First synthesis and full characterization of mexiletine N-carbonyloxy β-d-glucuronide

Article abstract of DOI:10.1016/j.tetlet.2010.07.150

A simple, convergent synthesis of the N-carbonyloxy β-d-glucuronide of mexiletine (sodium salt) in moderate yield is described. The compound is now available as an authentic reference standard for analytical studies, enabling more detailed investigation on the metabolism of mexiletine.

Full text of DOI:10.1016/j.tetlet.2010.07.150

Tetrahedron Letters 51 (2010) 5265–5268
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
First synthesis and full characterization of mexiletine N-carbonyloxy
b-D-glucuronide
*
Maria Maddalena Cavalluzzi, Giovanni Lentini , Angelo Lovece, Claudio Bruno, Alessia Catalano,
Alessia Carocci, Carlo Franchini
Dipartimento Farmaco-Chimico, Università degli Studi di Bari, via E. Orabona n. 4, 70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple, convergent synthesis of the N-carbonyloxy b-D-glucuronide of mexiletine (sodium salt) in mod-
Received 13 May 2010
Revised 23 July 2010
Accepted 26 July 2010
Available online 1 August 2010
erate yield is described. The compound is now available as an authentic reference standard for analytical
studies, enabling more detailed investigation on the metabolism of mexiletine.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Curtius rearrangement
Glycosidic linkage
Stereochemistry
Ring-opening reaction
1. Introduction
Recently, Senda et al. reported a novel chemical structure of the
major mexiletine glucuronide in human urine, sowing a seed of
Mexiletine, 1-(2,6-dimethylphenoxy)propan-2-amine (Fig. 1), is
an orally effective class IB antiarrhythmic agent.1,2 It has also been
shown to be effective in the treatment of myotonic syndromes3–6
and for neuropathic pain resulting from both cancer7 and diabetes
mellitus.8,9 Its analgesic efficacy for neuropathic pain has been re-
cently confirmed in a meta-analysis.10 In human, mexiletine
undergoes extensive liver metabolism (<10% of an administered
oral dose is recovered unchanged in urine) by numerous oxidative
pathways (phase I metabolism) involving aliphatic and aromatic
hydroxylation, dealkylation, deamination, and N-oxidation.11–13
The major oxidation metabolites, namely p-hydroxymexiletine
(PHM), hydroxymethylmexiletine (HMM), m-hydroxymexiletine
(MHM), and N-hydroxymexiletine (NHM) are shown in Figure 1.
Phase I metabolites, as well as mexiletine, undergo a further
glucuronidation reaction (phase II metabolism) which plays a piv-
otal role in the elimination of the drug from the body. In fact,
approximatively 30% of the dose is excreted as glucuronide metab-
olites.14,15 However, there is some confusion about the chemical
structure of the main glucuronic acid conjugate. For a long time,
N-hydroxymexiletine glucuronide has been indicated as the major
conjugated metabolite of mexiletine, representing one of the major
metabolic pathway in the disposition of the drug.16–18
doubt on the expected in vivo formation of N-hydroxymexiletine
conjugate.19 By using LC/MS/MS and HPLC analyses, they estab-
lished that the glucuronide contained a carbonyloxy moiety in its
structure, besides mexiletine and glucuronic acid moieties. Their
results indicated that the structure of the isolated metabolite
was the b-D-glucuronic acid conjugate of N-carbonyloxymexiletine
(1, Scheme 1) which appears to be one of the major metabolites of
mexiletine in human urine. In fact, its estimated urinary recovery
corresponded to about 80% of total mexiletine glucuronide and
over 25% of the dose of the drug.19 On the other hand, the forma-
tion of such carbamyl O-b-
an amino group has already been described before.20–23 Since
authentic reference standard for mexiletine carbonyloxy b- -glu-
curonide was unavailable,19 a chemical synthesis was indeed re-
quired to provide reliable analytical standard useful for
D-glucuronides from drugs containing
D
a
unambiguous identification of the main metabolic product of mex-
iletine conjugation. In particular, being mexiletine glucuronidation
enantioselective, favoring the R isomer,14,17,18,24,25 and since the R
isomer of mexiletine performs as the eutomer,26–30 we focused
our efforts on the synthesis of carbonyloxy b-D-glucuronide of
(R)-mexiletine (1). The voltage-gated sodium channels’ blocking
activity of the newly synthesized compound (sodium salt, 1ꢀNa,
Scheme 1) has already been evaluated31 as a part of a program
aimed at the study of mexiletine metabolite effects on skeletal
muscle.32,33 1ꢀNa does not retain the parent drug’s activity.31 The
synthesis and full characterization of 1ꢀNa are reported herein.
* Corresponding author. Tel.: +39 080 5442744; fax: +39 080 5442724.
E-mail address: glentini@farmchim.uniba.it (G. Lentini).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.07.150

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M. M. Cavalluzzi et al. / Tetrahedron Letters 51 (2010) 5265–5268
Figure 1. Phase I metabolic pathways of mexiletine in human.
Scheme 1. Convergent synthesis of mexiletine N-carbonyloxy b-
D-glucuronide as its sodium salt (1ꢀNa).
ether, 8 was obtained as the pure b anomer. The b-linkage of the
glucuronate moiety was assessed on the basis of the trans-diaxial
coupling of 8.0 Hz found for H-1 doublet. The b epimer, compared
2. Results and discussion
The sodium salt of (R)-mexiletine carbonyloxy b-D-glucuronide
with the
hemiacetal
a
-form, was desirable since it was hydrolyzed faster to
(1ꢀNa, Scheme 1) was prepared following a convergent synthetic
route based on the coupling between two building blocks, the car-
boxylic acid (R)-2 and the hemiacetal 3, as the key step.
3
in the next step, giving higher yields. The
hydrolysis was carried out by treating 8 with an equimolar amount
of tributyltin methoxide in anhydrous THF affording methyl 2,3,4-
(R)-2 was prepared by modifying a procedure used in the liter-
tri-O-acetyl-D
-glucopyranuronate (3) as an anomeric mixture.36
ature (Scheme 2).34 The preparation of 3 started from commer-
The prepared synthons (R)-2 and 3 were coupled according to
the Curtius rearrangement, in the presence of diphenylphosphoryl
azide and Et3N,37 giving the corresponding carbamate 4 exclusively
as the b-anomer (Scheme 1). The stereochemistry of the glycosidic
linkage of 4 was determined on the basis of the value of the
coupling constant of the anomeric proton (JH1–H2 = 8.0 Hz). Thus,
cially available D-glucuronolactone (7) which was submitted to a
ring-opening reaction with NaOH in methanol followed by acyla-
tion with acetic anhydride in pyridine at 4 °C to give methyl
tetra-O-acetyl-D
-glucopyranuronate (8) (Scheme 3).35
Low temperature was necessary to obtain the b isomer of 8 in
high percentage. After recrystallization from EtOAc/petroleum
Scheme 2. Preparation of synthon (R)-2.

M. M. Cavalluzzi et al. / Tetrahedron Letters 51 (2010) 5265–5268
5267
Scheme 3. Preparation of synthon 3.
22. Gipple, K. J.; Chan, K. T.; Elvin, A. T.; Lalka, D.; Axelson, J. E. J. Pharm. Sci. 1982,
71, 1011–1014.
23. Delbressine, L. P. C.; Funke, C. W.; van Tilborg, M.; Kaspersen, F. M. Xenobiotica
1990, 20, 133–134.
b-stereospecificity of the coupling reaction might stem from the
well-known higher nucleophilicity of the 1-OH group in the b- than
in the
a
-configuration.38 Deprotection of the acetyl groups and
24. Grech-Bélanger, O.; Turgeon, J.; Gilbert, M. Br. J. Clin. Pharmacol. 1986, 21, 481–
487.
hydrolysis of the methyl ester of compound 4 were performed
using 1 N NaOH in MeOH, followed by an acidic work-up with
Amberlyst 15 ion-exchange resin.39 At odds with what reported
for other glucuronides,39 this work-up gave the desired product 1
as its sodium salt 1ꢀNa. The reaction was stereochemically reliable,
giving 1ꢀNa with exclusive b-stereochemistry at C-1, as confirmed
by 1H NMR analysis (JH1–H2 = 8.0 Hz).
25. Knoche, B.; Gehrcke, B.; König, W. A.; Wainer, I. W. Chirality 1996, 8, 30–34.
26. De Luca, A.; Natuzzi, F.; Desaphy, J.-F.; Loni, G.; Lentini, G.; Franchini, C.;
Tortorella, V.; Conte Camerino, D. Mol. Pharmacol. 2000, 57, 268–277.
27. De Luca, A.; Natuzzi, F.; Falcone, G.; Duranti, A.; Lentini, G.; Franchini, C.;
Tortorella, V.; Conte Camerino, D. Naunyn Schmiedebergs Arch. Pharmacol. 1997,
356, 777–787.
28. Turgeon, J.; Uprichard, A. C.; Bélanger, P. M.; Harron, D. W.; Grech-Bélanger, O.
J. Pharm. Pharmacol. 1991, 43, 630–635.
29. Hill, R. J.; Duff, H. J.; Sheldon, R. S. Mol. Pharmacol. 1988, 34, 659–663.
30. De Luca, A.; Natuzzi, F.; Lentini, G.; Franchini, C.; Tortorella, V.; Conte
Camerino, D. Naunyn Schmiedebergs Arch. Pharmacol. 1995, 352, 653–661.
31. De Bellis, M.; De Luca, A.; Rana, F.; Cavalluzzi, M. M.; Catalano, A.; Lentini, G.;
Franchini, C.; Tortorella, V.; Conte Camerino, D. Br. J. Pharmacol. 2006, 149, 300–
310.
32. Catalano, A.; Carocci, A.; Fracchiolla, G.; Franchini, C.; Lentini, G.; Tortorella, V.;
De Luca, A.; De Bellis, M.; Desaphy, J.-F.; Conte Camerino, D. Chirality 2004, 16,
72–78.
33. Cavalluzzi, M. M.; Catalano, A.; Bruno, C.; Lovece, A.; Carocci, A.; Corbo, F.;
Franchini, C.; Lentini, G.; Tortorella, V. Tetrahedron: Asymmetry 2007, 18, 2409–
2417.
34. Franchini, C.; Cellucci, C.; Corbo, F.; Lentini, G.; Scilimati, A.; Tortorella, V.;
Stasi, F. Chirality 1994, 6, 590–595.
3. Conclusions
In summary, a facile convergent synthesis of (R)-mexiletine N-
carbonyloxy b-D-glucuronide (1)—the main phase II mexiletine
metabolite—as its sodium salt (1ꢀNa) has been developed.40 Ready
availability of 1ꢀNa should favor more detailed studies on the
metabolism of mexiletine. Investigation on the possible extension
of the route described herein to deuterated analogs of 1 has been
undertaken.
Acknowledgment
35. Bollenback, G. N.; Long, J. W.; Benjamin, D. G.; Lindquist, J. A. J. Am. Chem. Soc.
1955, 77, 3310–3315.
36. Nudelman, A.; Herzig, J.; Gottlieb, H. E.; Keinan, E.; Sterling, J. Carbohydr. Res.
1987, 162, 145–152.
37. Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron 1974, 30, 2151–2157.
38. Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212–235.
39. Learmonth, D. A.; Freitas, A. P. Bioconjug. Chem. 2002, 13, 1112–1118.
40. Procedures for key compounds.
This work was accomplished, thanks to the financial support of
the Italian Ministero dell’Istruzione, dell’Università e della Ricerca
(MIUR 2005033023_001).
References and notes
Methyl tetra-O-acetyl-b-D-glucopyranuronate (8).
Prepared as reported in the literature.35 Yield: 57%; mp 177–178 °C, lit.35 177–
178 °C; ½a 2D0
ꢁ
+8.6 (c 1, CHCl3), lit.35 a D23 +7.4 (c 2, CHCl3); 13C NMR (CDCl3) d
½ ꢁ
1. Yamauchi, M.; Watanabe, E.; Yasui, K.; Takeuchi, H.; Terasawa, T.; Sawada, K.;
Hishida, H.; Kodama, I. Int. J. Cardiol. 2005, 103, 92–97.
20.7 (1C), 20.8 (2C), 21.0 (1C), 53.2 (1C), 69.1 (1C), 70.3 (1C), 72.0 (1C), 73.2
(1C), 91.5 (1C), 167.0 (1C), 169.1 (1C), 169.4 (1C), 169.7 (1C), 170.1 (1C); MS
(70 eV) m/z (%) 334 (M+ꢂ43, <1), 43 (100). 1H NMR spectrum was in agreement
with that reported in the literature.41
2. Fenster, P. E.; Comess, K. A. Pharmacotherapy 1986, 6, 1–9.
3. Rüdel, R.; Lehmann-Horn, F. Physiol. Rev. 1985, 65, 310–356.
4. Lehmann-Horn, F.; Jurkat-Rott, K. Physiol. Rev. 1999, 79, 1317–1372.
5. Chrestian, N.; Puymirat, J.; Bouchard, J.-P.; Dupré, N. Neurology 2006, 2, 393–
399.
6. Gay, S.; Dupuis, D.; Faivre, L.; Masurel-Paulet, A.; Labenne, M.; Colombani, M.;
Soichot, P.; Huet, F.; Hainque, B.; Sternberg, D.; Fontaine, B.; Gouyon, J.-B.;
Thauvin-Robinet, C. Am. J. Med. Genet. A 2008, 146A, 380–383.
7. Sloan, P.; Basta, M.; Storey, P.; von Gunten, C. Anesth. Analg. 1999, 89, 760–761.
8. Jarvis, B.; Coukell, A. J. Drugs 1998, 56, 691–707.
Methyl 2,3,4-tri-O-acetyl-a-D-glucopyranuronate (3).
Prepared as reported in the literature.36 Yield: 86%; mp 103–105 °C (EtOAc/
hexane), lit.42 98–100 °C (Et2O); ½a 2D0
ꢁ
+86.0 (c 2, CHCl3), lit.42 a 2D0
½ ꢁ +81.2 (c
0.105, CHCl3); IR (KBr): 3474 (OH), 1753 (C@O) cmꢂ1; MS (70 eV) m/z (%) 275
(M+ꢂ59, <1), 43 (100). 1H NMR and 13C NMR spectra were in agreement with
those reported in the literature.42
Methyl (10R,2S,3R,4R,5R,6R)-3,4,5-tri-O-acetyl-6-[2-(2,6-dimethylphenoxy)-1-methyl
ethylaminocarbonyloxy]tetrahydro-2H-pyran-2-carboxylate (4).43
A solution of (ꢂ)-(R)-3-(2,6-dimethylphenoxy)-2-methylpropanoic acid [(R)-2]
9. Oskarsson, P.; Ljunggren, J.-G.; Lins, P.-E. Diabetes Care 1997, 20, 1594–1597.
10. Tremont-Lukats, I. W.; Challapalli, V.; McNicol, E. D.; Lau, J.; Carr, D. B. Anesth.
Analg. 2005, 101, 1738–1749.
(0.65 g, 3.10 mmol), methyl 2,3,4-tri-O-acetyl-a-D-glucopyranuronate (1.55 g,
4.65 mmol) (3), diphenylphosphoryl azide (DPPA, 1.28 g, 4.65 mmol), and Et3N
(0.95 mL, 6.82 mmol) in 40 mL of dioxane was stirred under reflux for 19 h. After
evaporation of the solvent, the residue was taken up with EtOAc, washed with
2 N HCl, and the aqueous phase was washed with EtOAc. The two organic phases
were washed, separately, with 2 N NaOH and then combined and dried over
anhydrous Na2SO4. The solvent was concentrated in vacuo to afford 1.07 g of a
brown solid. The crude product was purified by flash chromatography (EtOAc/
hexane 4:6) to give 0.89 g (53%) of the desired product as a yellow solid which
11. Beckett, A. H.; Chidomere, E. C. Postgrad. Med. J. 1977, 53, 60–66.
12. Beckett, A. H.; Chidomere, E. C. J. Pharm. Pharmacol. 1977, 29, 281–285.
13. Grech-Bélanger, O.; Turgeon, J.; Lalande, M.; Bélanger, P. M. Drug Metab. Dispos.
1991, 19, 458–461.
14. Abolfathi, Z.; Fiset, C.; Gilbert, M.; Moerike, K.; Bélanger, P. M.; Turgeon, J. J.
Pharmacol. Exp. Ther. 1993, 266, 1196–1201.
15. Ueno, K.; Tamamura, A.; Matsumoto, K.; Komamura, K.; Kamakura, S.;
Miyatake, K.; Shibakawa, M. Ann. Pharmacother. 2002, 36, 241–245.
16. Lanchote, V. L.; Santos, V. J.; Cesarino, E. J.; Dreossi, S. A. C.; Mere, Y., Jr.; Santos,
S. R. C. J. Chirality 1999, 11, 85–90.
was recrystallized from EtOAc/hexane(0.49 g, 29%): mp 156–158 °C; ½a D20
ꢁ
+8.4 (c
1, CHCl3); IR (KBr): 3343 (NH), 1748, 1726 (C@O) cmꢂ1 1H NMR (CDCl3) d 1.40 (d,
;
J = 6.9 Hz, 3H), 2.02 (s, 6H), 2.04 (s, 3H), 2.23 (s, 6H), 3.65–3.85 (m overlapping s
at 3.72, 2H), 3.72 (s overlapping m at 3.65–3.85, 3H), 3.95–4.10 (m, 1H), 4.16 (d,
J = 9.9 Hz, 1H), 5.15 (toverlapping t at 5.21, J = 9.2 Hz, 1H), 5.21 (t overlapping t at
5.15, J = 9.5 Hz, 1H), 5.32 (t overlapping d at 5.36, J = 9.4 Hz, 1H), 5.36 (d
overlapping t at 5.32, J = 8.2 Hz, 1H), 5.72 (d, J = 8.0 Hz, 1H), 6.85–7.05 (m, 3H);
13C NMR (CDCl3) d 16.5 (2C), 17.9 (1C), 20.7 (1C), 20.8 (1C), 20.9 (1C), 47.8 (1C),
53.2 (1C), 69.4 (1C), 70.2 (1C), 72.2 (1C), 73.0 (1C), 73.6 (1C), 92.7 (1C), 124.4 (1C),
129.2 (2C), 131.0 (2C), 153.3 (1C), 155.0 (1C), 167.0 (1C), 169.7 (2C), 170.2 (1C);
MS (70 eV) m/z (%) 205 (M+ꢂ333, 56), 122 (100). Anal. Calcd for (C25H33NO12.0.5
H2O): C, 54.74; H, 6.25; N, 2.55. Found: C, 54.45; H, 6.03; N, 2.76.
17. Lanchote, V. L.; Cesarino, E. J.; Santos, V. J.; Mere, Y., Jr.; Santos, S. R. C. J.
Chirality 1999, 11, 29–32.
18. Turgeon, J.; Parè, J. R.; Lalande, M.; Grech-Bélanger, O.; Bélanger, P. M. Drug
Metab. Dispos. 1992, 20, 762–769.
19. Senda, C.; Toda, S.; Tateishi, M.; Kobayashi, K.; Igarashi, T.; Chiba, K. Xenobiotica
2003, 33, 871–884.
20. Elvin, A. T.; Keenaghan, J. B.; Byrnes, E. W.; Tenthorey, P. A.; McMaster, P. D.;
Takman, B. H.; Lalka, D.; Manion, C. V.; Baer, D. T.; Wolshin, E. M.; Meyer, M. B.;
Ronfeld, R. A. J. Pharm. Sci. 1980, 69, 47–49.
21. Straub, K.; Davis, M.; Hwang, B. Drug Metab. Dispos. 1988, 16, 359–366.

5268
M. M. Cavalluzzi et al. / Tetrahedron Letters 51 (2010) 5265–5268
Sodium (1’R,2S,3R,4R,5R,6R)-3,4,5-trihydroxy-6-[2-(2,6-dimethylphenoxy)-1-methyl
3H), 3.50–3.95 (m, 4H), 5.27 (d, J = 8.0 Hz, 1H), 6.80–7.00 (m, 3H); 13C NMR
(CD3OD) d 15.3 (2C), 16.7 (1C), 60.4 (1C), 72.2 (1C), 72.7 (1C), 74.1 (1C), 75.4 (1C),
76.7 (1C), 95.4 (1C), 123.9 (1C), 128.7 (2C), 130.7 (2C), 155.4 (1C), 155.6 (1C),
175.1 (1C); LC/MS m/z 398 [Mꢂ]. Anal. Calcd for (C18H24NNaO9.1.5 H2O): C, 48.21;
H, 6.07; N, 3.12. Found: C, 47.92; H, 5.81; N, 3.11.
ethylaminocarbonyloxy]tetrahydro-2H-pyran-2-carboxylate (1ꢀNa).44
To a cooled and stirred suspension of compound 4 (0.54 g, 1.00 mmol) in
methanol (13 mL), 1 N NaOH solution (10.1 mL, 10.1 mmol) was added
dropwise. The mixture was stirred in an ice bath for 30 min and then left at
room temperature for 30 min. The undissolved material was removed by
filtration and the filtrate was added to cold water containing Amberlyst 15 ion-
exchange resin so as to reach pH 2. The mixture was filtered and the resin was
washed with water/methanol (1:1). The filtrate was concentrated under vacuum
and water was azeotropically removed. Crystallization of the residue from
MeOH/i-PrOH gave 0.15 g (38%) of the title product as a yellow solid: mp 198–
41. Root, Y. Y.; Wagner, T. R.; Norris, P. Carbohydr. Res. 2002, 337, 2343–2346.
´
42. Trynda, A.; Madaj, J.; Konitz, A.; Wisniewski, A. Carbohydr. Res. 2000, 329, 249–
252.
43. Compound 4 may be also referred to as methyl 5-[2-(2,6-dimethylphenoxy)-1-
methylethylaminocarbonyloxy]-2,3,4-tri-O-acetyl-b-
44. Compound 1ꢀNa may be also referred to as sodium 5-[2-(2,6-
dimethylphenoxy)-1-methylethylaminocarbonyloxy]-b- -glucopyranuronate.
D-glucopyranuronate.
200 °C; ½a 2D0
ꢁ
+0.9 (c 1, MeOH); IR (KBr): 3342 (NH, OH), 1720, 1614 (C@O), 1415
;
D
(C–O–C) cmꢂ1
1H NMR (D2O) d 1.11 (d, J = 6.9 Hz, 3H), 2.06 (s, 6H), 3.25–3.50 (m,