Synthesis of deuterium-labeled pregabalin

Article abstract of DOI:10.1002/jlcr.1936

This paper describes the synthesis of deuterium-labeled pregabalin. The stable isotopic-labeled compound was obtained in nine steps starting from the commercially available 4-[²H₁₁]methylvaleric acid as the stable-labeled reagent. It

Full text of DOI:10.1002/jlcr.1936

Research Article
Received 12 July 2011,
Revised 27 August 2011,
Accepted 1 September 2011
Published online 13 October 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1936
Synthesis of deuterium-labeled pregabalin
a
b
a
c
*
Zucheng Shen, Wenchao Wu, Wei Wang, and Liqin Chen
This paper describes the synthesis of deuterium-labeled pregabalin. The stable isotopic-labeled compound was obtained in
nine steps starting from the commercially available 4-[2H11]methylvaleric acid as the stable-labeled reagent. It is used as an
internal standard for analysis and metabolic studies.
2
Pregabalin labeled with H was obtained in nine steps using the commercially available 4-[2H11]methylvaleric acid as the
stable-labeled reagent. The synthesis prevents deuterium from scrambling and offers the labeled compound with over
99% isotopic enrichment.
Keywords: deuterium; labeled; synthesis; pregabalin
diisopropylamide below À35ꢀC, which was then alkylated with
Introduction
tert-butyl bromoacetate to produce [4R-(3S*,4a,5a)]-4-methyl-b-(2-
methylpropyl-g,2-dioxo-5-phenyl-3-[2H10]oxazolidine butanoic acid,
Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, is an
anticonvulsant drug used for neuropathic pain and as an adjunct
1,1-dimethylethyl ester (4). The one deuterium removed does not
therapy for partial seizures.1 It is marketed by Pfizer (New York,
scramble the protons on the a position of t-butyl ester. The yield
NY, USA) under the trade name Lyrica. As a follow-up compound
for this reaction was found to be highly dependent on the careful
to gabapentin, pregabalin was found to be more potent and
temperature control (if the reaction temperature was allowed to
rise above À35ꢀC, yields decreased significantly).8 The retention
robust in preclinical models of epilepsy and neuropathic pain.
Recent studies have shown that pregabalin is effective in treat-
factor of the starting material (compound 3) and product (com-
ing chronic pain in disorders such as fibromyalgia and spinal
pound 4) was identical. The reaction can be continued to the next
cord injury. In June 2007, pregabalin became the first medication
reaction without isolation. Compound (4) was treated with the
approved by the US Food and Drug Administration specifically
mixture of LiOH and H2O2 (30%) to form (S)-2-(2-methylpropyl)-
1,4-[2H10]butanedioic acid, 4-(1,1-dimethylethyl) ester (5). Reduction
for the treatment of fibromyalgia. However, as an alkylated
analog of g-aminobutyric acid (GABA), pregabalin is inactive at
of compound (5) with borane/dimethyl sulfide produced (S)-3-
(hydroxymethyl)-5-[2H10]methylhexanoic acid, 1,1-dimethylethyl
GABAA and GABAB receptors, is not converted metabolically into
a GABAA or a GABAB antagonist, and does not block GABA
ester (6). Compound (6) was treated with p-toluenesulfonyl
uptake or alter GABA degradation.2 Accordingly, pharmacoki-
chloride to form (S)-5-methyl-3-[[[(4-methylphenyl)sulfonyl]
oxy]methyl]-[2H10]hexanoic acid, 1,1-dimethyl ester (7). Treat-
netics and absorption, distribution, metabolism, and excretion
investigations on pregabalin should be extensively performed
to support clinical studies. Thus, a stable isotope-labeled stan-
dard is required. Although A.W. Czarnik has described the preg-
ment of (7) with sodium azide in dry dimethyl sulfoxide gave
(S)-3-(azidomethyl)-5-[2H10]methylhexanoic acid, 1,1-dimethylethyl
ester (8). Hydrolysis of compound (8) in a mixture of formic acid
and sulfuric acid gave (S)-3-(azidomethyl)-5-[2H10]methylhexanoic
abalin labeled at different positions with various numbers of
deuterium,3 it lacked detailed procedures and data. The present
acid (9). Reduction of compound (9) with hydrogen (50psi) and
paper describes the preparation of [2H10]pregabalin in detail
(Figure 1).
Pd/C (10%) afforded [2H10]pregabalin (10).
After purification by recrystallization, the desired product (10)
was obtained with 99.8% chemical purity. Mass spectrometry
Results and discussion
Although pregabalin has been readily prepared via several syn-
thetic routes,4–8 the details of the synthesis of deuterium-labeled
pregabalin have not been described. Scheme 1 presents a concise
and efficient synthetic scheme for preparing [2H10]pregabalin (10).8
aSchool of Pharmaceutical Sciences, Nanjing University of Technology, 5 Xinmofan
Road, Nanjing, Jiangsu, 210009, China
bSchool of Biotechnology and Pharmaceutical Engineering, Nanjing University of
Technology, 5 Xinmofan Road, Nanjing, Jiangsu, 210009, China
Treatment of compound (1) with SOCl2 produced 4-[2H11
]
methylpentanoyl chloride (2). (4R,5S)-4-methyl-5-phenyl-2-
oxazolidinone was treated with n-BuLi in dry THF and then
cHi-Tech Research Institute and State Key Laboratory of Materials-Oriented
Chemical Engineering, Nanjing University of Technology, 5 Xinmofan Road,
alkylated with compound (2) to give (4R,5S)-4-methyl-3-(1-oxo-4- Nanjing, Jiangsu, 210009, China
[2H11]methylpentyl)-5-phenyl-2-oxazolidinone (3). n-BuLi must be
*Correspondence to: Liqin Chen, Hi-Tech Research Institute and State Key
Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of
Technology, 5 Xinmofan Road, Nanjing, Jiangsu, 210009, China.
added carefully to the reaction mixture and stopped once the red
color forms to prevent epimerization and provide high yield for
compound (3).8 The anion of (3) was readily formed with lithium E-mail: liqin_chen@hotmail.com
J. Label Compd. Radiopharm 2011, 54 809–812
Copyright © 2011 John Wiley & Sons, Ltd.

Z. Shen et al.
Agilent 1200 HPLC (Agilent Technologies, Santa Clara, CA, USA)
with an XDB-C18 column, 5 mm, 4.6 Â 150 mm.
4-[2H11]methylpentanoyl chloride (2)
To a solution of SOCl2 (3.12 g, 26.22 mmol) was added 4-[2H11]
methylvaleric acid (2.50 g, 19.68 mmol) slowly. The resulting solu-
tion was stirred at 35ꢀC for 1 h and then stirred at 75ꢀC for
30 min. The reaction mixture was concentrated under reduced
pressure to afford (2) as a colorless liquid (2.48 g, 86.7%). Crude
product (2) was used without further purification.
Figure 1. Chemical structure of pregabalin.
(4R,5S)-4-methyl-3-(1-oxo-4-[2H11]methylpentyl)-5-phenyl-
2-oxazolidinone (3)
analysis of compound (10) revealed that the compound has over
99% deuterium enrichment. The compound provided an excel-
lent internal standard for LC-MS-MS studies.
A solution of oxazolidinone (3.10 g, 17.50 mmol) in dry THF
(26 ml) was cooled to À5ꢀC under an argon atmosphere. n-BuLi
in hexane (7.9 ml 2.2 M, 17.50 mmol) was added while maintain-
ing an internal temperature below À5ꢀC at all times. The solution
of compound (2) (2.48 g, 17.00 mmol) in dry THF was added
slowly. The reaction mixture was stirred at À5ꢀC for 2 h and then
Experimental
General
All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) quenched with water (6 ml). The product was extracted with
and CDN Isotopes Inc. (Pointe-Claire, Quebec, Canada). Mass heptane (3 Â 20 ml). The combined organic layers were washed
spectra were recorded using a Quattro micro API mass spectro- with saturated NaHCO3 solution (10 ml), water (3 Â 10 ml), and
meter (Waters Corporation, Milford, MA, USA). 1H NMR spectra brine (10 ml), dried over anhydrous Na2SO4, filtered and concen-
were recorded on a Bruker 300-MHz instrument (Bruker Daltonics trated under reduced pressure to give a brown oil. The crude
Inc., Billerica, MA, USA). Chemical purities were determined by an product was purified by chromatography on silica gel (120 g)
Scheme 1. Synthesis route of deuterium-labelled pregabalin.
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 809–812

Z. Shen et al.
column, eluted with hexanes/EtOAc (20:1) to afford (3) as a white then cooled to below 5ꢀC in an ice bath. Borane/dimethyl sulfide
solid (4.20 g, 86.0%).
(3.4 ml 2 M, 6.70 mmol) was added slowly. The reaction mixture
1H NMR (300 MHz, CDCl3): d 7.42-7.30 (m, 5H), 5.67 (d, 1H), 4.77 was stirred at 5ꢀC for 3 h, warmed to about 25ꢀC and then stirred
(m, 1H), 0.89 (d, 3H).
for overnight. Thin layer chromatography analysis showed that
the reaction was complete. The reaction mixture was cooled to
0ꢀC and quenched with water (2 ml). The resulting mixture was
diluted with ether (30 ml) and then washed with saturated
NaHCO3 solution (3 Â 10 ml), water (3 Â 10 ml), dried over
Na2SO4, filtered and concentrated under vacuum to dryness to
give an oil (0.70 g, 53.0%), which was used without further
purification.
[4R-(3S*,4a,5a)]-4-methyl-b-(2-methylpropyl-g,2-dioxo-5-
phenyl-3-[2H10]oxazolidinebutanoic acid, 1,1-dimethylethyl
ester (4)
Flask A
• The solution of (3) (4.20 g, 14.66 mmol) in dry THF (12 ml) was
cooled to below À40ꢀC under nitrogen.
1H NMR (300 MHz, CDCl3): d 3.66 (dd, 1H), 3.50 (dd, 1H), 2.33
(m, 1H), 2.27 (dd, 1H), 1.46 (s, 9H).
Flask B
(S)-5-methyl-3-[[[(4-methylphenyl)sulfonyl]oxy]methyl]-
[2H10]hexanoic acid-1,1-dimethyl ester (7)
• The solution of diisopropylamine (1.48 g, 14.66 mmol) in dry THF
(2 ml) was cooled to below 0ꢀC. n-BuLi in hexane (6.7 ml 2.2M,
14.66 mmol) was added slowly to the amine/tetrahydrofuran
solution. The solution was stirred for 30 min below 0ꢀC. The result-
ing lithium diisopropylamide solution was transferred slowly to
flask A.
To a solution of (6) (0.70 g, 3.09 mmol) in heptane (10 ml) at 0ꢀC
was added p-toluenesulfonyl chloride (0.71 g, 3.70 mmol) and
triethylamine. The reaction mixture was stirred at 27ꢀC over-
night. 1 M HCl (2 ml) was added, and the solution was extracted
with Et2O (3 Â 20 ml). The combined organic layers were concen-
trated under reduced pressure to give a yellow oil. The crude
product was purified by chromatography on silica gel (50 g),
eluted with hexanes/EtOAc (20:1) to afford (7) as an oil (0.25 g,
21.2%).
After the addition, the resulting solution was stirred for 30 min
below À35ꢀC. tert-butyl bromoacetate (2.86 g, 14.66 mmol) was
added slowly to the solution of flask A. The reaction mixture
was stirred for 30 min below À35ꢀC. The temperature was then
allowed to rise slowly to À15ꢀC over 2.5–3.5 h. The reaction
was quenched with saturated NH4Cl solution (8 ml). The mixture
was warmed up to 25ꢀC and then extracted with EtOAc
(3 Â 20 ml). The combined organic layers were washed with
water (3 Â 10 ml) and brine (10 ml), dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure to give a light
yellow solid. The crude product was purified by chromatography
on silica gel (200 g) column, eluted with hexanes/EtOAc (20:1), to
afford (4) as an off-white solid (3.20 g, 54.6%).
1H NMR (300 MHz, CDCl3): d 7.80 (d, 2H), 7.36 (t, 2H), 4.01
(dd, 1H), 3.93 (dd, 1H), 2.45 (s, 3H), 2.29 (dd, 1H), 2.16 (dd, 1H),
1.40 (s, 9H).
(S)-3-(azidomethyl)-5-[2H10]methylhexanoic acid,
1,1-dimethylethyl ester (8)
To a stirred solution of compound (7) (0.25 g, 0.65 mmol) in
dry dimethyl sulfoxide (1 ml) was added sodium azide
(0.042 g, 0.65 mmol) and stirred at 60ꢀC overnight. The
reaction mixture was cooled to 20ꢀC and diluted with
water (10 ml) and heptane (20 ml). The organic layer was
separated, and the aqueous layer was back extracted with
heptane (3 Â 20 ml). The combined organic layers were
washed with brine (10 ml), dried over Na2SO4, filtered and
concentrated under vacuum to give (8) as a light yellow
oil. The crude (8) (0.12 g, 72.7%) was used without further
purification.
1H NMR (300 MHz, CDCl3): d 7.61-7.29 (m, 5H), 5.66 (d, 1H), 4.86
(m, 1H), 2.70 (dd, 1H), 2.42 (dd, 1H), 1.41 (s, 9H), 0.91 (d, 3H).
(S)-2-(2-methylpropyl)-1,4-[2H10]butanedioic acid,
4-(1,1-dimethylethyl) ester (5)
Compound (4) (3.20 g, 8.00 mmol) was dissolved in dry THF
(10 ml) and then cooled to 0ꢀC. Separately, a solution of
LiOH•H2O (3.36 g, 8.00 mmol) in water (3.5 ml) was cooled to
0ꢀC. H2O2 (1.5 ml, 30%) was added slowly to the LiOH solution.
The peroxide solution was slowly added to the aforementioned
THF solution. The resulting solution was stirred at 0ꢀC for 4 h
and then quenched through the addition of saturated Na2SO3
solution (9 ml). The mixture was extracted with Et2O (3 Â 20 ml).
The aqueous layer was back washed with Et2O (20 ml). The com-
bined organic layers were washed with brine (30 ml), dried over
anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give a light yellow oil. The crude product was purified
by chromatography on silica gel (100 g), eluted with hexanes/
EtOAc (15:1) to afford (5) as a colorless oil (1.40 g, 72.7%).
1H NMR (300 MHz, CDCl3): d 2.58 (dd, 1H), 2.33 (dd, 1H), 1.42
(s, 9H).
1H NMR (300 MHz, CDCl3): d 3.37 (dd, 1H), 3.29 (dd, 1H), 2.29
(dd, 1H), 2.22 (dd, 1H), 1.46 (m, 9H).
(S)-3-(azidomethyl)-5-[2H10]methylhexanoic acid (9)
To a solution of compound (8) (0.12 g, 0.48 mmol) in ice
bath was added slowly the mixture of formic acid (0.48 g,
10.40 mmol) and sulfuric acid (5 mg, 0.05 mmol). The reac-
tion mixture was stirred at room temperature for 2 h
and quenched with saturated NaCl solution (2 ml). The
reaction was diluted with Et2O (20 ml). The organic layer
was separated, and the aqueous layer was extracted with
Et2O (3 Â 10 ml). The combined organic layers were washed
with water (5 ml) and brine (5 ml), dried over Na2SO4, fil-
tered and concentrated under vacuum to give a light yel-
(S)-3-(hydroxymethyl)-5-[2H10]methylhexanoic acid,
1,1-dimethylethyl ester (6)
After drying by azeotropic distillation with toluene, compound low oil (0.07 g, 75.0%), which was carried directly into the
(5) (1.40 g, 5.80 mmol) was dissolved in dry Et2O (3.5 ml), and next step.
J. Label Compd. Radiopharm 2011, 54 809–812
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

Z. Shen et al.
1H NMR (300 MHz, CDCl3): d 3.39 (dd, 1H), 3.33 (dd, 1H) 2.36
(dd, 1H) 2.28 (dd, 1H).
Acknowledgements
This work is financially supported by the San Chuang Joint
Project of Nanjing University of Technology and Nanjing New &
High Technology Industry Development Zone.
[2H10]pregabalin (10)
To a solution of (9) (0.40 g, 2.00 mmol) in isopropyl alcohol, Pd/C
(10%, 40 mg) was added. The solution was stirred at room tem-
perature under 50 psi of hydrogen overnight. The mixture was
heated to 80ꢀC and filtered through a bed of Celite to remove
catalyst, and then washed with a hot (80ꢀC) solution of water
and isopropyl alcohol (1:1). The combined filtrate was concen-
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www.jlcr.org
Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 809–812