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Electrochemical Oxidation of 5-Hydroxytryptophol.I: Studies in Acid Solution
Electrochemical Oxidation of 5-Hydroxytryptophol. I: Studies in Acid Solution GLENNDRYHURS? Received December 9, 1988, from the Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019. Accepted for publlcation June 8, 1989.
Fu-CHOUCHENG
AND
vent 5 was prepared by adding 3.0 mL of concentrated formic acid to 1 L of MeOH:H,O (10:90, v/v) and adjusting the pH to 3.40 with concentrated ammonium hydroxide. Solvent 6 consisted of 10% dioxane:20% acetonitri1e:lW methanol in water. Spectroscopic equipment used has been described elsewhere.14 Isolation and Identification of Products-The electrochemical behavior of 5-HTOL was the same in pH 2.0 phosphate buffer ( p = 0.5) and dilute HCl solution at the same pH. Analytical HPLC studies showed that the products of controlled-potential electro-oxidationsof 5-HTOL in the latter supporting electrolytes were the same. However, when semipreparative HPLC was attempted, large amounta of inorganic phosphate seriously degraded separations, wherear, dilute HCl did not cause this effect. Accordingly, all controlled-potential electrolyses, which were subsequently followed by HPLC separations, were performed in dilute HC1 solution at pH 2.0. Typically, repetitive 2-mL injections of product solutions were chromatographed and each individual HPLC peak was collected. The biological reasons for studying the oxidation chemistry Chromatograms of product solutions obtained following controlledand biochemistry of endogenous central nervous system electro-oxidations of 0.1 mM 5-HTOL in 0.01 M HCl are (CNS)indoles have been summarized elsewhere.14 The elec- potential shown in Figure 1. Chromatographic peak C is due to unreacted trochemical oxidation of 5-hydroxytryptamine (5-HT11-3 and 5-HTOL. The following sections will describe how components A, B, its precursor in the CNS, 5-hydroxytryptophan (5-HTPP1,' D, E,F, G, H,and I were isolated and purified and present spectral have recently been described. 5-Hydroxytryptophol(5-HTOL) and electrochemical evidence for their structures. is a normal metabolite of the latter indoleamines.5 The 4,4'-Bi-(5-hydroxytryptophol) (AbAliquots (2 mL) of A diseolved electrochemicaloxidation of 5-HTOL has been studied in acid in solvent 1 were injected onto the reversed-phase column using solvent 5 as the mobile phase (4 mL .min-') to completely separate solution to permit comparisons with the behaviors of 5-HT unreacted 5-HTOL. Then 10-20 mL of A in solvent 5 were pumped and 5-HTPP. onto the same column, which had been equilibrated with water, followed by 40 mL of water. Under the latter conditions, ammonium Experimental Section formate was eluted from the column while A was strongly retained. Compound A was subsequently eluted by changing the mobile phase 5-Hydroxytryptophol (5-HTOL) was obtained from Sigma (St. to solvent 6. The resulting eluant was collected and freeze dried. Louis, MO).Equipment and techniques employed for electrochemical Compound A was a white solid (Amx at pH 2.0 304,277.219 nm). studies have been described.l-4teA pyrolytic graphite electrode (PGE; Analysis by FAJLMS (dithioerythrito1:dithiothreitolmatrix) gave an P f h r Minerals, Pigmenta and Metals Division, Easton, PA), having an approximate surface area of 4 mm2, was used for voltammetric intense pseudomolecular ion (MH') at mle = 353.1543 (Co,,Hz1N~04; calc. m/e = 353.1501).Before discussing the 'H NMR spectrum of A, measurements. Controlled-potentialelectrolyses employed plates of it is of value to summarize that of 5-HTOL which has not previously pyrolytic graphite as the working electrode. All potentials are been reported (structures and numbering of all compounds are referred to the saturated calomel reference electrode (SCE)at ambipresented in Schemes 1-111 and elsewhere in the text): 'H NMR ent temperature (22 f 2 "C). High-performance liquid chromatography ( H P L C P employed a (Me,SO-$; 300 MHz): 8 11.49 (9, lH, N(l)-H), 8.56 (8, lH, 0(5)-H), reversed-phase column (Brownlee Laboratories, RP-18, 5 pm, 25 x 7.10 (d, J = 8.7 Hz,lH, C(7)-H),7.02 (8, lH, C(2)-H),6.79 (d, J = 2.4 Hz,lH, C(4)-H),6.57 (dd, J = 8.7 Hz,J = 2.4 Hz, 1H. C(6)-H),4.69 0.7 cm). For the initial separation of products, a binary gradient (t, lH, O(y)-H, 3.62 (m, 2H, C(/3)-H2),and 2.74 ppm (t, 2H, C(a)-Hp). HPLC method was employed. Solvent 1 was prepared by adding 10 Assignments of the various resonances in the spectrum of 5-WCOL, mL of concentratedformic acid to 1L of 2.5% dioxane:5% acetonitrile and indeed for other compounds reported, were based on comparisons in water and then adjusting the pH of the resulting solution to 3.40 with the spectra of 5-HT' and 5-HTPP and homonuclear decoupling with concentrated ammonium hydroxide. Solvent 2 was prepared by experiments. Addition of a few drops of D,O caused the resonances at adding 3.5 mL of concentrated formic acid to 1L of 10%dioxane:20% 11.49, 8.56, and 4.59 ppm to disappear. The 'H NMR spectrum of A acetonitrile:lO%methanol in water and then adjusting the pH of the is as follows: 'H NMR (Me,SO-$): S 10.37 (d, J = 2.1 Hz,2H, NU)-H resulting solution to 3.40 by addition of concentrated ammonium and N(l')-H),7.57 (s,2H, O(5)-Hand 0(5')-H),7.10 ( d , J = 8.6 Hz,2H, hydroxide. The following gradients were employed between 0 and 18 C(7)-H and C(7')-H), 6.86 (8, 2H, C(2)-H and C(2')-H), 3.87 (t, 2H, min, a linear gradient from 100%solvent 2 to 90% solvent 2 and 10% O(y)-H and O(y')-H), 3.11 (m, 4H, C(/3)-H, and C(F)-H,), and 1.96 solvent 1; between 18 and 30 min, a linear gradient to 55% solvent 2 ppm (m, 4H, C(a)-H2 and C(a')-H,). Addition of D,O caused the and 45% solvent 1;between 30 and 45 min, a linear gradient to 100% resonances at 10.37,7.57, and 3.87 ppm to disappear. Compared with solvent 1;this 100%solvent was then passed for an additional 15min. the spectrum of 5-HTOL, A lacks a signal for a C(U-proton. The A flow rate of 2 mL * min-I was used at all times. Four other HPLC C(6)-Hresonance of 5-HTOL appears ar, a doublet of doubleta owing solvents were used for purification and desalting of products. Solvent to spin couplings with C(7)-H and C(4)-H (long range). The C(6)-H 3 was 30% methanol in water. Solvent 4 was prepared by adding 3.5 resonances of A exhibit no long-range spin coupling and appear 88 a mL of concentrated formic acid to 1 L of 6% dioxane:12% doublet, indicating that the C(4)-H protons are missing in both acetonitrile:5%methanol in water. The pH of the resulting solution 5-HTOL residues. was adusted to 3.40 with concentrated ammonium hydroxide. Sol-
Abatrrct 0 The oxidation chemistry of the endogenous central nervous system indole 5-hydroxytryptophol(5-HTOL)has been studied at pH 2 using electrochemical methods. The first voltammetric oxidation peak (I) appears to involve an initial one-electron abstraction giving a transient radical cation that, in the rate-controlling step, deprotonates to give a neutral radical. A radical-substrate reaction then occurs to give a dimer radical which is further oxidized to yield three simple dimers (4,4'-,4,6'-, and 2,4'-linked).The neutral radical can be further oxidized (le) to a quinone imine that, as a result of very fast follow-up chemistry and electrochemistry,yields tryptophol-4,5-dione(B) which has been isolated in pure form. Reactions between intermediate species also resutt in three dimers containing residues of 5-HTOL and B and an unusual oxygenbridged trimer.
286 I Journal of Pharmaceutical Sciences Vol. 79, No. 3, March 1990
OO22-3549/90/03OO-0266$01 .ooJo
8 1990, American PharmaceuticalAssociation
t
(MH') of B. Accurate ma88 measurements on this ion gave mle = 192.0663 (C,,HloN,O3, calc. mle = 192.0661; 'H NMR spectrum (Me,SO-d,): S 11.80 (bs, lH, N(l)-H),7.29 (d, J = 9.9 Hz, lH, C(7)-H), 6.82 (8, lH, C(2)-H), 5.88 (d, J = 9.9 Hz, lH, C(6)-H), 4.59 (bs, lH, O(y)-H), 3.55 (m, 2H, C(P)-H,), and 2.75 ppm (t, 2H, C(a)-H2). Addition of D,O caused the resonances a t 11.80 and 4.59 pprn to disappear. The NMR results indicate that, compared with the spectrum of 5-HTOL, the C(4)-H and O(5)-H resonances are absent in B. The shiR of the C(6)-H resonance from 6.57 ppm (dd) in 5-HTOL to 5.88 ppm (d) in B supports the conclusion that in B this proton is located adjacent to a carbonyl group' and that B is, therefore, tryptophol4,5-dione. Confirmation of this structure was obtained by reacting B with a-phenylenediamine.2 The resulting orange product (Arn= at pH 2.0 533, 419, 365(sh), 280, 223(sh), and 207 nm) was purified by HPLC using solvent 2 as the mobile phase (2 mL min-' flow rate, tR = 30 min). Analysis by EI-MS (70 eV, 270 "C) gave mle = 263 (1192, M+), 233 (loo%, M+-CHOH), and 232 (77%, M+-CH,OH); 'H NMR (Me,SO-4): S 11.87 (6, lH,N(l)-H),8.23 (m, 2H, aromatic H, ring a), 8.01 (d, J = 9.0 Hz,lH, C(7)-H),7.87 (m, 2H, aromatic H, ring a), 7.74 ( d , J = 9.0 Hz, lH, C(6)-H), 7.37 (s, l H , C(2)-H),4.68 (t, lH, O(y)-H), 3.90 (m, 2H, C(P)-H,), and 3.52 ppm (t, 2H, C(a)-H2).These spectral data are in accord with structure J formed by condensation of B with o-phenylenediamine. 4-[7'-(Tryptophol-4',5'-dione)l-5-hydroxytryptophol (D)Compound D (10-20 mL), dissolved in solvent 1,was pumped onto the HPLC column (previously equilibrated with water) and then 40 mL of water was pumped through the column (2 mL . min-') to elute ammonium formate. The mobile phase was then changed to solvent 6 (2 mL * min - l ) to elute D. The resulting solution was freeze dried. Compound D was a purple solid (Amm at pH 2.00: 548, 368, 300(sh), 283, and 223 nm). The F A E M S (dithioerythrito1:dithiothreitol matrix) analysis gave intense ions a t mle = 367(57%),368(100%),and 369(11%). The purple color of D faded in the FAB-MS matrix indicating that it was partially reduced. Thus, the ion at mle = 367 represents the pseudomolecular ion (MH'). Exact mass measurements on this ion gave mle = 367.1255 (C,oH,7N,0,; calc. d e = 367.1294); 'H NMR (Me,SO-d,): S 11.20 (bs, l H , N(l')-H), 10.82 ( 8 , lH, N(1)-H),8.87 (bs, lH, 0(5)-H),7.27 (d, J = 8.7 Hz,lH, C(7)-H), 7.10 (8, 1H, C(2)-H), 6.78 (d, J = 8.7 Hz, lH, C(6)-H), 6.61 (8, lH, C(2')-H), 5.67 (9, lH, C(6')-H), 4.61 (t, lH, O($-H), 4.42 (t, lH, O(y')-H), 3.60 (m, 2H, C(P)-H,), 2.80 (m, 2H, C(a)-H2),and 2.60 ppm (m, 2H, C(a'-H,). The C(P')-H, resonance was obscured by a large HOD signal. Addition of D,O caused the resonances at 11.20, 10.82, 8.87, 4.61, and 4.42 ppm to disappear. The F A E M S analysis indicates that D is a dimer containing one residue of 5-HTOL and another having a dione structure. The 'H NMR spectra indicate only a single C(7)-H residue and no C(4)-Hresonances. The singlet a t 5.67 ppm has been assigned to the C(6')-H proton in a tryptophol-4,5-dione residue by analogy with similar structures (e.g., C(6)-H in 5hydroxytryptamine-4,7-dioneresonates at 5.73 ppm'). The structure of D was confirmed by reacting it with o-phenylenediamine? a t pH 2.00: 550,431,374(sh), 277, The yellow-orange product [A, and 237(sh) nml was purified by HPLC using solvent 2 (2 mL * min-', tR = 25 rnin): FAB-MS (glycerol matrix) gave MH+(lOO%)a t mle = 439.1772 (CZ6Hz1N4O3;calc. rnle = 439.1770); 'H NMR (Me,SO&): 11.00 (bs, 1H, NU')-H), 10.76 (9, lH, N(l)-H), 8.25 (dd, 2H, aromatic H from ring a), 7.87 (m, 2H, aromatic H from ring a), 7.50 ( 8 , lH, C(6')-H), 7.32 (d, J = 8.6 Hz, C(7)-H), 7.16 (8, lH, C(2)-H),7.03 (8, lH, C(2')-H), 6.87 (d, J = 8.6 Hz, l H , C(6)-H), 4.73 (bs, lH, O(y)-H), 3.94 (m, 3H, C(P)-H,) and O(y')-H), 3.66 (m, 2H, C(a)-H2),3.03 (m, 2H, C(P')-H,), and 2.01 ppm (m, 2H, C(a')-H,). The NMR and MS spectral data are in accord with structure K which would be expected to be formed by reaction of D with o-phenylenediamine. 4,6'-Bi-(5-hydroxytryptophol)(EkCompound E (10-20 mL) die-
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Time I min
0
1
C
0.3
l0.N
2o.m
l
a.m
Ea.N
Time I min
Flgure 1-High-pressure
liquid chromatograms of the products formed
upon controlled-potential electro-oxidation of 0.10 rnM 5-hydroxytryptophol in dilute HCI (pH 2.0) at (A) 0.45 V and (B) 0.70 V. The HPLC conditions are given in the Experimental Section.
Tryptophol-4,5-dione (BbCompound B in solvent 1 (10-20 mL) was pumped onto the reversed-phase column that had previously been equilibrated with water; this was followed by the addition of 30-60 mL of water. Under these conditions, B was retained on the column, whereas all components of solvent 1 were eluted. The mobile phase was then changed to solvent 6 (2-3 mL * min-'), which caused B to elute. The resulting solution was freeze dried. Component B was a blue-purple solid (A,= in dilute HC1, pH 2.0: 550, 358, 233 nm). Component B was not very stable in solution. Therefore, during the purification and isolation procedures, solutions containing this compound were stored a t -80 "C to minimize decomposition. Analysis by FAB-MS (thioglycerol matrix) gave a cluster of three intense ions a t mle = 192 (17%), 193 (59%), and 194 (40%). However, it was noted that when B was mixed with the FAB-MS matrix its blue-purple color faded, indicating partial reduction of the compound. Similar effects have been noted by other workers with easily reducible compounds.7-9 Hence, the ion at mle = 192 is the pseudomolecular ion
J Journal of Pharmaceutical Sciences I 267 Vol. 79, No. 3, March 7990
K solved in solvent 1 was pumped onto the column followed by 40 mL of water (2 mL . min-’) to elute ammonium formate. Compound E was eluted using solvent 6 as the mobile phase (2 mL . min-I). The solution containing E was then freeze dried. Compound E was a white solid (A,,- at pH 2.00: 305,280, and 226 nm). Analysis by EI-MS (70 eV, 270 “C)gave mle = 352 (69%,M+),321 (48%,M+-CH,OH). Exact mass measurements on M’ gave mle = 352.1453 ( C 2 ~ , o N 2 0 , ,calc. mle = 352.1423); IH NMR (Me,SO-$): 6 10.46 (d, J = 2.4 Hz,lH, N(1)-H), 10.38 (d, J = 1.5 Hz, lH, N(l’)-H), 7.09 (d, J = 8.4 HZ, lH, C(7)-H), 7.02 (8, lH, C(4‘)-H), 6.94 ( 8 , lH, C(2)-H), 6.93 ( 8 , lH, C(7‘)-H),6.91 (9, l H , C(2’)-H),6.69 (d, J = 8.7 Hz, lH, C(6)-H), 4.64 (m, lH, O(y)-H),4.10 (m, 1H) O(y’)-H),4.04 (m, 2H, C(p)-H,), 3.67 (m, 2H, C(P’)-H,), 2.79 (m, 2H, C(a-H2),and 2.17 ppm (m, 2 H, C(a’)-H2). Addition of D 2 0 caused the resonances a t 10.46,10.38,4.64, and 4.10 ppm to disappear. 2,4’-Bi-(5.hydroxytryptophol)(FbCompound F (10-20 mL) was pumped onto the column followed by 40 mL of water (2 mL * min- ’) to remove ammonium formate. Compound F eluted using solvent 6 as the mobile phase (2 mL min -’). The resulting solution was freeze dried. Compound F waa a white solid (Amax at pH 2.0: 300 and 226 nm). The FAB-MS (dithioerythrito1:dithiothreitolmatrix) analysis gave MH+(53%) a t mle = 353.1548 (C2,H1,N,O,; calc. m/e = 353.1501); ’H NMR (Me,SO&): 6 10.63 (s, lH, N(l)-H),10.52 ( 8 , lH, N(1’)-H), 8.50 (8, lH, 0(5)-H), 8.30 (6, IH, 0(5’)-H), 7.22 (d, J = 8.7 Hz, l H , C(7’)-H), 7.06 (d, J = 8.7 Hz, l H , C(7)-H), 7.02 (8, 1H, C(2’)*H),6.83 (d, J = 2.1 Hz, lH, C(4)-H), 6.74 (d, J = 8.7 Hz, lH, C(6’)-H),6.55 (dd, J = 8.6 Hz, J = 2.4 Hz, lH, C(6)-H), 4.49 ( 8 , lH, O(y)-H),and 4.11 ppm (t, lH, O(y’)-H).The region below 4.0 ppm was not well defined owing to the interfering signals of HOD and Me,SO. Nevertheless, comparison of the NMR spectra of F and 5-HTOL clearly shows the absence of a C(2)-H in one 5-HTOL residue of F and the absence of C(4)-H in the other. Compounds G, H, and I-Compounds G, H, and I were initially collected together in solvent 2. The combined solutions were then freeze dried. The resulting solid was dissolved in the minimum volume of solvent 2 and diluted five times with water. Aliquots (2 mL) of this solution were then chromatographed using solvent 4 as the mobile phase (2 mL min-’). The tR values for G, H,and I were 11, 16, and 21 min, respectively. Each of the solutions containing the latter products was diluted five times by the addition of water. The solutions containing C,H, or I were desalted by pumping 10-20 mL onto the column and then passing water through the column (2 mL min-l, -40 mL). Solvent 6 was used to elute the appropriate product. Each product solution was then freeze dried. 2-[7’-(Tryptophol-4’,5’-dione)]-5-hydroxytryptophol(GICompound G was a deep brown solid (Arnm at pH 2.0: 506(sh), 450, 380(sh), 275, and 212 nm). The FAELMS(dithioerythrito1:dithiothreitol matrix) analysis gave mle = 367(31%), 368(100%),and 369(86%). Compound C is a very easily reduced compound and hence was partially reduced by the FAELMS matrix giving the ions at mle = 368 and 369. The pseudomolecular ion (MH’) had an exact mass of mie = 367.1305 (C;oHlgN206; calc. mle = 367.1294); ’H NMR(Me,SO-d,): 6 11.67 (8. 1H. N(l’)-H). 11.16 (8. 1H. N(1)-H), 8.88 (8, 1H. 0(5)-H), 7.22 (d, J = 8.7 Hz, l H , C(7)-H),6.92 ( d , J = 2.7 Hz, lH, C(4)-H),6.83 (6, 1H, C(2’)-H), 6.76 (dd, J = 8.7 HZ,J = 2.4 HZ, lH, C(6)-H), 6.08 (a, lH, C(6‘)-H),5.04 (bs, lH, O(y’)-H),4.61 (t, lH, O(y)-H), 3.69 (m, 2H, C(p)-H,), 3.59 (m, 2H, C(P’)-H2),2.91 (t, 2H, Cb)-HJ, 2.82 (t, 2H, C(a’)-H2). Addition of DzO caused the resonances at 11.67, 11.16, 8.88,5.04, and 4.61 ppm to disappear. The sharp singlet (1H) observed
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268 I Journal of Pharmaceutical Sciences Vol. 79, No. 3, March 1990
at 6.08 ppm is indicative of the presence of one residue of tryptophol4,5-dione in G. The characteristic peaks for C(4)-H (6.92 ppm, d), C(6)-H (6.76 ppm, dd), and C(7)-H (7.22 ppm, d) indicate that G contains one residue of 5-HTOL. The absence of one C(2)-H resonance confirms that this residue is linked to the tryptophol-4,6-dione through the C(2)-position. The absence of a C(7)-H resonance 8880ciated with the tryptophol-4,5-dione moiety indicates that this residue is linked a t (37). l-[7’-(Tryptophol-4,5-dione)]-5-hydr0xytryptophol ( H r C o m pound H was a deep brown solid (&= at pH 2.0: 595(sh), 462,313(sh), 280, and 235 nm). Compound H is a very easily reducible compound. Thus, FAB-MS (dithioerythrito1:dithiothreitolmatrix) exhibited intense ions at m/e = 367(44%, MH’), 368(100%),and 369(58%). Exact mass measurements (MH’) gave m!e = 367.1316(C,&$J,06; calc. mle = 367.1294); ’H NMR (Me2SO-d6):6 11.83 ( 8 , lH, N(l’)-H), 9.24 (s,lH, O(5)-H), 7.43 (s, IH, C(2)-H), 7.41 (d, J = 9.0 HZ, lH, C(7)-H), 6.96 (d, J = 2.4 Hz, lH, C(4)-H), 6.94 (9, lH, C(2’)-H), 6.78 (dd, J = 9.0 Hz, J = 2.4 Hz, l H , C(6)-H), 5.94 ( 6 , l H , C(6’)-H), 4.71 (t, lH, O(f)-H), 4.63 (t, lH, O(y)-H), 3.72 (m, 2H, C(p’)-H,), 3.61 (m,2H, C(P)-H,), 2.85 (t, 2H, C(a‘)-H,), and 2.80 ppm (t, 2H, C(a)-H2). Addition of D,O caused the peaks a t 11.83, 9.24,4.71, and 4.63 ppm to disappear. The NMR data indicate that only one N-H is present in H and that the tryptophol-4,5-dione residue is linked a t (37‘). 5-044’4 5’-Hydroxytryptophol)]-1’[7“-(tryptophol-4”,~~ione)~ tryptophol (I&Compound I was a deep brown solid (Ama at pH 2.0: 560(sh), 458, 304(sh), 277, and 228 nm). The FAB-MS(dithioerythrito1:dithiothreitol matrix) analysis gave mle = 542(3.4%, MH’), 543(3.6%), and 544(3.1%).The matrix thus caused partial reduction of I. Exact mass measurements on MH+ gave rnle = 542.1968 (C3JIz8N30,; calc. mle = 542.1927); ‘H NMR (Me,SO-&): 6 11.10 (8, lH, N(l)-H),10.98 (bs, l H , N(l”)-H),9.54 (6, lH, 0(5’)-H), 7.34 (8, 1H,C(2’)-H),7.21 (d, J = 8.7 Hz, lH,C(7’)-H),7.05 ( d , J = 9.0 Hz,lH, C(7)-H), 7.00 (8, l H , C(2)-H), 6.78 (d, J = 7.8 Hz,lH, C(6’)-H), 6.76 (d, J = 2.1 Hz, 1H, C(4)-H),6.74 (8, lH, C(2)-H),6.66 (dd, J = 8.4 Hz; J = 1.8 Hz, lH, C(6)-H), 5.92 ( 8 , lH, C(6)-H), 4.82 (t, lH, O(V)-H), 4.62 (t, lH, O(y’)-H), 4.60 (t, lH, O(y)-H),3.85 (m, 2H, C(p”)-H,), 3.61 (m, 2H, C(p)-H,), 3.49 (m, 2H, C(p’)-H2), 3.09 (t, 2H, C(a)-H,), and 2.86 ppm (t, 2H, C(a‘)-H2).The C(d’)-H, resonance was obscured by the signal due to HOD at 3.30 pprn. Addition of D,O caused the resonances at 11.10, 10.98, 9.54, 4.82, 4.62, and 4.60 ppm to dieappear. These NMR results indicate that in trimer I, one 5-HTOL residue is linked through its N(1) position and C(4) positions to other residues. Only one 0(5)-H resonance appears in the spectrum, suggesting, therefore, that one of the two 5-HTOL residues ie linked via its 0(5)-position. The singlet at 5.92 ppm indicates that a tryptophol-4,5-dione residue is present in I. Furthermore, the latter residue lacks a proton at the C(7)-position which, therefore, must be the site a t which it is linked to a 5-HTOL residue.
Results and Discussion Cyclic Voltammetry-A cyclic voltammogram of 5-HTOL at pH 2.0 shows oxidation peaks I, (E, = 0.495 V at a sweep rate of 200 mV s-’) and 11, (E, -0.80 V; Figure 2A). After scan reversal, reduction peaks R,, R,, and R3 appear, the former two peaks being extensively overlapped. On the second anodic sweep, oxidation peaks 0, and 0, appear. The rounded shape of peak I, suggests that it does not represent a simple oxidation process and peak clipping experiments confirmed this suggestion. When the initial anodic scan was reversed at the foot of peak I,, only a small reduction peak (R,) appeared (Figure 2B). Scan reversal at the E , for peak I, resulted in a considerable growth in peak R, and the first appearance of peak R,, and, on the second anodic sweep, oxidation peak O2 appeared (Figure 2C).This behavior indicates that peaks and O2 constitute a reversible couple. Scan reversal at more positive potentials resulted in the growth of the R2/02 couple and appearance of the R,/O, couple (Figure 2D). The peak current function (i,,/ACV”2)lo for peak I, increased with increasing Y. At a given Y, the peak current function for peak I, decreased with increasing concentration of 5-HTOL. Such behaviors indicate that 5-HTOL is adsorbed at the PGE.11 With increasing concentrations of 5-HTOL,reductionpeak R, and, particularly, the R,/O, couple peaks grew relative to
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6-3
B
T
0.0
-0.2
c
A.
? . A , -. 0.2
I/
1
R, R, n
F
aEda log C = 9 mV (0.2-1 mM 5-HTOL; 5 mV * s-'); aE,/apH = -30 mV (1 mM 5-HTOL; 5mV s-'). Controlled-Potential Electrolysis and Coulometry-At 5-HTOL concentrations between 0.03 and 1 mM, the coulometric n-value increased as the applied potential was made more positive (Table I). At a given potential, however, the n-value decreased as the concentration of 5-HTOL was increased. High-Performance Liquid Chromatography AnalysesThe HPLC analysis of the product solution obtained following incomplete electro-oxidation of 0.1 mM 5-HTOL a t 0.45 V showed three major peaks due to 4,4'-bi-(5-hydroxytryptophol)(A), 4-[7'-(tryptophol-4',5'-dione)]-5-hydroxytryptophol (D), and 2-[7'-(tryptophol-4',5'-dione)l-5-hydroxytryptophol (GI. Minor products were tryptophol-4,5-dione (B), 4,6'bi-(5-hydroxytryptophol)(El,2,4'-bi-(5-hydroxytryptophol) (F), l-17'-~tryptophol-4,5-dione)l-5-hydroxytryptophol (HI, and 5-0-[4'-(5'-hydroxtryptophol)]-l'-[7"-(tryptophol-4",5"dione)]-tryptophol (I; Figure 1A). Chromatographic peak C is due to unreacted 5-HTOL. Electrolysis a t more positive potential (0.70 V) for the same length of time caused 5-HTOL to be oxidized more rapidly (e.g., peak C is much smaller in Figure 1B than in Figure 1A). Compounds B, D, and G were the major products and the yields of E and F increased significantly. Compounds H and I were minor and A virtually disappeared. Electro-oxidation of higher concentrations of 5-HTOL (0.5 mM) gave dimer A as the major product a t all applied potentials employed (0.45-0.70 V), along with D, G, H, and I. Compound B was a significant product only when 5-HTOL was electrolyzed at high applied potentials (0.6-0.7 V). Electrolyses of low concentrations of 5-HTOL (33pM) gave D and G as the major products at low applied potentials; but, with increasingly positive potentials, B became the major product. Dimers A, E, and F were minor products and H and I were not formed. Electro-oxidation of 10 pM 5-HTOL at 0.70 V gave B in almost quantitative yield. Thus, dimers A, G, and H and trimer 1 are favored products when high concentrations (-0.5 mM) of 5-HTOL are oxidized a t low applied potentials (0.45 V). Dione B is favored by oxidation of low 5-HTOL concentrations at high potentials (0.70 V). Formation of B requires, overall, transfer of 4e per molecule of 5-HTOL oxidized. Trimer I, dimers D, G, and H, and dimers A, E, and F require 2.7e, 3e, and l e per molecule oxidations of 5-HTOL, respectively. The coulometric n-values reported in Table I reflect the effects of 5-HTOL concentration and applied potential on the product distribution. Cyclic Voltammetry and Spectra of P r o d u c t d y c l i c voltammograms of the oxidation products of 5-HTOL are shown in Figure 3. The features of all these voltammograms will not be discussed. However, it is of interest to note that dimer A exhibits a voltammetric oxidation peak at potentials very close to peak I, of 5-HTOL. After scan reversal, a reversible couple appears a t 0.30 V; that is, at the potential as the R,/O, couple observed in cyclic voltammograms of 5-HTOL (Figure 2). Dimer F shows similar behavior. Dione B shows a reversible couple at 0.10 V; that is, at the same potential as the RJO, couple observed in cyclic voltammograms of 5-HTOL (Figure 2). Dimers D, G, and H and trimer I, all of which contain a residue of B, show reversible couples at 0.10 V characteristic of the latter species. The UV-vis spectra of oxidation products of 5-HTOL are shown in Figure 4. Reaction Pathways-The characteristics of peak I, of 5-HTOL at pH 2 under conditions where the effects of adsorption are minimized (slow sweep rates and relatively high concentrations of 5-HTOL) are close to those predicted for a group of four dimerization reactions, all of which involve a n initial reversible one-electron abstraction to form a radical 5-200 mV s-');
R
tl I"J ,1
Potential I Volt vs. SCE Figure 24yclic voltammograms at the PGE of A-D (0.1 mM 5-HTOL), E (0.5mM 5-HTOL), and F (1.O mM 5-HTOL) in pH 2.0 phosphate buffer ( p = 0.5).Sweep rate: 200 mV s'.Switching potentials (mV): (A) 800, -230; ( 8 )410, -250; (C) 500, -200; (D) 610, -140; (E) 629, -220; (F) 610, -300.
peak I,, whereas the R,/O, couple peaks systematically decreased. Owing to a shift of peak 11, towards negative potentials with increasing 5-HTOL concentration, peaks I, and 11, merge at concentrations >1mM. Conversely, peak I, shifts to more positive potentials with increasing Y, with the result that peaks I, and 11, merge at Y values 2 500 mV.s-'. However, within the concentration and Y limits where peak I, can be clearly observed, the following peak characteristics were measured: aEJa log Y = 30 mV (1mM 5-HTOL; v = Table Woulometrlc n-Values Obtalned followlng Controlled-Potentlal Electro-oxldatlon of 5Hydroxytryptophol In 0.01 M HCI at Peak I, Potsntlals
n-Value for 5-HTOLn.b
Applied Potential, Volt versus SCE
0.035 mM
0.1 00 mM
0.500 mM
0.45 0.50 0.60 0.70
2.13 2.47 3.14 3.60
2.06 2.54 2.54 3.09
1.21 1.33 1.48 1.65
Average of three replicate measurements. Electrolyses were terminated when 30% of 5-HTOL was oxidized in order to minimize secondary electrochemical oxidations.
Journal of Pharmaceutical Sciences I 269 Vol. 79, No. 3, March 7990
r
\
A
I
A
1
D
I
L
C
2L 3
0 i
F
Potential I Volt vs. SCE Figure 3-Cyclic voltammograms at the PGE of A, 6 , D, E, F,G, H, and I in pH 2.0 phosphate buffer ( p = 0.5). Sweep rate: 200 mV-s '. Switching potentials (mv): (A) 1200,-200;( 6 )-200,1200; (D) -500,1300; (E)1100,-100;(F) 1200,-700;(G) -300,1100;(H) -300,1100;and (I)
-200,1200.
cation (5-HTOLt) that deprotonates, in a rate-controlling step, to a neutral radical (5-HTOL; Scheme 11.12 "he four possible mechanisms differ in the chemical/electrochemical reaction of 5-HTPP to form dimeric products. These mechanisms are predicated on the assumption that all chemical reactions are very rapid and all electron transfers are Nernstian. Because of very rapid reaction of the initial radical intermediate, no reverse reduction peak should be observed in cyclic voltammograms. In agreement with this prediction, cyclic voltammograms of 5-HTOL at u values 2 50 V s-l showed no evidence for a reduction peak reversibly coupled to peak I,. Formation of dimers A, E, and F upon electrooxidation of 5-HTOL a t peak I, potentials provides important support for a reactive radical intermediate. One mechanism which would result in the experimentally observed characteristics for peak I, involves radical-radical coupling. However, in each of the simple dimers formed (A, E, F), at least one 5-HTOL residue is linked at C(4). This suggests that the unpaired electron in 5-HTOLis localized at C(4) rather than being delocalized over the indole ring system. In the latter case, dimers linked at other sites (e.g., 6+6,2-*2,2+6, etc.) would be expected products but are not, in fact, formed. This leads to the conclusion that 5-HTOL, 270 I Journal of Pharmaceutical Sciences Vol. 79, No. 3, March 1990
Wavelength / nm Flgure &The UV-vis spectra of A, 9, D, E, F,G, H, and I in pH 2.0 phosphate buffer ( p = 0.5).
with the unpaired electron located at C(4), is attacked by 5-HTOL (i.e., a radica1:substrate reaction) to yield dimer radicals 1, 2, and 3 which are further oxidized (le,lH+) to dimers 4, 5, and 6 , respectively. Enolation of these dimers yields the isolated dimers A, E, and F, respectively (Scheme I). Mechanistic nuances associated with the radica1:substrate reaction outlined in Scheme I have been considered,lz but with the available experimental information we are unable to distinguish between these. Under both cyclic voltammetric and controlled-potential electrolysis conditions, dione B emerges as a major product at increasingly positive potentials, largely at the expense of dimers A, E, and F. This leads to the conclusion that 5-HTOL is further oxidized ( l e )to quinone imine species 7 (Scheme II). Nucleophilic attack by water yields 4,5-dihydroxytrytryptotrytr phol (10) which is immediately oxidized (2e,2H') to B (Scheme 11). In principle, 7 could react with 5-HTOL to yield a simple dimeric product (i.e., an ionmbstrate dimerization12). However, since B can only be derived from 7 and formation of €3 as a major product is favored by potentials considerably more positive than E, for peak I,, it seems unlikely that such a dimerization mechanism occurs. Disproportionation of 5-HTOL into 7 and 5-HTOL does not appear to occur to any appreciable extent, otherwise significant
H
H
5-HTOL
H
5-HTOL?
5-HTOL.
1 yCH
I-"'
5-HTOL
H
H
OH @OH
H
OH
goH 1 1
Q H
Peak 0,
0
-2H*-2e
H
F1 HF H
H
H
+2H++2e
N
loH
Peak R,
0
H
H O + H
P
N H
H
11
H
1
l-H'-e
Scheme II HO
I C H N
O
I
4
H
precursor 10 (the yield of G is estimated to be 5 16%of B), and only trace amounts of D and H.However, oxidations of higher concentrations of 5-HTOL (0.5-1mM) result in formation of G in higher yield than B, along with significant amounts of D and H.These observations provide support for Scheme III because when high concentrations of 5-HTOL are oxidized at high potential, 10 must be formed very rapidly in high yield
H
OH
HO
HNOH
F
E
N H
A
Scheme I amounts of B should be formed even when 5-HTOL is electrolyzed at low potentials. However, under such conditions, only small yields of B are formed. Nevertheless, the latter observation indicates that oxidation of 5-HTOL to 7 occurs at only slightly more positive potentials than that at which 5-HTOLis oxidized. Dimers D, G, and H contain residues of 5-HTOL and B. The 4,7'-, 2,7'-, and 1,7'-linked dimers of 5-HTOL are not known and hence it is not possible to ascertain if these compounds are oxidized to D, G, and H,respectively. However, dimers A, E,and F cannot be electro-oxidized to the corresponding analogues of these compounds at peak I, potentials. Addition of 6-HTOL to solutions of B at pH 2 does not yield D,G, or H.Hence, it may be concludedthat the latter dimers are formed during electro-oxidation of 5-HTOL by coupling of reactive intermediates. Accordingly, we propose that D, G, and H are formed by reactions between quinone imine 7 and 4,5-dihydroxytryptamine (10) as conceptualized in Scheme 111. Of the three dimers, G is always formed in highest yield. Oxidation of low concentrations of 5-HTOL (10-20 @) a t high applied potential (0.70' V) yields only a small amount of G compared with B and, hence, its immediate
11
13
fg+ -2H'-2e
+ HO
OH
HN \
\
H
HO
/
OH
D
G
H
Scheme 111 Journal of PharmaceuticalSciences I 271 Vol. 79, No. 3, March 1990
by the pathway shown in Scheme I1 and hence could effectively compete with water as a nucleophile for 7. Trimer I is a minor product formed in significant amounts only when relatively high concentrations of 5-HTOL are electro-oxidized. In view of the complex structure of I and the fact that many potential pathways and intermediates might be involved, it is not possible a t this time to propose a reaction pathway for its formation. Trimer I is the only product isolated from the electro-oxidation of 5-HTOL, 5-HT,ZS3and 5-HTPP4 at low pH which contains an oxygen bridge between two indolic residues. However, oligomers containing such linkages are formed at higher pH values.13 Cyclic voltammograms of 5-HTOL (Figure 2) reveal that at peak I, dimers A and F must be partially oxidized to the products responsible for the R,/O, couple. The identity of the latter species awaits a n investigation of the oxidation chemistry of A and F. The R3/Oz couple is due to B/10. Cyclic voltammograms of dimers A and F, which show the R,/O, couple, do not show reduction peak R,. Hence, it is unlikely that R, is an adsorption prepeak of peak R,. This implies, therefore, that R, is due to species which are precursors of A, F, B, or other identified products. Unfortunately, it is not possible at this time to definitively identify the unstable species responsible for reduction peak R,.
Conclusions The electrochemical oxidation of 5-HTOL proceeds through initial formation of the radical 5-HTOL. In this respect, the oxidation chemistry of 5-HTOL is similar to that of the biogenic indoleamines 5-HT2.3 and 5-HTPP.4 Based solely on the fact that every simple dimer formed upon oxidation of 5-HTOL, 5-HT, and 5-HTPP contains at least one indole residue linked at C(4), it has been concluded that the initial radical intermediates have the unpaired electron localized at C(4) and that dimerization proceeds through a radica1:substrate mechanism. The radical 5-HTOL can be further oxidized to quinone imine 7 which reacts with water to yield, ultimately, dione B or with 10 to yield a family of dimers (D, G, € containing I) one 5-HTOL residue and one residue of B. Compound B has been isolated in pure form. This is the first instance that the 4,5-dione of a biogenic indole has been isolated. There are several significant differences between the oxidation chemistry of 5-HTOL and that of 5-HT2.3and 5-HTPP.4
H
14 For example, a major oxidation product from 5-HT at pH 2 is the 3,4'-indolenine-indoledimer 14.3 A similar dimer is also formed upon oxidation of 5-HTPP, but it rapidly reacts further to yield a quinoline-indole dimer.4 Products analogous to 14 are not formed in the oxidation of 5-HTOL. Dimers containing 5-HTOL and B residues (e.g., D, G, and €are I)important oxidation products of 5-HTOL but analogous compounds are not formed from 5-HT293 and 5-HTPP.4 Similarly, one oxidatrimer I; I tion product of 5-HTOL is N(l)-C(4) linked dimer € contains a C(5)-0-C(4') linkage in addition to a n N(l')-C(7") linkage. Analogous structures have not been found as oxidation products of 5-HT and 5-HTPP. It is not yet known whether under pathological conditions 5-HTOL undergoes any oxidation reactions in the CNS. However, this study reveals that 5-HTOL is a n easily oxidized endogenous indole that yields a complex array of products via highly reactive intermediates. It is intriguing to speculate, if oxidation reactions similar to those reported here do occur under certain conditions in the CNS, that one or more of intermediates/products might possess neurodegenerative activity.
References and Notes Wrona, M. Z.; Dryhurst, G. J . Med. Chem. 1986,29, 499-505. Wrona, M. Z.; Dryhurst, G. J . Org. Chem. 1987,52,2817-2825. Wrona, M. Z.; Dryhurst, G. J . Org. Chem. 1989, 54, 2718-2721. Humphries, K.; Dryhurst, G. J . Pharm. Sci. 1987, 76, 839-847. Kveder, S.;Iskric, S.;Keglevic, D.Biochem. J . 1962,85,447-449. Owens, J. L.; Marsh, H. A.; Dryhurst, G. J . Electroanul. Chem. InteTfacMl Electrochem. 1978,91, 231-247. 7. Fenselau, C.; Cotter, R. J . Chem. Rev. 1987, 501412. 8. Gale, P. J.; Bentz, B. L.; Chait, B.; Field, F. H.;Cotter, R. J. A d . Chem. 1986.58, 1070-1076. 9. Pelzer, G.; DePauw, E.; Dung, D. V.; Marien, J. J . Phys. Chem. 1984,88,5065-5068. 10. In this function i is the eak current,A is the electrode area, C is the concentr&&n of 5-hTOL and v is the voltage sweep rate. 11. Wopschall, R. H.; Shain, I. Anal. Chem. 1967,39, 1514-1527. 12. Na ' 0 , L.; Savbant, J. M. J . Electroanal. Chem. Interfacial Electroc m. 1973.44.327-366. -,-- ---13. Wrona, M. Z.; Cheng, F-C.;Humphries, K.;Dryhurst, G., unpublished results. 1. 2. 3. 4. 5. 6.
L
Acknowledgments This work was sup rted by NIH Grant No: GM 32367. Additional support was provideaOby the Research Council and Vice Provost for Research Administration of the University of Oklahoma.
I
272 I Journal of Pharmaceutical Sciences Vol. 79, No. 3, March 1990
Fu-CHOUCHENG
AND
vent 5 was prepared by adding 3.0 mL of concentrated formic acid to 1 L of MeOH:H,O (10:90, v/v) and adjusting the pH to 3.40 with concentrated ammonium hydroxide. Solvent 6 consisted of 10% dioxane:20% acetonitri1e:lW methanol in water. Spectroscopic equipment used has been described elsewhere.14 Isolation and Identification of Products-The electrochemical behavior of 5-HTOL was the same in pH 2.0 phosphate buffer ( p = 0.5) and dilute HCl solution at the same pH. Analytical HPLC studies showed that the products of controlled-potential electro-oxidationsof 5-HTOL in the latter supporting electrolytes were the same. However, when semipreparative HPLC was attempted, large amounta of inorganic phosphate seriously degraded separations, wherear, dilute HCl did not cause this effect. Accordingly, all controlled-potential electrolyses, which were subsequently followed by HPLC separations, were performed in dilute HC1 solution at pH 2.0. Typically, repetitive 2-mL injections of product solutions were chromatographed and each individual HPLC peak was collected. The biological reasons for studying the oxidation chemistry Chromatograms of product solutions obtained following controlledand biochemistry of endogenous central nervous system electro-oxidations of 0.1 mM 5-HTOL in 0.01 M HCl are (CNS)indoles have been summarized elsewhere.14 The elec- potential shown in Figure 1. Chromatographic peak C is due to unreacted trochemical oxidation of 5-hydroxytryptamine (5-HT11-3 and 5-HTOL. The following sections will describe how components A, B, its precursor in the CNS, 5-hydroxytryptophan (5-HTPP1,' D, E,F, G, H,and I were isolated and purified and present spectral have recently been described. 5-Hydroxytryptophol(5-HTOL) and electrochemical evidence for their structures. is a normal metabolite of the latter indoleamines.5 The 4,4'-Bi-(5-hydroxytryptophol) (AbAliquots (2 mL) of A diseolved electrochemicaloxidation of 5-HTOL has been studied in acid in solvent 1 were injected onto the reversed-phase column using solvent 5 as the mobile phase (4 mL .min-') to completely separate solution to permit comparisons with the behaviors of 5-HT unreacted 5-HTOL. Then 10-20 mL of A in solvent 5 were pumped and 5-HTPP. onto the same column, which had been equilibrated with water, followed by 40 mL of water. Under the latter conditions, ammonium Experimental Section formate was eluted from the column while A was strongly retained. Compound A was subsequently eluted by changing the mobile phase 5-Hydroxytryptophol (5-HTOL) was obtained from Sigma (St. to solvent 6. The resulting eluant was collected and freeze dried. Louis, MO).Equipment and techniques employed for electrochemical Compound A was a white solid (Amx at pH 2.0 304,277.219 nm). studies have been described.l-4teA pyrolytic graphite electrode (PGE; Analysis by FAJLMS (dithioerythrito1:dithiothreitolmatrix) gave an P f h r Minerals, Pigmenta and Metals Division, Easton, PA), having an approximate surface area of 4 mm2, was used for voltammetric intense pseudomolecular ion (MH') at mle = 353.1543 (Co,,Hz1N~04; calc. m/e = 353.1501).Before discussing the 'H NMR spectrum of A, measurements. Controlled-potentialelectrolyses employed plates of it is of value to summarize that of 5-HTOL which has not previously pyrolytic graphite as the working electrode. All potentials are been reported (structures and numbering of all compounds are referred to the saturated calomel reference electrode (SCE)at ambipresented in Schemes 1-111 and elsewhere in the text): 'H NMR ent temperature (22 f 2 "C). High-performance liquid chromatography ( H P L C P employed a (Me,SO-$; 300 MHz): 8 11.49 (9, lH, N(l)-H), 8.56 (8, lH, 0(5)-H), reversed-phase column (Brownlee Laboratories, RP-18, 5 pm, 25 x 7.10 (d, J = 8.7 Hz,lH, C(7)-H),7.02 (8, lH, C(2)-H),6.79 (d, J = 2.4 Hz,lH, C(4)-H),6.57 (dd, J = 8.7 Hz,J = 2.4 Hz, 1H. C(6)-H),4.69 0.7 cm). For the initial separation of products, a binary gradient (t, lH, O(y)-H, 3.62 (m, 2H, C(/3)-H2),and 2.74 ppm (t, 2H, C(a)-Hp). HPLC method was employed. Solvent 1 was prepared by adding 10 Assignments of the various resonances in the spectrum of 5-WCOL, mL of concentratedformic acid to 1L of 2.5% dioxane:5% acetonitrile and indeed for other compounds reported, were based on comparisons in water and then adjusting the pH of the resulting solution to 3.40 with the spectra of 5-HT' and 5-HTPP and homonuclear decoupling with concentrated ammonium hydroxide. Solvent 2 was prepared by experiments. Addition of a few drops of D,O caused the resonances at adding 3.5 mL of concentrated formic acid to 1L of 10%dioxane:20% 11.49, 8.56, and 4.59 ppm to disappear. The 'H NMR spectrum of A acetonitrile:lO%methanol in water and then adjusting the pH of the is as follows: 'H NMR (Me,SO-$): S 10.37 (d, J = 2.1 Hz,2H, NU)-H resulting solution to 3.40 by addition of concentrated ammonium and N(l')-H),7.57 (s,2H, O(5)-Hand 0(5')-H),7.10 ( d , J = 8.6 Hz,2H, hydroxide. The following gradients were employed between 0 and 18 C(7)-H and C(7')-H), 6.86 (8, 2H, C(2)-H and C(2')-H), 3.87 (t, 2H, min, a linear gradient from 100%solvent 2 to 90% solvent 2 and 10% O(y)-H and O(y')-H), 3.11 (m, 4H, C(/3)-H, and C(F)-H,), and 1.96 solvent 1; between 18 and 30 min, a linear gradient to 55% solvent 2 ppm (m, 4H, C(a)-H2 and C(a')-H,). Addition of D,O caused the and 45% solvent 1;between 30 and 45 min, a linear gradient to 100% resonances at 10.37,7.57, and 3.87 ppm to disappear. Compared with solvent 1;this 100%solvent was then passed for an additional 15min. the spectrum of 5-HTOL, A lacks a signal for a C(U-proton. The A flow rate of 2 mL * min-I was used at all times. Four other HPLC C(6)-Hresonance of 5-HTOL appears ar, a doublet of doubleta owing solvents were used for purification and desalting of products. Solvent to spin couplings with C(7)-H and C(4)-H (long range). The C(6)-H 3 was 30% methanol in water. Solvent 4 was prepared by adding 3.5 resonances of A exhibit no long-range spin coupling and appear 88 a mL of concentrated formic acid to 1 L of 6% dioxane:12% doublet, indicating that the C(4)-H protons are missing in both acetonitrile:5%methanol in water. The pH of the resulting solution 5-HTOL residues. was adusted to 3.40 with concentrated ammonium hydroxide. Sol-
Abatrrct 0 The oxidation chemistry of the endogenous central nervous system indole 5-hydroxytryptophol(5-HTOL)has been studied at pH 2 using electrochemical methods. The first voltammetric oxidation peak (I) appears to involve an initial one-electron abstraction giving a transient radical cation that, in the rate-controlling step, deprotonates to give a neutral radical. A radical-substrate reaction then occurs to give a dimer radical which is further oxidized to yield three simple dimers (4,4'-,4,6'-, and 2,4'-linked).The neutral radical can be further oxidized (le) to a quinone imine that, as a result of very fast follow-up chemistry and electrochemistry,yields tryptophol-4,5-dione(B) which has been isolated in pure form. Reactions between intermediate species also resutt in three dimers containing residues of 5-HTOL and B and an unusual oxygenbridged trimer.
286 I Journal of Pharmaceutical Sciences Vol. 79, No. 3, March 1990
OO22-3549/90/03OO-0266$01 .ooJo
8 1990, American PharmaceuticalAssociation
t
(MH') of B. Accurate ma88 measurements on this ion gave mle = 192.0663 (C,,HloN,O3, calc. mle = 192.0661; 'H NMR spectrum (Me,SO-d,): S 11.80 (bs, lH, N(l)-H),7.29 (d, J = 9.9 Hz, lH, C(7)-H), 6.82 (8, lH, C(2)-H), 5.88 (d, J = 9.9 Hz, lH, C(6)-H), 4.59 (bs, lH, O(y)-H), 3.55 (m, 2H, C(P)-H,), and 2.75 ppm (t, 2H, C(a)-H2). Addition of D,O caused the resonances a t 11.80 and 4.59 pprn to disappear. The NMR results indicate that, compared with the spectrum of 5-HTOL, the C(4)-H and O(5)-H resonances are absent in B. The shiR of the C(6)-H resonance from 6.57 ppm (dd) in 5-HTOL to 5.88 ppm (d) in B supports the conclusion that in B this proton is located adjacent to a carbonyl group' and that B is, therefore, tryptophol4,5-dione. Confirmation of this structure was obtained by reacting B with a-phenylenediamine.2 The resulting orange product (Arn= at pH 2.0 533, 419, 365(sh), 280, 223(sh), and 207 nm) was purified by HPLC using solvent 2 as the mobile phase (2 mL min-' flow rate, tR = 30 min). Analysis by EI-MS (70 eV, 270 "C) gave mle = 263 (1192, M+), 233 (loo%, M+-CHOH), and 232 (77%, M+-CH,OH); 'H NMR (Me,SO-4): S 11.87 (6, lH,N(l)-H),8.23 (m, 2H, aromatic H, ring a), 8.01 (d, J = 9.0 Hz,lH, C(7)-H),7.87 (m, 2H, aromatic H, ring a), 7.74 ( d , J = 9.0 Hz, lH, C(6)-H), 7.37 (s, l H , C(2)-H),4.68 (t, lH, O(y)-H), 3.90 (m, 2H, C(P)-H,), and 3.52 ppm (t, 2H, C(a)-H2).These spectral data are in accord with structure J formed by condensation of B with o-phenylenediamine. 4-[7'-(Tryptophol-4',5'-dione)l-5-hydroxytryptophol (D)Compound D (10-20 mL), dissolved in solvent 1,was pumped onto the HPLC column (previously equilibrated with water) and then 40 mL of water was pumped through the column (2 mL . min-') to elute ammonium formate. The mobile phase was then changed to solvent 6 (2 mL * min - l ) to elute D. The resulting solution was freeze dried. Compound D was a purple solid (Amm at pH 2.00: 548, 368, 300(sh), 283, and 223 nm). The F A E M S (dithioerythrito1:dithiothreitol matrix) analysis gave intense ions a t mle = 367(57%),368(100%),and 369(11%). The purple color of D faded in the FAB-MS matrix indicating that it was partially reduced. Thus, the ion at mle = 367 represents the pseudomolecular ion (MH'). Exact mass measurements on this ion gave mle = 367.1255 (C,oH,7N,0,; calc. d e = 367.1294); 'H NMR (Me,SO-d,): S 11.20 (bs, l H , N(l')-H), 10.82 ( 8 , lH, N(1)-H),8.87 (bs, lH, 0(5)-H),7.27 (d, J = 8.7 Hz,lH, C(7)-H), 7.10 (8, 1H, C(2)-H), 6.78 (d, J = 8.7 Hz, lH, C(6)-H), 6.61 (8, lH, C(2')-H), 5.67 (9, lH, C(6')-H), 4.61 (t, lH, O($-H), 4.42 (t, lH, O(y')-H), 3.60 (m, 2H, C(P)-H,), 2.80 (m, 2H, C(a)-H2),and 2.60 ppm (m, 2H, C(a'-H,). The C(P')-H, resonance was obscured by a large HOD signal. Addition of D,O caused the resonances at 11.20, 10.82, 8.87, 4.61, and 4.42 ppm to disappear. The F A E M S analysis indicates that D is a dimer containing one residue of 5-HTOL and another having a dione structure. The 'H NMR spectra indicate only a single C(7)-H residue and no C(4)-Hresonances. The singlet a t 5.67 ppm has been assigned to the C(6')-H proton in a tryptophol-4,5-dione residue by analogy with similar structures (e.g., C(6)-H in 5hydroxytryptamine-4,7-dioneresonates at 5.73 ppm'). The structure of D was confirmed by reacting it with o-phenylenediamine? a t pH 2.00: 550,431,374(sh), 277, The yellow-orange product [A, and 237(sh) nml was purified by HPLC using solvent 2 (2 mL * min-', tR = 25 rnin): FAB-MS (glycerol matrix) gave MH+(lOO%)a t mle = 439.1772 (CZ6Hz1N4O3;calc. rnle = 439.1770); 'H NMR (Me,SO&): 11.00 (bs, 1H, NU')-H), 10.76 (9, lH, N(l)-H), 8.25 (dd, 2H, aromatic H from ring a), 7.87 (m, 2H, aromatic H from ring a), 7.50 ( 8 , lH, C(6')-H), 7.32 (d, J = 8.6 Hz, C(7)-H), 7.16 (8, lH, C(2)-H),7.03 (8, lH, C(2')-H), 6.87 (d, J = 8.6 Hz, l H , C(6)-H), 4.73 (bs, lH, O(y)-H), 3.94 (m, 3H, C(P)-H,) and O(y')-H), 3.66 (m, 2H, C(a)-H2),3.03 (m, 2H, C(P')-H,), and 2.01 ppm (m, 2H, C(a')-H,). The NMR and MS spectral data are in accord with structure K which would be expected to be formed by reaction of D with o-phenylenediamine. 4,6'-Bi-(5-hydroxytryptophol)(EkCompound E (10-20 mL) die-
-
Time I min
0
1
C
0.3
l0.N
2o.m
l
a.m
Ea.N
Time I min
Flgure 1-High-pressure
liquid chromatograms of the products formed
upon controlled-potential electro-oxidation of 0.10 rnM 5-hydroxytryptophol in dilute HCI (pH 2.0) at (A) 0.45 V and (B) 0.70 V. The HPLC conditions are given in the Experimental Section.
Tryptophol-4,5-dione (BbCompound B in solvent 1 (10-20 mL) was pumped onto the reversed-phase column that had previously been equilibrated with water; this was followed by the addition of 30-60 mL of water. Under these conditions, B was retained on the column, whereas all components of solvent 1 were eluted. The mobile phase was then changed to solvent 6 (2-3 mL * min-'), which caused B to elute. The resulting solution was freeze dried. Component B was a blue-purple solid (A,= in dilute HC1, pH 2.0: 550, 358, 233 nm). Component B was not very stable in solution. Therefore, during the purification and isolation procedures, solutions containing this compound were stored a t -80 "C to minimize decomposition. Analysis by FAB-MS (thioglycerol matrix) gave a cluster of three intense ions a t mle = 192 (17%), 193 (59%), and 194 (40%). However, it was noted that when B was mixed with the FAB-MS matrix its blue-purple color faded, indicating partial reduction of the compound. Similar effects have been noted by other workers with easily reducible compounds.7-9 Hence, the ion at mle = 192 is the pseudomolecular ion
J Journal of Pharmaceutical Sciences I 267 Vol. 79, No. 3, March 7990
K solved in solvent 1 was pumped onto the column followed by 40 mL of water (2 mL . min-’) to elute ammonium formate. Compound E was eluted using solvent 6 as the mobile phase (2 mL . min-I). The solution containing E was then freeze dried. Compound E was a white solid (A,,- at pH 2.00: 305,280, and 226 nm). Analysis by EI-MS (70 eV, 270 “C)gave mle = 352 (69%,M+),321 (48%,M+-CH,OH). Exact mass measurements on M’ gave mle = 352.1453 ( C 2 ~ , o N 2 0 , ,calc. mle = 352.1423); IH NMR (Me,SO-$): 6 10.46 (d, J = 2.4 Hz,lH, N(1)-H), 10.38 (d, J = 1.5 Hz, lH, N(l’)-H), 7.09 (d, J = 8.4 HZ, lH, C(7)-H), 7.02 (8, lH, C(4‘)-H), 6.94 ( 8 , lH, C(2)-H), 6.93 ( 8 , lH, C(7‘)-H),6.91 (9, l H , C(2’)-H),6.69 (d, J = 8.7 Hz, lH, C(6)-H), 4.64 (m, lH, O(y)-H),4.10 (m, 1H) O(y’)-H),4.04 (m, 2H, C(p)-H,), 3.67 (m, 2H, C(P’)-H,), 2.79 (m, 2H, C(a-H2),and 2.17 ppm (m, 2 H, C(a’)-H2). Addition of D 2 0 caused the resonances a t 10.46,10.38,4.64, and 4.10 ppm to disappear. 2,4’-Bi-(5.hydroxytryptophol)(FbCompound F (10-20 mL) was pumped onto the column followed by 40 mL of water (2 mL * min- ’) to remove ammonium formate. Compound F eluted using solvent 6 as the mobile phase (2 mL min -’). The resulting solution was freeze dried. Compound F waa a white solid (Amax at pH 2.0: 300 and 226 nm). The FAB-MS (dithioerythrito1:dithiothreitolmatrix) analysis gave MH+(53%) a t mle = 353.1548 (C2,H1,N,O,; calc. m/e = 353.1501); ’H NMR (Me,SO&): 6 10.63 (s, lH, N(l)-H),10.52 ( 8 , lH, N(1’)-H), 8.50 (8, lH, 0(5)-H), 8.30 (6, IH, 0(5’)-H), 7.22 (d, J = 8.7 Hz, l H , C(7’)-H), 7.06 (d, J = 8.7 Hz, l H , C(7)-H), 7.02 (8, 1H, C(2’)*H),6.83 (d, J = 2.1 Hz, lH, C(4)-H), 6.74 (d, J = 8.7 Hz, lH, C(6’)-H),6.55 (dd, J = 8.6 Hz, J = 2.4 Hz, lH, C(6)-H), 4.49 ( 8 , lH, O(y)-H),and 4.11 ppm (t, lH, O(y’)-H).The region below 4.0 ppm was not well defined owing to the interfering signals of HOD and Me,SO. Nevertheless, comparison of the NMR spectra of F and 5-HTOL clearly shows the absence of a C(2)-H in one 5-HTOL residue of F and the absence of C(4)-H in the other. Compounds G, H, and I-Compounds G, H, and I were initially collected together in solvent 2. The combined solutions were then freeze dried. The resulting solid was dissolved in the minimum volume of solvent 2 and diluted five times with water. Aliquots (2 mL) of this solution were then chromatographed using solvent 4 as the mobile phase (2 mL min-’). The tR values for G, H,and I were 11, 16, and 21 min, respectively. Each of the solutions containing the latter products was diluted five times by the addition of water. The solutions containing C,H, or I were desalted by pumping 10-20 mL onto the column and then passing water through the column (2 mL min-l, -40 mL). Solvent 6 was used to elute the appropriate product. Each product solution was then freeze dried. 2-[7’-(Tryptophol-4’,5’-dione)]-5-hydroxytryptophol(GICompound G was a deep brown solid (Arnm at pH 2.0: 506(sh), 450, 380(sh), 275, and 212 nm). The FAELMS(dithioerythrito1:dithiothreitol matrix) analysis gave mle = 367(31%), 368(100%),and 369(86%). Compound C is a very easily reduced compound and hence was partially reduced by the FAELMS matrix giving the ions at mle = 368 and 369. The pseudomolecular ion (MH’) had an exact mass of mie = 367.1305 (C;oHlgN206; calc. mle = 367.1294); ’H NMR(Me,SO-d,): 6 11.67 (8. 1H. N(l’)-H). 11.16 (8. 1H. N(1)-H), 8.88 (8, 1H. 0(5)-H), 7.22 (d, J = 8.7 Hz, l H , C(7)-H),6.92 ( d , J = 2.7 Hz, lH, C(4)-H),6.83 (6, 1H, C(2’)-H), 6.76 (dd, J = 8.7 HZ,J = 2.4 HZ, lH, C(6)-H), 6.08 (a, lH, C(6‘)-H),5.04 (bs, lH, O(y’)-H),4.61 (t, lH, O(y)-H), 3.69 (m, 2H, C(p)-H,), 3.59 (m, 2H, C(P’)-H2),2.91 (t, 2H, Cb)-HJ, 2.82 (t, 2H, C(a’)-H2). Addition of DzO caused the resonances at 11.67, 11.16, 8.88,5.04, and 4.61 ppm to disappear. The sharp singlet (1H) observed
-
268 I Journal of Pharmaceutical Sciences Vol. 79, No. 3, March 1990
at 6.08 ppm is indicative of the presence of one residue of tryptophol4,5-dione in G. The characteristic peaks for C(4)-H (6.92 ppm, d), C(6)-H (6.76 ppm, dd), and C(7)-H (7.22 ppm, d) indicate that G contains one residue of 5-HTOL. The absence of one C(2)-H resonance confirms that this residue is linked to the tryptophol-4,6-dione through the C(2)-position. The absence of a C(7)-H resonance 8880ciated with the tryptophol-4,5-dione moiety indicates that this residue is linked a t (37). l-[7’-(Tryptophol-4,5-dione)]-5-hydr0xytryptophol ( H r C o m pound H was a deep brown solid (&= at pH 2.0: 595(sh), 462,313(sh), 280, and 235 nm). Compound H is a very easily reducible compound. Thus, FAB-MS (dithioerythrito1:dithiothreitolmatrix) exhibited intense ions at m/e = 367(44%, MH’), 368(100%),and 369(58%). Exact mass measurements (MH’) gave m!e = 367.1316(C,&$J,06; calc. mle = 367.1294); ’H NMR (Me2SO-d6):6 11.83 ( 8 , lH, N(l’)-H), 9.24 (s,lH, O(5)-H), 7.43 (s, IH, C(2)-H), 7.41 (d, J = 9.0 HZ, lH, C(7)-H), 6.96 (d, J = 2.4 Hz, lH, C(4)-H), 6.94 (9, lH, C(2’)-H), 6.78 (dd, J = 9.0 Hz, J = 2.4 Hz, l H , C(6)-H), 5.94 ( 6 , l H , C(6’)-H), 4.71 (t, lH, O(f)-H), 4.63 (t, lH, O(y)-H), 3.72 (m, 2H, C(p’)-H,), 3.61 (m,2H, C(P)-H,), 2.85 (t, 2H, C(a‘)-H,), and 2.80 ppm (t, 2H, C(a)-H2). Addition of D,O caused the peaks a t 11.83, 9.24,4.71, and 4.63 ppm to disappear. The NMR data indicate that only one N-H is present in H and that the tryptophol-4,5-dione residue is linked a t (37‘). 5-044’4 5’-Hydroxytryptophol)]-1’[7“-(tryptophol-4”,~~ione)~ tryptophol (I&Compound I was a deep brown solid (Ama at pH 2.0: 560(sh), 458, 304(sh), 277, and 228 nm). The FAB-MS(dithioerythrito1:dithiothreitol matrix) analysis gave mle = 542(3.4%, MH’), 543(3.6%), and 544(3.1%).The matrix thus caused partial reduction of I. Exact mass measurements on MH+ gave rnle = 542.1968 (C3JIz8N30,; calc. mle = 542.1927); ‘H NMR (Me,SO-&): 6 11.10 (8, lH, N(l)-H),10.98 (bs, l H , N(l”)-H),9.54 (6, lH, 0(5’)-H), 7.34 (8, 1H,C(2’)-H),7.21 (d, J = 8.7 Hz, lH,C(7’)-H),7.05 ( d , J = 9.0 Hz,lH, C(7)-H), 7.00 (8, l H , C(2)-H), 6.78 (d, J = 7.8 Hz,lH, C(6’)-H), 6.76 (d, J = 2.1 Hz, 1H, C(4)-H),6.74 (8, lH, C(2)-H),6.66 (dd, J = 8.4 Hz; J = 1.8 Hz, lH, C(6)-H), 5.92 ( 8 , lH, C(6)-H), 4.82 (t, lH, O(V)-H), 4.62 (t, lH, O(y’)-H), 4.60 (t, lH, O(y)-H),3.85 (m, 2H, C(p”)-H,), 3.61 (m, 2H, C(p)-H,), 3.49 (m, 2H, C(p’)-H2), 3.09 (t, 2H, C(a)-H,), and 2.86 ppm (t, 2H, C(a‘)-H2).The C(d’)-H, resonance was obscured by the signal due to HOD at 3.30 pprn. Addition of D,O caused the resonances at 11.10, 10.98, 9.54, 4.82, 4.62, and 4.60 ppm to dieappear. These NMR results indicate that in trimer I, one 5-HTOL residue is linked through its N(1) position and C(4) positions to other residues. Only one 0(5)-H resonance appears in the spectrum, suggesting, therefore, that one of the two 5-HTOL residues ie linked via its 0(5)-position. The singlet at 5.92 ppm indicates that a tryptophol-4,5-dione residue is present in I. Furthermore, the latter residue lacks a proton at the C(7)-position which, therefore, must be the site a t which it is linked to a 5-HTOL residue.
Results and Discussion Cyclic Voltammetry-A cyclic voltammogram of 5-HTOL at pH 2.0 shows oxidation peaks I, (E, = 0.495 V at a sweep rate of 200 mV s-’) and 11, (E, -0.80 V; Figure 2A). After scan reversal, reduction peaks R,, R,, and R3 appear, the former two peaks being extensively overlapped. On the second anodic sweep, oxidation peaks 0, and 0, appear. The rounded shape of peak I, suggests that it does not represent a simple oxidation process and peak clipping experiments confirmed this suggestion. When the initial anodic scan was reversed at the foot of peak I,, only a small reduction peak (R,) appeared (Figure 2B). Scan reversal at the E , for peak I, resulted in a considerable growth in peak R, and the first appearance of peak R,, and, on the second anodic sweep, oxidation peak O2 appeared (Figure 2C).This behavior indicates that peaks and O2 constitute a reversible couple. Scan reversal at more positive potentials resulted in the growth of the R2/02 couple and appearance of the R,/O, couple (Figure 2D). The peak current function (i,,/ACV”2)lo for peak I, increased with increasing Y. At a given Y, the peak current function for peak I, decreased with increasing concentration of 5-HTOL. Such behaviors indicate that 5-HTOL is adsorbed at the PGE.11 With increasing concentrations of 5-HTOL,reductionpeak R, and, particularly, the R,/O, couple peaks grew relative to
-
6-3
B
T
0.0
-0.2
c
A.
? . A , -. 0.2
I/
1
R, R, n
F
aEda log C = 9 mV (0.2-1 mM 5-HTOL; 5 mV * s-'); aE,/apH = -30 mV (1 mM 5-HTOL; 5mV s-'). Controlled-Potential Electrolysis and Coulometry-At 5-HTOL concentrations between 0.03 and 1 mM, the coulometric n-value increased as the applied potential was made more positive (Table I). At a given potential, however, the n-value decreased as the concentration of 5-HTOL was increased. High-Performance Liquid Chromatography AnalysesThe HPLC analysis of the product solution obtained following incomplete electro-oxidation of 0.1 mM 5-HTOL a t 0.45 V showed three major peaks due to 4,4'-bi-(5-hydroxytryptophol)(A), 4-[7'-(tryptophol-4',5'-dione)]-5-hydroxytryptophol (D), and 2-[7'-(tryptophol-4',5'-dione)l-5-hydroxytryptophol (GI. Minor products were tryptophol-4,5-dione (B), 4,6'bi-(5-hydroxytryptophol)(El,2,4'-bi-(5-hydroxytryptophol) (F), l-17'-~tryptophol-4,5-dione)l-5-hydroxytryptophol (HI, and 5-0-[4'-(5'-hydroxtryptophol)]-l'-[7"-(tryptophol-4",5"dione)]-tryptophol (I; Figure 1A). Chromatographic peak C is due to unreacted 5-HTOL. Electrolysis a t more positive potential (0.70 V) for the same length of time caused 5-HTOL to be oxidized more rapidly (e.g., peak C is much smaller in Figure 1B than in Figure 1A). Compounds B, D, and G were the major products and the yields of E and F increased significantly. Compounds H and I were minor and A virtually disappeared. Electro-oxidation of higher concentrations of 5-HTOL (0.5 mM) gave dimer A as the major product a t all applied potentials employed (0.45-0.70 V), along with D, G, H, and I. Compound B was a significant product only when 5-HTOL was electrolyzed at high applied potentials (0.6-0.7 V). Electrolyses of low concentrations of 5-HTOL (33pM) gave D and G as the major products at low applied potentials; but, with increasingly positive potentials, B became the major product. Dimers A, E, and F were minor products and H and I were not formed. Electro-oxidation of 10 pM 5-HTOL at 0.70 V gave B in almost quantitative yield. Thus, dimers A, G, and H and trimer 1 are favored products when high concentrations (-0.5 mM) of 5-HTOL are oxidized a t low applied potentials (0.45 V). Dione B is favored by oxidation of low 5-HTOL concentrations at high potentials (0.70 V). Formation of B requires, overall, transfer of 4e per molecule of 5-HTOL oxidized. Trimer I, dimers D, G, and H, and dimers A, E, and F require 2.7e, 3e, and l e per molecule oxidations of 5-HTOL, respectively. The coulometric n-values reported in Table I reflect the effects of 5-HTOL concentration and applied potential on the product distribution. Cyclic Voltammetry and Spectra of P r o d u c t d y c l i c voltammograms of the oxidation products of 5-HTOL are shown in Figure 3. The features of all these voltammograms will not be discussed. However, it is of interest to note that dimer A exhibits a voltammetric oxidation peak at potentials very close to peak I, of 5-HTOL. After scan reversal, a reversible couple appears a t 0.30 V; that is, at the potential as the R,/O, couple observed in cyclic voltammograms of 5-HTOL (Figure 2). Dimer F shows similar behavior. Dione B shows a reversible couple at 0.10 V; that is, at the same potential as the RJO, couple observed in cyclic voltammograms of 5-HTOL (Figure 2). Dimers D, G, and H and trimer I, all of which contain a residue of B, show reversible couples at 0.10 V characteristic of the latter species. The UV-vis spectra of oxidation products of 5-HTOL are shown in Figure 4. Reaction Pathways-The characteristics of peak I, of 5-HTOL at pH 2 under conditions where the effects of adsorption are minimized (slow sweep rates and relatively high concentrations of 5-HTOL) are close to those predicted for a group of four dimerization reactions, all of which involve a n initial reversible one-electron abstraction to form a radical 5-200 mV s-');
R
tl I"J ,1
Potential I Volt vs. SCE Figure 24yclic voltammograms at the PGE of A-D (0.1 mM 5-HTOL), E (0.5mM 5-HTOL), and F (1.O mM 5-HTOL) in pH 2.0 phosphate buffer ( p = 0.5).Sweep rate: 200 mV s'.Switching potentials (mV): (A) 800, -230; ( 8 )410, -250; (C) 500, -200; (D) 610, -140; (E) 629, -220; (F) 610, -300.
peak I,, whereas the R,/O, couple peaks systematically decreased. Owing to a shift of peak 11, towards negative potentials with increasing 5-HTOL concentration, peaks I, and 11, merge at concentrations >1mM. Conversely, peak I, shifts to more positive potentials with increasing Y, with the result that peaks I, and 11, merge at Y values 2 500 mV.s-'. However, within the concentration and Y limits where peak I, can be clearly observed, the following peak characteristics were measured: aEJa log Y = 30 mV (1mM 5-HTOL; v = Table Woulometrlc n-Values Obtalned followlng Controlled-Potentlal Electro-oxldatlon of 5Hydroxytryptophol In 0.01 M HCI at Peak I, Potsntlals
n-Value for 5-HTOLn.b
Applied Potential, Volt versus SCE
0.035 mM
0.1 00 mM
0.500 mM
0.45 0.50 0.60 0.70
2.13 2.47 3.14 3.60
2.06 2.54 2.54 3.09
1.21 1.33 1.48 1.65
Average of three replicate measurements. Electrolyses were terminated when 30% of 5-HTOL was oxidized in order to minimize secondary electrochemical oxidations.
Journal of Pharmaceutical Sciences I 269 Vol. 79, No. 3, March 7990
r
\
A
I
A
1
D
I
L
C
2L 3
0 i
F
Potential I Volt vs. SCE Figure 3-Cyclic voltammograms at the PGE of A, 6 , D, E, F,G, H, and I in pH 2.0 phosphate buffer ( p = 0.5). Sweep rate: 200 mV-s '. Switching potentials (mv): (A) 1200,-200;( 6 )-200,1200; (D) -500,1300; (E)1100,-100;(F) 1200,-700;(G) -300,1100;(H) -300,1100;and (I)
-200,1200.
cation (5-HTOLt) that deprotonates, in a rate-controlling step, to a neutral radical (5-HTOL; Scheme 11.12 "he four possible mechanisms differ in the chemical/electrochemical reaction of 5-HTPP to form dimeric products. These mechanisms are predicated on the assumption that all chemical reactions are very rapid and all electron transfers are Nernstian. Because of very rapid reaction of the initial radical intermediate, no reverse reduction peak should be observed in cyclic voltammograms. In agreement with this prediction, cyclic voltammograms of 5-HTOL at u values 2 50 V s-l showed no evidence for a reduction peak reversibly coupled to peak I,. Formation of dimers A, E, and F upon electrooxidation of 5-HTOL a t peak I, potentials provides important support for a reactive radical intermediate. One mechanism which would result in the experimentally observed characteristics for peak I, involves radical-radical coupling. However, in each of the simple dimers formed (A, E, F), at least one 5-HTOL residue is linked at C(4). This suggests that the unpaired electron in 5-HTOLis localized at C(4) rather than being delocalized over the indole ring system. In the latter case, dimers linked at other sites (e.g., 6+6,2-*2,2+6, etc.) would be expected products but are not, in fact, formed. This leads to the conclusion that 5-HTOL, 270 I Journal of Pharmaceutical Sciences Vol. 79, No. 3, March 1990
Wavelength / nm Flgure &The UV-vis spectra of A, 9, D, E, F,G, H, and I in pH 2.0 phosphate buffer ( p = 0.5).
with the unpaired electron located at C(4), is attacked by 5-HTOL (i.e., a radica1:substrate reaction) to yield dimer radicals 1, 2, and 3 which are further oxidized (le,lH+) to dimers 4, 5, and 6 , respectively. Enolation of these dimers yields the isolated dimers A, E, and F, respectively (Scheme I). Mechanistic nuances associated with the radica1:substrate reaction outlined in Scheme I have been considered,lz but with the available experimental information we are unable to distinguish between these. Under both cyclic voltammetric and controlled-potential electrolysis conditions, dione B emerges as a major product at increasingly positive potentials, largely at the expense of dimers A, E, and F. This leads to the conclusion that 5-HTOL is further oxidized ( l e )to quinone imine species 7 (Scheme II). Nucleophilic attack by water yields 4,5-dihydroxytrytryptotrytr phol (10) which is immediately oxidized (2e,2H') to B (Scheme 11). In principle, 7 could react with 5-HTOL to yield a simple dimeric product (i.e., an ionmbstrate dimerization12). However, since B can only be derived from 7 and formation of €3 as a major product is favored by potentials considerably more positive than E, for peak I,, it seems unlikely that such a dimerization mechanism occurs. Disproportionation of 5-HTOL into 7 and 5-HTOL does not appear to occur to any appreciable extent, otherwise significant
H
H
5-HTOL
H
5-HTOL?
5-HTOL.
1 yCH
I-"'
5-HTOL
H
H
OH @OH
H
OH
goH 1 1
Q H
Peak 0,
0
-2H*-2e
H
F1 HF H
H
H
+2H++2e
N
loH
Peak R,
0
H
H O + H
P
N H
H
11
H
1
l-H'-e
Scheme II HO
I C H N
O
I
4
H
precursor 10 (the yield of G is estimated to be 5 16%of B), and only trace amounts of D and H.However, oxidations of higher concentrations of 5-HTOL (0.5-1mM) result in formation of G in higher yield than B, along with significant amounts of D and H.These observations provide support for Scheme III because when high concentrations of 5-HTOL are oxidized at high potential, 10 must be formed very rapidly in high yield
H
OH
HO
HNOH
F
E
N H
A
Scheme I amounts of B should be formed even when 5-HTOL is electrolyzed at low potentials. However, under such conditions, only small yields of B are formed. Nevertheless, the latter observation indicates that oxidation of 5-HTOL to 7 occurs at only slightly more positive potentials than that at which 5-HTOLis oxidized. Dimers D, G, and H contain residues of 5-HTOL and B. The 4,7'-, 2,7'-, and 1,7'-linked dimers of 5-HTOL are not known and hence it is not possible to ascertain if these compounds are oxidized to D, G, and H,respectively. However, dimers A, E,and F cannot be electro-oxidized to the corresponding analogues of these compounds at peak I, potentials. Addition of 6-HTOL to solutions of B at pH 2 does not yield D,G, or H.Hence, it may be concludedthat the latter dimers are formed during electro-oxidation of 5-HTOL by coupling of reactive intermediates. Accordingly, we propose that D, G, and H are formed by reactions between quinone imine 7 and 4,5-dihydroxytryptamine (10) as conceptualized in Scheme 111. Of the three dimers, G is always formed in highest yield. Oxidation of low concentrations of 5-HTOL (10-20 @) a t high applied potential (0.70' V) yields only a small amount of G compared with B and, hence, its immediate
11
13
fg+ -2H'-2e
+ HO
OH
HN \
\
H
HO
/
OH
D
G
H
Scheme 111 Journal of PharmaceuticalSciences I 271 Vol. 79, No. 3, March 1990
by the pathway shown in Scheme I1 and hence could effectively compete with water as a nucleophile for 7. Trimer I is a minor product formed in significant amounts only when relatively high concentrations of 5-HTOL are electro-oxidized. In view of the complex structure of I and the fact that many potential pathways and intermediates might be involved, it is not possible a t this time to propose a reaction pathway for its formation. Trimer I is the only product isolated from the electro-oxidation of 5-HTOL, 5-HT,ZS3and 5-HTPP4 at low pH which contains an oxygen bridge between two indolic residues. However, oligomers containing such linkages are formed at higher pH values.13 Cyclic voltammograms of 5-HTOL (Figure 2) reveal that at peak I, dimers A and F must be partially oxidized to the products responsible for the R,/O, couple. The identity of the latter species awaits a n investigation of the oxidation chemistry of A and F. The R3/Oz couple is due to B/10. Cyclic voltammograms of dimers A and F, which show the R,/O, couple, do not show reduction peak R,. Hence, it is unlikely that R, is an adsorption prepeak of peak R,. This implies, therefore, that R, is due to species which are precursors of A, F, B, or other identified products. Unfortunately, it is not possible at this time to definitively identify the unstable species responsible for reduction peak R,.
Conclusions The electrochemical oxidation of 5-HTOL proceeds through initial formation of the radical 5-HTOL. In this respect, the oxidation chemistry of 5-HTOL is similar to that of the biogenic indoleamines 5-HT2.3 and 5-HTPP.4 Based solely on the fact that every simple dimer formed upon oxidation of 5-HTOL, 5-HT, and 5-HTPP contains at least one indole residue linked at C(4), it has been concluded that the initial radical intermediates have the unpaired electron localized at C(4) and that dimerization proceeds through a radica1:substrate mechanism. The radical 5-HTOL can be further oxidized to quinone imine 7 which reacts with water to yield, ultimately, dione B or with 10 to yield a family of dimers (D, G, € containing I) one 5-HTOL residue and one residue of B. Compound B has been isolated in pure form. This is the first instance that the 4,5-dione of a biogenic indole has been isolated. There are several significant differences between the oxidation chemistry of 5-HTOL and that of 5-HT2.3and 5-HTPP.4
H
14 For example, a major oxidation product from 5-HT at pH 2 is the 3,4'-indolenine-indoledimer 14.3 A similar dimer is also formed upon oxidation of 5-HTPP, but it rapidly reacts further to yield a quinoline-indole dimer.4 Products analogous to 14 are not formed in the oxidation of 5-HTOL. Dimers containing 5-HTOL and B residues (e.g., D, G, and €are I)important oxidation products of 5-HTOL but analogous compounds are not formed from 5-HT293 and 5-HTPP.4 Similarly, one oxidatrimer I; I tion product of 5-HTOL is N(l)-C(4) linked dimer € contains a C(5)-0-C(4') linkage in addition to a n N(l')-C(7") linkage. Analogous structures have not been found as oxidation products of 5-HT and 5-HTPP. It is not yet known whether under pathological conditions 5-HTOL undergoes any oxidation reactions in the CNS. However, this study reveals that 5-HTOL is a n easily oxidized endogenous indole that yields a complex array of products via highly reactive intermediates. It is intriguing to speculate, if oxidation reactions similar to those reported here do occur under certain conditions in the CNS, that one or more of intermediates/products might possess neurodegenerative activity.
References and Notes Wrona, M. Z.; Dryhurst, G. J . Med. Chem. 1986,29, 499-505. Wrona, M. Z.; Dryhurst, G. J . Org. Chem. 1987,52,2817-2825. Wrona, M. Z.; Dryhurst, G. J . Org. Chem. 1989, 54, 2718-2721. Humphries, K.; Dryhurst, G. J . Pharm. Sci. 1987, 76, 839-847. Kveder, S.;Iskric, S.;Keglevic, D.Biochem. J . 1962,85,447-449. Owens, J. L.; Marsh, H. A.; Dryhurst, G. J . Electroanul. Chem. InteTfacMl Electrochem. 1978,91, 231-247. 7. Fenselau, C.; Cotter, R. J . Chem. Rev. 1987, 501412. 8. Gale, P. J.; Bentz, B. L.; Chait, B.; Field, F. H.;Cotter, R. J. A d . Chem. 1986.58, 1070-1076. 9. Pelzer, G.; DePauw, E.; Dung, D. V.; Marien, J. J . Phys. Chem. 1984,88,5065-5068. 10. In this function i is the eak current,A is the electrode area, C is the concentr&&n of 5-hTOL and v is the voltage sweep rate. 11. Wopschall, R. H.; Shain, I. Anal. Chem. 1967,39, 1514-1527. 12. Na ' 0 , L.; Savbant, J. M. J . Electroanal. Chem. Interfacial Electroc m. 1973.44.327-366. -,-- ---13. Wrona, M. Z.; Cheng, F-C.;Humphries, K.;Dryhurst, G., unpublished results. 1. 2. 3. 4. 5. 6.
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Acknowledgments This work was sup rted by NIH Grant No: GM 32367. Additional support was provideaOby the Research Council and Vice Provost for Research Administration of the University of Oklahoma.
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