Evaluation of α-hydroxycinnamic acids as pyruvate carboxylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.07.027

Through a structure-based drug design project (SBDD), potent small molecule inhibitors of pyruvate carboxylase (PC) have been discovered. A series of α-keto acids (7) and α-hydroxycinnamic acids (8) were prepared and evaluated for inhibition of PC in two assays. The two most potent inhibitors were 3,3′-(1,4-phenylene)bis[2-hydroxy-2-propenoic acid] (8u) and 2-hydroxy-3-(quinoline-2-yl)propenoic acid (8v) with IC₅₀ values of 3.0 ± 1.0 μM and 4.3 ± 1.5 μM respectively. Compound 8v is a competitive inhibitor with respect to pyruvate (K_i = 0.74 μM) and a mixed-type inhibitor with respect to ATP, indicating that it targets the unique carboxyltransferase (CT) domain of PC. Furthermore, compound 8v does not significantly inhibit human carbonic anhydrase II, matrix metalloproteinase-2, malate dehydrogenase or lactate dehydrogenase.

Full text of DOI:10.1016/j.bmc.2019.07.027

Accepted Manuscript
Evaluation of α-hydroxycinnamic acids as pyruvate carboxylase inhibitors
Daniel J. Burkett, Brittney N. Wyatt, Mallory Mews, Anson Bautista, Ryan
Engel, Chris Dockendorff, William A. Donaldson, Martin St. Maurice
PII:
S0968-0896(19)30705-9
https://doi.org/10.1016/j.bmc.2019.07.027
BMC 15011
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 May 2019
10 July 2019
14 July 2019
Please cite this article as: Burkett, D.J., Wyatt, B.N., Mews, M., Bautista, A., Engel, R., Dockendorff, C., Donaldson,
W.A., St. Maurice, M., Evaluation of α-hydroxycinnamic acids as pyruvate carboxylase inhibitors, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.027
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Evaluation of -hydroxycinnamic acids as
pyruvate carboxylase inhibitors
Daniel J. Burkett, Brittney N. Wyatt, Mallory Mews, Anson Bautista, Ryan Engel, Chris Dockendorff, William
A. Donaldson and Martin St. Maurice
Department of Biological Sciences, Marquette University, P. O. Box 1881, Milwaukee, WI 53201-1881, USA
and Department of Chemistry, Marquette University, P. O. Box 1881, Milwaukee, WI 53201-1881, USA

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Evaluation of -hydroxycinnamic acids as pyruvate carboxylase inhibitors
Daniel J. Burketta‡, Brittney N. Wyattb‡, Mallory Mewsb, Anson Bautistaa, Ryan Engela, Chris
Dockendorffa, William A. Donaldsona,* and Martin St. Mauriceb,*
a Department of Chemistry, Marquette University, P. O. Box 1881, Milwaukee, WI 53201-1881, USA
b Department of Biological Sciences, Marquette University, P. O. Box 1881, Milwaukee, WI 53201-1881, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Through a structure-based drug design project (SBDD), potent small molecule inhibitors of
pyruvate carboxylase (PC) have been discovered. A series of -keto acids (7) and -
hydroxycinnamic acids (8) were prepared and evaluated for inhibition of PC in two assays. The
two most potent inhibitors were 3,3’-(1,4-phenylene)bis[2-hydroxy-2-propenoic acid] (8u) and
2-hydroxy-3-(quinoline-2-yl)propenoic acid (8v) with IC50 values of 3.0 ± 1.0 M and 4.3 ± 1.5
M respectively. Compound 8v is a competitive inhibitor with respect to pyruvate (Ki = 0.74
M) and a mixed-type inhibitor with respect to ATP, indicating that it targets the unique
carboxyltransferase (CT) domain of PC. Furthermore, compound 8v does not significantly
inhibit human carbonic anhydrase II, matrix metalloproteinase-2, malate dehydrogenase or
lactate dehydrogenase.
Available online
Keywords:
pyruvate carboxylase
carboxyltransferase
-hydroxycinnamic acids
-keto acids
2009 Elsevier Ltd. All rights reserved.
1. Introduction
covalently attached to the biotin carboxyl carrier protein (BCCP)
domain, is carboxylated in the BC domain, and subsequently
Pyruvate carboxylase (PC) is an important anaplerotic enzyme
that catalyzes the conversion of pyruvate to oxaloacetate (OAA)
in a wide range of organisms.1 Studies have linked PC activity to
a number of diseases and infections including certain types of
cancer,2a type 2 diabetes,2b and listeriosis2c that are sustained as a
result of aberrant or essential PC expression in cells. Genetic
manipulations of PC have validated it as a potential target for the
treatment of these diseases and infections, but genetic tools alone
are insufficient to identify the precise mechanistic role that PC
plays in vivo. Specific and potent small molecule effectors of PC
can alter the catalytic activity without reducing or eliminating the
gene product from the cellular system, thus precluding
unintentional effects on associated metabolic and signaling
enzymes in the cell. To date, attempts to use small molecule
effectors to study PC directly in cellular studies have made use of
unselective inhibitors of PC expression/activity, all of which
have the potential to interact with multiple cellular systems.3 For
example phenylacetate has been applied as a PC inhibitor in
studies of insulin release.4 However, the high concentrations ( 5
mM) required to partially inhibit PC are expected to impact many
other cellular processes, including the induction of cell cycle
arrest,5a,b and inhibition of nitric oxide synthase.5c
transferred to the CT domain where pyruvate is carboxylated to
form OAA. A structure-based project was initiated to develop
chemical probes that will target the CT domain of PC. We
describe here the discovery of promising first generation small
molecule PC inhibitors and their kinetic characterization.
2. Results and Discussion
Both substrates and inhibitors of the CT domain of PC can
promote carboxybiotin decarboxylation.8 Binding of CT domain
ligands facilitates a structural rearrangement which allows
carboxybiotin to access the CT domain active site and
decarboxylate.9 Fluoropyruvate (1a, Figure 1), hydroxypyruvate
(1b), oxamate (2) and glyoxylate (3) all promote carboxybiotin
decarboxylation,8 and crystal structures of PC with pyruvate, 3-
bromopyruvate (1c), oxalate (4), 1b, and 2 reveal that these
molecules bind with overlapping orientations of their -keto acid
functional groups in the CT domain active site.10
–
Figure 1. Known inhibitors of pyruvate carboxylase.
The MgATP-dependent carboxylation of pyruvate by HCO3
to form OAA occurs in two distinct active sites of PC: the biotin
carboxylation (BC) domain that is conserved across all biotin
dependent carboxylases,6 and the unique carboxyltransferase
(CT) domain that, outside of PC, is found in only a few bacterial
oxaloacetate decarboxylases.7 The biotin cofactor, which is
In parallel with efforts to screen compound libraries,11 we
endeavored to identify inhibitors by taking the classical approach
of modifying the enzymatic substrate. We reasoned that the
range of known -keto acid PC inhibitors and their related X-ray

–
structures would also support a structure-based approach that
could help to identify larger -keto acids inhibitors of PC, but
with higher potency and selectivity. Our overall objective is to
identify probes with suitable potency, selectivity and
physico/physiochemical properties for in vivo studies.
pyruvate + HCO3
pyruvate carboxylase
–
–
MeO
MeO
CO2
CO2
N
–
–
N
N
CO2
CO2
FVB
N
2.1. Compound synthesis
O
OAA
O
BzHN
BzHN
Cl–
Me

max = 530 nm
Me
Two routes were employed for the preparation of -keto
acids/substituted -hydroxycinnamic acids (Scheme 1). Addition
of one equivalent of a Grignard reagent to diethyl oxylate in
ether/THF at –78 oC, followed by aqueous quench at low
temperature afforded the corresponding -ketoesters 5a-b.12
Alternatively, heating a mixture of hydantoin and cyclohexane-
carboxaldehyde or substituted benzaldehydes in ethanol/water
afforded the corresponding 5-methylene-2,4-imidazolidinediones
6c-u, which generally precipitated during the Knoevenagel
condensation.13 Base hydrolysis of -ketoesters 5a-b or of 5-
methylene-2,4-imidazolidinediones 6c-u gave the corresponding
-keto acids (7) or substituted -hydroxycinnamic acids (8).
Scheme 2. Detection of OAA production by reaction with FVB.
–
–
CO2
CO2
pyruvate
carboxylase
malate
–
–
CO2
dehydrogenase
CO2
pyruvate
+ HCO3
–
O
OAA
OH
malate
NAD+
NADH

max = 340 nm
Scheme 3. Detection of OAA production by
dehydrogenase (MDH) coupled enzyme assay.
a malate
observed by measuring the change in absorbance of NADH at
340 nm.
Compounds which existed predominantly as the -keto acids
(7a-d, Figure 2; 7f and 7l, Table 1) as well as 3-phenyllactic acid
(9) had negligible inhibitory activity (IC50 > 1000 M). In
contrast, those compounds which existed as enols (-
hydroxycinnamic acids), such as phenylpyruvic acid (8e, IC50
=
240 M, Table 1), exhibited promising inhibitory activity in the
FVB assay. The higher inhibition of enols over -keto acids is
consistent with the predicted pyruvate enolate intermediate in the
CT domain reaction mechanism, suggesting that the high affinity
of these compounds comes from their ability to serve as
intermediate analogs. As such, these results are consistent with
the CT domain as the target of these compounds. While -
Scheme 1. Syntheses of -ketoacids/-hydroxycinnamic acids
The parent, m- and p-substituted 3-arylpyruvic acids 8e, 8g-k,
and 8m-v were found to exist predominantly in the enol form
(i.e. -hydroxycinnamic acid), while the alkyl substituted pyruvic
acids 7a-c and the o-substituted 3-arylpyruvic acids 7f and 7l
exist predominantly in the keto form. These assignments are
based on NMR spectral data. In particular, signals at ca.  107
hydroxycinnamic acid 8g bearing
a
strongly electron
withdrawing para-nitro group exhibited only weak inhibitory
activity, the corresponding -hydroxycinnamic acid bearing a
para-CF3 group (8k) inhibited pyruvate with IC50 = 130 M in
the FVB assay. There does not appear to be a strong correlation
between the electron withdrawing/electron donating ability of
para-substituents and inhibitory activity. The two most potent
inhibitors were 3,3’-(1,4-phenylene)bis[2-hydroxy-2-propenoic
acid] (8u) and 2-hydroxy-3-(quinoline-2-yl)propenoic acid (8v)
with IC50 values of 3.0 ± 1.0 M and 4.3 ± 1.5 M respectively.
IC50 values determined in the MDH coupled assay were generally
slightly lower than those determined in the FVB assay, but the
values from these two assays correlated well.
1
and at ca.  6.4 ppm (s, 1H) in the 13C and H NMR spectra of
compounds 8 (in d6-DMSO) correspond to C3 and its attached
proton of the enol structures, while signals at ca.  30 and at ca. 
1
3 ppm in the 13C and H NMR spectra of compounds 7 (in d6-
DMSO) correspond to C3 and its attached proton of the keto
structures. Similar keto-enol distribution has been previously
observed in CD3CN.14
2.2. PC inhibition assays
While human PC represents the ideal target for these studies,
it has proven to be extremely difficult to obtain workable yields
of full-length, recombinant human PC.9a Instead, compounds
were initially screened for inhibition of Staphylococcus aureus
PC (SaPC) using an optimized fixed-time assay11 that is based on
the reaction of OAA, the product of the PC-catalyzed reaction,
with Fast Violet B (FVB), a diazonium salt which produces a
colored adduct with an absorbance maximum at 530 nm (Scheme
2).
As a secondary assay to confirm SaPC inhibition, the
carboxylation of pyruvate in full-length SaPC was measured by
coupling the formation of OAA to the reaction catalyzed by
malate dehydrogenase (MDH), which converts OAA to malate
and oxidizes NADH to NAD+ (Scheme 3). This can be readily
Figure 2. Compounds which exist predominantly in the -keto
acid form (7a-7d) and 3-phenyllactic acid (9) exhibit weak
inhibitor activity against SaPC (FVB assay).

Table 1. S. aureus pyruvate carboxylase inhibition activity (IC50
values in M).
R2
H
R3
H
R4
H
FVB IC50 MDH IC50
8e
7f
240
>2000
>1000
360
130
>2000
>1000
23
Figure 3. Compound 8v is a competitive inhibitor with respect to
pyruvate and a mixed-type inhibitor with respect to ATP. A) A
double-reciprocal plot demonstrating competitive inhibition of 8v
with respect to pyruvate, Ki value = 0.74 M. B) A double-
reciprocal plot demonstrating mixed-type inhibition of 8v with
respect to ATP, Ki value = 5.2 M. Initial velocities were
determined at the following concentrations of 8v: 0 M (circles),
1.5 M (squares), 3 M (upright triangles) and 6 M (inverse
triangles).
NO2
H
H
H
8g
8h
8i
H
NO2
H
H
Me
H
H
Me
Me
CF3
H
210
140
38
8j
H
Me
H
98
8k
7l
H
130
96
2.4 Docking studies
Br
H
H
>1000
200
>1000
81
Computational docking was conducted using the CT domain
structure from S. aureus PC (pdb code 3BG5) to predict the
relative binding affinity and poses of the most potent inhibitors
for the CT domain of PC, and to visualize the predicted binding
site. Upon the binding of pyruvate, the CT domain adopts a
closed conformation where the carboxy moiety of pyruvate forms
a salt bridge with the guanidinium side-chain of Arg644 (human
PC numberings are used throughout) and the carbonyl oxygen of
pyruvate is within hydrogen bonding distance of Arg571 and
8m
8n
8o
8p
8q
8r
8s
H
Br
H
H
Cl
280
220
30
H
H
F
100
H
H
OMe
OH
3-thienyl
170
56
H
H
67
22
H
H
48.0
58.6
–
–
8t
118
–
8u
3.0±1.0
11
8v
4.3±1.5
3.2±0.5
2.3 Determination of mode of inhibition
Steady-state kinetic analysis was used to determine the Ki
value and mode of inhibition for one of the most potent
compounds (8v). It was determined to be a competitive inhibitor
with respect to pyruvate with a Ki = 0.74 M, and a
noncompetitive inhibitor with respect to ATP with Ki = 5.2 M
(Figure 3). As an additional measure to assess whether our
procedure for determining Ki values was accurate, predictive Ki
values were determined with the Cheng-Prussoff equation for
competitive inhibition for 8v (0.46 M) and oxalate (0.43 M).
The close match of the predictive and kinetically determined Ki
values, as well as the unaltered binding affinity with increased
concentration of ATP is consistent with direct competition of 8v
with pyruvate in the CT domain active site. Identical modes of
inhibition were observed against ATP and pyruvate for oxalate
and for 8i, suggesting that -hydroxycinnamic acids specifically
target the CT domain for inhibition (Supplementary Figure S1).
Figure 4. A) Docking pose of 8u in the closed conformation of
the CT domain without biotin present. The Mn2+/Zn2+ metal ion
is represented by the grey sphere. B) Overlay of docked structure
with the crystal structure showing the -enolic carboxyl moiety
of 8u that is not predicted to coordinate the metal ion occupies
the binding location of biotin. C) Docking pose of 8v in the
closed conformation of the CT domain without biotin present. D)
Overlay of docked structure with the crystal structure showing
the quinolinyl ring 8v occupies the binding location of biotin.
Residues are numbered according to the sequence of human PC.

Gln575. These residues contribute to stabilizing the enolpyruvate
intermediate. In the closed conformation, Tyr651 hydrogen
bonds with Asp613.15 Due to the predicted importance of the enol
moiety of the -hydroxy-3-arylcinnamic acids, all compounds
were docked in their enol form. In the docking pose of 8u and 8v,
with PC in the closed conformation, the carboxyl moieties are not
positioned such that they form salt bridges with Arg644, but
rather both are oriented such that the carboxyl groups are
coordinated with the metal ion in the center of the CT domain
active site (Figure 4A and 4C). This interaction with Mn2+
contributes to an octahedral coordination, which is consistent
with what has been observed in multiple crystal structures of PC.
The carboxy moiety coordinated to the metal ion of both 8u and
8v is within hydrogen bonding distance of Arg571. Notably,
docking studies predict both the -enolic carboxyl moiety of 8u
that is not predicted to coordinate the metal ion and the fused
quinolinyl ring of 8v occupy the space that biotin has been shown
to occupy in several X-ray crystal structures (see Figure 4B and
4D).9
for the continued development of small molecule probes directed
against PC. Such probes will be enormously useful in evaluating
the metabolic role of PC in the onset and progression of diseases
such as type 2 diabetes and cancer. Future structure-based efforts
will seek to further improve the design of molecular properties en
route to producing the first class of potent and selective inhibitors
of this important metabolic enzyme.
4. Experimental
4.1 Chemistry
4.1.1 General experimental
All reactions involving moisture or air sensitive reagents were
carried out under a nitrogen atmosphere in oven-dried glassware
with anhydrous solvents. THF and ether were distilled from
sodium/benzophenone. Purifications by chromatography were
carried out using flash silica gel (32-63 ). NMR spectra were
recorded on either a Varian Mercury+ 300 MHz or a Varian
UnityInova 400 MHz instrument. CDCl3 and d6-DMSO was
purchased from Cambridge Isotope Laboratories. 1H NMR
spectra were calibrated to 7.27 ppm for residual CHCl3 or 2.50
ppm for d5-DMSO. 13C NMR spectra were calibrated from the
central peak at 77.23 ppm for CDCl3 or 39.52 ppm for d6-DMSO.
Coupling constants are reported in Hz. Elemental analyses were
obtained from Midwest Microlabs, Ltd., Indianapolis, IN, and
high-resolution mass spectra were obtained from the COSMIC
lab at Old Dominion University.
2.5 Evaluation of 8u and 8v against hCAII, MMP-2, MDH and
LDH.
The docking studies predicted that the -keto acid moiety
directly coordinates with the metal ion, raising questions about
the selectivity of this class of compounds for PC inhibition.
Selectivity was evaluated using two common metalloenzymes,
human carbonic anhydrase
2
(hCAII) and matrix
metalloproteinase 2 (MMP-2). While the potent PC inhibitor 8v
did not appreciably inhibit hCAII at 200 M or MMP-2 activity
at 50 M (Figure 5), compounds 8u and 8s increased the activity
of hCAII, while 8u, 8s, and 8r exhibited substantial inhibition of
MMP-2 at 50 M. It is reassuring that 8v also did not inhibit
enzymes that act on similar substrates: MDH (Supplementary
Table S1, Supplementary Figure S2) and lactate dehydrogenase
(LDH) (Supplementary Figure S2, Supplementary Figure S3),
nor is it used as a substrate of LDH (Supplementary Figure S4).
Ethyl 2-oxo-5-hexenoate (5a)12a and ethyl 2-oxo-4-
phenylbutanoate (5b)12b were prepared by literature procedures.
Methyl 4’-formylcinnamate and methyl 3’-formylcinnamate were
prepared by Pd-catalyzed Heck coupling of methyl acrylate with
either 4-bromobenzaldehyde or 3-bromobenzaldehyde, and were
identified by comparison to literature spectral data.17a 4-(3-
Thienyl)benzaldehyde was prepared by Pd-catalyzed Suzuki
coupling of 3-thiophene boronic acid with 4-bromobenzaldehyde,
and was identified by comparison to literature spectral data.17b
Phenylglyoxylic acid (7d), (2-nitrophenyl)pyruvic acid (7f), 2-
hydroxy-3-(4-hydroxyphenyl)-2-propenoic acid (8q) and 3-
phenyllactic acid (9) were purchased from Sigma-Aldrich, and 2-
hydroxy-3-(quinolin-2-yl)propenoic acid (8v) was purchased
from Enamine Building Blocks.
4.1.2 General procedure for hydrolysis of -ketoesters
To compounds 5a-b, a solution of excess 2.5 N NaOH was
added. The aqueous mixture was stirred at room temperature
until the initially emulsified mixture reached uniformity, at least
12 hours. The reaction mixture was quenched with 1 N HCl, and
the resulting -keto acids were extracted several times with ether,
and the combined organic layers washed with brine, dried
(MgSO4) and concentrated to give the corresponding product
Figure 5. Activity of human carbonic anhydrase II (hCAII =
black bars) in the presence of compounds (200 M) or matrix
metalloproteinase-2 (MMP-2 = grey bars) in the presence of
compounds (50 M). (a) negative control, (b) known inhibitor
(acetazolamide, 5 M for hCAII; PD166793, 100 M for MMP-
2), (c) 8v, (d) 8u, (e) 8s, (f) 8r. Error bars represent standard
error of the mean for four determinations each.
4.1.3 2-Oxohex-5-enoic acid (7a)
The hydrolysis of 5a (60 mg, 0.38 mmol) by the general
procedure gave 7a as a yellow oil (14 mg, 29%). H NMR (400
MHz, CDCl3) δ 5.80-5.65 (m, 1H), 5.04-4.91 (m, 2H), 2.98 (t, J
= 7.2 Hz, 2H), 2.44-2.30 (m, 2H) 13C NMR (100 MHz, CDCl3) δ
195.1, 160.0, 135.8, 116.2, 36.7, 26.6 ppm.
1
4.1.4 2-Oxo-4-phenylbutanoic acid (7b)
3. Conclusions
The hydrolysis of 5b (52 mg, 0.25 mmol) by the general
procedure gave 7b as a yellow oil (29 mg, 56%). H NMR (400
MHz, CDCl3) δ 7.33-7.17 (m, 5H), 3.21 (t, J = 7.6 Hz, 2H), 2.96
(t, J = 7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 195.4, 161.1,
140.1, 128.6, 128.4, 126.4, 40.1, 29.0 ppm. The spectral data for
this compound was consistent with the literature values.18
Building off the common binding motif observed for -keto
acids in the CT domain active site of PC, we synthesized and
evaluated a series of -hydroxycinnamic acids as a new class of
PC inhibitors. Compared to previously characterized inhibitors
of PC, these demonstrate improved potency and, for 8v,
promising initial selectivity that serves as a good starting point
1

o
1
4.1.5 General procedure for preparation of substituted 5-
methylenehydantoins
30%) as a white solid. mp > 215 C; H NMR (300 MHz, d6-
DMSO)  10.89 (br s, 4 H), 7.63 (s, 4H), 6.40 (s, 2H).
Hydantoin (500 mg, 5 mmol) was dissolved in H2O (50 mL)
at 70 °C with stirring. Ethanolamine (0.61 mL) was added to the
mixture, which was then heated to reflux. An equimolar quantity
of the appropriate aldehyde solution (5 mmol in 5 mL ethanol)
was added dropwise. The reaction mixture was heated at reflux
for 5 h. The mixture was cooled to room temperature, during
which time the product precipitated. The precipitate was
collected via vacuum filtration, washed several times with H2O
and dried in vacuo. Compounds 6c, 6e-i, and 6k-p were prepared
by the procedure and their NMR spectral data was consistent
with the literature values.13a
4.1.11 General procedure for hydrolysis of benzylhydantions
To a suspension of the benzylhydantoin in H2O (25 mL) was
added solid NaOH (15 equivalents). The mixture was heated at
reflux for 12 h under an N2 atmosphere, during which time the
material went into solution. After cooling to room temperature,
the stirred reaction mixture was quenched with concentrated HCl.
The mixture was extracted several times with ether. The
combined extracts were dried (MgSO4) and concentrated to give
the -keto acid/2-hydroxy-3-arylcinnamic acid. Compounds 7c,
7l14, 8e13c, 8g13d, 8h13c, 8i13c, 8k13f, 8n13f, 8o13e, 8p13f, were
prepared by the procedure and their NMR spectral data was
consistent with the literature values.
4.1.6 5-(3,4-Dimethylphenyl)methylene-2,4-imidazolidinedione
(6j)
The following compounds were prepared by this procedure:
Condensation of 3,4-dimethylbenzaldehyde (671 mg, 5.00
4.1.12 3-(3,4-Dimethylphenyl)-2-hydroxy-2-propenoic acid (8j)
mmol) by the general procedure gave 6j (653 mg, 60%) as a
o
1
white solid. mp > 215 C; H NMR (400 MHz, d6-DMSO) δ
11.08-10.47 (br s, 2H), 7.38 (s, 1H), 7.27 (d, J = 7.9 Hz, 1H),
7.10 (d, J = 7.9 Hz, 1H), 6.30 (s, 1H), 2.19 (s, 3H), 2.17 (s, 3H);
13C NMR (100 MHz, d6-DMSO) δ 166.2, 156.1, 137.5, 137.2,
130.8, 130.4, 130.3, 127.7, 127.4, 109.2, 19.7, 19.5 ppm. Anal.
Calcd. For C12H12N2O2: C, 66.65; H, 5.59; N, 12.93. Found: C,
66.87; H, 5.54; N, 13.10.
Hydrolysis of 6j (424 mg, 1.99 mmol) by the general
procedure gave 8j (206 mg, 55%) as an orange solid. mp 134-136
1
oC; H NMR (400 MHz, d6-DMSO): δ 9.90 (s, 1H), 7.60 (d, J =
7.6 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 6.64 (s, 1H), 2.23 (s, 3H),
2.18 (s, 3H); 13C NMR (100 MHz, d6-DMSO) δ 167.0, 141.4,
136.4, 136.0, 132.9, 130.8, 129.9, 127.4, 110.4, 19.9, 19.7 ppm.
HRMS (FAB): M + Na+, found 215.0675. C11H12O3Na requires
215.0678.
4.1.7 5-[4-(3-Thienyl)phenyl]methylene-2,4-imidazolidinedione
(6r)
4.1.13 3-(4-Bromophenyl)-2-hydroxy-2-propenoic acid (8m)
Condensation of 4-(3-thienyl)benzaldehyde (498 mg, 2.65
mmol) by the general procedure gave 6r (560 mg, 78%) as a
beige solid. mp > 215 oC; 1H NMR (300 MHz, d6-DMSO) δ 8.28
(s, 1H), 7.93 (s, 1H), 7.76 (d, J = 3.9 Hz, 1H), 7.74-7.70 (m, 2H),
7.64 (s, 1H), 7.62-7.57 (m, 2H), 6.34 (s, 1H); 13C NMR (75 MHz,
d6-DMSO) δ 162.0, 157.3, 141.7, 135.6, 132.5, 130.3, 129.1,
128.1, 126.5, 122.6, 122.3, 107.9.
Hydrolysis of 6m (403 mg, 1.51 mmol) by the general
procedure gave 8m (99 mg, 27%) as a white solid. mp 194-196
1
oC; H NMR (400 MHz, d6-DMSO): δ 9.49 (s, 1H), 7.69 (d, J =
8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 6.36 (s, 1H); 13C NMR
(100 MHz, d6-DMSO) δ 166.5, 143.1, 134.7, 131.7, 131.6, 120.5,
108.5 ppm. HRMS (FAB):
C9H7O3BrNa requires 264.9471.
M +
Na+, found 264.9470.
4.1.8
5-(4-(2-Methoxycarbonyl)ethenylphenyl)methylene-2,4-
4.1.14 2-Hydroxy-3-[4-(3-thienyl)phenyl]-2-propenoic acid (8r)
imidazolidinedione (6s)
Hydrolysis of 6r (493 mg, 1.83 mmol) by the general
procedure gave 8r (49 mg, 13%) as a pale yellow solid. mp 194-
196 oC; 1H NMR (400 MHz, d6-DMSO): 9.29 (s, 1H), 7.92 (q, J=
5.5 Hz, 1H), 7.87-7.83 (m, 1H), 7.81 (s, 1H), 7.76 (d, J = 8.2 Hz,
2H), 7.20 (d, J = 8.2 Hz, 2H), 6.39 (s, 1H) 13C NMR (100 MHz,
d6-DMSO): 167.0, 142.5, 141.8, 134.5, 134.4, 130.6, 127.8,
126.7, 126.6, 121.7, 110.0. HRMS (FAB): M + Na+, found
269.0241. C13H10O2SNa+ requires 269.0243.
Condensation of methyl 4’-formylcinnamate (554 mg, 2.91
mmol) by the general procedure gave 6s (369 mg, 47%) as a pale
o
1
yellow solid. mp > 215 C; H NMR (400 MHz, d6-DMSO) δ
11.31 (s, 1H), 10.69 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.61 (d, J =
8.0 Hz, 2H), 7.53 (d, J = 15.4 Hz, 1H), 6.55 (d, J = 15.4 Hz, 1H),
6.37 (s, 1H), 3.53 (s, 3H); 13C NMR (100 MHz, d6-DMSO) δ
168.1, 165.9, 156.1, 143.6, 135.2, 134.4, 130.2, 129.0, 128.3,
120.1, 107.8, 57.8.
4.1.15
3-[4-(2-Carboxyethenyl)phenyl]-2-hydroxy2-propenoic
4.1.9
5-(3-(2-Methoxycarbonyl)ethenylphenyl)methylene-2,4-
acid (8s)
imidazolidinedione (6t)
Hydrolysis of 6s (369 mg, 1.34 mmol) by the general
procedure gave 8s (43 mg, 14%) as a mustard brown solid. mp >
Condensation of methyl 3’-formylcinnamate (537 mg, 2.44
mmol) by the general procedure gave 6t (409 mg, 53%) as a
o
1
220 C; H NMR (400 MHz, d6-DMSO): 9.55 (s, 1H), 7.75 (d, J
= 8.6 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 17.2 Hz,
1H), 6.45 (d, J = 17.2 Hz, 1H), 6.38 (s, 1H); 13C NMR (100
MHz, d6-DMSO): δ 168.1, 166.5, 144.0, 143.2, 137.4, 130.0,
128.7, 128.5, 119.4, 108.9. HRMS (FAB): (M – H+) found
233.0453. (C12H10O5 – H+) requires 233.0455.
o
1
yellow solid. mp 232-235 C; H NMR (400 MHz, d6-DMSO) δ
8.27-8.14 (m, 1H), 7.84 (s, 1H), 7.77 (s, 1H), 7.59 (d, J = 16.4
Hz, 1H) 7.57-7.47 (m, 1H), 7.41 (t, J = 6.8 Hz, 1H), 7.25 (t, J =
6.8 Hz), 6.65 (d, J = 16.4 Hz, 1H), 6.39 (s, 1H), 3.20 (s, 3H); 13
C
NMR (100 MHz, d6-DMSO) δ 166.3, 165.8, 156.3, 139.0, 136.5,
134.0, 132.6, 129.8, 129.0, 127.2, 123.4, 121.7, 108.0, 60.3.
HRMS (FAB): (M2 + Na+) found 567.1487. (C14H12N2O4)2Na
requires 567.1486.
4.1.16
3-[3-(2-Carboxyethenyl)phenyl]-2-hydroxy2-propenoic
acid (8t)
From 6t (389 mg, 1.29 mmol), gave 8t (80 mg, 24%) as a
yellow solid. mp 189 oC (dec.); 1H NMR (400 MHz, d6-DMSO):
9.41 (s, 1H), 7.94 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.53 (d, J =
8.6 Hz, 1H), 7.50 (s, 1H), 7.36 (t, J= 7.7 Hz, 1H), 7.22 (d, J =
16.1 Hz, 1H), 6.46 (d, J = 16.2 Hz, 1H), 6.40 (s, 1H); 13C NMR
(100 MHz, d6-DMSO): 168.0, 166.6, 144.3, 143.0, 138.6, 136.2,
4.1.10
5,5’-(1,4-Phenylenedimethylidyne)bis[2,4-
imidazolidinedione] (6u)
Condensation of 1,4-benzenedicarboxaldehyde (671 mg, 5.00
mmol) by the general procedure was modified by the use of two
equivalents of hydantoin (1.011g, 10.10 mmol) gave 6u (442 mg,

134.6, 131.1, 129.4, 127.1, 126.0, 119.7. HRMS (FAB): (M +
Na+) found 257.0421. C12H10O5Na+ requires 257.0420.
an assay modified from Day and Cohen.18 All assay reagents and
solutions were prepared separately and maintained at 22 °C until
warmed to 30 °C. All substrates, effectors, and enzyme were
freshly dissolved and diluted in assay buffer containing 50mM
Tris (pH 8.0). The final concentration of the human carbonic
anhydrase II enzyme was 200 nM. Where noted, acetazolamide
was included at a final concentration of 10 μM. The substrate, p-
nitrophenyl acetate, was added at a final concentration of 504
μM. For all experiments, 20 μL of enzyme stock solution was
added to each well in a 96-well, flat-bottom, polystyrene
microplate (Santa Cruz Biotechnology) followed by the addition
of 10μL of the effectors (assay buffer for the uninhibited
reaction, acetazolamide for inhibition). The 96-well plate was
then incubated in a plate reader at 30 °C for 10 min;
concurrently, the substrate solution was also warmed to 30 °C for
10 min. The enzymatic reaction was initiated by adding 70 μL of
the substrate solution (p-nitrophenyl acetate). The absorbance
values were then measured at 405 nm every 30 s over a period of
20 min for a total of 40 measurements.
4.1.17 3,3’-(1,4-Phenylene)bis[2-hydroxy-2-propenoic acid] (8u)
Hydrolysis of 6u (443 mg, 1.77 mmol) by the general
procedure gave 8u (122 mg, 33%) as a dark yellow solid. mp 210
oC (dec.); 1H NMR (400 MHz, , d6-DMSO): 9.29 (s, 2H), 7.69 (s,
2H), 7.14 (d, J = 8.0 Hz, 2H), 6.35 (s, 2H); 13C NMR (100 MHz,
d6-DMSO) δ 166.7, 142.4, 134.3, 129.6, 109.8. HRMS (FAB): M
+ Na+, found 273.0369. C12H10O6Na+ requires 273.0370.
4.2. Enzyme inhibition assays
4.2.1 Screening compounds for inhibition using Fast Violet B
Compounds were initially screened for inhibition of S. aureus
PC (SaPC) with an optimized assay that is based on the reaction
of OAA, the product of the PC-catalyzed reaction, with Fast
Violet B, a diazonium salt which produces a colored adduct with
an absorbance maximum at 530 nm.11 The published 96-well
plate procedure was modified to a 384-well plate format such that
all of the steps were identical except that the volumes were
decreased by half to a final assay volume of 60 L. Assay
conditions consisted of 50 mM Bis-Tris (pH 7.7), 3 mM MgCl2,
150 mM KCl, 1% DMSO and 0.5% Triton x-100. To determine
whether the compounds interfered with formation of the FVB-
OAA colored adduct itself, the procedure was modified slightly:
15 L of OAA was added to a final concentration of 200 M in
in place of 15 L enzyme. This functioned to assess for possible
interference of the compounds with the formation of the FVB-
OAA adduct. None of the described compounds interfered with
the FVB assay signal at 200 M (Supplementary Table S1).
4.2.4 Evaluation against matrix metalloproteinase-2 (MMP-2)
Assays for human matrix metalloproteinase-2 activity were
performed in a 96-well plate format for a total reaction volume of
100 L, in an assay modified from Day and Cohen.19 All assay
o
reagents were prepared and maintained at 22 C until warmed to
37 oC. The omniMMP fluorogenic substrate and MMP-2 enzyme
were prepared in assay buffer (50 mM HEPES, 10 mM CaCl2,
0.05% Brij-35, pH of 7.52), and small molecule effectors were
prepared as a solution in 50% DMSO, 50% assay buffer. The
final concentration of enzyme in the 100 L enzymatic reaction
was 0.0083 U/L.
A known inhibitor, N-[(4'-bromo[1,1'-
biphenyl]-4-yl)sulfonyl]-L-valine (PD166793), was included
where noted at a final concentration of 100 M. OmniMMP
fluorogenic substrate was added to the 100 L enzymatic
reaction at a final concentration of 4 M. For all experiments, 30
L of enzyme stock solution was added to each well in a 96-well,
4.2.2 Measurement of OAA formation with malate
dehydrogenase
Malate dehydrogenase coupled enzyme assays were
performed at 22 C in a 96-well plate format for a total reaction
volume of 200 L. Assay conditions consisted of 100 mM Tris
(pH 7.8), 7 mM MgCl2, 150 mM KCl and 0.5% Triton x-100.
First, 20 L PC was added such that the final concentration in the
assay was 10 g/mL. Subsequently, 20 L of compound was
added to obtain the desired final concentration, 20 L of MDH
was added such that the final concentration in the assay was 20
U/mL, and lastly 140 L of substrates were added to initiate the
flat-bottom,
black
polystyrene
microplate (SantaCruz
Biotechnology), followed by the addition of 10 L of the
effectors (50% assay buffer, 50% DMSO for the uninhibited
reaction, PD166793 for inhibition). The 96-well plate was then
incubated in a plate reader at 37 oC for 30 min; concurrently, the
o
substrate solution was also warmed to 37 C for 30 min. The
enzymatic reaction was initiated by adding 60 L of the
omniMMP fluorogenic substrate solution. The change in
fluorescence was then monitored for 30 min with excitation and
emission wavelengths at 320 and 400 nm, respectively every 46
seconds for a total of 40 measurements.
-
reaction (pyruvate, HCO3 , ATP and NADH to a final assay
concentration of 12 mM, 25 mM, 2.5 mM and 0.25 mM,
respectively). To assess the inhibition with respect to pyruvate,
the procedure was modified such that 20 L of pyruvate was
added to the desired final concentrations prior to the addition of
-
4.4 Computational docking
120 L of substrates to initiate the reaction (HCO3 , ATP and
NADH to a final assay concentration of 25 mM, 2.5 mM and
0.25 mM, respectively). To assess the inhibition with respect to
ATP, the procedure was modified such that 20 M of ATP was
added to the desired final concentrations prior to the addition of
The ligand structures were built using ChemDraw
(PerkinElmer) and saved as MDL Molefiles. The docking studies
were performed using the docking program FITTED within the
web-based FORECASTER platform,20 using the workflow
“Docking-2D Molecules to a Rigid Protein (pdb)” which
converts the 2D MDL Molefile to a 3D molecule and the protein
pdb file (pdb code 3BG5) to a mol2 file. Protein Rotamer
Elaboration and Protonation based on Accurate Residue Energy,
or PREPARE, was utilized as part of the FITTED workflow. This
software prepared all the atom files from the pdb files for suitable
mol2 files. In addition to preparing the pdb files, PREPARE
identified pyruvate in the pdb file by the residue name (PYR),
chain location (A for open or C for closed conformation), and
residue number (2001). The protein (as a mol2 file) was
subsequently processed by PROtein Conformational Ensemble
System Setup, or PROCESS, in the FITTED workflow to dock
the 3D compounds in the binding site of the CT domain. The
–
120 L of substrates to initiate the reaction (HCO3 , ATP and
NADH to a final assay concentration of 25 mM, 12 mM and 0.25
mM, respectively). To account for possible compound inhibition
of MDH (see Supplementary Table S1), the procedure was
modified such that 20 L OAA was added to a final
concentration of 30 mM, in place of PC. Reagents were
dispensed manually by a hand-held, multi-channel micropipette
and absorbance measurements were recorded at 340 nm with a
Multiskan Ascent spectrophotometer (Thermo).
4.2.3 Evaluation against human carbonic anhydrase (hCAII)
Assays for human carbonic anhydrase activity were performed
in a 96-well plate format for a total reaction volume of 100 L, in

7.
8.
9.
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center of the binding site was defined by the center of pyruvate
present in the active site. After the removal of pyruvate from the
active site, a sphere with a radius of 15 Å was applied as the
default for establishing the binding site cavity. FITTED utilized a
genetic algorithm/hybrid matching algorithm to dock the
compounds within the established cavity site. Docking scores
were reported from FITTED as the predicted binding energy
(G) and following computational docking, the lowest energy
pose was visualized in Pymol.
10. (a) Lietzan, A. D.; St. Maurice, M. Biochem. Biophys. Res.
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Acknowledgments
Financial support for this research was provided by the
Marquette University Strategic Innovation Fund. We thank Prof.
Nicolas Moitessier (McGill University) for providing access to
the FITTED docking software in the FORECASTER platform.
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‡These authors contributed equally.
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Supplementary Material
Supplementary data associated with this article can be found,
in the online version, at
6.
Tong, L. Cell. Mol. Life Sci. 2013, 70, 863-891.
8i
8j
H
H
H
Br
H
H
H
H
H
H
H
Me
H
Me
Me
210
98
140
38
Table 1. S. aureus pyruvate carboxylase inhibition activity (IC50
values in M).
8k
7l
CF3
H
130
>1000
200
280
100
170
67
96
H
>1000
81
8m
8n
8o
8p
8q
8r
H
Br
H
Cl
220
30
R2
H
R3
H
R4
H
FVB IC50 MDH IC50
H
F
8e
7f
240
>2000
>1000
360
130
>2000
>1000
23
H
OMe
OH
3-thienyl
56
NO2
H
H
H
H
22
8g
8h
H
NO2
H
H
48.0
–
H
Me

8s
58.6
–
8t
118
–
8u
3.0±1.0
11
8v
4.3±1.5
3.2±0.5
Evaluation of alpha-hydroxycinnamic acids as
pyruvate carboxylase inhibitors
Daniel J. Burkett, Brittney N. Wyatt, Mallory
Mews, Anson Bautista, Ryan Engel,
Chris Dockendorff, William A. Donaldson and
Martin St. Maurice
TOC graphic