Microsomal metabolism of N,N-diethyl-m-toluamide (DEET, DET): The extended network of metabolites

Article abstract of DOI:10.1080/004982599238588

The aim was to set out to establish the complete network of metabolites arising from the phenobarbital-treated rat liver microsomal oxidation of N,N-diethyl-m-toluamide (DEET). The products formed from DEET and all its subsequent metabolites were identifi

Full text of DOI:10.1080/004982599238588

xenobiotica , 1999, vol. 29, no. 4, 409±416
M icrosomal metabolism of
,
-diethyl-
-
m
N N
toluamide (DEET, DET) : the extended network of
m etabolites
LUIS CONSTANTINO‹ and JIM ILEYŒ*
‹
Œ
CECF, Faculdade de Farmacia, Avenida das For as Armadas, 1600 Lisbon, Portugal
Chemistry Department, The Open University, Milton Keynes, MK7 6AA, UK
Received 25 August 1998
1. The aim was to set out to establish the complete network of metabolites arising from
the phenobarbital-treated rat liver microsomal oxidation of N,N-diethyl-m-toluamide
(DEET). The products formed from DEET and all its subsequent metabolites were
identi®ed by HPLC retention times, UV spectroscopy, mass spectrometry and by
comparison with authentic standards.
1a
1b
and N-ethyl-m-(hydroxymethyl)benzamide
), and, at low substrate concentrations or extended reaction times, two minor
1c 3a 1b 2a
2. DEET ( ) produces three major metabolites, N-ethyl-m-toluamide ( ), N,N-
2a
)
diethyl-m-(hydroxymethyl)benzamide
2b
(
(
metabolites, toluamide ( ) and N,N-diethyl-m-formylbenzamide ( ).
and
are
primary metabolites and their formation follows Michaelis-Menten-type kinetics. At low
DEET concentrations, ring methyl group oxidation is favoured ; at saturation concen-
trations, methyl group oxidation and N-deethylation proceed at similar rates. The rate of
2b
2b
formation of
metabolite of DEET and DEET acts as a competitive inhibitor of the metabolism of
2a
decreases with increasing DEET concentration ;
is therefore a secondary
1b
and
.
3. Except for the primary amides, where N-dealkylation is impossible, metabolism of
1b,c 2a-c 3a-c 4a,b
all subsequent compounds,
,
,
and
, involves an N-deethylation (NEt
#
! NHEt or NHEt ! NH ) competitive with a ring substituent oxidation (CH ! CH OH,
#
$
are also reduced to
#
3a-c
CH OH ! CHO or CHO ! CO H). Surprisingly, the aldehydes
#
#
2a-c
the corresponding alcohols
3a-c 2a-c
continues uninhibited.
(CHO ! CH OH); CO inhibits the oxidative metabolism
#
of
, but reduction to
4. The outcomes of this work are that (1) previously unreported aldehydes
form part of the DEET network of metabolites, (2) the reduction of the aldehydes
3b
3a-c
3c
has
and
the potential to inhibit the formation of the more highly oxidized DEET metabolites, (3)
amide hydrolysis was not observed for any substrate and (4) no evidence was obtained for
N-(1-hydroxyethyl)amide intermediates.
Introduction
N,N-diethyl-m-toluamide (DEET)
1a
(®gure 1) is generally considered the
most eåective insect repellent in world-wide use (Robbins and Cherniack 1986), and
is active against the yellow fever mosquito (Aedes aegypti) and the malaria mosquito
(Anopheles quadrimaculatus) (Gilbert 1966). DEET has been in extensive use for 40
years, and is generally considered of low toxicity. However, there have been several
reports of toxic eåects, including encephalopathy and anaphylaxis, related to the use
of DEET (Robbins and Cherniack 1986), though it is unclear whether these eåects
are elicited by DEET or by one or more of its metabolites. DEET has at least two
diåerent, readily oxidizable groups, namely the amide N,N-diethyl substituents and
the aromatic methyl substituent. Previous studies involving rat liver microsomes
!
* Author for correspondence : E-mail: j.n.iley open.ac.uk
‹
Reprint requests to : Dr J. Iley, Chemistry Department, The Open University, Walton Hall,
Milton Keynes MK7 6AA UK.
Xenobiotica ISSN 0049±8254 print}ISSN 1366±5928 online ’ 1999 Taylor & Francis Ltd
http:}}www.tandf.co.uk}JNLS}xen.htm http:}}www.taylorandfrancis.com}JNLS}xen.htm

410
L. Constantino and J. Iley
Figure 1. Pathways of DEET metabolism. The horizontal direction represents N-dealkylation ; the
vertical direction represents ring-methyl oxidation.
1b 1c 2a 2b
3a
(®gure 1) as metabolites (Taylor
have identi®ed compounds
1986, Yeung and Taylor 1988), and an in vivo study (Taylor and Spooner 1990)
4a c
,
,
,
and
identi®ed
- , as well as m-toluic acid, as urinary metabolites.
Our understanding of DEET metabolism, and of the metabolites that may
contribute to its toxicological eåects, remains incomplete. Consequently, to
elucidate further the net of pathways of DEET metabolism, we have undertaken a
study of the metabolism of DEET and each of its subsequent metabolites by
phenobarbital-treated rat liver microsomes. Phenobarbital is a known inducer of

Microsomal metabolism of DEET
411
P450 isozymes that catalyse both amide dealkylation (Hall and Hanzlik 1990) and
toluene benzylic oxidation (Wang and Nakajima 1991). The method has involved
incubation of DEET with rat liver microsomes, identi®cation of the metabolites so-
formed, synthesis of the metabolites, subsequent incubation of each metabolite with
rat liver microsomes and identi®cation of the products formed. This process was
repeated with each secondary metabolites until no further metabolism was observed.
This approach has enabled us to identify (1) metabolites that are formed directly
from DEET, (2) compounds that are formed from the subsequent transformation of
DEET metabolites and (3) previously unknown metabolites. It has also enabled us
to establish the diåerent pathways of metabolite formation.
M aterials and m ethods
Substrates and metabolites
All compounds were puri®ed prior to use and were analytically pure by HPLC and gave satisfactory
1a
microanalyses (C, H, N‰ 0.3%). N,N-Diethyl-m-toluamide , m-toluamide
1c
and m-toluic acid were
1b
Schotten-Baumann method (Furniss et al. 1978). N,N-Diethyl-m-(hydroxymethyl)benzamide
purchased from Aldrich Chemical Co. (Dorset, UK). N-Ethyl-m-toluamide
, was synthesized by the
2a
and
2b
N-ethyl-m-(hydroxymethyl)benzamide
Research Station, Alberta) and puri®ed by semipreparative reverse-phase HPLC. m-(Hydroxy-
2c 3c
.
were a kind gift from Dr Wesley Taylor (Canada Agriculture
methyl)benzamide
was synthesized by sodium borohydride reduction of m-formylbenzamide
3a 3b 3c
N,N-Diethyl-m-formylbenzamide , N-ethyl-m-formylbenzamide
and m-formylbenzamide
were
obtained from m-formylbenzoic acid by activation with dicyclohexylcarbodiimide and subsequent
addition of the corresponding amine (Taylor and Spooner 1990). m-Formylbenzoic acid was synthesized
from m-toluic acid (Irrevedere et al. 1961).
4a
m-(Diethylaminocarbonyl)benzoic acid
1a
was synthesized by the oxidation of N,N-diethyl-m-
(7.65 g, 0.04 mol) in water (125 ml), KMnO (3.17 g,
%
1a
toluamide
as follows. To a suspension of
0.02 mol) was added. The suspension was heated at 80 ÊC for 1 h, after which the addition of
permanganate and the heating were repeated. The suspension was then cooled and NaOH was added to
dissolve the product. After ®ltration, the solution was extracted with dichloromethane and the aqueous
phase was acidi®ed. The product was extracted with chloroform, and the extract washed with water, dried
cm- (KBr disc)
"
4a
m
(magnesium sulphate), and evaporated to aåord
d
in 31 % yield : m.p. 128±129 ÊC ;
}
max
3090, 1716, 1609, 1434, 734, 708 ;
}
(CDCl ) 1.23 (6H, t), 3.43 (4H, m), 7.33±8.23 (4H, m), 11.8 (1H,
$
H
s) ; m z (%) 221 (29) (M+ ), 220 (63), 150 (15), 149 (100), 121 (20), 76 (12), 65 (30). Found: C 65.3 ; H 6.8;
N 6.1 % C
H NO requires: C 65.1 ; H 6.8 ; N 6.3 %.
"# "& $
4b
m-(Ethylaminocarbonyl)benzoic acid
was isolated in 10 % yield as a secondary product from the
4a
preferentially soluble in chloroform whereas
4a
is
synthesis of
. Separation of the two from the reaction mixture proved possible because
is soluble in ethyl acetate : m.p. 227±230 ÊC ;
cm-
"
max/
4b
m
d
3301, 1690, 1638, 1545, 1421, 730, 690 ;
(%) 193 (24) (M+ ), 192 (33), 166 (18), 165 (6), 149 (100), 121 (23), 76 (11), 65 (28), 50 (11), 28 (16).
Found: C, 61.9 ; H, 5.4 ; N, 7.05 %. C NO requires: C, 62.2 ; H, 5.7 ; N, 7.25 %.
(CDCl ) 1.16 (3H, t), 3.33 (2H, m), 7.33±8.66 (4H, m); m z
}
H
$
H
"!
4c
""
$
m-(Aminocarbonyl)benzoic acid
m
was synthesized in 41 % yield by analogous KMnO oxidation of
"
%
1c
d
H
: m.p. 287±289 ÊC (decomp.);
cm- 3474, 3190, 1700, 1634, 1558, 1398, 747, 690 ;
(CDCl )
$
}
m ax
3.52 (2H, s br), 7.57±8.54 (4H, m), 13.22 (1H, s) ; m z (%) 165 (71) (M+ ), 150 (11), 149 (100), 121 (40),
}
76 (16), 75 (12), 65 (48), 50 (24), 44 (17). Found: C, 58.1 ; H, 4.3 ; N, 8.0%. C H NO requires : C, 58.1;
)
(
$
H, 4.3 ; N 8.5 %.
1b
4c
4b
4a
Compounds
and
were recrystallized from ethanol. The acid
was recrystallized from
2a 2b
were
chloroform n-hexane ;
was recrystallized from ethyl acetate :n-hexane. Compounds
puri®ed by semi-preparative HPLC using a C-18 column and an eluant comprising acetonitrile-water
2c
and
}
(85 :25). Compound
was puri®ed by chromatography on silica using ethyl acetate as an eluant. The
3a-c
aldehydes
eluant.
were puri®ed by chromatography on silica using dichloromethane-ethyl acetate (4: 1) as
Microsomes
Ten-week-old male Wistar rats were injected intraperitoneally with phenobarbital (80 mg.kg- day
-
"
"
)
for 4 days, starved for 1 day and killed by decapitation 24 h after the last injection. The livers were
m
m
removed, immersed in ice-cold Tris-HCl buåer (50 m ) containing KCl (154 m ) minced, and washed
with the same solution. The mince was homogenized with a Te¯on-glass homogenizer immersed in ice,
using 3 ml washing solution per g liver. The homogenate was centrifuged at 10 000g for 20 min at 4 ÊC.

412
L. Constantino and J. Iley
The pellet was discarded and the supernatant centrifuged again at 100 000 g for 1 h at 4 ÊC. The resultant
m
supernatant was discarded and the pellet resuspended in pH 7.4 phosphate buåer (68 m ) containing
–
m
NaCl (74 m ) (PBS) and stored at 80 ÊC. Protein concentration was determined by the method of
Lowry et al. (1951). A cytochrome P450 content of 1.50 nmol (mg protein)- was determined using the
CO diåerence method (Omura and Sato 1964a, b).
"
Product quanti®cation
Microsomal incubations were performed in pH 7.4 PBS using 3.1 mg protein ml- microsomes,
"
+
6.25 m glucose 6-phosphate, 1.25 m NADP , 5 m magnesium chloride and 2.5 U ml- glucose 6-
"
m
m
m
phosphate dehydrogenase. Reactions were initiated by the addition of the substrate dissolved in pH 7.4
m
PBS. Initial substrate concentrations varied between 0.1 and 10 m . Reactions were quenched by the
m
m
barium chloride and centrifuged. The amide
sequential addition of 0.20
content of the supernatant was determined by HPLC using a Jones C-18 chromatography column (5
particle size, 25 cm) and an eluant consisting of either (1) 15 % acetonitrile in pH 2.6 phosphate buåer
1c 2b 2c 3c 4c
zinc sulphate and 0.16
l
m
m
(0.05 ) for compounds
,
,
,
and , or (2) a linear gradient starting with 15 % acetonitrile in
m
m
pH 2.6 phosphate buåer (0.05 ) reaching 42 % acetonitrile in pH 2.6 phosphate buåer (0.05 ) after
22 min for the remaining compounds. Detection was achieved using a diode array detector, and
quanti®cation performed at 229 nm.
Reaction kinetics
Triplicate incubations were performed as above. Aliquots (100 l) were withdrawn at timed intervals
l
m
l
m
and quenched by the sequential addition of 0.2 m zinc sulphate (400 l) and of 0.16 m barium chloride
l
(400 l). After centrifugation, the supernatant (750 l) was treated with 1 NaOH (200 l) for 10 min,
acidi®ed with
l
m
l
m l
HCl (250 l) and analysed by HPLC. Alternatively, for the presence of N-
1
hydroxyalkyl-N-alkylamides, the supernatant was analysed directly by HPLC without NaOH treatment.
Initial rates were determined from the linear phase of the reaction (the ®rst 16 min) and correlated with
substrate concentration via the Michaelis-Menten equation using a non-linear least-squares method. For
reactions carried out in the presence of CO, CO was generated from formic and sulphuric acids (Furniss
et al. 1978) and bubbled into the incubation mixture for 1 min prior to the addition of substrate.
Isolation of microsomal metabolites for mass spectrometric identi®cation
m
Substrates (10 m ) were subjected to microsomal metabolism as described above. Reactions were
monitored by HPLC and every 2 h further aliquots of glucose 6-phosphate, NADP+ and glucose 6-
phosphate dehydrogenase were added until 50 % of the substrate had been consumed (between 6 and
8 h). After termination, by the addition of zinc sulphate and barium chloride and centrifugation, the
supernatant solutions were extracted with ethyl acetate (33 10 ml), the organic phase dried (sodium
sulphate), evaporated, the residue redissolved in acetonitrile, and the metabolites separated by preparative
HPLC using a C18 column with acetonitrile-water (85 :15) as eluant.
Results and discussion
The microsomal incubation of DEET gives rise to three metabolites, the
1b
2a
, derived from
monoethylamide
oxidation of the ring methyl group, and , derived from both ring methyl oxidation
1b, 2a 2b
, derived from N-deethylation, the alcohol
2b
and N-deethylation. A plot of initial rate, vi , for the formation of
1b 2a
and
interpolation of
in table 1. Clearly, at low DEET
versus DEET concentration is shown in ®gure 2. For
the data gives rise to the Vmax and Vmax
and
K
}
m
concentrations oxidation of the methyl group is preferred, whereas at saturating
concentrations of DEET both ring methyl oxidation and N-deethylation proceed at
1b
2a
2b
2b
similar rates. In contrast with
a decrease of vi with increasing DEET concentration, consistent with
1b 2a
and
, the pro®le for the formation of
exhibits
being a
secondary metabolite of DEET that can arise from
,
or from them both.
1b 2a
Independent microsomal incubation of authentic samples of
and
is indeed formed from both compounds. The kinetic constants for the formation
2b 1b 2a
reveals that
2b
of
from
and from
are shown in table 1. Either in terms of Vmax or Vmax
K
}
m
2a
1b
or . Thus
these data reveal that DEET is metabolized more readily than either

Microsomal metabolism of DEET
413
Table 1. Kinetic constants obtained from the microsomal oxidation of DEET.
Product
2a
1b
2b
2b
2a
1b
)
(from DEET)
(from DEET)
(from
)
(from
Vmax (nmol min mg protein)
}
10.3‰ 0.1
1.5‰ 0.2
6.8
11.8‰ 0.1
3.8‰ 0.5
3.1
2.5‰ 0.2
10.2‰ 1.6
0.25
7.7‰ 0.5
12.1‰ 0.2
0.6
}
km (10-
Vmax
m
"
m
)
k
(10- min mg protein)
&
}
}
}
m
Data are the mean ‰ SEM for three determinations.
1b
2a
2b
) and (inset) (E ) from DEET.
Figure 2. Substrate-dependency for the formation of
At low DEET concentrations oxidation of the methyl group is preferred ; at saturating
(+ ),
(
U
concentrations of DEET, ring methyl oxidation and N-deethylation proceed at similar rates.
2b
Increasing DEET concentration inhibits the formation of
.
DEET eåectively acts as a competitive inhibitor of the subsequent oxidation
1b 2a 2b
with
reactions of
increasing DEET concentration.
At low substrate concentrations ( 0.5 mm), or with longer incubations, two
3a 1c,
and
. Hence the decrease in the rate of formation of
!
further minor metabolites, the aldehyde
and the toluamide
were identi®ed in
1b
microsomal incubations of DEET. In comparison with the oxidation of both
and
, these compounds can arise from the subsequent microsomal oxidation of
1b
2a
2b
to
2a
and
respectively. Indeed, the incubation of an authentic sample of the alcohol
3a 2b
2a
leads to the formation of
as well as
. Similar incubation of the
1c 2b
as well as
1b
monoalkylamide
produces the primary amide
. Thus, the origin
of the three minor metabolites in the microsomal incubation of DEET involves the
1b 2a
. Independent
sequential metabolism of the primary metabolites
experiments revealed that DEET was metabolized at least three times faster than
1b 2a 1c 3a
and
either
or
and the absence of
and
at the higher concentrations of DEET
1b 2a
can be attributed to competitive inhibition of
and
by DEET itself. At low
DEET concentrations, DEET metabolism depletes its concentration such that its
ability to inhibit further metabolism of the primary metabolites is diminished.
4a-c,
Since the urinary metabolites of DEET are the acids,
we were interested to
ascertain the pathways leading to these highly oxidized products, especially as the

414
L. Constantino and J. Iley
Figure 3. N-Ethyl-N-(1-hydroxyethyl)-m-toluamide.
2a
3a
in the
‹
m
Table 2. Initial rates obtained from the independent incubations of 3.5 m DEET,
and
Œ
absence or presence of CO
.
Initial rates (nmol product min mg protein)
}
}
Ring substituent
oxidation
Ring substituent
reduction
Dealkylation
–
–
–
Substrate
CO
1CO
CO
1CO
CO
±
1CO
1a
10.3
(‰ 0.4)
0.2
(‰ 0.04)
8.7
(‰ 0.6)
1.9
(‰ 0.2)
±
±
2a
3a
1.9
(‰ 0.2)
0.08
(‰ 0.02)
4.6
(‰ 0.5)
0.2
(‰ 0.03)
±
0.8
(‰ 0.2)
0.1
(‰ 0.03)
8.1
(‰ 1.7)
0.73
(‰ 0.1)
7.6
(‰ 0.7)
6.8
(‰ 0.6)
‹
Œ
Mean (‰ SD) of three determinations.
Independent incubations were also performed in the absence of NADPH and all reactions,
including the reduction reaction, were inhibited (data not shown).
3b
3c
have not previously been reported as DEET metabolites.
aldehydes
and
Therefore we studied the incubation of authentic samples of the secondary
1c 2b
3a
2c 3b 3c 4a
4b
2c
metabolites
,
and
as well as subsequent metabolites
,
,
,
and
.
.
1c
Incubation of the primary amide
gave only one product, the alcohol
Taylor and Spooner (1990) have reported m-toluic acid in the urine of rat treated
with DEET. Under the conditions of our experiments, we did not observe formation
1a-c
of this product, which could arise from hydrolysis of any of the amides
, even
when the cytochrome P450-catalysed reactions were inhibited by CO. We have
previously reported that, for certain N,N-dialkylbenzamides, hydrolysis is a minor,
competing pathway observed during microsomal incubations (Iley and Constantino
1994). However, the only amides that were found to hydrolyse at rates comparable
with the microsomal oxidation were N-cyclopropyl-N-methylbenzamide and N-
cyclopropylbenzamide.
2b
2c 3b
(from further N-dealkylation) and
Incubation of
(from oxidation of the hydroxymethyl group to aldehyde). Incubation of
3c 3b 3c
are identi®ed for the
gave two products;
2c
gave
only one metabolite, the aldehyde . The aldehydes
and
®rst time as potential DEET metabolites; the compounds isolated from the
microsomal incubations had identical characteristics, (namely HPLC retention
time, UV spectra and EI mass spectra, data not shown) to authentic samples.
3a
2a 3b
4a 3b
.
Microsomal incubation of the aldehyde
gave three products,
, the products of N-dealkylation and aldehyde oxidation respectively, were
2a
,
and
4a
and
expected. However, , the product of aldehyde reduction, was not. This reduction
3b 3c
,
pathway was also observed during separate incubations of the aldehydes
and
with microsomes in the
2b 2c 3a
the products being
and
respectively. Incubation of
presence of CO reveals that the reduction pathway remains unaåected, whereas the
rates of the N-dealkylation and ring methyl oxidation reactions are signi®cantly
reduced (table 2), as expected for cytochrome P450-mediated reactions. Aldo-keto

Microsomal metabolism of DEET
415
reductases are a class of cytoplasmic enzymes present in microsomes that possess
broad substrate speci®cities; they require NADPH as a cofactor but, unlike
cytochrome P450, are not inhibited by CO (Low and Castagnoli 1979). This
reduction reaction may play an important role in vivo because it has the potential to
2a c
are transformed into the corresponding
retard the rate at which the alcohols
-
carboxylic acids. Moreover, these alcohols are polar compounds that increases the
possibility of their excretion either unchanged or in a conjugated form. Alternatively,
if the alcohols are not excreted rapidly, then the partial reversal of the ring methyl
oxidation route aåords greater importance to the N-dealkylation pathway.
3b
3c
4b
Oxidation of
yields
(from aldehyde reduction). Similarly, microsomal oxidation of
4c 2c
(from N-dealkylation),
(from aldehyde oxidation)
2b
3c
and
carboxylic acid
via N-dealkylation while
4c
yields the
4a 4b
and the alcohol . Finally, microsomal incubation of
yields
. No further
upon incubation with microsomes was apparent.
4b 4c
itself was further dealkylated to
transformations of
Figure 1 summarizes the complete network of pathways for the oxidative
metabolism of DEET by phenobarbital-treated rat liver microsomes obtained from
this study. DEET metabolism is characterized by two main pathways : one (vertical)
due to sequential oxidation of the aromatic substituent ; the other (horizontal), due
to amide dealkylation. We did not ®nd any products resulting from the hydrolysis of
4c
the amides in our study, even from
which might be expected to be a substrate for
any hydrolytic pathway since the competing oxidation reactions are avoided.
Wu et al. (1979) proposed N-ethyl-N-(1-hydroxyethyl)-m-toluamide (®gure 3)
as an intermediate of DEET metabolism, but subsequently Taylor (1986) was
unable to identify this compound in the microsomal incubation of DEET. We, too,
have been unable to identify this or any other carbinolamide either as metabolites of
2a 3a
4a
or . Indeed, carbinolamides have only
DEET or of the N,N-diethylamides
,
been observed as intermediates in the microsomal oxidation of N,N-dimethylamides
(Constantino et al. 1992). For amides in which the N-alkyl groups are larger than
methyl the derived carbinolamide is too unstable to be isolated (Ross et al. 1983,
Bundgaard and Johansen 1984) and rapidly decomposes to the N-dealkylated
amide.
Acknow ledgem ents
The authors thank Dr Wesley Taylor from the Canada Agriculture Research
Station, Alberta, for the kind gift of N,N-diethyl-m-(hydroxymethyl)benzamide
and N-ethyl-m-(hydroxymethyl)benzamide. L. C. thanks JNICT FCT Portugal
}
for ®nancial support through grant BD 1061 ID.
}
}
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