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Mycologia, 94(1), 2002, pp. 1–5.
᭧ 2002 by The Mycological Society of America, Lawrence, KS 66044-8897Issued 28 January 2002 Formation of conjugates from ciprofloxacin and norfloxacin in cultures of
Trichoderma viride
desethylene-N-acetyl, and N-formyl metabolites as well as to metabolites in which an amino group has replaced the piperazine ring (Parshikov et al 2001b).
Recently, the formation of two new products in cul- tures of Trichoderma viride was noted after dosing with ciprofloxacin and norfloxacin. The strain had been isolated during the screening of soil fungi for National Center for Toxicological Research, Food and the ability to metabolize fluoroquinolones. Both cip- Drug Administration, Jefferson, Arkansas 72079-9502 rofloxacin and norfloxacin appeared to be conjugat-ed with an unstable secondary metabolite, which hadbeen previously reported in other Trichoderma spp.
tibacterial fluoroquinolone drugs, ciprofloxacin andnorfloxacin, was observed in cultures of Trichodermaviride that had been grown in sucrose-peptone broth and extracted 16 d after dosing with the drugs. Both Strain T-58, isolated from a fruiting body of Trametes versi- conjugates were purified by high-performance liquid color collected in a forest in Jefferson County, Arkansas, was chromatography and found to be optically active.
identified as Trichoderma viride by Dr. S. N. Lekomtseva, They were identified by mass and proton nuclear Department of Mycology and Algology, Moscow State Uni- magnetic resonance spectra as 4-hydroxy-3-oxo-4-vi- versity, Moscow, Russia. Triplicate experimental cultures in nylcyclopent-1-enyl ciprofloxacin and 4-hydroxy-3- flasks containing sucrose-peptone broth (Parshikov et al oxo-4-vinylcyclopent-1-enyl norfloxacin. The transfor- 1999) were incubated at 28 C with rotary shaking at 180 mation of veterinary fluoroquinolones in the pres- rpm. After 2 d, the cultures were dosed with 300 ␮M cip- ence of fungi may have ecological significance.
rofloxacin or 313 ␮M norfloxacin (Parshikov et al 2001b); Key Words:
in one experiment, 300 ␮M piperidine hydrochloride (Al-drich Chemical Co.) was substituted. The dosed cultures,control cultures, and noninoculated controls were incubat-ed with shaking for another 16 d.
After harvesting, cultures were extracted with ethyl ace- tate (Parshikov et al 1999) and the residues were dissolved At present, limited information is available about the in methanol: acetonitrile: acetic acid (10:10:2) for analysis.
ability of fungi to transform fluoroquinolones, such Compounds were purified by collecting the peaks separated as the widely used antibacterial agents ciprofloxacin by high-performance liquid chromatography (HPLC), us- (FIG. 1A) and norfloxacin (FIG. 1B). Several fungi ing the gradient described previously (Parshikov et al transform the fluoroquinolones enrofloxacin, dano- 2001a), and the relative concentrations were estimated floxacin, and sarafloxacin to various metabolites from the peak areas at 280 nm. Circular dichroism spectra (Martens et al 1996, Chen et al 1997, Wetzstein et al were obtained in methanol with a Jasco model 500A spec- 1997, Parshikov et al 2000, 2001a). Mucor ramanni- anus transforms ciprofloxacin to N-acetylciprofloxa- Direct exposure probe (DEP) mass spectrometry (MS) experiments were performed as previously described (Par- cin (Parshikov et al 1999). Gloeophyllum striatum and shikov et al 1999), using the single quadrupole (Q1) and other wood-decaying basidiomycetes convert cipro- product-ion modes. The ion-source pressure for chemical floxacin to at least 16 metabolites, including CO2 ionization was 5.0–5.5 Torr, uncorrected. Product ions were (Wetzstein et al 1999). Pestalotiopsis guepini trans- generated with a collision-cell pressure of 0.5 mTorr of ar- forms both ciprofloxacin and norfloxacin to N-acetyl, gon and a collision energy of 100 eV. Electrospray ioniza-tion (ESI) MS experiments (Parshikov et al 1999) were per- Accepted for publication June 28, 2001.
formed by either flow injection or LC/ESI MS. For flow 1 Present address: Department of Medicinal Chemistry, University injection, the mobile phase was 50% methanol with 0.1% trifluoroacetic acid. For LC/ESI MS, a procedure described 2 Corresponding author, Email: jsutherland@nctr.fda.gov previously (Parshikov et al 2000) was used except that the Structures of compounds used for dosing cul- tures. A. Ciprofloxacin. B. Norfloxacin. C. Piperidine.
percent solvent B was 50% from 0 to 3 min and then wasincreased to 90% in a 15-min linear gradient. LC/ESI MS/MS experiments (Parshikov et al 1999) were performed 1H NMR spectrum, obtained in CDCl3 at 500 with a collision energy of 25–50 eV.
MHz, of the ciprofloxacin metabolite (4-hydroxy-3-oxo-4-vi- 1H nuclear magnetic resonance (NMR) spectroscopy was nylcyclopent-1-enyl ciprofloxacin) produced by T. viride.
performed at 500 MHz (Parshikov et al 1999) with the com-pounds dissolved in deuterated chloroform. 13C NMR spec-troscopy was performed on one compound at 125.77 MHz.
spectrum had a positive Cotton effect at 295 nm, in-dicating that the compound was optically active.
The DEP/NICI mass spectrum of the ciprofloxacin product (TABLE I) consisted of a molecular anion [MϪ.] at m/z 453 and an oxygen adduct [M ϩ O2]Ϫ.
tracts from cultures of T. viride dosed with ciproflox- at m/z 485. The product-ion (NICI MS/MS) mass acin showed residual ciprofloxacin eluting at 11.1 spectrum (TABLE I) for the ion at m/z 453 had sig- min and an apparent metabolite at 21.7 min. Other nificant fragment ions at m/z 412 [M Ϫ 41]Ϫ, 368 [M peaks were found but were also detected in the con- Ϫ 85]Ϫ, and 246 [M Ϫ 207]Ϫ. The LC/ESI MS/MS trols. After 16 d, as shown by the peak areas at 280 mass spectrum (not shown) had an intense fragment nm, 31% of the ciprofloxacin had been transformed ion at m/z 436 [MH–H2O]ϩ and several smaller ions.
to the product and 69% remained unchanged. The The 1H NMR spectrum for the ciprofloxacin prod- ciprofloxacin product had a UV absorption spectrum uct is shown in FIG. 2; proton resonances that cor- responded to those found in the spectrum of cipro- Mass spectral data for ciprofloxacin, norfloxacin, and the conjugates found in cultures of T. viride Mass spectral significant ions, m/z (% relative intensity) 453, 412, 368, 350, 287, 246, 243, 203, 189, 182, 176, 441, 412, 368, 350, 275, 246, 203, 201, 189, 182, 176, PARSHIKOV ET AL: FLUOROQUINOLONE CONJUGATION BY T. VIRIDE 1H NMR spectral data for ciprofloxacin, norfloxacin, and the conjugates found in cultures of T. viride a 3.79 (H␣), 3.65 (Hf), 3.48 (H␤), 1.47, 3.66 (H␤), 3.52 (Hf), 3.41 (H␣), 2.87(Ha), 1.40, 1.20 (Hg) (Hf), 3.94, 3.84 (H␣), 3.31 (H␤), 1.34 4.31 (Hf), 3.66 (H␤), 3.39 (H␣), 2.86(Ha), 1.59 (Hg) floxacin were assigned accordingly (TABLE II). Five and one, respectively. Irradiation of each resonance additional resonances (Ha–He) appeared to be part resulted in a nuclear Overhauser effect (NOE) at the of an additional 123-Dalton moiety that had been de- piperazine ␤ resonance; irradiation of the resonance tected by mass spectrometry (FIG. 3A). The reso- at 2.87 ppm (Ha) produced an NOE at 5.88 pm nances at 5.22, 5.44, and 5.88 (Hb–d) had multiplic- (Hb), even though these protons were not coupled ities and coupling constants consistent with a vinyl to one another. A proton-decoupled 13C NMR spec- group. In addition, there were two singlets at 2.87 trum (not shown) was acquired from the ciproflox- and 5.13 ppm (Ha and He) that integrated as two acin conjugate and showed the same resonances(within 1.44 ppm) as those reported for a 4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl moiety (Mukhopadhyayet al 1996), as well as those consistent with the car-bons of the molecule. The NMR data show that theprotons are arranged on a five-membered carbonring. Based on the MS and NMR results, the cipro-floxacin product (FIG. 3A) was identified as a conju-gate, 1-cyclopropyl-6-fluoro-7-[4-(4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl)piperazinyl]-4-oxohydroquino-line-3-carboxylic acid (ϭ 4-hydroxy-3-oxo-4-vinylcyclo-pent-1-enyl ciprofloxacin).
tracts from cultures of T. viride dosed with norflox-acin showed residual norfloxacin eluting at 10.9 minand an apparent metabolite at 21.5 min. Other peakswere found but were also detected in the controls.
After 16 d, as shown by the peak areas at 280 nm,42% of the norfloxacin had been transformed intothe product and 58% remained unchanged. The nor-floxacin product had a UV absorption spectrum with 286, 321 and 330 nm. The circular dichroism spectrum had a positive Cotton effect at 292 nm, in- Structures of metabolites produced by T. viride dicating that the compound was optically active.
from fluoroquinolones. A. 4-Hydroxy-3-oxo-4-vinylcyclo-pent-1-enyl ciprofloxacin. B. 4-Hydroxy-3-oxo-4-vinylcyclo- The DEP/NICI mass spectrum of the norfloxacin pent-1-enyl norfloxacin. The carbon atoms are numbered product (TABLE I) consisted of the molecular anion as shown in the NMR data and the asymmetric carbon atom [MϪ.] at m/z 441 and an oxygen adduct [M ϩ O2]Ϫ.
at m/z 473. The product-ion (NICI MS/MS) mass spectrum (TABLE I) for the ion at m/z 441 had sig- tabolite produced by Trichoderma spp. We suspect nificant fragment ions at m/z 412 [M Ϫ 29]Ϫ and 368 that the conjugation is a chemical process, because [M Ϫ 73]Ϫ. The LC/ESI MS/MS mass spectrum (not an enzymatic process would not be likely to work with shown) had an intense fragment ion at m/z 424 [MH–H2O]ϩ and several smaller ions.
Although the antibacterial activities of the conju- The 1H NMR spectrum of the norfloxacin product gates produced by T. viride have not yet been inves- (TABLE II) was similar to that of norfloxacin for the tigated due to the minuscule amounts that have been H2, H5, H8, ethyl (Hf–g), and piperazine (H␣–␤) available so far, those ciprofloxacin metabolites that resonances. It also showed five additional resonances have been tested have generally had significantly low- (Ha–e) with chemical shifts (FIG. 3B) that were the er antibacterial activities than the parent drug (Zeiler same as those of the substituted cyclopentenyl ring et al 1987). Since species of Trichoderma and similar in the ciprofloxacin conjugate. Based on the MS and fungi are widespread on straw and other cellulose- NMR results, the norfloxacin product was identified rich debris in the environment (Cooke and Rayner as a conjugate, 1-ethyl-6-fluoro-7-[4-(4-hydroxy-3-oxo- 1984), the conjugation of fluoroquinolone residues 4-vinylcyclopent-1-enyl)piperazinyl]-4-oxohydroquin- with fungal metabolites may be ecologically impor- oline-3-carboxylic acid (ϭ 4-hydroxy-3-oxo-4-vinylcy- tant where these drugs are used for treatment of live- 3-Dimethylamino-5-hydroxy-5-vinyl-2-cyclopenten- 1-one has no apparent antibacterial or antifungal ac- gates could be produced from other secondary tivity (Mukhopadhyay et al 1996). However, the ad- amines, cultures of T. viride were grown with 300 ␮M dition of the unstable Trichoderma spp. metabolite to piperidine (FIG. 1C). Metabolites were extracted and other compounds may potentially be a useful reac- analyzed directly by LC/ESI MS; one peak was found tion for the modification of secondary amines that that was consistent with a conjugate similar to those are being investigated as antimicrobial agents, anti- seen for ciprofloxacin and norfloxacin. A positive-ion ESI mass spectrum with collision-induced dissocia-tion showed ions at m/z 208 (3) [MH]ϩ, 190 (29)[MH-H 2O]ϩ, 162 (100) [MH-H2O-C2H4]ϩ, and 134 (9) [MH-H2O-2C2H4]ϩ. This spectrum is consistent We thank C. E. Cerniglia and E. B. Hansen, Jr., for their with the structure of 4-hydroxy-3-oxo-4-vinylcyclo- useful suggestions, P. P. Fu and L. S. von Tungeln for cir- cular dichroism spectroscopy, S. N. Lekomtseva for theidentification of T. viride, and J. V. Pothuluri for commentson the manuscript. We also thank Bayer Corp. for kindly providing us with the ciprofloxacin.
This work was supported in part by an appointment to Sulfate, formyl, and acetyl conjugates of ciprofloxa- the Postgraduate Research Program at the National Center cin (Zeiler et al 1987, Parshikov et al 1999, 2001b) for Toxicological Research administered by the Oak Ridge and formyl, acetyl, and glucuronide conjugates of Institute for Science and Education through an interagency norfloxacin (Pauliukonis et al 1984, Parshikov et al agreement between the U.S. Department of Energy and the 2001b) have previously been detected as products of various biological reactions. The compounds pro-duced from these two fluoroquinolones in cultures of T. viride, in contrast, were optically active 4-hy-droxy-3-oxo-4-vinylcyclopent-1-enyl conjugates.
Chen Y, Rosazza JPN, Reese CP, Chang H-Y, Nowakowski The structures of the fluoroquinolone conjugates MA, Kiplinger JP. 1997. Microbial models of soil me- are reminiscent of 5-hydroxy-3-methoxy-5-vinyl-2-cy- tabolism: biotransformations of danofloxacin. J Ind Mi- clopenten-1-one, derived from cultures of T. album (Strunz et al 1977), and 3-dimethylamino-5-hydroxy- Cooke RC, Rayner ADM. 1984. Ecology of saprotrophic fun- 5-vinyl-2-cyclopenten-1-one, derived from cultures of Martens R, Wetzstein H-G, Zadrazil F, Capelari M, Hoff- T. koningii (Mukhopadhyay et al 1996). When we mann P, Schmeer N. 1996. Degradation of the fluoro- dosed cultures of T. viride with piperidine, a second- quinolone enrofloxacin by wood-rotting fungi. Appl ary amine similar to the piperazine moiety of cipro- floxacin and norfloxacin, we found evidence of its Mukhopadhyay T, Roy K, Sawant SN, Deshmukh SK, Gan- conjugation with the same unstable fungal metabo- guli BN, Fehlhaber HW. 1996. On an unstable antifun- lite found by Mukhopadhyay et al. This showed that gal metabolite from Trichoderma koningii: isolation and other secondary amines may also react with the me- structure elucidation of a new cyclopentenone deriva- PARSHIKOV ET AL: FLUOROQUINOLONE CONJUGATION BY T. VIRIDE tive (3-dimethylamino-5-hydroxy-5-vinyl-2-cyclopenten- Pauliukonis LT, Musson DG, Bayne WF. 1984. Quantitation of norfloxacin, a new antibacterial agent in human Parshikov IA, Freeman JP, Lay JO, Beger RD, Williams AJ, plasma and urine by ion-pair reverse-phase chromatog- Sutherland JB. 1999. Regioselective transformation of ciprofloxacin to N-acetylciprofloxacin by the fungus Strunz GM, Ren W-Y, Stillwell MA, Valenta Z. 1977. Struc- Mucor ramannianus. FEMS Microbiol Lett 177:131– ture and synthesis of a new cyclopentenone derivative from Trichoderma album. Can J Chem 55:2610–2612.
———, ———, ———, ———, ———, ———. 2000. Mi- Wetzstein H-G, Schmeer N, Karl W. 1997. Degradation of the fluoroquinolone enrofloxacin by the brown rot crobiological transformation of enrofloxacin by the fungus Gloeophyllum striatum: identification of metab- fungus Mucor ramannianus. Appl Environ Microbiol olites. Appl Environ Microbiol 63:4272–4281.
———, Stadler M, Tichy H-V, Dalhoff A, Karl W. 1999. Deg- ———, ———, ———, Moody JD, Williams AJ, Beger RD, radation of ciprofloxacin by basidiomycetes and iden- Sutherland JB. 2001a. Metabolism of the veterinary flu- tification of metabolites generated by the brown rot oroquinolone sarafloxacin by the fungus Mucor raman- fungus Gloeophyllum striatum. Appl Environ Microbiol nianus. J Ind Microbiol Biotechnol 26:140–144.
———, Heinze TM, Moody JD, Freeman JP, Williams AJ, Zeiler H-J, Petersen U, Gau W, Ploschke HJ. 1987. Antibac- Sutherland JB. 2001b. The fungus Pestalotiopsis guepini terial activity of the metabolites of ciprofloxacin and its as a model for biotransformation of ciprofloxacin and significance in the bioassay. Arzneim-Forsch/Drug Res norfloxacin. Appl Microbiol Biotechnol 56:474–477.

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