Synthesis and characterization of N-demethylated metabolites of malachite green and leucomalachite green

Article abstract of DOI:10.1021/tx0256679

Malachite green (MG), a triphenylmethane dye used to treat fungal and protozoan infections in fish, undergoes sequential oxidation to produce various N-demethylated derivatives (monodes-, dides(sym)-, dides(unsym)-, trides-, and tetrades-) both before and after reduction to leucomalachite green (LMG). The close structure resemblance of the metabolites with aromatic amine carcinogens implicates a potential genotoxicity from exposure to MG. The availability of the synthetic standards is important for metabolic and DNA adduct studies of MG. This paper describes a simple and versatile method for the synthesis of MG, LMG, and their N-demethylated metabolites. The synthesis involves a coupling of 4-(dimethylamino)-benzophenone or 4-nitrobenzophenone with the aryllithium reagents derived from appropriately substituted 4-bromoaniline derivatives, followed by treatment with HCl in methanol. The resulting cationic MG and their leuco analogues showed systematic UV/vis spectral and tandem mass fragmentation patterns consistent with sequential N-demethylation. The extensive ¹H and ¹³C spectral assignments of the metabolites were aided by the availability of ¹³C₇-labeled MG and LMG. The results indicate the existence of a resonance structure with the cationic charge located in the central methane carbon (C₇). The synthetic procedure is general in scope so that it can be extended to the preparation of N-demethylated metabolites of other structurally related N-methylated triphenylmethane dyes.

Full text of DOI:10.1021/tx0256679

Chem. Res. Toxicol. 2003, 16, 285-294
285
Syn th esis a n d Ch a r a cter iza tion of N-Dem eth yla ted
Meta bolites of Ma la ch ite Gr een a n d Leu com a la ch ite
Gr een
Bongsup P. Cho,*,† Tianle Yang,† Lonnie R. Blankenship,‡ J oanna D. Moody,§
Mona Churchwell,‡ Frederick A. Beland,‡ and Sandra J . Culp‡
Department of Biomedical Sciences, College of Pharmacy, University of Rhode Island,
Kingston, Rhode Island 02881, Division of Biochemical Toxicology, and Division of Microbiology,
National Center for Toxicological Research, J efferson, Arkansas 72079
Received November 27, 2002
Malachite green (MG), a triphenylmethane dye used to treat fungal and protozoan infections
in fish, undergoes sequential oxidation to produce various N-demethylated derivatives
(monodes-, dides(sym)-, dides(unsym)-, trides-, and tetrades-) both before and after reduction
to leucomalachite green (LMG). The close structure resemblance of the metabolites with
aromatic amine carcinogens implicates a potential genotoxicity from exposure to MG. The
availability of the synthetic standards is important for metabolic and DNA adduct studies of
MG. This paper describes a simple and versatile method for the synthesis of MG, LMG, and
their N-demethylated metabolites. The synthesis involves a coupling of 4-(dimethylamino)-
benzophenone or 4-nitrobenzophenone with the aryllithium reagents derived from appropriately
substituted 4-bromoaniline derivatives, followed by treatment with HCl in methanol. The
resulting cationic MG and their leuco analogues showed systematic UV/vis spectral and tandem
1
mass fragmentation patterns consistent with sequential N-demethylation. The extensive H
and 13C spectral assignments of the metabolites were aided by the availability of 13C7-labeled
MG and LMG. The results indicate the existence of a resonance structure with the cationic
charge located in the central methane carbon (C7). The synthetic procedure is general in scope
so that it can be extended to the preparation of N-demethylated metabolites of other structurally
related N-methylated triphenylmethane dyes.
analogue, gentian violet, has been shown to induce liver
tumors in mice (7). Thyroid peroxidase-catalyzed oxida-
tion of LMG results in the formation of various N-
demethylated primary and secondary aromatic amines,
whose structures are similar to aromatic amine carcino-
gens (8). These facts, together with our recent findings
of DNA adducts in MG-treated rats, imply a potential
genotoxicity from exposure to MG (9). Because of con-
cerns regarding its use in commercial fisheries, MG was
designated by the National Toxicology Program as a
priority chemical for carcinogenicity testing, and results
from a 2 year feeding study are currently being evalu-
ated.
The metabolic fates of MG and LMG have been studied
both in vivo and in vitro. In rats and mice, MG undergoes
systematic sequential oxidation to produce N-demethyl-
ated metabolites both before and after reduction to LMG
(Figure 1) (9). Small amounts of the N-oxides derivatives
of MG and monodes-MG have also been detected. Similar
metabolic profiles have been observed in incubations of
MG and LMG with the microsomal fractions of intestinal
microflora (10) and the fungus Cunninghamella elegans
(11) as well as thyroid peroxidase-catalyzed reactions (8)
of LMG. The availability of these metabolites as stan-
dards is necessary for future DNA adduct studies and
for use as biomarkers in metabolism studies.
In tr od u ction
MG1 (Figure 1), a member of the N-methylated triphen-
ylmethane class of dyes, has long been used to control
fungal and protozoan infections in fish (1, 2). Although
prohibited in the U.S., the antifungal use of MG in
commercial fisheries has continued worldwide due to its
ready accessibility and low cost (3).
After treatment, MG is readily reduced in the tissues
of fish to the corresponding base, LMG (Figure 1) (3).
Because of its lipophilic nature, LMG excretes very slowly
(t1/2 ∼ 40 days) in certain fatty fish tissues. A similarly
persistent accumulation of LMG has been documented
in mammalian systems. MG and LMG are cytotoxic to
both bacterial and mammalian cells but showed no
significant mutagenic and clastogenic activities regard-
less of the presence of S9 activation (4, 5). MG has been
reported to be a tumor promoter, but very little is known
about the carcinogenicity of MG (6), although a structural
* To whom correspondence should be addressed. Tel: 401-874-5024.
Fax: 401-874-5048. E-mail: bcho@uri.edu.
† University of Rhode Island.
‡ Division of Biochemical Toxicology, National Center for Toxicologi-
cal Research.
§ Division of Microbiology, National Center for Toxicological Re-
search.
1
Abbreviations: APCI, atmospheric pressure chemical ionization;
COSY, correlation spectroscopy; DDQ, 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone; HRMS, high-resolution mass spectrometry; LMG, leu-
comalachite green; MG, malachite green; NOE, nuclear Overhauser
effect.
We have previously reported the preparation of the 4′-
amino and 4′-nitro derivatives of N,N-dimethylamino-
triphenylmethane as precursors for presumed DNA ad-
10.1021/tx0256679 CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/04/2003

286 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Cho et al.
min gradient, 10-90% acetonitrile/100 mM ammonium acetate,
pH 4.5, 1 mL/min.
LC/MS. The HPLC separation for mass spectrometry was
performed using a Prodigy ODS-3 column (5 µm, 4.6 mm × 250
mm, Phenomenex Co., Torrance, CA) at a flow rate of 1.0 mL/
min, with a mobile phase gradient consisting of 50% acetonitrile
and 50% aqueous ammonium acetate (20 mM, pH 4.5) to 100%
acetonitrile over a 10 min linear ramp period delivered by a
GP40 pump (Dionex Co., Sunneyvale, CA). The entire mobile
phase flow was introduced into the mass spectrometer. For
direct infusion studies, the solvent was delivered at ca. 10 µL/
min using a syringe pump (KDS-100, KD Scientific Co., Boston,
MA).
A Quattro Ultima triple quadrupole mass spectrometer
(Micromass, Manchester, U.K.) equipped with an APCI interface
with heated nebulizer probe at 450 °C was used with an ion
source temperature of 130 °C. For MS/MS measurements, direct
infusion of pure analytes, or LC/MS, experiments were per-
formed using a collision cell gas pressure (Ar) of 1.8 × 10-3 mbar
and a collision energy of 30 eV. The mass spectrometer was
calibrated over the mass range m/z 85-1200 using a solution
of poly(ethylene glycol).
Gen er a l P r oced u r e for P a th w a y A. (1) Syn th esis of MG
a n d LMG. Butyllithium (4.8 mL, 2.5 M in hexane, 12 mmol)
was added via a syringe to a solution of 4-bromo-N,N-dimethyl-
aniline (3.15 g, 10 mmol) in dry THF (100 mL) at -78 °C under
an argon atmosphere. The reaction mixture was stirred at the
same temperature for 2 h, and then, a solution of 4-(dimethyl-
amino)benzophenone (4.5 g, 10 mmol) in 80 mL of THF was
added dropwise via a dropping funnel. The mixture was warmed
slowly to room temperature, and stirring was continued over-
night. The reaction was quenched by addition of H2O, and the
residual THF was evaporated. The aqueous residue was then
extracted with ether, and the combined ether extracts were
washed with H2O and dried over anhydrous MgSO4. Concentra-
tion by a rotoevaporator gave 4.26 g of a blue-greenish viscous
residue containing the carbinolic intermediate. A small portion
of the residue was purified by column chromatography on silica
to give pure MG carbinol, whose NMR characteristics match
those of the standard. 1H NMR (DMSO-d6): δ 2.83 (s, 12 H, 2
-N(CH3)2), 5.87 (s, 1H, D2O exchangeable OH), 6.60 (d, 4H,
H3,5,3′,5′, J ) 8.9 Hz), 6.96 (d, 4H, H2,6,2′,6′, J ) 8.9 Hz), 7.15 (m,
1H, H4′′), 7.18 (m, 2H, H3′′,5′′), 7.25 (m, 2H, H2′′,6′′). 13C NMR
(DMSO-d6): δ 42.84 (-N(CH3)2), 82.52 (C7), 113.93 (C3,5,3′,5′),
131.06 (C2,6,2′,6′). The remaining carbinolic residue was trans-
ferred to a flask containing 100 mL of methanol and 1 mL of
concentrated HCl. The light green solution turned dark blue
immediately upon contact with the acid solution. The mixture
was refluxed for 1 h. After it was cooled, the solution was
concentrated in vacuo to give a viscous residue that was
subsequently partitioned between H2O and ether. The aqueous
layer was washed repeatedly with ether until TLC of the ether
washings showed no organic residues. The green aqueous layer
was concentrated overnight to dryness using a Speedvac con-
centrator. The residue was purified by column chromatography
on silica using a 9:1 ratio of chloroform:methanol, which afforded
1.94 g of MG (53%) as a dark green residue. The UV, MS, 1H
and 13C NMR data of this sample match those of the standard
(see Tables 1-3).
F igu r e 1. Structures of MG, LMG, and their N-methylated
derivatives.
ducts of MG (12). Here, we describe a simple and versatile
synthesis of MG and LMG and their N-demethylated
metabolites (Figure 1). We also provide a detailed spec-
tral characterization of all of the compounds.
Exp er im en ta l P r oced u r es
Ma ter ia ls a n d Meth od s. THF was distilled fresh from
sodium/benzophenone prior to use. 13C-labeled benzaldehyde
was purchased from Isotec (Miamisburg, OH). All reagents and
solvents were purchased from Aldrich, Inc. (Milwaukee, WI).
Melting points were determined on a Thermoelectrical melting
point apparatus and are uncorrected. Elemental analyses were
obtained from M-H-W Laboratories, Phoenix, AZ. HRMS were
obtained in the University of Illinois Mass Spectrometry Labo-
ratory, School of Chemical Science, Urbana-Champaign, IL. 1H
and 13C NMR spectra were recorded on a Bruker AM500
spectrometer operating at 500 and 126 MHz, respectively.
Chemical shifts are expressed in ppm with respect to the
internal standard tetramethylsilane. Analytical HPLC analyses
MG (1.63 g, 4.8 mmol) was treated with NH2NH2‚H2O (800
µL, 15.5 mmol) and 10% Pd/C (250 mg) in absolute ethanol (150
mL), and the mixture was heated to reflux. The green solution
turned colorless upon refluxing for about 10 min. The refluxing
was continued for 1 h, and the solution was cooled and filtered
through a Celite bed in a Buchner funnel to remove the catalyst.
The filtrate was concentrated and partitioned between H2O and
ether. The aqueous layer was extracted with ether several times,
and the combined ether extracts were back-washed with water.
The organic layer was dried over anhydrous MgSO4, filtered,
and evaporated. The resulting oily residue was applied to a silica
gel column and eluted with ethyl acetate and hexane (1:4).
Appropriate portions were pooled, concentrated, and recrystal-
were conducted with
a photodiode array detector using a
Spherisorb column (CN, 4.6 mm × 250 mm, 5 µm) with a 30

Synthesis of Malachite Green Metabolites
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 287
lized from ethyl acetate and hexane to give 1.44 g (88%) of pure
mmol) in dry THF (100 mL) was added dropwise to the reaction
mixture, which turned to orange initially and then dark brown.
The mixture was warmed slowly to room temperature and
stirred overnight. The reaction was quenched with H2O and
extracted with CH2Cl2. The combined extracts were evaporated,
and the resulting residue was refluxed for 30 min with 50 mL
of methanol containing 3 mL of concentrated HCl. The solution
turned to blue immediately upon contact with the acid solution.
The solvent was evaporated and partitioned between H2O and
ether. The aqueous layer was repeatedly washed with ether.
The ether layer, which contained mostly the unreacted starting
materials, was discarded. Concentration of the H2O layer by
Speedvac afforded ∼1.5 g of a highly hygroscopic dark blue
residue, whose TLC (chloroform:emthanol ) 9:1) exhibited a
mixture of a major (Rf ) 0.21) and a minor (Rf ) 0.17) spot on
silica TLC, which showed characteristic green and blue colors,
respectively, upon standing. The crude cationic mixture was
treated with excess NH2NH2‚H2O and 10% Pd/C in refluxing
ethanol for 2 h. A usual work up with ether gave a mixture of
dides(unsym)-LMG and 6, which was separated on silica using
ethyl acetate and hexane as an eluting solvent system. The
chromatographic and spectral properties of the polar fraction
(Rf ) 0.24, hexane:benzene:ether:triethylamine ) 10:5:3:0.5) (50
mg, 1.7%) match those of dides(unsym)-LMG prepared via
pathway A (12). The major nonpolar product 6 (Rf ) 0.31) was
recrystallized from ethyl acetate and hexane as fluffy needles
(504 mg, 12.0%, mp 155-156 °C) and had the following
properties: TLC: Rf ) 0.31 (benzene:hexane:ether:triethylamine
1
LMG (mp 86-88 °C) as a beige-colored solid. The UV, MS, H
and 13C NMR data of this sample match those of standard (see
Tables 1 and 2).
(2) Syn th esis of Mon od es-MG a n d Mon od es-LMG. (a )
N-Meth yl-4-br om oa n ilin e. N-Methylaniline (1.07 g, 0.01 mol)
in CH2Cl2 (20 mL) was cooled to -78 °C under an argon
atmosphere. A solution of tetrabromo-2,5-cyclohexadienone (4.10
g, 0.01 mol) in CH2Cl2 (20 mL) was added dropwise (13). The
solution underwent a series of color changes from colorless to
dark gray, blue, and finally to dark blue. The mixture was allow-
ed to warm to room temperature over a period of 2 h, at which
time TLC indicated that the reaction was complete. The mixture
was treated with 20 mL of 1 N NaOH and stirred for 10 min to
remove the tribromophenol byproduct. The organic layer was
washed with additional NaOH and H2O and then dried over
anhydrous MgSO4. Evaporation of the solvent gave crude
product as a dark purple liquid. Vacuum distillation (4 mmHg)
gave 1.82 g (bp 85-93 °C, 99%) of pure product as a dark purple
liquid; Rf ) 0.50 (benzene:hexane:ether:triethylamine ) 10:5:
1
3:0.5). H NMR (CDCl3): δ 2.47 (s, -NCH3), 3.38 (bs, 1 H, NH,
D2O exchangeable), 6.13 (d, 2H, H2,6, J ) 8.1 Hz), 6.86 (d, 2H,
H3,5, J ) 8.1 Hz). Anal. calcd for C7H8NBr: C, 45.19; H, 4.33;
N, 7.53. Found: C, 44.92; H, 4.16; N, 7.76.
(b) N-Meth yl-N-tr im eth ylsilyl-4-br om oa n ilin e (2). Bu-
tyllithium (13 mL, 2.5 M in hexane, 32.4 mmol) was added to a
solution of N-methyl-4-bromoaniline (5.06 g, 27 mmol) in dry
THF (50 mL) at 0 °C under an argon atmosphere. The dark blue
solution turned sequentially to brown, yellow brown, and finally
a turbid colloidal solution upon the addition of butyllithium. The
solution was warmed to room temperature over 1 h and then
cooled in an ice bath, and chlorotrimethylsilane (3.52 g, 32.4
mmol) was added slowly via a syringe. The reaction, which was
slightly exothermic, was stirred overnight at room temperature.
The colloidal solution was filtered through a sintered glass to
remove LiCl and centrifuged for 15 min. The supernatant was
transferred to a distillation flask and distilled under vacuum
to give 3.7 g (66%) of product as a colorless liquid; bp 60-62 °C
(1 mmHg); Rf ) 0.72 (benzene:hexane:ether:triethylamine ) 10:
) 10:15:3:0.5), Rf ) 0.75 (CHCl3:methanol ) 95:5). HPLC: tR
)
23.7 min (acetonitrile in 0.1 mM pH 4.5 ammonium acetate 10-
90%, a 30 min gradient). UV (methanol): 297 nm (log 4.60),
263 nm (log 4.57). 1H NMR (DMSO-d6): δ 2.80 (s, 6H, -N(CH3)2),
2.83 (s, 6H, -N(CH3)2), 5.29 (s, 1H, methane proton), 6.64 (d,
2H, J ) 8.8 Hz), 6.67 (d, 2H, J ) 9.0 Hz), 6.77 (d, 2H, J ) 8.6
Hz), 6.84 (d, 2H, J ) 8.6 Hz), 6.90 (d, 2H, J ) 8.7 Hz), 6.94 (d,
2H, J ) 8.9 Hz), 7.08 (d, 2H, J ) 7.2 Hz), 7.15 (dd, 1H, J ) 7.3,
7.3 Hz), 7.26 (dd, 2H, J ) 7.5, 7.6 Hz), 7.55 (s, 1H, -NH, D2O
exchangeable). 13C NMR (DMSO-d6): δ 40.22 (-N(CH3)2), 40.85
(-N(CH3)2), 54.40 (methane carbon), 112.35, 113.85, 114.10,
120.87, 125.71, 128.04, 128.84, 129.38, 129.47, 132.11 (q), 132.11
(q), 133.03 (q), 133.64 (q), 143.89 (q), 145.27 (q), 145.86 (q),
148.74 (q). ESI-MS m/z 422 (M + 1)+. HRMS Calcd for C29H31N3,
421.2510; found, 421.2518. Anal. calcd for C29H31N3: C, 82.62;
H, 7.41; N, 9.97. Found: C, 82.70; H, 7.66; N, 9.89.
1
15:3:0.5). H NMR (acetone-d6) (14): δ 0.15 (s, 9H, -Si(CH3)3),
2.75 (s, 3H, -NCH3), 6.65 (d, 2H, H2,6, J ) 8.0 Hz), 7.18 (d, 2H,
H3,5, J ) 8.0 Hz).
Monodes-MG and monodes-LMG were obtained via pathway
A using N-methyl-N-trimethylsilyl-4-bromoaniline (2, 1.06 g, 5
mmol) and 4-(dimethylamino)benzophenone (1.13 g, 5 mmol).
Monodes-MG (0.84 g, 59%): 1H NMR (acetone-d6): δ 3.09 (s,
3H, -NCH3), 3.30 (s, 6H, -N(CH3)2), 7.01 (d, 2H, H3,5, J ) 9.3
Hz), 7.34 (d, 2H, H2,6, J ) 9.3 Hz), 7.34 (d, 2H, H3′,5′), 7.38 (m,
4H, H2′,6′,2′′6′′), 7.61 (m, 2H, H3′′,5′′), 7.72 (m, 1H, H4′′). Monodes-
LMG: 0.374 g (44%) as a white solid; mp 68-69 °C (ethyl
acetate and hexane). 1H NMR (acetone-d6): δ 2.74 (s, 3H,
-NCH3), 2.88 (s, 6H, -N(CH3)2), 4.72 (bs, 1H, -NH, D2O
exchangeable), 5.33 (s, 1H, H7), 6.52 (d, 2H, H3,5, J ) 8.6 Hz),
6.66 (d, 2H, H2,6, J ) 8.6 Hz), 6.88 (d, 2H, H3′,5′, J ) 8.6 Hz),
6.95 (d, 2H, H2′,6′, J ) 8.6 Hz), 7.13 (m, 3H, H2′′,6′′,4′′), 7.24 (m,
2H, H3′′,5′′). 13C NMR (acetone-d6): δ 30.63 (-NCH3), 40.74
(-N(CH3)2), 56.00 (C7), 112.53, 113.22, 126.44, 128.74, 130.03,
130.53, 130.58, 133.24, 133.65, 146.76, 149.17, 150.00. Anal.
calcd for C22H24N2: C, 83.50; H, 7.65; N, 8.85. Found: C, 83.57;
H, 7.64; N, 8.78.
Gen er a l DDQ Oxid a tion P r oced u r e. DDQ (51.3 mg, 0.226
mmol) was added to a solution of LMG (50 mg, 0.152 mmol) in
benzene (2 mL). After it was stirred for 30 min at room
temperature, the reaction mixture was poured onto a short
column containing aluminum oxide. Elution with CH2Cl2:
methanol (9:1) furnished a green residue. The residue was
treated with 50 mL of methanol containing 3 drops of concen-
trated HCl and refluxed for 1 h. The solvent was evaporated by
a rotoevaporator. The resulting dark residue was purified by
column chromatography on silica using CH2Cl2:methanol (9:1)
to give pure 45.3 mg (82%) of pure MG.
Gen er a l P bO2 Oxid a tion P r oced u r e. LMG (50 mg, 0.152
mmol) in H2O (1 mL) was treated with PbO2 (50 mg, 0.193
mmol) and 5 drops of concentrated HCl, and the reaction was
stirred at room temperature overnight. The mixture was diluted
with methanol (30 mL) and filtrated through a Celite bed in a
glass-fritted funnel, followed by chromatography on silica (CH2-
Cl2:methanol ) 9:1), affording pure MG (46 mg, 99%).
Dides(unsym)-MG and dides(unsym)-LMG were prepared in
a similar manner using 4-bromo-N,N-bis(trimethylsilyl)aniline
(3, 15) and 4-(dimethylamino)benzophenone as starting materi-
als. Details of the synthesis and the spectroscopic characteriza-
tion were described previously (12).
Tr id es-LMG a n d Tr id es-MG. Pathway B, using 4-nitroben-
zophenone (1.14 g, 5 mmol), N-methyl-N-trimethylsilyl-4-bro-
moaniline (2, 1.06 g, 5 mmol), and butyllithium (2.5 M in
hexane, 1.91 mL, 5 mmol) gave 0.214 g of blue color residue.
The crude mixture was reduced (NH2NH2‚H2O, 10% Pd/C) in
the usual manner, and a portion was isolated using silica gel
column chromatography (ethyl acetate:hexane ) 1:4) to give
Gen er a l P r oced u r e for P a th w a y B. (1) Syn th esis of
Did es(u n sym )-LMG. Butyllithium (4.8 mL, 2.5 M in hexane,
12 mmol) was added via a syringe to a solution of N,N-
(dimethylamino)-4-bromobenzene (2.40 g, 12 mmol) in dry THF
(100 mL) at -78 °C under an argon atmosphere. The resulting
turbid solution became clear after stirring at the same temper-
ature for 2 h. A solution of 4-nitrobenzophenone (2.27 g, 10
1
trides-LMG (59 mg, 6% overall yield). H NMR (acetone-d6): δ
2.78 (s, 3H, -NCH3), 4.43 (bs, 2H, -NH2, D2O exchangeable),
4.75 (bs, 1H, -NH, D2O exchangeable), 5.27 (s, 1H, H7), 6.50

288 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Cho et al.
(d, 2H, H3,5, J ) 9.0 Hz), 6.57 (d, 2H, H2,6, J ) 9.0 Hz), 6.79 (d,
2H, H3′,5′, J ) 8.9 Hz), 6.85 (d, 2H, H2′,6′, J ) 8.9 Hz), 7.13 (m,
3H, H2′′,6′′,4′′), 7.24 (m, 2H, H3′′,5′′). HRMS calcd for C20H20N2,
288.1627; found, 288.1630. Anal. calcd for C20H20N2: C, 83.50;
H, 7.65; N, 8.85. Found: C, 83.57; H, 7.64; N, 8.78. Trides-MG
was obtained by DDQ oxidation of trides-LMG. 1H NMR
(acetone-d6): δ 3.15 (s, 3H, -NCH3), 7.00 (d, 2H, H3,5, J ) 9.0
Hz), 7.08 (broad d, 2H, H3′,5′, J ) 9.0 Hz), 7.33 (d, 2H, H2,6, J )
9.0 Hz), 7.42 (d, 2H, H2′,6′, J ) 9.0 Hz), 7.62 (m, 2H, H3′′,5′′), 7.77
(m, 1H, H4′′).
Resu lts a n d Discu ssion
The structures and the numbering system of MG and
LMG and their N-demethylated derivatives are shown
in Figure 1. The N-demethylated compounds are labeled
to indicate the extent of N-demethylation (i.e., monodes,
dides, trides, and tetrades). For the dides derivatives, two
possibilities exist, symmetric or unsymmetric. The car-
bons in the molecules are numbered as shown for
identification. The carbon in the center of the molecule
is labeled as C7. Because of the symmetric nature, the
numberings of the substituted aromatic rings of MG,
LMG, and their dides(sym) and tetrades derivatives are
identical (i.e., C1 and C1′).
Tetrades-LMG and Tetrades-MG were prepared similarly via
pathway B using 4-nitrobenzophenone and N,N-bis(trimethyl-
silyl)-4-bromoaniline (3). Tetrades-LMG (overall yield 4%): 1H
NMR (acetone-d6): δ 4.43 (bs, 4H, 2 NH2, D2O exchangeable),
5.26 (s, 1H, H7), 6.57 (d, 4H, H3,5,3′,5′, J ) 8.5 Hz), 6.79 (d, 4H,
H
2,6,2′,6′, J ) 8.5 Hz), 7.11 (d, 2H, H2′′,6′′, J ) 8.4 Hz), 7.14 (m,
Syn th esis. Literature reports on the preparation of
MG and LMG are scarce (1, 2), and no information is
available on the systematic synthesis of the N-demeth-
ylated analogues of MG and LMG. The original synthesis
of LMG entailed a condensation reaction of benzaldehyde
with N,N-dimethylaniline (16). Aqueous PbO2 oxidation
of LMG gave a C7-carbinolic intermediate, which was
then subsequently treated with concentrated HCl to form
the cationic MG as a chloride salt in quantitative yield.
This procedure, although reported to be of good yield,
suffers from a rather laborious salt-induced precipitation
procedure and certain side reactions (16). The chloride
salt can be converted to its oxalate salt by oxalic acid
treatment of the carbinolic base, which is generated by
reaction of the chloride salt with NaOH (17). Doerge et
al. (18) have prepared 13C6-MG as an internal mass
spectral standard. The reaction involved an initial con-
densation of lithiated 13C6-benzene derived from 13C6-
bromobenzene and Michler’s ketone, followed by treat-
ment with anhydrous HCl; however, no synthetic details
were provided. DeFina and Dieckmann (19) have recently
reported selectively 15N- or 13CH3-labeled MG as model
ligands for structural studies of RNA aptamer/ligand
complexes. Their synthesis entails a coupling reaction of
methylbenzoate with the Grignard reagent derived from
an appropriately labeled 4-bromo-N,N-dimethylaniline.
Figure 2 shows our MG synthesis, which is general in
scope and can be used to prepare N-demethylated deriva-
tives. The overall synthetic strategy is similar to that of
the preparation of gentian violet metabolites by Mc-
Donald and co-workers (20); however, major modifica-
tions, including the isolation procedure, have been made.
MG was prepared via pathway A, which involves a
coupling of 4-(dimethylamino)benzophenone with the
aryllithium reagent derived from 4-bromo-N,N-dimethyl-
aniline. The resulting crude carbinolic intermediate was
subsequently treated with concentrated HCl in refluxing
methanol. After the solvent was evaporated, the dark
greenish residue was partitioned between ether and H2O.
The cationic MG was isolated from the aqueous layer and
purified by column chromatography on silica using a 9:1
mixture of chloroform and methanol as an eluant. The
overall yield of MG via this new procedure was in the
range of 50-65%.
1H, H4′′), 7.23 (m, 2H, H3′′,5′′). HRMS calcd for C19H18N2,
274.1470; found, 274.1470. Tetrades-MG was obtained by DDQ
oxidation of Tetrades-LMG. 1H NMR (acetone-d6): δ 6.58 (d,
4H, H2,6,2′,6′, J ) 8.6 Hz), 6.71 (d, 4H, H3,5,3′,5′, J ) 8.6 Hz), 7.14
(m, 3H, H2′′,6′′,4′′), 7.25 (m, 2H, H3′′,5′′).
13C7-LMG. 13C7-LMG was synthesized following the literature
procedure (16). A mixture of dimethylaniline (1.72 g, 14.2 mmol),
13CHO-benzaldehyde (492 mg, 4.6 mmol, Isotec), and concen-
trated HCl (1.0 mL) was refluxed overnight under an argon
atmosphere. To the cooled greenish solution was added 10 mL
of 1.0 N NaOH, and the mixture was extracted three times with
ether. The combined ether extracts were washed with H2O and
dried over anhydrous MgSO4. A yellow viscous residue resulted
upon concentration. Silica column chromatography (ethyl ac-
etate and hexane) followed by recrystallization from the same
solvent system afforded 1.32 g (87%) of a white solid; mp 87-
89 °C. UV: λmax 261.9 nm. ESI-MS: 332.1 (M + H)+. See Tables
1 and 2 for 1H and 13C NMR data.
13C7-MG. 13C7-LMG (0.25 g, 0.075 mmol) was suspended in
a H2O solution containing 0.25 mL of concentrated HCl. To the
solution was added PbO2 (0.18 g, 0.075 mmol) over a period of
10 min and stirred overnight at ambient temperature. The dark
viscous residue was filtered through a Celite bed and concen-
trated and chromatographed using chloroform and methanol to
give the cationic MG (100 mg). According to 1H NMR, this
product contains over 92% of MG and 7% of the leuco starting
material. Repeated chromatography gave a pure MG (>98%
purity). UV: λmax 614.4 nm (methanol); see Tables 1 and 2 for
1H and 13C NMR data. ESI-MS: 330 (M+). Anal. calcd for
C2213C1H26N2: C, 83.64; H, 7.39; N, 8.45. Found. C, 83.88; H,
7.87; N, 8.67.
Did es(sym )-LMG. A mixture of methylaniline (4.3 g, 40
mmol), benzaldehyde (1.4 g, 13 mmol), and 30% HCl (5.0 mL)
was refluxed under an argon atmosphere. The color of the
solution changed from yellow to light green and finally dark
green. After it was refluxed for 18 h, the condenser was removed
and the mixture was heated for about 1 h to boil the excess
N-methylaniline. The reaction mixture was cooled, 20 mL of 1.0
M NaOH was added, and the mixture was extracted with ether.
The combined ether extracts were washed with H2O and dried
over anhydrous MgSO4. Concentration of the organic solvent
gave crude product as an oily residue. Silica gel column
chromatography using ethyl acetate and hexane (1:4) as an
eluant gave the product as a foamy and glassy residue (1.20 g,
31%). 1H NMR (acetone-d6): δ 2.74 (s, 6H, 2 NCH3), 4.83 (bs,
2H, 2 NH, D2O exchangeable), 5.29 (s, 1H, H7), 6.51 (d, 4H,
H3,5,3′,5′, J ) 8.6 Hz), 6.86 (d, 4H, H2,6,2′,6′, J ) 8.5 Hz), 7.12 (d,
2H, H2′′,6′′, J ) 8.9 Hz), 7.13 (m, 1H, H4′′), 7.24 (m, 2H, H3′′,5′′).
13C NMR (acetone-d6): δ 30.58 (-NCH3), 56.11 (C7), 112.50,
126.39, 128.70, 130.02, 130.67, 133.37, 146.88, 149.13. Anal.
calcd for C21H22N2: C, 83.40; H, 7.33; N, 9.26. Found. C, 83.38;
H, 7.41; N, 9.09. The DDQ oxidation of dides(sym)-LMG gave
The new MG synthesis affords cleaner MG, in one pot,
and less contamination from byproducts. This is a major
improvement from the original condensation method,
which involved extra steps and a laborious salt-induced
precipitation (16). Conversion of the chromatic MG to the
leuco base LMG took place smoothly with hydrazine in
the presence of 10% Pd/C in refluxing ethanol. The
products’ identities were confirmed by chromatographic
1
dides(sym)-MG. H NMR (acetone-d6): δ 3.29 (s, 6H, 2 NCH3),
6.56 (d, 4H, H3,5,3′,5′, J ) 8.5 Hz), 6.87 (d, 2H, H2,6,2′,6′, J ) 8.5
Hz), 7.10 (d, 2H, H2′′,6′′, J ) 7.2 Hz), 7.12 (m, 1H, H4′′), 7.24 (m,
2H, H3′′,5′′).

Synthesis of Malachite Green Metabolites
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 289
F igu r e 2. Synthesis of MG, LMG, and their N-demethylated derivatives.
and spectral comparison with those of available stan-
two protons and the other one with one proton), all of
which were well-resolved. Additional features included
a characteristic methane proton and carbon signals at
5.29 and 54.40 ppm, respectively, and a D2O exchange-
able proton at 7.55 ppm. The formation of 6 appears to
be the consequence of a direct reaction between the
aromatic nitro functionality and the lithiated 4-(N,N-
dimethyl)aniline. Work is currently underway to eluci-
date the mechanism of this unusual reaction. The overall
yields for dides(unsym)-LMG and the major unknown 6
from this three step reaction sequence were 1.7 and
12.0%, respectively.
1
dards. Complete H and 13C NMR spectral characteriza-
tion of LMG and MG was made with the use of 13C7-
labeled analogues (see Structure Characterization).
Monodes- and dides(unsym)-MG and -LMG derivatives
were prepared in a similar manner using 2 and 3 (Figure
2), respectively, as starting lithiating reagents (Figure
2).
An analogous sequence (pathway B), which involved
the use of 4-nitrobenzophenone as a condensating agent
(Figure 2), was devised for the trides and tetrades
derivatives. We first attempted this procedure by reacting
the lithiated 1 with 4-nitrobenzophenone, which in theory
should give dides(unsym)-LMG as a product. The initial
condensation, followed by a HCl treatment, afforded a
highly hygroscopic inky residue, whose TLC (chloroform:
methanol ) 9:1) exhibited two spots (Rf ) 0.21 and 0.17),
with the former being the major one. Separation of the
products proved to be difficult; so we proceeded to reduce
the crude mixture directly (NH2NH2, 10% Pd/C). The
resulting leuco products were readily separated on a silica
gel column using ethyl acetate and hexane. The minor
product derived from the polar intermediate above was
found to be the desired dides(unsym)-LMG, whose struc-
ture was confirmed by spectral comparison with the
standard sample prepared by pathway A (12). This
implied that the polar residue before reduction was the
expected nitro intermediate (4).
The reduction product of the major nonpolar compound
(Rf ) 0.21) was obtained as fluffy needles from ethyl
acetate and hexane and gave an elemental composition
of C29H31N3 (high-resolution mass and elemental analy-
sis) and produced a protonated molecule at m/z 422 in
an electrospray mass spectrum. The material was ini-
tially thought to be the tandem condensation product 5;
however, analysis of NMR data indicated the isomeric
Analogous condensation reactions using 2 and 3 (Fig-
ure 2) afforded trides- and tetrades-LMG as minor
products (6 and 4% overall yield), respectively. We did
not characterize the major coupling adducts for trides and
tetrades derivatives, which appear to be complex.
Pathway B afforded leuco bases as initial products,
which required oxidation to the cationic MG derivatives
using aqueous PbO2 in concentrated HCl. Pure cationic
1
structure 6. The 500 MHz H NMR spectrum in DMSO-
d6 of the major product exhibited a total of seven doublets
(two protons each) and two doublet of doublets (one with

290 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Cho et al.
F igu r e 3. Synthesis of 13C7-MG, 13C7-LMG, and dides(sym)-MG and -LMG.
F igu r e 4. Expanded 1H NMR spectra of (a) LMG and (b) 13C7-LMG in DMSO-d6.
compounds were obtained by filtering through Celite,
followed by silica column chromatography. A similar
oxidation was achieved by treatment of the leuco deriva-
tives with an excess amount of DDQ (21) for about 30
min at room temperature. Removal of excess DDQ via
an aluminum oxide column and subsequent treatment
with methanolic HCl gave pure cationic derivatives.
We prepared the symmetrical dides-MG and dides-
LMG using N-methylaniline and benzaldehyde by a
modification of the original MG synthesis procedure
(Figure 3) (16). The same procedure was also employed
for the preparation of 13C7-labeled LMG using 13CHO-
labeled benzaldehyde and N,N-dimethylaniline. Both
dides(sym)-LMG and 13C7-LMG were converted to the
cationic MG derivatives by DDQ or PbO2 oxidation. As
expected, 13C7-LMG and 13C7-MG exhibited a protonated
molecule at m/z 332 and a cationic molecule at m/z 330,
respectively. While 13C7-LMG showed an identical UV/
vis λmax (261.9 nm) as LMG, the corresponding cationic
13C7-MG exhibited a λmax at 614.4 nm, which represented
a sizable hypsochromic shift (-3.6 nm) from the nonla-
beled MG (618.0 nm). The labeled compounds were used
derivatives listed in Figure 1 have previously been
observed in biological systems and their structures have
been examined primarily by mass spectrometry (8, 9, 11,
12, 22-24). Now, with the synthetic standards in hand,
we were able to analyze their chromatographic (TLC,
HPLC) and spectral (UV/vis, mass, 1H and 13C NMR) data
in more detail and found them to be consistent with the
previously assigned structures. We used 13C7-LMG and
13C7-MG to make spectral assignments for LMG and MG,
whose full characterizations were necessary in order to
elucidate the structures of their N-demethylated ana-
logues. Wherever appropriate, 1H and 13C NMR spectral
assignments were aided by “off-resonance” decoupling
and one-dimensional NOE experiments, considerations
of relative signal intensities and molecular symmetry.
1
The aromatic region of 500 MHz H NMR spectrum of
13C7-LMG in DMSO-d6 was virtually identical to that of
the unlabeled LMG except for the multiplets at 7.07, 6.88,
and 5.29 ppm, which arise from the couplings with 13C7
(Figure 4b). The 5.29 ppm doublet was readily recognized
as that of H7 due to a large one bond coupling (1J C-H
)
125.98 Hz) with 13C7. The remaining signals at 6.88 and
7.07 were assigned to H2,6,2′,6′ (four protons) and H2′′,6′′ (two
protons), respectively, which are three bonds away from
13C7. Because of the symmetry, all 13 aromatic protons
of the molecule appeared in five groups of signal in a 2:1:
2:4:4 ratio. The most upfield high intensity doublet at
1
extensively to aid the H and 13C spectral assignments
of MG and LMG, and their N-demethylated metabolites
(see below).
NMR Str u ctu r e Ch a r a cter iza tion . All of the N-
demethylated LMG and some of the corresponding MG

Synthesis of Malachite Green Metabolites
Ta ble 1. 1H NMR Ch em ica l Sh ifts (p p m ) a n d Cou p lin g Con sta n ts (Hz) of 13C7-LMG a n d 13C7-MGa
H2,6,2′,6′ H3,5,3′,5′ H2′′,6′′ H3′′,5′′ H4′′ H7
6.88 6.63
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 291
N(CH3)2
2.82
13C7-LMG
7.07
7.25
7.15
5.29
(J C7-H2 ) 2.0)
(J H3-H2 ) 9.2)
(J H2′′-H3′′ ) 7.6)
(J C7-H2′′ ) 4.1)
(J H2′′-H4′′ ) 1.4)
7.36c
(J H3′′-2′′ ) 7.6)
(J H4′′-H3′′ ) 7.4)
(J C7-H7 ) 125.98)
(J H2-H3 ) 9.2)
(J H3′′-H4′′ ) 7.4)
13C7-MGb
13C7-MGd
7.36
7.09
7.62
7.75
3.28
3.42
(J C7-H2 ) 3.1)
(J H2-H3 ) 9.4)
7.46
(J H3-H2 ) 9.4)
(J H2′′-H3′′ ) 7.4)
(J H3′′-H4′′ ) 7.4)
7.65
(J H4′′-H3′′ ) 7.4)
(J H4′′-H2′′ ) 1.2)
7.79
7.14
7.42
(J C7-H2 ) 3.2)
(J H2-H3 ) 9.3)
(J H3-H2 ) 9.3)
(J H2′′-H3′′ ) 7.9)
(J C7-H2′′ ) 3.7)
(J H2′′-H4′′ ) 1.3)
(J H3′′-H2′′ ) 7.9)
(J H3′′-H4′′ ) 7.4)
(J H4′′-H3′′ ) 7.4)
a
b
See Figure 1 for numbering system. Wherever symmetry is involved, only one relevant coupling constant is listed. Measured in
DMSO-d6. c Complete overlap with H2,6,2′6′. No coupling constants could be measured. Measured in acetone-d6.
d
Ta ble 2. 13C NMR Ch em ica l Sh ifts (p p m ) a n d Cou p lin g Con sta n ts (Hz) of 13C7-LMG a n d 13C7-MGa in DMSO-d 6
C1,1′
C2,6,2′,6′
129.34
(J C7-C2 ) 2.2)
139.96
C3,5,3′,5′
112.38
(J C7-C3 ) 3.2)
114.05
C4,4′
C1′′
132.16
(J C7-C1′′ ) 45.1)
139.15
13C7-LMG
13C7-MG
145.33
148.71
(J C7-C1 ) 42.6)a
126.33
156.37
13C7
(J C7-C1 ) 60.1)
(J C7-C2 ) 1.6)
(J C7-C3 ) 4.3)
(J C7-C1′′ ) 51.7)
C2′′,6′′
C3′′,5′′
C4′′
N(CH3)2
41.33
13C7-LMG
13C7-MG
128.00
128.82
125.66
54.19
(J C7-C2′′ ) 2.5)
128.62
(J C7-C3′′ ) 3.0)
134.11
(J C7-H7 ) 126.6)
174.92
132.79
40.62
(J C7-C2′′ ) 3.7)
(J C7-C3′′ ) 4.3)
a
See Figure 1 for numbering system. Wherever symmetry is involved, only one relevant coupling constant is listed.
6.63 ppm was narrowed upon irradiation of the N,N-
dimethyl signals at 2.82 ppm and was thus readily
identified as H3,5,3′,5′. Additional decoupling and COSY
experiments established the connectivities for the AX
(H2,3,5,6; H2′,3′,5′,6′) and AMX networks (H2′′,3′′,4′′,5′′,6′′), thus
completing the 1H assignments. The 1H NMR data of
13C7-MG and 13C7-LMG are summarized in Table 1.
The symmetric nature of 13C7-LMG was also apparent
in the 1H-decoupled 13C NMR spectrum that exhibited
only five protonated and three quaternary aromatic
carbons in addition to the singlet methyl signal at 40.71
ppm (not shown). The assignments are presented in
Table 2. As expected, the 13C7-methane carbon at 54.19
by decoupling experiments, and the results are sum-
marized in Table 2.
Essentially, all of the carbon signals, except for C1,1′
,
were shifted downfield going from the neutral LMG to
the cationic MG (Table 2). The 13C chemical shifts in the
triphenylmethyl cations were interpreted in terms of
charge densities (25-27). The deshielding effect was
largest (-120.73 ppm) on C7, as expected from the
behavior of para-substituted triphenyl cation systems
(25-27). A substantial shielding (+19 ppm) on the
aromatic point of attachment (C1,1′) indicates a buildup
of negative charge, which is a general feature predicted
by theoretical calculations (Table 1) (25, 27). A similar
trend, but more general deshielding effect, was observed
throughout the molecule in the 1H NMR spectrum (Table
1). These results indicate the existence of a resonance
delocalization in the substituted aromatic rings with the
cationic charge density primarily located in the central
C7 as depicted in structure 7.
ppm exhibited a large one bond C-H coupling (1J C-H
)
126.6 Hz) in the 1H-coupled 13C NMR spectrum. The most
downfield signal at 148.71 ppm was affected upon ir-
radiation of the methyl protons and, thus, was assigned
to C4,4′. The two signals at 145.33 and 132.16 ppm were
assigned to C1,1′ and C1′′, respectively, on the basis of their
one bond (1J 13C7-C1,1′ ) 42.6 Hz and J 13C7-C1′′ ) 45.1 Hz)
couplings with 13C7. The remaining aromatic carbon
signals, except for the 125.66 ppm signal, showed small
couplings ranging from 2.1 to 3.2 Hz (Table 2). Irradiation
of the methyl protons collapsed the most shielded doublet
at 112.32 ppm, which was assigned to C3,5,3′,5′. The
observed coupling (J ) 3.2 Hz), therefore, must be due
to a three bond 13C7-C3,5,3′,5′ coupling. The 128.82 ppm
signal, which showed a similar three bond 13C7-C3′′,5′′
coupling (3.0 Hz), was then assigned to C3′′5′′. The signals
at 129.34 and 128.00 ppm showed smaller couplings (2.2
and 2.5 Hz, respectively), with greater intensity for the
former, and were assigned to C2,6,2′6′ and C2′′,6′′, respec-
tively, because two bond C-C couplings are typically
smaller than three bond ones. The remaining low inten-
sity signal at 125.66 ppm was not split and, therefore,
was attributed to C4′′. The signals in the 13C NMR
spectrum of 13C7-MG were assigned in a similar fashion
The NMR assignments of LMG and MG helped in the
elucidation of the structures of their N-demethylated
derivatives. The spectral patterns of the metabolites were
classified into two groups based on their molecular
symmetry. For example, the symmetric dides(sym) and
tetrades derivatives showed simple spectral patterns,
similar to those of MG and LMG (i.e., five magnetically

292 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Cho et al.
equivalent signals for a total of 13 aromatic protons and
five carbon signals for a total of five equivalent proto-
nated aromatic carbons). The unsymmetric monodes,
dides(unsym), and trides derivatives exhibited seven sets
of proton signals for 13 aromatic protons and seven
signals for the seven protonated aromatic carbons. In all
cases, numbers of the methyl and exchangeable protons
were in good agreement with the assigned structures. For
example, the 1H and 13C NMR spectral patterns of dides-
(sym)-LMG were similar to those of LMG, except for the
presence of a D2O exchangeable singlet at 4.83 ppm and
a methyl singlet at 2.74 ppm, which integrated as two
1
and six protons, respectively. The H NMR spectrum of
monodes-LMG, however, showed two separate methyl
signals at 2.74 and 2.88 ppm in a ratio of 1:2 and a broad
singlet D2O exchangeable signal at 4.72 ppm for the NH
proton. The mono- and dimethyl signals appeared sepa-
rately at 30.63 and 40.74 ppm in the 13C NMR spectrum.
Trides-LMG showed the presence of two broad D2O
exchangeable signals at 4.75 and 4.43 ppm in a ratio of
1:2 for the NH and NH2 protons, respectively. As ex-
pected, tetrades-LMG showed a broad singlet at 4.43 ppm
that represented four protons. The structures of the
cationic N-demethylated MG derivatives were elucidated
similarly using the symmetry arguments.
Ma ss Sp ectr a l An a lysis. The synthesis and charac-
terization of the entire series of N-demethylated leuco
and cationic derivatives of MG permitted a systematic
examination of fragmentation patterns using tandem MS
that was not previously possible using single quadrupole
instrumentation. Figure 5 shows the positive APCI
daughter ion mass spectra for the leuco (a) and cationic
(b) series, all obtained at a common collision energy (30
eV). The fragmentation of the leuco species was domi-
nated by consecutive losses of methyl and phenyl moi-
eties. The isomeric dides-LMG species showed identical
mass spectra at all collision energies (18). The mass
spectra for cationic species show individual losses of all
pendant groups; for example, MG lost methyl (m/z 313),
benzene (m/z 251), and N,N-dimethylaniline (m/z 208)
moieties. With the N-demethylated cationic derivatives,
losses of each possible substituted aniline moiety were
also observed; for example, monodes-MG lost the N-
methylaniline and N,N-dimethylaniline moieties to give
m/z 208 and m/z 194, respectively (Figure 5b). Because
of this property, it was possible to assign unambiguously
the structures for isomeric dides-MG. The unsymmetrical
isomer dides(unsym)-MG lost N,N-dimethylaniline to
yield m/z 180 and aniline to yield m/z 208 whereas the
symmetrical isomer lost only N-methylaniline to yield m/z
194. The APCI mass spectra of the cationic species thus
provided more structural information than the corre-
sponding leuco species. Therefore, the use of on-line
postcolumn oxidation of chromatographically separated
leuco species with the PbO2 postcolumn (see below) in
conjunction with APCI/MS can provide unambiguous
identification of all MG-derived products.
F igu r e 5. APCI mass spectra of (a) LMG, (b) MG, and their
N-demethylated derivatives at collision energy 30 eV.
was attached to a column reactor containing 10% PbO2
on Celite. This postcolumn technique, which was first
developed by Allen and Meinertz (28), has been used
widely since LMG and MG can be analyzed simulta-
neously with increased specificity (3, 29).
The overall absorption characteristics of the N-meth-
ylated LMG and MG derivatives are very similar to those
of the parent compounds but showed a systematic hyp-
sochromic shift upon sequential N-demethylation. The
effect appears to follow the general additivity rules (30);
for example, a hypsochromic shift was observed going
from N,N-dimethyaniline (251 nm) to N-methylaniline
(245 nm) and to aniline (233 nm) in methanol (not
shown). A similar systematic, although small, hypsochro-
mic shift (3.5-4.8 nm per methyl) was observed going
from LMG to the fully N-demethylated tetrades-LMG
(Figure 6b). The effect was much greater for the cationic
MG derivatives (Figure 6a, 12.2-13.5 nm per methyl),
indicating the importance of resonance contributions to
UV/vis Sp ectr a l Ch a r a cter istics. The chromato-
graphic and UV/vis absorption characteristics of LMG
and MG derivatives are in good agreement with sequen-
tial N-demethylation, and the details are summarized in
Table 3. Figure 6 shows the normalized on-line UV/vis
spectral overlays of all six MG and LMG derivatives
(Figure 6a,b, respectively). On-line spectra of the cationic
MG derivatives were generated by injecting the corre-
sponding LMG derivatives into a HPLC system, which

Synthesis of Malachite Green Metabolites
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 293
Ta ble 3. Su m m a r y of Ch r om a togr a p h ic a n d Sp ectr a l P r op er ties of LMG a n d MG a n d Th eir N-Dem eth yla ted
Der iva tivesa
compd
LMG (MG)
monodes-LMG (-MG)
dides(sym)-LMG (-MG)
dides(unsym)-LMG (-MG)
trides-LMG (-MG)
TLC Rf valuesb
HPLC tR (min)c
λmax for LMG (nm)
λmax for MG (nm)
ESI-MSd (m/z)
0.70 [0.53, green]
22.17 [24.54]c
21.08 [23.04]
19.85 [21.36]
19.81 [21.55]
18.44 [19.71]
16.87 [17.96]
261.9, 308.0(sh)
258.4, 298.7(sh)
253.6, 299.9(sh)
252.5, 298.0(sh)
248.9, 288.6(sh)
244.2, 289.2(sh)
618.0, 424.6, 316.6
605.8, 418.4, 314.2
593.6, 412.3, 310.6
592.3, 409.9, 310.6
578.9, 405.1, 307.1
566.7, 403.9, 305.9
331 [329]
317 [315]
303 [301]
303 [301]
289 [287]
276 [274]
0.44 [0.47, light green]
0.29 [0.37, dark purple]
0.24 [0.37, purple]
0.11 [0.34, dark pink]
0.05 [0.32, pink]
tetrads-LMG (-MG)
a
b
The values in brackets indicate those of the cationic MG derivatives. TLC solvent system ) hexane:benzene:ether:triethylamine
(10:10:3:0.5) for LMG derivatives; dichloromethane:methanol (9:1) for MG derivatives (colors appeared upon standing on the TLC plate).
c HPLC conditions: a 30 min gradient 10-95% acetonitrile and ammonium acetate (pH 4.5), 1.0 mL/min. Spherisorb CN, 5 µm, 4.6 mm
× 250 mm. A postcolumn containing 10% PbO2 in Celite was used for the cationic MG derivatives. (M + H)+ for LMG derivatives; M+
d
for MG derivatives.
Ack n ow led gm en t. We thank D. R. Doerge for valu-
able comments and reading the manuscript. This re-
search was supported in part by an appointment of B.P.C.
to the Faculty Research Participation 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.
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a
toxicological Review. J . Am. Coll. Toxicol. 15, 219-238.
(2) Alderman, D. J . (1985) Malachite green: a review. J . Fish Dis.
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F igu r e 6. On-line HPLC-UV/vis spectral overlays of (a) MG,
(b) LMG, and their N-demethylated metabolites: MG and LMG
(dark green), monodes- (green), dides(unsym)- (cyan), dides-
(sym)- (black), trides- (red), and tetrades- (magenta). The inset
in panel c shows the UV spectra of dides(sym)-LMG and dides-
(unsym)-LMG. HPLC separation of the cationic MG derivatives
was obtained with a photodiode array detector using a Spher-
isorb column (CN, 4.6 mm × 250 mm, 4.6 mm × 250 mm, 5
µm) with a 30 min gradient, 10-90% acetonitrile/100 mM
ammonium acetate, pH 4.5, 1.0 mL/min.
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the cationic MG structure (see above). It is well-known
that the interaction between the nonbonding and the
π-orbitals is most effective in the para-substituted aniline
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While the HPLC retention times of the isomeric dides-
MG and -LMG derivatives (sym and unsym) were very
similar (Table 3), their UV/vis absorption characteristics
were different; for example, the λmax of the symmetric
dides-MG was slightly more red-shifted (1.3 nm) as
compared to the unsymmetric one (Figure 6a). The shift
difference was smaller (<1.1 nm) for the corresponding
leuco derivatives (Figure 6b). The dides(unsym)-LMG
isomer also exhibited a flattened absorption maximum,
which was noticeably different from the sym one (Figure
6c) and other derivatives. As discussed above, the APCI
mass fragmentation could differentiate the isomeric
dides-MG derivatives but not the corresponding leuco
ones.
In summary, we have described convenient synthetic
procedures for preparing MG and LMG and their N-
demethylated derivatives. The availability of the syn-
thetic standards and their thorough spectral character-
ization are important for future metabolic and DNA
adduct studies. The synthetic procedure is general in
scope so that it can be extended to the preparation of
N-demethylated metabolites of other structurally related
triphenylmethane dyes.
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