Synthesis of the neurotransmitter 4-aminobutanoic acid (GABA) from diethyl cyanomalonate

Article abstract of DOI:10.2174/157018010789869361

GABA was synthesized by deethoxycarbonylation, ester hydrolysis and nitrile reduction of a highly functionalized intermediate obtained by alkylation of diethyl cyanomalonate with ethyl bromoacetate. By judicious employment of D ₂O or NaBD₄ in one of the three functional group transformation steps, deuterium was selectively introduced into each of the three possible sites in GABA.

Full text of DOI:10.2174/157018010789869361

Letters in Drug Design & Discovery, 2010, 7, 9-13
9
Synthesis of the Neurotransmitter 4-Aminobutanoic Acid (GABA) from Diethyl
Cyanomalonate
Matthew C. Cook, Ross D. Witherell and Robert L. White*
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, B3H 4J3, Canada
Received July 15, 2009: Revised September 13, 2009: Accepted September 14, 2009
Abstract: GABA was synthesized by deethoxycarbonylation, ester hydrolysis and nitrile reduction of a highly functionalized intermedi-
ate obtained by alkylation of diethyl cyanomalonate with ethyl bromoacetate. By judicious employment of D2O or NaBD4 in one of the
three functional group transformation steps, deuterium was selectively introduced into each of the three possible sites in GABA.
Keywords: GABA, Diethyl cyanomalonate, Neurotransmitter, Deuterium labeling, Deethoxycarbonylation.
O
INTRODUCTION
NH3
4-Aminobutanoic acid (ꢀ-aminobutyric acid, GABA, 1), the
most widespread neurotransmitter in mammals, is responsible for
synaptic inhibition in the central nervous system [1], thereby play-
ing an important role in neurological function and disease. Hypo- or
hyperactivities of the GABAergic neurotransmitter system lead to
epilepsy, schizophrenia and other neurological conditions [2,3], and
drugs affecting GABAergic activity are clinically useful for the
treatment of neurological illnesses [3,4]. As a result, the detection
and quantification of GABA (1) continues to be an important focus
[5], and deuterium-labeled GABA is particularly sought after as an
internal standard for the mass spectral analysis of GABA in human
plasma and cerebrospinal fluid [5-7]. Isotopically labeled GABA
has been used in drug synthesis (e.g., progabide [8]) and, currently,
there is interest in improving drug pharmacokinetics by selective
deuterium substitution [9]. Labeled GABA also has been employed
as a substrate for NMR molecular dynamics [10], and investigations
of the stereochemistry [11] and mechanism [12] of enzyme-
catalyzed reactions.
O
1
O
R
R'
EtO
C
N
2
O
R
R'
R
R'
+
EtO
C
H
C
Previously, isotopically labeled versions of GABA have been
synthesized via multi-step reaction sequences. Typically, [2-
2H2]GABA was obtained from unlabeled GABA after several suc-
cessive exchanges in DCl/D2O [7,8,10,12] or NaOD/D2O [6]. Simi-
larly, successive exchange reactions on succinimide (pyridine/D2O)
[13], followed by imide reduction and lactam hydrolysis in
DCl/D2O, provided GABA deuterated at C-2 and C-3. Alterna-
tively, hydrogenation of a triple bond in a synthetic precursor has
been used to place tritium at C-2,3 of GABA [14]. Selective deu-
teration at C-4 of GABA has been achieved by LiAlD4 reduction of
ester [8,12] and imide [13] groups in synthetic precursors. Enzyme-
catalyzed reactions, placing one deuterium at C-3 [15] or C-4 [11],
have introduced chirality, producing single isomers of monodeuter-
ated GABA.
N
N
Br
3
4
5
a R = R' = H
b R = H; R' = CO2Et
c R = R' = CO2Et
Scheme 1.
electrophilic and nucleophilic synthons, such as a monohalogenated
acetate ester (e.g., 3) and the anion of acetonitrile (4a) or equivalent
(4b, 4c). The ethoxycarbonyl group introduced by using a stabilized
anion as the nucleophile can be removed selectively from the in-
termediates 2b and 2c under neutral conditions via the Krapcho
reaction [16]; subsequent ester hydrolysis and nitrile reduction fur-
nishes GABA.
While several multi-step sequences and exchange processes are
available, the route devised in the present investigation offers
greater flexibility, providing a single precursor into which deute-
rium can be introduced specifically into any combination of three
different locations, including C-3, a position less accessible from
known routes.
For the route outlined above, carbon isotopes would be
introduced most easily from commercially available 13C-labeled
ethyl bromoacetate, whereas the use of deuterated reagents during
the conversion of the highly functionalized intermediate (2) to
GABA (1) would likely lead to the introduction of hydrogen iso-
topes at specific locations in GABA. For example, the use of D2O
in the Krapcho deethoxycarbonylation of diethyl ethylmalonate
provided ethyl [2-2H2]butanoate containing 80-90% deuterium [17].
RESULTS AND DISCUSSION
Synthesis of GABA
Initially, the carbon-carbon bond forming step was attempted in
acetone using the K2CO3-mediated reaction of methyl bromoacetate
(6) with ethyl cyanoacetate (5b) (Scheme 2), but an equimolar mix-
ture of dimethyl 3-cyano-3-ethoxycarbonylpentanedioate (7) and
unreacted ethyl cyanoacetate (5b) was isolated. Based on the initial
electrophile, the dialkylation product 7 was obtained in nearly
From a retrosynthetic perspective (Scheme 1), dissection of the
central carbon-carbon bond in GABA (1) leads to readily available
*Address correspondence to this author at the Department of Chemistry, Dalhousie
University, Halifax, Nova Scotia, B3H 4J3, Canada; Tel: 1-902-494-6403; Fax: 1-902-
494-1310; E-mail: robert.white@dal.ca
1570-1808/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

10 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 1
Cook et al.
quantitative yield, reflecting the fact that the anion of the monoal-
kylated product is formed in preference to that of ethyl cyanoace-
tate and is readily alkylated. Dialkylation of ethyl cyanoacetate has
been observed when the anion is generated from sodium hydride
[18], but selective monoalkylation also has been reported [19].
prominent resonances accompanied by an equal number of lower
intensity resonances (ca. 10:1 ratio). After addition of triethylamine
to the NMR solution, only one set of ethyl ester resonances (shifted
slightly to lower frequency) was observed, suggesting the deproto-
nation of both components and the formation of anion 4c, the con-
1
jugate base of 5c (pKa 1.3 [23]). The H NMR spectrum of 5c ex-
CO2Et
MeO2C
hibited a very broad signal at ꢀ 13.2, attributed to an enolic hydro-
gen in the minor component. Enolization of 5c in acetonitrile was
indicated in a study of proton transfer reactions [24] and is prece-
dented by the high enol content documented for 20 structurally
analogous cyanomalonamides [25].
+
C
Br
N
5b
6
K2CO3
acetone
Solvent had a large effect on the enol content of diethyl cya-
nomalonate (5c). Only one set of ethyl ester resonances was ob-
served in the 1H NMR spectra of 5c recorded in CD3CN and
CD3OD, and the measured UV extinction coefficient (log ꢁ) was
larger in hexane (3.23) than in acetonitrile (2.97). The larger pro-
portion of enol observed for 5c in nonpolar solvents is consistent
with the relationship between enol content and solvent polarity
documented for cyanomalonamides [25] and ꢂ-diketones [26]. The
larger UV extinction coefficients measured for diethyl cya-
nomalonate (5c) in methanol (3.93) and water (4.28) were attributed
to ionization in polar protic solvents, forming 4c, the conjugate base
of the strong carbon acid 5c [23].
N
CO2Et
C
5b
+
MeO2C
CO2Me
7
Scheme 2.
To avoid similar dialkylation of diethyl malonate
((EtO2C)2CH2), a third ethoxycarbonyl group was introduced to
limit the reaction to the monoalkylation of a tertiary carbanion
((EtO2C)3C-) [20]. Using this strategy and conditions adapted from
literature procedures [21,22], the analogous trisubstituted substrate,
diethyl cyanomalonate (5c), was prepared by acylation of ethyl
cyanoacetate (5b) (Scheme 3). Under optimized conditions (dry
acetone, vigorous stirring and anhydrous, powdered K2CO3), repro-
ducible yields (> 80% after distillation) were obtained.
When more than two equivalents of ethyl chloroformate (8)
were included in the acylation reaction mixture (Scheme 3), a sec-
ond product was detected by NMR spectroscopy. Repetition of the
reaction using 10 equivalents of 8, led to the isolation of the second
product in high yield, and its identification as the cyano triester 9.
Diethyl cyanomalonate (5c), formed by the initial acylation, is read-
ily deprotonated under basic conditions. The conjugate base 4c,
thus formed, undergoes acylation, to yield the cyano triester 9, ef-
fectively demonstrating the nucleophilicity of 4c. As well, several
literature reports [22,27] of the alkylation of the diethyl cya-
nomalonate anion (4c) reinforce the inherent nucleophilicity of this
stabilized anion.
EtO2C CO2Et
CO2Et
H
C
C
K2CO3
ClCO2Et, 8
N
5c
N
5b
Using the above K2CO3/acetone conditions, the diethyl cya-
nomalonate anion (4c) reacted with ethyl bromoacetate (3). Upon
addition of a catalytic amount of KI, a higher yield of diethyl 2-
cyano-2-ethoxycarbonylbutanedioate (2c) was obtained. In acetone,
displacement of chloride by iodide is well known [28], suggesting
the intermediacy of ethyl iodoacetate, an electrophile with a better
leaving group.
K2CO3
acetone
HCl
acetone
excess
EtO2C CO2Et
C
EtO2C CO2Et
EtO2C
ClCO2Et, 8
C
N
N
acetone
9
4c
For the Krapcho deethoxycarbonylation reaction [16] of 2c, re-
1
action mixtures in DMSO-d6 were monitored using H NMR spec-
EtO2C
troscopy to optimize reaction times and temperatures. Deethoxy-
carbonylation was complete after 16 h at 140 °C or after only 0.5 h
at 180 °C, yielding ethyl 3-cyanopropanoate (10) and ethanol.
Products requiring no further purification were isolated in good
yield (87% at 140 °C and 71% at 180 °C) from the DMSO reaction
mixtures, simply by addition of water and extraction into ether. If
necessary, residual DMSO was removed by back extraction into
water.
Br
3
H2O
NaCl
EtO2C CO2Et
EtO2C
EtO2C
C
C
N
10
heat
DMSO
N
2c
Ester hydrolysis was accomplished by heating ethyl 3-
cyanopropanoate (10) in aqueous NaOH at reflux for 1.5 h. Nitrile
hydrolysis was minimized by using only 10% excess base, and
nitrile reduction [29] was achieved by carefully adding NaBH4 and
CoCl2 directly to the aqueous reaction mixture after ester hydroly-
sis. Following cation-exchange chromatography, GABA (1) was
obtained as a colorless powder in an overall yield of 32% from
ethyl cyanoacetate (5b).
NaOH
H2O
reflux
NaBH4
CoCl2
O2C
NH3
O2C
C
Na
N
11
1
H2O
Scheme 3.
The presence of two components in purified diethyl cya-
nomalonate (5c) was supported by spectral evidence. Two nitrile
bands (2262, 2225 cm-1) were apparent in the IR spectrum (neat),
Deuterium Labeling
In principle, Krapcho deethoxycarbonylation [17] of 2c in the
presence of D2O would form ethyl [3,3-2H2]-3-cyanopropanoate
1
while the H and 13C NMR spectra of 5c in CDCl3 each displayed

Synthesis of GABA
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 1 11
upon loss of the geminal ester groups from the quaternary carbon.
Under conditions (140 °C, 16 h) similar to those employed in the
synthetic sequence, deethoxycarbonylation of 2c yielded deuterated
ethyl 3-cyanopropanoate; the reduced intensity of the AA'BB' pro-
tons (ꢀ 2.72-2.62) relative to the ethyl CH2 signal (ꢀ 4.20) indicated
about 80% deuteration at C-2 and C-3. In the 13C NMR spectrum of
the isolated product, the signal at ꢀ 13.0 (C-3, ꢁ to the nitrile) was
absent, and the intensity of the signal at ꢀ 30.0 (C-2, ꢁ to the ester)
was reduced significantly because of the relaxation, NOE and mul-
tiplicity effects of deuterium [30]. These observations are consistent
with complete and partial (about 60%) deuteration at C-3 and C-2,
respectively.
(ppm) relative to tetramethylsilane (TMS, ꢀ = 0) or residual solvent
signal (D2O, ꢀ 4.79). All coupling constants (J) are reported in Hz;
splitting patterns are designated as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet) and br (broad). Ultraviolet (UV) and in-
frared (IR) spectra were recorded on Hewlett-Packard 8452A UV-
Vis and Bomem Michelson spectrometers, respectively. Electron
ionization mass spectra (EI-MS) and accurate mass determinations
(HRMS) were obtained on a CEC-110B sector instrument, and
electrospray ionization mass spectra (ESI-MS) were collected on a
Finnigan LCQ Duo ion trap using flow injection [32].
Dimethyl 3-Cyano-3-ethoxycarbonylpentanedioate (7)
The introduction of deuterium during Krapcho deethoxycar-
A mixture of ethyl cyanoacetate (5b, 0.74 g, 6.5 mmol), methyl
bromoacetate (6, 1.0 g, 6.5 mmol) and anhydrous K2CO3 (1.8 g, 13
mmol) in acetone (12 mL) was stirred in the dark at -18 °C for 19 h.
Ether (100 mL) was added, and the solution was dried (anhydrous
MgSO4). Rotary evaporation afforded a colorless oil (1.2 g), com-
posed of equimolar amounts of starting material (5b) and dialky-
lated product (7).
bonylation of 2c was evaluated in a series of experiments monitored
by H NMR spectroscopy. Deuterium incorporation at 140 °C was
1
not influenced by the use of DMSO-d6 and/or a longer reaction time
(24 h). At a higher temperature (180 °C) in DMSO-d6, similar lev-
els of deuterium incorporation were observed at 0.5 and 2 h; at 17 h
90% deuteration was obtained, but the isolated yield of deuterated
product was only 43%.
5b: 1H NMR (CDCl3) ꢀ: 4.27 (2H, q, J = 7.1), 3.50 (2H, s), 1.33
The lack of substantially higher deuterium incorporation at
longer reaction times (i.e., > 16 h at 140 °C and > 0.5 h at 180 °C)
suggests that exchange does not occur readily after deethoxycar-
bonylation is complete. Also, no exchange was detected when 10,
the product of deethoxycarbonylation, was subjected to Krapcho
reaction conditions (D2O/DMSO-d6, 140 °C, 18 h). Together these
results strongly suggest that exchange leading to deuterium incor-
poration at C-2 of 10 occurs prior to deethoxycarbonylation. On the
other hand, the corresponding protons in the starting cyano triester
2c and the monodeethoxycarbonylated intermediate 2b are more
acidic because of the additional electron-withdrawing inductive
effect provided by the ꢂ-ester substituents on C-3. Thus, at the tem-
peratures investigated, exchange and deethoxycarbonylation occur
as competing reactions. The high level of deuterium incorporation
suggests that exchange in 2c is more rapid than deethoxycarbonyla-
tion.
(3H, t, J = 7.1); 13C NMR (CDCl3) ꢀ: 163.0, 113.3, 62.9, 24.7, 13.8.
1
7: (98% from 6). H NMR (CDCl3) ꢀ: 4.33 (2H, q, J = 7.1),
3.75 (6H, s), 3.10 and 3.07 (4H, AB, J = 16.9), 1.35 (3H, t, J = 7.1);
13C NMR (CDCl3) ꢀ: 168.7, 166.9, 117.4, 63.6, 52.4, 42.2, 39.3,
13.9.
Diethyl Cyanomalonate (5c)
Ethyl cyanoacetate (5b, 5.70 mL, 54 mmol) was added to a
stirred mixture of K2CO3 (24 g, 190 mmol, pre-treated by heating at
o
130 C for 16 h) and acetone (70 mL, dried over K2CO3 for 16 h).
After 5 min, ethyl chloroformate (8, 10.2 mL, 108 mmol) was
added and the reaction mixture was heated at reflux for 6 h. The
reaction was allowed to cool to room temperature, water (100 mL)
was added and the acetone was removed in vacuo. Additional water
(50 mL) was added and the basic solution was extracted with di-
chloromethane (3 x 20 mL). The aqueous layer was acidified to pH
< 0 with concentrated HCl (30 mL) and extracted with diethyl ether
(4 x 25 mL). The ether layers were combined, dried over anhydrous
MgSO4, and evaporated in vacuo to yield a crude orange oil (8.86
g). The crude products from three replicate reactions (8.72 g, 7.74 g
and 8.86 g) were combined (25.32 g) and 22.5 g of the product was
vacuum distilled (b.p. 115-116 °C) yielding 5c as a clear, colorless
oil (20.5 g, 81%). IR (neat) cm-1: 2262, 2225, 1751; UV (solvent
ꢃmax (log ꢄ)): acetonitrile 248 nm (2.97), dioxane 252 nm (2.42),
hexane 256 nm (3.23), methanol 246 nm (3.93), water 246 nm
When C-2,3-deuterated ethyl 3-cyanopropanoate was subjected
to ester hydrolysis in base and subsequent nitrile reduction, the
resulting GABA contained deuterium (4% d3, 60% d2, 30% d1 and
6% d0 by ESI-MS) located predominantly at C-2 (1H NMR). The
significant exchange adjacent to the cyano group in deuterated 3-
cyanopropanoate most likely occurred during base-promoted ester
hydrolysis, a result noted previously under basic conditions [31].
With this observation, the ester hydrolysis of 10 was carried out
in D2O; subsequent nitrile reduction yielded GABA with a high
1
level of deuterium (97%). The H NMR spectrum exhibited two
broad singlets at ꢀ 2.96 and 2.25, consistent with deuterium at C-3.
While the ESI mass spectrum confirmed the high deuterium con-
tent, it also revealed about 20% incorporation of a third deuterium,
presumably by exchange of protons adjacent to the ester carbonyl
group prior to hydrolysis.
1
(4.28); Major tautomer: H NMR (CDCl3) ꢀ: 4.59 (s), 4.34 (q, J =
7.3), 1.34 (t, J = 7.3); 13C NMR (CDCl3) ꢀ: 160.6, 111.5, 63.8, 44.5,
13.5. Minor tautomer: 1H NMR (CDCl3) ꢀ: 13.2 (br s), 4.42 (q, J =
7.3), 1.40 (t, J = 7.3); 13C NMR (CDCl3) ꢀ: 176.0, 113.6, 64.2, 60.3,
13.9. 1H NMR (CD3CN) ꢀ: 4.81 (0.7H, s), 4.30 (4H, q, J = 7), 1.29
(6H, t, J = 7); 1H NMR (CD3OD) ꢀ: 4.73 (0.4H, s), 4.31 (4H, q, J =
7.3), 1.31 (6H, t, J = 7.3); ESI-MS (negative mode) m/z 184(100%)
[M - H]-, 66(12): CID (29%) of m/z 184: m/z 156(10), 66(100);
HRMS (70 eV) calculated for C8H11NO4 = 185.0688 amu, found =
185.0696 ± 0.0008 amu.
To place deuterium at C-4 in GABA, the third possible position,
NaBD4 was used in the nitrile reduction step; product containing
about 70% deuterium at C-4 was isolated. The low incorporation of
deuterium is consistent with partial exchange of deuterium during
the initial reaction of borodeuteride with CoCl2 and water [29], and
preferential transfer of hydride to the nitrile. The latter is indicated
by the kinetic isotope effect of 3.3 observed for the reduction of
benzonitrile [29].
Diethyl 2-Cyano-2-ethoxycarbonylpropanedioate (9)
Anhydrous K2CO3 (4.84 g, 35 mmol, pre-treated by heating at
o
130 C for 16 h) was added to acetone (15 mL, dried over K2CO3
EXPERIMENTAL SECTION
for 16 h) and the mixture was stirred for 15 min. Ethyl cyanoacetate
(5b, 1.06 mL, 10 mmol) was added, followed after 5 min by ethyl
chloroformate (8, 9.56 mL, 100 mmol). The reaction mixture was
refluxed with stirring for 6 h, allowed to cool, water (100 mL) was
1H and 13C NMR spectra were acquired on a Bruker/Tecmag
AC-250 MHz NMR spectrometer operating at 250.13 and 62.9
MHz, respectively. Chemical shifts are given in parts per million

12 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 1
Cook et al.
added and solution was acidified with concentrated HCl (10 mL) to
a pH < 0. The product was extracted with diethyl ether (3 x 25 mL).
The ether layers were combined and, dried over anhydrous MgSO4;
the solvent was removed in vacuo. A portion (1.51 g) of the crude
product (2.51 g, 98%) was dissolved in ether (50 mL) and washed
with saturated sodium bicarbonate (3 x 25 mL). The ether layer was
dried over anhydrous MgSO4 and, upon evaporation in vacuo, 9
was obtained as colorless oil (1.06 g, 70%). IR (neat) cm-1: 2267,
1778, 1753; 1H NMR (CDCl3) ꢀ: 4.40 (2H, q, J = 7.1), 1.37 (3H, t,
J = 7.1); 13C NMR (CDCl3)+ ꢀ: 159.6, 111.3, 64.9, 61.7, 13.8; EI-
34.6, 24.1; ESI-MS (positive mode) m/z 105(5), 104(100%) [M +
H]+, 87(13), 86(13): CID (17%) of m/z 104: m/z 87(100), 86(56).
Deuterated GABA
In trial experiments to assess deuterium incorporation during
Krapcho
deethoxycarbonylation,
diethyl
2-cyano-2-
ethoxycarbonylbutanedioate (2c, 0.75 mmol), NaCl (0.19 mmol)
and D2O (22.5 mmol) in DMSO (or DMSO-d6) were heated in a
capped tube. Product was isolated by extraction into ether (4 x 20
mL) after addition of water (15 mL). Deuterium incorporation was
calculated from the 1H NMR spectra of isolated products.
•
MS (70 eV): 257(10%) [M ], 185(9), 156(10), 139(14), 128(18),
43(15), 28(100); HRMS (70 eV) calculated for C11H15NO6
=
257.0899 amu, found = 257.0881 ± 0.0008 amu.
Ethyl [2,2,3,3-2H4]-3-Cyanopropanoate
Diethyl 2-Cyano-2-ethoxycarbonylbutanedioate (2c)
Diethyl 2-cyano-2-ethoxycarbonylbutanedioate (2c, 0.54 g, 2.0
A mixture of diethyl cyanomalonate (5c, 7.15 g, 38.6 mmol)
and anhydrous K2CO3 (2.69 g, 19.5 mmol) in acetone (50 mL) was
stirred at reflux for 20 min. Ethyl bromoacetate (3, 6.46 g, 38.6
mmol) and KI (0.32 g, 1.9 mmol) were added, and the stirred mix-
ture was heated at reflux for 14 h. The reaction mixture was mixed
with chloroform (300 mL); the mixture was dried (anhydrous
CaCl2), filtered, and solvent was removed by rotary evaporation.
The residue was dissolved in ether (130 mL) and the solution was
washed with aqueous NaHSO3 (5 x 100 mL). The ether layer was
dried (anhydrous CaCl2), decolorized over activated carbon, and
rotary evaporated to yield 2c as a pale yellow oil (9.56 g, 91%). IR
(neat) cm-1: 2256, 1746; 1H NMR (CDCl3) ꢀ: 4.35 (4H, q, J = 7.3),
4.22 (2H, q, J = 7.3), 3.27 (2H, s), 1.35 (6H, t, J = 7.3), 1.29 (3H, t,
J = 7.3); 13C NMR (CDCl3) ꢀ: 167.9, 162.8, 114.6, 64.3, 61.8, 51.7,
mmol), D2O (1.08 mL, 60 mmol) and sodium chloride (0.029 g,
0.50 mmol) were heated in DMSO-d6 (2 mL) at 180 C for 14 h.
o
The reaction mixture was allowed to cool to room temperature,
saturated brine was added (50 mL) and the aqueous layer was ex-
tracted with ether (2 x 25 mL). The ether layers were combined and
washed with brine (25 mL), which was back extracted with ether
(25 mL). The ether layers were combined, treated with charcoal for
1 h and filtered through Celite. After drying over MgSO4, the sol-
vent was removed in vacuo giving ethyl [2,2,3,3-2H4]-3-
1
cyanopropanoate as an oil (0.18 g, 62%). H NMR (CDCl3) ꢀ: 4.20
(2H, q, J = 7.1), 2.67 (0.58H, m), 1.29 (3H, t, J = 7.0); 13C NMR
(CDCl3) ꢀ: 170.1, 118.7, 61.5, 14.2.
+
•
[2,2-2H2]GABA
38.5, 13.8; EI-MS (70 eV) 271(1%) [M ], 197(2), 169(2), 225(3),
152(10), 124(12), 97(23), 43(18), 28(100); HRMS (70 eV): calcula-
ted for C12H17NO6 = 271.1056 amu, found = 271.1043 ± 0.0008
amu.
Ethyl [2,2,3,3-2H4]-3-cyanopropanoate (3-53, 0.18 g, 1.4 mmol)
and NaOH (0.120 g, 3.01 mmol) in water (15 mL) were heated at
reflux for 1.5 h. Reduction with CoCl2·6H2O (0.654 g, 2.75 mmol)
and NaBH4 (0.52 g, 14 mmol) followed by ion-exchange chroma-
tography gave [2,2-2H2]GABA as a white powder (0.07 g, 48%). 1H
NMR (D2O) ꢀ: 3.00 (2H, t, J = 7.3), 2.28 (0.4H, m), 1.88 (2H, br t,
J = 7); ESI-MS [M + H]+: m/z 106(49%) 105 (24), 104(4).
Ethyl 3-Cyanopropanoate (10)
Duplicate reaction mixtures, each consisting of a solution of di-
ethyl 2-cyano-2-ethoxycarbonylbutanedioate (2c, 1.36 g, 5.0
mmol), water (2.70 mL, 150 mmol) and sodium chloride (0.073 g,
1.2 mmol) in DMSO (5 mL), were heated at 140 °C for 18 h. Upon
cooling to room temperature, the reaction mixtures were combined;
water was added (100 mL), and the product was extracted into ether
(4 x 50 mL). The combined ether layers were dried over MgSO4
and the solvent was removed in vacuo, giving 10 as an oil (1.10 g,
87%). 1H NMR (CDCl3) ꢀ: 4.20 (2H, q, J = 7.1), 2.72-2.62 (4H, m),
1.29 (3H, t, J = 7.1); 13C NMR (CDCl3) ꢀ: 170.1, 118.6, 61.4, 30.0,
[3,3-2H2]GABA
As described above, ethyl 3-cyanopropanoate (10, 0.635 g, 5.0
mmol) was hydrolyzed in D2O (15 mL) containing NaOH (0.228 g,
5.7 mmol) and reduced using CoCl2·6H2O (2.38 g, 10.0 mmol) and
NaBH4 (1.89 g, 50 mmol). Ion-exchange chromatography yielded
1
[3,3-2H2]GABA as a white powder (0.24 g, 46%). H NMR (D2O)
+
+
•
ꢀ: 2.96 (2H, br s), 2.25 (2H, br s); ESI-MS [M + H]+: m/z 108(4%),
14.1, 13.0; EI-MS (70 eV) 128(2%) [M + H] [33], 127(5) [M ],
100(99), 82(91), 56(39), 55(100).
107(28), 106(100), 105(8), 104(3).
[4,4-2H2]GABA
GABA (1)
As described above, ethyl 3-cyanopropanoate (10, 0.255 g, 2.0
mmol) was hydrolyzed in aqueous NaOH (0.106 g, 2.6 mmol; 15
mL H2O) and reduced using CoCl2·6H2O (0.952 g, 4.0 mmol) and
NaBD4 (0.837 g, 20 mmol). Ion-exchange chromatography yielded
Ethyl 3-cyanopropanoate (10, 0.258 g, 2.0 mmol) and NaOH
(1.27 g, 3.2 mmol) were heated at reflux in water (15 mL) for 1.5 h.
Upon cooling to room temperature, CoCl2·6H2O (0.966 g, 4.1
mmol) was added to the reaction mixture, followed by the addition
of solid NaBH4 (0.768 g, 20 mmol) in small portions. After stirring
at room temperature for 4 h, water (100 mL) was added and the pH
was adjusted to 4.5 using conc. HCl. The mixture was filtered, and
the filtrate was applied to an Amberlite IR-120 ion-exchange col-
umn (30 x 2 cm, H+ form). The column was eluted with 0.5 M
aqueous ammonia and 200-mL fractions were collected. Fractions
3–8 were concentrated in vacuo and treated with activated charcoal
for 1 h. The charcoal was removed by filtration through Celite and
the filtrate was freeze dried, giving GABA as a white powder (0.10
1
[4,4-2H2]GABA as a white powder (0.18 g, 87%). H NMR (D2O)
ꢀ: 2.95 (0.5H, m), 2.27 (2H, t, J = 7.0), 1.85 (2H, m); ESI-MS [M +
H]+: m/z 107(8%), 106(100), 105(85), 104(21).
CONCLUSIONS
GABA (1) was synthesized in five steps, consisting of the in-
troduction of a blocking group, carbon-carbon bond formation, and
three functional group modifications. The utility of the reaction
sequence was demonstrated by the preparation of deuterated
GABA. The isotopic label was introduced predominantly at C-2, C-
1
g, 50%). H NMR (D2O) ꢀ: 3.00 (2H, t, J = 7.6), 2.29 (2H, t, J =
7.3), 1.89 (2H, quintet, J = 7.3); 13C NMR (D2O) ꢀ: 181.8, 39.5,

Synthesis of GABA
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 1 13
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ACKNOWLEDGEMENTS
We thank the Natural Sciences and Engineering Research
Council of Canada (NSERC) for research funding; scholarships to
RDW and MCC were kindly provided by NSERC and the Walter
C. Sumner Foundation, respectively. We are indebted to M.
Gardner for assistance with initial experiments, and NMR-3 and
MMSLab for providing NMR and mass spectra.
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