238
Chem. Res, Toricol. 1988, 1, 238-242
Leurosine Biotransformations: An Unusual Ring-Fission Reaction Catalyzed by Peroxidase Animesh Goswami,? T i m o t h y L. Macdonald,x Claire Hubbard,* Michael W. Duffel, and John P. N. Rosazza” Division of Medicinal Chemistry and Natural Products, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242 Received March 18, 1988
T h e dimeric V i n c a alkaloid leurosine undergoes a n unusual fission of the piperidine ring of the Iboga substructure when reacted with horseradish peroxidase and hydrogen peroxide. A preparative-scale oxidation of leurosine provided 15’-hydroxycatharinine, which was identified by infrared and proton and carbon-13 NMR spectroscopies and high-resolution mass spectrometry. A proposed pathway for the formation of 15’-hydroxycatharinine involves radical and iminium intermediates and cleavages of a putative diol. The enzymatic transformation product is 3 orders of magnitude less active than leurosine or vinblastine in vitro, in inhibiting the polymerization of tubulin.
Introduction Unsymmetrical “dimeric” alkaloids leurosine (l),vincristine (2), and vinblastine (3) were originally isolated from the plant Catharanthus roseus G. Don (Figure 1). All of the compounds display antineoplastic activity, and 2 and 3 have been widely used for the treatment of human cancers for nearly three decades. The one proven mechanism of action of the Vinca alkaloids involves specific binding to tubulin to cause metaphase arrest and ultimate cell death. Ample evidence suggests that the alkaloids are metabolized in living systems (1-3), but nothing is known about biotransformation pathways or the structural changes catalyzed by metabolizing enzymes or of the possible roles of enzymatic transformations in mechanism(s) of action and/or dose-limiting toxicities (4-6). Previous investigations from this laboratory have elaborated the mechanisms of oxidative transformations of monomeric and dimeric Vinca alkaloids catalyzed by different types of enzymes. For example, the Aspidosperma monomeric alkaloid vindoline (6) is transformed through the same oxidative pathway by peroxidase (7), copper oxidases (8,9), cytochrome P-450(IO),and enzyme systems of the bacterium Streptomyces griseus (11). Detailed investigations established that these alkaloid transformations involved one-electron oxidation steps (7, 8), which led to the formation of enamine and iminium intermediates in the oxidation pathway. Relatively stable carbinolamine products have been isolated when 14,15dihydrovindoline (12) and leurosine were oxidized by copper oxidases (13, 10). These types of alkaloid biotransformation reactions lead to the formation of reactive intermediates which could be implicated in new mechanisms of action or the toxicities well-known for the Vinca alkaloids. Biotransformation experiments with leurosine resulted in the identification of the phenolic product 10’hydroxyleurosine (4) (14) and the 5’-carbinolamine (5) (13). Aromatic hydroxylation by the bacterium, Streptomyces Present address: Rhone Poulenc Research Center, 8510 Corridor Road, Savage, MD 20763. *Department of Chemistry, The University of Virginia, Charlottesville, VA 22901.
0893-228x/88/2701-0238$01.50/0
griseus is probably catalyzed by the cytochrome P-450 monooxygenase enzyme system of this microorganism (15). Copper oxidases and two Aspergillus species oxidized leurosine to the stable carbinolamine, 5’-hydroxyleurosine (5) (13). These studies revealed that the pathways of dimeric Vinca alkaloid oxidation are highly dependent upon the nature of the oxidizing enzyme. The present report describes the unusual ring-fission reaction which occurs when leurosine is oxidized with horseradish peroxidase and hydrogen peroxide.
Materials and Methods Ultraviolet spectra were recorded in methanol solution using a Hewlett-Packard 8450 spectrophotometer. Infrared spectra were recorded in KBr disks with a Beckman IR 4240 spectrophotometer. Proton ‘H, 360 MHz) and carbon (13C, 90 MHz) NMR spectra were recorded in deuteriochloroform solution with a Bruker WH-360 Ft spectrophotometer using tetramethylsilane ( 6 = 0) and deuteriochloroform (6 = 77.0) as internal reference standards. Carbon multiplicities were determined by a modification of the INEPT spectral editing method. High-resolution mass spectra were obtained on a Kratos AEIU MS-50k instrument at the Midwest Center for mass spectrometry, University of Nebraska, Lincoln, NE, and on a DS-55 instrument at the Mayo Clinic, Rochester, MN. Leurosine sulfate was obtained from Eli Lilly and Company, Indianapolis, IN, and from Omnichem PRB, Brussels, Belgium. Before use, leurosine was fully characterized by mass spectrometry, ‘H and 13C NMR spectroscopies, and its purity was verified by TLC and HPLC. Horseradish peroxidase (EC 1.11.1.7, Type VI; 300 purpurogallin units per mg, Rz 3.0) was obtained from Sigma Chemical Co., St. Louis, MO. Piperazine-N-N’-bis[2-ethenesulfonic acid] (Pipes), ethylene glycol bis(beta-aminoethyl ether)-N,N,N’JV’-tetraaceticacid (EGTA), guanosine triphosphate (GTP), and MgS04 were obtained from the Sigma Chemical Company. Dimethyl sulfoxide (DMSO) was from Aldrich Chemical Company. Chromatography. Thin-layer chromatography (TLC) was performed on 0.25 mm thick silica gel GFZs4(Merck) plates prepared as needed with a Quickfit Industries spreader. Plates were air-dried and oven-activated a t 110 OC for 30 min prior to use. The solvent system used to develop chromatograms was benzene/methanol(Exl). Spots were visualized by W fluorescence quenching a t 254 nm and by spraying developed plates with Dragendorffs reagent (16). In this system, the various compounds gave the following R, values: leurosine (0.4), 15’-hydroxy-
0 1988 American Chemical Society
Chem. Res. Toxicol., Vol. 1, No. 4, 1988 239
Oxidation of Leurosine by Peroxidase
Table I. Carbon-13 NMR Spectral Comparisons of the Iboga (Upper Half) of Leurosine (I), 15'-Hydroxycatharinine (7), and Catharinine (8) chem shift, ppm 1
C
ref 25
16'-COOMe 16'-C OOMe
130.7 42.3 49.6 24.6 116.7 129.1 118.1 122.2 118.4 110.3 134.6 33.5 60.3 55.3 30.7 8.6 28.0 59.9 54.0 174.1 52.3
2'
I,
CHO
3' 5' 6' 7' 8' 9' 10' 11' 12'
13' 14' 15' 16' 17' 18' 19' 20' 21'
F i g u r e 1. The structures of leurosine (l),various leurosine derivatives, 15'-hydroxycatharinine (7), and related Vinca alkaloids. catharinine (0.35), traces of an unknown (0.5), and leurosine Nb-oxide (0.2). Column chromatography was performed with Baker 60-200 mesh (3404) silica gel, which was oven activated at 110 "C for 30 min prior to use. High-performance liquid chromatography (HPLC) was performed with a Waters Associates ALC/GPC 202 instrument equipped with a U6K universal injector and a 280-nm differential UV detector. Separations were achieved a t a flow rate of 1 mL/min a t operating pressures of about 1000 psi by using an Altech C-18 column (0.46 X 25 cm, 10 gm) eluted with methanol-10 mM potassium dihydrogen phosphate (pH 4.82) (70:30). Retention volumes in this system were as follows: leurosine (20 mL), 15'-hydroxycatharinine (6.9 mL),leurosine Nb-oxide (10 mL), and the trace product (11.3 mL). Horseradish Peroxidase Reactions. Analytical-scale experiments were performed in 50-mL DeLong flasks containing 8 mL of 0.1 M sodium phosphate buffer, pH 6.8, 1 mg of horseradish peroxidase, and 1mL of 170 mM hydrogen peroxide. A solution of 2.5 mg of leurosine sulfate in 1mL of water was added to initiate the reaction, which was shaken at 250 rpm on a New Brunswick G-24 gyrotory shaker held at 25 "C. Reactions were monitored by withdrawing 1-mL samples at various time intervals, adjusting these to pH 10 with 2 drops of 28% ammonium hydroxide solution, extracting with 1 mL of ethyl acetate, and spotting 30 gL of the extract on TLC plates. Control reactions consisted of entire mixtures plus boiled enzyme and of incubations minus peroxidase. Preparative-scale reactions were conducted in two, 1-L DeLong flasks (Bellco) each containing 175 mL of the same phosphate buffer, 40 mg of horseradish peroxidase, and 20 mL of 170 mM hydrogen peroxide. A solution of 100 mg of leurosine sulfate equivalent to 89.2 mg of leurosine base in 5 mL of water was added to each flask, and the reactions were shaken at 250 rpm a t 25 "C for 6 h. At this time, all of the leurosine had been transformed in the reaction. The reactions were combined, adjusted to pH 10 with ammonium hydroxide, and extracted with four portions (400 mL each) of ethyl acetate. The organic extracts were combined, dried over anhydrous sodium sulfate, and evaporated under vacuum to about 200 mg of a brown gummy solid. The extract was purified by column chromatography over 65 g of silica gel (3 X 30 cm) eluted with benzene/methanol (95:5). Volumes 242-296 mL gave traces of an unknown minor product, 296-460 mL gave mg 13 of a mixture of the unknown compound, leurosine,
obsd 7 iboga halves 130.804 133.527 42.279 52.298 49.839 49.634 24.892 24.774 116.832 111.259 129.166 127.941 118.122 117.510 122.172 122.486 118.791 119.301 110.299 110.880 134.580 135.452 33.491 32.043 59.965" 80.368 55.223 55.863 31.108 34.868 8.499 8.248 28.020 29.926 60.291" 210.909 53.943 163.679 174.107 173.773 52.204 52.885
8 ref 26
132.6 49.5 50.4 25.2 111.5 128.4 117.7 122.5 119.4 110.9 135.4 29.6 50.4 55.8 34.8 8.3 37.5 210.4 163.6 174.2 53.2
"The assignments for the 15'- and 20'-carbon atoms of leurosine are reversed from their reported values on the basis of our delayed decoupling experiments which clearly showed the peak at 60.2911 ppm is due to a quaternary carbon and the resonance at 59.965 is due to a methine carbon. and 7, and fractions between 460-655 mL gave 18 mg (10% yield) of 15'-hydroxycatharinine (7). Elution of the column with methanol afforded a mixture of 76 mg of leurosine Nb-oxide plus intractable polar material. Identification of 15'-Hydroxycatharinine (7). The peroxidase transformation product 7 exhibited the following physical (log e) 270 (4.255), 287 (4.211), and 297 nm properties: UV,,,A (4.152); IR (cm-') 3375 (OH), 1740 (COOR), 1720 (C=O), 1655 (N-CHO); 'H NMR (ppm) 0.6628 (3 H, br, 18'-CH3), 0.7750 (3 H, t, J = 6.5 Hz, 18-CH3), 2.1140 (3 H, s, 17-OCOCH3),2.7142 (3 H, s, NCH3), 3.5192 (3 H, s, 11-OCH3), 3.7391 (3 H, s, 16'COOCH,), 3.8069 (3 H, s, 16-COOCH3), 5.3598 (1 H, br, 15-H), 5.4710 (1 H, S, 17-H), 5.8425-5.8820 (1 H, dd, J = 4,lO Hz, 14-H), 6.0031 (1 H, s, 12-H), 6.715 (1H, s, 9-H), 7.0724-7.1682 (3 H, m, 10',11',12'-H), 7.3079 (1H, S, 21'-NCHO), 7.4944 (1H, d, J = 7.5 H, Y-H), 7.8696 (1H, brs, NH); 13C NMR, Table I; high-resolution mass spectrum, obsd 840.3935, calcd for C46H56N4011 840.3946 (M - CzH5),obsd 811.3398, calcd for C44H51N4011 811.3555. Other high-resolution mass spectral fragments are presented in a table submitted as supplementary data. Inhibition of Tubulin Polymerization. Microtubule protein (MTP) was prepared from bovine brain by three cycles of assembly/disassembly as described previously ( I 7). The third cycle pellet was suspended in P E M buffer (100 mM Pipes, 2 mM EGTA, 1mM MgSO,) and stored in aliquots at -176 "C. Protein concentrations were determined by the method of Bradford (18), calibrated using tubulin as a standard (19). All compounds tested were initially dissolved in DMSO a t a concentration of 0.01 M. The final DMSO concentration was never allowed to exceed 2% volume/volume. All tubulin polymerizaton assays were carried out in P E M buffer and contained 1.7 mg of MTP/mL, 1 mM GTP, and varying concentrations of the inhibitor. Samples were kept a t 0 "C prior to polymerization. Tubulin polymerization was monitored turbidimetrically a t 351 nm by using a Varian DMS 90 UV/vis spectrophotometer, equipped with a water-jacketed cell holder maintained a t 37 "C. The Z50 value for each compound was determined, and it is defined as the concentration of compound required to decrease by 50% the change in absorbance found for controls with no compound added. The results are presented in Table 11.
240 Chem. Res. Toaicol., Vol. 1, No. 4, 1988 Table 11. The Inhibitory 50% (Zao) Values of Leurosine and Various Products of Leurosine Formed by Enzymatic Transformation As Measured by Tubulin Polymerization comDd Zm X lo7. M vinblastine (2) 3.6 leurosine (1) 9.8 5’-hydroxyleurosine (5) 11 10’-hydroxyleurosine (4’) 32 15’-hydroxycatharinine (7) 4500
Results and Discussion Horseradish peroxidase catalyzes one-electron oxidations of a wide variety of substrates through its well-defined oxidation/reduction cycle which involves the powerful oxidant form of the enzyme known as compound I (Fe4+, cation radical) (7,20-23). Initial leurosine oxidation experiments using horseradish peroxidase as catalyst provided a completely different transformation product (TLC, HPLC) than those observed with other oxidants including the copper oxidases, DDQ, and benzoquinone (10,13). The new product was consistently formed only in complete reaction mixtures containing enzyme, hydrogen peroxide, and leurosine, thus ruling out the possibility that it arose as an artifact. Enzyme reactions always afforded some leurosine Nb-oxide which was also formed in control reaction mixtures containing leurosine and hydrogen peroxide and no enzyme. The identity of leurosine Nb-oxide was confirmed by comparing (TLC, HPLC) the product obtained in enzyme reactions with that obtained by reaction of leurosine with m-chloroperbenzoic acid. Considerable exploration of reaction conditions indicated that the leurosine oxidation product was formed rapidly (easily detectable by TLC within 30 min). Preparative reactions using the complete reaction mixture followed by chromatography gave an analytical sample of the major oxidation product in about 10% overall yield. Traces of another minor unidentified product were also obtained. The structure of the major horseradish peroxidase product was established as 15’-hydroxycatharinine (7) on the basis of its spectral properties. The ultraviolet spectrum of 7 was similar to that of leurosine suggesting the existence of the same chromophoric groups in both compounds. Infrared spectra presented some striking differences between the two compounds and revealed the presence of ketone (1720 cm-’) and N-formyl (1667, 1655 cm-’) functional groups in addition to ester carbonyl groups (1740 cm-’) in the leurosine product. The proton NMR spectrum also indicated the presence of an N-formyl group a t 7.31 ppm. In addition, the signal for the 18’-methyl group was shifted upfield by about 0.3 ppm relative to leurosine (24), indicating that the structural change probably occurred in close proximity to this carbon atom. Otherwise, the proton NMR spectrum was remarkably similar to that of leurosine. When the carbon-13 NMR spectrum of 7 is compared to the spectrum for leurosine (25) (Table I), all carbons of the “vindoline” portion of the dimeric alkaloid were essentially the same, indicating that the Aspidosperma portion of the molecule was unaffected in the peroxidase oxidation reaction. The carbon signals for the aliphatic carbon atoms of the “iboga” portion were quite different. The 15’,20’epoxide carbons and the 21‘-aminoethylene carbon signals of leurosine (1)were absent in the spectrum of 7 and were replaced by a ketone carbonyl signal at 210.9 ppm, a secondary hydroxyl group signal at 80.37 ppm, and an Nformyl carbon signal at 163.68 ppm. The carbon-13 NMR spectrum for 7 was very similar to that for the known alkaloid catharinine ( 8 ) (26), also known as vinamidine (27),except that the spectrum for 7 contains an additional
Goswami et al.
secondary hydroxyl group in place of a simple methylene carbon at position 15’. Among the aminomethylenes of the iboga portion of leurosine (l), that at position 21’ is missing in the spectrum of 7, indicating that the newly formed N-formyl group must be at position 21’. Comparison of the carbon-13 NMR spectra of leurosine (l), catharinine (8)(26), and 7 also indicated that the ketone carbonyl was at position 20’ in 7 and that the secondary hydroxyl group was at position 15’. The slight deshielding effect on carbon 14’ @-effect) and shielding of the 19’carbon atom (y-gauche effect) in 7 when compared to 8 supports these assignments. High-resolution mass spectral data also support the structure of the oxidation product as 15’-hydroxycatharinine (7). The molecular ion at m / z 840 gave C46H56N4011, and the loss of an ethyl fragment confirmed the existence of an ethyl ketone. Other fragments are included in a table provided as supplementary material. This is the first demonstration of an enzymatically catalyzed ring-fission reaction with a dimeric Vinca alkaloid. 15’-Hydroxycatharinine has never been identified as a metabolic or natural product. However, the compound has been prepared by potassium permanganate oxidation of anhydrovinblastine or leurosine (28). The structurally related alkaloid catharinine (8) is a natural product isolated from both Catharanthus longifolius and C. ovalis (26). Vinamidine isolated from C. roseus (27) was later found to be identical with catharinine. Speculative biogenetic pathways suggested that catharinine can be formed from leurosine and vinblastine (26,29). Our results with horseradish peroxidase indicate that leurosine cannot be a precursor for catharinine (8) in plants, a t least by way of a peroxidative transformation pathway. Our results would also suggest that if leurosine is a natural alkaloid (30), and not an artifact (31),15’-hydroxycatharinine (7) will likely be found as a natural product. Peroxidases are common enzymes in Catharanthus plant material (30,32). Using a crude peroxidase preparation from mature Catharanthus roseus leaves, Kutney et al. (30) reported the conversion of anhydrovinblastine and leurosine to Catharine (15) (Figure 3), a compound formed by a completely different type of ring-fission process. Enzymatic transformations with leaf homogenates were conducted on an analytical scale, and Catharine was apparently identified only by HPLC. Our data clearly rule out the possibility that the compound formed by horseradish peroxidase oxidation is the unsaturated N-formyl alkaloid 15. It is possible that different peroxidase(s) from C. roseus plant material would afford 15 instead of 7,but that point remains to be proven. It is interesting and surprising that horseradish peroxidase accomplishes oxidation only on the iboga portion of leurosine and that the pathway and direction of oxidation is different than that obtained by numerous chemical and enzymatic oxidizing systems in the formation of the carbinolamine 5. Likely, these differences in pathways can be explained by the occurrence of two different initial oxidation steps catalyzed by different oxidizing agents and influenced by structural features of the alkaloid starting material. With leurosine, these possibilities are outlined in Figure 2. Peroxidase is well-known to catalyze oxidations of nitrogenous substrates, and the first oxidation step with this enzyme may involve either the elimination of hydrogen from carbons adjacent to nitrogen or the formation of a cation radical centered at nitrogen (7,33). With leurosine, the generation of a carbon-centered radical (11) at the 21’-position (as in path B, Figure 2) could be influenced
Chem. Res. Toxicol., Vol. 1, No. 4, 1988 241
Oxidation of Leurosine by Peroxidase
".,
U /
I I
Figure 2. Pathways for the oxidation of the piperidine ring moiety of iboga alkaloids like leurosine.
CH300C'
'OCOCH,
Figure 3. The structure of Catharine (15).
by the 15',20'-epoxide oxygen. Subsequent reaction steps are speculative, and no intermediates have been observed under any experimental condition. Probable intermediates would include an iminium species like 12 and addition of a hydroxyl group to carbinol 13 to give the putative triol 14. Periodate-like cleavage of 14 by peroxidase would provide 7 by a process analogous to that observed in the cytochrome P-450 mediated diol cleavage in cholesterol side chain degradations (34) and in the cleavage of diols with iron-porphyrin catalysts (35). The proposed pathway fixes the stereochemistry of the 15'-alcohol functional group of 7 as shown, since this pathway maintains the oxygen stereochemistry as found in leurosine. We believe this t o be the first example of vicinal diol cleavage by a peroxidase. The alternative oxidation pathway involving initial oxidation at the Nb-nitrogen (9, path A, Figure 2) would give an unstable cation radical. The subsequent elimination of a hydrogen atom from one of the three adjacent methylene carbons at 3', 5', or 21' to form an iminium ion would likely proceed through the path which provides the most stable iminium intermediate. Two coincidental factors probably favor the formation of 10 by this route: the rigidity of the bicyclic system consisting of the six-membered piperidine ring and the epoxide and the much more flexible nine-membered ring. Elimination of the hydrogen atom from position 5' of 9 and subsequent hydration of 10 would explain the ready formation of the carbinolamine 5 by copper oxidases and other enzymes (13). Metabolic alterations in the structures of biologically active materials can positively or negatively influence their effects on living systems. The Vinca alkaloids are wellknown for their abilities to inhibit the polymerization of tubulin protein in eukaryotic cells (36,37). The inhibition
of tubulin polymerization of leurosine and its various metabolites such as 15'-hydroxycatharinine (7),the phenol 4, and carbinolamine 5 were compared, and vinblastine sulfate was used as a standard for comparison. The results in Table I1 present the Im (inhibitory 50%) values for each compound. With the exception of 7,all compounds are one-third to one-tenth as active as vinblastine sulfate in the inhibition of tubulin polymerization. 15'-Hydroxycatharinine is strikingly less active, with an 15,, value requiring concentrations nearly 3 orders of magnitude higher than for vinblastine sulfate. The results indicate that the peroxidative ring-fission reaction is a deleterious and inactivating reaction and that similar biotransformations occurring in vivo when drugs like leurosine or vinblastine are administered (38) will result in loss of activity. It is also interesting to note that while 5'-hydroxyleurosine ( 5 ) is effective in inhibiting tubulin polymerization in vitro, it lacks cytotoxicity vs the Vinca alkaloid sensitive CCRF-CFM cell culture system a t much higher doses of 0.02-20 pg/mL (13). We suggest that this discrepancy is probably due to the inability of the carbinolamine/iminium derivative to enter cells to inhibit cell growth. This work sheds new light on the involvement of interesting steric and electronic factors which apparently influence the direction of enzymatic oxidations with structurally complicated Vinca alkaloids. It demonstrates the first enzymatically catalyzed bioinactivation reaction of this widely used class of antineoplastic drug. Furthermore, the characterization of 7 provides a basis for understanding new types of metabolic transformations possible with other Vinca alkaloids such as vinblastine and vincristine. Acknowledgment. We express our grateful appreciation for financial support for this work through NIH Grant CA-13786-14 and to Dr. Ian Jardine, Department of Pharmacology, Mayo Clinic, Rochester, MN, for highresolution mass spectral analyses. Supplementary Material Available: A table of high-resolution mass spectral peaks for compound 4 (1page). Ordering information is given on any current masthead page.
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