This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Parshikov, I. A. Articles by Sutherland, J. B. Search for Related Content PubMed Articles by Parshikov, I. A. Articles by Sutherland, J. B. Agricola Articles by Parshikov, I. A. Articles by Sutherland, J. B.

Formation of conjugates from ciprofloxacin and norfloxacin in cultures of Trichoderma viride

     National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079-9502

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

The formation of conjugates from two antibacterial fluoroquinolone drugs, ciprofloxacin and norfloxacin, was observed in cultures of Trichoderma viride that had been grown in sucrose-peptone broth and extracted 16 d after dosing with the drugs. Both conjugates were purified by high-performance liquid chromatography and found to be optically active. They were identified by mass and proton nuclear magnetic resonance spectra as 4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl ciprofloxacin and 4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl norfloxacin. The transformation of veterinary fluoroquinolones in the presence of fungi may have ecological significance.

Key words: biotransformation, fluoroquinolones

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
At present, limited information is available about the ability of fungi to transform fluoroquinolones, such as the widely used antibacterial agents ciprofloxacin (Fig. 1A ) and norfloxacin (Fig. 1B ). Several fungi transform the fluoroquinolones enrofloxacin, danofloxacin, and sarafloxacin to various metabolites (Martens et al 1996 , Chen et al 1997 , Wetzstein et al 1997 , Parshikov et al 2000, 2001a ). Mucor ramannianus transforms ciprofloxacin to N-acetylciprofloxacin (Parshikov et al 1999 ). Gloeophyllum striatum and other wood-decaying basidiomycetes convert ciprofloxacin to at least 16 metabolites, including CO₂ (Wetzstein et al 1999 ). Pestalotiopsis guepini transforms both ciprofloxacin and norfloxacin to N-acetyl, 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 ).

View larger version (18K): [in this window] [in a new window]    FIG. 1. Structures of compounds used for dosing cultures. A. Ciprofloxacin. B. Norfloxacin. C. Piperidine.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Strain T-58, isolated from a fruiting body of Trametes versicolor collected in a forest in Jefferson County, Arkansas, was identified as Trichoderma viride by Dr. S. N. Lekomtseva, Department of Mycology and Algology, Moscow State University, Moscow, Russia. Triplicate experimental cultures in flasks containing sucrose-peptone broth (Parshikov et al 1999 ) were incubated at 28 C with rotary shaking at 180 rpm. After 2 d, the cultures were dosed with 300 µM ciprofloxacin or 313 µM norfloxacin (Parshikov et al 2001b ); in one experiment, 300 µM piperidine hydrochloride (Aldrich Chemical Co.) was substituted. The dosed cultures, control cultures, and noninoculated controls were incubated with shaking for another 16 d.

After harvesting, cultures were extracted with ethyl acetate (Parshikov et al 1999 ) and the residues were dissolved in methanol: acetonitrile: acetic acid (10:10:2) for analysis. Compounds were purified by collecting the peaks separated by high-performance liquid chromatography (HPLC), using the gradient described previously (Parshikov et al 2001a ), and the relative concentrations were estimated from the peak areas at 280 nm. Circular dichroism spectra were obtained in methanol with a Jasco model 500A spectropolarimeter.

Direct exposure probe (DEP) mass spectrometry (MS) experiments were performed as previously described (Parshikov et al 1999 ), using the single quadrupole (Q1) and product-ion modes. The ion-source pressure for chemical ionization was 5.0–5.5 Torr, uncorrected. Product ions were generated with a collision-cell pressure of 0.5 mTorr of argon and a collision energy of 100 eV. Electrospray ionization (ESI) MS experiments (Parshikov et al 1999 ) were performed by either flow injection or LC/ESI MS. For flow injection, the mobile phase was 50% methanol with 0.1% trifluoroacetic acid. For LC/ESI MS, a procedure described previously (Parshikov et al 2000 ) was used except that the percent solvent B was 50% from 0 to 3 min and then was increased to 90% in a 15-min linear gradient. LC/ESI MS/MS experiments (Parshikov et al 1999 ) were performed with a collision energy of 25–50 eV.

¹H nuclear magnetic resonance (NMR) spectroscopy was performed at 500 MHz (Parshikov et al 1999 ) with the compounds dissolved in deuterated chloroform. ¹³C NMR spectroscopy was performed on one compound at 125.77 MHz.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Ciprofloxacin – HPLC analysis of the ethyl acetate extracts from cultures of T. viride dosed with ciprofloxacin showed residual ciprofloxacin eluting at 11.1 min and an apparent metabolite at 21.7 min. Other peaks were found but were also detected in the controls. After 16 d, as shown by the peak areas at 280 nm, 31% of the ciprofloxacin had been transformed to the product and 69% remained unchanged. The ciprofloxacin product had a UV absorption spectrum with _max = 291 and 332 nm. The circular dichroism spectrum had a positive Cotton effect at 295 nm, indicating that the compound was optically active.

The DEP/NICI mass spectrum of the ciprofloxacin product (Table I ) consisted of a molecular anion [M^-.] at m/z 453 and an oxygen adduct [M + O₂]^-. at m/z 485. The product-ion (NICI MS/MS) mass spectrum (Table I ) for the ion at m/z 453 had significant fragment ions at m/z 412 [M - 41]^-, 368 [M - 85]^-, and 246 [M - 207]^-. The LC/ESI MS/MS mass spectrum (not shown) had an intense fragment ion at m/z 436 [MH–H₂O]⁺ and several smaller ions.

View larger version (16K): [in this window] [in a new window]    FIG. 2. 1H NMR spectrum, obtained in CDCl3 at 500 MHz, of the ciprofloxacin metabolite (4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl ciprofloxacin) produced by T. viride

View larger version (17K): [in this window] [in a new window]    FIG. 3. Structures of metabolites produced by T. viride from fluoroquinolones. A. 4-Hydroxy-3-oxo-4-vinylcyclopent-1-enyl ciprofloxacin. B. 4-Hydroxy-3-oxo-4-vinylcyclopent-1-enyl norfloxacin. The carbon atoms are numbered as shown in the NMR data and the asymmetric carbon atom is shown by an asterisk

The DEP/NICI mass spectrum of the norfloxacin product (Table I ) consisted of the molecular anion [M^-.] at m/z 441 and an oxygen adduct [M + O₂]^-. at m/z 473. The product-ion (NICI MS/MS) mass spectrum (Table I ) for the ion at m/z 441 had significant fragment ions at m/z 412 [M - 29]^- and 368 [M - 73]^-. The LC/ESI MS/MS mass spectrum (not shown) had an intense fragment ion at m/z 424 [MH–H₂O]⁺ and several smaller ions.

The ¹H NMR spectrum of the norfloxacin product (Table II ) was similar to that of norfloxacin for the H2, H5, H8, ethyl (Hf–g), and piperazine (H–ß) resonances. It also showed five additional resonances (Ha–e) with chemical shifts (Fig. 3B ) that were the same as those of the substituted cyclopentenyl ring in the ciprofloxacin conjugate. Based on the MS and NMR results, the norfloxacin product was identified as a conjugate, 1-ethyl-6-fluoro-7-[4-(4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl)piperazinyl]-4-oxohydroquinoline-3-carboxylic acid (= 4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl norfloxacin).

Piperidine – To determine whether similar conjugates could be produced from other secondary amines, cultures of T. viride were grown with 300 µM piperidine (Fig. 1C ). Metabolites were extracted and analyzed directly by LC/ESI MS; one peak was found that was consistent with a conjugate similar to those seen for ciprofloxacin and norfloxacin. A positive-ion ESI mass spectrum with collision-induced dissociation showed ions at m/z 208 (3) [MH]⁺, 190 (29) [MH-H₂O]⁺, 162 (100) [MH-H₂O-C₂H₄]⁺, and 134 (9) [MH-H₂O-2C₂H₄]⁺. This spectrum is consistent with the structure of 4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl piperidine.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Sulfate, formyl, and acetyl conjugates of ciprofloxacin (Zeiler et al 1987 , Parshikov et al 1999, 2001b ) and formyl, acetyl, and glucuronide conjugates of norfloxacin (Pauliukonis et al 1984 , Parshikov et al 2001b ) have previously been detected as products of various biological reactions. The compounds produced from these two fluoroquinolones in cultures of T. viride, in contrast, were optically active 4-hydroxy-3-oxo-4-vinylcyclopent-1-enyl conjugates.

The structures of the fluoroquinolone conjugates are reminiscent of 5-hydroxy-3-methoxy-5-vinyl-2-cyclopenten-1-one, derived from cultures of T. album (Strunz et al 1977 ), and 3-dimethylamino-5-hydroxy-5-vinyl-2-cyclopenten-1-one, derived from cultures of T. koningii (Mukhopadhyay et al 1996 ). When we dosed cultures of T. viride with piperidine, a secondary amine similar to the piperazine moiety of ciprofloxacin and norfloxacin, we found evidence of its conjugation with the same unstable fungal metabolite found by Mukhopadhyay et al. This showed that other secondary amines may also react with the metabolite produced by Trichoderma spp. We suspect that the conjugation is a chemical process, because an enzymatic process would not be likely to work with all secondary amines.

Although the antibacterial activities of the conjugates produced by T. viride have not yet been investigated due to the minuscule amounts that have been available so far, those ciprofloxacin metabolites that have been tested have generally had significantly lower antibacterial activities than the parent drug (Zeiler et al 1987 ). Since species of Trichoderma and similar fungi are widespread on straw and other cellulose-rich debris in the environment (Cooke and Rayner 1984 ), the conjugation of fluoroquinolone residues with fungal metabolites may be ecologically important where these drugs are used for treatment of livestock and poultry.

3-Dimethylamino-5-hydroxy-5-vinyl-2-cyclopenten-1-one has no apparent antibacterial or antifungal activity (Mukhopadhyay et al 1996 ). However, the addition of the unstable Trichoderma spp. metabolite to other compounds may potentially be a useful reaction for the modification of secondary amines that are being investigated as antimicrobial agents, antidepressants, or anticancer drugs.

	ACKNOWLEDGMENTS

 
We thank C. E. Cerniglia and E. B. Hansen, Jr., for their useful suggestions, P. P. Fu and L. S. von Tungeln for circular dichroism spectroscopy, S. N. Lekomtseva for the identification of T. viride, and J. V. Pothuluri for comments on the manuscript. We also thank Bayer Corp. for kindly providing us with the ciprofloxacin.

This work was supported in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

	FOOTNOTES

 
¹ Present address: Department of Medicinal Chemistry, University of Mississippi, Oxford, MS 38677.

² Corresponding author, Email: jsutherland{at}nctr.fda.gov

Accepted for publication June 28, 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Chen Y, Rosazza JPN, Reese CP, Chang H-Y, Nowakowski MA, Kiplinger JP., 1997 Microbial models of soil metabolism: biotransformations of danofloxacin J Ind Microbiol Biotechnol 19:378-384[Medline]

Cooke RC, Rayner ADM., 1984 Ecology of saprotrophic fungi London: Longman Group. 415 p

Martens R, Wetzstein H-G, Zadrazil F, Capelari M, Hoffmann P, Schmeer N., 1996 Degradation of the fluoroquinolone enrofloxacin by wood-rotting fungi Appl Environ Microbiol 62:4206-4209[Abstract]

Mukhopadhyay T, Roy K, Sawant SN, Deshmukh SK, Ganguli BN, Fehlhaber HW., 1996 On an unstable antifungal metabolite from Trichoderma koningii: isolation and structure elucidation of a new cyclopentenone derivative (3-dimethylamino-5-hydroxy-5-vinyl-2-cyclopenten-1-one) J Antibiot 49:210-211[Medline]

Parshikov IA, Freeman JP, Lay JO, Beger RD, Williams AJ, Sutherland JB., 1999 Regioselective transformation of ciprofloxacin to N-acetylciprofloxacin by the fungus Mucor ramannianus FEMS Microbiol Lett 177:131-135[Medline]

Parshikov IA, Freeman JP, Lay JO, Beger RD, Williams AJ, Sutherland JB., 2000 Microbiological transformation of enrofloxacin by the fungus Mucor ramannianus Appl Environ Microbiol 66:2664-2667[Abstract/Free Full Text]

Parshikov IA, Freeman JP, Lay JO, Moody JD, Williams AJ, Beger RD, Sutherland JB., 2001a Metabolism of the veterinary fluoroquinolone sarafloxacin by the fungus Mucor ramannianus J Ind Microbiol Biotechnol 26:140-144[Medline]

Parshikov IA, Heinze TM, Moody JD, Freeman JP, Williams AJ, Sutherland JB., 2001b The fungus Pestalotiopsis guepini as a model for biotransformation of ciprofloxacin and norfloxacin Appl Microbiol Biotechnol 56:474–477

Pauliukonis LT, Musson DG, Bayne WF., 1984 Quantitation of norfloxacin, a new antibacterial agent in human plasma and urine by ion-pair reverse-phase chromatography J Pharm Sci 73:99-102[Medline]

Strunz GM, Ren W-Y, Stillwell MA, Valenta Z., 1977 Structure and synthesis of a new cyclopentenone derivative from Trichoderma album Can J Chem 55:2610-2612

Wetzstein H-G, Schmeer N, Karl W., 1997 Degradation of the fluoroquinolone enrofloxacin by the brown rot fungus Gloeophyllum striatum: identification of metabolites Appl Environ Microbiol 63:4272-4281[Abstract]

Wetzstein H-G, Stadler M, Tichy H-V, Dalhoff A, Karl W., 1999 Degradation of ciprofloxacin by basidiomycetes and identification of metabolites generated by the brown rot fungus Gloeophyllum striatum Appl Environ Microbiol 65:1556-1563[Abstract/Free Full Text]

Zeiler H-J, Petersen U, Gau W, Ploschke HJ., 1987 Antibacterial activity of the metabolites of ciprofloxacin and its significance in the bioassay Arzneim-Forsch/Drug Res 37:131-134

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Parshikov, I. A. Articles by Sutherland, J. B. Search for Related Content PubMed Articles by Parshikov, I. A. Articles by Sutherland, J. B. Agricola Articles by Parshikov, I. A. Articles by Sutherland, J. B.