Design and optimization of N-acylhydrazone pyrimidine derivatives as E. coli PDHc E1 inhibitors: Structure-activity relationship analysis, biological evaluation and molecular docking study

Article abstract of DOI:10.1016/j.bmc.2017.08.038

By targeting the thiamin diphosphate (ThDP) binding site of Escherichia coli (E. coli) pyruvate dehydrogenase multienzyme complex E1 (PDHc E1), a series of novel ‘open-chain’ classes of ThDP analogs A, B, and C with N-acylhydrazone moieties was designed and synthesized to explore their activities against E. coli PHDc E1 in vitro and their inhibitory activity against microbial diseases were further evaluated in vivo. As a result, A1–23 exhibited moderate to potent inhibitory activities against E. coli PDHc E1 (IC₅₀ = 0.15–23.55 μM). The potent inhibitors A13, A14, A15, C2, had strong inhibitory activities with IC₅₀ values of 0.60, 0.15, 0.39 and 0.34 μM against E. coli PDHc E1 and with good enzyme-selective inhibition between microorganisms and mammals. Especially, the most powerful inhibitor A14 could 99.37% control Xanthimonas oryzae pv. Oryzae. Furthermore, the binding features of compound A14 within E. coli PDHc E1 were investigated to provide useful insights for the further construction of new inhibitor by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that A14 had most powerful inhibition against E. coli PDHc E1 due to the establishment of stronger interaction with Glu571, Met194, Glu522, Leu264 and Phe602 at active site of E.coli PDHc E1. It could be used as a lead compound for further optimization, and may have potential as a new microbicide.

Full text of DOI:10.1016/j.bmc.2017.08.038

Accepted Manuscript
Design and optimization of N-acylhydrazone pyrimidine derivatives as E. coli
PDHc E1 inhibitors: structure-activity relationship analysis, biological evalua-
tion and molecular docking study
Haifeng He, Hongying Xia, Qin Xia, Yanliang Ren, Hongwu He
PII:
S0968-0896(17)31537-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.038
BMC 13942
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Bioorganic & Medicinal Chemistry
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18 August 2017
22 August 2017
Please cite this article as: He, H., Xia, H., Xia, Q., Ren, Y., He, H., Design and optimization of N-acylhydrazone
pyrimidine derivatives as E. coli PDHc E1 inhibitors: structure-activity relationship analysis, biological evaluation
and molecular docking study, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.08.038
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Design and optimization of N-acylhydrazone pyrimidine derivatives as E. coli
PDHc E1 inhibitors: structure-activity relationship analysis, biological
evaluation and molecular docking study
Haifeng Hea,b, Hongying Xiab, Qin Xiaa, Yanliang Rena, Hongwu He*a
aKey Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, P. R. China
b Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology Normal
University, Fenglin Street, Nanchang, Jiangxi 330013, PR China
* To whom correspondence should be addressed. Tel./fax: +86 (0)27 67867960
E-mail addresses: he1208@mail.ccnu.edu.cn (H. He)
1

Abstract
By targeting the thiamin diphosphate (ThDP) binding site of Escherichia coli (E. coli) pyruvate
dehydrogenase multienzyme complex E1 (PDHc E1), a series of novel ‘open-chain’ classes of
ThDP analogs A, B, and C with N-acylhydrazone moieties was designed and synthesized to
explore their activities against E. coli PHDc E1 in vitro and their inhibitory activity against
microbial diseases were further evaluated in vivo. As a result, A1~23 exhibited moderate to potent
inhibitory activities against E. coli PDHc E1 (IC50 = 0.15–23.55 µM). The potent inhibitors A13,
A14, A15, C2, had strong inhibitory activities with IC50 values of 0.60, 0.15, 0.39 and 0.34 µM
against E. coli PDHc E1 and with good enzyme-selective inhibition between microorganisms and
mammals. Especially, the most powerful inhibitor A14 could 99.37% control Xanthimonas oryzae
pv. Oryzae. Furthermore, the binding features of compound A14 within E. coli PDHc E1 were
investigated to provide useful insights for the further construction of new inhibitor by molecular
docking, site-directed mutagenesis, and enzymatic assays. The results indicated that A14 had most
powerful inhibition against E. coli PDHc E1 due to the establishment of stronger interaction with
Glu571, Met194, Glu522, Leu264 and Phe602 at active site of E.coli PDHc E1. It could be used as
a lead compound for further optimization, and may have potential as a new microbicide.
Keywords ThDP; E. coli PHDc E1; enzyme-selective; molecular docking.
2

1. Introduction
The pyruvate dehydrogenase complex (PDHc) is an exquisite enzyme that catalyzes the
oxidative decarboxylation of pyruvate，and the subsequent acetylation of coenzyme A (CoA) to
acetyl-CoA.1,2 The overall reaction of oxidative decarboxylation can be simply exhibited in Fig. 1.
Fig.1 Oxidative decarboxylation catalyzed by PDHc.
The above reactions are carried out by PDHc complex, which is comprised of three different
enzyme components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2),
dihydrolipoamide dehydrogenase (E3).3 The pyruvate dehydrogenase complex E1 component
(PDHc E1) catalyzes the first step (rate-limiting and irreversible step) of multistep process, under
condition of using thiamine diphosphate (ThDP) and Mg2+ as cofactors.4–6 The cofactor of ThDP
plays an important role in the enzyme reaction and the catalysis mechanism.7 As would be
expected, blocking the active site of cofactor, for example, by replacing it with ThDP analogue,
inactivates the PDHc. Therefore, we selected PDHc E1 as the target enzyme to design new
cofactor ThDP analogs as PDHc E1 inhibitors.
Currently, much effort has been made to design and synthesize ThDP analogs (such as ThTDP,
ThTTDP, and triazole-ThDP in Fig. 2) 8-12 as PDHc E1 inhibitors. These ThDP analog inhibitors,
such as ThTDP and ThTTDP, can block the ThDP binding site, and exhibit significantly stronger
binding affinities for PDHc E1 than ThDP.13-14However, these ThDP analogs exhibit no potential
utility and display poor bioavailability, due to their complex structure with highly charged
pyrophosphate groups. They also showed poor enzyme-selective inhibition between
microorganisms and mammals.15
3

To the best of our knowledge, the crystal structures of the PDHc E1/ThDP or inhibitor
complex from E. coli (PDB ID:1L8A and 1RP7) have been determined.16 The crystal structures
enable structure-based design of novel inhibitors against PDHc E1. On that basis, we selected E.
coli PDHc E1 as the target pattern to design new cofactor ThDP analogs as inhibitors of PDHc E1.
In our laboratory, we recently had got preliminary progress for finding an effective E. coli PDHc
E1 inhibitor I by structure-based rational design.17,18 The mechanism was predicted with theory.
Base on this work, the linker of I was further optimized affording ThDP analogs II, III, IV, V and
VI as E. coli PDHc E1 inhibitors (Fig. 2).19–23 Some of them were demonstrated to be effective
inhibition (IC50>0.65 µM) of E. coli PDHc E1 with moderate antibacterial or antifungal activity.
The results convinced us that the linker (Fig. 2) of parent structure I plays a vital role in the
biological activity of these ThDP analogs. The findings encouraged us to further find useful PDHc
E1 inhibitors out with antibacterial or antifungal activity by further optimization of lead structure
I.
Fig.2 Structures of known PDHc E1 inhibitors.
Structure–activity relationship (SAR) indicated the introduction of hydrogen bond acceptor or
hydrogen bond donor structural unit into the linker in parent structure I could increase the
4

inhibitory activity against E. coli PDHc E1 and antifungal activity. 22,23 Aiming to explore more
optimizing linkages, the N-acylhydrazone moiety (-CONHN=CH-), which not only had
well-known biological activities,24–26 but also included hydrogen bond acceptor and hydrogen
bond donor structural unit, was introduced to the structure I as a ‘open-chain’ linker to form a new
structural class A as potential E. coli PDHc E1 inhibitors. Furthermore, the effect of Me group on
pyrimidine ring was firstly studied, which was replaced by H or NH2 group. Therefore, three new
structural classes of PDHc E1 inhibitors were formed (Fig. 3). Considering the binding site of
diphosphate of ThDP in the active site of E. coli PDHc E1, various chemical groups as Ar were
incorporated into the parent compounds A, B, and C to investigate their preliminary SAR.
Fig.3 Design of the new N-acylhydrazone pyrimidine derivatives A, B, and C
Herein, the chemical synthesis of these new N-acylhydrazone pyrimidine derivatives A, B, and
C as E. coli PDHc E1 inhibitors is described in details. The inhibitory activities on E. coli PDHc
E1 are presented along with their SAR analysis as follows. 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. The enzyme-selectivity of representative compounds between
microorganisms and mammals was also examined.
2. Chemistry
The synthetic route of A, B, and C is depicted in Scheme 1.
5

Scheme 1. Reagents and conditions (a) Na, EtOH, rt, 2h; (b) Raney Nickel, HCOOH, 80℃; (c)
H2SO4, EtOH, reflux; (d) NH2NH2.H2O, EtOH, reflux; (e) EtOH, CH3COOH, reflux;
Substituted-amidine and 2-(ethoxymethylene)malononitrile as starting material was used to
prepare 4-amino-2-substitutedpyrimidine-5-carbaldehyde 2 according to the literature method.27 2
is the key intermediate for the preparation of title compounds A, B, and C. The title compounds
could be synthesized by a five-step sequence starting from starting material. Various substituted
benzoic acid reacted with ethanol in the presence of concentrated sulfuric acid to produce
substituted ethyl benzoate 3, which reacted with hydrazine hydrate in ethanol to produce
corresponding hydrazide 4. Finally, the new N-acylhydrazone pyrimidine derivatives A, B, and C
were prepared in good yields by condensing compound 2 with a variety of substituted benzoyl
1
hydrazine in ethanol at reflux. All synthesized compounds were characterized by H NMR, 13C
6

NMR, mass spectrometry and elementary analysis.
3. Results and discussion
3.1. In vitro inhibition of E. coli PDHc E1
In order to enhance the inhibitory potency against E. coli PDHc E1, lead structure I was
revised by replacing linker with N-acylhydrazone moiety (Fig.3). Several N-acylhydrazone
pyrimidine derivatives A1~23 were firstly synthesized and their inhibitory potency against E. coli
PDHc E1 was evaluated. The IC50 values of A1~23 are summarized in Table 1. It was observed
that most of the compounds showed moderate to potent inhibitory activity with IC50 values in the
range of 0.15–23.55 µM. A14 was the most potent inhibitor in this series with the activity of IC50
at 0.15 µM. All A compounds displayed higher inhibitory activity than that of I with the same R
on the benzene ring (Table 2). Especially the inhibitory activity of A22 was 34-folds higher than
that of I22. The results revealed that the inhibitory potency could be meaningfully improved by
replacing linker of I with N-acylhydrazone moiety.
7

Table 1 The inhibitory activity against E. coli PDHc E1 of N-acylhydrazone pyrimidine
derivatives A
Compd.
A1
R
H
IC50a(µM)
6.29 0.27
8.53 0.43
21.42 0.76
3.28 0.29
4.92 0.32
20.95 0.79
20.95 0.79
2.09 0.14
3.17 0.17
7.43 0.51
23.55 0.52
2.75 0.26
Compd.
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
R
3-NO2
IC50a(µM)
0.60 0.03
0.15 0.01
0.39 0.01
3.60 0.27
19.56 0.72
9.34 0.40
6.01 0.18
5.87 0.25
13.57 0.46
2.36 0.16
12.85 0.67
4-F
3,5-(NO2)2
3-NO2-5-NH2
4-OH
A2
2-Cl
A3
3-Cl
A4
A5
4-Cl
2-OH
2,4-Cl2
2-Br
2-Me
A6
3-Me
A7
3-Br
4-Me
A8
A9
4-Br
3,5-Me2
4-OCH3
3-OCH3
4-NH2
2-Cl-4-NH2
4-NO2
A10
A11
A12
aIC50 (µM) value is defined as the micromolar concentration required for 50% inhibition against PDHc E1 from E.
coli in vitro.
Table 2 The relative activity of compounds A and I
Compd.
A1
R
H
IC50a(µM)
6.29
Relative activity
8.76
H
55.15
4.92
I1
4-Cl
A5
5.37
3.20
4-Cl
26.44
2.75
I5
4-NO2
4-NO2
4-OCH3
4-OCH3
A12
I12
8.80
2.36
A22
I122
34.58
81.62
aIC50 (µM) value is defined as the micromolar concentration required for 50% inhibition against PDHc E1 from E.
coli in vitro
8

As shown in Table 1, R on the benzene ring also had great influence on the inhibitory activity
base on the structure skeleton of A. Firstly, compounds with NO2 group as R exhibited better
inhibitory activity than that of compounds with other substituents, such as A14 (R=3,5-diNO2,
IC50 = 0.15 µM) was the most potent inhibitor in this series, followed by A15 (R=3-NO2-5-NH2,
IC50 = 0.39 µM) and A13 (R=3-NO2, IC50 = 0.60 µM). Secondly, compounds A with hydrogen
bonding acceptor group (NO2) as R exhibited better inhibitory activity than that of compounds
with hydrogen bonding donor group (NH2) substituents, such as A12 (R=4-NO2, IC50=2.75 µM) >
A10 (R=4-NH2, IC50=7.43 µM), A14 (R=3,5-(NO2)2, IC50=0.15 µM) > A15 (R=3-NO2-5-NH2,
IC50=0.39 µM). Finally, when substituted with the halogen groups on benzene ring, the activity
sequence is meta > para > ortho, such as A4 (R=3-Cl, IC50 = 3.28 µM) > A5 (R=4-Cl, IC50 = 4.92
µM)> A3 (R=2-Cl, IC50 = 21.42 µM); A8 (R=3-Br, IC50 = 2.09 µM) > A9 (R=4-Br, IC50 = 3.17
µM)> A7 (R=2-Br, IC50 = 20.95 µM). The results suggest that the 3-position group on the benzene
ring is favorable for the PDHc E1 inhibitory activity. When comparing the PDHc E1 inhibitory
activity of compounds A2, A5, and A9, the activity sequence is Br > Cl > F, which indicates the
larger substituent is better. In the other hand, R at 2-position on the benzene ring was unfavorable
to inhibitory activity. Inhibitory activity against E. coli PDHc E1 decreased in the following order:
A5 (R=4-Cl, IC50 = 4.95 µM) > A6 (R=2,4-diCl, IC50 = 20.95 µM) > A3 (R=2-Cl, IC50 = 21.42
µM); A10 (R=4-NH2, IC50 = 7.43 µM) > A11 (R=2-Cl-4-NH2, IC50 = 23.55 µM); A16 (R=4-OH,
IC50 = 3.60 µM) > A17 (R=2-OH, IC50 = 19.56 µM); A20 (R=4-Me, IC50 = 5.87 µM) > A19
(R=3-Me, IC50 = 6.01 µM)> A18 (R=2-Me, IC50 = 9.34 µM). Above results indicated that the
inhibitory activity of A was also dependent upon the structure, position and size of R on the
benzene ring.
9

Table 3 The inhibitory activity against E. coli PDHc E1 of compounds B
Compd.
R
Inhibition ratio
Compd.
R
Inhibition ratio（%）
(100 µM)a
14.44 0.23
0
（%）(100 µM)a
19.70 1.23
4.02 0.32
26.50 1.21
B1
B2
B3
B4
B5
B6
B7
H
2-Me
3,5-Me2
2,4-Cl2
4-F
12.07 0.98
16.80 0.98
4-Br
2-Cl
a Inhibitory potency (%) of compounds against enzyme in vitro at 100 µM as average of triplicate.
Table 4 The inhibitory activity against E. coli PDHc E1 of N-acylhydrazone pyrimidine
derivatives C
Compd.
C5
R
IC50(µM)
30.49 0.97
20.95 0.79
11.89 0.62
3.28 0.29
6.17 0.24
4.92 0.32
30.44 0.67
3.17 0.17
Compd
C1
R
H
IC50a(µM)
9.81 0.50
2,4-Cl2
2,4-Cl2
3-Cl
A6
H
6.29 0.27
0.34 0.02
0.60 0.03
5.76 0.25
7.43 0.51
10.30 0.79
23.55 0.52
A1
C2
C6
3-NO2
3-NO2
4-NH2
4-NH2
2-Cl-4-NH2
2-Cl-4-NH2
A13
C3
A4
3-Cl
C7
4-Cl
A10
C4
A5
4-Cl
C8
4-Br
A11
A9
4-Br
aIC50 (µM) value is defined as the micromolar concentration required for 50% inhibition against PDHc E1 from E.
coli in vitro.
In order to examine the effect of Me group on the pyrimidine ring on inhibitory activity
against E.coli PDHc E1, A was modified to produce B and C by replacing Me group on the
pyrimidine ring in A with H or NH2 moiety and its inhibitory activity against E. coli PDHc E1 was
tested (Table 3 and 4). The result showed that the substituting of Me group with H group led to a
decrease in inhibitory activity against E. coli PDHc E1. As show in Table 3, all title compounds B
only showed <30% inhibitory potency against E. coli PDHc E1 at 100 µM. It was found that the
10

inhibitory activity was slight enhanced by changing Me group into NH2 group on the pyrimidine
ring, when R included NO2 or NH2 group on the benzene ring. As shown in Table 4, C2
(R=3-NO2, IC50 = 0.34 µM), C3 (R=4-NH2, IC50 = 5.76 µM) and C4 (R=2-Cl-4-NH2, IC50 = 10.30
µM) displayed better inhibitory activity than A13 (R=3-NO2, IC50 = 0.60 µM), A10 (R=4-NH2,
IC50 = 7.43 µM) and A11 (R=2-Cl-4-NH2, IC50 = 23.55 µM). But when R was substituted by
halogen (Cl or Br) or H group, C5 (R=2,4-Cl2, IC50 = 30.49 µM), C6 (R=3-Cl, IC50 = 11.89 µM),
C7 (R=4-Cl, IC50 = 6.17 µM) and C8 (R=4-Br, IC50 = 30.44 µM) with NH2 group substituted on
the pyrimidine ring displayed lower inhibitory activity than that of corresponding A6 (R=2,4-Cl2,
IC50 = 20.95 µM), A4 (R=3-Cl, IC50 = 3.28 µM), A5 (R=4-Cl, IC50 = 4.92 µM) and A9 (R=4-Br,
IC50 = 3.17 µM) with Me group substituted on the pyrimidine ring. Especially, C8 showed 9-folds
activity lower than that of A9. Above observation showed the substituted group on the pyrimidine
ring in parent structure I also played a very important role in inhibitory potency against E. coli
PDHc E1. Compared with H or NH2 group, Me as substituent group on the pyrimidine ring was
much beneficial to inhibitory activity. These results suggest that the structure skeleton of A is
better than both B, C and lead structure I for finding more powerful PDHc E1 inhibitor.
3.2. Enzyme-Selective
The literature reported ThDP analogs as PDHc E1 inhibitors exhibited poor enzyme-selective
inhibition between mammals and microorganism.28 In this work, pig PDHc E1 and E. coli PDHc
E1 were chosen as the target pattern of mammalian and microorganism, respectively. A13, A14,
A15, C2 with good inhibition against E. coli PDHc E1 (IC50 = 0.15–0.60 µM) were selected to test
their inhibition against Pig heart PDHc E1. As shown in Table 5, A13, A14, A15, C2 exhibited
weak inhibitory potency (inhibition ratio<20%) against pig PDHc E1. These findings showed that
11

A13, A14, A15, C2 exhibited better enzyme-selective inhibition between microorganism and
mammal.
Table 5 Inhibition of A and C against E. coli and pig PDHc E1
Porcine PDHc E1
E. coli PDHc E1
Inhibitory potencyb (%)
Compd.
IC50a (µM)
0.60 0.03
0.15 0.01
0.39 0.01
0.34 0.02
Inhibitory potencyb (%)
18.46 1.23
15.46 1.34
9.11 0.03
100.00 0.19
100.00 1.39
100.00 2.73
100.00 3.21
A13
A14
A15
C2
0
a IC50 (µM) value is defined as the micromolar concentration required for 50% inhibition against E. coli PDHc E1
in vitro.
b Inhibitory potency (%) of compounds against enzyme in vitro at 100 µM as average of triplicate.
3.3. Antibacterial activity (In vivo)
In order to find useful PDHc E1 inhibitors with antibacterial activity, A13~15 and C2 with
higher inhibitory activity against E. coli PDHc E1 were chosen to further evaluate their
antibacterial activity against Acidovorax avenae subsp. Avenae and Xanthimonas oryzae
pv．Oryzae at 500 µg/mL. As shown in Table 6, A14, with the best E. coil PDHc E1 activity (IC50
= 0.15 µM), could 99.37% control Xanthimonas oryzae pv. Oryzae. The effect of A14 was
comparable to commercial bactericide streptomycin sulfate (97.58%) as a positive control. Its
inhibition against Xanthimonas oryzae pv. Oryzae is worth further examination.
Table 6 Antibacterial activity of A13~15 and C2
Inhibitory potencya (%) 500 µg/mL
Compd.
Acidovorax avenae subsp. Avenae;
Xanthimonas oryzae pv．Oryzae
13.66 0.27
25.52 0.34
12.34 0.33
10.73 0.25
96.54 0.27
40.56 0.51
99.37 0.30
14.25 0.30
19.50 0.24
97.58 0.35
A13
A14
A15
C2
Streptomycin Sulfate
aInhibitory potency (%) against the growth of pathogenic fungi at 500 µg/mL, 0 (no effect), 100% (completely
kill).
3.4. Analyses of the interaction between inhibitors and E.coil PDHc E1
12

To explore the interaction mode of N-acylhydrazone pyrimidine derivative A, B, and C with
the active site of PDHc E1. The SURFLEX module of SYBYL package was used to perform
molecular docking simulation of N-acylhydrazone pyrimidine derivative A, B, and C.17 A14 as the
best PDHc E1 inhibitor (IC50 = 0.15 µM) was selected to analyses their interaction with E. coil
PDHc E1 by molecular docking.
As shown in Fig. 4, A14 could occupy the ThDP-binding pocket with a ‘V’ conformation. On
the right side of the‘V’ conformation, the 4-aminopyrimidine ring of A14 not just displays a π-π
stacking with the side chain ring of Phe602，and the nitrogen atom of the pyrimidine ring establish
two key hydrogen bonding interaction with the side chain of Glu571 and Met194. The amino
group connected to the pyrimidine ring forms a strong hydrogen bond with Glu522, which is an
important residue in the stabilization of the enzyme bound LThDP.10 The oxygen atom of the
N-acylhydrazone moiety also establishes a hydrogen bond with Leu264. On the left side of the ‘V’
conformation of A14, three strong hydrogen bonds can be formed by the two nitryl groups on
benzene ring with His106, Lys392, and Asn260, meanwhile the two nitryl groups coordinate with
the Mg2+ in the active site. These docking results offered us a reasonable explanation for why
compound A14 had the highest PDHc E1 inhibitory activity.
Fig. 4 Optimal binding model for compound A14 into active site of PDHc E1 from E. coli docked
13

by SURFLEC module, ligand and some key residues are shown in stick, hydrogen bonds are
shown in red lines.
In order to verify the prediction of molecular docking, site-directed mutagenesis and
enzymatic assays were further performed. As shown in Table. 7, the IC50 value of A14 against
mutants E571A (36.11 µM), M194A (112.71 µM), E522A (13.31 µM), L264A (2.71 µM), H106A
(0.92 µM), K392A (0.50 µM), or F602A (4.82µM) was about 240 times, 751 times, 88 times, 18
times, 6 times, 3 times or 32 times higher than its value against wild-type PDHc E1 (0.15 µM),
respectively. The results suggests that the interaction between A14 and residue Glu571, Met194,
Glu522, Leu264, or Phe602 by forming hydrogen bond has a significant contribution for its
inhibitory activity against E. coli PDHc E1.
Table 7 The IC50 values of compound A14 against wild and mutant E. coli PDHc E1
IC50 (µM)
Wild
E571A
36.11 2.38 112.71 4.80 13.31 0.52 2.71 0.05 0.92 0.06 0.50 0.01 4.82 0.09
240.7 751.4 88.7 18.1 6.1 3.3 32.1
M194A
E522A
L264A
H106A
K392A
F602A
0.15 0.01
Relative activity
aIC50 (µM) value is defined as the micromolar concentration required for 50% inhibition against PDHc E1 from E.
coli in vitro.
4. Conclusion
In the current study, a series of novel ‘open-chain’ classes of E. coli PDHc E1 inhibitors,
N-acylhydrazone pyrimidine derivatives A, B, and C were designed and synthesized. As novel
ThDP analogs, all A displayed moderate to powerful inhibitory activity with IC50 values in the
range of 0.15 to 23.55 µM against E. coli PHDc E1. The inhibitory potency of compounds against
E. coli PDHc E1 could be greatly enhanced when the linkage (between pyrimidine and benzene
ring moiety) of lead compound I was replaced with N-acylhydrazone moiety. Moreover the
substituted on the pyrimidine ring in parent structure also played a very important role in
14

inhibitory potency against E. coli PDHc E1. The Me group as substituent group on the pyrimidine
ring was much beneficial to inhibitory activity, compared with H or NH2 group. These results
recommend that the structure skeleton of A is better than both B, C and lead structure I for finding
more powerful PDHc E1 inhibitor. Among these title compounds, compounds A13, A14, A15, and
C2 were found to be very effective inhibitors of E. coli PDHc E1, with IC50 values ranging from
0.15 to 0.60 µM. They also exhibited good enzyme-selective inhibition between microorganisms
and mammals. Compound A14, with inhibition rates of 99.37% at 500 µgmL−1 against
Xanthimonas oryzae pv. Oryzae, was the most powerful inhibitor of E. coli PDHc E1 among title
compounds. Binding mode analysis revealed that A14 displays a π-π stacking with the side chain
ring of Phe602，and four key hydrogen bonding interaction is made by the nitrogen and oxygen
atom with the side chain of Glu571, Met194, Glu522, Leu264.The two NO2 groups not only could
form three strong hydrogen bonds with Lys392, Asn260, and His106, but also could establish a
coordinate-bond with the Mg2+. The above hydrogen bonding interaction in turn seems to be
important for enhancing its inhibitory potency. The site-directed mutagenesis and enzymatic
assays further verified that the interaction between A14 with Glu571, Met194, Glu522, Leu264
and Phe602 had a significant contribution for its inhibitory activity against E.coli PDHc E1.
Therefore, the skeleton of N-acylhydrazone pyrimidine derivatives A could be as the novel lead
structure for further optimization.
5. Experimental
5.1 Chemistry
Melting points (mp) were measured on an electrothermal melting point apparatus and were
1
uncorrected. H NMR spectra were recorded at 400 MHz, in DMSO-d6 solution on a Varian
15

Mercury-Plus 400 spectrometer and chemical shifts were recorded in parts per million (ppm) with
TMS as the internal reference. Mass spectra (MS) were obtained on a QTRAP LC/MS/MS system
(API2000; Applied Biosystems, Foster City, CA, USA), and signals were given in m/z. Unless
otherwise noted, reagents were purchased from commercial suppliers and used without further
purification.
5.2 General procedure for preparation of 1a-c.
A solution of sodium ethoxide (1.15g, 50 mmol), and acetamidine hydrochloride (4.7 g, 50
mmol) in ethanol solution (60 mL) was stirred at room temperature for 1h. After this, the reaction
mixture was filtered out affording filtrate. Then 2-(ethoxymethylene)malononitrile (6.1 g, 50mmol)
was added to the filtrate, and the mixture was stirred at room temperature for 3h. After this, the
reaction mixture was filtered and washed with 10 mL ethanol and dried to give the desired
compound 4-amino-2-methylpyrimidine-5-carbonitrile 1a, which was used directly for the next
step. Under the same condition, the intermediate compounds 1b and 1c were also prepared.
5.3 General procedure for preparation of 2a-c.
A solution of 4-amino-2-methylpyrimidine-5-carbonitrile 1a (3.04 g, 30 mmol) and raney nickel
0
(3.0 g) in formic acid (20 mL) was stirred at 80 C for 4 h. After this, the reaction mixture was
filtered and washed with 10 mL formic acid. The filtrate and washings were collected together and
concentrated under reduced pressure. The residue was purified by column chromatography on
silica gel and eluted with ethyl acetate/petroleum ether (1:1, v/v) to give white solid 2a, which was
used directly for the next step. Under this same condition, the intermediate compounds 2a and 2c
were also prepared.
5.4 General procedure for preparation of 4.
16

We added dense H2SO4 (0.098g, 1 mmol) to a solution of substituted benzoic acid (5 mmol) in
dry ethyl alcohol (10 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 Na2CO3, brine, dried and concentrated, respectively.
The crude products (intermediate 3) were used directly for the next step.
5 mL of hydrazine monohydrate (80%) was added to a solution of intermediate 3 (5 mmol) in
ethanol (5 mL). The reaction mixture was maintained under reflux for 5 h. was then concentrated
under reduced pressure and the resulting solid was collected by filtration, washed with cold water
and dried to give the desired intermediate 4 as a white solid.
5.5 General procedure for preparation of target compounds A, B, and C.
Appropriate substituted benzoyl hydrazine 4 (1 equiv) was added to a solution of intermediate 2 (1
mmol) in ethanol (10 mL). The reaction mixture was stirred for 3 h at reflux under the condition of
the presence of acetic acid as catalyzer, then poured into cold water and the resulting solid was
collected by filtrated, washed with EtOH (10 mL), and dried in the atmospheric pressure to give
the desired title compounds A, B, and C.
5.5.1. N'-((4-amino-2-methylpyrimidine-5-yl)methylene)benzohydrazide (A1)
White solid; Yield 78%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 3H, CH3),
7.53 (s, 2H, NH2), 7.58 (s, 1H, Ar-H), 7.92 (d, 2H, Ar-H, J=4.9Hz), 8.07 (s, 1H, pyrimidine-H),
8.42 (s, 1H, CH=N), 8.42 (s, 2H, Ar-H), 12.08 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
δ(ppm): 167.0, 163.2, 160.0, 158.3, 146.3, 133.2, 132.1, 128.7, 127.8, 107.3, 25.8; ESI-MS m/z:
256.2 [M+1]+; Elemental Anal. Calcd for C13H13N5O: C, 61.17; H, 5.13; N, 27.43. Found: C,
61.03; H, 5.22; N, 27.26 .
17

5.6.2. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-4-fluorobenzohydrazide (A2)
White solid; Yield 89%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 3H, CH3),
7.38 (s, 2H, NH2), 7.99 (s, 1H, pyrimidine-H), 7.99 (s, 2H, Ar-H), 8.41 (s, 1H, CH=N), 8.41 (s, 2H,
Ar-H), 12.08 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 166.9, 163.1, 161.9, 159.9,
158.3, 146.2, 130.5, 129.5, 115.7, 115.5, 107.1, 25.7; ESI-MS m/z: 274.3 [M+1]+; Elemental Anal.
Calcd for C13H12FN5O: C, 57.14; H, 4.43; N, 25.63. Found: C, 57.04; H, 4.20; N, 25.91 .
5.6.3. N'-((4-amino-2-methylpyrimidine-5-yl)methylene)-2-chlorobenzohydrazide (A3)
Light yellow solid; Yield 68%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.35 (d, 3H,
CH3, J=34.5Hz), 7.46 (s, 2H, NH2), 7.52 (d, 1H, Ar-H, J=6.4Hz), 7.58 (d, 1H, Ar-H, J=6.4Hz),
8.03 (s, 1H, pyrimidine-H), 8.19 (s, 1H, CH=N), 8.25 (s, 2H, Ar-H), 8.29 (s, 2H, Ar-H), 12.14 (s,
1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 162.5, 159.9, 159.4, 158.6, 146.6, 135.0,
131.7, 130.6, 129.9, 129.6, 127.5, 106.9, 25.8; ESI-MS m/z: 290.1 [M+1]+; Elemental Anal. Calcd
for C13H12ClN5O: C, 53.89; H, 4.17; N, 24.17. Found: C, 53.60; H, 4.13; N, 24.25 .
5.6.4. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-3-chlorobenzohydrazide (A4)
White solid; Yield 75%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H, CH3),
7.59 (d, 1H, Ar-H, J=7.2Hz), 7.67 (s, 1H, Ar-H), 7.88 (d, 1H, Ar-H, J=6.6Hz), 7.96 (s, 1H, Ar-H),
8.07 (s, 1H, pyrimidine-H), 8.32 (s, 1H, CH=N), 8.41 (s, 2H, NH2), 12.15 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) δ(ppm): 167.0, 161.6, 159.9, 158.4, 146.7, 135.0, 133.4, 131.8, 130.6,
127.4, 126.5, 107.0, 25.7; ESI-MS m/z: 290.2 [M+1]+; Elemental Anal. Calcd for C13H12ClN5O: C,
53.89; H, 4.17; N, 24.17. Found: C, 54.02; H, 3.96; N, 24.50 .
5.6.5. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-4-chlorobenzohydrazide (A5)
White solid; Yield 79%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 3H, CH3),
18

7.63 (s, 2H, NH2), 7.94 (d, 2H, Ar-H, J=7.2Hz), 8.07 (s, 1H, pyrimidine-H), 8.30 (s, 1H, CH=N),
8.41 (s, 2H, Ar-H), 12.14 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 167.0, 162.0,
159.9, 158.4, 146.5, 136.9, 131.7, 129.7, 128.8, 107.2, 25.7; ESI-MS m/z: 290.1 [M+1]+;
Elemental Anal. Calcd for C13H12ClN5O: C, 53.89; H, 4.17; N, 24.17. Found: C, 53.86; H, 3.98; N,
24.34.
5.6.6. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-2,4-dichlorobenzohydrazide (A6)
Light yellow solid; Yield 76%; m.p 244-246oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.35 (d,
3H, CH3, J=30.2Hz), 7.53-7.65 (m, 2H, NH2), 7.75 (d, 1H, Ar-H, J=27.0Hz), 8.05 (s, 1H,
pyrimidine-H), 8.24 (s, 2H, Ar-H), 8.30 (s, 1H, CH=N), 12.17 (s, 1H, NH); ESI-MS m/z: 324.3
[M+1]+; Elemental Anal. Calcd for C13H11Cl2N5O: C, 48.17; H, 3.42; N, 21.60. Found: C, 48.12;
H, 3.22; N, 21.89 .
5.6.7. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-2-bromobenzohydrazide (A7)
1
White solid; Yield 69%; m.p 250-251oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.35 (d, 3H,
CH3, J=34.9Hz), 7.44 (s, 2H, NH2), 7.46-7.56 (m, 1H, Ar-H), 7.70 (dd, 1H, Ar-H, J=7.6, 7.5Hz),
8.03 (s, 1H, pyrimidine-H), 8.19 (s, 1H, CH=N), 8.24 (s, 1H, Ar-H), 8.29 (s, 1H, Ar-H), 12.12 (s,
1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 167.1, 163.3, 159.9, 158.5, 146.5, 137.1,
133.0, 131.8, 129.5, 127.9, 119.6, 106.9, 25.7; ESI-MS m/z: 334.3[M+1]+; Elemental Anal. Calcd
for C13H12BrN5O: C,46.72; H, 3.62; N, 20.96. Found: C, 46.85; H, 3.98; N, 20.80 .
5.6.8. N'-((4-amino-2-methylpyrimidine-5-yl)methylene)-3-bromobenzohydrazide (A8)
White solid; Yield 80%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H, CH3),
7.53 (d, 1H, Ar-H, J=7.4Hz), 7.82 (d, 1H, Ar-H, J=6.8Hz), 7.92 (d, 1H, Ar-H, J=6.8Hz), 8.10 (s,
2H, NH2), 8.33 (s, 1H, pyrimidine-H), 8.41 (s, 1H, Ar-H), 8.41 (s, 1H, CH=N), 12.16 (s, 1H, NH);
19

13C NMR (100 MHz, DMSO-d6) δ(ppm): 161.5, 159.9, 146.8, 135.2, 134.8, 130.9, 130.3, 127.0,
121.9, 106.1, 25.8; ESI-MS m/z: 334.2 [M+1]+; Elemental Anal. Calcd for C13H12BrN5O: C, 46.72;
H, 3.62; N, 20.96. Found: C, 46.65; H, 3.64; N, 20.83 .
5.6.9. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-4-bromobenzohydrazide (A9)
White solid; Yield 78%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H, CH3),
7.77 (s, 2H, NH2), 7.86 (s, 2H, Ar-H), 8.06 (s, 1H, pyrimidine-H), 8.34 (s, 1H, CH=N), 8.41 (s, 2H,
Ar-H), 12.13 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 167.0, 162.1, 159.9, 158.4,
146.6, 132.1, 131.7, 129.8, 125.9, 107.2, 25.8; ESI-MS m/z: 334.2 [M+1]+; Elemental Anal. Calcd
for C13H12BrN5O: C, 46.72; H, 3.62; N, 20.96. Found: C, 46.87; H, 3.40; N, 20.71 .
5.6.10. N'-((4-amino-2-methylpyrimidine-5-yl)methylene)-4-amino-benzohydrazide (A10)
White solid; Yield 83%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 3H, CH3),
5.83 (s, 2H, NH2), 6.60 (d, 2H, NH2, J=8.1Hz), 7.66 (d, 2H, Ar-H, J=7.8Hz), 7.96 (s, 1H,
pyrimidine-H), 8.25 (s, 1H, CH=N), 8.36 (s, 2H, Ar-H), 11.66 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ(ppm): 166.6, 163.0, 159.9, 157.7, 152.7, 144.3, 129.6, 119.1, 112.8, 107.6, 25.7;
ESI-MS m/z: 271.4 [M+1]+; Elemental Anal. Calcd for C13H14N6O: C, 57.77; H, 5.22; N, 31.09.
Found: C, 57.74; H, 4.91; N, 31.15 .
5.6.11. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-2-chloro-4-amino-benzohydrazide
(A11)
1
White solid; Yield 79%; m.p 234-236oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 3H,
CH3), 5.87 (s, 2H, NH2), 6.54 (d, 1H, Ar-H, J=8.2Hz), 6.65 (s, 1H, Ar-H), 7.27 (d, 1H, Ar-H,
J=8.3Hz), 8.00 (s, 1H, pyrimidine-H), 8.23 (s, 1H, CH=N), 8.23 (s, 2H, NH2), 11.77 (s, 1H, NH);
13C NMR (100 MHz, DMSO-d6) δ(ppm): 166.8, 162.9, 159.9, 158.0, 152.0, 145.3, 132.1, 131.0,
20

120.6, 114.0, 111.8, 107.3, 25.6; ESI-MS m/z: 305.3 [M+1]+; Elemental Anal. Calcd for
C13H13ClN6O: C, 51.24; H, 4.30; N, 27.58. Found: C, 51.36; H, 4.24; N, 27.74 .
5.6.12. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-4-nitrobenzohydrazide (A12)
White solid; Yield 78%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H, CH3),
8.14 (s, 1H, pyrimidine-H), 8.16 (s, 2H, NH2), 8.32 (s, 1H, CH=N), 8.39 (d, 2H, Ar-H, J=8.0Hz),
8.43 (s, 2H, Ar-H), 12.36 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 167.1, 161.4,
159.9, 158.6, 149.4, 147.3, 138.7, 129.2, 123.8, 106.9, 25.7; ESI-MS m/z: 301.2 [M+1]+;
Elemental Anal. Calcd for C13H12N6O3: C, 52.00; H, 4.03; N, 27.90. Found: C, 52.02; H, 3.90; N,
27.89 .
5.6.13. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-3-nitrobenzohydrazide (A13)
1
Red solid; Yield 67%; m.p >260oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H, CH3),
7.85 (t, 1H, Ar-H, J=7.6Hz), 8.10 (s, 1H, pyrimidine-H), 8.32 (s, 2H, NH2), 8.36 (d, 1H, Ar-H,
J=7.3Hz), 8.44 (s, 1H, CH=N), 8.44 (s, 1H, Ar-H), 8.76 (s, 1H, Ar-H), 12.38 (s, 1H, NH); 13C
NMR (100 MHz, DMSO-d6) δ(ppm): 167.2, 160.9, 159.9, 158.63, 147.9, 147.3, 134.4, 134.3,
130.5, 126.6, 122.4, 107.0, 25.8; ESI-MS m/z: 301.1 [M+1]+; Elemental Anal. Calcd for
C13H12N6O3: C, 52.00; H, 4.03; N, 27.99. Found: C, 51.85; H, 4.05; N, 27.69 .
5.6.14. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-3,5-dinitrobenzohydrazide (A14)
1
Red solid; Yield 78%; m.p >260oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.40(s, 3H, CH3),
8.58 (s, 1H, Ar-H), 8.75 (s, 1H, Ar-H), 8.89 (s, 1H, Ar-H), 9.06 (s, 2H, NH2), 9.48 (s, 1H,
pyrimidine-H), 8.80(s, 1H, CH=N), 13.35(s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm):
160.9, 160.4, 159.6, 148.2, 146.6, 144.8, 135.0, 128.3, 121.7, 107.7, 21.3; ESI-MS m/z: 346.4
[M+1]+; Elemental Anal. Calcd for C13H11N7O5: C, 45.22; H, 3.21; N, 28.40. Found: C, 45.15; H,
21

3.18; N, 28.54 .
5.6.15.
(A15)
N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-3-amino-5-nitrobenzohydrazide
1
Red solid; Yield 65%; m.p >260oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H, CH3),
6.16 (s, 2H, NH2), 7.48 (s, 1H, Ar-H), 7.56 (s, 1H, Ar-H), 7.88 (s, 1H, Ar-H), 8.08 (s, 1H,
pyrimidine-H), 8.30 (s, 2H, NH2), 8.44 (s, 1H, CH=N), 12.22 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ(ppm): 167.1, 161.7, 159.9, 158.5, 150.5, 148.9, 146.9, 134.9, 119.0, 110.1, 108.5,
107.1, 25.8; ESI-MS m/z: 316.1 [M+1]+; Elemental Anal. Calcd for C13H13N7O3: C, 49.52; H, 4.16;
N, 31.10. Found: C, 49.72; H, 4.28; N, 31.02 .
5.6.16. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-4-hydroxybenzohydrazide (A16)
White solid; Yield 65%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 3H, CH3),
6.86 (s, 2H, NH2), 7.80 (d, 2H, Ar-H, J=6.7Hz), 8.01 (s, 1H, pyrimidine-H), 8.38 (s, 1H, CH=N),
8.38 (s, 2H, Ar-H), 10.20 (s, 1H, OH), 11.86 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm):
166.7, 162.7, 161.0, 159.9, 157.9, 145.2, 129.8, 123.5, 115.2, 107.4, 25.7; ESI-MS m/z: 272.3
[M+1]+; Elemental Anal. Calcd for C13H13N5O2: C, 57.56; H, 4.83; N, 25.82. Found: C, 57.72; H,
4.84; N, 25.97 .
5.6.17. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-2-hydroxybenzohydrazide (A17)
White solid; Yield 82%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H, CH3),
6.97 (s, 2H, NH2), 7.44 (s, 1H, Ar-H), 7.87 (d, 1H, Ar-H, J=6.7Hz), 8.08 (s, 1H, pyrimidine-H),
8.31 (s, 2H, Ar-H), 8.45 (s, 1H, CH=N), 12.00 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
δ(ppm): 167.1, 164.7, 160.0, 159.2, 158.5, 147.1, 134.1, 128.7, 119.1, 117.5, 115.8, 107.1, 25.8;
ESI-MS m/z: 272.2 [M+1]+; Elemental Anal. Calcd for C13H13N5O2: C, 57.56; H, 4.83; N, 25.82.
22

Found: C, 57.73; H, 4.61; N, 26.04 .
5.6.18. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-2-methylbenzohydrazide (A18)
White solid; Yield 76%; m.p. >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H, CH3),
2.50 (s, 3H, CH3), 7.77 (s, 2H, NH2), 7.86 (s, 2H, Ar-H), 8.16 (s, 1H, pyrimidine-H), 8.25 (s, 1H,
CH=N), 8.42 (s, 2H, Ar-H), 12.16 (s, 1H, NH); 13C NMR(100 MHz, DMSO-d6) δ(ppm): 166.6,
162.1, 159.9, 157.6, 146.3, 136.2, 13.3, 131.7, 129.8, 127.7, 125.9, 107.1, 25.5, 20.9; ESI-MS m/z:
270.2 [M+1]+; Elemental Anal. Calcd for C13H13N5O: C,62.44; H, 5.61; N, 26.01. Found: C, 62.16;
H, 5.47; N, 26.06 .
5.6.19. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-3-methylbenzohydrazide (A19)
White solid; Yield 81%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 6H, 2CH3),
7.41 (s, 2H, NH2), 7.73 (s, 2H, Ar-H), 8.05 (s, 1H, pyrimidine-H), 8.28 (s, 1H, CH=N), 8.41 (s, 2H,
Ar-H), 12.03 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 166.9, 163.2, 159.9, 158.2,
146.1, 138.0, 133.1, 132.6, 128.5, 128.2, 124.9, 107.2, 25.7, 21.1; ESI-MS m/z: 270.2 [M+1]+;
Elemental Anal. Calcd for C14H15N5O: C, 62.44; H, 5.61; N, 26.01. Found: C, 62.05; H, 5.44; N,
25.84 .
5.6.20. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-4-methylbenzohydrazide (A20)
White solid; Yield 78%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 6H, 2CH3),
7.33 (s, 2H, NH2), 7.83 (d, 2H, Ar-H, J=6.5Hz), 8.04 (s, 1H, pyrimidine-H), 8.28 (s, 1H, CH=N),
8.41 (s, 2H, Ar-H), 12.00 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 166.9, 162.9,
159.9, 158.2, 145.9, 142.2, 130.2, 129.2, 127.8, 107.3, 25.8, 21.2; ESI-MS m/z: 270.2 [M+1]+;
Elemental Anal. Calcd for C14H15N5O: C, 62.44; H, 5.61; N, 26.01. Found: C, 62.23; H, 5.94; N,
26.38 .
23

5.6.21. N'-((4-amino-2-methylpyrimidine-5-yl)methylene)-3,5-dimethylbenzohydrazide (A21)
1
White solid; Yield 70%; m.p 246-248oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.34 (s, 6H,
2CH3), 2.38 (s, 3H, CH3), 7.23 (s, 1H, Ar-H), 7.52 (s, 2H, NH2), , 8.14 (s, 1H, pyrimidine-H), 8.23
(s, 1H, CH=N), 8.41 (s, 2H, Ar-H), 12.00 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm):
166.7, 163.2, 159.9, 157.8, 145.8, 137.8, 133.3, 133.0, 125.4, 107.2, 25.6, 20.9; ESI-MS m/z:
284.1 [M+1]+; Elemental Anal. Calcd for C15H17N5O: C, 63.59; H, 6.05; N, 24.72. Found: C,
63.64; H, 5.73; N, 24.62 .
5.6.22. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-4-methoxybenzohydrazide (A22)
White solid; Yield 79%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.38 (s, 3H, CH3),
3.83 (s, 3H, OCH3), 7.06 (s, 2H, NH2), 7.91 (d, 2H, Ar-H, J=6.3Hz), 8.00 (s, 1H, pyrimidine-H),
8.27 (s, 1H, CH=N), 8.40 (s, 2H, Ar-H), 11.92 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
δ(ppm): 166.8, 162.6, 162.3, 159.9, 158.1, 145.6, 129.7, 125.1, 114.0, 107.4, 55.6, 25.8; ESI-MS
m/z: 286.1 [M+1]+; Elemental Anal. Calcd for C14H15N5O2: C, 58.94; H, 5.30; N, 24.55. Found: C,
59.02; H, 5.10; N, 24.68 .
5.6.23. N'-((4-amino-2-methylpyrimidin-5-yl)methylene)-3-methoxybenzohydrazide (A23)
1
White solid; Yield 79%; m.p 235-237oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H,
CH3), 3.83 (s, 3H, OCH3), 7.17 (d, 1H, Ar-H, J=5.6Hz), 7.45 (s, 2H, NH2), 7.48 (d, 1H, Ar-H,
J=6.6Hz), 8.06 (s, 1H, pyrimidine-H), 8.29 (s, 1H, CH=N), 8.42 (s, 2H, Ar-H), 12.04 (s, 1H, NH);
13C NMR (100 MHz, DMSO-d6) δ(ppm): 167.0, 162.9, 160.0, 159.4, 158.3, 146.3, 134.5, 129.9,
120.0, 117.7, 113.1, 107.2, 55.5, 25.8; ESI-MS m/z: 286.1 [M+1]+; Elemental Anal. Calcd for
C14H15N5O2: C, 58.94; H, 5.30; N, 24.55. Found: C, 58.74; H, 5.09; N, 24.40 .
5.6.24. N'-((4-aminopyrimidine-5-yl)methylene)benzohydrazide (B1)
24

White solid; Yield 64%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 7.55 (s, 2H, NH2),
7.60 (s, 1H, Ar-H), 7.92 (s, 2H, Ar-H), 8.15 (s, 1H, Ar-H), 8.42 (s, 1H, Ar-H), 8.42 (s, 1H,
pyrimidine-6-H), 8.45 (s, 1H, CH=N), 8.45 (s, 1H, pyrimidine-2-H), 8.87 (s, 1H, Ar-H), 12.15 (s,
1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 163.1, 159.7, 157.9, 157.8, 145.9, 132.9,
132.0, 128.6, 127.7, 109.8; ESI-MS m/z: 242.4 [M+1]+; Elemental Anal. Calcd for C12H11N5O: C,
59.74; H, 4.60; N, 29.03. Found: C, 59.63; H, 4.68; N, 29.40.
5.6.25. N'-((4-aminopyrimidine-5-yl)methylene)-4-fluorobenzohydrazide (B2)
White solid; Yield 74%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 7.40 (s, 2H, NH2),
8.01 (s, 2H, Ar-H), 8.17 (s, 1H, pyrimidine-6-H), 8.44 (s, 1H, CH=N), 8.44 (s, 1H,
pyrimidine-2-H), 8.44 (s, 2H, Ar-H), 12.18 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm):
165.6, 163.1, 162.0, 159.7, 157.8, 146.0, 130.5, 129.3, 115.7, 115.5, 109.7; ESI-MS m/z: 260.4
[M+1]+; Elemental Anal. Calcd for C12H10FN5O: C, 55.60; H, 3.89; N, 27.02. Found: C, 55.83; H,
3.93; N, 27.07 .
5.6.26. N'-((4-aminopyrimidine-5-yl)methylene)-4-bromobenzohydrazide (B3)
White solid; Yield 64%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 7.78 (s, 2H, NH2),
7.81 (s, 2H, Ar-H), 8.17 (s, 1H, pyrimidine-6-H), 8.42 (s, 1H, CH=N), 8.42 (s, 1H,
pyrimidine-2-H), 8.44 (s, 2H, Ar-H), 12.22 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm):
161.9, 159.6, 157.8, 146.1, 131.8, 131.5, 129.6, 125.7, 109.5.; ESI-MS m/z: 320.4 [M+1]+;
Elemental Anal. Calcd for C12H10BrN5O: C, 45.02; H, 3.15; N, 21.88. Found: C, 45.30; H, 3.29; N,
21.87 .
5.6.27. N'-((4-aminopyrimidine-5-yl)methylene)-2-chlorobenzohydrazide (B4)
1
White solid; Yield 56%; m.p 239-240oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 7.47 (s, 2H,
25

NH2), 7.53 (s, 1H, Ar-H), 7.59 (d, 2H, Ar-H, J=7.4Hz), 8.28 (s, 1H, pyrimidine-6-H), 8.31 (s, 1H,
CH=N), 8.39 (s, 1H, Ar-H), 8.43 (s, 1H, pyrimidine-2-H), 12.21 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ(ppm): 168.4, 162.5, 159.8, 158.1, 146.4, 143.2, 134.8, 131.7, 130.5, 129.9, 129.5,
127.4, 109.5; ESI-MS m/z: 276.2 [M+1]+; Elemental Anal. Calcd for C12H10ClN5O: C, 52.28; H,
3.66; N, 25.40. Found: C, 52.31; H, 3.91; N, 25.75 .
5.6.28. N'-((4-aminopyrimidine-5-yl)methylene)-2-methylbenzohydrazide (B5)
1
White solid; Yield 51%; m.p 236-238oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.39 (s, 3H,
CH3), 7.29 (s, 1H, Ar-H), 7.32 (s, 2H, NH2), 7.41 (d, 1H, Ar-H, J= 6.9Hz), 7.47 (d, 1H, Ar-H, J=
7.0Hz), 8.30 (s, 1H, Ar-H), 8.30 (s, 1H, pyrimidine-6-H), 8.36 (s, 1H, CH=N), 8.41 (s, 1H,
pyrimidine-2-H), 12.05 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 165.2, 159.7, 157.9,
145.6, 136.2, 134.7, 130.8, 130.2, 127.6, 126.4, 125.8, 109.7, 19.5; ESI-MS m/z: 256.3 [M+1]+;
Elemental Anal. Calcd for C13H13N5O: C, 61.17; H, 5.13; N, 27.43. Found: C, 61.24; H, 4.95; N,
27.77 .
5.6.29. N'-((4-aminopyrimidine-5-yl)methylene)-3,5-dimethylbenzohydrazide (B6)
White solid; Yield 76%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.35 (s, 6H, 2CH3),
7.24 (s, 1H, Ar-H), 7.53 (s, 2H, NH2), 8.15 (s, 1H, pyrimidine-6-H), 8.44 (s, 1H, CH=N), 8.44 (s,
1H, pyrimidine-2-H), 8.44 (s, 2H, Ar-H), 12.06 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
δ(ppm): 163.2, 159.7, 157.8, 157.7, 145.7, 137.8, 133.3, 132.9, 125.4, 109.8, 20.9; ESI-MS m/z:
270.4 [M+1]+; Elemental Anal. Calcd for C14H15N5O: C, 62.44; H, 5.61; N, 26.01. Found: C,
62.35; H, 5.56; N, 26.15 .
5.6.30. N'-((4-aminopyrimidine-5-yl)methylene)-2,4-dichlorobenzohydrazide (B7)
1
White solid; Yield 77%; m.p 235-236oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 7.57 (s, 2H,
26

NH2), 7.65 (s, 1H, Ar-H), 7.77 (d, 1H, Ar-H, J=26.5Hz), 8.27 (s, 1H, pyrimidine-6-H), 8.27 (s, 1H,
CH=N), 8.27 (s, 1H, Ar-H), 8.44 (s, 1H, pyrimidine-2-H), 8.44 (s, 2H, Ar-H), 12.06 (s, 1H, NH);
13C NMR (100 MHz, DMSO-d6) δ(ppm): 161.6, 159.7, 158.0, 146.5, 134.8, 133.4, 131.9, 130.6,
127.4, 126.5, 109.6; ESI-MS m/z: 310.3 [M+1]+; Elemental Anal. Calcd for C12H9Cl2N5O: C,
46.47; H, 2.92; N, 22.58. Found: C, 46.43; H, 2.82; N, 22.67 .
5.6.31. N'-((2,4-diaminopyrimidine-5-yl)methylene)benzohydrazide (C1)
1
Light yellow solid; Yield 79%; m.p 250-252oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 6.55 (s,
2H, NH2), 7.51 (s, 2H, NH2), 7.53 (s, 1H, pyrimidine-H), 7.57 (d, 1H, Ar-H, J=6.5Hz), 7.89 (d, 2H,
Ar-H, J=6.7Hz), 7.95 (s, 1H, CH=N), 8.26 (s, 2H, Ar-H), 11.73 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ(ppm): 162.6, 161.0, 160.4, 147.5, 133.4, 131.7, 128.5, 127.5, 101.1; ESI-MS m/z:
257.3 [M+1]+; Elemental Anal. Calcd for C12H12N6O: C, 56.24; H, 4.72; N, 32.79. Found: C,
56.23; H, 4.56; N, 32.69 .
5.6.32. N'-((2,4-diaminopyrimidine-5-yl)methylene)-3-nitrobenzohydrazide (C2)
Light yellow solid; Yield 79%; m.p>260 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 6.60 (s, 2H,
NH2), 7.50 (s, 1H, pyrimidine-H), 7.83 (s, H, NH2), 7.99 (s, H, NH2), 8.17 (s, 1H, CH=N), 8.29 (s,
1H, Ar-H), 8.34 (s, 1H, Ar-H), 8.43 (s, 2H, Ar-H), 8.74 (s, 1H, Ar-H), 12.05 (s, 1H, NH); 13C
NMR (100 MHz, DMSO-d6) δ(ppm): 162.9, 161.1, 160.9, 160.4, 148.6, 147.9, 134.9, 134.1, 130.4,
126.3, 122.3, 101.0; ESI-MS m/z: 302.2 [M+1]+; Elemental Anal. Calcd for C12H11N7O3: C,
47.84; H, 3.68; N, 32.55. Found: C, 47.60; H, 3.76; N, 32.56 .
5.6.33. N'-((2,4-diaminopyrimidine-5-yl)methylene)-4-amino-benzohydrazide (C3)
1
Yellow solid; Yield 87%; m.p 238-240oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 5.76 (s, 2H,
NH2), 6.48 (s, 2H, NH2), 6.58 (s, 2H, NH2), 7.39 (s, 1H, pyrimidine-H), 7.63 (d, 2H, Ar-H,
27

J=5.4Hz), 7.91 (s, 1H, CH=N), 8.20 (s, 2H, Ar-H), 11.32 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ(ppm): 162.6, 162.3, 160.9, 159.6, 152.2, 145.7, 129.2, 119.5, 112.7, 101.4; ESI-MS
m/z: 272.2 [M+1]+; Elemental Anal. Calcd for C12H13N7O: C, 53.13; H, 4.83; N, 36.14. Found: C,
53.43; H, 4.55; N, 36.46 .
5.6.34. N'-((2,4-diaminopyrimidine-5-yl)methylene)-4-amino-2-chloro-benzohydrazide (C4)
Yellow solid; Yield 87%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 5.82 (s, 2H, NH2),
6.52 (s, 2H, NH2), 6.52 (s, 1H, NH2), 6.63 (s, 1H, NH2), 7.22 (d, 1H, Ar-H, J=7.8Hz), 7.42 (s, 1H,
pyrimidine-H), 7.89 (s, 1H, CH=N), 8.07 (s, 2H, Ar-H), 11.42 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ(ppm): 162.6, 162.3, 161.0, 160.3, 151.6, 146.4, 131.8, 130.8, 121.0, 113.7, 111.7,
101.0; ESI-MS m/z: 306.2 [M+1]+; Elemental Anal. Calcd for C12H12ClN7O: C, 47.14; H, 3.96;
N, 32.07. Found: C, 47.28; H, 3.75; N, 32.14.
5.6.35. N'-((2,4-diaminopyrimidine-5-yl)methylene)-2,4-dichloro-benzohydrazide (C5)
1
Light yellow solid; Yield 77%; m.p 149-151oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 6.60 (s,
2H, NH2), 7.50-7.57 (m, 2H, NH2), 7.61 (d, 1H, Ar-H, J=8.1Hz), 7.77 (s, 1H, pyrimidine-H), 7.88
(d, 2H, Ar-H, J=8.2Hz), 7.96 (s, 1H, CH=N), 8.07 (s, 2H, Ar-H), 11.81 (s, 1H, NH); ESI-MS m/z:
325.1 [M+1]+; Elemental Anal. Calcd for C12H10Cl2N6O: C, 44.33; H, 3.10; N, 25.85. Found: C,
44.39; H, 3.27; N, 25.82.
5.6.36. N'-((2,4-diaminopyrimidine-5-yl)methylene)-3-chloro-benzohydrazide (C6)
Light yellow solid; Yield 79%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 6.58 (s, 2H,
NH2), 7.49-7.58 (m, 2H, NH2), 7.65 (d, 1H, Ar-H, J=7.1Hz), 7.86 (d, 1H, Ar-H, J=6.8Hz), 7.94 (s,
1H, pyrimidine-H), 7.97 (s, 1H, Ar-H), 8.16 (s, 1H, CH=N), 8.25 (s, 1H, Ar-H), 11.81 (s, 1H, NH);
13C NMR (100 MHz, DMSO-d6) δ(ppm): 162.8, 161.1, 160.7, 158.9, 148.0, 135.4, 133.3, 131.5,
28

130.6, 127.3, 126.4, 100.9; ESI-MS m/z: 291.2 [M+1]+; Elemental Anal. Calcd for C12H11ClN6O:
C, 49.58; H, 3.81; N, 28.91. Found: C, 49.74; H, 3.61; N, 28.97.
5.6.37. N'-((2,4-diaminopyrimidine-5-yl)methylene)-4-chloro-benzohydrazide (C7)
Light yellow solid; Yield 87%; m.p >260oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 6.57 (s, 2H,
NH2), 6.65 (s, 1H, Ar-H), 7.50 (s, 1H, pyrimidine-H), 7.60 (d, 2H, NH2, J=8.0Hz), 7.92 (d, 2H,
Ar-H, J=7.9Hz), 8.13 (s, 1H, CH=N), 8.25 (s, 1H, Ar-H), 11.79 (s, 1H, NH); ESI-MS m/z: 291.3
[M+1]+; Elemental Anal. Calcd for C12H11ClN6O: C, 49.58; H, 3.81; N, 28.91. Found: C, 49.57; H,
3.98; N, 28.53.
5.6.38. N'-((2,4-diaminopyrimidine-5-yl)methylene)-4-bromo-benzohydrazide (C8)
1
Light yellow solid; Yield 80%; m.p 249-251oC; H NMR (400 MHz, DMSO-d6) δ(ppm): 6.56 (s,
2H, NH2), 7.75 (s, 2H, NH2), 7.47 (s, 1H, pyrimidine-H), 7.84 (d, 2H, Ar-H, J=6.9Hz), 7.95 (s, 1H,
CH=N), 8.25 (s, 2H, Ar-H), 11.80 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 163.2,
159.7, 157.8, 157.7, 145.7, 137.8, 133.3, 132.9, 125.4, 109.8; ESI-MS m/z: 335.3 [M+1]+;
Elemental Anal. Calcd for C12H11BrN6O: C, 43.00; H, 3.31; N, 25.07. Found: C, 42.85; H, 3.56; N,
24.83 .
5.7. Assay of E.coli PDHc E1 (in vitro) and site-directed mutagenesis of PDHc E1
The half maximal inhibitory concentration (IC50) values of the title compounds were determined at
the PDHc E1enzyme level in vitro. The cloning, expression, purification and activity of PDHc E1
were carried out according the literature by the method of Bradford or Ren.17, 29 The enzymatic
assay was used to measure the inhibitory activities of synthesized compounds. PDHc E1 activity
was measured by monitoring the reduction of 2,6-DichloroPhenolindophenol (2,6-DCPIP) at 600
nm using a microplate reader (BioTek Synergy 2, USA).30 Each experiment was performed at least
29