-HTT inhibitors administered before or after MA or MDMA block irreversible decreases of TPH activity and serotonergic neurotoxicity (23-25) without affecting the hyperthermia evoked by these drugs, another important factor associated with their neurotoxic mechanisms (26, 27). Several studies have concluded that released DA plays an important role in the degeneration of both serotonergic terminals evoked by MA and MDMA (28, 29) and of dopaminergic terminals by MA (23, 30, 31). Thus, it has been suggested that DA released by MA and MDMA (32) competes with released 5-HT for the 5-HTT, enters serotonergic neurons, and, once inside, in some way contributes to resultant damage (28, 33). Indeed, because MA and MDMA mediate intraneuronal generation of O2•- (12, 13) and ONOO- (14-17), these oxidants might oxidize DA to dopamine-o-quinone (DAQ) (34, 35), which could irreversibly inactivate TPH (36), tyrosine hydroxylase (37), and other essential proteins by covalent modification of active site cysteine (CySH) residues (38) and hence contribute to resultant neurotoxicity. However, other lines of evidence argue against an essential role for released DA in the serotonergic neurotoxicity evoked by MA and MDMA. For example,

10.1021/tx020084k CCC: $27.50 © 2004 American Chemical Society Published on Web 02/03/2004

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profound depletion of brain DA levels fails to block the serotonergic neurotoxicity of MDMA in rats provided that the ambient temperature is raised (39). Furthermore, the 5-HTT is probably unable to transport DA into serotonergic terminals (23). An alternative possibility is that the 5-HTT-mediated release and subsequent reuptake of 5-HT and its intraneuronal oxidation by O2•- and/or ONOO- might form a toxic metabolite that plays a key role in the inactivation of TPH and the serotonergic neurotoxicity of MA and MDMA. A product of the O2•- and ONOO--mediated oxidation of 5-HT is tryptamine4,5-dione (1) (40), which in vitro irreversibly inhibits TPH (41) and several mitochondrial respiratory enzyme complexes (42, 43) probably by covalent modification of active site CySH residues. However, GSH reacts with 1 (44) and, hence, attenuates the inhibition of TPH and mitochondrial enzymes by the dione (41-43). Hence, in order for intraneuronal 1 to inactivate these and other essential enzymes in vivo, the O2•-/ONOO--mediated oxidation of 5-HT would necessarily have to occur in the absence of high concentrations of GSH. It is of relevance, therefore, that energy impairments evoked, for example, by transient cerebral ischemia (45, 46) or the dopaminergic neurotoxin MPP+ (47) evoke a massive release of GSH. In recent microdialysis studies, we have observed that MA also evokes a massive rise of extracellular GSH in the rat striatum (unpublished studies). The release of GSH evoked by ischemia, MPP+, and MA is followed by a rise of extracellular CySH, an effect that is blocked by the γ-GT inhibitor acivicin (47, 48). As the energy impairments evoked by transient ischemia, MPP+, and MA subside, extracellular levels of GSH and CySH decline (47, 48). Because neurons are unable to import GSH, to replenish that released by ischemia-, MPP+-, and MA-induced energy impairments, these effects may be indicative of the degradation of extracellular GSH by γ-GT and dipeptidases to CySH, which can be transported into neurons where, normally, it is the ratelimiting substrate for intraneuronal GSH biosynthesis (49, 50). Thus, as a MA- or MDMA-induced serotonergic energy impairment subsides, the 5-HTT-mediated reuptake of released 5-HT and its oxidation by O2•-/ONOOin the cytoplasm of serotonergic terminals might form 1, which reacts not only with proteins but also with translocated free CySH. It is of relevance that the oxidation of CySH and similar thiols by O2•- (34, 51) and ONOO- (52) is slow. The possibility of intraneuronal reactions between 1 and CySH is of interest because the resultant products might possess biological properties of relevance to the serotonergic neurotoxicity of MA and MDMA. In this communication, the reactions of 1 with CySH and other thiols are described with a particular emphasis on identification of the major products formed at physiological pH.

Materials and Methods Chemicals. 5-HT (creatinine sulfate complex), TFA, CySH, cysteamine, GSH, CME, S-methyl cysteine, and N-acetylcysteine (NAC) were obtained from Sigma (St. Louis, MO). HPLC grade MeOH and MeCN were obtained from Fisher Scientific (Springfield, NJ). Tryptamine-4,5-dione (1) (43) and 7-S-glutathionyl-tryptamine-4,5-dione (3) (44) were synthesized as described elsewhere. Other chemicals used were of the highest purity commercially available. Spectroscopy. NMR spectra were obtained with Varian (Palo Alto, CA) Mercury VX 300 MHz or Varian INOVA 500 or

Jiang et al. 600 MHz spectrometer. Fast atom bombardment mass spectra (FAB-MS) were recorded on a VG Instruments (Manchester, U.K.) ZAB-E spectrometer. Electrospray ionization mass spectra (ESI-MS) were obtained with a Micromass (Wythenshaw, U.K.) Q-ToF mass spectrometer. UV-visible spectra were recorded on a Hewlett-Packard (Palo Alto, CA) 8452A diode array spectrophotometer. Preparative HPLC Methods. Two preparative HPLC methods were employed to separate, isolate, and purify reaction products. A Gilson (Middleton, WI) model 712 HPLC system was used equipped with a UV detector (254 nm) and a J. T. Baker (Phillipsburg, NJ) preparative reversed phase column (Bakerbond C-18, 10 µm particle size, 25 cm × 2.1 cm) protected by a guard column (5 cm × 0.9 cm) packed with the same stationary phase. Method I employed two mobile phase solvents. Solvent A was prepared by adding 50 mL of MeCN to 950 mL of deionized water, the apparent pH of the resulting solution being adjusted to 2.1 with concentrated TFA. Solvent B was prepared by adding 400 mL of MeCN to 600 mL of deionized water, the apparent pH being adjusted to 2.1 with TFA. The binary gradient system was employed as follows: 0-35 min, linear gradient from 100% solvent A to 25% solvent B; 35-40 min, linear gradient to 100% solvent B; 40-45 min, 100% solvent B. The mobile phase was then returned to 100% solvent A over 5 min before another sample was injected. The flow rate was 10 mL min-1. Method II employed the same mobile phase solvents as method I except that the pH was adjusted to 3.0 with TFA. The binary gradient was employed as follows: 0-50 min, linear gradient from 100% solvent A to 25% solvent B; 50-59 min, linear gradient to 100% B; 59-65 min, 100% solvent B. The flow rate was 10 mL min-1. Analytical HPLC. Analytical HPLC was employed to monitor reactions of interest and employed a Gilson system equipped with a UV detector (254 nm) and a Phenomenex (Torrence, CA) reversed phase column (Primesphere C-18, 5 µm particle size, 250 mm × 3.2 mm). Two mobile phase solvents were employed. Solvent C was prepared by mixing 1900 mL of deionized water, 100 mL of MeOH, and 1 mL of concentrated ammonium hydroxide (NH4OH). The resultant solution was then adjusted to an apparent pH of 3.0 with TFA. Solvent D was prepared by mixing 900 mL of water, 900 mL of MeCN, 200 mL of MeOH, and 1 mL of NH4OH. The apparent pH was then adjusted to 3.0 with TFA. The binary gradient was employed as follows: 0-15 min, linear gradient from 100% solvent C to 12% solvent D; 15-18 min, linear gradient to 30% solvent D; 18-21 min, linear gradient to 100% solvent D; 21-25 min, 100% solvent D. The flow rate was 0.7 mL min-1. Controlled Potential Electrolysis. Controlled potential electrolyses employed a Princeton Applied Research Corporation (Princeton, NJ) model 174 Polarographic Analyzer and a three compartment cell in which the working, counter, and reference electrode compartments were separated by a Nafion membrane (Type 117, DuPont Co., Wilmington, DE). The working electrode compartment had a capacity of 50 mL. The working electrode consisted of several plates of pyrolytic graphite (Pfizer Metals, Minerals and Pigments Division, Easton, PA) having a total surface area of approximately 40 cm2. The counter electrode was platinum gauze. Potentials were referenced to the SCE at ambient temperature (22 ( 2 °C). Synthetic Procedures. Procedures employed to synthesize compounds of interest are described below together with spectroscopic evidence bearing on their structures. Purification of compounds was accomplished using preparative HPLC. Generally, at least two HPLC purification steps were employed in order to obtain chromatographically pure compounds. While chemical names are provided for each compound, for simplicity, assignments of NMR resonances employed atom numbering systems shown for structures in Schemes 1-5. Assignments of 1H and 13C NMR resonances were confirmed by two-dimensional (2D) correlated spectroscopy (COSY), double-pulsed field gradient selective excitation nuclear Overhauser effect (NOE) and

Reactions of Tryptamine-4,5-dione with Cysteine gHMQC, gHMBC, gHSQC, and dfqCOSY together with onedimensional (1D) and 2D rotating frame NOE spectroscopy (ROESY) experiments. 7-S-Glutathionyl-4,5-dihydroxytryptamine (2). Dione 1 (0.01 mmol) was dissolved in 20 mL of aqueous 0.01 M HCl in a 50 mL round-bottomed flask containing a Teflon-coated magnetic stirring bar. The bright purple solution of 1 was thoroughly deoxygenated by N2 sparging. Then, maintaining an atmosphere of N2 over the solution, 5.0 mg (0.016 mmol) of GSH was added. The solution was stirred at ambient temperature until it became colorless. Following filtration, the resulting solution was pumped directly onto the preparative reversed phase column and chromatography was carried out using method I, the mobile phase solvents being thoroughly deoxygenated by N2 sparging prior to use. The chromatographic peak corresponding to 2 eluted at a retention time (tR) of 24 min. The solution eluted under this peak was collected, immediately frozen, and freeze-dried to give a white fluffy solid. A freshly chromatographed solution of 2 dissolved in the HPLC mobile phase (pH 2.1) exhibited a UV spectrum with λmax ) 316, 272, and 216 nm. FAB-MS (glycerol matrix) gave m/z ) 498.1639 (MH+, 9%; C20H28N5O8S; calcd m/z ) 498.1658). 1H NMR (D2O; 300 MHz) gave δ 7.17 (s, 1H, C(2)-H), 7.03 (s, 1H, C(6)-H), 4.29 (dd, 1H, J ) 8.4, 4.8 Hz, C(9)-H), 3.73 (t, 1H, J ) 6.3 Hz, C(13)H), 3.69 (s, 2H, C(16)-H2), 3.30 (t, 2H, J ) 6.5 Hz, C(β)-H2), 3.27 (dd, 1H, J ) 14.8, 4.8 Hz, C(8)-H), 3.16 (t, 2H, J ) 6.5 Hz, C(R)H2), 3.11 (dd, 1H, J ) 14.8, 8.4 Hz, C(8)-H), 2.30 (dd, 2H, J ) 7.8, 7.8 Hz, C(11)-H2), 2.00 (dt, 2H, J ) 7.8, 6.3 Hz, C(12)H2). 7-S-Glutathionyl-tryptamine-4,5-dione (3). The experimental procedure employed to synthesize and isolate 3 has been described in detail elsewhere (44). The results of 1D and 2D NMR and ROESY experiments on 3 are shown in Tables 1 and 2, respectively, and will be discussed subsequently. Spectral data are also available as Supporting Information. 7-S-(N-Acetylcysteinyl)-4,5-dihydroxytryptamine (4). The same basic procedure described above to synthesize 2 was employed to prepare 4 except that NAC (0.018 mmol) was used instead of GSH. Using preparative HPLC method I, 4 eluted at tR ) 31 min. After it was freeze-dried, 4 was isolated as a fluffy white solid. Dissolved in the HPLC mobile phase (pH 2.1) 4 exhibited a UV-visible spectrum with λmax ) 316, 272, and 226 nm. FAB-MS (glycerol matrix) gave m/z ) 354.1119 (MH+, 100%, C15H20N3O5S; calcd m/z ) 354.1123). 1H NMR (D2O; 300 MHz) gave δ 7.14 (s, 1H, C(2)-H), 7.02 (s, 1H, C(6)-H), 4.23 (dd, 1H, J ) 8.7, 3.6 Hz, C(b)-H), 3.31 (m, 3H, C(β)-H2, C(a)-H), 3.16 (m, 3H, C(R)-H2, C(a)-H), 1.73 (s, 3H, CH3). 7-S-(N-Acetylcysteinyl)tryptamine-4,5-dione (5). The synthesis and spectroscopic evidence bearing on the structure of this compound have been described in detail elsewhere (53). 7-S-Cysteinyl-4,5-dihydroxytryptamine (6) and Tryptamine-4,5-diol-7-S-cysteine Methyl Ester (6a). The procedure employed to synthesize 6 and 6a was the same as that used to synthesize 2 except that CySH or CME (0.02 mmol), respectively, replaced GSH. Using preparative HPLC method I, 6 and 6a eluted at tR ) 18 and 22 min, respectively. Compound 6 was isolated as a white fluffy solid. Dissolved in the HPLC mobile phase (pH 2.1), 6 exhibited a UV-visible spectrum with λmax ) 314, 270, and 224 nm. FAB-MS (glycerol matrix) gave m/z ) 312.1020 (MH+, 63%, C13H18N3O4S; calcd m/z ) 312.1018). 1H NMR (D2O; 300 MHz) gave δ 7.17 (s, 1H, C(2)-H), 7.08 (s, 1H, C(6)-H), 3.79 (dd, 1H, J ) 7.4, 4.1 Hz, C(b)-H), 3.31 (m, 3H, C(β)-H2, C(a)-H), 3.18 (m, 3H, C(R)-H2, C(a)-H). Compound 6a was also isolated as a white solid, which, dissolved in the HPLC mobile phase (pH 2.1), exhibited a UV spectrum with bands at λmax ) 316, 274, and 218 nm. ESI-MS (H2O) gave m/z ) 326.1183 (MH+, 100%, C14H20N3O4S; calcd m/z ) 326.1175). 1H NMR (D2O, 300 MHz) gave δ 7.05 (s, 1H, C(2)-H), 6.89 (s, 1H, C(6)H), 4.16 (t, 1H, J ) 4.5 Hz, C(b)-H), 3.46 (dd, 1H, J ) 13.0, 4.5 Hz, C(a)-H), 3.17 (t, 2H, J ) 6.9 Hz, C(β)-H2), 3.14 (dd, 1H, J ) 13.0, 4.8 Hz, C(a)-H), 3.04 (t, 2H, J ) 6.6 Hz, C(R)-H2), 2.98 (s, 3H, CH3).

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 359 7-S-Cysteinyl-tryptamine-4,5-dione (7). Diol 6 (0.007 mmol) was dissolved in 40 mL of aqueous 0.01 M HCl, and the resultant stirred solution was oxidized by controlled potential electrolysis (+200 mV vs SCE; pyrolytic graphite electrode) for 30 min. The resulting solution was chromatographed using preparative HPLC method I; 7 eluted at tR ) 26 min. The purple solution eluted under this peak was collected and freeze-dried to give a brown/purple solid. Dissolved in the HPLC mobile phase (pH 2.1), 7 exhibited a UV-visible spectrum with λmax ) 540, 330, and 240 nm. FAB-MS (glycerol matrix) gave m/z ) 312.1031 (MH2H+, 16%, C13H18N3O4S; calcd m/z ) 312.1018). 1H NMR (D O; 300 MHz) gave δ 6.83 (s, 1H, C(2)-H), 5.89 (s, 2 1H, C(6)-H), 4.18 (dd, 1H, J ) 6.6, 4.8 Hz, C(b)-H), 3.63 (dd, 1H, J ) 14.3, 4.8 Hz, C(a)-H), 3.54 (dd, 1H, J ) 14.3, 6.6 Hz, C(a)-H), 3.18 (t, 2H, J ) 6.9 Hz, C(β)-H2), 2.89 (t, 2H, J ) 6.9 Hz, C(R)-H2). 2-[3-(2-Aminoethyl)-4,5-dioxo-4,5-dihydro-1H-indol-7ylamino]-3-{2-[3-(2-aminoethyl)-4,5-dioxo-4,5-dihydro-1Hindol-7-ylamino]-2-carboxyethyldisulfanyl}propionic Acid (10). A 0.5 mM solution of 7 in aqueous 0.01 M HCl was adjusted to pH 7.4 by addition of 0.5 M Na2HPO4 with vigorous stirring, and the reaction vessel was open to the atmosphere. After 30 min, the resulting solution was introduced onto the preparative HPLC column and chromatographed using method II. Compound 10 eluted at tR ) 37 min. After it was freezedried, the solution was collected under this peak, and 10 was isolated as a red solid. Dissolved in the HPLC mobile phase (pH 3.0), 10 exhibited a UV-visible spectrum with λmax ) 532, 360, 318, and 238 nm. ESI-MS (H2O) gave m/z ) 617.1483 (MH+, 76%, C26H29N6O8S2) calcd m/z ) 617.1488). 1H NMR (D2O, 400 MHz) gave δ 6.70 (s, 2H, C(2)-H, C(2′)-H), 5.00 (s, 2H, C(6)-H, C(6′)-H), 4.50 (t, 2H, J ) 6.8 Hz, C(a)-H, C(a′)-H), 3.31 (dd, 2H, J ) 13.0, 7.4 Hz, C(b)-H, C(b′)-H), 3.03 (m, 6H, C(β)-H2, C(β′)H2, C(b)-H, C(b′)-H), 2.70 (m, 4H, C(R)-H2, C(R′)-H2). 2-[3-(2-Aminoethyl)-4,5-dioxo-4,5-dihydro-1H-indol-7ylamino]-3-{2-[3-(2-aminoethyl)-4,5-dioxo-4,5-dihydro-1Hindol-7-ylamino]-2-methoxycarbonylethyldisulfanyl}propionic Acid Methyl Ester (10a). The procedure employed to synthesize 10a was the same as that for 10 except that CME replaced CySH. Using preparative HPLC method II, 10a eluted at tR ) 56.5 min. Compound 10a was isolated as a red solid, which, dissolved in the HPLC mobile phase (pH 3.0), exhibited a UV-visible spectrum with λmax ) 534, 354, 318, and 238 nm. ESI-MS (D2O) gave m/z ) 645.1820 (MH+, 27%, C28H33N6O8S2; calcd m/z ) 645.1801). 1H NMR (Me2SO-d6, 500 MHz) gave δ 2.84 (m, 2H, C(β)-H2), 3.06 (m, 2H, C(R)-H2), 3.37 (m, 2H, C(b)H2), 3.67 (s, 3H, -OCH3), 4.53 (br s, 1H, C(a)-H), 4.99 (s, 1H, C(6)-H), 6.95 (s, 1H, C(2)-H), 7.91 (s, 3H, NH3+), 9.17 (br s, 1H, C(7)-NH), 12.85 (brs, 1H, N(1)-H). 13C NMR (Me2SO-d6) gave δ 23.7 C(β), 38.1 C(R), 38.7 C(b), 52.8 (OCH3), 55.1 C(a), 92.8 C(6), 118.9 C(3a), 120.9 C(3), 122.6 C(2), 131.9 C(7a), 150.0 C(7), 169.5 C(d), 177.2 C(5), and 177.9 C(4). Two-dimensional COSY experiments indicated H-H coupling between N(1)-H (δ ) 12.85) and C(2)-H but not between C(6)-H (4.99) and N(1)-H. A 3% enhancement of the N(1)-H NOE resonance was observed when C(2)-H was irradiated. However, no NOE enhancement of the N(1)-H resonance was observed when the C(6)-H resonance was irradiated. HMBC experiments demonstrated coupling between C(6)-H (s, 4.99 ppm) and both C(5) and C(4). Taken together, these results indicate that the C(7)-position of the residues of 1 in 10a is bound to the N(c) residue of GSH. 2-[3-(2-Aminoethyl)-4,5-dioxo-4,5-dihydro-1H-indol-7ylamino]-3-[3-(2-aminoethyl)-4,5-dioxo-4,5-dihydro-1H-indol-7-ylsulfanyl]propionic Acid (14). Ten milliliters of a 0.1 mM solution of CySH in deionized water was added to 10 mL of a 0.2 mM solution of 1 in aqueous 0.01 M HCl. The pH of this solution was adjusted to 7.4 by careful addition of aqueous 0.5 M Na2HPO4 with vigorous stirring, and the reaction vessel was open to the atmosphere. After 30 min, the solution was pumped into the preparative HPLC system and chromatographed using method II. Compound 14 eluted at tR ) 41 min. The solution collected under this peak was freeze-dried to give

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Table 1. One- and Two-Dimensional Spectra of 3a

chemical shifts

HMQC (attached C-H) (ppm)

HMBC [C (or N)-B,2,3, and 4 bonds] H

C

dfqCOSY (H-H coupling)

C

ppm

H

H

C

H

H

β 12 11 8 R

23.8 25.9 30.7 32.4 38.1

2.03 (m, 2H) 2.39 (m, 2H) 2.91 (m, 2H) 2.99 (m, 2H) 3.26 (dd, 1H)

25.9 (C-12) 30.7 (C-11) 23.8 (C-β) 38.1 (C-R) 32.4 (C-8)

C(12)-H C(11)-H C(β)-H2 C(8)-H C(8)-H′

11, 13, 14 10, 12, 13 R, 3a, 3, 2 β, 3 7, 9, 15b

C(12)-H C(11)-H C(β)-H2 C(R)-H2 C(8)-H

C(11)-H, C(13)-H C(12)-H C(R)-H2, C(R)-NH3+ C(β)-H2, C(R)-NH3+ C(8)-H′, C(9)-H

16

40.9

3.45 (dd, 1H)

32.4 (C-8)

C(16)-H

7, 9

C(8)-H′

C(8)-H, C(9)-H

9 13 6 3a 3 2 7a 7

50.9 51.5 114.5 118.7 122.6 123.1 134.3 149.2

3.76 (m, 2H) 3.86 (m, 1H) 4.64 (m, 1H) 6.03 (s, 1H) 6.88 (s, 1H)

40.9 (C-16) 51.5 (C-13) 50.9 (C-9) 114.5 (C-6) 123.1 (C-2)

C(9)-H C(6)-H C(2)-H C(R)-NH3+ C(2)-H C(R)-NH3+ C(13)-NH2 N(18)-H

15, 17 11.12, 14 8, 10,b 15 3a,b 4, 7, 7a, 5 β, 3, 3a, 7a R, β 12, 13, 14 15, 16

C(16)-H2 C(13)-H C(9)-H C(6)-H C(2)-H C(R)-NH2 C(13)-NH2 N(18)-H

N(18)-H C(12)-H2, C(13)-NH2 C(8)-H, C(8)-H′, N(19)-H

15

169.5

N(19)-H

8, 9, 10

N(19)-H

C(9)-H

14 17 10 4 5

170.7 170.8 171.4 174.8 179.1

C(12)-H, 2.03 (m, 2H) C(11)-H2, 2.39 (m, 2H) C(β)-H2, 2.91 (m, 2H) C(R)-H2, 2.99 (m, 2H) C(8)-H, 3.26 (dd, 1H, J ) 9, 14 Hz) C(8)-H, 3.45 (dd, 1H, J ) 5, 14 Hz C(16)-H2, 3.76 (m, 2H) C(13)-H, 3.86 (m, 1H) C(9)-H, 4.64 (s, 1H) C(6)-H, 6.03 (s, 1H) C(2)-H, 6.88 (s, 1H) C(R)-NH3+, 8.05 (s, 3H) C(13)-NH2, 8.51 (s, 2H) N(18)-H, 8.58 (t, 1H, J ) 6 Hz) N(19)-H, 8.64 (d, 1H, J ) 9 Hz) N(1)-H, 12.34 (brs, 1H)

N(1)-H

2, 3b, 3a, 7a

N(1)-H

C(2)-H

N(1)-H C(R)-H2, C(β)-H2 C(13)-H C(16)-H2

a Compound 3 was dissolved in Me SO-d , and spectra were recorded on a 500 MHz NMR spectrometer. b Very weak coupling (signal 2 6 intensity).

a red solid. The UV-visible spectrum of 14 dissolved in the HPLC mobile phase (pH 3.0) exhibited bands at λmax ) 546, 360, 324, and 224 nm. ESI-MS (H2O) gave m/z ) 498.1410 (MH+, 100%, C23H24N5O6S; calcd m/z ) 498.1447). 1H NMR (D2O; 400 MHz) gave δ 6.64 (s, 1H, C(2)-H), 6.55 (s, 1H, C(2′)-H), 5.69 (s, 1H, C(6′)-H), 5.00 (s, 1H, C(6)-H), 4.81 (dd, 1H, J ) 7.8, 3.0 Hz, C(a)-H), 3.64 (dd, 1H, J ) 15.4, 3.0 Hz, C(b)-H), 3.50 (dd, 1H, J ) 15.4, 7.8 Hz, C(b)-H), 2.93 (m, 4H, C(β)-H2, C(β′)-H2), 2.62 (t, 2H, J ) 7.2 Hz, C(R)-H2), 2.55 (t, 2H, J ) 7.2 Hz, C(R′)-H2). 2-[3-(2-Aminoethyl)-4,5-dioxo-4,5-dihydro-1H-indol-7ylamino]-3-[3-(2-aminoethyl)-4,5-dioxo-4,5-dihydro-1H-indol-7-ylsulfanyl]propionic Acid Methyl Ester (14a). The procedure for synthesis of 14a was identical to that described for 14 except that CME replaced CySH. Using preparative HPLC method II, 14a eluted at tR ) 60 min. After the solution that eluted under this peak was freeze-dried, 14a was isolated as a red solid. When dissolved in the HPLC mobile phase (pH 3.0), 14a exhibited a UV-visible spectrum with λmax ) 546, 360, 324, and 226 nm. ESI-MS (H2O) gave m/z ) 512.1668 (MH+, 71%, C24H26N5O6S; calcd m/z ) 512.1664). 1H NMR (D2O; 300 MHz) gave δ 6.72 (s, 1H, C(2)-H), 6.64 (s, 1H, C(2′)-H), 5.90 (s, 1H, C(6′)-H), 5.21 (s, 1H, C(6)-H), 4.98 (dd, 1H, J ) 7.2, 3.6 Hz, C(a)-H), 3.71 (s, 3H, CH3), 3.76-3.56 (m, 2H, C(b)-H2), 3.02 (m, 4H, C(β)-H2, C(β′)-H2), 2.75 (t, 2H, J ) 6.9 Hz, C(R)-H2), 2.68 (t,

2H, J ) 7.0 Hz, C(R′)-H2). 1H NMR (Me2SO-d6) gave δ 12.7 (bs, 1H, N-H), 12.3 (bs, 1H, N-H), 8.9 (d, 1H, J ) 7.5 Hz, N(a′)-H), 7.8 (bs, 6H, 2 × NH3+), 6.97 (d, 1H, J ) 2.4 Hz, C(2)-H), 6.85 (d, 1H, J ) 2.4 Hz, C(2′)-H), 6.01 (s, 1H, C(6′)-H), 5.13 (s, 1H, C(6)-H), 4.75 (m, 1H, C(a)-H), 3.82-3.67 (m, 2H, C(b)-H2), 3.73 (s, 3H, CH3), 2.99 (m, 4H, C(β)-H2, C(β′)-H2), 2.87 (m, 4H, C(R)H2, C(R′)-H2). 3-(-2-Aminoethyl-2-carboxyethyldisulfanyl)-2-[3-(2-aminoethyl)-4,5-dioxo-4,5-dihydro-1H-indol-7-ylamino]propionic Acid (15) and 3-(2-Aminoethyl)-4,5-dioxo-1,4,5,7,8,9hexahydro-6-thia-1,9-diaza-cyclopenta[a]naphthalene-8carboxylic Acid (12). A 6-fold molar excess of CySH was added to 200 mL of a freshly prepared solution of 1 (200 µM) in phosphate buffer (pH 7.4). The resulting solution was stirred for ca. 12 h at ambient temperature; the reaction vessel was open to the atmosphere. After filtration, the brown-green solution was introduced into the preparative HPLC system (method I). Compounds 15 and 12 eluted at tR ) 26.5 and 32.6 min, respectively. The solutions eluted under these peaks were collected separately and freeze-dried. Compound 15 was isolated as a red-brown fluffy solid that when dissolved in the analytical HPLC mobile phase (pH 3.0) exhibited a UV-visible spectrum with λmax ) 530, 364, 318, and 242 nm. In phosphate buffer (pH 7.4), the spectrum of 15 exhibited bands at λmax ) 532, 356, 314,

Reactions of Tryptamine-4,5-dione with Cysteine Table 2. ROESY Correlations for Compound 3a,b

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 361 457.1227 (MH+, 100%, C18H25N4O6S2; calcd m/z ) 457.1215). NMR (Me2SO-d6; 300 MHz) gave δ 12.48 (bs, 1H, N(1)-H), 8.70 (d, 1H, J ) 5.4 Hz, N(e)-H), 7.75 (bs, 6H, 2 x NH3+), 7.10 (s, 1H, C(2)-H), 5.09 (s, 1H, C(6)-H), 4.57 (dd, 1H, J ) 11.0, 6.0 Hz, C(a)-H), 4.33 (t, 1H, J ) 6.0 Hz, C(d)-H), 3.74-3.59 (m, 2H, C(b)-H), 3.70 (s, 3H, CH3), 3.40 (s, 3H, CH3), 3.24-3.16 (m, 2H, C(c)-H2), 3.01 (m, 2H, C(β)-H2), 2.89 (m, 2H, C(R)-H2). Compound 12a was a green solid, which, when dissolved in the analytical HPLC mobile phase (pH 3.0), gave a UV-visible spectrum with λmax ) 628, 362, 326, 276, and 220 nm. ESI-MS (H2O) gave m/z ) 322.0912 (MH+, 100%, C14H16N3O4S; calcd m/z ) 322.0862). 1H NMR (D2O, 300 MHz) gave δ 6.69 (s, 1H, C(2)-H), 4.72 (t, 1H, J ) 3.9 Hz, C(9)-H), 3.66 (s, 3H, CH3), 3.15 (dd, 1H, J ) 13.5, 4.5 Hz, C(8)-H), 3.04 (t, 2H, J ) 7.2 Hz, C(β)H2), 2.99 (dd, 1H, J ) 13.5, 3.3 Hz, C(8)-H′), 2.76 (t, 2H, J ) 7.2 Hz, C(R)-H2). 1H NMR (Me2SO-d6; 500 MHz) gave δ 12.55 (s, 1H, N(1)-H), 9.13 (d, 1H, J ) 5.3 Hz, N(10)-H), 7.81 (brs, 3H, C(R)-NH3+), 6.93 (s, 1H, C(2)-H), 4.89 (m, 1H, C(9)-H), 3.69 (s, 3H, C(13)-H3), 3.22 (dd, 1H, J ) 13.0, 3.0 Hz, C(8)-H′), 3.00 (m, 2H, C(R)-H2), 2.97 (dd, 1H, J ) 13.0, 3.0 Hz, C(8)-H), 2.87 (m, 2H, C(β)-H2). 13C NMR (Me2SO-d6) gave δ 23.7 (C-β), 25.0 (C8), 38.5 (C-R), 52.7 (C-9), 52.85 (C-13), 96.6 (C-6), 117.6 (C-3a), 121.1 (C-2), 121.2 (C-3), 132.6 (C-11a), 142.2 (C-11), 169.4 (C12), 174.1 (C-5), 175.9 (C-4). NOE experiments on 12a (Me2SOd6) showed that irradiation at 12.6 ppm (N(1)-H) resulted in NOE peaks at 9.20 (N(9)-H), 6.92 (C(2)-H), and 3.43 (-CH3) ppm. Two-dimensional NMR data (COSY, HMQC, HMBC) for 12a are available as Supporting Information. 7-S-Cysteaminyl-tryptamine-4,5-dione (16) and 3-(2Aminoethyl)-4,5-dioxo-1,4,5,7,8,9-hexahydro-6-thia-1,9-diaza-cyclopenta[a]naphthalene (17) 1. HCl (2 mg, ∼0.01 mmol) was dissolved in 20 mL of aqueous 0.01 M HCl, and then 3 mg (∼0.027 mmol) of cysteamine was added and the solution was stirred at room temperature. The pH of the solution was adjusted to 4.0 by addition of 0.5 M Na2HPO4 with vigorous stirring, and the reaction vessel was open to the atmosphere. After 60 min, the resulting solution was introduced onto the preparative HPLC column and chromatographed using method I. Red/purple compound 16 eluted at tR ) 32 min followed by green compound 17 at tR ) 42 min. After lyophilization of the solution eluted under the peak at tR ) 32 min, 16 was isolated as a red-brown solid. Dissolved in the HPLC mobile phase (pH 2.1), 16 exhibited a UV-visible spectrum with λmax ) 540, 336, and 242 nm. ESI-MS (H2O) gave m/z ) 266.0942 (MH+, C12H16N3SO2; calcd m/z ) 266.0963). 1H NMR (300 MHz, D2O) gave δ 6.6 (s, 1H, C(2)-H), 5.6 (s, 1H, C(6)-H), 3.1 (t, 4H, J ) 3 Hz), 2.9 (t, 2H, J ) 7.2 Hz), 2.6 (t, 2H, J ) 7.2 Hz). After it was freeze-dried, 17 was isolated as a green solid, which, dissolved in HPLC mobile phase (pH 2.1), gave a spectrum with λmax ) 618, 370, 278, and 222 nm. ESI-MS (H2O) gave m/z ) 264.0808 (MH+, 10%, C12H14N3O2S; calcd m/z ) 264.0878). 1H NMR (300 MHz, D2O) gave δ 12.5 (s, 1H), 9.1 (bs, 1H), 7.8 (brs, 3H), 6.9 (d, 1H, J ) 2.7 Hz), 3.6 (t, 2H, J ) 4.6 Hz), 2.99 (dd, 2H, J ) 10.1, 6.9 Hz), 2.87 (m, 4H). 1H

H

ROESY correlation

C(12)-H2 C(11)-H2 C(β)-H2 C(R)-H2 C(8)-H C(8)-H′ C(16)-H2 C(13)-H C(9)-H C(6)-H C(2)-H C(R)-NH3 C(13)-NH2 N(18)-H N(19)-H N(1)-H

C(11)-H, C(13)-H C(12)-H, N(19)-H C(R)-H, C(2)-H, C(R)-NH3c C(β)-H, C(R)-NH3, C(2)-Hc C(6)-H, C(8)-H′, C(9)-H,d N(19)-H C(6)-H, C(9)-H, C(8)-H N(18)-H C(12)-H, C(13)-NH2 C(6)-H, C(8)-H′, C(8)-Hd C(8)-H, C(8′)-H, C(9)-H N(1)-H, C(β)-H2, C(R)-H2c C(R)-H2, C(β)-H2 C(12)-H, C(13)-H C(9)-H, C(16)-H2 C(9)-H, C(11)-H2, C(8)-H, C(8′)-H C(2)-H

a Compound 3 was dissolved in Me SO-d , and spectra were 2 6 recorded on a 600 MHz NMR spectrometer. Both 1D and 2D ROESY experiments were carried out. b ROESY experiments measure NOEs under spin-locked conditions. This results in all NOE peaks being positive. Artifactual negative peaks are not reported. c Weak peak/correlation. d Very weak peak observed only in 1D ROESY experiments.

and 240 nm. ESI-MS (H2O) gave m/z ) 429.0890 (MH+, 20%, C16H21N4O6S2; calcd m/z ) 429.0903). 1H NMR (D2O; 400 MHz) gave δ 6.65 (s, 1H, C(2)-H), 5.07 (s, 1H, C(6)-H), 4.31 (dd, 1H, J ) 9.2, 3.6 Hz, C(a)-H), 3.70 (dd, 1H, J ) 7.6, 4.1 Hz, C(d)-H), 3.25 (dd, 1H, J ) 13.8, 3.6 Hz, C(b)-H), 3.08 (dd, 1H, J ) 14.4, 4.1 Hz, C(c)-H), 3.01 (m, 3H, C(β)-H2), C(b)-H), 2.87 (dd, 1H, J ) 14.4, 7.6 Hz, C(c)-H), 2.70 (m, 2H, C(R)-H2). Compound 12 was a dark green solid that, dissolved in the analytical HPLC mobile phase (pH 3.0), exhibited a UV-visible spectrum with bands at λmax ) 630, 370, 318, 278, and 224 nm. In phosphate buffer (pH 7.4), the spectrum of 12 showed bands at λmax ) 638, 370, 318 (shoulder), 278, and 224 nm. ESI-MS (H2O) gave m/z ) 308.0710 (MH+, 32%, C13H14N3O4S; calcd m/z ) 308.0705). 1H NMR (D2O; 300 MHz) gave δ 6.82 (s, 1H, C(2)H), 5.44 (dd, 1H, J ) 7.2, 2.4 Hz, C(9)-H), 3.17 (t, 2H, J ) 7.2 Hz, C(8)-H2), 2.92 (m, 4H, C(R)-H2, C(β)-H2). 2-[3- (2 - Aminoethyl)-4,5-dioxo-4,5-dihydro-1H - indol7-ylamino]-3-(2-amino-2-methoxycarbonyl-ethyl-disulfanyl))propionic Acid Methyl Ester (15a) and 3-(2-Aminoethyl)-4,5-dioxo-1,4,5,7,8,9-hexahydro-6-thia-1,9-diaza-cyclopenta[a]naphthalene-8-carboxylic Acid Methyl Ester (12a). The procedure employed to synthesize 15a and 12a was identical to that for preparation of 15 and 12 except that CySH was replaced by CME. Using HPLC method I, 15a and 12a eluted at tR ) 44 and 53 min, respectively. Compound 15a was a red-brown solid, which, when dissolved in the analytical HPLC mobile phase (pH 3.0), gave a UV-visible spectrum with λmax ) 536, 360, 320, and 238 nm. ESI-MS (H2O) gave m/z )

Results Site of Initial Nucleophilic Attack by Thiols on 1. In principle, the sulfhydryl residue of CySH, GSH, and the related thiols studied could initially attack dione 1 at either the C(6) or the C(7) positions, i.e., 1,6- or 1,4Michael additions, respectively. Indeed, nucleophilic addition of CySH to 1 forming 6-S-cysteinyl-tryptamine4,5-dione would provide a straightforward route to cyclized compound 12 (Figure 2; see later discussion). However, several lines of evidence suggest that it is the C(7) position of 1 that is the site of nucleophilic attack by CySH and other compounds containing cysteinyl or similar residues. To illustrate, the 1H NMR spectrum of dione 1 in Me2SO-d6 exhibits a singlet at 6.93 ppm (C(2)H) and doublets (J ) 9.9 Hz) at 5.80 (1H) and 7.08 (1H)

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Figure 1. Analytical HPLC chromatograms of (A) 0.2 mM 1 immediately after dissolving in 50 mM phosphate buffer (pH 7.4) and (B) 60 min after adding 1 mM GSH. Insets show the UV-visible spectra of 1 and 2 dissolved in the HPLC mobile phase (pH 3.0). For chromatography, the mobile phase solvents were thoroughly deoxygenated by N2 sparging.

ppm, which must correspond to the protons located at C(6) and C(7) (43). Previous studies of many R,β-unsaturated ketones such as 1 have shown that resonance deshields the β-proton. Thus, the 1H NMR peak for the β-proton appears downfield (g7.0 ppm) of the R-proton (e6.0 ppm) (54-56). Thus, in an earlier report (44), we concluded that in the 1NMR spectrum of 1 the upfield doublet at 5.80 ppm corresponded to C(6)-H, which is R to a carbonyl group, and the doublet at 7.08 ppm to C(7)H. The 1H NMR spectra of 3, 5, and 7, all monosubstituted thioethers of 1, exhibit upfield resonances (1H) at 6.03 (Table 1), 5.82 (53), and 5.89 ppm, respectively, but as singlets rather than as the doublet observed for 1. As discussed earlier, the upfield location of the singlets at 6.03, 5.82, and 5.89 ppm in the 1H NMR spectra of 3, 5, and 7, respectively, are indicative of a proton R to a carbonyl group; hence, these peaks probably correspond to C(6)-H. This, in turn, implies that the sulfhydryl residues of GSH, NAC, and CySH are substituted at the C(7) positions of 3, 5, and 7, respectively. Further NMR experiments designed to confirm this assignment were carried out with 3, the most stable of the monosubstituted thioethers of dione 1. The 1D/2D NMR spectra of 3 (Table 1) are not inconsistent with this compound being the 7-Sglutathionyl conjugate of 1. However, HMBC correlations between the upfield proton at 6.03 ppm and the five carbon atoms in the ring containing the carbonyl groups preclude a definitive conclusion concerning the site of substitution of the glutathionyl residue. This ambiguity was largely resolved using 1D and 2D ROESY experiments on compound 3 (Table 2). Previous studies have established that indoles with protons at C(2) and C(7) exhibit NOE enhancements of the signals for both of these protons when N(1)-H is irradiated (57-59). Fur-

thermore, when a substituent is present at C(7) and protons occupy the C(2) and C(6) positions, there is a NOE enhancement of only the C(2)-H signal when N(1)-H is irradiated (60). One-dimensional NOESY and 1D and 2D ROESY experiments on compound 3 failed to show any NOE enhancement between N(1)-H (12.38 ppm) and the proton at 6.03 ppm but a strong enhancement between N(1)-H and C(2)-H (6.88 ppm) (Table 2). These observations are consistent with the singlet at 6.03 ppm corresponding to C(6)-H and, hence, with the glutathionyl residue being substituted at the C(7) position. NOE enhancements in compound 3 were observed between C(6)-H (6.03 ppm) and C(8)-H, C(8′)-H, and C(9)-H (Table 2). However, irradiation of N(1)-H failed to cause enhancements of the latter signals. These observations imply that the peptide chain attached to the C(7) position in 3 is orientated away from N(1)-H as conceptualized in the structures shown in Tables 1 and 2. Reactions of 1 with GSH and NAC at pH 7.4. Addition of excess GSH or NAC to dione 1 in stirred deoxygenated (N2 sparging) phosphate buffer (50 mM; pH 7.4) caused the bright purple color of the dione to disappear over the course of several minutes giving a colorless solution. Figure 1A shows an analytical HPLC chromatogram of 1 prior to the addition of GSH; the inset shows the UV-visible spectrum of 1 in the HPLC mobile phase (pH 3.0). Figure 1B is a chromatogram of the product formed 60 min after addition of a 5-fold molar excess of GSH to 1 indicating formation of diol 2 (Scheme 1), the UV spectrum of which is shown in the inset. The reaction of 1 with NAC under identical conditions resulted in formation of diol 4 (Scheme 1). When dissolved in oxygenated phosphate buffer (pH 7.4), diols 2 or 4 were almost immediately autoxidized to diones 3 and 5,

Reactions of Tryptamine-4,5-dione with Cysteine Scheme 1

respectively (Scheme 1). In the absence of free GSH or NAC, diones 3 and 5 were stable in solution at pH 7.4. However, in the presence of excess GSH in oxygenated phosphate buffer (pH 7.4), 3 slowly reacts further to form more highly substituted glutathionyl conjugates of 1 (44). Similarly, 4 reacts with free NAC to form more highly substituted N-acetylcysteinyl conjugates of 1 (data not shown). Synthesis of 6 and 7. Efforts to prepare and isolate diol 6 following reaction of 1 with excess CySH in N2sparged phosphate buffer (pH 7.4) were complicated because of its extremely facile oxidation by trace levels of molecular oxygen and subsequent reactions of the resultant product, dione 7 (see subsequent discussion). Thus, diol 6 was more conveniently synthesized and isolated by addition of excess CySH to a solution of 1 dissolved in deoxygenated aqueous 0.01 M HCl (see

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 363

Materials and Methods). Controlled potential electrooxidation (+200 mV vs SCE; pyrolytic graphite electrodes) of this solution of 6 resulted in formation of dione 7, which could be isolated following purification by preparative HPLC. Stability of 6 and 7 in Solution at pH 7.4. Diol 6 and dione 7 were extremely unstable in phosphate buffer (pH 7.4) in the presence of molecular oxygen. To illustrate, Figure 2 presents an analytical HPLC chromatogram of the product solution formed immediately after 7 (or 6) was dissolved in air-saturated pH 7.4 phosphate buffer showing peaks corresponding to the disulfide-linked dimer 10 and the tricyclic compound 12. Reactions of 1 with CySH at pH 7.4. The reaction of 1 with CySH at mole ratios up to 1:0.5 formed 14 as the sole product (Figure 3). At higher mole ratios of CySH relative to 1, additional products appeared. For example, Figure 4A is an analytical HPLC chromatogram of the product solution formed by reaction of equimolar (0.2 mM) 1 and CySH in pH 7.4 phosphate buffer and shows peaks corresponding to 14 and the disulfide-linked dimer 10. As the molar excess of CySH relative to 1 was increased further, formation of 14 decreased, that of dimer 10 initially increased, and compounds 12 and 15 appeared as products (Figure 4B). Furthermore, with time, particularly with a large (g5-fold molar excess of CySH over 1) formation of 10 and 15 decreased and, correspondingly, that of 12 increased (Figure 4C). Reactions of 14 and 10 with CySH at pH 7.4. Equimolar concentrations of 14 and CySH in pH 7.4 phosphate buffer reacted to form primarily 10 (Figure 5A). In the presence of excess CySH, the transformation of 14 into 10 was followed by conversion of the latter dimer to 12 (Figure 5B). Reactions of 1 with CME at pH 7.4. Compounds 10, 12, and 14, formed by reactions of 1 and CySH at pH 7.4 were only sparingly soluble in water and particularly organic solvents. As a consequence, it was difficult to obtain the highest quality NMR spectra of these compounds. However, reactions of 1 with CME both in 0.01 M HCl and in pH 7.4 phosphate buffer were identical to those described previously between 1 and CySH. Furthermore, 10a, 12a, 14a, and 15a, the methyl ester

Figure 2. Analytical HPLC chromatogram of the product mixture formed immediately after dissolving 7 (0.2 mM) in 50 mM phosphate buffer (pH 7.4). Insets show the UV-visible spectra of 10 and 12 in the HPLC mobile phase (pH 3.0).

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Figure 3. Analytical HPLC chromatogram of the product mixture formed following reaction of 1 (0.2 mM) with CySH (0.05 mM) for 20 min in 50 mM phosphate buffer (pH 7.4). Inset shows the UV-visible spectrum of 14 in the HPLC mobile phase (pH 3.0).

Figure 4. Analytical HPLC chromatograms of the product mixtures obtained following reaction of 0.2 mM 1 and (A) 0.2 mM CySH and (B) 1 mM CySH for 10 min in 50 mM phosphate buffer (pH 7.4) and (C) same conditions as in panel B except the reaction mixture was sampled after 24 h. The inset shows the UV-visible spectrum of 15 in the HPLC mobile phase (pH 3.0).

derivatives of 10, 12, 14, and 15, respectively, were more soluble than the latter compounds. Thus, the spectro-

scopic properties of the cysteine methyl ester derivatives 10a, 12a, 14a, and 15a, particularly NMR and NOE

Reactions of Tryptamine-4,5-dione with Cysteine

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 365 Scheme 2

Figure 5. Analytical HPLC chromatograms of the product mixtures obtained following reaction of (A) 0.2 mM 14 and 0.2 mM CySH and (B) 0.2 mM 10 and 0.2 mM CySH for 300 min in 50 mM phosphate buffer (pH 7.4).

spectra, provided important support for the structures proposed for 10, 12, 14, and 15. Reaction Pathways. The NMR data obtained for glutathionyl conjugate 3 (Tables 1 and 2) support the conclusion that the cysteinyl sulfhydryl residue of GSH and other thiols initially attacks the C-(7) position of the dione. Nucleophilic addition of GSH or NAC to 1 in deoxygenated buffer initially form diols 2 or 4, respectively (Scheme 1). In the presence of molecular oxygen, 2 and 4 are readily autoxidized to diones 3 and 5 (Scheme 1), respectively. At low pH, nucleophilic addition of CySH or CME to 1 initially forms diols 6 or 6a, respectively. These diols are readily oxidized to diones 7 and 7a, respectively, by controlled potential electrolysis (Scheme 2). In the presence of molecular oxygen at pH 7.4, 6/6a are rapidly autoxidized to 7/7a, which, in turn, react further to form disulfide-linked dimers 10/10a and the tricyclic compounds 12/12a (Figure 2). A plausible pathway leading to the latter products involves an intramolecular cyclization in which nucleophilic attack by the deprotonated cysteinyl amino group of 7/7a forms spiro thiazolidine intermediate 8/8a that upon ring opening, forms 9/9a and its Schiff base tautomer 9′/9a′ (Scheme 3) (61). Oxidation of the free sulfhydryl group of 9/9a by molecular oxygen would then form thiyl radicals that dimerize to 10/10a. Alternatively, nucleophilic attack of the free sulfhydryl residue, particularly of 9′/9a′, at the C(6) position would form diols 11/11a, which are oxidized by molecular oxygen to 12/12a (Scheme 3). Glutathionyl conjugate 3 and N-acetyl conjugate 5, in which the amino group of the 7-S-cysteinyl substituent is blocked by a glutamyl or acetyl residue, respectively, exhibit no tendency to form products analogous to 10/10a and 12/12a at pH 7.4. This

provides some support for the proposed intramolecular cyclization of the unsubstituted cysteinyl amino groups of 7 and 7a to form 8/8a and thence 9/9a, the precursor of 10/10a and 12/12a. Addition of the sulfhydryl residue of CySH or CME to the C-(6) position of 1 followed by attack of the amino group of the attached cysteinyl group at C-(7) would seem to provide a more logical and direct route to 11/11a and 12/12a as shown in parentheses in Scheme 3. However, the NMR and other studies on compound 3, in our judgment, better support the conclusion that it is the C-(7) position of 1 that is most susceptible to attack by thiols and other nucleophiles (62) including water (63). Nevertheless, in the absence of a single-crystal X-ray data to absolutley confirm the structure of 3, there is a small possibility that the CySH, CME, and GSH initially attack at the C-(6) position of 1. At neutral or acidic pH, a carbonyl oxygen is often a better kinetic nucleophile than an amine nitrogen. This raised the possibility that an intermediate in the reaction might involve cyclization through a carboxylic acid oxygen. However, the reaction of cysteamine with 1 in aerated solution resulted in the initial formation of 7-S-cysteaminyl-tryptamine-4,5-dione (16) and then the cyclized compound 17, the spectra for which were very similar to those of 7/7a and 12/12a, respectively (Scheme 5). At pH 7.4, the reaction between 1 and CySH or CME at mole ratios of 1:e0.5 forms a single product, 14 (Figure 3) or 14a, respectively. The latter compounds could be formed by nucleophilic addition of the free sulfhydryl residue of putative intermediate 9/9a to the C(7) position of 1 (present in molar excess) to form 13/13a, which is oxidized by molecular oxygen to 14/14a (Scheme 4). Equimolar concentrations of 7/7a (which presumably rearrange to 9/9a at pH 7.4) and 1 also react rapidly to form 14/14a as the sole product (data not shown). An alternative pathway that might lead to 14/14a involves nucleophilic addition of the deprotonated cysteinyl amino group of 7/7a to the C(7) position of 1 (present in molar excess). However, this pathway seems unlikely in view of the fact that there is no significant reaction between S-methylcysteine (0.2 mM) and dione 1 (0.2 mM) after incubation for 1 h in pH 7.4 phosphate buffer. When the mole ratio of 1:CySH or CME is increased to 1:1, formation of 14/14a decreases while that of 10/ 10a increases (Figure 4A). Furthermore, equimolar con-

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Jiang et al. Scheme 3

Scheme 4

centrations of 14/14a and CySH or CME in aerated pH 7.4 phosphate buffer react to form 10/10a (Figure 5A). The latter observation suggests that 14/14a is attacked by CySH or CME in a nucleophilic displacement reaction to form 9/9a and 7/7a (Scheme 5). Intramolecular cyclization of 7/7a formed by this reaction and direct attack of CySH or 1 would then form 8/8a and thence 9/9a (Scheme 5). Oxidation of the free sulfhydryl group of 9/9a by molecular oxygen then forms the disulfide-bridged dimer 10/10a. In the presence of equimolar or small molar excesses of CySH or CME relative to 1 (or 14/14a), autoxidation of the sulfhydryl residue of these thiols and of 9/9a forms thiyl radicals, which couple to form 15/15a (Figure 4B; Scheme 5). However, large molar excesses of CySH or CME relative to 1 reduce the disulfide bond of 10/10a forming 9/9a and thence 11/11a, which is autoxidized to 12/12a (Figure 4B,C; Scheme 5). Indeed, incubation of 10/10a with a g5-fold molar excess of CySH/CME in aerated pH 7.4 phosphate buffer results in formation of 12/12a (Figure 5B). Similarly, reduction of the disulfide bond of 15/15a in the presence of a large molar excess of CySH/CME forms 9/9a and hence 12/12a (Scheme 5).

Reactions of Tryptamine-4,5-dione with Cysteine

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 367 Scheme 5

Discussion The neuroprotection afforded by 5-HTT inhibitors against the serotonergic neurotoxicity of MA and MDMA (23-25) suggests that one or more compounds released and/or taken up via this transporter play a key role in the underlying pathological mechanism. One such compound is 5-HT, which is released by MA- and MDMAinduced energy impairments (7-9) and subsequently returns to the cytoplasm of serotonergic terminals as the energy impairment subsides, both 5-HTT-mediated processes. Because increased intraneuronal production of O2•- (12, 13) and ONOO- (14-18) are involved in the neurotoxicity evoked by MA and MDMA and rapidly oxidize 5-HT to 1 (40), it is conceivable that the latter dione may contribute to the inhibition of TPH (41) and other essential enzymes (42, 43, 62) by covalent modification of active site CySH residues. However, similar to neuronal energy impairments evoked by transient cerebral ischemia (45, 46, 48) and MPP+ (47), MA also evokes a massive release of GSH, which is then degraded by γ-GT and dipeptidases to CySH. The subsequent decline of extracellular CySH levels as these energy impairments subside may be indicative of its transporter-mediated

uptake by neurons in an effort to replenish depleted intraneuronal GSH (49, 50). Thus, as MA- or MDMAinduced energy impairments subside, the simultaneous 5-HTT-mediated reuptake of released 5-HT and intraneuronal generation of O2•- and ONOO- would provide ideal conditions for formation of 1, which might react not only with cysteinyl residues of proteins but also with translocated free CySH. The results of earlier studies (44) and the present investigation indicate that 1 reacts readily with sulfhydryl nucleophiles such as GSH, NAC, CySH, and CME that selectively attack at the C(7) position of the dione (63) forming diols 2, 4, and 6, which, at pH 7.4, are very easily oxidized by molecular oxygen to diones 3, 5, and 7, respectively (Schemes 1 and 2). However, unlike compounds 3 and 5 in which the cysteinyl amino group is blocked and hence are stable at pH 7.4, dione 7 rapidly reacts to form the symmetrical disulfide-linked dimer 10 and tricyclic heterocycle 12 (Scheme 3). The present study indicates that formation of 1 in the presence of a g 2-fold molar excess over CySH leads to formation of 14 (Scheme 4). However, as CySH concentrations relative to those of 1 increase, the reaction shifts toward formation of 10 together with 15. In the

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Chem. Res. Toxicol., Vol. 17, No. 3, 2004

presence of large molar excesses of CySH, these disulfides are reduced leading to formation of 12 (Scheme 5). There is presently no experimental evidence that in vivo the reuptake and intraneuronal oxidation of 5-HT, released by MA and MDMA, do in fact form 1 or that this dione plays a role in the irreversible inhibition of TPH, other essential enzymes, and resultant serotonergic neurotoxicity. The high reactivity of 1 particularly toward sulfhydryl nucleophiles almost certainly precludes direct detection of this putative metabolite of 5-HT in the brains of animals exposed to MA or MDMA. However, the present study identifies a number of potential metabolites that might be formed by reaction of 1 with CySH. It remains to be established whether such metabolites are indeed present in brain tissue of animals exposed to neurotoxic doses of MA or MDMA. However, it is of interest that large (binge) doses of MA evoke decreased GSH levels in rat brain (64), which might in part be a consequence of the reaction of its precursor CySH with 1. A decrease in GSH in dopaminergic nigral cells in Parkinson’s disease has similarly been suggested to result from the oxidation of DA to DAQ, which reacts with CySH forming cysteinyl conjugates that may contribute to neuron death in this disorder (65). It has recently been reported that profound reduction of brain DA levels by AMT and reserpine fails to block the serotonergic neurotoxicity of MDMA in rats provided the ambient temperature is raised (39). This, together with the inability of the 5-HTT to transport DA into serotonergic terminals (23), argues against an essential role for intraneuronal DA in the serotonergic neurotoxicity of MDMA and, perhaps, MA. Reserpine also reduce 5-HT levels in rat brain, although this reduction is significantly less than that of DA evoked by AMT and reserpine (39). Thus, sufficient cytoplasmic 5-HT may be available for MDMA-induced release, subsequent reuptake, and intraneuronal oxidation to 1, which reacts with protein-bound and free CySH.

Acknowledgment. This work was supported by Grant GM32367 from the National Institutes of Health. The help of Professor Francis J. Schmitz in interpreting 2D and NOE NMR spectra is greatly appreciated. We also thank Dr. Carlos Amezcula at the Southwestern Medical Center in Dallas who provided the facilities to record 2D NMR spectra (500 MHz). The 1D and 2D ROESY experiments were carried out at Oklahoma State University using a 600 MHz NMR spectrometer. The help of Ms. Teresa Hackney in preparing this manuscript is appreciated.

Jiang et al.

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Supporting Information Available: Two-dimensional NMR data for compounds 3 and 12a (500 MHz) and 1D and 2D ROESY results (600 MHz) on compound 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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References

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