Synthesis, cytotoxicity for mimics of catalase: Inhibitors of lactate dehydrogenase and hypoxia inducible factor

Article abstract of DOI:10.1016/j.ejmech.2014.04.035

Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. Two Mn(II) complexes with ligand containing di(pyridylmethyl) amine and pyrrol-ketone were used to attenuate the activity of LDH-A. The inhibition of the manganese(II) complexes on the proliferation of HepG-2 cells is related to their ability to disproportionate H₂O₂. Importantly, the synthesized mimic of catalase can decrease the expression of hypoxia inducible factor (HIF-1α) in HepG-2 cells. So we envision that the multifunctional mimics of catalase could attenuate the activity of LDH-A signaling the cancer cells to death through HIF-1α involved path.

Full text of DOI:10.1016/j.ejmech.2014.04.035

European Journal of Medicinal Chemistry 80 (2014) 1e7
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, cytotoxicity for mimics of catalase: Inhibitors of lactate
dehydrogenase and hypoxia inducible factor
,
a
a
b
Juan-Juan Xue a, Qiu-Yun Chen a , Meng-Yun Kong , Chun-Yin Zhu , Zhi-Rong Gen ,
*
Zhi-Lin Wang b
a School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China
b State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the
highly activated glycolysis pathway in cancer cells. Two Mn(II) complexes with ligand containing
di(pyridylmethyl) amine and pyrrol-ketone were used to attenuate the activity of LDH-A. The inhibition
of the manganese(II) complexes on the proliferation of HepG-2 cells is related to their ability to
disproportionate H2O2. Importantly, the synthesized mimic of catalase can decrease the expression of
Received 31 October 2013
Received in revised form
12 March 2014
Accepted 10 April 2014
Available online 13 April 2014
hypoxia inducible factor (HIF-1a) in HepG-2 cells. So we envision that the multifunctional mimics of
catalase could attenuate the activity of LDH-A signaling the cancer cells to death through HIF-1
path.
a involved
Keywords:
Mimics of catalase
Cancer cells
LDH
Ó 2014 Elsevier Masson SAS. All rights reserved.
HIF
Manganese
1. Introduction
the transformation of pyruvate to lactate. LDH-A is a potentially
important metabolic target for inhibition of the highly activated
glycolysis pathway in cancer cells [7]. Most reported LDH-A in-
hibitors, such as oxamate, 3-hydroxyisoxazole-4-carboxylic acid
and N-hydroxyindole, are competitive with the pyruvate or NADH
Cancer cells typically display altered glucose metabolism char-
acterized by a preference of aerobic glycolysis, known as the War-
burg effect, which facilitates cell proliferation [1]. Glycolysis allows
for continued ATP production without the need of O2-dependent
oxidative phosphorylation, and is thus an important pathway un-
der hypoxic conditions [2]. Altered cancer cell energy metabolism
has been categorized as an emerging hallmark of cancer, and thus,
inhibition of metabolic processes associated with cancer cell
growth constitutes a promising approach in the search for new
cancer therapies [3,4]. One molecular target in the glycolytic
pathway is the lactate dehydrogenase (LDH) which catalyzes the
interconversion of lactate and pyruvate [5,6]. In mammalian cells,
there are four major isoforms of LDH, LDH-A, LDH-b, LDH-c and
LDH-h, with the A form having higher intrinsic activity to catalyze
substrate binding [7e9]. Moreover, targeted inhibition of HIF-1
another new pathway to cancer therapy. Hypoxia-inducible factor
(HIF-1 ) regulates the energy metabolism by triggering a switch
a is
a
from mitochondrial oxidative phosphorylation to anaerobic
glycolysis, increasing the expression of genes that encode glycolytic
enzymes and glucose transporters [10]. Cancer cells activate
glycolysis to meet their energy demands and use O2 to generate
excessive levels of the reactive oxygen species (ROS), superoxide
anion (O$2ꢀ) and hydrogen peroxide (H2O2). H2O2, a kind of ROS
generated in mitochondria, is considered a mediator of apoptotic
cancer cell, and can be eliminated by mitochondrial catalase [11,12].
With the over-expression of H2O2-detoxifying enzymes or human
catalase in vivo, H2O2 concentration will decrease, and cancer cells
could revert to normal appearance [13]. Thus, H2O2 is also an
important signal molecule to attenuate the metabolite of oxygen in
Abbreviations: LDH-A, Lactate dehydrogenase; HIF, hypoxia inducible factor;
dpa, Di(pyridine-2-yl)methyl)amine; PPMdpa, 4-(Chloromethyl)phenyl)(3,5-
cancer cells and the catalase could be a modulator of HIF-1a signals.
dimethyl-1H-pyrrol-2-yl)methanone);
idylmethyl)amine; phdpa, N-Benzyl-di(pyridylmethyl)amine; Mn1, [(PPMdpa)
MnCl2]; Mn2, [(PPMdpa)MnAc2]; Hepes, 4-(2-Hydroxyethyl)-1-
pierazineethanesulfonic acid; DMEM, Dulbecoo’s modified Eagle’s medium.
* Corresponding author.
pydpa,
N-(3-Pyridyl)methyl-di(pyr-
It is reported that the manganese(III) porphyrin complexes
could decrease the mitochondrial H2O2 [14]. Previously we found
that the carboxylate-bridged dimanganese(II) systems could be
used as functional models of catalase, which could inhibit the
E-mail address: chenqy@ujs.edu.cn (Q.-Y. Chen).
http://dx.doi.org/10.1016/j.ejmech.2014.04.035
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

2
J.-J. Xue et al. / European Journal of Medicinal Chemistry 80 (2014) 1e7
proliferation of HeLa cells through ROS signal induced apoptosis
[15]. These lipophilic complexes can accumulate in cancer cells
through perturbing the mitochondrial membrane potential or
inhibiting the swelling of over calcium(II) loaded mitochondria
[16,17]. Thus, we propose that the conjugation of pyrrol-conjugated
C]O groups (3,5-dimethyl-1H-pyrrole-2-carbonyl portion,
Scheme 1 and Scheme S1) with Mn(II) complexes of di(picolyl)
amine would produce a new kind of multifunctional complexes
indicating that the PPMdpa is stable in the ES-MS condition
(Fig. S5). The main peak for Mn2 at m/z ¼ 524.29 (100) corresponds
to species [(PPMdpa)Mn(m
-Ac)2Mn(PPMdpa)]2þ, which indicates
the existence of Mn2 (Fig. S6). The 50.58% weight loss in the range
of 109e540 ꢁC corresponds to the loss of di(pyridylmethyl)amine
groups from the ligand and two chlorine ions in Mn1 (calcd 50.37%)
(Fig. S7A). Thermal analysis (TG) curves of Mn2 in the range of
temperature from 0 to 1000 ꢁC are shown in Fig. S7B. The weight
loss of 54.25% at 98e460 ꢁC for Mn2 is attributed to the loss of four
Acꢀ ions and two dpa groups from the ligand (calcd 54.37%).
with activity of inhibiting LDH-A and HIF-1a. Here, we report the
synthesis, characterization and bioactivities of Mn(II) complexes of
PPMdpa.
Thermal analysis results confirm the formation of [(PPMdpa)Mn(m-
Ac)2Mn(PPMdpa)Ac2] (Mn2). The paramagnetic resonance of the
powder solid state complex Mn2 was carried out at 110 K. The EPR
spectrum recorded for the Mn2 displays a strong signal centered at
g ¼ 2.01 with no hyperfine splitting indicating no MneMn coupling
interaction in Mn2, which is consistent with that of those reported
dimanganese(II) species (Fig. S8) [22].
2. Results and discussion
2.1. Synthesis and characterization of ligand (PPMdpa) and
complexes
Di(pyridine-2-ylmethyl)amine (dpa) and 4-(chloromethyl)
phenyl)(3,5-dimethyl-1H-pyrrol-2-yl)methanone (PPM) were
refluxed in acetonitrile under nitrogen producing PPMdpa in 66%
yield (Scheme S1). The 1H NMR spectra of the PPM and PPMdpa
confirm the existence of PPMdpa (Figs. S1 and S2). Subsequent
reaction of PPMdpa with MnCl2$4H2O or MnAc2$6H2O resulted in
the formation of complexes Mn1 or Mn2. The structures of com-
plexes Mn1 and Mn2 were characterized by elemental analyses, IR,
UV, ES-MS and EPR. The elemental analyses show that the ratio of
metal:L in all complexes is 1:1. The PPMdpa and Mn1 are unsoluble
2.2. Crystal structure of [(PPMdpa)2Mn2(
m
-Cl)2Cl2]2 (Mn1)
The molecular structure of [(PPMdpa)2Mn2(
with the atomic labeling scheme is shown Fig. 1, and the selected
bond lengths and angles are listed in Table S3. The Mn1 atom in
[(PPMdpa)2Mn2(m-Cl)2Cl2]2 (Mn1) is coordinated by three N atoms
(N2, N3, N4), one chloride atom (Cl1), and two bridged chloride
anions (Cl2, Cl2A), resulting in a six coordinate dinuclear Mn(II)
m-Cl)2Cl2]2 (Mn1)
complex. The complex [(PPMdpa)2Mn2(m-Cl)2Cl2]2 thus shows a
distorted octahedral geometry. Atoms N2, N3, Cl2 and Cl2A form
the equatorial tetragonal plane (mean deviation ¼ 0.0828), while
N4 and Cl1 occupy the apical positions. The Mn1 atom is shifted by
in water, while Mn2 is slightly soluble in water. The d(CH) vibration
of pyridyl ring is at approximately 760 cmꢀ1 [18]. In contrast, these
vibrations in the complexes Mn1 and Mn2 are all shifted to 762 and
765 cmꢀ1, respectively. These shifts can be explained by the fact
that the nitrogen atoms of pyridyl ring of the ligands donate a pair
of electrons each to the central metal ions, forming coordinate
ꢀ
0.3419 A out of the equatorial plane toward Cl2. The Mn1eN4 and
ꢀ
Mn1eCl1 bond distances are 2.387(3) and 2.4257(13) A, respec-
tively. The N4eMn1eCl1 angle is 159.28(9) A. Atoms N2A, N3A, Cl2
and Cl2A form the equatorial tetragonal plane (mean
ꢀ
covalent bond [19]. The n(C]O) bands of the complexes Mn1 and
deviation ¼ 0.0828), while N4A and Cl1A occupy the apical posi-
Mn2 appear at approximately 1610 cmꢀ1 and 1606 cmꢀ1, respec-
tively, indicating the existence of the pyrrol-conjugated carbonyl
group in PPMdpa (Fig. S3) [20]. The strong peak at 3237 cmꢀ1 for
ꢀ
tions. The Mn1A atom is shifted by 0.3419 A out of the equatorial
plane toward Cl2. The Mn1eN4 and Mn1eCl1 bond distances are
ꢀ
2.387(3) and 2.4257(13) A, respectively. The N4eMn1eCl1 angle is
Mn1 and Mn2 in infrared spectra was assigned to
n(NeH) stretch-
ꢀ
159.28(9) A.
ing frequencies showing the existence of pyrrole in PPMdpa. The
stretching frequencies of n
(COOꢀ) at 1570 cmꢀ1 and 1016 cmꢀ1 for
2.3. Catalase-like activity measured by O2 evolution e kinetics
studies
complex Mn2, indicates the existence of COOꢀ [21].
The UV spectra of PPMdpa had bands at 206, 248 and 323 nm
(Fig. S4 and Table S1). The band at 248 nm can be attributed to the
The progresses of the reactions between complexes and H2O2 at
various conditions were monitored by UVevis spectroscopy. When
H2O2 (0.5 mL, 30%) was added to the white-yellow solution of Mn2,
the color of the solution became dark-yellow and poorly resolved
absorption bands in the range of 400e800 nm appeared, which
indicate that complex Mn2 could bind with H2O2 (Fig. S9). The O2
evolution volume was used for the kinetic study of H2O2 dispro-
portionation. The H2O2 disproportionation promoted by complexes
was carried out in MeCN and buffered solutions ((Tris/TriseHCl
0.1 M, NaCl 0.1 M, pH 7.4) at 0 and 37 ꢁC. It was found that less gas
evolution could be monitored when Mn1 was incubated with H2O2,
even after 3 h under the conditions described above, demonstrating
that Mn1 could not catalyze the disproportionation of dihydrogen
peroxide (Fig. 2a). In contrast, complex Mn2 can disproportionate
dihydrogen peroxide to generate dioxygen. The complex Mn2 could
disproportionate 0.5 mL H2O2 aqueous to liberate 1 mmol O2. The
obtained plot of the initial rate vs the concentration of dihydrogen
peroxide is fitted by using the Hill equation (V0 ¼ Vmax [s]n/
(Km þ [s]n)) (Fig. S10, Fig. S11). The parameter Kcat is calculated from
the equation Kcat ¼ Vmax/[Et] [23,24]. The maximum O2 evolution
rates were about 4.74 mM sꢀ1 and 0.94 mM sꢀ1 for Mn2 in the
MeCN and TriseHCl solution, respectively. The Hill constants (n)
nep* transition of the pyrrole-conjugated C]O. This band for
complexes Mn1 and Mn2 was red shift to 257 nm and 256 nm,
respectively. Nearly no free Mn(II) ions could be measured for the
solution of Mn1 and Mn2 in water-DMSO (1:1) by ICP methods,
indicating Mn1 and Mn2 are stable in solution. The main peak for
PPMdpa m/z ¼ 411.36(100) corresponds to species [(PPMdpa)H]þ
Scheme 1. The chemical structure of PPMdpa.

J.-J. Xue et al. / European Journal of Medicinal Chemistry 80 (2014) 1e7
3
Fig. 1. Crystal structure of [(PPMdpa)2Mn2(m-Cl)2Cl2]2 (Mn1). Hydrogen atoms were omitted.
and turnover number Kcat for Mn2 in both MeCN and buffered
solutions are 1.46 ꢂ 0.3 and 2.86 ꢂ 0.2, and 9.95 and 1.40, respec-
tively (Table S4). The turnover numbers of Mn2 in MeCN was higher
than that of [(Adpa)Mn(m2-O)2Mn(Adpa)]PF6∙8H2O, [(Adpa)2M-
characterized manganese catalases Thermus thermophilus catalase
and Thermoleophilum album catalase showed much higher catalytic
activity for the disproportionation of dihydrogen peroxide than
Mn2, the activities of the synthetic mimics (Mn2) of catalase were
remarkable [27e29]. According to the results, the intrinsic H2O2
disproportionation activities of complexes were in the order
Mn2 >> Mn1. The catalase activity of Mn2 under different condi-
tions is shown in Fig. 2b. The condition of the reaction carried out at
37 ꢁC in TriseHCl solution was the mimetic condition of the cell
environment. Thus, we deduce that complex Mn2 would also show
good catalase activity in vitro.
n2(Ac)(H2O)2](Ac) and [TPA2Mn2(
that of manganese complexes with bpia ligand and N,N0-bis(3-
selenomethyl-salicyladehyde-5-sulfonate) bis(1,3)-diamino-2-
m-O)2](ClO4)3 but was lower than
hydroxypropane) Schiff base [25,26]. Though the two
2.4. Evaluation of enzyme activity for inhibitors of LDH-A
The LDH inhibitory activities of the new compounds were
measured by standard enzyme kinetic experiments on rabbit LDH-
A purified isoform [8]. Initially, we verified the percentage inhibi-
tion relative to control at 30e120 mM for all compounds with LDH-
A (Fig. 3). It is observed that the inhibiting effect for the compounds
is dependent on the concentration. We were pleased to find a
dramatic increase in the LDH-A inhibition level when the concen-
tration increase to 75
Mn1 inhibition, respectively. However, previously reported Mn(II)
complex [(phdpa)2Mn2( -Cl)2(Cl)2] [15] (75 M) have no inhibition
on the activity of LDH-A in the same condition (Table S5). The
difference between [(phdpa)2Mn2( -Cl)2(Cl)2] and Mn1 is that
mM, with a 10% PPMdpa, a 60% Mn2 and a 99%
m
m
m
Fig. 2. (a) Rates of the O2 evolution of the complexes at 37 ꢁC in MeCN,
Ccomplexes ¼ 0.5 mM, 0.5 mL of 30% H2O2 aqueous solution, VMeCN ¼ 3 mL Mn1 (B),
Mn2 (-). (b) Rates of the O2 evolution of the complex Mn2 at different conditions,
CMn2 ¼ 0.5 mM, 0.5 mL of 30% H2O2 aqueous solution, Vsolvent ¼ 3 mL. 37 ꢁC in MeCN
Fig. 3. Colorimetric measurement of inhibition (% relative to control) of the enzymatic
activity of LDH-A in the presence of PPMdpa, Mn1 and Mn2 (concentration were
PPMdpa and Mn1 and Mn2). Values are reported as the mean ꢂ the SD of three in-
dependent experiments, the SD < 0.05.
(-), 37 ꢁC in TriseHCl (B), 0 ꢁC in TriseHCl (
6).

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J.-J. Xue et al. / European Journal of Medicinal Chemistry 80 (2014) 1e7
Table 1
there are pyrrol-conjugated C]O groups in Mn1, so we deduce that
pyrrol-conjugated C]O groups (3,5-dimethyl-1H-pyrrole-2-
carbonyl portion) give great contribution to the inhibition for
Mn1 to LDH-A. The apparent MichaeliseMenten constants (Km) of
NADH and pyruvate for LDH-A were measured by Lineweavere
Burk plots. From the values of Km0 so obtained, Ki values for each
single inhibitor were determined using double-reciprocal Line-
Inhibition data (Ki for LDH-A) obtained with Compounds.a
Compound
LDH-a (Ki, mM)
[NADH]b
[Pyruvate]c
PPMdpa
Mn1
Mn2
414.9 ꢂ 1.7
85.7 ꢂ 1.3
41.7 ꢂ 2.3
114.9 ꢂ 1.5
30.9 ꢂ 2.6
21.4 ꢂ 0.7
weavereBurk plots (Fig. 4, Table 1). For Mn2, Ki value is 41.7
mM vs
a
Ki determination was performed in presence of compounds PPMdpa, Mn1 and
Mn2 as described in the Experimental Section. Values are reported as mean ꢂ SD of
three or more independent experiments.
NADH and 21.4 M vs pyruvate. Therefore, we deduce that both
m
compounds were found to be more competitive inhibitors of LDH-A
with respect to NADH than pyruvate in the conversion of pyruvate
to lactate catalyzed by this enzyme, whereby NADH is converted to
NADþ.
b
Saturating concentration (2 mM) of pyruvate and competitive increasing con-
centrations (60e180
m
M) of NADH.
Saturating concentration (180
centrations (0.67e2 mM) of pyruvate.
c
mM) of NADH and competitive increasing con-
2.5. Cytotoxicity assay and effect on the expression of HIF-1
a
effects for Mn1 and Mn2 on HepG-2 cells are dependent on their
concentration (Fig. S12 and Fig. 5). The anticancer property, good
solubility and mimic of catalase make Mn2 a potential multifunc-
tional complex. The inhibiting rate of Mn2 is slightly smaller than
that of Mn1 in 24 h. This indicates that H2O2 discriminating prop-
erties may have some effect on the metabolite of oxygen or the ROS
signaling apoptosis in cancer cells. Oxygen is an important factor to
maintain cell life and the final electron acceptor in oxidative
phosphorylation for energy production [30]. Hypoxia-inducible
factor (HIF) regulates the energy metabolism by triggering a
switch from mitochondrial oxidative phosphorylation to anaerobic
glycolysis, increasing the expression of genes that encode glycolytic
enzymes and glucose transporters [31]. HIF is a common link be-
tween O2 availability, malignant progression, and changes in cancer
metabolism. Mn2 is a good mimic of catalase and inhibitor of LDH-
The in vitro cytotoxicity of complexes (Mn1 and Mn2) was
evaluated using MTT assay by measuring anti-proliferation on
HepG-2 cells. 5-Fluorouracial was used as a positive control for
cytotoxicity. The IC50 values are shown in Table 2. Complexes Mn1
and Mn2 can inhibit the proliferation of the HepG-2 cells with IC50
0.95 and 2.59
than previous reported Mn(II) complexes [(pydpa)2Mn3(-
H2O)2(Cl6)] and [(phdpa)2Mn2( -Cl)2(Cl)2] [15]. The inhibiting
mM. These indicate Mn1 and Mn2 are more active
m
A, it may have some effect on the expression of HIF-1
cells. Experimental results show that the Mn2 obviously de-
creases the expression of HIF-1 in 24 h (Fig. 6). Therefore, this
result demonstrates that Mn2 is a multifunctional complex to
a in HepG-2
a
attenuate the expression of HIF-1a in cancer cells.
3. Conclusion
Mn(II) complexes with (4-((bis(pyridin-2-lmethyl)amino)
methyl) phenyl)(3,5-dimethyl-1H-pyrrol-2-yl) methanone
(PPMdpa) were characterized and used as mimics of catalase and
inhibitors of LDH-A. This is the first report that functional mimics of
catalase (Mn(II) complexes) can be as inhibitor of LDH-A. The
anticancer activity of Mn2, a good mimic of catalase, is smaller than
that of Mn1, demonstrating that the inhibition of these man-
ganese(II) complexes on tumor cells in vitro is related to their H2O2
disproportionating activity. Importantly, Mn2 can decrease the
expression of HIF-1a in HepG-2 cells. Therefore, Mn2 can be used as
a chemical sensitizer to explore the cross-talk mechanism of HIF-1
a
related oxygen and energy metabolism in cancer cells in the future.
Given these advantages, we envision that combination of potential
inhibitors of LDH-A and mimics of catalase would promote the
development of compounds that can attenuate the metabolism of
oxygen and energy signaling the cancer cells to death through HIF-
1a involved path.
4. Experimental
4.1. General methods
The C, H and N microanalyses were performed on Vario EL
elemental analyzer. The electronic absorption spectra were recor-
ded in the 200e800 nm regions using a Varian Cary 50-BIO UVevis
spectrophotometer. The IR spectra were recorded on a Nicolet-470
Fig. 4. LineweavereBurk plots determined from triplicate experiments with inhibitors
PPMdpa (90 M), Mn1 (45 M) and Mn2 (45 M) using average activities: competition
experiments with NADH (a) and pyruvate (b).
m
m
m

J.-J. Xue et al. / European Journal of Medicinal Chemistry 80 (2014) 1e7
5
Table 2
was applied on the basis of multiscan. The structure was solved by
direct methods and refined on F2 by the full-matrix least-squares
methods, using SHELXTL version 5.10 [32]. All non-hydrogen atoms
were refined anisotropically. Hydrogen atoms were introduced in
their calculated positions. All computations were carried out using
the SHELXTL-PC program package.
IC50 values of the complexes on HepG-2 cell lines.
Complex
HepG-2
Mn1
Mn2
PPMdpa
MnCl2
0.95 ꢂ 0.2
2.59 ꢂ 0.3
21.5 ꢂ 0.3
>100
5-Fluorouracil**
32 ꢂ 0.8
4.3. Catalase-like activity
*All IC50 values were expressed as mean values of the three
experiments. **5-Fluorouracil was used as
control.
a positive
All of the reactions between the complexes and dihydrogen
peroxide were performed in buffered (Tris/TriseHCl 0.1 M, NaCl
0.1 M, pH 7.4) at 0 and 37 ꢁC. The reactivity of the complexes with
H2O2 was first investigated in water via UVevis spectroscopy
titration at 37 ꢁC. After the solution (10 mL) of complexes (0.5 mM)
was stirred at 37 ꢁC for 30 min, 0.5 mL of H2O2 aqueous solution
(30%) was added, and the spectra were recorded at 5 min intervals
at 37 ꢁC. The volumetric measurements of the evolved dioxygen
produced during the reaction of the complexes with H2O2 were
performed in triplicate as follows: a 25 mL round-bottom flask
containing a complex (0.5 ꢃ 10ꢀ3 M, 3.0 mL) in MeCN solvent (or a
buffered system) was placed in an ice (0 ꢂ 0.1 ꢁC) bath. The flask
was closed with a rubber septum, and a cannula was used to con-
nect the reaction flask to an inverted graduated pipet, filled with
water. While the solution containing the complex was being stirred,
a solution of 0.5 mL of H2O2 aqueous solution was added through
the septum using a microsyringe. The volume of oxygen produced
was measured in the pipet. The kinetics measurements of com-
plexes, Mn1 and Mn2 were performed in MeCN solution at 37 ꢁC.
Different concentrations of dihydrogen peroxide were prepared by
diluting the 30% H2O2 aqueous solution with acetonitrile solution
or TriseHCl buffer solution. The optimum reaction order of the
substrate with respect to the complexes was determined by
reacting different concentrations of complexes with a constant
concentration of substrate. Similarly, the optimum reaction order of
the complexes with respect to the substrate was determined by
reacting different concentrations of substrate with a constant
concentration of complexes.
Fig. 5. The concentration dependence of Mn2 on HepG-2 cells. Cell proliferation was
determined by MTT assay every 12 h. Each column represents the mean of the data
from three independent experiments.
spectrophotometer in the range 4000e400 cmꢀ1. The 1H NMR
spectra were measured on a Bruker 400 MHz spectrometer. The
electrospray mass spectra (ES-MS) were determined on a Finnigan
LCQ mass spectrograph, and the concentration of samples was
about 1.0 m
mol Lꢀ1. The diluted solution was electrosprayed at a
flow rate of 5 ꢃ 10ꢀ6 Lꢀ1 minꢀ1 with a needled voltage of þ4.5 kV.
The mobile phase was methanol. X-band EPR spectra were recor-
ded on a Bruker BioSpin GmbH instrument in liquid nitrogen
(110 K).
4.4. Inhibition of lactate dehydrogenase
Lactate dehydrogenase (LDH-A), an enzyme is the glycolysis
pathway, is considered a significant small-molecule oncology
target in cancer metabolism. The oxidation of NADH is observed at
340 nm, which is a direct measure of the reduction of pyruvate to
lactate in the absence of interfering reactions. The apparent
MichaeliseMenten constants (Km) of LDH-A was measured from
LineweavereBurk plots. The reaction velocity of purified LDH-A
was determined by a decrease in absorbance at 340 nm of NADH,
and the value of optical density was determined at 30 s intervals for
4 min. LDH-A activity was determined at 37 ꢁC by measuring the
oxidation of NADH spectrophotometrically at 340 nm as a function
of time. The Km values of LDH-A for NADH and pyruvate were
determined in saturating conditions as described below. In 100 mM
sodium phosphate buffer (pH 7.4), 0.5 units of LDH-A were com-
4.2. X-ray crystal structure determination
Crystallographic data for [(PPMdpa)2Mn2(m-Cl)2Cl2]2 (Mn1) is
listed in Table S2. The crystal of the complex was selected for lattice
parameter determination and collection of intensity data at 293 K
on a Rigaku Mercury2 CCD area detector (MSC Inc., 2005) with
ꢀ
monochromatized Mo K
corrected for Lorenz and polarization effects during the data
reduction. A semiempirical absorption correction from equivalents
a
radiation (
l
¼ 0.71073 A). The data was
bined with 2 mM pyruvate (pyruvate-saturated) and 60e180
mM
NADH, or 180 M NADH (NADH-saturated) and 0.67 mMe2 mM
m
pyruvate. These conditions were used for all assays. The reaction
velocity of LDH was determined spectrophotometrically (UVevis
spectrophotometer) by a decrease in absorbance at 340 nm
(ε ¼ 6.2 mMꢀ1 cmꢀ1) of NADH. MichaeliseMenten constants for
substrates were determined from initial rate measurements at
37 ꢁC by nonlinear regression analysis with the Origin 8.0. Under
these conditions NADH showed a Km of 36
mol/min)/mg, whereas the pyruvate showed a Km of 115
Vmax of 55 ( mol/min)/mg. Compounds PPMdpa, Mn1 and Mn2
m
M and a Vmax of 55
Fig. 6. The expression of HIF-1
(2.6 M) for 24 h. Control (C) (10
times).
a
in HepG-2 cells after incubated with Mn2 (M)
(m
mM and a
m
mM DMSO). (The experiment was repeated for three
m

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J.-J. Xue et al. / European Journal of Medicinal Chemistry 80 (2014) 1e7
were dissolved in stock solutions of DMSO (concentrations of
DMSO during the initial rate measurements did not exceed 0.5%).
Using 180 mM NADH and 2 mM pyruvate, we initially evaluated the
percent inhibition of the compounds. We evaluated the apparent
Km0 values in the presence of inhibitors. From the values of Km0 so
obtained, Ki values for each single inhibitor were determined using
doubleereciprocal plots [33].
O). 1H NMR (CDCl3, 400 MHz, 25 ꢁC, TMS),
NeH); 7.65 (d, J ¼ 8.0 Hz, 2H; BzH); 7.48 (d, J ¼ 8.0 Hz, 2H; BzH);
5.89 (s, 1H; pyrrole-H); 4.65 (s, 2H; eCH2e); 2.32 (s, 3H; eCH3);
2.00 (s, 3H; eCH3).
d
¼ 9.54 (s, 1H; pyrrole,
4.9. Synthesis of (4-((bis(pyridin-2-ylmethyl)amino)methyl)
phenyl)(3,5-dimethyl-1H-pyrrol-2-yl) methanone (PPMdpa)
4.5. Cytotoxicity testing
Bis(pyridine-2-ylmethyl)amine (dpa, 600 mg, 3 mmol) was
dissolved in acetonitrile (20 mL) under nitrogen at room temper-
ature. KI (107 mg, 0.64 mmol) was added with stirring. PPM
(605.2 mg, 2.4 mmol) in 25 ml acetonitrile was added slowly from a
dropping funnel in 1 h. The reaction was stirred at 80 ꢁC for 8 h. The
solvent was removed in vacuum and residue was dissolved in
chloroform. The organic layer was washed with water (3 ꢃ 50 ml),
dried with sodium sulfate and concentrated to give oil liquid after
column chromatography on silica gel, dark yellow powder PPMdpa
was obtained. Yield: 650 mg (66%); elemental analysis calcd (%) for
The cytotoxicity assay was in one kind of HepG-2 cells. Cells
were cultured in RMPI 1640 medium containing 4.8 g/L of Hepes,
2.2 g/L NaHCO3 and supplemented ith penicillin/streptomycin
(1000 units/ml), and 10% calf serum. HepG-2 cells were cultured in
DMEM medium containing 10% fetal bovine serum. All cells were
grown at 37 ꢁC in a humidified atmosphere in the presence of 5%
CO2. HepG-2 cells were seeded at a density of 4 ꢃ 104 cells/mL into
sterile 96-well plates and grown in 5% CO2 at 37 ꢁC. Test compounds
were dissolved in DMSO and diluted with culture media. After 24 h,
compounds were added, treated for 48 h. Cell viability was deter-
mined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenpyltetra-
zolium bromide (MTT) assay by measuring the absorbance at
570 nm with ELISA reader. IC50 data are calculated by the software
provided by Nanjing University. All experiments were repeated at
least three times.
C
26H26N4O: C, 76.07; H, 6.38; N, 13.65. Found: C, 75.76; H, 6.15; N,
13.87. IR (KBr,
n
/cmꢀ1):
n
¼ 3251 (]NH), 1596 (C]O), 761 (CH,
pyridine). UVevis (MeOH/nm) (ε ꢃ 10ꢀ4/dm3 molꢀ1 cmꢀ1): 206
(ε ¼ 2.61), 248 (ε ¼ 1.90), 323 (ε ¼ 2.59). 1H NMR (CDCl3, 400 MHz,
25 ꢁC, TMS),
d
¼ 9.18 (s, 1H; pyrrole, NeH); 8.54 (d, J ¼ 4.8 Hz, 2H;
PyH); 7.70 (td, J ¼ 8.0 Hz, 1.6 Hz, 2H; BzH); 7.60 (t, J ¼ 8.0 Hz, 4H;
PyH); 7.51 (d, J ¼ 8.0 Hz, 2H; BzH); 7.17 (dd, 3J ¼ 6.8 Hz, 5.2 Hz, 2H;
PyH); 5.87 (d, J ¼ 2.4 Hz, 1H; pyrrole-H); 3.84 (s, 4H; CH2e); 3.77 (s,
2H; eCH2e); 2.3 (s, 3H; eCH3); 1.95 (s, 3H; eCH3). ES-MS(ESIþ,
MeOH): calcd for [C26H27N4O]þ: 411.15, found 411.36.
4.6. Western blot analysis on expression level of HIF-1
cells
a in HepG-2
Nuclear protein extracts were prepared from 100 mm dishes,
except for experiments performed on 60 mm gas permeable dishes
(Greiner Bio One, Frickenhausen, Germany), protein was isolated to
perform Western analysis on Mini-Proten Tetra System (Molecular
Devices, Bio-RAD). A monoclonal mouse antibody for human HIF-
4.10. Synthesis of [(PPMdpa)2Mn2(m-Cl)2Cl2]2 (Mn1)
MnCl2$4H2O (150 mg, 0.757 mmol) in ethanol (20 mL) was
mixed with PPMdpa (250 mg, 0.609 mmol). The mixture was
heated to 70 ꢁC for 2 h, and then it was cooled to room temperature.
Diethyl ether (20.0 mL) was added to precipitate the products. After
filtration, a light yellow solid was obtained. Yield: 251 mg (77%).
Elemental analysis calcd. (%) for C26H26N4OMnCl2: C, 58.21; H, 4.85;
1a (2015-1, Epitomics) and a monoclonal mouse GAPDH Antibody
(AP0063, Bioworld) were used. Horseradish peroxidase-conjugated
goat anti-rabbit IgG-HRP: (BS13278, Bioworld) was used as sec-
ondary antibodies. Image was performed using ChemiDoc
XRS þ System (Bio-RAD).
N, 10.45. Found: C, 58.03; H, 4.95; N, 10.83. IR (KBr,n
/cmꢀ1):
n
¼ 3239 (]NH), 1610 (C]O), 762 (CH, pyridine). UVevis(MeOH/
nm) (ε ꢃ 10ꢀ4/dm3 molꢀ1 cmꢀ1): 208 (ε ¼ 2.10), 257 (ε ¼ 1.43), 321
4.7. Chemicals and starting materials
(ε ¼ 1.57).
Bis(pyridine-2-ylmethyl)amine (dpa, purity 99%), Tris (tris(hy-
droxymethyl)amino-methane) (tris), LDH-A, NADH, pyruvate, 4-
(chloromethyl)benzoyl chloride and 4-dimethyl-1H-pyrrole were
purchased from SigmaeAldrich. All chemicals used for synthesis
were of reagent grade and used without further purification
(Sinopharm Chemical Reagent Co. Ltd., China). Water was purified
with a Millipore Milli-Q system. Column chromatography was
carried out using silica FCP (200e300 mesh).
4.11. Synthesis of [(PPMdpa)2Mn2(m-Ac)2(Ac)2] (Mn2)
A solution of PPMdpa (84.4 mg, 0.20 mmol) in acetonitrile
(10 mL) was mixed with MnAc2$6H2O (50 mg, 0.2 mmol), and then
the mixture was refluxed at 80 ꢁC for 2 h. After the solution was
cooled to room temperature, an excess amount of diethyl ether was
added, and the obtained yellow brown precipitate was dried under
vacuum. Yield: 99 mg (85%). elemental analysis calcd. (%) for
C
30H32N4O5Mn: C, 61.75; H, 5.49; N, 9.61. Found: C, 61.86; H, 5.45;
4.8. Synthesis of (4-(chloromethyl)phenyl)(3,5-dimethyl-1H-
pyrrol-2-yl)methanone (PPM)
N, 9.87. IR(KBr,
n
/cmꢀ1):
n
¼ 3264 (¼NH), 1606 (C]O), 765 (CH,
pyridine). UVevis(MeOH/nm) (ε ꢃ 10ꢀ4/dm3 molꢀ1 cmꢀ1): 206
(ε ¼ 2.02), 256 (ε ¼ 1.20), 321 (ε ¼ 1.33).
4-(Chloromethyl)benzoyl chloride (1.5 g, 7.9 mmol) and triethyl
amine (1.061 g, 10.51 mmol) were dissolved in dichloromethane
(40 mL) under nitrogen at 0 ꢁC, then 2,4-Dimethyl-1H-pyrrole
(523 mg, 25 mmol) was added. After that, the mixture was stirred
for 6 h at room temperature; the solution was washed with 1 M
NaCl (20 mL) solution, dried with sodium sulfate and concentrated
to give oil liquid. After column chromatography on silica gel, light
yellow powder 4-(Chloromethyl)phenyl)(3,5-dimethyl-1H-pyrrol-
2-yl)methanone was obtained. Yield: 668 mg (70%); elemental
analysis calcd (%) for C14H14ClNO: C, 67.88; H, 5.7; N, 5.65. Found: C,
Acknowledgments
Financial support from National Natural Science Foundation of
China (20971059, 21271090) and Coordination Chemistry State key
Laboratory Foundation of Nanjing University.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.035.
67.58; H, 5.32; N, 5.85; IR (KBr,
n
/cmꢀ1):
n
¼ 3230 (]NH), 1611 (C]

J.-J. Xue et al. / European Journal of Medicinal Chemistry 80 (2014) 1e7
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