Isotopic effect study of propofol deuteration on the metabolism, activity, and toxicity of the anesthetic

Article abstract of DOI:10.1021/jm020864q

The use of isotopic substitution to delay the oxidative metabolism of the anesthetic propofol 1 was studied. The aromatic hydrogens of propofol 1 were replaced by deuterium to produce the mono- and trideuterated derivatives 4 and 5. In vitro metabolic stu

Full text of DOI:10.1021/jm020864q

5806
J . Med. Chem. 2002, 45, 5806-5808
Isotop ic Effect Stu d y of P r op ofol Deu ter a tion on th e Meta bolism , Activity, a n d
Toxicity of th e An esth etic
J . Helfenbein,*,† C. Lartigue,‡ E. Noirault,‡ E. Azim,† J . Legailliard,† M. J . Galmier,† and J . C. Madelmont‡
ORPHACHEM, Rue Montalembert, BP 184, 63005 Clermont-Ferrand, France, and UMR INSERM 484,
Rue Montalembert, BP 184, 63005 Clermont-Ferrand, France
Received February 25, 2002
The use of isotopic substitution to delay the oxidative metabolism of the anesthetic propofol 1
was studied. The aromatic hydrogens of propofol 1 were replaced by deuterium to produce the
mono- and trideuterated derivatives 4 and 5. In vitro metabolic studies on human hepatic
microsomes showed no isotopic effect in the para hydroxylation of propofol, and 1, 4, and 5
display similar hypnotic activity and toxicity in mice.
Sch em e 1a
In tr od u ction
Propofol 1 (2,6-diisopropylphenol) is a short-acting
hypnotic agent used for inducing and maintaining
anesthesia. It is intravenously administered either by
repeated bolus injections or by continuous injection.1
The exact neurochemical mechanism of action of pro-
pofol remains unclear. However, it is well-known that
the general neurochemical mechanism of anesthetics
undergoes interactions between the anesthetic and the
GABA receptor.2
The propofol is quickly and widely distributed into
the body, intensively metabolized, and eliminated. Thus,
88% of the initial dose is found in the urine within 5
days and 2% in the feces.3 The major metabolic pathway
of propofol is its glucuronidation consuming 50-60% of
the total dose. The second metabolic pathway is the para
hydroxylation of propofol producing compound 2 (Scheme
1). The final metabolic pathways are the 1- and 4-glu-
curonidation or the 4-sulfation of the metabolite 2.4 All
these metabolic pathways reduce or even suppress the
propofol activity.
The oxidative metabolism of propofol at low concen-
trations involves cytochrome CYP2C9 (at least 50%) and
other isoforms such as CYP2A6, 2C8, 2C18, 2C19, and
1A2. The role of the latter group of cytochromes is
amplified with increasing concentrations of propofol and
decreasing concentrations of CYP2C9. For this reason,
the metabolism of propofol has low interindividual
variability and low interactions with other drugs.5
Drugs labeled with stable isotopes can be used as
ideal internal standards in quantitative studies, and
stable isotopes having no isotopic effect such as 13C or
15N are then preferred. However, the deuterium isotope
is often employed. Indeed, it may induce some modifica-
tions of the chemical and physicochemical properties of
the labeled drugs (polarity, molar volume, electron
donation, van der Waals forces, dipolar moment, and
lipophilicity) and therefore may contribute to the modi-
fication of metabolism kinetics and biological properties
such as biodistribution and affinity for the receptors.
a
Reagents: (a) ICl/AcOH; (b) D2/Pd; (c) DCl/D2O; (d) HNO3/
AcOH; (e) Sn/HCl; (f) H2SO4/NaNO2; (g) Na2S2O4/NaOH; (h) DCl/
D2O.
Various biological consequences of deuterium labeling
can be observed. Thus, lower metabolism kinetics has
been established for the N-deethylation of deuterated
lidocaine6 and for the debenzylation of deuterated
1-benzyl-4-cyano-4-phenylpiperidine7 while the para
hydroxylation of the phenytoin8 (5,5-diphenylhydantoin)
is not modified by deuteration of the metabolism site.
Concerning the in vivo biological properties, the
deuterated amphetamines (phenyl-2-aminopropane) have
a lower locomotor activity9 than the protio compound,
the deuterated 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso-
urea (CCNU) has the same cytotoxic activity10 as the
protio compound, whereas the inhibitory activity on the
gastric secretion of N,N-dimethyl-N′-[2-(diisopropylami-
no)ethyl]-N′-(4,6-dimethyl-2-pyridyl)urea is increased by
deuterium substitution.11 Accordingly, we set out to
investigate the influence of deuterium labeling on the
metabolism and the pharmacological activity and toxic-
ity of propofol.
Ch em istr y
Monodeuterated propofol 4 was synthesized by a
catalyzed (Pd/C) exchange reaction under D2 atmo-
sphere from 4-iodo-2,6-diisopropylphenol 3, which was
obtained as previously described12 by direct iodination
* To whom correspondence should be addressed. Phone: (33) 4 73
27 29 52. Fax: (33)
clermont1.fr.
4 73 27 98 61. helfenbein@inserm484.u-
† ORPHACHEM.
‡ UMR INSERM 484.
10.1021/jm020864q CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/20/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5807
Ta ble 1. Km and Vm on Human Hepatic Microsomes, HD50 (Hypnotic Dose), LD50 (Lethality Dose), and TI (Therapeutic Index) on
Mice, Effects of a 24 mg/kg (2 × HD50) Dose on Mice, LRR (Loss of Righting Reflex), RW (Recovery to Walking), Loss of Sensibility
(LS), and Loss of Painful Sensibility (LPS)
Vmax
,
Km,
µM
HD50
,
LD50
,
LRR,
min
RW,
min
LS,
min
LPS,
min
nmol mg-1 min-1
mg kg-1
mg kg-1
TI
1
4
5
1.6 ( 0.2a
1.6 ( 0.3a
1.7 ( 0.3a
12.3 ( 3.8a
16.7 ( 4.1a
16.4 ( 2.8a
15
13
13
32
39
35
2.1
3
2.7
6.8 ( 1.6b
5.2 ( 1.4b
5.4 ( 1.8b
8.8 ( 3.2b
6.4 ( 1.9b
7.3 ( 3.0b
4.4 ( 0.7b
3.3 ( 1.0b
3.8 ( 1.3b
3.1 ( 0.6b
2.1 ( 0.7b
2.3 ( 0.9b
a
The results are expressed as means and standard deviations of four determinations from four separate microsomal experiments. The
b
differences in the Km and Vm among 1, 4, and 5 are not statistically significant (Student’s t test). The results are expressed as means
and standard deviations of 10 determinations.
of propofol 1 (Scheme 1). The trideuterated propofol 5
was synthesized by an exchange reaction in DCl/D2O
at 140 °C under pressure. The synthesis of 4-hydroxy-
2,6-diisopropylphenol 2 was undertaken by a Sandm-
eyer reaction from 4-amino-2,6-diisopropylphenol 7,
which was prepared as previously described12 by nitra-
tion of propofol 1 and reduction of 2,6-diisopropyl-4-
nitrophenol 6. The Sandmeyer reaction leads to a
mixture of 4-hydroxy-2,6-diisopropylphenol 2 and 2,6-
diisopropyl-1,4-quinone 8, which was reduced to 4-hy-
droxy-2,6-diisopropylphenol 2. 3,5-Dideuterium-4-hy-
droxy-2,6-diisopropylphenol 9 was synthesized by an
F igu r e 1. Arene oxidative pathway in microsomal oxidation
of aromatic substrate.
exchange reaction in DCl/D2O at 140 °C under pressure.
The monodeuterated propofol 4, the trideuterated pro-
pofol 5, and 3,5-dideuterium-4-hydroxy-2,6-diisopropy-
tion (following the characteristic ion at m/z 339), owing
lphenol 9 were obtained at low isotopic dilutions (3%,
to a NIH-shift mechanism17 (Figure 1), was not found.
The incubation of propofol-d3 5 likewise produced only
6%, and 3%, respectively). The compounds were fully
1
characterized by H NMR, 13C NMR, mass spectrom-
one metabolite, 3,5-dideuterium-4-hydroxy-2,6-diisopro-
etry, and microanalysis data.
pylphenol 9.
Propofol 1 and its deuterated derivatives 4 and 5
displayed similar kinetics of metabolism. The Michae-
lis-Menten plots gave mean values of Vm ) 1.6 ( 0.3
nmol mg-1 min-1 and Km ) 15.1 ( 3.6 µM (Table 1).
In Vitr o Exp er im en ts a n d GC-MS An a lysis
Gas chromatography-mass spectrometry (GC-MS)
has previously been used13-16 for propofol kinetic studies
in plasma16 or whole blood13 and for metabolic studies
on human hepatic microsomes.14 The propofol metabo-
lism was followed by measuring the propofol decrease13
or followed indirectly14 by measuring 2,6-diisopropyl-
1,4-quinone 8, produced from 4-hydroxy-2,6-diisopro-
pylphenol 2 in alkaline conditions. The GC-MS method
selected in the present work includes a derivatization
by silylation16 in order to improve the stability and peak
shapes of compounds in the course of gas chromatog-
raphy.
The metabolism of propofol 1, propofol-d1 4, and
propofol-d3 5 was followed using the appearance of the
related hydroxylated metabolites. A quadratic regres-
sion analysis was carried out between 10 and 10 000
ng/mL 4-hydroxy-2,6-diisopropylphenol 2 or 3,5-dideu-
terium-4-hydroxy-2,6-diisopropylphenol 9, and equa-
tions of the mean plots (n ) 4) were y ) (2.71 × 10-8)x2
+ (1.73 × 10-4)x + 1.47 ×10-2 (r ) 0.999) for the
4-hydroxy-2,6-diisopropylphenol 2 and y ) (6.15 ×
10-9)x2 + (2.55 × 10-4)x + 3.44 × 10-2 (r ) 0.999) for
the 3,5-dideuterium-4-hydroxy-2,6-diisopropylphenol 9.
The incubation of propofol 1 with human microsomes
and NADPH produced only one metabolite, 4-hydroxy-
2,6-diisopropylphenol 2. Indeed, the occurrence of the
metabolite 2,6-diisopropyl-1,4-quinone 8, systematically
followed using the characteristic ion at m/z 149, has
In Vivo Exp er im en ts
The hypnotic activity (HD50) and the toxicity (LD50)
of propofol 1 and of the two deuterated compounds
propofol-d1 4 and propofol-d3 5 were compared on mice.
The speed of induction, the sleeping time, and the
recovery time were noted and joined to a quantitative
assessment of the analgesia for a 24 mg/kg dose (about
2 × HD50) (Table 1). Concerning the hypnotic activity,
propofol-d1 4 and propofol-d3 5 presented slightly lower
HD50 compared to those obtained for the standard
propofol 1. Concerning the toxicity, the deuterated
compounds and mainly propofol-d1 4 were found to be
less toxic than propofol (LD50propofol < LD50propofol-d3
<
LD50propofol-d1). Further experiments performed in mice
at a 24 mg/kg dose showed that the induction times,
the sleeping times, and the recovery times of the three
compounds were similar and that the quality of the
analgesia was preserved. Times of loss of sensibility and
times of loss of painful sensibility were equivalent.
Discu ssion
The in vitro experiments of the three compounds give
mean values of Vm and Km (Vm ) 1.6 ( 0.3 nmol mg-1
min-1 and Km ) 15.1 ( 3.6 µM), which are similar and
in good agreement with those obtained for propofol 1
by Guitton et al.5 The deuterium substitution of the
aromatic hydrogens of propofol 1 does not postpone the
metabolism of the drug. The lack of evidence of an
never been observed. The incubation of propofol-d1
4
produced a single metabolite, 4-hydroxy-2,6-diisopro-
pylphenol 2. The occurrence of 3-deuterium-4-hydroxy-
2,6-diisopropylphenol 10 after propofol-d1 4 administra-

5808 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
Brief Articles
(6) Nelson, S. D.; Pohl, L. R.; Trager, W. F. Primary and â-secondary
deuterium isotope effects in N-deethylation reactions. J . Med.
Chem. 1975, 18, 1062-1065.
(7) McMahon, R. E.; Culp, H. W. Mechanism of the dealkylation of
tertiary amines by hepatic oxygenases. Stable isotope studies
with 1-benzyl-4-cyano-4-phenylpiperidine. J . Med. Chem. 1979,
22, 1100-1103.
(8) Mamada, K.; Kasuya, Y.; Baba, S. Pharmacokinetic equivalence
of deuterium-labeled and unlabeled-phenytoin. Drug Metab.
Dispos. 1986, 14, 509-511.
(9) Najjar, S. E.; Blake, M. I.; Benoit, P. A.; Lu, M. C. Effects of
deuteration on locomotor activity of amphetamine. J . Med.
Chem. 1978, 21, 555-558.
(10) Farmer, P. B.; Foster, A. B.; J arman, M.; Oddy, M. R.; Reed, D.
J . Synthesis, metabolism, and antitumor activity of deuterated
analogues of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. J . Med.
Chem. 1978, 21, 514-520.
(11) Hoffman, J . M.; Habecker, C. N.; Pietruszkiewicz, A. M.;
Bolhofer, W. A.; Cragoe, E. J .; Torchiana, M. L.; Hirschmann,
R. A Deuterium isotope effect on the inhibition of gastric
secretion by N,N-dimethyl-N′-[2-(diisopropylamino)ethyl]-N′-
(4,6-dimethyl-2-pyridyl)urea. Synthesis of metabolites. J . Med.
Chem. 1983, 26, 1650-1653.
(12) Trapani, G.; Latrofa, A.; Massimo, F.; Altomare, C.; Sanna, E.;
Usala, M.; Biggio, G.; Liso, G. Propofol analogues. Synthesis,
relationships between structure and affinity at GABAA receptor
in rat brain, and differential electrophysiological profile at
recombinant human GABAA receptors. J . Med. Chem. 1998, 41,
1846-1854.
(13) Guitton, J .; Desage, M.; Lepape, A.; Degoute, C. S.; Manchon,
M.; Brazier, J . L. Quantitation of propofol in whole blood by gas
chromatography-mass spectrometry. J . Chromatogr. B 1995,
669, 358-365.
isotopic effect in the aromatic hydroxylation of the
propofol indicates that the cleavage of the C-H bond is
not the rate-limiting step.
Concerning the metabolism of the propofol-d1 4, for
any deuterated metabolite detected, the mechanism of
hydroxylation of the propofol avoids using the NIH shift
pathway (Figure 1), which has previously been described
for the aromatic hydroxylation of different molecules
such as phenytoin,18 warfarin,19 and oxprenolol.20
Slight differences between the three analogues of
propofol are observed in the in vivo experiments. The
deuterated compounds (more particularly propofol-d1 4)
seem to have lower HD50, higher LD50, and as a result
higher therapeutic index. However, these differences are
not statistically significant and it may be partly related
to the low sensibility of the pharmacological tests.
Concerning the propofol anesthetic activity (HD50),
our results are in agreement with those obtained by
Anderson et al.21 We also point out that lower toxicity
has already been observed with deuterated compounds
compared to their protio analogues (i.e, amphetamines9
and 2,6-di-tert-butyl-4-methylphenol22), which is inter-
esting for a such a category of short-acting anesthetics
requiring repeated injections.
Con clu sion
(14) Guitton, J .; Burronfosse, T.; Sanchez, M.; Desage, M. Quanti-
tation of propofol metabolite, 2,6-diisopropyl-1,4-quinol, by gas
chromatography-mass spectrometry. Anal. Lett. 1997, 30,
1369-1378.
(15) Elbast, W.; Guitton, M.; Desage, M.; Deruaz, D.; Manchon, M.;
Brazier, J . L. Comparison between gas chromatography-atomic
emission detection and gas chromatography-mass spectrometry
for assay of propofol. J . Chromatogr. B 1996, 686, 97-102.
(16) Stetson, P. L.; Domino, E. F.; Sneyd, J . R. Determination of
plasma propofol levels using gas chromatography-mass spec-
trometry with selected-ion monitoring. J . Chromatogr. B 1993,
620, 260-267.
This work demonstrates that the deuteration proce-
dure, commonly used in quantitative studies with
internal standards or for the identification of metabolic
pathways, does not delay the metabolism kinetics of
propofol. The cleavage of the C-H bond is not the rate-
limiting step in the mechanism of para hydroxylation
of propofol, and this mechanism avoids using the NIH
shift pathway. Additionally, in vivo experiments show
that the deuteration does not abolish anesthetic proper-
ties of propofol.
(17) Langenhove, A. V. Isotope effects: definitions and consequences
for pharmacological studies. J . Clin. Pharmacol. 1986, 26, 383-
389.
(18) Claesen, M.; Moustafa, M. A. A.; Adline, J .; Vandervorst, D.;
Poupaert, J . H. Evidence for an arene oxide-NIH shift pathway
in the metabolic conversion of phenytoin to 5-(4-hydroxyphenyl)-
5-phenylhydantoin in the rat and in man. Drug Metab. Dispos.
1982, 6, 667-671.
(19) Darbyshire, J . F.; Iyer, K. R.; Grogan, J .; Korzekwa, K. R.;
Trager, W. F. Selectively deuterated Warfarin. Substate probe
for the mechanism of aromatic hydroxylation catalyzed by
cytochrome P450. Drug Metab. Dispos. 1996, 24, 1038-1045.
(20) Nelson, W. L.; Bru¨ck, R. T. Metabolism of â-adrenergic antago-
nists. Evidence for arene oxide-NIH shift pathway in the
aromatic hydroxylation of oxprenolol. J . Med. Chem. 1988, 22,
1088-1092.
(21) Anderson, A.; Belelli, D.; Bennett, D. J .; Buchanan, K. I.; Casula,
A.; Cooke, A.; Feilden, H.; Gemmell, D. K.; Hamilton, N. M.;
Hutchinson, E. J .; Lambert, J . J .; Maidment, M. S.; McGuire,
R.; McPhail, P.; Miller, S.; Muntoni, A.; Peters, J . A.; Sansbury,
F. H.; Stevenson, D.; Sundaram, H. R-Amino acid phenolic ester
derivatives: novel water-soluble general anesthetic agents which
allosterically modulate gaba(a) receptors. J . Med. Chem. 2001,
44, 3582-3591.
(22) Mizutani, T.; Yamamoto, K.; Tajima, K. Isotope effects on the
metabolism and pulmonary toxicity of butylated hydroxytoluene
in mice by deuteration of the 4-methyl group. Toxicol. Appl.
Pharmacol. 1983, 69, 283-290.
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal procedure for preparation, physical and spectral charac-
terization (1H and 13C NMR, mass spectrometry, IR spectros-
copy, and elemental analysis) of the synthesized compounds,
and general experimental procedures for in vitro biological
studies on human hepatic microsomes and in vivo biological
studies in mice. This material is available free of charge via
the Internet at http://pubs.acs.org.
Refer en ces
(1) Gepts, A.; Trenchant A. Propofol. Anaesth. Pharmacol. Rev. 1995,
3, 46-56.
(2) Franks, N. P.; Lieb, W. R. Molecular and cellular mechanisms
of general anesthesia. Nature 1994, 367, 607-614.
(3) Simons, P. J .; Cockshott, E. J .; Douglas, E. J .; Gordon, E. A.;
Hopkins, K.; Rowland, M. Disposition in male volunteers of a
subanaesthetic intravenous dose of an oil in water emulsion of
14C-propofol. Xenobiotica 1988, 18, 429-440.
(4) Trapani, G.; Altomare, C.; Sanna, E.; Biggio, J .; Liso, G. Propofol
in anesthesia. Mechanism of action, structure-activity relation-
ships and drug delivery. Curr. Med. Chem. 2000, 7, 249-271.
(5) Guitton, J .; Buronfosse, T.; Desage, M.; Flinois, J . P.; Perdrix,
J . P.; Brazier, J . L.; Beaune, P. Possible involvement of multiple
human cytochrome P450 in the liver metabolism of propofol. Br.
J . Anaesth. 1998, 80, 788-795.
J M020864Q