Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase

Article abstract of DOI:10.1016/j.bmc.2012.08.009

Two principal neurotransmitters are involved in the regulation of mammalian neuronal activity, namely, γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, and l-glutamic acid, an excitatory neurotransmitter. Low GABA levels in the brain have been implicated in epilepsy and several other neurological diseases. Because of GABA's poor ability to cross the blood-brain barrier (BBB), a successful strategy to raise brain GABA concentrations is the use of a compound that does cross the BBB and inhibits or inactivates GABA aminotransferase (GABA-AT), the enzyme responsible for GABA catabolism. Vigabatrin, a mechanism-based inactivator of GABA-AT, is currently a successful therapeutic for epilepsy, but has harmful side effects, leaving a need for improved GABA-AT inactivators. Here, we report the synthesis and evaluation of a series of heteroaromatic GABA analogues as substrates of GABA-AT, which will be used as the basis for the design of novel enzyme inactivators.

Full text of DOI:10.1016/j.bmc.2012.08.009

Bioorganic & Medicinal Chemistry 20 (2012) 5763–5773
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of novel heteroaromatic substrates
of GABA aminotransferase
⇑
Dustin D. Hawker, Richard B. Silverman
Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, and Center for Molecular Innovation and Drug Discovery,
Northwestern University, Evanston, IL 60208-3113, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two principal neurotransmitters are involved in the regulation of mammalian neuronal activity, namely,
Received 20 June 2012
Revised 2 August 2012
Accepted 8 August 2012
Available online 16 August 2012
c
-aminobutyric acid (GABA), an inhibitory neurotransmitter, and L-glutamic acid, an excitatory neuro-
transmitter. Low GABA levels in the brain have been implicated in epilepsy and several other neurological
diseases. Because of GABA’s poor ability to cross the blood–brain barrier (BBB), a successful strategy to
raise brain GABA concentrations is the use of a compound that does cross the BBB and inhibits or inac-
tivates GABA aminotransferase (GABA-AT), the enzyme responsible for GABA catabolism. Vigabatrin, a
mechanism-based inactivator of GABA-AT, is currently a successful therapeutic for epilepsy, but has
harmful side effects, leaving a need for improved GABA-AT inactivators. Here, we report the synthesis
and evaluation of a series of heteroaromatic GABA analogues as substrates of GABA-AT, which will be
used as the basis for the design of novel enzyme inactivators.
Keywords:
c
-Aminobutyric acid
GABA aminotransferase
Enzyme inhibition
Synthetic substrates
Ó 2012 Elsevier Ltd. All rights reserved.
Heteroaromatic compounds
1. Introduction
that has been effective in raising brain GABA concentrations is the
use of a compound that crosses the blood–brain barrier and inhib-
The two major neurotransmitters involved in the regulation of
brain neuronal activity are -aminobutyric acid (GABA), an inhibi-
tory neurotransmitter, and -glutamic acid, an excitatory neuro-
transmitter.1 GABA concentration is regulated by two pyridoxal
50-phosphate (PLP)-dependent enzymes:
-glutamic acid decarbox-
ylase (GAD), which catalyzes the conversion of -glutamate to
GABA, and GABA aminotransferase (GABA-AT), which degrades
GABA to succinic semialdehyde (SSA). GABA-AT catabolizes GABA
via transamination while converting PLP to pyridoxamine 50-phos-
its or inactivates GABA-AT.
c
L
The FDA approved a selective mechanism-based inactivator12,13
of GABA-AT, vigabatrin (1; 4-aminohex-5-enoic acid; c-vinyl
GABA), which is currently marketed for the treatment of epi-
lepsy.14–18 In addition, vigabatrin has been shown to be effective
in the treatment of addiction.19,20 Unfortunately, treatment with
vigabatrin has the potential for side effects, with 25–50% of patients
exhibiting irreversible abnormalities in peripherial vision following
chronic administration.21 Vigabatrin also suffers from low potency
(KI = 10 mM)14 and poor BBB permeability, resulting in doses of
1–3 g/day, leaving a clear need for improved GABA-AT inactiva-
tors.12,13,22 Vigabatrin was determined to inactivate GABA-AT
through two mechanistic pathways (Scheme 2): a Michael addition
mechanism (pathway a) and an enamine mechanism (pathway
b).23 Both inactivation pathways have the possibility of generating
potentially bioactive side products (7 and 8) via hydrolysis prior to
inactivation. These side products could be related to vigabatrin’s
side effects; however, the biochemical mechanisms underlying
them are still currently unknown.24
Previously, in an effort to increase potency and eliminate the
generation of side products, conformationally restricted analogues
of vigabatrin were synthesized and evaluated as potential inactiva-
tors of GABA-AT.25–29 Of those tested, 2 was found to be a very po-
tent GABA-AT inactivator with a potency that is 187 times greater
than that of vigabatrin.26 Because of the low lipophilicity of this
amino acid compound, a series of benzene-containing amino acid
L
L
phate (PMP), which is restored to PLP by conversion of
a-ketoglu-
tarate to
L
-glutamate (GABA-AT, E.C. 2.6.1.19; Scheme 1).2 When
the concentration of GABA diminishes below a certain level in
the brain, convulsions result; raising the brain GABA levels termi-
nates the seizure.3–5 A reduction in the concentrations of GABA and
of the enzyme GAD has been implicated not only in the symptoms
associated with epilepsy6,7 but also with several other neurological
diseases such as Huntington’s chorea8 Parkinson’s disease,9
Alzheimer’s disease,10 and tardive dyskinesia.11 GABA brain levels
cannot be increased by direct administration of GABA, however,
because it does not cross the blood-brain barrier (BBB). A strategy
Abbreviations: GABA,
c-aminobutyric acid; GABA-AT, GABA aminotransferase;
GAD,
L
-glutamic acid decarboxylase; PLP, Pyridoxal 50-phosphate; SSA, succinic
semialdehyde; PMP, pyridoxamine 5’-phosphate; BBB, Blood–brain barrier.
⇑
Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491 7713.
E-mail address: Agman@chem.northwestern.edu (R.B. Silverman).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.009

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D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
Scheme 1. Catalytic mechanism for GABA-AT.
Scheme 2. Mechanisms of inactivation of GABA-AT by vigabatrin (1). Pyr stands for the pyridoxal 50-phosphate ring system.
F
F
CO2H
H2N
H2N
CO2H
H2N
H2N
CO2H
CO2H
1
2
3a
3b
X
a. X = O
b. X = S
c. X = NH
X
X
H2N
H2N
CO2H
CO2H
H2N
CO2H
6
4
5
Figure 1. Previously described GABA-AT inactivators (1 and 2), aromatic substrates (3), and the new series of heteroaromatic analogues (4–6).
analogues of GABA (3, Fig. 1) were evaluated as substrates that
could be used as the basis for the development of inactivators with
improved lipophilicity30 and, possibly, bioavailability, and it was
found that some of these compounds were substrates of GABA-
AT.31 On the basis of this result, the remarkable potency of 2, and
the generally better activity observed for exocyclic-functionalized
cyclopentyl analogues over cyclohexyl analogues,27,32 a series of
five-membered heteroaromatic analogues was designed from
structures 3 as potential substrates for GABA-AT (4–6, Fig. 1). Be-
cause mechanism-based inactivators must undergo a catalytic

D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
5765
transformation by the enzyme to a species that inactivates the en-
zyme,13 identification of substrates is imperative for the develop-
ment of new inactivator scaffolds. Here we report the synthesis
and evaluation of 4–6 as substrates and inhibitors of GABA-AT,
the first step toward the design of mechanism-based inactivators.
formylation of 30 generated aldehydes 31a and 31b as a mixture
that was readily separated with flash chromatography. Treatment
of either with hydroxylamine provided the corresponding aldoxi-
mes (32a and b).35 Catalytic hydrogenation of the aldoximes fol-
lowed by Boc protection gave 33a or b. Saponification of the
ester36 followed by Boc deprotection provided desired products
4c and 5c as hydrochloride salts.
Despite having minimal literature precedent, the synthesis of
3,4-substituted pyrrole 6c was attempted but initial efforts were
unsuccessful. This was attributed to the instability of this substitu-
tion pattern, leading to decomposition under ambient conditions,
and was not pursued further.
2. Results and discussion
2.1. Chemistry
Compounds 4a and 4b were commercially available but were
converted to their corresponding hydrochloride salts prior to anal-
ysis. Compounds 5a and 5b were not commercially available and
were synthesized as shown in Scheme 3. The synthesis of 5a began
with 4,5-dibromofuran-2-carboxylic acid (7). Selective dehalogen-
ation of 9 using zinc metal, followed by esterification, provided 10
in good yields. Palladium-catalyzed cyanation of 10 generated ni-
trile 11, which was then reduced and Boc protected to yield 12.
Saponification of the ester followed by Boc deprotection of 12 gave
the desired amino acid product (5a) as the hydrochloride salt.
Compound 5b was synthesized starting with methyl 4-methylthi-
ophene-2-carboxylic acid (14). Radical bromination of 14 provided
bromomethyl intermediate 15 in good yields.33 Displacement of
the bromide with azide followed by catalytic hydrogenation and
Boc protection provided 16. Saponification of the ester followed
by Boc deprotection yielded the desired amino acid (5b) as the
hydrochloride salt.
The 3,4-disubstituted furan and thiophene analogues (6a and
6b) were synthesized as shown in Scheme 4. Synthesis of 6a began
with dimethyl ester 18. Monosaponification provided the ester-
acid 19. Selective reduction of the carboxylic acid gave alcohol
2034 that was then converted to the chloromethyl intermediate
21. Substitution of the chloride with azide followed by catalytic
hydrogenation and Boc protection generated 22. Saponification of
the ester followed by Boc deprotection yielded the desired amino
acid (6a) as the hydrochloride salt. Compound 6b was synthesized
starting with the bis-esterification of 3,4-thiophenedicarboxylic
acid (24) followed by a synthetic route similar to the one used to
generate 6a.
2.2. Enzymatic assay results
All of the synthesized compounds (4–6) were tested as compet-
itive inhibitors of GABA-AT using a coupled enzyme assay.37 None
of the compounds, at 5 mM concentration, inhibited any of the
coupling enzymes in the assays, namely, L-glutamate oxidase,
horseradish peroxidase, and succinic semialdehyde dehydroge-
nase. All of the compounds have Ki values (Table 1) similar to the
Km of GABA, which suggests binding to the active site with similar
affinity. Next, the compounds were evaluated as substrates, with
kinetic constants Km and kcat determined by Hanes and Woolf plots
(kinetic constants determined by direct Michaelis–Menten fitting
were not significantly different).38,39 Compounds 4a, 5b, 5c, and
6a exhibit Km values similar to GABA while the Km values of com-
pounds 4b and 4c were much higher. Transamination was ob-
served for 6b indicating substrate activity; however, a Km could
not be accurately determined. Compound 5a presented the lowest
Km at 0.31 0.07 mM, approximately 4-fold lower than that of
GABA. All of the compounds presented very slow turnover rates,
having kcat values that are orders of magnitude lower than that
of GABA. Compound 4a has the highest kcat/Km value indicating it
is the most efficient GABA-AT substrate of the series. The clogP val-
ues, a measure of lipophilicity, indicates that almost all of the com-
pounds are more lipophilic than 1 and 2, but not as lipophilic as 3a.
The normal catalytic mechanism of GABA-AT involves Schiff
base formation between GABA and the lysine-bound PLP coenzyme
followed by active-site base removal of the
c-proton to form a res-
The pyrrole amino acids (4c and 5c) were synthesized in paral-
lel from a common precursor (30) as shown in Scheme 5. Vilsmeier
onance-stabilized carbanion (Scheme 1). Reprotonation on the
Scheme 3. Synthesis of the 2,4-disubstituted furan and thiophene amino acids. Reagents and conditions: (a) (i) Zn (s), NH4OH, H2O, (ii) H2SO4, MeOH, reflux; (b) Pd(PPh3)4,
Zn(CN)2, DMF; (c) Boc2O, NaBH4, NiCl2ꢀ6H2O (cat.), MeOH; (d) LiOHꢀH2O, MeOH, H2O; (e) 6 N HCl (aq); (f) NBS, benzoyl peroxide, CCl4; (g) (i) NaN3, DMF, (ii) Boc2O, H2 (H), Pd-
C, EtOAc.

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Scheme 4. Synthesis of 3,4-disubstituted furan and thiophene amino acids. Reagents and conditions: (a) 1 equiv NaOH, MeOH; (b) BH3ꢀTHF, THF; (c) PPh3, CCl4, CH2Cl2; (d)
(i)NaN3, DMF, (ii) Boc2O, H2 (g), Pd-C (cat.), EtOAc; (e) LiOHꢀH2O, MeOH, H2O; (f) 6 N HCl (aq); (g) H2SO4, MeOH; (h) PPh3, CBr4, CH2Cl2.
Scheme 5. Synthesis of 2,4- and 2,5-disubstituted pyrrole amino acids. Reagents and conditions: (a) POCl3, DMF, CH2Cl2; (b) NH2OHꢀHCl, K2CO3, H2O; (c) Boc2O, H2 (g), Pd-C
(cat.), EtOAc; (d) LiOHꢀH2O, MeOH, H2O; (e) 6 N HCl (aq).
coenzyme followed by hydrolysis provides SSA and PMP, which is
converted back to PLP with conversion of -ketoglutarate to -glu-
responsible for deprotonation, the rate-determining step for turn-
over of GABA,40 and therefore decreasing turnover rates.
a
L
tamate. Despite having Ki values that suggest similar active-site
binding affinity, all compounds exhibited markedly low substrate
activity when compared with GABA. It was hypothesized that their
rigid structure may require a binding conformation that orients the
requisite proton unfavorably relative to the active-site base
To identify the rate-determining step for turnover of these ana-
logues, a kinetic isotope effect experiment was conducted using
deuterium labeled 5a, the analogue exhibiting the lowest Km value
of the series. The labeled analogue ([D2]-5a) was synthesized from
11 (Scheme 3) as shown in Scheme 6, and evaluated as a substrate

D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
5767
Table 1
Kinetic constants for substrate activity and inhibition of GABA-AT for 4–6 and GABA
Compound
Ki (mM)
Km (mM)
kcat (minꢁ1
)
kcat/Km (minꢁ1/mMꢁ1
)
clogPa
4a
4b
4c
5a
5b
5c
6a
6b
GABA
1.40 0.14
2.49 0.28
2.14 0.31
1.64 0.44
1.35 0.08
1.59 0.14
2.27 0.51
2.86 0.51
—
2.1 0.1
6.3 0.2
9.5 0.3
0.31 0.07
1.7 0.3
1.4 0.3
2.8 0.5
>10
3.4 ꢂ 10ꢁ2 4 ꢂ 10-3
1.0 ꢂ 10ꢁ2 3 ꢂ 10ꢁ3
1.5 ꢂ 10ꢁ3 2 ꢂ 10ꢁ4
2.3 ꢂ 10ꢁ3 1 ꢂ 10ꢁ4
5.2 ꢂ 10ꢁ3 5 ꢂ 10ꢁ4
3.4 ꢂ 10ꢁ3 3 ꢂ 10ꢁ4
8.3 ꢂ 10ꢁ4 5 ꢂ 10ꢁ5
n.d.b
0.016
0.19
0.83
0.09
0.0016
0.00016
0.0074
0.00031
0.0024
0.00030
n.d.b
ꢁ0.12
0.52
ꢁ0.22
ꢁ0.43
0.21
1.3 0.3
49
5
38.6
a
clogP, the calculated logP, was determined using molinspiration.com software. The clogP for 1 was ꢁ0.39, for 2 was ꢁ0.37, and for 3a was 1.01.
b
n.d. = Not determined.
Scheme 6. Synthesis of deuterated 5a. Reagents and conditions: (a) Boc2O, NaBD4, NiCl2ꢀ6H2O (cat.), MeOH; (b) LiOHꢀH2O, MeOH, H2O; (c) 6 N HCl (aq).
Scheme 7. Presumed substrate mechanism of 5a or [D2]-5a.
alongside unlabeled 5a. The Hk/Dk value for substrate turnover was
determined to be 1.22 0.07, which is considerably lower than
would be expected for a primary isotope effect if deprotonation
were rate limiting.41 Alternatively, a Hk/Dk of 1.22 0.07 is sugges-
tive of a secondary kinetic isotope effect and is typically observed
primary isotope effect from C–H(D) bond cleavage.51 While these
results are intriguing and somewhat unexpected, further work
would be required to truly identify the substrate mechanism for
these analogs; it is not unreasonable that the isotope effect is asso-
ciated with a nonchemical step. Ultimately, for a compound to be
an effective mechanism-based inactivator of an enzyme, large rate
constants (kinact) are not necessary. Therefore, we are currently
working on the syntheses of potential mechanism-based inactiva-
tors using the structure of the substrates identified in this study as
the basis for design.
with a change in hydridization
a- or b- to the heavy atom label,
with a Hk/Dk >1 indicative of a transition from sp3 to sp2 (or sp2
to sp) hybridization.
In the catalytic mechanism of GABA-AT with substrate GABA,
and presumably with this series of compounds, product release is
achieved by hydrolysis of the PMP-product Schiff base formed after
transamination. Hydrolysis of Schiff bases is known to proceed
through the formation of a hemiaminal intermediate followed by
elimination of the amine to form a carbonyl (Scheme 7). Hemiami-
nals, like hemiacetals, are known to be stabilized by electron-with-
drawing substituents.42–46 Hemiaminals have also been shown to
be stabilized by both intermolecular and intramolecular hydrogen
bonding.47–50 The hemiaminal formed during product hydrolysis
(35, Scheme 7) is stabilized by the electron-withdrawing nature
of the heterocyles and potentially favorable interactions within
the active site. On the basis of the secondary isotope effect ob-
served for [D2]-5a, and the known stability of aryl substituted
hemiaminals, the breakdown of this intermediate may be rate lim-
iting, leading to a normal secondary isotope effect (change in
hybridization from sp3 to sp2), which may supercede the expected
3. Conclusion
A series of heteroaromatic compounds were designed as poten-
tial substrates and inhibitors of GABA-AT. All analogues were found
to act as competitive inhibitors having Ki values near the Km of
GABA, suggesting similar active site binding affinity. In addition,
all analogues were observed to act as substrates; however, turnover
rates were found to be much lower than that of GABA. An isotope
effect experiment with one of the analogues suggests that the
hemiaminal formed during hydrolysis of the Schiff base between
the product and coenzyme PMP is rate limiting as a result of
increased stabilization by the heterocycle. These results are intrigu-
ing because, as shown previously in Scheme 2, vigabatrin has the
potential to produce metabolites (7 and 8) generated from

5768
D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
unwanted Schiff base hydrolysis prior to inactivation. The results
reported here will be useful in the design of improved vigabatrin
analogues, as well as other novel mechanism-based inactivators of
GABA-AT, which do not generate potentially harmfully metabolites.
4.2.3. Methyl 4-bromofuran-2-carboxylate (10)
4,5-Dibromofuran-2-carboxylic acid (9, 1.00 g, 3.7 mmol) was
suspended in water (11 mL) and NH4OH (3.5 mL) with vigorous
stirring at ambient temperature. Powdered zinc metal (1.30 g,
20.3 mmol) was added, and the mixture was allowed to stir at
ambient temperature for 3 h. The reaction mixture was filtered
through a pad of Celite and acidified (pH 2) with 2 N HCl. The fil-
trate was extracted with EtOAc (4 ꢂ 50 mL), dried (Na2SO4), and
concentrated to dryness under reduced pressure to provide
665 mg of a white powder. To this crude intermediate dissolved
4. Experimental procedures
4.1. General methods
Analogues 4a and 4b were purchased from Matrix Scientific and
ChemBridge, respectively. All syntheses were conducted under
anhydrous conditions in an atmosphere of argon, using flame-dried
apparatus and employing standard techniques in handling air-sen-
sitive materials, unless otherwise noted. All solvents were distilled
and stored under an argon or nitrogen atmosphere before use. All
reagents were used as received. Analytical thin layer chromatogra-
phy was visualized by ultraviolet light. Flash column chromatogra-
phy was carried out under a positive pressure of nitrogen.1H NMR
in MeOH (12 mL) was added concentrated sulfuric acid (80 lL)
while stirring. The resulting solution was heated to reflux and stir-
red overnight. The reaction mixture was allowed to cool to room
temperature followed by concentration under vacuum. The result-
ing crude residue was then partitioned between saturated aqueous
NaHCO3 and diethyl ether, and the aqueous layer was further ex-
tracted with diethyl ether (2 ꢂ 40 mL). The combined ether solu-
tions were washed with brine (20 mL), dried (Na2SO4) and
concentrated to dryness under reduced pressure to provide
652 mg (3.18 mmol, 86%) of the desired product as a clear oil. 1H
spectra were recorded on 500 MHz spectrometers. 1H NMR and 13
C
NMR spectra were obtained with a Bruker AVANCE III 500 spec-
trometer. Data are presented as follows: chemical shift (in ppm
on the d scale relative to d = 0.00 ppm for the protons in TMS), inte-
gration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, br = broad), coupling constant (J;Hz). Coupling con-
NMR (500 MHz, CDCl3)
d 7.61 (d, J = 1.0 Hz, 1H), 7.18 (d,
J = 1.0 Hz, 1H), 3.91 (s, 3H). 13C NMR (126 MHz, CDCl3) d 158.05,
144.89, 144.44, 120.20, 101.18, 52.19. HRMS (ESI): Calcd for
C6H5BrO3 [M+Na]+ 226.9314; found 226.932.
stants were taken directly from the spectra and are uncorrected.13
C
4.2.4. Methyl 4-cyanofuran-2-carboxylate (11)
NMR spectra were recorded at 125 MHz, and all chemical shift val-
ues are reported in ppm on the d scale, with an internal reference
of d 77.16 or 49.0 for CDCl3 or CD3OD, respectively. High resolution
mass spectra (HRMS) were measured with an Agilent 6210 LC-TOF
(ESI, APCI, APPI) mass spectrometer. The purity of the synthesized
final compounds was determined by HPLC analysis to be P95%.
Methyl 4-bromofuran-2-carboxylate (10, 141 mg, 0.74 mmol),
Pd(PPh3)4 (85 mg, 0.07 mmol), and Zn(CN)2 (52 mg, 0.44 mmol)
were suspended in anhydrous DMF (5 mL) under an argon atmo-
sphere. The resulting mixture was heated to 80 °C and stirred over-
night. After cooling to room temperature the reaction mixture was
partitioned between water and diethyl ether. The aqueous layer
was extracted with ether (3 ꢂ 10 mL). The combined organics were
washed with brine (10 mL), dried (Na2SO4), and concentrated un-
der reduced pressure to give a yellow oil, which was chromato-
graphed (ethyl acetate/hexanes, 1:6) to afford a white solid
(85 mg, 76%). 1H NMR (500 MHz, CDCl3) d 8.06 (d, J = 0.9 Hz, 1H),
7.33 (d, J = 0.9 Hz, 1H), 3.94 (s, 3H). 13C NMR (126 MHz, CDCl3) d
157.59, 151.84, 145.92, 117.76, 111.60, 100.03, 52.69. HRMS (LC-
TOF): Calcd for C7H5NO3 151.0269; found 151.0285.
The column used was
a Phenomenex Luna 5 lm 200 Å,
4.6 ꢂ 250 mm. The column was thoroughly equilibrated at 100%
solvent A, minimum 5 volumes. The compounds were eluted with
a gradient from solvent A (90:10, CH3CN: 50 mM NH4OAc pH 5.8)
to solvent B (50:50, CH3CN:10 mM NH4OAc pH 5.8), 0–2.5 min, 0%
B isocratic; 2.5–10 min, 0–100% B; 10–20 min, 100% B isocratic.
The compounds had the following retention times: 4c,
11.57 min; 5a, 11.24 min; 5b, 11.25 min; 5c, 11.57 min; 6a,
11.20 min; 6b, 11.27 min; [D2-5a], 11.47 min.
4.2.5. Methyl 4-((tert-butoxycarbonyl)aminomethyl)furan-2-
carboxylate (12)
4.2. Preparation and characterization of new compounds
Methyl 4-cyanofuran-2-carboxylate (11, 100 mg, 0.66 mmol),
Boc2O (289 mg, 1.32 mmol), and NiCl2ꢀ6H2O (16 mg, 0.07 mmol)
were dissolved in methanol and cooled to 0 °C with stirring. So-
dium borohydride (176 mg, 4.63 mmol) was added in portions
over 30 min. After addition of sodium borohydride, the reaction
was allowed to warm to room temperature, and to stir for 1 h, at
4.2.1. 5-(Aminomethyl)furan-2-carboxylic acid hydrochloride
(4a)
5-(Aminomethyl)furan-2-carboxylic acid (25.0 mg, 0.18 mmol)
was dissolved in 2 N HCl (5 mL) followed by solvent removal under
reduced pressure. This was repeated twice to yield 5-(amino-
methyl)furan-2-carboxylic acid hydrochloride as an off white pow-
der (31.5 mg, 100%). 1H NMR (500 MHz, MeOD) d 7.22 (d, J = 3.3 Hz,
1H), 6.71 (d, J = 3.3 Hz, 1H), 4.25 (s, 2H). 13C NMR (126 MHz,
MeOD) d 161.27, 152.05, 147.49, 119.76, 113.69, 36.80. HRMS
(ESI): Calcd for C6H7NO3 [MꢁH]ꢁ 140.0353; found 140.0343.
which point diethylenetriamine (72 lL, 0.66 mmol) was added.
The mixture was allowed to stir for an additional 30 min before
solvent evaporation. The residue was dissolved in EtOAc and
washed with saturated aqueous NaHCO3 (2 ꢂ 30 mL), dried
(Na2SO4), concentrated under reduced pressure, and chromato-
graphed (ethyl acetate/hexanes, 1:5) to afford the desired product
as a white solid (86 mg, 51%). 1H NMR (500 MHz, CDCl3) d 7.47 (s,
1H), 7.12 (s, 1H), 4.90 (br s, 1H), 4.14 (m, 2H), 3.86 (s, 3H), 1.42 (s,
9H). 13C NMR (126 MHz, CDCl3) d 159.03, 155.75, 144.89, 143.51,
125.52, 118.07, 79.81, 52.00, 35.28, 28.35, 28.21. HRMS (ESI): Calcd
for C12H17NO5 [M+Na]+ 278.0999; found 278.1001.
4.2.2. 5-(Aminomethyl)thiophene-2-carboxylic acid
hydrochloride (4b)
5-(Aminomethyl)thiophene-2-carboxylic
acid
(20.0 mg,
0.13 mmol) was dissolved in 2 N HCl (5 mL) followed by solvent re-
moval under reduced pressure. This was repeated twice to yield 5-
(aminomethyl)thiophene-2-carboxylic acid hydrochloride as
a
light yellow powder (24.6 mg, 100%). 1H NMR (500 MHz, MeOD)
d 7.71 (d, J = 3.7 Hz, 1H), 7.26 (d, J = 3.7 Hz, 1H), 4.37 (s, 2H). 13C
NMR (126 MHz, MeOD) d 164.70, 142.48, 137.95, 134.51, 131.04,
38.68. HRMS (ESI): Calcd for C6H7NO2S [MꢁH]ꢁ 156.0125; found
156.0122.
4.2.6. 4-((tert-Butoxycarbonyl)aminomethyl)furan-2-carboxylic
acid (13)
To
a
solution of methyl 4-((tert-butoxycarbonyl)amino-
methyl)furan-2-carboxylate (12, 86 mg, 0.34 mmol) in MeOH
(2 mL) and water (2 mL) was added LiOH H2O (16 mg, 0.37 mmol)

D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
5769
and allowed to stir overnight. The reaction mixture was diluted in
water (10 mL) and extracted with EtOAc (2 ꢂ 10 mL), which was
discarded. The aqueous phase was adjusted to pH 1 with hydro-
chloric acid (2 N), and the suspension was washed with ethyl ace-
tate (3 ꢂ 10 mL). The combined organics were washed with brine
(10 mL), dried (Na2SO4), concentrated under reduced pressure,
and chromatographed (5% methanol with 0.5% acetic acid in
dichloromethane) to afford a white solid (72 mg, 89%). 1H NMR
(500 MHz, MeOD) d 7.60 (s, 1H), 7.15 (s, 1H), 4.08 (s, 2H), 1.44 (s,
9H). HRSM (ESI): Calcd for C11H14NO5 [MꢁH]ꢁ 240.0877; found
240.0868.
130.19, 79.96, 39.75, 28.36. HRMS (ESI): calcd for C11H15NO4S
[MꢁH]ꢁ 256.0649; found 256.0640.
4.2.11. 4-(Aminomethyl)thiophene-2-carboxylic acid
hydrochloride (5b)
Compound 5b was synthesized using a similar procedure to
that of 5a (84%). 1H NMR (500 MHz, MeOD) d 7.86 (s, 1H), 7.84
(s, 1H), 4.16 (s, 2H). 13C NMR (126 MHz, MeOD) d 164.65, 137.39,
135.76, 134.66, 133.81, 38.94. HRMS (ESI): Calcd for C6H6NO2S
[MꢁH]ꢁ 156.0125; found 156.0117.
4.2.12. 4-(Methoxycarbonyl)furan-3-carboxylic acid (19)
To a solution of dimethyl furan-3,4-dicarboxylate (18, 2.00 g,
10.9 mmol) in methanol (10 mL) was added sodium hydroxide
(434 mg, 10.9 mmol) and stirred overnight at room temperature.
Solvent was removed, and the crude product was partitioned be-
tween diethyl ether (30 mL) and water (30 mL). The aqueous phase
was extracted with ether (3 ꢂ 20 mL), and the combined organic
layers were discarded. The aqueous phase was adjusted to pH 1
with hydrochloric acid (2 N) and extracted with EtOAc
(3 ꢂ 20 mL). The combined organics were washed with brine
(10 mL), dried (Na2SO4), and concentrated under reduced pressure
to yield the desired product as a white solid (1.72 g, 93%). 1H NMR
(500 MHz, CDCl3) d 8.26 (d, J = 1.8 Hz, 1H), 8.13 (d, J = 1.8 Hz, 1H),
4.01 (s, 3H). 13C NMR (126 MHz, CDCl3) d 167.13, 161.30, 152.67,
150.73, 118.92, 115.08, 77.30, 53.64. HRMS (ESI): Calcd for
C6H7O5 [M+Na]+ 193.0107; found 193.0109.
4.2.7. 4-(Aminomethyl)furan-2-carboxylic acid hydrochloride
(5a)
To
4-((tert-butoxycarbonyl)aminomethyl)furan-2-carboxylic
acid (13, 50 mg, 0.21 mmol) was added 6 N HCl (8 mL). The solu-
tion was stirred at room temp for 4 h. The solvent was then evap-
orated under reduced pressure followed by recrystallized from
ethanol/ethyl ether to give the product as white crystals (31 mg,
82%). 1H NMR (500 MHz, MeOD) d 7.89 (s, 1H), 7.34 (s, 1H), 4.06
(s, 2H). 13C NMR (126 MHz, MeOD) d 161.22, 147.55, 147.36,
121.28, 118.95, 34.85. HRMS (ESI): Calcd for C6H7NO3 [MꢁH]ꢁ
140.0353; found 140.0351.
4.2.8. Methyl 4-(bromomethyl)thiophene-2-carboxylate (15)30
Methyl 4-methylthiophene-2-carboxylate (14, 1 g, 6.4 mmol),
N-bromosuccinimide (1.25 g, 7.0 mmol), and benzoyl peroxide
(155 mg, 0.64 mmol) were dissolved in carbon tetrachloride
(30 mL). The reaction mixture was refluxed with stirring for 2 h,
followed by cooling to 0 °C and filtered. The reaction mixture
was then concentrated and chromatographed (ethyl acetate/hex-
anes, 1:6) to provide the desired product as a clear oil (1.34 g,
89%). 1H NMR (500 MHz, CDCl3) d 7.77 (d, J = 1.6 Hz, 1H), 7.47 (d,
J = 1.5 Hz, 1H), 4.44 (s, 2H), 3.86 (s, 3H). 13C NMR (126 MHz, CDCl3)
d 162.18, 138.87, 134.62, 134.06, 130.85, 52.37, 26.43. HRMS (ESI):
Calcd for C7H7BrO2S [MꢁBr]⁄+ 155.0161; found 155.0160.
4.2.13. Methyl 4-(hydroxymethyl)furan-3-carboxylate (20)34
A solution of 4-(methoxycarbonyl)furan-3-carboxylic acid (18,
2.0 g, 11.8 mmol) in anhydrous THF (50 mL) under an argon atmo-
sphere was cooled to 0 °C. Borane tetrahydrofuran (12.3 mL, 1.0 M
solution in THF, 12.3 mmol) was added dropwise via syringe with
stirring. The reaction was allowed to warm to ambient tempera-
ture and stir for 4 h, after which it was cooled to 0 °C and quenched
with the dropwise addition of water until gas evolution had ceased.
After bulk solvent removal, the resulting crude residue was then
partitioned between saturated aqueous NaHCO3 and diethyl ether,
and the aqueous layer was further extracted with diethyl ether
(2 ꢂ 40 mL). The combined ether solutions were washed with
brine (20 mL) and dried (Na2SO4), concentrated to dryness under
reduced pressure and chromatographed (ethyl acetate/hexanes,
2:5) to afford a clear oil (1.58 g, 86%). 1H NMR (500 MHz, CDCl3)
d 7.98 (s, 1H), 7.42 (s, 1H), 4.64 (s, 2H), 3.84 (s, 3H). 13C NMR
(126 MHz, CDCl3) d 164.60, 149.18, 141.22, 125.16, 117.44, 55.21,
51.69. HRMS (ESI): Calcd for C7H8O4 [MꢁH]ꢁ 155.0350; found
155.0339.
4.2.9. Methyl 4-(((tert-butoxycarbonyl)amino)methyl)-
thiophene-2-carboxylate (16)
Methyl 4-(bromomethyl)thiophene-2-carboxylate (14, 1 g,
4.25 mmol) and sodium azide (1.38 g, 21.3 mmol) were dissolved
in dimethylformamide (5 mL) and stirred at 60 °C under argon
atmosphere for 1 h. After cooling, the reaction was partitioned be-
tween diethyl ether (30 mL) and water (30 mL). The aqueous phase
was extracted with ether (3 ꢂ 20 mL), washed with brine (20 mL),
dried (Na2SO4), and concentrated to dryness under reduced pres-
sure. The crude product was dissolved in ethyl acetate (50 mL) in
the presence of 10% Pd/C (100 mg) and Boc2O (563 mg,
2.58 mmol). The mixture was placed under a H2 atmosphere and
vigorously stirred overnight at room temperature. The reaction
mixture was then filtered through a Celite pad, concentrated in va-
cuo and chromatographed (ethyl acetate/hexanes, 1:5) to yield the
desired product as a clear oil (738 mg, 64%). 1H NMR (500 MHz,
CDCl3) d 7.69 (s, 1H), 7.34 (s, 1H), 4.95 (br s, 1H), 4.27 (m, 2H),
3.86 (s, 2H), 1.44 (s, 7H). 13C NMR (126 MHz, CDCl3) d 162.52,
155.74, 140.84, 134.05, 133.19, 128.76, 79.79, 52.24, 39.79, 28.38.
HRMS (ESI): Calcd for C12H17NO4S [M+Na]+ 294.0770; found
294.0779.
4.2.14. Methyl 4-(chloromethyl)furan-3-carboxylate (21)
To a solution of methyl 4-(hydroxymethyl)furan-3-carboxylate
(19, 500 mg, 3.2 mmol) and triphenylphospine (1.26 g, 4.8 mmol)
in anhydrous CH2Cl2 was added carbon tetrachloride (739 mg,
4.8 mmol). The reaction was stirred under an argon atmosphere
at room temp for 18 h. The solvent was removed in vacuo, and
the crude product was chromatographed (ethyl acetate/hexanes,
2:5) to afford a clear oil (356 mg, 64%). 1H NMR (500 MHz, CDCl3)
d 8.14 (d, J = 3.4 Hz, 1H), 7.22 (d, J = 3.3 Hz, 1H), 4.74 (d, J = 6.8 Hz,
2H), 3.90 (s, 3H). 13C NMR (126 MHz, CDCl3) d 163.12, 149.20,
143.04, 122.62, 117.20, 51.57, 35.97. HRMS (LC-TOF): Calcd for
C7H7O3Cl 174.0084; found 174.0078.
4.2.10. 4-((tert-Butoxycarbonyl)aminomethyl)thiophene-2-
carboxylic acid (17)
Compound 17 was synthesized using a similar procedure to that
of 13 (90%). 1H NMR (500 MHz, CDCl3) d 7.78 (d, J = 1.6 Hz, 1H),
7.44 (s, 1H), 4.92 (s, 1H), 4.32 (d, J = 6.1 Hz, 2H), 1.47 (s, 9H). 13C
NMR (126 MHz, CDCl3) d 166.67, 155.78, 141.10, 134.41, 133.53,
4.2.15. Methyl 4-(((tert-butoxycarbonyl)amino)methyl)furan-3-
carboxylate (22)
Methyl 4-(chloromethyl)furan-3-carboxylate (21, 300 mg,
1.72 mmol) and sodium azide (560 mg, 8.60 mmol) were dissolved
in dimethylformamide (5 mL) and stirred at 60 °C under argon

5770
D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
atmosphere for 1 h. After cooling, the reaction was partitioned be-
tween diethyl ether (30 mL) and water (30 mL). The aqueous phase
was extracted with ether (3 ꢂ 20 mL), washed with brine (20 mL),
dried (Na2SO4), and concentrated to dryness under reduced pres-
sure. The crude product was dissolved in ethyl acetate (20 mL) in
the presence of 10% Pd/C (30 mg) and Boc2O (563 mg, 2.58 mmol).
The mixture was placed under a H2 atmosphere and was vigor-
ously stirred overnight at room temp. The reaction mixture was
then filtered through a Celite pad, concentrated in vacuo, and chro-
matographed (ethyl acetate/hexanes, 1:5) to yield the desired
product as a clear oil (303 mg, 69%). 1H NMR (500 MHz, CDCl3) d
7.96 (d, J = 1.7 Hz, 1H), 7.43 (s, 1H), 5.55 (t, J = 6.5 Hz, 1H), 4.29
(d, J = 6.4 Hz, 2H), 3.84 (s, 3H), 1.43 (br s, 9H). 13C NMR
4.2.20. Methyl 4-(hydroxymethyl)thiophene-3-carboxylate (27)
A solution of 4-(methoxycarbonyl)thiophene-3-carboxylic acid
(25, 595 mg, 3.2 mmol) in anhydrous THF (20 mL) under argon
atmosphere was cooled to 0 °C. Borane tetrahydrofuran (3.4 mL,
1.0 M solution in THF, 3.4 mmol) was added dropwise via syringe
with stirring. The reaction was allowed to warm to ambient tem-
perature and to stir for 4 h after which it was cooled to 0 °C and
quenched with dropwise addition of water until gas evolution
had ceased. After bulk solvent removal, the resulting crude residue
was then partitioned between saturated aqueous NaHCO3 and
diethyl ether, and the aqueous layer was further extracted with
diethyl ether (2 ꢂ 15 mL). The combined ether solutions were
washed with brine (15 mL), dried (Na2SO4), concentrated to dry-
ness under reduced pressure, and chromatographed (ethyl ace-
tate/hexanes, 2:5) to afford a clear oil (342 mg, 62%). 1H NMR
(500 MHz, CDCl3) d 8.13 (d, J = 3.4 Hz, 1H), 7.22 (d, J = 3.4 Hz, 1H),
4.74 (s, 2H), 3.88 (s, 3H). 13C NMR (126 MHz, CDCl3) d 164.30,
142.94, 135.67, 130.70, 124.12, 60.03, 52.11. HRMS (ESI): Calcd
for C7H8O3S [MꢁH]ꢁ 171.0121; found 171.0108.
(126 MHz, CDCl3)
117.49, 79.21, 51.52, 34.24, 28.33. HRMS (ESI): Calcd for
12H17NO5 [M+Na]+ 278.0999; found 278.0999.
d 164.02, 155.72, 149.03, 141.88, 122.75,
C
4.2.16. 4-((tert-Butoxycarbonyl)aminomethyl)furan-3-
carboxylic acid (23)
Compound 23 was synthesized using a similar procedure to that
of 13 (88%). 1H NMR (500 MHz, CDCl3) d 8.07 (d, J = 1.8 Hz, 1H),
7.44 (d, J = 2.1 Hz, 1H), 4.98 (br s, 1H), 4.27 (s, 2H), 1.43 (s, 9H).
13C NMR (126 MHz, CDCl3) d 166.43, 158.13, 150.83, 142.98,
124.79, 119.18, 80.30, 35.84, 28.72. HRSM (ESI): Calcd for
4.2.21. Methyl 4-(bromomethyl)thiophene-3-carboxylate (28)
Carbon tetrabromide (769 mg, 2.32 mmol) was added to a stir-
red solution of methyl 4-(hydroxymethyl)thiophene-3-carboxylate
(27, 265 mg, 1.54 mmol) in dry CH2Cl2 (10 mL) under argon atmo-
sphere. After stirring for 10 min, triphenylphosphine (608 mg,
2.32 mmol) was added and the resulting reaction mixture was stir-
red for 1 h at room temperature. Solvent was removed and the
crude product was separated between diethyl ether (15 mL) and
water (15 mL). Aqueous phase was extracted with ether
(3 ꢂ 10 mL). The combined ether solutions were washed with
brine (15 mL), dried (Na2SO4), concentrated to dryness under re-
duced pressure, and chromatographed (ethyl acetate/hexanes,
1:5) to afford a clear oil (232 mg, 64%). 1H NMR (500 MHz, CDCl3)
d 8.14 (d, J = 3.4 Hz, 1H), 7.39 (d, J = 3.3 Hz, 1H), 4.84 (s, 2H), 3.89 (s,
4H). 13C NMR (126 MHz, CDCl3) d 162.75, 138.74, 135.27, 130.28,
127.08, 51.85, 26.81. HRMS (ESI): Calcd for C7H7BrO2S [MꢁBr]+
155.0161; found 155.0162.
C
11H14NO5 [MꢁH]ꢁ 240.0877; found 240.0866.
4.2.17. 4-(Aminomethyl)furan-3-carboxylic acid hydrochloride
(6a)
Compound 6a was synthesized using a similar procedure to that
of 5a (78%).1H NMR (500 MHz, MeOD) d 8.23 (s, 1H), 7.81 (s, 1H),
4.19 (s, 2H). 13C NMR (126 MHz, MeOD) d 166.66, 151.40, 145.78,
119.50, 118.94, 34.45. HRMS (ESI): Calcd for C6H7NO3 [MꢁH]ꢁ
140.0353; found 140.0347.
4.2.18. Dimethyl thiophene-3,4-dicarboxylate (25)
To a stirred solution of thiophene-3,4-dicarboxylic acid (24,
1.5 g, 8.71 mmol) in methanol (15 mL) was added conc. sulfuric
acid (1 mL) dropwise. The reaction was heated to reflux and stirred
under nitrogen overnight. The reaction mixture was allowed to
cool to room temperature followed by concentration under vac-
uum. The resulting crude residue was then partitioned between
saturated aqueous NaHCO3 and diethyl ether, and the aqueous
layer was further extracted with diethyl ether (2 ꢂ 40 mL). The
combined ether solutions were washed with brine (20 mL), dried
(Na2SO4), and concentrated to dryness under reduced pressure to
provide the desired product as a clear oil (1.46 g, 84%). 1H NMR
(500 MHz, CDCl3) d 7.88 (s, 2H), 3.88 (s, 6H). 13C NMR (126 MHz,
CDCl3) d 163.41, 133.11, 131.90, 52.33. HRMS (LC-TOF): Calcd for
C8H8O4S 200.0143; found 200.0130.
4.2.22. Methyl 4-(((tert-butoxycarbonyl)amino)methyl)-
thiophene-3-carboxylate (29)
Compound 29 was synthesized using a similar procedure to that
of 22 (72%). 1H NMR (500 MHz, CDCl3) d 8.11 (d, J = 3.4 Hz, 1H),
7.25 (d, J = 3.4 Hz, 1H), 5.56 (t, J = 6.7 Hz, 1H), 4.45 (d, J = 6.6 Hz,
2H), 3.87 (s, 3H), 1.43 (s, 9H). 13C NMR (126 MHz, CDCl3) d
163.62, 155.78, 140.35, 135.16, 130.65, 124.81, 79.24, 51.83,
39.30, 28.44. HRMS (ESI): Calcd for
294.0770; found 294.0775.
C
12H17NO4S [M+Na]+
4.2.23. 4-((tert-Butoxycarbonyl)aminomethyl)thiophene-3-
carboxylic acid (30)
4.2.19. 4-(Methoxycarbonyl)thiophene-3-carboxylic acid (26)
To a solution of dimethyl thiophene-3,4-dicarboxylate (25,
825 mg, 4.12 mmol) in methanol (10 mL) was added sodium
hydroxide (165 mg, 4.12 mmol) and stirred overnight at room tem-
perature. Solvent was removed, and the crude product was parti-
tioned between diethyl ether (15 mL) and water (15 mL). The
aqueous phase was extracted with ether (3 ꢂ 10 mL), and the com-
bined organic layers were discarded. The aqueous phase was ad-
justed to pH 1 with hydrochloric acid (2 N) and extracted with
EtOAc (3 ꢂ 10 mL). The combined organics were washed with
brine (10 mL), dried (Na2SO4), and concentrated under reduced
pressure to yield the desired product as a white solid (667 mg,
87%). 1H NMR (500 MHz, CDCl3) d 8.49 (d, J = 3.7 Hz, 1H), 8.37 (d,
J = 3.7 Hz, 1H), 4.04 (s, 4H). 13C NMR (126 MHz, CDCl3) d 166.95,
161.38, 139.26, 138.45, 133.68, 128.05, 53.96. HRMS (ESI): Calcd
for C7H6O4S [MꢁH]ꢁ 184.9914; found 184.9900.
Compound 30 was synthesized using a similar procedure to that
of 13 (92%). 1H NMR (500 MHz, MeOD) d 8.21 (d, J = 3.4 Hz, 1H),
7.22 (d, J = 3.4 Hz, 1H), 4.94 (s, 1H), 4.44 (s, 2H), 1.44 (s, 9H). 13C
NMR (126 MHz, MeOD) d 166.05, 158.31, 142.29, 136.60, 132.45,
124.26, 80.32, 40.93, 28.78. HRMS (ESI): Calcd for C11H15NO4S
[MꢁH]ꢁ 256.049; found 256.044.
4.2.24. 4-(Aminomethyl)thiophene-3-carboxylic acid
hydrochloride (6b)
Compound 6b was synthesized using a similar procedure to
that of 5a (82%). 1H NMR (500 MHz, MeOD)
d
8.37 (d,
J = 3.2 Hz, 1H), 7.66 (d, J = 3.1 Hz, 1H), 4.32 (s, 2H). 13C NMR
(126 MHz, MeOD)
d 166.30, 137.67, 135.13, 132.94, 129.99,
38.99. HRMS (ESI): Calcd for C6H7NO2S [MꢁH]ꢁ 156.0125; found
156.0113.

D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
5771
4.2.25. Methyl 5-formyl-1H-pyrrole-2-carboxylate (31a) and
methyl 4-formyl-1H-pyrrole-2-carboxylate (31b)
to those used to prepare methyl 5-((hydroxyimino)methyl)-1H-
pyrrole-2-carboxylate from methyl 5-formyl-1H-pyrrole-2-car-
boxylate. 1H NMR (500 MHz, MeOD) d 8.00/7.26 (s, 1H), 7.67/
7.22 (d, J = 1.5 Hz, 1H), 7.20/7.06 (d, J = 1.7 Hz, 1H), 3.84/3.83 (s,
3H). 13C NMR (126 MHz, MeOD) d 162.99/162.88, 145.50/142.15,
129.01/125.20, 124.79/123.59, 119.92/118.28, 117.80/113.65,
52.00. HRMS (ESI): Calcd for C7H8N2O3 [MꢁH]ꢁ 167.0462; found
167.0463.
Prepared as described in the literature from methyl pyrrole-2-
carboxylate as a 5:2 ratio of 31a and 31b, respectively.52
Compound 31a. 1H NMR (500 MHz, CDCl3) d 9.85 (s, 1H), 9.78
(br s, 1H), 7.58 (dd, J = 3.3, 1.5 Hz, 1H), 7.32 (dd, J = 2.5, 1.5 Hz,
1H). 13C NMR (126 MHz, CDCl3) d 185.75, 161.37, 128.53, 127.79,
124.91, 114.31, 52.24.
Compound 31b. 1H NMR (500 MHz, CDCl3) d 10.24 (br s, 1H),
9.67 (s, 1H), 6.95–6.91 (m, 2H), 3.91 (s, 3H). 13C NMR (126 MHz,
CDCl3) d 180.64, 161.04, 134.65, 128.31, 119.89, 115.82, 52.41.
4.2.31. 4-(((tert-Butoxycarbonyl)amino)methyl)-1H-pyrrole-2-
carboxylate (33b)
Methyl
4-((hydroxyimino)methyl)-1H-pyrrole-2-carboxylate
4.2.26. Methyl 5-((hydroxyimino)methyl)-1H-pyrrole-2-
carboxylate (32a)35
(32b, 167 mg, 0.99 mmol) was converted to methyl 4-(((tert-butox-
ycarbonyl)amino)methyl)-1H-pyrrole-2-carboxylate (207 mg, 82%)
using conditions identical to those used to prepare 5-(((tert-butox-
ycarbonyl)amino)methyl)-1H-pyrrole-2-carboxylate from methyl
5-((hydroxyimino)methyl)-1H-pyrrole-2-carboxylate. 1H NMR
(500 MHz, MeOD) d 6.87 (d, J = 1.6 Hz, 1H), 6.78 (d, J = 1.8 Hz,
1H), 4.06 (s, 2H), 3.80 (s, 3H), 1.44 (s, 9H). 13C NMR (126 MHz,
MeOD) d 163.18, 158.47, 124.81, 123.35, 123.00, 115.81, 80.04,
51.72, 37.91, 28.81. HRMS (ESI): Calcd for C12H18N2O4 [M+Na]+
277.1159; found 277.1158.
Methyl 5-formyl-1H-pyrrole-2-carboxylate (31a, 2.50 g,
16.3 mmol) was dissolved in water at 65 °C, and a solution of
hydroxylamine hydrochloride (1.70 g, 24.5 mmol) and potassium
carbonate (1.40 g, 10.1 mmol) in water (30 mL) was added drop-
wise. Upon cooling a white precipitate formed, which was filtered
and recrystallized from water/ethyl acetate (3:1) to give the de-
sired product in a 3:5 mixture of the two diastereomers as a white
powder (1.56 g, 57%). 1H NMR (500 MHz, CDCl3) d 10.58/9.98 (br s,
1H), 8.24/8.20 (br s, 1H), 6.90 (m, 1H), 6.49/6.41 (dd J = 3.9, 2.5 Hz,
1H), 3.89 (s, 3H). 13C NMR (126 MHz, CDCl3) d 162.34/161.24,
142.17/138.02, 129.29/127.53, 124.86/124.64, 116.51/115.12,
115.57/114.56, 52.14/51.94. HRMS (ESI): Calcd for C7H8N2O3
[M+H]+ 169.0608; found 169.0610.
4.2.32. 4-(((tert-Butoxycarbonyl)amino)methyl)-1H-pyrrole-2-
carboxylic acid (34b)36
Compound 34b was synthesized using a similar procedure to
that of 13 (86%) 1H NMR (500 MHz, MeOD) d 6.86 (d, J = 1.7 Hz,
1H), 6.78 (d, J = 1.8 Hz, 1H), 4.07 (s, 2H), 1.44 (s, 9H). 13C NMR
4.2.27. Methyl 5-(((tert-butoxycarbonyl)amino)methyl)-1H-
(126 MHz, MeOD) d 164.33, 158.49, 124.70, 123.97, 122.77,
pyrrole-2-carboxylate (33a)
115.84, 80.03, 37.97, 28.80. HRMS (ESI): Calcd for C11H16N2O4
Methyl
5-((hydroxyimino)methyl)-1H-pyrrole-2-carboxylate
[MꢁH]ꢁ 239.1037; found 239.1026.
(32a, 3.0 g, 17.8 mmol) was dissolved in ethyl acetate (100 mL) in
the presence of 10% Pd/C (300 mg) and Boc2O (4.28 g, 19.6 mmol).
The mixture was placed under a H2 atmosphere and vigorously
stirred overnight at room temperature. The reaction mixture was
filtered through a Celite pad, concentrated in vacuo, and chromato-
graphed (ethyl acetate/hexanes, 1:5) to yield the desired product
as a clear oil (3.88 g, 84%). 1H NMR (500 MHz, CDCl3) d 9.92 (s,
1H), 6.81 (dd, J = 3.7, 2.5 Hz, 1H), 6.07 (dd, J = 3.2 Hz, 1H), 5.17 (s,
1H), 4.26 (d, J = 6.1 Hz, 2H), 3.84 (s, 3H), 1.45 (s, 9H). 13C NMR
4.2.33. 4-(Aminomethyl)-1H-pyrrole-2-carboxylic acid
hydrochloride (5c)
Compound 5c was synthesized using a similar procedure to that
of 5a (82%) 1H NMR (500 MHz, MeOD) d 7.10 (d, J = 1.8 Hz, 1H),
6.95 (d, J = 1.8 Hz, 1H), 4.00 (s, 2H). 13C NMR (126 MHz, MeOD) d
163.86, 125.28, 124.57, 117.94, 116.33, 37.13. HRMS (ESI): Calcd
for C6H8N2O2 [MꢁH]ꢁ 139.0513; found 139.0503.
(126 MHz, CDCl3)
108.69, 80.19, 51.45, 37.49, 28.34. HRMS (ESI): Calcd for
12H18N2O4 [M+H] 255.1339; found 255.1342.
d
161.51, 156.68, 135.29, 122.43, 115.34,
4.2.34. Methyl 4-((tert-
butoxycarbonyl)amino(2H2)methyl)furan-2-carboxylate ([D2]–
12)
C
Methyl 4-cyanofuran-2-carboxylate (11, 190 mg, 1.26 mmol)
was converted to methyl 4-((tert-butoxycarbonyl)amino(2H2)
methyl)furan-2-carboxylate (130 mg, 40%) by treatment with so-
dium borodeuteride (368 mg, 8.80 mmol), Boc2O (550 mg,
2.52 mmol), and NiCl2ꢀ6H2O (31 mg, 0.13 mmol), following the
same procedure used to generate methyl 4-((tert-butoxycar-
bonyl)aminomethyl)furan-2-carboxylate (12). 1H NMR (500 MHz,
CDCl3) d 7.45 (s, 1H), 7.10 (s, 1H), 4.99 (s, 1H), 3.83 (s, 3H), 1.39
(s, 9H). 13C NMR (126 MHz, CDCl3) d 159.06, 155.84, 144.88,
143.57, 125.56, 125.49, 118.14, 79.75, 52.01, 35.29, 35.04, 34.87,
28.38. HRMS (ESI): Calcd for C12H15D2NO5 [M+Na]+ 280.1124;
found 280.1116.
4.2.28. 5-(((tert-Butoxycarbonyl)amino)methyl)-1H-pyrrole-2-
carboxylic acid (34a)36
Compound 34a was synthesized using a similar procedure to
that of 13 (82%). 1H NMR (500 MHz, MeOD) d 6.77 (d, J = 3.7 Hz,
1H), 6.05 (d, J = 3.7 Hz, 1H), 4.19 (s, 2H), 1.44 (s, 10H). 13C NMR
(126 MHz, MeOD)
d 164.37, 158.57, 137.15, 123.41, 117.03,
109.13, 80.45, 38.12, 28.75. HRMS (ESI): Calcd for C11H15N2O4
[MꢁH]ꢁ 239.1037; found 239.1025.
4.2.29. 5-(Amino)methyl)-1H-pyrrole-2-carboxylic acid
hydrochloride (4c)
Compound 4c was synthesized using a similar procedure to that
of 5a (74%). 1H NMR (500 MHz, MeOD) d 6.84 (d, J = 3.8 Hz, 1H),
6.33 (d, J = 3.8 Hz, 1H), 4.14 (s, 2H). 13C NMR (126 MHz, MeOD) d
163.85, 129.69, 125.46, 116.96, 111.69, 36.77. HRMS (ESI): Calcd
for C6H7N2O2 [MꢁH]ꢁ 139.0513; found 139.0512.
4.2.35. 4-((tert-Butoxycarbonyl)amino(2H2)methyl)furan-2-
carboxylic acid ([D2]-13)
Methyl
4-((tert-butoxycarbonyl)amino(2H2)methyl)furan-2-
carboxylate ([D2]-12, 130 mg, 0.51 mmol) was converted to 4-
((tert-butoxycarbonyl)amino(2H2)methyl)furan-2-carboxylic acid
(85 mg, 69%) using conditions identical to those used to convert
methyl 4-((tert-butoxycarbonyl)aminomethyl)furan-2-carboxylate
(12) to 4-((tert-butoxycarbonyl)aminomethyl)furan-2-carboxylic
acid (13). 1H NMR (500 MHz, MeOD) d 7.60 (s, 1H), 7.16 (s, 1H),
1.44 (s, 9H). 13C NMR (126 MHz, MeOD) d 161.71, 158.38, 146.53,
4.2.30. Methyl 4-((hydroxyimino)methyl)-1H-pyrrole-2-
35
carboxylate (32b)
Methyl 4-formyl-1H-pyrrole-2-carboxylate (31b, 500 mg,
3.26 mmol) was converted to methyl 4-((hydroxyimino)methyl)-
1H-pyrrole-2-carboxylate (280 mg, 51%) using conditions identical

5772
D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
145.05, 127.46, 127.40, 127.34, 119.19, 80.34, 35.78, 35.69, 35.53,
35.36, 28.72. HRMS (ESI): Calcd for
266.0968; found 266.0954.
(GraphPad Prism). Cornish-Bowden replots indicated that the com-
pounds were competitive inhibitors.
C
11H13D2NO5 [M+Na]+
Acknowledgments
4.2.36. 4-Amino(2H2)methyl)furan-2-carboxylic acid
hydrochloride ([D2]-5a)
The authors are grateful to the National Institutes of Health for
financial support (GM066132 and DA030604). The authors would
also like to thank Park Packing Co. (Chicago, IL) for generously pro-
viding fresh pig brains for this study.
4-((tert-Butoxycarbonyl)amino(2H2)methyl)furan-2-carboxylic
acid ([D2]-13, 85 mg, 0.35 mmol) was converted to 4-amino(2H2)-
methyl)furan-2-carboxylic acid hydrochloride (63 mg, 100%) using
conditions identical to those used to convert 4-((tert-butoxycar-
bonyl)aminomethyl)furan-2-carboxylic acid (13) to 4-amino-
methyl)furan-2-carboxylic acid hydrochloride (5b) with P95%
isotopic purity by 1H NMR spectroscopy. 1H NMR (500 MHz, D2O)
d 7.81 (s, 1H), 7.30 (s, 1H). 13C NMR (126 MHz, D2O) d 158.59,
143.51, 142.27, 116.42, 116.36, 116.30, 115.55, 30.53, 30.30,
30.12. HRMS (ESI): Calcd for C6H5D2NO3 [MꢁH]ꢁ 142.0479; found
142.0471.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.009.
References and notes
1. Krnjevic, K. Physiol. Rev. 1974, 54, 418.
4.3. Enzyme and assays
2. Baxter, C. F.; Roberts, E. J. J. Biol. Chem. 1958, 233, 1135.
3. Karlsson, A.; Fonnum, F.; Malthe-Sørenssen, D.; Storm-Mathisen, J. Biochem.
Pharmacol. 1974, 23, 3053.
4.3.1. Purification of GABA-AT from pig brain
4. Gale, K. Epilepsia 1989, 30, S1.
GABA-AT was isolated and purified from pig brain by a modified
procedure.53 The purified GABA-AT used in these experiments was
found to have a concentration of 6.41 mg/mL with a specific activ-
ity of 1.84 units/mg.
5. Van Gelder, N. M.; Elliott, K. A. C. J. Neurochem. 1958, 3, 139.
6. Bakay, R. A. E.; Harris, A. B. Brain Res. 1981, 206, 387.
7. Ribak, C. E.; Harris, A. B.; Vaugh, J. E.; Roberts, E. Science 1979, 205, 211.
8. Wu, J. Y.; Bird, E. D.; Chen, M. S.; Huang, W. M. Neurochem. Res. 1979, 4, 575.
9. Lloyd, K. G.; Shemen, L.; Hornykiewicz, O. Brain Res. 1977, 127, 269.
10. Sherif, F. M.; Ahmed, S. S. Clin. Biochem. 1995, 28, 145.
11. Gunne, L. M.; Haeggstroem, J. E.; Sjoequist, B. Nature 1984, 309, 347.
12. Silverman, R.B. Mechanism-Based Enzyme Inactivation: Chemistry and
Enzymology, Vols. I and II; CRC Press:Boca Raton, FL; 1988.
13. Silverman, R. B. Methods Enzymol. 1995, 249, 240.
4.3.2. Evaluation of compounds as inhibitors of GABA-AT
Inhibition constants were determined by monitoring GABA-AT
activity in the presence of 0–5 mM concentrations of synthesized
analogues using a coupled assay with the enzyme succinic semial-
dehyde dehydrogenase (SSADH). The assay solution consisted of
14. Lippert, B.; Metcalf, B. W.; Jung, M. J.; Casara, P. Eur. J. Biochem. 1977, 74, 441.
15. Loscher, W. Neuropharmacology 1982, 21, 803.
16. Tassinari, C. A.; Micheluccia, R.; Ambrosetto, G.; Salvi, F. Arch. Neurol. 1987, 44,
907.
10 mM GABA, 5 mM
a
-ketoglutarate, 1 mM NADP+, 5 mM b-
17. Brown, T. R.; Mattson, R. J.; Penry, J. K.; Smith, D. B.; Treiman, D. M.; Wilder, B.
J.; Ben-Menachem, E.; Miketta, R. M.; Sherry, K. M.; Szabo, G. K. Br. J. Clin.
Pharmacol. 1989, 27, 95S.
18. Sivenius, M. R.; Ylinen, A.; Murros, K.; Matilainen, R.; Riekkinen, P. Epilepsia
1987, 28, 688.
19. Karila, L.; Gorelick, D.; Weinstein, A.; Noble, F.; Benyamina, A.; Coscas, S.;
Blecha, L.; Lowenstein, W.; Martinot, J. L.; Reynaud, M.; Lepine, J. P. Int. J.
Neuropsychopharmacol. 2008, 11, 425.
20. Peng, X.-Q.; Li, X.; Gilbert, J. G.; Pak, A. C.; Ashby, C. R., Jr.; Brodie, J. D.; Dewey,
S. L.; Gardner, E. L.; Xi, Z.-X. Drug Alcohol Depend. 2008, 97, 216.
21. Maguire, M. J.; Hemming, K.; Wild, J. M.; Hutton, J. L.; Marson, A. G. Epilepsia
2010, 51, 2423.
mercaptoethanol, and excess SSADH in 50 mM potassium pyro-
phosphate buffer, pH 8.5. Enzyme activity was determined by
observing the change in absorbance at 340 nm at 25 °C.37 Compet-
itive inhibition constants were determined by Dixon plots of ob-
tained data. Prior to their evaluation, initial experiments were
performed to confirm the synthesized analogues (at 5 mM concen-
tration) do not inhibit the coupling enzymes utilized in the sub-
strate and inhibition assays.
4.3.3. Evaluation of compounds as substrates for GABA-AT
22. Highlights of Prescribing Information (for Sabril). http://www.lundbeck.com/
upload/us/files/pdf/Products/Sabril_PI-IS_US_EN.pdf.
23. Nanavati, S. M.; Silverman, R. B. J. Am. Chem. Soc. 1991, 113, 9341.
24. Heim, M. K.; Gidal, B. E. Acta Neurol. Scand. 2012. http://dx.doi.org/10.1111/
j.1600-0404.2012.01684.x.
25. Qiu, J.; Silverman, R. B. J. Med. Chem. 2000, 43, 706.
26. Pan, Y.; Qiu, J.; Silverman, R. B. J. Med. Chem. 2003, 46, 5292.
27. Pan, Y.; Calvert, K.; Silverman, R. B. Bioorg. Med. Chem. 2004, 12, 5719.
28. Lu, H.; Silverman, R. B. J. Med. Chem. 2006, 49, 7404.
29. Yuan, H.; Silverman, R. B. Bioorg. Med. Chem. Lett. 2007, 17, 1651.
30. Ritchie, T. J.; Macdonald, S. J. F.; Young, R. J.; Pickett, S. D. Drug Discov. Today
2011, 16, 164.
31. Clift, M. D.; Silverman, R. B. Bioorg. Med. Chem. Lett. 2008, 18, 3122.
32. Wang, Z.; Silverman, R. B. Bioorg. Med. Chem. 2006, 14, 2242.
33. Peschke, B.; Madsen, K.; Hansen, B. S.; Johansen, N. L. Bioorg. Med. Chem. Lett.
1997, 7, 1969.
Compounds were tested using an experiment in which the con-
version of
a-ketoglutarate (a-KG) to L-glutamic acid was moni-
tored as an indication of the rate of PLP reduction to PMP, which
in turn corresponds to the rate of amine oxidation to the corre-
sponding aldehyde. Enzyme reactions were prepared at varying
concentrations of compounds in 100
(50 mM, pH 8.5) containing 5 mM
mercaptoethanol, and 0.13 mg/mL purified GABA-AT and allowed
to incubate at room temperature for 16 h. The -glutamic acid con-
tent was determined by combining 50 L of each incubation mix-
ture with 50 L of Tris-HCl buffer (100 mM, pH 7.5) containing
100 M Ampliflu™ Red (Sigma-Aldrich), 0.25 units/mL horseradish
peroxidase and 0.08 units/mL -glutamate oxidase in a 96-well
lL pyrophosphate buffer
a-ketoglutarate, 2 mM 2-
L
l
l
l
34. McCoy, L. L.; Mal, D. J. Org. Chem. 1984, 49, 939.
L
35. Anderson, H. J.; Lee, S. F. Can. J. Chem. 1965, 43, 409.
36. Zhang, Y.; Yin, Z.; He, J.; Cheng, J. P. Tetrahedron Lett. 2007, 48, 6039.
37. Silverman, R. B.; Levy, M. A. Biochemistry 1981, 20, 1197.
38. Woolf, B. Algemeine Chemie der Enzyme In S Haldane, J. B., Stern, K. G, Eds.;
Steinkopf: Dresden, 1932; pp 119–120.
black walled plate. After incubation at 37 °C for 30 min fluores-
cence was recorded with the aid of a microplate reader (BioTek
Synergy H1) with 530 nm excitation and 590 nm emission wave-
39. Hanes, C. S. Biochem. J. 1932, 26, 1406.
lengths, where fluorescence is proportional to the
centration. Compounds 4a, 4b, 5a, 5c, and GABA were evaluated in
triplicate at 90 M, 225 M, 450 M, 900 M, 2.25 mM and
4.5 mM concentrations. Compounds 4c, 5b, 6a and 6b were evalu-
ated in triplicate at 900 M, 2.25 mM, 4.5 mM, 9.0 mM and
L-glutamate con-
40. Yu, P. H.; Duren, D. A.; Davis, B. A.; Boulton, A. A. Neurochem 1987, 48, 440.
41. Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic Chemistry; University
Science Books, 2006 pp 428–431.
42. Forlani, L.; Marianucci, E.; Todesco, P. E. J. Chem. Res. 1984, 126.
43. Chudeck, J. A.; Foster, R.; Young, D. J. Chem. Soc. Perkin Trans. 2 1985, 1285.
44. Billard, T.; Langlois, B. R.; Blond, G. Eur. J. Org. Chem. 2001, 1467.
45. Matsui, M.; Yamada, K.; Funabiki, K. Tetrahedron 2005, 61, 4671.
46. Barys, M.; Ciunik, Z.; Drabnet, K.; Kwiecien, A. New. J. Chem. 2010, 34, 2605.
l
l
l
l
l
13.5 mM concentrations. Substrate kinetic constants were deter-
mined by Hanes–Woolf plots using linear regression analysis

D. D. Hawker, R. B. Silverman / Bioorg. Med. Chem. 20 (2012) 5763–5773
5773
47. Velázquez, M.; Salgado-Zamora, H.; Pérez, C.; Campos-A, M. E.; Mendoza, P.; 50. Hooley, R. J.; Iwasawa, T.; Rebek, J., Jr. J. Am. Chem. Soc. 2007, 129, 15330.
Jiménez, H.; Jiménez, R. J. Mol. Struc. 2010, 979, 56.
48. Heine, A.; DeSantis, G.; Luz, J. G.; Mitchell, M.; Wong, C. H.; Wilson, I. A. Science
2001, 294, 369.
51. Bordwell, F. G.; Boyle, W. J., Jr. J. Am. Chem. Soc. 1975, 97, 3447.
52. Schmuck, C. Tetrahedron 2001, 57, 3063.
53. Koo, Y. K.; Nandi, D.; Silverman, R. B. Arch. Biochem. Biophys. 2000, 374, 248.
49. Iwasawa, T.; Hooley, R. J.; Rebek, J., Jr. Science 2007, 317, 493.