Synthesis of ¹³C-Lidocaine as a Probe of Breath Test for the Evaluation of Cytochrome P450 Activity

Abstract

¹³C-Labeled lidocaine, 2-di[1-¹³C]ethylamino-N-(2,6-dimethylphenyl)acetamide (1), was synthesized from [1-¹³C]acetic acid in six steps, as a probe for a breath test to evaluate in vivo cytochrome P450 activity. The measurement of ¹³CO₂ in breath was successfully performed following oral administration of ¹³C-lidocaine 1 to mice.

Cytochrome P450s (CYPs) play a crucial role in drug metabolism and bioactivation.^1,2) CYPs have the following two major characteristics: (1) The quantity and the activity of CYPs are considerably different between patients³⁾; (2) CYPs are induced or inhibited by certain medications and food intakes.⁴⁾ Because of these characteristics, to assess CYP activity prior to or during pharmacotherapy has an important role to avoid risks for subtherapeutic or toxic responses to medications.

A large number of selective substrates for distinct CYP enzymes have been investigated as probes to assess enzyme activity in vitro and in vivo.^5,6) In general, the assessment of in vivo CYP activity is performed by administration of a selective probe for the target enzyme and the subsequent determination of the appropriate pharmacokinetic parameters, or by using the metabolism of endogenous substrates.^7,8) Above all, breath tests, where a ¹³C- or ¹⁴C-labeled drug is administered as a probe followed by measurement of the labeled CO₂ in expiration, are advantageous in that the procedures are simple and convenient, particularly in clinical settings. Although such breath tests have been studied for various CYP isoforms,⁹⁾ a breath test for CYP3A4 is of great value because the isoform is the most abundant and versatile. The ¹⁴C-labeled erythromycin (EM) breath test has been well-known as a promising non-invasive method for the quantitative measurement of in vivo CYP3A4 activity.^10–12) In this method, the ¹⁴C-labeled N-methyl group of ¹⁴C-EM is eliminated oxidatively by CYP3A4 and finally exhaled as ¹⁴CO₂ in breath. However, this method has a safety problem for clinical application due to its radioactivity. Thus, the method to use ¹³C-EM instead of ¹⁴C-EM as a probe was developed, where the exhaled ¹³CO₂ in breath is measured with an infrared (IR) spectrometer.¹³⁾ The CYP3A inhibition and induction were quantitatively evaluated by this breath test, in which ¹³C-EM was intravenously administrated to rats. However, the method requires a high dose because of the large molecular weight of ¹³C-EM. In addition, ¹³C-EM is not suitable for oral administration because the drug is susceptible to decomposition by gastric acid, which may hinder the quantitative evaluation of CYP activity. Recently, attention has been paid to the small intestine as an extrahepatic drug-metabolizing organ. CYP3A4 has been reported to be a predominant CYP isoform in human small intestine.^14,15) Thus, another ¹³C-labeled probes are desired for a breath test to evaluate CYP3A4 activity by oral administration .

Lidocaine, a commonly used local anesthetic and antiarrhythmic agent, is rapidly absorbed and metabolized to monoethylglycinexylidide (MEGX) after oral administration. In this biotransformation, the N-ethyl group is oxidatively cleaved by CYPs. As such, a dynamic liver function test based on hepatic CYP activity has been reported.¹⁶⁾ Although two isozymes, CYP3A4 and 1A2, are responsible for the N-deethylation of lidocaine, the biotransformation seems to be exclusively mediated by the former isozyme in the human small intestine where the latter isozyme is virtually undetectable.^14,17) The N-ethyl group eliminated by the CYPs is finally exhaled as CO₂ in breath. Thus, lidocaine, having a ¹³C-label in the N-ethyl group, is a possible substrate for a breath test to measure the CYP activities. However, such ¹³C-lidocaine is not commercially available, and no synthetic method has been reported to date.

The present study was thus undertaken to establish a method for the synthesis of ¹³C-lidocaine. The feasibility of the ¹³C-lidocaine breath test was examined by the oral administration to mice.

Results and Discussion

Breath tests require ¹³C labeling at the position whose carbon is rapidly and efficiently transformed to CO₂ and excreted in breath. Kajiwara et al. synthesized ¹³C-labeled phenacetin for a breath test to evaluate liver function.¹⁸⁾ The labeled compound had ¹³C-label at the C-1 position of O-ethyl group, which was rapidly transformed to ¹³CO₂. As the N-ethyl groups of lidocaine are considered to be metabolized in a similar manner, the C-1 position of the N-ethyl groups was selected as the labeling position for the synthesis of ¹³C-lidocaine, as shown in Chart 1.

Chart 1. Synthesis of ¹³C-Lidocaine 1

The positions of labeling are marked with *.

Lidocaine is generally prepared by the reaction of 2,6-xylidine with chloroacetyl chloride, followed by reaction with diethylamine.^19,20) According to this process, the synthesis of the ¹³C-lidocaine 1 should be conducted using di[1-¹³C]ethylamine as a synthetic intermediate. The di[1-¹³C]ethylamine can be synthesized from an ammonia-equivalent compound by N-diethylation using [1-¹³C]ethyliodide. However, N-diethylation using ethylhalide has a risk of forming polyethylated byproducts and seems to require an excessive amount of relatively expensive [1-¹³C]ethyliodide. Thus, the alternative synthetic process shown in Chart 1 was designed, in which the precursor of di [1-¹³C]ethylamine is synthesized using [1-¹³C]acetic acid as a labeling substrate.

4-Methoxybenzylamine was first converted into amide 2 by condensation with [1-¹³C]acetic acid using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as a coupling reagent.²¹⁾ Reduction of amide 2 with LiAlH₄ proceeded under reflux condition to give corresponding amine 3, and the second condensation with [1-¹³C]acetic acid and reduction gave amide 4 as a mixture of amide rotamers. Amine 5 was then smoothly obtained by LiAlH₄ reduction of amide 4 at room temperature, as the precursor of di[1-¹³C]ethylamine. The total yield of amine 5 was 58% based on the amount of [1-¹³C]acetic acid used in the synthesis of amide 2. The synthesis of ¹³C-lidocaine 1 was achieved by cleavage of the 4-methoxybenzyl group of amine 5 by hydrogenolysis and direct trapping of resulting volatile diethylamine with 2-chloro-N-(2,6-dimethylphenyl)acetamide.

The breath test was performed by oral administration of ¹³C-lidocaine to mice (5 mg/kg). When ¹³CO₂ exhaled in breath was measured after administration using an IR spectrophotometer, the Δ¹³CO₂ values based on the ¹³CO₂/¹²CO₂ ratios promptly increased (T_max=15 min) and gradually decreased, as shown in Fig. 1. These results indicate that ¹³CO₂ was generated by the rapid metabolism of ¹³C-lidocaine. Thus, ¹³C-lidocaine is applicable to the breath test aiming to evaluate CYP activity.

Fig. 1. Time Course of Δ¹³CO₂ (‰) Value after Oral Administration of ¹³C-Lidocaine 1 to Mice (5.0 mg/kg)

Data are expressed as the means with S.D. (n=4).

Conclusion

The synthesis of ¹³C-lidocaine was successfully carried out using [1-¹³C]acetic acid as a labeling reagent. The ¹³CO₂ formed by the metabolism of ¹³C-lidocaine administered to mice was detected by an IR spectrophotometer. The breath test using the ¹³C-lidocaine can be considered to be a promising method to evaluate CYP activity. The effects of selective CYP inhibitors or inducers on the breath response after oral ¹³C-lidocaine administration are presently under investigation.

Experimental

Materials

[1-¹³C]Acetic acid (99.2 atom% ¹³C) was purchased from Cambridge Isotope Laboratories, Inc. and used as received. DMT-MM was prepared from 2-chloro-4,6-dimethoxy-1,3,5-triazine and 4-methylmorpholine according to the method of Kunishima et al.²²⁾

Instruments

Melting points were measured with a Yanako MP-J3 melting point apparatus and was uncorrected. IR spectra of the synthesized compounds were recorded with a JASCO FT-IR-4100 type A spectrometer. ¹H- and ¹³C-NMR spectra were taken with a Bruker BioSpin AVANCE 500 spectrometer. Chemical shifts were given on a δ (ppm) scale with tetramethylsilane as an internal standard (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad). High-resolution electrospray ionization mass spectrum (ESI-MS) was obtained with a Bruker Daltonics micro TOF-Q spectrometer. Measurements of ¹³CO₂ in the breath test were performed by an Otsuka Electronics POCone IR spectrophotometer.

N-(4-Methoxybenzyl)-[1-¹³C]acetamide (2)

To a solution of [1-¹³C]acetic acid (1.0 g, 16.4 mmol) in methanol (35 mL) were added 4-methoxybenzylamine (2.2 mL, 17.0 mmol) and DMT-MM (5.4 g, 19.5 mmol), followed by stirring at room temperature overnight. The reaction mixture was diluted with ethyl acetate and washed successively with water (twice), 1.0 mol/L hydrochloric acid, water, and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with ethyl acetate to give amide 2 (2.75 g, 93% yield from [1-¹³C]acetic acid) as a colorless needles. mp: 95–96°C. IR (KBr) cm⁻¹: 3288, 1597, 1544, 1513. ¹H-NMR (CDCl₃) δ: 7.21 (2H, d, J=8.7 Hz), 6.87 (2H, d, J=8.7 Hz), 5.64 (1H, br s, NH), 4.37 (2H, dd, ³J_C,H=3.0, J=5.6 Hz), 3.80 (3H, s), 2.01 (3H, d, ²J_C,H=6.0 Hz). ¹³C-NMR (CDCl₃) δ: 169.7 (strong), 159.1, 130.3 (d, ³J_C,C=1.6 Hz), 129.2, 114.1, 55.3, 43.2, 23.2 (d, ¹J_C,C=51.5 Hz). ESI-MS (positive mode) m/z: 203.0893 (Calcd for C₉¹³CH₁₃NNaO₂⁺: 203.0872).

N-[1-¹³C]Ethyl-N-(4-methoxybenzyl)-[1-¹³C]acetamide (4)

To a cold (0°C) solution of amide 2 (2.7 g, 15.0 mmol) in dry tetrahydrofuran (75 mL) under an argon atmosphere was carefully added lithium aluminum hydride (850 mg, 22.5 mmol) with stirring. The reaction mixture was allowed to warm slowly to room temperature, and stirred overnight under reflux. The reaction mixture was kept at room temperature, diluted with tetrahydrofuran and brine was carefully added as well as a 10 mol/L sodium hydroxide solution (4.5 mL) with vigorous stirring. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude amine 3 was used for subsequent reaction without further purification.; ¹H-NMR (CDCl₃) δ: 7.23 (2H, d, J=8.6 Hz), 6.86 (2H, d, J=8.6 Hz), 3.80 (3H, s), 3.73 (2H, d, J=4.0 Hz), 2.67 (2H, qd, J=7.2, ¹J_C,H=133.1 Hz), 1.12 (3H, dt, ²J_C,H=4.5, J=7.2 Hz). ¹³C-NMR (CDCl₃) δ: 161.4, 158.6, 132.7, 129.3, 113.8, 55.3, 53.3, 43.5 (strong), 15.2 (d, ¹J_C,C=36.9 Hz).

To a solution of the above amine 3 in methanol (30 mL) were added [1-¹³C]acetic acid (1.0 g, 16.4 mmol) and DMT-MM (5.0 g, 16.4 mmol), followed by stirring at room temperature overnight. The reaction mixture was diluted with ethyl acetate and washed successively with water (twice), 1.0 mol/L hydrochloric acid, water, and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with hexane–ethyl acetate (1 : 3) to give amide 4 (2.3 g, 74% yield in 2 steps) as a colorless viscous oil.; IR (neat) cm⁻¹: 1602, 1513. ¹H-NMR (CDCl₃, 4 : 3 mixture of rotamers at 300K) δ: (major rotamer) 7.18 (2H, d, J=8.6 Hz), 6.84 (2H, d, J=8.6 Hz), 4.52 (2H, brt, ³J_C,H=3.4 Hz), 3.23 (2H, dqd, ³J_C,H=4.2, J=7.1, ¹J_C,H=136.6 Hz), 2.16 (3H, d, ²J_C,H=5.9 Hz), 1.12 (3H, dt, ²J_C,H=4.5, J=7.1 Hz); (minor rotamer) 7.10 (2H, d, J=8.7 Hz), 6.89 (2H, d, J=8.7 Hz), 4.45 (2H, br t, ³J_C,H=3.9 Hz), 3.40 (2H, dqd, ³J_C,H=3.4, J=7.1, ¹J_C,H=138.1 Hz), 2.12 (3H, d, ²J_C,H=6.0 Hz), 1.09 (3H, dt, ²J_C,H=4.5, J=7.1 Hz). ¹³C-NMR (CDCl₃, 4 : 3 mixture of rotamers at 300 K) δ: (major rotamer) 170.2 (strong, d, ²J_C,C=2.4 Hz), 158.9, 130.0, 129.4, 113.9, 55.2, 47.0, 42.1 (strong, d, ²J_C,C=2.4 Hz), 21.3 (d, ¹J_C,C=52.7 Hz), 13.5 (d, ¹J_C,C=36.4 Hz); (minor rotamer) 170.5 (strong), 159.1, 128.9, 127.5, 114.2, 55.3, 51.0, 40.5 (strong), 21.8 (d, ¹J_C,C=52.1 Hz), 12.6 (d, ¹J_C,C=36.5 Hz). ESI-MS (positive mode) m/z: 232.1239 (Calcd for C₁₀¹³C₂H₁₇NNaO₂⁺: 232.1219).

N,N-Di[1-¹³C]ethyl-N-(4-methoxybenzyl)amine (5)

To a cold (0°C) solution of amide 4 (2.3 g, 11.0 mmol) in dry diethyl ether (50 mL) under an argon atmosphere was carefully added lithium aluminum hydride (420 mg, 11.0 mmol) with stirring. The reaction mixture was allowed to warm slowly to room temperature, and stirred overnight at this temperature. The reaction mixture was diluted with tetrahydrofuran and brine was carefully added as well as a 1.0 mol/L sodium hydroxide solution (4.0 mL) with vigorous stirring. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with ethyl acetate to give amine 5 (1.8 g, 84% yield) as a colorless oil. ¹H-NMR (CDCl₃) δ: 7.23 (2H, d, J=8.7 Hz), 6.84 (2H, d, J=8.7 Hz), 3.80 (3H, s), 3.51 (2H, t, ³J_C,H=4.4 Hz), 2.50 (4H, dqd, ³J_C,H=4.3, J=7.1, ¹J_C,H=132.1 Hz), 1.03 (6H, dt, ²J_C,H=4.4, J=7.1 Hz). ¹³C-NMR (CDCl₃) δ: 158.5, 131.8, 130.0, 113.5, 58.8, 56.8, 46.5 (strong), 11.6 (dd, ³J_C,C=2.5, ¹J_C,C=37.7 Hz). ESI-MS (positive mode) m/z: 196.1612 (Calcd for C₁₀¹³C₂H₂₀NO⁺: 196.1607).

2-Di[1-¹³C]ethylamino-N-(2,6-dimethylphenyl)acetamide (1)

To a solution of amine 5 (1.8 g, 9.2 mmol) in ethanol (20 mL) under an argon atmosphere, was added 10% palladium on charcoal (80 mg). The mixture was stirred vigorously under a hydrogen atmosphere overnight at room temperature, followed by filtration through celite. The filter medium was washed with a minimum amount of ethanol. To the combined filtrate were added 2-chloro-N-(2,6-dimethylphenyl)acetamide (2.0 g, 10.1 mmol) and sodium bicarbonate (850 mg), and the mixture was stirred for one day under reflux. The reaction mixture was kept at room temperature, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and extracted with 1.0 mol/L hydrochloric acid. The aqueous layer was basified with 1.0 mol/L sodium hydroxide solution, followed by extraction with chloroform. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with hexane–ethyl acetate (1 : 1) to give ¹³C-lidocaine 1 (1.4 g, 65% yield in 2 steps) as colorless needles.; mp: 66–67°C. IR (KBr) cm⁻¹: 3252, 1664, 1497. ¹H-NMR (CDCl₃) δ: 8.91 (1H, br s, NH), 7.08 (3H, m), 3.22 (2H, t, ³J_C,H=4.6 Hz), 2.69 (4H, dqd, ³J_C,H=3.9, J=7.1, ¹J_C,H=133.2 Hz), 2.23 (6H, s), 1.03 (6H, dt, ²J_C,H=4.4, J=7.1 Hz). ¹³C-NMR (CDCl₃) δ: 170.3, 135.1, 134.0, 128.2, 127.1, 27.6, 49.0 (strong), 18.6, 12.7 (dd, ³J_C,C=2.6, ¹J_C,C=37.3 Hz). ESI-MS (positive mode) m/z: 237.1891 (Calcd for C₁₂¹³C₂H₂₃N₂O⁺: 237.1872).

Animal Experiments

Male ddY mice weighing 25–30 g were obtained from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan). All experiments were conducted according to the Showa University Guidelines for Animal Care and Use. The mice were housed in a plastic cage and given free access to standard laboratory chow and tap water. ¹³C-Lidocaine was dissolved in distilled water containing an equimolar amount of hydrochloric acid, and the resulting solution was orally administered to mice (5.0 mg/kg) after an overnight fast. The expired ¹³CO₂ was collected and measured by an IR spectrophotometer according to the procedures previously reported.¹³⁾

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