Design, synthesis and molecular modeling of novel N-acylhydrazone derivatives as pyruvate dehydrogenase complex E1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.11.051

As potential inhibitors of pyruvate dehydrogenase complex E1 (PDHc-E1), a series of 19 1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-methyl-N′- (substituent)benzylidene-1H-1,2,3-triazole-4-carbohydrazide 4 has been synthesized and tested for their PDHc-E1 inhibitory activity in vitro. Some of these compounds such as 4a, 4g, 4l, 4o, 4p, and 4q were demonstrated to be effective inhibitors by the bioassay of Escherichia coli PDHc-E1. SAR analysis indicated that the PDHc-E1 inhibitory activity could be further enhanced by optimizing the substituted groups in the parent compound. Molecular modeling study with compound 4o as a model was performed to evaluate docking. The results of modeling study suggested a probable inhibition mechanism.

Full text of DOI:10.1016/j.bmc.2013.11.051

Bioorganic & Medicinal Chemistry 22 (2014) 89–94
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and molecular modeling of novel N-acylhydrazone
derivatives as pyruvate dehydrogenase complex E1 inhibitors
⇑
⇑
Jun-Bo He , Ling-Ling Feng , Jing Li, Rui-Juan Tao, Yan-Liang Ren, Jian Wan , Hong-Wu He
Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
As potential inhibitors of pyruvate dehydrogenase complex E1 (PDHc-E1), a series of 19 1-((4-amino-2-
methylpyrimidin-5-yl)methyl)-5-methyl-N0-(substituent)benzylidene-1H-1,2,3-triazole-4-carbohyd-
razide 4 has been synthesized and tested for their PDHc-E1 inhibitory activity in vitro. Some of these
compounds such as 4a, 4g, 4l, 4o, 4p, and 4q were demonstrated to be effective inhibitors by the bioassay
of Escherichia coli PDHc-E1. SAR analysis indicated that the PDHc-E1 inhibitory activity could be further
enhanced by optimizing the substituted groups in the parent compound. Molecular modeling study with
compound 4o as a model was performed to evaluate docking. The results of modeling study suggested a
probable inhibition mechanism.
Received 23 October 2013
Revised 29 November 2013
Accepted 30 November 2013
Available online 8 December 2013
Keywords:
PDHc-E1 inhibitors
N-Acylhydrazone derivatives
Synthesis
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Molecular modeling
1. Introduction
I).16,17 Compound I was designed based on the characteristics of
active site occupied by ThDP in E. coli PDHc-E1. These results
The pyruvate dehydrogenase complex (PDHc) is the key regula-
tory site in cellular metabolism, in which it catalyzes the irrevers-
ible oxidative of pyruvate to acyl CoA.1 Therefore, it has been
reported as a promising target for antimicrobials,2 agrochemicals,3
and age-related diseases.4 The pyruvate dehydrogenase complex is
comprised of three different enzymes and a number of cofactors.
Pyruvate dehydrogenase complex E1 component (PDHc-E1, EC
1.2.4.1) is the initial member of PDHc, which catalyzes the first
step of multistep process. Accordingly, blocking the activity of
PDHc-E1 will inactivate the PDHc.
There had been some reports about synthesis and inhibition of
PDHc-E1 inhibitors as analogs of substrate (pyruvate).5–9 Cur-
rently, some compounds as analogs of PDHc-E1 cofactor, thiamine
diphosphate (ThDP), had been chemically synthesized and also
reported as effective inhibitors against Escherichia coli (E. coli)
PDHc-E1 in vitro (Fig. 1), but no reports about the study on their
antifungal or antibacterial activities in vivo.10–13 We expected to
find a potential fungicide or bactericide by designing inhibitor
against PHDc-E1 in microorganisms. On the basis of our study to
design plant PHDc-E1 inhibitors as practical herbicide,14,15 we
made an attempt to design novel PHDc-E1 inhibitors as potential
fungicide or bactericide. We recently had got preliminary progress
for finding an effective E. coli PDHc-E1 inhibitor I with antifungal
activity through structure-based rational design (Fig. 1, compound
encouraged us to further design and study E. coli PDHc-E1
inhibitors.
In order to find additional PDHc-E1 inhibitors, structurally
diversified inhibitors should be investigated. In the present work,
the synthesis strategy focused on the N-acylhydrazone moiety
(R1CONHN@CHR2) scaffold due to its well-known biological
activities.18–21 N-Acylhydrazone moiety was introduced into the
compound I to form a new structural class 4 as potential E. coli
PDHc-E1 inhibitors (Fig. 2). Different from the study on PHDc-E1
inhibitors reported in literatures, an integration study including
structure-based rational design by molecular docking, synthesis,
and bioassay for PDHc-E1 inhibition were carried out in this work.
Considering the binding site of diphosphate of ThDP in the active
site of E. coli PDHc-E1, various chemical groups as Ar were incorpo-
rated into the parent compounds 4 to investigate their preliminary
structural–activity relationship.
Herein, we describe the synthesis of nineteen 1-((4-amino-2-
methylpyrimidin-5-yl)methyl)-5-methyl-N0-(substituent)benzyli-
dene-1H-1,2,3-triazole-4-carbohydrazides 4a–s. These 19 title
compounds were tested for their inhibition activity against E. coli
PDHc-E1. Interaction between mode of inhibitor and target
PDHc-E1 was also explored by molecular docking study to identify
the critical binding sites of the target PDHc-E1.
2. Chemistry
⇑
Corresponding authors. Tel./fax: +86 (0)27 67867960.
E-mail addresses: jianwan@mail.ccnu.edu.cn (J. Wan), he1208@mail.ccnu.edu.cn
(H.-W. He).
The synthetic strategy followed for the preparation of
the target compounds (4a–s) is depicted in Scheme 1. The
These authors equally contributed to this work.
0968-0896/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.051

90
J.-B. He et al. / Bioorg. Med. Chem. 22 (2014) 89–94
NH2
N
NH2
NH2
N
CH3
S
CH3
NH2
N
CH3
H
3-
3-
3-
NO2
OP O6
OP2O6
OP2O6
2
O
N
N
N
N
N
N
N
N
N
S
S
N
H3C
H3C
N
H3C
H3C
S
O
ThTDP
tetrahydro-ThDP
ThTTDP
I
Figure 1. Structures of known PDHc-E1 inhibitors.
with a variety of commercially available aromatic aldehydes in eth-
anol at reflux.
NH2
N
NH2
N
CH3
N
O
Ar
N
N
N
N
N
N
The structural assignments to new compounds 4a–s were based
on their elemental analysis and spectral data (1H, 13C NMR, and
MS). The 1H NMR spectra of all the compounds showed two singlet
for three (2CH3) protons at d (2.31–2.33, and 2.26–2.58, respec-
tively), another singlet for two (CH2) protons at d (5.40–5.43), a
singlet for two (NH2) protons at d (6.88–6.91), and a singlet of
imine (CH@N) proton at d (8.42–9.02). A detailed analysis of the
1H NMR spectra of these new compounds 4a–s showed only one
N–H signal, which was deduced as the (E)-diastereomer.23 13C
NMR spectra of all these compounds were taken in DMSO-d6 and
the signal obtained further confirmed the proposed structures.
The mass spectrum of all the target compounds is in agreement
with the molecular formula. The spectral values for all the com-
pounds and C, H, N analyses are given in the experimental section.
NO2
N
HN
O
N
H3C
H3C
Figure 2. Design of the new N-acylhydrazone derivatives 4.
Table 1
Structures and IC50 values against E. coli PDHc-E1 of compounds 4a–s
NH2
CH3
O
Ar
N
N
N
N
HN
N
H3C
N
Compd
Ar
IC50 (lM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4-Cl–Ph
2-Cl–Ph
2,4-2Cl–Ph
2,3-2Cl–Ph
3,4-2Cl–Ph
2-Br–Ph
3-NO2-4-Cl–Ph
4-CH3–Ph
4-F–Ph
3-CH3–Ph
3,4-OCH2O–Ph
4-Br–Ph
2.35 2.35
13.64 0.38
NTa
3. Results and discussion
NTa
NTa
3.1. In vitro inhibition of E. coli PDHc E1
53.83 3.63
1.71 0.08
50.30 2.50
8.00 0.48
20.88 1.41
14.46 1.02
1.89 0.38
110.40 2.36
11.64 1.35
0.65 0.10
3.43 0.52
1.92 0.54
23.35 1.77
22.76 1.27
All the synthesized compounds 4a–s were subjected to be eval-
uated for their inhibitory activity against PDHc-E1 from E. coli. The
IC50 values are summarized in Table 1. It was observed that most of
the compounds showed moderate to potent inhibitory activity
H–Ph
with IC50 values in the range of 14.46–0.65
was the most potent inhibitor in this series with the activity of
IC50 at 0.65 0.10 M, followed by 4g, 4l, 4q, 4a, and 4p.
lM. Compound 4o
2-OH–Ph
4-NO2–Ph
4-OH–Ph
4-CF3–Ph
3-OH–Ph
2-Pyridine
l
To explore the structure–activity relationship (SAR) of these
new N-acylhydrazone compounds, the modifications are focused
on the right terminal Ar, in which the benzene ring locates in the
binding site of diphosphate of ThDP at the active site of PDHc-E1.
Compound 4m with unsubstituted benzene ring as Ar showed
4s
a
NT = not tested.
the lowest activity (IC50 = 110.40 2.36 lM). After replacing the
5-azidomethyl-2-methylpyrimidine-4-ylamine
1
was prepared
benzene ring with a 2-pyridine ring as Ar led to compound 4s,
we noticed a fivefold increase in 4s’s inhibitory activity. This sug-
gested that the nitrogen atom on pyridine moiety in the structure
of 4s may interact with the binding site of the receptor. We also
noticed that the introduction of a variety of substituent groups
would increase the inhibitory activity against E. coli PDHc-E1. In
this case, compounds with electron-withdrawing groups on the
benzene ring as Ar seem to be more effective than these
readily from thiamine hydrochloride according to literature.22 It
was converted into compound 2 with ethyl acetoacetate in the
presence of potassium carbonate, using DMF as solvent. The carbo-
hydrazide 3 was easily synthesized via the hydrazinolysis of the
ethoxycarbonyl group by treatment with hydrazine hydrate in
refluxing ethanol. Finally, the new N-acylhydrazone compounds
(4a–s) were prepared in good yields by condensing compound 3
NH2
N
NH2
CH3
NH2
CH3
S
c
a
b
N
N
N
N
N
N
N3
CO2Et
OH
N
H3C
H3C
N
H3C
N
2
1
NH2
CH3
NH2
CH3
O
d
N
N
N
Ar
N
N
N
CONHNH2
N
N
HN
N
H3C
N
H3C
N
4
3
Scheme 1. Reagents and conditions (a) NaN3, Na2SO3, H2O, 60–65 °C, 6 h, 63%; (b) ethyl acetoacetate, DMF, 80 °C, 8 h, 70%; (c) N2H4ꢀH2O, EtOH, reflux, 5 h, 85%; (d) Ar-CHO,
EtOH, reflux, 5 h, 29–96%.

J.-B. He et al. / Bioorg. Med. Chem. 22 (2014) 89–94
91
compounds with electron-donating alkyl counterparts as Ar. The
inhibitory activity is also definitely related to the position of sub-
stituent groups at the benzene ring. Among the active compounds
(4n, 4p, and 4r) with a hydroxyl group at the benzene ring, the best
compound was para-OH (4p), followed by ortho-OH (4n), and
meta-OH (4r). When substituted with the halogen groups, the
activity sequence is para-Cl (4a) > ortho-Cl (4b), para-Br
(4l) > ortho-Br (4f). These results suggest that the 4-position group
on the benzene ring is favorable for the PDHc-E1 inhibitory activ-
ity. When comparing the PDHc-E1 inhibitory activity of com-
pounds 4a, 4i, and 4l, the activity sequence is Br > Cl > F, which
indicates the larger substituent is better. Base on this SAR analyses,
we modified compound 4 by introducing a larger electron-with-
nitrogen atom of the imine group of the N-acylhydrazone moi-
ety establishes a hydrogen bond with His106.
These docking results provided us a reasonable explanation for
why compound 4o had the highest PDHc-E1 inhibitory activity and
some hints for future optimization synthetic directions.
4. Conclusion
We have designed and synthesized a series of 19 1-((4-amino-2-
methylpyrimidin-5-yl)methyl)-5-methyl-N0-(substituent)benzyli-
dene-1H-1,2,3-triazole-4-carbohydrazide as E. coli PDHc-E1
inhibitors. Among the group of 19 compounds, compounds 4a, 4g,
4l, 4o, 4p, and 4q were found to be very effective inhibitors of E. coli
drawing groups such as nitryl (compound 4o, IC50 = 0.65 0.10
lM)
PDHc-E1, with IC50 values ranging from 0.65 to 3.43 lM. Compound
and trifluoromethyl (compound 4q, IC50 = 1.92 0.54 M) groups
l
4o, with 4-NO2 in the benzene ring as Ar, was the best inhibitor of
E. coli PDHc-E1 among the group. The molecular docking results re-
vealed that the nitro group on compound 4o not only could form two
strong hydrogen bonds with Gly231 and Glu232, but also could form
acoordinate-bondwiththeMg2+,whichinturnseemstobeimportant
for enhancing its inhibitory potency. The SAR analyses and modeling
study by molecular docking suggested a probable inhibition mecha-
nism. These results provided us important hints for future optimiza-
tion to design even better PDHc-E1 inhibitors.
on the 4-position of benzene ring as Ar. As we expected, inhibitory
activity of both compounds 4o and 4q against E. coli PDHc-E1were
greatly increased.
3.2. Molecular docking studies
To explore the interaction mode of N-acylhydrazone com-
pounds with the active site of PDHc-E1, several molecular docking
simulation studies were carried out by using the SURFLEX module
of SYBYL package.16 Based on the in vitro inhibition results, we se-
lected compound 4o, which was found to be best PDHc-E1 inhibi-
5. Experimental
5.1. Chemistry
tor (IC50 = 0.65 0.10 lM) at our present work, as ligand example.
As shown in Figure 3, compound 4o is bound in the active
site of PDHc-E1, which had been reported as the ThDP-binding
pocket, with the ‘V’ conformation.16 On the right side of the
‘V’ conformation, it was observed that the pyrimidine ring of
Meltingpoints(mp)weremeasuredonanelectrothermalmelting
pointapparatusandwereuncorrected.1HNMRspectrawererecorded
in DMSO-d6 solution on a Varian Mercury-Plus 600 spectrometer at
600 MHz, 13C NMR spectra were recorded in DMSO-d6 solution on a
Varian Mercury-Plus 400 spectrometer at 100 MHz and chemical
shiftswererecordedinpartspermillion(ppm)withTMSastheinter-
nalreference.Massspectra(MS)wererunonaQTRAPLC/MS/MSsys-
tem(API2000;AppliedBiosystems,FosterCity,CA,USA)oraTraceMS
2000 organic mass spectrometry, and signals were given in m/z. Ele-
mentalanalyses(EA)weremeasuredonaVarioELIIICHNSOelemental
analyzer. Unless otherwise noted, reagents were purchased from
commercialsuppliersandusedwithoutfurtherpurification.Interme-
diate 1 was synthesized according to the existing methods.22
compound 4o displayed a strong
p–p stacking with the side
chain ring of Phe602. Also observed was that, three hydrogen
bonds participate in binding the aminopyrimidine ring. The
4-amino group of the pyrimidine ring forms a hydrogen bond
with the main chain oxygen of Val192. Other two key hydrogen
bonding interaction are made by the N10 and N20 of the pyrim-
idine ring with the residues Glu571 and Met194, respectively.
On the middle of the ‘V’ conformation, the triazole in compound
4o forms two hydrogen bond with the residues Glu522 and
His640. It should be noted that the binding modes of the right
and middle of the ‘V’ conformation were in perfect agreement
with the results in recently publication.16 However, on the left
side of the ‘V’ conformation, the docking results indicate that
the nitryl group on benzene ring not only can form two strong
hydrogen bonds with Gly231 and Glu232, but also coordinate
with the Mg2+ in the active site. It should be noticed that the
5.2. Preparation of ethyl 1-((4-amino-2-methylpyrimidin-5-
yl)methyl)-5-methyl-1H-1,2,3-triazole-4-carboxylate (2)
A
solution of 5-azidomethyl-2-methylpyrimidine-4-ylamine
(4.92 g, 30 mmol), ethyl acetoacetate (7.80 g, 60 mmol) and
K2CO3 (7.28 g, 60 mmol) in DMF (60 mL) was stirred at 80 °C for
8 h. After this, the reaction mixture was poured into cold water
and the resulting precipitate was filtered out affording compound
2 (5.79 g, 70%) as a white solid. Mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 1.27 (t, 3H, J = 7.2 Hz, CH3), 2.32 (s, 3H, CH3), 2.48 (s,
3H, CH3), 4.27 (q, 2H, J = 7.2 Hz, CH2), 5.36 (s, 2H, CH2), 6.85 (s,
2H, NH2), 7.80 (s, 1H, pyrimidine CH).
5.3. Preparation of 1-((4-amino-2-methylpyrimidin-5-
yl)methyl)-5-methyl-1H-1,2,3-triazole-4-carbohydrazide (3)
To a solution of compound 2 (5.52 g, 20 mmol) in ethanol
(80 mL) was added 20 mL of hydrazine monohydrate (80%). The
reaction mixture was maintained under reflux for 5 h. The reaction
mixture was then concentrated under reduced pressure and the
resulting solid was collected by filtration, washed with cold water
and dried to give the desired compound 3 (4.45 g, 85%) as a white
solid. Mp >260 °C; 1H NMR (DMSO-d6, 600 MHz): d 2.30 (s, 3H,
Figure 3. Optimal binding model for compound 4o into active site of PDHc-E1 from
E. coli docked by SURFLEX module, ligand and some key residues are shown in stick,
hydrogen bonds are shown in dashed lines.

92
J.-B. He et al. / Bioorg. Med. Chem. 22 (2014) 89–94
CH3), 2.47 (s, 3H, CH3), 4.41 (s, 2H, NH2), 5.35 (s, 2H, CH2), 6.85 (s,
2H, NH2), 7.78 (s, 1H, pyrimidine CH), 9.59 (s, 1H, NH).
5.4.5. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(3,4-
dichlorobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4e)
5.4. General procedure for preparation of 1-((4-amino-2-
methylpyrimidin-5-yl)methyl)-N-(substituented)benzylidene-
5-methyl-1H-1,2,3-triazole-4-carbohydrazide derivatives (4a–s)
White solid, yield: 76%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 2.58 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.88 (s, 2H, NH2), 7.68 (d, 1H, J = 7.8 Hz, Ar-H), 7.72 (d, 1H,
J = 8.4 Hz, Ar-H), 7.84 (s, 1H, pyrimidine CH), 7.91 (s, 1H, Ar-H),
8.51 (s, 1H, CH@N), 12.23 (s, 1H, NH); 13C NMR (DMSO-d6,
100 MHz): d 8.50, 24.48, 45.04, 107.47, 126.96, 128.35, 131.11,
131.74, 132.19, 135.29, 137.31, 137.85, 145.24, 153.24, 157.50,
161.89, 165.86; ESI-MS m/z: 419 (M+1)+; Anal. Calcd for C17H16Cl2-
N8O (%): C, 48.70; H, 3.85; N, 26.73. Found: C, 48.36; H, 3.72; N,
26.54.
To a solution of compound 3 (1 mmol) in ethanol (10 mL) was
added appropriate aromatic aldehyde (1.2 equiv). The reaction
mixture was stirred for 3 h at reflux, and then the mixture was
poured into cold water and the resulting precipitate was collected
by filtration, washed with CH2Cl2 (10 mL), and dried in the atmo-
spheric pressure to give the desired compounds 4a–s.
5.4.1. ((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(4-
chlorobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4a)
5.4.6. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(2-
bromobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4f)
White solid, yield: 76%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.88 (s, 2H, NH2), 7.52–7.53 (d, 2H, J = 8.4 Hz, Ar-H), 7.71–7.72 (d,
2H, J = 8.4 Hz, Ar-H), 7.84 (s, 1H, pyrimidine CH), 8.53 (s, 1H,
CH@N); 13C NMR (DMSO-d6, 100 MHz): d 8.46, 24.92, 45.10,
107.30, 128.67, 128.94, 133.39, 134.45, 137.39, 137.65, 146.59,
154.52, 157.38, 161.69, 166.45; ESI-MS m/z: 385 (M+1)+; Anal.
Calcd for C17H17ClN8O (%): C, 53.06; H, 4.45; N, 29.12. Found: C,
52.89; H, 4.76; N, 28.83.
White solid, yield: 77%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.88 (s, 2H, NH2), 7.36 (t, 1H, J = 7.8 Hz, Ar-H), 7.46–7.48 (t, 1H,
J = 7.2 Hz, Ar-H), 7.68 (d, 1H, J = 8.4 Hz, Ar-H), 7.84 (s, 1H, pyrimi-
dine CH), 7.98 (d, 1H, J = 7.8 Hz, Ar-H), 8.92 (s, 1H, CH = N), 12.39
(s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d 8.49, 24.50, 45.00,
107.47, 123.63, 127.24, 128.06, 131.66, 133.33, 137.38, 137.80,
146.36, 153.26, 157.53, 161.88, 165.87; ESI-MS m/z: 429 (M+1)+;
Anal. Calcd for C17H17BrN8O (%): C, 47.56; H, 3.99; N, 26.10. Found:
C, 47.89; H, 4.12; N, 25.69.
5.4.2. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(2-
chlorobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4b)
5.4.7. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(4-
chloro-3-nitrobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4g)
White solid, yield: 77%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.58 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.88 (s, 2H, NH2), 7.44–7.46 (m, 2H, Ar-H), 7.52 (t, 1H, J = 7.2 Hz,
Ar-H), 7.84 (s, 1H, pyrimidine CH), 8.01 (d, 1H, J = 7.2 Hz, Ar-H),
8.98 (s, 1H, CH = N); 13C NMR (DMSO-d6, 100 MHz): d 8.50, 24.73,
45.08, 107.40, 126.86, 127.55, 129.93, 131.40, 131.83, 133.31,
137.37, 137.80, 144.04, 153.93, 157.53, 161.79, 166.19; ESI-MS
m/z: 385 (M+1)+; Anal. Calcd for C17H17ClN8O (%): C, 53.06; H,
4.45; N, 29.12. Found: C, 52.91; H, 4.56; N, 28.76.
White solid, yield: 81%, mp 208–209 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.41 (s, 2H, CH2),
6.89 (s, 2H, NH2), 7.84 (d, 1H, J = 9.0 Hz, Ar-H), 7.84 (s, 1H, pyrim-
idine CH), 7.99 (d, 1H, J = 7.8 Hz, Ar-H), 8.34 (s, 1H, Ar-H), 8.56 (s,
1H, CH@N), 12.35 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d
8.48, 25.22, 45.20, 107.15, 123.48, 125.78, 131.58, 132.24, 135.11,
137.24, 137.90, 144.45, 147.84, 155.48, 157.57, 161.58, 166.91;
ESI-MS m/z: 430 (M+1)+; Anal. Calcd for C17H16ClN9O3 (%): C,
47.50; H, 3.75; N, 29.33. Found: C, 47.50; H, 4.06; N, 29.66.
5.4.3. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(2,4-
dichlorobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4c)
5.4.8. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-5-methyl-
N0-(4-methylbenzylidene)-1H-1,2,3-triazole-4-carbohydrazide
(4h)
White solid, yield: 62%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.58 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.88 (s, 2H, NH2), 7.53 (d, 1H, J = 8.4 Hz, Ar-H), 7.72 (s, 1H, Ar-H),
7.84 (s, 1H, pyrimidine CH), 7.99 (d, 1H, J = 8.4 Hz, Ar-H), 8.94 (s,
1H, CH@N), 12.40 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d
8.52, 24.04, 44.96, 107.64, 128.02, 129.36, 130.95, 132.29, 133.95,
134.99, 137.29, 137.96, 142.95, 151.95, 157.51, 162.07, 165.26;
ESI-MS m/z: 419 (M+1)+; Anal. Calcd for C17H16Cl2N8O (%): C,
48.70; H, 3.85; N, 26.73. Found: C, 48.75; H, 3.90; N, 26.40.
White solid, yield: 69%, mp 249–251 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.56 (s, 3H, CH3),
5.40 (s, 2H, CH2), 6.89 (s, 2H, NH2), 7.27 (d, 2H, J = 7.8 Hz, Ar-H),
7.58 (d, 2H, J = 7.8 Hz, Ar-H), 7.83 (s, 1H, pyrimidine CH), 8.48 (s,
1H, CH@N), 11.96 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d
8.45, 21.03, 25.24, 45.17, 107.21, 127.05, 129.44, 131.75, 137.45,
137.53, 139.82, 147.98, 155.46, 157.28, 161.57, 166.89; ESI-MS
m/z: 365 (M+1)+; Anal. Calcd for C18H20N8O (%): C, 59.33; H,
5.53; N, 30.75. Found: C, 59.70; H, 5.52; N, 31.13.
5.4.4. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(2,3-
dichlorobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4d)
5.4.9. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(4-
fluorobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4i)
White solid, yield: 76%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 2.58 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.88 (s, 2H, NH2), 7.46 (t, 1H, J = 7.8 Hz, Ar-H), 7.72 (d, 1H,
J = 7.2 Hz, Ar-H), 7.84 (s, 1H, pyrimidine CH), 7.97 (d, 1H,
J = 8.4 Hz, Ar-H), 9.02 (s, 1H, CH = N), 12.43 (s, 1H, NH); 13C NMR
White solid, yield: 73%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.56 (s, 3H, CH3), 5.41 (s, 2H, CH2),
6.89 (s, 2H, NH2), 7.29 (d, 2H, J = 7.8 Hz, Ar-H), 7.75 (d, 2H,
J = 7.8 Hz, Ar-H), 7.83 (s, 1H, pyrimidine CH), 8.51 (s, 1H, CH@N),
12.03 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d 8.45, 25.23,
45.15, 101.58, 105.00, 107.21, 108.46, 123.38, 128.87, 137.40,
137.53, 147.72, 148.01, 149.07, 155.45, 157.22, 161.57, 166.89;
ESI-MS m/z: 369 (M+1)+; Anal. Calcd for C17H17FN8O (%): C,
55.43; H, 4.65; N, 30.42. Found: C, 55.79; H, 5.09; N, 29.90.
(DMSO-d6, 100 MHz):
d 8.56, 22.90, 44.74, 108.07, 125.44,
128.43, 131.10, 131.47, 132.38, 134.28, 137.27, 138.14, 143.82,
148.56, 157.55, 162.54, 163.71; ESI-MS m/z: 419 (M+1)+; Anal.
Calcd for C17H16Cl2N8O (%): C, 48.70; H, 3.85; N, 26.73. Found: C,
48.26; H, 3.80; N, 26.44.

J.-B. He et al. / Bioorg. Med. Chem. 22 (2014) 89–94
93
5.4.10. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-5-methyl-
N0-(3-methylbenzylidene)-1H-1,2,3-triazole-4-carbohydrazide
(4j)
5.4.15. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-5-methyl-
N0-(4-nitrobenzylidene)-1H-1,2,3-triazole-4-carbohydrazide
(4o)
White solid, yield: 93%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.56 (s, 3H, CH3),
5.41 (s, 2H, CH2), 6.89 (s, 2H, NH2), 7.24 (d, 1H, J = 7.8 Hz, Ar-H),
7.33 (d, 2H, J = 7.8 Hz, Ar-H), 7.47 (d, 1H, J = 7.8 Hz, Ar-H), 7.52 (s,
1H, Ar-H), 7.83 (s, 1H, pyrimidine CH), 8.48 (s, 1H, CH@N), 12.01
(s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d 8.46, 20.89, 25.24,
45.17, 107.22, 124.48, 127.39, 128.74, 130.76, 134.42, 137.52,
138.08, 148.03, 155.45, 157.37, 161.58, 166.90; ESI-MS m/z: 365
(M+1)+; Anal. Calcd for C18H20N8O (%): C, 59.33; H, 5.53; N,
30.75. Found: C, 59.67; H, 5.47; N, 30.51.
Light yellow solid, yield: 79%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.33 (s, 3H, CH3), 2.60 (s, 3H, CH3), 5.43 (s, 2H, CH2),
6.90 (s, 2H, NH2), 7.86 (s, 1H, pyrimidine CH), 7.96 (d, 2H,
J = 8.4 Hz, Ar-H), 8.32 (d, 2H, J = 8.4 Hz, Ar-H), 8.65 (s, 1H, CH@N),
12.37 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d 8.50, 25.22,
45.19, 107.15, 124.08, 127.94, 137.24, 137.94, 140.77, 145.42,
147.79, 155.48, 157.59, 161.57, 166.90; MS (EI) (m/z, %): 395.16
(M+, 3.37); Anal. Calcd for C17H17N9O3 (%): C, 51.64; H, 4.33; N,
31.88. Found: C, 51.99; H, 4.33; N, 32.37.
5.4.16. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(4-
hydroxybenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4p)
5.4.11. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-
(benzo[d][1,3]dioxol-5-ylmethylene)-5-methyl-1H-1,2,3-
triazole-4-carbohydrazide (4k)
White solid, yield: 71%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.33 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.83–6.84 (d, 2H, J = 8.4 Hz, Ar-H), 6.88 (s, 2H, NH2), 7.52–7.53 (d,
2H, J = 8.4 Hz, Ar-H), 7.84 (s, 1H, pyrimidine CH), 8.42 (s, 1H,
CH@N), 9.94 (s, 1H, OH); 13C NMR (DMSO-d6, 100 MHz): d 8.50,
25.26, 45.22, 107.32, 115.81, 125.49, 128.97, 137.37, 137.68,
148.40, 155.47, 157.23, 159.47, 161.65, 166.98; MS (EI) (m/z, %):
367.24 (M++1, 2.24), 366.21 (M+, 11.73); Anal. Calcd for
White solid, yield: 29%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.56 (s, 3H, CH3), 5.40 (s, 2H, CH2),
6.08 (s, 2H, OCH2O), 6.88 (s, 2H, NH2), 6.98 (d, 1H, J = 7.8 Hz, Ar-
H), 7.11 (d, 1H, J = 7.8 Hz, Ar-H), 7.27 (s, 1H, Ar-H), 7.83 (s, 1H,
pyrimidine CH), 8.42 (s, 1H, CH@N), 11.92 (s, 1H, NH); 13C NMR
(DMSO-d6, 100 MHz):
d 8.44, 25.23, 45.15, 101.58, 105.00,
107.21, 108.46, 123.37, 128.87, 137.40, 137.53, 147.72,
148.01, 149.07, 155.45, 157.21, 161.57, 166.89; MS (EI) (m/z, %):
395.15 (M++1, 2.26), 394.14 (M+, 22.93); Anal. Calcd for
C17H18N8O2 (%): C, 55.73; H, 4.95; N, 30.58. Found: C, 55.55; H,
5.38; N, 30.26.
C18H20N8O (%): C, 54.82; H, 4.60; N, 28.41. Found: C, 54.60; H,
4.61; N, 28.89.
5.4.17. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-5-methyl-
N0-(4-(trifluoromethyl)benzylidene)-1H-1,2,3-triazole-4-
carbohydrazide (4q)
5.4.12. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(4-
bromobenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4l)
White solid, yield: 96%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.41 (s, 2H, CH2),
6.88 (s, 2H, NH2), 7.81 (d, 2H, J = 7.8 Hz, Ar-H), 7.84 (s, 1H, pyrim-
idine CH), 7.90 (s, 2H, J = 7.8 Hz, Ar-H), 8.60 (s, 1H, CH@N), 12.24 (s,
1H, NH); 13C NMR (DMSO-d6, 100 MHz): d 8.48, 25.22, 45.19,
107.17, 122.78, 125.73, 127.62, 137.32, 137.80, 138.43, 146.15,
155.48, 157.53, 161.58, 166.90; MS (EI) (m/z, %): 418.26 (M+,
4.31); Anal. Calcd for C18H17F3N8O (%): C, 51.67; H, 4.10; N,
26.78. Found: C, 51.55; H, 4.38; N, 27.06.
White solid, yield: 84%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.56 (s, 3H, CH3), 5.40 (s, 2H, CH2),
6.89 (s, 2H, NH2), 7.65 (s, 4H, Ar-H), 7.83 (s, 1H, pyrimidine CH),
8.49 (s, 1H, CH@N), 12.01 (s, 1H, NH); 13C NMR (DMSO-d6,
100 MHz): d 8.47, 25.24, 45.17, 107.19, 123.26, 128.92, 131.87,
133.73, 137.39, 137.65, 146.70, 155.46, 157.40, 161.57, 166.90;
ESI-MS m/z: 431 (M+3)+; Anal. Calcd for C17H17BrN8O (%): C,
47.56; H, 3.99; N, 26.10. Found: C, 47.72; H, 4.46; N, 26.26.
5.4.18. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(3-
hydroxybenzylidene)-5-methyl-1H-1,2,3-triazole-4-
carbohydrazide (4r)
5.4.13. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-
benzylidene-5-methyl-1H-1,2,3-triazole-4-carbohydrazide (4m)
White solid, yield: 54%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 2.56 (s, 3H, CH3), 5.41 (s, 2H, CH2),
6.89 (s, 2H, NH2), 7.44 (t, 3H, J = 8.4 Hz, Ar-H), 7.69 (d, 2H,
J = 7.8 Hz, Ar-H), 7.83 (s, 1H, pyrimidine CH), 8.52 (s, 1H, CH@N),
12.02 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d 8.44, 25.24,
45.14, 107.18, 127.05, 128.82, 130.01, 134.43, 137.46, 147.90,
155.44, 157.34, 161.55, 166.87; ESI-MS m/z: 351 (M+1)+; Anal.
Calcd for C17H18N8O (%): C, 58.27; H, 5.18; N, 31.98. Found: C,
58.35; H, 5.14; N, 32.01.
White solid, yield: 80%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.83 (d, 1H, J = 7.8 Hz, Ar-H), 6.91 (s, 2H, NH2), 7.05 (d, 1H,
J = 7.8 Hz, Ar-H), 7.16 (s, 1H, Ar-H), 7.25 (t, 1H, J = 7.8 Hz, Ar-H),
7.84 (s, 1H, pyrimidine CH), 8.44 (s, 1H, CH@N), 9.66 (s, 1H, OH),
11.98 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz): d 8.48, 25.25,
45.16, 107.21, 112.70, 117.41, 118.77, 129.91, 135.72, 137.49,
148.06, 155.44, 157.32, 157.68, 161.57, 166.89; MS (EI) (m/z, %):
367.16 (M++1, 4.29), 366.21 (M+, 16.29); Anal. Calcd for
C
17H18N8O2 (%): C, 55.73; H, 4.95; N, 30.58. Found: C, 55.91; H,
5.4.14. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-N0-(2-
hydroxybenzylidene)-5-methyl-1H-1,2,3-triazole-4-
5.20; N, 30.77.
carbohydrazide (4n)
5.4.19. 1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-5-methyl-
N0-(pyridin-2-ylmethylene)-1H-1,2,3-triazole-4-carbohydrazide
(4s)
White solid, yield: 67%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.41 (s, 2H, CH2),
6.90 (s, 2H, NH2), 6.91 (t, 2H, J = 8.4 Hz, Ar-H), 7.29 (s, 1H, Ar-H),
7.45 (d, 1H, J = 7.8 Hz, Ar-H), 7.84 (s, 1H, pyrimidine CH), 8.69 (s,
1H, CH@N), 12.03 (s, 2H, NH+OH); 13C NMR (DMSO-d6,
100 MHz): d 8.46, 25.42, 45.18, 107.14, 116.47, 118.58, 119.34,
129.91, 131.32, 137.04, 137.81, 148.96, 155.48, 157.17, 157.56,
161.56, 166.88; ESI-MS m/z: 367 (M+1)+; Anal. Calcd for
White solid, yield: 83%, mp >260 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 2.57 (s, 3H, CH3), 5.42 (s, 2H, CH2),
6.91 (s, 2H, NH2), 7.41 (t, 1H, J = 6.0 Hz, Ar-H), 7.83 (s, 1H, pyrimi-
dine CH), 7.87 (t, 1H, J = 7.8 Hz, Ar-H), 7.94 (d, 1H, J = 7.8 Hz, Ar-H),
8.56 (s, 1H, CH@N), 8.59 (d, 1H, J = 4.2 Hz, Ar-H), 12.36 (s, 1H, NH);
13C NMR (DMSO-d6, 100 MHz): d 8.51, 25.24, 45.23, 107.19, 119.88,
124.33, 136.83, 137.34, 137.89, 148.18, 149.48, 153.48, 155.48,
157.62, 161.59, 166.92; MS (EI) (m/z, %): 351.20 (M+, 10.85); Anal.
C17H18N8O2 (%): C, 55.73; H, 4.95; N, 30.58. Found: C, 55.91; H,
4.94; N, 30.84.

94
J.-B. He et al. / Bioorg. Med. Chem. 22 (2014) 89–94
Calcd for C16H17N9O (%): C, 54.69; H, 4.88; N, 35.88. Found: C,
54.20; H, 4.94; N, 36.17.
to explore the interaction model for the active site of PDHc-E1 with
its ligand. All atoms located within the range of 6.5 Å from any
atom of the cofactor ThDP were selected into the active site, and
the corresponding amino acid residue was, therefore, involved into
the active site if only one of its atoms was selected. Other default
parameters were adopted in the SURFLEX-docking calculations.
5.5. Assay of E. coli PDHc-E1 (in vitro)
The expressing plasmid pMal-C2X-PDHc-E1 was transformed
into E. coli strain TB1 and inoculated in Luria–Bertani (LB) broth
containing 2% glucose and 30 mg/ml ampicillin at 37 °C until
reaching a cell density to A600 of 0.6–0.8. Then cells were induced
with a final concentration of 0.5 mM IPTG for 7 h at 25 °C before
harvesting. Purification of the fusion protein was carried out using
a MBP affinity column attached to an AKTA purifier 10 (UPC-F920,
GE Healthcare Life Sciences). The concentrations of purified pro-
teins were determined by the method of Bradford24 using bovine
serum albumin (Tiangen) as standard. The final purity (>95%) of
the sample was verified by SDS–PAGE and then the purified protein
was stored in 50% (v/v) glycerol at ꢁ20 °C.
All calculations were performed on
a
CCNUGrid-based
computational environment (CCNUGrid website http://www.
202.114.32.71:8090/ccnu/chem/platform.xml).
Acknowledgments
The work was supported in part by the National Basic Research
Program of China (Nos. 2003CB114400 and 2010CB126100), the
National Natural Science Foundation of China (Nos. 20772042
and 21172090) and the PCSIRT (No. IRT0953).
The inhibitory activities of synthesized compounds were mea-
sured by the enzymatic assay. PDHc-E1 activity was assayed by a
modified method of N. Nemeria,11 and measured by monitoring
the reduction of 2,6-DichloroPhenolindophenol (2,6-DCPIP) at
600 nm using a microplate reader (BioTek Synergy2, USA). The to-
References and notes
1. Koike, M.; Reed, L. J.; Carroll, W. R. J. Biol. Chem. 1960, 235, 1924.
2. Birkenstock, T.; Liebeke, M.; Winstel, V.; Krismer, B.; Gekeler, C.; Niemiec, M. J.;
Bisswanger, H.; Lalk, M.; Peschel, A. J. Biol. Chem. 2012, 287, 2887.
3. Baillie, A.; Wright, K.; Wright, B.; Earnshaw, C. Pestic. Biochem. Physiol. 1988, 30,
103.
tal volume of 100
7.2, 2.0 mM sodium pyruvate as substrate, 0.8 mM 2,6-DCPIP,
7.1 M enzyme and different concentration of inhibitors. The reac-
tion mixtures were incubated for 3 min at 37 °C, then different
concentrations of ThDP (ranging from 0 to 200 M) were added
lL reaction mixture contained 50 mM K3PO4, pH
4. Stacpoole, P. W. Aging Cell 2012, 11, 371.
5. Lowe, P. N.; Perham, R. N. Biochemistry 1984, 23, 91.
l
6. Flournoy, D. S.; Frey, P. A. Biochemistry 1989, 28, 9594.
7. Bisswanger, H. Biochem. Biophys. Res. Commun. 1980, 95, 513.
8. Kluger, R.; Pike, D. C. J. Am. Soc. Chem. 1977, 99, 4504.
9. Schoenbrunn-Hanebeck, E.; Laber, B.; Amrhein, N. Biochemistry 1990, 29, 4880.
10. Lowe, P.; Leeper, F. J.; Perham, R. N. Biochemistry 1983, 22, 150.
11. Nemeria, N.; Yan, Y.; Zhang, Z.; Brown, A. M.; Arjunan, P.; Furey, W.; Guest, J. R.;
Jordan, F. J. Biol. Chem. 2001, 276, 45969.
12. Arjunan, P.; Chandrasekhar, K.; Sax, M.; Brunskill, A.; Nemeria, N.; Jordan, F.;
Furey, W. Biochemistry 2004, 43, 2405.
13. Arjunan, P.; Sax, M.; Brunskill, A.; Chandrasekhar, K.; Nemeria, N.; Zhang, S.;
Jordan, F.; Furey, W. J. Biol. Chem. 2006, 281, 15296.
14. He, H. W.; Yuan, J. L.; Peng, H.; Chen, T.; Shen, P.; Wan, S. Q.; Li, Y.; Tan, H. L.;
He, Y. H.; He, J. B.; Li, Y. J. Agric. Food Chem. 2011, 59, 4801.
15. He, H. W.; Peng, H.; Wang, T.; Wang, C. B.; Yuan, J. L.; Chen, T.; He, J. B.; Tan, X.
S. J. Agric. Food Chem. 2013, 61, 2479.
16. Ren, Y. L.; He, J. B.; Feng, L. L.; Liao, X.; Jin, J.; Li, Y. J.; Cao, Y.; Wan, J.; He, H. W.
Bioorg. Med. Chem. 2011, 19, 7501.
17. He, J. B.; Feng, L. L.; Li, J.; Tao, R. J.; Wang, F.; Liao, X.; Sun, Q. S.; Long, Q. W.;
Ren, Y. L.; Wan, J.; He, H. W. Bioorg. Med. Chem. 2012, 20, 1665.
18. Barbosa, V. A.; Formagio, A. S. N.; Savariz, F. C.; Foglio, M. A.; Spindola, H. M.;
Carvalho, J. E.; Meyer, E.; Sarragiotto, M. H. Bioorg. Med. Chem. 2011, 19, 6400.
19. Jordão, A. J.; Ferreira, V. F.; Lima, E. S.; de Souza, M. C. B. V.; Carlos, E. C. L.;
Castro, H. C.; Geraldo, R. B.; Rodrigues, C. R.; Almerida, M. C. B.; Cunha, A. C.
Bioorg. Med. Chem. 2009, 17, 3713.
l
to the initiate reaction. To determine the inhibitor concentration
of synthesized compounds at 50% inhibition (IC50), initial rate data
taken at saturating substrate, fixed effectors, and systematically
varied inhibitor concentrations were fit to Hill equation,
V = V0 ꢁ (V0ꢁV )/((IC50/I)n + 1),25 Where V, V0, and V are the
1
1
velocity, maximum velocity (at I = 0), and the limiting velocity
(at I saturating); n is the Hill coefficient associated with the
inhibitor; and IC50 is the inhibition concentration of synthesized
compounds at 50% inhibition. Each experiment was performed at
least three times. All kinetic data were fit to the growth/sigmoidal
model from origin 7.0 software. One unit of activity is defined as
the amount of 2,6-DCPIP reduced (lmol/min/mg of PDHc-E1).
5.6. Molecular docking
For docking purposes, the crystallographic coordinates of the
PDHc-E1 with bound ThDP from E. coli (PDB code: 1L8A) were ob-
tained from Brookhaven Data Bank. Hydrogen atoms were added
to the structure allowing for appropriate ionization at physiologi-
cal pH. The protonated state of several important residues, such
as His106, His142, Tyr599, Glu751 and His640, were adjusted by
using SYBYL7.3 (Tripos, St. Louis, USA) in favor of forming
reasonable hydrogen bond with the ligand. Molecular docking
analysis was carried out by the SURFLEX module of SYBYL package
20. Romeiro, N. C.; Aguirre, G.; Hernández, P.; González, M.; Cerecetto, H.; Aldana,
I.; Pérez-Silanes, S.; Monge, A.; Barreiro, E. J.; Lima, L. M. Bioorg. Med. Chem.
2009, 17, 641.
21. Tripathi, L.; Singh, R.; Stables, J. P. Eur. J. Med. Chem. 2011, 46, 509.
22. Erixon, K. M.; Dabalos, C. L.; Leeper, F. J. Org. Biomol. Chem. 2008, 6, 3561.
23. Cunha, A. C.; Figueiredo, J. M.; Tributino, J. M.; Miranda, A. L. P.; Castro, H. C.;
Zingali, R. B.; Fraga, C. A. M.; Souza, M. C. B. V.; Ferreira, V. F.; Barreiro, E. J.
Bioorg. Med. Chem. 2003, 11, 2051.
24. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
25. Hines, J. K.; Kruesel, C. E.; Fromm, H. J.; Honzatko, R. B. J. Biol. Chem. 2007, 282,
24697.