Chemical models of cytochrome P450 catalyzed insecticide metabolism. Application to the oxidative metabolism of carbamate insecticides

Article abstract of DOI:10.1021/jf980347+

Cytochrome P450 (CP450) catalyzed oxidative metabolism of carbofuran (1), carbaryl (2), and pirimicarb (3) has been modeled using biomimetic oxidations catalyzed by iron(III) tetraarylporphyrins. Oxidation products of 1 were identified by comparison of HPLC retention times measured under standardized conditions for metabolites synthesized and characterized by ¹H and ¹³C NMR spectroscopy. Comparison of product distributions to in vivo metabolic profiles revealed that the H₂O₂/meso- tetrakis(pentafluorophenyl)porphyrin iron(III) chloride [Fe(TF₂₀PP)] system mimics the action of insect CP450s against carbofuran. The effectiveness of this system was further demonstrated by the biomimetic oxidation of other carbamate insecticides (2 and 3) monitored by HPLC/electrospray MS. The predictive power of this biomimetic model was compared to that of knowledgebased expert systems. Although similar models were recently applied in pharmaceutical research, the usefulness of this approach has first been demonstrated for the prediction of metabolic profiles of agrochemicals.

Full text of DOI:10.1021/jf980347+

762
J. Agric. Food Chem. 1999, 47, 762−769
Ch em ica l Mod els of Cytoch r om e P 450 Ca ta lyzed In secticid e
Meta bolism . Ap p lica tion to th e Oxid a tive Meta bolism of Ca r ba m a te
In secticid es
Gyo¨rgy M. Keseru¨,*,† Gyo¨rgy Balogh,† Ire´n Czudor,† Tama´s Karancsi,‡ Andra´s Fehe´r,† and
Be´la Berto´k†
Department of Chemical Research, CHINOIN AGCHEM Business Unit, P.O. Box 49,
H-1780 Budapest, Hungary, and Central Research Institute of Chemistry, Hungarian Academy of Sciences,
P.O. Box 21, H-1521 Budapest, Hungary
Cytochrome P450 (CP450) catalyzed oxidative metabolism of carbofuran (1), carbaryl (2), and
pirimicarb (3) has been modeled using biomimetic oxidations catalyzed by iron(III) tetraarylpor-
phyrins. Oxidation products of 1 were identified by comparison of HPLC retention times measured
1
under standardized conditions for metabolites synthesized and characterized by H and 13C NMR
spectroscopy. Comparison of product distributions to in vivo metabolic profiles revealed that the
H2O2/meso-tetrakis(pentafluorophenyl)porphyrin iron(III) chloride [Fe(TF20PP)] system mimics the
action of insect CP450s against carbofuran. The effectiveness of this system was further demonstrated
by the biomimetic oxidation of other carbamate insecticides (2 and 3) monitored by HPLC/
electrospray MS. The predictive power of this biomimetic model was compared to that of knowledge-
based expert systems. Although similar models were recently applied in pharmaceutical research,
the usefulness of this approach has first been demonstrated for the prediction of metabolic profiles
of agrochemicals.
Keyw or d s: Oxidative metabolism; cytochrome P450; biomimetic oxidation; iron(III) tetraarylpor-
phyrins; carbamate insecticides; metabolite synthesis; HPLC/ electrospray MS
INTRODUCTION
metabolic processes. Simultaneous action of different
isoforms observed in vivo and in vitro provides a
macroscopic view to the system, whereas structural and
theoretical biochemistry, as well as bioorganic chemis-
try, enables an insight at the molecular level. Theoreti-
cal investigations of CP450-catalyzed insecticide me-
tabolism have recently been published from our labora-
tory (Keseru¨ et al., 1997; Keseru¨, 1998).
Cytochrome P450 (CP450) is one of the most impor-
tant systems responsible for the oxidative metabolism
of insecticides (Hodgson et al., 1993). Increased activity
of this enzyme has been linked to the development of
metabolic resistance against formerly active agrochemi-
cals (Feyereisen, 1995; Mulin and Scott, 1992). The first
observation of the involvement of P450 in the resistance
to pesticides was reported by Eldefrawi et al. (1960),
who counteracted carbaryl resistance with the methyl-
enedioxyphenyl P450 inhibitor, Sesamex. Studies on
carbamate-selected houseflies also revealed that resist-
ant strains possessed increased hydroxylation, dealkyl-
ation, and epoxidation potencies relative to susceptible
strains (Hassal, 1990). Resistance management tech-
niques involve the application of new active ingredients,
special formulations, or genetically immunized crops.
In addition to these methods, it has been recognized that
the synergistic action of selective metabolic inhibitors
might be also beneficial to decrease resistance (Lam-
oureux and Rusness, 1995).
Synthetic metalloporphyrins (Montanari and Casella,
1994) were found to be useful as chemical mimics of
peroxidases, whereas the peroxide shunt observed in the
catalytic cycle of CP450 represents a similar transfor-
mation that can also be modeled using synthetic met-
alloporphyrins. Although several biomimetic transfor-
mations for drugs have been successfully performed
using these catalysts (Andersen et al., 1994; Nagatsu
et al., 1990), agrochemical applications of synthetic
metalloporphyrins have been rather limited. To the best
of our knowledge, modeling of the oxidative metabolism
of carbamate insecticides is the first example.
In this work, we studied the biomimetic oxidation of
carbamate insecticides to find answers to the following
questions: (1) Can chemical models be used to mimic
the CP450-catalyzed oxidative metabolism of carbamate
insecticides? (2) Can metabolically sensitive functional
groups be identified in this way? (3) Do these model
systems adequately reproduce experimental metabolic
profiles? To clarify the above problems, metallopro-
phyrin-catalyzed oxidations of carbofuran (1), carbaryl
(2), and pirimicarb (3) in the presence of several
co-oxidants were carried out.
Development of metabolic inhibitors requires an
extensive knowledge of the molecular mechanism of
CP450-catalyzed oxidative reactions. Due to the com-
plexity of reactions catalyzed by P450 mono-oxygenases,
modeling studies are recognized as an important tool
for the understanding of molecular mechanisms of these
* Author to whom correspondence should be addressed (fax
+36-1-4241200; e-mail gykeseru@goliat.eik.bme.hu).
† CHINOIN AGCHEM Business Unit.
‡ Hungarian Academy of Sciences.
10.1021/jf980347+ CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/26/1999

Chemical Models for CP450
J. Agric. Food Chem., Vol. 47, No. 2, 1999 763
EXPERIMENTAL PROCEDURES
6), 127.92, (C-7a), 130.72 (C-7), 137.95 (C-3a); IR νmax 3345,
1730, 1225, 1131 cm-1 (KBr); TLC (diethyl ether/hexane, 2:1)
Rf 0.54; HPLC retention time (min) tR 3.15. Anal. Calcd for
1H and 13C NMR spectra were obtained by a Bruker AC-
200 spectrometer in CDCl3 using tetramethylsilane as an
internal standard. IR spectra were recorded on a Perkin-Elmer
398 spectrometer. UV absorption spectra were recorded on a
Shimadzu UV-160 spectrometer. Reverse-phase HPLC was
used to obtain purity data and retention times for all metabo-
lites of 1. These analyses were performed using a Hewlett-
Packard 1050 system equipped with a Spherisorb 5 ODS-1
column. An acetonitrile/water (40:60) gradient was used with
photometric detection at 220 nm by setting the flow rate to 1
mL/min. Metabolite profiles of 2 and 3 were obtained by HPLC/
MS measurements. An HPLC system equipped with a Sym-
metry C8 (Waters) 3.9 × 150 column, a Spectraphysics P200
gradient pump, a UV100 detector, and a 10 µL Rheodyne
injector was used for these analyses using the eluent system
of eluent A (acetonitrile/water/formic acid, 5:95:0.1) and eluent
B (acetonitrile/water/formic acid, 95:5:0.1) in a gradient of
100% A to 100% B in 20 min. Metabolites were photometrically
detected at 254 nm by setting the flow rate to 1 mL/min. Mass
spectrometry studies were performed by a VG QUATTRO
triplequad spectrometer (Micromass, Manchester, U.K.) with
electrospray ionization. An electronspray source temperature
of 120 °C, a cone voltage of 15 V, and an eluent split of 1:10
were applied. Scan parameters were set to scan over a range
of m/z 10-100 in 1 min. Centroid data were acquired.
Merck Kieselgel F254 TLC plates were used for the moni-
toring of reactions.
Ch em ica ls. All reagents as well as meso-tetrakis(penta-
fluorophenyl)porphyrin iron(III) chloride [Fe(TF20PP)] were
obtained from Aldrich Chemical Co. (Milwaukee, WI). Solvents
were of analytical grade and purchased from Chemolab Ltd.
(Hungary).
7-Hydr oxy-2,2-dim eth yl-2,3-dih ydr oben zofu r an (4). Car-
bofuran (1) was hydrolyzed according to the procedure of Balba
et al. (1968) (3.31 g, 15 mmol) using alcoholic potassium
hydroxide (1.01 g, 18 mmol) to give 4 (1.85 g, 77%): bp 70 °C
(80 Pa);1H NMR δ 1.50 (s, 6H, 2 × CH3), 3.05 (s, 2H, CH2),
5.19 (br s, 1H, OH), 6.73 (s, 3H, Ar); 13C NMR δ 28.52 (2 ×
CH3), 43.92 (C-3), 88.21 (C-2), 114.83 (C-4), 116.91 (C-5), 120.91
(C-6), 127.92 (C-7a), 140.23 (C-7), 146.12 (C-3a); IR νmax 3567,
1625, 1606, 1479, 1300, 1253 cm-1 (CHCl3); TLC [eluent,
benzene/diethyl ether (1:3)] Rf 0.63; HPLC retention time (min)
tR 6.86. Anal. Calcd for C10H12O2: C, 73.14; H, 7.37. Found:
C, 72.84; H, 7.83.
Ben zyloxym eth ylca r ba m ic Acid 2,2-Dim eth yl-2,3-d i-
h yd r oben zofu r a n -7-yl Ester (5). To a solution of 0.82 g (5
mmol) of 4 in dry benzene (25 mL) and triethylamine (0.1 mL)
was added 0.82 g (5 mmol) of benzyloxymethyl isocyanate. The
mixture was refluxed for 18 h and then extracted with diethyl
ether. The organic phase was dried over MgSO4 and evapo-
rated to yield 5 as a colorless solid (1.05 g, 66%): mp 60-62
°C; 1H NMR δ 1.49 (s, 6H, 2 × CH3), 3.05 (s, 2H, CH2), 4.66 (s,
2H, OCH2), 4.85 (d, 2H, NCH2), 5.97 (t, 1H, NH), 6.77, 7.42
(m, 8H, Ar); 13C NMR δ 28.13 (2 × CH3), 43.07 (C-3), 70.26
(NCH2), 72.02 (BzCH2), 88.34 (C-2), 120.11, 121.61, 122.39,
127.74, 127.95, 128.42, 129.54, 137.82 (C-Ar), 134.50 (C-7a),
150.18 (C-3a), 153.34 (CO); IR νmax 3443, 1752, 1482, 1231,
1136 cm-1 (CHCl3). Anal. Calcd for C19H21NO4: C, 69.70; H,
6.43; N, 4.27. Found: C, 69.56; H, 6.32; N, 4.15.
Hyd r oxym eth ylca r ba m ic Acid 2,2-Dim eth yl-2,3-d ih y-
d r oben zofu r a n -7-yl Ester (6). To a solution of 0.32 g (1
mmol) of 5 with CCl4 (20 mL) was added 1 g (5 mmol) of
trimethylsilyl iodide, and the reaction mixture was stirred for
1 h and filtered. The filtrate was quenched with MeOH (4-
fold excess) and stirred overnight. The solvent was then
evaporated and the residue dissolved in diethyl ether, ex-
tracted with aqueous sodium bisulfite, aqueous sodium bicar-
bonate, and aqueous NaCl and dried over MgSO4. Evaporation
yielded 0.14 g (50%) of 6 as colorless crystals: mp 133-135
°C; 1H NMR δ 1.50 (s, 6H, 2 × CH3), 3.04 (s, 2H, CH2), 3.41 (s,
1H, OH), 4.35 (d, 2H, NCH2), 5.38 (s, 1H, NH), 6.74-7.37 (m,
3H, Ar);13C NMR δ 22.92 (2 × CH3), 43.86 (C-3), 73.61
(CH2OH), 89.08 (C-2), 120.91 (C-4), 121.83 (C-5), 121.91 (C-
C
12H15NO4: C, 60.74; H, 6.32; N, 5.90. Found: C, 61.21; H,
6.23; N, 5.81.
2,2-Dim e t h yl-7-m e t h ylca r b a m oyloxyb e n zofu r a n -3-
on e (7). A solution of 1 (4.4 g, 20 mmol) in acetic acid (20 mL)
was oxidized with chromium trioxide (12.16 g, 80 mmol) at
25-30 °C for 16 h with continuous stirring. The reaction
mixture was then quenched with water (30 mL) and extracted
with diethyl ether. Evaporation of the solvent gave 7 (3.00 g,
65%) as colorless crystals: mp 163-165 °C (ethanol);1H NMR
δ 1.48 (s, 6H,2 × CH3), 2.92 (d, 3H, NCH3), 5.19 (s, 1H, NH),
7.00-7.54 (m, 3H, Ar); 13C NMR δ 22.99 (2 × CH3), 27.90
(NCH3), 89.08 (C-2), 121.64 (C-4), 121.79 (C-5), 131.03 (C-6),
123.57 (C-7a), 137.26 (C-7), 153.90 (C-3a), 161.12 (CO), 204.33
[(C-3)O]; IR νmax 3358, 1717, 1620, 1500, 1271 cm-1(KBr); TLC
(diethyl ether/benzene, 3:1) Rf 0.27; HPLC retention time (min)
tR 5.51. Anal. Calcd for C12H15NO4: C, 61.27; H, 5.57; N, 5.95.
Found: C, 60.8; H, 5.2; N, 6.1.
3,7-Dih yd r oxy-2,2-d im eth ylben zofu r a n (8). To a solu-
tion of 5 (0.3 g 1.2 mmol) in dry methanol was added 0.094 g
(2.5 mmol) of sodium borohydride. After the total conversion
of the starting material, the reaction mixture was evaporated
and the residue chromatographed using silica gel 60 [eluent,
diethyl ether/benzene (3:1)] to give 8 (0.08 g, 40%) as a colorless
oil: 1H NMR δ 1.35 (s, 3H, CH3), 1.49 (d, 3H, CH3), 2.11 (d,
1H, COH), 4.76 (d, 1H, CH), 5.75 (s, 1H, ArOH), 6.75, 6.98
(m, 3H, Ar); 13C NMR δ 20.84 (2 × CH3), 79.04 (C-2), 90.66
[(C-3)OH], 117.00 (C-4), 117.72 (C-5), 121.59 (C-6), 128.33 (C-
7a), 140.91 (C-7), 146.12 (C-3a); TLC (diethyl ether/benzene,
3:1) Rf 0.49; HPLC retention time (min) tR 3.46. Anal. Calcd
for C10H12O3: C, 67.11; H, 5.66. Found: C, 66.96; H, 5.51.
Meth ylca r ba m ic Acid 2,2-Dim eth yl-3-h yd r oxy-2,3-d i-
h yd r oben zofu r a n -7-yl Ester (9). To a solution of 7 (0.47 g,
2 mmol) in methanol (10 mL) was added sodium cyanoboro-
hydride (0.16 g, 2.5 mmol). pH was maintained between 3.1
and 3.4 using 5 N aqueous HCl at 45 °C for 3 h. The reaction
mixture was then quenched with water (5 mL), filtered, and
extracted with diethyl ether. The ether extract was dried over
MgSO4, and the solvent was evaporated. The residue was
chromatographed on silica gel 60 using diethyl ether/benzene
(3:1) as gradient. 9 was obtained as colorless crystals (0.16 g,
1
35%): mp 105-108 °C; H NMR δ 1.34 (s, 3H, CH3), 1.48 (s,
3H, CH3), 2.52 (d, 1H, OH), 2.87 (d, 3H, NCH3), 4.71 (d, 1H,
CH), 5.08 (s, 1H, NH), 6.83, 7.25 (m, 3H, Ar); 13C NMR δ 20.74
(2 × CH3), 26.17 (NCH3), 78.54 (C-2), 90.84 [(C-3)OH], 120.79
(C-4), 123.84 (C-5), 124.08 (C-6), 130.87 (C-7a), 135.26 (C-7),
150.90 (C-3a), 154.52 (CO); IR νmax 3574, 1718, 1295, 1124,
910 cm-1 (CHCl3); TLC [eluent, benzene/methanol (5:1)] Rf
0.43; HPLC retention time (min) tR 3.7. Anal. Calcd for C12H15
-
NO4: C, 60.74; H, 6.36; N, 5.90. Found: C, 60.9; H, 6.1; N,
5.7.
7-H yd r oxy-2,2-d im et h ylb en zofu r a n -3-on e (10). Car-
bamate 7 (3.52 g, 15 mmol) was hydrolyzed with alcoholic
potassium hydroxide (1.01 g, 18 mmol in 40 mL of ethanol) at
40-45 °C for 2 h. The reaction mixture was acidified using 5
N aqueous HCl and extracted with diethyl ether. The extract
was then dried over MgSO4, and the solvent was evaporated
to give 10 (2.55 g, 96%) as colorless crystals: mp 162-165 °C;
1H NMR δ 1.49 (s, 6H, 2 × CH3), 5.86 (s, 1H, OH), 6.93, 7.36
(m, 3H, Ar); 13C NMR δ 23.10 (2 × CH3), 89.15 (C-2), 116.20
(C-4), 120.37 (C-7a), 122.52 (C-5), 122.83 (C-6), 142.73 (C-7),
159.20 (C-3a), 204.33 [(C-3)O]; IR νmax 3566, 1716, 1621, 1453,
1288, 1257, 1124, 918 cm-1 (CHCl3); TLC (diethyl ether/
benzene, 3:1) Rf 0.58; HPLC retention time (min) tR 4.62. Anal.
Calcd for C10H10O3: C, 67.41; H, 5.66. Found: C, 67.16; H,
5.51.
Ben zyloxym eth ylca r ba m ic Acid 2,2-Dim eth yl-3-oxo-
2,3-d ih yd r oben zofu r a n -7-yl Ester (11). To a solution of 10
(0.89 g, 5 mmol) in dry benzene (25 mL) and triethylamine
(0.1 mL) was added 0.82 g (5 mmol) of benzyloxymethyl
isocyanate. The mixture was refluxed for 18 h and extracted
with diethyl ether. The organic phase was dried over MgSO4
and evaporated to yield 11 as a colorless solid (1.53 g, 90%):

764 J. Agric. Food Chem., Vol. 47, No. 2, 1999
Keseru¨ et al.
mp 119-121 °C; 1H NMR δ 1.48 (s, 6H, 2 × CH3), 4.67 (s, 2H,
ArCH2), 4.85 (d, 2H, NCH2), 5.99 (s, 1H, NH), 6.02, 7.57 (m,
8H, Ar); 13C NMR δ 22.99 (2 × CH3), 70.50 (NCH2), 72.12
(BzCH2), 89.21 (C-2), 121.83, 122.02, 122.39, 127.89, 128.52,
130.79, 137.58 (Ar), 136.93 (C-7a), 150.18 (C-3a), 153.64 [(C-
3)O], 203.41 [(C-3)O]. Anal. Calcd for C19H19NO5: C, 66.84;
H, 5.56; N, 4.10. Found: C, 66.4; H, 5.3; N, 4.1.
Ch a r t 1
Hyd r oxym eth ylca r ba m ic Acid 2,2-Dim eth yl-3-oxo-2,3-
d ih yd r oben zofu r a n -7-yl Ester (12). To a solution of 0.52 g
(1.5 mmol) of 5 in CCl4 (20 mL) was added trimethylsilyl iodide
(1.5 g, 7.5 mmol), and the reaction mixture was stirred for 1 h
and filtered. The filtrate was quenched with MeOH (4-fold
excess) and stirred overnight under argon atmosphere. The
solvent was then evaporated, and the residue was dissolved
in diethyl ether, extracted with aqueous sodium bisulfite,
aqueous sodium bicarbonate, and aqueous NaCl, and dried
over MgSO4. Evaporation of the solvent yielded 0.2 g (60%)
1
12 as colorless crystals: mp 141-145 °C; H NMR δ 1.48 (s,
6H, 2 × CH3), 3.43 (s, 1H, OH), 4.75 (d, 2H, NCH2), 5.98 (s,
1H, NH), 7.02, 7.57 (m, 3H, Ar);13C NMR δ 22.95 (2 × CH3),
73.91 (CH2OH), 89.17 (C-2), 121.79 (C-4), 121.97 (C-5), 127.98
(C-6), 130.79 (C-7), 120.91 (C-7a), 137.85 (C-3a), 153.85 [(C-
3)O], 204.16 [(C-3)O]; IR νmax 3348, 1752, 1405, 1095 cm-1
(CHCl3); TLC [eluent, diethyl ether/benzene (3:1)] Rf 0.47;
HPLC retention time (min) tR 6.85. Anal. Calcd C12H13NO5:
C, 57.36; H, 5.17; N, 5.57. Found: C, 57.2; H, 5.2; N, 5.6.
m eso-Tetr a k is(2,6-d ich lor op h en yl)p or p h yr in Ir on (III)
Ch lor id e [F e(TCl8P P )]. A solution of 2,6-dichlorobenzalde-
hyde (13.5 g, 77.1 mmol), pyrrole (5.35 mL, 77.1 mmol), and
zinc acetate (5.35 g, 23.2 mmol) in 2,6-lutidine (116 mL) was
placed in a 250 mL round-bottom flask equipped with a Soxhlet
extractor. The extractor was filled with dried Na2SO4, and the
reaction mixture was refluxed for 5 h. The solvent was
evaporated, and the residue was dissolved in toluene and kept
at 0 °C overnight. The purple precipitate was collected to give
Zn(TCl8PP) (14.88 g, 20%). The free porphyrin base was
obtained by treating the solution of Zn(TCl8PP) (1.86 g, 2
mmol) in dichloromethane (250 mL) with trifluoroacetic acid
(19 mL) for 6 h at room temperature. The reaction mixture
was extracted with aqueous NaHCO3 and water, the organic
layer was separated, and 50 mL of MeOH was added and
evaporated. The residue was recrystallized from MeOH to give
the free base (0.41 g, 24%), which was transformed to Fe(TCl8-
PP) by refluxing its solution in degassed DMF (270 mL) with
FeCl2‚4H2O (2.04 g, 2 mmol) for 4 h. Air was bubbled through
the resulting brown solution for 10 h followed by evaporation.
The residue was dissolved in 1 N HCl, and the dark brown
participate was filtered off to yield Fe(TCl8PP) (0.18 g, 57%).
All intermediates and the product were identified by compar-
ing their UV absorption spectra to literature data.
Gen er a l P r oced u r e of Biom im etic Oxid a tion s. Car-
bamate insecticide (1.1 mmol) and 0.01 molar equiv of the
catalyst were dissolved in 30 mL of the solvent, and 0.4 molar
equiv of the oxidizing agent was added. The reaction mixture
was stirred for 30 min at room temperature and the solvent
evaporated. The residue was directly analyzed by HPLC.
Oxidations effected by mCPBA were performed in dichloro-
methane; NaOCl and H2O2 were used in methanol/dichloro-
ethane (1:1).
Ch a r a cter iza tion of Meta bolites Obta in ed by th e Bio-
m im etic Oxid a tion of P ir im ica r b (3). 5,6-Dimethyl-2-
(methylformamido)-4-pyrimidinyl dimethylcarbamate (15) and
5,6-dimethyl-2-(methylamino)-4-pyrimidinyl dimethylcarbam-
ate (17) were isolated from the mCPBA-mediated biomimetic
oxidation of 3 catalyzed by Fe(TF20PP). Preparative TLC
[eluent, diethyl ether/benzene (3:1)] was used for the purifica-
tion of samples identified by MS and 1H and 13C NMR
spectroscopy.
CON(CH3)2], 7.37 (br m, 1H, NH-CH3); 13C NMR δ 10.49 (5-
CH3), 22.44 (6-CH3), 25.94 (CONHCH3), 36.79 [N(CH3)2],
107.10 (5-C), 152.81 (CONHCH3), 160.62 (2-C), 168.01 (4-C),
177.97 (6-C).
RESULTS AND DISCUSSION
Oxidative metabolism of carbamate insecticides has
been studied in plants, insects, and mammals (Kuhr and
Dorough, 1976). In this study we selected the com-
mercially most important carbamate insecticides, which
are shown in Chart 1.
On the basis of radiolabeling studies, the major
metabolite of carbofuran (1) was identified as 3-hy-
droxycarbofuran (7), along with N-hydroxymethyl (6)
and 7-hydroxy analogues (4) as minor components
(Metcalf et al., 1968). These compounds can be further
transformed to the corresponding 3-keto (7), 3,7-dihy-
droxy (8), and 3-keto-7-hydroxy (9) metabolites before
conjugation and excretion (Figures 1 and 2). Oxidative
metabolites of carbaryl (2) were identified as the cor-
responding N-hydroxymethyl carbamate and metabo-
lites hydroxylated at the aromatic ring (Figure 3)
(Dorough and Casida, 1964; Knaak et al., 1965). Oxida-
tive biotransformation of pirimicarb (3) involves de-
methylation of the dimethylamine moiety and oxidation
of the methyl group at C-6 (Figure 4) (The Pesticide
Manual, 1997).
Because the two other commercially most important
carbamate insecticides, furathiocarb and carbosulfan
(Chart 1), are also transformed to 1 along their detoxi-
fication pathway (The Pesticide Manual, 1997) oxidative
metabolism of carbofuran is of outstanding importance.
In addition to this, the huge amount of experimental
data published on the metabolism of carbofuran also
suggested that 1 be selected as the first test compound
15: MS (M + 1), 253; 1H NMR δ 2.12 (s, 3H, 5-CH3), 2.47 (s,
3H, 6-CH3), 3.05 and 3.11 [s, 3H, CON(CH3)2], 3.35 (s, 3H,
NH-CH3), 9.75 (s, 1H, CHO); 13C NMR δ 10.50 (5-CH3), 22.43
(6-CH3), 36.77 [CON(CH3)2], 115.00 (5-C), 152.80 [CON(CH3)2],
163.84 (NCHO), 164.22 (2-C), 168.87 (4-C), 178.15 (6-C).
17: MS (M + 1), 225; 1H NMR δ 2.04 (s, 3H, 5-CH3), 2.34 (s,
3H, 6-CH3), 2.93 (br d, 3H, NHCH3), 3.04 and 3.12 [s, 3H,

Chemical Models for CP450
J. Agric. Food Chem., Vol. 47, No. 2, 1999 765
under standardized conditions were used to identify
products obtained from biomimetic oxidations of 1.
Biom im etic Oxid a tion s. Halogenated meso-tetra-
arylporphyrins are known as efficient chemical models
mimicking CP450-catalyzed oxidative transformations
(Dolphin et al., 1997). It has been demonstrated by
Dolphin et al. that meso-tetrakis(2,6-dichlorophenyl)-
porphyrin iron(III) chloride [Fe(TCl8PP)] is one of the
most effective catalysts for biomimetic oxidations (Tray-
lor et al., 1984). Fe(TCl8PP) was therefore synthesized
using an improved procedure recently published by
J ohnstone and Chorghade (J ohnstone et al., 1996;
Chorghade et al., 1996). The reactive ferryloxy inter-
mediate was generated using a number of oxidizing
agents such as alkyl peroxides, peracides, iodosylarenes,
and inorganic oxidants (potassium peroxosulfate, so-
dium hypochlorite, hydrogen peroxide). Investigating
the mechanism of alkyl peroxide mediated oxidations
MacFaul et al. (1997) showed recently that it was the
RO• radical rather than the iron(IV)oxy porphyrin which
oxidized the substrate. Considering the high oxidation
potential of iodosylarenes, these compounds are very
likely to oxidize carbamates even in the absence of
catalysts. Biomimetic oxidation of 1-3 was therefore
carried out using m-chloroperbenzoic acid (mCPBA),
sodium hypochlorite, and hydrogen peroxide in the
presence of Fe(TCl8PP). For 1, reaction mixtures were
analyzed by HPLC and oxidation products were identi-
fied using carbofuran metabolites as internal standards
(Table 1). For 2 and 3, reaction mixtures were injected
to an HPLC/MS system and metabolites were identified
on the basis of molecular ions and fragmentations. In
the case of 3, the structures of main metabolites were
F igu r e 1. Metabolism of 1 in insects.
for the evaluation of our chemical models. An improved
synthesis of carbofuran metabolites has been developed
to obtain analytical standards for HPLC measurements.
1
also verified by H and 13C NMR spectroscopy. Product
distributions were calculated as a difference between
oxidations performed with and without the catalyst. All
reactions were repeated three to five times using the
protocol described under Experimental Procedures.
Data collected in the tables were calculated as the
average of experiments. The error is (0.5% of the
reported value.
Biomimetic Oxidation of 1. Comparing the results
with the metabolite profile measured in houseflies
(Musca domestica), we found that in contrast to in vivo
experiments, hydrolysis of the carbamate side chain
took place in all systems. In the case of NaOCl, due to
the alkaline medium, this hydrolysis became dominant.
In addition to the major metabolite 9, oxidation at the
C3 center yielded the 3-keto metabolite (7) as well.
Oxidations associated with the simultaneous hydrolysis
of the carbamate group led to the formation of products
(8 and 12) derived by multistep transformations.
It has been recently reported that fluorinated metallo-
porphyrins are even more efficient and more selective
catalysts in biomimetic oxidations (Chorghade et al.,
1994). Oxidations catalyzed by the most popular meso-
tetrakis(pentafluorophenyl)porphyrin iron(III) chloride
[Fe(TF20PP)] were therefore carried out to improve the
performance of our model (Table 1). Use of mCPBA as
oxidant resulted in the almost selective formation of 12.
Oxidations with H2O2, however, reproduced rather well
the in vivo profile. Fair agreement between our model
and the in vivo process is possibly due to the fact that
in biological systems H2O2 could also substitute for
NADPH and molecular oxygen in the catalytic cycle of
P450s (the peroxide shunt). Increased resistance of
Fe(TF20PP) against oxidative degradation may be re-
Syn th esis of Ca r bofu r a n Meta bolites. The first
synthesis of carbofuran metabolites has been reported
by Balba et al. (1968). Our synthetic strategy is depicted
in Figure 2. 4 was obtained by hydrolysis of 1 using
alcoholic KOH and was transformed to 5 by reacting it
with benzyloxyisocyanate (Balba et al., 1968). Removal
of the benzyloxy protecting group was first carried out
by hydrogenation using 10% Pd/C, which yielded only
13 corresponding to the cleavage of the N-CH2 bond.
Debenzylations effected by 5 N HCl or BF3‚Et2O/EtSH
(Fuji et al., 1979) also led to 13 selectively. Breaking
the O-CH2 bond was finally achieved by trimethylsilyl
iodide (J ung and Lyster, 1977) to give 6.
Metabolites 8-10 and 12 were prepared from 3-keto-
carbofuran (7) obtained by the regioselective oxidation
of 1 with chromium trioxide. Hydrolysis of 7 gave the
3-keto-7-hydroxy metabolite (10), which was trans-
formed to 12 via the corresponding benzyloxy derivative
11 by treatment with trimethylsilyl iodide. In contrast
to the original synthesis (Metcalf et al., 1968), the
treatment of 1 with acetic acid gave the corresponding
3-acetoxycarbofuran in rather low yield. The major
metabolite (9) was therefore synthesized from the 3-keto
analogue (7) by reducing the carbonyl group with
sodium cyanoborohydride under acidic conditions. Re-
duction of 7 with sodium borohydride resulted in the
simultaneous hydrolysis of the carbamate side chain to
give 8 as the major product. In conclusion, our improved
strategy allowed the preparation of all 3-oxy metabolites
from a single precursor. Metabolites were identified on
the basis of their 1H and 13C NMR spectra and were
analyzed by HPLC. HPLC retention times measured

766 J. Agric. Food Chem., Vol. 47, No. 2, 1999
Keseru¨ et al.
F igu r e 2. Synthesis of the oxidative metabolites of 1.
sponsible for differences between product distributions
observed in Fe(TCl8PP)- and Fe(TF20PP)-catalyzed reac-
tions. The success achieved in the Fe(TF20PP)-catalyzed
biomimetic oxidation of 1 prompted us to use this
catalyst for the oxidative transformation of 2 and 3 as
well.
Biomimetic Oxidation of 2. Fe(TF20PP)-catalyzed oxi-
dation of 2 was performed by H2O2 and mCPBA.
Reactions performed in the presence of mCPBA gave
only traces of oxidation products; however, monitoring
H2O2-mediated reactions revealed the conversion of the
starting material. Oxidation products were identified by
direct comparison of TLC results [eluent, diethyl ether/
hexane (4:1)] to that of the known metabolites (Dorough
and Casida, 1964). In addition to the significant amount
of 2, the main product was identified as the correspond-
ing N-hydroxycarbamate (14), whereas ring hydroxyl-
ated products were detected only in traces by MS
(Figure 3). Comparing the in vivo metabolite profile to
that obtained by the chemical model, it is apparent that
ring hydroxylated metabolites (4-hydroxy, 5-hydroxy,
and 5,6-dihydroxy) were produced only in trace by the
biomimetic system. The limited rate of aromatic hy-
droxylations can be interpreted by the fact that in
metalloporphyrin-catalyzed oxidations the aromatic moi-
ety remains usually unchanged. Because only activated
aromatics can undergo biomimetic oxidation (Artaud et
al., 1993; Keseru¨ et al., 1999), the reduced formation of
ring hydroxylated product is a limitation of the model
system with respect to enzymatic processes.
Biomimetic Oxidation of 3. Product distributions
detected in H2O2- and mCPBA-promoted oxidation of 3
catalyzed by Fe(TF20PP) are presented in Table 2.
Oxidation products were identified by HPLC/electro-

Chemical Models for CP450
J. Agric. Food Chem., Vol. 47, No. 2, 1999 767
Ta ble 2. P r od u ct Distr ibu tion s (P er cen t) Obta in ed by
th e Biom im etic Oxid a tion of 3a
oxidizing agent
M + 1
metabolite
mCPBA
H2O2
253
255
255
241
225
271
239
15
19
18
20
17
21
3
47.5
4.9
23.8
-
13.9
13.0
20.7
-
-
-
-
9.1
67.1
F igu r e 3. Products obtained by the biomimetic oxidation of
2.
-
a
Data were calculated as the average of three to five experi-
ments. Dashes stand for compounds not detected.
Ta ble 1. P r od u ct Distr ibu tion s (P er cen t) Obta in ed by
th e Biom im etic Oxid a tion of 1a
(16) (Benezet and Matsumura, 1977), a carbamate
insecticide substituted by a dimethylamino group at the
aromatic ring. Oxidative metabolism of 16 leads to the
corresponding N-demethylated analogue via the forma-
tion of this N-formyl intermediate. Identification of the
N-formyl metabolite of 3 suggests this mechanism to
be involved in the oxidative demethylation of the N,N-
dimethylamino group of 3 in vivo. Our proposal was
supported by the mCPBA/Fe(TF20PP)-catalyzed oxida-
tion of 3. In addition to the previously described 15, the
corresponding N-demethylated compound (17) could also
be detected. Because 15 and 17 are of crucial importance
with respect to the metabolism of 3, these compounds
were isolated by preparative TLC and were identified
by their 1H and 13C NMR spectra. In conclusion, we
suggest that demethylation of the N,N-dimethyl moiety
in 3 proceeds via 15, which has not yet been detected
in vivo. From a comparison of the structures of 3 and
16, it is clear that the N,N-dimethyl group is the
metabolically most sensitive function in 3, whereas the
monomethyl group at the carbamate nitrogen of 16
represents an alternative site for metabolic oxidation.
The N,N-dimethyl group of 3 is therefore rapidly de-
methylated in vivo, which is indicated by the lack of 15
from the in vivo metabolite profile.
Fe(TCl8PP)
Fe(TF20PP)
metab-
olite
M. domestica
tR
NaOCl mCPBA H2O2 mCPBA H2O2
in vivo
3.1
3.4
3.7
4.6
5.5
6.8
7.8
6
8
9
10
7
12
1
35.7
12.9
14.8
-
-
3.7
0.0
1.2
9.1
6.2
15.7
-
-
-
-
0.5
0.2
9.7
-
23.7
-
5.4
-
22.1
-
3.4
-
42.3
18.3
14.0
6.8
-
-
54.7
-
34.1
64.0
4.6
2.2
52.8
-
67.0
a
All data are differences between product distributions obtained
with and without the catalysts and were calculated as the average
of three to five experiments. Dashes stand for compounds not
detected.
spray MS, which was found to be useful in residue
analysis (Volmer, 1998). In addition to its sensitivity
and selectivity, electrospray ionization MS was found
to be optimal for the analysis of oxidation products.
Because degradation of these compunds might occur
using CI and EI techniques, atmospheric pressure
chemical ionization (APCI) was first applied. Degrada-
tion observed using this relatively soft ionization tech-
nique prompted us to use electrospray ionization for the
MS analysis of reaction mixtures obtained by the
biomimetic oxidation of 3. Oxidations performed by the
H2O2/Fe(TF20PP) system yielded the corresponding N-
formyl derivative (15) as the main component (Figure
4). In accordance with in vivo data, the dimethyl
carbamate group was unchanged. Formation of 15 was
rationalized by the oxidation of a methyl group at the
amine moiety. Although 15 has not yet been isolated
as a metabolite of 3, similar N-formyl compounds were
detected in the oxidative metabolism of mexacarbate
The formation of pyrimidine N-oxides (18 and 19) was
also revealed by their characteristic MS fragmentation.
This reaction was more characteristic in the presence
of mCPBA, where the corresponding N-demethylated
N-oxide (19) could also be isolated. Because mCPBA is
known as an oxidizing agent used for the N-oxidation
of heterocycles, the formation of N-oxides is not unex-
pected (Brown, 1994). Note, however, that the formation
F igu r e 4. Products obtained by the biomimetic oxidation of 3.

768 J. Agric. Food Chem., Vol. 47, No. 2, 1999
Keseru¨ et al.
of 1,3-N-oxides was not detected in these reactions.
Although N-oxidations cannot be affected by H2O2, a
considerable amount of 1,3-N-oxide was identified in
H2O2/Fe(TF20PP)-mediated reactions. This unexpected
reaction is related to the catalytic activity of the
Fe(TF20PP) metalloporphyrin catalyst and can be used
for the preparation of pyrimidine 1,3-N-oxides.
cally susceptible functional groups, this system mimics
the action of insect CP450s on carbamate insecticides.
From a comparison of our results to knowledge-based
predictions, it is apparent that our methodology can be
considered as an equally useful tool for predicting
oxidative metabolites and, furthermore, permits the
modeling of in vivo metabolic profiles semiquantita-
tively.
Com p a r ison t o Kn ow led ge-Ba sed P r ed ict ion s.
Knowledge-based metabolic information systems were
recognized as useful tools for the analysis of metabolic
data. In addition to Metabolite (Metabolite, 1997), the
complete metabolism information system of MDL used
for metabolite data management prediction of metabolic
pathways is also possible. MetabolExpert, a unique
knowledge-based expert system for the metabolite pre-
diction of drugs, pesticides, and xenobiotics, can be used
at the initial stage of metabolic research (Darvas, 1988).
Maps of metabolites obtained by this program could help
in metabolite searching and interpretation of metabolite
profiles. Testing our chemical model system, the oxida-
tive metabolites of 1-3 were predicted by Metabol-
Expert implemented to the Pallas package (Pallas,
1996). Predicted metabolites of 1 involved 3-OH (9),
4-OH and 6-OH derivatives, and also a derivative
demethylated at the carbamate nitrogen. The formation
of 9 is in accordance with in vivo data; other hydroxyl-
ated products were isolated only in vitro. In contrast to
knowledge-based predictions, demethylation at the car-
bamate nitrogen was not detected either in vivo or in
biomimetic oxidations. Furthermore, the corresponding
N-hydroxycarbamate (6) was not predicted by Metabol-
Expert; however, it was identified as a major oxidation
product both in vivo and in model systems.
2-OH, 4-OH, 6-OH, and 7-OH analogues, as well as
3,4-, 5,6-, and 7,8-epoxides, were predicted to be the
metabolites of 2. 2- and 4-naphthols were also detected
in biological systems. Epoxides were expected to form
corresponding diols in the next stage of metabolism.
Although the chemical model showed only limited
performance on aromatic hydroxylation, a mixture of
these diols could also be isolated from biomimetic
oxidations. This is in accordance with the formation of
the 5,6-dihydroxy metabolite observed in vivo. Predicted
demethylation of the N-methylcarbamate moiety is in
contrast to in vivo data; the nonpredicted in vivo
hydroxylation at this N-methyl group, however, was
reproduced by the biomimetic system.
ABBREVIATIONS USED
mCPBA, m-chloroperbenzoic acid; [Fe(TF20PP)], meso-
tetrakis(pentafluorophenyl)porphyrin iron(III) chloride;
[Fe(TCl8PP)],
meso-tetrakis(2,6-dichlorophenyl)por-
phyrin iron(III) chloride.
ACKNOWLEDGMENT
We are grateful to Dr. F. Darvas (ComGenex) for an
evaluation copy of the Pallas 2.0 package.
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