Identification of metabolites produced from N-phenylpiperazine by Mycobacterium spp
- First Online:
- Cite this article as:
- Adjei, M.D., Deck, J., Heinze, T.M. et al. J Ind Microbiol Biotechnol (2007) 34: 219. doi:10.1007/s10295-006-0189-x
- 76 Views
Mycobacterium sp. 7E1B1W and seven other mycobacterial strains known to degrade hydrocarbons were investigated to determine their ability to metabolize the piperazine ring, a substructure found in many drugs. Cultures were grown at 30°C in tryptic soy broth and dosed with 3.1 mM N-phenylpiperazine hydrochloride; samples were removed at intervals and extracted with ethyl acetate. Two metabolites were purified from each of the extracts by high-performance liquid chromatography; they were identified by mass spectrometry and 1H nuclear magnetic resonance spectroscopy as N-(2-anilinoethyl)acetamide and N-acetyl-N′-phenylpiperazine. The results show that mycobacteria have the ability to acetylate piperazine rings and cleave carbon-nitrogen bonds.
Many synthetic drugs, including antibacterial and antifungal agents, anthelmintics, anxiolytics, and antidepressants, contain a piperazine ring that plays a significant role in potency [14, 16, 18, 29]. For example, the antibacterial activity of some fluoroquinolone drugs is reduced or lost upon the substitution or partial removal of the piperazinyl moiety . Also, because piperazinyl compounds are used in textile dyeing and analytical chemistry , they have been found in industrial wastewater .
Piperazine is biodegraded by several bacteria in the genera Arthrobacter and Mycobacterium [13, 15]. Mycobacterium smegmatis, Nocardia sp., Pseudomonas aeruginosa, P. fluorescens, and Streptomyces griseus demethylate the piperazine ring of the fluoroquinolone drug danofloxacin to produce N-desmethyldanofloxacin; and M. smegmatis and P.fluorescens degrade the piperazine ring of danofloxacin to produce 1-cyclopropyl-6-fluoro-7-amino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid . The ability of mycobacteria to biotransform piperazine rings [1, 2] may be especially important because piperazinyl fluoroquinolones are used in combination therapy to treat drug-resistant tuberculosis .
Several fungi metabolize piperazine rings in fluoroquinolones. For instance, Penicillium spp. strains cleave the piperazine ring of danofloxacin  and Gloeophyllumstriatum mineralizes carbon from the piperazine rings of enrofloxacin and ciprofloxacin to CO2 [31, 32]. Umbelopsisramanniana (Mucorramannianus) produces N-acetylated metabolites and piperazine ring-cleavage products from enrofloxacin and sarafloxacin [20, 21] and Pestalotiopsisguepini similarly metabolizes the piperazine rings of ciprofloxacin and norfloxacin .
N-Phenylpiperazine, a model compound structurally related to piperazinyl fluoroquinolones , is also known to have adrenergic blocking properties . Mycobacterium sp. 7E1B1W (ATCC 29676), a non-pathogenic, fast-growing soil bacterium that degrades propane and n-tetradecane but not phenanthrene or pyrene [3–5], and seven other mycobacteria were investigated for the potential to metabolize N-phenylpiperazine.
Materials and methods
Cultures of Mycobacterium spp. were grown for 3 days in 125-ml flasks containing 30 ml tryptic soy broth (TSB) (Remel Inc., Lenexa, KS). They were then dosed with 3.1 mM (final concentration) N-phenylpiperazine hydrochloride (99%, Aldrich Chemical Co., Milwaukee, WI) that had been dissolved in water and filter-sterilized. Controls consisted of cultures that were not dosed and non-inoculated flasks of TSB dosed with N-phenylpiperazine. Cultures and controls were incubated aerobically at 30°C with shaking at 200 rpm. After 4 days, they were extracted with ethyl acetate for analysis by HPLC.
For kinetic studies, cultures and controls were incubated for 3 days in 500 ml flasks containing 100 ml TSB and then were dosed with 3.1 mM N-phenylpiperazine. At various intervals from 0 to 8 days, samples were withdrawn aseptically and extracted with ethyl acetate for analysis.
To determine the effect of N-phenylpiperazine concentration on metabolite production, cultures were grown for 3 days and then were dosed with N-phenylpiperazine (0, 0.4, 0.8, 1.5, or 3.1 mM). They were incubated as described above, harvested 4 days after dosing, and extracted with ethyl acetate for analysis.
Extraction and HPLC analysis
Cultures were centrifuged at 15,000 g for 10 min at 4°C and the supernatants were extracted three times with equal volumes of ethyl acetate. The extracts were dried in vacuo and dissolved in methanol. Metabolites were detected and purified by high-performance liquid chromatography (HPLC) using an Agilent Technologies (Palo Alto, CA) 1100 Series chromatograph; the diode array detector was monitored at 280 nm. A Phenomenex (Torrance, CA) Prodigy 5-μm ODS-3 column (250 × 4.6 mm) was used with a solvent system consisting of A (methanol: water: acetic acid, 10:90:0.2) and B (methanol: water: acetic acid, 90:10:0.2) at a flow rate of 2.0 ml/min. Solvent B was increased from 10 to 95% in a 20-min linear gradient and held at 95% for 10 min.
Direct exposure probe/electron ionization mass spectrometry (DEP/EI-MS) was performed on a ThermoFinnigan (San Jose, CA) TSQ700 mass spectrometer in the electron-ionization (EI) single quadrupole mode. The ion source temperature was 150°C and the electron energy was 70 eV (uncorrected). The first quadrupole analyzer was scanned from m/z 50 to 550 in 0.7 s. The rhenium wire of the DEP was heated from 0 to 800 mA with a linear ramp of 5 mA/s.
Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analyses were performed on a ThermoFinnigan Quantum Ultra mass spectrometer equipped with an Agilent Technologies 1100 Series HPLC. The mass spectrometer was operated in the positive-ion ESI mode with an in-source collision-induced dissociation (CID) offset of −15 or −20 V. Other ESI conditions were: spray voltage 3.0 kV, capillary temperature 350°C, sheath gas pressure 40 psi, ion sweep gas 10, and auxiliary gas 15. MS/MS conditions were: argon collision pressure 1.5 mTorr, collision energy 15 eV, parent set masses (m/z) 163, 179, and 205, and Q3 scanned m/z 30–250 per 0.5 s. HPLC was performed with a Phenomenex Prodigy 5-μm ODS-3 column (2.0 × 250 mm). The mobile phase was a linear gradient from 5% acetonitrile to 95% acetonitrile in 40 min, with constant 0.1% formic acid, at a flow rate of 0.2 ml/min.
Samples were dissolved in deuterated methanol and 1H nuclear magnetic resonance (NMR) spectroscopy was performed at 500 MHz using a Bruker Instruments (Billerica, MA) AM500 NMR spectrometer .
A one-way analysis of variance (anova) was performed to determine the effect of substrate concentration on metabolite production, using the statistical software JMP v 5.1 (SAS Institute). Differences among concentration means were compared using Tukey’s test.
During the HPLC analysis of extracts from dosed Mycobacterium sp. 7E1B1W cultures, N-phenylpiperazine eluted at 9.8 min and two small peaks (I and II) not found in control cultures eluted at 16.8 and 22.5 min, respectively. In extracts from cultures harvested 4 days after dosing, peaks I and II formed about 1.7 and 0.9%, respectively, of the total integrated peak area at 280 nm.
1H NMR data for N-phenylpiperazine and metabolites I and II produced by Mycobacterium sp. 7E1B1W
Chemical shifts (ppm)b
Coupling constants (Hz)b
J2,3 = 5.2, J2′,3′ = 8.6, J2′,4′ = 1.1, J3′,4′ = 7.3
J2,3 = 6.5, J2′,3’ = 8.6, J2′,4′ = 1.1, J3′,4′ = 7.3
J2,3 = 5.2, J2′,3′ = 8.6, J2′,4’ = 1.1, J3′,4’ = 7.3
A standard for N-(2-anilinoethyl)acetamide was synthesized by dissolving 50 mg N-phenylethylenediamine (Fisher Scientific, Pittsburgh, PA) in 4.5 ml water and adding 0.3 ml acetic anhydride (Eastman Chemical, Kingsport, TN). Of the two products formed, one was identified by LC/ESI-MS/MS as N-(2-anilinoethyl)acetamide (MH+ = m/z 179) by the prominent product ion at m/z 86. Because the HPLC retention times, DEP/EI and product-ion mass spectra, and NMR spectra were identical for metabolite I and the synthetic N-(2-anilinoethyl)acetamide standard, metabolite I was identified as N-(2-anilinoethyl)acetamide (Fig. 1b).
The DEP/EI mass spectrum of metabolite II had ions at m/z 204 (48) [M + .], 189 (4), 161 (14), 132 (100) [C6H5-N(CH2)CH = CH2] + , 120 (21), 119 (26) [C6H5-NH-CH = CH2] + , 106 (12) [C6H5-NH-CH2] + , 105 (21), 104 (21), 77 (15) [C6H5] + , and 56 (18). The mass spectrum matched that of 1-(4-phenyl-1-piperazinyl)ethanone (CAS# 2155713-1, National Institute of Standards and Technology reference library) with a reverse fit of 94%. Metabolite II was analyzed by LC/ESI-MS/MS. Fragmentation of the protonated molecule at m/z 205 gave the product-ion mass spectrum shown (Fig. 1c). Ions included the residual protonated molecule at m/z 205, a major N-phenylpiperazinium ion at m/z 163, an N-vinylbenzenaminium ion at m/z 120, and an N-acetyl-N-vinylethylenaminium ion at m/z 112. The NMR data for metabolite II (Table 1) showed a downfield chemical shift for the H3 resonance when compared to that of N-phenylpiperazine. A resonance representing the three protons of an acetyl group had been added. The H2, H6 and H3, H5 resonances of the piperazine ring were split into two triplet resonances each due to the asymmetry of the acetyl group.
A standard for N-acetyl-N′-phenylpiperazine was synthesized by dissolving 50 mg N-phenylpiperazine hydrochloride in 4.5 ml water and adding 0.3 ml acetic anhydride. Because the HPLC retention times, DEP/EI and product-ion mass spectra, and NMR spectra were identical for metabolite II and the synthetic standard and matched the data from the reference library, metabolite II was identified as N-acetyl-N′-phenylpiperazine (Fig. 1c).
To find whether other mycobacteria would produce metabolites from N-phenylpiperazine, seven other strains were tested, including M. frederiksbergense FAn9 (DSM 44346), M. gilvum ATCC 43909, M. gilvum BB1 (DSM 9487), M. gilvum PYR-GCK (ATCC 700033) , M. smegmatis mc2155 (ATCC 700084), Mycobacterium sp. PYR100 , and Mycobacterium sp. RJGII-135 . The same two metabolites (I and II) were detected in extracts from dosed cultures of all strains; the identities were confirmed by HPLC retention times and product-ion spectra (data not shown).
Eight different Mycobacterium spp. strains, when tested for the ability to metabolize N-phenylpiperazine, produced N-acetylated metabolites. The piperazine rings of fluoroquinolone drugs may be N-acetylated by fungi [20–22] and mycobacteria [1, 2]; similar reactions also occur in experimental animals  and in humans . The acetylated drug metabolites found in animals and human urine could also result from microbial metabolism. N-Acetyl-N′-phenylpiperazine has been synthesized previously for use as an activator in the radical polymerization of methyl methacrylate .
Although arylamine N-acetyltransferases from bacteria metabolize arylamines, arylhydrazines, and arylhydroxylamines [6, 24, 25], these enzymes are not known to acetylate piperazine rings. Mycobacterium sp. 7E1B1W also N-acetylates the piperazine rings of norfloxacin and ciprofloxacin [1, 2] but it did not acetylate p-aminobenzoic acid (data not shown).
Carbon–nitrogen bond cleavage, which occurred in the formation of N-(2-anilinoethyl)acetamide, is known in the metabolism of other cyclic secondary amines by mycobacteria [10, 12, 27]. It is typical of the metabolism of piperazine-containing fluoroquinolones by fungi [20–22], animals , and humans .
The metabolism of N-phenylpiperazine by Mycobacterium sp. 7E1B1W may proceed in two possible sequences, depending on whether N-acetylation precedes or follows C–N bond cleavage. Because of the simultaneous appearance of the two metabolites, no evidence is available at this time to show the sequence.
In summary, eight strains of Mycobacterium spp. modified the piperazine ring of N-phenylpiperazine, forming N-(2-anilinoethyl)acetamide and N-acetyl-N′-phenylpiperazine as products.
We thank Dr. C. E. Cerniglia for providing the Mycobacterium strains and also thank him and Dr. F. Rafii for helpful comments on the manuscript. 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 US Department of Energy and the US Food and Drug Administration. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.