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Chem. Res. Toxicol. 1997, 10, 414-419
Oxidation of Diclofenac to Reactive Intermediates by Neutrophils, Myeloperoxidase, and Hypochlorous Acid Gohachiro Miyamoto,§,‡ Nasir Zahid,§ and Jack P. Uetrecht*,|,†,§ Faculties of Pharmacy and Medicine and University of Toronto Drug Safety Research Group, University of Toronto, Toronto, Ontario M5S 2S2, Canada Received November 19, 1996X
Diclofenac is associated with a low, but significant, incidence of hepatotoxicity and bone marrow toxicity. It has been suggested that this could be due to a reactive acyl glucuronide. An alternative hypothesis is that an oxidative reactive metabolite could be responsible for such reactions and such metabolites formed by the enzymes present in neutrophils could be responsible for bone marrow toxicity. Others had reported the formation of 2,2′-dihydroxyazobenzene during the oxidation of diclofenac by myeloperoxidase/hydrogen peroxide. In contrast, in similar experiments we did not find evidence for the formation of 2,2′-dihydroxyazobenzene, but we did find several products, including a reactive iminoquinone. The same iminoquinone was formed by the oxidation of 5-hydroxydiclofenac. This iminoquinone was also formed by oxidation of diclofenac by HOCl or by activated neutrophils. It reacted with glutathione to form a conjugate. 5-Hydroxydiclofenac is also a major hepatic metabolite of diclofenac, and we found that rat hepatic microsomes oxidized 5-hydroxydiclofenac to the iminoquinone which was trapped with glutathione. This reactive metabolite represents another possible cause of the idiosyncratic reactions associated with the use of diclofenac.
Introduction Diclofenac is a widely used nonsteroidal antiinflammatory drug (NSAID)1. All NSAIDs are associated with a relatively high incidence of adverse reactions, especially gastrointestinal bleeding. Although uncommon, diclofenac appears to be associated with hepatitis (1, 2) and hematological toxicity, such as aplastic anemia, neutropenia (3-6), hemolytic anemia, and thrombocytopenia (5, 7-9). There is circumstantial evidence that idiosyncratic reactions are often due to chemically reactive metabolites (10, 11). One type of reactive metabolite associated with many NSAIDs is an acyl glucuronide, and it has been proposed that these acyl glucuronides are responsible for many of the idiosyncratic reactions associated with NSAIDs (12). It has been demonstrated that diclofenac forms an acyl glucuronide, and covalent binding of diclofenac, apparently due to the acyl glucuronide, has been observed in the liver (13-16). Although NSAIDs, as a class, are associated with a relatively high incidence of idiosyncratic reactions, only a few, such as phenylbutazone, indomethacin, and diclofenac, appear to be associated with a low, but statistically significant incidence of bone marrow toxicity (4). Although diclofenacassociated bone marrow toxicity may be due to the reactive acyl glucuronide, based on its structure, the glucuronide of phenylbutazone presumably is not reactive. However, phenylbutazone is oxidized to reactive intermediates by neutrophil-derived peroxidases (17, 18). * Address correspondence to this author at the Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ontario, Canada, M5S 2S2. Telephone: 416 978 8939. E-mail:
[email protected]. † University of Toronto Drug Safety Research Group. ‡ Present address: Otsuka Pharmaceutical Co., Tokushima, Japan. § Faculty of Pharmacy. | Faculty of Medicine. X Abstract published in Advance ACS Abstracts, March 15, 1997. 1 Abbreviations: MPO, myeloperoxidase; PMA, phorbol 12-myristate 13-acetate; HBSS, Hanks’ balanced salt solution; NSAID, nonsteroidal antiinflammatory drug.
S0893-228x(96)00190-7 CCC: $14.00
Acyl glucuronides have relatively long biological halflives and can reach sites distant from where they are formed, but this is not true for most reactive metabolites. Therefore, it is logical to examine the formation of reactive metabolites by the target of an idiosyncratic reaction. In the case of bone marrow toxicity, the target is the bone marrow cells, which include neutrophils and neutrophil precursors. We have demonstrated that essentially all of the drugs associated with the highest incidence of agranulocytosis are oxidized to reactive metabolites by activated neutrophils or by HOCl that is generated by the myeloperoxidase (MPO) system of neutrophils (19-21). Many of the drugs associated with agranulocytosis are arylamines, and diclofenac is a secondary arylamine. Although the hepatotoxicity of diclofenac may be due to an acyl glucuronide formed in the liver, agranulocytosis may be due to a reactive metabolite formed by activated neutrophils. Zuurbier et al. studied the oxidation of diclofenac by MPO/H2O2 and found strong evidence for the production of 2,2′-dihydroxyazobenzene (22); however, it is difficult to understand how such a product could be formed. In this study, we set out to determine if diclofenac is oxidized by activated neutrophils as well as simply by HOCl or MPO and, if it is, to determine if a reactive intermediate is involved.
Materials and Methods Materials. Diclofenac was purchased from Sigma Chemical Co. (St. Louis, MO). NaOCl and 2,2′-dihydroxyazobenzene were obtained from Aldrich Chemical Co. Inc. (Milwaukee, WI), and the concentration of NaOCl was determined spectrophotometrically (23). MPO was obtained from Cortex Biochem Inc. (San Leandrow, CA), and 1 unit of MPO was defined as the amount of enzyme that would decompose 1 µM H2O2/min at 25 °C and pH 6. 5-Hydroxydiclofenac was a generous gift from Ciba-Geigy Canada Ltd. (Calgary, Alberta). Analytical Procedures. HPLC was performed with UV detection at a wavelength of 254 nm. The conditions utilized
© 1997 American Chemical Society
Oxidation of Diclofenac for most of the HPLC consisted of a column packed with Ultracarb 5 ODS 30 (100 mm × 2.0 mm i.d.; Phenomenex; Torrance, CA) and a mobile phase of CH3CN/H2O/CH3COOH (50:50:1, v/v) at a flow rate of 0.2 mL/min. A mobile phase containing 35% acetonitrile was occasionally used to increase the resolution of compounds with short retention times. For preparative HPLC, the column had the same packing material but the dimensions were 150 × 10 mm. Ultraviolet/visible spectra were obtained with a Hewlett Packard 8452A diode array spectrophotometer (Hewlett Packard Co.; Palo Alto, CA) or, if done in conjunction with HPLC, with a Hewlett Packard 1040A diode array HPLC detector. Mass spectrometry was performed with a PE SCIEX API-III Biomolecular Mass Analyzer in the ion spray mode (Sciex, Toronto, Ontario). Most of the studies were done in the LC/ MS mode using the same HPLC column and solvent as described above for simple HPLC. NMR spectra of M5 and M6 were obtained with a Varian Unity Plus 500 MHz spectrometer (Varian Associates Inc., Palo Alto, CA). NMR spectra of M1 and M2 were obtained with a Bruker 500 MHz NMR spectrometer (Brucker Canada; Milton, Ontario). Metabolites were dissolved in CD3OD. Oxidation of Diclofenac by HOCl. Diclofenac, dissolved in methanol, was reacted with equimolar concentrations of HOCl dissolved in water at final concentrations from 1 mM to 10 mM for 30 min, and the products were analyzed by HPLC. Diclofenac was not sufficiently soluble in water to achieve this concentration; however, when the oxidation was carried out in the absence of methanol at lower concentrations, the pattern of products was essentially the same (data not shown). For isolation of larger quantities of products, diclofenac (100 mM) was oxidized with an equal volume of HOCl (0.7 M), and the products were purified by preparative HPLC and analyzed by MS/MS and NMR. The initial products of the reaction of diclofenac with HOCl were analyzed with a flow system in which the reactants were continuously pumped (Harvard infusion pumps, Model 55-2222, Harvard Apparatus, South-Natick, MA) into a mixing chamber (dead volume 3.1 µL; Upchurch Mixing Tee; Upchurch Scientific; Oak Harbor, WA) and from there the products were fed into the Sciex mass spectrometer. Infusion rates of each pump were 2-20 µL/min, and the flow rates of each pump were the same. The concentration of diclofenac was 1 mM, and that of HOCl was 100 mM. Diclofenac was dissolved in methanol, and HOCl was diluted with water. Oxidation of Diclofenac by MPO/H2O2/Cl-. Diclofenac (from 0.1 to 1 mM) was incubated with MPO (2, 4, and 6 units) and hydrogen peroxide (10 µL, 0.8 mM) in 1 mL of 0.1 M phosphate buffer (pH 5 or pH 6) containing 150 mM KCl for 1.5 h. The products were analyzed by HPLC. In order to duplicate the experiment of Zuurbier et al. (22), the oxidation of diclofenac [200 µM in phosphate buffer, pH 7.2, (without chloride)] by H2O2 (150 µM) in the presence of MPO (3 µM) was followed with a photodiode array spectrophotometer. Oxidation of Diclofenac by Activated Neutrophils. Blood was drawn from healthy subjects into a heparinized syringe. Neutrophils were isolated by differential centrifugation with Ficoll-Hypaque as described previously (24). Cell viability was greater than 95% as determined by trypan blue exclusion. Diclofenac (1, 5, 10, 50 and 100 µM) was incubated with neutrophils [2 × 106, 4 × 106, and 6 × 106 cells/mL and 20 nmol of phorbol myristate acetate (PMA) in 1 mL of Hanks’ balanced salt solution (HBSS)]. Metabolites were identified by LC/MS and comparison with the products produced by oxidation with HOCl. We did not have enough material for a standard curve, and so the concentrations of the metabolites were approximated by using the same extinction coefficient as that for diclofenac. This is only a gross approximation to the true concentration. Intermediates Trapped by Glutathione. Diclofenac (1 mM in methanol) was oxidized with HOCl (100 mM in water), and after 10 min, glutathione (1 mL of a 100 mM solution in PBS, pH 7.4) was added, and the products were analyzed by
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 415
Figure 1. HPLC of the products of diclofenac oxidation by HOCl. The concentration of diclofenac was 10 mM, and that of HOCl was 100 mM. LC/MS. A standard of 5-hydroxydiclofenac was also oxidized with HOCl and glutathione added in the same manner. Oxidation of 5-Hydroxydiclofenac by Rat Hepatic Microsomes. Rat hepatic microsomes (Sprague Dawley, 1 mg/ mL) were suspended in 1 mL of phosphate buffer (pH 7.4), and 5-hydroxydiclofenac (final concentration 1 mM), glutathione (final concentration 5 mM), and finally NADPH (1 mM) were added. After incubation at 37 °C for 30 min, the mixture was passed through a Supelclean tube (3 mL, LC-18 SPE; Supelco, Mississauga, Ontario) and washed with water. The compounds were then eluted with 50% methanol in water, and the solvent was removed under a stream of nitrogen. The residue was redisolved in 0.1 mL of 50% ethanol in water, and 5 µL of this was analyzed by LC/MS as described above.
Results Oxidation of Diclofenac by HOCl. The reaction of diclofenac with HOCl was slow relative to the analogous reaction of many other arylamines that we have studied, and an excess of HOCl was utilized in order to obtain products in a short period of time. A search was made for 2,2′-dihydroxyazobenzene, which has a retention time with our HPLC system of 29 min, but no peak with this retention time was observed. We also used LC/MS with selected ion monitoring at m/z 215 in case a small peak was hidden in the base line, but this product was not observed. Oxidation of diclofenac by HOCl led to several products as shown by HPLC in Figure 1. Some of these (M1, M2, M3, M4, M5, M6) were tentatively identified by LC/MS and/or NMR as described below, and this led to the proposed pathway shown in Figure 2. M1 had a retention time of 19 min on HPLC, and its MS revealed a M+1 ion at m/z 330 which represents substitution of a chlorine for a hydrogen atom on diclofenac. The proton NMR spectra of this product (Table 1) indicate that the chlorine is in the 3 position as shown in Figure 2. M2 was the major product and had a retention time of 25 min on HPLC. Its mass spectrum was similar to that of M1 with a M+1 ion at m/z 330. The NMR spectra (Table 1) indicate that the chlorine was in the 5 position. M3, with a retention time of 39 min on HPLC, had a M+1 ion on mass spectrometry at m/z 366, indicating the substitution of two chlorines. From the structures of M1 and M2, it is likely that the chlorines were in the 3 and 5 positions; however, we did not purify sufficient material to prove this by NMR. M4 had a retention time of 5 min, and its M+1 ion on mass spectrometry was at m/z 310 which corresponds to the addition of oxygen and loss of two hydrogens. MS/ MS gave fragments at m/z 292 (100%), 264 (14%), 235 (18%), 229 (23%), 201 (34%), 194 (30%), and 166 (20%). The λmax of this metabolite was 455.5 nm. When formed
416 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Figure 2. Proposed pathways for the oxidation of diclofenac by HOCl. The proposed intermediates in brackets were not observed.
by oxidation of diclofenac with HOCl, its concentration peaked after about 10 min and then decreased (data not shown). When the peak for M4 was collected from preparative HPLC and attempts were made to isolate it, it decomposed; however, if left in the acidic HPLC solvent it spontaneously converted to M6 as well as a trace of M5 over a period of days (data not shown). Its proposed chemical structure is shown in Figure 2. Addition of ascorbate to the reaction mixture led to the disappearance of M4 and the appearance of 5-hydroxydiclofenac, confirmed by comparison with the synthetic standard, and it had a M+1 ion at m/z 312. When 5-hydroxydiclofenac was oxidized with HOCl, the major product had the same retention time on HPLC, the same M+1 ion at m/z 310, and the same fragmentation pattern on MS/MS as M4 produced from diclofenac. M5 had a retention time of 7 min, and on mass spectrometry, its M+1 ion was at m/z 282 with a chlorine isotope peak at m/z 284 (69% of the M+1 ion). The fragment ions were at m/z 264 (80%, 282-H2O), 229 (32%, 282-H2O-Cl), 201 (22%, 282-H2O,-CO,-Cl), and 194 (37%, 282-H2O-2Cl). The NMR data are shown in Table 1. The COSY spectrum was consistent with the proton assignments. The proposed structure of M5 is shown in Figure 2. M6 has a retention time of 9 min and also has a M+1 ion of m/z 282 (57%) and a chlorine isotope peak at m/z 284 (66% of the M+1 ion). Its fragment ions were at m/z 264 (90%, 282-H2O), 247 (72%, 282-Cl), and 230 (100%, 282-HOCl). Its NMR spectral data are shown in Table 1, and its proposed chemical structure is shown in Figure 2. The COSY spectrum was consistent with the proton assignments. A C13 NMR spectrum had a major peak at 195.75 ppm, which is consistent with the presence of an aldehyde carbon. (The carboxylate carbon in diclofenac is at 180.62 ppm.)
Miyamoto et al.
Other products were observed on the chromatogram (Figure 1), but some of these products appear to be unstable because the chromatographic pattern changed with time although part of this was likely due to the excess of HOCl causing further oxidation. A flow system was utilized in which diclofenac and HOCl were mixed and the products went directly into the mass spectrometer in order to try to identify the earliest intermediates in the reaction. By varying the flow rate, one can get a crude estimate of the rate of the reaction and the half-life of the intermediates, and in this case, the range used was 1-20 µL/min. At a rate of 1 µL/min (in which case the reaction time was about 70 s), a chlorinated product was observed; however, at faster flow rates, the amount of product was not sufficient for detection by MS. Thus, the reaction rate was too slow for this method to provide further information about the nature of initial products of the reaction. Oxidation of Diclofenac by MPO/H2O2/Cl-. Diclofenac was also oxidized by the combination of MPO/ H2O2/Cl-, and the major products were M4 and M6 as shown by HPLC. The amount of M4 relative to M6 was greater at pH 5 than at pH 6 (data not shown). The chlorinated products formed by oxidation with HOCl were not observed. We again searched for the presence of 2,2′dihydroxyazobenzene by HPLC using a synthetic standard of the compound to determine the retention time, but none was observed. When we tried to duplicate the conditions used by Zuurbier, which did not include chloride, we also obtained an absorption band at about 452 nm (Figure 3) that increased with time, and it looks very similar to what they observed; however, an overlay of the absorption spectrum of 2,2′-dihydroxyazobenzene (dashed line) demonstrates that there are significant differences between the observed spectra generated during the oxidation of diclofenac with MPO/H2O2 and that of 2,2′-dihydroxyazobenzene. Although M4 has a λmax close to 450 nm, it also absorbs significantly between 300 and 400 nm, and so it is difficult to ascribe this peak solely to M4. On addition of ascorbic acid to the reaction mixture, the 452 nm peak very rapidly disappeared. In contrast, addition of ascorbic acid to 2,2′-dihydroxyazobenzene did not eliminate the absorption band at ∼450 nm. We were also unable to detect 2,2′-dihydroxyazobenzene in this incubation using HPLC. Oxidation of Diclofenac by Activated Neutrophils. Using the neutrophil system, metabolites of diclofenac were detected by HPLC (Figure 4). The major product had a retention time of 5 min. Using a mobile phase containing 35% rather than 50% acetonitrile, the retention time was 15.8 min, and when it was mixed with the product of oxidation of 5-hydroxydiclofenac by HOCl, there was only one peak on the chromatogram, indicating that the major metabolite must be M4. LC/MS confirmed that this product had a M+1 ion at m/z 310. The other two products both had M+1 ions at m/z 282, and they were presumably M5 and M6. M4 spontaneously converted to the two products with M+1 ions at m/z 282. Other metabolites were detected on the chromatogram at an earlier retention time than M4, and these appear to be unstable intermediates. Chlorinated metabolites, M1, M2, and M3, which were detected by oxidation of diclofenac by HOCl, were present in trace amounts or nil. The concentration of major metabolite, M4, was dependent on the concentration of diclofenac and the number of neutrophils (Figures 5, 6).
Oxidation of Diclofenac
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 417 Table 1.1H-NMR Spectra of Diclofenac, M1, M2, M5, and M6
1H
position
diclofenac δ (ppm) multiplicity J (Hz) integration M1 δ (ppm) multiplicity J (Hz) integration M2 δ (ppm) multiplicity J (Hz) integration M5 δ (ppm) multiplicity J (Hz) integration M6 δ (ppm) multiplicity J (Hz) integration
3
4
5
6
3′ & 5′
4′
CH2
6.355 d,d 8.06, 1.1 1H
6.959 t,d 8.06, 1.65 1H
6.815 t,d 7.32, 1.28 1H
7.186 d,d 7.32, 1.29 1H
7.355 d 8.05 2H
6.966 t 7.87 1H
3.623 s
-
7.240 d,d 8.06, 1.47 1H
6.831 t 8.05 1H
7.214 d,d 7.69, 1.47 1H
7.242 d 8.06 2H
7.068 t 7.78 1H
3.766 s
6.369 d 8.06 1H
7.052 d,d 8.61, 2.56 1H
-
7.233 d 2.38 1H
7.404 d 8.06 2H
7.084 t 8.06 1H
3.738 s
6.679 d 10.07 1H
6.468 d,d 10.07, 2.19 1H
-
6.795 q 2.20 1H
7.472 d 8.05 2H
7.167 t 8.05 1H
6.215 d 8.97 1H
6.904 d,d 8.97, 2.93 1H
-
7.112 d 2.93 1H
7.490 d 8.24 2H
7.246 t 8.24 1H
4.762 d 2.02 2H CHO 9.886 s
Figure 3. Absorption spectra from the oxidation of diclofenac with MPO/H2O2. The concentration of diclofenac was 200 µM, that of MPO was 3 µM, and that of H2O2 was 150 µM. The time between spectra was 1 min. The first spectrum is the bottom spectrum. The spectrum represented by the dotted line is that of authentic 2,2′-dihydroxyazobenzene.
Figure 4. HPLC of the products of diclofenac oxidation by activated neutrophils. The concentration of diclofenac was 100 µM, and the number of neutrophils was 6 × 106 cells/mL.
GSH Adduct with Reactive Metabolite. When glutathione was added to the products of the reaction of diclofenac with HOCl, M4 (retention time 5 min) disappeared, and a new peak with a retention time of 2 min was observed. LC/MS of this product indicated that this
2H
2H
2H
1H
Figure 5. Formation of M4 by oxidation of diclofenac with activated neutrophils as a function of time and at different concentrations of diclofenac. The absolute concentrations of M4 are only approximate and are based on the approximation that the extinction coefficient of M4 is equal to that of diclofenac.
new peak was a glutathione adduct with a M+1 ion at m/z 617. This corresponds to the adduct that would be expected from the reaction between M4 and glutathione. In addition, LC/MS/MS gave fragments at m/z 542 (19%, 617-Gly), 488 (11%, 617-Glu), 385 (33%, 617-GluCys), 342 (100%, 617-GSH+S), 324 (34%, 342-H2O), and 310 (6%, 617-GSH). The major product formed by oxidation of 5-hydroxydiclofenac with HOCl followed by addition of glutathione also had a M+1 ion at m/z 617, and by MS/MS, it also had the same fragmentation pattern as that of the glutathione adduct formed from oxidation of diclofenac. Oxidation of 5-Hydroxydiclofenac by Rat Hepatic Microsomes. We suspected that M4 might not be detected in the incubation of 5-hydroxydiclofenac with hepatic microsomes because of reaction with the microsomes; therefore, we added glutathione to trap it. We found the glutathione adduct with a M+1 ion at m/z 617, and it had the same fragementation pattern on MS/MS as the M4-glutathione conjugate formed by oxidation of diclofenac with HOCl.
418 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Figure 6. Formation of M4 by oxidation of diclofenac with activated neutrophils as a function of time and at different concentrations of neutrophils. The absolute concentrations of M4 are only approximate and are based on the approximation that theextinction coefficient of M4 is equal to that of diclofenac.
Discussion We failed to confirm the production of 2,2′-dihydroxyazobenzene in the oxidation of diclofenac by the MPO/ H2O2 system. Although the evidence provided in a previous report appeared substantial, it is very difficult to imagine a mechanism by which this product could be formed. However, we did find a series of products. Oxidation of diclofenac by HOCl produced primarily chlorination of the benzene ring but also significant amounts of other products. In contrast, M4 was the major product observed with oxidation by MPO or activated neutrophils. Both chlorination of the ring and formation of the quinone-type products could involve N-chlorination followed by loss of a chloride anion to form a nitrenium ion that could react with either water or chloride ion. The putative nitrenium ion may be stabilized by the formation of an intramolecular ion pair as shown in Figure 2. The reason for the difference in the products observed in the MPO and neutrophil incubation compared with oxidation by HOCl is unknown. Although it seems as if neutophils generate free HOCl, the MPO system also appears to be able to oxidize compounds without the involvement of HOCl (25). It could be that oxidation by the MPO system does not involve an N-chloro intermediate. Alternatively, it could be that the different environment of the putative N-chloro intermediate in the MPO and neutrophil systems leads to preferential formation of the phenol rather than reaction with chloride or rearrangement to form M1 or M2. Despite the differences in the pattern of products formed, M4 is a significant reactive intermediate formed by all three systems. It is likely that the reactions observed in this study are involved in the ability of diclofenac to inhibit HOCl formation by the MPO that was described by others (22). Monocytes from patients receiving diclofenac have also been shown to produce less superoxide when stimulated (26). On the other hand, the reaction of diclofenac with HOCl is relatively slow, and another study suggests that the in vivo scavenging effects of diclofenac would be small (27). Despite the observation that the reaction of diclofenac with HOCl is relatively slow, it is likely that a small amount of diclofenac could be oxidized by activated neutrophils or monocytes in vivo, and the reactive
Miyamoto et al.
metabolites generated may be responsible for some of the idiosyncratic reactions associated with diclofenac, especially those involving the bone marrow. Although it appears that several reactive species are formed during the oxidation of diclofenac, including a presumed nitrenium ion intermediate, it is likely that M4 would make the largest contribution to covalent binding, and it is likely to react with protein sulfhydryl groups analogous to its reaction with glutathione. The phenolic precursor to M4, 5-hydroxydiclofenac, is also a significant hepatic metabolite of diclofenac (28), and, therefore, M4 may be formed by oxidation of this phenol in the bone marrow. In addition, we found that M4 was formed by the oxidation of 5-hydroxydiclofenac with rat hepatic microsomes, so it is likely that M4 is also formed by oxidation of 5-hydroxydiclofenac in the liver of humans. Hargus et al. found evidence for P-450-mediated covalent binding of diclofenac in the liver (29); therefore, it is possible that M4 could also be responsible for the hepatotoxicity associated with the diclofenac.
Acknowledgment. This work was supported by a grant from the Medical Research Council of Canada (MT13478). We thank Dr. Henrianna Pang for her expert analysis of the mass spectra and Dr. R. A. McClelland for help with the structural analysis.
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