The design, synthesis and biological evaluation of novel thiamin diphosphate analog inhibitors against the pyruvate dehydrogenase multienzyme complex E1 from Escherichia coli

Article abstract of DOI:10.1039/c4ob01347f

Pyruvate dehydrogenase multienzyme complex E1 (PDHc E1) is a potential target enzyme when looking for inhibitors to combat microbial disease. In this study, we designed and synthesized a series of novel thiamin diphosphate (ThDP) analogs with triazole rin

Full text of DOI:10.1039/c4ob01347f

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The design, synthesis and biological evaluation
of novel thiamin diphosphate analog inhibitors
against the pyruvate dehydrogenase multienzyme
complex E1 from Escherichia coli†
Cite this: Org. Biomol. Chem., 2014,
12, 8911
Lingling Feng,a Junbo He,a,b Haifeng He,a Lulu Zhao,a Lingfu Deng,c Li Zhang,a
Lin Zhang,a Yanliang Ren,a Jian Wan*a and Hongwu He*a
Pyruvate dehydrogenase multienzyme complex E1 (PDHc E1) is a potential target enzyme when looking
for inhibitors to combat microbial disease. In this study, we designed and synthesized a series of novel
thiamin diphosphate (ThDP) analogs with triazole ring and oxime ether moieties as potential inhibitors of
PDHc E1. Their inhibitory activities against PDHc E1 were examined both in vitro and in vivo. Most of the
tested compounds exhibited moderate inhibitory activities against PDHc E1 (IC50 = 6.1–75.5 μM). The
potent inhibitors 4g, 4h and 4j, had strong inhibitory activities with IC50 values of 6.7, 6.9 and 6.1 μM
against PDHc E1 in vitro and with inhibition rates of 35%, 50% and 33% at 100 μg mL−1 against Gibberella
zeae in vivo, respectively. The binding mode of 4j to PDHc E1 was analyzed by a molecular docking
method. Furthermore, the possible interactions of the important residues of PDHc E1 with compound 4j
were examined by site-directed mutagenesis, enzymatic assays and spectral fluorescence studies. The
theoretical and experimental results are in good agreement and suggest that compound 4j could be used
as a lead compound for further optimization, and may have potential as a new microbicide.
Received 28th June 2014,
Accepted 18th September 2014
DOI: 10.1039/c4ob01347f
www.rsc.org/obc
acetylation of coenzyme A (CoA) to acetyl-CoA, during the
tricarboxylic acid metabolic pathway.5 PDHc contains three
1. Introduction
Pathogenic microbes such as Botrytis cinerea, Gibberella zeae, enzymatic components: pyruvate dehydrogenase (E1), dihydro-
Rhizoctonia solani, and Escherichia coli are hazardous to lipoamide acetyltransferase (E2), and dihydrolipoamide de-
modern agriculture and human health.1,2 Although there are hydrogenase (E3).5 The pyruvate dehydrogenase (PDHc E1)
microbicides (carbendazim, pyrimethanil, and ciprofloxacin) portion has two active sites: the substrate binding site and the
that can be used for these pathogenic microbial infections, the cofactor thiamin diphosphate (ThDP) binding site. PDHc E1
severe side effects and toxicity of these microbicides and the catalyzes the first step of this complex process and is an impor-
increasing resistance to these microbicides have been recorded tant target enzyme when finding inhibitors.6
worldwide and are important factors resulting in treatment
Much effort has been made to design and synthesize inhibi-
failure.3,4 Therefore, it is of general interest to develop a new tors of PDHc E1 including the PDHc E1 substrate analogs and
and efficient microbicide with novel structures or modes of action, cofactor ThDP analogs (Fig. 1).7–12 These ThDP analog inhibi-
or even new drug targets to help overcome microbial disease.
tors, such as thiamin thiazolone diphosphate (ThTDP) and
The pyruvate dehydrogenase multienzyme complex (PDHc) thiamine thiothiazolone diphosphate (ThTTDP), can block the
plays a vital role in cellular metabolism and catalyzes the formation of a covalent adduct between the substrate pyruvate
oxidative decarboxylation of pyruvate, and the subsequent and cofactor ThDP through the C2 atom of the thiazolium ring
in the reaction pathway for PDHc E1.11–15 These ThDP analog
inhibitors exhibit significantly stronger binding affinities for
PDHc E1 than ThDP and have received increasing attention.13
aKey Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education,
However, due to their complex structure with highly charged
pyrophosphate groups, these ThDP analogs exhibit no poten-
tial utility and display poor bioavailability.11–15
The crystal structures of the PDHc E1/ThDP complex and
the PDHc E1/inhibitor (ThTDP) complex from E. coli (PDB ID:
1L8A and 1RP7) have been determined.14,15 These structures
College of Chemistry, Central China Normal University, Wuhan 430079, China.
E-mail: jianwan@mail.ccnu.edu.cn, he1208@mail.ccnu.edu.cn;
Fax: +86-27-67862022, +86-27-67867960; Tel: +86-27-67862022, +86-27-67867960
bCollege of Food Science and Engineering, Wuhan Polytechnic University,
Wuhan 430023, China
cCollege of Life Science, Central China Normal University, Wuhan 430079, China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ob01347f
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site of PDHc E1 and facilitates our design of new ThDP inhibi-
tors targeted to the active site of PDHc E1.14,15 The ThDP con-
sists of three parts: diphosphate group, aminopyrimidine, and
thiazolium moiety, which are bound in a “V” conformation
involving the N-terminal and middle domains of PDHc E1.14
Due to the excellent antibacterial, antifungal, and antimicro-
bial activities of the oxime ether as well as the high charge of
the pyrophosphate group, we expect that the oxime ether
moiety of the new inhibitor (shown in red) can substitute for
the diphosphate group and avoid the high charge of the pyro-
phosphate group (Fig. 2).13,21–24
Fig. 1 Structures of ThDP and the known potential ThDP analog inhibi-
PDHc E1 is initiated by the formation of a covalent adduct
between the substrate and the cofactor through the C2 atom of
the thiazolium ring in the reaction pathway of PDHc E1.14,15
The triazole ring moiety has a surprisingly stable structure
with three adjacent nitrogen atoms, has a wide range of bio-
logical activities, and also plays an important role in bio-conju-
gation.19,20 We expected that the new inhibitor with the
triazole ring moieties would block this site by replacing the
proton on C2 with a nitrogen atom in the triazole ring moi-
eties (shown in blue) of the novel ThDP analog inhibitors
(Fig. 2). The aminopyrimidine ring was maintained for the
proper orientation of the newly designed inhibitors because
the aminopyrimidine ring hydrogen bonds to the active site of
PDHc E1, which is partly responsible for the proper orien-
tation of the cofactor ThDP.14 The structure of the new inhibi-
tor is shown as Fig. 2.
The synthetic route used for compounds 4a–4l is depicted
in Scheme 1. Intermediates 1a–1l were synthesized using
various substituted benzaldehydes or acetophenones and react
with hydroxylamine hydrochloride in the presence of potass-
ium carbonate.25 Compounds 1a–1l were then converted into
intermediates 2a–2l in the presence of 3-bromopropyne using
NaOH as the base.26 The 5-azidomethyl-2-methylpyrimidine-4-
ylamine 3 was readily prepared from thiamine hydrochloride
(ThDP) according to a literature report.27 Finally, the Cu-cata-
lyzed 1,3-dipolar cycloaddition reaction was used to assemble
the oxime ether 4a–4l employing copper(I) iodide-triethyl-
amine (TEA) in THF.28 The oxime-ether compounds are stable
in the solid and solution states. The final compounds 4a–4l
were purified by recrystallization from dimethyl formamide-
water and characterized by 1H NMR, 13C NMR, mass spec-
trometry (MS), and elemental analysis.
tors of PDHc E1.
reveal detailed characterization of the active site of PDHc E1,
and provide a solid basis for understanding the structure and
mechanism. They enable structure-based design of novel
inhibitors against PDHc E1. In our laboratory, we carried out a
systematic study for the design and synthesis of inhibitors
against PDHc E1 as a herbicide (HW02) (Fig. 1).16 The mechan-
ism was predicted with theory.17,18 The herbicide HW02 was
found to be an effective inhibitor against PDHc E1. Docking
studies were employed to shed light on the binding mode of
the interactions.
The triazole ring moiety can be tuned to form powerful
pharmacophores and plays an important role in bio-conju-
gation. Furthermore, it is a useful building block in organic
synthesis because of its wide range of biological activities.19,20
The oxime ether derivatives have attracted recent attention in
medicinal chemistry research due to their excellent antibacter-
ial, antifungal or antimicrobial activities.21–24 These results
encouraged us to explore and design novel ThDP analog
inhibitors against PDHc E1 that possess microbicide activity
by introducing the triazole ring and oxime ether moieties.
Here we report the design and synthesis of a series of new
compounds incorporating the active triazole ring and oxime
ether pharmacophores and evaluate their inhibitory activities
against PDHc E1 from E. coli. Furthermore, the mode of inter-
action of the important residues of PDHc E1 with the hit com-
pound was examined by molecular docking experiments, site-
directed mutagenesis, and enzymatic assays.
2. Results and discussion
2.1 Design and synthesis of new ThDP analog inhibitors (4a–4l) 2.2 Inhibitory activities of the new ThDP analog inhibitors
(4a–4l) against E. coli PDHc E1
The crystal structures of the PDHc E1/ThDP complex and the
PDHc E1/inhibitor (ThTDP) complex from E. coli (PDB ID: The synthesized ThDP analog inhibitors (4a–4l) were evalu-
1L8A and 1RP7) provides detailed characterization of the active ated for their inhibitory activities (half maximal inhibitory
Fig. 2 Design of novel thiamin diphosphate (ThDP) inhibitors of PDHc E1.
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Scheme 1 Synthetic pathway of novel thiamin diphosphate (ThDP) 4a–4l. Reagents and conditions: (a) NH2OH·HCl, K2CO3, EtOH, reflux, 7 h;
(b) 3-Bromopropyne, NaOH, CH3CN, reflux, 8 h; (c) NaN3, Na2S2O3, H2O, 60–65 °C, 6 h; (d) 2a–2l, CuI, Et3N, THF, rt, 10–15 h.
concentration, IC50) against E. coli PDHc E1 (Table 1). The R2 = 4-Cl (compound 4i) showed a higher inhibitory activity
compounds had moderate inhibitory activities (IC50
=
(IC50 6.9
1.2 μM) compared to ortho- or meta-substituted
6.1–75.5 μM), apart for compound 4b (IC50 > 100 μM). Com- compounds (R1 = CH3, R2 = 2-Cl (compound 4l) or R1 = CH3,
pounds 4h, 4i and 4j proved to be potent inhibitors with IC50 R2 = 3-Cl (compound 4g). This implies that para-substitution is
values of 6.7, 6.9 and 6.1 μM against PDHc E1 in vitro, respect- important for the inhibitory activity of the new oxime ether
ively. Therefore, the inhibitory rates against Gibberella zeae derivatives. In this case, compounds with electron-withdrawing
in vivo of compounds 4h, 4i and 4j were further examined. By groups on the aromatic ring (compounds 4h, 4i, and 4j) were
biological evaluation, compounds 4h, 4i and 4j had inhibition more effective that those bearing the electron-donating
rates of 35%, 50% and 33% at 100 μg mL−1 against Gibberella methoxy group (compound 4k).
zeae in vivo, respectively.
To explore the structure–activity relationships (SAR), we
2.3 Analyses of the interaction between the novel ThDP
first changed the R1 group by using H (compounds 4a–4f) or
analog inhibitors and PDHc E1
CH3 (compounds 4g–4l). The modifications are focused on R1,
Compound 4j was selected for the study of the interaction
mode of the novel ThDP analog inhibitors (4a–4l) with E. coli
PDHc E1, because it exhibited the highest inhibitory activity
(IC50 6.1 0.5 μM) against PDHc E1.
We performed molecular docking studies via site-directed
mutagenesis, enzymatic assays, and fluorescence spectral ana-
lyses. As shown in Fig. 3, compound 4j is bound to the active
site of PDHc E1, which has the ‘V’ conformation.14,15
in which the oxime ether group locates in the active site of
PDHc E1. The inhibitory potencies of the two groups were
obviously different (Table 1). In all categories, the inhibitory
activities of the compounds in which the R1 group is substi-
tuted with methyl (R1 = CH3, compounds 4g–4l) are higher
that those substituted with H (R1 = H, compounds 4a–4f). Sub-
sequently, we fixed the R1 group as methyl (R1 = CH3) and
changed the R2 group. The para-substituted analogs R1 = CH3,
The aminopyrimidine ring of compound 4j has similar
interactions with the amino acid residues to ThDP or ThTDP
in the ‘V’ conformation, i.e., there is strong π–π stacking with
the side chain ring of F602, and there are hydrogen bond inter-
actions between the main chain oxygen of V192 and two key
Table 1 Structures and inhibitory activities (IC50) of novel thiamin
diphosphate (ThDP) analog inhibitors (4a–4l) against E. coli PDHc E1
and inhibition rates (%) of 4h, 4i and 4j at 100 μg mL−1 against Gibberella
zeae
hydrogen atoms. These hydrogen bonds are between the N′ of
1
the pyrimidine ring and the side chain of E571, and the N′ of
2
the pyrimidine ring with the side chain of M194. The hydrogen
bonds and the π–π stacking are responsible for the proper
orientation of aminopyrimidine of the compounds to the
active site of PDHc E1 (Fig. 3). The packing contacts with
M194 are important in stabilizing the cofactor ThDP construc-
tion, as shown in the corresponding I415 in PDC.29 In line
with our docking prediction, the IC50 values of compound 4j
against the M194A mutant (69.9 μM) and V192A (57.8 μM)
were about 11.5-fold and 9.5-fold higher than the value
against the wild-type enzyme (6.1 μM), respectively (Fig. 4).
These results also reveal that compound 4j has stronger hydro-
gen bonding interactions with residues M194 (26.8-fold) and
V192 (29-fold) than ThDP, based on the lower IC50 values for
compound 4j with M194A (69.9 μM) and V192A (57.8 μM). The
Inhibition
rate (%)
Number
Compound
R1
R2
IC50 (μM)
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
H
3-Cl
4-NO2
4-Cl
4-Br
4-OCH3
3-Cl
4-NO2
4-Cl
4-Br
4-OCH3
2-Cl
75.5 0.02
>100
N/A
N/A
N/A
N/A
N/A
N/A
N/A
35
50
33
N/A
N/A
17.8 1.86
59.4 5.75
16.2 1.76
31.4 1.59
57.8 3.16
6.7 0.48
6.9 1.19
6.1 0.48
30.2 3.45
14.9 1.25
9
10
11
12
4j
4k
4l
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Fig. 3 Optimal binding model for compound 4j into the active site of PDHc-E1 from E. coli docked by the SURFLEX module. The ligand and some
key residues are presented as a stick model; hydrogen bonds are shown as dashed lines.
mutagenesis and enzymatic assays showed that the IC50 values
of compound 4j against the Y177A (150.7 3.8 μM) and H640A
(53.9 2.7 μM) mutants are about 39.5-fold and 8.8 higher
than the value against the wild type enzyme (6.1 μM) (Fig. 4).
This suggests that the hydrogen bond interaction between
compound 4j and the Y177 or H640 residue is also an impor-
tant pharmacophoric feature.
Our docking results also showed that the oxime ether group
attached to the aromatic ring in compound 4l not only forms
hydrogen bonds with H106, H142, and N260, but also coordi-
nates with the metal ion (Mg2+) in the active site. Site-directed
mutagenesis and enzymatic assays showed that the IC50 values
Fig. 4 The IC50 values of compound 4j against the wild type (WT) and
mutants of PDHc E1. The substrate is pyruvate acid, and the cofactor is
of compound 4j against the H106 (52.7 2.8 μM) and H142A
ThDP.
(32.1 1.4 μM) mutants are about 8.6 and 5.3-fold higher than
the value against the wild-type enzyme (6.1 μM) (Fig. 4),
Kd for ThDP with M194A is 2.6 μM and 2.0 μM for V192A.
These results suggest that the hydrogen bonding interaction
between compound 4j and M194 or V192 is an important phar-
macophoric feature.
suggesting that H106 and H142 play important roles in the
binding of compound 4j. However, compound 4j has stronger
interactions with H106 (66.7-fold) and similar interactions
with H142 (0.8-fold) than ThDP according to the IC50 values
for compound 4j with H106A (52.7 2.8 μM) and H142A (32.1
1.4 μM) compared to the Kd for ThDP with H106A (0.8
0.02 μM) and H142A (38.1 1.7 μM), respectively. These results
imply that H106 is one of the most important residues
affecting the binding of compound 4j to the active site of
PDHc E1.
It is impossible to understand the theoretically predicted
binding model directly through the enzymatic assay of the
N260A mutant, because the mutant exhibits much less enzy-
matic activity. To further validate the interaction between com-
pound 4j and N260A, the binding constant (Kb) values of
N260A were investigated using fluorescence spectral data. The
Kb value of N260A (149 M−1) is over 14-fold lower than the Kb
value of the wild type enzyme (2108 M−1) (Fig. 5), suggesting
that there is a stronger interaction between N260 and com-
pound 4j than with the wild-type enzyme. These results further
In the middle of the ‘V’ conformation, the thiazolium ring
of ThDP mainly interacts with residues I569 and D521.15
During turnover, the C2 of the thiazolium ring reacts with
pyruvate to form 2-α-lactyl ThDP, which in turn undergoes decar-
boxylation to 2-α-hydroxyethylidene-ThDP—an intermediate in
the PDHc E1 reaction pathway. The Y177 residue might inter-
act with the reaction intermediate.12 In the vicinity of the C2
of the thiazolium ring, there is a cluster of histidine residues,
including H640, H142, and H106, which likely attract and
orientate the negatively charged substrate pyruvate. Residue
H640 can form hydrogen bonds with pyruvate acid and is
partly responsible for its proper orientation.14,15 In compound
4j, we substituted the thiazolium ring with the triazole ring,
which forms two hydrogen bonds with residues Y177 and H640.
In our model docking study, compound 4j exhibits a strong
interaction with residues Y177 and H640. Site-directed
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ethanol (10 mL) was heated under reflux until the reaction was
complete, based on TLC monitoring. The solvent was then
removed under reduced pressure. The residue was dissolved in
water (10 mL), and the solid was filtered and recrystallized
from ethanol to yield compounds 1a–1l, which were used
directly for the next step.
4.1.2 General procedure for the preparation of compounds
2a–2l. We added sodium hydroxide (0.2 g, 5 mmol) to a solu-
tion of 1a–1l (5 mmol) and 3-bromopropyne (0.6 g, 5 mmol) in
dry acetonitrile (20 mL). The mixture was heated under reflux
until completion (as monitored via TLC), and the solvent was
removed under reduced pressure. The residue was dissolved in
ethyl acetate (50 mL) and washed with 0.1 M HCl, and brine
and then dried and concentrated. The crude product was
recrystallized with acetone to give the pure compounds 2a–2l,
which were used directly for the next step.
Fig. 5 Binding constants (Kb) determined by fluorescence spectral ana-
lyses for the binding of compound 4j to the wild type (WT) and mutants
of PDHc E1.
validate and explain the binding-mode between the inhibitors
and the active site of PDHc E1.
4.1.3 General procedure for the preparation of compounds
4a–4l. We added CuI (0.04 g, 2 mmol) to a stirred solution of
5-azidomethyl-2-methylpyrimidine-4-ylamine
3
(0.33 g,
3. Conclusion
2 mmol) and 2a–2l (2 mmol) in THF (10 mL) followed by Et3N
(0.4 g, 4 mmol). After overnight stirring at room temperature,
the reaction mixture was poured into water (50 mL), and the
precipitate was collected by filtration and dried under atmos-
pheric pressure. Recrystallization with DMF–H2O afforded
compounds 4a–4l.
In this study, we designed and synthesized a series of novel
ThDP analogs as potential inhibitors of PDHc E1. Most of the
compounds exhibited moderate inhibitory activities (IC50
6.1–75.5 μM). Compounds 4h, 4i, and 4j were identified as
potent inhibitors with IC50 values of 6.7, 6.9 and 6.1 μM
against PDHc E1 in vitro, and with inhibition rates of 35%,
=
4.1.3.1 Benzaldehyde O-((1-((4-amino-2-methylpyrimidin-5-yl)
50% and 33% at 100 μg mL−1 against Gibberella zeae in vivo, methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4a). Yellow solid;
respectively. The possible interactions of the important resi- 0.59 g; Yield 92%; m.p. 170–172 °C; 1H NMR (600 MHz,
dues of PDHc E1 with compound 4j were analyzed by mole- DMSO-d6) δ (ppm): 2.29 (s, 3H, CH3), 5.18 (s, 2H, CH2), 5.44 (s,
cular docking methods, site-directed mutagenesis, enzymatic 2H, CH2), 6.95 (s, 2H, NH2), 7.41–7.59 (m, 5H, Ar-H), 8.02 (s,
assays, and fluorescence spectral analyses. The theoretical and 1H, CH), 8.19 (s, 1H, CH), 8.24 (s, 1H, CH); 13C NMR
experimental results are in good agreement, indicating that (100 MHz, DMSO-d6) δ (ppm): 25.22, 46.74, 66.91, 108.45,
compound 4j could be used as a lead compound for further 124.64, 126.94, 128.78, 130.03, 131.76, 143.22, 149.34, 156.16,
optimization, and may have potential as a new microbicide.
161.47, 167.11; MS (EI) (m/z, %): 323.4 (M+, 5.34); Elemental
Analysis for C16H17N7O (323.15): C, 59.43; H, 5.30; N, 30.32.
Found: C, 59.65; H, 5.44; N, 30.24.
4.1.3.2 3-Chlorobenzaldehyde O-((1-((4-amino-2-methylpyri-
midin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4b). Yellow
solid; 0.33 g; Yield 46%; mp 154–155 °C; H NMR (DMSO-d6,
4. Experimental procedures
4.1 General procedures
1
Melting points (mp) were measured on an electrothermal
melting point apparatus and were uncorrected. The 1H NMR
spectra were recorded in DMSO-d6 on a Varian Mercury-Plus
600 spectrometer at 600 MHz. The 13C NMR spectra were
recorded in DMSO-d6 on a Varian Mercury-Plus 400 spectro-
meter at 100 MHz, and the chemical shifts were recorded in
parts per million (ppm) with TMS as the internal reference.
Mass spectra (MS) were obtained on a Trace MS 2000 instru-
ment. Elemental analyses (EA) were measured on a Vario ELIII
CHNSO elemental analyzer. Unless otherwise noted, reagents
were purchased from commercial suppliers and used without
further purification. Intermediate 3 was synthesized according
to existing methods.27
600 MHz): δ 2.29 (s, 3H, CH3), 5.21 (s, 2H, CH2), 5.44 (s, 2H,
CH2), 6.94 (s, 2H, NH), 7.44–7.50 (m, 2H, Ar-H), 7.57 (d, 1H, J =
9.0 Hz, Ar-H), 7.65 (s, 2H, CH), 7.99 (s, 1H, CH), 8.20 (s, 1H,
CH), 8.26 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.23,
46.83, 61.94, 100.68, 113.46, 116.36, 121.38, 124.96, 125.71,
133.81, 134.99, 135.80, 141.82, 156.38, 159.55, 161.53; MS (EI)
(m/z, %): 357.2 (M+, 2.74); Elemental Analysis for C16H16ClN7O
(%): C, 53.71; H, 4.51; N, 27.40. Found: C, 53.51; H, 4.58;
N, 27.69.
4.1.3.3 4-Nitrobenzaldehyde O-((1-((4-amino-2-methylpyrimi-
din-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4c). Yellow
1
solid; 0.48 g; Yield 65%; mp 214–216 °C; H NMR (DMSO-d6,
600 MHz): δ 2.29 (s, 3H, CH3), 5.26 (s, 2H, CH2), 5.43 (s, 2H,
CH2), 6.96 (s, 2H, NH), 7.86 (d, 2H, J = 9.0 Hz, Ar-H), 8.22 (s,
1H, CH), 8.27 (d, 2H, J = 8.4 Hz, CH), 8.42 (s, 1H, CH); 13C
NMR (DMSO-d6, 100 MHz): δ 25.29, 46.78, 67.44, 124.02,
124.85, 127.92, 128.61, 138.05, 142.97, 147.95, 156.28, 161.43,
4.1.1 General procedure for the preparation of compounds
1a–1l. A solution of benzaldehydes or acetophenones
(10 mmol), hydroxylamine hydrochloride (1.39 g, 20 mmol),
and anhydrous potassium carbonate (2.8 g, 20 mmol) in
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164.42, 167.42; MS (EI) (m/z, %): 368.1 (M+, 2.21); Elemental 123.11, 124.31, 126.56, 141.35, 142.76, 147.15, 152.98, 155.63,
Analysis for C16H16N8O3 (%): C, 52.17; H, 4.38; N, 30.42. 160.98, 167.12; MS (EI) (m/z, %): 383.3 (M+ + 1, 1.28), 382.3
Found: C, 51.72; H, 4.23; N, 30.89.
4.1.3.4 4-Chlorobenzaldehyde O-((1-((4-amino-2-methylpyri-
(M+, 8.05); Elemental Analysis for C17H18N8O3 (%): C, 53.40;
H, 4.74; N, 29.30. Found: C, 53.61; H, 4.31; N, 29.12.
midin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4d). Yellow
solid; 0.56 g; Yield 78%; mp 185–187 °C; H NMR (DMSO-d6, pyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4i). Yellow
600 MHz): δ 2.28 (s, 3H, CH3), 5.18 (s, 2H, CH2), 5.43 (s, 2H, solid; 0.67 g; yield 90%; mp 184–186 °C; H NMR (DMSO-d6,
4.1.3.9 1-(4-Chlorophenyl)ethanone O-((1-((4-amino-2-methyl-
1
1
CH2), 6.94 (s, 2H, NH), 7.48 (d, 2H, J = 8.4 Hz, Ar-H), 7.61 600 MHz): δ 2.14 (s, 3H, CH3), 2.29 (s, 3H, CH3), 5.20 (s, 2H,
(d, 2H, J = 8.4 Hz, Ar-H), 7.94 (s, 2H, CH), 8.19 (s, 1H, CH), CH2), 5.44 (s, 2H, CH2), 6.98 (s, 2H, NH), 7.47 (s, 2H, Ar-H),
8.25 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.31, 7.64 (s, 2H, Ar-H), 8.19 (s, 1H, CH); 13C NMR (DMSO-d6,
46.97, 67.19, 125.00, 128.83, 129.16, 130.87, 134.89, 143.39, 100 MHz): δ 12.25, 25.50, 46.74, 66.91, 122.69, 124.68, 127.85,
148.63, 161.72, 162.77; MS (EI) (m/z, %): 357.3 (M+, 3.98); 130.06, 131.31, 134.88, 143.51, 153.80, 155.72, 161.31, 168.79;
Elemental Analysis for C16H16ClN7O (%): C, 53.71; H, 4.51; MS (EI) (m/z, %): 373.2 (M+ + 2, 2.06), 371.3 (M+, 7.27); Elemental
N, 27.40. Found: C, 53.32; H, 4.56; N, 27.02.
4.1.3.5 4-Bromobenzaldehyde O-((1-((4-amino-2-methylpyrimi-
Analysis for C17H18ClN7O (%): C, 54.91; H, 4.88; N, 26.37.
Found: C, 54.45; H, 4.75; N, 26.18.
din-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4e). Yellow
solid; 0.69 g; Yield 86%; mp 179–180 °C; H NMR (DMSO-d6, methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime
4.1.3.10 1-(4-Bromophenyl)ethanone O-((1-((4-amino-2-
1
1
(4j). Yellow solid; 0.56 g; Yield 66%; mp 180–183 °C; H NMR
(DMSO-d6, 600 MHz): δ 2.13 (s, 3H, CH3), 2.30 (s, 3H, CH3),
5.20 (s, 2H, CH2), 5.49 (s, 2H, CH2), 7.02 (s, 2H, NH), 7.57
(d, 2H, J = 7.2 Hz, Ar-H), 7.60 (d, 2H, J = 7.8 Hz, Ar-H), 8.19
(s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 12.25, 25.50,
46.74, 66.91, 122.69, 124.68, 127.85, 130.06, 131.31, 134.88,
143.51, 153.80, 155.72, 161.31, 168.79; MS (EI) (m/z, %): 417.2
(M+ + 2, 5.79), 415.2 (M+, 6.78); Elemental Analysis for
C17H18BrN7O (%): C, 49.05; H, 4.36; N, 23.55. Found: C, 49.23;
600 MHz): δ 2.31 (s, 3H, CH3), 3.78 (s, 3H, CH3), 5.15 (s, 2H,
CH2), 5.47 (s, 2H, CH2), 6.98 (s, 2H, NH), 6.98 (s, 4H, Ar-H),
7.62 (d, 2H, J = 7.8 Hz, Ar-H), 7.94 (s, 1H, CH), 8.19 (s, 1H,
CH), 8.24 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 25.29,
46.86, 67.11, 123.44, 124.81, 128.85, 131.08, 131.87, 143.25,
148.51, 156.11, 161.52, 162.44; MS (EI) (m/z, %): 403.3 (M+ + 2,
2.57), 401.2 (M+, 1.94); Elemental Analysis for C16H16BrN7O
(%): C, 47.44; H, 4.01; N, 24.37. Found: C, 46.95; H, 4.10; N,
24.21.
4.1.3.6 4-Methoxybenzaldehyde O-((1-((4-amino-2-methylpyri- H, 4.54; N, 23.91.
midin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4f). Yellow
solid; 0.21 g; Yield 30%, mp 100–101 °C; H NMR (DMSO-d6,
4.1.3.11 1-(4-Methoxyphenyl)ethanone O-((1-((4-amino-2-methyl-
1
pyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4k). Yellow
1
solid; 0.47 g; Yield 64%; mp 154–156 °C; H NMR (DMSO-d6,
600 MHz): δ 2.31 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 5.15 (s, 2H,
CH2), 5.47 (s, 2H, CH2), 6.98 (s, 4H, Ar + NH), 7.54 (s, 2H,
Ar-H), 8.17 (s, 2H, CH); 13C NMR (DMSO-d6, 100 MHz):
δ 25.31, 36.09, 46.96, 67.19, 108.62, 125.00, 128.84, 129.16,
130.87, 134.89, 143.39, 148.63, 156.33, 161.72, 162.77; MS (EI)
(m/z, %): 354.4 (M+ + 1, 1.37), 353.3 (M+, 6.40); Elemental
Analysis for C17H19N7O2 (%): C, 57.78; H, 5.42; N, 27.75.
Found: C, 57.30; H, 5.41; N, 27.56.
600 MHz): δ 2.12 (s, 3H, CH3), 2.29 (s, 3H, CH3), 3.77 (s, 3H,
OCH3), 5.16 (s, 2H, CH2), 5.43 (s, 2H, CH2), 6.95 (d, 4H, J =
9.0 Hz, Ar-H + NH), 7.58 (d, 2H, J = 9.0 Hz, Ar-H), 8.01 (s, 1H,
CH), 8.17 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz): δ 12.43,
25.22, 46.69, 55.19, 66.71, 108.40, 113.81, 124.54, 127.37,
128.22, 143.64, 154.21, 156.28, 160.17, 161.51, 167.09; MS (EI)
(m/z, %): 367.4 (M+, 10.04); Elemental Analysis for C18H21N7O
4.1.3.7 1-(3-Chlorophenyl)ethanone O-((1-((4-amino-2-methyl- (%): C, 58.84; H, 5.76; N, 26.69. Found: C, 58.58; H, 5.62;
pyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4g). Yellow
solid; 0.47 g; Yield 64%; mp 131–133 °C; H NMR (DMSO-d6,
N, 26.13.
4.1.3.12 1-(2-Chlorophenyl)ethanone O-((1-((4-amino-2-
1
methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime
(4l). Yellow solid, 0.49 g; Yield 67%; mp 180–182 °C; H NMR
600 MHz): δ 2.15 (s, 3H, CH3), 2.29 (s, 3H, CH3), 5.22 (s, 2H,
CH2), 5.44 (s, 2H, CH2), 6.95 (s, 2H, NH), 7.42–7.48 (m, 2H,
Ar-H), 7.60 (d, 1H, J = 7.2 Hz, Ar-H), 7.67 (s, 1H, Ar-H), 8.00
(s, 1H, CH), 8.19 (s, 1H, CH); 13C NMR (DMSO-d6, 100 MHz):
δ 12.43, 25.25, 46.74, 67.08, 108.38, 108.30, 124.63, 125.59,
129.10, 130.32, 133.34, 137.91, 143.34, 153.69, 156.31, 161.49,
167.04; MS (EI) (m/z, %): 373.3 (M+ + 2, 1.96), 371.3 (M+, 8.28);
Elemental Analysis for C17H18ClN7O (%): C, 54.91; H, 4.88;
N, 26.37. Found: C, 54.48; H, 4.62; N, 26.26.
1
(DMSO-d6, 600 MHz): δ 2.14 (s, 3H, CH3), 2.29 (s, 3H, CH3),
5.20 (s, 2H, CH2), 5.45 (s, 2H, CH2), 6.98 (s, 2H, NH), 7.46
(s, 2H, Ar-H), 7.64 (s, 2H, Ar-H), 8.19 (s, 1H, CH); 13C NMR
(DMSO-d6, 100 MHz): δ 12.35, 25.22, 46.73, 66.94, 109.30,
124.63, 127.67, 128.46, 134.01, 134.60, 143.46, 153.76, 155.94,
158.09, 161.45, 167.35; MS (EI) (m/z, %): 373.3 (M+ + 2, 2.78),
371.3 (M+, 7.22); Elemental Analysis for C17H18ClN7O (%):
C, 54.91; H, 4.88; N, 26.37. Found: C, 54.18; H, 4.72; N, 26.46.
4.1.3.8 1-(4-Nitrophenyl)ethanone O-((1-((4-amino-2-methyl-
pyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)oxime (4h). Yellow
4.2 Structure-based docking
1
solid; 0.46 g; yield 60%; mp 222–224 °C; H NMR (DMSO-d6,
The crystallographic coordinates of PDHc-E1 with ThDP from
E. coli (PDB code: 1L8A) were obtained from the Brookhaven
Data Bank for structure-based docking rational analyses.
Hydrogen atoms were added to the structure to allow for
600 MHz): δ 2.21 (s, 3H, CH3), 2.30 (s, 3H, CH3), 5.27 (s, 2H,
CH2), 5.47 (s, 2H, CH2), 6.98 (s, 2H, NH), 7.89 (s, 2H, Ar-H),
8.21 (s, 1H, CH), 8.25 (d, 2H, J = 7.2 Hz, Ar-H); 13C NMR
(DMSO-d6, 100 MHz): δ 11.87, 24.83, 46.27, 66.81, 108.68,
8916 | Org. Biomol. Chem., 2014, 12, 8911–8918
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Paper
appropriate ionization at physiological pH. The protonated settlements was measured after 48 h of culture on potato
state of several important residues such as His106, His142, glucose solid medium. The final concentration of the com-
Tyr177, Met194, Glu571, and His640 were adjusted with pound in medium was 100 μg mL−1. Three replicates of each
SYBYL7.3 (Tripos, St. Louis, MO, USA) to form hydrogen bonds test were carried out. The growth inhibition rates (I) were cal-
with the ligand. Molecular docking analysis was performed culated using the equation: I = [(C − T)/C] × 100%, where C is
with the SURFLEX module of the SYBYL package to explore the average diameter of mycelia in the presence of these com-
the interaction model for the active site of PDHc-E1 with its pounds. The inhibition ratio of these compounds, at the dose
ligand, especially with cofactor ThDP. All atoms located within of 100 μg mL−1, are shown in Table 1.
6.5 Å from any atom of the cofactor ThDP were selected
into the active site, and the corresponding amino acid
4.5 Fluorescence spectral analyses
residue was thus involved with the active site only if one of
Fluorescence spectral analyses were carried out as described
its atoms were selected. Other default parameters were
previously.32 All fluorescent measurements were carried out on
adopted in the SURFLEX-docking calculations. All calcula-
a
FluoreMax-P fluorescence spectrophotometer (HORIBA
tions were performed on the CCNU Grid website http://
www.202.114.32.71:8090/ccnu/chem/platform.xml.
JOBIN YVON, France) equipped with a xenon lamp source and
1.0 cm quartz cell. The emission spectrum was recorded in the
305–500 nm range with excitation at 290 nm. The fluorescence
quenching experiments of PDHc E1 or its mutants (2 μM)
were performed at different concentrations of compounds
by applying a 1 cm path length cuvette. The appropriate
blank measurement corresponding to the buffer was sub-
tracted to correct the background fluorescence. The binding
constant (K) was calculated according to the equation:
4.3 Inhibitory enzymatic activity evaluation and site-directed
mutagenesis of PDHc E1
To evaluate the inhibitory activity of the compounds against
PDHc E1, the half maximal inhibitory concentration (IC50)
values of the compounds were determined at the PDHc E1
enzyme level in vitro. The cloning, expression, purification and
activity of PDHc E1 were carried out as described previously.18
ThDP and pyruvate were purchased from Sigma. Enzyme activi-
ties were measured as described previously.18 For IC50 determi-
nation, we used a standard reaction mixture containing
50 mM K3PO4 (pH 6.4), 0.4 mM 2, 6-dichlorophenolindo-
phenol (DCIP), 50 μM sodium pyruvate as the substrate, 5 μg
PDHc E1 purified enzyme and 50 µM ThDP. The compounds
from 0 to 200 μM were incubated for 5 min with PDHc E1 at
37 °C before the substrate (pyruvate) was added. The IC50
values were determined by nonlinear least-squares fitting of
the data using the Hill kinetic equations in the Growth/sigmoi-
dal model from origin 7.0 software as described previously.30
Site-directed mutagenesis of PDHc E1 was accomplished by
the introduction of specific base changes into a double-
stranded DNA plasmid, as described previously.30 DNA encod-
ing of the wild-type PDHc E1 cloned into the pMAL-C2x-PDHc-
E1 was used as a template for mutagenesis. The standard PCR
mixture contained 50–100 ng of template DNA and 100–200 ng
of each mutagenizing primer. The methylated plasmid was
digested with DpnI, and 4 µL of each reaction was used to
transform the DH5α competent cells. All mutations were con-
firmed by DNA sequencing. Verified plasmids containing the
desired mutations were transformed into the E. coli TB1 strain.
The mutant PDHc E1 proteins were purified in the same
manner as the wild-type PDHc E1.
ln F ꢀF ¼ ln K þ n ln½Qꢁ, where F0 and F are the fluorescence
0
F
intensities without and with the ligand, respectively. Term [Q]
denotes the concentration of the quencher. A plot of ln[(F0 −
F)/F] vs. ln[Q] gave a straight line using least squares analysis.
The Y-intercept was equal to ln K (where K is equal to the
binding constant).
Acknowledgements
This work was supported by the National Basic Research
Program of China (no. 2010CB126100), the Natural Science
Foundation of China (no. 21472061, 21373094, 21272089,
21172089, 21172090, 21072073, 20873049, 20872044,
30900054, and 30800169), the Program for New Century Excel-
lent Talents in University (no. NCET-06-0673), the Program for
Changjiang Scholars and Innovative Research Team in Univer-
sity (PCSIRT no. IRT0953), the Program for Academic Leader
in Wuhan Municipality (no. 201150530150), the Special Fund
for Basic Scientific Research of Central Colleges of Ministry of
Education (no. CCNU09A01010, CCNU10C01002, and
CCNU10A02006), and the Research Fund for the Doctoral
Program of Higher Education of China (200805581146).
4.4 Inhibitory activity evaluation of compounds against
Gibberella zeae in vivo
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