Major metabolites of Zolpidem: Expeditious synthesis and mass spectra

Article abstract of DOI:10.1248/cpb.54.1318

An expeditious route to the two major metabolites of Zolpidem - and readily applicable to the synthesis of the drug - was established via a cyclization reaction between a 2-aminopyridine and a suitable α-bromoacetophenone. The structures of the target compounds were confirmed from a 2D ¹H- ¹⁵N NMR correlation. Their mass spectra contribute to a reliable toxicological identification of the drug in the case of drug-facilitated crimes.

Full text of DOI:10.1248/cpb.54.1318

1318
Notes
Chem. Pharm. Bull. 54(9) 1318—1321 (2006)
Vol. 54, No. 9
Major Metabolites of Zolpidem: Expeditious Synthesis and Mass Spectra
a
Frédérique KLUPSCH, Raymond HOUSSIN, ,a Luc HUMBERT,b Michel IMBENOTTE,c
*
Jean-Pierre HÉNICHART,a and Michel LHERMITTE
b,c
a Institut de Chimie Pharmaceutique Albert Lespagnol, EA 2692, Université de Lille 2; 3 rue du Professeur J. Laguesse, BP
83, 59006 Lille, France: b Laboratoire de Toxicologie et Génopathies, Hôpital A. Calmette, CHRU; avenue du Professeur J.
c
Leclercq, 59037 Lille, France: and Laboratoire de Toxicologie, Faculté des Sciences Pharmaceutiques et Biologiques,
Université de Lille 2; 3 rue du Professeur J. Laguesse, BP 83, 59006 Lille, France.
Received March 16, 2006; accepted May 30, 2006
An expeditious route to the two major metabolites of Zolpidem—and readily applicable to the synthesis of
the drug—was established via a cyclization reaction between a 2-aminopyridine and a suitable a-bromoace-
1
tophenone. The structures of the target compounds were confirmed from a 2D H–15N NMR correlation. Their
mass spectra contribute to a reliable toxicological identification of the drug in the case of drug-facilitated crimes.
Key words Zolpidem; metabolite; regioselective cyclization; drug-facilitated crime; mass spectra
Zolpidem (Ambien®, Stilnox®) is a short-action non-ben- also been developed for hair analysis.6,7) Nevertheless, identi-
zodiazepine hypnotic drug which binds with a high affinity fication of metabolites is also of interest since convenient
as an allosteric modulator to a1 subunit-containing GABAA analysis could lead to specific quantifications and to unequiv-
receptors.1) The a1-GABAA-preferring property of this imi- ocally conclusive elements as to the origin of poisoning.
dazo[1,2-a]pyridine, which may be turned into an a3 partial
The aim of this work was to conclusively define an expedi-
agonism for imidazo[1,2-a]pyrimidines,2) has been docu- tious route to synthesize the two well-known major metabo-
mented as being responsible for some behavioural effects lites of Zolpidem8) (Chart 1), so as to characterize its crimi-
consistent with sedative properties such as suppression of lo- nal administration through its HPLC-MS parameters.
comotor activity and procumbent posture in animal models,3)
whereas stimulation of a2- and a3-GABAA subunits resulted
in little or no sedation.4)
Moreover, reports dealing with “drug-facilitated crimes”
(robbery, mugging, sexual assault) related to benzodi-
azepines and benzodiazepine-like hypnotics5) are tending to
increase because of their short half-life, their rapid excretion
and/or relatively long delay between the intake and urinary
sampling. If classical techniques (GC/MS, RIA, CE, HPLC,
DPV) have been used to identify and quantify the presence
of Zolpidem in body fluids, an approach by LC-MS/MS has Chart 1. Zolpidem Metabolites
Reagents and Conditions: (a) EtOH, reflux; (b) N2CH2COOC(Me)3, Cu, toluene, reflux; (c) 50% TFA, CH2Cl2, rt; (d) HBTU, HOBt, DIEA, (Me)2NH, rt; (e) 0.1 N LiOH, THF, rt.
Chart 2
∗ To whom correspondence should be addressed. e-mail: raymond.houssin@univ-lille2.fr
© 2006 Pharmaceutical Society of Japan

September 2006
1319
Results and Discussion
nucleophilic character of ring nitrogen with regard to a bro-
A synthetic route to the carboxylic metabolites 1 and 2 of momethylketone group.
Zolpidem was previously reported9) and was obtained
In conclusion, a flexible and rapid method to prepare the
through a scheme inspired from the preparation of the 14C- two main carboxyl-containing metabolites of Zolpidem has
labelled drug.10) As it is rather long, we decided to establish been developed and may be applied to the synthesis of the
a shorter access to these molecules via two analogous path- drug. Their mass spectra (Chart 4) provide a specific clue as
ways involving easily accessible reagents. The chosen proce- to their identification in biological fluids, particularly in
dure (Chart 2) reduced by half the reactional sequence and cases of drug-facilitated crimes: both compounds 1 and 2
led to the creation of the carboxylic and tertiary amide func- gave rise to peaks at m/zꢀ266 and 265 corresponding to the
tions of 1 and 2 via their corresponding cross-protected es- loss of the fragment (Me)2NCO (m/zꢀ72) and of the frag-
ters.
ment (Me)2NCOꢁH• (m/zꢀ73) from the quasimolecular ion
Following the classical procedure for the preparation of (MHꢁꢀ338). Characteristic peaks of each molecule can be
imidazo[1,2-a]pyridines,11) the cyclization of a-bromo-4- observed, such as fragmentation at m/zꢀ92—indicative of a
methylacetophenone 3 with methyl 2-aminopyridine-5-car- CH2 substituent on pyridine—for metabolite 2.
boxylate 4 gave the imidazopyridine 5. Substitution on C-3
Moreover, as reliable concentrations could be achieved for
by a tert-butyl acetate group was completed with tert-butyl standard solutions of 1 and 2, their quantitative analysis is
diazoacetate/Cu in toluene at reflux.12,13) By chimioselective now available to estimate the intake of Zolpidem and pro-
acidolysis of the tert-butyl ester of 6 (50% TFA/CH2Cl2)14) vides a valuable tool in forensic toxicology since this drug
carboxylic acid 7 was obtained, and this, in turn, reacted with leads to anterograde amnesia.5)
dimethylamine (peptidic coupling conditions: HBTU, HOBt,
DIEA) to give tertiary amide 8. Target carboxylic acid 1 was
Experimental
Melting points were determined with a Büchi 535 capillary melting point
apparatus. Analytical thin-layer chromatography was performed on pre-
coated Kieselgel 60F254 plates (Merck); the spots were located by UV (254,
366 nm); Rf values are given for guidance. Silica gel 60 230—400 mesh or
obtained by saponification of compound 8 with 0.1 N
LiOH/THF.15) An analogous pathway using a-bromo-4-(car-
boethoxy)acetophenone 9 and 2-amino-5-methylpyridine 10
as starting materials readily gave imidazopyridine 11. Substi- 5—40 mm purchased from Merck were used for column or preparative thick
tution on C-3 by N,N-dimethylaminoacetamide (compound
14) and final saponification into compound 2 were obtained
as previously described for imidazopyridine 1.
The structural identification of the regioisomer formed
during the cyclization step (2-arylimidazo[1,2-a]pyridines 5
and 11, respectively) was performed on final compounds 1
and 2. The 2D 1H–15N NMR correlation (Chart 3), optimized
2
3
on long range couplings (HMBC, JN–H and/or JN–H), dis-
played cross peaks between N-4 at 203 ppm and protons H5,
H8 and exocyclic CH2, excluding the formation of the 3-aryl
isomers: the presence of a withdrawing group (carboxylic
ester) on b-carbon of pyridine did not evidently modify the
Chart 3. Selected HMBC Correlations for 1 and 2 (Solvent: DMSO-d6)
Chart 4. Mass Spectra of 1 and 2

1320
Vol. 54, No. 9
layer chromatography. The structures of all compounds were supported by (98%, 1 g, 5.2 mmol) in acetic acid (15 ml) in a nitrogen atmosphere. The so-
IR (neat, FT-Bruker Vector 22 instrument) and by NMR on a Bruker lution was stirred at room temperature for 18 h, then poured into water
1
AVANCE 300 spectrometer operating at 300.13, 75 and 31.42 MHz for H, (200 ml) and the precipitate was successively washed with water and cyclo-
13C (HSQC and HMBC) and 15N, respectively, with a 5 mm broad band in- hexane. The solid was recrystallized (0.9 g, 64%): mp 75—76 °C (hexane)
verse gradient probe. All chemical shifts were referenced with TMS and (lit.16) 71 °C). Rfꢀ0.70 (ethyl acetate–cyclohexane, 7/3). IR cmꢂ1: 1706. 1H-
NH4Cl signal at dꢀ0 ppm for 1H, 13C and 15N, respectively; the splitting pat- NMR (CDCl3) d: 1.43 (3H, t), 4.43 (2H, q), 4.48 (2H, s), 8.04 (2H, d), 8.11
terns were designated as follows: s, singlet; d, doublet; t, triplet; q, quadru- (2H, d).
plet; m, multiplet. APCIꢁ mass spectra were obtained on an LC-MS system
Ethyl 4-[(6-Methyl)imidazo[1,2-a]pyridin-2-yl]benzoate (11) 2-
Thermo Electron Surveyor MSQ. HPLC-ESI-MS analyses were performed Amino-5-methylpyridine (95%, 1.5 g, 13.2 mmol) was added to a stirred so-
on a Waters 2695 HPLC apparatus interfaced with a Waters ZQ quadrupole lution of bromomethylketone (9) (3 g, 11 mmol) in ethanol (40 ml) in a dry
MS spectrometer: spectra were recorded in full scan mode between 50 and atmosphere. After 24 h, the precipitate was filtered, washed with ethanol and
450 m/z with positive electrospray at 30 V. Commercially available reagents recrystallized (2.7 g, 87%): mp 229—230 °C (ethanol). Rfꢀ0.47 (ethyl ac-
1
and solvents were used throughout without further purification.
etate–cyclohexane, 3/2). IR cmꢂ1: 1708. H-NMR (DMSO-d6) d: 1.36 (3H,
Methyl 2-(4-Methylphenyl)imidazo[1,2-a]pyridine-6-carboxylate (5) t), 2.42 (3H, s), 4.35 (2H, q), 7.78 (1H, d), 7.81 (1H, d), 8.15 (4H, m), 8.71
Bromomethylketone (3) (98%, 2 g, 9.3 mmol) was added while stirring to a
hot solution of ester (4) (97%, 1.44 g, 9.3 mmol) in ethanol (40 ml). After re-
(1H, s), 8.87 (1H, s). LC-MS (APCIꢁ) m/z: 281 (MHꢁ).
Ethyl 4-[(3-tert-Butoxycarbonylmethyl-6-methyl)imidazo[1,2-a]-
fluxing for 24 h, the solution was cooled and the solid that precipitated was pyridin-2-yl]benzoate (12) Commercial tert-butyl diazoacetate (1 ml,
filtered and washed with ethanol (1.86 g, 75%). Rfꢀ0.64 (ethyl acetate–cy- 7.03 mmol) was added in small amounts to a boiling solution of ester (11)
1
clohexane, 3/2). IR cmꢂ1: 1720. H-NMR (DMSO-d6) d: 2.39 (3H, s), 3.96 (1 g, 3.55 mmol) in 30 ml of dry toluene, followed by small portions of cop-
(3H, s), 7.41 (2H, d), 7.85 (2H, d), 8.17 (1H, d), 8.20 (1H, d), 8.80 (1H, s), per powder (total amount: 500 mg). When the additions were completed,
9.52 (1H, s). LC-MS (APCIꢁ) m/z: 267 (MHꢁ).
boiling was continued for a further 3 h. The mixture was cooled down to
Methyl 3-tert-Butoxycarbonylmethyl-2-(4-methylphenyl)imidazo[1,2- room temperature, the suspension was filtered on Celite and the filtrate was
a]pyridine-6-carboxylate (6) Commercial tert-butyl diazoacetate (0.5 ml,
3.57 mmol) was added via a syringe to a suspension of ester (5) (931 mg,
concentrated under reduced pressure. Purification was carried out by column
chromatography on silica gel (ethyl acetate–cyclohexane, 1/1) and gave
3.5 mmol) in 30 ml of dry toluene, this was followed by small portions of 600 mg (43%) of tert-butyl ester (12) which was immediately used in the
copper powder (total amount: 250 mg). After adding another portion of tert- next reaction. LC-MS (APCIꢁ) m/z: 395 (MHꢁ).
butyl diazoacetate (0.5 ml, 3.57 mmol) and of copper powder (total amount:
Ethyl 4-[(3-Carboxymethyl-6-methyl)imidazo[1,2-a]pyridin-2-yl]ben-
250 mg), the mixture was heated at reflux for 18 h. The mixture was cooled zoate (13) A solution of tert-butyl ester (12) (650 mg, 1.65 mmol) and tri-
down to room temperature and the solvent was evaporated under reduced fluoroacetic acid (5 ml) in dichloromethane (5 ml) was stirred for 1 h at room
pressure. Ethyl acetate was added to the residue, the suspension was filtered temperature and concentrated without heating. The aqueous phase resulting
on Celite and the filtrate was concentrated and used as such in the next reac-
tion (480 mg, 36%): Rfꢀ0.63 (ethyl acetate–cyclohexane, 3/2). LC-MS
(APCIꢁ) m/z: 381 (MHꢁ).
from adding a 10% potassium carbonate solution (50 ml) was washed twice
with diethyl ether and acidified (pH 6) with 6 N HCl. The resulting precipi-
tate was filtered, then washed with diethyl ether and recrystallized (200 mg,
Methyl 3-Carboxymethyl-2-(4-methylphenyl)imidazo[1,2-a]pyridine- 36%): mp 177—178 °C (acetonitrile). Rfꢀ0.05 (ethyl acetate). IR cmꢂ1
:
1
6-carboxylate (7) A solution of tert-butyl ester (6) (480 mg, 1.26 mmol)
and trifluoroacetic acid (10 ml) in dichloromethane (10 ml) was stirred for
12 h at room temperature and concentrated without heating. The aqueous
phase, resulting from the addition of a 10% potassium carbonate solution
(50 ml), was washed with ethyl acetate and acidified (pH 6) with 6 N HCl.
Further extractions with ethyl acetate and chloroform gave after evaporation
1719, 1701, 1661. H-NMR (DMSO-d6) d: 1.36 (3H, t), 2.46 (3H, s), 4.30
(2H, s), 4.38 (2H, q), 7.83 (4H, m), 8.18 (2H, d), 8.78 (1H, s), 13.20 (1H,
m).
Ethyl 4-[(3-Dimethylcarbamoylmethyl-6-methyl)imidazo[1,2-a]-
pyridin-2-yl]benzoate (14) Amide (14) was prepared from carboxylic
acid (13) (200 mg, 0.59 mmol), O-(1H-benzotriazol-1-yl)-N,N,Nꢃ,Nꢃ-tetra-
a white solid which was washed with ethyl acetate and recrystallized methyluronium hexafluorophosphate (335 mg, 0.88 mmol), 1-hydroxybenzo-
(240 mg, 59%): mp 240—241 °C (ethyl acetate). Rfꢀ0.05 (ethyl acetate–cy- triazole (40 mg, 0.29 mmol), N,N-ethyldiisopropylamine (152 mg, 1.18
clohexane, 3/2). IR cmꢂ1: 1712, 1636. 1H-NMR (DMSO-d6) d: 2.37 (3H, s),
mmol) and dimethylamine (33%, 0.18 ml, 1.18 mmol) in a similar manner to
that described for the synthesis of 8: 163 mg, 76%. Rfꢀ0.58 (ethyl
3.89 (3H, s), 4.15 (2H, s), 7.31 (2H, d), 7.67 (4H, m), 9.02 (1H, s).
Methyl 3-Dimethylcarbamoylmethyl-2-(4-methylphenyl)imidazo[1,2- acetate–cyclohexane, 4/1). IR cmꢂ1: 1711, 1610. 1H-NMR (CD3OD) d: 1.39
a]pyridine-6-carboxylate (8) O-(1H-Benzotriazol-1-yl)-N,N,Nꢃ,Nꢃ- (3H, t), 2.41 (3H, s), 3.04 (3H, s), 3.22 (3H, s), 4.27 (2H, s), 4.42 (2H, q),
tetramethyluronium hexafluorophosphate (969 mg, 2.56 mmol), 1-hydroxy- 7.31 (1H, d), 7.54 (1H, d), 7.74 (2H, d), 8.08 (1H, s), 8.14 (2H, d). LC-MS
benzotriazole (115 mg, 0.85 mmol), and N,N-ethyldiisopropylamine (0.6 ml, (APCIꢁ) m/z: 366 (MHꢁ).
3.47 mmol) were added to a suspension of carboxylic acid (7) (550 mg,
1.7 mmol) in dry dichloromethane (20 ml). The solution was stirred for
30 min in a dry atmosphere before dimethylamine (33%, 0.54 ml, 3.5 mmol)
was added. After a further addition of dichloromethane (30 ml), the solution
was washed with 0.5 N HCl, with a saturated aqueous NaCl solution, then
with an aqueous 10% sodium bicarbonate solution. The organic phase was
4-[(3-Dimethylcarbamoylmethyl-6-methyl)imidazo[1,2-a]pyridin-2-
yl]benzoic Acid (2) An aqueous solution of 0.1 N lithium hydroxide
(14 ml) was added dropwise to an ice-cold stirred solution of amide 14
(250 mg, 0.68 mmol) in tetrahydrofuran (14 ml). After 24 h, the solution was
diluted with 10% aqueous potassium carbonate (20 ml), washed with ethyl
acetate, then acidified (pH 5) with 1 N HCl to give a solid which was washed
dried over MgSO4 before the solvent was evaporated to give an oil which with ethyl acetate and chromatographed on silica gel (CH2Cl2–CH3OH, 9/1).
was chromatographed on silica gel (dichloromethane–methanol, 9/1) to give An analytical sample was recrystallized (149 mg, 65%): mp ꢄ250 °C (2-
1
amide (8) (500 mg, 84%). Rfꢀ0.41 (ethyl acetate–cyclohexane, 4/1). IR propanol). Rfꢀ0.44 (dichloromethane–methanol, 9/1). IR cmꢂ1: 1655. H-
cmꢂ1: 1718, 1618. 1H-NMR (CDCl3) d: 2.41 (3H, s), 2.97 (3H, s), 3.09 (3H, NMR (CD3OD) d: 2.40 (3H, s), 3.04 (3H, s), 3.20 (3H, s), 4.12 (2H, s), 7.26
s), 4.04 (3H, s), 4.27 (2H, s), 7.35 (2H, d), 7.52 (2H, d), 8.18 (1H, d), 8.28 (1H, d), 7.51 (1H, d), 7.63 (2H, d), 8.06 (1H, s), 8.07 (2H, d). 13C-NMR
(1H, d), 8.93 (1H, s). LC-MS (APCIꢁ) m/z: 352 (MHꢁ).
(DMSO-d6) d: 17.8, 28.9, 35.3, 37.0, 115.6, 115.9, 120.6, 122.4, 126.5,
3-Dimethylcarbamoylmethyl-2-(4-methylphenyl)imidazo[1,2-a]pyri- 127.0, 129.2, 135.0, 139.5, 142.6, 142.9, 168.1, 169.0. HPLC-ESI-MS m/z:
dine-6-carboxylic Acid (1) 0.1 N Aqueous lithium hydroxide (28 ml, 338 (M+Hꢁ).
2.8 mmol) was added dropwise to a stirred solution of amide (8) (500 mg,
1.42 mmol) in tetrahydrofuran (28 ml). After 18 h, the solvent was evapo- References
rated and the aqueous phase was successively washed with chloroform, acid-
ified (pH 5) with 1 N HCl and extracted with 1-butanol. After removal of the
organic solvent under reduced pressure, the resulting solid was washed with
ethyl acetate and purified by thick layer chromatography (dichloromethane–
methanol, 4/1): 72 mg, 15%. Rfꢀ0.13 (dichloromethane–methanol, 9/1). 1H-
NMR (CD3OD) d: 2.46 (3H, s), 3.06 (3H, s), 3.24 (3H, s), 4.35 (2H, s), 7.35
(1H, d), 7.55 (1H, d), 7.85 (2H, m), 8.65 (1H, s). HPLC-ESI-MS m/z: 338
(MꢁHꢁ).
1) Crestani F., Martin J. R., Möhler H., Rudolph U., Br. J. Pharmacol.,
131, 1251—1254 (2000).
2) Blackaby W. P., Atack J. R., Bromidge F., Castro J. L., Goodacre S. C.,
Hallett D. J., Lewis R. T., Marshall G. R., Pike A., Smith A. J., Street
L. J., Tattersall D. F. D., Wafford K. A., Bioorg. Med. Chem. Lett., 16,
1175—1179 (2006).
3) Rowlett J. K., Platt D. M., Lelas S., Atack J. R., Dawson G. R., Proc.
Natl. Acad. Sci. U.S.A., 102, 915—920 (2005).
a-Bromo-4-(carboethoxy)acetophenone (9) Bromine (0.27 ml, 5.2
mmol) was added dropwise to a stirred solution of ethyl 4-acetylbenzoate
4) Rudolph U., Crestani F., Möhler H., Trends Pharmacol. Sci., 22, 188—
194 (2001).

September 2006
1321
5) Goullé J.-P., Anger J.-P., Ther. Drug Monit., 26, 206—210 (2004).
6) Kintz P., Villain M., Ludes B., Ther. Drug Monit., 26, 211—214
(2004).
7) Chèze M., Duffort G., Deveaux M., Pépin G., Forensic Sci. Int., 153,
3—10 (2005).
8) Baselt R. C., “Disposition of Toxic Drugs and Chemicals in Man,” 6th
ed., Biomedical Publications, Foster City, California, 2002, pp. 1115—
1117.
1894—1897 (1994).
13) Trapani G., Franco M., Ricciardi L., Latrofa A., Genchi G., Sanna E.,
Tuveri F., Cagetti E., Biggio G., Liso G., J. Med. Chem., 40, 3109—
3118 (1997).
14) Ohkanda J., Buckner F. S., Lockman J. W., Yokoyama K., Carrico D.,
Eastman R., de Luca-Fradley K., Davies W., Croft S. L., Van Voorhis
W. C., Gelb M. H., Sebti S. M., Hamilton A. D., J. Med. Chem., 47,
432—445 (2004).
9) George P., Allen J., Fr. Patent 2581646 (1986) [Chem. Abstr., 107, 15) Li Q., Woods K. W., Claiborne A., Gwaltney S. L., II, Barr K. J., Liu
59031s (1987)].
G., Gehrke L., Credo R. B., Hui Y.-H., Lee J., Warner R. B., Kovar P.,
Nukkala M. A., Zielinski N. A., Tahir S. K., Fitzgerald M., Kim K. H.,
Marsh K., Frost D., Ng S.-C., Rosenberg S., Sham H. L., Bioorg. Med.
Chem. Lett., 12, 465—469 (2002).
10) Allen J., Tizot A., J. Labelled Compd. Radiopharm., 23, 393—400
(1986).
11) Almirante L., Mugnaini A., Rugarli P., Gamba A., Zefelippo E., De
Toma N., Murmann W., J. Med. Chem., 12, 122—126 (1969).
12) Laufer S. A., Augustin J., Dannhardt G., Kiefer W., J. Med. Chem., 37,
16) Diaz P., Thoreau E., Fr. Patent 2750426 (1998) [Chem. Abstr., 128,
192541h (1998)].