XENOBIOTICA,1991,VOL.
21, NO. 6, 737-750
Metabolism of levamisole, an anti-colon cancer drug, by human intestinal bacteria? Y.-2. SHU$, D. G. I . KINGSTON$§, R. L. VAN TASSELLII and T. D. WILKINSII 1Department of Chemistry and (:Departmentof Anaerobic Microbiology, Xenobiotica Downloaded from informahealthcare.com by National University of Singapore on 06/13/14 For personal use only.
Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Received 24 October 1990; accepted 1 January 1991
1. Anaerobic incubation of levamisole with human intestinal flora resulted in the formation of three thiazole ring-opened metabolites, namely, levametabol-I, I1 and 111. These new hydroxamic lactam-type metabolites were isolated and characterized by various spectroscopic methods. 2. Various pure cultures of human intestinal bacterial strains were shown, by quantitative h.p.1.c. analysis, to have ring-opening ability. Strong metabolizers include Bacteroides and CZostridium spp. Bacterial mixtures prepared from human faeces showed much greater ability to transform levamisole (74% in 48 h) than any pure strain culture. 3. Greatly decreased levamisole-transforming activity was observed with autoclaved bacterial cultures, and no activity was found with broth medium alone. This indicates that metabolism requires the presence of anaerobic bacteria and involves, at least in part, a nonenzymic process.
Introduction Levamisole (1) has been extensively used as an anthelmintic drug in veterinary and human medicine for over 20 years (Janssen 1976).Another major characteristic of this drug is its immunostimulatory activity (Symoens and Rosenthal 1977, Renoux 1980, Amery and Gough 1981). Several reports have described the metabolism of levamisole in animals and humans (Janssen 1976, Graziani and De Martin 1977, Koyama et al. 1983, Kouassi et al. 1986). It has been shown that levamisole is extensively metabolized in viuo, undergoing thiazole ring scission and aromatic hydroxylation to give a variety of metabolites. T h e most interesting major metabolite is 2-oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine(OMPI, 2, figure l), since it has been reported to: (1) interact with microtubules (De Brabander et al. 1978);( 2 ) be more potent than levamisole in enhancing the clearance of i.v. injected colloidal carbon in mice (Van Ginckel and De Brabander 1979);(3)protect cultured cells against necrosis induced by glutathione depletion (De Brabander et al. 1979), and (4)prevent peroxidase-mediated inhibition of neutrophil motility and lymphocyte transformation (Anderson et al. 1981). A prodrug hypothesis has thus been proposed for levamisole, and it is thought that most pharmacological effects of levamisole are mediated by the in viwo formation of OMPI (Z),the active form of the drug (Amery and Gough 1981). Very recently, as a result of a series of clinical studies of surgical adjuvant drug therapy in patients with resected stage C colorectal carcinoma, it was reported that therapy with levamisole plus 5-fluorouracil decreased the risk of cancer recurrence Y.Z.S. wishes to dedicate this paper to Professor Tsuneo Namba, of Toyama Medical and Pharmaceutical University, Japan, on the occasion of his 60th birthday. 9: Author to whom correspondence should be addressed. 0049-8254/91 $3.000 1991 Taylor & Francis Ltd.
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738
1. levamisole
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4. levametabol-I
2. OMPI
3. (OMPI),
5. Ievametabol-I1
6. levametabol-111
7. R= CH,CO-, R'zH-
8. R:R'= Figure 1 .
CH,CO-
Structures of levamisole, levamisole metabolites formed by human intestinal bacteria and related compounds.
by 41% and the overall death rate by 33% (Moertel et al. 1990). This discovery has been seen as the first successful drug therapy and an important intellectual breakthrough against colon cancer, a common type of malignancy in the USA. This combined-drug therapy has therefore been suggested as standard treatment for stage C colon carcinoma. Results from animal experiments have shown that following oral administration of 3H- or '4C-labelled levamisole a considerable portion (1 7-3573 of the radioactive dose was excreted in the faeces of rats (Graziani and De Martin 1977, Galtier et al. 1983), indicating the presence of levamisole and/or its metabolites in the intestines and colon. No in vivo or in vitro studies, however, have been performed on its metabolism by intestinal bacteria. Because of the significance of the novel anti-colon cancer activity of combined levamisole/fluorouracil therapy, it is important to know whether levamisole is metabolized in the colon and, if so, whether any OMPI-like metabolites are formed. We have, therefore, investigated the metabolism of levamisole by intestinal bacteria using human faecal mixtures as well as various pure bacterial cultures. In this paper we report the metabolic formation of several new thiazole ring-opened metabolites.
Materials and methods Chemicals Levamisole hydrochloride (1) was purchased from Aldrich Chem. Co. (Milwaukee, WI, U S A ) . 2~>xo-3-(2-mercaptoethyl)-5-phenylimidazolelidine (OMPI, 2) and its disulphide ((OMPI),, 3) were prepared by the reaction of levamisole with glyoxal by modified procedures of Van Belle and Janssen (1979). All other chemicals were of analytical grade and the solvents were of h.p.1.c. grade.
Levamisole metabolism by intestinal bacteria
739
All bacteriological media and supplements used in this study were prereduced and anaerobically sterilized following the methods described in the VPI Anaerobe Laboratory Manual (Holdeman et al. 1977).
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Instruments Specific rotations ([MID) were measured with a Perkin-Elmer 241 polarimeter. Infrared (i.r.) spectra were recorded with a Perkin-Elmer 710B infrared spectrophotometer. Ultraviolet (u.v.) spectra were recorded with a Beckman DU-70 spectrophotometer. Proton and carbon- 13 nuclear magnetic resonance ('H-n.m.r. and 13C-n.m.r.)spectra were obtained with a Varian Unity 400 ('H, 400 MHz, I3C, 100 MHz) and a Bruker WP-270 ('H, 270MHz, I3C, 68 MHz) spectrometer. Chemical shifts are given as 6 values relative to the signal of internal standard tetramethylsilane (TMS). Mass spectra were determined with a V G 7070 instrument operating in the electron ionization (EI), chemical ionization (CI, reagent gas, isobutane), or fast atom bombardment (FAB, matrix, 1-thioglycerol) mode. Chromatography Merck Kieselgel 60F254(silica) plates were used for t.1.c. and preparative t.1.c.; spots were detected under a U.V.lamp. Column chromatography was carried out on Merck silica gel (230-400 mesh). H.p.1.c. analysis was performed with a Waters 501 solvent delivery system equipped with a Biorad 1306 U . V . monitor under the following conditions: column, RCM Nova-Pak C,, Cartridge (4pm. 8 m m int. diam. x l00mm) in a Waters Radical Compression Module; mobile phase, acetonitrile-0.1 M ammonium acetate (46 : 54); flow rate, 1.3ml/min; pressure, c. 8280 kPa; detection at 245 nm with a range of 0.04 a.u.f.s. Under these conditions calibration curves for quantification of levamisole (l),levametabol-I (4) and levametabol-I1 ( 5 ) were prepared by using standard samples, and they were linear in the range of 5-1 20 pg/ml. Incubation of levamisole wiih human faecal bacterial mixtures, Bacteroides thetaiotaomicron or B. uniformis Human faeces from five non-smoking healthy donors (four male, one female) were anaerobically pooled and mixed under argon as previously described (Bashir et al. 1987 a). T h e pooled faeces (5 g) were diluted 20-fold in pre-reduced, anaerobically sterilized dilution fluid, and 0.5 ml was inoculated into 1OOOml of prereduced brain heart infusion (BHI) broth (Difco Lab, Detroit, M I , USA). T h e broth was incubated anaerobically for 18 h at 37°C. T o the broth was added levamisole hydrochloride (700mg) dissolved in distilled water (2 ml). The supplemented culture was incubated anaerobically for 48 h at 37°C and then extracted with water-saturated ethyl acetate (three times, 1000ml each). The combined organic layer was washed with 5% NaCl solution and concentrated to give a residue. A small portion of the residue (70mg) was immediately applied onto preparative t.1.c. plates, which were then developed in a solvent mixture of chloroform-zthyl acetate-methanol (10 : 1 : 0.2, by vol.). An unstable metabolite (levametabolI, 4, R, 0.70, 4mg) (figure l), and a stable metabolite (levametabol-11, 5,R , 0.61, 11 mg) were isolated. T h e rest of the residue (1.1 g ) was chromagographed on silica gel; the column was washed thoroughly with hexane and hexane-methylene dichloride (1 : 1, v/v), and eluted with methylene dichloride. This eluant gave a chromatographically homogeneous major metabolite (5,145mg, yield 21%) and a crude minor metabolite. The fraction containing the latter metabolite was further purified by preparative t.1.c. in chloroform-ethyl acetate-methanol (10: 3 : 0.5, by vol.) to afford pure metabolite (levametabol-111, 6 , 12mg, 1.8%). By using the procedure described above, we repeated the experiment with selected pure bacterial cultures. Levamisole hydrochloride (25&500 mg) was incubated with the BHI broths of Bacteroides thetaiotaomicron VPI 5482 and B. uniformis VPI TI-1. Preparative amounts of metabolites 5 (yield range, 7.3-10.5%) and 6 (yield range, 05-09%) were isolated from the metabolic mixtures; in addition, unchanged levamisole (yield range, 18.5-25.0%) was recovered from the same mixtures. Levametabol-I ( 4 ) A colourless oil, [a]2SD+22.2 (c=0.09, chloroform); U.V.I,,, (methanol) nm 242 (log E 2.80); high resolution mass spectrum: Found 238.0765, Calc. for [MI+,C, IH14N,02S,238.0776; EI mass spectrum m / z (relative intensity): 238(M+, 32), 206(22), 205(M+ -SH, loo), 146(80), 105(35); for 'H-n.m.r., see table 1 and figure 3; for "C-n.m.r., see table 2. Hydrogenation of metabolite 4. A stirred heterogeneous mixture of metabolite 4 (4mg) and 10% Pd/C (40 mg) in methanol (5 ml) was hydrogenated at room temp. for 18 h. T h e catalyst was filtered off, and the filtrate was concentrated in vacuo. Immediate purification by preparative t.1.c. in chloroform-zthyl acetateemethanol (10 : 1 : 0.2, by vol.) yielded an oily product (1 mg), which was identified as OMPI (2) by t.1.c. and by comparison of its 'H-n.m.r. spectrum (table 1) with that of an authentic sample. Levametabol-II ( 5 ) A colourless oil, [a]6'+43.3 (c=0.24, chloroform); U.V.I,,, (methanol) nm 242 (log E 4.33); i.r. v,,, (KBr) cm-': 3430 (br OH), 1634(C=O), 1491(arom C X ) , 1450, 1265, 1155; CI mass spectrum m / z (relative intensity): 476(M+ 2H, 0.4), 409(0.7), 408(0.5), 295(35), 281(43), 240(92), 239(100), 207(85), 203(72); for 'H-n.m.r., see table 1 and figure 4; for '3C-n.m.r., see table 2.
+
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740
Y.-2. Shu et al.
Acetylation of metabolite 5. To a solution of 5 (8 mg) in pyridine (0.8 ml) acetic anhydride (0.5 ml) was added dropwise at 0°C. The mixture was stirred at 70°C for 5 h, and then methanol (50ml) was added, and the solvent was removed in vacuo. Purification by'preparative t.1.c. in chloroform-ethyl acetate (5 : 1, v / v ) afforded acetates 7 (2.5mg) and 8 (l.Omg), and unreacted 5 (3mg). Compound 7, a colourless oil, 'Hn.m.r. (270MHz, CDCI,) 6 : 2.82(3H, s , CH,-CO-), 2.95-3.10(4H, m, 7-H x 2,7'-H x 2), 3.55 and 4.18 3.62and4.18(each l H , d d a n d t , J = Y . 9 , (each l H , d d a n d t , J=9.9,2.9HzandJ=9.9Hz,4-I-I,and4-Hb), 7.6 Hz, and J = 9.9 Hz, $-Ha and 4'-H,), 3.85 and 3.98 (each 1H, dt, J = 13.6.6.8 Hz, &-Haand 6'-H,), 3.99 and 4.04 (each l H , dt, J=9.6, 6.8Hz, 6-H, and 6-H,), 4.94(1H, dd, J = 9 . 9 , 7.6I-12, 5'..H), 5.58(1H, dd, J=9.9, 2.9Hz, 5-H), 6.08(1 H,brs,-N-Ou), 7.2&7.45(10H, m, arom. H); CI mass spectrum m / z (relative intensity): 281(92), 239(95), 205(100), 173(42), 145(40), 104(58). Compound 8, a colourless oil, 'H-n.m.r. (270 MHz, CDCI,) 6: 2.82(6H, s , CH,-CO- x 2), 3.00 and 3.03 (each 2H, dt, 13.1, 6.7 Hz, 7H, x 2, 7-H, x 2), 3.54 and 4.16 (each 2H, dd and t, J = 10.2, 3.0 Hz and J = 10.2 Hz, 4-Ha x 2 and 4I{,, x 2). 3.98 and 4.04 (each 2H, dt, J=9.6,6.7 Hz, 6-Ha x 2 and 6-H, x 2), 558(2H,dd, J = 102.3.0 Hz, 5H x 2), 7.20-7.45(10 H, m, arom. H); CI massspectrumm/z(relative intensity): 281(22), 249(17), 205(85), 189(40), 173(35), SS(l00); EI mass spectrum m / z (relative intensity): 279(32), 247(63), 237(100), 205(50), 173(35), 148(72), 146(75). Reduction of metabolite 5 with NaBH,. Compound 5 ( 5 mg) was dissolved in methylene dichloridemethanol (2 : 1, v/v, 3 ml), and NaBH, (15 mg) was added. The mixture was refluxed for 4 h in an argon atmosphere, and then poured into ice water and extracted with methylene dichloride, and the organic layer was concentrated. Purification by preparative t.1.c. in chloroform-ethyl acetate (5 : 1, v / v ) gave an oily compound (2mg), which was characterized as levametabol-I (4) by 'H-n.m.r. analysis (table 1). Reduction of metabolite 5 with aqueous TiCl,. T o a solution of 5 (1 5 mg, 0.032 mmol) in methanol-1 M sodium acetate (4 : 1, v/v, 10ml), a freshly prepared 20% w/v TiCI, solution ( 0 4 ml, 0 5 8 mmol) was added dropwise. The mixture was stirred in an argon atmosphere for 8 h at room temp. and then hasified with 2 M NaOII to pII 9, and extracted with ethyl acetate. T h e ethyl acetate layer was dried over anhydr. Na,SO, and evaporated, and the crude product was immediately purified by preparative t.1.c. in chloroform-ethyl acetate (5 : 1, v / v ) to yield a pure compound. T h e compound was identified as levametabol-I (4) on the basis of 'H-n.m.r. (table l), '3C-n.m.r. (table 2) and EI mass spectral data. Levametabol-III ( 6 ) A colourless oil, [a];'+ 50.5(c =0.19, chloroform); U.V.I,,, (methanol) nm 244 (log E 4.58); FAR mass spectrum m / z (relative intensity): 45Y(M+ + H , 2.7), 410(3.3), 391(2.6), 278(50), 239(100), 206(33), 203(72); CI mass spectrum m / z (relative intensity): 239(42), 223(92), 205(100), 189(20), 175(85), 148(40), 132(55), lOS(35); for 'II-n.m.r., see table 1 and figure 5; for I3C-n.m.r., see table 2. Reduction of metabolite 6 with NaBH,. Compound 6 (8 mg) was reduced with NaBH, as described for compound 5 . After work-up, immediate purification by prep-t.1.c. (CHCI,/EtOAc/MeOH, 10 : 1 :0.2) gave two major products (c. 2mg each), which were identified as kvametdbol-I (4) and OMPI (2), respectively,by comparing 'H-n.m.r. spectra with those of authentic samples. Two minor products in the reaction mixture were identified as levametabol-I1 ( 5 ) and (OMPI), (3) based on co-t.1.c. analysis. Screening of intestinal bacteria capable of transforming levamisole to levametabol-II ( 5 ) The 28 bacterial strains used in the study were obtained from the culture collection of the Department of Anaerobic Microbiology; the cultures were stored in chopped meat broth at room temperature. Each culturewas inoculated intoRH1 broth(l0ml)andgrownanerobicallyfor 1 8 h a t 37°C. Analiquot (1OOp1) o f levamisole hydrochloride (3 mg, 12.47pmol) in sterilized water was added to the bacterial culture. Incubation was carried out under a CO, atmosphere at 37°C for 48 h. A portion (2 ml) of the metabolic mixture was loaded onto a Sep-Ilak C,, cartridge (Waters Assoc.), which had been conditioned with methanol (10ml) and water (20ml). T h e cartridge was washed with water (20ml), and levamisole and its metabolites were eluted from the cartridge with methanol-water (8 : 2, v/v, 10 ml). A portion (1Opl of the resultant eluate was injected directly into the h.p.1.c. system described above. Metabolic time-course of levamisole in human faecal bacterial mixture A n 18h BHI broth seeded with human faeces was divided into tuhes (10ml each) under a stream of CO, and supplemented with levamisole hydrochloride (6mg in loop1 of sterilized water). The supplemented bacterial broths were incubated anaerobically at 37°C. After periods of 1 h, 3 h, 6 h, 12 h, 24 h, 36 h and 48 h, three of the tubes were chilled in an acetone-dry ice hath. The metabolic mixtures were thawed, processed and analysed by h.p.1.c. in the same fashion as described in the above bacterial screening experiment.
Levamisole metabolism by intestinal bacteria
741
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Results Analysis of Levamisole metabolites formed by human intestinal bacteria Preliminary t.1.c. analysis of a 36 h levamisole (1) metabolic mixture incubated with human faecal bacteria indicated the transformation of levamisole into three metabolites. These metabolites were not produced in a control experiment in which levamisole was incubated with BHI broth alone. T h e compounds were designated in order of their R, values as levametabols I (RF0-70), I1 (RF0.61) and 111 ( R , 024). Two reported metabolites (Van Belle and Janssen, 1979) of levamisole, OMPI (RF0.27) and (OMPI), (RF015), were not observed. In order to examine metabolite formation quantitatively, a convenient sample preparation procedure and an h.p.1.c. method were established. Prior to the h.p.1.c. analysis, bacterial samples were cleaned up and prepared by using Sep-Pak C,, cartridges rather than by a solvent extraction method. T h e recoveries of levamisole (l),levametabol-I (4), and levametabol-I1 ( 5 ) through this clean-up process were 95%, 92% and SO%, respectively (data not shown). As shown in figure 2 ( c ) , the chromatographic conditions provided a good separation of the six compounds OMPI (peak 2), levamisole (peak I), (OMPI, (peak 3), levametabol-111 (peak 6), levametabol-I (peak 4) and levametabol-I1 (peak 5). Four peaks corresponding to substrate and metabolites were observed in the chromatogram of the levamisole metabolic mixture (figure 2 ( b ) ) .Their retention times were identical with those of authentic levamisole (peak l), levametabol-111 (peak 6), levametabol-I (peak 4) and levametabol-I I (peak S), respectively. A bacterial component peak (peak 7) detected in this chromatogram was isolated and then identified as indole by spectroscopic analysis. However, no metabolite peaks except an unchanged substrate peak (peak 1) were seen in the incubation mixture of levamisole with BHI broth (figure 2 ( a ) ) .In agreement with the t.1.c. analysis results, no detectable amounts of OMPI or (OMPI), were present in the bacterial metabolic mixture.
5 9
id
4 2
si
L
Time (min) (4 Figure 2 .
Time (min) (b)
Time (min) (4
II.p.1.c. chromatograms of levamisole metabolites formed by human intestinal bacteria.
( a ) Sample of levamisole incubated with BHI broth. ( b ) Sample of levamisole incubated with human intestinal bacterial mixtures. (c) Mixture of standard samples. 1, Levamisole; 2, OMPI; 3, (OMPI),; 4, levametabol-I; 5, levametabol-11; 6, levametabol-111;7, indole.
Y.-2. Shu et al.
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742
Characterization of metabolites Levametabol-I, metabolite 4, a colourless oil, was carefully isolated by preparative t.1.c. from the incubated bacterial mixtures of levamisole. T h e metabolite decomposed rapidly after solvent (ethyl acetate) extraction even if the extract was maintained at 4°C rather than at room temperature. However, when isolated in pure form and stored in the absence of air, the compound was relatively stable. Levametabol-I showed a chemical composition of C, ,H1,N20,S by high resolution mass spectrometry, indicating that it possesses an extra oxygen atom compared to OMPI (2). This metabolite exhibited a U.V. absorption maximum at 242 nm, which is quite different from that of the parent drug levamisole (A,,, 226 nm). T h e 'Hn.m.r. (figure 3 , table I ) and 13C-n.m.r. spectra (table 2) of metabolite 4 differed in overall appearance from those of levamisole except for the signal due to the phenyl moiety, indicating that the structural changes through bacterial metabolism take place in the tetrahydroimidazothiazole ring system. T h e spectra on the other hand showed an identical number of methylene, methine and carbonyl groups to those of OMPI (2). Similarly to that of OMPI, the 'H-n.m.r. spectrum of metabolite 4 displayed a characteristic triplet (J= 7-1 Hz) at 6 1.50 due to a DzO exchangeable proton, which showed spin correlation with the 7-methylene protons (6 2.82, q , J=7.1 Hz) in a 'H-double resonance experiment, indicative of the presence of a free thiol group. That metabolite 4 was a free thiol-bearing compound was supported by mass spectral data; the base peak appearing at mjz 205 was ascribed by the loss of an -SH fragment from the molecular ion at m / z 238. T h e most distinct difference between the corresponding 13C-n.m.r. signals of metabolite 4 and OMPI (2) was observed at the C-2 carbonyl carbon (6 183.4 in 4 and 6 161-8 in 2). It is also important to note that the coupling of 5-H with the vicinal 1 - N H proton ( J = 1.5 Hz) in OMPI (2)was absent in metabolite 4. This indicated that the extra hydroxy group in the levametabol-I molecule might be located on N-I to form a hydroxamic acid partial structure. This was confirmed by chemical conversion; upon catalytic
X
I
I
/-
__r_
I
-,
7 ,
6 Figure 3 .
- T
5
-4
L
J
T-7,
3
,
,
,
2
,
,
,
,
1
,
,
(PPm)
'H-n rn r spectrum of levarnetabol-I (4) (400MHz, in CDC13).
, -1
0
-
Levamisole metabolism by intestinal bacteria
743
hydrogenation metabolite 4 was transformed into the lactam OMPI (2). T h e structure of levametabol-I was therefore established as 1-hydroxy-2-oxo-3-(2mercaptoethy1)-5-phenyl-imidazolidine(4). Levametabol-I1 (5) was isolated as the second major bacterial' metabolite of levamisole. T h e chemical ionization mass spectrum exhibited a quasi molecular ion (M 2H') at m / z 476 and a diagnostic base ion peak at m / z 239 resulting from the fragmentation of the quasi molecular ion into one half, implying that levametabol-I I ( 5 ) might have a symmetric dimeric structure. T h e presence of two homogeneous units in the molecule was also clear from the 'H- and 'jC-n.m.r. spectra (figure 4, tables 1, 2), since only 'H- and 13C-signals due to one monomeric portion were observed. Acetylation of metabolite 5 yielded the monoacetate 7 and the diacetate 8, demonstrating that metabolite 5 has two hydroxyl groups. Comparison of n.m.r. spectral data of metabolite 5 with metabolite 4 showed a close similarity except for the C-7 and C-8 methylene signals; in contrast with the resonances of C-7 (6 22.4)
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+
Table 1. 'H-n.m.r. spectral data of the levametabols and related compounds (p.p.m., in CDCI,).
4-Ha 4-Hb
LevarnetabolI (4)
LevametabolI1 (5)
Levametabol111 (6)
3.63, dd (9.9, 7.4) 4.18, t (9.9)
3.60, dd (9.9, 7.6) 4.19, t (9.9)
494, dd (9.9, 7.5)
OMPI
(OWI),
(2)
13)
3.61, dd (9.9, 7.6) 420, t (9.9) 3.29, dd (8.5, 7.6) 3.88, t (8.5)
3.28, dd (8.6, 7.4) 3.87, t (8.6)
3.26, dd (8.6, 7.5) 3.87, t (8.6)
4.94, dd (9.9, 7.8)
4.93, dd (9.9, 7.6) 4.79, ddd (8.5, 7.6, 1.5)
4.77, ddd (8.6, 7.4, 1.5)
4.76, ddd (8.6, 7.5, 1.5)
3.78, d t (14.0, 7.1) 3.83, d t (14.0, 7.1)
3.82, d t (13.9, 6.8) 3.98, dt (13.9, 6.8)
3.85, d t (14.1, 7.0) 3.99, d t (14.1, 7.0) 3.47, d t (140, 6.7) 3.64, d t (14.0, 6.7)
3.33, d t (13.9, 7.1) 3.45, d t (13.9, 7.1)
3.42, d t (13.8, 6.7) 3.60, d t (13.8, 6.7)
2.82, q (7.1) 2.82, q (7.1)
2.98, d t (13.6, 6.8) 3.02, d t (13.6, 9.8)
2.67, q (7.1) 2.67, q (7.1)
2.85, t (6.7) 2.85, t (6.7)
7.30-7.45, m 6.17, br s
7.28-7.45, m 6.48, b r s
2.98, d t (13.9, 7.0) 3.03, d t (13.9, 7.0) 2.89, t (6.7) 2.89, t (6.7) 7.30-7.42, m 6.08, b r s 4.68, br s
7.25-7.50, m
725-7.50, m
5 40, br s 1.42, t (7.1)
5 17, br n
4-Ha 4-Hb 5-H 5'-H 6-Ha
6-IIb G-Ha 6'-Hb 7-Ha 7-Hb 7'-Ha 7'-Hh Ph-H -N-OH -NII -SH
1.50, t (7.1)
Coupling constants ( J , Hz) are shown in parentheses.
Y.-2. Shu
744 Table 2.
al.
I3C-n.m.r. specttral data of the levametabols and related compounds (p.p.m., in CDCI,). 1,evametabol-
I
Carbon no.
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et
Levametabol-
Levametabol-
I1 (5)
(4)
111 (6)
(2)
(OMPI), (3)
OMPI
C-2
183.4(s)
183.4(s)
183.4(s) 161.0(s)
161.8(s)
161.2(s)
C-4 c-4'
58.0(t)
58.3(t)
58,2(t) 54,2(t)
54.0(t)
54-2(t)
C-5 C-5'
57.4(d)
57.5(d)
57.4(d) 53,9(d)
53.7(d)
53.8(d)
C-6 C-6'
49,6(t)
45.9(t)
45.9(t) 42.6(t)
46.6(t)
42.6(t)
c-7
22,4(t)
35+3(t)
35.8(t) 36,8(t)
22.8(t)
36.8(t)
C-1" C-1"'
140.1(s)
140.1(s)
14 0 3s) 141.5(s)
141.5(s)
141.5(s )
C-2", 6" C.2"', t,,,!
126,2(d)
126.1 (d)
126.0(d) 126.1(d)
1264(d)
126.1(d)
C-3", 5" C-3"', 5"'
129.2(d)
129.l(d)
129.1(d) 128.9(d)
128,9(d)
128,9(d)
c-4"
128,7(d)
128,7(d)
128,2(d) 128.6(d)
128.2(d)
128.2(d)
(2-7'
(2-4"'
Abbreviations given in parentheses indicate the signal patterns observed in the off-resonance decoupling technique, which were confirmed by I N E P T (insensitive nuclei enhanced by polarization transfer) experiment. s, Singlet; d, doublet; t, triplet. The assignment of phenyl moiety carbons is bascd on the calculation method (Stothers 1972) of substituted benzenes.
r
TMS
I
X
7--,-v-
8
-, r---
m7.,-r--l----l
3
,
,
~
6
Figure 4
'H-n m r. spectrum of levametabol-I1 ( 5 ) (400MIIz, in CDCI,)
5
4
2
1
,
(PPm)
7
,
,
0
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Levamisole metabolism by intestinal bacteria
745
and C-6 (6 49.6) in metabolite 4, the corresponding signals in metabolite 5 occurred at 6 3 5 4 (C-7) and 6 45.9 (C-6), respectively. Similar shifts of the signals for C-7 and C-6 were observed between the monomeric compound OMPI (2) and dimeric compound (OMPI), (3) (table 2). These findings indicated that levametabol-I1 (5) may consist of two monomeric units of levametabol-I (4), and that dimerization is most likely through a disulphide linkage. Final confirmation of the structure of metabolite 5 was achieved by chemical interconversion; reduction of metabolite 5 with NaBH, gave compound 4, while air oxidation of metabolite 4 yielded the dimeric product 5 (data not shown). Structure levametabol-I1 (5) was thus conclusively determined to be the disulphide of levametabol-I (4). Mattingly and Miller (1980) have reported a mild method for reduction of the N-0 bond in hydroxamic acids with aqueous titanium trichloride. Unfortunately, the reduction of levametabol-I1 (5) with the same reagent predominantly afforded the monomer 4 rather than the N-hydroxy reduced products, i.e. (OMPI), (3) or OMPI (2). After a prolonged reaction period, traces of compounds 3 and 2 were observed on t.l.c., but it proved impossible to isolate these minor products. Similarly, the attempted conversion of metabolite 5 into compounds 3 or 2 with Zn5% HC1 (data not shown) led to the formation of compound 4 as major product. Levametabol-111 (6) appeared to be a minor metabolite. T h e positive ion FAB mass spectrum of 6 gave a quasi molecular ion (MH') at m / z 459, and the CI mass spectrum demonstrated two prominent fragment ions at m l z 239 and m l z 223, characteristic of the levametabol-I (4) and OMPI (2) skeletons, respectively. T h e 'H- and 13C-n.m.r. spectra (figure 5, tables 1, 2) comprised pairs of signals clearly assignable to the monomeric portions in the levametabol-I1 (5) and (OMPI), (3), respectively. Moreover, when treated with NaBH,, levametabol-111 (6) released compounds 4 and 2. The above evidence indicates that levametabol-111 (6) is a heterogeneous disulphide consisting of monomeric units of levametabol-I (4) and
OMPI (2).
2HCb
x
rL-
,
8
--y-,, ,, , I , ,
i
,
I
'
"
1 (PPd
7
6
Figure 5 .
'H-n.m.r. spectrum of levametabol-111 (6) (400MHz, in CDCI,).
5
4
3
2
'
0
746
Y.-2. Shu et al.
Table 3. The capacity of various bacterial strains to transform levamisole to levametabol-I1 under anaerobic conditions. Levametabol-I I formed (mol/ml)
”/, Transformation
BHI broth alone Human bacterial mixture
0 0.459
0 73.6
Bacteroides distasonis VPI T3-25 B. distasonis VPI 17297 B. fragilis VPI 3390 B. fragilis VPI B2-22 B. ovatus VPI 3049 B . ovatus VPI GMH B. thetuiotaomicron VPI 5482 R. thetaiotaomicron VPI 5482 (autoclaved) B. thetaiotaomicron VI’I 5482 (aerobic) R. thetaiotaomicron VPI J19-34B R. uniformis VPI 0909 B.uniformis VPI T1-1 B. erulgatus VPI 5710 Rarteroides group VPI 3452A 2308
0.246 0.189 0.183 0,097 0.046 0.160 0.1 90 0.032 0 0,029 0115 0.201 0.096 0.097
39.4 30.3 29.4 15.5 7.4 25.7 30.5 5.1 0 4.6 18.4 32.2 15.4 15.6
Clostridium barkeri VPI 5359-1 C‘. radaveris VPI 2718 C. dostridiforme VPI 0316 C . dostridiforme VPI 12706 C,’. dificile VPI 10463 C. innocuum VPI A14-39A C. innocuum VPI 1614-1 C. oroticum VPI 056JA C. sordellii VPI 6356 C. sordellii VPI 9048 C . sphenoides VPI 10627 C. sporogenes VPI 8654E C . septicum VPI 2017 C . septicum VPI 1526
0.092 0.075 0.143 0.166 0.01 1 0.075 0073 0.097 0.1 17 0.344 0.029 0.241 0.01 1 0.010
14.8 12.0 22.9 26.7 1.8 12.0 11.7 15.6 18.8 55.2 4.6 38.4 1.8 1.6
E . moniliforme VPI 12566
0.069
11.1
Peptostreptococcus productus VPI 4299
0.057
9.1
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Bacteria
-
Each tube (10ml) contained 3 mg of levamisole hydrochloride (1,247 pmol/ml), and the incubation was carried out at 37°C for 48 h.
Screening of intestinal bacteria capable of transforming levamisole to the stable metabolite levametabol-11 ( 5 ) T h e levamisole-metabolizing activity of various intestinal strains was examined by quantitative h.p.1.c. analysis of the stable major metabolite levametabol-I1 ( 5 ) formed from the incubation. All 28 strains examined showed metabolic ability at different levels (table 3). Strong metabolizers having more than 20% conversion percentages included the Bacteroides and Clostridium spp. Bacterial mixtures prepared from human faeces demonstrated much greater transformation activity (74%)than any of the individual pure strain cultures. In addition, data (table 3) from the experiment with a common anaerobe Bacteroides thetaiotaomicron VPI 5482 show appreciable transforming activity (30.5%) after an anaerobic incubation, but no formation of metabolite 5 after an aerobic incubation. Surprisingly, a low level of the activity (5.1%) was found even with the autoclaved cells.
Levamisole metabolism by intestinal bacteria
z
h
747
1
2*5
'= 0
Ea
W
C
0
. I
Y
2
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Y
E
Q)
u
E
u" 0
6
12
18
24
30
36
42
48
Time of incubation (h) Figure 6. Time-course of levamisole metabolism by human intestinal bacterial mixtures. -0-, Levamisole; -0-, levametabol-11; ...., sum of levametabol-I and levametabol-I1 x 2.
Time-course of levamisole metabolism by human faecal bacterial mixture Figure 6 shows the time-course of transformation of levamisole by human faecal bacterial mixtures into its stable major metabolite, levametabol-I1 ( 5 ) , during anaerobic incubation. T h e formation of metabolite 5 was observed 3 h after the start of incubation, and its amount increased gradually with a corresponding decrease of levamisole. Levametabol-I (4) was not observed until 24 h, perhaps because of its lability and easy dimerization; after 24 h it appeared to accumulate in small amounts, and the other minor metabolite, levametabol-I11 (6), gave tiny peaks in the h.p.1.c. chromatogram. Unfortunately, the low concentrations of metabolites 4 and 6 did not permit accurate quantification by the h.p.1.c. method. Since compound 5 could be considered to be formed through the dimerization of levametabol-I (4), the net amount of metabolite 4 produced at 48 h of incubation was expected to be about 70% of added substrate, as represented by the dotted line in figure 6.
Discussion T h e present study demonstrates that the anti-colon cancer drug levamisole is converted to three thiazole ring-opened metabolites by human faecal bacteria. For the first time hydroxamic lactam derivatives were isolated and characterized as levamisole metabolites. T h e major metabolites obtained from the incubated bacterial mixtures are levametabol-I (4) and its disulphide (levametabol-11, S), which could be explained as an artifact of metabolite 4 resulting from spontaneous oxidation during processing. We were unable to obtain O M P I (2),a reported major active metabolite of levamisole present in animal plasma and urine (Janssen 1976, Graziani and De Martin 1977, Koyama et al. 1983), from our in vitro system, but we isolated a minor bacterial metabolite levametabol-I11 (6) which has OMPI (2) as a structural unit. These observations indicate that the formation of hydroxamic lactam-type metabolites by intestinal bacteria occurs preferentially to that of simple
748
Y.-2. Shu et al.
lactam derivatives. Since no conversion from compounds 415 to 213, or vice versa, was observed either by the bacterial incubation or by air oxidation, the formations of levametabol-I (4) and O M P I ( 2 ) are likely to proceed by different pathways. Although OMPI has been claimed as a major active metabolite of levamisole for many years, the experimental data describing its isolation from biological fluids and its structural characterization have not yet been published. In the present study we carried out various detailed spectroscopic comparisons between OMPI ( 2 ) , levametabol-I (4) and their analogues to confirm the structure of the new metabolites. All three bacterial metabolites obtained in the present investigation are dextrorotatory isomers: levametabol-I (4, [u]i5 22.2 (c = 0.09, CHCI,)), levametabol-I1 (5, [u]i5+43.3 (c=0.24, CHCI,)) and levametabol-111 (6, [c(]i5 + 50.5 (c = 0.19, CHCI,)). Similarly, OMPI ( 2 , 25.6 (c = 0.32, CHCl,)) and (OMPI), ( 3 , [u];’ +46.5 (c=0.23, CHCl,)) show dextrorotory properties. Since the racemization of asymmetric carbon C-5 is unlikely through the ring-opening process, the levametabols thus are most likely to have the same 5s-configuration as that in the parent levamisole. Compared with the structures of OMPI ( 2 ) and (OMPI), ( 3 ) , the presence of extra hydroxyl group(s) on N-1 of levametabol-I (4) and levametabol-II(5) would be expected to increase their polarity. Nevertheless, higher R, values for compounds 4 ( R , 0.70) and 5 (R, 0.61) were observed on normal phase t.1.c. than for compounds 2 ( R , 0.27) and 3 (R, 0.1 5 ) . This is probably due to intramolecular hydrogen bonding between the N-1 -hydroxyl hydrogen and the 2-carbonyl oxygen in compounds 4 and 5, leading to a greatly decreased polarity for the latter compounds. Investigations on the formation of O M P I from levamisole (Van Belle and Janssen 1979) revealed that some u-ketoaldehydes such as methylglyoxal, which may be formed metabolically from the common cell constituent glyceraldehyde-3phosphate, specifically catalyse the reaction. It is thus suggested that such nonenzymic reactions involving small molecules play an important role in the in vivo metabolism of levamisole into OMPI. As the present study has shown, the formation of levametabol-I1 from levamisole was not observed in BHI broth alone, but was observed in autoclaved cultures of Bacteroides thetaiotaomicron and faecal bacterial mixtures (data not shown), albeit at a much decreased level. This fact indicates that some bacterial cell constituent(s), most likely a heat-stable small molecule, accounts at least in part for the formation of levametabol derivatives. There was however no convincing evidence for the formation of levametabols when glyceraldehyde, methylglyoxal or glyoxal were incubated with levamisole in BHI broth. Further studies on the characterization of such ‘key’ small molecule species are under investigation. It is interesting to note that the formation of the hydroxamic lactam functionality from levamisole must involve an oxidation step, despite the anaerobic conditions required for the bacterial viability. T h e mechanism for this oxidation is at present unknown. One possibility is that the reaction might require the reduced form cofactor or the reduced form ‘key’ small molecule described above. Although the examples of oxidationldehydrogenationconversion mediated by anaerobic bacteria are relatively few, one precedent we observed in our series of studies on the anaerobic metabolism of food mutagens is that an imidazoquinoline-type dietary carcinogen, IQ, was hydrated and then oxidized to yield 7-hydroxy IQ (Bashir et al. 1987 a, b). This showed that intestinal bacteria may have a wide variety of unexplored enzyme sources that catalyse reactions beyond our current conventional knowledge.
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+
+
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Levamisole metabolism by intestinal bacteria
749
T h e recent discovery of unique anti-colon cancer efficacy by levamisole plus 5fluorouracil is beginning to received much attention. Although we could not detect direct chemical interaction of these two drugs in our incubation system which mimics the human colon environment, our observation that levamisole is metabolized in vitro by human intestinal flora to give the major metabolite levametabol-I, a close structural analogue to OMPI, implies that this metabolite may be formed in the human colon. This would further raise some very important questions, i.e. whether levametabol-I possesses in vitro or in vivo anti-colon cancer activity, and if so, whether its activity is even stronger than levamisole. Since OMPI has demonstrated enhanced immunomodulating and other biological activities as compared with levamisole, and has been proposed as the active drug form of levamisole (Amery and Gough 1981), we believe that detailed investigations on the in vivo formation of levametabol-I, particularly in the colon, and its related pharmacological activities could be of great significance to our understanding of the anti-colon cancer effect of levamisole.
Acknowledgements Financial support for this work was provided by the National Cancer Institute, National Institutes of Health, through grant number CA40821.
References AMEHY, W. K., and COUGH,D. A,, 1981, Levamisole and immunotherapy: some theoretic and practical considerations and their relevance to human disease. Oncology, 38, 168-181. ANDERSON, R., OSASHUIZEN, R., and GRABOW, G., 1981, Prevention of peroxidase mediated inhibition of neutrophil motility and lymphocyte transformation by levamisole, OMPI, sodium aurothiomalate, indomethacin and tolmetin in vitro. International Journal of Immunopharmacology, 3,123132. BASHIR, M., KINGSTON, D. G. I., CARMAN, R. L., VANTASSELI., R. L., and WILKINS, T . D., 1987a, Anaerobic metabolism of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) by human fecal flora. Mutation Research, 190, 187-190. BASIIIR, M., KINGSTON, D. G. I., CARMAN, R. L., VAN TASSELL, R. L., and WII.KINS,T. D., 1987b, Biological formation and chemical synthesis of 2-amino-3,6-dihydro-3-methyl-7Himidazolo[4,5-f]quinoline,the major metabolite of dietary carcinogen 2-amino-3-methyl-3Himidazolo[4,5-f]quinoline (IQ) by normal intestinal bacteria. Heterocycles, 26, 2877-2886. DE BRABANDER, M., AERTS, F., GEUENS, G., VANGINCKEL, R., VANDE VEIRE,R., and VAN BELLE,H., 1978, DL-2-0xo-3-(2-mercaptoethyl)-5-phenylimidazolelidine. A sulfhydryl metabolite of levamisole that interacts with microtubules. Chemico-Biological Interaction, 23, 45-63. DEBRABANDER, M., VANBELLE,H., AERTS,F., VANDEVEIRE, R., and GEUENS, G., 1979, Protective effect of levamisole and its sulfhydryl metabolite OMPI against cell death induced by glutathione depletion. International Journal of Immunopharmacology, 1, 93-100. GALTIER, P., CVCHE, Y., and ALVINERIE, M., 1983, Tissue distribution and elimination of [3H]levamisole in the rat after oral and intramuscular administration. Xenobiotica, 13, 4 0 7 4 1 3. GRAZIANI, G., and DE MARTIN,G. L., 1977, Pharmacokinetic studjes on levamisole, absorption, distribution, excretion and metabolism of levamisole in animals-a review. Drugs under Experimental and Clinical Research, 2, 221-233. HOLDEMAN, L. V., CATV,E. P., and MOORE,W. E. C., 1977, Anaerobe Laboratory Manual, 4th edition (Blacksburg: Virginia Polytechnic Institute and State University, Anaerobe Laboratory), pp. 141148. JANSSEN, P. A. J., 1976, T h e levamisole story. In Progress in Drug Research, vol. 20, edited by E. Jucker (Basel: Birkhauser Verlag), pp. 347-370. KOUASSI, E., CAII.I.E, G., LERY,L., LARIVIERE, L., and VERINA,M., 1986, Novel assay and pharmacokinetics of levamisole and P-hydroxylevamisole in human plasma and urine. Biopharmaceutics and Drug Disposition, 7 , 71-89. KVYAMA, K., OISHI,T., ISHII,A,, and DEGUCHI, T., 1983, Metabolic fate of levamisole in rats, dogs and monkeys. Plzarmacometrics (Tokyo), 26, 869-876. MATTINGLY, P. G., and MILLER, M. J., 1980, Titanium trichloride reduction of substituted N-hydroxy2-azetidinones and other hydroxamic acids. Journal of Organic Chemistry, 45, 410-41 5.
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MOERTEL, C . G . , FLEMING, T. R., MACDONALD, J . S., HALLER, D. G., LAURIE, J. A,, GOODMAN, P. J., ~JNGERLEIDER,J. S., EMERSON, W. A , , TORMEY, D. C., CLICK, J. H., VEEDER, M. H., and MAII.I.IARD, J. A , , 1990, Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma. New England Journal of Medicine, 322, 352-358. RENOCJX, G . , 1980, The general immunopharmacology of levamisole. Drugs, 19, 89-99. STOTHERS, J . H., 1972, Carbon-13 N M R spectroscopy (New York: Academic Press). pp. 196-205. SYMOENS, J . , and ROSENTHAL, M., 1977, 1,evamisole in the modulation of the immune response: the current experimental and clinical state. Journal of the Reticuloendothelial Society, 21, 175-221. VANBELLE, H., and JANSSEN, P. A. J . , 1979, a-Ketoaldehydes, specific catalysts for thiol formation from levamisole. Biochemical Pharmacology, 28, 1313-1 318. VANGINCKBI., R., and DEBRABANDER, M., 1979, T h e influence of a levamisole metabolite ~ ~ - 2 - o x 0 - 3 - ( 2 rnercaptoethyl)-5-phenylimidazolelidineon carbon clearance in mice. Journal of the Reticuloendothelial Society, 25, 125-1 31.
21, NO. 6, 737-750
Metabolism of levamisole, an anti-colon cancer drug, by human intestinal bacteria? Y.-2. SHU$, D. G. I . KINGSTON$§, R. L. VAN TASSELLII and T. D. WILKINSII 1Department of Chemistry and (:Departmentof Anaerobic Microbiology, Xenobiotica Downloaded from informahealthcare.com by National University of Singapore on 06/13/14 For personal use only.
Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Received 24 October 1990; accepted 1 January 1991
1. Anaerobic incubation of levamisole with human intestinal flora resulted in the formation of three thiazole ring-opened metabolites, namely, levametabol-I, I1 and 111. These new hydroxamic lactam-type metabolites were isolated and characterized by various spectroscopic methods. 2. Various pure cultures of human intestinal bacterial strains were shown, by quantitative h.p.1.c. analysis, to have ring-opening ability. Strong metabolizers include Bacteroides and CZostridium spp. Bacterial mixtures prepared from human faeces showed much greater ability to transform levamisole (74% in 48 h) than any pure strain culture. 3. Greatly decreased levamisole-transforming activity was observed with autoclaved bacterial cultures, and no activity was found with broth medium alone. This indicates that metabolism requires the presence of anaerobic bacteria and involves, at least in part, a nonenzymic process.
Introduction Levamisole (1) has been extensively used as an anthelmintic drug in veterinary and human medicine for over 20 years (Janssen 1976).Another major characteristic of this drug is its immunostimulatory activity (Symoens and Rosenthal 1977, Renoux 1980, Amery and Gough 1981). Several reports have described the metabolism of levamisole in animals and humans (Janssen 1976, Graziani and De Martin 1977, Koyama et al. 1983, Kouassi et al. 1986). It has been shown that levamisole is extensively metabolized in viuo, undergoing thiazole ring scission and aromatic hydroxylation to give a variety of metabolites. T h e most interesting major metabolite is 2-oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine(OMPI, 2, figure l), since it has been reported to: (1) interact with microtubules (De Brabander et al. 1978);( 2 ) be more potent than levamisole in enhancing the clearance of i.v. injected colloidal carbon in mice (Van Ginckel and De Brabander 1979);(3)protect cultured cells against necrosis induced by glutathione depletion (De Brabander et al. 1979), and (4)prevent peroxidase-mediated inhibition of neutrophil motility and lymphocyte transformation (Anderson et al. 1981). A prodrug hypothesis has thus been proposed for levamisole, and it is thought that most pharmacological effects of levamisole are mediated by the in viwo formation of OMPI (Z),the active form of the drug (Amery and Gough 1981). Very recently, as a result of a series of clinical studies of surgical adjuvant drug therapy in patients with resected stage C colorectal carcinoma, it was reported that therapy with levamisole plus 5-fluorouracil decreased the risk of cancer recurrence Y.Z.S. wishes to dedicate this paper to Professor Tsuneo Namba, of Toyama Medical and Pharmaceutical University, Japan, on the occasion of his 60th birthday. 9: Author to whom correspondence should be addressed. 0049-8254/91 $3.000 1991 Taylor & Francis Ltd.
Y.-2. Shu et al.
738
1. levamisole
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4. levametabol-I
2. OMPI
3. (OMPI),
5. Ievametabol-I1
6. levametabol-111
7. R= CH,CO-, R'zH-
8. R:R'= Figure 1 .
CH,CO-
Structures of levamisole, levamisole metabolites formed by human intestinal bacteria and related compounds.
by 41% and the overall death rate by 33% (Moertel et al. 1990). This discovery has been seen as the first successful drug therapy and an important intellectual breakthrough against colon cancer, a common type of malignancy in the USA. This combined-drug therapy has therefore been suggested as standard treatment for stage C colon carcinoma. Results from animal experiments have shown that following oral administration of 3H- or '4C-labelled levamisole a considerable portion (1 7-3573 of the radioactive dose was excreted in the faeces of rats (Graziani and De Martin 1977, Galtier et al. 1983), indicating the presence of levamisole and/or its metabolites in the intestines and colon. No in vivo or in vitro studies, however, have been performed on its metabolism by intestinal bacteria. Because of the significance of the novel anti-colon cancer activity of combined levamisole/fluorouracil therapy, it is important to know whether levamisole is metabolized in the colon and, if so, whether any OMPI-like metabolites are formed. We have, therefore, investigated the metabolism of levamisole by intestinal bacteria using human faecal mixtures as well as various pure bacterial cultures. In this paper we report the metabolic formation of several new thiazole ring-opened metabolites.
Materials and methods Chemicals Levamisole hydrochloride (1) was purchased from Aldrich Chem. Co. (Milwaukee, WI, U S A ) . 2~>xo-3-(2-mercaptoethyl)-5-phenylimidazolelidine (OMPI, 2) and its disulphide ((OMPI),, 3) were prepared by the reaction of levamisole with glyoxal by modified procedures of Van Belle and Janssen (1979). All other chemicals were of analytical grade and the solvents were of h.p.1.c. grade.
Levamisole metabolism by intestinal bacteria
739
All bacteriological media and supplements used in this study were prereduced and anaerobically sterilized following the methods described in the VPI Anaerobe Laboratory Manual (Holdeman et al. 1977).
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Instruments Specific rotations ([MID) were measured with a Perkin-Elmer 241 polarimeter. Infrared (i.r.) spectra were recorded with a Perkin-Elmer 710B infrared spectrophotometer. Ultraviolet (u.v.) spectra were recorded with a Beckman DU-70 spectrophotometer. Proton and carbon- 13 nuclear magnetic resonance ('H-n.m.r. and 13C-n.m.r.)spectra were obtained with a Varian Unity 400 ('H, 400 MHz, I3C, 100 MHz) and a Bruker WP-270 ('H, 270MHz, I3C, 68 MHz) spectrometer. Chemical shifts are given as 6 values relative to the signal of internal standard tetramethylsilane (TMS). Mass spectra were determined with a V G 7070 instrument operating in the electron ionization (EI), chemical ionization (CI, reagent gas, isobutane), or fast atom bombardment (FAB, matrix, 1-thioglycerol) mode. Chromatography Merck Kieselgel 60F254(silica) plates were used for t.1.c. and preparative t.1.c.; spots were detected under a U.V.lamp. Column chromatography was carried out on Merck silica gel (230-400 mesh). H.p.1.c. analysis was performed with a Waters 501 solvent delivery system equipped with a Biorad 1306 U . V . monitor under the following conditions: column, RCM Nova-Pak C,, Cartridge (4pm. 8 m m int. diam. x l00mm) in a Waters Radical Compression Module; mobile phase, acetonitrile-0.1 M ammonium acetate (46 : 54); flow rate, 1.3ml/min; pressure, c. 8280 kPa; detection at 245 nm with a range of 0.04 a.u.f.s. Under these conditions calibration curves for quantification of levamisole (l),levametabol-I (4) and levametabol-I1 ( 5 ) were prepared by using standard samples, and they were linear in the range of 5-1 20 pg/ml. Incubation of levamisole wiih human faecal bacterial mixtures, Bacteroides thetaiotaomicron or B. uniformis Human faeces from five non-smoking healthy donors (four male, one female) were anaerobically pooled and mixed under argon as previously described (Bashir et al. 1987 a). T h e pooled faeces (5 g) were diluted 20-fold in pre-reduced, anaerobically sterilized dilution fluid, and 0.5 ml was inoculated into 1OOOml of prereduced brain heart infusion (BHI) broth (Difco Lab, Detroit, M I , USA). T h e broth was incubated anaerobically for 18 h at 37°C. T o the broth was added levamisole hydrochloride (700mg) dissolved in distilled water (2 ml). The supplemented culture was incubated anaerobically for 48 h at 37°C and then extracted with water-saturated ethyl acetate (three times, 1000ml each). The combined organic layer was washed with 5% NaCl solution and concentrated to give a residue. A small portion of the residue (70mg) was immediately applied onto preparative t.1.c. plates, which were then developed in a solvent mixture of chloroform-zthyl acetate-methanol (10 : 1 : 0.2, by vol.). An unstable metabolite (levametabolI, 4, R, 0.70, 4mg) (figure l), and a stable metabolite (levametabol-11, 5,R , 0.61, 11 mg) were isolated. T h e rest of the residue (1.1 g ) was chromagographed on silica gel; the column was washed thoroughly with hexane and hexane-methylene dichloride (1 : 1, v/v), and eluted with methylene dichloride. This eluant gave a chromatographically homogeneous major metabolite (5,145mg, yield 21%) and a crude minor metabolite. The fraction containing the latter metabolite was further purified by preparative t.1.c. in chloroform-ethyl acetate-methanol (10: 3 : 0.5, by vol.) to afford pure metabolite (levametabol-111, 6 , 12mg, 1.8%). By using the procedure described above, we repeated the experiment with selected pure bacterial cultures. Levamisole hydrochloride (25&500 mg) was incubated with the BHI broths of Bacteroides thetaiotaomicron VPI 5482 and B. uniformis VPI TI-1. Preparative amounts of metabolites 5 (yield range, 7.3-10.5%) and 6 (yield range, 05-09%) were isolated from the metabolic mixtures; in addition, unchanged levamisole (yield range, 18.5-25.0%) was recovered from the same mixtures. Levametabol-I ( 4 ) A colourless oil, [a]2SD+22.2 (c=0.09, chloroform); U.V.I,,, (methanol) nm 242 (log E 2.80); high resolution mass spectrum: Found 238.0765, Calc. for [MI+,C, IH14N,02S,238.0776; EI mass spectrum m / z (relative intensity): 238(M+, 32), 206(22), 205(M+ -SH, loo), 146(80), 105(35); for 'H-n.m.r., see table 1 and figure 3; for "C-n.m.r., see table 2. Hydrogenation of metabolite 4. A stirred heterogeneous mixture of metabolite 4 (4mg) and 10% Pd/C (40 mg) in methanol (5 ml) was hydrogenated at room temp. for 18 h. T h e catalyst was filtered off, and the filtrate was concentrated in vacuo. Immediate purification by preparative t.1.c. in chloroform-zthyl acetateemethanol (10 : 1 : 0.2, by vol.) yielded an oily product (1 mg), which was identified as OMPI (2) by t.1.c. and by comparison of its 'H-n.m.r. spectrum (table 1) with that of an authentic sample. Levametabol-II ( 5 ) A colourless oil, [a]6'+43.3 (c=0.24, chloroform); U.V.I,,, (methanol) nm 242 (log E 4.33); i.r. v,,, (KBr) cm-': 3430 (br OH), 1634(C=O), 1491(arom C X ) , 1450, 1265, 1155; CI mass spectrum m / z (relative intensity): 476(M+ 2H, 0.4), 409(0.7), 408(0.5), 295(35), 281(43), 240(92), 239(100), 207(85), 203(72); for 'H-n.m.r., see table 1 and figure 4; for '3C-n.m.r., see table 2.
+
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740
Y.-2. Shu et al.
Acetylation of metabolite 5. To a solution of 5 (8 mg) in pyridine (0.8 ml) acetic anhydride (0.5 ml) was added dropwise at 0°C. The mixture was stirred at 70°C for 5 h, and then methanol (50ml) was added, and the solvent was removed in vacuo. Purification by'preparative t.1.c. in chloroform-ethyl acetate (5 : 1, v / v ) afforded acetates 7 (2.5mg) and 8 (l.Omg), and unreacted 5 (3mg). Compound 7, a colourless oil, 'Hn.m.r. (270MHz, CDCI,) 6 : 2.82(3H, s , CH,-CO-), 2.95-3.10(4H, m, 7-H x 2,7'-H x 2), 3.55 and 4.18 3.62and4.18(each l H , d d a n d t , J = Y . 9 , (each l H , d d a n d t , J=9.9,2.9HzandJ=9.9Hz,4-I-I,and4-Hb), 7.6 Hz, and J = 9.9 Hz, $-Ha and 4'-H,), 3.85 and 3.98 (each 1H, dt, J = 13.6.6.8 Hz, &-Haand 6'-H,), 3.99 and 4.04 (each l H , dt, J=9.6, 6.8Hz, 6-H, and 6-H,), 4.94(1H, dd, J = 9 . 9 , 7.6I-12, 5'..H), 5.58(1H, dd, J=9.9, 2.9Hz, 5-H), 6.08(1 H,brs,-N-Ou), 7.2&7.45(10H, m, arom. H); CI mass spectrum m / z (relative intensity): 281(92), 239(95), 205(100), 173(42), 145(40), 104(58). Compound 8, a colourless oil, 'H-n.m.r. (270 MHz, CDCI,) 6: 2.82(6H, s , CH,-CO- x 2), 3.00 and 3.03 (each 2H, dt, 13.1, 6.7 Hz, 7H, x 2, 7-H, x 2), 3.54 and 4.16 (each 2H, dd and t, J = 10.2, 3.0 Hz and J = 10.2 Hz, 4-Ha x 2 and 4I{,, x 2). 3.98 and 4.04 (each 2H, dt, J=9.6,6.7 Hz, 6-Ha x 2 and 6-H, x 2), 558(2H,dd, J = 102.3.0 Hz, 5H x 2), 7.20-7.45(10 H, m, arom. H); CI massspectrumm/z(relative intensity): 281(22), 249(17), 205(85), 189(40), 173(35), SS(l00); EI mass spectrum m / z (relative intensity): 279(32), 247(63), 237(100), 205(50), 173(35), 148(72), 146(75). Reduction of metabolite 5 with NaBH,. Compound 5 ( 5 mg) was dissolved in methylene dichloridemethanol (2 : 1, v/v, 3 ml), and NaBH, (15 mg) was added. The mixture was refluxed for 4 h in an argon atmosphere, and then poured into ice water and extracted with methylene dichloride, and the organic layer was concentrated. Purification by preparative t.1.c. in chloroform-ethyl acetate (5 : 1, v / v ) gave an oily compound (2mg), which was characterized as levametabol-I (4) by 'H-n.m.r. analysis (table 1). Reduction of metabolite 5 with aqueous TiCl,. T o a solution of 5 (1 5 mg, 0.032 mmol) in methanol-1 M sodium acetate (4 : 1, v/v, 10ml), a freshly prepared 20% w/v TiCI, solution ( 0 4 ml, 0 5 8 mmol) was added dropwise. The mixture was stirred in an argon atmosphere for 8 h at room temp. and then hasified with 2 M NaOII to pII 9, and extracted with ethyl acetate. T h e ethyl acetate layer was dried over anhydr. Na,SO, and evaporated, and the crude product was immediately purified by preparative t.1.c. in chloroform-ethyl acetate (5 : 1, v / v ) to yield a pure compound. T h e compound was identified as levametabol-I (4) on the basis of 'H-n.m.r. (table l), '3C-n.m.r. (table 2) and EI mass spectral data. Levametabol-III ( 6 ) A colourless oil, [a];'+ 50.5(c =0.19, chloroform); U.V.I,,, (methanol) nm 244 (log E 4.58); FAR mass spectrum m / z (relative intensity): 45Y(M+ + H , 2.7), 410(3.3), 391(2.6), 278(50), 239(100), 206(33), 203(72); CI mass spectrum m / z (relative intensity): 239(42), 223(92), 205(100), 189(20), 175(85), 148(40), 132(55), lOS(35); for 'II-n.m.r., see table 1 and figure 5; for I3C-n.m.r., see table 2. Reduction of metabolite 6 with NaBH,. Compound 6 (8 mg) was reduced with NaBH, as described for compound 5 . After work-up, immediate purification by prep-t.1.c. (CHCI,/EtOAc/MeOH, 10 : 1 :0.2) gave two major products (c. 2mg each), which were identified as kvametdbol-I (4) and OMPI (2), respectively,by comparing 'H-n.m.r. spectra with those of authentic samples. Two minor products in the reaction mixture were identified as levametabol-I1 ( 5 ) and (OMPI), (3) based on co-t.1.c. analysis. Screening of intestinal bacteria capable of transforming levamisole to levametabol-II ( 5 ) The 28 bacterial strains used in the study were obtained from the culture collection of the Department of Anaerobic Microbiology; the cultures were stored in chopped meat broth at room temperature. Each culturewas inoculated intoRH1 broth(l0ml)andgrownanerobicallyfor 1 8 h a t 37°C. Analiquot (1OOp1) o f levamisole hydrochloride (3 mg, 12.47pmol) in sterilized water was added to the bacterial culture. Incubation was carried out under a CO, atmosphere at 37°C for 48 h. A portion (2 ml) of the metabolic mixture was loaded onto a Sep-Ilak C,, cartridge (Waters Assoc.), which had been conditioned with methanol (10ml) and water (20ml). T h e cartridge was washed with water (20ml), and levamisole and its metabolites were eluted from the cartridge with methanol-water (8 : 2, v/v, 10 ml). A portion (1Opl of the resultant eluate was injected directly into the h.p.1.c. system described above. Metabolic time-course of levamisole in human faecal bacterial mixture A n 18h BHI broth seeded with human faeces was divided into tuhes (10ml each) under a stream of CO, and supplemented with levamisole hydrochloride (6mg in loop1 of sterilized water). The supplemented bacterial broths were incubated anaerobically at 37°C. After periods of 1 h, 3 h, 6 h, 12 h, 24 h, 36 h and 48 h, three of the tubes were chilled in an acetone-dry ice hath. The metabolic mixtures were thawed, processed and analysed by h.p.1.c. in the same fashion as described in the above bacterial screening experiment.
Levamisole metabolism by intestinal bacteria
741
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Results Analysis of Levamisole metabolites formed by human intestinal bacteria Preliminary t.1.c. analysis of a 36 h levamisole (1) metabolic mixture incubated with human faecal bacteria indicated the transformation of levamisole into three metabolites. These metabolites were not produced in a control experiment in which levamisole was incubated with BHI broth alone. T h e compounds were designated in order of their R, values as levametabols I (RF0-70), I1 (RF0.61) and 111 ( R , 024). Two reported metabolites (Van Belle and Janssen, 1979) of levamisole, OMPI (RF0.27) and (OMPI), (RF015), were not observed. In order to examine metabolite formation quantitatively, a convenient sample preparation procedure and an h.p.1.c. method were established. Prior to the h.p.1.c. analysis, bacterial samples were cleaned up and prepared by using Sep-Pak C,, cartridges rather than by a solvent extraction method. T h e recoveries of levamisole (l),levametabol-I (4), and levametabol-I1 ( 5 ) through this clean-up process were 95%, 92% and SO%, respectively (data not shown). As shown in figure 2 ( c ) , the chromatographic conditions provided a good separation of the six compounds OMPI (peak 2), levamisole (peak I), (OMPI, (peak 3), levametabol-111 (peak 6), levametabol-I (peak 4) and levametabol-I1 (peak 5). Four peaks corresponding to substrate and metabolites were observed in the chromatogram of the levamisole metabolic mixture (figure 2 ( b ) ) .Their retention times were identical with those of authentic levamisole (peak l), levametabol-111 (peak 6), levametabol-I (peak 4) and levametabol-I I (peak S), respectively. A bacterial component peak (peak 7) detected in this chromatogram was isolated and then identified as indole by spectroscopic analysis. However, no metabolite peaks except an unchanged substrate peak (peak 1) were seen in the incubation mixture of levamisole with BHI broth (figure 2 ( a ) ) .In agreement with the t.1.c. analysis results, no detectable amounts of OMPI or (OMPI), were present in the bacterial metabolic mixture.
5 9
id
4 2
si
L
Time (min) (4 Figure 2 .
Time (min) (b)
Time (min) (4
II.p.1.c. chromatograms of levamisole metabolites formed by human intestinal bacteria.
( a ) Sample of levamisole incubated with BHI broth. ( b ) Sample of levamisole incubated with human intestinal bacterial mixtures. (c) Mixture of standard samples. 1, Levamisole; 2, OMPI; 3, (OMPI),; 4, levametabol-I; 5, levametabol-11; 6, levametabol-111;7, indole.
Y.-2. Shu et al.
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742
Characterization of metabolites Levametabol-I, metabolite 4, a colourless oil, was carefully isolated by preparative t.1.c. from the incubated bacterial mixtures of levamisole. T h e metabolite decomposed rapidly after solvent (ethyl acetate) extraction even if the extract was maintained at 4°C rather than at room temperature. However, when isolated in pure form and stored in the absence of air, the compound was relatively stable. Levametabol-I showed a chemical composition of C, ,H1,N20,S by high resolution mass spectrometry, indicating that it possesses an extra oxygen atom compared to OMPI (2). This metabolite exhibited a U.V. absorption maximum at 242 nm, which is quite different from that of the parent drug levamisole (A,,, 226 nm). T h e 'Hn.m.r. (figure 3 , table I ) and 13C-n.m.r. spectra (table 2) of metabolite 4 differed in overall appearance from those of levamisole except for the signal due to the phenyl moiety, indicating that the structural changes through bacterial metabolism take place in the tetrahydroimidazothiazole ring system. T h e spectra on the other hand showed an identical number of methylene, methine and carbonyl groups to those of OMPI (2). Similarly to that of OMPI, the 'H-n.m.r. spectrum of metabolite 4 displayed a characteristic triplet (J= 7-1 Hz) at 6 1.50 due to a DzO exchangeable proton, which showed spin correlation with the 7-methylene protons (6 2.82, q , J=7.1 Hz) in a 'H-double resonance experiment, indicative of the presence of a free thiol group. That metabolite 4 was a free thiol-bearing compound was supported by mass spectral data; the base peak appearing at mjz 205 was ascribed by the loss of an -SH fragment from the molecular ion at m / z 238. T h e most distinct difference between the corresponding 13C-n.m.r. signals of metabolite 4 and OMPI (2) was observed at the C-2 carbonyl carbon (6 183.4 in 4 and 6 161-8 in 2). It is also important to note that the coupling of 5-H with the vicinal 1 - N H proton ( J = 1.5 Hz) in OMPI (2)was absent in metabolite 4. This indicated that the extra hydroxy group in the levametabol-I molecule might be located on N-I to form a hydroxamic acid partial structure. This was confirmed by chemical conversion; upon catalytic
X
I
I
/-
__r_
I
-,
7 ,
6 Figure 3 .
- T
5
-4
L
J
T-7,
3
,
,
,
2
,
,
,
,
1
,
,
(PPm)
'H-n rn r spectrum of levarnetabol-I (4) (400MHz, in CDC13).
, -1
0
-
Levamisole metabolism by intestinal bacteria
743
hydrogenation metabolite 4 was transformed into the lactam OMPI (2). T h e structure of levametabol-I was therefore established as 1-hydroxy-2-oxo-3-(2mercaptoethy1)-5-phenyl-imidazolidine(4). Levametabol-I1 (5) was isolated as the second major bacterial' metabolite of levamisole. T h e chemical ionization mass spectrum exhibited a quasi molecular ion (M 2H') at m / z 476 and a diagnostic base ion peak at m / z 239 resulting from the fragmentation of the quasi molecular ion into one half, implying that levametabol-I I ( 5 ) might have a symmetric dimeric structure. T h e presence of two homogeneous units in the molecule was also clear from the 'H- and 'jC-n.m.r. spectra (figure 4, tables 1, 2), since only 'H- and 13C-signals due to one monomeric portion were observed. Acetylation of metabolite 5 yielded the monoacetate 7 and the diacetate 8, demonstrating that metabolite 5 has two hydroxyl groups. Comparison of n.m.r. spectral data of metabolite 5 with metabolite 4 showed a close similarity except for the C-7 and C-8 methylene signals; in contrast with the resonances of C-7 (6 22.4)
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+
Table 1. 'H-n.m.r. spectral data of the levametabols and related compounds (p.p.m., in CDCI,).
4-Ha 4-Hb
LevarnetabolI (4)
LevametabolI1 (5)
Levametabol111 (6)
3.63, dd (9.9, 7.4) 4.18, t (9.9)
3.60, dd (9.9, 7.6) 4.19, t (9.9)
494, dd (9.9, 7.5)
OMPI
(OWI),
(2)
13)
3.61, dd (9.9, 7.6) 420, t (9.9) 3.29, dd (8.5, 7.6) 3.88, t (8.5)
3.28, dd (8.6, 7.4) 3.87, t (8.6)
3.26, dd (8.6, 7.5) 3.87, t (8.6)
4.94, dd (9.9, 7.8)
4.93, dd (9.9, 7.6) 4.79, ddd (8.5, 7.6, 1.5)
4.77, ddd (8.6, 7.4, 1.5)
4.76, ddd (8.6, 7.5, 1.5)
3.78, d t (14.0, 7.1) 3.83, d t (14.0, 7.1)
3.82, d t (13.9, 6.8) 3.98, dt (13.9, 6.8)
3.85, d t (14.1, 7.0) 3.99, d t (14.1, 7.0) 3.47, d t (140, 6.7) 3.64, d t (14.0, 6.7)
3.33, d t (13.9, 7.1) 3.45, d t (13.9, 7.1)
3.42, d t (13.8, 6.7) 3.60, d t (13.8, 6.7)
2.82, q (7.1) 2.82, q (7.1)
2.98, d t (13.6, 6.8) 3.02, d t (13.6, 9.8)
2.67, q (7.1) 2.67, q (7.1)
2.85, t (6.7) 2.85, t (6.7)
7.30-7.45, m 6.17, br s
7.28-7.45, m 6.48, b r s
2.98, d t (13.9, 7.0) 3.03, d t (13.9, 7.0) 2.89, t (6.7) 2.89, t (6.7) 7.30-7.42, m 6.08, b r s 4.68, br s
7.25-7.50, m
725-7.50, m
5 40, br s 1.42, t (7.1)
5 17, br n
4-Ha 4-Hb 5-H 5'-H 6-Ha
6-IIb G-Ha 6'-Hb 7-Ha 7-Hb 7'-Ha 7'-Hh Ph-H -N-OH -NII -SH
1.50, t (7.1)
Coupling constants ( J , Hz) are shown in parentheses.
Y.-2. Shu
744 Table 2.
al.
I3C-n.m.r. specttral data of the levametabols and related compounds (p.p.m., in CDCI,). 1,evametabol-
I
Carbon no.
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et
Levametabol-
Levametabol-
I1 (5)
(4)
111 (6)
(2)
(OMPI), (3)
OMPI
C-2
183.4(s)
183.4(s)
183.4(s) 161.0(s)
161.8(s)
161.2(s)
C-4 c-4'
58.0(t)
58.3(t)
58,2(t) 54,2(t)
54.0(t)
54-2(t)
C-5 C-5'
57.4(d)
57.5(d)
57.4(d) 53,9(d)
53.7(d)
53.8(d)
C-6 C-6'
49,6(t)
45.9(t)
45.9(t) 42.6(t)
46.6(t)
42.6(t)
c-7
22,4(t)
35+3(t)
35.8(t) 36,8(t)
22.8(t)
36.8(t)
C-1" C-1"'
140.1(s)
140.1(s)
14 0 3s) 141.5(s)
141.5(s)
141.5(s )
C-2", 6" C.2"', t,,,!
126,2(d)
126.1 (d)
126.0(d) 126.1(d)
1264(d)
126.1(d)
C-3", 5" C-3"', 5"'
129.2(d)
129.l(d)
129.1(d) 128.9(d)
128,9(d)
128,9(d)
c-4"
128,7(d)
128,7(d)
128,2(d) 128.6(d)
128.2(d)
128.2(d)
(2-7'
(2-4"'
Abbreviations given in parentheses indicate the signal patterns observed in the off-resonance decoupling technique, which were confirmed by I N E P T (insensitive nuclei enhanced by polarization transfer) experiment. s, Singlet; d, doublet; t, triplet. The assignment of phenyl moiety carbons is bascd on the calculation method (Stothers 1972) of substituted benzenes.
r
TMS
I
X
7--,-v-
8
-, r---
m7.,-r--l----l
3
,
,
~
6
Figure 4
'H-n m r. spectrum of levametabol-I1 ( 5 ) (400MIIz, in CDCI,)
5
4
2
1
,
(PPm)
7
,
,
0
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Levamisole metabolism by intestinal bacteria
745
and C-6 (6 49.6) in metabolite 4, the corresponding signals in metabolite 5 occurred at 6 3 5 4 (C-7) and 6 45.9 (C-6), respectively. Similar shifts of the signals for C-7 and C-6 were observed between the monomeric compound OMPI (2) and dimeric compound (OMPI), (3) (table 2). These findings indicated that levametabol-I1 (5) may consist of two monomeric units of levametabol-I (4), and that dimerization is most likely through a disulphide linkage. Final confirmation of the structure of metabolite 5 was achieved by chemical interconversion; reduction of metabolite 5 with NaBH, gave compound 4, while air oxidation of metabolite 4 yielded the dimeric product 5 (data not shown). Structure levametabol-I1 (5) was thus conclusively determined to be the disulphide of levametabol-I (4). Mattingly and Miller (1980) have reported a mild method for reduction of the N-0 bond in hydroxamic acids with aqueous titanium trichloride. Unfortunately, the reduction of levametabol-I1 (5) with the same reagent predominantly afforded the monomer 4 rather than the N-hydroxy reduced products, i.e. (OMPI), (3) or OMPI (2). After a prolonged reaction period, traces of compounds 3 and 2 were observed on t.l.c., but it proved impossible to isolate these minor products. Similarly, the attempted conversion of metabolite 5 into compounds 3 or 2 with Zn5% HC1 (data not shown) led to the formation of compound 4 as major product. Levametabol-111 (6) appeared to be a minor metabolite. T h e positive ion FAB mass spectrum of 6 gave a quasi molecular ion (MH') at m / z 459, and the CI mass spectrum demonstrated two prominent fragment ions at m l z 239 and m l z 223, characteristic of the levametabol-I (4) and OMPI (2) skeletons, respectively. T h e 'H- and 13C-n.m.r. spectra (figure 5, tables 1, 2) comprised pairs of signals clearly assignable to the monomeric portions in the levametabol-I1 (5) and (OMPI), (3), respectively. Moreover, when treated with NaBH,, levametabol-111 (6) released compounds 4 and 2. The above evidence indicates that levametabol-111 (6) is a heterogeneous disulphide consisting of monomeric units of levametabol-I (4) and
OMPI (2).
2HCb
x
rL-
,
8
--y-,, ,, , I , ,
i
,
I
'
"
1 (PPd
7
6
Figure 5 .
'H-n.m.r. spectrum of levametabol-111 (6) (400MHz, in CDCI,).
5
4
3
2
'
0
746
Y.-2. Shu et al.
Table 3. The capacity of various bacterial strains to transform levamisole to levametabol-I1 under anaerobic conditions. Levametabol-I I formed (mol/ml)
”/, Transformation
BHI broth alone Human bacterial mixture
0 0.459
0 73.6
Bacteroides distasonis VPI T3-25 B. distasonis VPI 17297 B. fragilis VPI 3390 B. fragilis VPI B2-22 B. ovatus VPI 3049 B . ovatus VPI GMH B. thetuiotaomicron VPI 5482 R. thetaiotaomicron VPI 5482 (autoclaved) B. thetaiotaomicron VI’I 5482 (aerobic) R. thetaiotaomicron VPI J19-34B R. uniformis VPI 0909 B.uniformis VPI T1-1 B. erulgatus VPI 5710 Rarteroides group VPI 3452A 2308
0.246 0.189 0.183 0,097 0.046 0.160 0.1 90 0.032 0 0,029 0115 0.201 0.096 0.097
39.4 30.3 29.4 15.5 7.4 25.7 30.5 5.1 0 4.6 18.4 32.2 15.4 15.6
Clostridium barkeri VPI 5359-1 C‘. radaveris VPI 2718 C. dostridiforme VPI 0316 C . dostridiforme VPI 12706 C,’. dificile VPI 10463 C. innocuum VPI A14-39A C. innocuum VPI 1614-1 C. oroticum VPI 056JA C. sordellii VPI 6356 C. sordellii VPI 9048 C . sphenoides VPI 10627 C. sporogenes VPI 8654E C . septicum VPI 2017 C . septicum VPI 1526
0.092 0.075 0.143 0.166 0.01 1 0.075 0073 0.097 0.1 17 0.344 0.029 0.241 0.01 1 0.010
14.8 12.0 22.9 26.7 1.8 12.0 11.7 15.6 18.8 55.2 4.6 38.4 1.8 1.6
E . moniliforme VPI 12566
0.069
11.1
Peptostreptococcus productus VPI 4299
0.057
9.1
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Bacteria
-
Each tube (10ml) contained 3 mg of levamisole hydrochloride (1,247 pmol/ml), and the incubation was carried out at 37°C for 48 h.
Screening of intestinal bacteria capable of transforming levamisole to the stable metabolite levametabol-11 ( 5 ) T h e levamisole-metabolizing activity of various intestinal strains was examined by quantitative h.p.1.c. analysis of the stable major metabolite levametabol-I1 ( 5 ) formed from the incubation. All 28 strains examined showed metabolic ability at different levels (table 3). Strong metabolizers having more than 20% conversion percentages included the Bacteroides and Clostridium spp. Bacterial mixtures prepared from human faeces demonstrated much greater transformation activity (74%)than any of the individual pure strain cultures. In addition, data (table 3) from the experiment with a common anaerobe Bacteroides thetaiotaomicron VPI 5482 show appreciable transforming activity (30.5%) after an anaerobic incubation, but no formation of metabolite 5 after an aerobic incubation. Surprisingly, a low level of the activity (5.1%) was found even with the autoclaved cells.
Levamisole metabolism by intestinal bacteria
z
h
747
1
2*5
'= 0
Ea
W
C
0
. I
Y
2
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Y
E
Q)
u
E
u" 0
6
12
18
24
30
36
42
48
Time of incubation (h) Figure 6. Time-course of levamisole metabolism by human intestinal bacterial mixtures. -0-, Levamisole; -0-, levametabol-11; ...., sum of levametabol-I and levametabol-I1 x 2.
Time-course of levamisole metabolism by human faecal bacterial mixture Figure 6 shows the time-course of transformation of levamisole by human faecal bacterial mixtures into its stable major metabolite, levametabol-I1 ( 5 ) , during anaerobic incubation. T h e formation of metabolite 5 was observed 3 h after the start of incubation, and its amount increased gradually with a corresponding decrease of levamisole. Levametabol-I (4) was not observed until 24 h, perhaps because of its lability and easy dimerization; after 24 h it appeared to accumulate in small amounts, and the other minor metabolite, levametabol-I11 (6), gave tiny peaks in the h.p.1.c. chromatogram. Unfortunately, the low concentrations of metabolites 4 and 6 did not permit accurate quantification by the h.p.1.c. method. Since compound 5 could be considered to be formed through the dimerization of levametabol-I (4), the net amount of metabolite 4 produced at 48 h of incubation was expected to be about 70% of added substrate, as represented by the dotted line in figure 6.
Discussion T h e present study demonstrates that the anti-colon cancer drug levamisole is converted to three thiazole ring-opened metabolites by human faecal bacteria. For the first time hydroxamic lactam derivatives were isolated and characterized as levamisole metabolites. T h e major metabolites obtained from the incubated bacterial mixtures are levametabol-I (4) and its disulphide (levametabol-11, S), which could be explained as an artifact of metabolite 4 resulting from spontaneous oxidation during processing. We were unable to obtain O M P I (2),a reported major active metabolite of levamisole present in animal plasma and urine (Janssen 1976, Graziani and De Martin 1977, Koyama et al. 1983), from our in vitro system, but we isolated a minor bacterial metabolite levametabol-I11 (6) which has OMPI (2) as a structural unit. These observations indicate that the formation of hydroxamic lactam-type metabolites by intestinal bacteria occurs preferentially to that of simple
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lactam derivatives. Since no conversion from compounds 415 to 213, or vice versa, was observed either by the bacterial incubation or by air oxidation, the formations of levametabol-I (4) and O M P I ( 2 ) are likely to proceed by different pathways. Although OMPI has been claimed as a major active metabolite of levamisole for many years, the experimental data describing its isolation from biological fluids and its structural characterization have not yet been published. In the present study we carried out various detailed spectroscopic comparisons between OMPI ( 2 ) , levametabol-I (4) and their analogues to confirm the structure of the new metabolites. All three bacterial metabolites obtained in the present investigation are dextrorotatory isomers: levametabol-I (4, [u]i5 22.2 (c = 0.09, CHCI,)), levametabol-I1 (5, [u]i5+43.3 (c=0.24, CHCI,)) and levametabol-111 (6, [c(]i5 + 50.5 (c = 0.19, CHCI,)). Similarly, OMPI ( 2 , 25.6 (c = 0.32, CHCl,)) and (OMPI), ( 3 , [u];’ +46.5 (c=0.23, CHCl,)) show dextrorotory properties. Since the racemization of asymmetric carbon C-5 is unlikely through the ring-opening process, the levametabols thus are most likely to have the same 5s-configuration as that in the parent levamisole. Compared with the structures of OMPI ( 2 ) and (OMPI), ( 3 ) , the presence of extra hydroxyl group(s) on N-1 of levametabol-I (4) and levametabol-II(5) would be expected to increase their polarity. Nevertheless, higher R, values for compounds 4 ( R , 0.70) and 5 (R, 0.61) were observed on normal phase t.1.c. than for compounds 2 ( R , 0.27) and 3 (R, 0.1 5 ) . This is probably due to intramolecular hydrogen bonding between the N-1 -hydroxyl hydrogen and the 2-carbonyl oxygen in compounds 4 and 5, leading to a greatly decreased polarity for the latter compounds. Investigations on the formation of O M P I from levamisole (Van Belle and Janssen 1979) revealed that some u-ketoaldehydes such as methylglyoxal, which may be formed metabolically from the common cell constituent glyceraldehyde-3phosphate, specifically catalyse the reaction. It is thus suggested that such nonenzymic reactions involving small molecules play an important role in the in vivo metabolism of levamisole into OMPI. As the present study has shown, the formation of levametabol-I1 from levamisole was not observed in BHI broth alone, but was observed in autoclaved cultures of Bacteroides thetaiotaomicron and faecal bacterial mixtures (data not shown), albeit at a much decreased level. This fact indicates that some bacterial cell constituent(s), most likely a heat-stable small molecule, accounts at least in part for the formation of levametabol derivatives. There was however no convincing evidence for the formation of levametabols when glyceraldehyde, methylglyoxal or glyoxal were incubated with levamisole in BHI broth. Further studies on the characterization of such ‘key’ small molecule species are under investigation. It is interesting to note that the formation of the hydroxamic lactam functionality from levamisole must involve an oxidation step, despite the anaerobic conditions required for the bacterial viability. T h e mechanism for this oxidation is at present unknown. One possibility is that the reaction might require the reduced form cofactor or the reduced form ‘key’ small molecule described above. Although the examples of oxidationldehydrogenationconversion mediated by anaerobic bacteria are relatively few, one precedent we observed in our series of studies on the anaerobic metabolism of food mutagens is that an imidazoquinoline-type dietary carcinogen, IQ, was hydrated and then oxidized to yield 7-hydroxy IQ (Bashir et al. 1987 a, b). This showed that intestinal bacteria may have a wide variety of unexplored enzyme sources that catalyse reactions beyond our current conventional knowledge.
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T h e recent discovery of unique anti-colon cancer efficacy by levamisole plus 5fluorouracil is beginning to received much attention. Although we could not detect direct chemical interaction of these two drugs in our incubation system which mimics the human colon environment, our observation that levamisole is metabolized in vitro by human intestinal flora to give the major metabolite levametabol-I, a close structural analogue to OMPI, implies that this metabolite may be formed in the human colon. This would further raise some very important questions, i.e. whether levametabol-I possesses in vitro or in vivo anti-colon cancer activity, and if so, whether its activity is even stronger than levamisole. Since OMPI has demonstrated enhanced immunomodulating and other biological activities as compared with levamisole, and has been proposed as the active drug form of levamisole (Amery and Gough 1981), we believe that detailed investigations on the in vivo formation of levametabol-I, particularly in the colon, and its related pharmacological activities could be of great significance to our understanding of the anti-colon cancer effect of levamisole.
Acknowledgements Financial support for this work was provided by the National Cancer Institute, National Institutes of Health, through grant number CA40821.
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