5806
`
`J. Med. Chem. 2002, 45, 5806-5808
`
`Isotopic Effect Study of Propofol Deuteration on the Metabolism, Activity, and
`Toxicity of the Anesthetic
`
`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.
`
`Introduction
`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.
`
`* To whom correspondence should be addressed. Phone: (33) 4 73
`27 29 52. Fax:
`(33) 4 73 27 98 61. helfenbein@inserm484.u-
`clermont1.fr.
`† ORPHACHEM.
`‡ UMR INSERM 484.
`
`Scheme 1a
`
`(a) ICl/AcOH; (b) D2/Pd; (c) DCl/D2O; (d) HNO3/
`a Reagents:
`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.
`
`Chemistry
`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
`
`10.1021/jm020864q CCC: $22.00 © 2002 American Chemical Society
`Published on Web 11/20/2002
`
`Downloaded via REPRINTS DESK INC on July 23, 2019 at 15:36:10 (UTC).
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`Brief Articles
`
`Journal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5807
`
`Table 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 (cid:2) HD50) Dose on Mice, LRR (Loss of Righting Reflex), RW (Recovery to Walking), Loss of Sensibility
`(LS), and Loss of Painful Sensibility (LPS)
`LD50,
`HD50,
`Vmax,
`Km,
`LPS,
`LS,
`RW,
`LRR,
`mg kg-1
`mg kg-1
`nmol mg-1 min-1
`(cid:237)M
`min
`min
`min
`min
`TI
`3.1 ( 0.6b
`4.4 ( 0.7b
`8.8 ( 3.2b
`6.8 ( 1.6b
`12.3 ( 3.8a
`1.6 ( 0.2a
`1
`2.1
`32
`15
`2.1 ( 0.7b
`3.3 ( 1.0b
`6.4 ( 1.9b
`5.2 ( 1.4b
`1.6 ( 0.3a
`16.7 ( 4.1a
`4
`3
`39
`13
`2.3 ( 0.9b
`3.8 ( 1.3b
`7.3 ( 3.0b
`5.4 ( 1.8b
`1.7 ( 0.3a
`16.4 ( 2.8a
`5
`2.7
`35
`13
`a The results are expressed as means and standard deviations of four determinations from four separate microsomal experiments. The
`differences in the Km and Vm among 1, 4, and 5 are not statistically significant (Student’s t test). b 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
`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-
`lphenol 9 were obtained at low isotopic dilutions (3%,
`6%, and 3%, respectively). The compounds were fully
`characterized by 1H NMR, 13C NMR, mass spectrom-
`etry, and microanalysis data.
`In Vitro Experiments and GC-MS Analysis
`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 (cid:2) 10-8)x2
`+ (1.73 (cid:2) 10-4)x + 1.47 (cid:2)10-2 (r ) 0.999) for the
`4-hydroxy-2,6-diisopropylphenol 2 and y ) (6.15 (cid:2)
`10-9)x2 + (2.55 (cid:2) 10-4)x + 3.44 (cid:2) 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
`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-
`
`Figure 1. Arene oxidative pathway in microsomal oxidation
`of aromatic substrate.
`
`tion (following the characteristic ion at m/z 339), owing
`to a NIH-shift mechanism17 (Figure 1), was not found.
`The incubation of propofol-d3 5 likewise produced only
`one metabolite, 3,5-dideuterium-4-hydroxy-2,6-diisopro-
`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 (cid:237)M (Table 1).
`
`In Vivo Experiments
`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 (cid: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.
`
`Discussion
`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 (cid:237)M), 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
`
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`5808 Journal of Medicinal Chemistry, 2002, Vol. 45, No. 26
`
`Brief Articles
`
`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.
`
`Conclusion
`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.
`
`Supporting Information Available: 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.
`
`References
`(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.
`
`(6) Nelson, S. D.; Pohl, L. R.; Trager, W. F. Primary and (cid:226)-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.; Jarman, 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.
`(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.
`(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 (cid:226)-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.
`JM020864Q
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